First Report of Rice Seedling Blight Caused by Burkholderia plantarii in North and Southeast China.

Burkholderia plantarii is the causal agent of rice seedling blight. It was first discovered in a rice nursery box in Chiba Prefecture, Japan, in 1985 (Azegami et al. 1985). B. plantarii produces the virulence factor tropolone, which causes seedling blights such as chlorosis, stunting, and root growth-inhibition (Wang et al. 2013). Since 1954, efforts have been made to monitor rice pathogens in China (Xie et al. 2002). However, B. plantarii has not been included in the quarantined organisms of China and introduction of this pathogen has not been monitored due to no use nursery box for the seedling production (Wang et al. 2014). To determine whether B. plantarii is present in China, 797 samples, including rice seedlings, rhizosphere soil, paddy water, and rice florets, were collected from 2013 to 2015 from four rice-production regions: Heilongjiang, Zhejiang, Fujian, and Hunan Provinces. Through GC-MS/MS analysis (Agilent 7000C), tropolone production was detected in 21 samples, which were further subjected to incubation in a tropolone-supplemented (100 ppm) potato dextrose (PD) agar plate at 25°C for 72 h. The 17 tropolone-tolerant bacterial strains obtained were subsequently shake-cultured at 25°C for 36 h in PD broth. GC-MS/MS analysis found that 3 strains, including ZJ17, FJ066, and HLJ338, produced tropolone (36.3 to 86.7 ppm). These strains were oxidase-positive (N,N-dimethyl-p-phenylenediamine assay), nonspore forming, nonencapsulated straight rods, motile with 1 to 7 polar flagella, and measured 0.7 to 1.0 μm × 1.5 to 2.0 μm. Cells occurred singly, in pairs, or in short chains. Genomic DNA was extracted from each strain to amplify the 1.5-kbp 16S rRNA gene using the primer-pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). Sequences were deposited in DNA Data Bank of Japan (Accession Nos. LC020026, LC020027, and LC020028). Nucleotide BLAST showed that the sequences of 16S rRNA gene of the strains had high relative sequence identity, ranging from 99 to 100%, with several B. plantarii strains, such as NBRC 104886 (Accession Nos. AB682220), CIP 105769 (NR_116151), and 9411(AB183678). Phylogenetic analysis (Mega 5.05) showed that three strains clustered with B. plantarii strains. Surface-sterilized intact rice seeds (Oryza sativa ‘Xiushui 09’) were inoculated with cell suspensions of ZJ171, FJ066, and HLJ338 at 103 CFU/ml (sterilized water for the control), and incubated at 25°C for 2 days until germination. They were then transplanted into Hoagland’s No. 2 medium. Unlike the controls, the treated rice seedlings showed chlorosis, stunting, and root growth-inhibition after 5 days of growth (25°C, 12-h photoperiod). Tropolone production was detected in the roots of treated rice seedlings, from which B. plantarii was reisolated and verified by the sequence of the 16S rRNA gene. Hence, ZJ171, FJ066, and HLJ338 were identified as B. plantarii using their metabolic, biochemical, morphological, molecular, and pathogenic properties, as described above. This is the first report of B. plantarii being isolated from a rice paddy in China, which broadens the geographical area of B. plantarii and suggests that B. plantarii has already spread to China. As in Japan, greenhouses have been adopted for the transplantation and production of rice seedlings in many rice production areas in China (Gu et al. 2011). The relatively high humidity and temperature in these greenhouses may foster outbreaks of seedling blight. © 2016, American Phytopathological Society. All rights reserved.