In order to fulfil Koch’s postulate we wanted to obtain an axenic culture of Ca. Liberibacter asiaticus (LAS) )- Vietnamese strain maintained in greenhouse on Citrus spp. and periwinkles – from primo-cultures of the bacteria in insect cell cultures used as feeder cells. An optimized protocol of bacteria extraction from infected citrus trees or periwinkles has been set up to inoculate various insect cell cultures, in various culture media. Antibiotics were used to select for Gram negative bacteria. Among nine different cell lines tested so far, we selected two Drosophila and one Aedes cell lines that were able to sustain LAS survival and growth the most consistently. About LAS/Drosophila co-cultures: the major challenge remains to decrease the insect cell population. We tested new culture media to contain their growth. Two media were selected for their ability to contain drosophila cells proliferation. However they didn’t give satisfactory primo-cultures. We are now looking into specific molecules that will affect drosophila cells growth without affecting LAS. LAS primocultures in Aedes cells were obtained at a low insect cell concentration. Different co-cultures were continuously grown over several months with successive transfers (transfers periods of~10 days). One of these cultures could be maintained over 10 months. We succeeded in axenising the cultures from the insect cells after 10 successive transfers (the presence of insect cell was checked by light microscopy and by PCR using primers within the cytochrome oxidase gene). A peak of concentration of 1.106 cells/ml was obtained at transfer 16 (as quantified by qPCR). To verify that the LAS culture was really axenic, we checked if LAS was the only bacteria in our cultures. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced antibiotic selection has been applied to get rid of gram positive contaminants and we are looking into a solid medium formulation to isolate LAS clones. To improve LAS fitness in culture, we complemented the primo-cultures with various nutrients based on data from healthy or infected Citrus phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. We reached a bacteria titer of ~1.107cells/ml in a LAS/Aedes co-culture complemented with proline and ethanolamine. We are also testing other insect cell lines. Two Diaphorina citri cell lines produced by the Keyhani’s lab are now routinely maintained in our lab and we are analyzing their capacity to assist LAS growth. The very next step is to inoculate the cultured bacteria into healthy Citrus in order to verify Koch’s postulates.
‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus greening also known as Huanglongbing (HLB) is a fastidious, Gram-negative, phloem-limited, alpha-proteobacterium, is one of the most destructive diseases of citrus worldwide. Based on the sequence information number of putative effectors unique to HLB present in prophage related gene clusters and/or pathogenicity islands have been identified and are believed to contain bacterial pathogenicity, multiplication and virulence determinants. The present proposal envisages the expression of these putative effectors in the phloem of citrus, the site of the pathogen’s natural habitat, using citrus tristeza virus (CTV) vector. As outlined in our progress report of January 2011, we have cloned number of putative effectors as additional cistrons between the two coat proteins in the CTV vector under a heterologous, beet yellows virus (BYV) coat protein promoter and agro-inoculated leaves of Nicotiana benthamiana. Partially purified virions of recombinant CTV containing HLB effectors from the infiltrated leaves and /or the systemic leaves of N. benthamiana were used to inoculate citrus plants. Thus, we have inoculated citrus with 7-8 HLB effectors and the remaining are at different stages of transfer to citrus. We have also cloned some of the effectors into the 3′ end of CTV between p23 ORF and the 3′ non-translated region of CTV based on the recent finding of augmented expression of an inserted foreign gene. One of the important effectors or virulence factors is hemolysin, a 50 kDa protein secreted by type I secretion system. The hypothesis is that the hemolysin might interfere with metal ion transportation leading to host metabolic imbalance potentially resulting in disease symptoms. We have submitted an abstract entitled ‘Functional studies of hemolysin (exotoxin) of ‘Candidatus Liberibacter asiaticus’ expression in citrus using Citrus Tristeza Virus vector’ to the 2011 American Phytopathological Society meeting. Additionally, we are also using a much faster approach to screen for virulence genes of HLB. Potato virus x (PVX) vector system has been used extensively to screen for virulence genes in herbaceous hosts and this system can be used much faster to screen for virulence genes of HLB. We have noticed that in the genome of CLas, there is only one copy of flagellin-encoding gene in contrast to four copies of them in Sinorhizobium meliloti. Transient expression of the Las-flagellin using PVX expression vector induces an immunity response with cell death in Nicotiana benthamiana. The flagellin consists of a 22 amino acids near N-terminus (-DRVSSGLRVSDAADNAAYWSIA-), sharing the conserved Flg22 domain. By comparison with the nonfunctional homologue from S. meliloti, three divergent amino acids were identified. This suggests that these three amino acids may be required for Candidatus Liberibacter asiaticus flagellin to induce cell death in tobacco plants. To test potential interaction between Candidatus Liberibacter asiaticus and citrus, 40 ‘M commercial Flg22 (RP19986, GenScript) and FLg22-Las were infiltrated into young tissue of citrus, respectively, and was observed to induce hypersensitive cell death in citrus leaves. An abstract entitled ‘Characterization of the host defense response induced by the flagellin protein of Candidatus Liberibacter asiaticus’ has been submitted detailing these findings to the American Phytopathological Society conference to be held at Hawaii in August 2011. Thus we are making progress in identifying the genes responsible for virulence of HLB and eventually developing strategies towards its control.
‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus greening, also known as Huanglongbing (HLB) was successfully detected from citrus tissues harboring the pathogen using PCR probes. Thirty gene specific primer-pairs for the t-RNA methyltransferase, elongation factor (EF-TU) proteins, outer membrane protein (OMP), RNA polymerase .-subunit, DNA polymerase region, the rDNA region, and the 23S and 16S ribosomal RNA intergenic regions etc., were developed. Of these 30 primer pairs, 18 primer pairs amplified specific amplicons from the DNA isolated from HLB infected plants and no products were amplified from the healthy plants. We used these primer pairs to prepare non-radioactive Digoxigenin (DIG) labeled DNA probes. Additionally, based on the Ca. L. asiaticus sequence we also identified several putative effector genes encoding hypothetical proteins present as part of pathogenicity island gene clusters that probably are involved in HLB pathogenesis . These effector genes are highly specific to Ca. L. asiaticus and putatively encode proteins which have not been annotated to any specific function. Non-radiactive probes were developed for these amplicons and tested for the detection of HLB infected citrus from the green house. Different tissue parts of citrus trees by tissue blotting on the nylon membrane, and psyllids, and psyllid nymphs as ‘squash blots’ were examined. Results indicated that hybridization observed with probes for EFTU, were much greater than any other probe tested in detecting HLB even in non-symptomatic branches with in an infected tree. Probes developed for putative effector genes were not optimal for detection of HLB since they are present in very low copy numbers in the HLB genome compared to EFTU or the outer membrane proteins. We are in the process of confirming these observations with symptomatic and asymptomatic field samples in new flush during spring and summer months. The results of these observations are being prepared for publication.
The Southern Gardens Diagnostic Laboratory (SGDL) has been in operation since October 2006. To date, the SGDL has received and assayed 168,469 grower samples, and approximately 35,600 additional samples from various sources (research samples, psyllid samples, and samples from Southern Gardens Citrus) for a total of over 204,000 samples since the SGDL began receiving samples. For the period of time covered by this report (July 1, 2010 to March 31, 2011), a total of 25,247 grower samples, 3,337 psyllid samples and 9,588 research and Southern Gardens samples were received and analyzed for a total of 38,172 samples for the period. Based on the number of samples received to date, it is expected that the sample load will approach the budgeted sample load of 45,000 allocated for the period of the funding. Although the total sample volume may not be changing drastically, one thing that is changing, however, is the mix of sample type/sample submitter. Although the number of grower samples is declining slightly, the number of research samples is increasing. The research samples are being submitted by private, state, and federal researchers in support of their research work. The research samples tend to be from large-scale field trials, survey projects, and from chemical/nutritional management projects. We expect this trend to continue. Among the reasons for this are continuity in testing and the fact that the cost of testing is being supported by the industry. Progress has been made in automating a portion of the DNA extraction process. Programming of the liquid handling robot has largely been completed and several hundred samples have been run using the automated extraction process and compared to the standard extraction protocol. Although generally the results from both methods are similar, we have had some problems with consistency using the automated method. Improving the consistency will be the major focus going forward. Plans are being made for the national psyllid ring test. It is hoped that the samples will be sent out during the final quarter of the funding period. It is expected that a large portion of the US labs currently doing HLB testing will participate as well as labs in Mexico and possibly elsewhere.
In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have nearly completed the genomic sequencing of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. The draft genomic DNA sequence of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle (Vinca) in Brazil has been assembled and annotation begun. A total of 1,743,660 shotgun library reads of 1.2 kb average insert size were assembled into 6,293 contigs of avg. size > 2.8 kb. Following gap closure by PCR, the Lam genome now has a total of 1,240,227 bps in 30 contigs. The average length of the contigs is 29,956 bps, with the largest being 332,541 bps. All contigs have been extensively confirmed by PCR. The average GC content of Lam is 30% (Min: 21%, Max: 36%). Approximately 80% of this DNA encodes genes, and 1,096 total proteins have been predicted. The overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 845 genes common to Lam, Lso and Las, 26 genes found in Lam and Lso but not Las, and only 6 genes common to Lam and Las but not found in Lso. The latter included a DNA uptake competence gene. Lam phage sequences corresponding to both SC1 and SC2 of Las were found, although SC1 (and not SC2 as in Las) appears to replicate as an integrative plasmid prophage. Las phage were found to become lytic in plant infections (Zhang et al. 2011); Lam appears to have same potential (ie., Las and Lam both carry “suicide program” or lytic cycle genes). SC2 in Lam Sao Paulo has clearly degenerated to a nearly nonfunctional state (in terms of lytic cycle, but its replication was detected in circular form. SC1 in Lam appears to more assume the role played by SC2 in Las, since its replication is high, and it carries two peroxidase genes that are likely to be needed for pathogenicity of plants and possibly transmission in psyllids. Notably, 2 of 4 peroxidase genes were missing on the SC2 phage, as well as 2 adhesins (surface anchor proteins). The reduced number of adhesins may be relevant to transmission. The reduced number of peroxidase genes may explain why Lam symptoms in infected citrus varieties are more severe than symptoms elicited by Las affecting the same variety, since reactive oxygen production by plants mimics many of the chlorotic symptoms of HLB. Also missing from both Lam phage were the possible colicin and anticolicin genes found in Las; this may render Lam sensitive to the presence of Las.
In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have continued sequencing and constructed a fosmid DNA library of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. The Lam genome now has a total of 1,373,269 bps in 198 contigs. Since the last report, we have closed 157 contigs by PCR and new sequencing. Nearly all contigs have now been organized on a predicted “scaffold” and gaps continue to be closed by PCR. The contigs currently have an average length of 6,935 bp (up from 3,390 bps at last report), with the largest contig at 63,439 bps. The overall gene organization and structure of the Lam genome is more similar to Ca. Liberibacter solanacearum (Lso) than to Las. The complete Lam phage DNA sequences corresponding to both SC1 and SC2 of Las and including putative lysogenic conversion genes) are now completed, and the integration sites of the prophage have been determined. We previously reported a potential colicin IA and potential cognate colicin immunity protein were identified on Las SC1 and Las SC2, respectively. Colicins are anti-bacterial proteins that kill any competitor cells lacking the cognate colicin immunity protein. Interestingly, although Las phage carry genes encoding both a colicin and a colicin immunity protein, no such genes have been found on the Lam phage (which are very similar to the Las phage) or in the Lam genome. This indicates that Lam might be sensitive to Las colicin, and may explain why Lam is disappearing in Brazil, since mixed Lam and Las infections in the same field situations have been documented. It is possible that the Las colicin kills Lam. Further investigation of Liberibacter colicins and activation of the phage lytic cycle seems warranted as potential control strategies for Liberibacters generally.
Over the past two seasons, a total of nine experiments were conducted on ‘Fallglo’, ‘Sunburst’, ‘Ruby’ red grapefruit, and navel oranges evaluating the effect of different washing methods on inhibition of degreening and ease of grading out fruit with canker lesions. The results will likely be applicable to other grade or quarantine defects as well. The washing methods tested included: 1) full wash (brush bed + high-pressure wash [HPW]) plus waxing, 2) full wash (brush bed + HPW), 3) HPW only, 4) HPW without the normal brushes, 5) brush wash only, 6) only running over a PVC roller conveyer, or 7) an untreated control. All wash treatments were conducted on commercial wash lines with wash durations of approximately 70 sec. on the brush bed, and 20 sec. on the HPW. Normal brush rotation speed was ~100 rpm. After washing, early season fruit were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and peel color of the fruit was measured almost daily. The fruit were subsequently stored under ambient conditions (~70-75F) and evaluated weekly for the development of decay and physiological disorders. Results showed that with more brushing on the packingline, the greater the inhibition of degreening. Specifically, degreening was most inhibited by fruit receiving the full wash, followed by brush wash or HPW only, and least in fruit that passed under the HPW nozzles without being brushed. Fruit color development was almost completely inhibited if the fruit were also waxed after washing, whereas fruit that were not washed at all, but simply run on a PVC roller conveyor experienced almost no inhibition of degreening. The method of washing had no significant effect on the development of postharvest decay or disorders. These results were observed on all citrus varieties tested. As expected, the method of washing affected how clean the fruit became and how well graders could identify blemishes on the fruit. On a 1 to 9 scale, with higher scores indicating fruit that were easier to grade, fruit receiving the full washing were the easiest to grade, whereas unwashed fruit (the control) were the most difficult. An experiment with 2400 fruit gave similar results. However, the benefits of washing on grading ability was not significant when the fruit were naturally clean at harvest, i.e., early in the season. Therefore, if fruit need to be pre-graded for defects of quarantine significance early in the season when the fruit is relatively clean, very minimal or even no washing is needed so that effects on degreening will be minimal. As the season progresses, fruit become dirtier, but the fruit have much better natural color, and pre-washing can be initiated and intensified as needed to allow adequate pre-grading.
A comparative study involving two copper loaded silica nanogel (CuSiNG) formulations and a series of commercially available Cu biocides (such as Kocide 2000, Kocide 3000, Champ 30DP, Cuprofix Ultra 40, Kentan DF, Badge X2 and Magna-Bon) was performed during 2010 field trial season (Grapefruit trial; Ft. Pierce, St. Lucie County; conducted by James H. Graham group). This trial data clearly demonstrated superior adherence property of CuSiNG material to fruit surface (an assay performed by Dr. Graham’s group) and reasonably good protection against canker incidence when compared to other biocide controls. Based on the feedback from last year’s trial, we have developed two most promising all water-based copper loaded silica nanoformulations (‘Cankicide NP200’). Sixteen gallons of each formulation (metallic copper content per gallon = 16.4 gms; synthesized in Dr. Santra’s lab at UCF-NSTC) have been delivered to Fort Pierce this month. One of these nanoformulations is transparent, stable and highly soluble in water (observed no settling for 3 months). This formulation could be directly added to spray tank prior to applications. Dynamic light scattering (DLS) data confirmed formation of copper loaded silica nanoparticles in solution with particle size as small as 10 nm. These particles take part in gelation process, producing larger structures in the micron size range (also characterized by DLS study). Additional materials characterization data will be reported in future reports.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, April 2011. The Huanglongbing (HLB) Diagnostic lab has been in service since February 1, 2008 and has processed more than 25,000 samples submitted from citrus producers, approximately 14,000 samples from researchers, and 1,000 samples from the Citrus Foundation screenhouse located at SWFREC for a total of more than 40,000 samples. During the time period of January-April, more than 2000 samples were received and processed. Growers, extension personnel, and researchers from around the state are utilizing this service. The diagnostic testing is now being utilized to to an increasing number of producers throughout all of the citrus producing counties in Florida. The lab has received samples from growers throughout Florida, with the highest number of samples received from Collier, Highlands, Palm Beach, Polk, and Hendry counties. There is a slight seasonality to the sample submission volume with respect to harvesting and new growth (flushing) events. Last year, growers have submitted the most samples January-May and we are seeing an increase in sample submission March-April. From the currently accumulated data, the HLB lab has a 73% positive sample submission rate, and a 27% negative submission, so the ratio is relatively unchanged. As previously reported, the methodology is briefly: samples are processed using USDA guidelines for HLB detection (Anonymous, 2007) and results are typically available within two weeks of submission. The HLB Diagnostic lab currently has one full-time dedicated employees and four part-time employees responsible for logging in samples, performing the diagnostic assays, and sending reports on the results. The testing procedure employs amplification of specific regions of the pathogen’s DNA sequence and detection in real-time by use of a PCR machine designed for this application. This test is both equipment and personnel intensive. In addition, expendable lab equipment and reagents are consumed in performing the test. The specific methodology used in this laboratory is described in ‘Real-time PCR for diagnostic detection of citrus greening or HLB (Huanglongbing) from plant samples’, USDA, APHIS, PPQ, CPHST. DNC WI-B-T-D-2 (Anonymous. 2007). These disease detection rates are not directly indicative of the actual overall field disease levels for HLB since scouting and field sampling are usually selective. The lab also provides support to on-going research and extension programs at University of Florida. To support research, the HLB Laboratory extended its capabilities to include the detection of the bacterium Candidatus Liberibacter asiaticus the causal organism of the citrus disease huanglongbing (HLB) in the Asian citrus psyllid, Diaphorina citri. Detection has been carried out in approximately 1500 samples. In addition to detection, quantification of the bacterial load has been carried out in 80% samples. Samples comprise of individual adult and fifth instars nymphs or groups of first to fourth instars nymphs. We also have quantification of bacterial load in plant tissue protocol validated for use in field research projects.
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 (Las) 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 and published a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb. All BAC clones of Las were sequenced, and sequence analyses revealed a potential mechanism of GENOME REDUCTION. 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, confirming only one operon responsible for zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, sour 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.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is FIRST TIME that ONE SEED-TRANSMITTED HLB SEEDLING was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. Repeat testing from the HLB-affected sour orange seeds is in the process. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 to 100,000,00 cells/ml were obtained within 48 hrs based on qPCR estimation. The Las cells number in the cultures were staggering and decline when they reached to Ct value 22.00 (16S rDNA-based RT-PCR) in liquid media. We are looking into factors affecting further growth.
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 and published a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb. All BAC clones of Las were sequenced, and some new sequences will be added to the published genome. 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, confirming only one operon responsible for zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, sour 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.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is FIRST TIME that ONE SEED-TRANSMITTED HLB SEEDLING was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant is underway. Repeat testing from the HLB-affected sour orange seeds is in the process. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 to 100,000,00 cells/ml were obtained within 48 hrs based on qPCR estimation. The Las cells number in the cultures were staggering and decline when they reached to Ct value 22.00 (16S rDNA-based RT-PCR) in liquid media. We are looking into factors affecting further growth.
The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized. Sequencing analysis of the hyvI/hyvII genes from 40 Las DNA samples collected globally revealed sequence conservation within the individual repeats but an extensive variation regarding repeat numbers, their rearrangement, and the sequences outside of repeat region. These differences are found not only in samples with distinct geographical origins but also from a single origin and even from a single Las-infected sample. The Florida isolates contain both hyvI and hyvII while all other global isolates contain only one of the two. Interclade assignments of the putative HyvI/II proteins from Florida isolates with other global isolates in the phylogenic trees imply a multi-source introduction of the Las bacterium into Florida. We have developed a real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting nearly identical tandem repeats of 100bp hyvI and hyvII. Because of higher copy number of the tandem repeats per bacterial genome, these methods significantly improved detection capacity of the HLB bacterium. In comparable samples relative to 16S HLBaspr real-time PCR, these new methods reduced the relative detectable threshold by approximate 9 and 3 cycles for LJ900fr and LJ900fpr, respectively. From HLB samples with extreme low titer of Las bacterium, both LJ900fr and LJ900fpr detected Las from otherwise non-detectable samples by APHIS standard, HLBaspr. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative NLSs in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with GFP driven by CaMV35S promoter, and transformed Agrobacterium tumefaciens strain GV 2660 with the pCX-DG-hyvI constructs. The results indicate that the HyvI protein did not target in plant nucleus but located in cytoplasm when transient expression in tobacco. In addition,the results show that hyvI gene did not induce hypersensitive response(HR) on tobacco leaves. RT(Reverse transciptase) PCR confirmed the hyvI gene expression both in the host plant and psyllids though the HyvI protein was not detected with Western blot. The obstacles for detection of the antigen from HLB-infected plants remains to be further investigated.
Clonal Complex analysis and Phylogeny of HLB-associated ‘Ca. L. asiaticus’: Allele frequencies at seven microsatellite loci that have been developed were used to characterize allele patterns and to estimate the genetic diversity of ‘Ca. L. asiaticus’ isolates collected from several major citrus growing countries including US, Brazil, India as reported earlier. The average number of alleles and the number of effective alleles per locus per population ranged from 1.0 to 2.7 and 1.0 to 2.3, respectively. A comparison of the number of alleles within each geographic region revealed that isolates from China possessed the most alleles (6.6 alleles per locus), followed by India (5.6 alleles per locus), Florida (4.3 alleles per locus), Brazil (3.0 alleles per locus), Cambodia (2.6 alleles per locus), respectively. The highest genetic diversity was found in India which ranged from 0.22 to 0.54 followed by US from 0.07 to 0.39, China from 0.07 to 0.35, Cambodia from 0.30 to 0.34, and Brazil China from 0.05 to 0.32, respectively. Genetic differentiation among the overall sample populations of ‘Ca. L. asiaticus’ from different countries were evaluated with the assumption of a short divergence time by comparing pairwise FST values. Results showed that overall sample populations from India has higher FST values when compared with sample populations from all other countries. Similarly, high FST value was also observed in other Asian countries such as China and Cambodia. Presumably, high genetic differentiation of ‘Ca. Liberibacter asiaticus’ populations among Asian countries are likely due to mutation accumulation, selection and genetic drift over a long history evolutionary process. Recent introductions of HLB in American continents, US and Brazil sparked research interests to test a hypothesis of the origins of HLB and the genetic relationships between American and Asian ‘Ca. L. asiaticus’ sample populations. We employed the eBURST analysis approach to identify putative founder types and clonal complexes (CCs). This analysis resulted in identifying two major Clonal complexes, CC1 and CC2. CC1 consisted of 260 members (112 haplotypes) and included members from all countries represented in our sample set, the corresponding sample (Haplotype-14) originated from an southern region of China. CC2 consisted of 33 members (30 haplotypes). Haplotype-14 was predicted to be the primary founder of CC2; the samples that compose this Haplotype originated from Andhra Pradesh, India. The results from UPGMA tree with START2 analyses are consistent with analysis of this dataset using eBURST, indicating that two major groups of ‘Ca. L. asiaticus’ are present worldwide: one lineage (CC1) is composed of samples from all 9 countries, while the other lineage (CC2) is composed of samples harvested from HLB-positive Indian citrus trees. Fingerprinting virulence strains: ‘Ca. L. asiaticus’ strains vary in pathogenicity and the severity of symptom expression is in accordance to the degree of virulence of the pathotypes. The pathogenic variants among ‘Ca. L. asiaticus’ strains were observed in the field and greenhouse experiments. To identify and differentiate different pathotypes, a genome wide sequencing analysis had conducted to map putatively virulent-related genes. Given the fact that the bacteria have compact genomes, many of these repetitive loci are located within or near DNA coding regions and could have profound effects on gene expression and functions. The next research objective is to evaluate sequence variation in target loci among selected strains. Part of this study had summarized in 2010 annual report and presented in 2011 International Research Conference on Huanglongbing, Orland, Florida. This report covers the period from last annual report to the present.
Multiple sensing techniques were evaluated for HLB detection in citrus. The major findings are summarized below: (i) VISIBLE-NEAR INFRARED (NIR) SPECTROMETRY: Field data analysis indicated that algorithms, QDA & SIMCA could classify the spectral features with a classification accuracy of > 90% (Comput. Electron. Agric. In press). In addition, feature extraction indicated that few spectral bands & vegetation indices yielded an accuracy of > 80% (Precision Agric. Under review). Currently, efforts are being made to reduce the number of spectral bands & wavelength indicative to HLB. (ii) MID-INFRARED (MIR) SPECTROSCOPY: The MIR spectra of the citrus leaves showed two prominent peaks, with peak at 9 to 10.5 ‘m representing carbohydrate (starch) absorption peaks. Statistical algorithm, kNN yielded a high average healthy, nutrient-deficient & HLB class classification accuracies (> 95%) (Talanta, v. 83, pp. 574-581, 2010). (iii) FLUORESCENCE SPECTROSCOPY: Analysis of Multiplex’ fluorescence sensor lab & field data from HLB, healthy, & nutrient-deficient citrus leaves indicated that features such as yellow (UV excitation), far red (UV excitation) & yellow fluorescence (green excitation) contributed to the classification of stressed condition in leaves. Larger dataset from different varieties of citrus trees is being analyzed to validate some of these results. (iv) AERIAL HYPERSPECTRAL IMAGING: Aerial multi- & hyperspectral images acquired from multiple citrus groves have been geo-rectified & radiometrically corrected. First & second derivatives of the spectral reflectance data were found to be effective in classifying healthy & infected canopies. In the first derivative spectra, healthy & HLB infected canopies have peak locations at 723 & 702 nm, respectively. For the second derivative spectra, peaks were identified at 516 & 696 nm for healthy, and at 510 & 690 nm for HLB infected leaves (J. Appl. Remote Sens. Under review). During the next year, we will continue our detection algorithm development & focus on increasing the detection accuracy with low false positives. (v) GAS CHROMATOGRAPHY/DIFFERENTIAL MOBILITY SPECTROMETER (GC/DMS): In-field testing (Hamlin) with our mobile sensor platform was carried out from Nov 2010-Mar 2011 to account for weather & seasonal (blossoming, fruit ripening, harvest, etc.) variations. Accomplishments include: (1) We located 9 separable peaks on the GC/MS data between HLB & healthy samples. Using the leave-one-out validation, the classification accuracy by applying PLSR to these 9 peaks is 83.3% (manuscript in preparation); (2) Selected DMS biomarkers shows that HLB detection accuracies ranging from ~70-95% in different months, with an aggregate 71% accuracy for the combined data set. Considering the confounding factors, we developed more robust approaches to detect DMS HLB biomarkers; (3) Using wavelet analysis for feature extraction, we increased the HLB diagnosis accuracy up to 82% for the combined data set (manuscript in preparation). We are working with industry collaborators for transition to a commercial partner at the project conclusion. (vi) VOC BIOMARKER IDENTIFICATION USING GC/MS: Over several weeks, 20 samples from a control tree & 16 samples from a HLB tree were collected at 4 h sampling time. There were five peaks that were found only in the control & never in HLB samples and three peaks only in HLB & never in control leaf samples. The five of the ‘control only’ peaks included 1 aldehyde, 1 ester, 2 organic acids & 1 nitrogen compound. Of the three ‘HLB only’ peaks, one of them has been tentatively identified. Identification confirmation of this peak and 2 unknown peaks is in progress. We are working on data treatment procedures to unambiguously identify HLB & healthy leaves using these markers. We are also currently modifying the volatile collection procedure to shorten the sampling time using a dynamic volatile collection technique.
A proposal to continue this project was approved. We have developed an efficient means to prepare inoculum for attempts to culture Liberibacter asiaticus. This involves collecting psyllid adults (Diaphorina citri) from huanglongbing-infected citrus plants, disinfesting the surfaces of the psyllids, aseptically removing their alimentary canals, and putting the canals into culture medium. Five canals are used in one inoculation event. After mixing to wash bacteria from the canals, a sample of the medium is stained with Syto-13, a DNA-binding fluorochrome, and examined with an epifluorescence microscope for the presence of Liberibacter asiaticus. If present, the bacteria are counted, and the inoculated medium is incubated and re-examined periodically for growth of the bacterium. This procedure has been used to evaluate numerous culture medium formulations. Media have been developed which support increases in the number of bacteria counted 4-7 days after inoculation. These cultures were transferred to fresh media, but the numbers of bacteria declined until the bacteria were no longer detected after several transfers. We are continuing to test modifications of those media which appear to support limited growth of Liberibacter asiaticus in order to find the combination of ingredients required to support continuous cultivation.
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