【作者】 赵青云；

【导师】 徐阳春；

【作者基本信息】 南京农业大学 ， 植物营养学， 2011， 博士

【摘要】 甜瓜是世界十大水果之一,广泛种植于温带和热带地区,中国甜瓜种植面积和产量分别占世界的48.5%和56.3%。甜瓜枯萎病是一种维管束病害,由甜瓜专化型枯萎病菌尖孢镰刀菌(Fusarium oxysporum f sp melonis)引起,在甜瓜全生育期均可发生。近20年来,随着保护地栽培方式大范围推广,连作甜瓜枯萎病发生更趋严重。甜瓜枯萎病的防治是一种世界性的难题,传统的化学及农业防治措施通常会带来环境问题或成本高、劳动强度大等不能成功的应用于农业生产。目前,对于生物防治甜瓜枯萎病虽有报道,但仅局限于生防效果方面,对机理方面研究甚少,另外,可供商品化开发应用的生防产品并不多。本论文在分离获得5株对甜瓜枯萎病原菌具有高效拮抗作用细菌的基础上,研制出一种抗甜瓜枯萎病的专用生物有机肥,通过盆栽、大田及室内试验研究了其生防效果与机理。采用绿色荧光蛋白标记技术对生防菌株枯草芽孢杆菌Y-IVI进行了标记,并通过盆栽及室内检测试验研究了其对甜瓜生长的促生作用及在根际土壤及植株体内的定殖情况。主要结果如下：1.甜瓜枯萎病致病菌的分离是生物防治此病害的前提工作。从安徽和县采集甜瓜枯萎病发病植株,用尖孢镰刀菌选择性培养基分离获得致病菌。通过菌落形态及大、小型分生孢子,厚垣孢子显微观察结果表明分离到的菌株为尖孢镰刀菌。根据柯赫法则用孢子悬液接种甜瓜幼苗20 d后,枯萎病发病率达到80%。因此,可以确定分离得到的尖孢镰刀菌为甜瓜枯萎病致病菌。以其为靶标菌株,利用平板对峙法从健康甜瓜植株根际土壤中初筛获得对枯萎病病原菌有显著拮抗作用的5株细菌(Y-6,Y-8,Y-10, Y-12, Y-IVI)及2株真菌(A1,P1)。5株拮抗细菌发酵液对甜瓜种子萌发均无抑制作用。与对照相比,经菌株Y-10, Y-IVI处理的甜瓜种子发芽率分别提高了10%和5%,胚根长度显著高于其余3株拮抗细菌发酵液的处理。拮抗菌株发酵液对病原菌的生长均有显著的抑制作用,菌株Y-6、Y-8、Y-10、Y-12和Y-IVI发酵液浓度为10%时对病原菌的抑制率分别为54%、67%、75.3%、68%和72.4%,表明菌株Y-10, Y-IVI抑菌效果强于其它菌株。室内检测结果表明菌株Y-IVI可产生铁载体、吲哚乙酸和氨等促生类物质。经生理生化及16S rDNA分子鉴定,确定Y-10, Y-IVI分别为多粘类芽孢杆菌(Genbank accession number GQ849013)和枯草芽孢杆菌(Genbank accession number GQ475486).2.通过盆栽试验研究了堆肥和拮抗菌株Y-IVI、Y-10、Al和P1固体二次发酵生产的生物有机肥对甜瓜枯萎病的生防效果。结果表明：施用生物有机肥降低了枯萎病发病率并提高了植株生物量,营养钵育苗和盆钵移栽时均施用生物有机肥的处理甜瓜枯萎病发病率为20%,而对照的发病率为80%；施用生物有机肥显著降低了甜瓜植株茎和根系中病原菌的数量,营养钵育苗和盆钵移栽时均施用生物有机肥Ⅱ的处理甜瓜植株茎和根系中病原菌的数量分别为2.27×103和6.67 x 103CFUg-1FW,而对照植株茎和根系病原菌数量分别为8.17×104和3.67 x 104 CFU g-1FW,病原菌分别降低了97%和82%；施用生物有机肥改变了土壤微生物区系,根际土壤中细菌、放线菌数量与对照相比显著增加,而真菌和病原菌数量大大减少；营养钵育苗和盆钵移栽时均施用生物有机肥的甜瓜植株叶片中与抗病相关的酶活性低于对照。总之,营养钵育苗和大田移栽时均施用生物有机肥可显著降低甜瓜枯萎病发病率,提高产量并改善土壤微生物区系。之后,用绿色荧光蛋白标记Y-IVI(GY-IVI)研究了其对甜瓜生长的促生作用及其在甜瓜植株体内及根际土壤中定殖的情况。结果表明：种子和土壤均经过标记菌株GY-IVI处理(GY-IVIs+GY-IVIp)的甜瓜植株地上部、根系干重等显著高于对照；接种30 d后处理GY-IVIs+GY-IVIp根际土壤中标记菌株GY-IVI仍维持在108 CFU g’DW,与接种时的浓度相比并无显著性变化,而甜瓜植株茎及根系内部标记菌株GY-IVI的含量分别为106和107 CFU g-1DW,在对照处理的根际及植株体内均未检测到标记菌株。综上所述,菌株GY-IVI能促进甜瓜植株的生长并能够稳定的定殖在根际土壤和植株内部而对植物产生促生作用。3.室内试验结果表明：拮抗菌株B.subtilis Y-IVI发酵液稀释10倍后对病原菌仍具有拮抗作用；发酵液中产生的抗真菌活性物质耐酸,耐热,在pH 2处理24h或80℃水浴2h后仍有拮抗作用,并且对胃蛋白酶和蛋白酶K稳定。用HCl沉淀法从发酵液中提取出脂肽类粗提物,此粗提物对多种病原真菌具有抑制作用。用高效液相色谱反复纯化粗提物,最终收集到2个对病原真菌具有较强抑制作用的色谱峰。通过串联质谱仪测定其分子量分别为1028.7、1042.7、1056.7和1463、1477、1491 Da,这两组物质分别属于伊枯草菌素A和芬枯草菌素。菌株Y-IVI在Landy培养基中产伊枯草菌素最高浓度为89.75 mg-1。4.通过盆栽试验研究了菌株Y-IVI制成的生物有机肥(BIO)对甜瓜枯萎病的防效及其生防机理。结果表明,营养钵育苗和盆钵移栽时均施用BIO的处理与对照相比,甜瓜枯萎病发病率降低了91%,干重增加了3倍。施用BIO能显著降低病原菌在植株茎、根际土及非根际土中的数量,而且拮抗菌株Y-IVI能成功定殖在甜瓜植株茎及根际土壤中,移栽1 0-60 d内拮抗菌株Y-IVI在根际土壤中的数量维持在107CFUg-1土。营养钵育苗施用BIO的甜瓜根际土壤中抗菌脂肽类物质iturin A的含量为78μg·g-1FW。移栽10 d时施用BIO的甜瓜植株叶片中水杨酸含量为17.5μg·g-1FW,显著高于对照。综上所述,施用BIO能有效地防治甜瓜枯萎病,其主要机制是拮抗菌株Y-IVI能成功的定殖在甜瓜植株体内及根际土壤中,防止病原菌的入侵,且能在甜瓜植株根际产生抗病原真菌的活性物质iturin A以抑制病原菌生长繁殖。此外,Y-IVI能诱导甜瓜产生系统抗性。我们首次从植物-土壤-生防菌-病原菌相互作用的根际土壤中成功检测并定量了抗菌脂肽类物质。更多还原

【Abstract】 Muskmelon (Cucumis melo. L) is one of the ten most popular fruits in the world and has been grown over temperate and tropical lands. Muskmelon planting in China accounts for 48.5% of the total world muskmelon cropping area and 56.3% of its production. Muskmelon fusarium wilt, a vascular wilt, which is caused by soil-borne pathogen Fusarium oxysporum f.sp melonis that infects the crop in the whole growth periods, is always a disaster for farmers all over the world. Thus control of the disease is an urgent need worldwidely. In recent twenty years, the severity of the disease in China becomes heavier than ever due to wide replanting problems from intensive farming. While chemical control is challenged by environment and human safety, farming control such as crop rotation and field management is usually time-and labor-consuming. Therefore, biological control has gathered much attention across many research fields to replace chemical inputs with environment-friedly biotechnological products. However, few reports have been found on the biocontrol mechanisms. Biocontrol products are also rare in market.In the present study, we developed a new bio-organic fertilizer to control muskmelon fusarium wilt disease and then investigated its biocontrol mechanisms on muskmelon by pot experiments and lab tests. Furthermore, we tagged the biological agent of Bacillus subtilis Y-IVI by green fluorescent protein technique to facilitate enumeration of its real population from complex environments. Pot experiment and series of lab tests were carried out to investigate Y-IVI’s promotion effects on muskmelon growth and its colonization ability in the rhizosphere and the interior of plant tissues.The main results obtained were listed as follows:1. Isolation of Fusarium wilt pathogen is the fundamental prerequisite step for biological control of the wilt disease. The pathogen was isolated from tissue of a diseased muskmelon plant (collected from Hexian County, Anhui Province, China) using Fusarium-selective medium. Based on the mycelium, conidiophores and hlamydospore characteristics, the isolated fungus was identified as Fusarium spp. Furthermore, the isolated Fusarium spp was confirmed as the responsible pathogen by procedures described in Koch’s postulation. The wilt disease incidence was 80% after inoculation of the conidia suspension 20 days. Five bacteria (Y-6, Y-8, Y-10, Y-12 and Y-IVI) and two fungi (Al and Pl) with strong antagonistic activities against Fusarium oxysporum f.sp melonis (FOM) were isolated from healthy muskmelon rhizosphere soil by using in vitro antagonism tests. The filtrate suspensions of 5 antagonistic bacteria have no negative effect on muskmelon seed germination. The germination rate of seeds treated with filtrate suspension of Y-10 and Y-IVI were increased by 10% and 5% compared with control, respectively, in addition, the radical length in these two treatments was significantly higher compared with others. The culture filtrates of antagonistic bacteria can highly inhibit pathogen growth in vitro antagonistic tests. The pathogen inhibition rate was 54%,67%,75.3%,68% and 72.4% corresponding to 10% filtrate suspension concentration of microbes Y-6, Y-8, Y-10, Y-12 and Y-IVI, respectively. These results showed that microbes of Y-10 and Y-IVI have stronger inhibition effect on FOM and could be used as potential biocontrol agents. Laboratory tests showed that B. subtilis Y-IVI can produce indole acetic acid, siderophores and ammonia. Based on morphological and biochemical characteristics and 16S rDNA technology, Y-10 and Y-IVI were respectively identified as Paenibacillus polymyxa (Genbank accession number GQ849013) and Bacillus subtilis (Genbank accession number GQ475486). The two antagonistic fungi microbes Al and PI were tentatively identified as Aspergilis spp and penicillum spp, respectively.2. Pot experiments were performed to investigate the effects of different bio-organic fertilizers (BIOs) made from organic fertilizer and different antagonistic microbes(Y-IVI, Y-10, Al and PI). BIOs decreased the incidence of fusarium wilt disease and increased melon yield. The disease incidence of treatments with double application (BIOs applied both in the nursery and the pot soil) was 20%, much lower than control (80%). Application of BIOs strongly reduced the number of pathogen colony-forming units (CFU) in stems and roots of melon. Pathogen populations were 2.27×103 and 6.67×103 CFU g"1 FW (fresh weight) on BIOII-treated stems and roots, respectively, and 8.17×104 and 3.67 x 104 CFU g-1 FW on control stems and roots, respectively; i.e., CFUs were reduced by 97% and 82%, respectively. Microbial community structure was ameliorated by all BIOs. The number of bacteria and actinomycota in rhizosphere soil increased markedly under all BIO applications compared to control. In contrast, pathogen and fungal density was dramatically higher in the rhizosphere of control plants. The activities of defense enzymes in the leaves of melons receiving double application of BIOII were lower than those of control plants. In conclusion, the most effective treatment was double application of BIOII, which minimized the incidence of wilt disease, maximized biomass production, and altered microbial community structure. In addition, greenhouse experiments were carried out to investigate the abilities of Bacillus subtilis Y-IVI to promote plant growth and to colonize the rhizosphere and interior tissues of muskmelon. The inoculation of soil with green fluorescent protein-tagged Y-IVI (GY-IVI) significantly increased plant shoot and root dry weights as compared with the noninoculated soils. The inoculation of soil with B. subtilis GY-IVI maintained approximately 108 colony-forming-units (CFU) of GY-IVI per gram of dry rhizosphere soil for one month. The GY-IVI recovered from the interior of crowns and roots in the inoculated soil were 106 and 107 CFU g-1 dry weight, respectively, suggesting that GY-IVI acted as an endophyte.3. The culture filtrate of B. subtilis Y-IVI has antifungal activity even after diluted to 10% of its suspension. The compounds in extracts were aciduric by adjusting pH to 2 for 24 h and thermostable by heating at 80℃for 2 h. They were also stable to digestion by pepsin and Proteinase K. The crude lipopeptides from culture filtrate were further extracted by HCl precipitation and purified by high-performance liquid chromatography (HPLC). Two peaks that were detected by HPLC had antifungal activities. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis showed that the mass spectra of the two peaks were characterized by two series of homologous ion peaks, one with molecular weights of 1028.7,1042.7 and 1056.7 and the other with molecular weights of 1463,1477 and 1491. The two series of compounds were ascribed to iturin A and fengycin, respectively. The maximum production of iturin by strain Y-IVI inoculated in Landy medium was 89.75 mg L-1. Fengycin production was not measured due to lack of its standard reagent. In conclusion, we provided biochemical evidence that strain Y-IVI was able to producing antifungal compounds and hence had great potential to be used in biological control of plant diseases.4. A bio-organic fertilizer (BIO) secondarily fermented with antagonistic strain Bacillus subtilis Y-IVI was used to control this disease. Pot experiments were carried out to investigate the efficacy and elucidate the biocontrol mechanisms of the disease. Application of BIO reduced the incidence of muskmelon wilt disease by 91% and significantly increased plant dry weight by 3.1 times compared with the control amended with nothing. The BIO treatment significantly decreased FOM densities in plant shoots, rhizosphere soil and bulk soil. The colony-forming-unit (CFU) of FOM in rhizosphere soil of the BIO treatment was 1000-fold lower than that in the control. The previously lab-screened bacterial strain, Y-IVI, could effectively colonize rhizosphere soil and plant shoots. The logarithmic CFU of strain Y-IVI maintained between 7.6 and 6.7 in rhizosphere soil sampled from 10 to 60 days after transplanting into the BIO treatment. The average concentration of antifungal lipopeptide Iturin A in the BIO treatment was 78.1μg·g-1 of fresh rhizosphere samples. Ten days after transplanting, the content of salicylic acid in BIO treated plant leaves was 17.5μg·g-1 fresh weight, which was significantly higher than that in the control which showed that the BIO can induce plant systemic resistance. In conclusion, BIO can effectively control muskmelon Fusarium wilt, possibly because the antagonistic microbes in BIO effectively colonized the rhizosphere and plant shoots to preclude pathogen invasion. Furthermore, the antagonistic microbes in BIO produce antifungal lipopeptides in the rhizosphere and induce plant systemic resistance at an early stage of attack by pathogens. We first checked and quantified rhizosphere production of iturin and surfactin by biocontrol agents under interactions of plant-pathogen-biocontrol agents.更多还原