Growth promotion and inhibition of leaf spot disease in riceberry rice by Endophytic bacteria

Abstract

Rice (Oryza sativa Linn.) variety Riceberry is a highly nutrition black rice. It is increasingly popular as healthy food among consumers. Riceberry rice is susceptible to leaf spot disease caused by pathogenic fungi that has effect on decreasing quality and grain yield of rice. Using of chemical fungicides to control leaf spot disease can cause negative impacts to human and environment. The application of endophytic bacteria is an alternative method to enhance growth and disease control for rice farming. Objectives of this study were to select the effective endophytic bacteria for promoting the growth of Riceberry rice, and to evaluate the potential endophytic bacteria for suppressing leaf spot disease of Riceberry rice. The endophytic bacteria were isolated from roots, stems and leaves of five varieties of rice included Hom Nil, Riceberry, San-pah-tawng 1, Black glutinous and upland rice (Jow Khao Chiangmai) in Chiang Mai province. The total of 146 isolates of endophytic bacteria were obtained from the rice samples. The number of endophytic bacteria from roots, stems and leaves were 41, 69 and 36 isolates, respectively. It was found that the highest numbers of endophytic bacteria were isolated from Black glutinous rice (51 isolates), which were collected from organic farming.For isolation of causative agent of leaf spot disease, leaves of Riceberry rice with leaf spot symptom were collected and used for fungal isolation. The isolate RBJL was identified as Curvularia hominis. The pathogenicity of the isolate RBJL was test by Koch's postulation with Riceberry rice. The result was confirmed that the isolate RBJL, C. hominis, was a causative agent of leaf spot disease of Riceberry rice. Antagonistic ability of the isolated endophytic bacteria was evaluated against C. hominis by dual culture assay. There were 17 isolates of the endophytic bacteria that could inhibit the growth of C. hominis. The isolate KCL04 was the best strain for inhibition the growth of C. hominis and it was identified as Bacillus subtilis based on 16S rDNA sequencing data. The culture filtrate of the isolate B. subtilis KCL04 strongly suppressed mycelial growth and spore germination of C. hominis. Microscopic observations revealed that C. hominis treated with culture filtrate of B. subtilis KCL04 were swelled on hyphae and germ tubes germinated from spores of C. hominis. The partial metabolites extracted from B. subtilis KCL04 culture filtrate suppressed the growth of C. hominis. It showed band on SDS- PAGE corresponding to the molecular mass of 25 kDa, which may be chitinase protein. Disease incidence decreased (natural infection of C. hominis) up to 80% when B. subtilis KCL04 was treated with seeds of Riceberry rice. In pot experiment. 7 days after spraying spore suspension of C. hominis, the rice plants showed symptom of leaf spot disease. Riceberry rice plants treated with the bacterial isolate KCL04 significantly reduced disease incidence of leaf spot disease in Riceberry rice. Leaves of Riceberry rice inoculated with the isolate KCL04 had lower the disease symptom than the control treatment. Endophytic bacteria were screened for plant growth promoting activities by evaluation of total indole production and phosphate solubilization. The results showed that 16 isolates of the endophytic bacteria could produce total indole. The highest concentrations of total indole were produced by the isolates RBR04 and BGL12 at the levels of 69.89 and 66.40 µg/ml, respectively. For phosphate solubilization, the endophytic bacteria were screened on National Botanical Research Institute's phosphate (NBRIP) agar containing tricalcium phosphate. It was found that 26 isolates could solubilize tricalcium phosphate on the NBRIP agar. The isolate RBS09 could solubilize phosphate at the highest concentration (36.65 µg/ml) followed by the isolate STS02 (32.65 µg/ml) in NBRIP broth containing rock phosphate. Four endophytic bacterial isolates (BGL12, RBR04, RBS09 and STS02) were selected for evaluation of their effects on the growth of Riceberry rice. In bottles in the laboratory, rice plants inoculated with the selected endophytic bacteria significantly increased plant height, shoot dy weight and root dry weight of Riceberry rice compared with uninoculated treatment. In a greenhouse, seeds treated with the selected isolates of BGL12, RBR04, RBS09 and STS02 increased grain weight of Riceberry rice. The isolates BGL12 and RBR04 were identified as Pseudomonas otitidis based on 16S rDNA sequencing data. In addition, the selected endophytic bacteria showed positive root colonization capacity of germ-free Riceberry rice seedlings. ทมยนฺติ This research was concluded that, B. subtilis KCL04 was the effective endophytic bacteria on inhibition leaf spot disease in Riceberry rice. Pseudomonas otitidis RBR04 had potential to be used for growth of Riceberry rice. The selected endophytic bacteria can be used to promote growth of rice and control leaf spot disease of rice.