稻瘟病菌不同小种菌株间无性重组的研究

 将分别携带抗稻瘟灵对多菌灵敏感(MBC^sIPT^r)标记与携带抗多菌灵对稻瘟灵敏感(MBC^rIPT^s)标记的稻瘟病菌营养体亲和的不同小种菌株在白米玫瑰红培养基上,25℃、黑暗中对峙培养18d,配对菌株菌落交界处出现肉眼可见的菌丝融合带。4个小种间营养体可亲和菌株配对组合是:PO21-MBC^r-27(ZA₄₉)×PO3-IPT^r-54(ZF₁)、PO21-MBC^r-27(ZA₄₉)×PO2-IPT^r-51(ZF₁)、PO21-MBC^r-27(ZA₄₉)×PO33-IPT^r-69(ZD₁)和PO21-MBC^r-41(ZA₄₉)×PO3-IPT^r-54(ZF₁)。分别切取各配对组合产生的菌丝融合带,分别建立单菌丝片段群体,测定其对稻瘟灵、多菌灵的抗药性。结果是,从4个配对组合的单菌丝片段后代中,均检测到分别携带MBC^sIPT^r、MBC^rIPT^s标记和同时携带双亲抗性标记(MBC^rIPT^r)3种个体,其中MBC^rIPT^r个体的检出率为1.0%~3.6%。无性重组体的MBC^r和IPT^r性状在其菌丝块移植继代培养和单菌丝片段株后代发生分离。以配对组合PO21-MBC^r-27×PO3-IPT^r-54产生的菌丝融合带建立单孢无性系,从3178个单孢株中筛选到4株MBC^rIPT^r个体,其MBC^rIPT^r性状在单孢后代和单芽管分离株后代均能稳定遗传。结果表明,营养体亲和的稻瘟病菌不同小种菌株之间可通过菌丝融合产生无性重组体,同时提示无性重组体内可能发生了准性生殖。     

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。     

苎麻疫霉对棉苗致病力的遗传与变异研究

 以分离自江苏省棉铃疫病病组织的苎麻疫霉(Phytophthora boehmeriae Sawada)野生型菌株JS-5为亲本,采用菌丝块创伤接种法测定了苎麻疫霉对棉苗致病力在游动孢子无性系和卵孢子后代的遗传。结果表明,苎麻疫霉对棉苗的致病力在单游动孢子无性系连续两代稳定遗传,而在单卵孢第1代(OG₁)则发生连续性变异。从OG₁中选致病力强、弱2个单卵孢株为亲本,分别建立单卵孢第2代(OG₂)和单游动孢子无性系,并测定其对棉苗的致病力。结果为上述2个单卵孢株的游动孢子后代对棉苗的致病力均与其各自亲本相似,而在它们的单卵孢株群体(即OG₂)中对棉苗的致病力继续发生复杂的连续性变异。上述结果表明,苎麻疫霉对棉苗的致病力可能由细胞核杂合多基因控制。     

KMnO4和H2O2刺激掘氏疫霉卵孢子萌发对单卵孢株生物学性状的影响

 本文对经KMnO₄、H₂O₂刺激掘氏疫霉(Phytophthora drechsleri)卵孢子萌发建立的单卵孢株群体应用于掘氏疫霉遗传研究的可行性做了研究。2个掘氏疫霉菌株(A₁、A₂交配型各1株)经聚碳膜间隔配对产生的卵孢子用KMnO₄、H₂O₂以及不用上述氧化剂处理分别建立K、H、C 3个单卵孢株群体(卵孢子萌发率8-16%)。在群体水平上对来自同一亲本的上述3个群体生物学性状比较结果表明,H₂O₂处理刺激卵孢子萌发不影响生长速度、菌落形态、耐35℃高温生长、致病力及交配型的遗传稳定性,所建立的单卵孢株群体可用于上述性状的遗传研究;KMnO₄处理导致掘氏疫霉A₁和A₂交配型在自交后代各自分离为A₁、A₂、A₁A₂和A₂、A₁A₂,但不影响其他生物学性状的遗传稳定性,所建立的单卵孢株群体可用于除交配型性状以外的其他生物学性状的遗传研究,不宜用于交配型正常遗传规律的研究。用KMnO₄处理掘氏疫霉A₁或A₂交配型菌株单株自交产生的卵孢子,从A₁菌株自交后代获得A₂和A₁A₂以及从A₂菌株自交后代获得A₁A₂交配型单卵孢株的结果说明,掘氏疫霉异宗配合菌株体内可能同时包含控制A₁和A₂交配型的遗传因子,在正常条件下有1种交配型遗传因子不表达。KMnO₄处理可望作为揭示交配型遗传本质的有用试验手段。     

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。     

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。     

恶疫霉有性生殖交配行为的研究

 对同宗配合疫霉种——恶疫霉(Phytophthora cactorum Schroter)抗甲霜灵突变株和抗地茂散突变株的抗药性在游动孢子和卵孢子后代的遗传分析,筛选到抗性分别由细胞核纯合显性单基因控制的抗甲霜灵突变株和由细胞质可稳定遗传的线粒体基因控制的抗地茂散突变株。将携带抗甲霜灵对地茂散敏感(即Mt^rCn^s)标记的亲本与携带抗地茂散对甲霜灵敏感(即Mt^sCn^r)标记的亲本共同培养,在200个有性生殖单卵孢后代中检测到携带3种不同抗药性标记的单孢株,其中32株携带Mt^rCn^s标记,165株携带Mt^sCn^r标记,另有3株同时携带双亲的标记性状即MtrCnr。结果表明:恶疫霉种内不同菌株共同培养时,有性生殖以配对亲本的各自自交为主,同时配对亲本间也可发生一定频率的杂交。     

玉米大斑病菌有性杂交F1代菌株的生理小种鉴定和AFLP分析

玉米大斑病菌是异宗配合真菌，有性杂交有可能增强病菌的致病力，或形成新的致病小种，因此对该病菌有性杂交后代进行致病性测定和遗传多态性分析对控制该病菌的危害具有重要意义。对亲本菌株132、135和它们杂交产生的70个单子囊孢子F1代菌株进行了生理小种鉴定和AFLP（扩增性片段长度多态性）分析。生理小种鉴定结果表明，F1代菌株中与亲本菌株132（23N号小种）属于同一小种类型的占41．4％，与亲本菌株135（23号小种）相同的占20．0％，另外还出现了0、1、2、3、13、123、12N、13N和123N号小种，所占比例分别为2．9％、1．4％、2．9％、2．9％、4．3％、8．6％、1．4％、4．3％和10．0％，说明有性杂交可使后代菌株的致病性发生比较广泛的变异。AFLP分析表明，F1代菌株之间分子遗传相似系数在0．87～0．99之间，其中84．3％的F1代菌株与亲本菌株的遗传相似系数在0．878以上，但与亲本菌株132同源性较强的F1代菌株数目大约是与亲本菌株135的5倍，说明不同菌株具有不同的遗传传递能力。比较生理小种鉴定和AFLP分析结果，发现生理小种分化和AFLP分子遗传多态性间有一定的相关性，但不能完全对应，不存在遗传谱系就等于小种的简单对应关系。     

绿僵菌Ma55抗多菌灵突变株的诱变及抑菌作用研究

在室内条件下测定了供试绿僵菌对多菌灵的抗性。结果表明, 供试菌株对多菌灵均表现为敏感, 其中, Ma55菌株对多菌灵最敏感。采用分生孢子定向筛选的方法获得了金龟子绿僵菌抗多菌灵突变株, 测定了抗性突变株对多菌灵的抗性差异。结果显示, 在获得的抗性突变株中, MC-2的EC50最大, 达到397.064 3μg/mL, 对多菌灵的抗性水平最高, 抗性水平指数达到102.35。对Ma55抗多菌灵突变株的菌丝生长速率、产孢量及对棉花枯萎病菌和棉花红腐病菌的抑制效果的研究结果表明, 抗性突变株的菌丝生长速率均比亲本菌株Ma55小, 但产孢量均比Ma55大, 其中, MC-2的产孢量最大, 为5.12×106个/mL; MC-5次之, 其产孢量为4.81×106个/mL, MC-8的产孢量在抗性突变株中最低。平板对峙培养结果显示, 金龟子绿僵菌抗多菌灵突变株对棉花枯萎病菌和棉花红腐病菌菌丝生长的抑制作用均达到显著水平, 其中, MC-2对棉花枯萎病菌、棉花红腐病菌菌丝生长的抑制作用最强, 与亲本对照菌株Ma55差异不显著。     

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.     

Vegetative Compatibility of Fusarium graminearum Isolates and Genetic Study on Their Carbendazim-Resistance Recombination in China

ABSTRACT Monoconidial isolates of 33 carbendazim-sensitive isolates and 31 carbendazim-resistant isolates of Fusarium graminearum were selected from three regions of China for vegetative compatibility group (VCG) analysis. A total of 213 and 224 nit mutants were recovered from the 33 sensitive and the 31 resistant isolates, respectively. Of all the nit mutants, the frequency of the different phenotypes was 44.6, 46.5, 5.7, and 3.2% for nit1, nit3, nitM, and nitA, respectively. VCG analysis identified 30 different VCGs among the 33 sensitive- and the 31 carbendazim-resistant isolates, with VCG diversity 0.91 and 0.97, respectively. Both, a carbendazim-sensitive and a -resistant isolate from the same field belonged to the same VCG. In all then, a total of 59 VCGs were identified among the 64 isolates with an overall VCG diversity 0.92. Direct hyphal fusion was observed in six pairs of vegetatively compatible complements, which is evidence of heterokaryon formation. It was hypothesized that carbendazim resistance could not be transferred by hyphal fusion or there is a small chance to be transferred between two compatible isolates. Three stable sexual recombinants of F. graminearum were randomly chosen from each of the three genetic crosses to study their biological properties. There were no significant differences in mycelial linear growth and pathogenicity between recombinants and their parents, but they differ in sporulation ability and capacity to produce perithecia. We concluded that sexual recombination presumably played a role in the development of carbendazim resistance under field conditions.     

Progeny derived from Thanatephorus cucumeris (Rhizoctonia solani) AG-2-2 IV isolates on sugar beet foliage show more clonal diversity in somatic compatibility grouping than those from roots and petioles

Twenty-nine isolates of Thanatephorus cucumeris (Rhizoctonia solani) AG-2-2 IV were collected from the roots, petioles, and leaves of diseased sugar beets in Hokkaido, Japan. We examined the genetic variation of the field isolates using somatic compatibility grouping of progeny (cultured isolates derived from induced basidiospores) based on analysis of hyphal anastomosis reactions (i.e., hyphal perfect fusion) and inter-simple sequence repeats (ISSRs). The number of somatic compatibility groups (SCGs) of single basidiospore isolates was strongly correlated with the host plant tissue from which the parental isolates were obtained. Parental isolates from roots and petioles tended to be genetically heterogeneous and generated plural SCGs, whereas isolates (except for two non-self-anastomosing isolates) from leaves tended to be homogeneous and generated a single SCG. Heterogeneous sibling basidiospore isolates yielded homogeneous progeny within a few generations. These findings were supported by the results of ISSR analysis, which showed that the homogeneous isolates generated progeny with the same genotype, whereas heterogeneous isolates generated progeny with different genotypes. However, like SCGs, ISSR genotypes of heterogeneous progeny tended to be homogeneous within two or three generations. Additionally, we examined clonal diversity during the basidiospore infection process over a 2-year period. A heterogeneous isolate generated a large number of progeny SCGs, thereby increasing clonal diversity. In contrast, progeny SCGs were almost all the same in a homogeneous isolate plot with a few exceptions. These results indicate that infection of T. cucumeris basidiospores play a role in the clonal diversity of AG-2-2 IV isolates, as indicated by progeny SCGs in the field.     

An assessment of aesculin hydrolysis, vegetative compatibility and DNA polymorphism as criteria for characterizing pathogenic races within Fusarium oxysporum f.sp. vasinfectum

Fifteen strains, assigned to four pathogenic races of Fusarium oxysporum f.sp. vasinfectum on the basis of host range, were characterized by means of vegetative compatibility, restriction fragment length polymorphisms (RFLPs) and pigmentation on aesculin-containing medium. Isolates were divided into two major vegetative compatibility groups (VCGs) comprising Races 1 and 2 (VI) and Race 3 (V2). The two VCGs were represented by a single RFLP pattern. An additional RFLP pattern was observed for a single Race 4 isolate. Isolates received as belonging to Races 2, 3 and 4 blackened aesculin agar, while designated and presumed Race 1 isolates gave a negative reaction.     

Heterokaryon compatibility and genetic recombination within a host plant between hop wilt isolates of Verticillium albo–atrum

Heterokaryon compatibility was tested by pairing complementary auxotrophic mutants of three fluctuating (M18, M33 and M50) and three progressive (PV1, PV2 and PV3) hop wilt isolates of Verticillitim albo–atrum . The criteria of compatibility adopted were prototrophic growth on a glucose minimal medium at 26°C and the presence of diploid conidia. Most pairings produced at least some heterozygous diploids, showing there was no complete incompatibility barrier to hyphal and nuclear fusion on an agar medium. The incidence of prototrophic diploidy was greater within paired mutants of progressive isolates, and between PV1 and the three fluctuating isolates. A recombinant haploid prototroph was re–isolated following inoculation of Antirrhinum plants with a pair of complementary auxotrophs (M18 nic -4 cob -26 and PV3 arg -8 pyr -2). In hop, this recombinant was of intermediate pathogenicity compared with the two parental wild–type isolates. Four large–spored, diploid isolates were obtained from Antirrhinum following inoculation with heterozygous diploid conidia (M18 nic -4 cob -26/PV3 arg -8 pyr -2) from diploids synthesized on agar. All four diploids remained stable on agar and one showed moderately high pathogenicity in hop, from which it was re–isolated as a stable diploid 10 weeks later.     

Laboratory production of oospores of Peronospora parasitica (crucifer downy mildew) and the recovery and characterization of sexual progeny from crosses between isolates with different host specificity

The production of viable oospores of Peronospora parasitica under laboratory conditions and the recovery of isolates (referred to as sexual progeny) from these oospore populations are described. Oospores were produced when isolates of opposite sexual compatibility type, specialized to the same or different Brassica species, were grown together in seedling cotyledons of a host line capable of supporting growth of both isolates. Recovery of sexual progeny from oospore populations produced from two out of four pairings between isolates specialized to the same host species (homologous pairings) proved relatively easy. On the basis of their characterization with respect to virulence, response to phenylamide fungicides, sexual compatibility type and isoenzyme polymorphisms, there was evidence that the sexual progeny from these homologous pairings could be of hybrid origin. For the first time in a member of the Peronosporaceae, it proved possible to recover and successfully characterize a few sexual progeny from pairings between isolates specialized to different host species (heterologous pairings). However, the majority of such isolates sporulated weakly and as a consequence proved difficult to maintain and were lost. Nevertheless, some evidence for the hybrid nature of progeny from heterologous pairings was obtained.     

Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis

A single 3 kb segment of double-stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis . These dsRNA-containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA-free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence-associated traits as those of the original dsRNA-containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis .     

Barrage Zone Formation Between Vegetatively Incompatible Fusarium graminearum (Gibberella zeae) Isolates

ABSTRACT Vegetative compatibility has been used to assess the population biology of many fungal plant pathogens. However, for many species, including Fusarium graminearum, this has meant making auxotrophic mutants to force heterokaryon formation. A method was developed to observe barrage zones of thick, raised mycelium at the junctions of vegetatively incompatible F. graminearum isolates. The appearance of the barrage zones was influenced by the growth medium and the light. Barrage zones on V8 agar were thicker and better defined than those on potato dextrose agar, Spezieller Nahrstoffarmer agar, and water agar. The addition of ground wheat kernels to V8 agar enhanced barrage zone formation. Incubating the cultures under constant light at 2,150 lx produced more distinct barrage zones than constant light at 3,400 lx, constant darkness, or ambient room light. Forty-three F. graminearum isolates from 34 vegetative compatibility groups, determined previously using nit auxotrophic mutants, were paired in all combinations using these optimized conditions. Isolates in different vegetative compatibility groups typically formed distinct, thick barrage zones at their junctions. Pairs of isolates in the same vegetative compatibility group had a very slight or no visible reaction, or rarely, a distinct "line gap" of sparse mycelium. Subcultures from the same isolate typically had no visible reaction at their colony junctions; however, subcultures from some isolates had thin, slight barrage zones. This method was used to identify the proportion of each of four F. graminearum isolates from infected barley spikes in the field, inoculated previously with a mixture of conidia from these four isolates. Barrage zone formation represents a rapid method to screen vegetative compatibility groups in F. graminearum and may be useful for other Fusarium species.     

湖南稻瘟病菌致病力的初步分析

 本文记述1973-1977年对湖南的稻瘟病菌致病力的研究结果。1973年从湖南各地分离的48个单胞菌株,在9个初选的鉴别品种上测定,可分成8个小种群,25个小种,1975年分离的69个单胞菌株,在同一鉴别品种上测定可分成9个小种群,36个小种。1976年从早稻感病穗颈上分离的142个单胞菌株,接种于14个抗性谱较广的品种上,特特普、IR26、科印矮3号未发现致病菌株。讨论了稻瘟病菌小种研究中的一些问题。