木霉菌REMI转化体对番茄灰霉病的防治及其机理的研究

研究不同木霉菌转化体对番茄灰霉病防治效果及机理,为木霉菌生物防治的合理利用奠定基础。利用限制性内切酶介导基因整合技术(restriction enzyme-mediated integration,REMI),通过插入线性化质粒DNA获得了生物防治番茄灰霉病(Botrytis cinerea)效果优于出发菌T21菌株(出发菌)的3个木霉菌转化体Ttrm31、Ttrm34和Ttrm55,对侵染花器和叶片的灰霉病防效分别比原生物防治木霉菌株提高了16.9%和8%。木霉菌转化体的产孢能力、分生孢子的萌发率、对碳氮源的利用能力及对高温的抵抗能力都有所提高;木霉菌转化体本身产生的几丁质酶和β-1,3-葡聚糖酶的活性均比出发菌高,因此通过REMI技术可以获得新的有益木霉菌转化体,在一定程度上提高了生物菌株防治番茄灰霉病的水平。说明REMI技术可以用于改良生防木霉菌株的功能,提高生物防治效果。     

木霉菌REMI变异株与不同宿主的专化性互作研究

比较了木霉菌野生株及不同木霉REMI变异株与宿主植物间的相互关系。试验结果表明,供试木霉菌均不能促进黄瓜生长;Trm31对高羊茅草生长有抑制作用,其他菌株对高羊茅草生长无明显影响;T21、Trm47、Trm55对稗草的生长有促进作用,Trm31、Trm34对稗草的生长有明显的抑制作用。     

木霉REMI突变株主要生防酶活性改变及其基因部分序列差异的研究

本文探讨了来源于同一出发菌株木霉T21的不同木霉REMI突变株的主要生防酶活性的改变及其基因部分序列的差异,利用生化技术测定了木霉几丁质酶和β-1,3-葡聚糖酶酶活性,并对这两种酶的基因部分序列进行了分析.结果表明,木霉REMI突变株几丁质酶和β-1,3-葡聚糖酶活性变化较大;相对于出发菌株T21,菌株Ttrm68和Ttrm31酶活性极显著高于出发菌株T21,而Ttrm94则极显著低于T21.采用简并引物克隆几丁质酶和β-1,3-葡聚糖酶基因的部分序列,发现菌株Ttrm68、Ttrm31在几丁质酶基因中插入了GGTATCGATATCGAC片段,菌株Ttrm94在β-1,3-葡聚糖酶基因中插入了GTC片段.     

生防棘孢木霉T31菌株的分离筛选及其生物学特性

为评价具有生防潜力的棘孢木霉,采用平板对峙法、对扣培养法、圆盘滤膜法从长白山土样中分离筛选出木霉菌株T31,结合形态学及分子生物学分析对其进行种类鉴定,并采用菌丝生长法研究了该菌株的生物学特性。所分离的菌株T31被鉴定为棘孢木霉Trichoderma asperellum,该菌株对8种靶标菌的抑菌率在44.49%~77.91%之间,其中,对玉米大斑病菌Exserohilum turcicum、核盘菌Sclerotinia sclerotiorum、腐霉菌Pythiumuhtimum sp.及镰刀菌Fusarium sp.均有较强生长竞争优势,对玉米灰斑病菌Magnaporthe oryzae具明显重寄生作用。菌株T31的最适培养基为PDA培养基,最适生长温度为30 ℃、最适产孢温度为25 ℃,最适pH值为6。持续光照可以促进木霉菌孢子产生,Zn²⁺、Mn²⁺、Ca²⁺和Mg²⁺利于菌丝生长,但是高浓度的Fe²⁺和Cu²⁺抑制菌丝生长,Mn²⁺和Mg²⁺可促进产孢。此外,与普通木霉菌相比,该菌株具有耐寒性及耐盐性。     

防治甜瓜枯萎病木霉菌REMI变异株筛选

采用平皿对峙培养的方法从本实验室构建的22株木霉菌REMI变异株及出发菌株T21中筛选出5株抑制率较高的木霉菌变异株,并进行了防治甜瓜枯萎病盆栽试验,最终筛选出2株防治效果较好的木霉菌变异株Ttrm42和Ttrm26,防效分别达到78.66%和74.67%。然后进行田间防效测定,防效分别达到78.05%和73.04%。研究结果表明,木霉菌REMI变异株可以提高对甜瓜枯萎病的防效。     

木霉菌REMI变异株对部分生态因子的影响

采用常规微生物分离和植物生物量测定等方法,将木霉菌REMI变异菌株的孢子进行种子接菌、土壤接菌、水中接菌,研究木霉菌REMI变异菌株对农作物甜瓜和玉米及杂草等生长性状、甜瓜根际土壤微生物和鱼苗生长以及在水中存活等的影响,检测其生物安全性。结果表明,除Ttrm53外,其余木霉菌变异株和出发菌株T21对甜瓜和玉米种子安全,并且能够显著提高种子的出苗率、增加须根数和促进幼苗生长;部分变异菌株对杂草具有抑制作用;供试木霉菌变异株对甜瓜根际土壤真菌有抑制作用,对细菌和放线菌群体数量无显著影响;木霉菌孢子在河水及自来水中均不能萌发,分生孢子在水中存活约12～18天,厚垣孢子约18～27天,菌体自行消解;对鱼苗生长没有不良影响。     

常用土壤杀菌剂和肥料对绿色木霉菌T23的影响

采用常规土壤微生物分离和真菌生物量测定等方法,检测6种常用土壤杀菌剂多菌灵、甲基托布津、福美双、代森锰锌、百菌清和五氯硝基苯;3种肥料尿素、过磷酸钙和硫酸钾;以及8种营养元素铜、锌、铁、钼、钙、锰、镁和钾对绿色木霉菌菌株T23菌丝生长、产孢及其在土壤中定殖的影响.不同杀菌剂、肥料和营养元素及其不同处理浓度处理对木霉菌株T23菌丝生长、产孢和定殖的影响差异显著,多菌灵和甲基托布津、尿素、硫酸锌和硫酸镁具有明显的抑制作用,20μg/mL多菌灵、甲基托布津原药对菌株123菌丝生长抑制率分别为100.0%和79.0%;0.23%尿素处理土壤21天后对根际定殖抑制率为35.6%;100mg/L硫酸锌和1000mg/L硫酸镁处理土壤21天后对木霉菌定殖的抑制率分别为31.1%和15.9%.适量应用代森锰锌、硫酸钾及二钼酸铵、硫酸钙、磷酸二氢钾等有利于木霉菌根际定殖.     

烟草黑胫病菌培养特性的研究

研究了云南烟草黑胫病菌在不同培养基、不同温度的生长情况及不同菌龄与不同诱导时间孢子囊的产生数量。结果表明,菌株在8种供试培养基中,以燕麦培养基和选择性培养基上菌落生长最好,其次为胡萝卜培养基,在番茄汁和马铃薯培养基上生长最差。同一菌株在不同培养基上产孢量不同、产生的孢子囊大小亦有所不同。05P226菌株在玉米培养基上产孢量最高;05P55菌株在燕麦培养基上产孢量最高,在番茄汁培养基上产生的孢子囊最大,而05P62菌株在燕麦培养基上产孢量最高,在烟叶汁培养基上产生的孢子囊最大。在30℃菌丝生长速率最高,其次为28℃,以35℃生长最差。供试3个菌株间生长差异显著,以05P226生长最好,05P55菌株次之,05P62生长最差。05P226、05P55菌株皆以培养14d、诱导72h产孢量最高,最适宜试验中产孢培养。     

不同碳源对生防木霉菌T23、T56生长和生防活性的影响

采用基础培养基添加不同碳源培养木霉菌T23、T56的方法,研究了不同碳源对木霉菌生长、产孢量和生防活性的影响,以期获得木霉菌理想的碳源。同时,评价生物炭作为碳源或基质的效果。结果表明,葡萄糖、淀粉、乳糖、生物炭单独为碳源时,木霉菌生长和产孢以葡萄糖为较优;与生物炭复配后,以生物炭和葡萄糖的复配为最佳,T23与T56的菌丝干重分别为1.24、1.41 g,孢子产量分别高达177.5×107个/m L、172.5×107个/m L,第3天的生长直径分别为75.30、79.20 mm。T23与T56对镰孢菌的抑制作用以生物炭和葡萄糖复配的培养基最好,抑制率分别为83.42%、72.00%。     

短密木霉菌株GAS1-1的分离鉴定、拮抗作用及其生物学特性

为了解短密木霉Trichoderma brevicompactum对植物病害的生防作用及其生物学特性,利用稀释平板分离法从甘肃省景泰县马铃薯连作田植株根际土壤中分离获得1株木霉菌株GAS1-1,经形态观察、rDNA-ITS和EF-1α序列分析明确其分类地位;用生物学方法研究该菌的营养生长和产孢条件;采用对峙培养法测定该菌株对5种植物病原真菌的抑制作用。结果表明,基于形态学特征和基因序列分析结果,菌株GAS1-1被鉴定为短密木霉Trichoderma brevicompactum,为甘肃省木霉新记录种。该菌株对玉米镰孢茎腐病菌Fusarium graminearum、玉米穗腐病菌F. verticillioides、棉花枯萎病菌F. oxysporum、玉米腐霉茎腐病菌Pythium inflatum和灰葡萄孢霉Botrytis cinerea均具有较好的拮抗效果,尤其对玉米腐霉茎腐病菌的抑制作用最好,抑菌率达100.00%。该菌株营养生长和产孢的最适碳源和氮源分别为葡萄糖和酵母膏;其在15～35℃均可生长,菌丝最适生长温度为30℃,最佳产孢温度为25℃;在pH 5～12的培养基上菌丝均可生长,菌丝最适生长和产孢的pH均为5;24 h黑暗条件下菌丝营养生长最快,12 h光暗交替条件有利于产孢;孢子致死温度为69℃,10 min。表明短密木霉菌株GAS1-1具有较好的生防应用潜力。     

短短芽孢杆菌JK-2的GFP标记及其抑菌作用

本文采用gfp基因标记枯萎病生防菌短短芽孢杆菌Brevibacillus brevis菌株JK-2。标记菌株菌落圆形,菌体短杆状,与原始菌株相同;荧光显微观察发现菌落和菌体发绿色荧光。标记菌株起始期生长缓慢,进入对数生长期后生长迅速。在无选择压力条件下连续转接15次,GFP标记仅丢失11.7%。gfp标记菌株对不同植物枯萎病菌具有很强的抑菌能力,与野生型菌株相当。     

小麦纹枯病拮抗细菌的筛选

 从小麦纹枯病病田土壤中分离到722株细菌,通过对峙培养法筛选出拮抗细菌397株,拮抗菌率为55.0%。细菌对小麦纹枯病病原菌的拮抗作用分为两种类型:其一以竞争作用为主,表现为菌株生长快,抑菌率高但抑菌带较窄,如菌株BNT-25抑菌效果可达100.0%,抑菌带仅为2 mm;其二为抗生物质的产生,表现为菌株生长慢,菌落小,抑菌率高且抑菌带宽,如JJ-5菌株抑菌效果可达94.3%,抑菌带为12 mm。选用18株抑菌作用良好的拮抗细菌在室内测定其防治小麦苗期纹枯病的效果。结果发现,与空白对照相比,有9株拮抗细菌的防治效果达到了极显著水平,有15株拮抗细菌的防治效果达到了显著水平。在极显著水平中菌株NZT-47、MTT-8和DHT-12的防治效果分别为76.45%、64.03%和58.89%,对小麦纹枯病表现了较高的防治效果;菌株HNT-7-16、BDT-94和BNT-28的防治效果分别为43.47%、40.97%和40.97%,具有一定的防治作用。其中NZT-47的防治效果最好。     

Parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> and role of the gelatinous matrix

Trichoderma (T. asperellum-203, 44 and GH11; T. atroviride-IMI 206040 and T. harzianum-248) parasitism on Meloidogyne javanica life stages was examined in vitro. Conidium attachment and parasitism differed beween the fungi. Egg masses, their derived eggs and second-stage juveniles (J2) were parasitized by Trichoderma asperellum-203, 44, and T. atroviride following conidium attachment. Trichoderma asperellum-GH11 attached to the nematodes but exhibited reduced penetration, whereas growth of T. harzianum-248 attached to egg masses was inhibited. Only a few conidia of the different fungi were attached to eggs and J2s without gelatinous matrix; the eggs were penetrated and parasitized by few hyphae, while J2s were rarely parasitized by the fungi. The gelatinous matrix specifically induced J2 immobilization by T. asperellum-203, 44 and T. atroviride metabolites that immobilized the J2s. A constitutive-GFP-expressing T. asperellum-203 construct was used to visualize fungal penetration of the nematodes. Scanning electron microscopy revealed the formation of coiling and appressorium-like structures upon attachment and parasitism by T. asperellum-203 and T. atroviride. Gelatinous matrix agglutinated T. asperellum-203 and T. atroviride conidia, a process that was Ca²⁺-dependent. Conidium agglutination was inhibited by carbohydrates, including fucose, as was conidium attachment to the nematodes. All but T. harzianum could grow on the gelatinous matrix, which enhanced conidium germination. A biomimetic system based on gelatinous-matrix-coated nylon fibers demonstrated the role of the matrix in parasitism: T. asperellum-203 and T. atroviride conidia attached specifically to the gelatinous-matrix-coated fibers and parasitic growth patterns, such as coiling, branching and appressoria-like structures, were induced in both fungi, similarly to those observed during nematode parasitism. All Trichoderma isolates exhibited nematode biocontrol activity in pot experiments with tomato plants. Parasitic interactions were demonstrated in planta: females and egg masses dissected from tomato roots grown in T. asperellum-203-treated soil were examined and found to be parasitized by the fungus. This study demonstrates biocontrol activities of Trichoderma isolates and their parasitic capabilities on M. javanica, elucidating the importance of the gelatinous matrix in the fungal parasitism.     

木霉耐盐突变菌株的紫外诱变选育

为了获得高效耐盐木霉突变菌株,以盐碱土中分离的一株深绿木霉T-YM作为原始菌株进行紫外诱变处理,并筛选了突变菌株的最佳诱变条件,利用NaCl溶液模拟盐胁迫条件测定了突变菌株耐盐指标生长速率、产孢量和菌丝生长量。结果表明,与对照(野生型菌株)相比,当诱变时间为3 min时能够显著提高深绿木霉T-YM突变菌株菌落生长速度和产孢量,分别增加17.69%和28.82%;诱变时间为0.5 min和5 min时能够显著提高突变菌株菌丝生长量,分别增加42.22%和46.67%。利用10 mg·mL^-1 NaCl溶液模拟盐胁迫条件时,与对照相比突变菌株菌落生长速度、产孢量和菌丝干重整体高于野生型菌株,尤其当诱变时间为3 min时,不同浓度盐胁迫下其产孢量平均增加51.18%,诱变时间为0.5 min时其菌丝干重平均增加23.65%;NaCl胁迫(10 mg·mL^-1)下,诱变处理0.5 min获得的正突变菌株经传代接种培养后其耐盐性较为稳定。因此,紫外诱变可作为一种选育高效耐盐木霉突变菌株的有效方法。     

啶酰菌胺不同抗性类型多主棒孢的生物学特性

由多主棒孢Corynespora cassiicola引起的黄瓜棒孢叶斑病是黄瓜上的重要病害。本研究测定了对啶酰菌胺不同抗性类型多主棒孢的生物学特性差异和环境适合度,旨在为探明多主棒孢对啶酰菌胺的抗性变化机制提供生物学研究基础。随机选取不同地区24株对啶酰菌胺具有不同抗性类型的多主棒孢,分析了7种类型抗性突变体对几种不同琥珀酸脱氢酶抑制剂类 (SDHIs) 杀菌剂的交互抗性、不同抗性类型多主棒孢在无药剂选择压力下的抗性遗传稳定性,以及抗性突变体在不同碳源、氮源、温度等环境条件下的生物学特性及适合度。结果表明:除突变类型SdhB-H278Y及SdhB-H278R对啶酰菌胺与氟吡菌酰胺之间存在负交互抗性外,其他突变类型对啶酰菌胺与吡噻菌胺、氟吡菌酰胺及萎锈灵之间均表现为正交互抗性;所有突变类型菌株的抗药性均能稳定遗传;不同突变类型菌株之间致病性存在差异,其中SdhD-D95E突变体的致病力最强;利于所有突变类型菌株生长的碳源是麦芽糖,氮源种类则对突变体的生长影响不显著;各突变类型菌株的最适生长温度范围为25~30 ℃,其中突变体SdhD-D95E在高于30 ℃条件下菌丝生长速率大于其他突变体;耐热性研究中,抗性突变体经65 ℃高温处理45 min后无法存活,同时发现,60 ℃条件下突变体能正常生长,而敏感菌株不能生长;各质量浓度NaCl处理下,SdhD-D95E突变菌株菌丝生长速率快于其他突变菌株,而SdhB-H278Y突变菌株慢于其他突变菌株;葡萄糖对SdhB-H278R突变菌株的生长较为重要。研究表明,对啶酰菌胺不同抗性类型多主棒孢突变菌株的生物学特性及适合度存在差异,SdhD-D95E突变菌株适合度有所提高,表明该突变类型多主棒孢在田间具有较强的竞争力,容易形成优势种群。     

The effect of fungicides on fungal antagonists of onion white rot and selection of dicarboximide-resistant biotypes

The sensitivity of four fungal antagonists (Chaetomium globosum, Trichoderma harzianum, T. viride, Trichoderma sp.) to six fungicides was evaluated. The fungi were insensitive to captan, mancozeb and thiram but were sensitive to benomyl (EC₅₀ < 0 3/μg/ml) and the two dicarboximides iprodione and procymidone (EC₅₀ < 3 3/μg/ml). Conidia and mycelia of the four fungi were exposed to a series of ultraviolet irradiations in order to select biotypes resistant to benomyl and iprodione. Resistance to benomyl was not induced but a number of biotypes of each fungus were isolated from irradiated cultures that showed resistance to iprodione (EC₅₀ values of up to 56·4, 177·8, 171·5 and 216·4/μg/ml for Chaetomium globosum, Trichoderma harzianum, T. viride and Trichoderma sp., respectively). With three exceptions, all selected biotypes retained their fungicide-resistance after being extensively cultured on fungicide-free medium. In general, growth of the fungicide resistant biotypes on MEA plates and in MYE liquid medium was equal to that of the corresponding wild-type strains. One resistant biotype of Trichoderma sp. (C62UV3) grew significantly (P < 0·05) better both on solid and in liquid medium. This resistant biotype was also observed to have greatly enhanced conidial production on agar plates. All fungicide resistant-biotypes retained the ability to antagonize the pathogen Sclerotium cepivorum in dual culture and, in two instances (A53UV1, A53UV5), exhibited greater antagonism as evidenced by the production of larger inhibition zones. Similarly, with two exceptions, the resistant biotypes retained the ability to control onion white rot disease caused by S. cepivorum in the glasshouse. In particular, two fungicide-resistant biotypes (Trichoderma sp. C62UV3 and Chaetomium globosum A53UV1) reduced white rot of onion more effectively than did their respective wild-type parents.     

Strong resistance to the fungicide fenhexamid entails a fitness cost in Botrytis cinerea, as shown by comparisons of isogenic strains

BACKGROUND: Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3‐ketoreductase (Erg27) involved in C‐4 demethylation. Several fenhexamid‐resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS: Fenhexamid‐resistant mutants were generated by site‐directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild‐type erg27 allele was replaced by the three mutated alleles (erg27^F412S/I/V) by homologous recombination. These isogenic strains were shown to be fenhexamid‐resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild‐type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS: The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions. Copyright © 2011 Society of Chemical Industry     

野生酸枣内生细菌筛选、鉴定及其抑菌活性测定

为广泛开发植物内生菌资源,采用组织分离法从野生酸枣的根、茎、叶和果实中进行生防菌的分离和筛选,通过平板对峙法和发酵液法测定了所得菌株对8种植物病原菌的活性,并利用生物学和分子学方法对所得菌株进行了鉴定。结果显示,从野生酸枣的根、茎、叶和果实中共分离得到76株内生细菌;通过对所有菌株进行初筛和复筛,最终得到8株拮抗作用较强的菌株,菌株SZG-23、SZG-1、SZY-13和SZS-3对梨黑斑病菌Alternaria kikuchiana和黄瓜枯萎病菌Fusarium oxysporum f.sp.cucumerinum的抑制作用较强,抑制率均分别高于82.5%和71.1%,其中菌株SZG-23的抑制率最高,达90.3%和72.4%。菌株SZG-23发酵液对8种植物病原菌生长均有一定的抑制作用,其中对梨黑斑病菌和番茄灰霉病菌Bortytis cinerea的抑制作用最好,抑制率分别达92.2%和90.5%。菌株SZG-23的抗利福平突变菌株能在酸枣幼苗中定殖和移动。结合形态观察、生理生化特征及16S r DNA同源性分析将菌株SZG-23鉴定为解淀粉芽胞杆菌Bacillus amyloliquefaciens,16S r DNA序列在Gen Bank中登录号为KF483660。     

水稻恶苗病菌田间抗咪鲜胺菌株的适合度及其交互抗性

恶苗病是水稻生产上较为严重的种传真菌病害,咪唑类广谱内吸性杀菌剂咪鲜胺是目前防治该病害的主要药剂。以对咪鲜胺抗性及敏感的田间水稻恶苗病菌为试材,研究了其适合度及对几种常用杀菌剂的交互抗性。结果显示:抗性菌株的抗药性可稳定遗传,其温度敏感性与敏感菌株无明显差异,部分抗性菌株在菌丝生长速率、产孢量、孢子萌发率和致病力方面显著高于田间敏感菌株;咪鲜胺与三唑类及2-氰基丙烯酸酯类杀菌剂之间均无交互抗性。研究表明,对咪鲜胺产生抗性的水稻恶苗病菌具有较强的适合度,在田间自然条件下有可能形成优势群体,因此需合理轮换使用不同作用机制的杀菌剂,以延缓其抗药性的发展。