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Lou B.H.,Guangxi Citrus Research Institute | Song Y.Q.,Guangxi Citrus Research Institute | Deng C.L.,Guangxi Citrus Research Institute | Chen G.P.,Guangxi Citrus Research Institute
Journal of Plant Pathology | Year: 2012

"Candidatus Liberibacter asiaticus" (Las), one of the three known bacterial species associated with citrus Huanglongbing (HLB), is unevenly distributed in bark tissue, leaf midrib, roots, and different floral parts of infected citrus trees. In this study, 10 petal, 12 stamen, 12 pistil, 3 pollen, 20 seed hull, 20 seed coat and 20 endosperm samples were collected from HLB-infected Shatianyou pummelo trees [= Citrus maxima (Burm.) Merr. cv. Shatian Yu] in China and analyzed for the presence of Las. The bacterium was detected by conventional PCR using the Las-specific primer pair OI1/OI2c. Amplification products of the expected size (ca. 1100 bp) were obtained from 9 petal (90%), 11 stamen (91.7%), 12 pistil (100%), 2 pollen (66.7%), 18 seed coat (90%), 15 endosperm (75%) samples, but not from any of the seed hull samples. The sequence of a representative amplicon matched that of Las. To our knowledge, this is the first report of the presence of Las in pollen, seed coat and endosperm of pummelo in China.


Lou B.H.,Guangxi Citrus Research Institute | Deng C.L.,Guangxi Citrus Research Institute | Song Y.Q.,Guangxi Citrus Research Institute | Zhao X.L.,Guangxi Agricultural Vocational Technical College | And 2 more authors.
Journal of Plant Pathology | Year: 2013

In September 2010, erumpent, callus-like lesions with water- soaked margins, the typical citrus canker (CC) symptoms, were observed on leaves of Nanfeng tangerine [Citrus reticulata Blanco var. kinokuni (Tanaka) H.H. Hu] in Guangxi Province (China). Seven leaf samples of Nanfeng tangerine showing symptoms of CC were collected from three different groves. Pathogen isolations were done using Luria-Bertani medium, and yellow mucoid, Xanthomonas-like isolates were selected. Pathogenicity tests were carried out on Nanfeng tangerine, navel orange (C. sinensis), Mexican lime (C. aurantifolia), and Eureka lemon (C. limon). Four attached leaves of each one-year-old citrus cultivar were inoculated with each of seven isolated Xanthomonas-like strains and two previously identified X. citri subsp. citri pathotype A (Xcc-A) strains at 1 × 107 CFU/ml. After 21 days, all inoculated bacterial isolates induced clear-cut CC symptoms on each of the four tested citrus species and were successfully re-isolated from the symptomatic leaves of inoculated plants. The 16S rDNA and 16S/23S intergenic transcribed spacer (ITS) of the seven isolates were amplified with primer sets fD1/rP1 (1) and 1493f/23r (2), respectively, cloned and sequenced. Consensus sequences of 1469 bp 16S rDNA (accession No. KC820706) and 545 bp 16S/23S ITS (KC820707) were obtained. BLAST analysis showed both 16S rDNA and 16S/23S ITS from this study had the highest sequence identity (99.9% and 100%, respectively) with those of Xcc strain 306 (AE008923). These results confirmed that the pathogen inducing typical CC symptoms on Nanfeng tangerine was Xcc-A. To our knowledge, this is the first formal report of Xcc-A on Nanfeng tangerine in China.


Lin H.,San Joaquin Valley Agricultural science Center | Lou B.,San Joaquin Valley Agricultural science Center | Lou B.,Guangxi Citrus Research Institute | Glynn J.M.,San Joaquin Valley Agricultural science Center | And 6 more authors.
PLoS ONE | Year: 2011

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with 'Candidatus Liberibacter solanacearum', a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for 'Ca. L. solanacearum'. Here we present the sequence of the 1.26 Mbp metagenome of 'Ca. L. solanacearum', based on DNA isolated from potato psyllids. The coding inventory of the 'Ca. L. solanacearum' genome was analyzed and compared to related Rhizobiaceae to better understand 'Ca. L. solanacearum' physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, 'Ca. L. solanacearum' is related to 'Ca. L. asiaticus', a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to 'Ca. L. asiaticus', 'Ca. L. solanacearum' probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes.


Lin H.,U.S. Department of Agriculture | Chen C.,Guangxi Citrus Research Institute | Doddapaneni H.,University of Iowa | Duan Y.,U.S. Department of Agriculture | And 3 more authors.
Journal of Microbiological Methods | Year: 2010

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan® PCR in a single tube was developed for detection of "Candidatus Liberibacter asiaticus". The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan® is high because the fluorescent signal can only be generated from the TaqMan® probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan® PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.


Islam M.-S.,U.S. Department of Agriculture | Glynn J.M.,U.S. Department of Agriculture | Bai Y.,Guangxi Citrus Research Institute | Duan Y.-P.,U.S. Department of Agriculture | And 4 more authors.
BMC Microbiology | Year: 2012

Background: Huanglongbing (HLB) is one of the most destructive citrus diseases in the world. The disease is associated with the presence of a fastidious, phloem-limited - proteobacterium, 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus'. HLB-associated Liberibacters have spread to North America and South America in recent years. While the causal agents of HLB have been putatively identified, information regarding the worldwide population structure and epidemiological relationships for 'Ca. L. asiaticus' is limited. The availability of the 'Ca. L. asiaticus' genome sequence has facilitated development of molecular markers from this bacterium. The objectives of this study were to develop microsatellite markers and conduct genetic analyses of 'Ca. L. asiaticus' from a worldwide collection. Two hundred eighty seven isolates from USA (Florida), Brazil, China, India, Cambodia, Vietnam, Taiwan, Thailand, and Japan were analyzed. Results: A panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide. Conclusions: Our results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'. © 2012 Islam et al; licensee BioMed Central Ltd.


Lin H.,U.S. Department of Agriculture | Islam M.S.,U.S. Department of Agriculture | Bai Y.,U.S. Department of Agriculture | Bai Y.,Guangxi Citrus Research Institute | And 5 more authors.
European Journal of Plant Pathology | Year: 2012

'Candidatus Liberibacter solanacearum' is associated with the Zebra Chip (ZC) disorder of potatoes. A panel of eight simple sequence repeat (SSR) markers was developed and used to genetically characterize 'Ca. L. solanacearum' strains obtained from ZC-affected potato plants in the United States and Mexico. The multilocus SSR markers in this study effectively differentiated genotypes and estimated genetic diversity of 'Ca. L. solanacearum' strains. Genotype assignment analyses identified two major lineages of 'Ca. L. solanacearum' in the North American populations while only one lineage type was identified in Mexican population. No clear genetic structure was found among haplotypes based on geographical proximity or host. The high resolution power of the SSR marker system developed in this study provides a useful tool for genotyping closely related strains and tracking sources of the pathogen. Genotype information combined with epidemiological data will advance knowledge of ZC disease and will facilitate development of effective disease management. © 2011 KNPV.


Lou B.,Guangxi Citrus Research Institute | Lou B.,Southwest University | Bai X.,Guangxi Academy of Agricultural science | Bai Y.,Guangxi Citrus Research Institute | And 4 more authors.
Journal of Phytopathology | Year: 2014

In the year 2010, in a survey in Guangxi Province, China, to detect and characterize phytoplasmas in a huanglongbing (HLB)-infected grapefruit (Citrus paradisi) orchard, 87 leaf samples with symptoms of blotchy mottle were collected from symptomatic grapefruit trees, and 320 leaf samples from symptomless trees adjacent to the symptomatic trees. Nested polymerase chain reaction (PCR) using universal phytoplasma primer set P1/P7 followed by primer set fU5/rU3 identified 7 (8.0%) positive samples from symptomatic samples but none from symptomless samples. Of the 87 symptomatic samples, 77 (88.5%) were positive for 'Candidatus Liberibacter asiaticus' and 5 for both phytoplasma and 'Ca. L. asiaticus'. Sequence analysis indicated that seven 881-bp amplicons, amplified by nested phytoplasma primer sets P1/P7 and fU5/rU3, shared 100.0% sequence identity with each other. Genome walking was then performed based on the 881 bp known sequences, and 5111 bp of upstream and downstream sequences were obtained. The total 5992 bp sequences contained a complete rRNA operon, composed of a 16S rRNA gene, a tRNAIle gene, a 23S rRNA gene and a 5S rRNA gene followed by eight tRNA genes. Phylogenetic analysis and virtual restriction fragment length polymorphism analysis confirmed the phytoplasma was a variant (16SrII-A*) of phytoplasma subgroup 16SrII-A. As phytoplasmas were only detected in blotchy-mottle leaves, the 16SrII-A* phytoplasma identified was related to HLB-like symptoms. © 2013 Blackwell Verlag GmbH.

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