Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. As requested by CRDF and SAB, we have revised project and will focus on the following two objectives: to characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors; to test different compounds in controlling HLB and characterize their mechanisms in controlling HLB. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We are characterizing the two putative virulence genes sndA and stbA of Las, e.g. subcellular localization and host proteins interacting with them using yeast two hybrid system.
Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. How bacteria escape from infected plants is underexplored. Understanding the molecular determinants of lesion rupture, how Xcc survives in the intercellular spaces, and how Xcc releases from lesions of host plants will provide many fundamental and practical benefits. Despite the tremendous effort to eradicate citrus canker, the pathogen has spread to most citrus production areas in Florida and continues to spread. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes. Currently, complementation analysis of the mutants is underway. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. All the mutants were tested for affect in release from citrus canker lesions (dispersal assay) on grapefruit for 5, 7, 10 and 14 days as compared to wild type strain. We are repeating this assay to eliminate any false positives. Pectate lyase assay, proteinase assay and motility tests, and EPS assay have been completed for all the mutants. Characterization of the mutants on LPS, capsule and biofilm formation are complete. Currently, we are studying the release process by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. The total RNA of grapefruit infected with wild-type Xac and mutant were extracted and microarray analysis is being conducted. Quantitative reverse transcription PCR is being used to study expression of selected plant genes. Anatomical analysis of the inoculated tissues were conducted using transmission electron microscopy.
Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Ca. L. asiaticus in planta and to reduce the innoculum for psyllid transmission. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In our study, we expanded our previous study in identifying lead antimicrobial compounds with higher activities by targeting SecA using various computational techniques like homology modeling, virtual screening, molecular docking & minimization. Due to the uncultivable nature of Ca. L. asiaticus, we tested the potential inhibitory effect of the selected compounds against Agrobacterium tumefaciens, which is phylogenetically related to Ca. L. asiaticus. Twenty compounds were selected for biological activity study against SecA of Ca. L. asiaticus and A. tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 ug/ml to 256 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Ca. L. asiaticus. Those findings were published in the article entitled: Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design. on Eur J Med Chem (http://dx.doi.org/10.1016/j.ejmech.2012.05.035). To test the application potential of those compounds on plants, the phytotoxicity studies were performed on the five compounds against citrus. At higher concentrations (0.25mg/ml), all five compounds showed phytotoxicity. When the concentrations were diluted to 0.025mg/ml, the toxicity went down to mild to low. Currently, we are testing the inhibitory effects of the five compounds again Ca. L. asiaticus in planta. We are evaluating different solvents and adjuvants for the five compounds. Their antimicrobial activities against Liberibacter crescens, which is closely related Ca. L. asiaticus and could be cultured, are being tested. Currently, we are optimizing these five antimicrobial compounds to identify compounds higher antimicrobial activity. Utilizing similarity search methods on these five active structures, fourteen compounds were selected from commercially available compound database for antimicrobial activity study. Two of them showed similar antimicrobial activities as the five compounds identified previously. In collaboration with Killiny lab, four antimicrobial compounds were shown to affect psyllid transmission of Ca. L. asiaticus.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. We have developed a culture collection of approximately 400 bacteria initially isolated from the root and rhizosphere of citrus. These bacterial isolates have been screened for various beneficial traits . We are also evaluating the antagonistic activity of these bacterial strains against some well-known plant pathogenic fungi. Especially we have screened a bacterial isolate designated as 43A which possess multiple plant growth promoting activity and is also able to antagonize different fungi. We are also testing the plant growth promoting activity of 24 isolates using seed germination pouch in greenhouse. We have also selected several Bacillus spp. possessing multiple beneficial traits to develop bacterial consortium which can be further developed as carrier based bioformulation. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Three isolates could promote growth of grapefruit seedlings. Those isolates are being used to evaluate their potential to prevent citrus seedlings from Las infection vectored by psyllids in a greenhouse. The three strains seem to delay the development of both HLB symptoms and pathogen population on citrus leaves after root application. Several isolates also exhibit inhibitory activity again the citrus canker pathogen Xanthomonas citri ssp. citri. Currently, we are testing the effect of the selected isolates to control HLB in greenhouse using seedlings and in citrus groves. We have selected two groves and conducted a survey of HLB disease index. We are also evaluating the titers of Candidatus Liberibacter asiaticus in the selected trees.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. The hypotheses of the proposed research are (1) Biofilm formation and QS play important roles in X. citri ssp. citri infection of citrus fruit; (2) Control of citrus canker could be improved by interfering with biofilm formation and QS of X. citri ssp. citri. The hypotheses are based on previous studies and our preliminary studies. The specific objectives of this proposed research are as follows: Objective I: To understand the role of biofilm formation and QS in X. citri ssp. citri infection of citrus fruit. Objective II: To prevent X. citri ssp. citri infection of fruit by combining copper with inhibitors of biofilm formation and QS. Recently, we compared the attachment of the QS mutants on the citrus fruit surface. Compared with wild type stain Xac 306, the quorum sensing mutant ‘rpfF showed significantly reduced attachment to the fruit surface as revealed by CLSM (confocal laser scanning microscopy) observation with the GFP-labeled bacterial strains. We also evaluated the effect of nine compounds on Xac biofilm formation on abiotic surfaces using the crystal violet staining method. The data obtained showed that three compounds were active in inhibiting Xac biofilm formation in NB liquid medium at. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been submitted for publication consideration by Phytopathology. We begin to test those selected compounds in the field condition in controlling citrus canker as well as their effect on citrus production.
At the request of the CRB, as of 10-1-11, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major effort this last year has focused on helping the industry to deal with Fuller rose beetle in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that in 2012-13, loads found to be infested with viable egg masses may be denied entry into that country. Morse presented a summary of recent research on Fuller rose beetle at the Korea Export meeting held in Tulare the morning of 9-4-12 and two talks, one on bean thrips and a second on mite contaminants of export citrus, during the 9-4-12 afternoon Australia / New Zealand Export meeting. An update on the FRB situation was given to the CCM Marketing committee 2-20-13, to growers and PCAs in Exeter 2-21-13, and to the CCQC Board in Pomona 3-26-13. A meeting with packing house personnel, CCM, and CCQC will be held 4-12-13 in Exeter and a FRB field day at the Lindcove REC on 4-22-13 will provide an update and demonstration of trunk sprays using a hand operated “spray wand”. Drs. Spencer Walse (USDA-ARS, Fresno) and Beth Mitchum (UC Davis) are spearheading research on postharvest control of FRB eggs, Beth Grafton-Cardwell is doing research on foliar sprays useful in control of FRB adults, and Morse is focusing on trunk sprays on skirt-pruned trees. FMC and CCQC will soon be submitting a 24(c) application requesting that the present 2ee label allowing use of 0.5 lb ai/a/yr bifenthrin as a trunk spray be expanded to allow a total of 1.0 lb ai/acre. To support the 24(c) request, three field studies were conducted to determine fruit residues after either 4 applications of 0.25 lb ai/a spaced out every 6 weeks (4 week PHI) versus 2 applications of 0.5 lb ai/a spaced 12 weeks apart (9 week PHI). Studies were run in a commercial grove north of Bakersfield (Morse lab), at Lindcove (Grafton-Cardwell), and in Riverside (Morse). A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences) and is being used to analyze levels of residues on trunks at various dates post application with different application rates. Data from a FRB adult bioassay run at Lindcove concurrent with trunk residue analysis allows us to determine when trunk sprays are no longer effective and an additional study is in progress at Lindcove evaluating the impact of sprinkler irrigation impacting the trunk. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley. Citrus thrips resistance to abamectin was investigated 10-31-12 and failed to show significant resistance. Two products nearing registration on citrus that will be useful in control of citrus thrips (as well as other pests) are Bexar and Closer. Fall fruit scarring evaluations were taken at a spring 2012 mandarin field trial comparing the efficacy of abamectin, Delegate, Bexar, and Closer but citrus thrips levels were relatively low at location in 2012 (despite extreme levels in 2011), making it difficult to draw strong conclusions. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, data have been collected from 11 navel 1 Valencia, 1 pummelo, 1 grapefruit, and 2 mandarin loads (16 total).
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (January 1 to March 31, 2013), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Dr. Futch continues to evaluate trifoliate rootstocks for HLB tolerance. ‘ Applications continued in the ‘commercial scale’ nutritional trial. ‘ Harvest of all ‘Fresh Varieties’ plus Midsweet completed ‘ Conducting spring fertilizer and pest management programs for the groves o Established compost comparison on ‘commercial scale’ ‘ Planted 1 acre of pomegranate for Dr. Castle ‘ Rejuvinative pruning in Drs. Albrigo/Wong trials ‘ Applications and base line growth measurements made in MFCF replicated nutritional programs evaluation ‘ Mid Florida Citrus Foundation Field Day held on February 28, 2013 attended by 65 growers and allied industry members. Topics covered: o New Varieties Development and Management Corp. update o Dr. Grosser’s Seedless Valencia and establishment of the trial evaluating promising rootstocks for HLB tolerance o New replicated nutritional programs trial o Asian citrus psyllid and citrus leafminer management studies o New Citrus BMP Program ‘ Commercial Trials: o Eurofins evaluations continue o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o Keyplex established a nutritional trial o Bayer demonstration of Optiva programs for citrus canker management ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB
Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. We have surveyed three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. We selected the blight diseased and healthy citrus trees to be used for sampling. Root samples were collected from 24 trees. The first set of DNA and RNA samples have been purified and sent for deep sequencing to identify the microbes associated with blight diseased and healthy citrus. We have received the sequencing result for the first batch of samples and are currently analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Now we are aligning the reads from DNA samples to sweet orange genome and C. clementina genome (V1.0), about 30%-40% reads could not mapped on these three citrus genomes. Those unmapped reads which are not citrus sequences are being used for metagenomic analysis. Root samples were collected again from 12 trees in the selected citrus grove at St. Cloud in March 2014. The DNA and RNA samples are being purified and sent for deep sequencing to identify the changes in microbes associated with blight diseased and healthy citrus after six months. Meantime, those sampled trees were being tested for citrus blight.
RNA was extracted from a pool of 3-5 leaves harvested from each of three trees used for each of the therapeutic spray applications and controls, collected at 0 time and 3 days after the treatments. The six therapeutic treatments and the two controls have been previously described. We obtained enough quantity of RNA to analyze as many as 80 genes that correspond to potential targets and target pathways that maybe expected to respond to the therapeutic spray treatments. The studied genes are related to key pathways that are disrupted in citrus during HLB infection such as starch/sucrose, glycolysis, pentose phosphate pathways, photosynthesis, raffinose, MEP, polyphenols, terpenoid pathways, transcriptions factors (i.e. WRKYs, NACs, MYBs), hormone-related pathways and plant immune responses (salicylic acid, jasmonate mediated responses and their crosstalk with the other hormones). Our current analysis included around 26 genes selected and based on our previous analysis of the citrus transcriptome data obtained from HLB infected trees in orchards in Florida. The focus of our analysis was to analyze target gene responses to elucidate treatment effects on two set of genes: 1) the metabolic syndrome (starch and sucrose metabolism) induced by the source-sink disruption, 2) and the immune dysfunction induced by the disease (including hormone biosynthesis and metabolism, signaling, small molecule crosstalk, defense responses). The current analysis includes the following genes: alpha-amylase, water dikinase starch degradation, pectate lyase, AGD-2 (bacterial defense response), WRKY48, ATP translocase, phloem protein 2 (pp2), GPT2, gibberellin 2-beta-dioxygenase, gibberellin 3-oxidase, invertase, OPDA (Jasmonic biosynthesis), methylsalicylate, NPR1, jasmonic acid methyl transferase, PR-1, sucrose phosphate synthase, SR1, starch synthase, WRKY54, terpene synthase, phosphofructokinase, ent-kaurenoic acid oxidase 1, GASA5, ERF-1, EDS1. Our spray applications appear to improve the HLB induced metabolic syndrome with respect to starch and sucrose metabolism, we observed that alpha-amylase (HLB-down regulated in our previous data) was significantly induced in all treated trees and as well as induced in uninfected trees. The expression of the water dikinase starch degradation gene was down regulated by one of sucrose+atrazine and one of the benzyl adenine+gibberellin treatments. The sucrose synthase transcripts were in higher abundance in gibberellin and atrazine-treated plants. Interestingly, GPT, a key player in the HLB-mediated starch accumulation in leaves, was significantly down regulated in all three treatments down to levels observed in uninfected trees. Invertase was clearly repressed by atrazine+sucrose treatments in comparison to the untreated trees (Time 0). These findings showed that sucrose+atrazine treatments seem to be effective to partially regress HLB-regulation of key genes involved in symptom progression. This might restore the normal source-sink relation, affecting starch accumulation in leaves. Regarding plant immune responses, NPR1, key player in salicylic and jasmonic acid crosstalk was significantly down regulated in arginine treatments. We believe that this finding will allow infected plants to improve SAR responses. We observed up regulation of methyl salicylate (SAR signal) and the up regulation of WRKY54 in gibberellin- and atrazine- treated trees. Interestingly, gibberellin, atrazine and arginine seems to induce PR1 involved in SAR downstream defense pathways. OPDA, involved in the biosynthesis of jasmonic acid was repressed by all the treatments. Gibberellin and arginine seem to positively regulate key players in SAR response (such as SR1, EDS1 and PAD4). Taken together, these findings are encouraging for possible field trials. We are planning to continue qRT-PCR analysis of some more genes at both 24h and 3 day time points.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, December 2013. The lab has been in operation for more than 7 years, and as of December 2013, we have processed more than 62,300 grower samples. Additionally, more than 70,000 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers. Numbers specific to this report are 673 samples received from growers. This number represents a decline from previous years which was expected since disease incidence of HLB is near100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
Microscopic observations of leaf midrib cross-sections: The damage to the phloem observed in cross sections of midrib tissue show the phloem cell wall thickening, collapsed cambium, and phloem cell plugging in Las infected leaves with normal and high B fertilization rates. Only a single leaf sample from the boron deficient plants was both infected with Las and not suspected to have a recent change in fertilization. There is little of note of the Las infected boron deficient leaves. However, noninfected boron deficient leaves showed similar microscopic symptoms to HLB infection. Boron deficiency also results in phloem cell wall thickening, cambium collapse, and starch accumulation. Although less frequent, some phloem plugging can also be observed. Unlike the leaf midribs with HLB damage under normal fertilization in this experiment, there has been substantial phloem multiplication leading to a thickened phloem layer in the B deficient noninfected leaves. However, phloem multiplication is a common symptom of HLB in leaf midribs. In most cases the B deficiency symptoms resemble the damage observed in the early stages of HLB symptom development (personal observations). However, this damage would be uniform throughout the tree in B deficiency compared to the noncontinuous symptoms observed in HLB affected trees. It has previously been subjected that Las in damaged phloem tissue is mostly dead (Folimonova et al., 2010). This damage caused by B deficiency is most likely responsible for the loss of detection of Las in B deficient trees. The poor rate of infection could also be due to phloem damage limiting the spread of Las from the infected budwood. This would fit with the hypothesis that the active Las population exists in presymptomatic leaves. After symptoms develop and phloem damage occurs the bacteria appear to become trapped and die. At present there is little evidence of a significant effect of B fertilization on HLB with a lack of effect on leaf chlorophyll content, trunk caliper, or leaf nutrient status. However, Las population data suggest that phloem degeneration could be reduced at high B fertilization rates although this is not readily observed in leaf midrib cross sections. The most conclusive results will come from analysis of a B fertilization and HLB interaction of biomass measures taken at the end of the experiment. However, microscopy analysis does show similarities in phloem damage occurring from HLB and B deficiency. Although increased B fertilization does not appear to alleviate phloem damage, this does suggest that the damage is caused by similar mechanisms. B deficiency is known to inhibit proper cell wall formation resulting in physiological responses due to plant cell sensing of weakened cell wall structure. It is possible that as Las inhabits and grows in phloem tissue it causes damage to cell wall integrity, through an unknown mechanism and triggers a similar physiological response to that of B deficiency. Please refer to the final comprehensive report for final biomass analyses, with no page limitations and including figures and tables which better illustrate the microscopy results.
Currently no differences in tree caliper or leaf chlorophyll content as measured by SPAD have been observed among treatments (Table 1). However, tree vigor and growth differences are visually apparent. At termination of the experiment canopy and root biomass data will be collected to evaluate the effects of treatment on overall tree growth and vigor. B deficient trees have mostly stopped pushing new flush. When new shoots are observed they are small and few in number. Table 2. Caliper and SPAD measurements Caliper SPAD B Treatment Pre treat Posttreat Total Pre treat Posttreat Total Inoc Deficient 12.6 ‘ 1.0 12.4 ‘ 1.0 12.5 ‘ 1.0 71.2 ‘ 13.0 70.4 ‘ 5.6 70.8 ‘ 9.4 Normal 11.8 ‘ 1.2 13.3 ‘ 0.7 12.5 ‘ 1.2 66.7 ‘ 5.9 67.1 ‘ 7.0 66.9 ‘ 6.1 High 12.1 ‘ 0.8 12.9 ‘ 0.8 12.5 ‘ 0.9 68.7 ‘ 6.5 69.1 ‘ 5.0 68.9 ‘ 5.4 Infected Deficient 11.8 ‘ 0.7 65.1 ‘ 5.8 Normal 12.7 ‘ 1.2 66.1 ‘ 6.5 High 12.3 ‘ 1.0 68.7 ‘ 5.1 NonInoc Deficient 11.8 ‘ 1.4 12.5 ‘ 0.5 12.2 ‘ 1.1 62.8 ‘ 6.5 70.4 ‘ 5.6 67.4 ‘ 6.7 Normal 12.2 ‘ 1.3 12.6 ‘ 0.6 12.4 ‘ 1.0 67.7 ‘ 1.9 69.9 ‘ 5.2 68.8 ‘ 3.9 High 12.7 ‘ 0.2 12.1 ‘ 1.0 12.4 ‘ 0.7 70.7 ‘ 3.5 68.9 ‘ 3.5 69.9 ‘ 3.4 No significant differences were found in any treatment.
Microscopic observations of leaf midrib cross-sections: The damage to the phloem observed in cross sections of midrib tissue show the phloem cell wall thickening, collapsed cambium, and phloem cell plugging in Las infected leaves with normal and high B fertilization rates. Only a single leaf sample from the boron deficient plants was both infected with Las and not suspected to have a recent change in fertilization. There is little of note of the Las infected boron deficient leaves. However, noninfected boron deficient leaves showed similar microscopic symptoms to HLB infection. Boron deficiency also results in phloem cell wall thickening, cambium collapse, and starch accumulation. Although less frequent, some phloem plugging can also be observed. Unlike the leaf midribs with HLB damage under normal fertilization in this experiment, there has been substantial phloem multiplication leading to a thickened phloem layer in the B deficient noninfected leaves. However, phloem multiplication is a common symptom of HLB in leaf midribs. In most cases the B deficiency symptoms resemble the damage observed in the early stages of HLB symptom development (personal observations). However, this damage would be uniform throughout the tree in B deficiency compared to the noncontinuous symptoms observed in HLB affected trees. It has previously been subjected that Las in damaged phloem tissue is mostly dead (Folimonova et al., 2010). This damage caused by B deficiency is most likely responsible for the loss of detection of Las in B deficient trees. The poor rate of infection could also be due to phloem damage limiting the spread of Las from the infected budwood. This would fit with the hypothesis that the active Las population exists in presymptomatic leaves. After symptoms develop and phloem damage occurs the bacteria appear to become trapped and die. At present there is little evidence of a significant effect of B fertilization on HLB with a lack of effect on leaf chlorophyll content, trunk caliper, or leaf nutrient status. However, Las population data suggest that phloem degeneration could be reduced at high B fertilization rates although this is not readily observed in leaf midrib cross sections. The most conclusive results will come from analysis of a B fertilization and HLB interaction of biomass measures taken at the end of the experiment. However, microscopy analysis does show similarities in phloem damage occurring from HLB and B deficiency. Although increased B fertilization does not appear to alleviate phloem damage, this does suggest that the damage is caused by similar mechanisms. B deficiency is known to inhibit proper cell wall formation resulting in physiological responses due to plant cell sensing of weakened cell wall structure. It is possible that as Las inhabits and grows in phloem tissue it causes damage to cell wall integrity, through an unknown mechanism and triggers a similar physiological response to that of B deficiency. Please refer to the final comprehensive report for final biomass analyses, with no page limitations and including figures and tables which better illustrate the microscopy results.
The effects of B nutrient status on Las populations in leaf midribs was somewhat unexpected. Because Las populations in leaf midribs has a bimodal distribution a permutation based ANOVA (PERMANOVA) was used for statistical analysis. PERMANOVA does not make assumptions about normality of distributions, providing added statistical power with bimodal distributions. When all population data from trees with successful infection was considered together with repeated measures, no significant differences were detected. The mean Las titer beginning at 10 months post inoculation (mpi), after most infected trees tested positive by qPCR, are presented (Table 1). A dramatic reduction in mean Las titer is observed at 14 mpi. When analyzed alone there is a weak significance of B status on Las titer with lower Las populations as the concentration of B in the trees declines (Table 1). This reduction in Las titer as the B concentration in the tree declines appears to be due to a shift in the bimodal distribution towards low population leaves. A similar and highly significant trend is seen when the rate of successful inoculation is analyzed (Table 1). The reduced infection rate and Las titer at deficient B concentrations is suspected to be caused by less efficient phloem generation after graft inoculation and reduced phloem function caused by B deficiency. The infection rate is based on leaf sampling. It is possible that a higher infection rate occurred, but B deficiency induced phloem disfunction prevented spread of sufficient Las from the inoculation site on the stem to the tree canopy. Table 1. Las titer and infection success rate in leaf midribs from 3-4 pooled leaf samples at multiple months post inoculation (mpi) Log Las genomes/g midrib tissue* Presence of Las* B Treatment 10 mpi 12 mpi 14 mpi Deficient 4.1 ‘ 2.4 5.8 ‘ 2.3 2.7 ‘ 4.6a 3/10a Normal 3.9 ‘ 3.6 5.1 ‘ 3.0 6.3 ‘ 2.3ab 8/10b High 4.3 ‘ 2.9 7.0 ‘ 1.6 7.6 ‘ 1.4b 7/10b Pseudo-F p-value p-value** Pseudo-F p-value B level 1.61 0.21 0.0313 9.52 0.0003 MPI 2.39 0.102 0.16 0.77 B level x MPI 1.34 0.27 0.78 0.53 *Values with the same letter are not significantly different **p-values for analysis of only 14 mpi Samples of leaf midrib have been collected, fixed, and embedded. Sectioning and staining of the samples for light microscopy is currently underway. The general functionality of phloem and starch accumulation resulting from HLB infection will be visible using light microscopy. To attempt to observe effects of treatments on Las cells, especially in B deficient plants sectioning and observation by TEM will be done on representative samples for each treatment group identified by light microscopy.
Boron (B) deficiency causes phloem plugging and collapse and excess callose formation. These phloem disfunctions are similar to those caused by Candidatus Liberibacter asiaticus (Las) during the development of Huanglongbing (HLB). Degradation of phloem tissue in B deficient plants is attributed to the lack of sufficient B to participate in its essential role in proper cell wall formation. The purpose of this study was to compare the effects of B deficiency and Las on phloem tissue to determine if cell wall damage is a likely cause of symptoms in HLB and to determine if supplemental B would reduce symptoms or Las infection, phloem damage, or improve health and vigor of Las infected trees. Changes in these factors are monitored by 1) qPCR of Las DNA in leaf midribs, 2) microscopy of leaf midrib tissue, and 3) classic measurements of growth and health including caliper, SPAD, biomass, and nutrient analysis. Due to the extremely slow development of B deficiency in the trees, achieved only after 12 months, we requested a 9-month extension to complete the remaining measurements for this study. Tree nutrient status showed that leaf B concentrations had reached desired levels in each treatment, specifically: B-deficient, HLB-inoculated: 5.8 ‘ 0.8 ppm B-deficient, uninoculated: 7.2 ‘ 4.3 ppm Normal B, HLB-inoculated: 90.6 ‘ 42.8 ppm Normal B, uninoculated: 128.5 ‘ 27.0 ppm High-B, HLB-inoculated: 254.6 ‘ 120.5 ppm High-B, uninoculated: 361.2 ‘ 30.2 ppm Results were highly significant for boron fertilization, HLB inoculation, and there was also a significant BxHLB interaction. The BxHLB interaction was likely due to a greater suppression of leaf B concentrations by HLB when amounts were either high or normal, than when amounts were deficient. This may in part be due to the lower success of HLB inoculation by bud grafting on the B-deficient trees. The BxHLB interaction was the only significant leaf nutrient interaction. This lack of other nutrient interaction suggests that supplemental B fertilization is not reducing the effects of Las on nutrient distribution in the plant. Therefore, there is no evidence that B is altering the effects of Las on nutrient uptake or distribution in citrus trees. It is noteworthy that changes in concentrations of Ca in the leaf mirrored those of B, but that HLB inoculation in particular caused a reduction in leaf Ca concentrations. For most other nutrients the leaf concentrations are at optimum concentrations or above. Mg remains below optimum, which was likely caused by an imidacloprid application. The leaf Mg concentrations are the same among all treatments, so confounding effects should be minimized. Changes in B fertilization did also cause significant changes in leaf concentrations of N, P, K, and Fe.
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