Molecular and physiological mechanisms underlying the antifungal and nutrient acquisition activities of beneficial microbes
Endophytes are microbes that inhabit host plants without causing disease. Wild and ancient relatives of modern crops are grown without fungicides or synthetic fertilizers. I hypothesized that these genotypes possess beneficial endophytes that help their hosts to combat fungal pathogens and/or acquire nutrients. To test the first hypothesis, 190 bacterial endophytes isolated from wild, ancient and modern genotypes of Zea (corn grass family) were tested for their ability to combat fungal pathogens. For high-throughput disease screening, a relative of Zea, creeping bentgrass (Agrostis stolonifera L.) was used as a model system, as it can be grown rapidly in test tubes. Two pathogens of creeping bentgrass were selected as targets: Sclerotinia homoeocarpa and Rhizoctonia solani. Three endophyte strains (all Burkholderia gladioli) were found to combat S. homoeocarpa, while four strains (3 Burkholderia gladioli and 1 Paenibacillus polymyxa) could suppress R. solani. All the Burkholderia endophytes originated from wild or ancient maize, while the single Paenibacillus strain originated from modern maize. These results support the hypotheses that wild and ancient maize genotypes host microbes that can control fungal pathogens. B. gladioli strain 3A12 was selected for isolation of the underlying antifungal genes. Tn5-mutagenesis combined with whole genome sequencing revealed that the antifungal genes encode: YajQ (a c-di-GMP receptor), fatty acid desaturase, lysine-tRNA synthetase, tolR, arginine/ornithine/lysine decarboxylase and succinate dehydrogenase. The candidate mutants showed similar phenotypes (reduced motility, biofilm formation, attachment and microcolony formation around target fungi), suggesting they may belong to the same genetic network. To test the second hypothesis, 73 endophytes were screened for their ability to promote growth of annual ryegrass on rock-phosphate as the sole P source. One endophyte, isolated from a wild maize genotype, was able to promote root growth. This endophyte was found to secrete acids to help solubilize rock P in vitro, and synthesize auxin and promote lateral root and root hair growth, presumably to increase P-scavenging. The endophyte can localize to epidermal root surfaces including on root hairs, and remarkably, also near lateral root primordia. The results support the hypothesis that wild plants may be a reservoir of microbes that assist their hosts with nutrient acquisition.