Yanli Tian

The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon.

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 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00-1 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.

Dickeyafangzhongdai sp. nov., a plant-pathogenic bacterium isolated from pear trees (Pyrus pyrifolia).

Gram-stain-negative, pectinolytic bacteria were repeatedly isolated from pear trees displaying symptoms of bleeding canker in China. Three strains, JS5T, LN1 and QZH3, had identical 16S rRNA gene sequences that shared 99 % similarity to the type strain of Dickeya dadantii. Phylogenetic analysis of strains JS5T, LN1 and QZH3 with isolates representing all species of the genus Dickeya and related Pectobacterium species supported their affiliation to Dickeya. Multi-locus sequence typing employing concatenated sequences encoding recA, fusA, gapA, purA, rplB, dnaX and the intergenic spacer illustrated a phylogeny which placed strains JS5T, LN1 and QZH3 as a distinct clade, separate from all other species of the genus Dickeya. Average nucleotide identity values obtained in comparison with all species of the genus Dickeya supported the distinctiveness of strain JS5T within the genus Dickeya. Additionally, all three strains were phenotypically distinguished from other species of the genus Dickeya by failing to hydrolyse casein, and by producing acids from (-)-d-arabinose, (+)melibiose, (+)raffinose, mannitol and myo-inositol, but not from 5-keto-d-gluconate or β-gentiobiose. The name Dickeya fangzhongdai sp. nov. is proposed to accommodate these strains; the type strain is JS5T (=CGMCC 1.15464T=DSM 101947T).

Simultaneous Detection of Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola in Rice Seed Using a Padlock Probe-Based Assay

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.

Type VI secretion systems of Erwinia amylovora contribute to bacterial competition, virulence, and exopolysaccharide production

The type VI secretion system (T6SS) plays a major role in mediating interbacterial competition and might contribute to virulence in plant pathogenic bacteria. However, the role of T6SS in Erwinia amylovora remains unknown. In this study, 33 deletion mutants within three T6SS clusters were generated in E. amylovora strain NCPPB1665. Our results showed that all 33 mutants displayed reduced antibacterial activities against Escherichia coli as compared with that of the wild-type (WT) strain, indicating that Erwinia amylovora T6SS are functional. Of the 33 mutants, 19 exhibited reduced virulence on immature pear fruit as compared with that of the WT strain. Among them, 6, 1, and 12 genes belonged to T6SS-1, T6SS-2, and T6SS-3 clusters, respectively. Interestingly, these 19 mutants also produced less amylovoran or levan or both. These findings suggest that E. amylovora T6SS play a role in bacterial competition and virulence possibly by influencing exopolysaccharide production.

Reliable and Sensitive Detection of Acidovorax citrulli in Cucurbit Seed Using a Padlock-Probe-Based Assay

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.

First Report of Bacterial Leaf Blight of Tea (Camellia sinensis) Caused by Acidovorax avenae in China

Tea (Camellia sinensis L.) is an economically important crop widely cultivated for leaves in China. Leaf and young shoot blights were observed on some commercial gardens of tea (Camellia sinensis, cv. Yinghong IX) in Guangzhou in April from 2013 to 2015, China. The disease occurred initially on the young leaves producing water-soaked spots. When the disease further developed, the spots merged together into large patches and killed the leaf blade. The symptoms developed on the young shoots about two weeks later. Disease incidences ranged from 20 to 30% in the affected fields. Signs of bacterial streaming from all infected leaves were observed under microscope, and no fungal mycelium or spore was found from the diseased areas. Bacterial isolates were obtained by surface-sterilizing small fragments (5 × 5 mm) of symptomatic leaf tissues in 0.5% NaOCl, rinsing the sections in sterilized water twice, and streaking on Luria-Bertani (LB) plates. Bacterial colonies were convex, beige-tan colored, round and non-mu...

First report of Pectobacterium aroidearum causing soft rot of Chinese cabbage in China.

In June-October, 2015, we surveyed Chinese cabbage (Brassica rapa subsp. pekinensis) exhibiting soft rot symptoms in commercial field in Beijing, China. The lesions initiated at petiole base, expanded and rapidly covered entire plant. Analysis of 30 isolates from infected plants revealed that one of them, KC20, was an isolate of P. aroidearum, while the other 29 isolates were the common Chinese cabbage pathogens P. carotovorum. Bacterial strain KC20 was tested for pathogenicity by dropping a bacterial suspension (10 μL/inoculation site, 2×108 CFU/mL) onto sterilized Chinese cabbage petioles surface with sterile distilled water as control. Water-soaked lesions appeared on inoculated petioles in 24 h after incubation in growth chamber at 28 °C and strains were re-isolated successfully from symptomatic Chinese cabbages to complete Koch’s postulates. In a PCR assay, strain KC20 produced a 434 bp product with pel gene-specific primers Y1/Y2 for Pectobacterium spp. (Darrasse et al. 1994). Analysis of 16S-23S in...

Evidence for a Novel Phylotype of Pseudomonas syringae Causing Bacterial Leaf Blight of Cantaloupe in China

Bacterial leaf blight (BLB) has caused severe yield losses in cantaloupe (Cucumis melo L.) in the major melon-growing regions of China since the beginning of the twentieth century. Historically, Pseudomonas syringae pv. lachrymans was considered to be the causal agent of BLB of cantaloupe and angular leaf spot of cucumber. In the process of characterizing bacteria isolated from cantaloupe, we observed that putative P. syringae pv. lachrymans yielded negative results in P. syringae pv. lachrymans-specific PCR assays. This suggested that the P. syringae pv. lachrymans-like strains from cantaloupe were distinct from those recovered from cucumber. To investigate the differences between P. syringae pv. lachrymans-like strains isolated from cantaloupe and cucumber, 13 P. syringae strains isolated from cantaloupe [12 from China and 1 from Zimbabwe (NCPPB2916)] and 7 additional P. syringae reference strains were analyzed by catabolic profiling, phylogenetic analysis by multilocus sequence analysis (MLSA) and pathogenicity tests on cantaloupe leaflets. Catabolic profiling and MLSA based on 10 housekeeping genes and 2 hypersensitive response and pathogenicity (hrp) genes allowed us to differentiate strains isolated from cantaloupe and cucumber. Pseudomonas syringae pv. lachrymans strains isolated from cucumber clustered with genomospecies 2, and 13 P. syringae strains isolated from cantaloupe belonged to genomospecies 1. While all cantaloupe strains were closely related to P. syringae pv. aptata, they could be differentiated from this pathovar based on metabolic tests and MLSA. Pathogenicity tests showed that all strains isolated from cantaloupe and cucumber were only pathogenic on their original hosts. Based on these observations we conclude that P. syringae pv. lachrymans strains recovered from cantaloupe in China represent a novel phylotype.

First Report of Seedling Blight of Watermelon Caused by Acidovorax citrulli Transmitted from Rootstock of Pumpkin in China

Bacterial fruit blotch (BFB) is a devastating disease caused by Acidovorax citrulli, which was first observed in the United States in 1988 (3). A. citrulli can cause severe infection on a wide range of cucurbits, including watermelon, cantaloupe, and pumpkin. Cotyledon symptoms are brown, angular, necrotic spots or large necrotic lesions. The disease is seedborne, so seeds usually serve as the primary inoculum source for BFB outbreaks (2). In July 2012, seedling blight was observed by local farmers from Anhui province in China on watermelon seedlings grafted to pumpkin rootstocks; lesions were morphologically similar to those caused by A. citrulli. Presence of A. citrulli was detected in symptomatic samples by using species-specific primers BX-L1/BX-S-R2 (1). The seed company claimed seeds of watermelon (cv. Changfeng) were certified free of bacterial fruit blotch, but pumpkin seeds (cv. Kangkuxianfeng-1) had not been tested for A. citrulli. For investigating the inoculum source, the remaining seeds of watermelon (cv. Changfeng) and pumpkin (cv. Kangkuxianfeng-1) for seedling production were collected from the farmer and processed for pathogen extraction as described by Walcott and Gitaitis (2). Two microliters of seed wash was used as template for PCR using primers BX-L1/BX-S-R2 (1). The experiment was conducted three times. A 279-bp DNA fragment was consistently amplified by PCR from seed wash of pumpkin seeds, but not from the seed wash of watermelon seeds. Three Acidovorax-like strains (A1, A2, and A3) were isolated from pumpkin seed wash using TWZ semi-selective medium (0.5% peptone, 0.025% CaCl2, 1% Tween-80, 50 mg/liter berberine, 50 mg/liter cycloheximide, 50 mg/liter 2,3,5-triphenyltetrazolium chloride). PCR was performed on the 16S rDNA gene from isolate strain A1, A2, and A3 (1,492 bp; GenBank Accession Nos. JX875533, JX875534, and JX875535) with primers rp1/fd2 (4), and PCR products were sequenced. Results of sequence analysis showed the sequences of strains A1, A2, and A3 were 99% identical to that of the type strain of A. citrulli AAC00-1 (NC_008752). Pathogencity was confirmed by injection of pumpkin cotyledons with bacterial suspensions of each isolate. Collected pumpkin seeds (n = 100 seeds) and watermelon seeds (n = 100 seeds) were planted in plastic pots containing sterilized field soil at room temperature to detect A. citrulli by a wet chamber growing out test. Eight days later, brown, angular, necrotic spots or wilt developed in pumpkin seedlings, but no symptoms were noted on the watermelon seedlings. To our knowledge, this is the first report of A. citrulli causing watermelon seedling blight transmitted from pumpkin seeds by grafting in China. References: (1) O. Bahar et al. Plant Pathol. 57:754, 2008. (2) R. R. Walcott and R. D. Gitaitis. Plant Dis. 84:470, 2000. (3) G. C. Wall et al. Phytopathology 78:1605, 1988. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.