To date, 400 different media formulations have been tested for their ability to support growth of Candidatus Liberibacter asiaticus. Inoculum was prepared by washing the bacteria from biofilms formed on the surface of alimentary canals of the psyllid, Diaphorina citri, which were reared on citrus plants infected with huanglongbing. Two days ago, a four to six fold increase in the number of the bacterium was detected in one medium formulation (BM397) five days after it was inoculated. Several other medium formulations exhibited lesser degrees of increased numbers. The culture in medium BM397 was transferred to fresh medium and will be monitored for further growth. New cultures in medium BM397 and the other media that had promising results are being established to determine if the these results are repeatable. Medium BM 397 has an extremely complex formulation that will need to be optimized if it continues to support growth of Liberibacter asiaticus. In summary, our approach to culturing Liberibacter asiaticus appears to be working working.
This report will be present according to objectives of project, i.e., diagnosis of Liberibacter asiaticus by both, serological and molecular approaches. Firstly, from the molecular approach the Nested PCR protocol was finalized and definitively the double step is much more sensitive than the one step. We used DNA from a single psyllid or from inoculated citrus plants by psyllids as template for quantitative PCR (QPCR) and the results were compared against Nested PCR one step (NPCR-OS), double step (NPCR-DS), and traditional PCR. We verified that a hundred percent of matches between QPCR and NPCR-DS was obtained for the samples whose threshold cycle (Ct) values ranged from 22 to 32, as determined by QPCR. For the samples whose Ct values ranged from 32.1 to 34, and from 34.1 to 36 there were 98.11% and 96.61% of matches between NPCR-DS and QPCR, respectively. For those samples with Ct values above 36.1 only 54% of results were shared between QPCR and NPCR-DS. The number of positive samples determined by NPCR-OS was lower than determined by NPCR-DS or QPCR, but a higher when compared to traditional PCR for all comparison levels. Finalizing, based on the significant agreement between NPCR-DS and QPCR for the samples with Ct <36 as well as others facts, as the non-frequently Ct around of 38 reported for the negative controls, we used the 36th cycle as a threshold for decide if the samples are positive (Ct <36) or negative (Ct >36). For the serologic-based diagnosis we tested the eight potential antibodies (see last report) against Lib. asiatic symptomatic plant and negative controls. From the eight antibodies, four (T3699-4, T3699-5, T3699-6, T3699-7) shown reactivity against infected samples by ELISA (OD >0.456), but no reaction was observed against health plants or several bacteria species as Xylella fastidiosa, Xanthomonas, Pseudomonas, Agrobacterium, Escherichia coli, and Methilobacterium spp (OD<0.099). Previous tests shown that the reactivity of antibody T3699-7 was statistically more reactive to the others three antibodies, T3699-4, T3699-5, and T3699-6. The ELISA results of that antibody using different citrus species budding infected by Las were quit similar to real time PCR, with a moderate Cohen's Kappa index (0.583; SD 0.316). Both, the moderate value and the high standard deviation (SD) probably are consequence of low number of samples used in the tests. Also, the T3699-7 was used on Dot Blot methodology which shown potentially useful on diagnosis of Lib. asiaticus. Although this project was officially finish on November 2010, we are still working on set-up and optimize the serologic based protocols. The good specificity and reactivity of T3699-7 and the absence of a commercial serologic based test make us excite on continuous this work. The next steps will be to test the T3699-7 on a broad number of samples using both ELISA and DOT-BLOT methodologies.
Previously, a total of 293 ‘Ca. L. asiaticus’ isolates obtained from HLB-affected plants American and Asian countries were used for genetic analysis. Samples were successfully amplified by seven microsatellite primer pairs. The number of alleles and haploid genetic diversity per locus ranged from 2 to 30, and 0.204 to 0.881, respectively. In the clonal corrected data set, genotypic linkage disequilibrium was not detected by pairwise comparison of loci across the overall isolates (P.>.0.01). A total of 147 genotypes (haplotypes) were identified. Overall haploid genetic diversities were the highest in Asian countries. The haploid genetic diversity of the Florida (USA) isolates was lowest among all the geographic groupings. A UPGMA clustering analysis identified three major groups of ‘Ca. L. asiaticus’. Isolates from India were clustered in a distinct group. Most of the isolates from China and other Asian countries, and Brazil were generally grouped in group 1. While some isolates from Florida occurred in group 1, most isolates from Florida were clustered in group 2. The STRUCTURE analysis based on Bayesian modeling further assessed the genetic structure of ‘Ca. L. asiaticus’. This approach utilizes statistical methods to determine the relationships among the isolates without prior population information. In this analysis three different clusters (K) were identified based on the ad hoc statistic .K. The membership of each isolate in a group obtained from STRUCTURE analysis can be estimated as (q), the ancestry coefficient, which varies on a scale between 0’1.0, with 1.0 indicating full membership in a population. Individuals can be assigned to multiple clusters (with values of q summing to 1.0) indicating they are admixed. Individual sample with q.’.0.90 (ancestry coefficient) were considered as having single lineage and individuals with q.<.0.90 were considered as admixed lineage. The result of STRUCTURE analysis is consistent with UPGMA in which isolates from India were grouped in a distinct cluster. Brazilian and most east-southeast Asian isolates were clustered as a single lineage (q.'.0.90). Some isolates taken from central Florida (Polk, Pasco, and Lake Counties) shared the same lineage with east-southeast Asian and Brazilian isolates. Most Florida isolates, however, grouped in a different cluster. Some admixed isolates (q.<.0.90) were found in Florida as well as in Guangxi province in China, and in Cambodia. The seven microsatellites developed in this study are useful for detection, isolate differentiation, and genetic analysis of 'Ca. L. asiaticus'. Our results showed that current 'Ca. L. asiaticus' populations in HLB-affected citrus in Asia and the Americas are comprised of three distinct genetic groups: (1) Indian, (2) predominantly east-southeast Asian and South American (Brazil) and (3) predominantly North American (Florida, USA). While regional differences were observed from the distribution of dominant clusters, the similar genetic makeup of east-southeast Asian and Brazilian isolates lead us to hypothesize that 'Ca. L. asiaticus' populations in Brazilian groves were most likely introduced from east or southeast Asia. The precise sources of the dominant genetic group of 'Ca. L. asiaticus' retrieved from Florida are not clearly resolved from the present analysis. However, less-pervasive groups may have been introduced directly from Asia or via Brazil. While the results here provide some insights into the origins and genetic structure of HLB-associated 'Ca. L. asiaticus', it should be noted that more samples adding to this study will help further increase resolution for differentiation genetic relationships among 'Ca. L. asiaticus' worldwide.
The methods if this project, Laser Capture Microdissection (LCM) followed by 16sDNA and metagenomic 454 XLR pyrosequencing, failed to reveal the presence of L. asiaticus in infected tissue, despite the fact that qPCR controls on parallel infected tissues showed the presence of typical amounts of L. asiaticus. We speculate that this may be attributed to the non-uniform distribution of these bacteria in the infected plant. We attempted to compensate for this by using larger samples for sequence analysis in additional trials, but we still did not find Liberibacter. Other reasons for failing to find Liberibacter cannot be ruled out. The second unexpected finding of our study was that the infected tissues, but not the uninfected control tissues, showed the presence of a wide diversity of bacteria at very low abundance. Should this result prove reproducible, it would suggest that the Liberibacter infection results in increased susceptibility to secondary infections. We hasten to point out that this study was not designed to test that hypothesis, and thus less material was examined from uninfected leaves than from infected, and that DNA yields were disappointingly low throughout this study. Nonetheless, this is an intriguing observation that merits further examination.
The Southern Gardens Diagnostic Laboratory has been in operation since October 2006. To date, the SGDL has received approximately 152,000 grower samples, 17,100 samples from Southern Gardens, and approximately 6,000 individual psyllid samples. In addition, several thousand samples have been submitted by various University and USDA researchers on various projects. For the period covered by this report (July, 2010 to September, 2010), a total of 4,803 samples were processed. Of these, approximately 45.4% were grower samples (2,180), 92% were samples from Southern Gardens (442), and 45.4% were research or psyllid samples from various University, USDA, or private research projects (2,181). Although sample volume varies from year to year and also during a given year, it appears that the total grower sample load will be lower this year compared to the previous year. The total number of grower samples is projected to be in the 30,000 range compared to the previous year’s grower sample load of 44,985 grower samples. However the sample load of research related samples is increasing and combined we expect to be in the range of the samples used to prepare the budget for this project. In addition to the disease testing activities, the lab recently coordinated a blind panel test of infectious psyllids for many of the laboratories involved in psyllid testing in California. In addition to the various labs, a variety of methods were evaluated and the results of the panel testing will be presented at the International HLB meeting in Orlando in January, 2011. Currently we are in the process of organizing a similar infectious psyllid panel test that will be conducted on a national level. We are also in the process of automating a portion of the DNA extraction procedures which should increase the lab efficiency. Validation of the process is expected to be completed during the first quarter of 2011.
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 a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb . We have also obtained a draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. A comparative genomics of these close-related bacteria revealed useful information for understanding their pathogenesis and evolutions. We are continuing the screening of our BAC library, and results indicate that the BAC library contains most, if not all sequences of the Las genome with the insets up to 130 kb. Sequencing analysis confirmed our previous published Las psy62 genome sequence. However, in the hyper variable regions, genetic diversity was observed among the clones. 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.” 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. In vitro culture of Las bacterium appeared to be possible. Ten to thirty-five folds increase of Las bacterium in liquid media were observed. We are continuing to modify growth conditions for the improvement of the Las growth rate in media.
The objective of this project is to characterize the hypI 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 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 hypI (renamed as hyvI) was further characterized. Another homologous of the hyvI was also identified and named as hyvII. The hyvI (2760 bp) contains 12 nearly identical 132-bp repeats and 4 partial repeats while the hyvII (1026 bp) contains only one incomplete repeat and shares homology with hyvI. Frequent deletions or insertions of these repeats within the hyvI/II genes were observed, but none of which disrupted the open reading frame. Sequencing analysis of the hyvI/hyvII genes from 35 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 within two Las prophage genes, 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.
HLB detection using volatile profiling: Our primary objective has been to develop a highly reproducible method to trap field leaf samples in sufficient amounts to allow unambiguous identification and then to compare healthy and greening leaf volatile patterns. New sampling techniques involving large volume air sampling and solid phase volatile traps have also been developed and are currently being optimized. Various solid phase volatile trap materials were evaluated. The most efficient trap material was a combination of Tenax and graphite. Two liquid nitrogen dispensing devices were evaluated. The large volume (160L) 22 psi tank was the most reliable with shortest recycle times. Cryofocusing temperatures from 0 to -100’C, were evaluated with -100 ‘C being optimum. Over 72 chromatographic peaks (Carbowax column) could be reproducibly detected from a one hour air sample taken over the surface from a healthy Valencia flush leaf cluster. Identification of these peaks is in progress. In addition to the above activities, progress is being made on the development of a portable instrumentation platform for leaf volatile sensing, together with our partners in industry. We now have a miniature ‘suitcase’ instrument capable of taking direct measurements in the field from tree leaves within the citrus canopy. The instrument can acquire samples a time frame that ranges between 2 and 10 min (adjustable) and concentrates the VOCs released from the plants into an on-board sorbent trap. The trap is then heated to desorb the compounds into the DMS for in-field analysis. Concurrently, we have continued to work on determining the most robust HLB-associated VOC profiles released from citrus post infection. We have identified a few chemical peaks from Valencia and Hamlin samples (greenhouse plants), and we are in the process of robust chemical identification at this time. We also continue to make progress on algorithms needed for signal processing and identification of disease-relevant biomarkers from our field tests. By the end of October 2010, we will be using the developed instrument for a planned field trial and field testing of two additional suitcase instruments, during November and December of 2010. We continue to work on adapting these methods for real-time field detection and robust classification strategies. HLB detection using reflectance spectroscopy: A new Multiplex’ fluorescence sensor was acquired from Dynamax Inc. and a large set of laboratory and field data were collected from citrus leaves of HLB-infected, healthy, and nutrient-deficient trees. In addition, visible and near-infrared spectral reflectance data were also collected from HLB-infected, healthy, and nutrient-deficient leaf samples under laboratory and field conditions. Data from both non-symptomatic and symptomatic leaf samples from HLB-infected trees were collected. The data analysis on these sets of data is ongoing. The manuscript describing research on HLB-detection based on mid-infrared spectroscopy has been accepted for publication in ‘Talanta’ and is currently in press. HLB detection using airborne hyperspectral imaging: A new set of aerial images of HLB infected groves will be acquired between October and November, 2010 using three camera systems (color, multispectral and hyperspectral cameras) with the cooperation of Dr. Yang’s group at the USDA ARS in Weslaco, Texas. The grove is located at 26’19’ 2.9” N and 80’57’ 28.7” W. More image analyses are currently being conducted with the previous images acquired in 2007 and 2009. A journal article is currently being prepared from the previous research activities.
The goal of this project is to obtain the in vitro culture of the bacteria – Ca. Liberibacter asiaticus (LAS) – associated with the Citrus greening syndrome. The strategy consists of primo-cultures of the bacteria in insect cells cultures used as feeder cells. Inoculums from LAS infected citrus or periwinkle were macerated with various insect cell cultures, in different media. LAS presence in the inoculated cell cultures was checked by direct PCR and confirmed by sequencing of the amplicons. Nine different cell lines were tested from Mamestra, Spodoptera, Drosophila, Aedes, Diaphorina insects. Mamestra and Spodoptera cell lines were not suitable for LAS growth. Two Drosophila and one Aedes cell lines showed to sustain Liberibacter survival and growth. LAS/Drosophila co-cultures: the major challenge remains to maintain the insect cell population low. We are currently testing new culture media and conditions to contain their growth. Culture in tubes in different media helped in containing drosophila cells proliferation. LAS signal in those cultures is very low and we are currently monitoring and adapting the conditions for improved bacterial growth. LAS/Aedes co-cultures were obtained at a low insect cell concentration. One co-culture has been continuously growing for 9 months and 16 successive transfers. Axenization of this culture is progressing intriguingly along with the formation of biofilm-like assemblies in the flask. These assemblies are testing positive for LAS and we will further analyze them by microscopy. To reach higher bacterial concentrations, we analyzed metabolic pathways potentially encoded by the released Ca. Liberibacter genome sequences to define limiting factors and/or growth inhibitors and we complemented the primo-cultures with various additives (sugars, vitamins,’). Adding proline and ethanolamine in a LAS/Aedes co-culture allowed us to get higher concentrations of LAS (~1.107cells/ml) and are currently looking into best ways to maintain/increase those concentration levels and to stock those LAS cultures. Diaphorina cell lines were recently received and are under investigation regarding their capacity to maintain the bacteria. The Keyhani’s lab recently sent us three Diaphorina citri cell lines. The inoculated cell cultures tested positive after 5 to 34 days and two cell lines are positive after one transfer so far. So we now have three different insect cells that succeeded in getting a LAS positive signal after inoculation (Drosophila, Aedes, Diaphorina) and while still working on getting improved growth of bacteria, we continue to test additional insect cells for our co-cultures.
We have made some progress from the last quarter progress report in the detection of ‘Candidatus Liberibacter asiaticus’ (CLas) in citrus tissue blots. Digoxigenin based PCR probes seem to work better compared to riboprobes at present. We have been able to consistently obtain specific hybridization with the EF-Tu PCR probe. Other probes have been used with varying efficiency. Using this probe we have been able to do whole leaf blots. This is done by gently scraping the abaxial side of the leaf with a razor blade and making a tissue imprint on the hybridization membrane. The results indicated that a strong hybridization was observed in the mid-rib and lateral veins of older symptomatic leaves suggesting the presence of CLas. However, with young leaves from the same branch (non-symptomatic) hybridization was not observed but not as strong as the older symptomatic leaves. We have not overcome the non-specific hybridizations observed with psyllids using whole psyllid squashes. The healthy psyllids also show non-specific signal. However, using single psyllid extract in phosphate buffer and use of total extract (high speed) eliminated the non-specific hybridization. This eliminated the need for the preparation of nucleic acids from the psyllids. However, this is still time consuming, and our aim is to make the whole psyllid squash blot to work consistently without non-specific hybridization. As mentioned in the previous progress report we are preparing probes for the genomic regions of CLas which encode hypothetical proteins will and test them soon.
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 a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb, and the sequence has been released on NCBI. End sequencing of all BAC clones of Las revealed new clones containing different Las sequences and potential rearrangement of Las genome. Based on the variations within the Las prophages, eight different haprotypes (populations) have been identified, and their correlations with phenotypes and disease severity of HLB is under investigations. 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, 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 100,000 to 1,000,000 cells/ml were obtained within 72 hrs based on qPCR estimation. The Las cells number in the cultures were staggering and decline when they reached to Ct value 25.00 in liquid media. We are looking into the potential inhibition due to the prophage/phage conversion. Factors affecting the growth are being further evaluated using a spit-plot design.
The nutritional trials with the Boyd cocktail are beginning to identify those components most important for maintaining trees with respect to healthy foliage, yield and fruit quality. The trees are still responding with vigorous vegetative growth and an impressive fruit crop. The components groups of the cocktail are macronutrients, micronutrients, SARs (Salicylic acid and Serenade), and hydrogen peroxide. The highest Valencia yields the past two seasons have been from treatments that had the micronutrients plus a phosphite. We have expanded our investigation this year to use what we learned in 2008 and 2009 to better define the contributions of the SARs and micronutrients, and include other micronutrient formulations. We have trials comparing the Boyd cocktail with and without the Salicylic acid and the Serenade. Based on grower interest and suggestion we have included in this trail two programs using KeyPlex. We have also initiated a trial to evaluate the value of each micronutrient. We have individual treatments of the micronutrients as sulfates and the Zn and Mn as a phosphite. All plots are HLB positive at the beginning of the experiment. A 12-acre block of ‘Valencia’ orange on ‘Swingle’ citromelo planted in June 2001 was selected to evaluate effects of foliar nutritional treatments and ACP management on yield and ACP populations. A randomized complete block design was used with 4 replicates of a 2 x 2 factorial experiment and 4 treatments: 1) untreated, 2 Insecticides only, 3) Nutritionals only, 4) nutritional+insecticide. ACP control consisted of dormant sprays and applications based on scouting results during the growing season. Cumulative insect x day populations in the untreated plots averaged 9.6 times greater than in the treated plots. The experiment was initiated in March 2008 with a 29.9% HLB infection rate based on PCR which increased to 94.7% in 32 months. Insecticide treated plots have had higher CT values (lower bacterial titer) than untreated plots consistently over time with no effect of nutrition. In 2010, the ‘nutrition + insecticide’ treatment had the highest yield (6.1 ‘ 0.6 Lb solid/tree), brix/acid ratio (19.0 ‘ 1.1), and juice/fruit ratio (65.0 ‘ 1%), and the lowest fruit drop (17.4 ‘ 6.5 Lb dropped/tree) of all other treatments. Fruit drop appeared to be caused by an untimely herbicide application a few days before the harvest.
We made progress in developing the non-radioactive digoxigenin labeled probes for the detection of Candidatus Liberibacter asiaticus (CLas) in citrus tissues and psyllid vector using both DNA PCR probes as well as RNA probes. The probe that worked well with citrus tissues was the EF-TU probe. We made full-length probe as well as the truncated probes that worked with equal specificity. However, we have employed many probes with varying degree of success. Hybridizations were more specific in the tissue blots from the young flushes compared to the older parts of citrus. This is probably because of the relative high concentrations of the CLas pathogen in the new flush. Although the older parts of the infected citrus plants show clear symptoms of HLB (especially in the leaf), the hybridization signals were not very clear. Based on the CLas sequence we have identified several putative effector genes that probably are involved in pathogenesis. These effector genes are highly specific to CLas and putatively encode proteins which have been not annotated to any specific function. We are in the process of transferring them into citrus using the citrus tristeza virus vector. It is highly possible that the the probes made to these gene sequences would be highly specific to CLas. Towards this end, we have made primers to the end regions of these genes and have amplified them from total nucleic acids isolated from HLB infected citrus tissue. Amplifications were not observed with templates from healthy citrus plants. We will make probes for few of these genes and use as hybridization probes. We are also optimizing the hybridization using DNA and RNA probes for psyllids. As outlined earlier use of whole psyllids gave higher hybridization back ground in the ‘squash blots’. Therefore we intend to use the head and/or the thorax and the abdomen regions for making tissue (squash) blots of psyllids. As soon as the hybridization conditions are standardized for psyllids we will start using populations of psyllids from green house and subsequently from the field. The results of these experiments would provide information of the progression of the CLas spread in citrus and infection ratios in the psyllid population.
Proposed research involves expression of putative effectors of Candidatus Liberibacter asiaticus (Las) in citrus using citrus tristeza virus vector in an effort to understand the pathogenesis caused by Las. Recently, I have been able to recruit Dr. Subash Hajeri as Post-Doctoral Associate to initiate studies on this project. Based on the sequence information of Las, it is believed that many pathogenicity islands observed in the Las genome might contain bacterial effectors required for pathogen virulence, multiplication etc., and insect transmission determinants for the horizontal gene transfer. One way is to identify and express these putative effectors of Las in citrus and study their role in pathogenicity and the best approach currently available is our improved CTV-vector system, which expresses foreign genes in the citrus phloem. Towards this end, we have identified two prophage related gene-clusters of nearly 16 and 30 Kb which putatively encode many hypothetical proteins which are believed to play role in Las pathogenesis. At present we have amplified, by PCR using specific primers, some of these hypothetical protein genes from the total nucleic acids isolated from HLB infected citrus plants and cloned into our CTV vector. Additionally, two hypothetical proteins, hemolysin-like and serralysin-like proteins, have also been cloned into the CTV vector and all are in the process of being transferred into citrus.
The potential of mid-infrared spectroscopy for HLB detection was investigated as a part of this project. The mid-infrared spectral features were extracted and two prominent peaks: 5.5 to 6.5 ‘m (1,538 to 1,818 cm-1) and 9 to 10.5 ‘m (952 to 1,112 cm-1) representing water and carbohydrate absorption peaks, respectively were observed. The spectral data were preprocessed (baseline correction, negative offset correction, removal of water absorbance band, principal component analysis) before further analysis. Two different classification algorithms: k-nearest neighbor (kNN) and quadratic discriminant analysis (QDA) were used for distinguishing healthy, nutrient-deficient, and HLB-infected leaf samples. The kNN-based statistical algorithm yielded a higher average healthy, nutrient-deficient and HLB class classification accuracies (> 95%) than those of QDA. Future study will involve the detection of HLB-infected leaves at asymptomatic stages using mid-infrared spectroscopy. In another approach, efforts are being made to utilize plant volatile organic compounds (VOCs) or biomarkers that indicate the presence of HLB infection. Measurements of VOCs using gas chromatograph/mass spectrometer were made for healthy and HLB-infected plants of two citrus varieties (Hamlin and Valencia) in green house. Using the VOC profiles, we were able to identify the disease and control plants for each variety. Some of the potential biomarkers representing the disease were also identified. Further, based on the leave-one-out validating strategy, predictive models developed by combining auto-regression as feature extraction approach and principal component regression yielded an accuracy that ranged from 60 to 100%, which holds great promise for VOC based detection of plant pathogens. The overall accuracy of the predictive model was limited by the variation in sampling conditions over time which also resulted in plant aging. Efforts are ongoing to further improve accuracy by reducing the variability in the samples. Several manuscripts have been prepared for journal publication. The titles of some of the manuscripts are: Visible-near infrared spectroscopy for detection of Huanglongbing in citrus orchards, Mid-infrared spectroscopy for detection of Huanglongbing (greening) in citrus leaves, and Volatile emissions from citrus plants change after infection with Candidatus Liberibacter asiaticus. The articles summarize the results on HLB detection using visible-near infrared spectroscopy, mid-infrared spectroscopy, and VOCs profiling.
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