Control of Stripe Rust in Wheat (Cause by Puccinia Striiformis Tritici) Using Endophytic Bacteria

Abstract

Control of Stripe Rust in Wheat (Cause by Puccinia striiformis tritici) using Endophytic Bacteria In this study, endophytic bacteria isolated from root, stem, and leaf tissues of stripe rust– susceptible (Inqilab 91, Galaxy 2013, and 15BT023) and stripe rust–resistant (NARC 2011, Ujala 2015, TW1410) cultivars, were identified and characterized. Abundance of endophytes was found in roots as compared with stems and leaves. Resistant and susceptible cultivars significantly differed in abundance of endophytic bacteria. Restriction analysis of 16S rRNA genes amplified from 100 bacterial isolates produced 20 unique patterns. Representatives of each of the 20 unique patterns were sequenced and identified. Among the sequenced bacteria, 16 Operational Taxonomic Units (OTUs) belonged to resistant cultivars while 3 OTUs belonged to susceptible cultivars, and 1 OTUs belonged to both resistant and susceptible cultivars. Equal number of Firmicutes and Proteobacteria (45%) were isolated, while only 10% of the Actinobacteria were isolated from the wheat cultivars. Most of the isolates have PGP properties (plant growth–promoting) and a few have the potential of producing hydrolytic enzymes. Out of 16 identified bacteria, 5 endophytic bacterial strains Serratia marcescens (3A), Bacillus megaterium (6A), Paneibacillus xylanexedens (7A), Bacillus subtilis (11A), and Staphyloccus agentis (15A), isolated from resistant wheat cultivars, showed significant inhibition of Puccinia striiformis f. sp. tritici (Pst) urediniospore germination. Two formulations, fermented liquid with bacterial cell (FLBC) and fermented liquid without bacterial cells (FL) were used. Both formulations of 5 selected endophytic bacterial strains significantly inhibited the urediniospore germination in lab. This was confirmed on seedlings of Pakistani wheat cultivar Inqilab 91 and Chinese wheat cultivar Mingxian 169 in greenhouse. FLBC and FL formulations applied 24 hours before Pst inoculation (hbi) displayed a protective mode. For FLBC control efficacy was between 82.27% and 37.52% in Mingxian 169 while in Inqilab 91 the control efficiency was in the range of 84.47% and 32.59%. while for FL control efficacy was 84.44% when applied 24 hbi. xii Inqilab 91 inoculated with these selected bacterial strains and fungus was also tested under natural conditions conducted during 2017-2018 cropping season at adult plant stage. The strains Bacillus megaterium(6A) and Paneibacillus xylanexedens(7A) significantly reduced the disease severity of stripe rust with a control efficacy 65.16% and 61.11% for the FLBC, respectively in a protective effect while 46.07% and 44.47% in a curative effect. These bacterial treatments inhibit the growth of Pst urediniospore by changing the urediospoes morphology that resulted into nonfunctional fungal structures. Endophytic bacterial and fungal treated seedlings of Inqilab 91 and Mingxian 169 showed higher activities of antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL). The treated seedlings also showed high expression of Pathogenesis related (PR) protein genes, Antifungal protein (PR-1), β-1,3-endoglucanases (PR-2), endochitinases (PR-4), Peroxidase (PR-9) and Ribonuclease-like proteins (PR-10). Among the antioxidant enzymes SOD, POD and PPO exhibited their maximum activities at 72 hai while for PAL maximum induction was recorded at 24 and 48 hai, while PR proteins genes were found to be upregulated at 48 and 72hai. However, few bacterial treatments (B. subtilus in both wheat cultivars and B. megaterium in Inqilab 91) showed maximum inductions of PR-10 at 24 hai. These results concluded that endophytic bacteria have the biocontrol potential, which can be used to control Puccinia striiformis. High production of HCN, Siderophore, and antioxidant enzymes as well as high expression of PR protein genes might be crucial in triggering the host defense mechanism against Pst. This study can be useful in further defining and elaborating a possible mechanism of biocontrol for managing the stripe rust in detail.