新月弯孢菌营养体亲和群鉴定方法探讨

 采用菌株直接配对、显微镜观察菌丝融合和硝酸盐缺陷型(nit)突变体互补测试法,对44个来自不同地区的玉米弯孢菌叶斑病叶中的新月弯孢菌(Curvularia lunata)菌株进行了营养体亲和性分析。在野生型菌株的两两配对中,C.lunata菌株接触产生4种反应类型,但无明显的抗衡反应产生。在菌丝融合显微观察中,光学显微镜下只见大多数菌丝反应为一菌丝向另一菌丝无限靠近,未见融合,但在电子显微镜下,发现两菌丝细胞已不再是独立的。这些结果表明,因两菌落间的抗衡反应和菌丝融合特征都不明显,故不宜采用直接配对和显微观察来划分C.lunata的营养体亲和群。在nit突变体互补测试中,菌株在KClO₃浓度1.5%~3.0%的KPS中诱导表明,2.0%~3.0% KClO₃适宜于大多数菌株。抗氯酸盐突变体在Czapek培养基中能鉴定出nit突变体,但在MM上却不能。全部3640抗氯酸盐突变体在Czapek中共鉴定出2 40个稳定的nit突变体,其中nit1占59.2%,nit3占39.2%,NitM占0.8%,nit D (3种氮源都不能利用)占0.8%。44个菌株中,24个(55%)获得了nit突变体,20个(45%)还没有,仅2个菌株(124和155)各获得了1个NitM突变体。各菌株突变体类型间只有NitM与nit1或nit3是异核亲和的,其余均不能产生亲和反应。菌株124和155都是营养体自身亲和菌株。用2个NitM作测试菌株,初步划分出2个VCGs,5个归入VCG1,另有5个归入VCG2,其它菌株因未获得NitM或nit突变体还不能鉴定。以上结果表明,硝酸盐缺陷型突变体互补测试法可用于鉴定C.lunata菌株的营养体亲和群。     

新月弯孢变种的生物学特性及其对药剂的敏感性

为明确新月弯孢变种的发生和流行规律及有效防治方法，在不同碳源、氮源、温度、pH、光照等培养条件下，测定新月弯孢变种菌株HNWB-131、HNWB-185和模式菌株CX-3的生物学特性，并采用菌丝生长速率法测定这3株菌株对5种药剂的敏感性。结果表明，新月弯孢变种菌株HNWB-131和HNWB-185对碳、氮源的利用更敏感，在大部分碳、氮源培养基上其菌丝均为灰白色至黑色，与新月弯孢模式菌株的墨绿色存在明显区别；2株新月弯孢变种菌株的菌丝生长和产孢最适温度均为30℃，而新月弯孢模式菌株的菌丝生长和产孢最适温度分别为30℃和35℃；新月弯孢变种对光照条件更敏感，且黑暗条件对其分生孢子的产生和萌发有一定刺激作用；新月弯孢变种菌丝生长的最适pH为7~9，比新月弯孢模式菌株的最适pH范围窄（pH为5~11）。苯醚甲环唑对CX-3、HNWB-131、HNWB-185菌株的菌丝生长抑制作用最好，EC₅₀分别为0.39、0.58和0.52 μg/mL；其次为丙环唑，EC₅₀分别为0.79、1.97和1.64 μg/mL，表明2种新月弯孢变种对药剂的敏感性比模式菌株差。     

尖孢镰孢(Fusarium oxysporum)硝酸盐营养突变株及其营养体亲和性

 从荸荠、棉花、西瓜、黄瓜、胡瓜、大豆、花生等12种寄主作物上获得尖孢镰孢(F-usarium oxysporum)菌株20个,已知15个菌株分属于6个专化型。所有20个菌株在含有氯酸盐的培养基上产生抗氯酸盐的突变体,其突变体在以硝酸盐作为氮源的培养基上表现为无气生菌丝生长,即不能利用硝酸盐的突变株(nit),共获得nit突变株181个。同一专化型的nit突变株之间在以硝酸盐为氮源的培养基上配对时,在菌丝接触处可产生互补作用,即营养体亲和性,表现为旺盛气生菌丝生长。不同专化型的nit突变株之间则不能互补产生异核体而表现为非亲和性仍为突变型生长。每个菌株均获得能产生互补作用的nit突变株。根据营养体亲和性反应,20个菌株归入13个营养体亲和群(VCG),并显示出VCGs与菌株专化型之间的相关性。nit突变株在一般条件下是稳定的,而且某些nit株的亲和能力特强。     

尖孢镰孢(Fusarium oxysporum)硝酸盐营养突变株及其营养体亲和性

从荸荠、棉花、西瓜、黄瓜、胡瓜、大豆、花生等12种寄主作物上获得尖孢镰孢(F—usarium oxysporum)菌株20个,已知15个菌株分属于6个专化型。所有20个菌株在含有氯酸盐的培养基上产生抗氯酸盐的突变体,其突变体在以硝酸盐作为氮源的培养基上表现为无气生菌丝生长,即不能利用硝酸盐的突变株(nit),共获得nit突变株181个。同一专化型的nit突变株之间在以硝酸盐为氮源的培养基上配对时,在菌丝接触处可产生互补作用,即营养体亲和性,表现为旺盛气生菌丝生长。不同专化型的nit突变株之间则不能互补产生异核体而表现为非亲和性仍为突变型生长。每个菌株均获得能产生互补作用的nit突变株。根据营养体亲和性反应,20个菌株归入13个营养体亲和群(VCG),并显示出VCGs与菌株专化型之间的相关性。nit突变株在一般条件下是稳定的,而且某些nit株的亲和能力特强。     

新月弯孢菌及其近似菌菌落形态和可溶性蛋白电泳图谱比较分析

通过对分离自玉米及其他寄主的 12个新月弯孢菌菌株和 5个近似菌株菌落形态及可溶性蛋白电泳谱比较分析 ,表明菌落形态在不同种之间存在明显的差异 ,同种之间也存在一定差异 ,但不稳定和存在变异。 17个菌株可溶性蛋白SDS聚丙烯酰胺凝胶电泳显示 ,Curvularia属在Rf值为 0.177处有一条该属的特征蛋白带 ,新月弯孢菌在Rf值为0.225处有一条该种的特征蛋白带 ,但新月弯孢菌蛋白带存在多样性 ,聚类分析将未能确定的 13号菌株定为不等弯孢菌     

黄瓜枯萎病菌毒力、营养体亲和性及ISSR分析

 本研究对来自哈尔滨、长春、沈阳、北京、西宁5个城市的70个尖孢镰刀菌黄瓜专化型菌株进行了毒力、营养体亲和性及ISSR分析。毒力测定结果显示黄瓜枯萎病菌在东农803品种上存在明显的毒力分化。在营养体亲和群的测定中有8个菌株没有产生nit突变体,2个菌株经测定为异核体自身不亲和性菌株,不能进行营养体亲和群的测定;其余60个菌株可分为5个营养体亲和群。利用筛选的7个引物对70个菌株进行了ISSR分子标记,聚类分析可将70个菌株分为3个类群,其中IGⅠ的41个菌株均来自东北三省,IGⅡ的21个菌株均来自北京,IGⅢ的8个菌株全部来自西宁。VCGs和ISSRs与菌株的地理来源及毒力存在一定的相关性。     

玉米内生菌L10的分离、鉴定及拮抗活性

为获得对玉米茎腐病主要病原菌禾谷镰孢Fusarium graminearum有明显拮抗作用的玉米内生菌,采用平板对峙法从成熟健康玉米茎秆中筛选禾谷镰孢拮抗菌株,并分析其抗菌谱;通过形态特征、生理生化特性及16S rDNA序列分析进行菌种鉴定;利用盆栽生防试验检测其对玉米茎腐病的防治效果。结果表明,共分离获得了164株玉米内生细菌菌株,其中L10菌株对禾谷镰孢具有较好的抑制效果,抑菌圈半径达1.68 cm;该菌对玉米大斑病菌Setosphaeria turcica、层出镰孢F. proliferatum、禾谷镰孢F. graminearum、拟轮枝镰孢F. verticilliodes、玉米弯孢叶斑病菌Curvularia lunata、玉米小斑病菌Bipolaris maydis、立枯丝核菌Rhizoctonia solani、茄链格孢Alternaria solani共8种植物病原菌均有拮抗作用,尤其对禾谷镰孢抑制效果最佳;结合形态特征、生理生化性质及16S rDNA序列分析,将L10菌株鉴定为多粘类芽胞杆菌Paenibacillus polymyxa。L10菌株脂肽类物质对禾谷镰孢菌具有较好的抑制活性,且盆栽生防试验结果显示该菌株对玉米茎腐病具有一定的防治效果。表明菌株L10对玉米镰孢茎腐病的防治具有一定潜力。     

苹果树腐烂病菌VmMFS1~VmMFS3基因的功能分析

为绿色持久防控苹果树腐烂病,该研究分析苹果树腐烂病菌Valsa mali的3个主要协同转运蛋白超家族（major facilitator superfamily,MFS）编码基因的氨基酸序列特征,利用实时荧光定量PCR （quantitative real-time PCR,RT-qPCR）技术分析这3个基因在苹果树腐烂病菌侵染阶段的表达水平,通过构建这3个基因的缺失突变体和回补菌株分析其在病原菌营养生长、致病力和非生物胁迫应答等方面的功能。结果表明,这3个基因的氨基酸序列均具有MFS保守结构域,将其命名为VmMFS1~VmMFS3; VmMFS1和VmMFS2的进化距离较近,均与VmMFS3的进化距离较远;在苹果树腐烂病菌侵染过程中VmMFS1~VmMFS3基因表达均显著上调;与野生型03-8菌株相比,VmMFS1~VmMFS3基因缺失突变体的菌落形态无明显差异,但生长速度下降; VmMFS1~VmMFS3基因缺失突变体的致病力均显著降低; VmMFS1~VmMFS3基因缺失突变体对H₂O₂胁迫的敏感性无明显变化,但对NaCl胁迫更敏感;基因回补后基因缺失突变体的表型缺陷能恢复到野生型菌株的水平。     

蜡蚧轮枝菌生物学特性及其对温室白粉虱致病性的研究

通过对引进的 6个蜡蚧轮枝菌的生长、产孢量、分生孢子萌发和对温室白粉虱若虫侵染致病性的研究表明 ,6个菌株在 10～30℃下均可生长 ,菌丝生长和产孢的最适温度为 25℃ ,但菌株间存在显著差异。菌株V4和V5产孢量显著高于其他菌株 ,14d的产孢量分别为 2.6×10⁹个 /皿和 2.3×10⁹个 /皿。菌株V₄和V₁的菌落直径累计增长量和日增长量显著地高于其他菌株 ,分别为43.7、2.85mm和43.1、2.84mm。温度对孢子的萌发影响较大 ,15℃时孢子萌发缓慢 ,萌发达 90%以上时需 24h ,20～30℃下菌株V2 孢子萌发速率明显高于其他菌株 ,10h萌发率达 90%以上 ,30℃下 ,菌株V6萌发速度明显延缓。对温室白粉虱若虫的致病性 ,菌株间存在一定的差异。V₂ 、V₄接菌后第 5天若虫死亡率分别为 70.2%和 63.5% ,综合其菌丝生长、产孢量、萌发率和致病性表明 ,菌株V₂和V₄可作为生物制剂的生产菌株     

稻瘟病菌营养体亲和配对筛选及其亲和能力遗传

 在白米玫瑰红培养基上对江苏省的稻瘟病菌Magnaporthe grisea(Hebert) Barr.不同小种间营养体亲和菌株配对组合进行了筛选。当不同小种的菌株对峙接种,在21±1℃、黑暗中培养18 d后,部分配对组合的菌落交界处出现一条肉眼明显可见的菌丝融合带,且具有稳定的重复性。出现菌丝融合带的配对菌株在玻璃纸上对峙微培养2~3 d后,镜检观察到菌丝融合现象普遍存在;未出现明显菌丝融合带的菌株组合经对峙微培养,菌丝融合现象未见或较少。从ZA₄₉、ZG₁、ZF₁和ZD₁ 4个小种的12个菌株中筛选出3对营养体亲和性强的小种间菌株配对组合。以所选菌株为亲本建立单分生孢子无性系,测定营养体亲和能力在单孢后代的遗传。结果是,营养体亲和能力在单孢后代遗传不稳定,与另一亲本的单孢株配对产生融合带的百分率为53%~74%之间。进一步研究发现,来自同一个分生孢子不同细胞的单芽管株与配对菌株的营养体亲和能力相同;亲和能力在单芽管株的单孢株后代继续发生分离。     

Vegetative Compatibility Groups in Cercospora kikuchii, the Causal Agent of Cercospora Leaf Blight and Purple Seed Stain in Soybean

ABSTRACT Nitrogen nonutilizing (Nit) mutants were used to assess vegetative compatibility of 58 isolates of Cercospora kikuchii, 55 of which were isolated from soybean plants in Louisiana. Two isolates were vegetatively self-incompatible. Of 56 self-compatible isolates, 16 were assigned to six multimember vegetative compatibility groups (VCGs), 01 to 06, with 2 or 3 isolates in each VCG. The other 40 isolates each belonged to a distinct VCG. All six multimember VCGs contained isolates from different soy bean cultivars, and three included isolates from different locations. Only one of six multimember VCGs included isolates both from soybean leaves and seed, while the other five included isolates from only leaves or seed. The likelihood of tissue specificity or preference was discussed. All isolates and tested Nit mutants produced cercosporin on potato dextrose agar under light. Significantly different amounts of cercosporin were produced among wild-type isolates, and two Nit mutants produced significantly more cercosporin than their wild-type counterparts. All isolates produced typical Cercospora leaf blight symptoms on soybean plants in greenhouse pathogenicity tests.     

灰葡萄孢菌nit突变体的诱导及其在营养体亲和性测定中的应用初探

 从山西运城、临汾、长治、晋中、大同等地保护地黄瓜灰霉病病株上采集、分离的分属于3个不同菌丝融合群的8个灰葡萄孢菌单孢菌株,经氯酸盐诱导处理,共获得了抗氯酸盐的硝酸盐利用缺陷突变体(nit突变体)59株,其中nit1型38株,nit3型10株,nitM型11株。所有nit突变株分别在PDA斜面转管培养3次(21 d)后,除6株恢复成野生菌株外,其余多数nit突变菌株表现稳定。来源于同一野生菌株的不同类型nit突变体间或同一菌丝融合群不同野生菌株的nit突变体间可产生互补反应而形成异核体,其中以nitM型突变株互补性最好,在利用nit突变体测定灰葡萄孢菌营养体亲和性时应作为标准菌株。来源于不同菌丝融合群的nit突变体间不能产生互补反应。     

棉花落叶型黄萎病菌的检测技术

采用Nit硝酸还原酶缺陷型突变体技术，共得到棉花黄萎病菌Nit突变体301个，其中A型155个，B型97个，C型29个。共检测到落叶型棉花黄萎病菌11株，除以前报道的6株外，发现了5个新菌株。针刺接种后，在棉株上均引起典型的落叶型症状。通过聚丙烯酰受凝胶电泳比较落叶型和非落叶型的8个代表菌株的蛋白质电泳图谱的差异，发现在浓度为7.5%的凝胶中，落叶型菌株有两条特异性的蛋白，其迁移率分别为Rf5=0.135,Rf9=0.405。用非落叶型菌株DF4、落叶型菌株SY12菌体的可溶性蛋白制备获得两个抗血清。用间接ELISA法检测，即能区分棉花黄萎病菌的立枯、枯萎、炭疽病菌，落叶型和非落叶型黄萎病菌均呈阳性反应，但落叶型黄萎病菌的OD值高于非落叶型黄萎病菌。     

Characterization of Pyricularia grisea in the United States Using Independent Genetic and Molecular Markers

ABSTRACT A total of 540 isolates of Pyricularia grisea from rice in the United States were examined for vegetative compatibility, MGR586 DNA fingerprint diversity, and mating type based on hybridization with the mat1-1 and mat1-2 sexual mating type alleles. The collections contained both archived and contemporary field isolates representative of the known MGR586 lineages and races that occur throughout the United States. Complementary nitrate nonutilizing (nit) or sulfate nonutilizing (sul) mutants were used to assess vegetative compatibility in P. grisea. There was a complete correspondence between vegetative compatibility groups (VCGs), MGR586 lineage, and mating type among 527 contemporary isolates (collected between 1991 and 1997) from Arkansas, Louisiana, Missouri, Mississippi, and Texas; all isolates in MGR586 lineages A, B, C, and D belonged to VCGs US-01, US-02, US-03, and US-04, respectively. In addition, all isolates tested in VCGs US-01 and US-04 had the mat1-1 mating type allele whereas those in VCGs US-02 and US-03 had the mat1-2 allele. The strict association of independent markers during this sample period was consistent with a strictly asexual mode of reproduction. However, examination of archived isolates collected in the 1970s and 1980s and contemporary isolates revealed an incongruent relationship between the independent markers. MGR586 C and E isolates were vegetatively compatible which indicated that multiple robust MGR586 delineated lineages could be nested within certain VCGs. Although isolates in lineages C and E were vegetatively compatible, they were of opposite mating type. Several hypotheses, including recombination, could account for the incongruence between the various markers. Among the eight MGR586 lineages (A through H) that occur in the United States, all isolates in lineages A, D, E, G, and H had the mat1-1 allele, whereas isolates in lineages B, C, and F had the mat1-2 allele. Nit mutants can be recovered relatively easy from P. grisea and should allow large numbers of individuals within a population to be assessed for vegetative compatibility. VCGs may prove to be an effective multilocus marker in P. grisea. Thus, VCGs should be a useful means for characterizing genetic structure in populations of the rice blast fungus worldwide, provide a useful genetic framework to assist in interpreting molecular population data, and may provide insight into potential sexual or asexual recombination events.     

Reassessment of vegetative compatibility of Sclerotinia homoeocarpa using nitrate-nonutilizing mutants

Nitrate-nonutilizing (nit) mutants were recovered for the first time from 21 isolates of Sclerotinia homoeocarpa collected in the United States. Mutants were selected from shredded mycelium of each isolate when cultured on water agar medium amended with 4% (wt/vol) potassium chlorate. The mutants could be classified into three phenotypes: nit1, nit3, and NitM, based on their growth on minimal medium (Czapek solution agar) supplemented with NaNO(2) or hypoxanthine. Complementary heterokaryons were observed in pairings between different phenotypes of nit mutants derived from compatible isolates, but not in self-fusions or pairings between incompatible isolates. The vigor of prototrophic growth varied with isolates and mutant phenotypes. Strong and continuous heterokaryons, as well as weak and spontaneous ones, formed depending on pairings of nit mutants. Stable heterokaryons between compatible isolates, but apoptotic reactions between incompatible isolates, were observed immediately after hyphal fusion under the epifluorescence microscope. The 21 isolates used in this study, which were previously assigned into 11 different vegetative compatibility groups (VCGs) based on the formation of a barrage zone at the contact site of paired isolates on complete medium (potato dextrose agar), were regrouped into five VCGs based on heterokaryon formation between nit mutants on minimal medium.     

应用均匀设计法筛选稗草病原菌产孢最佳配方培养基

 筛选生防菌最佳产孢培养基是高效、大量生产生防菌孢子的基础。应用均匀设计法结合二次多项式回归分析,从众多因素中筛选出稗草病原菌弯孢菌(Curvularia lunata)和互格交链孢菌(Alternaria alternata)最佳产孢培养基组分,建立多元二次回归数学模型。弯孢菌最佳产孢组分为胡萝卜、芹菜、欧芹、水芹、莴苣、蔗糖和Vc;互格交链孢菌最佳产孢组分为胡萝卜、芹菜、水芹、莴苣和蔗糖。根据回归模型计算理论(拟和)产孢值。比较实测产孢量和理论产孢值,结果表明,稗草病原菌的实测产孢量和回归模型拟合产孢值非常接近,拟合误差小。说明采用均匀设计法从众多因素中筛选培养基最佳成分是可行的。     

Vegetative Compatibility Groups in Colletotrichum coccodes, the Causal Agent of Black Dot on Potato

ABSTRACT Black dot of potato, caused by Colletotrichum coccodes, is a disease of growing economic importance, but the degree of genetic diversity and pathogenic differentiation among isolates is unknown. Using nitrate auxotrophic (Nit) mutants, we characterized vegetative compatibility groups (VCG) diversity for C. coccodes for 110 isolates originating from Israel, The Netherlands, and France. We recovered frequencies of nit1 and NitM mutant classes at 38.5 and 7.2%, respectively, and selected 12 isolates as tester isolates. Using these testers, we defined four multimember VCGs at 7.3, 35.5, 20.0, and 10.0% frequency in this sample. Thirty isolates (27.3% of all tested isolates) could not be assigned to any of the major groups, and showed only self-compatibility. The frequency of recovery of Nit mutant sectors was highest in isolates from VCG4, with 50.9 and 13.6% recovery for nit1 and NitM, respectively. However, we did not detect differences in the frequency of mutant classes among the three countries of origin. In pathogenicity tests, isolates from VCG3 were the most aggressive to potato, as expressed by high stem colonization levels and sclerotia density on root and crown. These results suggest that there is significant VCG diversity in this species and that this VCG diversity may be correlated with pathogenic characteristics or specialization.     

玉米新月弯孢菌(Curvularia lunata)的RAPD分析

 对分离自玉米弯孢菌叶斑病标样中的77株新月弯孢菌和1株来自水稻的新月弯孢菌进行RAPD分析表明,菌株间具有丰富的遗传多样性,在相似系数约0.60处,所有菌株被聚为3个组,但88.0%的菌株聚入第Ⅰ组内,其余菌株被聚入另外2个组内。第Ⅰ组内共有69个菌株,包含来自不同区域的致病性较强的菌株,是玉米弯孢菌叶斑病的优势类群,其余2个类群主要是一些致病能力较弱或不致病的菌株。结果表明,新月弯孢菌种内菌株的遗传多样性与致病性相关,但与菌株地理来源无明显的直接关系。     

Mating Behaviour and Vegetative Compatibility in Spanish Populations of Botryotinia fuckeliana

Mating behaviour and vegetative compatibility were studied in Spanish populations of Botryotinia fuckeliana. Fifty-seven isolates out of the 61 tested were sexually fertile with one or more of the reference strains of known mating type (MAT1-1 or MAT1-2). Thirty-nine isolates were heterothallic, giving fertile crosses when mated with the MAT1-1 (24 isolates) or the MAT1-2 (15 isolates) reference strain. Eighteen isolates crossed successfully with both MAT1-1 and MAT1-2 reference strains, and were referred to as homothallic or MAT1-1/2. Both mating types were widespread, being represented in isolates from two regions, from the same and different greenhouses, from different hosts, and from different years of isolation. Isolates were paired on Malt Extract Agar + NaCl to evaluate vegetative compatibility. Most of the paired isolates were unable to fuse and showed a different reaction of incompatibility. Nitrate-non-utilising (nit) mutants were selected by growth on a medium amended with 30–50gl^–1 potassium chlorate. Over 600 chlorate-resistant sectors were recovered from 40 isolates at a mean frequency of 0.15–2.39 sectors per colony, but only 11% were identified as nit mutants by their thin growth with no aerial mycelium on minimal medium. However, most of these nit mutants reverted to wild type during six months of storage on chlorate-amended medium. Genetic complementation between nit mutants occurred only in two cases between mutants from the same isolate.