Characterization and Antagonistic Effect of Culturable Apple-Phyllosphere Endophytic Bacteria from the Cold Plateau in Yunnan, China

Abstract

The endophytic bacteria in apple leaves from apple-producing areas of Yunnan, China were isolated and identified on the basis of bacterial colony morphology and nucleotide sequences of 16S rRNA and rpoB genes. The endophytic bacterial isolates with nitrogen, phosphorus, and potassium utilization abilities were screened by culturing on functional media. A total of 5709 isolates of culturable endophytic bacteria (CEB) were isolated from 30 apple leaf samples collected from different regions. A total of 39 CEB representative isolates were identified as Bacillus velezensis, B. subtilis, B. licheniformis, B. safensis, B. pumilus, and Priestia megaterium. Among them, B. velezensis and B. subtilis were the main CEB, accounting for 55.00% and 34.37%, respectively, which exhibited potential inhibition on not only the main apple disease pathogens of Alternaria alternata, Valsa mali, Fusarium oxysporum, and Rosellinia necatrix, but also some important and uncontrollable phytopathogens, including F. oxysporum f. sp. cubense that causes banana Fusarium wilt, and Phytophthora nicotianae that causes tobacco black shank. Among these isolated endophytic bacteria species, a total of 10 strains, including b3, b4, b16, b17, b20, and b23 of B. subtilis, b7, b24, and b28 of B. licheniformis, and b38 of B. velezensis, can fix nitrogen; 8 strains, including b7 and b28 of B. licheniformis, b5, b10, and b23 of B. subtilis, b8 of B. safensis, and b6 of Priestia megaterium, could dissolve inorganic phosphorus; 11 strains, including b9, b12, b14, b30, b34, and b43 of B. velezensis, b6 of Priestia megaterium, and b17, b18, b20, and b26 of B. subtilis, could degrade organic phosphorus; and 5 strains, including b4, b5, and b26 of B. subtilis, and b7 and b28 of B. licheniformis could dissolve potassium. These strains are valuable resources of endophytic bacteria that have adapted to the ecological environment of the Cold Plateau apple-production area and could be used as plant disease biocontrol agents and biofertilizers of crops. The culturable phyllosphere endophytes in apple leaves relate to geographic locations, apple varieties, and environment, providing the basis to explain the mechanisms underlying the establishment of apple endophyte diversity and may help to devise apple disease management strategies.