| Damping-off is an important disease in seedling stage of Pinus massoniana,which seriously restricts the development of seedling industry of P.massoniana.In the past,the use of chemical agents for the control of damping-off has been unable to meet the requirements of production practice and social development.Therefore,it is necessary to develop more effective and environmentally friendly control measures.Biological control of diseases has the advantages of no pollution,long-lasting control effect and no resistance of pathogens,which is gradually replacing chemical control of diseases.In this study,after obtaining the pathogen of damping-off of P.massoniana,the healthy adult P.massoniana was taken as the object to separate the endophytic fungi at different time and different parts,and the diversity of endophytic fungi and the potential of endophytic fungi to control damping-off of P.massoniana were evaluated.At the same time,the mechanism of controlling damping-off of P.massoniana was clarified.The results are as follows:（1）In this study,the pathogenic fungi were isolated from the roots of 5 kinds of P.massoniana seedlings.The results showed that the pathogen of P.massoniana in Duyun nursery was F.oxysporum,and the pathogenicity test showed that the pathogenicity of F.oxysporum was the strongest（P<0.05）.The results showed that the content of hydrolytic nitrogen and organic matter in rhizosphere soil decreased significantly（P<0.05）.The results of high-throughput sequencing showed that the OTUs number and population diversity index of fungi and bacteria in the rhizosphere soil samples of diseased seedlings were lower than those of healthy seedlings,and the number of pathogens in the fungal community of diseased seedlings was significantly increased,while the composition of bacterial community was not significantly changed.（2）In this study,1099 endophytic fungi were isolated from 1328 tissues of P.massoniana.Through morphological identification,they were divided into 45 groups.After BLAST comparison,1084 endophytic fungi were identified,while 15 isolates were not.1084 endophytic fungi belong to 3 phyla,10 classes,21 orders,37 families,55 genera and 69 species.endophytic fungis belong to Ascomycetes（IR=79.82%,IF=96.47%）,Basidiomycetes（IR=0.69%,IF=0.81%）and Mucoromycota（IR=1.12%,IF=1.36%）;and the dominant genera were Trichoderma（IR=10.84%,IF=13.10%）,Pestalotiopsis（IR=9.55%,IF=11.57%）,Fusarium（IR=8.58%,IF=10.38%）.In different time,the endophytic fungi isolated in autumn are the most abundant.Trichoderma（IR=9.32%,IF=10.45%）,Fusarium（IR=8.83%,IF=9.88%）and Pestalotiopsis（IR=8.07%,IF=9.04%）were the dominant genera in this season.At the same time,there are similarities among the four seasons,with the highest similarity between winter and summer(Cs winter-summer=0.43).The diversity index of endophytic fungi in roots was 2.96,Fusarium（IR=11.20%,IF=13.16%）,Trichoderma（IR=9.60%,IF=11.20%）,Phoma（IR=7.41%,IF=8.54%）and Alternaria（IR=7.24%,IF=8.45%）.In terms of similarity,endophytic fungi isolated from stem and root had the highest similarity(Cs stem-root=0.45).（3）Four strains of endophytic fungi were screened out from 90 strains of endophytic fungi through the confrontation test.The inhibition rate of strains 3Y-G14（87.67%）>12Y-G7（75.00%）>6Y-J42（71.67%）>3Y-Y27（70.97%）.Four strains were identified as Trichoderma koningiopsis,Trichoderma virens,Trichoderma spirale and Trichoderma longibrachiatum.The results showed that strain 3Y-Y27 showed the best biological control effect,the control rate was82.52%,strain 6Y-J42 was the second,the control rate was 77.24%.（4）The results showed that 4 strains of endophytic fungi control F.oxysporum were confronted with each other under the microscope.They competed for nutrients to make their mycelia deform,bend and shrink.The results showed that the volatile metabolites of four strains of endophytic fungi had significant inhibitory effect on damping-off（P<0.05）,and the inhibitory rate was 30.34-36.11%,but there was no significant difference between the four strains（P>0.05）.Among the non-volatile metabolites,strain 12Y-G7 had the most significant inhibitory effect on pathogen（P<0.05）,and its inhibitory rate was 55.62%.Meanwhile,34 compounds were obtained by measuring the non-volatile metabolites of endophytic fungi,including ketones,alcohols,aldehydes,esters,phenols and organic acids.（5）After inoculation of endophytic fungi and damping-off pathogen for 60 days,the results showed that both endophytic fungi and pathogen could be planted in the roots of P.massoniana seedlings,and formed similar infection structure of hyphae invading the epidermis.The results showed that the seedlings inoculated with endophytic fungi had a significant effect on the growth of P.massoniana seedlings（P<0.05）,among them,the seedling of P.massoniana inoculated with strain 3Y-G14 reached a maximum of 19.20cm,56.99%higher than that of water seedling;the root length was 1.08 times higher than that of water seedling;the number of lateral roots was 4.12times higher than that of water seedling;the fresh weight and dry weight were also the highest,1.48 and 2.68 times higher than that of water seedling,respectively.A series of physiological and biochemical indexes of P.massoniana seedlings treated with different methods were determined.The results showed that,compared with the treatment with clear water,each index of the seedlings treated with endophyte control showed significant growth promoting effect,among which strain3Y-Y27 showed the most significant.Compared with the fresh water seedlings,the total chlorophyll content of the seedlings inoculated with strain 3Y-Y27 increased 1.14 times;the relative conductivity decreased 32.87%;the MDA content decreased 29.93%;in the leaves and roots,the soluble sugar content increased 23.65%and 52.74%,the proline content increased17.03%and 86.13%;the POD content increased 1.09 times and 56.04%,SOD content increased1.02 times and 88.80%,CAT content increased 97.83%and 1.05 times,respectively.Compared with the seedlings inoculated separately,the seedlings inoculated with four endophytes also showed a certain resistance effect on each index.The results showed that endophyte control could effectively control the damage of damping-off to seedlings through many ways. |
PDF Full Text Request