CLas Bacteria


Pre-Grading Fresh Citrus for Canker Prior to Dumping on the Main Packingline

Report Date: 04/15/2011   Project: 70555

Pre-Grading Fresh Citrus for Canker Prior to Dumping on the Main Packingline

Report Date: 04/15/2011
Project: 70555
Category: CLas Bacteria
Author: Mark Ritenour
Sponsor: Citrus Research and Development Foundation

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.



Copper loaded silica nanogel technology for long term prevention of citrus canker disease

Report Date: 04/15/2011   Project: 328

Copper loaded silica nanogel technology for long term prevention of citrus canker disease

Report Date: 04/15/2011
Project: 328
Category: CLas Bacteria
Author: Swadeshmukul Santra
Sponsor: Citrus Research and Development Foundation

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

Report Date: 04/15/2011   Project: 170

Diagnostic service for growers for detection of Huanglongbing to aid in management decisions

Report Date: 04/15/2011
Project: 170
Category: CLas Bacteria
Author: Pamela Roberts
Sponsor: Citrus Research and Development Foundation

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.



Dissecting The Disease Complex of Citrus Huanglongbing in Florida

Report Date: 04/14/2011   Project: Duan-162

Dissecting The Disease Complex of Citrus Huanglongbing in Florida

Report Date: 04/14/2011
Project: Duan-162
Category: CLas Bacteria
Author: Yongping Duan
Sponsor: Citrus Research and Development Foundation

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.



Dissecting The Disease Complex of Citrus Huanglongbing in Florida

Report Date: 04/14/2011   Project: Duan-162

Dissecting The Disease Complex of Citrus Huanglongbing in Florida

Report Date: 04/14/2011
Project: Duan-162
Category: CLas Bacteria
Author: Yongping Duan
Sponsor: Citrus Research and Development Foundation

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.



Characterization of a putative insect-transmission determinant/virulence gene (hypI) of Candidatus Liberibacter asiaticus(Las)

Report Date: 04/14/2011   Project: Duan-310

Characterization of a putative insect-transmission determinant/virulence gene (hypI) of Candidatus Liberibacter asiaticus(Las)

Report Date: 04/14/2011
Project: Duan-310
Category: CLas Bacteria
Author: Yongping Duan
Sponsor: Citrus Research and Development Foundation

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.



Development of SSR markers for detection, genotyping, phenotyping and genetic diversity assessment of Candidatus Liberibacter strains in Florida

Report Date: 04/13/2011   Project: 125

Development of SSR markers for detection, genotyping, phenotyping and genetic diversity assessment of Candidatus Liberibacter strains in Florida

Report Date: 04/13/2011
Project: 125
Category: CLas Bacteria
Author: Hong Lin
Sponsor: Citrus Research and Development Foundation

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.



Detecting citrus greening (HLB) using multiple sensors and sensor fusion approach

Report Date: 04/07/2011   Project: 57

Detecting citrus greening (HLB) using multiple sensors and sensor fusion approach

Report Date: 04/07/2011
Project: 57
Category: CLas Bacteria
Author: Reza Ehsani
Sponsor: Citrus Research and Development Foundation

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.



Culturing Liberibacter asiaticus

Report Date: 03/23/2011   Project: 77623

Culturing Liberibacter asiaticus

Report Date: 03/23/2011
Project: 77623
Category: CLas Bacteria
Author: Michael Davis
Sponsor: Citrus Research and Development Foundation

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.



Developing an Educational & Outreach Program for Increase Awareness of Citrus Diseases in Texas.

Report Date: 03/01/2011  

Developing an Educational & Outreach Program for Increase Awareness of Citrus Diseases in Texas.

Report Date: 03/01/2011
Category: CLas Bacteria
Author: Kevin Ong
Sponsor: Texas Citrus Producers Board

As of March 1, 2011, the following progress highlights are noted for this project: 1. Completed training of 65 Master Gardener Specialist in Citriculture (volunteers) in Citrus Greening survey sample collection and submission. MG have to complete exercise which include submitting at least 6 collected samples along with proper identifying information for the particular specimen. 2. Highlighted citrus diseases with particular emphasis of Citrus Greening regulation, survey and monitoring efforts in Texas to: a. Landscape & Nursery professionals (ornamental industry) – Tyler, TX [attendance =~150] b. General public – Citrus Enthusiast League – Galveston county, TX. [attendance =~70]



Genomic sequencing to closure of a curated Florida citrus greening strain of Candidatus Liberibacter asiaticus

Report Date: 02/20/2011   Project: 65

Genomic sequencing to closure of a curated Florida citrus greening strain of Candidatus Liberibacter asiaticus

Report Date: 02/20/2011
Project: 65
Category: CLas Bacteria
Author: Dean Gabriel
Sponsor: Citrus Research and Development Foundation

In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have now closed the circular genome of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. A draft circular genomic DNA sequence of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle (Vinca) in Brazil has been completed. The genome size is 1,195,167. The circular genome has been extensively confirmed by PCR. All weak assembly areas, predicted truncated or incomplete proteins and pseudogenes are currently being verified by PCR. The average GC content of Lam is 31.12%. So far, 1,071 genes have been called, with 1,015 encoding predicted proteins, and 56 encoding rRNA genes and 45 encoding tRNAs. Of the protein coding genes, 839 (78.34%) have a predicted function, while 176 (16.43%) have no predicted function. 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. As with both Liberibacter asiaticus (Las) and L. solanacearum (Lso), two prophage were found. As with Las, two circular forms were confirmed in Lam, with Lam-SC1 being 39,941 bp and Lam-SC2 being 16,398 bp in size. SC1 (and not SC2 as in Las) appears to replicate as an integrative plasmid prophage. This stable plasmid lysogen can be useful for lysogenic conversion (host range and pathogenicity enhancing), and indeed lysogenic conversion genes are found on these plasmid prophage—specificially peroxidases and adhesins. 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 a “suicide program” or lytic cycle genes). In the stable “lysogenic” state, the phage keeps its lytic genes dormant by its use of gene repressors. To ensure its own survival, however, phage can also carry anti-repressors that sense host cell stress and then activate the phage “lytic” cycle genes, thereby forming phage particles, killing the host cell and liberating infectious phage. Some phage hijack available host cell repressors, such as LexA, to maintain a stable lysogenic state by repressing phage antirepressors. Some phage also hijack available host cell anti-repressors to assist sensing of host stress, including host heat shock and other SOS responses. Externally applied chemicals, as well as heat shocks have been used in other bacterial systems to activate the SOS response and derepress various phage lytic cycle genes. Lam is heat sensitive and carries a unique cold shock gene; Las is missing this gene, is not heat sensitive, and encodes two likely lytic cycle repressors and four anti-repressors. Lam encodes only one putative repressor and a rearranged anti-repressor. Las and Lam both are missing LexA, found in Lso. Experiments designed to exploit these genomics findings and determine what stresses can activate the Las lytic response are currently underway.



Identification of Spiroplasma citri secreted proteins as detection markers for citrus stubborn disease

Report Date: 02/15/2011   Project: 5200-139

Identification of Spiroplasma citri secreted proteins as detection markers for citrus stubborn disease

Report Date: 02/15/2011
Project: 5200-139
Category: CLas Bacteria
Author: Wenbo Ma
Sponsor: Citrus Research and Development Foundation

The purpose of this project is to develop a robust ELISA-based detection method for the citrus stubborn disease. We used a comparative proteomic approach using mass spectrometry (MS) from which the proteins secreted from the causal agent, Spiroplasma citri, to citrus phloem were identified. The secreted protein profiles from S. citri cell cultures with or without the induction of citrus phloem extracts were determined by MS and several potential secreted proteins were identified. To facilitate with the analysis on the MS data, we obtained an additional funding from an UC Discovery Grant to fully sequence the genome of S. citri strain S616 using next-generation Illumina sequencing. Using these sequencing data, we were able to match most of the peptides from the MS analysis to the S. citri genome sequences and obtained the complete gene sequences. The deduced amino acid sequences of many of the identified proteins contain potential N-terminal secretion signals indicating that they are secreted in a Sec-dependent manner. Through these approaches, two suitable proteins (CAK99824 and CAK98563) were chosen for further detection marker development. CAK99824 and CAK98563 are being evaluated for their suitability as detection markers according to the following criteria: 1) high abundance and dispersal in phloem; 2) wide distribution in representative S. citri isolates. By comparing the genome sequences of S. citri strain S616 from our own sequencing and the available genome sequence of another S. citri strain, GII3, we confirmed that the selected protein candidates are present in both genomes with almost identical DNA sequences indicating that they are conserved genes in S. citri. In addition, none of these proteins have close homologs in other organisms according to a search in the Genbank database, suggesting that they are also unique for S. citri. These characteristics make them excellent detection markers for citrus stubborn disease. Polyclonal antibodies have been generated for CAK99824 and CAK98563, as well as for spiralin. Spiralin is a highly abundant membrane protein and will be used as a control for the presence of S. citri. Using Western blots, the antibodies were tested on S. citri cultures grown in the presence of citrus phloem extracts. CAK99824 and CAK98563 were present in both the cell pellets and the cell-free supernatants. Furthermore, both proteins were induced by the phloem extracts, which is evident by increased amounts in the supernatant of the induced samples. The antibodies are now being tested on samples from trees grafted with S. citri infected buds and from field trees confirmed to be infected with spiroplasma. Preliminary results indicate that the CAK98563 antibody is capable of detecting spiroplasma within the leaf tissue of the graft tree and within the phloem tissues dissected from the leaf, branch and fruit of the field trees. It has not been possible to detect CAK99824 using any citrus tissue. Further work is necessary to optimize the methods of extracting proteins from plant tissues and on the detection of the spiroplasma proteins.



An Integrated Low Cost Nucleic Acid Analysis Platform for the Rapid Detection of Plant Pathogens

Report Date: 02/12/2011   Project: 5300-141

An Integrated Low Cost Nucleic Acid Analysis Platform for the Rapid Detection of Plant Pathogens

Report Date: 02/12/2011
Project: 5300-141
Category: CLas Bacteria
Author: Bruce Cary
Sponsor: California Citrus Research Board

As concern regarding the spread of citrus greening or Huanglongbing (HLB) and other agricultural diseases has increased, the need for more accessible, easily used and rapid point-of-use diagnostic tests has become more critical. Molecular diagnostic tests (i.e. DNA and RNA-based assays) for citrus diseases potentially offer the most sensitive and specific means of detecting viral and bacterial infections, often capable of detecting the presence of a pathogenic agent prior to the overt exhibition of visually detectable symptoms. Unfortunately, existing molecular diagnostics rely on instrumentation dependent laboratory manipulations requiring highly trained technicians for execution. These constraints often limit the accessibility and utility of molecular methods due to the infrastructure, costs and complexities associated with their use. The reliance of molecular techniques on laboratory instrumentation and personnel must ultimately be eliminated to broaden the utility of such tests and enable their execution under field conditions. In past work, MTI developed a highly simplified means of purifying nucleic acids (NAs) from plant tissue and contaminating pathogens without human intervention or machine-based automation. The resulting technology enables the isolation of amplification ready NA in less than 15 minutes using a completely disposable device. To make use of this rapid and simple NA isolation technology to enable field deployable molecular diagnostics, we have focused our efforts in this period on the development of extremely low cost yet precise temperature control systems to support robust amplification of nucleic acids in a disposable test device. To support the thermal requirements of NA amplification reaction strategies within the context of a self-contained and disposable diagnostic device, we developed low cost microheaters capable of supporting rapid PCR (and reverse transcriptase PCR for RNA targets) as well as isothermal amplification methods. The design employs an inexpensive microcontroller to modulate temperatures in multiple low thermal mass reaction chambers interfaced to the microheater elements. The use of multiple independently controlled reaction chambers allows parallel amplification of multiple targets thus reducing the burden inherent to multiplexing a single amplification reaction while retaining the capacity to amplify multiple pathogen NA signatures in a single device. Detection of amplification reaction products can be accomplished in minutes using lateral flow microarrays, a rapid and inexpensive yet sensitive detection technology developed by the PI while at Los Alamos National Laboratory. The combination of instrumentation independent sample preparation technology with low cost battery operated microheater mediated amplification and lateral flow microarray-based detection offers a means to overcome the major hurdles facing field deployable molecular testing. We are now working to incorporate our innovative sample preparation, amplification and detection subsystems into a single low cost disposable device that is both easy to use and capable of providing molecular diagnostic data within one hour of sample introduction.



Investigating important diseases of citrus in California

Report Date: 02/11/2011   Project: 5300-140

Investigating important diseases of citrus in California

Report Date: 02/11/2011
Project: 5300-140
Category: CLas Bacteria
Author: Akif Eskalen
Sponsor: California Citrus Research Board

Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: Dothiorella gregaria was long believed to be the pathogen causing Dothiorella gummosis but recent data showed that this may not be valid. Our objectives were to (a) determine the Botryosphaeria species causing branch canker and dieback on citrus and their geographical distribution in California. In 2010, cankered branches were collected in six citrus growing counties in California, namely ‘ Fresno, Riverside, San Diego, San Luis Obispo, Tulare, and Ventura. The combination of morphology with three molecular markers (ITS, Beta Tubulin, and Elongation Factor) helped to distinguish among closely related isolates. Hence, 18 isolates were fully identified and tested indicating that multiple species of Botryospharia, including B.australis, B. iberica, B. lutea, B. species, B. parva, B. stevensii, B. viticola, and Neofusiccocum mediterraneum, were causing symptoms on citrus that were previously believed to be caused by Dothiorella gregaria (B. ribis). These multiple species were widely distributed within counties. To our knowledge, this is the first comprehensive study of Botryosphaeria gummosis on citrus. The alliance between Fusarium solani and other factors in citrus dry root rot: The main pathogen causing dry root rot (DRR) is Fusarium solani but its interactions with Phytophthora spp. and other biotic and abiotic factors is an unclear dynamic in DRR. We are studying the distribution, seasonal occurrence, and survival of F. solani and its interaction with P. nicotianae, P. citrophthora, other biotic (other Fusarium spp), and abiotic (soil) factors on citrus in California. Microorganism isolations were made from infected tree samples and soil were collected and analyzed across the state. About 60 Fusarium isolates and 18 Phytophthora isolates have been identified across the state. Detailed identification was done using molecular methods with 3 primers (ITS, BT, and TEF). Preliminary results indicate that in addition to F.solani and Phytophthora spp., F. proliferatum, and F. oxysporum are major organisms associated with DRR. Pathogenicity tests are currently being conducted for these specific isolates to be sure of their roles in DRR. Results will help towards better management of DRR. Understanding foamy bark rot of Fukumoto navel: Fukumoto navel orange has many desirable attributes but ‘foamy bark rot’ is causing tree decline. The objective of this study was to isolate fungal and bacterial pathogens associated with foamy bark rot. Several bacterial and fungal isolates have been collected from infected trees in Fresno, Kern, and Tulare counties and identified using molecular methods. Preliminary results support the hypothesis that foamy bark rot is initiated by scion-rootstock incompatibility, resulting in nutritional and/or physiological abnormalities, which provide opportunity for infection by certain pathogen(s) and foam production is possibly due to yeast activities. At a UC-Riverside greenhouse, Carrizo and Volkameriana rootstocks are almost ready for pathogenicity tests with the identified microorganisms. These experiments will fulfill the principle of Koch’s postulates and help to determine the pathogenicity and aggressiveness of the isolated organisms on Fukumoto navel. Findings will provide a baseline for the management of foamy bark rot.



Investigating important diseases of citrus in California

Report Date: 02/11/2011   Project: 5300-140

Investigating important diseases of citrus in California

Report Date: 02/11/2011
Project: 5300-140
Category: CLas Bacteria
Author: Akif Eskalen
Sponsor: California Citrus Research Board

Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: Dothiorella gregaria was long believed to be the pathogen causing Dothiorella gummosis but recent data showed that this may not be valid. Our objectives were to (a) determine the Botryosphaeria species causing branch canker and dieback on citrus and their geographical distribution in California. In 2010, cankered branches were collected in six citrus growing counties in California, namely ‘ Fresno, Riverside, San Diego, San Luis Obispo, Tulare, and Ventura. The combination of morphology with three molecular markers (ITS, Beta Tubulin, and Elongation Factor) helped to distinguish among closely related isolates. Hence, 18 isolates were fully identified and tested indicating that multiple species of Botryospharia, including B.australis, B. iberica, B. lutea, B. species, B. parva, B. stevensii, B. viticola, and Neofusiccocum mediterraneum, were causing symptoms on citrus that were previously believed to be caused by Dothiorella gregaria (B. ribis). These multiple species were widely distributed within counties. To our knowledge, this is the first comprehensive study of Botryosphaeria gummosis on citrus. The alliance between Fusarium solani and other factors in citrus dry root rot: The main pathogen causing dry root rot (DRR) is Fusarium solani but its interactions with Phytophthora spp. and other biotic and abiotic factors is an unclear dynamic in DRR. We are studying the distribution, seasonal occurrence, and survival of F. solani and its interaction with P. nicotianae, P. citrophthora, other biotic (other Fusarium spp), and abiotic (soil) factors on citrus in California. Microorganism isolations were made from infected tree samples and soil were collected and analyzed across the state. About 60 Fusarium isolates and 18 Phytophthora isolates have been identified across the state. Detailed identification was done using molecular methods with 3 primers (ITS, BT, and TEF). Preliminary results indicate that in addition to F.solani and Phytophthora spp., F. proliferatum, and F. oxysporum are major organisms associated with DRR. Pathogenicity tests are currently being conducted for these specific isolates to be sure of their roles in DRR. Results will help towards better management of DRR. Understanding foamy bark rot of Fukumoto navel: Fukumoto navel orange has many desirable attributes but ‘foamy bark rot’ is causing tree decline. The objective of this study was to isolate fungal and bacterial pathogens associated with foamy bark rot. Several bacterial and fungal isolates have been collected from infected trees in Fresno, Kern, and Tulare counties and identified using molecular methods. Preliminary results support the hypothesis that foamy bark rot is initiated by scion-rootstock incompatibility, resulting in nutritional and/or physiological abnormalities, which provide opportunity for infection by certain pathogen(s) and foam production is possibly due to yeast activities. At a UC-Riverside greenhouse, Carrizo and Volkameriana rootstocks are almost ready for pathogenicity tests with the identified microorganisms. These experiments will fulfill the principle of Koch’s postulates and help to determine the pathogenicity and aggressiveness of the isolated organisms on Fukumoto navel. Findings will provide a baseline for the management of foamy bark rot.