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Tian Y.,Nanjing Agricultural University | Zhao Y.,Shanghai Agricultural Technology Extension and Service Center | Xu R.,Nanjing Agricultural University | Liu F.,Nanjing Agricultural University | And 3 more authors.
Phytopathology | Year: 2014

Based on 16S-23S internal transcribed spacer ribosomal DNA sequence data, two padlock probes (PLPs), P-Xoo and P-Xoc, were designed and tested to detect Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively. These PLPs were combined with dot-blot hybridization to detect X. oryzae pv. oryzae and X. oryzae pv. oryzicola individually in rice seed. Using this technique, a detection sensitivity of 1 pg of X. oryzae pv. oryzae genomic DNA was observed. The technique also facilitated the detection of X. oryzae pv. oryzae in rice seedlots with 2% artificially infested seed. With regards to X. oryzae pv. oryzicola a detection threshold of 1 pg genomic DNA was observed and the pathogen was successful detected in rice seedlots with 0.2% artificially infested seed. The PLP assays detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 39.3% (13 of 33) and 21.3% (10 of 47) of naturally infested commercial rice seedlots, respectively. In contrast, conventional polymerase chain reaction using OSF1/OSR1 and XoocF/XoocR primers sets detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 9.1% (3 of 33) and 8.5% (4 of 47) of the same rice seedlots, respectively. We also detected both pathogens simultaneously in two seedlots, which successfully proved that PLPs (P-Xoo and P-Xoc) combined with reverse dotblot hybridization can be used to simultaneously detect multiple pathogens in naturally infested commercial rice seedlots. This approach has the potential to be an important tool for detecting multiple pathogens in seed and thereby preventing the spread of important pathogens. © 2014 The American Phytopathological Society. Source

Tian Y.,Nanjing Agricultural University | Zhao Y.,Shanghai Agricultural Technology Extension and Service Center | Wu X.,Nanjing Agricultural University | Liu F.,Nanjing Agricultural University | And 3 more authors.
Molecular Plant Pathology | Year: 2015

Summary: The type VI protein secretion system (T6SS) is essential for the virulence of several Gram-negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant-pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25kb in length and comprising 17 genes, was found in the A.citrulliAAC00-1 genome. Seventeen A.citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed-to-seedling transmission between wild-type A.citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed-to-seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed-to-seedling transmission of BFB on melon. Molecular Plant Pathology © 2015 BSPP and John Wiley & Sons Ltd. Source

Tian Y.,Nanjing Agricultural University | Zhao Y.,Shanghai Agricultural Technology Extension and Service Center | Bai S.,Nanjing Agricultural University | Walcott R.R.,University of Georgia | And 2 more authors.
Plant Disease | Year: 2013

A method was developed using a padlock probe (PLP) and dot-blot hybridization for detecting Acidovorax citrulli in cucurbit seed. The PLP was designed based on the 16S-23S internal transcribed spacer ribosomal DNA sequence from A. citrulli. The detection threshold for the PLP assay was 100 fg of genomic DNA, and A. citrulli was detected in 100% of artificially infested seedlots with 0.1% infestation or greater. In addition, using the PLP assay, 4 of 8 melon seedlots collected from Xinjang province and 15 of 47 watermelon seedlots collected from Ningxia province were positive for A. citrulli. In contrast, a conventional polymerase chain reaction (PCR) assay that relied on primers WFB1 and WFB2 facilitated A. citrulli detection in 1 of 8 and 5 of 47 seedlots from Xinjiang and Ningxia provinces, respectively. These data indicate that the PLP and dot-blot hybridization technique was more effective than conventional PCR for seed health testing. © 2013 The American Phytopathological Society. Source

Li L.,Shanghai JiaoTong University | Tian L.,Shanghai JiaoTong University | Wang T.-T.,Shanghai JiaoTong University | Jiang Q.-G.,Shanghai Qingpu District Agricultural Technology Extension and Service Center | And 5 more authors.
Zhiwu Shengli Xuebao/Plant Physiology Journal | Year: 2012

Waxy (Wx) gene, which is a key factor in regulating amylose content (AC), plays an important role in determining rice eating and cooking quality (ECQ). In this study, for further exploiting the underlying relationship and regulatory mechanism between the AC and ECQ, the Wx polymorphism was characterized in the high-quality rice varieties 'Qingxiangruanjing' and 'Nanjing46' with 8.7% and 10.2% AC respectively. Although all of the varieties we analyzed belong to the Wx-II japonica cultivars, the AC is different among them. Expression analysis of Wx gene in 'Qingxiangruanjing' and 'Nanjing46' indicates that their expression patterns are different, which might be correlated to the nucleotide polymorphism at nucleotide position -1773 from the start codon in 'Nanjing46' variety, and the AA/CT nucleotides locate in the transcription regulatory area. While the Arg158/His158 polymorphism in both 'Qingxiangruanjing' and 'Nanjing46' varieties might have sophisticated effect on AC since the amino acid is located in the pocket area of the Wx protein, which may change the catalytic activity of the starch synthase for substrate and affect the AC in rice, and this mutation site also have been reported to be the one of characterizations of the Wxmq gene. Therefore, our study enriches the research of Wx genetic polymorphism, and provides basis for further validating the relationship between rice AC and Wx gene polymorphism. Source

Lu Y.,Shanghai JiaoTong University | Cui X.,Shanghai JiaoTong University | Li R.,Shanghai JiaoTong University | Huang P.,Shanghai JiaoTong University | And 5 more authors.
Journal of Integrative Plant Biology | Year: 2015

DNA markers play important roles in plant breeding and genetics. The Insertion/Deletion (InDel) marker is one kind of co-dominant DNA markers widely used due to its low cost and high precision. However, the canonical way of searching for InDel markers is time-consuming and labor-intensive. We developed an end-to-end computational solution (InDel Markers Development Platform, IMDP) to identify genome-wide InDel markers under a graphic pipeline environment. IMDP constitutes assembled genome sequences alignment pipeline (AGA-pipe) and next-generation re-sequencing data mapping pipeline (NGS-pipe). With AGA-pipe we are able to identify 12,944 markers between the genome of rice cultivars Nipponbare and 93-11. Using NGS-pipe, we reported 34,794 InDels from re-sequencing data of rice cultivars Wu-Yun-Geng7 and Guang-Lu-Ai4. Combining AGA-pipe and NGS-pipe, we developed 205,659 InDels in eight japonica and nine indica cultivars and 2,681 InDels showed a subgroup-specific pattern. Polymerase chain reaction (PCR) analysis of subgroup-specific markers indicated that the precision reached 90% (86 of 95). Finally, to make them available to the public, we have integrated the InDels/markers information into a website (Rice InDel Marker Database, RIMD, The application of IMDP in rice will facilitate efficiency for development of genome-wide InDel markers, in addition it can be used in other species with reference genome sequences and NGS data. © 2015 Institute of Botany, Chinese Academy of Sciences. Source

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