Citrus Biotic and Abiotic Stress Management

#### **Chapter 4**

## Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based Agroforests Structural Characteristics

*Ndo Eunice Golda Danièle and Akoutou Mvondo Etienne*

### **Abstract**

The health and productivity of citrus are generally jeopardized by a host of diseases, for which the environmental conditions of the cropping system are critical drivers. Several studies conducted on various diseases of perennial crops have shown the involvement of the structural futures of the cocoa-based agroforestry system (CBAFS) in the spread of pathogens and the epidemics development. This chapter highlights the effect of the CBAFS's structural characteristics on the intensity of three citrus diseases in the humid forest zones of Cameroon. The involvement of CBAFS structural characteristics in diseases regulation is demonstrated. In particular, the spatial structure of citrus in agroforests shows an effect on the spread of diseases. Moreover, distribution of citrus in the CBAFS, with minimum spacing of 12 m between citrus trees, limits the damage caused by Pseudocercospora leaf and fruit spot disease (PLFSD) and citrus diseases caused by Phytophthora (CDP). Dense shading helps to minimize the intensity of diseases such as CDP and PLFSD and Citrus scab disease. This work may make it possible to contribute to the development of an integrated management tool for citrus diseases in an associated crop context.

**Keywords:** Integrated disease management, cultural practices, citrus, fungal diseases, shade trees, spatial structure

#### **1. Introduction**

Conventional and intensive agriculture has enabled a considerable increase in agricultural production since the 1950s. However, the resulting heavy ecological balance sheet discredits this unsustainable model of agriculture [1]. Thus, although the preferred strategies of intensive agriculture have shown undeniable benefits, their use is becoming increasingly worrying both for agriculture itself and for the environment and human health. This is partly due to the excessive use of pesticides and other chemicals [2–6]. The improvement of intensive production systems towards new models of sustainable agriculture, favoring the development of effective means of combating diseases that are sustainable and environmentally friendly, is becoming a matter of urgency. Tropical agroforestry systems, thanks to their high biodiversity and structural diversity, represent a privileged way out of the agroecological transition. Several studies have demonstrated the contribution of the structural characteristics of these systems in the integrated management of pests and diseases of perennial crops, particularly citrus [7–9].

Citrus represent a fruit crop of prime importance in socio-ecological terms in Cameroon [10–12]. Their significance lies in the fact of their high-quality nutritional value and their contribution to the diversification of producers' incomes in rural areas. Citrus are also important in the local pharmacopeia [13–15]. They are also known for their role in restoring ecological balances after deforestation [16]. However, despite the favorable agroecological conditions throughout the country, the number of production basins identified and even the density of trees in farms, production remains poor [12, 17, 18]. A diversity of diseases affecting citrus in the country humid zones is the main constraint to their production [8, 11, 19, 20].

Pseudocercospora leaf and fruit spot disease (PLFSD) caused by *Pseudocercospora angolensis*, citrus scab disease caused by *Elsinoe* spp.; and citrus diseases caused by Phytophthora (CDP) caused by *Phytophthora* spp., are the main soil diseases on citrus in Cameroon (**Figure 1**) [7, 21–25]. Damages caused by these diseases result in heavy crop losses [26]. Sorting deviations of up to 100% of the production can be recorded in the case of PLFSD, if no treatment measures are taken [27]. Concerning citrus scab, severe attacks on young *C. volcameriana* plants, for example in the nursery, result in their death [20, 28]. CDP significantly limits citrus production in the plots where it is present [29–34]. In addition to reduced yields from the beginning of infection, the economic viability of orchards is reduced following tree death [7, 32, 35]. This strong and constantly changing diseases pressure, which not only causes enormous economic losses, but also leads (in the case of PLFSD) to quarantine and a ban on the export of citrus products to other production areas [25].

A variety of strategies are used to control citrus diseases. These include the practice of sanitation measures, the use of resistant cultivars and varieties, grafting, organic or mineral soil amendments, the use of plant extracts, biological control in a systemic approach and, above all, chemical control through fungicides [32, 36–40]. However, the high costs of these methods, development of resistance to chemical inputs, emergence of new diseases and growing concerns about environmental and soil health make these methods inadequate [41–45]. In addition, most of these techniques are unsuitable for the socio-cultural and even technological context of small-scale producers in inter-tropical regions. The development of targeted control protocols, taking into account the local socio-ecological context and existing production systems is therefore imperative. This chapter highlights the effect of the

#### **Figure 1.**

*Symptoms of citrus diseases in Cameroon. C. paradisi fruit torn following a severe attack by Pseudocercospora angolensis (a), C. volcameriana fruit covered with scab spots due to Elsinoe spp. (b), lesions resulting from crown attacks due to Phytophthora spp. on* C. sinensis *tree(c) and dieback due to various diseases of an*  C. sinensis *(d).*

*Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*

structural characteristics of the cocoa-based agroforestry system on the intensity of the three main citrus diseases in the humid forest zones of Cameroon.

#### **2. Complex cocoa-based agroforest systems and structural characteristics**

In Cameroon, citrus are mainly grown in cocoa-based agroforestry systems (CBAFS) [7, 46]. These are complex, highly biodiverse, natural forest-like cropping systems (**Figure 2**) [30, 47, 48]. In this system, several interactions of different nature and intensity can take place depending on the species present, their sizes and their positions [9, 49, 50]. One of these interactions is the action of diseases. The structural characteristics of CBAFS can contribute to control of these [20, 30, 51, 52]. Studies in these cropping systems and on various pathosystems have shown that spatial structure of species is important in reducing diseases development [49, 52]. Indeed, spatial structure has a twofold effect on the pathogen: firstly, the high plant biodiversity into CBAFS makes it possible to dilute the pathogens resource and thus reduce their presence and damage [53–55]. Secondly, multi-species agroecosystems are recognized for the high diversity of vertical and horizontal structures that can be adopted by the plant population [56]. This diversity of plant spatial structure affects diseases mainly through the microclimatic weathering mechanism [51, 52].

Previous works have supported interactions between individuals of a host population of pathogen and associated plants within intercropping systems [7, 8]. This type of interaction is likely to influence the presence of diseases. The action of shade trees on the understory microclimate decreases with decreasing distance between trees and pathogen transmission decreases as the distance between host individuals increases [51, 52, 57–59].

**Figure 2.**

*Illustration of a cocoa-based agroforestry system planted with citrus trees (a) and the horizontal structure of its plant population (b). 1 = cocoa trees, 2 = various forest tree species, 3 = various other fruit tree species, 4 = citrus and 5 = palm trees.*

#### **3. Effect of spatial structure of citrus into cocoa based agroforest systems on citrus diseases**

The spatial structure of a plant community is the vertical and horizontal arrangement of constituent elements [51, 60, 61]. It reflects therefore the local environment around each individual [55, 62]. Within agroforest, non-host plants mainly perform a physical barrier effect on diseases [27, 51, 54]. The effect of the citrus spatial structure within CBAFS on the three main diseases affecting them in Cameroon has been assessed through various studies. A network of 27 plots in the three study sites was set up in Obala, Muyuka and Bokito sites. These are located in three ecological conditions into humid forest zone of Cameroon. CBAFS with at least 12 citrus trees in the plot area were selected. Each plot area was a square of at least 2500 m2 (50 X 50 m). Plots were chosen in villages among the most productive areas and also representative of the study zone in terms of system diversity and variability of citrus species produced.

The analysis of the spatial structure of the citrus sub-population was done by the Ripley method [62]. Following the method illustrated in Ngo Bieng [55], a typology of spatial structure was build based on the spatial structure of the citrus trees in the study plots. In a first step, the horizontal spatial structure was characterized on the citrus trees in each plot, using the L(r) modified Ripley function [20, 61, 63] . The L (r) function is based on the calculation of the expected number of neighbor trees (**Figure 2**), within a distance ≤ to r of any point of the study pattern. This method enables to distinguish three types of tree spatial patterns: regular when L (r) is <0, aggregated when L (r) is >0, and random when L (r) =0. This function characterizes the neighborhood structure around a point. It is used for a simple, homogeneous and isotropic point process of density λ [64].

$$\mathbf{K}(\mathbf{r}) = \boldsymbol{\lambda}^{-1} \mathbf{E}(\mathbf{r}) \quad L\_{(r)} = \sqrt{\frac{K(r)}{\pi}} - r \tag{1}$$

Subsequently, a hierarchical cluster analysis based on the Euclidean distance between the values of the L(r) function of the citrus trees in the different plots was made. It resulted in clusters of plots with a similar spatial structure, based on their trend to regular, random or aggregated spatial structure. This analysis was done with *ads* and *ade4* package R 3.2.2 software (**Figure 3**). Symptoms of CDP, PLFSD, and citrus scab were assessed by the visual recognition method. The intensity was assessed using a scale from 1 to 4.

From this study it emerges that, the spatial structure has a significant influence on the intensity of the diseases observed. The analysis of variance and the mean comparison test reveal that plots in which citrus have an aggregated spatial structure, have a high intensity of citrus scab disease and PLFSD. On the other hand, plots in which citrus fruits have a regular spatial structure show a significantly low intensity of these same diseases (**Table 1**).

**Figure 3.**

*Hierarchical classification of plots according to the spatial structure of citrus trees in the experimental plots.*

*Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*


### **Table 1.**

*Effect of the spatial structure of citrus trees in cocoa based agroforests systems on diseases.*

It is thus demonstrated that the aggregate spatial structure of citrus in CBAFS has a negative effect on diseases observed. These results are similar to those obtained by Ndo *et al*. [7, 8, 30] in the particular case of PLFSD. In addition to that, the involvement of spatial structure in the spread of various diseases has been shown [8, 53, 54]. Indeed, the aggregation of host populations favors the dispersion of diseases, while regularity would reduce it [65]. In addition, it is recognized that transmission of the pathogen decreases as the distance between host individuals increases [52, 58]. On the other hand, it has been shown that aggregation of host populations can reduce the incidence of pathogens [66]. Because the transmission of the pathogen between aggregates decreases with increasing distance between aggregates. These aspects would therefore explain the low intensity of CDP observed in plots where citrus fruits have an aggregated spatial structure.

#### **4. Effect of shade intensity management on CDP and PLFSD**

Depending on the situation of citrus in the CBAFS and in relation to the upper stratum, three levels of shading (dense, moderate and no shading) were defined. Shade trees play various roles in tropical agroforests. They can improve adverse weather conditions by modulating temperature variations [51, 52, 57, 59]. Shading has been recognized as one of the factors that can influence PLFSD dissemination [54, 67, 68]. Given that shading favors climatic conditions for the development of certain citrus pathogenic fungi such as *P. angolensis* or many *Phytophthora* spp. (high relative humidity (>60%) and cool temperature conditions (<25°C)) [18, 28, 69]. It is assumed that within a plot, trees under shade would have a higher incidence of the disease than those in full sunlight. On the other hand, given the role of shade trees in improving climatic and nutritional conditions, the growth of trees under these conditions can be improved, as well as their vigor and response to disease. In addition, shade trees can act as a barrier against wind and rain (the main factors in the spread of conidia) and slow the progress of the epidemic.

An experiment carried out in a fruit trees orchard in Foumbot in the Western region of the country demonstrated the effect of shade trees on the PLFSD epidemic. The trial was set up in the Institute of Agricultural Research for Development (IRAD) experimental orchard. This orchard comprises collection plots of mango (*Manguifera indica*), avocado (*Persea americana*) and various citrus trees separated from each other by fallow plots often reserved for annual crops. This experimental plot enabled to compare tree shading situations ie dense shade (under mango trees), light shade (under avocado trees) and full sun light (fallow plot). One-year old pomelo seedlings have been placed in three levels of shade i.e.: under mango trees (dense shade), under avocado trees (light shade) and on fallow land (no shade).

The results of this experiment showed that the higher the shade index (under mango trees), the lower the PLFSD severity. When the shade intensity is lower (under avocado trees), disease severity is also lower, however the differences are not significant (**Figure 4**). These results suggest that shading should be sufficient to significantly reduce PLFSD incidence. Otherwise too little shading will not have significant effect on disease severity. But in the meantime*,* the plant must receive sufficient sun radiation for good growth. So, it is necessary to determine an optimum shading that allows a good compromise between plant growth and reduces PLFSD incidence. This optimum can vary according to the climatic and sanitary conditions of the plantations under consideration [18, 20, 52].

*Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*

#### **Figure 4.**

*Graphical representation of the percentage of diseased leaves for each pomelo plant and the shade indices of the different shade trees during the first observation date (A) and the second date (B) in the Foumbot plot.*


*In the same column, values with same letter are not significantly different (Tukey HSD test P < 0.05). \*\*\* indicates highly significant.*

#### **Table 2.**

*Effect of shade on citrus diseases.*

Another experiment conducted in 26 cocoa-based agroforestry systems showed the effect of shading on the spread of CDP and citrus scab disease. In this study, a total set of 476 citrus were observed under three shading conditions. Depending on the tree diversity and population, the various scenarios of shade density have been coded as follows: (1) "*dense shade*" when the citrus tree were placed under a direct and thick shading of the upper stratum; (2) "*light shade*" when the citrus tree received a mean shading of a higher stratum and finally (3) "*full sun*" when the citrus fruit did not receive a shade of a top stratum.

Results showed that, there is a variation in the citrus diseases intensity depending on whether they are located under dense shade, light shade or in full sunlight. The mean comparison test thus revealed significant differences in the intensities of CDP, according to the three citrus trees situations depending on shade. Citrus trees under dense shade are significantly less affected by CDP compared to those under light shade and those in full sunlight (**Table 2**).

Results therefore showed that, the shade had a significant effect on diseases. This shading effect was positive on the intensity of CDP. In general, in CBAFS, shade reduces diseases intensity [52, 70]. Indeed, for pathogens, spore dispersal and germination are the two main phases of their life cycle. Shading promotes spore germination while the sensitivity of the dispersion of pathogen spores to microclimate depends on how it is dispersed [7, 59].

#### **5. Mode of dispersal of citrus infectious pathogens and CBAFS structural characteristics**

The majority of fungi require moisture for infection and production of conidia [9, 71, 72]. These conidia may be disseminated by wind or soil water runoff for telluric fungi like *Phytophthora*. Local dispersal is primarily favored by rain-splash as well as some insects moving on trees [24]. This mode of dissemination determines the spatial distribution of each disease.

In the case of PLFSD, the analysis of its spatial distribution indicates that the disease is distributed in clusters, and that above 12 m there is no spatial dependency. In fact, the disease spreads from one tree to its closest neighbors depending on wind speed and/or rainfall intensity. Infection will depend on the presence and quality of the host. If neighbors are susceptible hosts, infection continues and the epidemiological cycle continues. Otherwise, the course of the disease can be circumscribed. This may explain the aggregated spatial structure of diseases that usually have this mode of spread. However, Brown and Bolker [65] pointed out that the aggregation of host populations favors the dispersal of the diseases while its regulation reduces it. That is, the further away the trees are from each other, the slower the transmission of the disease [22].

With regard to CDP, the spread of *Phytophthora* in the field is primarily ensured by the use of infected plant material [32]. However, mechanical means of dispersal of *Phytophthora* have been illustrated. Cases of transmission from an infected root to a healthy root following their respective growing zones have been reported. The inoculum can also be spread by run-off water. Splashes can promote the spread of the inoculum from the soil to the aerial parts of the plant. This mode of disease spread may be promoted by aggregation of the host species of this pathogen. This hypothesis was confirmed by Akoutou *et al*. [7, 30]. These studies showed that citrus with an aggregated spatial structure were more attacked by CDP in contrast to those with a regular spatial structure. In fact, root diversity in the rhizosphere could limit contact between roots of the same species. Host trees planted at wide spacings and having non-host trees between them are less likely to come into contact and this would help to limit the spread of the inoculum. This effect of dilution of the pathogen's resource can also be applied to the mode of transmission through diseased fruits and contact with parts of the plant close to the soil.

In addition, it was shown that environmental factors play a critical role in the development, severity, dispersal and conservation of inoculum in the epidemiology of *Phytophthora* disease. The increase in temperature favors population growth of species such as *P. parasitica* and *P. palmivora*. Hot, dry climates are favorable to *P. parasitica*. These observations corroborate the conclusions drawn on the effect of shading on CDP development. Indeed, the cooler environmental conditions in the understory created by shade trees would make the habitat unfavorable for the pathogen. Citrus planted under dense shade would therefore be less exposed to the inoculum, which is therefore more intense in plot areas of the plot where there is no shade.

#### **6. Conclusion**

This study highlighted the effect of shading trees on citrus in agroforestry plots. In such plots citrus trees are mixed with plants belonging to different tree. Spatial structure has a significant influence on the observed diseases intensity. Plots in which citrus have an aggregated spatial structure have a high intensity of studied diseases, while plots in which citrus have a regular spatial structure are significantly *Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*

less attacked by these diseases. Optimizing the structural characteristics of CBAFS could lead to the development of integrated control strategies against fungal diseases. These management strategies will be adapted to local agroecological contexts, respectful of the environment, and applicable by smallholders.

#### **Author details**

Ndo Eunice Golda Danièle1 and Akoutou Mvondo Etienne1,2\*

1 Institute of Agricultural Research for Development (IRAD), Yaounde, Cameroun

2 University of Yaounde I, Yaounde, Cameroon

\*Address all correspondence to: etienneakoutou@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Tilman, D., Cassman, K. G., Matson, P. A., Naylor, R. and Polasky, S. (2002), 'Agricultural sustainability and intensive production practices', Nature 418(6898), 671-677.

[2] Dornbush, M.E., von Haden, A.C., 2017. Intensified agroecosystems and their effects on soil biodiversity and soil functions. In: Al-Kaisi, Mahdi M. (Ed.), Soil Health and Intensification of Agroecosytems. Iowa State University, Ames, IA, United States, pp. 173-193. https://doi.org/10.1016/ B978-0-12-805317-1.00008-7.

[3] Eddleston, M., Karalliedde, L., Buckley, N., Fernando, R., Hutchinson, G., Isbister, G., Konradsen, F., Murray, D., Piola, J. C., Senanyake, N., Sheriff, R., Singh, S., Siwach, S. B. and Smit, L., 2002. Pesticide poisoning in the developing world - a minimum pesticides list', The Lancet 360 (9340), 1163-1167.

[4] Leibee, G. L. and Capinera, J. L., 1995. Pesticide Resistance in Florida Insects Limits Management Options', Florida Entomologist 78(3), 386-399.

[5] Martin, E.A., Feit, B., Requier, F., Friberg, H., Jonsson, M., 2019. Assessing the resilience of biodiversity-driven functions in agroecosystems under environmental change. In: Bohan, D.A., Dumbrell, A.J. (Eds.), In Resilience in Complex Socio-Ecological Systems, vol. 60, pp. 59-123. https://doi.org/10.1016/ bs.aecr.2019.02.003.

[6] Schreinemachers, P. and Tipraqsa, P. (2012), 'Agricultural pesticides and land use intensification in high, middle- and low-income countries', Food policy 37 (6), 616-626.

[7] Akoutou Mvondo, E., Ndo, E.G.D, Tsouga Manga, M.L., Abaane, C.L., Abondo Bitoumou, J., Bella Manga,

Bidzanga Nomo, L., Ambang, Z., Cilas, C., 2019. Effects of complex cocoa-based agroforests on citrus tree decline. *Crop Protection* Crop Protection 130 (2020) 105051. DOI: https://doi.org/10.1016/j. cropro.2019.105051

[8] Ndo E.G.D., Akoutou M.E., Ambang Z., Manga B., Cilas C., Nomo L.B., Gidoin C., Ngo Bieng M.A., 2019a. Spatial organization influences citrus Pseudocercospora leaf and fruit spot disease severity in cocoa-based agroforestry systems. Am. J. Plant Sci. 10, 221-235. https://doi.org/10.4236/ ajps.2019.101017

[9] Winans, K.S., Tardif, A.S., Lteif, A.E., Whalen, J.K., 2015. Carbon sequestration potential and costbenefit analysis of hybrid poplar, grain corn and hay cultivation in southern Quebec. Canada. Agroforest. Syst. 89, 421-433. https://doi.org/10.1007/ s10457-014-9776-4.

[10] Economos, C., and Clay, W.D., 1998. *Nutritional and health benefits of citrus fruits*. Paper presented at the Twelth Session of the IntergovernmentalGroup on Citrus Fruit.

[11] Kuate, J., Bella-Manga, Damesse, F., Kouodiekong, L., Ndindeng, S.A., David, O. and Parrot, L. (2006) Fruit Trees Cultivated in Family Farms in the Humid Zone of Cameroon: A Survey. Fruits, 61, 373-387. https://doi. org/10.1051/fruits:2006037

[12] Temple L., 2001. Quantification des productions et des échanges de fruits et légumes au Cameroun. Ca. Agri*, 10*: 87-94.

[13] Bayaga, H.N., Guedje, N.M., Biye, E.H., 2017. Ethnopharmacological and ethnobotanical approach of medicinal plants used in the traditional treatment of Buruli ulcer in Akonolinga *Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*

(Cameroon). IJBCS 11 (4), 1523-1541. https://www.ajol.info/index.php/ijbcs/ article/download/164026/153514

[14] Boeing, H.; Bechthold, A.; Bub, A.; Ellinger, S.; Haller, D.; Kroke, A.; Leschik-Bonnet, E.; Mueller, M.J.; Oberritter, H.; Schulze, M.; Stehle, P.; Watzl, B. Critical review: Vegetables and fruit in the prevention of chronic diseases. Eur. J. Nutr. 2012, *51*, 637-663.

[15] Radhika, G.; Sudha, V.; Sathya, R.M.; Ganesan, A.; Mohan, V., 2008, Association of fruit and vegetable intake with cardiovascular risk factors in urban South Indians. Brit. J. Nutr. *99*, 398-405.

[16] Westphal, E., Embrechts, J., Ferweda, J.D., van-Gils-Meeus, H.A.E., Mutsaers, H.W., & Wesphal-Stevels, J.M.C., 1985. *Cultures vivrières tropicales avec référence spéciale au Cameroun*. Wageningen, Netherlands.

[17] Ndo E. G. D. 2007. *Analyse du risque épidémiologique des populations d'agrumes vis à vis de la cercosporiose, du scab et de la gommose dans les zones humides du Cameroun.* Thèse de MSc, Faculté d'Agronomie et des Sciences Agricoles Université de Dschang, Cameroun, 85 p

[18] Ndo E.G.D., 2011. Evaluation des facteurs de risque épidémiologique de la Pseudocercosporiose des agrumes dans les zones humides du Cameroun. Thèse de doctorat, Sup Agro Montpelier, 202 pp. https://agritrop.cirad.fr/562661/1/do cument\_562661.pdf.

[19] Mariau, D., 1999. *Les Maladies des Cultures Pérennes Tropicales* (CIRAD ed.).

[20] Ndo, E.G.D., Kuate, J., Sidjeu Wonfa, C.S., Tchio, F., Ndzana Abanda, F.X., Mbieji Kemayou, C., Akoutou Mvondo E., Amele Ndjoumoui, C., Amang A Mbang, J., 2019. Tolerance of citrus genotypes towards *Pseudocercospora* leaf and fruit spot disease in western highlands zone of

Cameroon. Crop Protection 124. https:// doi.org/10.1016/j.cropro.2019.05.022

[21] Agostini, J.P., Bushong, P. M., Bahia, A., & Timmer, L. W., 2003. Influence of environmental factors on severity of citrus scab and melanose. Plant Disease*, 87*, 1102-1106.

[22] Boccas, B., and Laville, E. (Eds.). (1978). *Les maladies à Phytophthora* (IRFA Paris ed.).

[23] Graham, J.H., and Timmer, L.W. (2003). Phytophthora diseases of citrus.

[24] Kuate, J. (1998). Cercosporiose des agrumes causés par Phaeoramularia angolensis. Cahier Agriculture*, 7*(2), 121-129.

[25] Ndo, E.G.D., Bella-Manga, Ndoumbe-Nkeng, M., and Cilas C., 2019. Distribution of Pseudocercospora fruit and leaf spot, Phytophthora foot rot and scab diseases and their effect on Citrus tree decline prevalence in the humid zones of Cameroon. Fruits 74(5), 249-256; https://doi.org/10.17660/ th2019/74.5.5

[26] Yesuf, M., 2013. *Pseudocercospora* leaf and fruit spot disease of citrus: achievements and challenges in the citrus industry: a review. Agric. Sci. 4, 324-328.

[27] De Vallavieille-Pope C., Fraj M. B., Mille B., et Meynard J.-M., 2004. Les associations de variétés: accroître la biodiversité pour mieux maîtriser les maladies. Doss de l'environnement de. Dossiers de l'environnement de l'INRA, 30: 101-109.

[28] Kuate, J., Fouré, E., Foko, J., Ducelier, D., & Tchio, F., 2002. La phaeoramulariose des agrumes au Cameroun due à *Phaeoramularia angolensis*:expression parasitaire à différentes altitudes. Fruits*, 54*(04), 207-218.

[29] Adaskaveg, J.E., Hao, W., and Forster, H., 2015. Postharvest Strategies for Managing Phytophthora Brown Rot of Citrus using Potassium Phosphite in Combination with Heat Treatments. Plant Dis. 99:1477-1482. https://doi. org/10.1094/PDIS-01-15-0040-RE

[30] Akoutou Mvondo, E., Ndo, E. G. D., Ngo Bieng, M.A., Ambang, Z., Bella Manga, Cilas, C., Tsouga Manga, M. L., Bidzanga Nomo L., 2017. Assessment of the interaction between the spatial organization of citrus trees populations in cocoa agroforests and *Phytophthora* foot rot disease of citrus severity. *AgroForestry Systems*, 10 p. http://dx.doi. org/10.1007/s10457-017-0140-3

[31] Chaudhary, S., David, A. Laughlin, M.S.J.V., da Graça, M.K., O.J. Alabi, K.M., Crosby, K.L. Ong, and Veronica A., 2020. Incidence, Severity, and Characterization of Phytophthora Foot Rot of Citrus in Texas and Implications for Disease Management. APS, Published Online: 30 Jun 2020 https://doi.org/10.1094/ PDIS-07-19-1493-RE

[32] Graham, J.H., Timmer, L.W., 2008. Florida Citrus pest management guide: Phytophthora foot rot and root rot. Retrieved 15.02.2013, from University of Florida IFAS extension. http://edis.ifas. ufl.edu/cg009.

[33] Mekonen, M, Ayalew, A, Weldetsadik, K. and Seid, A., 2015. Assessing and Measuring of Citrus gummosis (*Phytophthora* spp.) in Major Citrus Growing Areas of Ethiopia. J Horticulture 2: 154. doi:10.4172/2376-0354.1000154

[34] Sakupwanya, M.N., Labuschagne, N., loots, T., and Apostolides, Z., 2018. Towards develoloping a metabolicmarker based predictive model for Phytophthora nicotianae tolerance in citrus rootstocks. Journal of Plant Pathology, 100(2), 269-277. Doi:1007/ s42161-018-0080-4

[35] Graham, J., and Feichtenberger, E., 2015. Citrus phytophthora diseases: Management challenges and successes. J. Cit. Pathol. iocv\_journalcitruspathology\_27203.

[36] Cacciola, S.O. and Di San Lio, G.M., 2008. Management of citrus diseases caused by Phytophthora spp. In: Ciancio A, Mukerji KG (eds) Integrated management of diseases caused by fungi, bacteria and Phytoplasma. Springer, Netherlands, pp. 61-84

[37] Gade and Lad, 2019. Strategies for management of *Phytophthora* diseases in citrus in India. Biointensive approaches: Application and effectiveness in plant diseases management (2019): 435-451

[38] Ippolito, A., Schena L., Nigro, F., Soleti Ligorio, V., & Yaseen, T., 2006. Real-time detection of *Phytophthora nicotianae* and *P. citrophthora* citrus roots and soil. European Journal of Plant Pathology, 110, 833-843.

[39] Ippolito, A., Schena, L., & Nigro F., 2002. Detection of *Phytophthora nicotianae* and *P. citrophthora* in citrus roots and soils by nested PCR. European Journal of Plant Pathology, 108, 855-868.

[40] Jazet-Dongmo P. M., Kuate J., Boyom F. F., Ducelier, D., Damesse, F. et Zollo, P.H. A., 2008. Composition chimique et activité antifongique in vitro des huiles essentielles de citrus sur la croissance mycélienne de Phaeoramularia angolensis. Fru. 57(2): 95-104.

[41] Eddleston, M., 2020. Poisoning by pesticides. Medicine 48 (3), 214-217. https://doi.org/10.1016/j. mpmed.2019.12.019.

[42] Jia, Z.-Q., Zhang, Y.-C., Huang, Q.-T., Jones, A.K., Han, Z.-J., Zhao, C.-Q., 2020. Acute toxicity, bioconcentration, elimination, action *Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*

mode and detoxification metabolism of broflanilide in zebrafish, *Danio rerio*. Journal of Hazardous Materials 394. https://doi.org/10.1016/j. jhazmat.2020.122521 122521.

[43] Mohamed, A. Hassaan, Ahmed El Nemr, 2020. Pesticides pollution: Classifications, human health impact, extraction and treatment techniques. Egyptian Journal of Aquatic Research 46 (2020) 207-220. https://doi. org/10.1016/j.ejar.2020.08.007

[44] Panth, S.C. Hassler and F. Baysal-Gurel., 2020. Methods for Management of Soilborne Diseases in Crop Production. Agriculture 2020, 10, 16; doi: 10.3390/agriculture10010016 www. mdpi.com/journal/agriculture

[45] Qiu, Y.-W., Zeng, E.Y., Qiu, H., Yu, K., Cai, S., 2017. Bioconcentration of polybrominated diphenyl ethers and organochlorine pesticides in algae is an important contaminant route to higher trophic levels. Science of The Total Environment 579, 1885-1893. https:// doi.org/10.1016/j.scitotenv.2016.11.192.

[46] Ndo E. G. D., Bella-Manga F., Atanga Ndindeng S., Ndoumbe-Nkeng M., Ajong Dominic Fontem A. D., et Cilas C., 2010. Altitude, tree species and soil type are the main factors influencing the severity of Phaeoramularia leaf and fruit spot disease of citrus in the humid zones of Cameroon. Eur J Plant Pathol., 128: 385-397

[47] Laird, S. A., Leke-Awung, G., & Lysinge, R. J., 2007. Cocoa farms in the Mount Cameroon region: biological and cultural diversity in local livelihoods. Biodiversity and Conservation*, 16*, 2401-2427.

[48] Sonwa DJ, Nkongmeneck BA, Weise SF, Tchatat M, Adesina AA, Janssens MJJ (2007) Diversity of plants in cocoa agroforests in humid forest areas of Southern Cameroon. Biodiv Conserv 16:2385-2400

[49] Cerda R., Allinne C., Gary C., Tixier P., Harvey C.A., Krolczyk L., Mathiot C. Clément E., Auberto J.N., Avelino J., 2017. Effects of shade, altitude and management on multiple ecosystem services in coffee agroecosystems. Europ. J. Agronomy. 82(2017) 308-319. http://dx.doi. org/10.1016/j.eja.2016.09.019

[50] Rao M. R., Nair P. K. R., et Ong, C. K., 1998. Biophysical interactions in tropical agroforestry systems. Agrofor. Syst.*,* 38: 3-50.

[51] Gidoin C., 2013. *Relations entre structure du peuplement végétal et bioagresseurs dans les agroforêts à cacaoyers. Application à trois bioagresseurs du cacaoyer: la moniliose au Costa Rica, la pourriture brune et les mirides au Cameroun*. Thèse de doctorat, Centre International d'Etudes Supérieures en Sciences Agronomiques de Montpellier. 189 p

[52] Schroth, G., Krauss, U., Gasparotto, L., Aguilar, J. A. D. and Vohland, K., 2000. Pests and diseases in agroforestry systems of the humid tropics', Agroforestry Systems 50(3), 199-241.

[53] Gidoin, C., Avelino, J., Deheuvels, O., Cilas, C. and Bieng, M.A.N., 2014. Shade Tree Spatial Structure and Pod Production Explain Frosty Pod Rot Intensity in Ca-cao Agroforests, Costa Rica. Phytopathology, 104, 275-281. https://doi.org/10.1094/ PHYTO-07-13-0216-R

[54] Ngo Bieng M. A., Laudine A. Chloé C. and Philippe T., 2017. Tree spacing impacts the individual incidence of *Moniliophthora roreri* disease in cacao agroforests. *Pest Manag Sci* (2017). DOI 10.1002/ps.4635. wileyonlinelibrary. com/journal/ps

[55] Ngo Bieng, M. A., Gidoin, C., Avelino, J., Cilas, C., Deheuvels, O. and Wery, J. (2013), 'Diversity and spatial clustering of shade trees affect cacao

yield and pathogen pressure in Costa Rican agroforests', Basic and Applied Ecology 14(4), 329-336.

[56] Malezieux, E., Crozat, Y., Dupraz, C., Laurans, M., Makowski, D., Ozier-Lafontaine, H., Rapidel, B., de Tourdonnet, S. and Valantin-Morison, M., 2009. Mixing plant species in cropping systems: concepts, tools and models. A review', *Agronomy for Sustainable Development* 29(1), 43-62.

[57] Guenat, S., Kaartinen, R., Jonsson, M., 2019. Shade trees decrease pest abundances on brassica crops in Kenya. Agrofor. Syst. 93, 641-652. https://doi. org/10.1007/s10457-017-0159-5.

[58] Ostfeld, R. S., Glass, G. E. and Keesing, F., 2005. Spatial epidemiology: an emerging (or re-emerging) discipline', Trends in Ecology & Evolution 20(6), 328-336.

[59] Somarriba, E. (2005), '¿Cómo evaluar y mejorar el dosel de sombra en cacaotales ?', *Agroforestería en las Américas* (41-42)

[60] Akoutou, M.E., 2015. Effect of the spatial structure of citrus on the severity of three fungal diseases (*Pseudocercospora angolensis*, *Phytophthora* disease and *Elsinoe*) in cocoa-based agroforestry systems in wetlands in Cameroon. Msc degree, Université de Yaoundé 1, Faculté des Sciences. Yaoundé, 71p.

[61] Goreaud F., 2000. *Apports de l'analyse de la structure spatiale en forêt tempérée à l'étude et à la modélisation des peuplements complexes*., PhD thesis, L'ENGREF, centre de Nancy, France, 526 p.

[62] Ngo Bieng, M. A., Ginisty, C. and Goreaud, F. (2011), 'Point process models for mixed sessile forest stands', Annals of Forest Science 68(2), 267-274.

[63] Ripley, B. D. (1977), 'Modeling Spatial Patterns', Journal of the Royal Statistical Society Series B-Methodological 39 (2), 172-212.

[64] Walter J.M.N., 2001. La méthode de ripley pour l'analyse des structures spatiales ponctuelles en écologie. DEA de Géographie Physique et Aménagement, Université Louis Pasteur, Strasbourg, 2000-2001

[65] Brown, D. H. and Bolker, B. M., 2004. The effects of disease dispersal and host clustering on the epidemic threshold in plants. Bulletin of Mathematical Biology 66 (2), 341-371.

[66] Watve, M. G. and Jog, M. M., 1997. Epidemic diseases and host clustering: An optimum cluster size ensures maximum survival. Journal of Theoretical Biology 184(2), 165-169.

[67] Abdulai, I., Jassogne, L., Graefe, S., Asare, R., Van Asten, P., La Ederach, P., 2018. Characterization of cocoa production, income diversification and shade tree management along a climate gradient in Ghana. PLoS One 13 (4), e0195777. https://doi.org/10.1371/ journal.pone.0195777.

[68] Barrios, E., Valencia, V., Jonsson, M., Brauman, A., Hairiah, K., Mortimer, P.E., Okubo, S., 2018. Contribution of trees to the conservation of biodiversity and ecosystem services in agricultural landscapes. IJBESM 14 (1), 1-16. https:// doi.org/10.1080/21513732.2017.1399167.

[69] Seif A.A., Hillocks R.J., 1998. Some factors affecting infection of citrus by *Phaeoramularia angolensis*. Journal of phytopathology 146 (8-9), 385-391.

[70] Babin, R., Anikwe, J. C., Dibog, L. and Lumaret, J. P., 2011. Effects of cocoa tree phenology and canopy microclimate on the performance of the mirid bug *Sahlbergella singularis*. Entomologia Experimentalis Et Applicata 141(1), 25-34.

*Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based… DOI: http://dx.doi.org/10.5772/intechopen.95571*

[71] Diallo, M.T.S., 2003. Vers une lutte contre la cercosporiose des agrumes en Guinée. Fruits*, 58*(6), 329-344.

[72] Pretorius, M. C., Crous, P. W., Groenewald, J. Z., & Braun, U., 2003. Phylogeny of some cercosporoid fungi from Citrus. Sydowia*, 55*, 286-305.

#### **Chapter 5**

## A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid and Hop Stunt Viroid

*Zineb Belabess, Nabil Radouane, Tourya Sagouti, Abdessalem Tahiri and Rachid Lahlali*

#### **Abstract**

Citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) are the main viroids circulating in all citrus-growing areas worldwide, and causing two well-known diseases on citrus trees; exocortis and cachexia, respectively. These viroids are small, covalently closed single-stranded RNA, allocated to the *Pospiviroidae* family. CEVd is the first viroid being described on citrus trees in 1948 in California. It is considered the largest citrus viroid at 371 nucleotides. It causes bark scaling disorder on the rootstock of citrus trees grafted on trifoliate orange and its hybrids and can cause dwarfing of trees grown on these rootstocks. HSVd was first observed in 1945 in Florida. It consists of 299 nucleotides. Stunting, chlorosis, bark gumming, stem pitting, decline, and depressions in the wood are the main symptoms of HSVd in mandarin and its hybrids. The introduction and propagation of infected budwoods are the main causes of viroids spread in citrus orchards. These agents are mechanically sap-transmissible and spread by contaminated tools. Neither seed transmission nor vectors have been reported for both viroids. Root transmission, though possible, would be overshadowed by mechanical transmission. Rapid and sensitive molecular-based detection methods specific to both viroids are available. Both diseases are controlled by using viroids-free budwoods for new plantations, launching budwood certification programs, and establishing a quarantine system for new citrus varieties introduction. The most important achievements in CEVd and HSVd researches are outlined in this chapter. This would help to provide a clearer understanding of the diseases they cause and contribute to the development of better control strategies.

**Keywords:** CEVd, HSVd, citrus, *Pospiviroidae*, transmission, diagnostic, interactions, synergy, antagonism

#### **1. Introduction**

Viroids are circular, highly structured, single-stranded RNA (ssRNA) phytopathogens. Although they do not code for any peptide, these enigmatic pathogens have evolved the capacity to replicate within cellular organella, the nucleus and chloroplast for *Pospiviroidae* and *Avsunviroidae*, respectively [1–4]. Viroid replication is ensured through an RNA-based rolling-circle mechanism [1]. Intriguingly, viroids can induce severe diseases in susceptible host plants similar to those caused by numerous plant viruses [5–7]. From the seven known citrus viroids only, two, namely, CEVd and HSVd, have been reported to be associated with citrus diseases that can pose significant economic risks to global citrus production. These diseases are exocortis and cachexia, respectively [8]. Since their original description in 1948 and 1950, respectively, both diseases have been reported to be present in almost all citrus-growing areas of the world, as well as in early citrus budwood registration programs [9, 10]. Given the importance of and rapid research progress in citrus virology in recent years, this review emphasizes recent findings related to CEVd and HSVd, the most serious viroids associated with citrus. It comprises reviews and research articles covering broad research areas on the characterization of both viroids and their symptoms, the development of reliable and rapid diagnosis methods, and management strategies. A brief snapshot of the present situation of CEVd and HSVd in the Mediterranean region, with an emphasis on their spread in citrus-growing areas of Morocco, is included.

#### **2. Citrus exocortis viroid**

#### **2.1 Taxonomy**

Citrus exocortis is a destructive disease infecting citrus species [9, 11]. The agent of this disease, citrus exocortis viroid [*Pospiviroidae*; *Pospiviroid*; CEVd], is a small, covalently closed ssRNA of about 371 nucleotides (nts) [12–14]. CEVd molecules can exist as either linear or circular [9]. As all viroids allocating to the *Pospiviroid* genus, CEVd lacks RNA self-cleavage activity and has a central conserved region (CCR), composed of two sets of conserved nucleotides in the upper and lower strands of its rod-like secondary structure, and a terminal conserved region (TCR) [15]. The rod-like secondary structure of CEVd takes the form of a model of five structural-functional domains. The latter is the Central (C), the Pathogenic (P), the Variable (V), the Terminal Left (TL), and the Terminal Right (TR) domains [16]. Based on their biological properties, CEVd sequences have been classified into two groups by using tomato as an experimental host: severe "Class A" and mild "Class B". Both classes of sequences differ by a minimum of 26 nts. These mutations affect two genomic regions, designated PL and PR, located respectively within the P and the V domains [17–19]. It is important to emphasize that CEVd strains of both classes cause distinct symptoms in gynura (*Gynura aurantiaca* (Blume) DC.) [20]. However, they induce only subtle differences in trifoliate orange (*Poncirus trifoliate* L. Raf.) used as a rootstock and a similar overall performance of the infected trees [21]. Sequencing of additional CEVd isolates revealed that further strains different from those of "Class A" and "Class B" existed. Furthermore, it seems that the sequence/pathogenicity relationship was more complex than originally anticipated [22]. Infectivity assays carried out with chimeric cDNA clones suggested that PL is the pathogenicity-modulating domain. Although it remains to be explored how this domain modulates pathogenicity (i.e. stunting and epinasty). The role of the PR domain is not known. However, infectivity assays suggested that it may influence the efficiency of viroid infection or replication in the plant [18]. Further infectivity assays of CEVd chimeras and another viroid of the *Pospiviroid* genus, tomato apical stunt viroid [TASVd], have been done to identify the role of individual structural domains. Firstly, it has been demonstrated that symptom severity is modulated by the TL and the P domains. Secondly, it has been shown that the V and the TR domains are involved in regulating viroid replication and/or accumulation [23].

*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

#### **2.2 Symptoms and economical impact**

Citrus exocortis could affect a various part of the tree including the rootstock (at bark and wood levels), scion, leaves, and fruits, thus causing different types of damages such as bark scaling and cracking, bumps, severe stunting, low fruit-bearing, the poor appearance of the canopy [21, 24–27], and poor tree performance [28]. CEVd-infected trees in the orchard show typical symptoms. The most characteristic one is bark scaling on trifoliate orange rootstock, yellow stem blotch on trifoliate orange and its hybrids and Rangpur lime (*Citrus limonia* Osb.), and stunting on trifoliate orange or its hybrid rootstocks [9, 11]. It is important to note that classic exocortis symptoms are not always closely associated with all CEVd isolates. For instance, only transient flaking (Washington Navel orange L) or a fine reticulum of surface cracks (Washington Navel orange 3536) on the trifoliate orange rootstock have been observed on two CEVd-infected trees in Australia [28]. Additionally, no bark scaling symptoms have been observed in CEVd-infected Washington Navel orange trees grafted on Carrizo citrange although these trees presented lesions and blisters in the roots [26]. It is important to emphasize that bark scaling symptoms could be caused by viroids other than CEVd. Indeed, it has been proved that, in certain viroid combinations, synergistic effects occur and cause exocortis scaling symptoms in the absence of CEVd [24]. Furthermore, in Australia, a large number of trees showing exocortis-like symptoms including dwarfing and/or bud union abnormalities produced only mild epinasty when grafted on the indicator plant Etrog citron. The presence of viroids other than CEVd has been highlighted [28]. Bark scaling symptoms could be also the consequence of tree exposure to abiotic stresses such as sunburn [29]. A reduction in vegetative growth has been observed on commune clementine (*Citrus clementine* Hort. ex Tanaka) trees infected by CEVd as it has been determined by the height and rootstock and scion circumferences [21]. Similar but milder symptoms have been reported in CEVd-infected Washington Navel orange trees grafted on Carrizo citrange [26].

The major susceptible citrus rootstocks, which show exocortis bark scaling symptoms, are trifoliate orange and its hybrids, Palestine sweet lime (*Citrus limettioedes* Tan.) or Rangpur lime [11]. Trees grown on trifoliate orange are the most severely affected, with symptoms of bark scaling and severe stunting usually developing when the trees are around 4 years old [29, 30]. Cracking and peeling of the bark below the bud union appear when bark scaling occurs on these rootstocks [30]. Symptoms of exocortis have been also reported on citrange and Swingle citrumelo rootstocks. However, unlike trifoliate orange, bark scaling symptom does not always occur on trees grown on citrange rootstocks. Trees grafted on these rootstocks exhibit symptoms somewhat late and the level of tree stunting is usually less severe than that on trifoliate orange. On another susceptible rootstock, CEVd-infected trees showed symptoms of stunting, yellowing of the canopy, and general tree decline, and occasional flaking of the rootstock bark. On these trees, fruit quality is not affected. However, tree yield is severely reduced since the viroid causes tree stunting [30]. The time required for disease expression by citron scions is believed to be directly associated with the inherent vigor of the rootstock, the environmental temperature, and cultural practices [31]. CEVd does not induce any symptoms in most sweet orange (*Citrus sinensis* (L.) Osbeck), mandarin (*Citrus reticulate* Blanco), and grapefruit (*Citrus paradisi* Macfad) scion cultivars. However, when CEVd-infected budwoods are grafted on one of the previous susceptible rootstocks, distinct symptoms may appear [11].

The type and severity of symptoms induced by citrus exocortis disease depend not just on the selected rootstock as described above, but also on the amount of viroid present in the scion and the infection with other citrus viroids. High

temperatures can also accelerate the development of symptoms [30]. The results of a long-term field trial carried out with clementine trees grafted on the trifoliate orange rootstock revealed that CEVd-induced effects might be both reduced or increased when CEVd-infected trees were exposed to mixed viroid infections. In other words, several interactions among viroids including CEVd have been revealed through comparative assays between symptoms developed on trees infected with CEVd alone or co-infected with other viroids. The most clear-cut interaction occurs between CEVd and Citrus viroid IV [*Pospiviroidae*; *Cocadviroid*; CVd-IV]. This interaction is manifested by the attenuation of bark-scaling or bark-cracking symptoms as a result of the occurrence of antagonism between both viroids. CVd-IV limits the negative effects of CEVd on tree performance. The reduction of tree size and fruit yield occurs mainly in trees infected with combinations containing CEVd or CVd-III and, to a lesser extent, those containing Citrus bent leaf viroid [*Pospiviroidae*; *Apscaviroid*; CBLVd] [24].

Numerous field trials have been conducted on different citrus species, varieties, and rootstocks under three different agroecosystems, to evaluate the effect of CEVd on vegetative growth and yield (**Table 1**). The first field trial has been conducted to assess the effect of CEVd infection on commune clementine trees grafted on Pomeroy trifoliate orange. CEVd-infected trees have been periodically monitored for a period of 12 years (from 1990 to 2002) for symptom expression, growth, and fruit yield. CEVd-infected trees showed a significant reduction of growth and yield, which became increasingly apparent over time with infection. Cumulative yield varied from 291,1 to 570,3 kg in 2001 for CEVd and the control, respectively. This equated to 50% cumulative yield lost. This yield attenuation was associated mainly with the loss of large fruit production. Indeed, it has been shown that CEVd reduced fruit production significantly for calibers 2 to 5. Cumulative weights were smaller than the control for caliber 0–1 and small calibers 6 and 7–8, with some significant difference [21]. The quality of fruits from CEVd-infected orange trees (Washington Navel) grafted on Carrizo citrange rootstock has been evaluated from 2004 to 2007. The results of this experiment showed that the quality of the fruit was not affected by CEVd infection [26].

#### **2.3 Transmission and epidemiology**

All citrus viroids are distributed primarily by the introduction and propagation of infected budwoods and subsequently by mechanical transmission, and CEVd is no exception [11]. Mechanical transmission of CEVd has been already reported. It took place on secateurs, tools, knives, and hedging equipment [9, 11, 27, 29] especially from lemon (*Citrus lemon* Bum. f.) to lemon [11]. Further, it has been shown that CEVd can survive for 8 days on steel knife blades. CEVd infectivity was not affected over a wide range of time intervals between knife contamination and transfer to citron or by 2 sequential transfers by this method. CEVd spread to susceptible hosts by contaminated tools was accomplished from numerous tested citrus species of great economic importance such as lemon, sweet orange, grapefruit, tangerine, and a trifoliate hybrid [31]. Another transmission assay carried out under greenhouse conditions showed that CEVd can be mechanically transmitted from citron to healthy citron [32, 33] and gynura herbaceous plant [33] by a single slash with a knife blade [32, 33]. CEVd-retransmission from infected gynura back to citron was successful [33]. Natural grafts of citrus roots seem to be associated with CEVd propagation. That is the case for example for a budwood multiplication block of an Australian nursery where the propagation of CEVd by natural grafts of roots induced the infection of some healthy lemon mother trees on which neither hedging nor pruning operations took place before


*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*


*SE: Significant effect. NSE: No significant effect.*

*a Susceptible to citrus tristeza virus [Closteroviridae; Closterovirus; CTV] but viroids tolerant. b Susceptible to CTV stem-pitting and cachexia.*

*c CTV tolerant.*

*\* A function of the used viroid isolates.*

#### **Table 1.**

*Results summary of the known field trials carried out in three citrus-growing countries of the Mediterranean area to evaluate the effect of CEVd and HSVd on vegetative growth and yield of different citrus scion and rootstock combinations.*

their removal. This may be mainly linked to the fact that citrus trees were planted close to each other (within 2 m). The role of root grafting in CEVd transmission was assessed by excavating root systems [29]. CEVd root transmission, though possible, would be overshadowed by mechanical transmission. CEVd is not known to be a vector- or seed-transmitted [9, 11]. The role of gots as possible vectors of viroids, including CEVd, has been investigated. The experiment was carried out by rubbing healthy citrus plants with goat horns previously rubbed for 24 h on infected Etrog stems. Results highlighted the detection of CEVd in the tested plants. Therefore, transmission through gots could have facilitated the long-range spread of CEVd among both cultivated and wild plants and *vice versa* and also among graft-incompatible plants [34].

*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

#### **3. Hop stunt viroid**

#### **3.1 Taxonomy**

Cachexia is a destructive disease infecting citrus species [11]. The agent of this disease, hop stunt viroid [*Pospiviroidae*; *Hostuviroid*; HSVd], is a small covalently closed ssRNA of about 300 nts [3, 35]. HSVd is a single member of the genus *Hostuviroid* [36]. HSVd molecules can exist as either circular or linear [35]. HSVd isolates are divided into five groups: three major and two minor groups. The first groups, composed of "plum-type", "hop-type" and "citrus-type", are composed of isolates from a limited number of isolation hosts. As to the second group, it has been suggested that they are the results of the occurrence of recombination events between members of the main groups [37]. Like CEVd, HSVd takes the form of a model of five structural-functional domains within the rod-like secondary structure: C, P, V, TR, and TL [15]. However, HSVd has a genus-specific CCR and a terminal conserved hairpin (TCH) and lacks a TCR [10]. It is worth mentioning that two HSVd-related Group II citrus viroids that differ by a "cachexia expression motif" have been described. It includes a cachexia disease non-pathogenic variant (CVd-IIa) and two pathogenic variants (CVd-IIb and CVd-IIc) [38–40]. Electrophoretic profiles obtained with single-stranded polymorphism (SSCP) allowed deciphering the variability among and within cachexia-inducing sources of citrus isolates of HSVd. SSCP allowed discrimination between non-cachexia and cachexia sources of HSVd. Sequence analysis showed that the V domain was extremely conserved among all the cachexia variants. Indeed, 5 nts differences, affecting both the upper (3 nts) and the lower (2 nts) strands of the V domain, were identified as the most characteristic motif allowing the discrimination between cachexia and non-cachexia sequences. It has been suggested, therefore, that the 5 nts affect the organization of a short helical region and two flanking loops of the V domain, thus modifying the three-dimensional geometry of the molecule [41]. Subsequently, it has been shown that only a single change in HSVd modulates citrus cachexia symptoms [38].

#### **3.2 Symptoms and economical impact**

Cachexia could affect a various part of the tree including the trunk, bark, twigs, branches, leaves, and fruits, thus causing different types of damages such as bark and trunk gumming with a rough and rugose appearance, bark-cracking, moderate and severe tree stunting, chlorosis, decline and death of severely affected trees, brown stipple spotting on the underside of the leaves, and the appearance of small pits on the wood [9, 21, 24]. Cachexia disease mainly affects some mandarins and their hybrids such as tangelos, and *Citrus macrophylla* Wester. Most other citrus species seem to be symptomless unless grafted on susceptible rootstocks [10]. Cachexia-inducing variants were proven to cause gummy bark disease of sweet orange [42, 43] and split bark disorder of sweet lime (*Citrus limetta* Risso) [44]. HSVd variants have been reported to induce yellow corky vein disease of Kagzi lime (*Citrus aurantifolia* (Christm.) Swingle) [45] and sweet orange [44] in India and Iran, respectively. It was subsequently shown that cachexia and a similar disorder previously described in Palestine sweet lime, known as xyloporosis, are caused by the same type of HSVd variants [40].

As mentioned before, for CEVd, the type and severity of citrus cachexia symptoms depend also on the presence of other citrus viroids in the tree. The results of a long-term field trial carried out with clementine trees grafted on the trifoliate

orange rootstock revealed that HSVd-induced effects might be both reduced or increased when HSVd-infected trees were exposed to mixed viroid infections. The most clear-cut interaction occurs between HSVd and CVd-IV. This interaction is manifested by a slight increase in fruit yield and reduction of scion circumferences [24].

The same field trials described before to evaluate the effect of CEVd on vegetative growth and yield (**Table 1**) were used for the same purpose for HSVd. Little or no effect in vegetative growth has been observed on commune clementine trees infected by HSVd as it has been determined by the measure of height and rootstock and scion circumferences [21]. Cumulative yield varied from 377,6 to 570,3 kg in 2001 for HSVd (CVd-IIc isolate) and the control, respectively. This equated to 34% cumulative yield lost [21]. The negative impact of HSVd infection on cumulative yield has been reported in another study carried out on Orange Maltaise demi sanguine grafted on Alemow (*C. macrophylla*). HSVd-infected trees have been periodically monitored for a period of 12 years (from 2005 to 2017) for growth and fruit yield. HSVd-infected trees showed a significant reduction of yield of about 76% compared to healthy control [46]. As for CEVd, the effect of HSVd infection on the quality of fruit from Washington Navel orange trees grafted on Carrizo citrange rootstock has been evaluated from 2004 to 2007. The results of this experiment showed that the quality of the fruit was not affected by HSVd infection. However, a reduction occurred in the diameter of the harvested fruits [26].

#### **3.3 Transmission and epidemiology**

As pointed out before, for CEVd, propagation of infected budwoods and mechanical inoculations with contaminating tools were reported as the principal causes for the omnipresence of multiple viroid species, including HSVd, among citrus orchards [34]. Mechanical transmission of HSVd has been already reported. Indeed, the results of a transmission assay carried out under greenhouse conditions revealed that all HSVd strains are mechanically transmitted from citron to healthy citron by a single slash with a knife blade [32]. As for CEVd, the potential involvement of gots in HSVd spread has been shown under controlled conditions [34]. Top working, a common practice in Mediterranean countries, seems to have largely contributed to HSVd spread in Mediterranean citrus orchards [9]. HSVd is not known to be seed-borne [47] in citrus or to have natural vectors [11, 48].

#### **4. Signaling pathways in citrus exocortis and cachexia pathogenesis**

It is usually accepted that although the mechanisms through which viroids interact with their hosts are beginning to be dissected, the key triggering events and molecular mechanisms underlying viroid pathogenesis remain unclear [49, 50], and CEVd and HSVd are no exception. As demonstrated by various types of citrus pathogens [51, 52], further investigation of the molecular basis of viroid-host interactions is crucial to better understand the pathogenesis of viroids, and thus help to develop effective strategies to combat viroid diseases [50, 53]. Important changes occur in the chloroplast, cell wall, peroxidase, and symporter activities upon infection of Etrog citron with CEVd [54]. The CEVd-infected citron system has been subsequently used for studying the feedback regulation mechanism using transcriptomic analysis. The analysis of the woody host response to CEVd revealed the activation of basic defense and RNA-silencing mechanisms following CEVd infection. In other words, a large number of genes (about 1530) encoding key proteins involved in the RNA silencing pathway, and proteins related to basic defense

*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

responses are expressed following CEVd infection [53]. Furthermore, a recent study elucidates the role of phytohormone pathways, particularly those linked to ethylene, in disease development and ribosomal stress caused by CEVd infection by using tomato as an experimental host [55]. For HSVd, a small RNA-mediated gene silencing response has been highlighted upon the infection of lemon by HSVd. The large amounts of HSVd-small interfering RNA (siRNA) from both central and variant domains have been suggested to be involved in interference with host gene and symptom development [56].

#### **5. Detection methods**

#### **5.1 Biological indexing and cross protection**

Generally, biological assays based on indicator host plants expressing typical symptoms of infection and able to withstand higher levels of viroid replication played an essential role in both viroid detection and characterization [57]. CEVd and HSVd are viroid diseases present in citrus orchards around the world [11]. The biological diagnosis through indexing method is considered as an efficient tool to test the health status of a plant, regarding a disease by inoculation with the grafting of the budwood or any other infected tissue in indicator plants that allow viroid replication, symptoms expression [11, 58], and the enhancement of viroid concentration [59]. However, bioassays for CEVd and HSVd detection and identification may require a panel of indicator host plants [60]. Certain considerations need to be respected for the proper indexing of citrus viroids. These include the use of excellent plants, the work under warm temperatures, and the use of citron index plants grown one per container. As mentioned previously, citrus viroids are highly mechanically transmissible and tools must be disinfected to avoid their spread [9].

The citron test is a very sensitive and diagnostic index for determining the presence of CEVd [9]. However, indexing, *in vivo* for CEVd diagnosis is time-consuming, labor-intensive, and requires technician greenhouses [59]. It can take 90 days after inoculation or grafting onto indicator plants [61–63]. Symptoms of slight to severe epinasty leaves wrinkled and twisted to the reverse with light to dark brown cracks in petiole and branches, blisters in the petiole, corking of the midrib, and reduced growth are the main symptoms observed on Etrog citron Arizona 861-S indicator plants graft-inoculated with CEVd-infected budwoods [28, 29, 59, 63]. An *in vitro* indexing procedure has been developed to minimize the risks of epidemics caused by viroids including CEVd. It has been proved that the *in vitro* indexing of CEVd is efficient as well as the *in vivo* diagnosis, and requires between 20 and 40 days less to reach the maximum incidence after inoculation. Epinasty, growth reduction, and rugged leaves with dry tips, and reduced size are the main symptoms observed on the sprouts planted *in vitro* and grafted with CEVd-infected callus [59]. The same symptoms have been reported for sprouts grafted with CEVd infected cortex [62]. Cuban Shaddock (*Citrus maxima* (Burm.) Merr.) has been proved to be the best rootstock, compared to rough lemon (*Citrus jambhiri* Lush.) or Volkamer lemon (*Citrus volkameriana* Ten.), for symptom expression on Arizona 861 S-1 citron indicator plants for indexing exocortis [64]. Gynura is also considered as an excellent indicator for CEVd. This latter reacts strongly in this host plant [9].

As to cachexia, Parson's special mandarin budded on vigorous root-stock such as rough lemon or Volkamer lemon is reported as an excellent indicator for the disease [9, 65]. The biological indexing may take up to one year before symptoms are seen. The reaction of Parson's Special mandarin may differ depending on HSVd isolates. In other words, some isolates are very mild reacting, whereas others are quite severe in their reaction to the indicator plant. Indeed, a mild strain reaction consists of just a slight browning at the bud union or cut back region of the Parson's Special mandarin while a severe reaction consists of the appearance of gum in the wood that may extend via the entire plant [9]. Cuban Shaddock has been proved to be the best rootstock, compared to rough lemon or Volkamer lemon, for symptom expression on Clemeline 11–20 indicator plants for indexing cachexia. Furthermore, the application of 0,5% foliar urea sprays, alone or in combination with 20 ppm gibberellic acid showed to produce more intense expression of cachexia symptoms in the indicator Clemeline 11–20 than the unsprayed control [64].

Cross protection is a biological assay, in which the infection of a plant with a viroid strain ensures protection from infection with another strain of the same viroid. This bioassay can be used for indirect viroid biological indexing. It has been applied in the diagnosis of several viroids including CEVd and HSVd. Typically, the principle of this method is based on the infection of the plant with a mild strain of a viroid, followed by its inoculation with inoculum from a plant suspected to be infected with a severe strain of the same viroid. Positive indexing of the viroid is revealed by the non-expression of symptoms in the tested plants [60]. It has been shown in a cross-protection assay, performed with CEVd-129 as a "protecting" strain against the severe type strain of CEVd that a mild strain of CEVd could lead to apparent "protection" against challenge inoculation with the severe strain. However, it is important to highlight that variability has been shown in the induced protection effect. The latter varied from only a brief delay to almost total impairment of symptom expression. The level of protection depends on the length of the interval between the inoculations with the mild and severe strains [66].

#### **5.2 Nucleic acid-based methods**

Since viroids lack a protein capsid, serological techniques used routinely in plant viruses' detection are not applicable [67]. Nucleic acid-based methods, including polyacrylamide gel electrophoresis (PAGE), hybridization (dot- and northern-blots and micro−/macroarrays), amplification (reverse transcription-polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP)) and sequencing (next-generation sequencing and Sanger sequencing), offer rapid cost-effective, and reliable diagnosis of viroids [60].

PAGE is considered as the first molecular technique used for the rapid (2–3 day period) identification of viroid infected plants. This technique continues to play a crucial role in the identification of new viroids since it is the only diagnostic method that is sequence-independent. PAGE analysis under denaturing conditions showed that many *Citrus* species may harbor numerous viroids including CEVd and HSVd [57]. PAGE and ethidium bromide or silver staining is considered as the first molecular technique applied for CEVd detection [22, 68]. However, it seems that the sensitivity of this technique requires an adequate viroid accumulation level [22]. In other terms, the PAGE procedure was used successfully to directly detect CEVd from field-grown sweet orange and grapefruit trees. The key was reported to be the use of large (50 g) samples of succulent, expanding-flush tissue collected during the summer season. However, samples collected from field-grown trees in January and February did not give consistent detection in trees known to be CEVd-infected, presumably because lack of new growth and low temperatures do not favor CEVd replication [69]. PAGE analysis can routinely resolve as many as four different viroids in the same sample. For instance, it has been shown that this technique can resolve two HSVd variants differing in length by only four nucleotides i.e. 303 nts vs. 299 nts [57].

#### *A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

Since the beginning of their use in the 1980s, dot blot hybridization and hybridization of tissue imprints began to replace PAGE for routine viroid detection. This is mainly because these methods allow the processing of a large number of samples [57]. A northern hybridization protocol, which relied on the analysis of preparations from bark tissues, was proved to be more sensitive than PAGE to detect CEVd and HSVd from field-grown plants of different citrus species and cultivars [70]. A citrus viroids-multiprobe composed of full-length clones of HSVd, CEVd, and two other citrus viroids has been constructed for the simultaneous detection of viroids associated with citrus trees. All the tested viroids were effectively detected with this multiprobe when tested by both northern hybridization and dot blot methods. It is important to highlight that this multiprobe does not allow the identification of the viroid type species resulting in a positive signal [71].

Due to the small size of viroids, numerous RT-PCR approaches can be applied for both their detection and subsequent characterization. In the case of CEVd and HSVd, numerous RT-PCR and real-time RT-PCR approaches have been developed and proved to allow the detection of CEVd and HSVd in both singleplex or multiplex assays [63, 72–76]. A list of some RT-PCR and related tests developed to detect CEVd and HSVd are presented in **Table 2**. Some of these tests allow also the discrimination between mild and severe CEVd strains and the identification of HSVd isolates associated with cachexia symptoms [77]. The multiplex one-step RT-PCR assay developed by Wang et al. [75] is considered a good tool streamlining the simultaneous detection of up to five citrus viroids, including CEVd and HSVd. This enables to reduce time and labor without affecting sensitivity and specificity. Indeed, serial dilution experiments showed that the singleplex RT-PCR sensitivity was similar to that of multiplex RT-PCR for all the tested viroids [75]. This type of assay could be used in high throughput screenings of viroids associated with citrus in field surveys, germplasm banks, nurseries, as well as in other viroid disease management programs [74]. Similarly, the multiplex RT-TaqMan PCR assay developed by Papayiannis [76] enables accurate discrimination between CEVd and HSVd with a diagnostic sensitivity and specificity of 100%. It is important to emphasize that in conventional RT-PCR tests, the overall sensitivity and specificity were lower and varied between 97 and 98% for HSVd, and 94 and 95% for CEVd. Therefore, this essay presented 1000-fold more analytical sensitivity [76]. The specificity of the tests described previously was confirmed by including healthy controls and/or plant tissue infected with other citrus graft transmissible virus and bacteria pathogens and non-targeted citrus viroids. Both singleplex and multiplex assays did not cross-react with any non-inoculated negative controls or other citrus pathogens [63, 74, 76]. To date, PCR-based approaches have been proven efficacy on viroid direct detection. However, false positives and negatives due to amplicon contamination and failure to generate cDNA of suitable size during reverse transcription, respectively, are not uncommon and therefore preclude the application of RT-PCR for large scale indexing [70].

Next-generation sequencing (NGS) technologies are currently becoming routinely applied in different fields of virus and viroids studies. These advanced technologies have therefore contributed to a revolution in both the detection and discovery of plant viruses and viroids [78–81]. NGS has also provided an alternative method to identify viroids in the citrus cultivars. In other words, transcriptome sequencing has shown efficacy in citrus viroid diagnostics. Indeed, this method enabled the simultaneous identification of numerous viroids from various citrus samples, including CEVd and HSVd [82]. A deep sequencing approach, combined with bioinformatics analysis, is already being implemented for HSVd detection in *C. lemon* in China. This finding suggests that HSVd could infect this host and potentially be a pathogen that causes disease on *C. lemon* trees [56].


#### *Citrus - Research, Development and Biotechnology*


#### *A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*


### **Table 2.**

*Primer sequences and their annealing temperature (Tm), primer/probe location, and expected size of PCR products for each primer pair when used to amplify CEVd and HSVd by RT-PCR and related tests (this is not a full or exclusive list).* *A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

#### **6. Control strategies**

*In vitro* somatic embryogenesis, from both style and stigma cultures, has been proved to be a highly effective sanitation method leading to the complete elimination of the main virus and virus-like diseases associated with citrus. Furthermore, it has been shown efficacy to eliminate diseases induced by viroids, and the production of healthy citrus plants [83–85]. This method was applied to eliminate CEVd and HSVd from some *Citrus* species [83]. For example, somatic embryogenesis has been tested on 13 genotypes, belonging to the Algerian germplasm collection of two different *Citrus* species, lemon and sweet orange, infected by at least one grafttransmissible agent, including CEVd and HSVd. This method has shown efficacy to eliminate CEVd from 12/13 tested genotypes. However, HSVd was proved to be the most infectious viroid since it has been eradicated only from 5/13 tested genotypes. It is important to emphasize that no somaclonal variability has been highlighted in lemon regenerated plants. However, a genetic instability has been observed in some of the regenerated orange plants Washington navel 251 [83]. Sanitation by *in vitro* shoot-tip grafting has also been proved to be a very effective method for citrus graft-transmissible diseases eradication including citrus viroids (success rate of about 100%) [86–88]. For instance, it has been reported that CEVd and HSVd can be routinely eliminated from citrus by shoot-tip grafting. Since citrus viroids are extremely tolerant of heat, the use of thermotherapy as a sanitary method is not effective in eliminating viroids from citrus budwoods [9].

No naturally occurring durable resistance has been observed in most species, despite non-hosts for viroids exist. Therefore, the effective control methods for viroid diseases consist mainly of detection and eradication, and cultural controls [50].

#### **7. Viroids situation in the mediterranean region: focus on Morocco**

Exocortis and cachexia are widespread diseases in the Mediterranean region. CEVd and HSVd have been reported in most Mediterranean countries and are among the most prevalent citrus viroids in the region [9]. The development of reliable diagnostic methods facilitated extensive surveys for CEVd and HSVd in different parts of the region. Both viroids were successively identified in many countries, including Morocco [89–92], Cyprus [33], Spain [26], Egypt [43, 93], Italy [61, 94], Tunisia [46, 95], France [21], Syria [96], and Turkey [42].

In Morocco, exocortis and cachexia are among the major citrus viroid diseases [90, 91]. These diseases are prevalent in citrus orchards and can be found in all *Citrus* species and varieties [89–92, 97]. Mechanical transmission of citrus viroids, including CEVd and HSVd, via working tools seems to be behind the widespread of these phytopathogens and their detection in both old and young plantings in all surveyed citrus orchards [92]. Research, recently completed from 2008 to 2018, to monitor CEVd and HSVd prevalence, in the main citrus-growing areas of Morocco (Gharb, Haouz, Loukkos, Moulouya, Souss, and Tadla), showed that CEVd and HSVd are omnipresent in almost all citrus-growing areas of the country with relatively high prevalence. That is the case for example for the Gharb area where CEVd and HSVd were detected at a prevalence of 85% [89] and 21% [92], respectively. Concerning genetic analysis, a first sequence comparison among six Moroccan HSVd isolates collected in the six main citrus-growing areas of Morocco has been recently reported by Afechtal et al. [92]. Phylogenetic analysis showed that the six HSVd isolates are clustered into one group within the "citrus-type". Furthermore, it seems that sequence variability is neither a function of host plant nor a function of the symptoms [92].

#### **8. Conclusions**

Citrus viroids, including CEVd and HSVd, are distributed mainly by the introduction and propagation of infected budwoods, by top working, and by mechanical transmission [9, 11]. Both viroids are known for their ability to infect a large number of host plants [36]. CEVd and HSVd are destructive to certain citrus varieties and, can cause yield losses that may be as high as 34 to 76 percent depending on the combination viroid-rootstock-scion [21, 46]. The mechanisms through which CEVd and HSVd interact with their hosts and induce pathogenesis are beginning to be deciphered. In other words, the involvement of RNA-silencing and basic defense mechanisms following CEVd and HSVd infection has been highlighted [54, 56].

Once introduced and established in a country, both viroids can spread relatively rapidly because of their ability to be transmitted via mechanical means [9, 11]. Since CEVd and HSVd have a high resistance to heat, the chemical treatment appears to be the best method to disinfect CEVd- and HSVd-contaminated tools. For instance, a 0,25 to 0,5 and a 1 percent solution of sodium hypochlorite appears to be the best option to eliminate CEVd and HSVd, respectively, from contaminated hedging and budwood cutting tools [11, 98]. Like all citrus viroids, CEVd and HSVd seem to be successively eliminated from propagative material by shoot-tip grafting or by the deployment of nucellar budlines. Being extremely tolerant of heat, CEVd and HSVd have not been successfully eliminated from budwood by applying thermotherapy [9]. Certification programs must include measures to control viroid spread in nurseries [32]. The majority of rootstocks that are tolerant to the citrus tristeza virus [*Closteroviridae*; *Closterovirus*; CTV] are susceptible to citrus viroids. Therefore, in the absence of a certification program, exocortis disease usually follows upon the replantation of these rootstocks [9]. Since no useful sources of natural resistance to viroid disease are known, diagnostic tests continue to play a key role in efforts to control viroid diseases [67]. Nowadays, several nucleic acid-based methods for detecting CEVd and HSVd exist, including PAGE, hybridization, amplification, and sequencing [60]. Although biological assay has several disadvantages, it will always play a pivotal role in viroid research. Indeed, Cuban Shaddock has been proved to be the best rootstock for symptom expression on Arizona 861 S-1 citron and Clemeline 11–20 indicator plants for indexing exocortis and cachexia, respectively [64]. Besides, gynura seems to be an excellent indicator for CEVd [9]. A combination of both molecular and biological assays should lead to the most effective means for viroid identification and characterization [60].

Complicated interactions, including antagonism and synergy, occur between viroids coinfecting the same citrus host. These interactions may lead to different symptoms, canopy volumes, fruit yields, and commercial performance. Although no obvious physiological changes in citrus hosts have been described in mixed infections of CEVd and HSVd and both viroids do not induce severe symptoms in citrus [24, 99], their interaction was intriguing because they are commonly found simultaneously infecting different citrus cultivars and they have identical biological properties within the same host. The relationship between the two viroids has been investigated over 3 years (from 2011 to 2013). Results showed a positive correlation between CEVd and HSVd in specific tissues of two citrus cultivars (blood orange and Murcott mandarin). This result has been supported by three findings: titer enhancement, localization similarity, and lack of symptom aggravation under mixed-infection conditions. Compared to their concentrations under single-infection conditions, a significant increase in the CEVd and HSVd population has been observed under mixed-infection during 6 and only 1 season of the 12 monitored seasons, respectively. This result is somewhat surprising because no competition phenomenon for host resources occurs between the two viroids although they have

#### *A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

the same biological functions and share identical cellular and subcellular spaces [27]. This issue merits consideration in future research.

Regarding the current situation of CEVd and HSVd in Morocco, this chapter provides a general overview of their spread in the Moroccan citrus-growing areas. Preventing the introduction and the establishment of exocortis and cachexia diseases in the Moroccan citrus orchards can be set up through the use of viroid-free (certified) planting material, disinfection of pruning tools, regular monitoring of citrus orchards to ensure early detection of both diseases, and by avoiding top working practice. This review pointers to new research avenues in exocortis and cachexia diseases in Morocco or elsewhere. These research fields could include for instance the characterization of CEVd and HSVd isolates, searching for secondary hosts, and developing sustainable control strategies. Investigating the prevalence of CEVd and HSVd infection in numerous natural host plants, and the characterization of the viroid sequence variants is valuable especially that a cross-transmission phenomenon between different hosts seems to be possible for HSVd [100].

Studying functional genomics through transcriptomic analysis and/or proteomic approaches in citrus-CEVd/HSVd interaction would be an interesting approach to shed more light on the full mechanisms underlying the complex and varied events associated with such interactome, and thus contribute to the development of novel diagnostic methods and plant protection strategies. This further advanced research will expand our understanding of CEVd and HSVd epidemiology and the mechanisms behind their spread across the world in general and Morocco in particular, and could potentially help in devising innovative management strategies of both viroids.

#### **Acknowledgements**

This research was financially supported by INRA Institute (CRRA de Oujda; Qualipole de Berkane) and the Phytopathology Unit of the Department of Plant Protection, Ecole Nationale d'Agriculture de Meknes.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Zineb Belabess1 , Nabil Radouane2,3, Tourya Sagouti4 , Abdessalem Tahiri<sup>2</sup> and Rachid Lahlali2 \*

1 Plant Protection Laboratory, INRA, Centre Régional de la Recherche Agronomique (CRRA), Berkane, Morocco

2 Phytopathology Unit, Department of Plant Protection, Ecole Nationale d'Agriculture de Meknès, Meknes, Morocco

3 Department of Biology, Laboratory of Functional Ecology and Environmental Engineering, FST-Fez, Sidi Mohamed Ben Abdellah University, Fez, Morocco

4 Faculty of Sciences and Technic of Mohammedia, Laboratory of Virology, Microbiology and Quality/Ecotoxicology and Biodiversity, Mohammedia, Morocco

\*Address all correspondence to: rlahlali@enameknes.ac.ma

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

#### **References**

[1] Flores, R.; Gas, M.E.; Molina-Serrano, D.; Nohales, M.A.; Carbonell, A.; Gago, S.; De la Peña, M.; Daròs, J.A. Viroid replication: Rolling-circles, enzymes and ribozymes. *Viruses* **2009**, *1*, 317-334, doi:10.3390/v1020317.

[2] Carbonell, A.; Martínez de Alba, Á.E.; Flores, R.; Gago, S. Doublestranded RNA interferes in a sequencespecific manner with the infection of representative members of the two viroid families. *Virology* **2008**, *371*, 44-53, doi:10.1016/j.virol.2007.09.031.

[3] Marquez-Molins, J.; Gomez, G.; Pallas, V. Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions. *Mol. Plant Pathol.* **2020**, 1-10, doi:10.1111/mpp.13022.

[4] Palukaitis, P. What has been happening with viroids? *Virus Genes* **2014**, *49*, 175-184, doi:10.1007/ s11262-014-1110-8.

[5] Adkar-Purushothama, C.R.; Perreault, J.P. Current overview on viroid–host interactions. *Wiley Interdiscip. Rev. RNA* **2019**, *11*, 1-21, doi:doi:10.1002/wrna.1570.

[6] Ding, B.; Itaya, A. Viroid: A useful model for studying the basic principles of infection and RNA biology. *Mol. Plant-Microbe Interact.* **2007**, *20*, 7-20, doi:10.1094/MPMI-20-0007.

[7] Bani Hashemian, S.M.; Serra, P.; Barbosa, C.J.; Juárez, J.; Aleza, P.; Corvera, J.M.; Lluch, A.; Pina, J.A.; Duran-Vila, N. Effect of a field-source mixture of citrus viroids on the performance of "nules" clementine and "navelina" sweet orange trees grafted on carrizo citrange. *Plant Dis.* **2009**, *93*, 699-707, doi:10.1094/PDIS-93-7-0699.

[8] Semancik, J.S.; Roistacher, C.N.; Rivera-Bustamante, R.; Duran-Vila, *N.*  *citrus* Cachexia Viroid, a New Viroid of Citrus: Relationship to Viroids of the Exocortis Disease Complex. *J. Gen. Virol.* **1988**, *69*, 3059-3068, doi:10.1099/0022-1317-69-12-3059.

[9] Roistacher, C.N. Diagnosis and Management of Virus and Virus like Diseases of Citrus. In *Diseases of Fruits and Vegetables*; Naqvi, S.A.M.H., Ed.; 2004; Vol. I, pp. 109-189.

[10] Hataya, T.; Tsushima, T.; Sano, T. Hop Stunt Viroid. In *Viroids and Satellites*; Hadidi, A., Flores, R., Randles, J.W., Palukkaitis, P., Eds.; Sara Tenny, 2017; pp. 199-210.

[11] Roistacher, C.N. Graft-transmissible diseases of citrus, hand book for detection and diagnosis. *Food Agric. Organ. United Nations* **1991**, p.285.

[12] Gross, H.J.; Krupp, G.; Domdey, H.; Raba, M.; Jank, P.; Lossow, C.; Alberty, H.; Ramm, K.; Sanger, H.L. Nucleotide Sequence and Secondary Structure of Citrus Exocortis and Chrysanthemum Stunt Viroid. *Eur. J. Biochem.* **1982**, *121*, 249-257, doi:10.1111/j.1432-1033.1982. tb05779.x.

[13] Visvader, J.E.; Gould, A.R.; Bruening, G.E.; Symons, R.H. Citrus exocortis viroid: nucleotide sequence and secondary structure of an Australian isolate. *FEBS Lett.* **1982**, *137*, 288-292, doi:10.1016/0014-5793(82)80369-4.

[14] Duran-Vila, N.; Roistacher, C.N.; Rivera-Bustamante, R.; Semancik, J.S. A Definition of Citrus Viroid Groups and Their Relationship to the Exocortis Disease. *J. Gen. Virol.* **1988**, *69*, 3069-3080, doi:https://doi. org/10.1099/0022-1317-69-12-3069.

[15] Flores, R.; Randles, J.W.; Ownes, R.A.; Bar-Joseph, M.; Diener, T.O. The Suviral Agents. In *Virus Taxonomy,* 

*Eighth Report of the International Committee on Taxonomy of Viruses*; Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A., Eds.; 2005; pp. 1145-1161 ISBN 0122499514.

[16] Keese, P.; Symons, R.H. Domains in viroids: Evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. *Proc. Natl. Acad. Sci. U. S. A.* **1985**, *82*, 4582- 4586, doi:10.1073/pnas.82.14.4582.

[17] Visvader, J.E.; Symons, R.H. Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. *Nucleic Acids Res.* **1985**, *13*, 2907-2920, doi:10.1093/nar/13.8.2907.

[18] Visvader, J.E.; Symons, R.H. Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid. *EMBO J.* **1986**, *5*, 2051-2055, doi:10.1002/j.1460-2075.1986. tb04465.x.

[19] Garcia-Arenal, F.; Pallas, V.; Flores, R. The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid. *Nucleic Acids Res.* **1987**, *15*, 4203-4210.

[20] Chaffai, M.; Serra, P.; Gandía, M.; Hernández, C.; Duran-Vila, N. Molecular characterization of CEVd strains that induce different phenotypes in *Gynura aurantiaca*: structure-pathogenicity relationships. *Arch. Virol.* **2007**, *152*, 1283-1294, doi:10.1007/s00705-007-0958-5.

[21] Vernière, C.; Perrier, X.; Dubois, C.; Dubois, A.; Botella, L.; Chabrier, C.; Bové, J.M.; Duran Vila, *N. Citrus* viroids: Symptom expression and effect on vegetative growth and yield of clementine trees grafted on trifoliate orange. *Plant Dis.* **2004**, *88*, 1189-1197, doi:10.1094/PDIS.2004.88.11.1189.

[22] Duran-Vila, *N. Citrus* Exocortis Viroid. In *Viroids and Satellites*; 2017; pp. 169-179.

[23] Sano, T.; Candresse, T.; Hammond, R.W.; Diener, T.O.; Owens, R.A. Identification of multiple structural domains regulating viroid pathogenicity. *Proc. Natl. Acad. Sci. U. S. A.* **1992**, *89*, 10104-10108, doi:10.1073/ pnas.89.21.10104.

[24] Vernière, C.; Perrier, X.; Dubois, C.; Dubois, A.; Botella, L.; Chabrier, C.; Bové, J.M.; Duran Vila, N. Interactions Between Citrus Viroids Affect Symptom Expression and Field Performance of Clementine Trees Grafted on Trifoliate Orange. *Virology* **2006**, *96*, 356-368, doi:10.1094/PDIS.2004.88.11.1189.

[25] Vernière, C.; Botella, L.; Dubois, A.; Chabrier, C.; Duran-Vila, N. Properties of Citrus Viroids: Symptom Expression and Dwarfing. In Proceedings of the Fifteenth International Organization of Citrus Virologists Conference Proceedings-Viroids; 2002; pp. 240-248.

[26] Murcia, N.; Bani Hashemian, S.M.; Serra, P.; Pina, J.A.; Duran-Vila, *N. Citrus* viroids: Symptom expression and performance of washington navel sweet orange trees grafted on carrizo citrange. *Plant Dis.* **2015**, *99*, 125-136, doi:10.1094/PDIS-05-14-0457-RE.

[27] Lin, C.Y.; Wu, M.L.; Shen, T.L.; Hung, T.H. A mutual titer-enhancing relationship and similar localization patterns between Citrus exocortis viroid and Hop stunt viroid co-infecting two citrus cultivars. *Virol. J.* **2015**, *12*, 1-11, doi:10.1186/s12985-015-0357-6.

[28] Gillings, M.R.; Broadbent, P.; Gollnow, B.I. Viroids in Australian Citrus: Relationship to Exocortis, Cachexia and Citrus Dwarfing. *Aust. J. Plant Physiol.* **1991**, *18*, 559-570, doi:10.1071/PP9910559.

*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

[29] Broadbent, P.; Gollnow, B.I.; Gillings, M.R.; Bevington, K.B. Root Grafting and Mechanical Transmission of Citrus Exocortis Viroid Within a Citrus Budwood Multiplication Block. In Proceedings of the International Organization of Citrus Virologists Conference; 1988.

[30] Hardy, S.; Donovan, N.; Barkley, P.B. Citrus Exocortis. *Primefact* **2008**, *772*, 1-3.

[31] Allen, R. Mechanical transmission of exocortis virus. *Citograph* **1970**, *55*, 145-148.

[32] Barbosa, C.J.; Pina, J.A.; Pérez-Panadés, J.; Bernad, L.; Serra, P.; Navarro, L.; Duran-Vila, N. Mechanical Transmission of Citrus Viroids. *Plant Dis.* **2005**, *89*, 749-754, doi:10.1094 / PD-89-0749.

[33] Kyriakou, A.P. Incidence in Cyprus of citrus exocortis viroid and its mechanical transmission. *Plant Pathol.* **1992**, *41*, 20-24, doi:10.1111/j.1365-3059.1992.tb02310.x.

[34] Cohen, O.; Batuman, O.; Moskowits, Y.; Rozov, A.; Gootwine, E.; Mawassi, M.; Bar-Joseph, M. Goat horns: Platforms for viroid transmission to fruit trees? *Phytoparasitica* **2005**, *33*, 141-148, doi:10.1007/BF03029972.

[35] Semancik, J.S.; Roistacher, C.N.; Duran-Vila, N. A New Viroid is the Causal Agent of the Citrus Cachexia Disease. In Proceedings of the Tenth International Organization of Citrus Virologists Conference; 1988; pp. 125-135.

[36] Eiras, M.; Targon, M.L.P.N.; Fajardo, T.V.M.; Flores, R.; Kitajima, E.W. Citrus exocortis viroid and Hop Stunt viroid doubly infecting grapevines in Brazil. *Fitopatol. Bras.* **2006**, *31*, 440-446, doi:https://doi.org/10.1590/ S0100-41582006000500002.

[37] Amari, K.; Gomez, G.; Myrta, A.; Di Terlizzi, B.; Pallás, V. The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a conserved hammerhead-like structure on the viroid molecule. *J. Gen. Virol.* **2001**, *82*, 953-962, doi:10.1099/0022-1317-82-4-953.

[38] Serra, P.; Gago, S.; Duran-Vila, N. A single nucleotide change in Hop stunt viroid modulates citrus cachexia symptoms. *Virus Res.* **2008**, *138*, 130- 134, doi:10.1016/j.virusres.2008.08.003.

[39] Reanwarakorn, K.; Semancik, J.S. Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids. *J. Gen. Virol.* **1998**, *79*, 3163-3171, doi:10.1099/0022-1317-79-12-3163.

[40] Reanwarakorn, K.; Semancik, J.S. Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus. *Phytopathology* **1999**, *89*, 568-574, doi:10.1094/PHYTO.1999.89.7.568.

[41] Palacio-Bielsa, A.; Romero-Durbán, J.; Duran-Vila, N. Characterization of citrus HSVd isolates. *Arch. Virol.* **2004**, *149*, 537-552, doi:10.1007/ s00705-003-0223-5.

[42] Önelge, N.; Cinar, A.; Szychowski, J.A.; Vidalakis, G.; Semancik, J.S. Citrus viroid II variants associated with "Gummy Bark" disease. *Eur. J. Plant Pathol.* **2004**, *110*, 1047-1052, doi:10.1007/s10658-004-0815-2.

[43] Sofy, A.R.; Soliman, A.M.; Mousa, A.A.; Ghazal, S.A.; El-Dougdoug, K.A. First record of Citrus viroid II (CVd-II) associated with gummy bark disease in sweet orange (*Citrus sinensis*) in Egypt. *New Dis. Reports* **2010**, *21*, 24, doi:10.519 7/j.2044-0588.2010.021.024.

[44] Bagherian, S.A.A.; Izadpanah, K. Two novel variants of hop stunt viroid associated with yellow corky vein

disease of sweet orange and split bark disorder of sweet lime. In Proceedings of the 21st International Conference on Virus and other Graft Transmissible Deseases of Fruit Crops; 2010; pp. 105-113.

[45] Roy, A.; Ramachandran, P. Occurrence of a Hop stunt viroid (HSVd) variant in yellow corky vein disease of citrus in India. *Curr. Sci.* **2003**, *85*, 1608-1612.

[46] Najar, A.; Hamdi, I.; Varsani, A.; Duran-Vila, *N.* Citrus viroids in Tunisia: Prevalence and molecular characterization. *J. Plant Pathol.* **2017**, *99*, 787-792, doi:10.4454/jpp.v99i3.3989.

[47] Bar-Joseph, M. A Contribution to the Natural History of Viroids. In Proceedings of the Thirteenth International Organization of Citrus Virologists Conference Proceedings (1957-2010); 2010; pp. 226-229.

[48] Fuchs, M.; Almeyda, C. V.; Al Rwahnih, M.; Atallah, S.S.; Cieniewicz, E.J.; Farrar, K.; Foote, W.R.; Golino, D.A.; Gómez, M.I.; Harper, S.J.; et al. Economic Studies Reinforce Efforts to Safeguard Specialty Crops in the United States. *Plant Dis.* **2020**, PDIS-05-20- 1061, doi:10.1094/pdis-05-20-1061-fe.

[49] Thibaut, O.; Claude, B. Innate immunity activation and RNAi interplay in citrus exocortis viroid tomato pathosystem. *Viruses* **2018**, *10*, doi:10.3390/v10110587.

[50] Kovalskaya, N.; Hammond, R.W. Molecular biology of viroid-host interactions and disease control strategies. *Plant Sci.* **2014**, *228*, 48-60, doi:10.1016/j.plantsci.2014.05.006.

[51] Marmisolle, F.E.; Arizmendi, A.; Ribone, A.; Rivarola, M.; García, M.L.; Reyes, C.A. Up-regulation of microRNA targets correlates with symptom severity in *Citrus sinensis* plants infected with two different isolates of citrus psorosis

virus. *Planta* **2020**, *251*, 1-11, doi:https:// doi.org/10.1007/s00425-019-03294-0.

[52] Reyes, C.A.; Ocolotobiche, E.E.; Marmisollé, F.E.; Robles Luna, G.; Borniego, M.B.; Bazzini, A.A.; Asurmendi, S.; García, M.L. Citrus psorosis virus 24K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression. *Mol. Plant Pathol.* **2016**, *17*, 317-329, doi:10.1111/mpp.12282.

[53] Wang, Y.; Wu, J.; Qiu, Y.; Atta, S.; Zhou, C.; Cao, M. Global transcriptomic analysis reveals insights into the response of 'etrog' citron (*Citrus medica* L.) to Citrus Exocortis viroid infection. *Viruses* **2019**, *11*, doi:10.3390/ v11050453.

[54] Rizza, S.; Conesa, A.; Juarez, J.; Catara, A.; Navarro, L.; Duran-Vila, N.; Ancillo, G. Microarray analysis of etrog citron (*Citrus medica* l.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection. *Mol. Plant Pathol.* **2012**, *13*, 852-864, doi:10.1111/j.1364-3703.2012.00794.x.

[55] Vázquez Prol, F.; López-Gresa, M.P.; Rodrigo, I.; Bellés, J.M.; Lisón, P. Ethylene is involved in symptom development and ribosomal stress of tomato plants upon citrus exocortis viroid infection. *Plants* **2020**, *9*, 1-15, doi:10.3390/plants9050582.

[56] Su, X.; Fu, S.; Qian, Y.; Xu, Y.; Zhou, X. Identification of Hop stunt viroid infecting *Citrus limon* in China using small RNAs deep sequencing approach. *Virol. J.* **2015**, *12*, 1-5, doi:10.1186/s12985-015-0332-2.

[57] Owens, R.A.; Sano, T.; Duran-Vila, N. Methods in Molecular Biology. In *Antiviral Resistance in Plants-Methods and Protocols*; Watson, J.M., Wang, M.B., Eds.; 2012; pp. 253-271.

*A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

[58] Hajeri, S.; Ramadugu, C.; Manjunath, K.; Ng, J.; Lee, R.; Vidalakis, G. In vivo generated Citrus exocortis viroid progeny variants display a range of phenotypes with altered levels of replication, systemic accumulation and pathogenicity. *Virology* **2011**, *417*, 400- 409, doi:10.1016/j.virol.2011.06.013.

[59] Alcántara-Mendoza, S.; Alcántara-Mendoza, S.; García-Rubio, O.; Cambrón-San- doval, V.H.; Cambrón-San- doval, D.; Cambrón-San- doval, C. Characterization of Citrus exocortis viroid in different conditions of indexing. *Rev. Mex. Fitopatol.* **2017**, *35*, 284-303.

[60] Nie, X.; Singh, R.P. Viroid Detection and Identification by Bioassay. In *Viroids and Satellites*; 2017; pp. 347-356.

[61] Malfitano, M.; Barone, M.; Duran-Vila, N.; Alioto, D. Indexing of viroids in citrus orchards of Campania, Southern Italy. *J. Plant Pathol.* **2005**, *87*, 115-121.

[62] Kapari-Isaia, T.; Kyriakou, A.; Papayiannis, L.; Tsaltas, D.; Gregoriou, S.; Psaltis, I. Rapid in vitro microindexing of viroids in citrus. *Plant Pathol.* **2008**, *57*, 348-353, doi:10.1111/j.1365-3059.2007.01774.x.

[63] Lin, C.Y.; Wu, M.L.; Shen, T.L.; Yeh, H.H.; Hung, T.H. Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and citrus exocortis viroid infecting citrus in Taiwan. *Virol. J.* **2015**, *12*, 1-11, doi:10.1186/ s12985-015-0247-y.

[64] Pérez, R.; Correa, E.; del Valle, N.; Otero, O. Reduced Indexing Time for Cachexia and Exocortis Diseases in Citrus. In Proceedings of the Fourteenth International Organization of Citrus Virologists Conference; 2000; pp. 386-387.

[65] Ito, T.; Furuta, T.; Ito, T.; Isaka, M.; Ide, Y.; Kaneyoshi, J. Identification of

cachexia-inducible Hop stunt viroid variants in citrus orchards in Japan using biological indexing and improved reverse transcription polymerase chain reaction. *J. Gen. Plant Pathol.* **2006**, *72*, 378-382, doi:10.1007/ s10327-006-0303-y.

[66] Duran-Vila, N.; Semancik, J.S. Variations in the "cross protection" effect between two strains of citrus exocortis viroid. *Ann. Appl. Biol.* **1990**, *117*, 367-377.

[67] Flores, R.; Ownes, R.A. Encyclopedia of Virology. In *Encyclopedia of Virology*; Mahy, B., Regenmortel, M.H.V.V., Eds.; 2008; pp. 332-342.

[68] Duran-Vila, N. Viroids as companions of a professional career. *Viruses* **2019**, *11*, 1-10, doi:10.3390/ v11030245.

[69] Baksh, N.; Lee, R.F.; Gasnsey, S.M. Detection of Citrus Exocortis Viroid by Polyacrylamide Gel Electrophoresis. In Proceedings of the Ninth International Organization of Citrus Virologists Conference Proceedings (1957-2010); 1984; pp. 343-352.

[70] Murcia, N.; Serra, P.; Olmos, A.; Duran-Vila, N. A novel hybridization approach for detection of citrus viroids. *Mol. Cell. Probes* **2009**, *23*, 95-102, doi:10.1016/j.mcp.2008.12.007.

[71] Cohen, O.; Batuman, O.; Stanbekova, G.; Sano, T.; Mawassi, M.; Bar-Joseph, M. Construction of a multiprobe for the simultaneous detection of viroids infecting citrus trees. *Virus Genes* **2006**, *33*, 287-292, doi:10.1007/s11262-006-0067-7.

[72] Semancik, J.S.; Szychowski, J.A.; Rakowski, A.G.; Symons, R.H. Isolates of citrus exocortis viroid recovered by host and tissue selection. *J. Gen. Virol.* **1993**, *74*, 2427-2436, doi:10.1099/0022-1317-74-11-2427.

[73] Kofalvi, S.A.; Marcos, J.F.; Cañizares, M.C.; Pallás, V.; Candresse, T. Hop stunt viroid (HSVd) sequence variants from Prunes species: Evidence for recombination between HSVd isolates. *J. Gen. Virol.* **1997**, *78*, 3177- 3186, doi:10.1099/0022-1317-78-12-3177.

[74] Osman, F.; Dang, T.; Bodaghi, S.; Vidalakis, G. One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts. *J. Virol. Methods* **2017**, *245*, 40-52, doi:10.1016/j.jviromet.2017.03.007.

[75] Wang, X.; Zhou, C.; Tang, K.; Zhou, Y.; Li, Z. A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China. *Eur. J. Plant Pathol.* **2009**, *124*, 175-180, doi:10.1007/s10658-008-9386-y.

[76] Papayiannis, L.C. Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid. *J. Virol. Methods* **2014**, *196*, 93-99, doi:10.1016/j. jviromet.2013.11.001.

[77] Bernad, L.; Duran-Vila, N. A novel RT-PCR approach for detection and characterization of citrus viroids. *Mol. Cell. Probes* **2006**, *20*, 105-113, doi:10.1016/j.mcp.2005.11.001.

[78] Pecman, A.; Kutnjak, D.; Gutiérrez-Aguirre, I.; Adams, I.; Fox, A.; Boonham, N.; Ravnikar, M. Next generation sequencing for detection and discovery of plant viruses and viroids: Comparison of two approaches. *Front. Microbiol.* **2017**, *8*, 1-10, doi:10.3389/ fmicb.2017.01998.

[79] Wu, Q.; Ding, S.W.; Zhang, Y.; Zhu, S. Identification of Viruses and Viroids by Next-Generation Sequencing and Homology-Dependent and Homology-Independent Algorithms. *Annu. Rev. Phytopathol.* **2015**, *53*, 425-444, doi:10.1146/ annurev-phyto-080614-120030.

[80] Martinez, G.; Donaire, L.; Llave, C.; Pallas, V.; Gomez, G. High-throughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem. *Mol. Plant Pathol.* **2010**, *11*, 347-359, doi:10.1111/j.1364-3703.2009.00608.x.

[81] Adkar-Purushothama, C.R.; Perreault, J.P. Impact of nucleic acid sequencing on viroid biology. *Int. J. Mol. Sci.* **2020**, *21*, 1-27, doi:10.3390/ ijms21155532.

[82] Wang, Y.; Atta, S.; Wang, X.; Yang, F.; Zhou, C.; Cao, M. Transcriptome sequencing reveals novel Citrus bark cracking viroid (CBCVd) variants from citrus and their molecular characterization. *PLoS One* **2018**, *13*, 1-12, doi:10.1371/journal.pone.0198022.

[83] Meziane, M.; Frasheri, D.; Carra, A.; Boudjeniba, M.; D'Onghia, A.M.; Mercati, F.; Djelouah, K.; Carimi, F. Attempts to eradicate grafttransmissible infections through somatic embryogenesis in Citrus ssp. and analysis of genetic stability of regenerated plants. *Eur. J. Plant Pathol.* **2016**, *148*, 85-95, doi:10.1007/ s10658-016-1072-x.

[84] Meziane, M.; Boudjeniba, M.; Frasheri, D.; D'Onghia, A.M.; Carra, A.; Carimi, F.; Haddad, N.; Haddad, S.; Braneci, S. Regeneration of Algerian Citrus germplasm by stigma/style somatic embryogenesis. *African J. Biotechnol.* **2012**, *11*, 6666- 6672, doi:10.5897/ajb10.2485.

[85] Ben Mahmoud, K.; Najar, A.; Jedidi, E.; Hamdi, I.; Jemmali, A. Detection of two viroids in the Tunisian sweet orange (*Citrus sinensis* L) cv . Maltese and sanitation via somatic embryogenesis. *J. Chem. Pharm. Res.* **2017**, *9*.

[86] Roistacher, C.N.; Navarro, L.; Murashige, T. Recovery of citrus selections free of several viruses, exocortis viroid, and Spiroplasma *A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid… DOI: http://dx.doi.org/10.5772/intechopen.95914*

citri by shoot-tip grafting in vitro. In Proceedings of the Seventh International Organization of Citrus Virologists Conference Proceedings (1957-2010); 1976; pp. 186-193.

[87] Carimi, F.; De Pasquale, F.; Fiore, S.; D'Onghia, A.M. Sanitation of citrus germplasm by somatic embryogenesis and shoot-tip grafting. In *Options Méditerranéennes, Série B n. 33*; 2001; pp. 61-65.

[88] Kapari-Isaia, T.; Minas, G.J.; Polykarpou, D.; Iosephidou, E.; Arseni, S.; Kyriakou, A. Shoot-tip Grafting in vitro for Elimination of Viroids and Citrus psorosis virus in the Local Arakapas Mandarin in Cyprus. In Proceedings of the Fifteenth International Organization of Citrus Virologists Proceedings; 2002; pp. 417-419.

[89] Ouantar, M.; Bibi, I.; Chebli, B.; Ait Friha, A.T.; Afechtal, M. Prospection et première caractérisation moléculaire de l'exocortis (Citrus exocortis viroid, CEVd ) dans la région du Gharb. *Rev. Marocaine des Sci. Agron. Vétérinaires* **2018**, *6*, 14-18.

[90] Bibi, I.; Afechtal, M.; Chafik, Z.; Bamouh, A.; Benyazid, J.; Bousamid, A.; Kharmach, E. Occurrence and distribution of virus and virus-like Diseases of citrus in North-Est of Morocco - Moulouya perimeter. In Proceedings of the Onzième Congrès de l'Association Marocaine de Protection des plantes, 26-27 Mars 2019, Rabat, Maroc; 2019; pp. 119-134.

[91] Bibi, I.; Kharmach, E.; Chafik, Z.; Benyazid, J.; Bousamid, A. Incidence of Citrus exocortis viroid and Hop stund viroid in commercial citrus groves from Morocco. *Moroccan J. Agric. Sci.* **2020**, *1*, 142-144.

[92] Afechtal, M.; Kharmach, E.; Bibi, I. Survey and molecular characterization of Hop stunt viroid (HSVd) sequence

variants from citrus groves in Morocco. *Moroccan J. Agric. Sci.* **2020**, *1*, 145-148.

[93] Abdel-Salam, A.M.; Abdou, Y.A.; Abou-Zeid, A.A.; Abou-Elfotoh, M.A. Studies on Citrus Exocortis Viroid (CEVd) in Egypt. In *Options Méditerranéennes : Série B. Etudes et Recherches; n. 43*; D'Onghia, A.M., Djelouah, K., Roistacher, C.N., Eds.; 2002; pp. 105-108.

[94] Ragozzino, E.; Faggioli, F.; Barba, M. Distribution of citrus exocortis viroid and hop stunt viroid in citrus orchards of central Italy as revealed by one-tube one-step RT-PCR. *Phytopathol. Mediterr.* **2005**, *44*, 322-326.

[95] Najar, A.; Duran-Vila, N. Viroid Prevalence in Tunisian Citrus. *Plant Dis.* **2004**, *88*, 1286, doi:10.1094/ PDIS.2004.88.11.1286B.

[96] Abou Kubaa, R.; Saponari, M.; El-Khateeb, A.; Djelouah, K. First identification of citrus exocortis viroid (CEVd) and citrus dwarf viroid (CVd-III) in citrus orchards in Syria. *J. Plant Pathol.* **2016**, *98*, 171-185, doi:http:// dx.doi.org/10.4454/JPP.V98I1.045.

[97] Belabess, Z.; Afechtal, M.; Benyazid, J.; Sagouti, T.; Rhallabi, N. Prévalence des phytovirus associés aux agrumes dans la région de Berkane. *African Mediterr. Agric. Journal-Al Awamia* **2020**, *129*, 144-160.

[98] Garnsey, S.M.; Weathers, L.G. Factors Affecting Mechanical Spread of Exocortis Virus lnoculation Procedures. **1967**.

[99] Vidalakis, G.; Pagliaccia, D.; Bash, J.A.; Semancik, J.S. Effects of mixtures of citrus viroids as transmissible small nuclear RNA on tree dwarfing and commercial scion performance on Carrizo citrange rootstock. *Ann. Appl. Biol.* **2010**, *157*, 415-423, doi:10.1111/j.1744-7348.2010.00430.x.

*Citrus - Research, Development and Biotechnology*

[100] Zhang, Z.; Zhou, Y.; Guo, R.; Mu, L.; Yang, Y.; Li, S.; Wang, H. Molecular characterization of Chinese Hop stunt viroid isolates reveals a new phylogenetic group and possible cross transmission between grapevine and stone fruits. *Eur. J. Plant Pathol.* **2012**, *134*, 217-225, doi:10.1007/ s10658-012-9983-7.

#### **Chapter 6**

## Indexing Virus and Virus-Like Diseases of Citrus

*Yasir Iftikhar, Muhammad Zeeshan Majeed, Ganesan Vadamalai and Ashara Sajid*

#### **Abstract**

Citrus is a highly nutritive and prized fruit crop around the world. It contributes a substantial share in local consumption and exports of a nation to earn a handsome foreign exchange. The production of citrus is under the threat of citrus decline. Different factors are responsible for the citrus decline but virus and virus-like diseases have the major role in this decline. Virus and virus-like diseases alone or in association with other biotic and abiotic factors exist in the citrus orchards. Therefore, indexing of diseases caused by virus and virus-like pathogens is the key factor to manage these citrus diseases. Proper facilities and skilled personnel are the pre-requisite for the diseases indexing procedures. Biological, serological and molecular indexing is sensitive, reliable and durable strategy for managing different citrus virus and virus-like diseases under different conditions. Moreover, indexing of viruses and virus-like pathogens are very important for the production of disease free citrus nurseries. This chapter gives a brief review for the commonly used biological, serological and molecular assays for the detection of citrus virus and virus-like pathogens.

**Keywords:** citrus, detection, diseases indexing, viruses and virus-like pathogens, graft-transmissible diseases, viroids, RT-PCR, ELISA

#### **1. Introduction**

Citrus belongs to family Rutaceae and holds an important position among fruits all around the globe. It is the most cultivated fruit in the world after grapes. Citrus is believed to be originated from southeastern Asian region [1]. Northern hemisphere accounts for about 70% of the total citrus production and approximately 80 citrus species are native to India and other tropical and sub-tropical areas of Asia [2]. Citrus being a perennial fruit tree is usually produced through vegetative propagation of scion on rootstock. Combination and compatibility of scion and rootstock can result in high yielding citrus plants. The United States, China, Brazil and the Mediterranean countries contribute two third of global citrus production and are regarded as major citrus producing countries [3]. Citrus products and by-products provide the basis for local agricultural industries, which generate employment and raise income, and in many cases, this industry constitutes an important source of foreign revenue for developed and developing countries such as Pakistan.

A number of factors and certain conditions are collectively responsible for fluctuations in citrus production. Selection of rootstock, agronomic practices and management in citrus nurseries and orchards, propagation methods and biotic and abiotic factors contribute their share to some extent in reduced citrus production. Like other commercial crops, number of diseases, insect pests and genetic problems affect the citrus production. Diseases are one of the major limiting factors for the low citrus production and gives a serious threat to citrus industry. These diseases are caused by fungi, prokaryotes, nematodes, viroids, viruses and virus-like pathogens. Among these, viruses and virus-like pathogens play a major role in citrus decline. These pathogens incur varying degree of damages to citrus plants and make their life span shorter, causing low yield and deterioration of quality and ultimately loss of economy which leads towards the citrus decline [4].

Citrus decline is the matrix of all above mentioned factors and conditions. The common diseases, playing an important role in citrus decline are citrus gummosis caused by *Phytophthora* sp. and *Fusarium* sp., citrus canker caused *by Xanthomonas* sp., Huánglóngbìng caused by *Candidatus laberibactor* sp., citrus stubborn caused by *Spiroplasma citri* and one of the most devastating citrus viruses *i.e.* citrus tristeza virus. Citrus viruses play a vital role in its decline by using the prevailing conditions and many other factors as these are bud/graft-transmissible and have systemic infections. A variety of symptoms has been observed regarding the infection of citrus viruses resulting in systemic infection. No viral diseases on citrus was under discussion or the hot issue before 1940 but during and after 60 years, thirty economically important viruses and virus-like diseases of citrus were recognized as a cause of citrus decline in different parts of the world [5–7]. Unfortunately, citrus orchards are short lived and decline within 15 years as against their potential of 50 years or more. This is mainly attributed, among other factors, to the prevalence of graft-transmissible virus and virus-like diseases, faulty nursery operations and poor orchard management. However, most of the problems originate from nurseries.

Therefore, it is the time when citrus nurseries should operate on highly technical and scientific lines and start providing disease-free and certified plants to the growers. In the first instance, nurseries should be registered and indiscriminate multiplication and sale of uncertified citrus plants must end. For this purpose, the most imperative points such as the prevalence and detection of citrus viral diseases, selection of material, production of disease-free material and streamlined screening procedures are highlighted in this bulletin. If the guidelines are properly followed and certified bud-wood becomes available for producing disease-free citrus plants, the problem of citrus decline can be minimized.

#### **1.1 Citrus pathology**

Citrus pathology is the study of citrus diseases caused by biotic (pathogens) and abiotic factors. It is now being considered as a major part in the field of plant pathology. Being a major fruit crop in the world, citrus production always remains important for the citrus industry. Physiology, morphology, biochemistry and behavior of the citrus tree towards the prevailing climatic conditions are the key areas to be kept in mind while investigating the citrus diseases. Etiology of citrus diseases and their detection methods help to manage these diseases. A plenty of information regarding the diseases of citrus and their control has been published around the world.

#### **2. Virus and virus-like diseases of citrus**

Virus, viroids and virus-like diseases, however, infecting different citrus species could not receive due attention because of the lack of laboratories with proper facilities for their proper identification. These diseases are also known as

#### *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

'graft-transmissible diseases' (GTDs) and the term used for the casual agents is 'citrus graft-transmissible pathogens' (CGTPS) [8]. These are an emerging threat for citrus industry. Major viruses and virus-like pathogens include citrus tristeza virus (CTV), citrus yellow vein clearing virus (CYVCV), citrus variegation virus (CVV), concave gum, psorosis, cristacortis, ringspot, exocortis, *Cachexia-xyloprosis*, *Candidatus liberibacter asiaticus* and *Spiroplasma citri* [9, 10]. A brief description of these virus and virus-like pathogens is summarized below (**Table 1**).

Although plant pathologists have put their efforts for the identification and management of virus and virus-like diseases of citrus but there are some areas need to be investigated. A comprehensive book has been written by Roistacher in 1991 regarding the detection of virus and virus-like diseases of citrus. These diseases reduce the citrus yield and ultimately result in the loss of low foreign exchange. Diseases caused by viruses and virus-like pathogens are infectious, contagious and devastating due to their systemic nature. They are transmitted through different means in nature; through vegetative propagation, by insect vectors and horticultural tools used for the routine activities in citrus orchards and nurseries. These diseases have a considerable economic importance because of their involvement in


*Note: All diseases are graft-transmissible. No adequate information on vector transmission is available except their identity; viroids problems are favored by warm conditions.*

*\*The above summarized information is extracted from the work of [10–12].*

#### **Table 1.**

*Major virus and virus-like diseases of citrus in Pakistan, their transmission and hosts\* .*


*Note: "+" is the indication of presence of infection on the citrus varieties.*

*CTV = Citrus Tristeza Virus, IVV = Infection variegation, RS = Ring spot; Ex = Exocortis, CX = Cachexia xyloporsis, GR = Greening disease, ST = Stubburn Disease, BS = Bark Scaling, GB = Gummy Bark, BU = Bud Union Disease, PS = Psorosis, DE = Decline.*

*[10, 12].*

#### **Table 2.**

*Citrus species and presence of viruses and virus-like diseases.*

the citrus decline [4]. Millions of citrus trees have been died due to CTV. The CGTPS usually have two types of effects either quick decline or long term losses. These diseases are very difficult to control or manage unless or until by the application of integrated management practices. The appropriate diagnosis or indexing method plays an important role for the management of CGTPS [8].

The major symptoms due to virus and virus-like pathogens are vein clearing, bark cracking, yellowing of leaves, leaf dropping, gummosis, mosaic, rugosity, bark scaling, stem pitting, dwarfing, chlorosis and mottling [10, 13]. The virus and viruslike diseases, infecting different citrus species in Pakistan, have been neglected for a long time due to lack of proper facilitations in the research laboratories and skilled personnel for their detection and characterization. A brief description is presented in **Table 2** regarding the citrus species and viruses and virus-like diseases in Pakistan. Indexing facilities are very important for the diagnosis of plant pathogens. Similarly, unlike other pathogens viruses and virus-like pathogens are very sensitive to their indexing through different techniques. Pathogen detection system always played an important role in management of virus and virus-like pathogens. Proper indexing facilities help in the characterization and differentiation of different viruses and their isolates. Management of viruses and virus-like pathogens is only possible when appropriate indexing procedures and facilities are available.

#### **3. Insects as vectors of virus and virus-like pathogens**

Insect pests have always been key role players in the direct or indirect transmission of plant pathogens in agricultural and horticultural crops [14–16]. Citrus tristeza, cachexia-xyloporosis, greening or Huánglóngbìng, infectious variegation, vein *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

enation, yellow vein clearing, exocortis and stubborn are the most conspicuous viral diseases of citrus all over the world including Pakistan [11, 17]. These diseases are usually graft-transmissible and phloem-restricted. Although these diseases along with other fungal, bacterial or mycoplasmic infections of citrus are usually spread through unhealthy mechanical intrusions and by the use of infected uncertified bud, scion or rootstock in plant propagation, many type of sap-feeding insect pests play important role in the transmission of these diseases such as leafhoppers, aphids, psyllids, whiteflies and thrips [17–20].

Among the vector borne viral diseases of citrus, citrus tristeza (CTV) which is caused by a *Closterovirus* is the most dominant and widely studied viral diseases of citrus. It is transmitted by different aphid species primarily by black citrus aphid (*Toxoptera citricida* Kirk.) and cotton-melon aphid (*Aphis gossypii* Glov.) [17, 21]. Another emerging viral disease of citrus is the yellow vein clearing (CYVCV) caused by a *Mandarivirus*. It was first observed in Pakistan in 1988 in the orchards of sour orange (*C. aurantium* L.) and lemon (*C. limon* L.) [22], and later on it was reported in China, India, Iran and Turkey [23–26]. This CYVCV is reported to be vectored by e transmitted by whiteflies and aphids (*Aphis craccivora* and *A. spiraecola*) [25, 27]. Although not virus borne, citrus stubborn is a destructive disease being caused by a bacterium *Spiroplasma citri*. It is usually transmitted by many species of leafhoppers, primarily by *Scaphytopius nitridus* and Circulifer tenellus in citrus-growing suburbs of California and Arizona and by *Circulifer haematoceps* in the Mediterranean zones [17].

#### **4. Indexing strategies**

Indexing is an indispensable procedure to produce and diagnose disease-free plants. Different techniques or combination of techniques have been applied in this regard and the effectiveness of each depends upon the facilities available. Generally indexing can be divided into two types.


Commonly used indexing methods are tissue grafting, budding, insect transmission for biological indexing and enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) for quick indexing strategies. Although all viruses and virus-like pathogens can be detected through PCR and its derivatives, polyacrylamide gel electrophoresis (PAGE) is commonly used for the detection of viroids.

#### **4.1 Biological indexing**

Biological indexing is the inoculation or introduction of virus source (infected sample) into the indicator plants for detection and purification. It involves one of the common indexing methods such as vegetative propagation of infected scion (grafting/budding) to indicator plants, mechanical inoculation of indicator plants or transmission of virus through the insect vector (*e.g.* aphids for CTV, psyllids for greening and leaf hoppers for prokaryotic diseases). Biological indexing is usually


*Note: The above information is extracted from the work of [10, 12] and personal communication of Dr. S.M. Mughal. (Dr. Mughal was also in the team of Dr. Catara during the surveys of citrus growing areas of Pakistan in early 1980's).*

#### **Table 3.**

*Biological indexing of citrus graft-transmissible pathogens.*

time consuming, require glasshouse facilities and takes about 6–12 months for results. At least 3–4 plants are required per treatment. Biological indexing of grafttransmissible pathogens, indicator plants and symptoms in the indicator plants are summarized in **Table 3**.

Detailed methodology for biological indexing has been described much in literature [28–31]. Followings are the generalized and simplified steps to be kept in mind during the biological indexing on the basis of available literature.

i. Sow the seeds of test plants (usually Mexican lime or acid lime) in the sand in germinating tray. Transplant the seedlings in pots having potting media

(sand, soil and moss @ 1:1:1 ratio) after 17–28 days of germination, depending on the germinating conditions.


xv. Temperature range between 65 and 95°F helps the appearance of symptoms on indicator plants for viruses and virus-like pathogens. Observation time also varies from 3 to 16 months for different viruses, virus-like pathogens and viroids.

Laboratory indexing/advanced detection methods

There are rapid methods, highly specific, routinely applicable and some of which test large number of samples. These methods are summarized in **Table 4**. ELISA is the main laboratory indexing method used for the detection of CTV, PAGE for viroids and PCR for all diseases. Mother plants (plants recovered by nucellar embroyony *in-vivo* or *in-vitro*, by thermotherapy or micro-grafted plants or by microbudding may be indexed by any of the above methods. Although 'chromatography' is a useful in chemical indexing of certain virus and virus-like pathogens but it is less reliable than vegetative propagation indexing. Electron microscopy is also helpful for the detection of greening and stubborn diseases other than the viruses. Moreover, *S. citri* can be cultured on a specific medium. Now-a-days, commercial kits are available for the ELISA, PCR and other detection methods along with the instructions.

#### **4.2 Serological assays**

Serology involves the quick indexing of plant viruses, based on the antibody– antigen reaction. Enzyme-linked immunosorbent assay (ELISA) is one of the widely


*Note: Large scale screening of material is possible with any of the method(s) mentioned above. However, there are several limitations including time and availability of proper facilities and trained manpower.*

#### **Table 4.**

*Advanced methods for the detection of viral diseases of citrus.*

*Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

used in detection of plant viruses. It is relatively cheap and can test large number of samples.

ELISA with its derivatives, direct (DAS-ELISA) and indirect (DAC-ELISA), is the main serological indexing tool used for most of the citrus viruses at large scale samples.

Followings are some general steps followed during the ELISA based detection or indexing [35].


**Note:** Repeat the washing step after every step before adding the substrate. Stop the reaction in both types of ELISA with the help of 1 N NaOH.

#### **4.3 Molecular assays**

Molecular detection of citrus viruses and virus-like diseases has revolutionized the subject and provided the platform to detect the early stages of infection to reduce the economic losses. The molecular hybridization techniques supplemented with nucleic acid amplification methods based on PCR, in which high-throughput sequencing approaches can be adopted to identify the strains in relation to evolutionary history or phylogenetic assemblages [36, 37]. Although, nucleic acid based methods are highly sensitive and discriminatory allowing specific strain typing, but it bears the problems in reproducibility [38, 39]. Progressive efforts have been made to decrease the troubleshoots and hurdles to improve the amplification systems by improving the sensitivity and specificity of detection by limiting the high contents of plant related enzyme inhibitors. In contest, nested and multiplex PCR

provides high sensitivity and make the possible to detect several targets in single assay [40]. Moreover, highly sensitive technologies by conducting the amplification of nucleic acids in an isothermal reaction, nucleic acid sequencebased amplification (NASBA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides specific detection of viruses and virus-like diseases.

The addition of real-time PCR for high-throughput testing allows the automation of PCR by combing the fluorimeteric approaches to detect and quantify the targets simultaneously [41, 42]. The combination of different protocols including the serological techniques and molecular approaches will increase the accuracy and reliability of virus diagnostic. Furthermore, in future prospects, nucleic acid arrays and biosensors assisted by nanotechnology will open new corridors to revolutionize the detection of plant viruses and virus-like diseases.

Citrus tristeza virus (CTV) is the most dangerous citrus disease all over the world and is also known as quick decline disease reducing the population of citrus trees significantly [43–45]. However, the utilization of advanced diagnostic methods, such as, biological indexing, electron microscopy (EM), ELISA and PCR or reverse transcriptase PCR (RT-PCR) is providing promising detection of the virus particles and leading towards the management strategies of CTV [46]. The application of conventional PCR is sensitive and specific under optimized and controlled conditions. However, sometimes, it is not possible to judge the amount of pathogens in the samples. Therefore, researchers have to employ other subsequent techniques for complete detection and quantification. Meanwhile, with real-time PCR approach, users can monitor the reaction and also the quantification of the specific pathogen in the sample. While setting up the real-time reaction for virus detection, it is the basic requirement to adapt the specific conditions of the detection system and instrument, and the characteristics of the reaction reagents and cycling procedures in which the most important are primer design, reaction components and conditions. The real-time PCR works well with small amplicons (5–200 bp), while standard PCR allows amplification of several hundred bases without sensitivity and specificity. Moreover, concentrations of MgCl2, primers, and dNTPs are usually higher than conventional PCR [47].

The new developing chemistries are setting up the protocols with different characteristics depending upon the target and assay requirements. In addition to the most widely working chemistries (SYBRGreen, TaqMan, Scorpion, Molecular Beacons), there are more novel chemicals or technologies such as Amplifluor; Locked Nucleic Acid (LNA) Probes, Sigma Proligo; Cycling Probe Technology (CPT), Takara; Light Upon eXtension (Lux) Fluorogenic Primers, Invitrogen Corporation; Plexor Technology, Promega [48, 49]. Real-time technology is being used also in multiplex formatting for the specific detection and strain identification for several viruses [50–55]. Furthermore, real-time reaction in multiplex system is difficult to optimize due to different ratio between the targets and the reaction. The replacement of conventional PCR with real-time PCR is providing new horizons towards the multiple detection system of plant viruses especially of the citrus viruses and virus-like diseases.

#### **5. Detection of citrus viroids**

After the discovery of viroid group of pathogens as an infectious agent to the plants, new aspects in virology were come in front of researchers to be addressed. Viroids are the smallest pathogens which consist of 246 to 401 nucleotides. They are *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

low molecular weight, circular and single stranded RNAs. Viroids exist as free RNA because they lack protein coat [56]. Since viroids do not code for protein and enzyme, they rely on host enzyme for protein synthesis system and replication. To date, 38 viroids have been identified and they are classified into 2 families *i.e. Pospiviroidae* and *Avsunviroidae* [57].

The major economic important viroids in different plants are coconut viroids (CCCVd), citrus viroids (Exocortis and cachexia and variants), Hop stunt viroid and Potato spindle tuber viroids [57]. The origin of viroids is still questionable as they do not have natural host relationship [58, 59].

Citrus production is also affected by viroids. These are the emerging threat to citrus industry. To date, seven citrus viroids have been detected so far in citrus *viz*. *Citrus Exocortis viroid* (CEVd), *Citrus Bent Leaf viroid* (CBLVd), *Hop Stunt viroidcitrus* (HPSVd-cit), *Citrus Dwarfing viroid* (CDVd), *Citrus Bark Cracking viroid* (CBCVd), *Citrus viroid V* (CVd V) and *Citrus viroid VI* (CVd VI-OS). These have been distributed in different geographical areas as shown in **Table 5** [70]. Diseases caused by citrus viroids are citrus exocortis disease (CED), citrus cachexia disease (CCD), citrus leaf bending disease (CLBD), citrus bark cracking disease (CBCD) and citrus dwarfing disease (CDD). Among these, citrus exocortis and citrus cachexia-xyloporosis are the most devastating and widely distributed [57]. These diseases cause a reduction in yield, size of fruit and quality of production [8]*.* These are transmitted directly and through propagation [71]. Stunting, dwarfing, bark cracking, yellowing of leaves, backward leaf bent, pin holing, yield loss and ultimately tree decline are the common symptoms of citrus viroid diseases [63, 71, 72]. Citrus viroids alone or with other viruses or prokaryotes in the host contribute considerably in tree decline [73]. Exocortis and cachexia are the major viroids which are widely distributed in citrus orchards. Other citrus viroids have also been detected from citrus orchards in different parts of the world [73]. Unlike viruses, viroids do not have protein coat, therefore, these are very difficult to detect through serological methods. For this purpose, molecular techniques such as PCR, PAGE are available for the detection of citrus viroids. These are sensitive, sophisticated and rapid detection techniques. Molecular techniques not only help in the detection but also in the characterization of viroids.


#### **Table 5.**

*Geographical distribution of citrus viroids.*


*Note: The above information is extracted from the work of Hammond & Owens (2006) and King et al. (2011).*

#### **Table 6.**

*Classification of citrus viroids (king et al., 2011; Hammond & Owens, 2006).*

#### **5.1 Pathogen description and characterization**

All citrus viroids are classified in different genus under *Pospiviroidae* as mentioned in **Table 6**.

#### **5.2 Diagnostic methods for citrus viroids**

**Biological indexing** is done through graft inoculation in indicator plants. It is very suitable to check the symptoms produced by citrus viroids and their severity. The most important host for indexing CEVd is Etrog citron (*Citrus medica*, Arizona 861) because of its great sensibility and rapid symptom expression [74]. According to Nakahara et al*.* [75], bioassay on Etrog citron is the most sensitive technique in detection of viroids although it takes more time compared to other methods. **Molecular tools** are now widely being used in the detection of citrus viroids. Combinations of several molecular techniques are very useful for reducing the time and to allow large numbers of samples to be examined and to identify each citrus viroid species [75, 76]. **PAGE** is also used to separate variation based on molecular weight. PAGE is not suitable for indexing large number of samples because it is not cost-effective. It is used to test the circularity of viroid RNA by two-dimensional denaturing PAGE (2D-PAGE) [77–79]. Sequential-PAGE is also commonly used and capable of detecting all citrus viroids [71].

**Reverse transcription- polymerase chain reaction (RT-PCR)** is the most commonly used method to detect citrus viroids. It is also a reliable method for quick screening and detection of citrus viroids [80]. It is known for its high specificity and ability to detect unknown viroids or variants [57]. **Multiplex RT-PCR** is another approach to detect simultaneously more than a viroid by using several set of primers. For instance, CEVd, CBLVd, CVd 1-LSS, CVd-II, CVd-III, CVd-IV and CVd-VI were successfully detected simultaneously via multiplex RT-PCR [69]. **Real-time RT-PCR** is also used to detect citrus viroid. It is a quantitative PCR technology basically same as RT-PCR but it measures and quantifies products generated during each cycle of PCR [81]. **Molecular hybridization** is based on the specific interaction between complementary purine and pyrimidine bases forming A-U and G-C base pairs. According to Targon et al*.* [82], imprint hybridization technique is fast, sensitive and economic methods to be used as a routine for citrus viroid indexing in the certification programs. However, dot-blot technique is required an appropriate amount of extracted nucleic acids [75], and it is not suitable *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

for detection of new or unknown viroids. Another molecular approach to detect viroids is Northern blot hybridization. CEVd, CBLVd, CVd-II, CVd-III and CVd-VI were successfully detected by Northern blot hybridization using specific probes in inoculated Etrog citron [83].

#### *5.2.1 RT-PCR for detection of citrus viroids*

#### *5.2.1.1 Samples collection*

Collect the leave samples based on virus and viroids-like symptoms in the field. Bring the leaves samples to laboratory for processing and preservation until use as follows;


#### *5.2.1.2 Nucleic acid extraction*

Extract the nucleic acids from leave samples using the TESLP buffer [84] as follows;


Reverse Transcription Polymerase Chain Reaction [69]:

*5.2.1.3 Synthesis of cDNA*

The extracted RNA is used to run RT-PCR. Reverse Transcription process is carried out in two steps to synthesis cDNA as follows;

#### **Step 1: (1X)**

Experimental RNA = 5 μl Reverse primer = 1 μl Double distilled water = 2.5 μl Total Volume = 8.5 μl The reaction is incubated at 80°C for 12 min then immediately transferred to ice for 5 min.

#### **Step 2: (1X)**

```
AMV-RT = 1 μl
dNTPs = 2 μl
RNAse Inhibitor = 0.5 μl
MgCL2 = 4 μl
RT buffer = 4 μl
Total volume = 11.5 μl
```
The reaction is incubated at 55°C for 30 min. After 30 min, the process is stopped when it reaches to 10°C. The cDNA obtained is stored in 80°C freezer until use (or it can be used immediately).

*5.2.1.4 PCR protocol*

The final volume of PCR should be 25 μl which consists of 12.5 μl of PCR master mix, 5 μl of cDNA, 5.5 μl of sterile double distilled water, 1 μl of forward primer and 1 μl of reverse primer.

The conditions for PCR amplification (35 cycles) are as follows: a. Denaturation:

1.94°C for 10 min


*Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

b. Annealing at 60°C for 10 seconds.

c. Extension at 72°C for 10 seconds and then 5 min.

The list of specific primers used is given in **Table 7**.

#### *5.2.1.5 Agarose gel electrophoresis*

The amplified RT-PCR product is separated using 2% agarose gel as follows [85];


#### *5.2.1.6 PCR product purification*

Positive PCR products with expected size are purified using MinElute® Gel Extraction Kit according to the standard protocol provided with Kit.

1.The expected size of band is excised from the agarose gel with a sterile, sharp scalpel.


#### **Table 7.**

*List of specific primer for citrus viroids [69].*


*5.2.1.7 Molecular Cloning (TOPO TA cloning kit, Invitrogen)*

Positive PCR samples will be cloned using the TOPO TA cloning kit according to the standard protocol provided along with the Kit as follows;

Ligation


*Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

Transformation


**Note:** Strictly follow the incubation time and temperature in the protocol during cloning.

#### *5.2.1.8 Two Dimensional poly acrylamide gel electrophoresis (2D PAGE)*

2D PAGE is carried out to for the detection and to check the circularity of Viroid RNA. Following is the recipe and protocol for PAGE.

Gel Ingredients


#### **Non Denaturing Gel**:



#### **5% Non-denaturing Gel**



#### **Silver Staining**:


*Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*


#### **5.3 Citrus tristeza virus (Ctv): a case study**

#### *5.3.1 Introduction*

CTV belongs to the genus *Closterovirus* of the family *Clostervoiridae*. Virus particle is a monopartite, positive sense, comprising of ssRNA genome of approximately 20Kb in size. It is the largest known form of a plant virus and its genome is encapsulated in a flexuous rod 2000 nm long particles composed of coat protein subunits of 25KDA [86–89]. ssRNA genome comprised of 19,296 nucleotides that encode for 12 open reading frames [90]. CTV probably originated in Asia and has been spread to all citrus growing areas by infected plant material movement and now is widely distributed to all major citrus growing areas as summarized in **Table 8**. Over the two decades *i.e.* 1930–1950, millions of citrus trees were destroyed due to CTV infection and citrus orchards were almost wiped out in Brazil, Spain, and Argentina. This virus was the killer of three million citrus trees grafted on sour orange rootstock alone in south California [91–94]. The tristeza disease was first reported in Florida in 1959 and by 1980s became the serious threat to citrus industry [95]. By 1991, an estimation of total world loss of 100 million trees was recorded due to CTV in Argentina, Brazil, Spain, California, Venezuela and other areas [96, 97]. Several strains of CTV have been identified primarily on the basis of their biological reaction in several citrus species and indicator plant. The major groups of strains are mild that cause barely detectable clearing of leaf veins in Mexican lime; decline–inducing strains cause death of trees when propagated on sour orange rootstock. Stem pitting strains cause mild to severe pitting of stems and branches of grapefruit and orange resulting in low yield [95, 98]. Almost all the citrus varieties and hybrids have been infected with CTV [91]. Symptom expression of CTV in citrus hosts is highly variable and depends upon host species (rootstock and scion combination), virulence of CTV isolates and soil or environmental conditions. Characteristics symptoms of CTV are vein clearing, decline, stem pitting, seedling yellows, stunting and leaf corking on different citrus hosts like sweet orange, grapefruit, grafted on sour range root stock. Severity of infection and symptoms expression on cultivars vary from mild to severe isolates [99–101]. CTV is transmitted in nature by different species of aphids in a semi-persistent manner and through grafting [102, 103]. The most efficient vector involved in semipersistent manner is *T. citricida* Kirkaldy (brown or black citrus aphid) when compared with other aphids.

#### *5.3.2 Indexing*

**Serological and biological indexing:** Indexing includes biological, serological and molecular methods, which are the common procedures according to their reliability, sensitivity and duration to detect the CTV. During a survey in Spain, 22 CTV isolates were collected on the basis of geographical information, source tree and symptomology and then were characterized by biological indexing. Diversified


#### **Table 8.**

*Geographical Distribution of Citrus tristeza closterovirus.*

symptoms were produced on 9 indicator species. Mexican lime was found to be a good indicator host [104].

In Morocco, 14 diverse isolates were selected from samples during survey and then characterized on the basis of reaction pattern. Among these 14 isolates, four were severe and two were mild isolates. Isolates were also indexed against a series of monoclonal antibodies [105]. DAS-ELISA was used to detect the CTV from the samples collected during a survey in Western and Midwestern development regions of Nepal [106]. One hundred and eighty-eight samples were analyzed through biological indexing and DAS- ELISA to detect tristeza, psorosis and similar diseases like-symptoms including viroids in orange varieties in all the regions and the cachexia was detected as the most important and widespread disease [107]. Biological indexing is still considered as an important tool using for the characterization of CTV isolates. Different strains were identified through symptoms expression on differential hosts, including Mexican lime and sweet orange. Moreover, they

#### *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

observed visual symptoms of different strains on Mexican lime and sweet orange through biological indexing followed by ELISA [108]. Detection of CTV in Spain was compared by indexing using monoclonal and polyclonal antibodies [109].

**Molecular indexing:** Different nucleic acid based indexing methods have been developed for the quick detection of CTV. The adaptability of these methods depends upon the reliability, time duration and sensitivity. Alteration in protein patterns in rootstock bark from CTV infected tree were analyzed through PAGE [110]. There was a clear modification in protein pattern but not in CTV free trees. Similarly, Northern blot technique was used to compare dsDNAs extracted from CTV infected and CTV free plants. Two out of the three CTV isolates were detected by this method [111]. CTV was also detected in the three aphid species through RT-PCR. IC-RT-PCR was used to amplify the coat protein gene [112]. Sensitivity of cDNA probe was slightly better than hybridization with 32P-labeled probe. Similarly, hybridization with tissue print with DIG-probe could differentiate CTV isolates grown under green house or field conditions [113]. In Taiwan, RT-PCR was found to be a rapid and sensitive assay than other serological methods but one step RT-PCR, which is the combination of reverse transcriptase and polymerase chain reaction in one tube. It is more sensitive and detects the CTV when virus concentration is very low. Comparison between ELISA and RT-PCR revealed that ELISA was better than RT-PCR at detecting mild CTV strains as the virus was detected in all parishes, while RT-PCR detected CTV in only 8 parishes. It would appear that the primers used for RT-PCR are more specific for severe CTV isolates [114]. Some modifications were introduced in PCR-ELISA to increase its sensitivity and reduced the costs of detection. PCR-ELISA is the immune-detection of PCR products and effective for detection and differentiation of plant viral nucleic acids. PCR-ELISA being a laborious and expensive method was modified and simplified by using asymmetric PCR. It made PCR-ELISA more sensitive than TaqMAN™, a fluorescence-based detection method.

Three microscopy procedures for detecting CTV were compared which provided additional alternatives for very rapid CTV indexing, including the use of EM, SSEM and light microscopy. In light microscopy, inclusions were found in young phloem tissues of all CTV-infected hosts examined. Similarly, in SSEM virus particles were found on grids prepared with antiserum and extracts from infected tissue. CTV particles could be detected in pooled samples representing one in 100. Similarly, virus particle fragments were observed infrequently in samples representing one infected plant in 1,000 samples [32].

#### **6. Conclusion**

Citrus is an important fruit crop of the world and has a great potential for local consumption, export purposes and industrial uses. Unfortunately, citrus orchards are facing the problem of low productivity due to citrus decline. This is mainly attributed, among other factors to the prevalence of graft-transmissible virus and virus-like diseases, unhygienic nursery operations and poor orchard management. However, most of the problems arise from nurseries. It is the time that the nurseries should operate on highly technical and scientific lines and should work on providing disease-free and certified plants to the citrus growers. To establish the disease free nurseries, indexing of virus and virus-like diseases are the major area that needs to be focused. Implication of traditional and modern high-throughput biological, serological and molecular indexing techniques, such as ELISA, RT-PCR, PAGE, should be put in practice for the detection and indexing of virus and virus-like diseases of citrus plants. Moreover, citrus nurseries should be registered and indiscriminate multiplication and sale of uncertified citrus plants should be prohibited.

#### **Author details**

Yasir Iftikhar<sup>1</sup> \*, Muhammad Zeeshan Majeed<sup>2</sup> , Ganesan Vadamalai<sup>3</sup> and Ashara Sajid<sup>1</sup>

1 Department of Plant Pathology, College of Agriculture, University of Sargodha, Pakistan

2 Department of Entomology, College of Agriculture, University of Sargodha, Pakistan

3 Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, Selangor, Malaysia

\*Address all correspondence to: yasir.iftikhar@uos.edu.pk

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

#### **References**

[1] Davies FS, Albrigo LG. History, distribution and uses of citrus fruit. Citrus, CAB international. Printed in Great Britain by Redwood books. Trowbridges, Wiltshine, 1994;1–254.

[2] Hooker JD. 1872. Flora of British India. Reeve and co. London.

[3] Liu P. World markets for organic citrus and citrus juices. Food and Agriculture Organization of the United Nations, 2003; 6p.

[4] Khan MM, Y. Iftikhar Y, Abbas M, Naqvi SA, Iqbal, MT, Jaskani MJ, Khan IA. Disease free citrus nursery production system. In Proc. "Workshop on production of disease free citrus nursery plants". Institute of Horticultural Sciences, University of Agriculture, Faisalabad, September 12–13, 2007; 29–35.

[5] Iftikhar Y. Serological, Biochemical and Molecular characterization of Citrus tristeza virus. Thesis submitted in department of Plant Pathology, University of Agriculture, Faisalabad, 2009.

[6] Bove JM. 1995. Virus and virus-like diseases of citrus in the near east region. F.A.O. Rome. pp: 239–266.

[7] Khan IA. 1992. Virus and virus like diseases of citrus. In: Khan, I. A (Ed.), Proceedings of 1st International Seminar on Citriculture in Pakistan. University of Agriculture, Faisalabad, Dec 2–5, pp 343–352.

[8] Roistacher CN (Ed.). 1991. Graft transmissible diseases of citrus. F.A.O, Rome, 1991: 266.

[9] Iftikhar Y. Some Biological and physical properties of yellow vein clearing virus of lemon. Thesis submitted in department of Plant Pathology, University of Agriculture, Faisalabad, 2004.

[10] Mughal SM. Symptomatology, detection, distribution and management of virus and virus-like diseases of citrus in Pakistan. In: Proc. Int. Symp. Citriculture, U.A.F. Pakistan. 2004;106– 113.

[11] Arif, M., Ahmad, A., Ibrahim, M. and Hassan, S. (2005). Occurrence and distribution of virus and virus-like diseases of citrus in north-west frontier province of Pakistan. *Pakistan Journal of Botany*, 37(2), 407–421.

[12] Catara, A., Azzaro, A., Mughal SM., and Khan DA. (1988). Virus, viroids and prokaryotic diseases of citrus in Pakistan. In: *Proceedings 6th Int. Citrus Cong.* Mar.6-11. pp. 957-962.

[13] Iftikhar Y, Mughal SM, Khan MM, Khan MA, Nawaz MA, Hussain Z. Symptomatic expression of tristezainfected citrus plants in Pakistan. Archives of Phytopathology and Plant Protection. 2013; 46: 98–104.

[14] Kluth, S., Kruess, A. and Tscharntke, T. (2002). Insects as vectors of plant pathogens: mutualistic and antagonistic interactions. *Oecologia*, 133 (2), 193–199.

[15] Agrios, G.N. (2009). *Transmission of plant diseases by insects*. Available: http:// entomology.ifas.ufl.edu/capinera/e ny5236/pest1/content/03/3\_plant\_disea ses.pdf

[16] Wu, F., Qureshi, J. A., Huang, J., Fox, E. G. P., Deng, X., Wan, F., Liang, G. and Cen, Y. (2018). Host plantmediated interactions between '*Candidatus Liberibacter asiaticus*' and its vector *Diaphorina citri* Kuwayama (Hemiptera: Liviidae). *Journal of Economic Entomology*, 111(5), 2038–2045.

[17] Roistacher, C.N. (2004). Diagnosis and management of virus and virus like diseases of citrus. In: *Diseases of Fruits*

*and Vegetables* (Volume I), Springer, Dordrecht. pp. 109–189.

[18] Mitchel, P.L. (2004). Heteroptera as vectors of plant pathogens. *Neotropical Entomology*, 33, 519–545.

[19] Raccah, B. and Fereres, A. (2009). *Plant Virus Transmission by Insects*; John Wiley and Sons, Ltd.: Chichester, UK.

[20] Heck, M. (2018). Insect transmission of plant pathogens: A systems biology perspective. Msystems, 3(2). e00168–17.

[21] Bar-Joseph, M. and Nitzan, Y. (1991). The spread and distribution of citrus tristeza virus isolates in sour orange seedlings. In: *Proceedings of the 11th Conference International Organization of Citrus Virologists (IOCV)*, Riverside, CA. pp. 162–165.

[22] Catara, A., Azzaro, A., Davino, M., and Polizzi, G. (1993). Yellow vein clearing of lemon in Pakistan. In: *Proceedings of the 12th Conference International Organization of Citrus Virologists (IOCV)*, Riverside, CA. pp. 364–367.

[23] Önelge, N. (2002). First report of yellow vein clearing of lemons in Turkey*. Journal of Turkish Phytopathology,* 32, 53–55.

[24] Alshami, A., Ahlawat, Y.S. and Pant, R.P. (2003). A hitherto unreported yellow vein clearing disease of citrus in India and its viral etiology. *Indian Phytopathology*, 56(4), 422–427.

[25] Zhou, Y., Chen, H., Cao, M., Wang, X., Jin, X., Liu, K.H. and Zhou, C.Y. (2017). Occurrence, distribution, and molecular characterization of Citrus yellow vein clearing virus in China. *Plant Disease*, 101(1), 137–143.

[26] Hashmian, S.B. and Aghajanzadeh, S. (2017). Occurrence of citrus yellow vein clearing virus in citrus species in

Iran. *Journal of Plant Pathology*, 99(1), 290–297.

[27] Önelge, N., Satar, S., Elibuyuk, O., Bozan, O. and Kamberoolu, M. (2011). Transmission studies on citrus yellow vein clearing virus. In: I*nternational Organization of Citrus Virologists Conference Proceedings* (1957–2010) (Vol. 18, No. 18).

[28] Noordam, D. Identification of Plant viruses. Methods and Experiments. Centre of Agril. Publishing and documentation, Wageningen, 1973; 207p.

[29] Ashfaq M, Mughal SM, Iftikhar Y, Khan MA, Khan NA. Study on host range, serology and inclusion bodies of yellow vein clearing virus (YVCV) of lemon. Pakistan Journal of Phytopathology. 2004;**16:** 1–4.

[30] Iftikhar Y, Mughal SM, Ashfaq M, khan MA, Haq IU. Some biological and physical properties of yellow vein clearing virus of lemon. Pakistan Journal of Phytopathology. 2004; **16**: 5–8.

[31] Ahlawat YS. Diagnosis of Plant viruses and Allied Pathogens. Studium Press (India) Pvt. Ltd. 2010; 224p.

[32] Garnsey SM, Christie RG, Derrick KS, Bar-Joseph M. Detection of Citrus tristeza virus II. Light and Electron microscopy of inclusions and viral particles. In: Calavan, EC, Garnsey, SM and Timmer, LW (Eds.), Proceeding of 8th International organization of citrus virologists, conference, California, Riverside, 1979, U.S.A. pp.9–16.

[33] Garnsey SM., and Cambra M. (1991). Enzyme-linked immunosorbent assay (ELISA) for citrus pathogens. In: Roistacher CN ed. Graft transmissible diseases of citrusHandbook for detection and diagnosis. Rome, Italy, FAO. Pp. 193-216

[34] Semancik, JS., Duran-Vila, N. (1991). The Grouping of Citrus Viroids: *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

Additional Physical and Biological Determinants and Relationships with Diseases of Citrus. UC Riverside International Organization of Citrus Virologists Conference Proceedings, (1957-2010), 11(11) ISSN 2313-5123. https://escholarship.org/uc/item/ 64r9q712.

[35] Iftikhar Y, Khan MA, Rashid A, Mughal SM, Iqbal Z, Batool A, Abbas M, Khan MM, Muhammad S, Jaskani MJ. Occurrence and distribution of Citrus tristeza Closterovirus in the Punjab and NWFP, Pakistan. Pakistan Journal of Botany. 2009; 41: 373–380.

[36] Wylie, SJ, Wilson, CR, Jones, RAC, Jones, MGK. A polymerase chain reaction assay for *Cucumber mosaic virus* in lupin seeds. Australian Journal of Agricultural Research, 1993; **44:** 41–51.

[37] Olmos, A., Bertolini, E, Gil, M, Cambra, M. Real-time assay for quantitative detection of nonpersistently transmitted *Plum pox virus* RNA targets in single aphids. Journal of Virological Methods, 2005; **128:** 151–155.

[38] Bertolini, E, Olmos, A, Martínez, MC, Gorris, MT, Cambra, M. Singlestep multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees. Journal of Virological Methods, 2001; **96:**33–41.

[39] Olmos, A, Bertolini, E, Cambra, M. Isothermal amplification coupled with rapid flowthrough hybridisation for sensitive diagnosis of *Plum pox virus*. Journal of Virological Methods, 2007; **139:**111–115.

[40] Bertolini, E, Olmos, A, López, MM, Cambra, *M. multiplex* nested reverse transcriptionpolymerase chain reaction in a single closed tube for sensitive and simultaneous detection of four RNA viruses and *Pseudomonas savastanoi* pv. *savastanoi* in olive trees.

Phytopathology, 2003; **93:**286–292.

[41] Walsh, K, North, J, Barker, I, Boonham, N. Detection of different strains of *Potato virus Y* and their mixed infections using competitive fluorescent RT–PCR. Journal of Virological Methods, 2001; **91:** 167–173.

[42] López, MM, Bertolini, E, Marco-Noales, E, Llop, P, Cambra, M. Update on molecular tools for detection of plant pathogenic bacteria and viruses. In Molecular diagnostics: current technology and applications, J.R. Rao, C. C. Fleming, and J.E. Moore, eds. Horizon Bioscience, Wymondham, UK, 2006: 1–46.

[43] Rocha-Pena, MA, Lee, RF, Lastra, R, Niblett, CL, Ochoa-Corona, FM, Garnsey, SM, Yokomi, RK. Citrus tristeza virus and its aphid vector Toxoptera citricida: threats to citrus production in the carribean and central and North America. Plant Disease, 1995; **79(5):** 437–445.

[44] Ahlawat, YS. Viruses, greening bacterium and viroids associated with citrus (Citrus species) decline in India. Indian Journal of Agricultural Science, 1997; **67:**51–57.

[45] Bar-Joseph M, Marcus R, Lee RF. The continous challenges of citrus tristeza virus control. Annual Review of Phytopathology, 1989;**27:** 291–316.

[46] Biswas, KK. Molecular diagnosis of Citrus tristeza virus in mandarin (*Citrus reticulata*) orchards of Darjeeling hills of West Bengal. Indian Journal of Virology, 2008; **19:** 26–31.

[47] López, MM, Llop, P, Olmos, A, Marco-Noales, E, Cambra, M, Bertolini, E. Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses?. Current issues in molecular biology, 2009; **11(1):** 13.

[48] Lukhtanov, EA, Lokhov, SG, Gorn, VV, Podyminogin, MA, Mahoney, W.

Novel DNA probes with low background and high hybridizationtriggered fluorescence. Nucleic Acids Research, 2007; **35:** 5 e30. doi:10.1093/ nar/gkl1136.

[49] Gasparic, MB, Cankar, K, Zel, J, and Gruden, K. Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms. BMC Biotechnology, 2008; doi:10.1186/1472-6750-8-26.

[50] Korimbocus, J, Coates, D, Barker, I, and Boonham, N. Improved detection of *Sugarcane yellow leaf virus* using a realtime fluorescent (TaqMan) RT-PCR assay. Journal of Virological Methods, 2002; **103:** 109–120.

[51] Beuret, C. Simultaneous detection of enteric viruses by multiplex real-time RT-PCR. Journal of Virological Methods, 2004; **115:** 1–8.

[52] Munford, RA, Skelton, A, Metcalfe, E, Walsh, K, and Boonham, N. The reliable detection of Barley yellow and mild mosaic viruses using realtime PCR (TaqMan). Journal of Virological Methods, 2004; **117:** 153–159.

[53] Varga, A, and James, D. Use of reverse transcription loop-mediated isothermal amplification for the detection of *Plum pox virus*. Journal of Virological Methods, 2006; **138:** 184– 190.

[54] Agindotan, BO, Shiel, PJ, and Berger, PH. Simultaneou detection of potato virus, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan(®) real-time RT-PCR. Journal of Virological Methods, 2007; **142:** 1–9.

[55] Kogovsek, P, Gow, ., Pompe-Novak, M, Gruden, K, Foster, GD, Boonham, N, and Ravnikar, M. Single-step RT realtime PCR for sensitive detection and discrimination of Potato virus Y isolates. Journal of Virological Methods, 2008; **149:** 1–11.

[56] Agrios, G.N. (2005). Plant Pathology 5th Edition. *Department of Plant Pathology, University of Florida.*

[57] Hadidi, A., Flores, R., Randles, J.W., and Semancik, J.S. (2003). Viroids. *Science Publisher, Inc.*

[58] Bostan H, Nie X, Singh RP (2004). An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids. J. Virol. Methods, 116: 189–193

[59] Bar-Joseph, M. (1996) A contribution to the natural history of viroids.*Proc. 13th IOCV Conf.* (Riverside, CA, USA), pp. 226–229.

[60] Pagliano, G., Umaña, R., Pritsch, C., Rivas, F. and Duran-Vila, N. (2013). Occurrence, prevalence and distribution of citrus viroids in Uruguay. *Journal of Plant Pathology*. pp. 631-635

[61] Bani Hashemian, S.M., Taheri, H., Duran-Vila, N. and Serra, P. (2010). First Report of Citrus viroid V in Moro Blood Sweet Orange in Iran. *Plant Disease: An International Journal of Applied Plant Pathology*. pp. 129-129

[62] Semancik, J.S., Morris, T.J., Weathers, L.G., Rordorf, G.F., and Kearns, D.R. (1975). Physical properties of a minimal infectious RNA (viroid) associated with the exocortis disease. *Virology 63*: 160-167

[63] Duran-Vila, N., Pina, J.A., Ballester, J.F., Juarez, J., Roistacher, C.N., Rivera-Bustamante, R., and Semancik, J.S. (1988). The Citrus Exocortis Disease: *A complex of viroid-RNAs. Tenth IOCV.*

[64] Ashulin, L., Lachman, O., Hadas, R., and Bar-Joseph, M. (1991). Nucleotide sequence of a new viroid species, *Citrus bent leaf viroid* (CBLVd) *Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

isolated from grapefruit in Israel. *Nucleic Acids Res*.

[65] Cao, M. J., Atta, S., Liu, Y. Q., Wang, X. F., Zhou, C. Y.,Mustafa, A. and Iftikhar, Y. (2009). First report of Citrus bent leaf viroid and Citrus dwarfing viroid from citrus in Punjab, Pakistan. *Plant Disease 2009* Vol. 93 No. 8 pp. 840.

[66] Rakowski, A.G., Szychowski, J.A., Avena, Z.S. and Semancik, J.S. (1994). Nucleotide sequence and structural features of the group III citrus viroids. *J Gen Virol* 75:3581-3584.

[67] Putcha, H., Ramm, K., Luckinger, R., Haddas, R., Bar-Joseph, M. and Sanger, H.L. (1991). Primary and secondary structure of citrus viroid IV, a new chimeric viroid present in dwarfed grapefruit in Israel. Nucleic Acids Res 19:6640

[68] Serra, P., Eiras, M., Bani-Hashemian, S. M., Murcia, N., Kitajima, E. W., Daròs, J. A., Flores, R., and Duran-Vila, N. (2008). Citrus viroid V: Occurrence, host range, diagnosis, and identification of new variants. *Phytopathology 98*:1199-1204.

[69] Ito, T., Ieki, H. and Ozaki, K. (2002). Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction. *Journal of Virological Methods 106*, 235–239.

[70] Srivastava, S., & Prasad, V. (2020). Viroids: small entities with a mean punch. In Applied Plant Virology (pp. 209–226). Academic Press.

[71] Garnsey, S.M, Zies, D.L., Irey, M., Sieburth, J.S., Semancik, J.S., Levy, L. and Hilf, M.E. (2002). Practical field detection of citrus viroids in Florida by RT-PCR. *Fifteenth IOCV Conference.*

[72] Eiras, M., Silva, S.R., Stuchi, E.S., Carvalho, S.A., and Garcez, R.M.

(2013). Identification and characterization of viroids in 'Naveline ISA 315'sweet orange. *Tropical Plant Pathology, 38:* 058–062.

[73] Abubaker, M. Y. A., & Elhassan, S. M. (2010). Survey and molecular detection of two citrus viroids affecting commercial citrus orchards in the Northern part of Sudan. Agric. Biol. JN Am, 1(5), 930–937.

[74] Pagliano, G., Peyrou, M., Campo, R. D., Orlando, L., Gravina, A., Wettstein, R., and Francis, M. (2000). Detection and characterization of citrus viroids in Uruguay. *Fourteenth IOCV Conference.*

[75] Nakahara, K., Hataya, T., Uyeda, I., and Ieki, H. (1998). An Improved Procedure for Extracting Nucleic Acids from Citrus Tissues for Diagnosis of Citrus Viroids*. Ann. Phytopathol. Soc. Jpn. 64*: 532–538.

[76] Murcia, N., Serra, P., Olmos, A., and Duran-Vila, N. (2009). A novel hybridization approach for detection of citrus viroids. *Molecular and Cellular Probes* 23.

[77] Schumacher, J., Randles, J.W., and Riesner, D. (1983). A two dimensional electrophoretic technique for detection of circular viroids and virusoids. *Anal. Biochem.* 135:288

[78] Nakaune, R. and Nakano, M. (2006). Efficient methods for sample processing and cDNA synthesis by RT-PCR for the detection of grapevine viruses and viroids. *Journal of Virological Methods 134*: 244–249

[79] Jiang, D., Hou, W., Kang, N., Qin, L., Wu, Z., Li, S., and Xie, L. (2012). Rapid detection and identification of viroids in the genus Coleviroid using a universal probe. *Journal of Virological Methods.*

[80] Kunta, M., Gracxa, J.V.D., and Skaria, M. (2007). Molecular Detection and Prevalence of Citrus Viroids in Texas. *Hortscience* 42(3):600–604.

[81] Papayiannis, L.C. (2013). Diagnostic real-time RT-PCR for the simultaneous detection of *Citrus exocortis viroid* and *Hop stunt viroid*. *Virological Methods 196*: 93–99

[82] Targon, M.L.P.N., Carvalho, S.A.D., Stuchi, E.S., Souza, J.M., Muller, G.D. and Machado, K.M.B.M.A. (2005). Hybridization techniques for indexing of citrus viroids in Sao Paulo State, Brazil. *LARANJA, Cordeiropolis*: Volume 26, p25–38.

[83] Narayanasamy, P. (2010). Microbial Plant Pathogens-Detection and Disease Diagnosis: *Viral and Viroid Pathogens.*Vol 3. *Springer Science & Business Media.*

[84] Ito, T., Ieki, H. and Ozaki, K. (2000). A population of variants of a viroid closely related to citrus viroid-I in citrus plants*. Archives of Virology 145*: 2105–2114

[85] Bernard, L., and Duran-Vila, N. (2006). A novel RT-PCR approach for detection and characterization of citrus viroids. *Mollecular and Cellular Probes 20*: 105–113

[86] Mehta P, Brlansky RH, Gowda S. Reverse Transcription Polymerase Chain Reaction Detection of Citrus Tristiza Virus in Aphids. Plant Disease, 1997; **81:**1066–1069.

[87] Mathews DM, Riley KR, Dodds JA. Comparison of detection methods for citrus tristeza virus in field trees during months of non-optimal titer. Plant Disease, 1997; **81:** 525–529.

[88] Niblett CL, Genc H, Cevik B, Halbert S, Brown I, Nolasco G, Bonacalza B, Manjunath KL, Febres VJ, Pappu HR, Lee RF. Progress on strain differentiation of citrus tristeza virus and its application to the epidemiology

of citrus tristeza disease. Virus Research, 2000; **71:**97–106.

[89] Suastika G, Natsuaki T, Terui H, Kano T, Leki H, Okuda S. Nucleotide sequence of citrus tristeza virus seedlig yellows isolates. Journal of General Plant Pathology, 2001; **67:** 73–77.

[90] Bar-Joseph M, Lee RF. 1989. Citrus tristeza virus. AAB Descriptions of Plant Viruses no. 353. AAB, Wellesbourne (GB). http://www.ncbi.nlm.nih.gov/ ICTVdb/ICTVdB/

[91] Anonymous. 2004. Citrus Tristeza Closterovirus. Data sheets on Quarantine pests. EPPO A2 list, No.93. www.**eppo**.org/**QUARANTINE**/virus/ **Citrus**\_**tristeza**/

[92] Bar-Joseph M, Marcus R, Lee RF. The continous challenges of citrus tristeza virus control. Annual Review of Phytopathology, 1989;**27:** 291–316.

[93] Kallsen C. 2002. Controlling citrus tristeza virus in the San Joaquin valley of California. Citrus subtropical Horticulture/Pistachios. http://cekern. ucdavis.edu/Custom\_Program143/ Controlling\_Citrus\_Tristeza\_Virus\_in\_ the\_SJ\_Valley\_of\_California.htm.

[94] Mooney P, Harty A. 1992. Citrus tristeza virus. The Orchardist. http:// www.hortnet.co.nz/publications/scie nce/kk0992.htm.

[95] Futch SH, Brlansky RH. 2005. Field diagnosis of citrus tristeza virus. HS996, one of a series of the Horticultural services department, Florida cooperative extension service. IFAS, University of Florida, U.S.A. http://edis. ifas.ufl.edu.

[96] Bar-Joseph M, Roistacher CN, Garnsey SM, Gumpf DJ. A review of tristeza, an ongoing threat to citriculture. In: Proceeding of International Society of Citriculture, Tokyo, Japan, 1981; 419–423.

*Indexing Virus and Virus-Like Diseases of Citrus DOI: http://dx.doi.org/10.5772/intechopen.95897*

[97] Mooney P, Dawson T, Harty A. 1994. Citrus tristeza virus preimmunization strategies. The Orchadist. http://www.hortnet.co.nz/ publications/science/kk0894.htm

[98] Chung KR, Brlansky RH. Citrus diseases exotic to Florida: Citrus Tristeza Virus-Stem Pitting (CTV-SP). Fact Sheet. pp 227. Plant Pathology Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, 2006, Florida. http://edis.ifas.uf l.edu.

[99] Brlansky RH, Damsteegt VD, Howd DS, Roy A. Molecular analysis of citrus tristeza virus subisolates separated by aphid transmission. Plant Disease, 2003; **87:**397–401

[100] EPPO Bulletin. Protocol for the diagnosis of quarantine organism, citrus tristeza closterovirus. 2004; **34:** 239– 246.

[101] Lbida B, Bennani A, Serrhini MN, Zemzami M. Biological, serological and molecular characterization of three isolates of citrus tristeza *closterovirus* introduced into Morocco. OEPP/EPPO Bulletin 2005; **35:**511–517.

[102] Brown LG, Denmark HA, Yakomi RK. Citrus Tristeza Virus and its vectors. In; Florida. Plant Pathology circular no.311. Florida Department of Agriculture and Consumer Service. Division of Plant Industry. 1988.

[103] Komazaki S. Biology and virus transmission of citrus aphid. Akitsu Branch, Fruit and Tree Research Station, Ministry of Agriculture, Forestry and Fisheries, Hiroshima, Japan, 1993. http://www.fftc.agnet.org/ library/tb/136/

[104] Ballester OJF, Pina JA, Carbonell EA, Moreno P, Hermoso de Mandoza A, Cambra M, Navarro L. Biological diversity of citrus tristeza virus (CTV)

isolates in Spain. Plant Pathology, 1993; **42:**219–229.

[105] Zemzami M, Garnsey SM, Nadori EB, Hill JH. Biological and serological characterization of citrus tristeza virus (CTV) isolates from Morocco. Phytopathologia-Mediterranea, 1999; **38:** 95–100.

[106] Malla S, Sah DN. Severity and prevalence of citrus tristeza virus (CTV) in the western and Mid-Western regions of Nepal. Working paper lumle Agricultral Research Centre, 2001; 14.

[107] Besoain XA, Valenzuela M, Castro M, Ballester Olmos JF. Current status of some virus and virus like diseases of citrus in Chile. Fitopatologia. 2000;**35:** 98–104.

[108] Polek M, Gumpf DJ, C. M. Wallen CM, Riley KM. Biological Characterization of Naturally Occurring *Citrus tristeza virus* Strains in California Citrus. In: In: M.E. Hilf, N. Duran-Vila and M.A. Rocha-Pena (Eds.), Proceeding of the 16th International Organization of Citrus Virologist Conference, California, Riverside, 2005, USA:68–74.

[109] Cambra M, Serra J, Bonet JC, Moreno P. Present status of the citrus tristeza virus in the valencian community. In: L. W. Timmer, S. M. Garnsey and L. Navarro. (Eds), Proceeding of 10th International Organization of Citrus Virologists, California Riverside, 1998; 1–7.

[110] Moreno, P., J. Guerri and J. Ortiz. Alteration of bark proteins associated with citrus tristeza virus (CTV) infection on susceptible citrus species and scion-rootstock combinations. Phytopathology, 1989; **125**:55–66.

[111] Acikgoz S. Detection of citrus tristeza virus (CTV) isolates with northern blot hybridization. Doga Turk Tarim-ve-Ormancilik Dergisi, 1991;**15:** 836–840.

[112] Anfoka GH, Abhary MK, Fattash I, Nakhla MK. Occurrence and distribution of citrus tristeza virus in the Jordan valley. Phytopathologia Mediterranea, 2007;44:17–23.

[113] Narvaez G, Skander BS, Ayllon MA, Rubio L, Guerri J, Moreno P. A new procedure to differentiate citrus tristeza virus isolates by hybridization with digoxigenin-labelled cDNA probes. Journal of Virological Methods, 2000; **85:** 83–92.

[114] Fisher L, Tennant P, Molaughlin W. 2005. Detection and differentiation of Citrus Tristiza Virus (CTV) in Jamaica using ELISA, RT-PCR, DNA hybridization and RFLP. Ministry of Agriculture's citrus replanting projectresearch services. Conference, The Environment: Biodiversity, O-23, Faculty of Pure and Applied Sciences, University of West Indies. www.mona. uwi.edu/fpas/conference/fpas7.

#### **Chapter 7**

## *Xanthomonas citri* ssp. *citri* Pathogenicity, a Review

*Juan Carlos Caicedo and Sonia Villamizar*

#### **Abstract**

The infectious process of plant by bacteria is not a simple, isolated and fortuitous event. Instead, it requires a vast collection of molecular and cell singularities present in bacteria in order to reach target tissues and ensure successful cell thriving. The bacterium *Xanthomonas citri* ssp. *citri* is the etiological agent of citrus canker, this disease affects almost all types of commercial citrus crops. In this chapter we review the main structural and functional bacterial features at phenotypical and genotypical level that are responsible for the symptomatology and disease spread in a susceptible host. Biological features such as: bacterial attachment, antagonism, effector production, quorum sensing regulation and genetic plasticity are the main topics of this review.

**Keywords:** Biofilm, Secondary Metabolites, Antibiotic, Xanthomonadine, Quorum sensing

#### **1. Introduction**

The surface of the plants is one of the most hostile environments, prevailing factors at the phyllosphere such as: the low availability of nutrients, the high incidence of UV rays, the fluctuating periods of temperature and humidity, mechanical disruption by winds, antibacterial compounds produced by the host plant or by microorganisms member of leaf microbiome, among others, make the bacterial persistence and survival itself a pathogenicity strategy. Due the symptoms development ceases when one pathway involved in the bacterial epiphytic survival is seriously threatened [1]. In phytopathogenic bacteria whose infection route is the phyllosphere, it is important to understand how phenotypic traits upset to ensure survival and surface fitness, and how these traits interact with the phyllosphere microbiome in order to secure the onset of infection (**Figure 1**). Besides, over the time, on a large-scale, plant leaves will age and fall, thus, the phyllosphere bacteria must have to anticipate living outside of the leaf, for example in the air, soil or reach to young leaves [2].

Bacterium *Xanthomonas citri* ssp. *citri* (*Xcc*) is the etiological agent of bacterial citrus canker. This bacterium is equipped with a huge arsenal of cellular structures that allow its survival in the phyllosphere before it reaches the target mesophyll tissue. *Xcc* secretes toxins that directly affect the survival of its competitors. Once in the mesophilic tissue *Xcc* produces effectors that are responsible by the appearance of spongy and corky pathognomonic lesion of citrus canker. In this chapter we will review the both bacterial life style outside and inside of the host.

#### **Figure 1.**

*Phytopathogenic bacteria plant infection. A. Surface leaf survivor and biofilm formation. B. Bacterial movement to natural opening on leaf. C. Phytotoxins secretion to modulate stomatal closure. D. Effector secretion that affect the cell host behavior. E. Degrading cell wall plant protein secretion.*

#### **2.** *Xanthomonas citri* **ssp.** *citri* **taxonomy**

The bacterium *Xantomonas citri* ssp. *citri* is a gram negative rod shape bacteria with a single polar flagellum. *Xcc* belongs to the *Xanthomonas* genus from the gamma proteobacteria group. This genus is constituted by 28 species and more than 150 pathovars [3]. In the early 1900s, due to pathogenicity experiments, the bacterium was classified as *Pseudomonas citri* [4]. Subsequently, the bacterium was classified into different genus such as: *phytomonas bacterium* and finally at late 1930 classified as *Xanthomonas citri* [5]. The bacterium continued in *X. citri* until 1978, when it was classified in *X. campestris* pv. *citri* in order to reserve citri at the specific level [6]. In 1989 Gabriel suggest the replaced of bacterium as *X. citri* [7]. Using DNA–DNA hybridization approach and based on renaturation rates, the bacterium was classified as *X. axonopodis* pv. *citri* by Vauterin [8]. Lately, It was suggested major changes to Xanthomond taxonomy, it which were based on multilocus sequence analysis (MLSA) and digital DNA–DNA hybridization of whole genome nucleotide, the author has been recommend the names *Xanthomonas citri ssp citri* for the etiological agent of citrus cancer type A [9].

#### **3. Microbe- host interaction**

#### **3.1 Microbe -host interaction outside the susceptible host "epiphytic life style"**

Bacterial citrus canker disease cycle begins with the deposit of inoculum of *XCC* at the leaf surface by rain splash. Subsequently, the bacteria move toward the natural opening of leaves, the stomata, then, the bacteria reach the apoplastic space and start the infection process inside the host "endophytic lifestyle". In this section we are going to focus on the structures, toxins, molecules and extracellular substances that favor and promote the epiphytic interaction between XCC and susceptible citrus host.

Xanthomonas citri *ssp.* citri *Pathogenicity, a Review DOI: http://dx.doi.org/10.5772/intechopen.97776*

#### *3.1.1 Type IV pili*

Several bacterial genera are endowed with filamentous appendages called pili. These filamentous organelles include the chaperon- Usher pili, type IV pili (T4P) and gram-positive pili. All types of pili are homopolymers ensembled of thousands of units of pilin protein. The outstanding function of pili is the attachment to surfaces, besides, in *Xcc* pili type IV is also responsible for the twitching motility and biofilm formation [10]. Type 4 pili is unique in its dynamism, since, it polymerizes and depolymerizes in very fast cycles, which leads to instantaneous extension and retraction cycles producing considerable mechanical force [11], as a consequence, this organelle could attract several substrates like DNA or bacteriophages in order to internalize to periplasmic space, as well as to secrete protein across the membrane [12]. Twitching motility is a bacterial displacement that able to cell to move over humid on organic and inorganic surfaces on a fashion independent of flagella [13]. In the process of biofilm development, the T4P contributes in the initial steps exactly in the reversible attachment phase and subsequently, in the formation of mushroom microcolonies. Contribution of T4P in the pathogenicity in XCC is not completely demonstrated, however, the mutation of *pilM* gene responsible to encode a membrane protein that participate in the T4P pili ensemble reduce drastically the bacterial virulence [10].

#### *3.1.2 Type V secretion system (non fimbrial adhesins)*

Xanthomonads encode type V secretion system (T5SS), it which has a function as non fimbrial adhesins [14]. Compared with the other bacterial secretion systems, the secretion system 5 is one of the simplest complexities from the structural point of view; it is smaller and has only presence at the outer membrane of gram negative bacteria [15]. This T5SS do not have a direct energy source, there is no ATP accessible in the periplasm space neither proton gradient. Consequently, the name of autotransporter has been coined for the this T5SS [16]. The T5SS is comprised of two domains: the β barrel that is located at the out membrane and a secreted passenger. There are five subtypes of T5SS from Va-Ve and recently a new subtype the Vf has been discovered [17]. The bacterium *Xcc* is endowed with three subclasses of T5SS: Va, Vb and Vc. (**Figure 2**). Va is a classical auto-transporter, it which transport proteases, lipases and adhesins. The type Vb is a secretion system knowing as Two-Partner Secretion System (TPS), which is composed by a translocator protein and a cognate passenger protein. Translocation from the cytoplasm to the periplasm space occurs by Sec translocase pathway once the perception of amino terminal from signaling peptide is done. The passenger protein has effector function and is termed TpsA. It is transported by TpsB, which forms a pore in the outer membrane in order to enable the TpsA translocation. TpsB also comprise two periplasmic domains. TpsB typically contains a 16-stranded beta-barrel domain that forms the outer membrane pore and two periplasmic POTRA (Polypeptide transport associated). Its function is the recognition of the cognate partner via binding to a TPS domain in TpsA.

The T5SS subclass Vc have a trimeric transporter adhesin conformation, this surface exposed adhesin assembles as homotrimeric structure at the outer membrane [18]. Proteomic and functional studies involving T5SS have revealed roles in pathogenicity to host primarily implicated in the adhesion, especially in the initial steps of pathogenicity process [19, 20].

#### *3.1.3 Xanthomonadin pigment*

Xanthomonads bacteria produce a yellow pigment membrane bound known as Xanthomonadins. Several studies have shown that Xanthomonadin has a pivotal

#### **Figure 2.**

*Schematic representation of T5SS present in Xcc.* β *barrel domain and POTRA are characterized with blue, linker, passengers transported are represented in green and two partner secretion system domain are characterized with red.*

role in a epiphytic survival and in plant-pathogen interaction [21, 22]. In the early years this yellow pigment was associated with the carotenoids. However, it was only until its full characterization was achieved that this pigment represents a unique group of aryl-polyene, water insoluble new type of pigment [23]. Genomic analysis shows that a region near to 25.4 kb contains seven transcriptional units (*pigA*, *pigB, pigC, pigD, pigE, pigf* and *pigG).* This gene cluster encodes necessary elements for Xanthomonadin biosynthesis [24]. Biological roles of xanthomonadin in a pathogenicity context are: (i). *favor the bacterial epiphytic survival,* since, Xanthomonadin avoid the photodamage produced by UV light irradiation that results in ROS production. Similar as structural related carotenoids, Xanthomonadin absorbs wavelengths between UV-C to red light. This pigment gives the bacteria additional advantages against the other phyllosphere colonizer bacteria as it is to deal with stress related factors such as UV irradiation and consequently the photo oxidative damage. Xanthomonadin also offers protection against visible light in the presence of exogenous photosensitizers. Cellular location of Xanthomadin (outer membrane) strongly suggests that this pigment stabilizes cell membrane in the epiphytic phase of this phytopathogenic bacterium. Previous studies in which *Xcc* deletion mutants of the *pig* genes were used and which were inoculated using the needleless syringe pressure technique did not show a significant reduction in virulence compared to the wild type phenotype inoculated using the same technique. Instead, when using the spray

infection method, that resembles the natural infection method, it which involve the epiphytic fitness stage, the *Xcc* pig mutant strains display great reduction in the virulence compare with the wild type phenotypes [25]. (ii) *Antioxidant activity*, the oxidative stressors as ROS and H202 injury the membranes, DNA and proteins, the carotenoids pigments could efficiently quench the ROS.

#### *3.1.4 EPS xanthan and LPS*

The EPS in Xanthomonas is named as xanthan, this polysaccharide surrounds the outer membrane through non-covalent ligations [26]. Pathogenicity roles in Xanthomonas genus differ greatly depending on specie, e.g. in *Xanthomonas campestris*, xanthan suppresses induced innate immunity by calcium chelation [27]. In addition xanthan increases the plant susceptibility to *X. campestris* due to avoiding the callose deposition [28]. In Xac there is controversy regarding the direct participation of xanthan in the pathogenicity process, while some authors find just a discrete participation in the epiphytic survival [29], another study shows that xanthan deletion mutants reduce the surface leave colonization ability and consequently the severity of citrus canker disease was deeply reduced [30]. Xanthan is a key component in the biofilm formation. The gene cluster *gum* is responsible for the xanthan production and exportation. This gene cluster comprise 12 successive genes with one operon-like identical direction of transcription i.e. *gumB* to *gumM.* The first two genes of cluster *gumB* and *gumC* encode components of channel than spans the outmembrane and the periplasmic space and enable the xanthan secretion [31].

The LPS is the major component of the outer leaflet of the outer membrane. The LPS in *Xcc* have a classic conformation being a tripartite glycoconjugate forming by: lipid A that carries a core oligosaccharide and polysaccharide the O- antigen. LPS that lack the O-antigen are named as lipooligosaccharide (LOS) or rough-type LPS. LPS has an essential role in bacterial growth acting as a barrier for antibacterial compounds and delivering protection against stress as well contributing to the structural proprieties of outer membrane. Lipid A is fairly conserved in most gram-negative bacteria, however, in Xanthomonas genus there is variation in the core oligosaccharide and O antigen structures, there may even be variation between the different species of Xanthomonas [32]. Nowadays is has been established that LPS has a double role in plant-microbial interaction; (i) elicitor of immunity plant response and (ii) It has a role in the promotion of virulence, because it acts as a barrier against antimicrobial activity compounds produced by root hair. *Xcc* is able to overwhelming plant defense responses induced by LPS.

#### *3.1.5 Quorum sensing and biofilm formation*

One discovery in microbiology that completely changed the conception of microbial ecology in the last two decades was the establishment of cooperative behavior in bacterial populations. This social behavior allows members of the bacterial community to adapt to new ecological niches, colonize new habitats, gain a competitive advantage against potential competitors and resist or avoid the host defense [33]. This cooperative behavior is based on a cell to cell communication system known as Quorum Sensing. Quorum sensing (QS) is a system of bacterial cell–cell communication that enables the microorganism to sense a minimum number of cells (quorum) in order to respond to external stimuli in a concerted fashion [34]. The process of QS relies upon the production, release and detection of small signaling molecules called auto-inducers. Each bacterial cell produces a basal quantity of auto-inducers, which are exported to the extracellular environment and reflect bacterial population density. At high cell densities, the auto-inducers reach a critical concentration, at which point they are recognized by their cognate receptor, triggering a cascade of biological functions [35].

The autoinducer in *Xcc* is a short chain fatty acid molecule known as DSF (Diffusible Signal Factor). Once this DSF accumulates at the extracellular space up to a critical level, it is sensed by its cognate receptor and triggers a cascade of biological function via the internal second messenger cyclic di-GMP, which is involved in virulence, resistance and biofilm formation. The encoding genes for quorum sensing components in *Xcc* form a cluster termed as *rpf* (Regulation of Pathogenicity Factors). For detailed revision of DSF quorum sensing circuit in *Xcc* [36].

Once *Xcc* reaches a leaf surface, it begins the initial adhesion process that was mention above. This attachment is followed by the formation of biofilm-like structures. Biofilm classical definition is an aggregated composed by several bacterial communities, which are embedded in a self-produced matrix of EPS, these bacterial cells are attached to each other or/and to a surface [37]. Biofilm is composed by polysaccharides, nucleic acids (eDNA), proteins, and have a pivotal role in attachment and protection against biotic and abiotic factors. In *Xcc* the biofilm formation in leaf and fruit surfaces is a main virulence factor in the early stage of development of citrus canker disease. In *Xcc* biofilm formation and dispersion is modulated by the quorum sensing autoinducer molecule DSF. How it was mention before DSF autoinducer promotes the biofilm formation because it stimulates the EPS production and pilus ensemble. On the other hand, DSF negatively regulates the biofilm formation because; it upregulates β 1–4 mannanase, ManA, leading to EPS dispersion and disassembly of biofilm [38]. Our previous study shown that quorum sensing signaling plays an essential role in the epiphytic stage survival, which is crucial at the early phase of pathogenicity development. Since, quorum quenchers bacteria belonging to genus Pseudomonas and Bacillus, it which were isolated from leaves of susceptible citrus host, which displayed the ability to disrupt the DSF pathway in *Xcc* and reduce citrus canker severity in a high susceptible citrus host [34].

#### *3.1.6 T4SS and T6SS potentiates the Xcc antagonism with bacteria inhabiting the phylloplane and the soil amoeba*

Nutrient limitation in the phyllosphere additional to environmental changes conditions, make the surface of the leaves one of the most hostile, restrictive and competitive habitats [38]. The type IV protein secretion system is used by bacteria to inject proteins and/or DNA into the prokaryotes and eukaryotes targets. Xanthomonas are endowed with genes that encode components of T4SS, the encoding genes VirB7, VirB8 and VirB9 responsible for the outer membrane pore formation. Genes that encode for VirB3, VirB4, VirB6, VirB8, Vir11, VirD4 and VirB10, responsible for the pore formation at the inner membrane. Finally, the gene that encodes for the subunits VirB2 and VirB5 that form the extracellular pilus structure. Besides, the encoding gene for VirB1 subunit predicted as a periplasmic lytic transglycosylase that plays a role in peptidoglycan alteration throughout T4SS biogenesis [39].

A recent study shows that in *Xcc* there are near to 12 proteins that interact with inner membrane associated ATPase VirD4, that is responsible for the recognition of substrates to be secreted [40]. These proteins share a C terminal domain termed XVIPCDs (Xanthomonas VirD4-interacting proteins conserved domains). These proteins are translocated into the target bacteria cell resulting in the dead of the receptor cells [41]. This bactericidal T4SS is knowing as X-T4SS and the effectors secreted by this nanomachine are termed X-Tfes (Xanthomonadales likeceeae t4SS effectors). Finally, a recent study reported that T6SS protect *Xcc* against the predatory amoeba Dyctiostelium [42].

#### **3.2 Microbe -host interaction inside the susceptible host**

Once the bacterium *Xcc* reaches the mesophilic tissue, after of epiphytic fitness and survival events mention before, must have to face the host defense response and parallel to express the pathogenicity factors;

#### *3.2.1 T3SS the main pathogenicity determinant*

The type 3 Secretion systems T3SS is the main protein secretion system widely studied in relationship to the pathogenicity. This secretion system is shared with several pathogenic bacteria ranging from animal to plants. This system is known as the "needle" and it works by delivering effector proteins directly to the target cells and modifying their behavior. Effectors from *Xcc* strains determine the host range. i.e. avirulence factors limit the specificity at the pathogen race/cultivar level by triggering immunity reactions in hosts with a related specific resistance gene. [43]. The effector delivered by the T3SS in *Xcc* belongs to the AvrBs3/PthA family. *Xcc* contains four PthA genes that encode transcription activator-like effector (TALE); of these four genes, pthA4 is responsible for the formation of citrus canker lesions. In citrus host the gene known as CsLOB is targeted by the TALE encoded by the *Xcc* gene pthA4; this gene was assessed in two susceptible host to *Xcc* infection, i.e., grape fruit and sweet orange [44]. CsLOB1-specific function still remains unclear; some previous studies suggest that CsLOB1 is involved in the regulation of development of lateral organ and metabolism of nitrogen and anthocyanin. Some plant hormones such as auxin, gibberellin, and cytokines also have proven to exert an effect on CsLOB1 gene [45]. Therefore, TALE have been shown to promote host cell transcriptional reprogramming as a virulence strategy [46].

#### **4. Conclusions**

The bacterium *Xcc* uses various adaptation and colonization strategies, it which are mainly aimed at guaranteeing its epiphytic survival, either by overcoming stress factors of biotic origin (predators, competitors, nutrient limitation) and abiotic origin (UV radiation, humidity and temperature variability). Because, this phase of epiphytic adaptation is crucial for the subsequent development of citric cancer symptoms in the susceptible host. Despite, these mechanism not having a direct effect on the health of the host, they become virulence factors, since its abolition avoid the subsequent development of the characteristic symptoms of citric cancer. Already inside the host, the bacterium uses as the main direct pathogenicity factor, the inoculation of effector proteins TALE, this effector is responsible for inducing cell hyperplasia, leading to rupture of the leaf epidermis and resulting in raised corky and spongy lesions surrounded by a water-soaked margin, the pathognomonic lesson of bacterial citrus canker.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Juan Carlos Caicedo1 \* and Sonia Villamizar2

1 Universidad de Santander, Faculty of Exact, Natural and Agricultural Science, Research Group CIBAS, Bucaramanga, Colombia

2 School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, Brazil

\*Address all correspondence to: jua.caicedo@mail.udes.edu.co

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Xanthomonas citri *ssp.* citri *Pathogenicity, a Review DOI: http://dx.doi.org/10.5772/intechopen.97776*

#### **References**

[1] Pfeilmeier S, Caly DL, Malone JG. Bacterial pathogenesis of plants: future challenges from a microbial perspective: Challenges in Bacterial Molecular Plant Pathology. Mol Plant Pathol. 2016 Oct;17(8):1298-1313. doi: 10.1111/ mpp.12427.

[2] Vorholt JA. Microbial life in the phyllosphere. Nat Rev Microbiol. 2012 Dec;10(12):828-840. doi: 10.1038/ nrmicro2910. PMID: 23154261.

[3] Bull CT, De Boer SH, Denny TP et al (2012) List of new names of plant pathogenic bacteria (2008 2010). J Plant Pathol 94(1):21-27

[4] Hasse, C.H. (1915) Pseudomonas citri, the cause of citrus canker – a preliminary report. J. Agric. Res. 4, 97-100.

[5] Dowson, W.J. (1939) On the systematic position and generic names of the gram negative bacterial plant pathogens. Zentr. Bakteriol. Parasitenk. Abt. II. 100, 177-193.

[6] Young, J.M., Dye, D.W., Bradbury, J.F., Panagopoulos, C.G. and Robbs, C.F. (1978) Proposed nomenclature and classification for plant pathogenic bacteria. N. Z. J. Agric. Res. 21, 153-177

[7] Gabriel, D.W., Kingsley, M.T., Hunter, J.E. and Gottwald, T. (1989) Reinstate- ment of Xanthomonas citri (ex Hasse) and Xanthomonas phaseoli (ex Smith) to species and reclassification of all Xanthomonas campestris pv citri strains. Int. J. Syst. Bacteriol. 39, 14-22

[8] Vauterin, L., Hoste, B., Kersters, K. and Swings, J. (1995) Reclassification of Xanthomonas. Int. J. Syst. Bacteriol. 45, 472-489.

[9] Constantin, E.C., Cleenwerck, I., Maes, M., Baeyen, S., Van Malderghem, C., De Vos, P. and Cottyn, B. (2016) Genetic characterization of strains named as Xanthomonas axonopodis pv. dieffenbachiae leads to a taxonomic revision of the X. axonopodis species complex. Plant Pathol. 65, 792-806.

[10] German Dunger, Cristiane R. Guzzo, Maxuel O. Andrade, Jeffrey B. Jones, and Chuck S. Farah (2014) Xanthomonas citri subsp. citri Type IV Pilus Is Required for Twitching Motility, Biofilm Development, and Adherence Molecular Plant-Microbe Interactions 27:10, 1132-1147

[11] Ribbe, J., Baker, A. E., Euler, S., O'Toole, G. A. & Maier, (2017) B. Role of cyclic Di-GMP and exopolysaccharide in type IV pilus dynamics. J. Bacteriol. 199, e00859–e00816

[12] Ellison, C. K. et al (2018). Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural Sheetz, M. pili enables transformation in Vibrio cholerae. Nat. Microbiol. 3, 773-780.

[13] Henrichsen, J. 1983. Twitching motility. Annu. Rev. Microbiol. 37:81-93.

[14] Moreira LM, de Souza RF, Almeida Jr NF, Setubal JC, Oliveira JC, Furlan LR, Ferro JA, da Silva AC (2004) Comparative genomics analyses of citrus-associated bacteria. Annu Rev Phytopathol 42:163-184.

[15] Leo, J. C., Grin, I., and Linke, D. (2012). Type V secretion: mechanism(S) of autotransport through the bacterial outer membrane. Philos. Trans. R. Soc. B Biol. Sci. 367, 1088-1101. doi: 10.1098/ rstb.2011.0208

[16] Drobnak, I., Braselmann, E., Chaney, J. L., Leyton, D. L., Bernstein, H. D., Lithgow, T., et al. (2015). Of linkers and autochaperones: an unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion. Mol. Microbiol. 95, 1-16. doi: 10.1111/ mmi.12838

[17] Grijpstra, J., Arenas, J., Rutten, L., and Tommassen, J. (2013). Autotransporter secretion: varying on a theme. Res. Microbiol. 164, 562-582. doi: 10.1016/j. resmic.2013.03.010

[18] Fan E, Chauhan N, Udatha DBRKG, Leo JC, Linke D. Type V Secretion Systems in Bacteria. Microbiol Spectr 2016;4. https://doi.org/10.1128/ microbiolspec. VMBF-0009-2015.

[19] Bogdanove AJ, Koebnik R, Lu H, Furutani A, Angiuoli SV, Patil PB, et al. Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp. J Bacteriol 2011;193:5450-5464.

[20] Mhedbi-Hajri N, Darrasse A, Pigné S, Durand K, Fouteau S, Barbe V, et al. Sensing and adhesion are adaptive functions in the plant pathogenic xanthomonads. BMC Evol Biol 2011;11:67.

[21] Park, Y.J., Song, E.S., Noh, T.H., Kim, H., Yang, K.S., Hahn, J.H. et al. (2009) Virulence analysis and gene expression profiling of the pigmentdeficient mutant of Xanthomonas oryzae pathovar oryzae. FEMS Microbiol Lett 301: 149-155

[22] Rajagopal, L., Sundari, C.S., Balasubramanian, D., and Sonti, R.V. (1997) The bacterial pigment xanthomonadin offers protection against photodamage. FEBS Lett 415: 125-128.

[23] Andrewes, A.G., Jenkins, C.L., Starr, M.P., Shepherd, J., and Hope, H. (1976) Structure of xanthomonadin I, a novel di- brominated aryl-polyene pigment produced by the bacterium Xanthomonas juglandis. Tetrahedron Lett 17: 4023-4024

[24] Poplawsky, A.R., and Chun, W. (1997) pigB determines a diffusible factor needed for extracellular polysaccharide slime and xanthomonadin production in Xanthomonas campestris pv. campestris. J Bacteriol 179: 439-444.

[25] Poplawsky, A. R., Urban, S. C., & Chun, W. (2000). Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris. Applied and environmental microbiology, 66(12), 5123-5127.doi. org/10.1128/aem.66.12.5123-5127.2000

[26] BeckerA, Vorholter F-J. Xanthan biosynthesis by Xanthomonas bacteria: an overview of the current biochemical and genomic data. In: Bernd H. A. Rehm (ed). Microbial Production of Biopolymers and ·polymer Precursors: Applications and Perspectives. UK: Caister Academic Press, 2009, 1-12

[27] Aslam SN, Newman MA, Erbs G et al. Bacterial polysaccharides suppress induced innate immunity by calcium chelation. Curr Biol 2008;18:1078-1083.

[28] Yun MH. Xanthan induces plant susceptibility by suppressing callose deposition. PLANT Physiol 2006;141:178-187.

[29] Dunger G, Relling VM, Tondo ML et al. 2007. Xanthan is not essential for pathogenicity in citrus canker but contributes to Xanthomonas epiphytic survival. Arch Microbiol;188:127-135.

[30] Rigano LA, Siciliano F, Enrique R et al. 2007. Biofilm Formation, Epi- phytic Fitness, and Canker Development in Xanthomonas axonopodis pv. citri. Mol Plant-Microbe Interact;20:1222-30.

[31] Bianco MI, Jacobs M, Salinas SR et al. 2014. Biophysical characterizaion of the outer membrane polysaccharide export protein and the polysaccharide co-polymerase protein from Xanthomonas campestris. Protein Expr Purif;101:42-53.

Xanthomonas citri *ssp.* citri *Pathogenicity, a Review DOI: http://dx.doi.org/10.5772/intechopen.97776*

[32] Molinaro A, Silipo A, Lanzetta R et al. 2003. Structural elucidation of the O-chain of the lipopolysaccharide from Xanthomonas campestris strain 8004. Carbohydr Res;338:277-281

[33] Ng WL, Bassler BL, 2009. Bacterial quorum-sensing network architectures. Annual Review of Genetics 43, 197-222.

[34] Caicedo JC, Villamizar S, Ferro MIT, Kupper KC, Ferro JA. (2016). Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity. Plant Pathology.;65:782-791

[35] Federle MJ, Bassler BL, 2003. Interspecies communication in bacteria. Journal of Clinical Investigations 112, 1291-1299.

[36] Juan Carlos Caicedo, Sonia Villamizar and Jesus Aparecido Ferro (2017). Quorum Sensing, Its Role in Virulence and Symptomatology in Bacterial Citrus Canker, Citrus Pathology, Harsimran Gill and Harsh Garg, IntechOpen, DOI: 10.5772/66721.

[37] Vert, M. et al. Terminology for biorelated polymers and applications (IUPAC Recommendations 2012). Pure Appl. Chem. 84, 377-410 (2012).

[38] Lindow SE, Brandl MT, 2003. Microbiology of the phyllosphere. Applied and Environmental Microbiology 69, 1875-1883.

[39] Ilangovan A, Connery S, Waksman G. (2015). Structural biology of the Gram-negative bacterial conjugation systems. Trends Microbiol;23:301-310.

[40] Sgro GG, Oka GU, Souza DP, Cenens W, Bayer-Santos E, Matsuyama BY, et al. 2019. Bacteriakilling type IV secretion systems. Front Microbiol;10:1078.

[41] Alegria MC, Souza DP, Andrade MO, Docena C, Khater L, Ramos CHI, et al. 2005. Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri. J Bacteriol;187:2315-25.

[42] Bayer-Santos E, Lima L dos P, Ceseti L de M et al. Xanthomonas citri T6SS mediates resistance to Dictyostelium predation and is regulated by an ECF σ factor and cognate Ser/Thr kinase. Environ Microbiol 2018;20:1562-1575.

[43] He YQ, Zhang L, Jiang BL et al. 2007. Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris. Genome Biol;8:R218.

[44] Yang H, Junli Z, Hongge J, Davide S, Ting L, Wolf BF, et al (2014). Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proceedings of the National Academy of Sciences of the United States of America.;111:E521-E529

[45] Majer C, Hochholdinger F. (2011). Defining the boundaries: Structure and function of LOB domain proteins. Trends in Plant Science.;16(1):47-52. DOI: 10.1016/j.tplants.2010.09.009

[46] Peng Z, Hu Y, Zhang J, Huguet-Tapia JC, Block AK, Park S, et al. (2019 ). Xanthomonas translucens commandeers the host rate-limiting step in ABA biosynthesis for disease susceptibility. Proc Natl Acad Sci;116:20938-46. https://doi. org/10.1073/pnas.1911660116

#### **Chapter 8**

## Climate Change and Citrus

*Waqar Shafqat, Summar A. Naqvi, Rizwana Maqbool, Muhammad Salman Haider, Muhammad Jafar Jaskani and Iqrar A. Khan*

#### **Abstract**

Climate change is the change in the statistical distribution of weather patterns that lasts for an extended period. Climate change and agriculture are interrelated processes and affect in many ways. Citrus fruits are one of the largest fruit crops in the world. Yield loss at a drastic level due to abiotic stress annually in which temperature and water stress are the main environmental factors. These factors cause biochemical, anatomical, physiological, and genetic changes in plant structure and lead to defective growth, development, and reproduction, which ultimately cause a reduction in the economic yield of the crop. An increase in temperature and water stress at critical phenological stages of citrus results in reduced tree fruit set, decrease in fruit growth and size, increase in fruit acidity, low tree yield, reduced fruit peel thickness, and pre-harvest fruit drop. Stomatal conductance and net carbon dioxide assimilation in citrus leaves can be reduced by super optimal leaf temperature. Water deficit reduces the transpiration rate, stomatal conductance by stomatal closure associated with ABA content and causes an abrupt decrease in photosynthesis and CO2 assimilation in citrus which reduce trees overall growth and production. Interventions in agronomic practices, breeding strategies, and biotechnological approaches can mitigate climate change effects on citrus. The groundwork against climate change is compulsory for better global livelihood and food security.

**Keywords:** Citrus fruits, environment, global warming, abiotic stress, genetic improvement, climatic adaptation

#### **1. Introduction**

Citrus and its related genera i.e., Poncirus, Eremocitrus, Fortunella, and Microcitrus belong to the family Rutaceae [1, 2]. Citrus is a prominent fruit tree of tropical and sub-tropical regions that require a suitable climate for quality production. Citrus fruit quality and quantity are inclined by multiple factors including climatic conditions [3]. Change in optimum climate elements like low temperature/freezing, heat stress/heatwaves, CO2 assimilation, drought/water scarcity, intensive rainfall, and relative humidity, may affect directly and indirectly citrus production [4].

Citrus tree (rootstock and scion) growth, development, fruit production, and fruit quality is reduced under the biotic and abiotic stresses [5]. Citrus with tolerant rootstocks against biotic and abiotic factors improve the growth and productivity of the trees [6]. The potential citrus yield is 18–20 tones ha−1, which goes up to

25 tones ha−1 in the developed world; however, the citrus average yield in Pakistan is 10–12 tones ha−1 and is affected by abiotic and biotic stresses [7]. The yield gap is due to biotic, abiotic, and general factors, like agronomic practices in countries of climate risk [8].

The productivity and growth of plants are affected by climate change especially drought and high temperatures collectively [9]. Reactive oxygen species accumulate superoxide and hydrogen peroxide [10] is due to water stress and high-temperature stress which reduce the biochemical, physiological, and molecular regulation. Reduction in carbohydrate accumulation affects the flowering, fruit set, and fruit yield. However, to reduce the negative plant physiological stresses, there should be good management practices in citrus orchards. Choice of better scion enhances citrus trees to produce higher yield with good fruit quality [11].

Citrus has a phenological life cycle of the whole year, starting from February to next year January. Flowering starts during February–March in subtropical regions and is generally considered a critical period for citrus production. An increase in temperature and water stress after pollination inhibits ovule fertilization [12], which in return reduces tree fruit set, increases June fruit drop, and reduces tree yield [13–15]. Fruit growth phases from button size to mature fruit are more sensitive to heat stress and deficit irrigation. Citrus under water deficit conditions faces reduced fruit growth and ripening, which is associated with a decrease in fruit size, an increase in fruit acidity [16], and low tree yield [13]. Water stress at the pre-harvest stage in oranges develops fruit peel wrinkles [17]. An increase in optimum temperature at fruit ripening causes pre-harvest fruit drop and reduced yield (**Figure 1**).

To deal with heat and water deficit stress, there is a need to improve agronomic management practices and adopt breeding and molecular approaches. Agronomic management practices encompass factors like irrigation, nutrition, pruning, pests, diseases, and other injuries which have a key role in citrus fruits quantity and quality [19]. Breeding approaches need to search out/develop rootstocks that are tolerant/resistant against abiotic and biotic stresses. Based on breeding techniques, better rootstocks can be developed that can mitigate climate risk and other major biotic factors [20]. Molecular approaches are very helpful to deal with heat and

**Figure 1.** *Key phenological stages and management activities [18].*

#### *Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

water deficit conditions. Modulation in genes and gene expression related to stress help plants to cope and mitigate stress adversity. Henceforth, somatic hybridization, mutation, somaclonal variation, and genetic transformation techniques help to improve trees for thermotolerance [21].

Thus, in this chapter, we present an overview of climate change i.e., heat and drought stress impact on citrus and its management through agronomic, breeding, and molecular approaches.

### **2. Climate change**

The main reason behind climate change is greenhouse gasses; especially carbon dioxide accumulation in the environment. Fossil fuel burning is the primary source of greenhouse gasses emission. The use of pesticides in agriculture and cutting of forests are also contributing to the proliferation of such gasses that cause climate change. An optimum amount of these gasses is necessary for controlling the earth's temperature, but now their concentration is increasing dramatically. From the expected beginning of human civilization about thousand years ago to 1900, the carbon dioxide concentration in the environment was 0.03%, but now due to climate change, it has been reached to 0.04%, the highest in history [22].

#### **2.1 What is the effect of climate change?**

The earth's mean temperature has risen for the past hundred years [23]. The increase in temperature of the earth due to climate change can affect the environment adversely. Today, the average temperature is 4°F more in comparison to the last Ice Age [24]. Global warming is causing the melting of polar caps and warming the ocean's water, which is leading to greater storms and frequent floods along with heavy winds and rains. A heat rise is also enhancing the incidences of wildfires, which damage natural habitat and creatures [25]. Climate change threatens the world's population. The world is severely facing the issue of climate change, especially the third world countries. American and European countries are prepared well against climate change [26]; however, the countries of the

#### **Figure 3.**

*Global surface temperature anatomy 1880–2018 [28].*

**Figure 4.** *Top 17 countries facing the risk of extremely high water stress [29].*

Middle East, Asia, and Africa are more exposed to environmental changes due to less preparedness and technology to tackle these issues [27]. Norway would be the country likely to survive climate change due to its low vulnerability against climate change. The neighboring countries: Finland (third), Sweden (fourth), Denmark (sixth), and Iceland (eighth) are well prepared. The countries least likely to survive global warming change include the Central African Republic, South Africa, Eritrea, Chad, Somalia, and the Democratic Republic of the Congo. These countries have poor infrastructure, unstable governance, poor health, and food and water scarcity (**Figure 2**).

#### **2.2 Rise in temperature**

NASA center graph associated with climate (**Figure 3**) indicates the average global surface temperature during the era of 1880–2018. After 1940, an abrupt increase in temperature was noted for a duration of two years and then continuous high temperature was witnessed after 1980–2016 [28]. The researchers believe that global temperature will rise continuously over the next few decades, mainly due to humans generated greenhouse gases. The IPCC (The Intergovernmental Panel on Climate Change) predicts a rise of 2.5–10°F over the next century [29].

#### **2.3 Water scarcity**

Water is highly important for plants and its global importance is not difficult to understand. There is a frequent rise in water scarcity due to changes in climatic patterns. It is expected that the world will face a decrease of 66% in water availability up till 2050. The water cycle is adversely affected by climate change. Due to the changing climate, several areas are getting dry. There are 17 nations under the extremely high risk of water scarcity; out of which 12 are in North Africa and Middle East [29]. India and Pakistan, two Asian countries, fell in the list of 17 countries having a risk of water scarcity (**Figure 4**).

### **3. Effect of heat and water deficit on tree health**

The yield of any crop begins to decrease when the temperature exceeds the ideal temperature range and the water level falls below the ideal water demand of the crop. Temperature and precipitation variables of climate are described as diagrammatic sketch alternatively for intensity and duration of drought, which show a small portion of the climate space presently exceeding tree mortality threshold (**Figure 5**). It is predicted that there will be high temperature and drought due to extreme climate change, which can cause severe damage to agriculture and could become a risk for tree populations [31].

#### **3.1 Tree physiology**

Plant physiology includes all the dynamic processes of growth, metabolism, reproduction, defense, and communication responsible for plant survival [32, 33]. Heatwaves affect the plant's physiological processes and responses, their ability to tolerate heat, as well as the effectiveness of strategies used for thermotolerance

**Figure 5.** *Climatic variables, temperature, and precipitation, with a range of variability [30].*

improvement [34]. In citrus fruits, the temperature above than optimum causes a big difference in the leaf to air vapor ratio as well as high leaf temperature, but the shade conditions can relieve the water pressure and lower the temperature of the leaves [35]. Stomatal conductance and carbon dioxide uptake are reduced by superoptimal leaf temperature and water tension [36].

The gas exchange activity is reduced badly in citrus trees under deficient irrigation [37, 38]. CO2 assimilation, conductivity, and transpiration rate decrease under water stress, so these gas exchange parameters are a water stress indicator [39–41]. Citrus trees under water deficit conditions reduce the conductivity of stomata and increase photorespiration [42], which reduces the yield, size, and quality of fruits [43]. Under groundwater deficiency, citrus trees lead to stomata closure associated with high ABA content and result in a sudden decrease in photosynthesis [16, 44, 45] and production losses [14, 35]. Chlorophyll *a* is easily damaged compared to chlorophyll *b* due to lack of water. Genotypes/species that maintain stomata conduction under dry conditions also maintain chlorophyll fluorescence and high growth levels [46].

#### **3.2 Tree morphology**

Altered temperatures and water deficit conditions affect citrus leaf-to-air vapor pressure during the day in the early morning and midday [8], indicating a vapor pressure of 4.3 kPa at 37–40°C and 6.2 kPa at ≥40°C, respectively. Citrus exposed to the temperature above than optimum (37°C) and vapor pressure deficit (3.6 kPa) at 330 μmols−1 CO2 concentration during midday, shows depression in carbon dioxide exchange rate [47]. Swingle citrumelo, a citrus species increased its total biomass when kept under slightly high temperature [8]. Drought stress affects both plant vegetative and reproductive growth parameters [48, 49]. Citrus under water deficit lessens vegetative growth, fruit size and quality and orchards face a major economic loss [14]. Orange trees exposed to prolonged or excessive water deficit can lead to leaf drop, gradual drying of the tips of the branches, and a drastic decline in fruit production due to severe flower and fruit drop [50]. Young lemons exposed to water stress showed a decrease in daily stem diameter and water flow [51]. The growth of plant roots is dependent on the soil water availability. The roots of irrigated soils are well distributed and widespread compared to roots with less irrigation. Valencia orange roots on the Swingle citrumelo rootstock and a significant difference in root distribution between irrigated and non-irrigated trees were observed [52]. Water stress decreases the growth and metabolism of citrus fruits [43, 53] and increases the cost of extracting juice [21]. Dryness also reduces the thickness of fruit peel, making citrus fruits more sensitive to damage during handling and transportation [54]. Irrigation stoppage during initial and final growth phases of Lane Late orange (*Citrus sinensis* Osbeck) significantly reduced yield [55]. General studies of water stress in citrus show that the extent and duration of water stress at critical development stages are more vulnerable to the production of citrus. On the other hand, the cultivar and properties of orchards *i.e.*, soil, climate, and cultivation also play important role in success under deficit irrigation [56].

#### **3.3 Tree water status**

The transport of water is determined inside the plant by the availability of soil water and relative humidity. Plant physiological adjustments under changing environmental conditions maintain the turgor pressure of the plant cell. In perennial species, seasonal variations in environmental conditions can affect water relationship. In citrus fruits, the large crown and low hydraulic conductivity of the

#### *Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

trunk and roots contribute to severe water scarcity [57]. Transient water deficit in citrus at midday [58] reduces photosynthetic rates [47]. Root hydraulic conductivity decreases under drought stress to prevent the plants from mortality. High temperatures can also increase the loss of root moisture to a harmful level [59]. In plants, heat stress appears as the supply of water is insufficient to meet the evaporation requirement. Heat stress is linked to drought as the plant and soil quickly lose water at high temperatures. It is known that heat stress and drought reduce nutrient uptake and photosynthetic efficiency in plants [60].

#### **3.4 Tree biochemistry**

Citrus leaf water potential and leaf abscisic acid (ABA) are the indicator of water stress. Citrus rootstock Rangpur lime grafted with scion Pera orange resulted in decreased leaf water potential and decreased leaf ABA concentration when subjected to water stress [61]. Citrus trees produce endogenous hormones and their regulation by promoting synthesis and accumulation under severe water stress [43]. Plant phytohormones are found in a minor quantity but drought stress accumulates jasmonic acid [62]. Drought synthesizes roots ABA and leaves by transpiration stream [43, 63]. The amount of sugar, like non-reducing sugar (sucrose) and reducing sugar (fructose and glucose), in contrast to the sorbitol content, decreases dramatically over the drought period. Water stress accumulates proline contents, an important osmoprotective agent, and its concentration increases with increasing water deficit conditions in citrus orchard [64]. Proline levels in leaf were recorded in Gada dahi citrus rootstock on day 24 of the water stress in comparison to tolerant rootstocks, which indicated that the accumulation of proline was greater in susceptible genotypes than in tolerant genotypes due to higher stress. Lower accumulation of proline was due to its protective function, removing radicals, maintaining the redox balance, and reducing cell damage [65]. The total phenol content also increases in plants under drought stress, compared to normal irrigated plants [66, 67]. Proteins are involved in several processes that change the plant metabolism under stress conditions and activate the plant defense signal [19, 68]. The protein content of drought-tolerant genotypes is generally higher than that of drought-sensitive genotypes. Carrizo citrange, a tolerant genotype, shows notable soluble proteins in leaves and roots [69, 70]. Higher MDA and H2O2 contents observed in plants under water stress indicate greater oxidative damage, which determines the severity of the plant and indicates low efficacy of antioxidant machinery of Carrizo citrange drought-tolerant rootstock [43]. Plants produce several antioxidant enzymes, such as CAT, SOD, and POD to treat the cell damage caused by stress at the oxidative level. SOD is the main enzyme that is expressed under stress, especially under water stress conditions. Carrizo citrange has shown an excellent defense mechanism under water stress with high activity of CAT, SOD, and POD in roots and leaves [19, 68].

#### **3.5 Tree anatomy**

Alteration in anatomy by applying heat stress is established. Stress treatment at 40–45°C was given to similar size plants and anatomical changes (size of the epidermis, size of pith, cortex, leaf thickness, epidermal cells, parenchyma tissue) in root, stem, and rhizomes were studied. The thickness of mesophyll, epidermis, and cortex was increased in stressed plants [71]. Some common anatomical changes include increased densities of stomata and trachomatous, cell size reduction, stomata enclosure, and higher xylem vessels in roots and shoots [72]. It has been demonstrated that grafted plant size is reduced on dwarfing rootstock, and such plants

are unable to maintain drought or water-deficient conditions [73]. The researchers explain that the vessel density of root and stem are decreased with tree height [74]. The rootstock growth ability is dramatically affected by the number of xylem traits, xylem phloem ratio, vessel size, and vessel density [73, 75]. Maintaining hydraulic conductance of stem, root [74, 76], vessel size and number is the basic factor in hydraulic conductance maintenance [77]. Fewer small vessels may decrease hydraulic conductance, as a result, growth decrease in fruit trees [78]. Water stressed leaf spongy cells have a dense arrangement and reduce the conductivity of leaf diffusion. These results give an idea to understand the direct relationship between mesophilic conductivity and the porosity of the soils [79].

#### **3.6 Tree genetics**

Stress-related genes are activated through high temperature and drought, [80], and sugars, different functional proteins, amino acids, and amines are synthesized through these genes [81]. HSPs are the heat shock proteins consisting of a group of genes relevant to heat stress in plants and animals [82, 83]. Heat shock proteins play an important role in maintaining/removing ROS, cell membrane integrity, producing antioxidants, and osmolytes [84, 85]. Heat shock proteins protect plant cells/tissues from drought and heat stress [84]. Citrus HSP70 expression has been examined against water scarcity and high-temperature stress in the *Poncirus trifoliata* rootstock. In *P. trifoliata* HSP70 and HSP90 genes against abiotic stress are upregulated [86]. HSP90s play a vital role in signal transduction, cell cycle regulation, protein breakdown, genomic mutation, and protein trade [81, 87]. Aquaporins are transmembrane channel proteins found in tonoplasts, plasma membranes, and other intracellular membranes and are abundantly expressed in plant roots [88, 89]. Major intrinsic proteins (MIPs) are a superfamily of aquaporins that regulate intracellular water passage [90]. The plasma membrane proteins (PIPs) are the most important group of natural proteins that respond to water transport. Overexpression of PIP under abiotic stress conditions confirms the importance of PIP for heat and water stress tolerance [91] as the combination of heat stress and the scarcity of groundwater generally limits the physiology, growth, and productivity of plants [92].

#### **3.7 Tree productivity**

The citrus phenological cycle starts from February to next year January in subtropical regions. Flowering starts during February–March and is generally considered a critical period for fruit production. An increase in temperature and water stress after pollination inhibits ovule fertilization [12] which reduces tree fruit set, increases fruit drop, and reduces tree yield [13]. Phases of fruit growth from button size to mature fruit are more sensitive to deficit irrigation and heat stress. Hence, reduced fruit growth, and delayed ripening occur which are associated with a decrease in fruit size, increase in fruit acidity, and low tree yield [13]. Drought at a pre-harvest stage in oranges develops wrinkle on fruit peel [17]. An increase in optimum temperature at fruit pre-harvest causes fruits drop and reduced yield. Citrus under different phenological stages respond to deficit irrigation or water stress and contribute negatively to yield/production and fruit quality. In an experiment, eleven-year-old sweet orange scion grafted on Carrizo citrange were evaluated against water stress and revealed 10–12% relative yield decline. Gonzalez [37] compared Clementina (*Citrus clementina*) tree performance under 25–50% deficit irrigation during initial fruit enlargement and pre-maturation phases and recorded a significant negative effect on fruit yield [13]. Navelina sweet orange (*Citrus sinensis* Osbeck) yield reduced significantly

when irrigation was reduced at 55% with respect to crop water requirement during flowering and fruit set.

### **4. Management of citrus under climate change**

#### **4.1 Agronomic management**

Implementation of proper orchard management practices decreases the adverse effects of heat and drought stress. The management includes trees requirement based nutrition and irrigation techniques, organic and synthetic mulches, as well as selecting the most suitable cultivars/rootstocks that are resistant to various stresses.

The selection and development of new rootstocks tolerant to biotic and abiotic stress is inevitable for the stable production of citrus under the scenario of climate change. New and known diseases and environmental conditions also help to force developing new citrus rootstocks according to the demand [93]. Citrus rootstocks like Volkamer lemon (*C. volkameriana*), Rangpur lime (*C. limonia*), and Rough lemon (*C. jambhiri* Lush.) resist water stress and increase the production of cultivars grafted on these.

Fertilizer application can also be helpful to manage plants against abiotic stresses [94]. Application of Ca and K macronutrients and B and Mn micronutrients modify the function of stomata under heat/high-temperature stress [95]. K, Ca, B and Mn activates physiological and metabolic processes that help maintain a high water potential in tissues, which increases tolerance to heat stress [96]. The use of N, K, Ca and Mg also reduces the toxicity of ROS, thereby increase the levels of antioxidant enzymes in plant cells [96].

Plant growth regulators (PGRs) in managing water and heat stress also play an important role. PGRs like cytokinins, abscisic acid (ABA), and salicylic acid play role in resistance to heat and drought. The application of PGRs increases the water potential and chlorophyll content in citrus trees [97]. The exogenous use of ABA increases productivity in the absence of water [98]. ABA formulations are available with commercial manufacturers to improve the drought tolerance of trees [99, 100].

Mulching underneath the trees is often used as a technique for water conservation [101, 102]. Mulches are used to maintain moisture levels high in the soil, control soil temperature, and evaporation [93, 103]; thereby reduce the need for irrigation during growing seasons [104]. The need for water in the soil is decreased and the ability to withstand drought and heat is increased by using mulches [105, 106]. Plastic films are more effective than organic compost for groundwater protection [107].

#### **4.2 Breeding strategies**

Citrus rootstock breeding programs are aimed to combine biotic and abiotic tolerance/resistance in new rootstocks. However, conventional plant breeding (**Figure 6**) in mitigating the abiotic stresses has limited success against plant productivity [108]. Similarly, developing better rootstocks through breeding by the conventional method is a long-term approach due to many difficulties, particularly the complexity of citrus biology (high heterozygosity, long juvenility, polyembryony) [109, 110]. Typically, from a breeding program, it takes at least 15 years for a new standard variety to emerge in the citrus industry. Moreover, a sexual hybrid is difficult to identify at an early stage. In this case, trifoliate leaf (a morphological marker) is used as a male parent and unifoliate as a female for identification of sexual hybrid at the seedling stage [111]. The trifoliate trait is dominant, and the

#### **Figure 6.**

*Traditional cross-hybridization in citrus. (A) a large unopened bud, (B) emasculation, (C) pollination of the emasculated flower, (D) bagging of the pollinated flower, (E) bagged twig, (F) general view of the seed parent after crossing.*

seedlings showing the parental trifoliate pollen phenotype are considered hybrids. In absence of a trifoliate pollen parent, the hybrids can be identified by using SSR and RAPD molecular markers [112].

Besides citrus biological constraints, valuable traits like resistance to low/high temperature, root rot, viruses, nematodes, salinity, and drought are important to incorporate in rootstocks. A list of some important biotic and abiotic traits of citrus rootstocks are presented in **Table 1**, which can help in the development of new tolerant/resistant rootstocks.

Several rootstock hybrids have been released to the citrus industry worldwide. Swingle citrumelo rootstock is a hybrid of Duncan grapefruit and Trifoliate orange, crossed by Swingle in 1907 and released in 1974. Since then, it has been used successfully as the standard rootstock for better traits *i.e.*, moderate drought


*Abbreviations in the table: T; tolerant, TT; very tolerant, pT; poorly tolerant, S; susceptible, SS; very susceptible, G; good, L; low, H; high.*

#### **Table 1.**

*Some biotic and abiotic traits of selective citrus rootstocks [113].*

#### *Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

tolerance and high fruit quality. Citranges are hybrids of Washington Navel orange and *Poncirus trifoliata*, out of which Carrizo and Troyer are the two main citranges. These can tolerate water shortages and produce excellent quality fruits. Benton citrange is a cross of Ruby blood orange and trifoliate orange developed in late 1940 and is more tolerant to heat and water scarcity [114]. Brazilian sour orange also shows tolerance against heat, drought, and their combined stress [85].

#### **4.3 Biotechnological interventions**

Hybridization by somatic approaches is a protoplast fusion process that has become an important tool for plant production, combine (partially or totally) desired cultivars somatic cells, species, or genera, resulting in the development of new genetic combination. In addition to intergenerational mixtures in somatic hybridization, more emphasis has been placed on interspecific mixtures between *C. reticulata* and *C. maxima* [109, 110] to meet the specific needs of the citrus industry. Poncirus is drought-prone, while Citrange C-35 is more drought-tolerant. Among these rootstocks, 4475 citrumelo have the best ability to adapt to the environment. Cleopatra mandarin + *Poncirus trifoliata* and Cleopatra mandarin + C-35 Citrange somatic hybrids have resistance to CTV, tolerance to nematodes, and phytophthora. The Sweet orange + *Poncirus* and the Sweet orange + C-35, as well as the Sweet orange + Citrumelo 4475, can adapt to low moisture soils and tolerate biotic stresses. Macrophylla is a productive rootstock and adapts well to saltwater, limestone, and water stress [115].

*In vitro* mutagenesis and somaclonal variation are important tissue culture techniques being used in citrus improvement. Somaclonal variation, genetic and phenotypic variation between plants, can be used to improve citrus cultivars under conditions of water and heat stresses. Genetic improvement by *in vitro* selection of Satsuma mandarins (*Citrus unshiu* Marc.) has been made successfully; however, the frequency of somaclonal variation by factors, including genotypes, explant culture length, sources, and environmental composition [116, 117]. Cell lines success stories of some salt-tolerant cultivars are *C. sinensis* cv. "Shamouti" [118], *C. limonium* [119] and "Troyer citrange" [120].

Genetic transformation is an alternate technique for citrus genetic improvement. PEG-mediated genetic transformation of citrus fruits is a direct DNA transfer method [120] that seeks to express an aminoglycoside phosphotransferase II gene in isolated protoplasts from sweet orange (*Citrus sinensis* Osbeck) culture for suspension. The genetic transformation of citrus fruits has mainly been carried out from young materials such as embryogenic cells from the epicotyl segment of *in vitro* germinated seedlings. Excess protein for late embryogenesis (OHL), heat shock proteins, and certain transcription factors that affect the expression of various stress-related target genes have also been used to improve drought tolerance in transgenic plants. Drought-induced genes with different functions have been identified through molecular and genomic analyses in a variety of plant species such as the C/CBF family (Shinozaki) [121]. By regulating stress gene expression and signal transformation, plants indirectly become more stressresistant [97, 122]. The TDF genes have been identified as drought-induced and the proteins encoded include fructose aldols bisphosphate, a cold-like protein found in WCOR413. The PIP2 protein, an aquaporin specializing in a water channel to transport water across the plasma membrane, and the tonoplast have been observed in sweet orange. TDF21, TDF38 and TDF80 are involved in the regulation of signal transduction and expression of genes. These are sensitive to stress and also regulate the expression of stress-induced genes, possibly induced by drought.

#### **5. Future research strategies**

Soil microbiome research offers the opportunity to improve abiotic stress in plants. The mechanisms by which plants recover from drought and/or heat stress can be mediated by microbes surrounding the plant, particularly the roots, and these are involved in various stages of plant growth. Advances in the application of new molecular and genomic tools and technologies have paved the way for the study of plant microbiota, and these promising advances enable the study of the biological functions of various microorganisms both inside and outside the host tissue.

Significant advances in the characterization of the plant genome and the optimization of techniques for manipulating the plant genome have contributed and will further improve our knowledge and ability to develop stress-resistant plants. Ultimately, genetic engineering or transgenic methods must be combined with conventional breeding activities and supported by markers in order to obtain the desired improved varieties.

Plants have developed complex adaptive mechanisms to withstand diverse and complex abiotic stresses. With the advent of new technologies such as genomics and genetic transformation, significant advances have been made in understanding these complex traits in higher plants. However, the commercial application of successful research results requires additional validation of the products or prototypes in the field.

These efforts will lead to tangible practical outcomes that may help mitigate the effects of climate change, especially concerning drought and heat stresses, and will contribute to improved crop productivity and food security.

#### **6. Conclusion**

Plants adaptation is considered a striking strategy to manage the impacts of climate change. In climate change, the most important factors are fluctuating patterns of temperature and drought which have an adverse effect on plants physiology, morphology, water status, biochemistry, anatomy, genetics, and productivity. Hence the emphasis should be on the development of production systems for improved water-use efficiency and to adapt to the hot and dry conditions through agronomic practices. Development of climate-resilient citrus rootstocks and scion through genomics and biotechnology are essentially required.

#### **Acknowledgements**

The authors are thankful to the Higher Education Commission, Islamabad, Pakistan for research funding (NRPU-19-8781).

#### **Conflict of interest**

The authors have no conflict of interest with any person or institution.

*Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

### **Author details**

Waqar Shafqat1 , Summar A. Naqvi1 , Rizwana Maqbool<sup>2</sup> , Muhammad Salman Haider3 , Muhammad Jafar Jaskani1 \* and Iqrar A. Khan1

1 Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan

2 Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan

3 Key Laboratory of Genetics and Fruit Development, College of Horticulture, Nanjing Agricultural University, 210095, Nanjing, China

\*Address all correspondence to: jjaskani@uaf.edu.pk

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Ghada B, Amel O, Aymen M, Aymen A, Amel SH. Phylogenetic patterns and molecular evolution among 'True citrus fruit trees' group (Rutaceae family and Aurantioideae subfamily). Sci Hortic (Amsterdam). 2019;253:87- 98. https://doi.org/10.1016/j. scienta.2019.04.011

[2] Ristow M. Unraveling the truth about antioxidants: mitohormesis explains ROS-induced health benefits. Nat Med. 2014;20:709-711. https://doi. org/10.1038/nm.3624

[3] Nawaz R, Abbasi NA, Hafiz IA, Khalid A, Ahmad T, Aftab M. Impact of climate change on kinnow fruit industry of Pakistan. Agrotechnology. 2019;8:2. https://doi. org/10.35248/2168-9881.19.8.186

[4] Abobatta WF. Drought adaptive mechanisms of plants–a review. Adv Agr Env Sci. 2019;2:42-45.

[5] Liu G-H, Yang J-Y. Contentbased image retrieval using color difference histogram. Pattern Recognit. 2013;46:188-198. https://doi. org/10.1016/j.patcog.2012.06.001

[6] Rouphael NG, Stephens DS. Neisseria meningitidis: biology, microbiology, and epidemiology. In: Neisseria meningitidis. Springer; 2012. p. 1-20. https://doi. org/10.1007/978-1-61779-346-2\_1

[7] Naeem A, Ghafoor A, Farooq M. Suppression of cadmium concentration in wheat grains by silicon is related to its application rate and cadmium accumulating abilities of cultivars. J Sci Food Agric. 2015;95:2467-2472. https:// doi.org/10.1002/jsfa.6976

[8] Rosenzweig C, Jones JW, Hatfield JL, Ruane AC, Boote KJ, Thorburn P, et al. The agricultural model intercomparison and improvement project (AgMIP):

protocols and pilot studies. Agric For Meteorol. 2013;170:166-182. https://doi. org/10.1016/j.agrformet.2012.09.011

[9] Mittler R, Finka A, Goloubinoff P. How do plants feel the heat? Trends Biochem Sci. 2012;37:118-125. https:// doi.org/10.1016/j.tibs.2011.11.007

[10] Jaspers P, Kangasjärvi J. Reactive oxygen species in abiotic stress signaling. Physiol Plant. 2010;138:405- 413. https://doi.org/10.1016/j. tibs.2011.11.007

[11] Zekri M. Factors affecting citrus production and quality. Trees. 2011.

[12] Hsiao TC. Effects of drought and elevated CO 2 on plant water use efficiency and productivity. In: Interacting stresses on plants in a changing climate. Springer; 1993. p. 435-465. https://doi. org/10.1007/978-3-642-78533-7\_28

[13] García-Tejero I,

Romero-Vicente R, Jiménez-Bocanegra JA, Martínez-García G, Durán-Zuazo VH, Muriel-Fernández JL. Response of citrus trees to deficit irrigation during different phenological periods in relation to yield, fruit quality, and water productivity. Agric Water Manag. 2010;97:689-699. https://doi. org/10.1016/j.agwat.2009.12.012

[14] Guerra P, Romero PR. Pistol grip style elongated dissecting and dividing instrument. 2006.

[15] García O, Johnson SI, Seltzer K. The translanguaging classroom: Leveraging student bilingualism for learning. Caslon Philadelphia, PA; 2017. https:// doi.org/10.21283/2376905x.9.165

[16] Pérez-Pérez JG, Robles JM, Botía P. Influence of deficit irrigation in phase III of fruit growth on fruit quality in

*Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

'lane late'sweet orange. Agric Water Manag. 2009;96:969-974. https://doi. org/10.1016/j.agwat.2009.01.008

[17] Goldhamer DA, Salinas M. Evaluation of regulated deficit irrigation on mature orange trees grown under high evaporative demand. In: Proceedings of internat soc citriculture IX congress. 2000. p. 227-231.

[18] Khurshid T. Project, the enhancement of citrus value chains production in Pakistan and Australia through improved orchard management practices. 2015. https://research.aciar. gov.au/aik-saath/sites/\_co-lab.aciar.gov. au.pdf

[19] Zandalinas SI, Balfagón D, Arbona V, Gómez-Cadenas A. Modulation of antioxidant defense system is associated with combined drought and heat stress tolerance in citrus. Front Plant Sci. 2017;8:953. https://doi.org/10.3389/ fpls.2017.00953

[20] Cimen B, Yesiloglu T. Rootstock breeding for abiotic stress tolerance in citrus. In: Abiotic and Biotic Stress in Plants-Recent Advances and Future Perspectives. IntechOpen; 2016. https:// doi.org/10.5772/62047

[21] Hasanuzzaman M, Nahar K, Fujita M. Extreme temperature responses, oxidative stress and antioxidant defense in plants. Abiotic Stress responses Appl Agric. 2013;13:169-205. https://doi. org/10.5772/54833

[22] Buis A. A Degree of Concern: Why Global Temperatures Matter. NASA's Glob Clim Chang Website, June. 2019;19.

[23] Steffen W, Leinfelder R, Zalasiewicz J, Waters CN, Williams M, Summerhayes C, et al. Stratigraphic and Earth System approaches to defining the Anthropocene. Earth's

Futur. 2016;4:324-345. https://doi. org/10.1002/2016EF000379

[24] Wang H, Ni J, Prentice IC. Sensitivity of potential natural vegetation in China to projected changes in temperature, precipitation and atmospheric CO 2. Reg Environ Chang. 2011;11:715-727. https://doi.org/10.1007/ s10113-011-0204-2

[25] Pausas JG, Marañón T, Marañón T, Marañón T, Caldeira MC, Pons J. Natural regeneration. 2009.

[26] Gössling S, Scott D, Hall CM, Ceron J-P, Dubois G. Consumer behaviour and demand response of tourists to climate change. Ann Tour Res. 2012;39:36-58. https://doi.org/10.1016/j. annals.2011.11.002

[27] Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest. 1992;101:1644-1655. https://doi. org/10.1378/chest.101.6.1644

[28] Tomsits E, Pataki M, Tölgyesi A, Fekete G, Rischak K, Szollár L. Safety and efficacy of a lipid emulsion containing a mixture of soybean oil, medium-chain triglycerides, olive oil, and fish oil: a randomised, doubleblind clinical trial in premature infants requiring parenteral nutrition. J Pediatr Gastroenterol Nutr. 2010;51:514-521. https://doi.org/10.1378/chest.101.6.1644

[29] Ford JD, Cameron L, Rubis J, Maillet M, Nakashima D, Willox AC, et al. Including indigenous knowledge and experience in IPCC assessment reports. Nat Clim Chang. 2016;6:349-353.

[30] Garcia ES, Swann ALS, Villegas JC, Breshears DD, Law DJ, Saleska SR, et al. Synergistic ecoclimate teleconnections from forest loss in different regions structure global ecological responses.

PLoS One. 2016;11:e0165042. https:// doi.org/10.1371/journal.pone.0165042

[31] Allen GL. Principles and practices for communicating route knowledge. Appl Cogn Psychol Off J Soc Appl Res Mem Cogn. 2000;14:333-359. https://doi.org/10.1002/1099- 0720(200007/08)14:4<333::aidacp655>3.0.co;2-c

[32] Brenner ED, Stahlberg R, Mancuso S, Vivanco J, Baluška F, Van Volkenburgh E. Plant neurobiology: an integrated view of plant signaling. Trends Plant Sci. 2006;11:413- 419. https://doi.org/10.1016/j. tplants.2006.06.009

[33] Scott WR. Approaching adulthood: the maturing of institutional theory. Theory Soc. 2008;37:427. https://doi. org/10.1007/s11186-008-9067-z

[34] Wahid A. Physiological implications of metabolite biosynthesis for net assimilation and heat-stress tolerance of sugarcane (Saccharum officinarum) sprouts. J Plant Res. 2007;120:219- 228. https://doi.org/10.1007/ s10265-006-0040-5

[35] Syvertsen JP, Lloyd JJ. Citrus. Handb Environ Physiol fruit Crop. 1994;2:65-99. https://doi.org/10.17660/ ActaHortic.2012.928.44

[36] Kiefer AK, Sanchez DT, Kalinka CJ, Ybarra O. How women's nonconscious association of sex with submission relates to their subjective sexual arousability and ability to reach orgasm. Sex Roles. 2006;55:83-94. https://doi. org/10.1007/s11199-006-9060-9

[37] Ginestar C, Castel JR. Responses of young clementine citrus trees to water stress during different phenological periods. J Hortic Sci. 1996;71:551-559. https://doi.org/10.1080/14620316.1996. 11515435

[38] González-Altozano P, Castel JR. Regulated deficit irrigation in'Clementina de Nules' citrus trees. I. Yield and fruit quality effects. 1999. https://doi.org/10.17660/ actahortic.2000.537.89

[39] Sinclair TR, Allen Jr LH. Carbon dioxide and water vapour exchange of leaves on field-grown citrus trees. J Exp Bot. 1982;33:1166-1175. https://doi. org/10.1093/jxb/33.6.1166

[40] 40.Shalhevet J, Levy Y. Citrus trees. Agron. 1990. https://doi.org/10.21273/ HORTTECH.15.1.0095

[41] Chattha WS, Atif RM, Iqbal M, Shafqat W, Farooq MA, Shakeel A. Genome-wide identification and evolution of Dof transcription factor family in cultivated and ancestral cotton species. Genomics. 2020;112:4155-4170. https://doi. org/10.1016/j.ygeno.2020.07.006

[42] Flexas J, Medrano H. Droughtinhibition of photosynthesis in C3 plants: stomatal and non-stomatal limitations revisited. Ann Bot. 2002;89:183-189. https://doi. org/10.1093/aob/mcf027

[43] Arbona V, Iglesias DJ, Jacas J, Primo-Millo E, Talon M, Gómez-Cadenas A. Hydrogel substrate amendment alleviates drought effects on young citrus plants. Plant Soil. 2005;270:73-82. https://doi.org/10.1007/ s11104-004-1160-0

[44] Lopez-Medina J, Moore JN. Chilling enhances cane elongation and flowering in primocane-fruiting blackberries. HortScience. 1999;34:638- 640. https://doi.org/10.21273/ hortsci.34.4.638

[45] Adamis PDB, Gomes DS, Pinto MLCC, Panek AD, Eleutherio ECA. The role of glutathione transferases in cadmium stress. Toxicol Lett. 2004;154:81-88. https:// doi.org/10.1016/j.toxlet.2004.07.003

*Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

[46] Blum A. Drought resistance–is it really a complex trait? Funct Plant Biol. 2011;38:753-757. https://doi.org/10.1071/ fp11101

[47] Brakke M, Allen LH. Gas exchange of Citrus seedlings at different temperatures, vapor-pressure deficits, and soil water contents. J Am Soc Hortic Sci. 1995;120:497-504. https://doi. org/10.21273/jashs.120.3.497

[48] Xie Z, Miller GM. Trace amineassociated receptor 1 is a modulator of the dopamine transporter. J Pharmacol Exp Ther. 2007;321:128-136. https://doi. org/10.1124/jpet.106.117382

[49] Wu K, Horejsi T, Byrum J, Bringe N, Yang J, Pei D, et al. Agronomically elite soybeans with high beta-conglycinin content. US Patent Application 11/517186. Date issued: 11 September. Agron Elit soybeans with high betaconglycinin content US Pat Appl 11/517186 Date issued 11 Sept. 2007.

[50] Syvertsen T. The many uses of the "public service" concept. Nord Rev. 1999;20:5-12.

[51] Fauchere J-L, Ortuno J-C, Levens N, Chamorro S, Boutin J. Novel aminotriazolone compounds, method for preparing same and pharmaceutical compositions containing same. 2004.

[52] Carvalho-Junior O, Birolo AB, Macedo-Soares LCP de. Ecological aspects of neotropical otter (Lontra longicaudis) in Peri lagoon, south Brazil. IUCN Otter Spec Gr Bull. 2010;27:105- 115. http://dx.doi.org/10.1590/ S0001-37652013005000014

[53] Perez-Campo R, Lopez-Torres M, Cadenas S, Rojas C, Barja G. The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach. J Comp Physiol B. 1998;168:149-158. https://doi. org/10.1007/s003600050131

[54] Takeyama K, Dabbagh K, Lee H-M, Agustí C, Lausier JA, Ueki IF, et al. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci. 1999;96:3081-3086. https://doi.org/10.1073/pnas.96.6.3081

[55] Adie BAT, Pérez-Pérez J, Pérez-Pérez MM, Godoy M, Sánchez-Serrano J-J, Schmelz EA, et al. ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis. Plant Cell. 2007;19:1665- 1681. https://doi.org/10.1105/ tpc.106.048041

[56] Panigrahi P, Sharma RK, Hasan M, Parihar SS. Deficit irrigation scheduling and yield prediction of 'Kinnow'mandarin (Citrus reticulate Blanco) in a semiarid region. Agric Water Manag. 2014;140:48-60. https:// doi.org/10.1016/j.agwat.2014.03.018

[57] Moreshet S, Huck MG, Hesketh JD, Peters DB. Relationships between sap flow and hydraulic conductivity in soybean plants. 1990. https://doi. org/10.1051/agro:19900504

[58] Shapiro L, Cohen S. Novel alginate sponges for cell culture and transplantation. Biomaterials. 1997;18:583-590. https://doi. org/10.1016/s0142-9612(96)00181-0

[59] Berger B, Parent B, Tester M. High-throughput shoot imaging to study drought responses. J Exp Bot. 2010;61:3519-3528. https://doi. org/10.1093/jxb/erq201

[60] Lamaoui M, Jemo M, Datla R, Bekkaoui F. Heat and drought stresses in crops and approaches for their mitigation. Front Chem. 2018;6:26. https://doi.org/10.3389/ fchem.2018.00026

[61] Kavar T, Maras M, Kidrič M, Šuštar-Vozlič J, Meglič V. Identification of genes involved in the response

of leaves of Phaseolus vulgaris to drought stress. Mol Breed. 2008;21:159- 172. https://doi.org/10.1007/ s11032-007-9116-8

[62] Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, et al. (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol. 2009;5:344-350. https://doi. org/10.1038/nchembio.161

[63] ZHANG J, Davies WJ. Does ABA in the xylem control the rate of leaf growth in soil-dried maize and sunflower plants? J Exp Bot. 1990;41:1125-1132. https://doi.org/10.1093/jxb/41.9.1125

[64] Hayat S, Alyemeni MN, Hasan SA. Foliar spray of brassinosteroid enhances yield and quality of Solanum lycopersicum under cadmium stress. Saudi J Biol Sci. 2012;19:325-335. https:// doi.org/10.1016/j.sjbs.2012.03.005

[65] Naliwajski MR, Skłodowska M. Proline and its metabolism enzymes in cucumber cell cultures during acclimation to salinity. Protoplasma. 2014;251:201-209. https://doi. org/10.1007/s00709-013-0538-3

[66] Olmstead MA, Lang NS, Ewers FW, Owens SA. Xylem vessel anatomy of sweet cherries grafted onto dwarfing and nondwarfing rootstocks. J Am Soc Hortic Sci. 2006;131:577-585. https:// doi.org/10.21273/jashs.131.5.577

[67] Djoukeng JD, Arbona V, Argamasilla R, Gomez-Cadenas A. Flavonoid profiling in leaves of citrus genotypes under different environmental situations. J Agric Food Chem. 2008;56:11087-11097. https://doi. org/10.1021/jf802382y

[68] Sarkar C, Guenther AB, Park J-H, Seco R, Alves E, Batalha S, et al. PTR-TOF-MS eddy covariance measurements of isoprene and monoterpene fluxes from an eastern Amazonian rainforest. Atmos Chem Phys. 2020;20:7179-7191. https://doi. org/10.5194/acp-20-7179-2020

[69] Mathews H, Litz RE, Wilde HD, Merkle SA, Wetzstein HY. Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos. Vitr. 1992;28:172-178. https://doi. org/10.1007/bf02823312

[70] Chakraborty U, Pradhan B. Oxidative stress in five wheat varieties (Triticum aestivum L.) exposed to water stress and study of their antioxidant enzyme defense system, water stress responsive metabolites and H2O2 accumulation. Brazilian J Plant Physiol. 2012;24:117-130. https://doi. org/10.1590/s1677-04202012000200005

[71] Chaidir L, Annisa J, Dian S, Parwati I, Alisjahbana A, Purnama F, et al. Microbiological diagnosis of adult tuberculous meningitis in a ten-year cohort in Indonesia. Diagn Microbiol Infect Dis. 2018;91:42-46. https://doi. org/10.1016/j.diagmicrobio.2018.01.004

[72] Ribotta PD, Perez GT, Leon AE, Anon MC. Effect of emulsifier and guar gum on micro structural, rheological and baking performance of frozen bread dough. Food Hydrocoll. 2004;18:305- 313. https://doi.org/10.1016/ s0268-005x(03)00086-9

[73] Trifilò P, Gullo MA Lo, Nardini A, Pernice F, Salleo S. Rootstock effects on xylem conduit dimensions and vulnerability to cavitation of Olea europaea L. Trees. 2007;21:549- 556. https://doi.org/10.1007/ s00468-007-0148-9

[74] Zach C, Klopschitz M, Pollefeys M. Disambiguating visual relations using loop constraints. In: 2010 IEEE Computer Society Conference on Computer Vision and Pattern Recognition. IEEE; 2010. p. 1426-1433. https://doi.org/10.1109/ cvpr.2010.5539801

*Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

[75] Meland M. Efficacy of chemical bloom thinning agents to European plums. Acta Agric Scand Sect B-Soil Plant Sci. 2007;57:235-242. https://doi. org/10.1080/09064710600914236

[76] Tombesi F, Cappi M, Reeves JN, Palumbo GGC, Yaqoob T, Braito V, et al. Evidence for ultra-fast outflows in radio-quiet AGNs-I. Detection and statistical incidence of Fe K-shell absorption lines. Astron Astrophys. 2010;521:A57. https://doi. org/10.1051/0004-6361/200913440

[77] Tyree MT, Ewers FW. The hydraulic architecture of trees and other woody plants. New Phytol. 1991;119:345-360. https://doi.org/10.1111/j.1469-8137.1991. tb00035.x

[78] Olmstead RG, Bohs L. A summary of molecular systematic research in Solanaceae: 1982-2006. In: VI International Solanaceae Conference: Genomics Meets Biodiversity 745. 2006. p. 255-268. https://doi.org/10.17660/ actahortic.2007.745.11

[79] Loreto F, Harley PC, Di Marco G, Sharkey TD. Estimation of mesophyll conductance to CO2 flux by three different methods. Plant Physiol. 1992;98:1437-1443. https://doi. org/10.1104/pp.98.4.1437

[80] Shinozaki K, Yamaguchi-Shinozaki K. Gene networks involved in drought stress response and tolerance. J Exp Bot. 2007;58:221-227. https://doi. org/10.1093/jxb/erl164

[81] Wang W, Vinocur B, Altman A. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta. 2003;218:1-14. https://doi.org/10.1007/ s00425-003-1105-5

[82] Kotak S, Vierling E, Bäumlein H, von Koskull-Döring P. A novel transcriptional cascade regulating expression of heat stress proteins during seed development of Arabidopsis. Plant Cell. 2007;19:182-195. https://doi. org/10.1105/tpc.106.048165

[83] Shalchi A, Döring H. Velocity correlation functions of charged test particles. J Phys G Nucl Part Phys. 2007;34:859. https://doi. org/10.1088/0954-3899/34/5/007

[84] Khan Z, Shahwar D. Role of Heat Shock Proteins (HSPs) and Heat Stress Tolerance in Crop Plants. In: Sustainable Agriculture in the Era of Climate Change. Springer; 2020. p. 211-234. https://doi. org/10.1007/978-3-030-45669-6\_9

[85] Shafqat S, Kishwer S, Rasool RU, Qadir J, Amjad T, Ahmad HF. Big data analytics enhanced healthcare systems: a review. J Supercomput. 2020;76:1754- 1799. https://doi.org/10.1007/ s11227-017-2222-4

[86] Alves MS, Dadalto SP, Gonçalves AB, De Souza GB, Barros VA, Fietto LG. Plant bZIP transcription factors responsive to pathogens: a review. Int J Mol Sci. 2013;14:7815-7828. https://doi.org/10.3390/ijms14047815

[87] Kang B, Rancour DM, Bednarek SY. The dynamin-like protein ADL1C is essential for plasma membrane maintenance during pollen maturation. Plant J. 2003;35:1-15. https://doi. org/10.1046/j.1365-313x.2003.01775.x

[88] Javot H, Maurel C. The role of aquaporins in root water uptake. Ann Bot. 2002;90:301-313. https://doi. org/10.1093/aob/mcf199

[89] North RJ, Jung Y-J. Immunity to tuberculosis. Annu Rev Immunol. 2004;22:599-623. https://doi.org/10.1146/annurev. immunol.22.012703.104635

[90] Banerjee A, Samanta S, Singh A, Roychoudhury A. Deciphering the molecular mechanism behind

stimulated co-uptake of arsenic and fluoride from soil, associated toxicity, defence and glyoxalase machineries in arsenic-tolerant rice. J Hazard Mater. 2020;390:121978. https://doi. org/10.1016/j.jhazmat.2019.121978

[91] Kumar V, Kumar M, Sharma S, Varma A, Bhalla-Sarin N. Procedural Insights on In Vitro Propagation of Litchi chinensis (Sonn.). In: Lychee Disease Management. Springer; 2017. p. 217-35. https://doi. org/10.1007/978-981-10-4247-8\_13

[92] Barnabás B, Jäger K, Fehér A. The effect of drought and heat stress on reproductive processes in cereals. Plant Cell Environ. 2008;31:11-38. https://doi. org/10.1111/j.1365-3040.2007.01727.x

[93] Yang N, Sun Z-X, Feng L-S, Zheng M-Z, Chi D-C, Meng W-Z, et al. Plastic film mulching for water-efficient agricultural applications and degradable films materials development research. Mater Manuf Process. 2015;30:143-154. https://doi.org/10.1080/10426914.2014. 930958

[94] Prasad RN. Varietal Evaluation of Pomegranate under Arid Conditions. Ann Arid Zone. 2000;39:427-430. http://krishi.icar.gov.in/jspui/ handle/123456789/2370. Accessed 4 Feb 2020. http://doi. org/10.123456789/2370

[95] Samoliński B,

Raciborski F, Lipiec A, Tomaszewska A, Krzych-Fałta E, Samel-Kowalik P, et al. Epidemiologia chorób alergicznych w Polsce (ECAP). Alergol Pol J Allergol. 2014;1:10-18. https://doi.org/10.1016/j. alergo.2014.03.008

[96] Waraich EA, Ahmad R, Halim A, Aziz T. Alleviation of temperature stress by nutrient management in crop plants: a review. J soil Sci plant Nutr. 2012;12:221-244. https://doi. org/10.4067/s0718-95162012000200003 [97] Zhang B, Schmoyer D, Kirov S, Snoddy J. GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinformatics. 2004;5:1-8. https://doi. org/10.4067/10.1186/1471-2105-5-16

[98] Huang D. Dietary antioxidants and health promotion. 2018. https://dx.doi. org/10.3390%2Fantiox7010009

[99] Hu ZQ, Huang XM, Chen HB, Wang HC. Antioxidant capacity and phenolic compounds in litchi (Litchi chinensis Sonn.) pericarp. In: III International Symposium on Longan, Lychee, and other Fruit Trees in Sapindaceae Family 863. 2008. p. 567-574. https://doi.org/10.17660/ actahortic.2010.863.79

[100] Waterland NL, Finer JJ, Jones ML. Benzyladenine and gibberellic acid application prevents abscisic acid-induced leaf chlorosis in pansy and viola. HortScience. 2010;45:925-933. https://doi. org/10.21273/hortsci.45.6.925

[101] Chakraborty D, Nagarajan S, Aggarwal P, Gupta VK, Tomar RK, Garg RN, et al. Effect of mulching on soil and plant water status, and the growth and yield of wheat (Triticum aestivum L.) in a semi-arid environment. Agric water Manag. 2008;95:1323-1334. https://doi. org/10.1016/j.agwat.2008.06.001

[102] Zribi W, Aragüés R, Medina E, Faci JM. Efficiency of inorganic and organic mulching materials for soil evaporation control. Soil Tillage Res. 2015;148:40-45. https://doi. org/10.1016/j.still.2014.12.003

[103] Kader MA, Senge M, Mojid MA, Ito K. Recent advances in mulching materials and methods for modifying soil environment. Soil Tillage Res. 2017;168:155-166. https://doi. org/10.1016/j.still.2017.01.001

#### *Climate Change and Citrus DOI: http://dx.doi.org/10.5772/intechopen.95488*

[104] Zhang H, Huang Z, Xie B, Chen Q, Tian X, Zhang X, et al. The ethylene-, jasmonate-, abscisic acid-and NaClresponsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco. Planta. 2004;220:262- 270. https://doi.org/10.1007/ s00425-004-1347-x

[105] Li Q, Li H, Zhang L, Zhang S, Chen Y. Mulching improves yield and water-use efficiency of potato cropping in China: A meta-analysis. F Crop Res. 2018;221:50-60. https://doi. org/10.1016/j.fcr.2018.02.017

[106] Kader MA, Singha A, Begum MA, Jewel A, Khan FH, Khan NI. Mulching as water-saving technique in dryland agriculture. Bull Natl Res Cent. 2019;43:1-6. https://doi.org/10.1186/ s42269-019-0186-7

[107] Li SX, Wang ZH, Li SQ, Gao YJ, Tian XH. Effect of plastic sheet mulch, wheat straw mulch, and maize growth on water loss by evaporation in dryland areas of China. Agric water Manag. 2013;116:39-49. https://doi. org/10.1016/j.agwat.2012.10.004

[108] Parmar N, Singh KH, Sharma D, Singh L, Kumar P, Nanjundan J, et al. Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review. 3 Biotech. 2017;7:239. https://doi.org/10.1007/ s13205-017-0870-y

[109] Shen X, Gmitter FG, Grosser JW. Immature embryo rescue and culture. In: Plant Embryo Culture. Springer; 2011. p. 75-92. https://doi. org/10.1007/978-1-61737-988-8\_7

[110] Ananthakrishnan G, Ćalović M, Serrano P, Grosser JW. Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to

replace sour orange rootstock. Vitr Cell Dev Biol. 2006;42:367-371. https://doi. org/10.1079/ivp2006784

[111] De Oliveira AC, Garcia AN, Cristofani M, Machado MA. Identification of citrus hybrids through the combination of leaf apex morphology and SSR markers. Euphytica. 2002;128:397-403. https:// doi.org/10.1007/s10722-014-0188-0

[112] Kepiro JL, Roose ML. AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis) in Citrus maxima× Poncirus trifoliata. Tree Genet genomes. 2010;6:1-11. https://doi.org/10.1007/ s11295-009-0223-z

[113] Dambier D, Benyahia H, Pensabene-Bellavia G, Kaçar YA, Froelicher Y, Belfalah Z, et al. Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry. Plant Cell Rep. 2011;30:883-900. https://doi. org/10.1007/s00299-010-1000-z

[114] Uzun A, Yesiloglu T, Aka-Kacar Y, Tuzcu O, Gulsen O. Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs). Sci Hortic (Amsterdam). 2009;121:306-312. https://doi. org/10.1016/j.scienta.2009.02.018

[115] Grosser JW, Gmitter FG. Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell, Tissue Organ Cult. 2011;104:343- 357. https://doi.org/10.1007/ s11240-010-9823-4

[116] Skirvin RM, McPheeters KD, Norton M. Sources and frequency of somaclonal variation. HortScience. 1994;29:1232-1237. https://doi. org/10.21273/hortsci.29.11.1232

[117] Duncan RR, Waskom RM, Nabors MW. In vitro screening and field evaluation of tissue-cultureregenerated sorghum (Sorghum bicolor (L.) Moench) for soil stress tolerance. In: The Methodology of Plant Genetic Manipulation: Criteria for Decision Making. Springer; 1995. p. 373-380. https://doi. org/10.1007/978-94-011-0357-2\_46

[118] Kochba J, Ben-Hayyim G, Spiegel-Roy P, Saad S, Neumann H. Selection of stable salt-tolerant callus cell lines and embryos in Citrus sinensis and C. aurantium. Zeitschrift für Pflanzenphysiologie. 1982;106:111- 118. https://doi.org/10.1016/ s0044-328x(82)80073-1

[119] Piqueras Castillo A. Selección mediante cultivo" in vitro" y caracterización de una linea de callo embriogenico de (Citrus Limonum R.). 1992. http://hdl.handle.net/10201/31365

[120] El Yacoubi H, Rochdi A, Ayolie K, Rachidai A. Sélection et évaluation de lignées de cals stables et tolérantes vis-à-vis du stress salin chez le citrange 'Troyer'[Citrus sinensis (L.)× Poncirus trifoliata (L.) Raf.]. Fruits. 2004;59:325- 337. https://doi.org/10.1051/ fruits:2004031

[121] Matsukura S, Mizoi J, Yoshida T, Todaka D, Ito Y, Maruyama K, et al. Comprehensive analysis of rice DREB2 type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes. Mol Genet Genomics. 2010;283:185- 196. https://doi.org/10.1007/ s00438-009-0506-y

[122] Forner-Giner MA, Llosá MJ, Carrasco JL, Perez-Amador MA, Navarro L, Ancillo G. Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock Poncirus trifoliata (L.) Raf. J Exp Bot. 2010;61:483-490. https://doi. org/10.1093/jxb/erp328

## Section 3
