Gray blight disease, caused by Pestalotiopsis-like fungi, is one of the deadliest threats to tea (Camellia sinensis) production. However, little information is known about the traits and characteristics of this pathogen. Here, a systematic survey was performed, and a total of 20 representative isolates were obtained from the leaves of tea plants affected by gray blight in two main tea plantations located in Anhui Province, China. Further analyses showed that two isolates were identified as Neopestalotiopsis ellipsospora, three isolates were regarded as Pseudopestalotiopsis chinensis, one isolate was considered as Pseudopestalotiopsis camelliae-sinensis, and the remaining isolates belonged to Pseudopestalotiopsis spp., on the basis of morphological characteristics and multigene phylogenetic analyses of the internal transcribed spacer, β-tubulin, and translation elongation factor 1-α. Pathogenicity tests indicated that there were significant differences in virulence among the Neopestalotiopsis and Pseudopestalotiopsis isolates when inoculated on the leaves of the tea plant (C. sinensis ‘Shuchazao’). Furthermore, varied pathogenicity was also observed for the same isolate when inoculated on different varieties of tea plants. To our knowledge, this is the first record of Neopestalotiopsis ellipsospora and Pseudopestalotiopsis chinensis causing gray blight disease of tea plants in China.

The type VI secretion system (T6SS), a macromolecular machine, plays an important role in the pathogenicity of many Gram-negative bacteria. However, the role of T6SS in the pathogenicity of Pseudomonas syringae pv. actinidiae (Psa), the pathogen of kiwifruit bacterial canker, is yet to be studied. Here, we found a T6SS gene cluster consisting of 13 core genes (A-J) in the genome of Psa M228 based on a genome-wide analysis. To determine whether the T6SS gene cluster affects the pathogenicity of Psa M228, T6SS and its 13 core gene deletion mutants were constructed and their pathogenicity was determined. The deletion mutants showed different degrees of reduction in pathogenicity compared with the wild-type strain M228; in tssM and tssJ mutants, pathogenicity was significantly reduced by 78.7 and 71.3%, respectively. The pathogenicity results were also confirmed by electron microscopy. To further confirm that the reduction in pathogenicity is related to the function of T6SS, we selected the T6SS gene cluster, comprising tssM and tssJ, for further analyses. Western blot results revealed that tssM and tssJ were necessary for hemolytic co-regulatory protein secretion, indicating that they encode a functional T6SS. Further, we explored the mechanism by which T6SS affects the pathogenicity of Psa M228. The ability of bacterial competition, biofilm formation, hydrogen peroxide tolerance, and proteolytic activity were all weakened in the deletion mutants M228ΔT6SS, M228ΔtssM, and M228ΔtssJ. All these properties of the two gene complementation mutants were restored to the same levels as those of the wild-type strain, M228. Quantitative real-time results showed that during the interaction between the deletion mutant M228ΔT6SS and the host, expression levels of T3SS transcriptional regulatory gene hrpR, structural genes hrpZ, hrcC, hopP1, and effector genes hopH1 and hopM1 were down-regulated at different levels. Taken together, our data provide evidence for the first time that the T6SS plays an important role in the pathogenicity of Psa, probably via effects on bacterial competition, biofilm formation, and environmental adaptability. Moreover, a complicated relationship exists between T6SS and T3SS.

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During warm and humid periods in the winters from 2005 to 2008, head rot symptoms on broccoli (cv. Sijilv) (Brassica oleracea L. var italica Planch) were observed in commercial fields in Ningbo, Zhejiang Province, China. In agreement with the report of Cui and Harling (1), water-soaked lesions developed on the buds and then progressed into a brown-black soft rot. Longitudinal sections of the symptomatic inflorescences showed brown discoloration and rotting of the internal tissues. Broccoli production is hampered by the disease, with disease incidence ranging from 65 to 81%. Bacteria were isolated by streaking on nutrient agar (3) and individual colonies formed after 2 to 3 days of incubation at 28°C. Fifteen of thirty isolates induced hypersensitive reactions (HR) on tobacco leaves (Nicotiana tabacum cv. Samsun) within 48 h. All the HR-positive strains were fluorescent on King's medium B and the colonies were smooth, convex, entire, and round. Classical bacteriological tests indicated that the fluorescent strains were gram negative, obligate aerobes, arginine dihydrolase positive, and oxidase positive. Also, the fluorescent strains were positive for the production of levan from sucrose. Five representative strains were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc., Newark, DE) with the aerobic bacterial library (TSBA50). The five strains were identified as Pseudomonas fluorescens with Biolog and FAME similarity indexes of 0.61 to 0.68 and 0.52 to 0.58, respectively. The 16S rRNA gene sequence of broccoli strain PFB-01 (GenBank Accession No. GQ352649) was determined according to Li et al. (2). A subsequent GenBank search showed that this sequence had 98% nucleotide identity with the type strain of P. fluorescens (ATCC 17386T, GenBank Accession No. AF094726). Koch's postulates were completed by the inoculation of broccoli heads (cv. Sijilv) with cell suspensions (107 CFU/ml) of the above five strains by spraying on the surface of subcorymbs. Each treatment had five replicates. All strains induced head rot symptoms similar to those observed in natural infections. No symptoms were noted on the control plants inoculated with sterile water. Bacteria were successfully reisolated from symptomatic heads and confirmed by the cellular fatty acid composition. To our knowledge, this is the first report in China that P. fluorescens is the causal pathogen of bacterial head rot of broccoli. References: (1) X. Cui and R. Harling. Phytopathology 96:408, 2006. (2) B. Li et al. J. Phytopathol. 154:711, 2006. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

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