Biological control of rice blast disease caused by Pyricularia oryzae with Bacillus amyloliquefaciens, Epicoccum nigrum, and Penicillium citrinum

Abstract

The objectives of the experiments were to evaluate the biological control potential of Bacillus amyloliquefaciens, Epicoccum nigrum, and Penicillium citrinum for the control of rice blast caused by Pyricularia oryzae in the rice cultivar (Oryza sativa L.) BRSMG Caçula, in the ability to enhance plant growth and productivity, as well as to analyze the ability to stimulate plant defense mechanisms through microscopic and molecular analyses. The experiments were carried out in a greenhouse. Evaluations were based on the incidence and severity of rice blast, not only but also plant height, several tillers, panicles, and rice grain weight. Confocal laser microscope (CLM), scanning electron microscope (SEM), and Epi-fluorescence microscope (EFM) were used to analyze the interaction of rice plants with microbial biocontrol agents (BCAs). Soil and rice seeds were inoculated with Bacillus BMH and the seeds were sprayed with P. oryzae isolates 10965 and IA25. Bacillus BMH reduced rice blast severity by 53.1%, 50% and 37.5% when applied to soil or via preventive foliar treatment and pathogen-BMH mixture, respectively. The height of rice plants was higher in the treatment with Bacillus BMH applied in soil (28.5 cm) compared to the control treatment (25.7 cm) at 63 DAS. Likewise, the number of tillers was higher in plants grown in soil treated with Bacillus BMH (12.6) compared to the control (9.13). The grain weight was 10.2 g for soil treatments with Bacillus BMH (write which ones) versus 7.1 g for the untreated. Colonization of vascular tissues of rice roots and leaves by Bacillus BMH cells was observed in CLM and SEM indicating its endophytism. Bacillus BMH seed treatment stimulated plant defense mechanisms against blast through upregulation of rice β-1,3-glucanases (OsGLN1), OsPR1a, and OsWRKY28 genes. E. nigrum seed soaking and spraying on leaves preventively reduced blast incidence by 41.7% to 31.2%, respectively, and severity by 54.5% to 34.9%. Seed treatment with E. nigrum increased the number of tillers to 8.3 against 5.9 in the control treatment. SEM and EFM images showed the rice root surface colonization by E. nigrum. Penicillium citrinum GP1 and GP3 consortium in seed drenching treatment and sprayed on leaves, reduced blast severity by 33.3% to 37.4%, respectively. The consortium of P. citrinum isolates increased the number of rice tillers by 11.25 versus 10.17 in the control, respectively. The individual or combined use of these isolates did not affect plant height, panicle number, and plant yield compared to untreated plants. Both isolates colonized rice root tissues. Two strains of Epicoccum (from grapevine and maize), used in this study were identified as E. nigrum based on ITS, RPB2, β-TUB2, and LSU regions of rDNA sequence homology and phylogenetic analyses. In the same way, the two Penicillium from grapevine (GP1 and GP3) were identified as P. citrinum based on RPB2, β-TUB2, and LSU regions of rDNA sequence homology and phylogenetic analyses. The greenhouse experiments showed that Bacillus BMH, E. nigrum and P. citrinum reduced blast severity as well as stimulated plant growth. The beneficial effects suggest that these biocontrol agents have the potential to be used as biofungicides and biofertilizers in rice cultivation.