Screening of endophytic growth- promoting fungi and their characteristics in apple

Abstract:【Objective】Plant endophytes mostly include bacteria, fungi and actinomycetes, which are interacted with plants to form the plant holobiont. To make full use of the beneficial effects of endophytes on healthy development in apple production, the endophytic fungi in the root, branch and leaf of Malus pumila were isolated and identified. The auxin and siderophore producing ability, phosphate solubilizing activity, potassium solubilizing activity, and nitrogen fixation ability were studied for the possible mechanisms of plant growth promotion in vitro.【Methods】The endophytic fungi of the apple were isolated from the roots, branches and leaves by tissue separation. The plant materials were surface sterilized with 75% ethanol and 5% sodium hypochlorite. In order to obtain more endophytic fungi, three kinds of agar media (PDA, LB and Rose Bengal medium) were used simultaneously. Fungal genomic DNA was prepared with the Ezup Column Fungi Genomic DNA Purification Kit. The ITS sequence was amplifified by PCR with universal ITS4 and ITS5 primers. Then the PCR products were sent to Qingke Biotechnology for sequencing. Sequence similarity searches were performed for each representative fungal sequence against the non-redundant database maintained by the National Center for Biotechnology Information using the BLAST algorithm. The growth- promoting abilities of the strains were screened and compared, and the Salkowski reagent method was used for indole-3-acetic acid (IAA) production. The blue chrome azurol S (CAS) agar plate was used for siderophore detection. The screening of fungal isolates for their phosphate solubilizing activity was done in the inorganic phosphorus agar(NPA) and the organic phosphorus agar (OPA) of Monika. The fungal potassium solubilizing activity was screened by a halo zone formation method on Aleksandrov agar medium. Ashby’s nitrogen- free medium was used for enumerating nitrogen fixing ability. Further, apple tissue culture seedlings were inoculated with the potential endophytic fungal strains and growth parameters (plant height, leaf number, root length, leaf chlorophyll content and total nitrogen content in leaves) were assessed after 50 days. 【Results】A total of 51 endophytic fungi were isolated and identified as 29 species. All of the isolated fungal enophytes belonged to Ascomycota. Of them, 9 strains were classed into 5 different genera while 5 different species were obtained from healthy roots of M. pumila. The taxa Dactylonectria alcacerensis was the most frequently isolated from roots. 26 strains were classified into 9 different genera while 14 different species were obtained from healthy branches. The taxa Didymella prosopidis was the most frequently isolated. 16 strains were classified into 9 different genera while 10 different species were obtained from healthy leaves. The taxa Pseudochaetosphaeronema kunmingense was the most frequently isolated. The growth promoting function of 29 endophytic fungi was screened. The results indicated that all of the fungal strains showed the potential to produce IAA with PG06 (Clonostachys rosea f. catenulata) from the root was the highest IAA producer fungi. 10 strains of the isolates were confirmed as strains with high siderophore producing activity, of which 6 isolates were from branches, 2 isolates were from roots, and 2 isolates were from leaves. 3 strains of endophytic fungi turned out to have organophosphorus solubilization activities, and LG06(Dactylonectria alcacerensis) had the strongest phosphorus soluble ability. 26 of 29 strains of isolated endophytic fungi were shown to have nitrogen fixing ability. Then, 5 strains with higher IAA level and more kinds of growth- promoting functions (LG06 Dactylonectria alcacerensis, PG06 Clonostachys rosea f. catenulata, LG12 Chaetomium globosum, LY09 Arthrinium rasikravindrae and PJ09 Alternaria alstroemeriae) were selected and applied as bioinoculant to apple tissue culture seedlings. Through light microscope examination, mycelium was observed in the primary phloem of roots, which proved that the endophytic fungus had been colonized. Compared with the control, the number of leaves, chlorophyll content and total nitrogen content in leaves of the plant inoculated with strain PG06 had a significant difference (p＜0.05), which increased by 61.17%, 44.08% and 42.56%, respectively. The chlorophyll content and total nitrogen content in the leaves inoculated with LG12 strain increased by 42.92% and 40.67%, respectively. The height of plants inoculated with LY09 was the highest, which increased by 46.31% compared with the control. The root length of plants inoculated with LG06 was the largest, which increased by 67.77% compared with the control. The results showed that 5 endophytic strains were tested, of which 4 endophytic fungi exerted beneficial effects upon tissue-cultured apple seedlings growth.【Conclusion】The apple endophytic fungi PG06, LG12, LG06 and LY09 can significantly promote the growth of apple tissue culture seedlings. Different isolates expressed different aspects and levels of plant growth promotion. The apple tissue culture seedlings inoculated with strain PG06 had the highest survival rate and the best growth promotion efficacy. These screened endophytic fungal isolates have the potential to be used as a source of novel biofertilizers in the future. The field experiments can be further explored.