Significant progress was made in rearing, release, and evaluation of parasitism rates to enhance biological control of ACP in Florida as described under objective 1) Tamarixia radiata colonies from south China, North Vietnam and Pakistan were established in DPI quarantine and parasitoid releases initiated in October after approval by USDA-APHIS and DPI. So far, 23,571 (S. China), 18,783 (N. Vietnam), and 11,294 (Pakistan) wasps have been released in Zolfo springs, Lake Wales, and Immokalee, respectively. Psyllid populations were low at release sites in Zolfo springs and Lake Wales, and high in Immokalee particularly in a block of 1 m tall citrus plants at the SWFREC. During Oct-Nov, parasitism averaged 10, 2, and 60% at Zolfo springs, Lake Wales, and Immokalee, respectively. We also established an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville, and released 1,325, and 5,750 wasps during 2008 and 2009, respectively, in conventional and organic citrus groves and dooryard Muraya paniculata. A parasitism rate of 6-19% was observed from nymphs reared from M. paniculata in May 2008, although none were recovered in 2009. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata from our 3 new colonies, as well as from Florida and the Caribbean was completed and published. We will continue development of genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) We are consistently improving rearing methods and increasing the production of the previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 88,000 wasps between March and December 2009 that were used to initiate and help maintain the colony at OrangeCo, conduct laboratory experiments at SWFREC, Immokalee and CREC, Lake Alfred, and release in experimental, conventional, and organic groves. During Oct-Nov, parasitism averaged 8-50% in release blocks compared to 7-18% in blocks where parasitoids were not released. Parasitism on sentinel plants placed in a conventional grove averaged 60%, 26%, and 27% in Apr 09, Nov 09 and Jan 10, respectively, and 36% and 22% in Nov 09 and Jan 10, respectively, at SWFREC. We also released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom in an organic grove in Lake Wales, FL. Predatory mites, averaged 3 and 5 individuals per two tap samples per tree in March and April, respectively, and monthly average for psyllid adults was < 1 per tap sample for the year except May when 1.5 per tap sample were observed. 4) We assisted Orange Co. and DPI to establish their mass rearing facilities, made several statewide and national presentations and published our findings to reach the target clientele. Our efforts have generated much interest in the US and Latin America in mass release of T. radiata and the number of projects has mushroomed. For this reason, we organized with the USDA-APHIS and the California Department of Agriculture, the "International Tamarixia Workshop" in Feb 2010 at McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective was to improve the technology by sharing practical information. 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332. 2) Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3) Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by ACP Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136.
Significant progress was made in rearing, release, and evaluation of parasitism rates to enhance biological control of ACP in Florida as described under objective 1) Tamarixia radiata colonies from south China, North Vietnam and Pakistan were established in DPI quarantine and parasitoid releases initiated in October after approval by USDA-APHIS and DPI. So far, 23,571 (S. China), 18,783 (N. Vietnam), and 11,294 (Pakistan) wasps have been released in Zolfo springs, Lake Wales, and Immokalee, respectively. Psyllid populations were low at release sites in Zolfo springs and Lake Wales, and high in Immokalee particularly in a block of 1 m tall citrus plants at the SWFREC. During Oct-Nov, parasitism averaged 10, 2, and 60% at Zolfo springs, Lake Wales, and Immokalee, respectively. We also established an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville, and released 1,325, and 5,750 wasps during 2008 and 2009, respectively, in conventional and organic citrus groves and dooryard Muraya paniculata. A parasitism rate of 6-19% was observed from nymphs reared from M. paniculata in May 2008, although none were recovered in 2009. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata from our 3 new colonies, as well as from Florida and the Caribbean was completed and published. We will continue development of genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) We are consistently improving rearing methods and increasing the production of the previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 88,000 wasps between March and December 2009 that were used to initiate and help maintain the colony at OrangeCo, conduct laboratory experiments at SWFREC, Immokalee and CREC, Lake Alfred, and release in experimental, conventional, and organic groves. During Oct-Nov, parasitism averaged 8-50% in release blocks compared to 7-18% in blocks where parasitoids were not released. Parasitism on sentinel plants placed in a conventional grove averaged 60%, 26%, and 27% in Apr 09, Nov 09 and Jan 10, respectively, and 36% and 22% in Nov 09 and Jan 10, respectively, at SWFREC. We also released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom in an organic grove in Lake Wales, FL. Predatory mites, averaged 3 and 5 individuals per two tap samples per tree in March and April, respectively, and monthly average for psyllid adults was < 1 per tap sample for the year except May when 1.5 per tap sample were observed. 4) We assisted Orange Co. and DPI to establish their mass rearing facilities, made several statewide and national presentations and published our findings to reach the target clientele. Our efforts have generated much interest in the US and Latin America in mass release of T. radiata and the number of projects has mushroomed. For this reason, we organized with the USDA-APHIS and the California Department of Agriculture, the "International Tamarixia Workshop" in Feb 2010 at McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective was to improve the technology by sharing practical information. 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332. 2) Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3) Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by ACP Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136.
In the fourth quarter, we have primarily focused on selecting T1 Arabidopsis transformants expressing citrus SA homologs and testing some T2 transgenic plants in disease resistance. So far, preliminary data obtained from Arabidopsis expressing ctNDR1 look promising. The Arabidopsis ndr1-1 mutant was shown previously to lack a hypersensitive response (HR) when challenged with the Pseudomonas syringae avrRpt2 strain. We transformed ndr1-1 with ctNDR1 and obtained T2 seeds. The T2 plants were infected with the P. syringae avrRpt2 strain at OD=0.1 for a HR test. We found that T2 plants from 10 independently transformed lines were segregated into HR+ and HR-, consistent with the fact that the T2 transgenic plants are heterozygous in the transgene. In the control experiments, all T2 plants showed no HR when challenged with a virulent isogenic P. syringae strain. In addition, when we infected the T2 ndr1-1 plants expressing ctNDR1 with P. syringae avrRpt2 strain at OD=0.0001 for a disease resistance test, we observed a segregation of resistance and susceptibility among the T2 lines. As controls, WT plants were resistance and ndr1-1 plants were more susceptible to this strain. These data suggest that the ndr1-1 mutant is likely complemented by the ctNDR gene. We will isolate homozygotes of the transgenic plants and test them again for defense response in the next generation. In the meantime, the ctNDR1/pBINplusARS construct will be placed in the pipeline for citrus transformation. Summary for additional transgenic plants are in the following: 1. CtNPR1/pBINplusARS to Col or the npr1-1 mutant: we infected T2 transgenic npr1-1 plants expressing ctNPR1. So far we did not observe a drastic change of disease resistance in the T2 plants. We will isolate homozygous lines and plant them side by side with WT and the npr1-1 mutant for a disease resistance test in the next generation. 2. CtPAD4/pBINplusARS to Col or the pad4-1 mutant: We obtained T2 seeds from Col and the pad4-1 mutant expressing ctPAD4 and will infect the T2 plants soon with P. syringae. 3. CtEDS5/pBINplusARS to Col or the eds5-1 mutant: We screen T0 seeds for transgenic plants expressing ctEDS5 but were not able to obtain any lines. This is possibly due to the toxicity of overexpressing ctEDS5 and/or the failure of transformation. We are now repeating the transformation process. In the meantime, we will transiently express ctEDS5 in tobacco to see if overexpression of this gene could cause cell death or other damages to tobacco cells. In addition, we continue to clone additional SA genes from citrus. We are currently working on cloning several additional SA homologs from citrus, including ctEDS1, ctSID2, ctALD1, and ctWIN3. All these genes were previously shown to be involved in either SA biosynthesis or regulating SA levels in Arabidopsis. We already obtained 3′ end of ctEDS1 and in the process of cloning other genes.
We have proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. Objective 1 of our proposal intended to evaluate candidate genes for dsRNA-induced lethality of Diaphorina citri and our model organism, Myzus persicae, using artificial feeding assays. To date, we have cloned sequences at least 400 bp in length from nine Intellectual Property-Free (IP-Free) homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 and constitutively expressed S4e ribosomal protein from M. persicae, as well a control derived from green fluorescent protein sequence. We are currently repeating our feeding assay experiment to confirm our initial results, which indicated that dsRNA had a negative effect on both the lifespan of the insects and the number of offspring generated. Since our last report we have also made considerable headway with regards to objective 2: to evaluate the RNAi strategy in planta for its effects against our model insect, M. persicae. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, this approach may prove to be critical to the success of this study. This research requires the use of Gateway-based vectors that express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We previously cloned and confirmed the phloem-specificity of both the AtSUS1 and AtSUC2 promoters from Arabidopsis thaliana, and also cloned the putative promoter regions (approximately 1.5 kb upstream of the translational start site) of SUS1 (CsSUS1-1 and 2) and SUC2 (CsSUT1) homologues from Citrus sinensis cv. valencia. We have since confirmed the phloem-specificity of two separate alleles of the CsSUS1 promoter and have determined that the CsSUT1 promoter confers flower-specific expression in A. thaliana. Expression driven by the CsSUS1-1 and CsSUS1-2 promoters occurs at very high levels, bearing a closer resemblance to the robust AtSUC2 promoter than the weaker AtSUS1 promoter. To test the ability of our citrus promoters to drive phloem-specific expression in a wide range of species, both CsSUS1::GUS and CsSUT1::GUS cassettes have also been transformed into a variety of other plants, including Nicotiana tabacum (tobacco), Prunus domestica (plum) and Malus domestica (apple). GUS assays will be carried out on leaf tissues of potted plants in the coming weeks. To develop an IP-free system, we have successfully replaced the 35S promoter contained within our original Gateway vector with a multiple cloning site that will allow the introduction of our IP-Free efficient phloem-specific promoter to drive the expression of our insect-derived IP-Free dsRNAs for our in planta RNAi assays. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and are repeating our analysis of a subset of derived IP-Free dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned several IP-Free phloem-specific promoters from various organisms, including Citrus sinensis, and are in the process of evaluating their expression patterns in a number of plant systems. Finally, we are developing new IP-Free Gateway-derived vectors bearing a constitutive promoter and phloem-specific promoters (AtSUC2 and CsSUS1), respectively, for use in RNAi against sap-sucking insects in planta (objective 2).
This report covers the period October 1, 2009 through December 31, 2009. This project was funded July 1, 2009. Five coordinating meetings were held during this period. Seven meetings were held with field personnel to arrange, in particular, intensive trapping experiments. This program coordinated with the aerial application program through Southeastern Air and the Indian River Citrus League. Pesticide applications were mapped for the Indian River and St. Lucie counties for subsequent for time and space analyses of Asian citrus psyllid populations. Coordination with citrus growers was increased during this period in order to understand the relationship between grower programs to suppress Asian citrus psyllid populations with pesticide applications by ground and the areawide management program by aerial pesticide application. Trapping was adjusted by blocks to obtain more control(non-sprayed) areas within the area covered by the areawide aerial program. Traps set by county were: Indian River, 205; St. Lucie, 225; Martin, 72. Total traps set and retrieved during this period were: 7,070. Trees surveyed during this period were: Indian River, 2,870; St. Lucie, 3,150; Martin, 1,050. Total Asian citrus psyllids caught were: Indian River 3,413; St. Lucie, 2,510; Martin, 2,020. A new intensive trapping program was begun in Indian River County(the Marsh) and in St. Lucie and Okeechobee Counties(McArthur Farms) to provide baseline psyllid population data prior to applying pesticides by air for the areawide program. Citrus growers were contacted, new blocks for sampling were established by GPS and mapping and traps were set in these new areas. Data sets were now transferred weekly for statistical and other analyses. It is noted that 29 Caribbean fruit flies were caught on the yellow sticky traps in this areawide program during this period. This period established field procedures through experience and correction and provided a large data set for statistical analyses.
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.
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