| Juglans regia is a kind of economic tree species,and its fruit walnut isedible and can be used for medicine.It is a healthy product with rich nutritions,and is one of the most popular nuts.It is often praised as "longevity fruit".However,there are many diseases occurred during the growth of walnut,among which walnut anthracnose is one of the important diseases.This disease mainly harms the leaves,fruits,tender shoots,or buds of walnut,and lead to heavy loss of the yield and quality.Therefore,the control of walnut anthracnose has becoming one of the main measures to improve the quality and quantity of walnut.Currently,chemical agents are commonly used to control walnut anthracnose worldwide,but chemical control has significant risks on our ecological damage and environmental pollution.Some chemical residues can also cause certain damage to human bodies.Therefore,biological control is much better for plant disease prevention and control.The purpose of this study is to isolate and screen antagonistic fungi that have inhibitory effects on walnut anthracnose from healthy walnut tissues,and further explore the control effect and biological characteristics of the antagonistic fungi.Endophytic fungi were isolated from the roots,stems,and leaves of three different walnut varieties,including’Xiangling’,’Qingxiang’,and ’Yuxiang’,using tissue isolation methods.Antagonistic endophytic fungi were screened and tested for pathogenicity using a confrontation culture method.Antagonistic activities of endophytic fungi which had no pathogenicity and good antagonistic effects were selected.Then,biological characteristics of the antagonistic strains were studied,which can help for future research on biological control of walnut anthracnose.The main findings are as follows:(1)Isolation and molecular identification of endophytic fungi from walnut.In this study,52 endophytic fungi were isolated from the roots,stems,and leaves of three walnut varieties,including ’Xiangling’,’Qingxiang’,and ’Yuxiang’.The isolation rate of walnut stems was 17.1%,7.3% for roots,and 5.1% for leaves.The total DNA of 52 strains was extracted and analyzed by PCR amplification,DNA sequencing,and alignment of ITS sequences.Finally,52 endophytic fungi of walnut were identified which belonging to 18 different genera,including Trichoderma sp.,Diporthe sp.,Penicillium sp.,Cladosporium sp.,Fusarium sp.,and Alternaria sp.Among them,41.2% of the strains belong to Alternaria.(2)Screening of antagonistic fungi.The isolated endophytic fungi were cultured against Colletotrichum gloeosporioides.It was found that 52 strains of endophytic fungi from walnut had different inhibitory effects on C.gloeosporioides.Nine strains with better antagonistic effects against C.gloeosporioides were selected,six strains from walnut stems,two strains from walnut leaves,and one from walnut roots.Among them,strain Chen-P20 had the best antagonistic effect against C.gloeosporioides,with an inhibition rate of 83.3%.(3)Field pathogenicity of antagonistic strains.Field pathogenicity tests were conducted on 9 antagonistic strains,and it was found that two endophytic fungal strains Chen-P12(Botryospaeria dothidea)and Chen-P18(Fusarium sp.)have pathogenicity,and the other 7 antagonistic strains have no effect on walnut.(4)Rescreening of antagonistic fungi for Chen-P20.The strain Chen-P20 with the best antagonistic effect and no pathogenicity was selected for rescreening and purification.The inhibitory effect of spore suspension of the strain Chen-P20 was tested against Colletotrichum gloeosporum.It was found that the spore suspension of the strain had a high antagonistic effect against C.gloeosporum,with the inhibition rate of 84.7%.(5)Resistance in vivo of antagonistic strain Chen-P20 in walnut plants.Using fresh walnut leaves,stems,and seedlings,the antagonistic activity of strain Chen-P20 in walnut leaves,stems,and seedlings was tested.The antagonistic activity of strain Chen-P20 against Colletotrichum gloeosporioides was significant,and the inhibition rates were 62.6%,55.6%,and 66.8%,respectively.(6)Morphological characteristics and phylogenetic analysis of strain Chen-P20.The morphological characteristics of antagonistic strain Chen-P20 were observed using electron microscopy.The ITS sequence of strain Chen-P20 was obtained and aligned through BLAST searches in NCBI.A phylogenetic tree was constructed using the ITS and tef1 sequences,and the antagonistic strain Chen-P20 was identified as Trichoderma sp.(7)Biological characteristics of antagonistic strain Trichoderma sp.Chen-P20.Strain Chen-P20 was cultured in different media,carbon source,nitrogen source,temperature,and light conditions.The mycelial diameter after 48 hours and the conidiation after 7 days were studied.It was found that the best medium for Trichoderma sp.Chen-P20 was PDA.The optimal carbon source is maltose,the optimal nitrogen source is yeast powder,and the optimal conditions are 25℃,continuous illumination,and p H between 5-6.In this study,a new endophytic Trichoderma sp.Chen-P20 strain was screened from walnut stems for the first time.Through plate confrontation method,spore suspension rescreening,and resistance testing in vivo of walnut plants,it showed that the endophytic Trichoderma sp.Chen-P20 strain had good inhibition effects on walnut anthracnose.The study provides a reference for the future biological control of walnut anthracnose and the utilization of endophytic Trichoderma sp.as a biocontrol fungi. |
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