Safeguarding Citrus: Exploring State-of-the-art Management Strategies for Bacterial Citrus Diseases

Abstract

Bacterial diseases pose significant risks to the citrus industry, causing major economic losses and threatening global production. The most severe threats include citrus canker (Xanthomonas citri) which leads to visible lesions on leaves, fruit, and stems and Huanglongbing (HLB) (Candidatus Liberibacter spp.) which results in mottled leaves, stunted growth, and deformed, bitter fruit. Beyond these major diseases, citrus blast and citrus variegated chlorosis (CVC) are emerging concerns. Citrus blast, caused by Pseudomonas syringae pv. citri, results in leaf lesions, cankers, and defoliation, managing it involves copper-based bactericides, removing infected branches, and cultural practices to reduce spread. CVC, caused by Xylella fastidiosa, is a vascular disease leading to chlorosis, leaf scorch, and dieback. Management strategies for CVC include controlling insect vectors with insecticides and developing resistant citrus varieties. Integrated disease management is crucial, focusing on sustainable approaches that combine cultural practices, biological control agents, and resistant varieties. Advances in technology, such as molecular diagnostics, remote sensing, and precision agriculture, are improving early detection and monitoring. Public awareness and education are keys to encouraging growers to adopt best practices. Collaboration among researchers, growers, and policymakers remains essential to tackle the complex challenges of bacterial citrus diseases and ensure the citrus industry’s sustainability.

1. Introduction

Citriculture holds significant global economic importance, benefiting from the adaptability of citrus varieties to diverse regions within the intertropical range [1]. Engaging at least 140 countries, citrus cultivation yields an annual production exceeding 122 million tons of fruits. According to the United States Department of Agriculture (2023), China holds the top position as the leading citrus producer, particularly excelling in tangerines/mandarins, with an anticipated output of 26.5 million tons. Despite its economic prominence, citriculture faces threats from diseases that pose substantial risks to production due to their widespread occurrence. Bacterial diseases, such as citrus variegated chlorosis (CVC), Huanglongbing (HLB), citrus canker and citrus blast, contribute to significant economic impacts on the global citrus industry [2]. These diseases, each characterized by distinct pathogen disease cycles and reproductive strategies, exhibit broad host ranges, pathogenicity, and genomic variations, indicating horizontal gene transfer of pathogenicity factors. Monoculture practices in commercial citrus groves, where a limited number of varieties cover extensive areas, lack natural variation in resistance, further aggravating disease susceptibility [3]. To study these pathogens and to manage them, it is essential first to differentiate between parasitism and pathogenicity. While a parasite can derive nutrition from its host without necessarily causing disease, a pathogen induces disease but not always on all hosts. For instance, a Liberibacter-infected pear tree may display no symptoms [4]. Pathologists traditionally focus on pathogens affecting commercially vital crops, overlooking potential wider parasitic host ranges. Overlapping host ranges among bacterial species and the potential for insect vectors to disguise host range limitations contribute to emerging and severe disease threats. The introduction of vectors with broad host ranges, encompassing weeds or arboreal crops, can facilitate horizontal gene transfer events, intensifying recombination frequencies across species and genera. Transcontinental dispersal of pathogens and vectors further amplifies the risk of horizontal gene transfer [3]. In light of these concerns, this chapter comprehensively explores major diseases affecting global citriculture, including citrus canker, HLB, CVC, and citrus blast. The discussion covers topics ranging from the causes and symptoms of the disease to strategies for control, diagnostic methodologies, and recent advancements in alternative management approaches.

2. Etiology and symptoms of bacterial threats in citrus

Citriculture, a crucial economic activity in many countries, faces significant challenges arising from the prevalence of various diseases that pose a considerable threat to production. The frequency of occurrence and the extensive damage inflicted by these diseases have heightened concerns within the global citrus industry. Important bacterial diseases involved are mentioned in Table 1.

Table 1.

Major bacterial diseases of citrus.

2.1 Citrus canker

Citrus canker is an important bacterial disease of citrus generally linked to two bacterial species within the Xanthomonas genus. X. citri subsp. citri (Xcc) is responsible for causing citrus canker A, which is identified as the most aggressive strain, posing a significant threat in Asia and South America and will be discussed here whereas X. citri subsp. aurantifolii pathotypes B and C (XauB and XauC) causing citrus canker B and C, respectively, exhibit lower aggressiveness. Citrus canker B is prevalent in Paraguay, Argentina, and Uruguay whereas citrus canker C has been restricted to Sao Paulo state in Brazil [18, 19].

The formation of biofilms by Xcc bacteria is a critical virulence factor, playing a significant role in the initial stages of infection and the colonization of hosts [20, 21]. The characteristic symptoms of asiatic citrus canker include the development of necrotic lesions with moist edges, the presence of chlorotic rings in leaves, stems, and fruits [20, 22] and heightened mitotic cell division leading to the formation of cankers [23, 24]. Severe asiatic citrus canker cases can lead to defoliation, dieback, and fruit drop, resulting in reduced fruit value or complete unmarketability. While most commercial citrus varieties exhibit varying degrees of susceptibility, grapefruit stands out as the most vulnerable [25]. The transmission of the disease is facilitated by rain splash and wind, with the extent of bacterial aerosol spread ranging from a few centimeters in mild rain without wind to several kilometers during tropical storms and hurricanes, with the latter conditions intensifying the outbreak [26, 27]. The severity of Asiatic citrus canker outbreaks is heightened in regions where the Asian citrus leaf miner (Phyllocnistis citrella) and citrus canker overlap, due to the insect-feeding activities that result in wounds that expose leaf mesophyll tissues to splashed inoculum, thus significantly increasing the likelihood of infection [28, 29]. Asiatic citrus canker exhibits high contagion, often spreading through human-mediated means such as contaminated clothing, vehicles, equipment (e.g., pruners, hedgers, mowers, ladders, harvesters), or animals, whether in commercial or residential citrus environments; the bacteria is released from hyperplastic lesions on wetted foliage and can be transferred to susceptible foliar tissues on neighboring or distant trees when coming into contact with the bacterial ooze through mechanical devices, humans, or animals [3].

2.2 Huanglongbing

Generally known as Huanglongbing (HLB) in the Americas, it initially gained recognition under different names in various regions. In South Africa, it was referred to as Greening, while in the Philippines, it was identified as Mottle Leaf. In India, the disease was termed Dieback, and in Indonesia, it was recognized as Vein Phloem Degeneration. These diverse names reflect the global distribution and regional awareness of the same underlying threat to citrus crops, showcasing the widespread impact of this disease on citrus cultivation across different continents [30, 31].

Jagoueix et al. were pioneers in employing 16S rDNA sequence comparisons to establish that this citrus pathogen belongs to the alpha subdivision of proteobacteria and categorized it under the provisional taxon Candidatus. Liberibacters constitute a closely related cluster of alphaproteobacteria within the Rhizobiaceae family. HLB is induced by three Gram-negative bacteria which were named based on the continents where they were initially identified: Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter americanus (CLam), and Candidatus Liberibacter africanus (CLaf) [32, 33, 34]. Additionally, the bacteria growth in planta is systemic, phloem limited and circulative in its psyllid vectors Diaphorina citri and Trioza erytreae. Intriguingly, they display a higher degree of adaptation to their insect hosts than to plant hosts. They can spread both inter- and intracellularly across various tissues in insect hosts, including salivary glands, alimentary canal, midgut epithelium, basement membrane, Malpighian tubules, hemolymph, muscle and fat tissues, and ovaries. Remarkably, there is little to no evidence of pathogenic effects associated with this spread in insect vector [35]. The intensity of symptoms is linked to the duration of the plant’s infection. Following the initial infection, a notably extended incubation period, typically lasting at least 6 months, is minimal before any observable symptoms as CLas will try to suppress the immune response by its citrus host [3, 36].

The primary identifiable symptoms of HLB disease include the yellowing of branches, the distortion of small leaves accompanied by yellow spots, fruit deformities, seed abortion, premature leaf and fruit shedding, as well as the death of sprouts. The visual characteristics of symptomatic leaves closely resemble those resulting from deficiencies in zinc, manganese, and iron, as documented by various researchers [30, 31, 37, 38]. CLas and CLam in citrus exhibit notable differences in multiplication and symptom induction. CLas demonstrates a tenfold higher multiplication in citrus compared to CLam, yet CLam induces more severe symptoms of HLB despite having a lower titer [39]. Grafting budwood for CLas transmission achieves nearly 100% efficiency, while CLam transmission through grafting is typically below 50%. Comparable data for CLaf are currently unavailable. Natural transmission by psyllids is influenced by various factors, with bacterium multiplication and temperature being crucial. The transmission of CLas by D. citri is affected by CLas titer in citrus, with efficient transmission occurring from citrus to citrus, while CLam transmission is less efficient [3].

2.3 Citrus blast

Citrus blast, commonly referred to as black pit, is caused by Pseudomonas syringae pv. syringae (Pss) bacterium, a Gram-negative, chemoorganotrophic bacterium belonging to the Gammaproteobacteria. The Pss bacterium is naturally present in citrus leaves and various plant species. Elevated populations of Pss strains which are associated with blast and black pit, proliferate on the surfaces of citrus leaves and twigs during extended periods of fog, rain, and low temperatures [40].

Pss bacterium infects susceptible citrus tissues, including shoots, twigs, young petioles, or fruit, entering through wounds caused by hail, wind, or abrasions [10, 41]. Lesions initially form on the leaf petiole or wing of citrus and appear as small water-soaked or dark spots. These lesions rapidly expand towards the leaf midvein, twig, and axil tissues, causing the leaves to wither, curl, and turn brown, eventually dropping, often without petioles. Severe attacks can lead to the death of entire twigs, contributing to the tree’s blasted appearance, a condition sometimes mistaken for frost damage. Black pit symptoms are visible on citrus fruits, particularly lemons, which are highly susceptible. Affected fruits, especially those with physical injuries from hail or thorn and branch abrasion, exhibit light brown spots that progressively darken and become markedly sunken, forming black pits typically less than 1 cm in diameter. During postharvest storage, these pits may enlarge, resulting in fruit loss [13, 41, 42]. The occurrence of blast and black pit is prominent in regions characterized by frequent wet, cool, and windy conditions in winter and spring [40].

2.4 Citrus variegated chlorosis (CVC)

X. fastidiosa, the first plant pathogen to undergo complete genome sequencing [43], colonizes the xylem vessels of citrus plants, thus compromising plant development by blocking water and nutrients and leading to CVC [44]. Xylem colonization by Xfp initiates when the insect vector, carrying the pathogen, feeds on the xylem sap of a healthy tree. This colonization is facilitated by enzymes that degrade xylem pit membranes, preventing the obstruction of Xf movement [45]. Ultimately, Xfp colonization leads to the blockage of sap flow [46, 47], and infected trees exhibit characteristic disease symptoms first on leaves and later on fruits.

Although X. fastidiosa can colonize a broad spectrum of hosts, encompassing over 500 plant species, the disease itself is constrained to affecting sweet oranges (Citrus sinensis), tangerines, and their hybrids. Therefore, the range of host species susceptible to infection by Xfp is delimited by the phytopathogen’s genome [3, 48]. Xfp can be transmitted naturally through root grafts [49] or by sharpshooters [3]. The vectors of CVC are insects that feed on citrus xylem vessels, specifically leafhoppers of the Cicadellidae family, such as Bucephalogonia xanthophis and Macugonalia leucomelas, which are particularly effective in transmitting the pathogen. Additionally, transmission can occur through spittlebugs of the Aphrophoridae family [50, 51].

CVC affected leaves display small spots on both surfaces, progressing to orange gum in the centre of the lower surface spot, with yellow spots coalescing and turning necrotic in advanced stages. Highly affected leaves often abscise, leaving tufts of small leaves on affected branches resembling zinc deficiency. Fruits on affected branches mature earlier, are smaller and harder, with increased sugar content but higher acidity compared to fruits from asymptomatic branches of infected or healthy trees [52]. Smaller fruit size leads to direct losses, requiring more affected fruits to fill a standard commercial box. The harder fruits impede juice production, and their reduced size makes them less appealing to consumers in fresh markets. The progression of CVC is more rapid in younger trees and those exposed to seasonal water deficits. Under such conditions, tree development is significantly impacted by the disease, resulting in a substantial decrease in fruit production, reaching up to 75% compared to healthy or asymptomatic trees [53].

3. Detection and management strategies for tackling diseases

3.1 Citrus canker

Despite the economic significance of citrus canker, there has been a lack of effective and environmentally friendly treatments developed to date. The approach to controlling Asiatic citrus canker depends on whether the disease is newly introduced or if it has become endemic. In instances where the disease is recently introduced with low incidence, efforts are often directed towards eradication to restore the region to a disease-free state, aiming to avoid the expenses associated with quarantines and trade restrictions. The eradication process involves the prompt removal of infected plants at a rate faster than the rate of new infections, minimizing the chances of ongoing pathogen spread and hastening the extinction of the disease. However, practical implementation is intricate, as newly infected plants may not be readily recognizable due to a common several-week incubation period before disease expression. Subclinical infections, where plants are infected but not displaying symptoms, can persist for 10–14 days for Asiatic citrus canker, making timely detection challenging. Detection in residential areas has been estimated to take 108 days post-infection, allowing ample time for polycyclic reproduction, and spread. Moreover, diseased plants are not always grouped conveniently for eradication, often presenting a diffuse pattern within a healthy population. Achieving eradication, therefore, requires the removal of not only recognized infections but also asymptomatic yet potentially latently infected plants within large radii. These eradication programs, aimed at Asiatic citrus canker epidemics, have occasionally faced resistance from commercial growers and residential homeowners due to the removal of seemingly healthy trees, impacting the social tolerance threshold and creating a challenge in garnering support for the benefit of the commercial citrus industry [3].

In regions where Asiatic citrus canker is endemic, effective mitigation involves the integration of suitable cultural practices, including the implementation of windbreaks and control measures against leaf miners. Regular applications of copper sprays, such as copper hydroxide and copper oxychloride, are recommended [54]. Notably, grapefruit exhibits high susceptibility to Asiatic citrus canker, necessitating more frequent sprays for acceptable control compared to less susceptible varieties like oranges or mandarins. Antibiotics like gentamicin, kasugamycin, and streptomycin show efficacy in canker control when properly formulated, although concerns about bacterial resistance and potential health issues for humans and animals arise with widespread antibiotic use in agriculture [55, 56]. Addressing the challenge of resistant Xcc populations could involve the implementation of strategies designed to mitigate resistance to copper bactericides, ultimately enhancing their efficacy and minimizing the need for frequent application [20, 57]. One viable alternative is the adoption of the tree row volume (TRV) methodology, initially proposed by Byers et al., which optimizes spray volume and copper application rates based on estimated canopy volume in a specific area [58]. Previous research on citrus canker control using TRV demonstrated a remarkable 73% reduction in water volume and a 50% reduction in copper rates compared to standard applications [59]. More recent findings by Behlau et al. affirmed the efficacy of the TRV method, revealing that citrus canker control can be achieved with just 40 mL of water volume and a copper rate of 36.8 mg/m³ [60].

Employing resistant Xcc citrus varieties is an effective means of pathogen control however, developing these varieties is both expensive and time-intensive. An environmentally friendly alternative for managing citrus canker involves utilizing endophytic bacteria as a biological control method. The utilization of endophytic bacteria in biocontrol not only enhances resistance to diseases but also presents a promising substitute for chemical control strategies [61]. This approach offers a viable and ecologically sound option for combating citrus canker. An alternative approach with potential application involves the induction of Systemic Acquired Resistance (SAR) as a means of controlling phytopathogens [62]. SAR serves as an induced defense mechanism, triggered by salicylic acid, and leads to the accumulation of pathogenicity-related proteins, bolstering the plant’s resistance to various microorganisms. Molecules analogous to salicylic acid, such as isonicotinic acid (INA) and acibenzolar-S-methyl (ASM), can also induce SAR and serve as pathogen control agents. In a study on Swingle citrumelo, soil application of SAR inducers, including imidacloprid, INA, and ASM, reduced lesion numbers and altered their characteristics, resulting in smaller, darker, and less eruptive lesions. This reduction in lesions and a shift in phenotype were associated with a decrease in Xcc population, suggesting that inducing SAR response in susceptible citrus species holds promise for controlling Xcc [63]. A recent investigation into novel approaches for citrus canker disease control focused on the application of penicillin acid to citrus leaves, which showed a decrease in disease lesions indicating the potential of penicillin acid as an effective agent for Citrus Canker disease control [64].

Presently, the identification of citrus canker relies on bacterial culture methods and PCR techniques. While PCR offers rapid results, it may yield false negatives due to PCR inhibitors in samples, and culture techniques, although more reliable, are time-intensive [65, 66]. Consequently, there is a pressing need for the development of an accurate and early field detection method for citrus canker. Haji-Hashemi et al. investigated an electrochemical immunosensor targeting the effector protein PthA for citrus canker diagnosis, demonstrating high selectivity, stability, repeatability, and considerable potential for real sample applications [67]. Abdulridha et al. explored the application of remote sensing technology, employing an unmanned aerial vehicle and hyperspectral imaging, achieving high accuracy in detecting citrus canker in infected leaves during asymptomatic developmental stages [65].

3.2 Huanglongbing

At present, there are no citrus varieties displaying resistance to HLB, and no identified cure exists for this disease. Additionally, efforts to eradicate HLB from any specific area have not been successful [68]. HLB poses many challenges which include the similarity of its symptoms to those of other citrus diseases, a limited understanding of infection mechanisms, the uneven distribution of the pathogen within the host, and the inability to cultivate the bacterium in vitro; collectively presenting a significant hurdle for its study and thus management [30, 69, 70, 71]. Effective monitoring of psyllids in commercial areas and their control through insecticides is crucial for preventing the dissemination of HLB [72, 73] but persistent use of insecticides like imidacloprid, acetamiprid, nitenpyram, clothianidin, thiamethoxam, and cyantraniliprole, in excess, results in high production expenses, the emergence of resistant individuals, environmental pollution, and potential disruption of the ecological balance between pests and their natural adversaries, leading to outbreaks and pest resurgence [74, 75, 76, 77]. So regular general practices like removing infected trees from the orchard can be followed. Regular inspections are crucial to identify new symptomatic plants or those overlooked in prior assessments. Once visually identified, the removal of symptomatic plants is essential to reduce inoculum sources and prevent further spread [3]. Numerous alternative approaches have been explored for managing the issue, including the employment of chemotherapy, thermotherapy, the application of antibiotics, the utilization of natural enemies of the disease vector, and the injection of defense activators into the plant trunk [75, 76, 78]. Nevertheless, all these approaches have their limitations, thereby allowing citrus to persist as a source of inoculation for HLB disease [75, 78].

HLB detection using Polymerase Chain Reaction (PCR) [79] faces challenges due to the irregular distribution of CLas, leading to potential false-negative outcomes. Additionally, the method’s high cost and time-intensive sample preparation make its widespread implementation [38, 70]. Thus, no field-deployable device has been officially validated through PCR results for reliable use in diagnosing. Pagliai et al. demonstrated a potential therapeutic approach against CLas by selectively inhibiting the transcriptional activator LdtR, a key regulator of the ldtP gene encoding a transpeptidase. Through screening over 1300 small molecules, including phloretin, hexestrol, and benzbromarone, they identified compounds that hindered the binding of LdtR to DNA. In vitro assays revealed that these molecules induced morphological changes and osmotic stress sensitivity in CLas cells, suggesting their potential efficacy in controlling HLB [80]. Recent techniques used for detection involve Raman spectroscopy (effective in discerning HLB in citrus by comparing CLas-infected, healthy, and nutritionally deficient samples. Its high sensitivity relies on detecting molecules secreted in early infection stages, surpassing PCR’s detection limits for low CLas levels [81], unique biomarkers like sec-delivered effector 1 (SDE1) (expressed highly in early-stage infected citrus tissues, systemic distribution of which in trees minimizes false negatives and the required sample collection per tree) [70] and use of desorption electrospray ionization coupled to mass spectrometer imaging (DESI-MSI) in HLB monitoring by studying various metabolites, including organic acids, phytohormones, sugars, and amino acids, associated with the plant defense system [82].

Nevertheless, replicated field studies have shown that canines can achieve over 99% accuracy in detecting CLas- and CLaf-infected plants. Preliminary findings also suggest that canines can effectively alert trained human handlers to CLam-infected plants [83]. The urgent need for early detection methods for CLas transmission, given its brief latency period, is a primary focus of current research efforts.

3.3 Citrus blast

Reducing inoculum sources of Pss involves pruning dead or diseased twigs and shoots in spring and post-rainy seasons, which effectively diminishes the incidence and severity of citrus blast and black pit disease by curbing pathogen spread. Implementing wind protection measures, such as installing windbreaks to mitigate wind-induced injuries, is recommended, considering wind is a primary infection inducer. Monitoring tree nutritional status, coupled with adhering to a well-structured fertilization schedule and regular pruning, proves essential to forestall excessive susceptible new growth in the autumn [41]. Opting for resistant or tolerant cultivars stands as an effective approach to disease control [13]. While no citrus cultivar exhibits complete resistance to blast and black pit, certain cultivars demonstrate lower susceptibility. ‘Eureka’, lemon cultivar displays the least susceptibility to citrus blast disease, whereas clementine cultivars such as ‘MA3’ and ‘Cassar’ tend to be less susceptible to black pit [13]. Consequently, these cultivars are recommended for replanting affected citrus orchards, particularly in cold and wet environments conducive to citrus bacteriosis development. For new plantings, it is advisable to choose bushy cultivars with relatively few thorns to minimize vulnerability to injury during severe windstorms [13, 41].

The chemical management of Pss involves a preventive use of copper compounds [84]. The application of protective copper-containing sprays, such as Bordeaux mixture or fixed copper compounds, is advised in conditions conducive to the disease. It is recommended to apply these sprays timely during the fall and early winter, before the initial rains, to safeguard leaves and shoots from Pss infections during late winter and spring [41]. While copper products prove highly effective against bacterial pathogens, their prolonged use may pose significant environmental and health risks, including the emergence of copper-resistant bacterial strains. Considering these concerns, biological control methods are strongly advocated [85, 86]. Biological control involves employing living organisms and their byproducts to restrict or suppress pathogen activities and populations [87]. Plant extracts and certain biological agents, such as Bacillus spp., can play a significant role in suppressing or managing citrus bacteriosis [61]. As noted by noted by Mougou et al., garlic extract and Bacillus spp. exhibit robust antimicrobial activity against the bacterial agents responsible for citrus blast [40] in both in vitro and greenhouse conditions. Additionally, a preliminary study explored the use of phage treatment for controlling bacterial blast in citrus species [88]. Consequently, biological control methods hold significant potential for pathogen management, although further research is required to assess their economic feasibility.

3.4 Citrus variegated chlorosis (CVC)

Management of CVC has traditionally involved preventive measures, primarily the eradication of symptomatic citrus trees, to diminish inoculum sources and minimize the risk of recurrent infections. While weeds and certain arboreal plants may host Xfp, their limited bacterial titer and short life substantially reduce their significance as CVC sources [89]. The accurate identification of diseased trees is crucial, demanding frequent and meticulous inspections due to the continuous emergence of new symptomatic trees resulting from ongoing infections. Under specific conditions, the health of diseased trees can be restored through drastic pruning of symptomatic branches, but this approach is successful only for trees over 2.5 years old and exhibiting symptoms on the tip of a single secondary branch [90].

A novel mitigation measure “Scion substitution” strategy has been developed based on the observed high resistance to Xfp infection in the Cravo Rangpur lime rootstock [91]. Utilizing these rootstocks, where the bacteria do not move downward below the graft union, healthy scions can be generated by removing all infected scions, even from severely affected sweet orange trees, and grafting them with healthy budwood onto these rootstocks. Unfortunately, the rootstock’s immunity to CVC does not transfer to the scion. Nonetheless, the new shoots on the rootstock trunk produce fruits more rapidly and at lower costs than those on newly planted nursery trees [47]; but as there is inconsistent adoption of this technique by growers, certain areas may face elevated inoculum pressure from neighboring groves. Hence, effective control of the insect vector is vital for CVC control. Presently, this control involves the application of systemic insecticides to the trunk of younger trees designated for replanting [92].

Mauricio et al. conducted a 6-year study on the susceptibility of 264 Murcott tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) and Pera sweet orange (C. sinensis (L.) Osbeck) hybrids. Their research revealed that plants exhibiting high expression of isoflavone reductase displayed significant resistance to Xfp infection. Additionally, the study involved the assessment of 12 defense-related genes in both resistant and susceptible varieties to CVC disease. The findings indicated significantly elevated expression in resistant varieties, suggesting the involvement of these genes in conferring resistance against CVC. Among the identified resistance-related genes were those associated with disease resistance proteins (RGA2 and DRP), protein kinase activity (LRR-RK and FLS2), a nucleotide-binding protein (IFR2), and a transcription factor (b-Zip). This outcome underscores that the resistance of these citrus hybrids to Xfp is attributed not to their anatomical structure but rather to the defense mechanisms inherent in these hybrids [93]. Recently, Pereira et al. identified that the CrRAP2.2 gene in C. reticulata is linked to resistance against X. fastidiosa infection in this citrus variety. This gene shares homology with Arabidopsis thaliana AtRAP2.2, associated with the transcriptional factor RAP2.2 and resistance to the pathogen Botrytis cinerea in A. thaliana [94, 95]. The expression of the CrRAP2.2 gene was found to induce resistance to CVC in C. sinensis, subsequently reducing disease symptoms [95]. Developing diagnostic methods for CVC during its asymptomatic phase is crucial for early citrus removal, preventing it from becoming an inoculum source for the bacteria. In this context, Soares et al. employed Liquid Chromatography coupled with Atmospheric Pressure Chemical Ionization-Mass Spectrometry-Selected Reaction Monitoring (LC/APCI-MS-SRM) to detect CVC before observable symptoms appeared in affected trees. The study analyzed samples of C. sinensis grafted on C. limonia, revealing elevated flavonoid concentrations in leaves and coumarins in roots of symptomatic plants compared to asymptomatic and healthy plants. Based on these findings, Soares et al. suggested that the LC/APCI-MS-SRM method could effectively detect CVC before symptomatic manifestations, offering a simple and accurate diagnostic approach [96].

4. Conclusion and prospect for future

Understanding the pathogenicity mechanisms of these diseases remains an ongoing challenge. The substantial genetic variability of phytopathogens like X. fastidiosa and Xanthomonas spp., and difficulties in culturing the bacterium Candidatus Liberibacter spp. contribute to gaps in knowledge [14, 97, 98, 99]. Citrus canker, CVC, HLB and citrus blast have exerted a lasting impact on the global economy for decades [38, 100]. The rapid and imperceptible transmission of these diseases poses a significant challenge for citrus growers, intensified by the absence of effective treatments, ultimately resulting in the demise of infected plants [3, 93, 98, 101]. In response to these challenges, current research efforts are focused on unraveling the mechanisms of infection during the initial stages of the diseases, employing analytical approaches [70, 82, 96, 102]. The advancement of biochemical techniques and analytical platforms is anticipated to enhance our understanding of infection and pathogenicity mechanisms, potentially paving the way for the discovery of effective strategies for diagnosing and controlling major citrus diseases.

Conflict of interest

The authors declare no conflict of interest.