In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was...In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was used to evaluate five factors（template,Mg2＋,dNTP,Taq DNA polymerase,and primer） from four levels.Subsequently,the optimal annealing temperature,annealing time,extension time and cycle numbers were evaluated.The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer,30 ng soil DNA template,3.0 mmol·L-1 Mg2＋,0.2 mmol·L-1 dNTPs,0.1 μmol·L-1 each forward and reverse primer,and 2.0 U Taq enzyme in 50 μL reaction volume.The optimal thermal cycling protocol consisted of initial melting at 94℃ for 5 min,followed by 35 cycles at 94℃ for 30 s,56℃ for 30 s,72℃ for 90 s,and a final extension of 72℃ for 10 min.展开更多
【Objectives】Si and microbial application could relieve the crop replanting problems(CRPs).We further studied the change of key microorganisms that are related to the beneficial effects,aiming at provide reference fo...【Objectives】Si and microbial application could relieve the crop replanting problems(CRPs).We further studied the change of key microorganisms that are related to the beneficial effects,aiming at provide reference for the manufacture and application of both microbial agents and Si fertilizer in food lily production.【Methods】A field experiment was conducted over a three-year period,from March 2019 to March 2022.The experimental field had been continuously cultivated with lily for 9 years.Three treatments were established:silicon fertilizer(SF),microbial agents(“Special 8^(TM)”,MF),and combined application of silicon fertilizer and microbial agents(SMF).A control group with blank soil(CK)was also included.At seedling stage of Lanzhou lilies in 2020 and 2021,the shoot and bulb dry weight,and the plant height and stem diameter of Lanzhou lilies were investigated for calculation of seedling index.In July 2020,20 plants were selected in each plot,and root zone soils were sampled at a depth of 20 cm,10 cm away from the roots,and then mixed to form a composite sample.The soil available Si and organic matter content were analyzed,and the fungal community structure and some specific microbial groups in soils were determined with high-throughput sequencing of ITS.【Results】All the three treatments significantly enhanced the lily plant growth and the seedling index,compared to CK.Besides,SF and MF treatments increased the relative abundances(RA)and diversity of fungal communities,and altered the community structures.The RA of some specific groups were found to be significantly correlated with the seedling index and/or soil available Si.Of them,the RA of the genera Fusarium,Dactylonectria,Humicola,Stilbella,and the species Humicola_grisea showed a positive correlation,while that of the genera Mortierella,Stilbella,Holtermanniella,and the species Mortierella_fatshederae showed a negative correlation with seedling index.The genera Fusarium,Stilbella,the species Humicola_grisea,and Dactylonectria_estremocensis showed a positive correlation,while the genura Stilbella,and the species Mortierella fatshederae showed a negative correlation with available Si content.In the co-occurence network of top twenty fungal genera and top sixteen bacterial genera(RA>0.2%),Holtermanniella was the only genus that interacted with the bacteria and negatively correlated with bacterial genus Blastococcus.Holtermanniella was also the most densely connected genera,followed by the genus Fusarium,Didymella and Humicola.In addition,the genus Holtermanniella was the key species connecting fungal and bacterial community in soil.Fungal functional prediction revealed that SF,MF and SMF treatments decreased plant pathogens guilds and increased the beneficial guilds Ectomycorrhizal,plant saprophyte,leaf saprophyte,and arbuscular mycorrhizal compared to CK.【Conclusions】Combined application of silicon fertilizer and microbial agents can alleviate continuous replanting problems of Lanzhou lilies through restoring the fungal community diversity,and promoting plant residue depredation,thus reducing soil born disease incidence.The beneficial genus Humicola and its one species H.grisea acts as bioconversion,and the genus Acremonium acts as plant pathogen inhibitor.展开更多
基金Supported by National Natural Science Foundation of China (30571264)Nation Key Technology Program (2006BAD07B03)
文摘In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was used to evaluate five factors（template,Mg2＋,dNTP,Taq DNA polymerase,and primer） from four levels.Subsequently,the optimal annealing temperature,annealing time,extension time and cycle numbers were evaluated.The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer,30 ng soil DNA template,3.0 mmol·L-1 Mg2＋,0.2 mmol·L-1 dNTPs,0.1 μmol·L-1 each forward and reverse primer,and 2.0 U Taq enzyme in 50 μL reaction volume.The optimal thermal cycling protocol consisted of initial melting at 94℃ for 5 min,followed by 35 cycles at 94℃ for 30 s,56℃ for 30 s,72℃ for 90 s,and a final extension of 72℃ for 10 min.
基金Key Research project of Gansu Province of China(22YF7NA108)National Natural Science Foundation of China(31860549)+1 种基金Industry Supporting Project from Education Department of Gansu Province(2023CYZC-49)Major Science and Technology project of Gansu province(24ZDNA006)。
文摘【Objectives】Si and microbial application could relieve the crop replanting problems(CRPs).We further studied the change of key microorganisms that are related to the beneficial effects,aiming at provide reference for the manufacture and application of both microbial agents and Si fertilizer in food lily production.【Methods】A field experiment was conducted over a three-year period,from March 2019 to March 2022.The experimental field had been continuously cultivated with lily for 9 years.Three treatments were established:silicon fertilizer(SF),microbial agents(“Special 8^(TM)”,MF),and combined application of silicon fertilizer and microbial agents(SMF).A control group with blank soil(CK)was also included.At seedling stage of Lanzhou lilies in 2020 and 2021,the shoot and bulb dry weight,and the plant height and stem diameter of Lanzhou lilies were investigated for calculation of seedling index.In July 2020,20 plants were selected in each plot,and root zone soils were sampled at a depth of 20 cm,10 cm away from the roots,and then mixed to form a composite sample.The soil available Si and organic matter content were analyzed,and the fungal community structure and some specific microbial groups in soils were determined with high-throughput sequencing of ITS.【Results】All the three treatments significantly enhanced the lily plant growth and the seedling index,compared to CK.Besides,SF and MF treatments increased the relative abundances(RA)and diversity of fungal communities,and altered the community structures.The RA of some specific groups were found to be significantly correlated with the seedling index and/or soil available Si.Of them,the RA of the genera Fusarium,Dactylonectria,Humicola,Stilbella,and the species Humicola_grisea showed a positive correlation,while that of the genera Mortierella,Stilbella,Holtermanniella,and the species Mortierella_fatshederae showed a negative correlation with seedling index.The genera Fusarium,Stilbella,the species Humicola_grisea,and Dactylonectria_estremocensis showed a positive correlation,while the genura Stilbella,and the species Mortierella fatshederae showed a negative correlation with available Si content.In the co-occurence network of top twenty fungal genera and top sixteen bacterial genera(RA>0.2%),Holtermanniella was the only genus that interacted with the bacteria and negatively correlated with bacterial genus Blastococcus.Holtermanniella was also the most densely connected genera,followed by the genus Fusarium,Didymella and Humicola.In addition,the genus Holtermanniella was the key species connecting fungal and bacterial community in soil.Fungal functional prediction revealed that SF,MF and SMF treatments decreased plant pathogens guilds and increased the beneficial guilds Ectomycorrhizal,plant saprophyte,leaf saprophyte,and arbuscular mycorrhizal compared to CK.【Conclusions】Combined application of silicon fertilizer and microbial agents can alleviate continuous replanting problems of Lanzhou lilies through restoring the fungal community diversity,and promoting plant residue depredation,thus reducing soil born disease incidence.The beneficial genus Humicola and its one species H.grisea acts as bioconversion,and the genus Acremonium acts as plant pathogen inhibitor.
