【作者】 孙莉；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养学， 2015， 硕士

【摘要】 枯萎病是一种典型的土传病害,其病原菌为尖孢镰刀菌(Fusarium oxysporum),该病的防治一直是瓜类生产研究的重点与难点。目前,生物防治和土壤熏蒸已成为克服土传植物病害的重要途径之一,日益受到国内外研究者的重视。本研究首先评估了碳酸氢铵和石灰作为土壤熏蒸剂防治尖孢镰刀菌病害的可行性。扫描电子显微镜观察结果显示,碳酸氢铵对尖孢镰刀菌F.oxysporum f.sp.niveum(FON)和F.oxysporum f.sp.cucumerinum(FOC)菌丝生长具有一定的破坏作用。土壤培养试验进一步表明,碳酸氢铵对尖孢镰刀菌的生长具有抑制作用,土壤中施用熏蒸剂的最佳用量为:西瓜枯萎病:碳酸氢铵2g·kg-1,石灰2g·kg-1;黄瓜枯萎病:碳酸氢铵2 g·kg-1,石灰4 g·kg-1。碳酸氢铵和石灰混合物进行土壤熏蒸7天后,土壤中的尖孢镰刀菌FON和FOC的数量减少近90%。此外,土壤熏蒸剂在正常的室外温度和土壤含水量中都能较好的发挥其对尖孢镰刀菌的杀灭作用。利用拮抗菌SQR-9制成的微生物有机肥,联合石灰和碳铵对土壤的熏蒸,在盆栽和大田进行了防治西瓜和黄瓜枯萎病试验。盆栽和大田试验均设置6个处理:1)LAB-BOF:在移栽前对土壤用碳酸氢铵和石灰(西瓜:碳酸氢铵2 g·kg-1 +石灰2 g·kg-1;黄瓜:碳酸氢铵2 g·kg-1 +石灰4 g·kg-1)进行薄膜熏蒸处理10天,揭膜使土壤透气3天,然后施用微生物有机肥;2)LAB-OF:熏蒸处理同LAB-BOF,然后施用普通有机肥;3)AB-BOF:在移栽前对土壤施用碳酸氢铵(碳酸氢铵2 g·kg-1)进行薄膜熏蒸处理10天,揭膜使土壤透气3天,然后施用微生物有机肥;4)AB-OF:熏蒸处理同AB-BOF,然后施用普通有机肥;5)CK-BOF:不进行熏蒸处理,施用微生物有机肥;6)CK-OF:不进行熏蒸处理,施用普通有机肥。盆栽试验结果表明,单独施用微生物有机肥对西瓜和黄瓜枯萎病的防控率分别为22.22%和14.29%。土壤熏蒸联合微生物有机肥的施用,西瓜和黄瓜枯萎病的防控率较单独使用微生物有机肥显著提高,其中土壤经碳酸氢铵和石灰混合物熏蒸后施用微生物有机肥处理的防控率最高,分别达到88.88%和85.72%。可培养微生物数量统计结果表明,碳酸氢铵和石灰混合物熏蒸后土壤中的FOC和FON数量分别降低71.3 8%和95.50%;碳酸氢铵熏蒸后土壤中的FOC和FON数量分别降低64.3%和87.31%。熏蒸处理后土壤中可培养微生物的数量显著降低,并且碳酸氢铵和石灰混合物熏蒸处理后的土壤中可培养真菌、细菌和放线菌的数量最低。理化性质测定结果表明,熏蒸后土壤pH值升高,总氮、铵态氮和硝态氮含量均升高,而土壤中总碳含量降低。此外,土壤熏蒸对西瓜和黄瓜植株生长没有显著影响,而微生物有机肥还可以在一定程度上促进植株的生长。田间试验结果表明,单独施用微生物有机肥对西瓜和黄瓜枯萎病的防控率分别为20%和19.62%;碳酸氢铵和石灰熏蒸处理后再施用微生物有机肥对西瓜和黄瓜枯萎病的防控率分别为96%和58.83%。碳酸氢铵和石灰混合物熏蒸处理和碳酸氢铵熏蒸处理土壤中病原菌FON的数量分别显著性降低了 80.47%和63.42%。碳酸氢铵和石灰混合物熏蒸处理土壤中的FOC的数量降低了 73.13%。为进一步研究土壤熏蒸联合微生物有机肥的施用对西瓜和黄瓜大田土壤种植过程中土壤微生物区系的影响及研究所形成微生物区系和枯萎病之间的关系,MiSeq高通量测序技术被用来分析了微生物区系的变化。试验结果表明,黄瓜大田土壤熏蒸后,变形菌门(Proteobacteria)、芽单胞菌门(Gemmatimonadetes)、疣微菌门(Verrucomicrobia)、酸杆菌门(Acidobacteria)、厚壁菌门(Firmicutes)、担子菌门(Basidiomycota)和接合菌门(Zygomycota)的相对丰度较高,而拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、子囊菌门(Ascomycota)和壶菌门(Chytridiomycota)的相对丰度较低。西瓜大田土壤熏蒸后,土壤中拟杆菌门(Bacteroidetes)、芽单胞菌门(Gemmatimonadtes)、奇异球菌-栖热菌门(Deinococcus-Thermus)、Parcubacteria、接合菌门(Zygomycota)和担子菌门(Basidiomycota)的相对丰度显著增加,而变形菌门(Proteobacteris)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、子囊菌门(Ascomycota)和壶菌门(Chytridiomycota)的相对丰度显著降低。与黄瓜枯萎病发病率呈负相关的门类有芽单胞菌门(Gemmatimonadetes)、疣微菌门(Verrucomicrobia)和接合菌门(Zygomycota),呈正相关的门类有变形菌门(Proteobacteria)、衣原体门(Chlamydiae)、子囊菌门(Ascomycota)和担子菌门(Basidiomycota);呈负相关的属类有Gemmatimonas和Mortierella,呈正相关的属类有 Simkania、Ktedonobacter、Geomyces、Cladosporium、Fusarium、Thielavia、Sarocladium、Botryotinia、Scedosporium、Micromonosporineae、Phycisphaera和Wallemia。与西瓜枯萎病发病率呈负相关的门类有绿弯菌门(Chloroflexi)、芽单胞菌门(Gemmatimonadetes)、Parcubacteria、奇异球菌-栖热菌门(Deinococcus-Thermus)和厚壁菌门(Firmicutes),呈正相关的门类有酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)、浮霉菌门(Planctomycetes)和子囊菌门(Ascomycota),呈负相关的属类有Gemmatimonas、Bellilinea、Kriegella、Blastopirellula和Conexibacteraceae,呈正相关的属类有Polyanguaceae、Propionibacterineae、Skermanella、Emericellopsis、Fusarium、Gibberella、Sarocladium、Humicola、Gibellulopsis、Aspergillus、Diaporthe 和 Guehomyces。综上所述,土壤熏蒸联合微生物有机肥的施用能够有效抑制枯萎病的发生,并且改变土壤微生物群落结构和土壤理化性质。通过增加有益微生物,减少有害微生物,使得土壤向一个健康的方向发展。更多还原

【Abstract】 Fusarium wilt diseases,caused by formae speciales of the fungus Fusarium oxysporum are a serious problem for the production of cucumber,watermelon and other plants around the world.Soil pretreatment,such as soil fumigation,has been widely used to control weeds,plant parasitic nematodes,soil-borne fungi,insects,and other disease organisms in high-value agricultural production systems.Moreover,many studies have demonstrated that biocontrol is an effective strategy for controlling soil-borne diseases.In this study,effects of combined utilization of soil fumigation and bio-organic fertilizer on Fusarium wilt disease of cucumber and watermelon were investigated.The fumigation agent ammonium bicarbonate,which releases ammonia and showed an inhibitory effect on growth of Fusarium oxysporum in the cultural condition,was investigated when applied alone and with lime as a direct soil amendment under sealed conditions.For the Fusarium oxysporum f.sp.cucumerinum(FOC),and Fusarium oxysporum f.sp.niveum(FON),the suitable additive concentrations of ammonium bicarbonate was 2 g·kg-1 soil dry weight(DW),when applied alone.Ammonium bicarbonate had a stronger antifungal effect when mixed with lime(2 g·kg-1 for FOC;4 g·kg-1 for FON)than when applied alone in the pot experiments.In the field experiment,stronger antifungal effects were observed when the pH value of the soil was less than 7,and no significant difference between the two treatments was observed in alkaline soil.Moreover,these concentrations produced the best effects when short processing times,broad temperature ranges and wide soil moisture contents were used.Effects of combined utilization of the novel soil fumigation strategy and application of bio-organic fertilizer produced by Bacillus amyloliquefaciens SQR-9 on Fusarium wilt disease of cucumber and watermelon were investigated in the greenhouse and field experiments.Pot and field experiments were set up six treatments as following:1)LAB-BOF:the soil was treated with ammonium bicarbonate and lime before the application of the bio-organic fertilizer(BOF);2)LAB-OF:the soil was treated with ammonium bicarbonate and lime before the application of the common organic fertilizer(OF);3)AB-BOF:the soil was treated with ammonium bicarbonate before the application of the bio-organic fertilizer(BOF);4)AB-OF:the soil was treated with ammonium bicarbonate before the application of the common organic fertilizer(OF);5)CK-BOF:the soil was applied with bio-organic fertilizer(BOF);6)CK-OF:the soil was applied with common organic fertilizer(OF).Results from the pot experiment showed that compared to the control,disease control efficiency on watermelon and cucumber after application of bio-organic fertilizer was 22.22%and 14.29%,respectively,while combined application of bio-organic fertilizer and soil fumigation significantly improved the control efficiency,among which LAB-BOF treatments showed highest control efficiency of 88.88%for watermelon and 85.72%for cucumber,respectively.Based on cultural-dependent method,the number of FOC and FON were reduced by 71.38%and 95.50%after soil fumigation with ammonium bicarbonate and lime mixture which also showed the lowest number of culturable fungi,bacteria and actinomycetes and decreased by 64.3%and 87.31%after soil fumigation with ammonium bicarbonate.Results from physical and chemical properties showed that soil pH,total nitrogen,ammonium nitrogen and nitrate nitrogen contents were improved after fumigation while the content of total organic carbon reduced.In addition,no significant effects on the watermelon and cucumber plants growth were observed after soil fumigation and the bio-organic fertilizer can promote the growth of plants.Field experiment results showed that compared to the control,disease control efficiency on watermelon and cucumber after application of bio-organic fertilizer was were 20%and 19.62%respectively.Combined application of bio-organic fertilizer and soil fumigation with a mixture of ammonium bicarbonate and lime showed efficient control efficiency of 96%or watermelon and 58.83%for cucumber,respectively.The numbers of FON were reduced by 80.47%and 63.42%after soil fumigation with ammonium bicarbonate alone and mixed with lime,respectively.While the number of FOC decreased by 73.13%after fumigation with the mixture.In order to further study the relationship between soil microflora and Fusarium wilt disease,soil microflora in samples collected from the watermelon and cucumber fields were investigated by MiSeq high-throughput sequencing technology.Results showed that,in cucumber field,compared to the control,the relative abundances of Proteobacteria,Gemmatimonadetes,Verrucomicrobia,Acidobacteria,Firmicutes,Basidiomycota and Zygomycota were improved,while the value for Bacteroidetes,Actinobacteria,Ascomycota and Chytridiomycota were decreased after soil fumigation.In watermelon field,compared to the control,the relative abundances of Bacteroidetes,Gemmatimonadetes,Deinococcus-Thermus,Parcubacteria,Zygomycota and Basidiomycota were improved,while the value for Proteobacteria,Acidobacteria,Actinobacteria,Ascomycota and Chytridiomycota were decreased after soil fumigation.Among these phyla,a strong negative correlation between Gemmatimonadetes,Verrucomicrobia and Zygomycota and the disease incidence of cucumber and a clear positive correlation between Proteobacteria,Chlamydiae,Ascomycota and Basidiomycota and the disease incidence of cucumber were observed.Among these genus,a strong negative correlation between Gemmatimonas and Mortierella and the disease incidence of cucumber and a clear positive correlation between Simkania,Ktedonobacter,Geomyces,Cladosporium,Fusarium,Thielavia,Sarocladium,Botryotinia,Scedosporium,Micromonosporineae,Phycisphaera and Wallemia and the disease incidence of cucumber were observed.Among these phyla,a strong negative correlation between Chloroflexi,Gemmatimonadetes,Parcubacteria,Deinococcus-Thermus and Firmicutes and the disease incidence of watermelon and a clear positive correlation between Acidobacteria,Actinobacteria,Verrucomicrobia,Planctomycetes and Ascomycota and the disease incidence of watermelon were observed.Among these genus,a strong negative correlation between Gemmatimonas,Bellilinea,Kriegella,Blastopirellula and Conexibacteraceae and the disease incidence of watermelon and a clear positive correlation between Polyangiaceae,Propionibacterineae,Skermanella,Emericellopsis,Fusarium,Gibberella,Sarocladium,Humicola,Gibellulopsis,Aspergillus,Diaporthe and Guehomyces and the disease incidence of watermelon were observed.In summary,soil fumigation effectively suppressed the occurrence of Fusarium wilt disease for watermelon and cucumber and altered the soil microbial community structure and soil properties.更多还原