Fig 1.
Antibacterial and antifungal activities of RABs revealed by dual culture experiments.
The bacterial suspension of each RAB was spotted after Burkholderia glumae cells were spread-plated, or a mycelial plug of Rhizoctonia solani was placed on the center of a PDA plate. The plates of dual culture were incubated at 25C for 72 h in the dark. (A) Selected plates of dual culture showing antifungal activities of RABs against R. solani. (B) Selected plates of dual culture showing antibacterial activities of RABs against B. glumae. (C) Antifungal (upper graph) and antibacterial (lower graph) activities of RABs against R. solani and B. glumae, respectively. Each error bar indicates standard deviation from three replications. The letter above each column indicates significant difference from other data with P < 0.0001 based on Tukey’s test and each error bar indicates standard deviation from three replications.
Table 1.
Gram reactions and predicted bacterial species of rice associated bacteria (RABs).
Fig 2.
Phylogenetic tree based on 16S rDNA sequences of the 29 rice-associated bacteria (RABs) and other Bacillus species from GenBank nucleotide database.
GenBank accession numbers for the sequences obtained from the database are given in parentheses. The tree was generated using neighbor-joining method, and genetic distances were calculated using the Kimura 2-parameter method using MEGA5 [76]. Numbers at nodes indicate percentage of occurrence in 1000 bootstrapping. The bar at the bottom of the tree indicates the scale for genetic distances. Burkholderia glumae 336gr-1 was used as an out-group control.
Fig 3.
Phylogenetic tree based on gyrB (A) and recA (B) sequences of the five rice-associated bacteria (RABs) and other Bacillus species from GenBank nucleotide database.
GenBank accession numbers for the sequences obtained from the database are given in parentheses. The tree was generated using neighbor-joining method, and genetic distances were calculated using the Kimura 2-parameter method using MEGA5 [76]. Numbers at nodes indicate percentage of occurrence in 1000 bootstrapping. The bar at the bottom of the tree indicates the scale for genetic distances. Burkholderia glumae 336gr-1 was used as an out-group control.
Fig 4.
Inhibitory activities of antagonistic RABs on Rhizoctonia solani.
(A) Inhibition of sclerotial germination by RABs. Overnight cultured sclerotia with RAB inoculum in test tube were grown on PDA media and incubated at 25°C for 72 h. Sclerotia cultured only in LB broth were used as control. Sclerotial germination was inhibited by all RABs inoculum except in the control where the mycelial growth can be observed. (B) Inhibition of sheath blight lesion development by pretreatment of RABs: Examples of detached leaf assay revealing inhibitory activities of RABs against lesion development by R. solani. A detached rice leaf pretreated with a RAB was placed on paper towel moisturized with sterilized ddH2O and a sclerotium of R. solani was inoculated on the center of the detached leaf. Petri-dishes containing the inoculated rice leaves were incubated at 25°C for 7 d with 12 h of light period per day (upper); and Inhibition of sheath blight lesion development by the five selected RABs that showed highest antifungal activities in the dual-culture assay (lower). Each column of the graph represents the average of three replications and the letter above each column indicates significant difference with P < 0.05, which was determined by Tukey’s test.
Fig 5.
Biological control activities of selected RABs in the field obtained from the field trial in 2012.
(A) Suppression of sheath blight by RABs. R. solani was inoculated 24 h after the treatment of RABs at the tillering stage of rice plants (cv. Bengal). Rice plants treated with water instead of a RAB and those without inoculation of R. solani were included as controls. Disease severity was rated 27 days after inoculation based on a 0 to 9 scale scheme [79]. Each error bar indicates standard error from three replications and the letters above individual columns indicate significant differences with P < 0.05 according to Dunn’s test. (B) Suppression of bacterial panicle blight by RABs. B. glumae was inoculated 24 h after the treatment of RABs at the 30% heading stage of rice plants (cv. Bengal). Rice plants treated with water instead of a RAB and those without inoculation of B. glumae were included as controls. Disease severity on the rice panicles was rated 10 days after inoculation of B. glumae 336gr-1, using a 0 to 9 scale scheme [80]. Each error bar indicates standard error from three replications and the letters above individual columns indicate significant differences with P < 0.05 according to Dunn’s test.