【作者】 曹云；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养学， 2011， 博士

【摘要】 黄瓜枯萎病是一种土传病害,由致病性的尖孢镰刀菌黄瓜专化型Fusariutm. oxysporum f. sp. cucumerinum J. H. Owen (FOC)引起,发病严重时田间病株率达50%以上,已成为困扰设施农业的重要病害。目前防治此类病害主要用化学农药,但化学农药的防治易造成环境污染、农药残留等问题,所以采用安全、高效、环境友好型生物防治成为有前景的另一选择。单纯使用菌株制剂抑制土传病害的效果往往有限,利用优质有机肥做载体,复合几种拮抗菌二次发酵后生产的微生物有机肥料,对连作障碍中枯萎病的生防效果较好。本文从拮抗菌的筛选鉴定、抗菌物质的分离提纯、定殖情况、诱导植物产生系统抗性、土壤微生物区系的变化等方面对微生物有机肥防治黄瓜枯萎病进行了较为系统的研究。采用传统的形态、生理生化特性分析和分子生物学的方法对筛选的拮抗细菌SQR9进行了鉴定,确定其为枯草芽孢杆菌(Bacillus subtilis)。平板对峙实验表明菌株SQR9对黄瓜枯萎病致病菌尖孢镰刀菌的生长有抑制作用。以菌株SQR 9的基因组DNA为模板PCR扩增得到11个与脂肽抗生素surfactin, fengycin, iturin, bacillomycin, subtilosin, subtilisin的合成相关的基因。菌株SQR 9的发酵液进行酸沉淀、甲醇抽提后的粗提液稀释2倍,对尖孢镰刀菌菌丝的生长抑制率为54.9%,稀释1倍和未经稀释的粗提液,使黄瓜枯萎病病原菌孢子萌发率分别比对照降低了70.1%、83.6%。粗提液对多种土传病原菌有强烈的抑制作用。HPLC-ESI-MS分析表明,粗提液中检测出质荷比(M+H)+为1063.6、1017.5、1031.6、1045.6、1059.6以及1506.0、1435.0、1449.9、1463.9、1492.9、1477.9的两组物质,分别为bacillomycin和fengycin的同系物。将拮抗菌SQR 9与Trichoderma harzianum SQR T-037接种于合适的有机肥(OF)载体中,进行二次固体发酵,获得了3种分别含SQR 9 (BIO A)、含SQR T-037 (BIOB)、含SQR 9和SQR T-037 (BIO AB)的微生物有机肥。盆栽试验采用营养钵育苗和盆钵中同时添加肥料的方法,比较了有机肥与3种微生物有机肥防治黄瓜枯萎病的效果差异。结果表明,黄瓜移栽后60 d,3种生物有机肥处理的植株的地上部生物量提高了1.9-2.3倍,根系生物量平均提高了2.1倍,株高平均增加了72.7%。有机肥对黄瓜生长的促进作用不及微生物有机肥。对照黄瓜枯萎病的发病率最高(73%),BIOAB处理的发病率最低,仅为15%。施用有机肥处理的黄瓜发病率比对照降低了43.2%。采用Komada选择性培养基对根际土壤尖孢镰刀菌计数表明,黄瓜移栽后第60天,对照根际土的病原菌数量显著高于其它处理,达1.48×105CFU·g-1(根鲜重)。单施有机肥的黄瓜根际土的病原菌降低了84.6%,BIO AB处理的黄瓜根际土内的病原菌数量最低(1.2×103CFU·g-1(根鲜重))。用尖孢镰刀菌黄瓜转化型的特异引物扩增其基因组的一段DNA片段,对F. oxysporum数量进行Real-time PCR定量结果可知,各处理根际土的DNA拷贝数在3.4×105-1.32×107copy·g-1(根鲜重)之间,定量PCR的计数与传统的选择性培养基计数法所得到的结果呈现相同的规律,即施用微生物有机肥处理的根际土待测DNA拷贝数比对照显著降低,3种微生物有机肥处理的黄瓜根际土壤的病原菌DNA片段的拷贝数比对照低2个数量级。采用枯草芽孢杆菌选择性培养基在未接种的对照(CK)、有机肥(OF)、生物有机肥B (BIO B)处理的黄瓜根际土中没有分离出符合SQR 9特征的菌落,而在BIO A和BIO AB处理的植株根际能分别回收到6.03×104、1.62×104CFU·g-1(根鲜重)的SQR 9。用哈茨木霉的专用引物进行定量PCR扩增结果看来,未接种处理的土壤哈茨木霉数量为102-103copy·g-1(根鲜重),BIO B和BIO AB处理的木霉增殖至4.9-5.0 log copy·g-1 (根鲜重),表明B. subtilisSQR 9和T. harzianum SQR-T037已经在根际土成功定殖,因而能有效地阻止病原菌的入侵。盆栽试验还发现,待黄瓜植株枯萎病开始发病时(移栽后30 d),施用有机肥和生物有机肥处理的黄瓜叶片内的MDA含量比对照显著降低,生物有机肥处理的黄瓜过氧化氢酶(CAT)比对照提高了35.4-59.0%,过氧化物酶(POD)活性比对照提高了46.1-86.8%,超氧化物酶(SOD)活性平均提高了33.3%,苯丙氨酸解氨酶(PAL)活性提高了71.2-109.3%；微生物有机肥的施用还提高了黄瓜叶片中β-1,3-葡聚糖酶和几丁质酶的活性,两者比对照分别平均提高了58.5-86.5%、52.1-71.4%。定殖实验中,通过电转化的方法成功获得了具GFP标记和卡那霉素抗性的转化子SQR9-gfp,菌株SQR 9-gfp可以通过蘸根的方法在自然土壤中成功定殖,其在黄瓜根表的定殖数量最多(106-107CFU·g-1根),其次为根际土壤、根内,土体土中数量最少。菌体在根表和根际土的定殖有利于其在根表形成“防御层”抵御病原菌的侵染。主要定殖部位为主根伸长区和根毛区、主根与侧根的分岔处,根尖部分定殖很少,这可能是因为这些部位根系分泌物的组成与浓度有关,也表明菌体可能是在根形成后迁移过去的,而不是随根尖生长点扩散的。对BIO AB, OF和CK 3个土壤样品细菌16S rDNA进行454高通量测序,结果表明3个土壤样品的细菌群落构成相似,均以厚壁菌门(Firmicutes)含量最多、其次为变形菌门(Proteobacteria)、再次为放线菌门(Actinobacteria)。但不同处理间某些菌群数量有差异,样品BIO AB和OF中芽孢杆菌纲内的属含量最高的3个属都依次是Bacillaceae、Paenibacillaceae和Planococcaceae,而对照样品土壤中芽孢杆菌纲内的属含量最高的3个属依次是Bacillaceae. Paenibacillaceae和Clostridiaceae。BIO AB和OF中Bacillaceae的含量分别达37.82%和43.55%,而对照样品中Bacillaceae的含量则仅有20.51%。在属水平上,有机肥和生物有机肥中Bacillus、Methylocystaceae、Paenibacillus、Actinomadura和Actinoalloteichus属为优势菌,而对照土壤样品中的优势菌则为Bacillus, Asticcacaulis, Haliangium和Clostridium。BIO AB和OF中第一优势菌Bacillus的含量分别为35.24%、41.21%,对照中Bacillus的含量仅为13.06%。对全样品基于种群结构相似度指数Thetayc和种群重叠指数Jclass分别作的树状图。两种方法统计的结果表明,对照(CK)与有机肥(OF)、生物有机肥(BIO AB)处理的样品细菌群落有显著差异,而后两者之间的群落组成无差异。更多还原

【Abstract】 Vascular wilt of cucumber, caused by Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FOC) is an economically important disease throughout the world. The wilt incidence rate can reach 50% in field, leading to important losses of cucumber and limits production in many areas of the world. Chemical control of Fusarim wilt relies to a large extent on the use of fumigant methyl bromide and other fungicides. However, environmental pollution caused by the residues of these fungicides limited their use in many countries. Environmental-friendly biological control represents an alternative for protection of plants against Fusarium wilts. The introduction of antagonistic microorganisms into soil has been proposed for the biocontrol of soi-borne diseases. Currently, it is believed that a combination of antagonists with suitable mature compost may be more efficient in inhibiting disease than using single antagonistic microbes or compost alone. In this study, we screened a new biocontrol agent SQR 9 from cucumber rhizosphere soil. In order to exploit the mechanisms of Fusarim wilt control, we isolated and purified and the antifungal componds produced by SQR 9 and its rhizosphere colonization. The soil bacterial community responses and induced systemic resistance triggered by application of bio-organic fertilizers were also studied.The morphology, biochemical and physiological properties and the sequence of 16S rDNA indicated that SQR 9 was a Bacillus subtilis strain. The strain showed significantly depressed mycelium growth of Fusarium oxysporum f. sp. cucumerinum on PDA by the dual culture technique. A total of 11 gene fragments of the expected size correlated with antibiotic biosynthesis, including surfactin, fengycin, iturin, bacillomycin, subtilosin, subtilisin were efficiently amplified from B. subtilis SQR 9. The methanol-extracts from the culture broth supernant inhibited Fusarium oxysporum growth in in vitro assays. The mycelic growth inhibition rate was 54.9% when 2-fold diluted crude extract was used in PDA. When compared with the control, the spore germination rates were decreased by 70.1% and 83.6% by adding 1-fold diluted and original crude extract into the water-agar medium. The crude extact showed significant inhibitory activities against several soilborne pathogens in cylinder-plate assays. HPLC-ESI-MS analysis suggested that SQR 9 produced two groups of ion peaks with mass difference of 14 Da at m/z (M+H)+1063.6,1017.5, 1031.6,1045.6,1059.6 and 1506.0,1435.0,1449.9,1463.9,1492.9,1477.9. Concerning the literatures and our results, the two compounds might be bacillomycin and fengycin with structural analogs with different side chain length (-CH2-), respectively.The shuttle plasmid pHAPⅡwas used to introduce plasmid-borne gfp genes into SQR 9 by electroporation methods. The gyp-transformant SQR 9-gfp could be easily distinguished by their fluorescence when visualized by fluorescence microscopy. The SQR 9-gfp strain could be colonized on cucumber rhizosphere soil by root-dip inoculation. The strain colonized on cucumber root surfaces most (106-107CFU·g-1 root), followed by rhizosphere soil and internal roots, and the bulk soil. SQR 9-gfp strain was mainly distributed on elongation zone, the root hair zone of cucumber primer roots and the lateral root junctions. Very few cells were found in the root tips.Greenhouse experiments were carried out to evaluate the effect of organic fertilizer inoculated and solid-fermented with Bacillus subtilis SQR 9 and Trichoderma harzium SQR T-037 alone or in combination, hereby defined as bio-organic fertilizers BIO A, BIO B and BIO AB, on the control cucumber Fusarium wilt. The bioorganic fertilizer products were applied into soil during the nursery phase of cucumber seedlings followed by a second application to Fusarium-infested soil when cucumber seedlings were transplanted. Compared with the control treatment, the shoot and root biomass of bio-organic fertilizer-treated plants were promoted by 1.9 to 2.3-fold and 2.1-fold, respectively. Plant height was increased by 72.7%. The organic fertlizer also had a positive effect on plant growth, but the effect was less signifcantly than bio-organic fertilizers. The control treatment had the highest wilt disease incidence rate (73%) 60 d after transplantation. Biocontrol was also achieved by applying bioorganic fertilizers to Fusarium-infested soil. The best biocontrol was obtained by application of BIO AB, which significantly decreased the desease incidence (only 15%). And application of organic fertilizer also reduced the disease incidence (43.2%).Plate counting and Real-time PCR were used to detect the rhizosphere colonization by the pathogen and the two biological agents. Populations of FOC in BOF treated soil declined to 103 CFU·g-1 root after 60 days of inoculation, while those in control treatment remained high (2.48×10S CFU·g-1 root). Application of organic fertilizer alone reduced the FOC population by 84.6%, compared with the control. The treatment BIO AB had the lowest number of FOC (1.2×103 CFU·g-1 root). Real-time PCR analysis with SCAR primer FocF3 (F)/FocR7 (R) earlier designed to be F. oxysporum f. sp. cucumerinum specific was carried out to determine copy number of the target DNA present in cucumber rhizosphere. The DNA copy number varied from 3.4×105 to1.32x107 copy·g-1 root. In agreement with the plate counting, application of bio-organic fertilizers produced a suppressive effect on DNA copy number. The counts of FOC in BIO AB treatment were two orders of magnitude lower than the control. The level of rhizosphere colonization by B. subtilis SQR 9 was monitored in all treatments. Using B. subtilis-selective medium, No background Bacillus-like colonies were found on the non-inoculated plants (CK,OF and BIO B) roots, while the BIO A and BIO AB-treated roots were colonized by 6.03x104 and 1.62x104 CFU·g-1 fresh root B.subtilis 60 d after pathogen challenge.SQR-T037 ITS regions were amplified using primers and probe specific for Trichoderma harzium in Real-time Taqman PCR assay. The non-inoculated treatments had a backgroud growth of T. harzium of 102-103 copy·g-1 root, while in BIO B and BIO AB treatment, the number of T-037 reached 4.9-5.0 log copy·g-1 root, indicating that B. subtilis SQR 9 and T. harzianum SQR-T037 was successfully colonized in cucumber rhizosphere and thus effectively protected the plants from being attacked by pathogens.Before the outbreak of Fusarium wilt, the leaf MDA contents in organic fertilizer and bio-organic fertilizer treated cucumber plants were significantly lower than control 30d after transplantation. The activities of catalase (CAT), peroxidase (POD), superoxidase (SOD) and phenylalanine ammonia-lyase (PAL) were increased by 35.4-59.0%,46.1-86.8%, 33.3% and 71.2-109.3% by bio-organic fertilizer application, respectively, when compared with the control. The activities ofβ-1,3-glucanase and chitinase were also promoted by 58.5-86.5% and 52.1-71.4%, respectively.In order to study the bacterial community responses to application of organic fertilizer and bio-organic fertilizer, we analysed the bacterial richnesses, diversities and community compositons of soil samples in bio-organic fertilizer (BIO AB), organic fertilizer (OF) and the control (CK) treaments using 454-sequencing. The observed OTUs were distributed across three bacterial phyla, Firmicutes, Proteobacteria and Actinobacteria in all samples, suggesting the three samples may have similar bacterial composition. However, the number of OTUs of some groups within one phyla among each sample varied. In BIO AB and OF samples, Bacillaceae, Paenibacillaceae and Planococcaceae were the highest in OTUs in Bacilli class, while Bacillaceae, Paenibacillaceae and Clostridiaceae more abundant in CK treatment. The Bacillaceae accounted for 37.82% and 43.55% of OTUs in BIO AB and OF samples, while only 20.51% of OTUs in the control were assigned as Bacillaceae. On the genus level, Bacillus, Methylocystaceae, Paenibacillus, Actinomadura and Actinoalloteichus were the five most abundant genuses in BIO AB and OF soil samples, while Bacillus, Asticcacaulis, Haliangium and Clostridium were most abundant in the control. It is also noteworthy that Bacillus accounted for 35.24% and 41.21% in BIO AB and OF, respectively, while it only taken up for 13.06% in the control treatment. Moreover, the bacterial diversity of control was significantly different from BIO AB and OF treated soil. Analysis on Thetayc (community structure similarity) and Jclass (community overlap) estimators suggested that there were significant difference in bacterial community structure between BIO AB, OF and the control, while no significant difference was observed between the treatments of BIO AB and OF.更多还原