| Rapeseed(Brassica napus)is one of the important oil crops in China.Sclerotinia stem rot(SSR),caused by Sclerotinia sclerotiorum,is a major fungal disease that affects the yield,quality and economic benefits of rapeseed.S.sclerotiorum exists in the soil over summer and winter mainly in the form of the sclerotium.Therefore,the soil microbial environment has an important impact on the survival of S.sclerortiorum.Conothyrium minitans is a mycoparasite of S.sclerotiorum and has a similar temperature requirement to S.sclerotiorum,which makes it a potential application in the biocontrol of SSR.In previous study,the growth promotion effect of C.minitans was significant in the field with low SSR disease incidence.It indicates that C.minitans may also have unknown functions besides parasitic hyphae and sclerotia of S.sclerotiorum.In this study,the microbiome of sclerotium-sphere parasitized by C.minitans sclerotia parasitized by C.minitans were elucidated with microbial culture and amplicon sequencing.The microbial strains with significantly increased abundance and beneficial to rapeseed were further screened,and their promotion effect on the rot of sclerotia by C.minitans was verified.Soil from rapeseed field was selected and five treatments were set:(1)As a control,the soil was inoculated with neither C.minitans nor sclerotia;(2)The sclerotium-sphere soil treated with C.minitans;(3)Soil treated with C.minitans spores(1×10~6 spore/m L);(4)Sclerotium-sphere soil treated with C.minitans spores(1×10~6 spore/m L);(5)Sclerotium-sphere soil treated with C.minitans spores(1×10~7 spore/m L).The soil samples were then incubated at 20°C and 10 replicates were set up for each treatment.The sclerotium-sphere soil was obtained at the early(7 d),the middle(14 d),the late(30 d),and the last(40 d)stages.The DNA was extracted and libraries were constructed,sequenced,and analyzed to elucidate the effects of C.minitans on the soil microbiota around the sclerotia and the abundance of C.minitans in the soil.The results showed that in the soil of rapeseed fields inoculated with neither sclerotia nor C.minitans,there were no enriched soil fungi with higher abundance,and a total of 57 common genera of fungi were found at the four stages.Among them,the genera Talaromyces,Fusarium,and Setophoma were the common ones with higher abundance.The sclerotium-sphere soil inoculated with C.minitans enriched with different abundances of Sclerotiniaceae,Trichodema and Talaromyces at the early stage,at the middle stage enriched with different abundances of Trichodema,Clonostachys and Sclerotiniaceae,at the late stage enriched with different abundances of Sclerotiniaceae,Clonostachys and Talaromyces and at the last stage enriched with different abundances of Trichodema,Sclerotiniaceae and Talaromyces.A total of 59common genera of fungi were detected in the four stages.Among them,the Sclerotiniaceae,Trichodema and Clonostachys were the common groups with higher abundance.Soil treated with Coniothyrium 1×10~6 spore/m L enriched with different abundances of C.minitans,Talaromyces and Chaetomiaceae at the early stage,at the middle stage enriched with different abundances of C.minitans,unidentified fungi and Chaetomiaceae,and at the late and last stage enriched with different abundances of C.minitans and unidentified fungi.A total of 48 common genera of fungi were detected in the four stages.Among them,the genera C.minitans,unidentified fungi and Talaromyces were the common genera with higher abundance.Sclerotium-sphere soil inoculated with C.minitans 1×10~6 spore/m L and 1×10~7 spore/m L,respectively.At the early stage a large number of Trichoderma was enriched.Although Trichoderma and Clonostachys were enriched at the middle,late and last stages,Trichoderma was most enriched at the middle stage,the lowest abundance at the late stage and Clonostachys was enriched with the highest abundance at the late stage.A total of 60 common genera of fungi were found in the four stages.Among them,the genera Trichoderma,Clonostachys and Talaromyces were the common genera with higher abundance.In the structure of bacterial communities,the soil which was inoculated with neither C.minitans nor sclerotia enriched with different abundances of Sphingomonas,Comamonadaceae and SC-I-84 at the early stage.In the middle stage different abundances of Sphingomonas,Intrasporangium and SC-I-84 were enriched.In the late stage different abundances of Sphingomonas,Commatimonas and SC-I-84 were enriched.In the last stage different abundances of Sphingomonas and SC-I-84 were enriched.A total of 108 common genera of bacteria were found in the four stages.Among them,the genera SC-I-84,Sphingomonas,and Commatimonas were the common genera with higher abundance.The sclerotium-sphere soil inoculated with Coniothyrium enriched different abundances of Massili,Burkholderia-Caballeronia-Paraburkholderia and Sphingomonas in the early stage.In the middle stage different abundances of Burkholderia-Caballeronia-Paraburkholderia,Intrasporangium and Gemmatimonas were enriched.In the late stage different abundances of SC-I-84,Sphingomonas,Gemmatimonas were enriched.In the last stage,different abundances of Sphingomonas,Comamonadaceae and SC-I-84 were enriched.A total of 102common genera of bacteria were found in the four stages.Among them,the genera Commatimonas,Sphingomonas,and Burkholderia-Caballeronia-Paraburkholderia were the common genera with higher abundance.Sclerotium-sphere soil inoculated with Coniothyrium 1×10~6 spore/m L enriched with different abundances of Burkholderia-Caballeronia-Paraburkholderia in the early stage.In the middle stage,differentabundancesofBurkholderia-Caballeronia-Paraburkholderia,Comamonadaceae and Dyella were enriched.In the late stage different abundances of Burkholderia-Caballeronia-Paraburkholderia,Dyella and Sphingomonas were enriched.A total of 82 common genera of bacteria were found in the four stages.Among them,the genera Burkholderia-Caballeronia-Paraburkholderia,Sphingomonas and Grmmatimonas were the common genera with higher abundance.Whatever in the early,middle and late stages,Sclerotium-sphere soil inoculated with Coniothyrium1×10~6spore/m L and 1×10~7spore/m L,respectively was enriched with Burkholderia-Caballeronia-Paraburkholderia which was the most abundant compared to other enriched species.No apparently abundant genera were found in later stages.A total of104 common genera of bacteria were found in the four stages.Among them,the genera Burkholderia-Caballeronia-Paraburkholderia and Dyella were the common genera with higher abundance.The high-abundance microorganisms were isolated using plate dilution method and explore their effect on sclerotia,and the isolated high-abundance microorganisms were prepared into 1×10~6 spore/m L spores solution and bacterial solution,treated sclerotia with them,buried in the soil,and observed the degree of decay at 14 and 30days.It was found that the sclerotia treated with Burkholder’s bacteria had no significant difference on the rot index of the sclerotia at either 14 or 30 days.The sclerotium rot index was the highest when combining C.minitans with Trichoderma,which indicates that the soil microbiota of sclerotium-sphere may be altered by the mycoparasitism of C.minitans against sclerotia.Soil microbiota of sclerotium-sphere introduced with C.minitans can enrich higher beneficial microbial Trichoderma,and this change may accelerate the decay of sclerotia,which is beneficial to the prevention and control of SSR and promotes the formation of healthy rhizosphere microflora in rapeseed,thereby promoting the healthy growth of rapeseed.In summary,this study found that the parasitical process of S.sclerotiorum by C.minitans caused the enrichment of beneficial microorganisms.Strains of Trichoderma spp and Burkholderia spp were isolated,laying the foundation for creating a composite microbial agent for biological control of rapeseed stem rot. |
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