Diversity of endophytic fungi and screening of biocontrol strains against canker disease in walnut

Abstract: 【Objective】The present experiment was carried out in order to understand the endophytic fungi population diversity and community structure of walnut trees, and investigate the differences in endophytic fungal communities among different tissues (limb- barks, roots and branches) of walnut in southern Xinjiang. At the same time, another objective was also to screen out the biocontrol fungi that can not only prevent and control walnut canker disease, but also promote the growth of walnut seedlings.【Methods】The endophytic fungi were isolated from different tissues of walnut by conventional tissue separation method. The endophytic fungi were classified and identified by morphological and molecular biology techniques. The population distribution and diversity of endophytic fungi in walnut were analyzed according to Shannon-Weiner diversity index (H'), Pielou’s evenness index (E), Margalef’s index (M), separation rate, separation frequency and Srenson’s similarity coefficients (Cs). The plate confrontation method was used to screen the antagonistic strains of walnut canker disease, the indoor pot method was used to study the growth promotion of the strains, and the control effect of the biocontrol fungus on walnut canker disease was determined by the inoculation test of isolated branches. 【Results】A total of 129 endophytic fungi were isolated from 210 tissues of ten 50- year- old walnuttrees, of which 58 were isolated from limb-barks, 42 from roots and 29 from branches, with a total isolation rate of 61.40%. The 129 endophytic fungi belonged to three phyla and 18 genera, among which ascomycetes were the largest with 122 strains, accounting for 94.57% of the total number of fungi, five were zygomycetes, accounting for 3.88%, and two were basidiomycetes, accounting for 1.55%. The 18 genera of fungi were Fusarium, Alternaria, Nectria, Chaetomium, Phaeosphaeria, Leptosphaeria, Dactylonectria, Mortierella, Penicillium, Sarocladium, Talaromyces, Corynespora, Aspergillus, Acremonium, Rosellinia, Phoma Dothiorella and Ceratobasidium, among which Fusarium and Alternaria were the dominant populations, with the isolation rates of 18.10% and 12.30%, respectively. The Shannon index (H'), evenness index (E) and richness index (M) of stem endophytic fungi were the highest, which were 2.452, 0.603 and 8.129, respectively, followed by root, each index was 2.067, 0.553 and 5.847, while the Shannon index (H'), evenness index (E) and richness index (M) of root endophytic fungi were the lowest, 1.804, 0.535 and 3.993, respectively. The highest similarity coefficient between limb- bark and root endophytic fungi was 0.84, while the lowest similarity coefficient between limb- bark and branch was 0.66. On the whole, the endophytic fungal groups were different among different tissues. Some fungi, such as Phoma, Rosellinia and Chaetomium, were found only in limb- barks, but not in roots or branches. Three antagonistic strains (SF01, SF05 and SF08) were obtained by screening. Based on morphological characteristics, and molecular and biological identification results, SF01, SF05 and SF08 were identified as Dactylonectria torresensis, Chaetomium globosum and Penicillium rubens, respectively. The inhibitory rates of three antagonistic strains against Cytospora chrysosperma were 71.20%, 73.50% and 68.50%, respectively. Inoculation of isolated branches showed that SF01 had the best preventive effect on walnut canker disease (80.10%), while SF08 had the strongest therapeutic effect on walnut canker disease (83.40%). All three strains had the ability to produce iron carriers, among which SF05 had the strongest ability to produce iron carriers. SF01 and SF05 had the ability to solve phosphorus, SF01 and SF08 had the ability to dissolve phosphorus, and SF08 also had the ability to produce protease. Compared with the control, three antagonistic strains significantly promoted the root length, plant height, fresh weight, dry weight, stem diameter and other agronomic indexes of walnut seedlings (p＜0.05). However, there were differences in growth promoting function among the three strains. Therefore, whether the mixed bactericides of the three strains can improve the ability of disease prevention and growth promotion remains to be further studied.【Conclusion】Endophytic fungi of walnut in southern Xinjiang were abundant in diversity, but their distribution was uneven, and their distribution characteristics were tissue-specific. In general, the diversity of endophytic fungi in limb-bark was higher than that in roots, and the diversity of endophytic fungi in branches was the lowest. Limb-bark endophytes were most similar to root endophytes because of their spatial location. Because the branches were pruned every year, the number of endophytic fungi species was relatively small, and there was a great difference between limb- bark endophytic fungi and branch endophytic fungi. Ascomycetes had the highest frequency of isolation in different tissues of walnut because of its strong reproductive ability and adaptability. Three biocontrol strains including SF01, SF05 and SF08, which had good control effect on walnut canker disease and promoted the growth of walnut seedlings were screened out. The result showed that the three strains were different and complementary in both disease control and seedling growth promotion, which has laid a good foundation for further research and development of mixed fungi. The studied results have provided a new material and way for the biological control of walnut canker disease and the cultivation of strong seedlings.