Isolation of Trichoderma virens 6PS-2 and its effects on Fusarium proliferatum f. sp. Malus domestica MR5 related to apple replant disease in China

Apple replant disease(ARD) is primarily caused by biotic factors that seriously inhibits the development of apple industry. Therefore, the use of biological control measures to inhibit the main pathogens(such as Fusarium spp.) that cause ARD is of great significance to the sustainable development of the apple industry. Trichoderma virens 6PS-2, which exhibited antagonism toward a variety of pathogens, was screened from the rhizosphere soils of healthy apple trees(Malus robusta) in different replanted orchards in the Yantai and Zibo Cities, Shandong Province, China. Its fermentation extract inhibited the growth of pathogenic Fusarium proliferatum f. sp. Malus domestica MR5, which was proportional to the concentration. These substances also increased the hairy root volume and growth of Arabidopsis thaliana lateral roots. The phenotype of Malus hupehensis seedlings and microbial community structure in rhizosphere soils in greenhouse experiment using Highthroughput sequencing were analyzed, and the field experiment with grafted apple trees were used for further verification. Compared with the application of potato dextrose broth(PDB) medium, application of 6PS-2 spore suspension directly to replanted soils could improve the growth of M. hupehensis seedlings as well as the elongation of grafted apple trees. Concomitant decreases in the gene copy number of Fusarium and increases in the culturable bacteria/fungi were also observed in the greenhouse and field experiments. The abundance of Trichoderma,Bacillus, and Streptomyces increased significantly, but that of Fusarium, Pseudarthrobacter, and Humicola decreased. The content of esters, phenols,furans, and amino acids in root exudates of M. hupehensis seedlings increased, which significantly inhibited the multiplication of Fusarium, but was positively correlated with Bacillus and Trichoderma. In summary, T. virens 6PS-2 not only directly inhibits the activity of pathogenic Fusarium but also secrets secondary metabolites with antifungal and growth-promoting potential. In addition, 6PS-2 spore suspension can also promote the growth of plants to a certain extent, and change the soil microbial community structure of rhizosphere soils. It is believed that T. virens 6PS-2 has the potential for the alleviation of apple replant disease(ARD) in China.

Isolation,identification and artificial inoculation of Ustilago esculenta on Zizania latifolia

The smut Ustilago esculenta is an endophytic fungus that invades Zizania latifolia(Jiaobai)and plays an important role in inducing the host stem to form a swollen gall.The aims of this study were to investigate isolation,identification and inoculation methods of U.esculenta from Jiaobai.The spore suspension culturing method was the best method of isolating U.esculenta,and yeast-like fungal strains and septate hyphal strains were obtained.The universal primers ITS1 and ITS4 amplified a ribosomal DNA spacer element from all tested strains,while specific primers for Lam16A gene generated a 442 bp product only with septate hyphae strains.De novo transcriptome assembly results showed that the yeast-like fungal strains did not match Ustilago,and genomic analysis suggested the septate hyphae strains were highly similar to known smut fungi.Therefore,the septate hyphae strains were identified as U.esculenta,which indicated that the ITS primers were not species-specific but that Lam16A-specific primers can distinguish U.esculenta accurately and easily.In vitro,U.esculenta grew quickly on PDA medium at 28°C in darkness.Furthermore,an inoculation method was established to produce swollen stems by injecting a spore suspension of U.esculenta at OD600=2.5 into the basal culm internode of male Jiaobai.