The objectives of the first year’s research project are focusing on: 1. Conduct genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of ‘Candidatus Liberibacter asiaticus’ (Las). Design and develop PCR-based multi-locus SSR molecular diagnosis assay. 2. Analyzing Las population structures, assessing the genetic diversity of Las in Florida populations. We have conducted genome wide sequence analysis to identify (SSR) loci in Las. This resulted in the identification of ~175 total sequencing loci in Las containing mono, di-, tri-, tetra-, penta-, hex- type of perfects, in-perfect and compound types of simple repeats. Based on the criteria for primer design and BLAST analysis against available microbe sequences in the NCBI databases, 112 SSR loci were selected for primer design. We then designed and experimentally evaluated Las SSR primers. These evaluations were based on PCR experiments using 2-3 Las isolates collected from US Florida, India, China and Brazil, respectively. These efforts led to the successful validation and development of 10 SSR primers. While we continue working on more SSR marker development, these 10 SSR markers that showed to be useful for unambiguous detection and discrimination of Las genotypes were used for Las population genetic analysis. To adapt high through-put sample analysis platform, these SSR markers were labeled with fluorescent dyes (FAM, NET, VIC). These labeled SSR markers can be multiplexed and analyzed by an automatic ABI 3130 Genetic Analyzer. This Las genetic analysis system can process 4 x 96 samples in 2.5 hours. We assessed Florida Las population and compared it with a global genetic diversity of Las populations using a multi-locus SSR marker system. A total of 166 HLB isolates representing four major citrus production regions from US Florida, Brazil, India and China were analyzed using 5 SSR primers. Two of them (India and China) represent populations in Asian continent where HLB has been for centuries while another two (US Florida and Brazil) represent recently emerging populations in American continent. Based on allele frequencies of SSR loci among the four populations, a genetic similarity matrix that consisted of 71 alleles and 166 samples was generated. The genetic distance analyses were performed using Neil’s coefficient with 1,000 bootstrap and 95% confidential intervals. A pair wise population genetic analysis indicated that among four Las populations, the genetic distance between Florida vs China Las populations is 0.61, while the genetic distance between Florida vs India is 1.35, Florida vs Brazil is 0.96, China vs Brazil is 0.84, India vs Brazil is 0.95 and China vs India is 1.72, respectively. The results indicated that the genetic distance between Florida and China have the closest distance (0.61) as compared with other populations. High percentage of commonly-shared allele types detected in both populations suggests that Las populations in Florida and China are more related. This leads to a hypothesis that Las populations in Florida could be possibly derived from China. It is not clear if the introduction was from single or multiple times and/or from multiple sources as well. Our results demonstrate the useful of multi-locus SSR marker system for genetic analysis of Las. Bacterium has very compact genome. Sequencing variations of SSR loci located within or near the coding and/or gene regulatory regions could have profound effect on gene expression and functions. We have mapped SSR loci genome containing genes of interest. Our next step is to examine the functionality of the candidate genes using a standard in vitro heterologous expression system. This type of study will facilitate the link of DNA-based genotyping to phenotyping of Las. We have accomplished objective 1 and 2 during the first year of the project. Research had been summarized and reported in 2009 and 2010 American Phytopathological Society meetings.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained approximate 1.25Mb of sequence from Ca. L. psyllaurous. This nearly complete genome contains less than 10 contigs and has ca. 34X coverage . We have also obtained a draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. We are currently confirming the sequence of these contigs in both psyllids and host plants. A preliminary comparison revealed significant differences between Ca. L. asiaticus and Ca. L. americanus. The information from our genome sequence allowed us to design new primers and probes that target various regions of the bacterial genome. Using these new primers and probes, genetic diversity of Candidatus Liberibacter asiaticus (Las) samples collected from Florida, Brazil, China, Philippines, Thailand, India and Japan can be found. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect-transmission determinant gene was identified and the role of this gene is under investigation. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, with an overall aim of interfering with the ability of Las to regulate zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” A super sensitive qPCR detection technology has been developed, which increased the sensitivity of detection by 100-2000 fold, thereby eliminating the need for DNA isolation and increasing the throughput of the detection method. The cost savings can be up to 500%. Because the detection is based on HLB bacterium-specific primers, the detection data further confirmed our results on seed transmission and HLB disease phenotypes with low bacterial titers. The role(s) of the seed-transmitted Las is under investigation.
We have accomplished the first objective to duplicate at other grove sites the positive response in HLB infected trees by applying nutrient/SAR foliar sprays obtained by citrus grower Maury Boyd in his Orange Hammock grove in Felda. The second objective was to identify the important components in the foliar spray cocktail. This was approached by arranging the cocktail components into groups of micronutrients (Mg, Zn, Mn, Mo), SARs (Salicylic acid, Serenade Max WP), Phosphite, Hydrogen peroxide, and macronutrients. We have determined that the nutrients are giving the strongest results in rejuvenating and maintaining HLB infected trees. These nutrients include macros and micros, and phosphite. The role of the SARs is undetermined but a synergistic affect may be involved when added to the mix. In 2010 we will separate SAR products and evaluate their contribution (see budget justification). Leaf samples were collected and analyzed at selected intervals since summer 2008. Four treatments (i.e. 4, 5, 6, and 8) do not contain the micronutrient component of the complete HLB cocktail. Trees were consistently and significantly lower in manganese and zinc but not magnesium when compared with trees receiving micronutrients. Micronutrient deficiencies were only observed in treatments not containing micronutrients. Boron was found to be significantly lower after June 2009, presumably after reserves had been exhausted. Boron has been added to the mix. In 2010 we will compare the contribution of the micronutrients in both sulfate and phosphite forms (see budget justification). Yield in 2009 from the commercial grove was higher when trees received phosphite with the nutrients. Based on input prices prevalent during the latter part of 2009, the cost of the foliar nutrient program has been estimated to increase production costs by $200/acre/year over the ‘standard’ HLB management strategy that requires tree inspections and removal of symptomatic trees. A 15-year net present value (NPV) model determined that for a delivered-in price of $1.25 per p.s., annual HLB tree losses had to be greater than 3% before the foliar nutrient program would return a higher 15-year NPV. The cost and future value of resets will be included in this analysis. A first draft of the NPV model has been posted on the SWFREC website to allow growers to determine a HLB tree loss threshold based on their production cost data. Asian citrus psyllid (ACP) populations and Can. Libericacter asiaticus (CLas) titer in plants and psyllids, are being monitored. Two treatments: 1) micronutrients + systemic acquired resistance inducers (Micro+SAR), and 2) Psyllid chemical control applications are being evaluated. Adult ACP populations have been maintained 3 times lower, and up to 50 times lower in insecticide-treated plots compared to insecticide-free plots. The Micro+SAR treatment has had no effect on ACP populations. Overall PCR positive trees increased from 29% in November 2008 to 83% in January 2010. Treatments with insecticidal control had significantly higher Ct values (avg. 25.4) than non-insecticide treatments (avg. 24.1) indicating lower CLas titer in plants protected by insecticides. Despite the high percentage of infected trees, yield collected in March 2009 from trees receiving both insecticidal and nutritional treatments produced 1.32’0.15 boxes/tree, a 30%+ increase over 0.95-0.99 boxes/tree other treatments. No significant treatment effects on fruit or juice quality were observed. These results could be interpreted to mean that the Micro+SAR package is capable of reducing negative effects of HLB if CLas titer can be maintained below some threshold level through psyllid management. We will not feel comfortable with this conclusion until we see similar results from the 2009 crop harvested in spring 2010.
The main focus thus far has been to improve the current media to increase growth of the group of bacteria known as Candidatus Liberibacter. To quantify differences between media, microscopy is being used to calculate the number of cells/colonies present on the different media. We are also quantifying differences between different DNA extraction methods using phase contrast and light field microscopy from culture material. This methodology will also establish a correlation between cell/colony count and the Ct value of RT-PCR assays. In collaboration with Ft. Pierce, FL, the testing of Koch’s postulates under field conditions has continued this quarter. Symptoms have not appeared, and a second inoculation will be started this quarter. The second focus has been sequencing the genomes of the three Liberibacter species from pure culture. Using amplified DNA from cultured cells and Illumina Solexa second generation sequencing technology, we sequenced L. asiaticus strain China1 previously shown to be pathogenic. A collection of 21M trimmed paired-end 90mer reads obtained from this sequencing was aligned to all published bacterial genomes at NCBI using GSnap. Notably only 2,220 reads aligned to the current reference for L. asiaticus CP001677.2, primarily to regions identified as rDNA. Some alignment to chromosome 1 of Ralstonia pickettii and Ralstonia megaplasmid was observed. Reads also demonstrated alignment to the family Rhizobiaceae. Over 90 percent of reads were novel, not aligning to any bacterial genome at NCBI. The read set was used to construct a draft Phase 1 assembly (contigs unordered and unoriented) resulting in 481 contigs, indicating a genome size of 3.8M bp. These contigs demonstrate local similarity to bacterial sequences at NCBI, but contain much novel material and in many cases ORFs similar to known genes or conserved domains. Sequence-derived evidence was used to confirm the presence or absence of these sequence contigs in 8 culture samples and 34 diseased tissue samples. The presence of Rhizobiaceae-like sequence in all samples confirmed the successful sequencing of the genome from cultured cells of Liberibacter. Ralstonia plasmid sequence was found in 25% of Asian samples, but not in North and South American samples. Unique sequences showed two patterns; sequence present in all samples suggesting bacterial chromosomal DNA and sequence present in a percentage of the samples suggesting plasmid DNA. The genomes of L. asiaticus and L. americanus are currently being assembled and annotated. The genome of L. africanus is still in the sequencing phase.
This work on this project will determine if certain alternative plant species are better hosts for the suspected HLB bacterial pathogens (Ca. Liberibacter asiaticus (Ca. Las), Ca. Liberibacter americanus (Ca. Lam) and Ca. Liberibacter africanus (Ca. Laf)) and can serve as a reservoir hosts for infection to citrus. This work is a collaborative effort of five researchers located at four locations since part of the work involves exotic HLB associated pathogens. At the University of Florida, CREC Lake Alfred, quantitative real time PCR (qPCR) work focused on Severinia buxifolia, rough lemon, Calomondin and Murraya paniculata. Graft inoculations to Severinia buxifolia (orange boxwood) and showed that it was an excellent host for the Las bacterium. Asian citrus psyllids were allowed to feed on HLB infected S. buxifolia and 20-30% were found PCR positive. Transmission tests from S. buxifolia to sweet orange was done and the transmission rate was found to be high. This research was done by Hao Hu a graduate student studying with Dr. Brlansky on this project. Studies to determine the absolute quantification of live bacteria in the tissues has been done using two compounds to separate dead from live bacteria. We feel that this is necessary since previous results have shown high numbers of Las in infected tissues but microscopy has revealed few bacteria. Real time PCR results on rough lemon (C. jambhiri) showed infection of both symptomatic as well as asymptomatic shoots and leaves with similar PCR values for all shoots. Rough lemon continues to grow even when infected with Ca. Las. Other commercial rootstocks have been tested and results show that many of them continue to grow even when infected with Las. In field experiments rootstocks from clipped HLB positive trees have shown that rootstock sprouts are positive for Las and maybe a source for further spread. Various commercial sweet orange cultivars were inoculated with Florida Ca. Las and one cultivar tested PCR negative in multiple graft inoculation tests. We have expanded tests on this cultivar and plan to report these results at the Florida State Horticultural Society meetings in June. Experiments on the existence of alternative hosts where edge effects next to non- citrus plantings has begun. Zanthozylum species have been found but PCR tests are negative. At Texas A&M Citrus Center, psyllid feeding tests on eight indigeous rutaceous plants. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica and Zanthoxylum fragara, Casimiroa terameria were found to be feeding hosts for the psyllid. Egg laying was found on torchwood but the psyllids did not complete development. Egg laying and nymphal development to adults were found on C. ternata and C. arizonica. Feeding and egg laying on 3 of the others species but nymphs died. At the USDA, ARS, Beltsville quarantine greenhouse graft inoculations were done to M. paniculata with exotic Ca. Ca. Lam and Ca. Laf isolates and are being tested. Work with dodder as an alternative host was completed studying the plant infection process and for its use to transmit all Liberibacters to plants that are not graft compatible with citrus. A manuscript was submitted for publication in Phytopathology by Drs. Hartung and Brlansky. Dodder became infected and phloem necrosis occurred similar to that in citrus. We showed that Liberibacter exists in two morphological forms in the dodder similar to that seen in citrus and periwinkle and an intermediate between the two forms was discovered. At the USDA, ARS, FDWSRU, Ft. Detrick, MD a manuscript entitled ‘The relevance of Murraya paniculata and related species as potential hosts and inoculum reservoirs of ‘Candidatus Liberibacter asiaticus’, causal agent of Huanglongbing (HLB)’ was completed and accepted for publication. Authors from the grant include Drs. Damsteegt, Brlansky and Schneider. The details of this work were reported in earlier reports. Psyllid transmissions from HLB (Ca. Laf and Ca. Las (Thailand strain)) infected sweet orange to Severinia buxifolia have been done and testing will begin after incubation.
For the first year (2009) of research on healthy, asymptomatic and symptomatic HLB commercially-processed juice samples (healthy, HLB-AS and HLB-S, respectively) from two Valencia and two Hamlin harvests (15+ trees/sample), previous reports were given on sugar, acid, Brix, titratable acidity, ratio, and oil content as well as sensory perception of flavor for the first three of four harvests. The results for these three harvests showed that there were minimal differences between juice from healthy and HLB trees for HLB-AS fruit but that there were differences for HLB-S fruit, both chemically and in terms of sensory perception (difference from control tests) except for the bitter compounds limonin and nomilin. These compounds were higher in both HLB-AS and HLB-S juice compared to healthy, but the differences were much greater for HLB-S fruit for January Hamlin and April but not June Valencia. Trained panels detected more differences in some descriptors between healthy and HLB-AS and especially HLB-S juice. Generally, juice from later season fruit showed less differences due to HLB. Now we have data for the final December Hamlin harvest where the difference from control test did show differences between healthy and HLB-AS juice for taste this time, and even greater differences for HLB-S juice for taste and smell. The chemical data, which explained the sensory, showed that the healthy juice had the highest Brix (10.44) and ratio (26.1), while the HLB juice had lower Brix (9.12 and 9.91 for HLB-AS and HLB-S, respectively) and ratio (19.2 and 22.5) higher acid (0.44 and 0.48% titratable acidity, TA) than control juice (0.40% TA) with the biggest differences for HLB-S juice. Oil content ran between 0.17 ‘ 0.24%, being slightly higher in HLB-S fruit juice. Previously we had sent 2008 and 2009 samples for e-nose and e-tongue analyses off site, and had good e-tongue but not good e-nose separation (since our e-nose temporarily out of commission). Interestingly, our repaired e-nose could differentiate between all the 2009 Hamlin and Valencia harvests and between healthy, HLB-AS and HLB-S juice within each harvest based on juice volatiles. So this means that both the e-nose and e-tongue show promise for distinguishing between healthy, HLB-AS and HLB-S juice based on volatile and non-volatile compounds, respectively. Finally, several harvests of Hamlin and Valencia leaves have been analyzed for phytochemical content to look for HLB disease chemical markers. The analysis by HPLC-UV-MS allows for detection and quantification by UV absorbance, and by select ion mass detection. A tremendous amount of chromatographic and MS data has been collected, but only a small portion of it has been thoroughly processed. Thus far, the data has consistently shown that the alkaloid exhibiting a mass fragment at 188 amu and the compound, feruloyl putrescine, occur at much higher concentrations in HLB affected leaves compared to healthy control leaves. Several of the polar hydroxycinnamates occur at higher concentrations in HLB affected leaves compared to control leaves as well. Differences were also observed in the levels of several mid-polar hydroxycinnamtes in the HLB and control leaves, but these differences have not been thoroughly analyzed. Several flavone glycosides occur at lower concentrations in HLB than in healthy leaves. The main flavanone, hesperidin, does not appear to be affected by HLB, however. In several cases, the HLB leaves contained higher limonin glucoside levels than control leaves, but levels of the limonoid aglycones have been too low for detection. The polymethoxylated flavones do not seem to be consistently affected by HLB, although further analysis is in progress. The differences in the chemical compositions of control and HLB affected leaves are being further investigated by a LECO time of flight mass spectrometer with enhanced peak convolution and detection capabilities.
Plants infected with ‘Ca. Liberibacter asiaticus’ were established and propagated at Lake Alfred, Ft. Detrick and Beltsville. Some of these plants were used to raise large numbers of psyllids to be used to immunize mice. Others were grown and used as plant extracts to screen antibody libraries. Visiting scientists were recruited from Sigma-tau Pharmaceuticals SA (Rome, Italy; Dr. Minenkova, an international authority on scFv libraries) and from Luzhou Medical College, (Luzhou, PRC; Dr. Yuan, performing the scFv work in Beltsville). A large number of psyllids from Ft. Detrick and Lake Alfred have been screened for the presence of ‘Ca. Liberibacter asiaticus’, and the 3% of the psyllids with the highest titer of bacteria were used to inject BALB/c mice. This required the development of a rapid and efficient method to make psyllid extracts which could be assayed by q-PCR without DNA extraction so that intact bacteria were available for injection into the mice. Each injection contained extracts of 1-2 insects at 2-5 x 108 ‘Ca. Liberibacter asiaticus’ per insect mixed with adjuvant. Two sets of mice were immunized. One set was taken to Agdia after a series of four immunizations and used to create standard monoclonal antibodies using hybridoma technology. The second set of mice received an additional immunization and was used to create a scFv library in the bacteriophage vector pKM19. Promising antibody expressing cell lines have been identified at Agdia. These antibodies appear to react with the outer membrane protein of ‘Ca. Liberibacter asiaticus’ purified from Escherichia coli containing the OMP gene cloned in an expression vector. These antibodies are being purified and characterized further. While screening these hybridomas we observed cross reactions to phloem extracts from healthy sweet orange fruit. This was not expected since the mice were immunized with psyllid and not plant extracts. The nature of the cross reacting antigen is presently unknown. A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has also been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently in the initial stages of screening by ‘biopanning’against extracts of rough lemon plants. These extracts are confirmed to be high titer for ‘Ca. Liberibacter asiaticus’ by q-PCR. We anticipate phagemids encoding scFv with specificity against ‘Ca. Liberibacter asiaticus’ in the near future. These phagemids should recognize a diversity of epitopes in ‘Ca. Liberibacter asiaticus’, and will be characterized as they become available. Some other results should be noted. In the process of assaying the psyllids for the concentration of ‘Ca. Liberibacter asiaticus’ we obtained a dataset of the concentration of ‘Ca. Liberibacter asiaticus’ present in 686 psyllids. In separate research this dataset may lead to insights into relations among other commensal bacteria and ‘Ca. Liberibacter asiaticus’ and in the epidemiology of HLB disease. Also, at the beginning of the project, while plant materials were graft inoculated and insects were multiplying on ‘Ca. Liberibacter asiaticus’ infected plants, we immunized mice with Xylella fastidiosa strain 9a5c (citrus variegated chlorosis) mixed with psyllid extracts. We did this to become familiar with process of creating the scFv libraries and screening them for phagemids with desired scFv. Interestingly, we have identified phagemid expressing scFv which react strongly in ELISA tests to strain Xylella fastidiosa 9a5c but which do not react at all to strains of Xylella fastidiosa associated with Pierce’s disease of grapevine. These scFv fragments have interesting potential for diagnostics.
We want to obtain a pure culture of Ca. Liberibacter asiaticus (LAS) by first co-culturing the bacteria with insect cells. The strategy consists of primocultures of the bacteria in insect cell cultures used as feeder cells. For this annual report our project objectives are described and discussed below. Objective 1: LAS inoculum. LAS source materials are from infected symptomatic citrus trees from Vietnam. Transmissions from citrus to citrus or from citrus to periwinkles are performed via the insect psyllid Diaphorina citri and subsequent grafting. A maceration method was found to be the most appropriate way to release the bacteria in the insect cell cultures. Antibiotics are used to select for Gram negative bacteria. Objective 2: Primo-cultures. Various insect cell cultures in various culture media were tested. -We didn’t detect any Las in Mamestra (hemocyte, ovarian cells) or Spodoptera (hemocyte cells) cell lines after inoculation. -We detected LAS in two lines of drosophila cell cultures by direct PCR after inoculation. One line lost the detection after 6 subcultures. For the second line, positive PCR signal was obtained up to the 20th subculture, then LAS detection troubles occured due to high insect cell density and we detected the presence of another bacteria (Bradyrhizobium). We started new LAS primo-cultures with much less drosophila cells and got LAS positive cultures for some combinations of cell line/culture medium, still positive after 5 weeks and successive dilutions (confirmed by sequencing). -First attempt to co-culture LAS with an Aedes albopictus insect cell line failed. However, adding an antioxidant and osmoprotectant at the time of inoculation allowed us to get LAS positive Aedes cultures still positive after 9 weeks and successive dilutions (confirmed by sequencing). We are currently adapting conditions of described LAS qPCR detection to quantify LAS in insect cell cultures. We can detect LAS bacteria by qPCR but more efforts are needed to quantify bacteria:insect cell ratio with a multiplexed qPCR approach. Our objectives 3 and 4 are aiming to improve culture conditions to get a higher bacterial titer and to free the co-cultures of insect cells (axenization). Objective 3: Axenization. LAS/Aedes primo-cultures were obtained at a low insect cell concentration. We are progressively diluting the concentration of insect cells through each new passage . Objective 4: Medium optimization. To maintain the bacteria for a longer period of time and to reach higher bacterial concentration, we started complementing the primo-cultures with various sugars, vitamins described in citrus/periwinkle phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. Of the complements tested we selected sodium pyruvate, proline and fructose for their positive effect on the bacteria detection and they now are systematically assayed on our new inoculations. We are analyzing sugar, amino-acids and minerals and trace elements variations in insect cell culture media over culture time to identify potential LAS growth limiting factors. We are reaching our milestones for the first year of this project with significant progress on objectives 1, and 2 (inoculums, primo-cultures). For objective 2, we will test new insect cell lines. As at least two laboratories involved in the FCPRAC program established Diaphorina citri cell lines, we will request for these lines to trial our inoculation protocol and try to obtain LAS/ D. citri co-cultures. We are now fully involved in objectives 3 and 4 (axenization/medium improvement). If they succeed in time, we will attempt to inoculate cultivated bacteria through Diaphorina citri to Citrus trees (objective 5).
The goal of this proposal is to investigate whether Candidatus Liberibacter asiaticus (Ca. Las) is transmitted between infected and uninfected ACP adults in a sex-related manner to understand the potential role of this mechanism in disease spread in the field. Our investigations to date indicate that Ca. Las is sexually transmitted from Ca. Las-infected male psyllids to healthy females but not from infected females to healthy males or among psyllids of the same sex. Ca. Las was transmitted from Ca. Las-infected male psyllids to roughly 3% of healthy females. Ca. Las was not detected in the recipient sex immediately after mating but required a minimum incubation period of 2 weeks in psyllid bodies for PCR detection. These results also suggested multiplication of bacteria within psyllid bodies. No Ca. Las was detected in recipient psyllids when the recipient psyllids were maintained on HLB-resistant (Murraya koenigii) plants for longer than 4 weeks suggesting that psyllids may lose infectivity if they continuously live on HLB-resistant plants. We were able to detect Ca. Las bacteria in ACP ovaries of recipient females with PCR. However, we were unable to detect the presence of bacteria in genital parts of male and female psyllids with scanning and transmission electron microscopy perhaps due to washing of bacteria during sample preparation procedures. Ca. Las was also not detected in psyllid salivary glands using electron microscopy. More precise and accurate procedures such as in situ hybridization may be required to detect the presence of bacteria in psyllids. Also, we were able to detect Ca. Las in eggs of recipient females with PCR but not with electron microscopy. PCR detection of Ca. Las in psyllid ovaries suggested transovarial transmission of bacteria. Transovarial transmission was also confirmed in F2 generations of Ca. Las-recipient females which were produced on M. koenigii plants. We continue to evaluate if the Ca. Las-recipient females are capable of infecting new citrus plants. The experimental procedures for this have been completed; however, we are awaiting to collect the results because a minimum 10 week period is required for detection of HLB in newly infected plants.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We are determining whether this is due to plant genetics, Las variation, or randomness. Some Poncirus hybrids are more susceptible than others suggesting that resistance to Las is segregating. We are beginning experiments to map citrus genes that provide Las resistance. Las also appears to have difficulty spreading in Poncirus. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We already have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. Third, these results have led to the development of methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or antipsyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another large experiment is underway. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, are presently being tested against HLB; and, 4) a series of lemonine trees reported to be resistant to HLB for Gene Albrigo.
This is a continuing project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. We think that this approach could be used beginning 2-3 years from now and until probably 15 years from now when resistant trees should be available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 40 different antibacterial peptides have been tested in trees. We initially found three peptides that allow much better growth of trees that were grafted with HLB-infected buds. Some trees had no symptoms and no detectable Las, some trees had no symptoms and low levels of Las, and other trees had leaf symptoms but continued growth of the trees with normal levels of Las. Another result is that we found that leader peptides for the export of the peptide from the CTV-infected is not needed for HLB but is needed for citrus canker. Because we were concerned that graft inoculation of HLB into the trunks of small trees is a too severe challenge that might cause peptides that could work in the field to be missed, we developed a system that only allows inoculation by infected psyllids. We have established a containment plant growth room in which psyllids inoculate the plants expressing the peptides. Using this system, we have found two peptides that appear to effectively protect sweet orange trees from HLB. To speed up the search for effective anti-HLB genes, Falk (UC Davis) has developed a tobacco-tomato psyllid/liberibacter model to screen for effective genes against the similar bacterium. This system is working and screening is on-going. We also are improving the CTV-based vector to be able to produce 2-5 peptides at the same time. This will allow expression of genes against HLB and canker or multiple of genes against HLB. We have developed a vector that can be re-added to trees if the anti-Las gene is lost or a better gene becomes available. A major objective that we are pursuing is to make a vector that cannot be transmitted by aphids. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. We have received permission for USDA APHIS for the field test, but were delayed by EPA. We are now submitting a revised application to USDA APHIS to include EPA requirements and are expecting to establish the field test this spring. In addition, we are screening a series of transgenic sweet orange and grapefruit expressing antibacterial genes for Erik Mirkov of Texas A&M and Mike Irey of Southern Gardens.
A manuscript on the sequence of symptom development and the identification of callose and phloem protein 2 (PP2) as the amorphous and filamentous plugging materials was accepted: DS Achor, et al. 2010.Plant Pathology J. 9(2): in press. The upset of normal phloem translocation of carbohydrates to other plant parts and ultimately the starvation of the root system (Etxeberria et al, 2009, Physiol. Mol. Plant Pathol. 74:76-83) may be the main reason for tree decline from HLB infection. Field samples of several cultivars were collected and prepared to determine the amounts of amorphous versus filamentous plugs disrupting phloem sap flow. If one type of plugging is more prevalent, that pathway for plugging may be more important. Earlier work by a colleague indicated that three common rootstocks do not express HLB symptoms when infected with HLB and grown in a greenhouse. One of these, Swingle, was found infected in the field, but it developed typical leaf mottle symptoms and phloem plugging. Carrizo trees are being monitored until they become PCR positive. Poncirus trifoliate trees are being monitored in two field sites also as they may have HLB tolerance. Since insufficient bacteria are present to directly cause phloem plugging, work is underway to understand the mechanism by which the bacterial infection leads to this phloem plugging. To determine how the bacteria elicits the over expression of phloem plugging materials, one Agilent microarray was designed based on the genome sequence of Candidatus Liberibacter asiaticus. Bioinformatics analysis was performed to identify potential virulence factors. Six potential virulence factors were cloned into pGEMT-easy vector. The insertions were confirmed using PCR and three virulence factors were expressed in tobacco; one of these caused plant death. These factors and others will be inserted into citrus with a phloem promoter to study their potential roles in virulence. Transgenic approaches to achieve over-expression of the citrus ‘-1,3-glucanase gene using different promoters, though protoplast/GFP co-transformation and Agrobacterium-mediated transformation are underway in order to minimize HLB associated callose-plugging. Initial transgenic grapefruit trees that over-express 1, 3-. glucanase were grown out on Macrophylla rootstocks and challenged with HLB by bud inoculation. Only 4 of 44 plants had successful bud take and none of these show symptoms 4 months after the inoculation attempt. All these plants were re-inoculated with infected buds in February. The citrus ‘-1,3-glucanases gene from Valencia embryogenic callus and young leaves (McCollum et al., 1999) was cloned. Citrus .-1,3 glucanase cDNA (GenBank accession number AJ000081) was synthesized from Valencia leaf and embryogenic callus. A 1011 bp citrus .-1,3 glucanase gene fragment was amplified. To obtain the suitable restriction site and additional cMyc tag (to facilitate subsequent western analysis) on the cDNA, another PCR reaction was performed using a new primer set with a different restriction site including the cMyc tag. The final PCR product (BG3) was purified, cloned into the pGEM T-Easy vector (Promega) and sequenced. The cloned BG3 fragment was ligated into a vector (pUCLON) between the 35S promoter and 35S terminator, and was transformed into host E coli DH5. cells. Plasmid DNA was isolated using 5prime kit and checked through restriction digestion and PCR analysis. From the ligated plasmid ( pUBG3), the HindIII fragment was excised and ligated into the HindIII site on another vector pCIT101 holding the GFP/NPTII fusion gene. The final vector , pCITBG3, was transformed into Agrobact. Co-transformation experiments can now be done. Plasmid vector pARS108 with the ER-targeted GFP gene was used to make a new construct for efficient protoplast transformation. Another vector, pGASS, was constructed to target BG3 expression in phloem tissues only. Two new sweet orange callus lines are available to carry out transformations. >Antisense or other knockout methods for phloem protein 2 and callose genes will be used in year 2.
A RAPID SCREENING PROGRESS FOR CHEMICAL CONTROL OF HUANGLONGBING Annual Research Report-2009 In the second year, the project was mainly focused on: 1) Screening and evaluating anti-microbial molecules for suppression of liberibacter using the optimized screening systems in periwinkle; 2) The effective compounds were tested and evaluated in HLB-affected citrus in the greenhouse 1. Screening of chemical compounds that eliminate or suppress the Las bacteria in periwinkle using the optimized regeneration system. Antibiotics (penicillin G and streptomycin, oxytetracyclin and Metronidazole), a biocide (DBNPA), a fungicide (zineb), two peptides (D4E1 and D2A21) and three SARs (SA, antiguard, and ortho-phenylphenol) were tested for their ability to eliminate or suppress Las bacteria and to promote the growth of severe HLB-affected cuttings using the optimized regeneration system. More than 75% of plants could be regenerated from the severe HLB-affected periwinkle cuttings treated with a combination of penicillin and streptomycin (PS). All regenerated plants from the HLB-affected cuttings treated with PS tested negative for Las, even by nested PCR. The Las bacteria were undetected in these regenerated plants with average Ct value of 39.33. When treated with oxytetracyclin, metronidazole or peptides (D4E1 or D2A21), the regenerated plants also tested negative by nested PCR or qPCR with Ct values over 32. However, the regeneration percentage was lower than 30%. The regenerated control plants treated with water tested positive for Las by PCR and qPCR with low Ct values <26.0, indicating that the bacteria titers were at least 100-fold higher than those in the antibiotic or peptide-treated, regenerated periwinkle. DBNPA can also suppress the Las bacteria. The severe HLB-affected cuttings had a regeneration rate of 33.1 %. The Ct values of the regenerated plants ranged from 27 to 30. The fungicide zineb and three SARs (SA, antiguard and ortho-phenylphenol) were not effective in controlling Las bacteria. Whether treated with zineb or not, the Las bacteria can keep reproducing. The Ct value was lower in the zineb-treated, regenerated plants than those treated with antibiotics or peptides, and similar to the water control. Zineb and ortho-phenylphenol are not very soluble. The water solubility is only 10 mg/L. When PS was foliar-sprayed at one-week intervals for three consecutive weeks on the Las-infected periwinkle plants at different rates (1x, 5x and 10x), no significant differences were found among treatment rates, but each treatment eliminated the Las-bacteria in the infected periwinkle as compared to the water control. DMSO or Silwet L-7 as emulsifiers has no different effect on Las-infected periwinkle plants. 2. Effect of screened chemical compounds on HLB-affected citrus in the greenhouse. Nutrition and HLB development in citrus. The nutrients K, Zn, N, and Ca were tested for their ability to suppress HLB symptoms in citrus. Potassium and Zn, but not N or Ca could delay HLB symptoms. All-treated citrus remained infected by Las bacterium after 9 months. K and Zn reduced titers of Las bacteria at 5 months after treatment. Effect of SAR-inducer on HLB development. Seven months after treatment with SAR-inducer (SA and antiguard), HLB-affected citrus tested positive for Las by PCR with an average Ct value of lower than 28, indicating SAR was not effective in suppressing or inhibiting the Las bacterium. This had also been the case with a perwinkle host. Effect of antibiotics on HLB-affected citrus by root soaking or foliar-spray. HLB-affected citrus was soaked or foliar-spraye
This report covers the period of July 1, 2009 through September 30, 2009. This project was funded July 1, 2009. This project was organized and many activities were completed prior to July 1, 2009. There were four organizational meetings between FDACS, CHRP, USDA, University of Florida, the Indian River Citrus League and consulting personnel. There were 42 organizational meetings of the field personnel. The Indian River Citrus League held three meeting with their board to explain the areawide suppression program and to invite growers to participate. The project was organized into Martin, St. Lucie and Indian River Counties. Growers were contacted for access to groves, blocks were surveyed for trap placement and trees and blocks were mapped and GPSed. All reports were designed and supplies were ordered. The tracking and analysis of data was organized. Personnel were trained in procedures for trapping and identification of Asian citrus psyllid. This program cooperated in providing additional materials for aerial application for Asian citrus psyllid and Caribbean fruit fly control. Materials were provided by the pesticide industry and efficacy experiments were designed by the University of Florida. Three large, replicated efficacy experiments were conducted. One additional experiment was designed and organized, but not implemented due to weather. Materials were applied by air with psyllid monitoring before and after applications and comparisons with psyllid numbers in control plots. The efficacy of materials for Caribbean fruit fly control was assessed in the same experiments. Sterile Caribbean fruit flies were released before applications and fly numbers were monitored before and after application. Materials not previously registered are now moving toward label inclusion of aerial application for Asian citrus psyllid and for Caribbean fruit fly. For the period July 1-Sept 30, 2009: Traps placed by county were: Indian River, 205; St. Lucie, 225; Martin, 72. Trees surveyed were: Indian River, 2,665; St. Lucie, 2925; Martin, 975. Traps set and retrieved were 6,565. Asian citrus psyllids caught were: Indian River, 5,583; St. Lucie, 8,414; Martin, 4,672. This project was successfully organized and initiated during this period.
Objective 1: Sprays of copper (Cu) formulations, containing copper hydroxide (Kocide, Champ, Kentan, Badge) or copper sulfate (Cuprofix), were moderately to highly effective for control of canker on fruit of susceptible Ruby red grapefruit and Hamlin orange in the lack of high disease pressure due to early and late season wind-blown rain storms in 2009. A chelated Cu (Magna-Bon, copper pentahydrate) at a 50% lower rate of Cu per application than standard Cu formulations performed as well for reducing fruit disease incidence for grapefruit or canker-induced fruit drop for Hamlin. Early season infection and fruit drop of grapefruit and Hamlin was minimal because April was relatively dry and Cu treatments were initiated before significant rainfall occurred in May. Cumulative fruit drop due to early season infection of untreated Hamlin amounted to about 5%. Five sprays of Cu formulations reduced the incidence fruit drop due to canker by about 40-50% to 2.5% cumulative fruit loss. Objective 2: In Marsh grapefruit, canker control increased with number of Cu sprays from 3 to 11 (April to October), canker infection and copper burn occurred after rains commenced in July. In August, fruit were growing most rapidly which would produce a thinning of fruit cuticle and an increase in the rate that new stomates open due to more rapid expansion of the fruit surface. Season-long copper spray also gave the best control of late season scab and melanose on fruit. In Hamlin, sprays beyond mid-July provided additional canker control of fruit drop confirming that late season lesions do not cause fruit drop like early season lesions. Objective 3: In two grapefruit trials, Firewall (streptomycin[Sm]) applied alone or in combination with a reduced rate of Kocide 3000 in July and early August gave equivalent control on grapefruit to Kocide alone. The adjuvant, Polymer Delivery System (PDS) did not increase the residual activity of Cu on grapefruit or control efficacy of Cu. The residual activity of Cu on fruit was not affected by Kocide rate but decreased with time after application due to increase in fruit surface area over 21 days. This result supports the recommendation for use of 21 day interval Cu sprays for adequate canker control and explains the reduced efficacy of 28-day interval sprays. Objective 4: The Cu resistance gene was identified as CopL on a plasmid from a resistant Xanthomonas citri subsp. citri (Xcc) strain from Argentina that was exposed long-term to Cu for canker control. The identical resistance gene sequence was found in Xanthomonas spp. causing bacterial spot in tomato and pepper. Primers constructed based on the gene sequence were used to screen the remaining Cu resistant strains of Xcc from Argentina and Cu resistant strains of X. alfalfae. pv citrumelonis from Florida citrus nurseries with citrus bacterial spot. All strains screened thus far contain the CopL resistance gene. In addition, a non-pathogenic strain of Xanthomonas isolated from a citrus grove was found to be Cu resistant and may represent a pre-existing source of risk in citrus groves for horizontal transfer of CopL into Xcc. Cu and Sm resistance were monitored in Xcc and epiphytic bacterial populations on grapefruit trees sprayed with Cu or Sm every 21 days for two growing seasons (22 sprays total). Each season Cu and Sm sprays increased the ratio of epiphytic bacterial population with tolerance to these chemicals. Overall, the Sm resistant bacterial populations were proportionally lower than Cu tolerant bacterial population. No resistance to either Cu or Sm was verified in Xcc or epiphytic populations after two years of season long sprays. Objective 5: In 2009, canker management talks were given at county extension and other meetings. Updated 2010 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been made to the Florida Citrus Production Managers and at Florida Citrus Show.
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