香菇自然群体中个体间的空间分布及其遗传联系

应用体细胞不亲和性反应、交配型因子分析和基因组DNA的RAPD分析,研究了一个分布于方圆约1km的6根倒木上的18个香菇野生菌株间的遗传差异。结果表明,该群体大多数菌株间配对（80．4％）体细胞不亲和,而同一倒木菌株间配对的体细胞亲和率达62．5％。不同倒木菌株间未发现体细胞亲和的配对。该群体存在11个特异的A因子和7个特异的B因子。同一倒木的菌株有的交配型因子相同,有的则不同。不同倒木的菌株大多数交配型因子不同,未发现交配型因子完全相同的菌株。RAPD分析显示,体细胞亲和的菌株,交配型因子完全相同的菌株,在基于DNA相似系数的遗传相关聚类中,首先聚为小类。总起来看,在自然群体中,香菇个体间的遗传差异与其空间分布之间存在一定的联系,随着空间距离的增大,菌株间的异质性相应增高。     

香菇自然群体中个体间的空间分布及其遗传联系*

应用体细胞不亲和性反应、交配型因子分析和基因组ＤＮＡ的ＲＡＰＤ分析,研究了一个分布于方圆约１ｋｍ的６根倒木上的１８个香菇野生菌株间的遗传差异。结果表明,该群体大多数菌株间配对（８０．４％）体细胞不亲和,而同一倒木菌株间配对的体细胞亲和率达 ６２．５％。不同倒木菌株间未发现体细胞亲和的配对。该群体存在 １１个特异的Ａ因子和７个特异的Ｂ因子。同一倒木的菌株有的交配型因子相同,有的则不同。不同倒木的菌株大多数交配型因子不同,未发现交配型因子完全相同的菌株。ＲＡＰＤ分析显示,体细胞亲和的菌株,交配型因子完全相同的菌株,在基于ＤＮＡ相似系数的遗传相关聚类中,首先聚为小类。总起来看,在自然群体中,香菇个体间的遗传差异与其空间分布之间存在一定的联系,随着空间距离的增大,菌株间的异质性相应增高。     

球孢白僵菌营养亲和型多样性与生态背景的关系

本文将来源不同的球孢白僵菌菌株分别按相同地理来源和相同分离奇主进行配对培养,发现相同地理来源菌株的营养亲和型多样性要明显小于相同分离寄主的菌株,前者的P及Shannon-Wiener多样性指数分别为0.4286及0.8708,后者分别为0.7500及1.3321。这说明来源于同一地区的菌株属于同一VCG的概率要大于相同分离寄主的菌株,同时也说明相同地理来源菌株的遗传相似性要高于相同分离寄主菌株的遗传相似性。     

香菇交配型因子次级重组体的鉴定

对13个香菇菌株的担孢子后代进行了交配型分析,其中8个菌株非亲和反应与亲和反应之比与预期的3∶1的比例无显著差异。另外5个菌株非亲和反应与亲和反应之比不符合3∶1,其中4个菌株在0.05显著水平的X2值仅略高于理论值,而另一菌株HL01具有特殊的表现,其单核体132个随机配对的非亲和反应与亲和反应之比为82∶50,X2值显著偏离3∶1的临界值。用4个标准测试菌株鉴定了来自HL01同一子实体的189个孢子单核体的交配型,在189个单核体中,161个单核体归于4种正常交配型(A1B1,A2B2,A1B2,A2B1)之一。而另外28个可能源于次级重组的单核体可分成另外4个类群。通过以所有可能的组合进行配对杂交,进一步分析了28个单核体的交配型。结果表明,次级重组同时在A因子和B因子中发生,重组值分别为8.5%和11.6%。A因子至少由2个亚基组成而B因子可能由不止2个亚基组成。随后的出菇试验表明,至少含有1个重组体的所有可亲和配对均具有结实能力。     

球孢白僵菌营养亲和型多样性与生态背景的关系

本文将来源不同的球孢白僵菌菌株分别按相同地理来源和相同分离寄主进行配对培养,发现相同地理来源菌株的营养亲和型多样性要明显小于相同分离寄主的菌株前者的Ｐ及Ｓｈａｎｎｏｎ－Ｗｉｅｎｅｒ多样性指数分别为０．４２８６及０．８７０８,后者分别为０．７５００及１．３３２１。这说明来源于同一地区的菌株属于同一ＶＣＧ的概率要大于相同分离寄主的菌株册时也是来源菌株的遗传相似性要高于相同分离寄主菌株的遗传相似性。     

担孢子介导传播对香菇双分体病毒LePV1群体形成的影响

【背景】LePV1为中国香菇种质资源中携带的主要病毒之一。在前期研究中,根据分子序列特征将LePV1带毒菌株群体分为两个分子类群(亚型I和亚型II),亚型I包含大部分带毒香菇菌株,而亚型II仅包含少数几个供试带毒香菇菌株,在地理上相距遥远的不同遗传背景香菇菌株中发现了同一LePV1分子类型。【目的】探究担孢子介导传播对香菇双分体病毒LePV1群体形成的影响。【方法】比较不同亚型菌株的有性担孢子后代带毒率差异,并采用单单杂交和单双杂交方式分析杂交对LePV1群体形成的影响等。【结果】亚型I中栽培菌株ZP51和野生菌株YS94的担孢子带毒率分别为70%和100%,亚型II中栽培菌株ZP28和野生菌株YS5的担孢子带毒率分别为45%和55%,暗示亚型I菌株担孢子传毒效率高于亚型II;当杂交配对的任一亲本携带LePV1时,无论是单单杂交还是单双杂交,获得的杂交子带毒率为100%。【结论】不同亚型担孢子病毒携带率的不同和杂交在香菇双分体病毒LePV1群体形成中可能发挥着重要作用;此外,LePV1不能在杂交不亲和的单核体之间传播,也不能在不亲和的单双杂交配对中进行传播;在杂交可亲和的单双杂交配对中,杂交成功的杂交子在与亲本双核体菌丝接触一段时间后,可以将病毒LePV1通过胞质传播传给亲本双核体菌丝体。该研究为明确香菇双分体病毒LePV1传播规律及LePV1群体形成机制提供了线索。     

粉被虫草无性型单孢子株间和单孢子株内的营养亲和性

在含5％氨酸钾的KMM和KPS培养基上,粉被虫草无性型以3种途径产生不利用硝酸盐的突变株（nit突变株）。（1）由菌落基质菌丝形成的快速生长气生菌丝角变;（2）菌落表面快速生长的气生菌丝;（3）菌落基质菌丝缓慢生长形成的基质菌丝角变。来自18个单孢子株的94个nit突变株中,64.8％的突变株是稳定的。配对试验结果表明:在全部19个配对中,单孢子株内配对率为57.9％,单孢子株间配对率为42.1％。在全部nit突变株中,Cp-14c3突变株与其它突变株间的配对率最高（18.2％）。单孢子株间配对率高的孢子株是Cp-14Cp-7,Cp-5和Cp-6,将来自Cp-14同一单孢子株的Cpe-14C3分别与Cp-14cl和Cp-14c4nit突变株配对后发现,它们形成的浓密生长配接线的颜色是不相同的,前者橙色,后者白色。统计结果发现,所试全部的单泡子株可分成11个营养亲和群（VCGs）,那些含有易与其它菌株配对的nit突变株的单孢子株,如Cp-1,Cg-4,Cp-5,Cp-6,Cp-7,Cp-13,和Cp-14等皆在同一营养亲和群内。用Hochest33258荧光染色观察发现,野生型菌株的菌丝和分生孢子单核,nit突变株的少量分生孢子中可见双核,互补配对形成的浓密菌丝丛中的分生孢子则常见双核。     

玉米穗、茎腐病串珠镰孢菌nit突变体的筛选及营养亲和性测定

在真菌的生活史中,不同个体间常形成异核体,它能使单倍体菌丝享受双倍体菌丝在功能上的优越性,如杂种优势等(Leslie,1993)。菌丝间营养亲和的过程即是形成异核体的过程。菌丝间能否进行营养亲和形成异核体,受营养非亲和性基因(VIC)控制,VIC基因代表一种识别自身的机制,这种机制在生物界的大部分生物中均有存在。若不同菌株能相互亲和,形成稳定的营养异核体,表明菌株在每一个VIC位点的等位基因都是一致的,因此亲和与否能反应菌株在遗传进化过程中亲缘关系的远近。属于同一营养亲和群(VCG)的菌株,在遗传上是同源的(鲍建荣等,1992)。Puha…     

亚洲产Clavicorona的形态学及亲和性研究

中国产的Clavicorona pyxidata与北美的种样本间配对实验结果显示该种在不同的地理分布区内的种群之间完全性亲和或具有相同的交配型等位基因,表明中国产与北美产的该形态学种同属于一个生物种。日本产的标本与C. pyxidata在子实体外部形态及孢子尺度上有差异,虽然在日本被鉴定为该种。日本产的菌株与C. pyxidata菌株间配对实验证明二者之间完全不亲和,即属于不同的生物种。生殖隔离拌随着形态学变异。     

新月弯孢菌Curvularia lunata营养体亲和性的进一步研究

来自全国39个地区的70株新月弯孢菌Curvularialunata在WAC培养基上诱导培养后,随机挑取10197个抗KClO3突变体,经CDA鉴定获得2207株nit突变体,nit突变体频率为21.64%。在这些nit突变体中,1397个为nit1,占63.30%;734个为NitM,占33.26%;76个为nit3,占3.44%。70个菌株全部获得了稳定的nit突变体,其中52个菌株获得了NitM突变体。结果表明WAC比先前报道的KPS更适合用于C.lunatanit突变体的筛选。通过不同菌株间互补nit突变体配对测试,将其中的65个C.lunata菌株划分为22个营养体亲和群(VCGS),而另外5个菌株因未获得NitM突变体暂时无法确定其VCG。划分出的22个VCGs中,有11个VCGs是由多菌株组成的,VCG3为优势类群,含18个菌株,其地理来源最复杂,主要为致病性中等以上的菌株;其余11个VCGs内均仅有1个自身亲和的菌株。以上结果初步表明,在C.lunata群体内存在丰富的VCG多样性,VCG3可能是与致病性相关的优势VCG,但营养体亲和性与菌株地理来源没有明显的直接关系。     

细脚拟青霉田间分离菌株间的异核现象

本文报道细脚拟青霉(paecilomyces tenuipes)不同田间分离菌株单孢子后代间的异核现象。用来自荣园、菜地、水稻田三种生境的四个菌株(803、2801、1401和3101)的单孢子培养后代,在加有酵母膏和麦芽糖的改良萨氏培养基上进行配接实验,仅在803与1401两菌株间能形成异核体,其频率为13.5%。在配接实验中两亲和菌落交界处长出白色致密的菌丝簇组成的实线可推测为异核体。从来自菌丝簇组线的每个单菌丝尖端培养物中分离出30个以上的单孢子,井分别移接到 PDA 平板上。将来源于单孢子的菌落与两亲本菌落进行比较,有三个单菌丝尖端培养物(C_3、B_3、F_3)重现了两亲本类型或出现了新的菌落类型,从而异核现象得到证实。     

Vegetative compatibility groups of Japanese isolates ofVerticillium dahliae

Japanese isolates ofVerticillium dahliae were examined for vegetative compatibility relationships using nitrate-nonutilizing mutants. Four levels of vegetative compatibility were differentiated according to the degree of compatibility between the tester mutants ofnit1 and NitM. Wild-type growth with a complementation line greater than 5 mm wide was defined as “strong reaction (++)”, i.e., compatible. Ten out of 15 isolates showed compatibility and were separated into three groups, provisionally designated as VCGJ1, VCGJ2, and VCGJ3, depending upon their reactions. This method was used to estimate, genetic diversity within a local population ofV. dahliae. Another 12 isolates from Gunma Pref. were paired with tester isolates of the three vegetative compatibility groups proposed. Eight Gunma isolates were assigned to VCGJ1 or VCGJ2. Two isolates were incompatible with all testers. The remaining 2 isolates were self-incompatible. Thus, 18 out of 27 Japanese isolates ofV. dahliae were assigned to VCGs: 8 to VCGJ1, 7 to VCGJ2, and 3 to VCGJ3. VCGJ1 was compatible with both VCGJ2 and VCGJ3, but VCGJ2 and VCGJ3 showed a weak reaction with each other. Japanese isolates ofV. dahliae were thus demonstrated to form a VC group comprising three subgroups.     

Molecular differences distinguish clonal lineages within East African populations of Fusarium oxysporum f.sp. cubense

Molecular approaches for the assessment of intraspecific diversity within an economically important plant pathogen were compared with traditional physiological methods (vegetative compatibility testing). The vegetative compatibility groups (VCGs) of 14 isolates of Fusarium oxysporum f.sp. cubense (FOC) from Kenya were first assessed using nitrate non-utilizing mutants. Nine of these isolates, from different areas of the country, were compatible with one or more of VCGs 0124, 0125, 0128 and 01220, i.e. they formed a single clonal lineage. Three isolates, all originating from the banana growing district of Kisii, were compatible with the VCG 01212 and formed a second distinct clonal lineage. Mutants could not be recovered from one isolate (62) and two isolates (27 and 30) were not vegetatively compatible with any of the VCG testers and may represent two novel VCGs. Polymerase chain reaction (PCR) fingerprinting, especially when using the M13 derived primer, was found to produce banding patterns that correlated with clonal lineage and also distinguished isolates 27 and 30 when analysed by unweighted pair group method analysis and principle co-ordinate analysis. This approach also distinguished FOC from F. oxysporum IMI350438 isolated from Triticum sp. and from isolates of Colletotrichum gloeosporioides . Total protein profiles were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and although clonal lineages were not separated, isolates 27 and 30 were again distinguishable and FOC produced a different profile to F. oxysporum (IMI 350438) and C. gloeosporioides.     

Variability in Indian isolates of Sclerotium rolfsii

Variability among 26 isolates of Sclerotium rolfsii collected from various hosts/soil samples and localities in India is reported. The isolates varied in colony morphology, mycelial growth rate, sclerotium formation, teleomorph production and sclerotial size and color. Out of 26 isolates, only 4 produced the teleomorph stage on Cyperus rotundus rhizome meal agar medium. Mycelial incompatibility among the isolates was also seen, and out of 325 combinations, only 29 combinations (8.9%) showed compatible reactions. Based on mycelial compatibility, 13 vegetative incompatibility groups (VCG) were identified among the isolates. HPLC analysis of the ethyl acetate fraction of culture filtrates of the isolates revealed 10-22 peaks. Six peaks were identified as gallic, oxalic, ferulic, indole-3-acetic acid (IAA), chlorogenic, and cinnamic acids. Oxalic, IAA, and cinnamic acids were present in the culture filtrates of all the isolates in varying amounts. The other three phenolic acids were not detected in some of the isolates. A comparative HPLC analysis of sclerotial exudate, sclerotia, mycelia, and culture filtrates of two S. rolfsii isolates (leaf spot- and collar rot-causing) producing different symptoms on their respective hosts revealed variation in the content of phenolic acids, IAA, and oxalic acid.     

High diversity of vegetative compatibility types in Cryphonectria parasitica in Japan and China

We found high diversity of vegetative compatibility (vc) types in native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China; almost every isolate was in a unique vc type. In Japan we found 71 vc types in a sample of 79 isolates pooled from six populations. Within two populations in China, all isolates (n=28 and 11) had unique vc types; we found 15 vc types among 25 isolates in a third Chinese population where multiple isolates were collected from some trees. None of the isolates from China and only three isolates in the 71 vc types from Japan were compatible with any of 64 vc type testers from Europe, which have known vegetative incompatibility genotypes. To our knowledge this is the first report of vc type diversity for C. parasitica in Japan or of any comparisons of vc types between Asia and Europe. The most significant result of this survey is the identification fungal isolates for expanding knowledge of the genetics of vegetative incompatibility.     

Genetic and molecular characterization of pathogenic isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) in the state of Paraná, Brazil

Isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) spikes with blast symptoms were analyzed by classical (VCG) and molecular (RAPD) techniques. P. grisea mutants, unable to use sodium nitrate (nit) as nitrogen source, were obtained with potassium chlorate. For vegetative compatibility (VCG) tests, genetically complementary nit mutant pairs were inoculated in a medium with sodium nitrate as a single nitrogen source. P. grisea isolates were divided into two vegetative compatibility groups and two RAPD groups. Since vegetative compatible strains may mutually exchange genetic and cytoplasmatic material, the contribution of the parasexual cycle in the genetic variability of Brazilian P. grisea isolates is discussed.     

Association between vegetative compatibility and aflatoxin production by <Emphasis Type="Italic">Aspergillus</Emphasis> species during intraspecific competition

Intraspecific competition is the basis for biological control of aflatoxins, but there is little understanding of the mechanism(s) by which competing strains inhibit toxin production. Evidence is presented that demonstrates a relationship between strength of the vegetative compatibility reaction and aflatoxin production in Aspergillus flavus and A. parasiticus using the suspended disk culture method. Combining wild-type aflatoxin-producing isolates belonging to different vegetative compatibility groups (VCGs) resulted in a substantial reduction in aflatoxin yield. Pairs of aflatoxin-producing isolates within the same VCG, but showing weak compatibility reactions using complementary nitrate-nonutilizing mutants, also were associated with reduced levels of aflatoxin B₁. In contrast, pairings of isolates displaying a strong compatibility reaction typically produced high levels of aflatoxins. These results suggest that interactions between vegetatively compatible wild-type isolates of A. flavus and A. parasiticus are cooperative and result in more aflatoxin B₁ than pairings between isolates that are incompatible. Successful hyphal fusions among spore germlings produce a common mycelial network with a larger resource base to support aflatoxin biosynthesis. By comparison, vegetative incompatibility reactions might result in the death of those heterokaryotic cells composed of incompatible nuclei and thereby disrupt the formation of mycelial networks at the expense of aflatoxin biosynthesis. The content of this paper was presented at the 50th Anniversary Meeting of the Mycological Society of Japan, June 3–4, 2006, Chiba, Japan     

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ('vegetative isolates') with the majority possessing multiple delta-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified delta-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.     

粟胡麻斑病菌有性世代的研究

诱发粟胡麻斑病菌(Cochliobolus setariae)有性世代的条件:培养温度23—24℃,Sach培养基(pH 不予调整),培养基物稻草杆段。粟胡麻斑病菌有性生殖的方式为异宗配合,病菌菌株间有性杂交的亲和性较低。粟胡麻斑病菌和玉米小斑病菌(Cochliobolus heterostro-phus)的种间杂交,产生不育的子囊。     

How do Japanese isolates ofVerticillium dahliae correspond with standardized VCG testers?

We examined the vegetative compatibility of 56 Japanese isolates provisionally assigned to four subgroups ofV. dahliae to estimate the genetic relatedness with testers of the standardized VCGs. Subgroup J1 was assigned to VCG 2A/B as a new category of assignment. Subgroup J2, except isolate Vdt 110, was assigned to VCG 2A, and subgroup J3, except isolate Vdf 1, was assigned to VCG 2B. Isolates Vdf 1 and Vdt 110 were assigned to VCG 2A/B. Subgroup J4 was assigned to two subgroups, VCG 4B for Vde 1 and VCG 4A/B for FY 3 and HR 1. Four isolates were compatible with both VCG 2 and 4. Isolate U56 was compatible with VCG 2A/B and 4A. Isolates of VCG 2A, Vdt 9 and FF1, were compatible with either VCG 4A or 4A/B. One isolate of VCG 2B, Vdp-4, was compatible with VCG 4A. Three isolates of subgroup J2 showed weak reactions with the testers of VCG 4. These isolates may be “bridging strains”. Japanese isolates were composed of two VCGs, 2 and 4, “bridging strains” compatible with these VCGs, and some self-incopatible isolates. Testers of VCG 1 and VCG 3 did not show any reactions with the Japanese isolates.