Isolation and Characterization of Root-Associated Bacterial Endophytes and Their Biocontrol Potential against Major Fungal Phytopathogens of Rice (Oryza sativa L.)

Abstract

Rice (Oryza sativa L.) is a major cereal food crop worldwide, and its growth and yield are affected by several fungal phytopathogens, including Magnaporthe oryzae, Fusarium graminearum, F. moniliforme, and Rhizoctonia solani. In the present study, we have isolated and characterized root-associated bacterial endophytes that have antifungal activities against rice fungal phytopathogens. A total of 122 root-associated bacterial endophytes, belonging to six genera (Bacillus, Fictibacillus, Lysinibacillus, Paenibacillus, Cupriavidus, and Microbacterium) and 22 species were isolated from three rice cultivars. Furthermore, the 16S rRNA sequence-based phylogeny results revealed that Bacillus was the most dominant bacterial genera, and that there were 15 different species among the isolates. Moreover, 71 root-associated endophytes showed antagonistic effects against four major fungal phytopathogens, including M. oryzae, F. graminearum, F. moniliforme, and R. solani. Additionally, the biochemical, physiological, and PCR amplification results of the antibiotic-related genes further supported the endophytes as potential biocontrolling agents against the rice fungal pathogens. Consequently, the findings in this study suggested that the isolated bacterial endophytes might have beneficial roles in rice defense responses, including several bioactive compound syntheses. The outcomes of this study advocate the use of natural endophytes as an alternative strategy towards the rice resistance response.

Keywords: biological control; phytopathogens; rice; root-associated endophytes.

Figure 1

The 16S rRNA gene sequences-based phylogeny of all the 122 root-associated bacterial endophytes isolated from different rice cultivars. The 16S rRNA gene sequences were clustered to the MUSCLE alignment through the neighbor-joining method and the 16S rRNA gene sequences-based phylogeny was concatenated by inputting the 16S rRNA gene sequences into the MEGA 7 software. The above green, red, blue, yellow, pink, dark red and black, colored circle symbols represent different bacterial species such as; Bacillus ssp., Fictibacillus ssp., Lysinibacillus ssp., Cupriavidus ssp., Paenibacillus ssp., Microbacterium ssp., and reference type strains. The scale bar value indicates to the bootstrap analysis from (1000) replicates for the genetic distance.

Figure 2

Biocontrol potentials of different root-associated bacterial endophytes to rice blast (Magnaporthe oryzae). (A) The above plates represent in-vitro antifungal inhibitory activities by dual-culture antagonistic tests of different root-associated bacterial endophytes against rice blast (Magnaporthe oryzae) phytopathogen. (B) The above selected graphical bars indicate antifungal inhibition percentages of radial mycelial growth of (Magnaporthe oryzae) by the 10 root-associated bacterial endophytes, respectively. Values are denoted as mean ± SE (n=3). Values followed by different letters are significantly different (p≤0.05) according to the least significant difference (LSD) test.

Figure 3

Biocontrol potential of different root-associated bacterial endophytes to rice seedling blight (Fusarium graminearum), (A) the above plates represent in-vitro antifungal inhibitory activities by dual-culture antagonistic tests of different root-associated bacterial endophytes against rice seedling blight (Fusarium graminearum) phytopathogen, (B) the above selected graphical bars indicate antifungal inhibition percentages of radial mycelial growth of (Fusarium graminearum), by the 20 root-associated bacterial endophytes respectively. Values are denoted as mean ± SE (n = 3). Values followed by different letters are significantly different (p ≤ 0.05) according to the LSD test.

Figure 4

Biocontrol potential of different root-associated bacterial endophytes to rice bakanae (Fusarium moniliforme), (A) the above plates represent in-vitro antifungal inhibitory activities by dual-culture antagonistic tests of different root-associated bacterial endophytes against rice bakanae (Fusarium moniliforme), fungal phytopathogen of rice, (B) the above selected graphical bars indicate antifungal inhibition percentages of radial mycelial growth of (Fusarium moniliforme) by the 16 root-associated bacterial endophytes respectively. Values are denoted as mean ± SE (n = 3). Values followed by different letters are significantly different (p ≤ 0.05) according to the LSD test.

Figure 5

Biocontrol potential of different root-associated bacterial endophytes to rice sheath blight (Rhizoctonia solani), (A) the above plates represent in-vitro antifungal inhibitory activities by dual-culture antagonistic tests of different root-associated bacterial endophytes against rice sheath blight (Rhizoctonia solani), fungal phytopathogen of rice, (B) the above selected graphical bars indicate antifungal inhibition percentages of radial mycelial growth of (Rhizoctonia solani), by the 26 root-associated bacterial endophytes respectively. Values are denoted as mean ± SE (n = 3). Values followed by different letters are significantly different (p ≤ 0.05) according to the LSD test.