【作者】 张树生；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养， 2009， 博士

【摘要】 黄瓜镰刀霉菌枯萎病(Fusarium wilt)是黄瓜连作栽培中一种危害最大的土传病害,产生黄瓜连作障碍的主要原因是黄瓜连作导致土壤的微生物区系恶化,土壤有益微生物受抑制、有害微生物成为优势菌群,致使尖孢镰刀菌轻易侵染黄瓜根部的导管组织而发生。很多研究表明,有机肥料含有各种营养元素和生理活性物质,可改善作物根系微生态环境中的理化性状以及微生物活性,在一定程度上能提高作物的抗病害能力。黄瓜连作障碍的防治方法有很多种,有化学、物理和生物等方法,但生物防治是目前公认的最有前景的防治方法。本研究从黄瓜枯萎病的生物防治入手,进行了多方面的探索,得到如下结果：1、从枯萎病发病极为严重的黄瓜田块中的健康黄瓜植株根际土壤筛选到对黄瓜枯萎病致病菌尖孢镰刀菌具有强拮抗作用的拮抗菌二株(005号和021号),他们对病原菌的平板抑菌率大于70%,无菌培养液(胞外拮抗物质)的抑菌率大于40%,并且互相之间无拮抗现象,因此,可以将005号和021号二株拮抗菌复配应用。将二株高效拮抗菌进行常规的形态观察、生理生化鉴定和分子生物学鉴定,确定005号拮抗菌为炭疽芽孢杆菌(Bacillus anthracis),021号拮抗菌为多粘芽孢杆菌(Paenibacillus polymyxa)。2、施用生物酶解制成的氨基酸有机肥料(AAF)能使黄瓜生物学性状得到显著改善,黄瓜的叶绿素增加、叶面积增大、光合作用增强,干重提高；连作土壤的放线菌数量增加1.57-2.18倍,真菌数量增加2.22-8.43倍,尖孢镰刀菌明显减少,真菌／细菌比值显著提高,尖孢菌／真菌比值显著降低,对枯萎病的防治率最高达80%以上。说明AAF能通过激发土壤微生物活性而缓解黄瓜连作障碍的产生。3、连作土灭菌能消除土壤中所有的不利生物因素,改善黄瓜的生长。连作土灭菌增加黄瓜叶片的SOD、POD、CAT活性及可溶性蛋白含量,降低MDA含量。连作土灭菌改善土壤中的细菌、放线菌和真菌生长,提高其各种微生物的数量及其多样性,但消除了土壤中的尖孢镰刀菌,因而,黄瓜枯萎病也不致发生。这也说明生物因素是造成土壤连作障碍的主要因素。4、将吸附有混合微生物菌剂的有机肥(微生物有机肥)进行连续三年的田间试验(共四个田间试验)表明,普通有机肥处理,黄瓜枯萎病的发病率高达30.3%至90.4％,而微生物有机肥处理,黄瓜枯萎病的发病率则仅为2.9%至13.5%。在四个试验中,微生物有机肥的对黄瓜枯萎病的防治率分别达到73.6%、82.5%、96.3%和92.6%。黄瓜枯萎病发病率的下降,显著减少了黄瓜产量损失,微生物有机肥处理的产量分别比对照高98.8%、25.6%、73.3%和198.6%。5、土壤的DGGE和Biolog分析显示,施用微生物有机肥能改变土壤微生物群落结构,尖孢镰刀菌(Fusarium oxysporum)的数量显著减少。用扫描电镜结果显示,黄瓜发病部位的导管中分布着很多的类似菌丝的管状物,同时伴生大量无定形沉淀物,并且管状物越多,伴生的无定型沉淀物也越多,直至将导管全部堵塞。X射线电子探针元素显微分析发现,类似菌丝的管状物和伴生的沉淀物具有基本相同的成分,均含有较高的Ca和P,较低的C和O；与活的菌丝和植物的导管组织(两者也具有基本相同的元素组成)均含有较高的C和O,较低的Ca和P有显著区别。更多还原

【Abstract】 Fusarium wilt disease (FWD), a vascular wilt of cucumber plants caused by Fusarium oxysporum f. sp. Cucumerinum J. H. Owen, is one of the most devastating diseases. Organic amendments are routinely used to improve soil structure, soil biochemical and microbial properties and plant nutrition. Some chemical and physical way, can control soil borne pathogen to some extent, but biocontrol is a best promising way, The objectives in present study were to make and evaluate a bio-organic fertilizer containing Paenibacillus polymyxa and Bacillus anthracis for cucumber production in a continuously cropping soil where Fusarium wilt disease is a problem and to quantify Fusarium population after and before bio-organic fertilizer application, the results obtained were as follows.1. Two antagonistic strains (No.005 and No.021) were isolated from the rhizosphere soil of health cucumber plants in the field of severe diseased Fusarium wilt of cucumber plant. The inhibition rate of them to Fusarium oxysporum is more than 70%, and the inhibition rate of the Bacteria-free filtrate of antagonistic bacteria is more than 40%. No.005 and No.021 were Bacillus anthracis and Paenibacillus polymyxa by the ways of confirming of culture, physico-biochemical characteristics, microscopical evaluation and molecular identification. The co-culture test showed that the two strains had no antagonistic action if they were cultivated separately in the medium plate. Therefore, they should be applied in mixed culture of two strains.2. Pot experiments were carried out to investigate effect of application of amino acid fertilizer (AAF) on growth of cucumber and soil microorganism under continuous mono-cropping. The soil which had been planted with continuously mono-cropping cucumber for 4 seasonal crops and had a severe continuous obstacle, was used in the experiment. Results show that application of AAF increased chlorophyll content, leaf area, photosynthesis rate, and dry weight of the cucumber plants. Moreover, after the application of AAF with high-quality carbon and nitrogen, the number of actinomycetes was increased by 1.57-2.18 folds, fungi by 2.22-8.43 folds, while the number of Fusarium oxysporum, a witling disease causing fungi, was significantly decreased. The ratio of fungi/bacteria was significantly increased while the ratio of F.oxy. to fungi was significantly decreased. The controlling rate of Fusarium wilt was more than 80%. The results suggested that AAF application could significantly promote the growth of cucumber plants, stimulate microbial activities in the soils, and alleviate the adverse effect of continuous mono-cropping of cucumber.3. Soil sterilization can eliminate the adverse effect of soil borne pathogen on crop growth, Pot experiments were carried out to investigate effect of sterilization and application of AAF on growth of cucumber plants and soil microflora in a continuously mono-cropping soil. The results indicated that soil sterilization could improve growth of cucumber plants to greater extent, and SPAD value, plant height, leaf area and fresh weight of cucumber plants grown in sterilized soil, for example, were much higher than those in non-sterilized soil. The differences of cucumber growth between the sterilized soil and non-sterilized soil decreased with increase of AAF application, showing that the application of AAF could solve some problems coming from continuous mono-cropping system. SOD, POD and CAT activities in the leaves of cucumber plants grown in sterilized soil were higher than those in CK, and soluble protein contents of all treatments in sterilized soil were much higher than those in non-sterilized soil while MDAs were significantly lower. The number of bacteria and fungi in sterilized soil was increased by 1.89-3.03 folds and 6.96-2.43 folds, respectively, compared with those in non-sterilized soil and the ratio of fungi to bacteria was significantly increased. In addition, Actinomycetes were slightly enhanced. The analyses of PCR-DGGE profiles showed that the number of bands on the lanes of DGGE profiles in sterilized soil was more than those in non-sterilized soil, with low similarities of DGGE profiles, and the diversities of soil microorganism in sterilized soil were increased compared to those in non-sterilized soil. The incidence of Fusarium wilt of cucumber plants grown in non-sterilized soil was up to 31.2%-68.8%, but no incidence was found in sterilized soil.4. Four field experiments were conducted to evaluate the effect of organic fertilizer application either with or without antagonistic bacteria on the impact of Fusarium wilt disease in cucumber. The incidence of Fusarium wilt disease was 5.3-13.5% for cucumber plants treated with bio-organic fertilizer while it was 30.3-90.4% in controls applied only with organic fertilizer. The inhibition rates of Fusarium wilt disease were 73.6%,82.5%, 96.3% and 92.6% in the four independent field experiments and accordingly, cucumber yields treated with bio-organic fertilizer were increased by 98.8%,25.6%,73.3% and 198.6% respectively compared with CK. The application of Bio-organic fertilizer had a great potential for the control of Fusarium oxysporum wilt disease in cucumber plants.5. Denaturing gradient gel electrophoresis (DGGE) and Biolog analysis showed a significant change in soil bacterial community after bioorganic fertilizer application. The numbers of colony forming units (CFU) of Fusarium oxysporum in bio-organic fertilizer applied soils were significantly decreased compared with controls. Scanning electron micrographs of cucumber basal stems showed a presence of mycelia-like mini strands accompanied by an amorphous substance within the xylem vessels. This amorphous substance and mini strands were richer in calcium and phosphorus but had a lower carbon and oxygen than the living mycelia.更多还原