| Seed endophytic microorganisms settled in the seed coat,embryo,endosperm and other niches,which can not only directly promote seed germination and seedling establishment,but also prevent plant diseases by producing secondary metabolites,and a few microorganisms also have adverse effects on seed germination and plant growth.At present,research on seed endophytic microorganisms is mostly focused on crops such as rice,corn and wheat,while research on plant seed endophytic bacteria in rocky desertification areas is relatively rare.The area of rocky desertification in Yunnan Province ranks second in China.Soil inoculant is one of effective methods to control rocky desertification and promote ecosystem recovery.Soil inoculants contain seeds of a variety of plants,and microorganisms in the seeds play an important role in seed germination and seedling growth.In this study,the endophytic microbial diversity of plant seeds in secondary forests around seven rocky desertification regions were investigated,and the seed germination promotion and plant pathogenicity of dominant genera were evaluated.The main results are as follows:1.Diversity of endophytic fungi in seedsThe diversity of endophytic fungi in 34 species of 37 plant seeds was studied by culture-independent method.The results showed that 75.32% of the sequences can be identified to the level below the phylum fungi,and these sequences belong to 7 phyla:Ascomycota,Basidiomycota,Cryptomycota,Mortierellomycota,Chytridiomycota,Mucoromycota,Glomeromycota,26 classes,57 orders,108 families and 135 genera.The dominant genera were Eremothecium,Thermomyces,and Aureobasidium.Cryptomycota and Archaeorhizomycetes were first found in plant seeds,and Archaeorhizomycetes in seeds represented a brand-new branch of this class.Alpha diversity showed that Smilax glabra had the largest number of endophytic fungi species,while Nitraria sibirica had the lowest diversity.A total of 437 strains of fungi were isolated by culture method,belonging to 36 genera,23 families,14 orders,7 classes and 2 phyla: Ascomycota and Basidiomycota,and the dominant genus was Alternaria(130 strains)of Ascomycota.The isolation frequency and population diversity of endophytic fungi in different plant seeds are very different,and the isolation frequency of endophytic fungi in Rhamnus rhamnoides seeds is the highest,which is 7.88%.The separation rate of Solanum nigrum,Cynanchum komarovii and Cynanchum komarovii is the lowest,which is 0.23%.The number of endophytic fungi detected by the culture-independent method was about fourfold that by the culture independent method,and the five phyla detected by the culture-independent method were not obtained in the culture.In contrast,the fungi belonging to 11 families and 11 genera of the 2 classes(Agaricomycotin and Exobasidiomycetes)that could be isolated in culture were not detected in high-throughput sequencing,suggesting that the combination of the two methods was more accurate for the investigation of fungal diversity.2.Diversity of endophytic bacteria in seedUsing the culture-independent technique to study the endophytic bacteria of 37 plant seed,the results showed that 2.09% of Archaea and 97.91% of Bacteria.Archaea belonged to 2 phyla: Thaumarhaeota and Euryarchaeota,2 classes,4 orders,4 families and 2 genera.The Bacterial community belonged to 15 phyla,45 classes,91 orders,104 families,and 153 genera,among which Proteobacteria is the common dominant phyla of endophytic bacteria in different seed samples,Pantoea,Ralstonia and Pseudomonas are the common dominant genera.The Alpha diversity of Lycium barbarum is the highest,while that of Rhamnus rhamnoides collected from Qinglong town in the same place is the lowest.In addition,Patescibacteria is mostly isolated from water resources and air,and this phylum was identified from plant seeds for the first time in this study.3.Seed endophytic fungi can promote germinationIn order to evaluate the beneficial effects of seed endophytic fungi on plants,129 endophytic fungi belonging to the dominant genera were tested for promoting tomato seed germination.It was found that 69.77% strains had the potential of promoting germination,but the ability of promoting germination of various seeds was different.Only12.22% strains can promote the germination of cucumber and radish seeds,and 15.56%strains can promote the germination of Chinese cabbage seeds.These germination promoting strains are mainly distributed in Alternaria,Didymella,Epicoccum and Fusarium.At the same time,under the condition of mild drought(simulated by 5% PEG-6000solution),19 strains can promote the germination of tomato seeds,which belong to 9genera: Alternaria,Botrytis,Coniothyrium,Didymella,Epicoccum,Fusarium,Pestalotiopsis,Phoma and Sarocladium.Three strains with the best effect on promoting the germination of tomato seeds were selected for further study.The fermentation broth of Y4-4(D.coffeae-arabicae),L16-1(S.Zeae)and L13-6(Phoma sp.)with dilution times of 80,60 and 60 times respectively had the best effect on promoting the germination of tomato seeds,and the germination potential,germination index and vigor were the best.Meanwhile,the three strains could promote the germination of tomato seeds under different degrees of drought stress.The liquid fermentation products of these three endophytic fungi were analyzed by LCMS.Compared with the fermentation products of strains L13-5(D.americana)and Y2-6(P.crystalifera),the up-regulated compounds in the fermentation products of these three strains were mostly amino acids and their derivatives,ketones,ethers and other compounds which can promote germination.4.Pathogenicity of endophytic fungi in seeds129 strains of dominant genera were selected and tested for pathogenicity.Only 15strains(11.63%)had different degrees of pathogenicity on tomato leaves in vitro,belonging to Alternaria,Diaporthe,Epicoccum,Fusarium,Nothophoma and Phoma.At the same time,it shows that seed endophytic fungi have more beneficial effects on plants. |
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