Fungal Biodiversity Of Sand-fixed Plant Rhizosphere Soil And Antifungal Mechanisms Of Some Trichoderma Species In Tengger Desert

Rhizosphere soil fungi of sand-fixed plants is a kind of very important fungal resources,which plays an important role in the formation of sand soil structure,the balance of microflora,the utilization of beneficial microbial resources and promoting plant growth etc.However,there are few studies on community structure and diversity of rhizosphere soil fungi in sand-fixed plants,and the research of utilizing Trichoderma spp.resource in desert environment is also less.Therefore,this study aimed at the weak situation of research and utilization of rhizosphere soil fungi in sand-fixed plants,combining the random survey and the fixed point research method,using taxonomy of fungi and high-throughput sequencing technology to study community structure and diversity of rhizosphere soil fungi in sand-fixed plants in Tengger Desert in Ningxia;By isolating,screening,identifying,analyzing biological characters of Trichoderma spp.of rhizosphere soil in sand-fixed plants,unveiling the antagonism mechanisms of Trichoderma spp.against Rhizoctonia solani;evaluating the biological control of Trichoderma spp.against potato black scurf?R.solani?,and on the micro-ecosystem of rhizosphere soil.The purpose of this research is to reveal the change of community structure and diversity of rhizosphere soil fungi in sand-fixed plants,to clarify the antifungal mechanisms of Trichoderma spp.against potato black scurf.The results provides a basic theory which has a significant meaning in desert ecological management and remediation of western China,and it's also contribute to developing and applying beneficial microbial resources in extreme habitats.1.The cultivable fungi community structure and distribution characteristics of rhizosphere soil in sand-fixed plants.By combining the random survey and the fixed point research method,the structure and distribution characteristics of cultivable fungal community of rhizosphere soil in sand-fixed plants under different vegetation,time and habitat were studied.The results showed that the quantity of cultivable fungi was 6.38×10³²32.28×10³ CFU·g^-1 and the fungal species was 2⁹ genera from rhizosphere soil in 52 species of sand-fixed plants;the quantity and species of fungi in Peganum harmala were the highest,while those in Kalidium foliatum were the lowest.3176 fungal isolates belonging to 25 genera were isolated from rhizosphere soil in sand-fixed plants,of which species of Penicillium,Fusarium and Aspergillus were ubiquitous;the quantity and species of soil fungi reached a peak in summer;from 2013 to 2016,the quantity of soil fungi increased by 15.86%,while the species of soil fungi remained unchanged;the quantity of soil fungi in steppe region was the highest,while that in desert region was the lowest;the species of soil fungi in desert region was the highest,while that in closing protective region was the lowest;Trichoderma was the dominant genus in desert region,Penicillium was the dominant genus in semi-desert region and steppe region,and Penicillium and Monilia were the dominant genus in closing protective region.2.The diversity of cultivable fungal community of rhizosphere soil in sand-fixed plantsBased on the survey data of quantity and species of soil fungi,the diversity,ecological distribution and soil character correlation of cultivable fungi community of rhizosphere soil in sand-fixed plants were studied.The result showed that summer had the highest fungal diversity index and richness,and the lowest Pielou eveness,while autumn had the lowest fungal diversity index and richness,and the highest Pielou eveness;fungal diversity index,Pielou eveness and richness in 2013 were higher than those in 2016;fungal diversity index,Pielou eveness and richness in desert region were the highest,reaching 2.2390?0.8938 and3.0191 respectively,while those in steppe region were the lowest,being 1.6507?0.7184 and2.1729 respectively;similarity of fungal species within four habitats ranked on a medium dissimilar level,semi-desert region and closing protective region had the highest species similarity,while steppe region and desert region the lowest;species as Penicillium,Aspergillus,Fusarium,Phoma and Mucor had wider niche breadth and wider adaptability;N content and K content were the mainsoil characters affecting variation of cultivable fungal community in sand-fixed plants'rhizosphere soil.3.Analyze fungal diversity of rhizosphere soil in sand-fixed plants through high-throughput sequencing technologyThe fungal diversity in sand-fixed plants'rhizosphere soil in different habitats were investigated through high-throughput sequencing technology.2,212,338 effective sequence was obtained from four habitats,the sequence was into 4043 OTUs?Operational Taxonomic Units?,belonging to 14 phyla,44 classes,108 orders,231 families,442 genera and 471species;Ascomycota was the dominant fungal phylum out of four habitats,and the dominant fungal genus was Fusarium;relative abundance of saprotroph was the highest among functional types,and relative abundance of plant pathogen was the highest among the functional groups;species index,Chao1 index and diversity index in Steppe region were the highest,reaching 1057,1423.84 and 6.12 respectively,while those in desert region were the lowest,being 657.33,884.57 and 4.46 respectively;fungal community structure among four habitats was significantly different?P<0.01?,the difference is extremely significant between desert region and closing protective region?P<0.001?;organic matter,total nitrogen,total phosphorus,available nitrogen and available potassium were the main soil characters that could affect the diversity of fungal community in sand-fixed plants'rhizosphere soil;organic matter,total nitrogen and available nitrogen were more sensitive to fungi community of rhizosphere soil in sand-fixed plants in different habitats.4.Isolation,identification and biological characters of Trichoderma spp.of rhizosphere soil in sand-fixed plantsTrichoderma M-33 strain was isolated and screened from rhizosphere soil in sand-fixed plants by the methods of dual culture,cultivating face to face and cultivating in cellophane film.It was identified by morphological characteristics and DNA analysis,and its'biological characters were studied through mycelial growth and spore counting.The results showed that Trichoderma M-33 was identified as Trichoderma harzianum,and the inhibition rates of crude fermentation extract,volatile metabolites and non-volatile metabolites of Trichoderma M-33 to R.solani were 74.51%,58.43%and 92.75%respectively;Trichoderma M-33 had low demand for nutrient and strong adaptability to environment.5.Antagonism mechanisms of T.harzianum M-33 against R.solaniThe antagonistic effect of T.harzianum M-33 against R.solani was unveiled by microscopic observation and determination of enzyme activity.The results showed that the hyphae of T.harzianum M-33 could grow on the medium and occupy nutrient space rapidly before contacting with R.solani;after the two fungi coming into contact,the hyphae of T.harzianum M-33 was attached and enwound to hyphae of R.solani,meanwhile,the secretions produced by T.harzianum M-33 could cause cell wall of hyphae of R.solani break and dissolve,and then the hyphae and conidia of T.harzianum M-33 could invade into the hyphae and sclerotia of R.solani,colonize in R.solani;R.solani could induce T.harzianum M-33 producing enzyme activity;maximum activity of chitinase(7.31U·?min·mL?^-1)and?-1,3-glucanase(1.63 U·?min·mL?^-1)were recorded at 5 days,and maximum activity of neutral protease(0.155 U·?min·mL?^-1)and cellulase(0.899U·?min·mL?^-1)were recorded at 6 days after inoculation in medium supplemented with R.solani;chitinase was the key enzyme in the antagonistic effect of T.harzianum M-33against R.solani.6.Effects of biological control against potato black scurf?R.solani?and on rhizosphere soil micr-ecosystem in treatment of T.harzianum M-33The optimum straw additives and inoculation quantity of T.harzianum M-33 were screened by spread-plate method.Then,by pot and field experiments,the biological control of T.harzianum M-33 applied together with wheat straw against potato black scurf was studied.And the impact of micro-ecosystem of potato's rhizosphere soil was further analyzed by dilution-plate method and high-throughput sequencing technology.The results showed that wheat straw was the optimum straw additives,and 1×10⁸ unit·g^-1 was the optimal inoculation quantity of T.harzianum M-33.The pot experiment indicated that the emergence rate of the treatment was 100%,the disease control effectwas 70.26%,and the plant height,stem diameter and branch numbers of potato treated with the treatment were 43cm,0.82 cm and 3.89,respectively,and all the four parameters were significantly higher than those of CK?P<0.01?;after the treatment,the number of rhizosphere soil fungi were reduced,while the number of rhizosphere soil bacteria,actinomycetes and Trichoderma genus were increased;the treatment had great impacts on fungal community diversity and community structure composition,but had small impacts on those of bacteria;the treatment could promote the accumulation of beneficial microbes in potato's rhizosphere soil.Field experiments verified that the treatment had better control efficiency against potato black scurf,and promoted potato's growth,increased the yield;after the treatment,relative abundance of Trichoderma fungi increased and reached at the peak in the adult stage,while the pathogens showed a decreasing trend in the whole growth period of potato.