Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. From the screening of random mutants which had previously been performed, it became clear that we needed to screen a larger number of mutant clones to find recognition of elf18-CLas. In order to perform screening on complex EFR mutant libraries required to discover mutants which respond to elf18-CLas we have been developing a FACS based screen. To this end we have generated a number of reporter lines in both suspension cultures and transgenic Arabidopsis plants. The reporter lines are driven by the FRK1, WRKY30 and PER4 promoters. We have tested two PER4 cell suspension lines for responsiveness to elf18 and both of these give clear induction of the reporter gene following treatment. We will also test the WRKY30 and FRK1 cell suspension lines once the liquid cultures are established. In addition we have generated transgenic plants with these reporter constructs and are currently waiting to collect seed from primary transformants. We plan to test the activity of the pPER4:GFP cell suspension lines following protoplast transformation to ensure that the reporter is not activated during. If these tests are clear then we will proceed to FACS screen of mutant EFR libraries. In addition to the mutagenesis approach we are currently setting up to screen the Nordborg collection of Arabidopsis ecotypes for sensitivity to elf18-CLas or reciprocal chimeric peptides of elf18-Ecoli and elf18-CLas, in order to determine whether a natural variant of EFR capable of binding elf18-CLas can be isolated. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. These constructs have been constructed and tested and a manuscript is under revision. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Constructs containing EFR alone or in combination with XA21 or XA21-EFR were provided to the Moore lab, and epicotyl transformation experiments are ongoing (May to July 2014). To date a total of 2,945 ‘Duncan’ grapefruit and 879 sweet orange epicotyl segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). Preliminary GUS histochemical assays were carried out on 6 sweet orange shoots regenerated from transformation experiments with EFR, EFR-XA21 and EFR-XA21-EFRchim. Results indicate that no GUS positive shoots were obtained, however 1 shoot from the EFR-XA21-EFRchim construct was chimeric for GUS.
Citrus Huanglongbing (HLB), also known as citrus greening has been threatening the citrus industry since the early of 1900’s and up to this date there is no effective cure for this disease. Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detected AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. In fact the plant has another signals that bind to LuxR and then, the complex control CLas gene expression to be pathogenic (in susceptible varieties) or less pathogenic (tolerant variates). “Enhancement funding” In order to identify the molecules that influence CLas signals for movement and aggregation in plant, we compared the phloem sap composition of many varieties of citrus and citrus relatives. Phloem sap samples were collected from 14 varieties of healthy (not HLB) infected citrus and 4 varieties of non-citrus, which were derivatized using silylation reagents (TMS) as well as methylchloroformate (MCF) and then injected into the GC-MS. MCF is most sensitive toward free amino acids and some organic acids, whereas TMS is most sensitive toward organic acids and especially sensitive for mono- and disaccharide sugars. Therefore, all samples were analyzed with both derivatization methods. Phloem saps from citrus were found to have compounds such as malic, citric and quinic acids, the sugars glucose, fructose and sucrose, and 17 of the 22 amino acids found in nature. Sugar alcohols such as xylitol, mannitol and several forms of inositol were also found. Altogether, more than 85 different compounds were identified and about 50 have been confirmed using analytical standards purchased from Sigma Aldrich. The distribution of organic compounds in healthy citrus phloem sap in this study was between 22% and 62% sugars, 10-35% organic acids, 2-10% amino acids and 20-35% minor and trace compounds such as fatty acids, amino sugars, sugar acids, and sugar alcohols. in general, varieties can be well separated using principal component analysis to tolerant and suspectable groups. The data is in the final stages of analysis and the manuscript in is the writing stage.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. We reported cloning six new full-length cDNAs of different citrus genes into the entry vector last quarter. Now we have moved five of these clones into the binary vector pBINplusARS. Citrus transformation with these five clones shall be initiated shortly. In addition, a seventh new full-length cDNA was obtained in the entry vector for further DNA construction followed by plant transformation. In addition, we continue to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project has already been terminated.
This project aims to characterize conditions for optimal deployment of a superinfecting Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from the development of the HLB disease and to treat trees that already established the disease. In order to provide protection against HLB, the superinfecting CTV vector will be carrying an anti-HLB insert: a gene of an effective antimicrobial peptide or an RNAi-based insert that would target a psyllid gene. The majority of trees in Florida are already infected with a CTV isolate. The goal of this project to understand how these pre-existing isolates would affect the establishment of infection with the superinfecting vector and, thus, expression and the production level of an anti-HLB insert. I the experiments, we are examining how preexisting infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees. We are assaying the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We first graft-inoculated sweet orange trees with the T36,T30 and/or T68 isolate of CTV, singly or in mixtures (these isolates were propagated in our greenhouse) as well as with CTV-infected material obtained from the field trees (FS series isolates). In addition to wild type isolates, we also included several CTV constructs that could be used as vectors for expression of genes of interest in trees to see how they compete with wild type isolates. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Real time PCR quantification is also being employed to these tests. In these experiments we are using different citrus rootstock/scion combinations in order to find combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the anti-HLB protein of interest from this vector. These combinations include trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. Evaluation of results is ongoing. As a result of this activities, we mapped several genomic regions within the CTV genome that determine the ability of an isolate of CTV to block the secondary infection with another virus isolate. We also discovered how a CTV virus variant that is already established in a tree could be removed from a tree (replaced by a wild type naturally occurring CTV virus). This discovery could be important in the situation when a recombinant CTV vector has to be removed from field trees if its presence is no longer needed or desired.
This quarter we have continued to make progress on our transformation approaches: 1. Stable transformation in citrus using new vectors. We previously found that the original vector system used to create the Bs3 promoter constructs was contributing to low transformation efficiency in citrus, and switched to a pCAMBIA-based vector system. Two ProBs314EBE:avrGf2 transgenic plants created using the new vectors in citrus cultivar Carrizo have now been confirmed by PCR to contain the transgene, and these will be examined for appropriate gene expression by RT-PCR. An ongoing pipeline of transformants are being generated with the new vectors. To date a total of 2,056 putative transgenic shoots of grapefruit, sweet orange and Carrizo were screened for this period. Results show that no GUS positive has been observed for the sweet orange cultivar transformed with any of the constructs analyzed, however, 3 shoots were chimeric for the pCAMBIA2201:NosT:Bs3super::avrGF2 construct. In general, grapefruit had a combined total of 12 and 41 shoots being GUS positive and chimeric for GUS, respectively for all constructs anlayzed while Carrizo citrange had 185 and 180 shoots being GUS positive and chimeric for GUS, respectively. These GUS and chimeric shoots will later be screened via PCR once rooted and transferred to soil for acclimatization. 2. Stable transformation in tomato test system: A tomato test system was previously designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. T1 generation of Bonny Best and Large Red Cherry transformed with ProBs3_14EBE:avrBs4 were screened for pathogenicity reaction with X. euvesicatoria strain (Race 9). Promising resistant transgenics have been selected for T2 generation analysis to confirm that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance.
The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and is being analyzed. A study of CiFT3 transgenic plants treated with various growth regulators has been performed and all of the data have been collected except for the flowering dates of the nontransgenic control plants that have not yet flowered (they should soon). The growth hormones produced striking and individually different phenotypes in each treatment. The endogenous ciFT3 promoter was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. The complete construct is completed and is being tested in tobacco for a rapid test before citrus experiments are started. This research will be presented at the upcoming ASHS national meeting.
The main objective of this research was to study the CLa-flg22 triggered immune response (basal defense or PTI) in citrus genotypes with different levels of tolerance to HLB and the effect of chemicals associated with SAR priming and signaling (azelaic acid and salicylic acid) have on this response and determine if they could be used to enhance the tolerance against this important pathogen. We analyzed the response of ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) after inoculation with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) using comparative Ct real time PCR. The expression of 16 defense-associated genes was determined. In ‘Sun Chu Sha’ we observed that PBS1, NDR1, RAR1, SGT1, EDS1, EDR1, PAL1, AZI1, NPR2, NPR3 and RdRp were significantly induced compared to water controls. However, in ‘Duncan’ only PR1 was significantly induced compared to water controls. Our results suggested that PTI (PAMP-triggered immunity) was induced by CLas-flg22 in the more tolerant genotype but not the susceptible one. These results were presented during the Third International Research Conference on HLB held in Orlando, Florida on February 4-8 of 2013. Additionally, although not part of the original proposal, RNAseq samples were generated from ‘Sun Chu Sha’, ‘Nagami’, and ‘Duncan’, each treated with CLas-flg22 plus controls to further characterize gene expression in the different citrus types beyond the scope that could be analyzed using real time PCR. The use of RNASeq increased and the cost of this technique came down after we wrote the proposal and we felt it would generate important information and verify our results. The sequencing has been finished and we have analyzed the results partially; however, full analysis is still in progress and has not been completed yet. Regarding the chemical applications, we did not observe an increased immune response to CLas-flg22 by pre-treating with azelaic acid (in terms of heightened defense gene expression). However applications of salicylic acid did enhance the response of some defense genes. Subsequently ‘Sun Chu Sha’, ‘Nagami’ and ‘Duncan’ plants were pre-treated with CLas-flg22 peptide 24 hours prior to inoculation with Xanthomonas citri pv citri (Xcc) in an attempt to corroborate whether CLas-flg22 was capable of inducing PAMP-triggered immunity (PTI). The Xcc population/concentration in the inoculated leaves was determined via colony forming units (cfu) by a standard procedure in a time course of up to 2 days post inoculation (dpi). Bacterial growth in the CLas-flg22 pretreated leaves was lower than in those treated with water in both ‘Nagami’ and ‘Sun Chu Sha’ but not in ‘Duncan’, confirming that PTI was triggered. We used Xcc instead of CLas because of the impossibility of infiltrating the latter pathogen and shorter times to estimate the results. Similar experiments using Xcc-flg22 also triggered PTI in resistant genotypes but not susceptible ones. Taken together our results are encouraging and exciting: CLAs-flg22 induces PTI and the level of induction correlates with the levels of resistance observed in different genotypes. Thus, stronger induction of PTI could potentially lead to tolerant or more resistant genotypes to both HLB and citrus canker. A manuscript describing this research has been submitted for publication.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the lasA1 and lasA2 genes. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana and citrus. We have obtained transgenic lines with these constructs. These transgenic Arabidopsis lines were verified by PCR and RT-PCR and their segregation in T2 and T3 were analyzed. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and/or overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. We also expressed LasA1 protein using the Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E. coli. Purified LasA1 protein are used for antibody production and crystallization study. Immunoprecipitation and elution of FLAG-tagged autotransporters from Agro-infiltration in Nicotiana benthamiana yielded several protein candidates, indicating LasA1/LasA2 interacted with mitochondria and chloroplast proteins. To further confirm their interactions, constructs for yeast two hybridization assay have been made. The antibody against LasA1 protein are able to detect LasA1 protein from transient-expressed LasA1 and Las-infected plant tissue using Western blot. The successful generation of this antibody will enhance our research in several aspects. Meanwhile LasA1 sequence has been optimized for a variety of parameters that are critical for efficient gene expression in plants. 30 destabilizing elements, 2 potential destabilization sequences and 2 cryptic splice sites were found and optimized. Transient expression of the optimized lasA1gene yielded much higher level of expression. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein is able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants expressing this protein displayed HLB-like symptom, yellow shoot. Further characterization of this effector and identification of factors affecting its stability in plants is underway.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We have propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild stain status was maintained. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. Due to the slow hiring process of ARS, we have made a limited progress in this reporting period of time.
The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower’s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. We are continuing the field samples and data analysis on heat-treated HLB-affected trees. We are in the process of determining an algorithm that relates environmental conditions with decreases in Las titer. In term of transcriptome analyses, we have conducted a comparison study between heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the the citrus trees treated with heating. We also conducted heat-treatment with constant temperature at 40’C, 85 % relative humidity, and a 12 hr photoperiod for 4 days. RNA samples were prepared with this group of trees and control trees, and sent for RNA-sequencing. Currently, we are analyzing these RNA-seq data, and we are verifying the putative important genes with differential expression(DE) by RT-PCR.
Stress intolerance of HLB trees is a direct consequence of greater than 30 percent loss of fibrous root density compared to non-diseased trees. This root loss may be compounded an additional 20% by the interaction with bicarbonates in the rhizosphere as a result of irrigation with well water high in bicarbonates and/or over-liming with dolomite to avoid copper toxicity. HLB and bicarbonate stress also favor infection by root pathogens such as Phytophthora spp. In field surveys and greenhouse seedlings of different rootstocks with and without Liberibacter asiaticus (Las) inoculation, populations of Phytophthora spp. are often elevated in the rhizosphere of HLB infected plants. When Las interacts with Phytophthora, infection of fibrous roots may be temporarily greater than that caused by Las alone depending on the stage of Las infection and root damage. Las infection disrupts of root function by altering the concentration of soluble sugars and increasing root exudation that attracts and accelerates infection by P. nicotianae. This exacerbation continues until the Las causes root death at which point populations of P. nicotianae rapidly decline. Similar increases and decreases of Phytophthora populations have been observed in the statewide Phytophthora survey conducted Syngenta. Comparing rootstocks in the greenhouse, tolerance to Phytophthora is broken by the interaction with Las such that resistance to biotic or abiotic stress is greatly reduced which results in the premature root loss. This explains the reduction in root system capacity for water and nutrient uptake in field trees affected by HLB on all rootstocks. Moreover, normal annual cycles of fibrous root production appear to be disrupted on HLB affected trees. To confirm this, bimonthly changes in fibrous root mass density and rhizosphere populations of Phytophthora are currently being surveyed in 8 ridge and 4 flatwoods sites in south central Florida.
Installation and sampling with root cages to quantify seasonal changes in root density continues in two field sites. In the first site (Hamlins), 6 months of root growth and root density in healthy and infected trees has been assessed. Sampling revealed seasonal variation in root infection and apparent shifts in the root flush cycle caused by Liberibacter asiaticus (Las). The initial sampling of root cages at the second field site (Valencia) was completed. Installation of root cages in a second site was necessitated and delayed by efforts to find new healthy trees as the previously sampled trees became positive. The development of the Valencia site provides additional insight into how root growth dynamics differ between early season Hamlins and late season Valencias based on different seasonal carbohydrate allocation for fruit ripening. We are also investigating non-destructive ways to measure root growth in the field that will allow for more precise determination of fibrous root longevity. As more healthy trees become PCR positive for Las it is increasingly difficult to find sufficient putative healthy trees in the field. It is especially hard to find trees of more advanced age for seasonal root sampling. As field sampling proceeds, it will become more of a description of HLB-affected tree decline than a comparison to healthy trees. Therefore, emphasis will continue to shift to greenhouse experiments for valid healthy vs HLB comparisons. Sampling at a rootstock trial site is underway with a year and a half of data on the effects of HLB on new experimental rootstocks. This sampling has begun to reveal how these new rootstock lines respond to Las infection. Data for these rootstocks is currently being summarized to present results at upcoming grower meetings. The two most promising rootstock lines and Swingle as a standard rootstock control have been graft inoculated in the greenhouse and transplanted into rhizotrons to monitor root growth and death. Two months after transplant, Las was detected at similar incidence in roots (up to 50% of inoculated trees) for all rootstocks tested. Currently root growth and root longevity in response to Las infection is being assessed.
FireWall 50WP (65.8% streptomycin sulfate; Agrosource, Inc.) has been granted a second year of EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition of FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance and cross resistance to human antibiotics in treated groves. The objective of this survey is to comply with EPA’s protocol for evaluation of streptomycin resistance for Xcc and risk of cross resistance for other leaf and soil bacteria in the treated locations. This protocol is based on one developed by Dr. George Sundin at Michigan State Univ. for the section 18 use of the antibiotic kasugamycin against the Fire blight pathogen. I will be meeting with Dr. Sundin at the annual meeting of the American Phytopathological Society in August to discuss application of his protocol for our survey.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. An improved assay to evaluate the chemotactic response of Xanthomonads to different compounds was tested. A paper disk impregnated with the test compound was placed on semisolid agar plate to establish a chemical gradient. A disk soaked with sterile distilled water was positioned on the opposite side of the agar plate with a drop of a bacterial suspension placed in the middle. After three days incubation, asymmetric colonies developed when the compound produced a positive chemotactic effect. Assays with Xcc 306 strain confirmed a positive chemotactic response for serine and apoplastic fluid from sweet orange leaves. This assay is currently being used to compare the effect of apoplastic fluids from sweet orange, Key lime, kumquat, grapefruit, lemon, cabbage and Prunus spp. for strains Xcc A, A*, Aw, X. alfalfae subsp. citrumelonis, X. campestris pv. campestris, and X. arboricola pv. pruni. Objective 2. To investigate biofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads. Biofilm formation was evaluated for wide (Xcc 306, Xcc 62), and restricted (Xcc Aw, Xcc Iran2) host range strains of Xcc, X. alfalfae subsp. citrumelonis and X. campestris pv. campestris. Strains responded positively to apoplast fluids from sweet orange, Key lime, kumquat and cabbage on LB agar medium rich in nutrients or XVM2 media that models nutrient availability in the apoplast. Differences in biofilm formation were related to the nutrient availability of the culture media. In nutrient limited medium XVM2, apoplast fluid from sweet orange, Key lime and kumquat reduced biofilm formation for all the Xanthomonas strains evaluated. While in LB medium the response of Xcc strains was mostly an increase in biofilm formation. In contrast response of X. campestris pv. campestris was variable. Currently, the effect of DNAse treatments on surface movements on semisolid culture medium plates is being evaluated for all the strains mentioned above.
In the 6 year old block of Valencia on Swingle in Lake Placid, PCR positive trees with symptoms or pre-symptomatic were were treated with soil drenches of Magna-Bon (MB), Cop-R-Quik (CQ) and an experimental compound (EXP) with well demonstrated systemic activity against citrus canker (caused by Xanthomanas citri subsp. citri) as a soil drench. After two seasons of spring and fall soil drench applications with high and low rates of each of these compounds in 3 replicated blocks, visual tree health ratings on a scale of 1-5 (1=Vigorous, asymptomatic, 2=Slight decline; 3=Moderate decline; 4=Severe decline; 5=Non viable, won’t recover) were higher for treated trees than the untreated check. Tree responses indicate that these treatment were having phytotoxic effects on HLB infected trees rather than achieving reduction in bacterial infection and or reduction in HLB symptom expression.
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