The Bacteria Associated With Rice Seed And Its Biological Coating Agent

Knowledge of microbial ecology on plant seeds is the basis for effective biological control. Development of seed associated antagonistic bacteria to suppress plant diseases is one of the best ways in biocontrol. At present study the bacteria isolated from rice seeds of south China were identified and the pathogenic or antagonistic potential ones were further studied. A biological seed coating agent against bacterial blight and bacterial leaf streak was developed by the antagonistic bacteria screened from rice seeds as major component after its serial tests including physical, chemical and storage properties, mechanisms, colonization of the antagonistic bacteria in rice plants.One hundred and seven rice seed samples were collected from south China and 428 bacterial strains were isolated. All the strains isolated were identified first by colony morphology, whole-cell fatty acid analysis, pathogenicity test on rice seedlings and antagonistic tests against major rice bacterial pathogens in vitro. The pathogenic and antagonic bacteria were further identified by 16SrDNA gene sequences. The bacteria isolated from rice seeds roughly consisted of 65.2%gram-negative and 38.2% gram-positive bacteria. It involved in 40 species of 20 bacterial genera. Predominant bacterial genera were identified as Pseudomonas, Pantoea, Sphingomonas, Microbacterium, Bacilllus and Paenibacillus. The pathogenicity tests showed that about 88% of total bacterial strains isolated had no any lesion after inoculation on rice seedlings. About 7% of the total strains induced remarkable symptoms on the inoculated rice seedlings, and were considered as true pathogens, including Acidovorax avenae subsp. avenae, Burkholderia glumae, Pseudomonas fuscovaginae, Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Another 5%of the strains were identified as Bacilllu pumilus, Microbacterium barkeri, Pantoea ananatis, Pantoea agglomerans, Paenibacillus polymyxa, Pseudomonas aeruginosa, Pseudomonas syringae syringae, Xanthomonas sp., respectively, which produced small lesions on rice seedings. About 12.4%of total strains showed in vitro antagonism against Xanthomonas oryzae pv. oryzicola, which involved in 14 species. Pseudomonas putida A N51 and Paennibacillus lentimorbus N72 were the 2 best strains showing good performance with larger inhibition zones, which were developed for biological control agents. In this study, Pantoa ananatis were first isolated from rice seeds in China with 32.7%isolation frequency as one of the dominant species. About 14.3%of P. ananatis induced disease symptoms on rice seedlings. P. ananatis is regarded as an unconventional plant pathogen occurring on previously unrecorded hosts in different parts of the world. It needs to be further studied.Paennibacillus lentimorbus N72 and Pseudomonas putida A N51 mentioned above were used as major components for the biological seed coating agents in which their physical and chemical properties were examined in the experiment. Its pH, viscosity, sieve analysis, suspension rate, coating uniformity, and other indicators were in line with national quality standards of suspended seed coating product. Biological seed coating agent N72 showed excellent physical and chemical properties and storability. In the field experiment it significantly enhanced plant growth, and exhibited 72.71%control effectiveness against bacterial leaf streak of rice. The colonization experiment showed that both N72 and N51 could be successfully inoculated onto rice seeds, and colonized in the rhizosphere, roots, stems, and leaves of rice during the seed germination, emergence and plant growth. N72-Rif and N51-Rif could be found in the plant 60 days after sowing, and population of both strains were more stable and survived longer in rice rhizosphere than those in stem and leaves, indicating a good ability in rhizosphere colonization is one of the mechanisms of the antagonism strains in biocontrol. It was found in the mechanism research of biological seed coating agent that the activities of major defensive enzyme POD and POO were increased in rice leaves, and PAL did not changed after rice seed coated with biological agent N72 and N51. After inoculation of X. oryzae pv. oryzicola activities of the 3 enzymes were first decreased and then increased, reaching a peak then declining. The enzyme activities by biological seed coating treatment were higher than those of the control, indicating that induced disease resistance was acquired by rice plant after seed coating treatment. The biocontrol agent N72 used as major componet of biological seed coating agent were identified as P. lentimorbus which was no any report in China. The biological seed coating agent N72 have been applied for China innovation patent (Patent Name:A Paennibacillus lentimorbus strain and its seed coating agent; Patent Application Number: 200910154899.1).