亚麻立枯病拮抗菌的筛选、生防效果及发酵条件优化

【目的】从健康亚麻植株的根际土壤中筛选对亚麻立枯病菌具有较强抑菌作用的拮抗菌,优化其产生抑菌活性物质的发酵条件,为其生防利用奠定基础。【方法】采用稀释平板涂布法和对峙培养法进行拮抗菌的筛选;根据菌株形态学特征、生理生化特性以及16S r RNA基因序列分析对其进行鉴定;利用温室抗病实验确定其生防效果;通过单因素实验和均匀设计实验优化其发酵条件。【结果】分离筛选到一株对亚麻立枯病菌具有显著拮抗作用的细菌HXP-5,且其对另外7种植物病菌真菌均有拮抗作用;鉴定菌株HXP-5为枯草芽孢杆菌;温室抗病实验结果表明其生防效果可达71.22%;其产生抑菌活性物质的最佳发酵条件为:葡萄糖为2.3%,胰蛋白胨+酵母粉(3:1)为0.25%,Na Cl为0.18%,发酵时间为72 h,发酵温度为27°C,转速为210 r/min,250 m L摇瓶装液100 m L,接种量为1.7%。【结论】经鉴定,对亚麻立枯病病菌具拮抗作用的菌株HXP-5为枯草芽孢杆菌,且对亚麻立枯病具有较强的防治效果,发酵条件进行优化后其对亚麻立枯病病原菌显示出更强的拮抗作用。     

牡丹根腐病原菌拮抗细菌抑菌活性物质分析

前期研究发现解淀粉芽胞杆菌(Bacillus amyloliquefaciens)md8和md9对牡丹根腐病原菌具有较好的抑制作用,但其抑菌物质的组成尚不清楚。本文首先明确了2个菌株3种脂肽类物质合成的基因片段,利用酸沉淀和葡聚糖凝胶层析法进行抑菌物质的分离纯化,牛津杯对峙法检测脂肽类粗提物和凝胶层析分离组分的抑菌活性;进一步利用实时荧光定量PCR(RT-qPCR)检测菌株在拮抗根腐病原菌过程中脂肽类物质合成基因相对表达量的变化,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析抑菌物质的种类。结果表明,2个菌株的脂肽类粗提物和凝胶层析分离组分对根腐病原菌均具有良好的平板抑菌效果,RT-qPCR结果表明2个菌株合成伊枯草菌素(iturin)的基因ituD在对峙培养过程中相对表达量显著增加,芬荠素(fengycin)合成基因fenA也呈现上调表达。MALDI-TOF-MS分析表明2个菌株中具有抑菌作用的分离组分主要为伊枯草菌素类物质Iturin B和Bacillomycins D,结合实时荧光定量PCR结果,推测菌株md8和md9在拮抗根腐病原菌过程中发挥主要生防作用的物质为伊...     

The feeding preference of mycophagous Collembola varies with the ectomycorrhizal symbiont

The interactions of the collembolan insect Proisotoma minuta with ectomycorrhizal and/or pathogenic fungi was examined in three experiments: (1) in vitro analysis of feeding patterns, (2) in vitro food preference test, and (3) in situ analysis of ectomycorrhizal colonization in relation to population density. The ectomycorrhizal fungi Laccaria laccata, Pisolithus tinctorius, Suillus luteus, Thelephora terrestris and the pathogenic fungi Rhizoctonia solani were employed in all experiments. In vitro and in situ experiments revealed that Pr. minuta consumed all the ectomycorrhizal fungi tested but the feeding pattern and consumption varied with each isolate. In a comparative in vitro feeding preference test, where Pr. minuta was given a choice, R. solani was grazed more heavily than the ectomycorrhizal fungi. Among the ectomycorrhizal fungi examined, Pi. tinctorius was consumed significantly less than L. laccata, S. luteus or T. terrestris in the presence of R. solani. A 10-week in situ analysis of loblolly pine (Pinus taeda L.) seedling root systems inoculated with Pr. minuta revealed that ectomycorrhizal colonization was significantly less than that of control plants (without Pr. minuta). Collectively, these data suggest that mycophagous Collembola may play a major role in the distribution and biomass of ectomycorrhizal fungi in the rhizosphere of tree seedlings.     

小麦内生细菌的分离及其对小麦纹枯菌的拮抗作用

利用涂布平板法从小麦根系中分离出8株内生细菌,从中筛选出1株对小麦纹枯菌(Rhizoctonia cerealis)具有拮抗作用的内生菌。室内测定该菌株培养液对小麦纹枯病菌的抑制作用,结果发现,小麦纹枯病菌在培养液中生长缓慢,培养6d后菌丝量与对照相比下降了89％,同时发现病菌菌丝生长畸形,出现断裂和细胞壁瓦解。双抗标记法测定该拮抗菌在小麦根系中的定殖情况,发现该菌能够在根系中长期定殖。初步的鉴定结果表明该菌为蜡样芽孢杆菌。     

中国北方棉花主产区立枯丝核菌的融合群鉴定

从山东、河北、河南三省采集棉花立枯病样品和土壤200余份,经分离获得198个丝核菌Rhizoctonia solani分离物。菌丝融合测定及5.8S rDNA-ITS区序列分析结果表明,这些分离物分别属于多核丝核菌的AG4-HG-I和AG4-HG-III融合群以及双核丝核菌的AG-A、AG-F、AG-Fb融合群。其中AG4-HG-I是优势融合类群,占分离物总数的88.38%,其次是AG4-HG-III,占10.10%,AG-A、AG-F、AG-Fb各仅有1株。其中双核丝核菌AG-A、AG-F和AG-Fb融     

云南省水稻纹枯病菌系研究*

将来自云南省20多个县市的水稻纹枯病标样130多份,选代表性的标样分离得到54个菌株。按菌丝融合测定法, 将54个菌株分为5个菌系:R.solani的AG-1 IA ,AG-1 IC,AG-6GW 以及双核丝核菌的AG-Bb, AG-I II。经致病性测定表明,该菌系对水稻、玉米、小麦的苗期及成株期的致病性有显著差异。其中AG-1 IA, AG-1 IC, AG-I II 的致病力最强, AG-6GW的最弱。对这些菌系的酯酶同工酶进行比较研究发现,不同菌系间均存在明显差异, 而同菌系不同菌株间却具一致性。由此说明,按菌丝融合与否区分丝核菌种群较之现行的其它分类法更能反映其遗传本质和亲缘关系。     

Growth promotion and disease suppression ability of Pseudomonas fluorescens in acid lime

Biochemical analysis was carried out for 10 isolates of Pseudomonas fluorescens. All isolates were found positive for siderophore and indole acetic acid production (except Pf IX) and phosphate solubilisation (except Pf VII). Biochemically efficient strains (Pf I, Pf IV, Pf VII and Pf IX) were selected for management of root rot, collar rot and damping off caused by Phytophthora parasitica, Pythium sp. and Fusarium solani. The activity of defence-related enzymes like esterase, peroxidase and polyphenol oxidase was also detected by polyacrylamide gel electrophoresis (PAGE). Consistent appearance of esterase isozyme bands was visualised, in the range of 0.193–0.349. The highest Rm value 0.349 was observed on Pf IV. Whereas, for peroxidase, Rm value ranged between 0.302 and 0.373 and for polyphenol oxidase, it was in the range of 0.211–0.800. P. fluorescens Pf IV was found significantly effective to arrest the per cent mycelial growth of P. parasitica (55.20%), Pythium sp. (65.33%) and F. solani (64.67%). Among bioagents, seed treatment and soil drenching with Pf IV at 15 and 30 days after sowing were found effective to reduce per cent disease incidence (30.55%) at 120 days after emergence. Seed treatment with copper oxychloride at 3g/kg seed and metalaxyl at 2 g/kg seed were also found effective.     

浙皖鄂地区水稻纹枯病菌5个种群的遗传结构分析

水稻纹枯病是世界性的主要病害之一。目前对该病病原菌种群的遗传多样性研究不多,知之甚少,了解其种群的遗传结构可以增加对其进化历程的了解,以制定科学的防治策略。水稻纹枯病菌通常被认为是以无性克隆繁殖为主,但有研究报道它具有混合繁殖方式。有关我国浙皖鄂地区水稻纹枯病菌种群的遗传多样性研究尚未见报道。为了解该地区水稻纹枯病菌种群的遗传变异、基因流、繁育方式及其遗传背景,采用ITS-5.8SrDNA测序技术,分析了分离自浙江富阳(FY)、安徽绩溪(JX)和巢湖(CH)以及湖北荆州(JZ)和孝感(XG)的5个水稻纹枯病菌种群75个菌株的遗传多样性。RhizoctoniasolaniAG-1IA是采集地区水稻纹枯病菌的优势类群。ITS-5.8SrDNA序列经测定共检测到78个多态位点,碱基A、T、C、G的平均含量分别为25.4%、33.6%、21.0%和20.0%。序列的平均转换与颠换比(Ti/Tv)为1.65,其中密码子第3位点的变异最高。根据序列的核苷酸变异共定义了29种单倍型,其中单倍型H5为5个种群的共享单倍型,占样本数的61.33%。5个种群的单倍型多样性和核苷酸多样性分别为0.627和0.482%,显示水稻纹枯病菌种群具有较高的遗传多样性。种群间固定化指数Fst为-0.0253-0.0170,基因流Nm为5.56-11.12,说明种群间基因交流频繁,基因流抑制了由遗传漂变引起的遗传分化,菌丝或菌核短距离扩散和带菌种子远距离传播增加了种群间的基因交流。AMOVA分析显示,种群间的遗传变异仅占总变异的19.03%,而80.97%的变异存在于种群内部,种群间的遗传分化很低。Mantel检验发现,遗传距离与地理距离无显著相关性(r=-0.241,P=0.499)。采用UPGMA法构建的单倍型间的系统发育树表明,不同地点的单倍型分支混合分布,这进一步验证了Mantel检验的结果。单倍型的网状分析显示,水稻纹枯病菌种群曾经发生过种群暴发而不断扩散,因还未能获得足够的时间建立更加复杂的结构故而呈非典型"星状"。采用中性检验分析了水稻纹枯病菌种群遗传结构,结果表明,种群间存在很强的自然选择作用,群体符合Hardy-Weinberg遗传平衡,说明水稻纹枯病菌群体是一个随机交配群体,具有以担孢子进行有性繁殖和以菌丝或菌核进行无性繁殖的混合繁殖方式。这种生物学特性可能是导致其在较小生态范围内较高的遗传多样性水平和较低的种群遗传分化的原因。另外,水稻纹枯病菌经有性繁殖产生新的基因型,并通过无性繁殖在群体内固定繁殖,这种遗传模式极有可能导致其进化潜能提高,极易对杀菌剂产生抗性。因此,对水稻纹枯病菌的防治,除了施用化学药剂和种植抗性品种外,还需要防治农田灌水引起的病原菌(菌丝和菌核)在地区间的流动传播,减少带菌种子迁移或农用机械的交叉污染,对水稻、大豆和玉米等寄主作物的种子进行播前处理等等,这些对于水稻纹枯病菌的防治也是极为重要的。     

Effect of solarization of soil within plastic bags on root rot of gerbera (Gerbera jamesonii L.)

Solarization of soil, (potting mix = coarse sand:Eucalyptus marginata fines = 1∶1) infested with 3 fungi pathogenic to gerbera (Phytophthora cryptogea, Fusarium oxysporum andRhizoctonia solani), for 3 to 4 weeks within transparent polyethylene bags controlled root rot of gerbera. Solarization for 2 weeks however, was less effective. All plants grown in the infested potting mix which had been kept in shade for 2, 3 or 4 weeks were severely attacked. Solarization of soil within plastic bags for 4 weeks also increased availability of nutrients such as NH₄ ⁺-N, PO₄ ⁻ and K⁺ in comparison to bagged soil kept in the shade for the same period.     

实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础,本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列,Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani)AG1-IA融合群菌株GD118为对照,采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小,研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。