**5.2 Las bacterial control**

### *5.2.1 Antibiotics*

Antibiotics are crucial for controlling bacterial diseases in fruit-bearing trees, vegetables, and ornamentals. Although antibiotics can be detected on plant surfaces using delicate analytical chemistry techniques for up to a month after application, their ability to inhibit bacterial growth is lost within a week [120]. In-plant disease control, nearly 40 antibiotics were screened; only streptomycin and tetracycline were used extensively in fruit trees [121]. The only commercially applied treatment for HLB was tetracycline, which is bacteriostatic rather than bactericidal, in Reunion Island's orchards [122, 123]. Tetracycline was the only approved antibiotic injection in trees injected directly into the trunks of HLB-affected citrus trees in China, Indonesia, India, Taiwan, and South Africa during the 1970s [36, 117, 124]. Although the symptoms of HLB were considerably decreased, this antibiotic trunk injection method was not in practice owing to its phytotoxicity and labor costs. The use of penicillin-carbendazim antibiotics in citrus trees showed significant control of HLB disease. The antibiotic disadvantage is a reduction in the fruit size owing to phytotoxicity and the residues of the antibiotics in citrus fruits [125]. The development of therapeutic compounds and bactericidal agents to control devastating HLB could provide an additional solution for an effective integrated disease management program. However, other than selective antibiotics, nonselective bactericide is recommended for general use in most crops, particularly citrus [126]. The combination treatment of streptomycin with penicillin efficiently eliminated or repressed the Las bacterium compared with the separate administration of either antibiotic [126]. The treatment of penicillin combined with streptomycin also significantly reduced the bacterial titer of Las in greenhouse citrus plants. Kasugamycin and Oxytetracycline combination therapy *via* trunk injection significantly reduced HLB bacterial titer in the field. However, the combination of kasugamycin and streptomycin was not effective against the bacterium of Las [127]. Penicillin with oxytetracycline combination therapy has been more effective in controlling citrus pathogens [128] but may require annual treatment [20]. Among the 31 tested antibiotics, some were effective at reducing and eliminating Las bacterial titers in inoculated rootstock and the treated scions of citrus plants, such as ampicillin, carbenicillin, penicillin, cefalexin, rifampicin, and sulfadimethoxine [20]. Oxytetracycline has therefore been suggested to be used more frequently in combination treatment [129, 130] with penicillin or kasugamycin against HLB to control the progression of bacterial resistance and maximize the antibiotic efficacy against HLB pathogenic bacteria [131]. The Environmental Protection Agency (EPA) of the USA allows citrus growers to spray streptomycin and oxytetracycline as routine treatments in the citrus field several times per year [132]. Oxytetracycline (1 g/L) was delivered to leaves of HLB-infected trees through the

foliar application, and oxytetracycline was found in all leaves, although at reduced levels than in the directly applied leaves [132]. However, the phytotoxicity of tetracycline should be considered [20]. Antibiotics tested to combat HLB malady are tabulated in **Table 1**.


#### **Table 1.**

*Antibiotics effectiveness against CLas bacterium and phytotoxicity.*

*Devious Phloem Intruder* Candidatus *Liberibacter Species Causing Huanglongbing: History… DOI: http://dx.doi.org/10.5772/intechopen.105089*

#### *5.2.2 Thermotherapy*

Heat treatment or thermotherapy of planting material is a century-old disease control method that has proven effective against various pathogenic microorganisms. Thermotherapy, simple in principle, can eliminate the conserved pathogens depending on temperature/time regime and can cause mild injuries to the host during the treatment. Heat is mainly generated by water, vapor, or air [133]. The main advantage of thermotherapy treatment is that it is more environmentally friendly than harmful agrochemicals. Thermotherapy has proven to be an effective strategy against HLB that helps to enhance the vigor of citrus trees and promotes new root growth and development. The efficacy of thermotherapy against HLB pathogens depends on the temperature and citrus varieties [134]. Therapy could recuperate HLB-affected citrus plants by eliminating or suppressing Las bacterial titers at temperatures above 40°C [6, 134]. *Candidatus* Liberibacter asiaticus is a heat-tolerant phloem-limited bacteria that can withstand a temperature of about 35°C, while *Candidatus* Liberibacter americanus is heat-sensitive [135]. Thermotherapy could eliminate HLB pathogens from valuable horticultural trees associated with shoot tip grafting [136].

Lin opined on eliminating yellow shoot disease with water-saturated hot air treatment of graft wood 48–58°C with no loss of tissue viability [137]. In India, the thermotherapy of budwood at 47°C for 2 hours of diminished disease incidence, and more prolonged treatment eradicated the pathogen [138]. Heat treatment at temperatures around 38–40°C for 3 or 4 weeks killed HLB pathogens in young infected plants or citrus seedlings grafted with infected tissues [138, 139]. In South Africa, HLB-infected budwoods were treated with hot water baths at 51°C for 1 hour, 49°C for 2 hours, and 47°C for 4 hours, eliminating HLB pathogens with some loss of viability at higher temperatures [140]. In HLB-affected trees topped with polyethylene fiberglass sheets for 2 to 5 months, the number of diseased fruits decreased. However, this technique is not feasible for extensive use in citrus groves [27]. The HLB-affected citrus seedlings were continuously exposed to 40 to 42°C heat therapy for 7 to 10 days, significantly reducing titer or eliminating Las bacteria. This treatment can be helpful to combat HLB-affected plants in greenhouse and nursery settings [134]. Ehsani et al. [141] also postulated a decrease in HLB symptoms in groves of citrus trees after heat treatment. The combined thermo- and chemotherapy of sulfathiazole sodium or sulfadimethoxine sodium was more effective at 45°C than in thermotherapy alone, chemotherapy alone, or a combination of thermotherapy at 40°C and chemotherapy [142]. The temperature treatment at 45°C for 8 h per day for a week and a combination of ampicillin sodium, actidione, and validoxylamine A as a bark paint on grapefruits plant significantly reduced Las titer [143]. Two-year-old graft HLB-affected citrus reticulate treated with thermotherapy at 45°C and 48°C showed diminished HLB symptoms and Las titers 8 weeks after treatment in the greenhouse condition [144]. Commercial and residential citrus trees covered with portable plastic enclosures exposed to elevated temperatures through solarization showed vigorous growth in 3–6 weeks after treatment. Although commercial citrus trees showed Las after heat treatment, many trees generated extensive flushes and grew strongly for 2 to 3 years after therapy [145]. Inner bark heat treatment with 60°C–0.03 MPa-30s in 9-year-old citrus plants exhibited significantly reduced Las bacterial titer with vigorous plant growth from all treated HLB-affected trees [146]. Abdulridha et al. [147] reported that HLB-affected trees with canopy cover were treated with combined hot water and steam therapy at 55°C for 90 seconds. The temperature distribution inside the canopy cover was not uniform; the canopy temperatures were more significant than the trunk

temperatures. The mobile thermotherapy treatment needs to be improved to increase the temperatures around the tree trunk to nearly the same temperature as a canopy. Vincent et al. [132] postulated that heat treatment from 43 to 54°C for no longer than 45 s showed adverse effects on citrus tree growth.

HLB is a systemic disease. Efficient elimination of Las bacteria from the entire citrus tree, including roots, is vital to managing the disease. The current thermotherapy challenge is that although adequately elevated temperatures can reach the above-ground areas of the plant, killing temperatures are unlikely to be attained at the roots where the temperature is mitigated by the soil [148]. Therefore, heat treatment is unlikely to reduce the populations of HLB pathogens in the roots, which then acts as a site for canopy reinfection during flushes. The efficacy of heat treatment in eliminating Las bacterial populations in underground roots must be enhanced to become a feasible part of integrated citrus HLB management [15]. To overcome this barrier, Hoffman et al. [134] suggest that heat treatment, coupled with chemotherapy in HLBaffected plants, can lead to a potential future strategy for controlling citrus HLB.

#### *5.2.3 Plant defense activators to combat HLB*

Trunk injection is an alternative target-precise technique for efficiently delivering plant protective chemicals in tree fruit crops. It harnesses the rapid transportation ability of the xylem that enables therapeutic compounds' translocation and subsequent distribution into the canopy where plant protection is needed [149]. There has been limited research on the trunk injection of antibiotics and plant defense activators for better disease control. Several recent field studies have demonstrated the utility of trunk injection of bactericides and plant defense activators in disease management [150].

Treatments with β-aminobutyric acid (BABA), 2,1,3-benzothiadiazole (BTH), 2,6-dichloroisonicotinic acid (INA), ascorbic acid (AA), and the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DDG) plant defense inducers individually or in combination found effective in suppressing Las bacterial population in plants and sustaining fruit production to a certain extent. Treatment with BABA and BTH was the most effective in reducing the Las population in plant tissues compared with other plant defense inducers [151]. Hu and Wang proved that trunk injection of oxytetracycline in HLB-affected trees exhibited long-lasting suppression of Las populations. It also prevented the tree decline by promoting new growth without the disease [152]. Trunk injections of salicylic acid, potassium phosphate, acibenzolar-S-methyl, and oxalic acid in the HLB-affected tree significantly suppressed the Las titer and HLB disease progress [150].

Brassinosteroids (BRs) are a class of steroid hormones that regulate gene expression, growth, and developmental processes in response to biotic and abiotic stress [153]. The plant defense mechanism of brassinosteroids was mediated by leucinerich repeat receptor kinase (LRR-RK) BAK1, which serves as a coreceptor for both microbe-associated molecular patterns (MAMPs) and steroid hormone [154], which binds to BRs and FLS2 eliciting microbe-induced immunity. BR treatment showed increasing disease resistance against many pathogens [6]. Canales et al. [155] postulated that applying epibrassinolide as a foliar spray in HLB-infected plants improved immunity against *Candidatus* Liberibacter asiaticus in greenhouse and field citrus plants. *Candidatus* Liberibacter asiaticus titer was markedly reduced in epibrassinolide-treated plants due to the enhanced defense gene expression in the citrus leaves. However, the molecular mechanism of BRs in plant responses under normal and environmentally challenging conditions has remained unclear [155].

*Devious Phloem Intruder* Candidatus *Liberibacter Species Causing Huanglongbing: History… DOI: http://dx.doi.org/10.5772/intechopen.105089*

#### **5.3 Nanoemulsions to deliver chemicals against Las bacteria**

HLB is caused by Las proteobacteria that reside in the phloem of infected citrus trees. It is, therefore, challenging to deliver effective compounds into the phloem through a foliar spray. The presence of wax, cutin, and pectin in plant cuticles prevents the effective bactericidal compounds from entering the phloem through a foliar spraying method. The use of chemical adjuvant enhanced the foliar uptake of agrochemicals [156, 157]. However, foliar spray treatment, including the combination of antibiotic PS and adjuvants in dimethyl sulfoxide and Silwet L-77, did not significantly impact the HLB-affected citrus trees [128]. Therefore, there is a need for candidate adjuvants, which can potentially increase the permeability of citrus cuticles to deliver antimicrobial compounds into citrus phloem.

Nanoemulsions or submicron emulsions are colloidal dispersion systems with average droplets size ranging from 50 to 1000 nm that has extensively studied for delivering chemical compounds. Nanoemulsions were pondered as thermodynamically and kinetically stable isotropic dispersions, composed of two immiscible liquids such as water and oil, stabilized by an interfacial film composed of an appropriate surfactant and co-surfactant to form a single-phase [158]. However, the approach efficacy relies on nanoemulsions droplet characteristics, such as low surface tension, tiny size, ample surface area, and low interface tension [159]. Our research group postulated that water in oil nanoemulsions containing ampicillin coupled with adjuvant Brij 35 was used as a foliar spray to enhance the permeability through the citrus cuticle into the phloem and more efficiently eliminated Las bacteria in HLB-affected citrus *in planta* [160]. Ampicillin showed the lowest phytotoxicity to citrus trees infected with Las bacteria [20]. However, the US Environmental Protection Agency (EPA) has not approved the commercial use of ampicillin in crops due to the development of resistant bacterial strains [160]. In another study, oil in water nanoemulsions was formulated using a spontaneous emulsification method, where five different antimicrobial compounds alone combined with Cremophor EL (viscous oil), acetone, and Span 80/Tween 80, which formed tiny droplets, were effectively applied to the bark for efficiently control HLB [161].

Silver nanoparticles (AgNPs) are one of the most investigated and used in agricultural science to enhance the yield and sustainable development of the crop. This has long been reported to have significant antibacterial, antifungal, antiviral, and pesticide effects. AgNPs are used as foliar sprays to prevent the development of rot, mold, fungi, and other plant pathogens [162]. Stephano-Hornedo et al. [18] evaluated the commercially available AgNPs to directly eradicate *Candidatus* Liberibacter asiaticus (CLas), responsible for HLB in the citrus field. The 93 HLB-infected citrus trees administered foliar and trunk injections of silver nanoparticles showed a remarkable reduction of 80–90% in bacterial titer by both methods than control. Compared with other effective treatments involving b-lactam antibiotics, the effectiveness of AgNPs is 3- to 60-fold higher when administered by foliar spray and 75- to 750-fold higher when injected *via* tree trunk. Thus, the silver nanoparticles could be a sustainable method for mitigating citrus HLB. However, AgNPs toxicity to a citrus tree and the environment needs to be warranted before its commercial use.

#### **5.4 Transgenic approach to combat HLB**

Globally, insect pests are responsible for significant crop losses through direct harm and transmission of plant diseases [163]. The best long-term alternative strategy for managing citrus HLB is to develop disease-resistant cultivars in commercial citrus production. Due to the lack of resistant cultivars, developing HLB-resistant plants by conventional citrus breeding is difficult. Resistance occurs in citrus relatives, such as kumquat, where its genetic background influences the quality and yield of the fruit [164]. In addition, conventional citrus breeding is labor- and time-consuming, and very costly as citrus species are polygenic, extremely heterozygous plants with a long juvenile phase. The genetic transformation approach is an essential strategy that would aid in incorporating disease-resistant genes into citrus cultivars to combat the HLB disease. The progression of citrus breeding through genetic transformation is still early, indicating a lack of molecular pathogenesis understanding of innate disease resistance in citrus [165].

Systemic acquired resistance (SAR), a natural plant defense response mechanism, has been well characterized in *Arabidopsis thaliana*. SAR entails signal molecule salicylic acid (SA) to activate defense mechanisms. In response to SA, the non-expression of pathogenesis-related gene 1 (NPR1) is translocated to the nucleus, where it triggers the expression of pathogenic related (PR) genes by interacting with TGA transcription factors, thereby provoking SAR [166, 167]. *Arabidopsis* mutants contain deficiencies in the *NPR1* gene showing decreased PR gene expression induced by SA and SAR, leading to increased susceptibility to pathogens [167, 168]. Conversely, overexpression of the *NPR1* gene in *Arabidopsis* increased the disease resistance to bacteria and oomycete pathogens. Interestingly, the over-expression of *AtNPR1*gene in most plant species does not provoke noticeable adverse effects on plant growth and development [169]. Thus, *NPR1* is a target gene for the genetic transformation of nonspecific resistance in crop plants.

Dutt et al. [170] postulated that the overexpression of the *AtNPR1* gene in Hamlin and Valencia orange cultivars resulted in trees with normal phenotypes, and exhibited increased resistance to HLB. Transgenic trees showed reduced disease severity, and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field. The phloem-expressed *NPR1* gene was equally effective in increasing disease resistance by triggering several indigenous gene expressions involving plant defense mechanisms of signaling pathways. In addition to triggering resistance to HLB, the observed SAR response could protect citrus trees from other major fungal and bacterial diseases, such as black spots and citrus canker [170].
