| Soybean cyst nematode(Heterodera glycines, Ichinohe) was one of major yield-limiting factors in the soybean producing. It was an important soil borne pathogens of soybean. So soybean continuous cropping would accumulate the cysts in soil, resulting in increase of the harm of soybean cyst nematode. Soybean cyst nematode was one of the main factors that causing the continuous cropping obstacles. The nature decline of soybean cyst nematode was happened in more than five years of soybean continuous cropping fields. This soil was called as soybean cyst nematode suppressive soil. In suppressive soil, parasitic microorganism-SCN-soybean formed a special environment suppressive ecology system. In this ecological system, parasitic fungi of soybean cyst nematode were suppressive factors, and played an important role. Aim to discuss the diversity and effect of soybean cyst nematode parasitic fungi in suppressive soil, the field of long term rotation system was tested. The community of soybean cyst nematode parasitic fungi, dominant fungal species, and pathogenicity were investigated by traditional isolati on, morphological identification methods combined with sequencing analysis, Biolog and PCR-DGGE methods. Meanwhile, according to the analysis of parasitic fungi community and the effect of dominant fungi in suppressive soil, the mechanism of the soybean cyst nematode suppressive soil was explored, and it had important value and significance on the research of suppressive ecology system and nematode ecology.Through the investigation of soybean cyst nematode population and soybean growth, we found that the density was significantly decreased in soybean monoculture for 23 years.There were just 7 cysts in per 100 gram soil, it was significantly less than soybean monoculture for 2years and there was no significant difference with rotation cropping in soybean flower ing stage and seed filling stage. The ratio of empty cyst number to total cyst number was 70% at soybean flowering stage and seed filling stage, and significantly increased in 23 years soybean monoculture. In soybean growth periods, population of soybean cyst nematode increased in 2years soybean monoculture and rotation cropping. But the density of soybean cyst nematode was no significant change in 23 years soybean monoculture. Condition of soybean growth was better and yield of soybean increased in 23 years soybean monoculture. It proved that this soybean monoculture field was soybean cyst nematode suppressive soil, and soybean cyst nematode population was controlled, especially in soybean flowering stage and seed filling stage. Besides, the growth of soybean was also better in 23 years soybean monoculture.The function of soybean cyst nematode parasitic microorganism was studied by Biolog method. The results showed that AWCD values of soybean cyst nematode parasitic microorganism increased in 2years soybean monoculture. However, there was no significant difference in 23 years soybean monoculture and rotation cropping, and evenness of carbon substrate utilization have significant difference in 23 years soybean monoculture. RDA demonstrated that substrate utilization patterns of the cyst microorganism communities changed in different rotation systems. The result of principal component analysis(PCA) revealed that ability of carbon substrate utilization was clearly divided into three groups. The carbon types including carbohydrates, amino acids, carboxylic acids and polymers caused this difference. It showed that functional diversity of soybean cyst nematode was positively correlated with soybean cyst nematode population, and community of soybean cyst nematode parasitic microorganism has changed.The results of DGGE showed that there were some differences in community of soybean cyst nematode parasitic fungi, and brightness of DGGE band. There were special bands in soybean monoculture for 23 years. Diversity of cyst parasitic fungi community was significantly increased in 23 years soybean monoculture and rotation cropping. The major bands were seq uenced, and tentatively identified by comparison with sequences available in GenBank. The result showed that these parasitic fungi mainly belonged to phylum of Ascomycota and Basidiomycota. The result of PCA revealed that the community of cyst parasitic fungi has changed in soybean long-term continuous cropping. Parasitic fungi of 23 years soybean monoculture included Acremonium sclerotigenum, Paraphoma radicina, Mortierella elongate, Verticillium chlamydosporium and Galactomyces candidum. Parasitic fungi of 2 years soybean monoculture included Paraphoma chrysanthemicola, Cylindrocarpon sp., Neonectria ramulariae, Fusarium oxysporum and Galactomyces candidum. It was proof that the community of cyst parasitic fungi was significantly changed and dominant fungi were formed, after soybean long-term continuous cropping.The dominant and special cyst parasitic fungi were identified by morphological identification and sequencing analysis. The result showed that they were Fusarium oxysporum, Verticillium chlamydosporium, Alternaria alternate, Paraphoma radicina, Neonectria radicicola, Dendryphion nanum, Fusarium verticillioides, Trichocladium opacum, Mortierella elongate, Paecilomyces lilacinus, Penicillium polonicum, Aspergillus fumigatus, Aspergillus versicolor and Aspergillus niger. They were potential bio-control fungi of soybean cyst nematode.Fusarium spp., Verticillium chlamydosporium and Paecilomyces lilacinus were isolated from 23 years soybean monoculture in the whole soybean growth period, and the number of these parasitic fungi was significantly increased, when compared with 2 years soybean monoculture and rotation cropping. Isolation rates of egg parasitic fungi were 1.3% and 2.6% from 23 years soybean monoculture cropping in flowering stage and seed filling stage, respectively. There were no egg parasitic fungi isolated from 2years soybean monoculture in soybean flower ing stage, and just only 0.1% egg parasitic fungi was isolated in soybean seed filling stage. Isolation rate s of egg parasitic fungi were 0.4% and 0.8% from rotation cropping in flowering stage and seed filling stage, respectively. The number of soybean cyst nematode parasitic fungi was significantly increased from 23 years soybean monoculture cropping in flower ing stage and seed filling stage. Dominant parasitic fungi of soybean cyst nematode were negatively correlated with population of soybean cyst nematode. Therefore, soybean cyst nematode parasitic fungi were related to natur al decline of SCN.The pathogenicity of dominant parasitic fungi was tested. The result showed that there were significant differences among these parasitic fungi and different strains. The strains H-4-E-14, G-4-E-16 and 4-G-C-A of Fusarium spp. were better to control soybean cyst nematode, and the percentage of suppressive effect was more than 90%. The suppressive effect of G-4-E-16 was 90.6% on egg of SCN. The suppressive effect of 4-G-C-A was 94% on J2 of SCN. 4-H-C-12 strain of Verticillium chlamydosporium was better to control cyst of SCN, suppressive effect of 4-G-C-6 strain was 81.6% on egg of SCN, and suppressive rate of 4-H-C-12 was 96% for J2 of SCN. 17-HC-15 strain of Paecilomyces lilacinus was better(suppressive rate was96.8%) to control cyst of SCN, 4-G-C-10 strain was better to control egg of SCN, and the percentage of suppressive effect was 92% on J2 of SCN. These soybean cyst nematode parasitic fungi can inhibit the population of SCN.In conclusion, soybean long-term continuous cropping formed soybean cyst nematode suppressive soil, and could control the population of soybean cyst nematode. It also could improve the soybean growth and yield of soybean. After soybean long-term continuous cropping, community of SCN parasitic fungi was changed, parasitic fungi were accumulated, and dominant parasitic fungi were formed. |
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