【作者】 袁树忠；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 农药学， 2008， 博士

【摘要】 辣椒疫病是一种对甜椒、辣椒具有毁灭性的病害,在我国的辣椒产地均有发生为害。利用生物防治控制辣椒疫病能减少化学药剂的使用数量和缓解病菌的抗药性,但生防菌防治病害的不稳定性限制了生物防治的发展。采用生防菌与杀菌剂协同防治病害,既能克服化学药剂的问题,也能弥补生物防治的不足。本文以辣椒疫霉为靶标病原菌,开展了颉颃微生物的分离筛选、候选菌的鉴定、候选菌对辣椒疫霉的颉颃作用特点、与卵菌杀菌剂的协同作用以及重金属离子的影响研究。结果如下：以辣椒疫霉为靶标病原菌,采用对峙培养的方法筛选得到了105个颉颃菌株,以辣椒疫霉Phytophthora capsici、辣椒炭疽病菌Collectotrichum capsici、番茄灰霉病菌Botrytis cinerea、番茄早疫病菌Alternaria solani、番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici、小麦赤霉病菌Fusarium graminearum、黄瓜蔓枯病菌Mycosphaerella melonis等7种病原菌进行复筛,结果表明,放线菌中以A001菌株对辣椒疫霉菌的作用最强；细菌中以B100菌株最好,对7种植物病原菌的抑菌带达11.3-18.5mm,生长抑制率达50%-84.3%。在定殖试验和盆栽试验中,B100可在辣椒根表面的稳定定殖,在处理后20d根部的菌量仍能达到7.06×105cfu/g(根重),而B148的定殖能力则明显差于B100。在盆栽试验中,B148对辣椒疫病的防治效果最好,B100处理的次之,根据抑制力-定殖力双重筛选的方法,确定与卵菌杀菌剂进行协同防治辣椒疫病的菌株为B100。利用形态、生理生化特征以及16S rDNA序列扩增分析对B100进行了鉴定。结果表明,菌株B100是一株好氧或兼性厌氧的革兰氏阳性的芽孢杆菌,生理生化特性与多粘类芽孢杆菌最相似；16S rDNA序列与GenBank中登录号为AY359623的Paenibacillus polymyxa GB-465具有最高的同源性,达99.9%,在系统发育树上两者也最接近。由此将B100鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa)。在离体条件下研究了多粘类芽孢杆菌B100菌株的生长条件及对辣椒疫霉的抗菌作用,结果表明,马铃薯葡萄糖培养液、牛肉膏酵母膏蛋白胨蔗糖培养液以及NA是适合B100生长的培养液,培养条件为28℃、培养液初始pH为6.0-8.0时B100生长最好；B100活菌菌液、无菌滤液、灭菌菌液对辣椒疫霉抗菌作用的顺序为：活菌菌液>灭菌菌液>无菌滤液,灭菌后的菌液有抗菌活性表明抗菌物质具有耐热性能。B100能造成辣椒疫霉菌菌丝畸形、分枝增多,细胞质异常浓缩、菌丝破裂、原生质外溢等。用B100菌液浇灌处理甜椒根部,测定了其叶片和根部中防御酶的活性变化,结果表明,B100能使甜椒(茄门甜椒)幼苗叶片和根中过氧化物酶、多酚氧化酶活性明显提高,但植株中苯丙氨酸解氨酶的活性没有显著变化。离体测定的B100与嘧菌酯抑制辣椒疫霉的联合作用试验结果表明,当B100浓度高于103cfu/ml时,与嘧菌酯0.5-50μg a.i./ml混用,对辣椒疫霉菌丝的实际抑制率均高于理论抑制率,显示为增效作用,而在B100浓度为102cfu/ml时,与嘧菌酯混合对辣椒疫霉菌丝的实际抑制率则低于理论值,两者混用后则为颉颃作用；而采用等效线法判断,则明确表明菌-药混用具有增效作用。在盆栽试验中,处理后7d,嘧菌酯与B100混用对辣椒疫病的防效显著高于B100单用。在田间试验中,采用游动孢子悬浮液灌根接种,B100分别与嘧菌酯、甲霜灵混用对辣椒疫病的效果好且稳定,防效高于药剂单用；通过菌丝块田间接种试验,B100与这两种卵菌杀菌剂混用的防效也高于药剂单用。B100与嘧菌酯、甲霜灵可以协同防治辣椒疫病。离体下研究了化学因子对辣椒疫霉和B100生长繁殖的影响,结果表明,氟吗啉、甲霜灵对辣椒疫霉菌丝生长抑制力强,而嘧菌酯对辣椒疫霉的作用力弱,而三种药剂在试验浓度下均未对B100有明显的抑制作用；重金属离子中,只有10mg/L的Ag+和Cu2+对辣椒疫霉菌丝生长有明显的抑制作用,其他4种重金属离子在≤10 mg/L浓度下对辣椒疫霉菌丝生长没有明显的抑制作用。而对于B100,Cu2+、Bi3+、Pb2+处理的菌浓度与对照的没有显著差异,0.1-1 mg/L的Cd2+、0.5mg/L的Ag+处理,B100的浓度显著高于对照,只有0.5-1mg/L的Hg2+处理后B100的浓度显著低于对照。如果土壤受到了重金属离子的污染,那么生防菌B100对辣椒疫病的防治效果可能会受影响。更多还原

【Abstract】 Bell pepper blight caused by Phytophthora capsici L. is one of the most important diseases of pepper growing in open and greenhouse in China. Application of biocontrol agent could reduce application quantity of chemicals for controlling plant disease and delay the development of fungicide resistance in pathogen population. However, development of biocontrol was limited because of the instability of biocontrol efficacy. A new control measure, which combined fungicide with biocontrol agent for disease, was attempted to develop. The approach could solve both problem of chemicals and shortage of biocontrol. This paper carried out a series of experiments on Phytophthora capsici as a target pathogen suppressed by antagonistic microbes, which screened, identificated. Additionally, it was involved in mechanism of biocontrol, combination of antagonistic agent and fungicide, and impact of heavy metal ions on antagonistic agent. The results showed as followed:Antagonistic microbes from soil were screened with dual incubation in vitro. One hundred five isolates were collected. Thirteen antagonistic agents from these were bioassayed in dual incubation suppressing seven pathogens, such as Phytophthora capsici, Collectotrichum capsici, Botrytis cinerea, Alternaria solani, Fusarium oxysporum f. sp. lycopersici, Fusarium graminearum and Mycosphaerella melonis. The results indicated that antagonistic activitiy of A001 was the strongest among 5 antinomycetes and B100 was a best bacterium isolate.In colonization experiment, antagonistic bacterium B100 could colonize along root surfacees of pepper successfully though its efficiency controlling pepper blight was lower than B148. Colonies of regained from root of pepper reached to 7.06×105 cfu/g (root weight) after 20 days of inoculation. The colonization of B148 was much less than that of B100 isolate.Thereby, isolate B100 was determined as a candidate for biocontrol P. capsici according to a screening method of suppression-colonization. The identification of B100 was based on the morphological characteristics, physiological and biochemical characteristics and analysis of 16S rDNA sequence comparison. The results indicated that B100 was a spore producing bacilliform bacterium, which owed to aerobic or facultative anaerobic, gram positive. It was the most similar with Paenibacillus polymyxa on physiological and biochemical characteristics. The 16S rDNA sequence of B100 possessed high identities compared with Paenibacillus polymyxa GB-465 that its accession number was AY359623 on GenBank, and identities reached to 99.9%. Similarly, B100 mostly closed to Paenibacillus polymyxa in Phylogenetic tree. Therefore, B100 was identified as a bacterium of Paenibacillus polymyxa.The growth condition of B100 and its antagonism to P. capsici were studied in vitro. The results showed that the PD medium, beef extract-yeast extract peptone sucrose broth or NA broth were optimum to growth of B100. The optimum original pH values of the medium broth were from 6.0 to 8.0 in 28℃. In antagonism test, the antifungal rank as followed:B100 suspension> sterilized suspension> filtrate. Light microscopic studies clearly showed the effect of B100 on hyphal morphology of P. capsici. Exposure to B100, phytopathogen produced swelled or increased divaricator hyphae, abnormal contraction of cytoplasm, lysis of hyphae and cell.The variations in three defense enzymes activities were determined in leaf and root of the 5-6 leaf-old seedlings of bell pepper, which had been treated with B100 at concentration of 108 cfu/ml. The results indicated that the activities of peroxidase and polyphenol oxidase were significantly induced by B100 in both leaf and roots of bell pepper. However, the activities of phenylalanine ammonia-lyase in leaf and root were no obviously induced by B100 compared with untreated plant.The joint action between B100 and Amistar (azoxystrobin), against P. capsici was determined in vitro. The synergistic effect each of Azoxystrobin and B100, which concentration was beyond 102 cfu/ml, suppress hyphae growth of P. capsici in that the inhibitions of observed was higher than those of expected. The same effect was observed based on isobole of 90% inhibition. The combined Azoxystrobin or metalaxyl with B100 against bell pepper Phytophthora blight would provide more effectiveness than fungicide alone respectively in field.The impacts of three fungicides and six heavy metal ions on both mycelium growth of Phytophthora capsici and reproduction of B100 were determined in vitro. The results showed that inhibition of flumorph or mefenoxam on mycelium growth of P. capsici was much more than that of azoxystrobin, and there was no inhibition on B100. Only the inhibition of Ag+ or Cu2+ at concentration of 10mg/L amending PDA media on growth of P. capsici was observed in six heavy metal ions. In experiment for B100, There were no obviously differences in the bacteria concentration of B100, which had been respectively treated by Cu2+, Bi3+ and Pb2+, compared with control. The concentrations of B100 in nutrient broth amended 0.1-1 mg/L of Cd2+ and 0.5mg/L Ag+ were obviously higher than that of control. However, the concentrations of B100 in broth amended Hg2+ at 0.5-1.0 mg/L was obviously lower than that of control. Then, heavy metal ions may decrease efficacy of biocintrol agent B100 in the field soil polluted by the ions.更多还原