Faculty of Animal Sciences and Agricultural Technology, Silpakorn University, Phetchaburi IT campus, Cha-Am, Phetchaburi, Thailand

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1 Detection of heat-resistant endophytic Bacillus spp. associated with commercial and indigenous rice seeds in Thailand and its effect to rice seed germination and seedling growth Suebphankoy C 1, Sookanun P 1, Na Chiangmai P 1, Sawangsri P 2 and Kanjanamaneesathian M 1 1 Faculty of Animal Sciences and Agricultural Technology, Silpakorn University, Phetchaburi IT campus, Cha-Am, Phetchaburi, Thailand 2 Biotechnology Research and Development Office, Department of Agriculture, Patumthani,12110, Thailand

2 From 1st largest of rice exporter in the world, Thailand drop to 3rd

3 As if drought, flooding and insect infestations were not enough, Thailand's rice crop is now threatened by the risk of a fungus and bacteria spreading and wiping out crops. Seedling rot disease in nursery box (Curvularia lunata (Wakk.) Board., Helminthosporium oryzae Breda de haan) Brown spot disease (Helminthosporium oryzae Breda de Haan. orbipolaris oryzae (Brada de Haan) Shoemaker

4 The previous solutions to solve all of rice problems are enough? - Use resistant cultivars - Chemical pesticides or fungicides - Soil improvement - Soil drying - Rotation cropping with other plant species - Use fertilizer - Weed control - Bio-control Golden apple snail Although rice production has many problems induced to the reduction on yield, Thailand is one of many countries that found on rice genetic variation for breeding and research to find some advantages.

5 What is the situation of indigenous rice cultivars in Thailand? Various indigenous rice cultivars which have been cultivated by the Thai farmers in small areas all over the country, where water for irrigation is scarce. An adaptation of indigenous rice cultivars to specific ecology may be induced by some factors. Various beneficial microbes which play a role in sustaining growth and development of rice. And, bacteria inoculants were utilized in increasing rice productivity. Source: Kowalski (2013) [online]. Available: Ganesh Tree & Plant Health Care,

6 Reported effects of plant growth promoting rhizobacteria (PGPR) on rice. PGPR Proposed mode of action References Isolates from rice rhizosphere Azospirillum lipoferum Produce phytohormones and solubilise phosphate Phytohormone synthesis, siderophore production Ashrafuzzaman et al. (2009) Boyer et al. (2008) Azospirillum Increased N fixation Pedraza et al. (2009) Pseudomonas spp. Phytohormone synthesis Karnwal (2009) Azospirillum; Enterobacter; Aeromonas veronii Pseudomonas spp. Azospirillum Herbaspirillum sp. Strain B501 gfp1 Burkholderia vietnamiensis Pantoea sp. Source: Cummings (2009) Increased N fixation and phytohormone synthesis Increased N fixation and phytohormone synthesis Increased N fixation and phytohormone synthesis Hypothesis: increased N fixation and photohormone prodution natural biofertilizer and bioprotective against plant pathogen Mehnaz et al. (2001) Mirza et al. (2006) Zakria et al. (2007) Van et al. (2000) Ruiza et al. (2011)

7 OBJECTIVES This preliminary research is conducted to 1. detect the heat-resistant endophytic Bacillus spp. which has associated with commercial and indigenous rice seeds in Thailand. 2. the role of endophytic bacteria species to rice seed germination and seedling growth, to compare between the epiphytic bacteria isolated from the rice seeds.

8 Isolation of both epiphytic and endophytic Bacillus spp. Epiphytic bacteria From 50 rice seeds, epiphytic bacteria was isolated by submerged all seeds in 5 ml sterile water (in test tube), and was subjected to hot water (at 80 C) for 30 minutes. For isolating Bacillus spp., an aliquot from these tubes was streaked onto the PDA medium.

9 Isolation of endophytic Bacillus spp., 50 rice seeds (de-husked) were surface sterilized with 70% ethyl alcohol for 30 minutes. The remaining rice seeds were macerated with the mortar in 5 ml sterile water and in water bath (at 80 C) for 30 minutes. The aliquot was streaked onto the medium for bacterial isolation.

10 RESULTS Species of heat-resistant endophytic bacteria associated with rice seeds Bacillus licheniformis (Jow Haw), B. amyloliquefaciens (Chai Nat 1 and Suphan Buri 1), B. subtilis (Chai Nat 1, Reing Thong, and Suphan Buri 1), B. thuringiensis (Ni Kor) Epiphytic bacteria Alicyclobacillus pomorum (RD 31) Bacillus licheniformis (Ni Kor) Brevibacillus brevis (Roa Su Ya and Reing Thong) Bacillus sp. (Chai Nat 1)

11 Effect of selected bacteria to seed germination and seedling growth Percent seed germination (highly significant) B. amyloliquefaciens = 94.5% a Alicyclobacillus pomorum = 91.3% b non-treated control = 90.4% b Promoting seedling growth (plant height) (highly significant) Bacillus amyloliquefaciens = 6.9 cm a A. pomorum = 6.8 cm a non-treated control = 5.0 cm b Different rice cultivars responded differently to the bacteria. RD 31 had the greatest respond when was treated with B. amyloliquefaciens in both traits.

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13 ACKNOWLEDGEMENTS We would like to thank Silpakorn University, Phetchaburi IT campus, Cha-Am, Phetchaburi, Thailand, for financial support for attending and oral presentation in this research in the International Symposium on Plant Protection and Plant Health in Europe (PPPHE) 2013, Berlin, Germany.

14 REFERENCES Cottyn B; Debode J; Regalado E; Mew TW; Swings J Phenotypic and genetic diversity of rice seedassociated bacteria and their role in pathogenicity and biological control. Journal of Applied Microbiology.107, Garcia JAL; Probanza A; Ramos B; Palomino MR; Gutiérrez Mañero FJ Effect of inoculation of Bacillus licheniformis on tomato and pepper. Agronomie. 24, Hardoim PR; Andreote FD; Reinhold-Hurek B; Sessitsch A; van Overbeek LS; van Elsas JD Rice rootassociated bacteria: insight into community structures across 10 cultivars. FEMS Microbiology Ecology. 77, Nautiyal CS; Srivastava S; Chauhan PS; Seem K; Mishra A; Sopory SK Plant growth-promoting bacteria Bacillus amyloliquefaciens NBRISN 13 modulates gene expression profile of leaf and rhizosphere community in rice during salt stress. Plant Physiology and Biochemistry. 66, 1-9. Ruiza D; Agaras B; de Werrab P; Wall LG; Valverde C Characterization and screening of plant probiotic traits of bacteria isolated from rice seeds cultivated in Argentina. Journal of Microbiology. 49, Schisler DA; Slininger PJ; Behle RW; Jackson MA Formulation of Bacillus spp. for biological control of plant diseases. Phytopathology. 94,

15 Thank You Silpakorn University, Thailand