致病疫霉在中国云南的马铃薯田间形成卵孢子*

由致病疫霉Phytophthora infestans (Mont.)de Bary引起的马铃薯和番茄晚疫病是世界性的作物病害,每年均造成巨大的经济损失和社会影响。致病疫霉是异宗配合的卵菌,有两个已知交配型A1和A2,两个相对交配型互作时可进行有性生殖产生卵孢子( Gallegly & Galindo,1958)。过去许多年一直认为致病疫霉在除墨西哥以外的国家中只存在A1交配型,通过产生孢子囊进行无性繁殖。近年来,由于致病疫霉新致病群体的产生以及全球范围的迁移和替代,A2交配型菌株先后在欧洲、美洲、亚洲和非洲的许多国家被发现。A1、A2两种交配型菌株的同时存在,增…     

河北省番茄上致病疫霉Phytophthora infestans群体结构的分析

番茄晚疫病是河北省番茄生产上最具毁灭性的病害之一,对引起该病害的致病疫霉群体结构进行分析有利于病害的防治。利用对峙培养法和菌落直径法对2007-2008年采自河北省保定、沧州和唐山分离自番茄的49个致病疫霉菌株进行了交配型和甲霜灵抗性的表型测定,结果表明该群体所有菌株均为A1交配型,以甲霜灵敏感菌株为主,抗性菌株仅7株。利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)、简单序列重复(SSR)和扩增片段长度多态性(AFLP)等分子技术对该群体的基因型进行了分析,结果表明供试菌株线粒体基因型均为Ia型,共鉴定出了Ⅰ、Ⅱ和Ⅲ3种SSR基因型,AFLP聚类分析在相似系数0.87时可以形成α、β和γ等3个不同的分支。河北省所有番茄上致病疫霉菌株均分布在α分支上,该分支又可进一步分为7个亚分支。AFLP亚分支与甲霜灵抗性和地理来源均无明显相关性,但Ⅱ型SSR与甲霜灵抗性和地理来源有明显的相关性。综合表型和基因型数据说明河北省番茄上致病疫霉群体结构比较单一,遗传多样性程度较低。     

四川省马铃薯晚疫病菌群体表型和遗传变异的分析

致病疫霉Phytophthora infestans引起的晚疫病是马铃薯的一种毁灭性病害。为了对四川省近年马铃薯晚疫病菌进行系统多样性分析,本研究从表现型和基因型两个方面鉴定了四川省马铃薯晚疫病菌的群体多态性。交配型、甲霜灵敏感性和生理小种的鉴定结果发现A1交配型菌株只有2份,A2型有29份,自育型12份;甲霜灵敏感菌株3株,中抗菌株22株,高抗菌株18株;共测定出11个生理小种,其中全毒力小种发生频率最高,占供试菌株的25.58%。基因型鉴定结果表明,43份材料共发现了10种SSR基因型,5个SSR标记在该种群上共产生了16个SSR位点,每个标记平均有3.2个位点,多态性信息含量平均值为0.46,SSR4是多态性最丰富的标记。本试验中A2菌株和自育型的基因型SSR相同,其中全毒力小种是当地的优势致病菌,该研究为今后四川马铃薯晚疫病的有效防控提供理论依据。     

草菇子代单孢菌株的菌落类型与A因子分布规律研究

根据菌株在培养皿中的生长情况,草菇V23的124个单孢分离菌株可分为气生型和匍匐型两大类,气生型菌株为44株,匍匐型菌株为80株。根据草菇A因子相关特异性分子标记,PCR验证单孢萌发菌株的A因子中的A1、A2分子标记的分布情况,探讨了A因子与不同菌落形态的相关性。试验结果表明:124株菌株中,同核体101株,异核体为23株,所占比例分别为81.45%和18.55%。气生型的草菇单孢菌株A1因子为20株,占气生型菌株比例为45.45%,气生型的草菇单孢菌株A2因子为15株,其比例为34.09%;匍匐型的草菇单孢菌株A1因子为15株,占匍匐型菌株比例为18.75%,匍匐型的草菇菌株A2因子为51株,其比例为63.75%,未能发现A因子与菌落形态之间的明显相关性。选用不同A因子,不同菌落表型的草菇菌株相互交配,经PCR筛选,获得20株真正的杂交菌株,杂交菌株的菌落形态气生型与匍匐型占的比例为1:1。表明只要气生型菌丝参与杂交,其杂交菌株的菌落形态则是以气生型为主;匍匐型与匍匐型杂交后的菌丝也不全是气生型,而是以匍匐型为优势群体。选取8株杂交菌株进行岀菇,只有1株产生子实体。     

云南番茄致病疫霉的交配型、甲霜灵敏感性及毒力类型

对2003~2004年云南省番茄致病疫霉的交配型、甲霜灵敏感型、毒性因子及毒力类型进行了系统的测定和分析。结果表明:云南番茄致病疫霉只存在A1交配型,未发现A2交配型或自育型。甲霜灵抗性和中抗菌株是主要菌系,抗性、中抗、敏感菌株分别占测定菌株的51.2%、31.7%、17.1%。云南番茄致病疫霉居群对已知的抗性基因R1、R2、R3、R4、R6、R7、R8、R9、R10、R11有毒性,其毒性频率在9.8%~95.1%之间。测定的61个菌株中检测到26种不同的毒力类型,每种类型平均含有5.2个毒性因子,其中毒力类型1.3.4.7.9出现频率最高,为优势的毒力类型,出现频率为44.3%,其次为1.3.4.6.7.9、1.3.7.9,出现频率均为4.9%,其它23种毒力类型出现频率仅在1.6%~3.3%之间。云南番茄致病疫霉居群显示了复杂的表型特征。     

江苏省稻瘟病菌有性态的研究

用标准菌株对1997－1999年在江苏吴江市、宜兴市、通州市、高邮市和赣榆县采集的325个稻瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为22．77％,可育率仅为7．08％。不同年份、不同地区采集的稻瘟病菌菌株的性亲和力和交配型有较大的差异,三年的交配率分别为26．61％、8．26％和33．64％;通州地区和赣榆地区菌株的交本相对较高,分别为26．15％和25．42％,宜兴地区菌株的交配率较低,只有15．38％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,1997年29个可交配菌株中有21个菌株表现为MAT1－2交配型,而1999年36个可交配菌株均为MAT1－1交配型。用江苏省稻瘟病菌的可育菌株进行互交,25个组合中只有6个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

马铃薯晚疫病菌DK98-1、DX98-2和DX98-3的生物学特性及AFLP指纹分析

利用黑麦培养基和V8-蔬菜汁培养基研究了马铃薯晚疫病菌Phytophthora infestans特异菌株DK98-1、DX98-2和DX98-3 的生物学特性,发现该菌株与普通菌株相比菌落生长速度慢、孢子囊产生数量少、有性杂交后卵孢子产生量大（2047～75623个/cm²）;利用AFLP分子标记研究这3个菌株的DNA指纹图谱,发现用引物E+CG/M+CC扩增菌株DK98-1、DX98-2和DX98-3后,在330bp处与普通菌株相比各缺失一条谱带,用引物E+AC/M+CT扩增菌株DK98-1、DX98-2和DX98-3后,在370bp处比普通菌株增加1条谱带,说明这3个菌株与普通菌株在遗传上明显不同。同时可以利用上述2对特异性引物,鉴定在自然界的晚疫病菌群体中这类特异菌株的出现频率。     

马铃薯晚疫病菌DK98-1、DX98-2和DX98-3的生物学特性及AFLP指纹分析

利用黑麦培养基和V8-蔬菜汁培养基研究了马铃薯晚疫病菌Phytophthora infestans特异菌株DK98-1、DX98-2和DX98-3的生物学特性,发现该菌株与普通菌株相比菌落生长速度慢、孢子囊产生数量少、有性杂交后卵孢子产生量大(2047～75623个/cm2);利用AFLP分子标记研究这3个菌株的DNA指纹图谱,发现用引物E CG/M CC扩增菌株DK98-1、DX98-2和DX98-3后,在330bp处与普通菌株相比各缺失一条谱带,用引物E AC/M CT扩增菌株DK98-1、DX98-2和DX98-3后,在370bp处比普通菌株增加1条谱带,说明这3个菌株与普通菌株在遗传上明显不同。同时可以利用上述2对特异性引物,鉴定在自然界的晚疫病菌群体中这类特异菌株的出现频率。     

从天然蛹虫草不同部位分离菌株特性比较

以采集自泰山地区的6株野生蛹虫草为研究对象,分别通过菌核、子座组织分离和子囊孢子分离法共获得18个菌株,经ITS鉴定均为蛹虫草菌株后,进一步对18个菌株进行了菌丝生长速度、菌落形态、出草试验、主要活性物质（虫草素、腺苷、虫草多糖、β-类胡萝卜素、叶黄素）和交配型的测定分析。结果表明：在菌丝长势、出草产量和整齐度方面,从子囊孢子分离得到的菌株普遍优于组织分离菌株;而在活性物质含量方面,从子囊孢子和子座分离得到的菌株普遍优于菌核分离菌株。通过交配型测定发现,从菌核、子座和子囊孢子分离得到的18个菌株均不存在交配型缺失现象。     

桦纤孔菌有性生殖相关特征及交配型位点分析

对桦纤孔菌菌株MDJCBS88的显微形态、菌丝及担孢子核相进行了观察。采用棉籽壳培养基对担孢子萌发形成的菌株进行栽培试验,筛选出不形成子实体或子实体发育不完整的菌株,将这些菌株在平板上进行了亲和试验,分析桦纤孔菌的有性生殖方式;并基于基因组序列进行交配型基因克隆验证,分析桦纤孔菌的交配型位点结构。显微观察发现,桦纤孔菌菌丝没有锁状联合结构,菌丝细胞无核到多核;子实层担孢子可含0-4个不等的细胞核,不同时期弹射的担孢子含有的细胞核数量不同。桦纤孔菌担孢子萌发率极低,能萌发的担孢子多为早期弹射的担孢子;培养基也影响担孢子的萌发率,与PDA培养基和CYM培养基相比,桦木屑培养基最适合桦纤孔菌担孢子萌发,萌发率为4.55%。从担孢子萌发的96个菌株中获得了2个不结实菌株和9个结实不产孢菌株,占11.5%,这些菌株间亲和试验出现不同的表现特征,包括形成产孢子实体,产生菌丝纽结,相互融合和相互拮抗等现象,认为桦纤孔菌的有性生殖以次级同宗结合为主,并受交配型基因控制。交配型位点克隆测序后分析发现,桦纤孔菌交配型A位点共14 034 bp,含有一个MIP基因和两组HD1和HD2基因;交配型B位点包含3个疑似信息素受体基因和1个信息素前体编码基因。     

Phenotypic and Genotypic Characterization of Phytophthora infestans Isolates from China

A total of 288 (202 from potato and 86 from tomato) isolates of Phytophthora infestans were collected from 1998 to 2007 in China. The isolates were characterized based on mating type, in vitro metalaxyl sensitivity, virulence on potato differentials, allozymes of glucose-6-phosphate isomerase ( Gpi ), peptidase ( Pep ), and mitochondrial DNA (mtDNA) haplotype and examined by DNA-based simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) fingerprinting. The majority (283 of 288) of the isolates were of the A1 mating type, the other three were the A2 mating type and two were the A1A2 mating type. Resistance to metalaxyl was frequently observed, with 248 (86.1%) resistant, 21 (7.3%) intermediate and 19 (6.6%) sensitive isolates identified. Virulence was assessed for 125 isolates on a set of 11 potato differentials and 61 races were detected. Most isolates were virulent on the differential genotype with gene R3, and all known virulence genes were found, with race 3.4.7.11 being the most common. This pattern did not appear to be associated with geographic origin, sample type, mating type or metalaxyl sensitivity. The dominant banding patterns for Gpi were 100/100/111 (176 isolates) and 100/100 (109 isolates), but genotypes 86/100 and 100/111 were also identified. All isolates tested were homozygous (100/100) at the Pep locus. The majority (205 of 288) of isolates tested was of mtDNA haplotype IIb, 76 were haplotype IIa and seven were the rare Ib haplotype. The genetic diversity of 60 representative isolates from China was assayed by two types of molecular markers, RAPD and SSR. A high level of polymorphism was found. The results demonstrated the diverse phenotypic and genotypic structure of the current populations of P. infestans in China.     

Phytophthora infestans Field Isolates from Gansu Province,China are Genetically Highly Diverse and Show a High Frequency of Self Fertility

The genetic diversity of 85 isolates of Phytophthora infestans collected in 2007 from Gansu province in China was determined and compared with 21 isolates collected before 2004. Among them, 70 belonged to the A1 mating type and 15 were self‐fertile (SF). The mitochondrial DNA haplotypes revealed both Ia (25%) and IIa (75%) haplotypes. Metalaxyl resistance occurred with high frequency (54%) in Gansu. Simple sequence repeat (SSR) genotyping revealed 26 genotypes (13 from the Tianshui region) among the 85 isolates, and 18 genotypes among the 21 isolates collected before 2004, without overlap in genotypes detected in the two groups. Cluster analysis showed clear subdivisions within the different mating type isolates. Among Gansu's isolates, Nei's and Shannon's diversity indices were highest in isolates collected in Tianshui where both A1 and SF isolates were found. Analysis of molecular variance of isolates from Gansu indicated that 51% and 49% of the variance was explained by within‐area and among‐area variance, respectively. The results suggest that the occurrence of SF isolates increases the risk of sexual reproduction, the formation of oospore as initial inocula in the field, and affects the genotypic diversity in the population.     

麦根腐平脐蠕孢有性生殖生物学研究

本文报道麦根腐平脐蠕孢Bipolaris sorokiniana(有性型为Cochliobolus sativus)人工诱导形成子囊座,并获得该菌A和a两种不同交配型。子囊座形成的最佳条件是:以麦秆浸汁琼脂为培养基(pH6—6.5)。以透析袋为培养基物,24℃培养1周,移至20℃培养2周。培养中光照不是重要因素。在无培养基物的培养基上不形成子囊座。对不同地区、不同寄主植物上麦根腐平脐蠕孢72个菌株交配型测定结果,两个不同交配型在自然界分配比例为1:1,交配型菌株的分布与地理、寄主植物的差异无相关性。     

江苏省稻瘟病菌有性态的研究*

用标准菌株对１９９７～１９９９年在江苏省吴江市,宜兴市、通州市、高邮市和赣榆县采集的３２５个猪瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为２２．７７％,可育率仅为７．０８％。不同年份、不同地区采集的猪瘟病菌茵株的性亲和力和交配型有较大的差异,三年的交配率分别为２６．６１％、８．２６％和３３．６４％;温州地区和赣榆地区菌株的交配率相对较高,分别为２６．１５％和２５．４２％,宜兴地区菌株的交配率较低,只有１５．３８％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,１９９７年２９个可交配菌株中有２１个菌株表现为ＭＡＴ１－２ 交配型,而１９９９年３６个可交配茵株均为ＭＡＴ１－１交配型。用江苏省稻瘟病菌的可育菌株进行互交,２５个组合中只有６个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

Diversity of the Phytophthora infestans population in Flanders, Belgium

A total of 94 isolates of Phytophthora infestans were collected from disease outbreaks in commercial potato crops and private gardens in 2002 and 2003. The isolates were recovered successfully from single lesions of diseased potato foliage. Not from all isolates pure cultures were obtained due to contaminations with Fusarium species and bacteria. The structure of the population was analysed phenotypically. Characteristics of the isolates included in vitro growth rate, mating type, in vitro sensitivity to the phenylamide fungicide metalaxyl-M and allozyme genotype at glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) loci. Significant differences in in vitro growth rate were observed among the 52 isolates by comparing the main radial growth of the isolates after 7 days. Forty seven from the isolates tested were the Al mating type. Only one isolate was characterized as A2 mating type. Isolates with sensitive, intermediate and resistant responses to metalaxyl-M were detected in the populations. Forty isolates had a growth of less then 40 % at 5 ppm metalaxyl-M. Three isolates had a growth of less then 40 % at 100 ppm metalaxyl-M. Eight isolates had a growth of more then 40 % at 5 and 100 ppm metalaxyl-M. Cellulose acetate electrophoresis was used to examine Gpi and Pep banding pattern of the population of P. infestans attacking potato in Flanders. All the isolates tested produced the 100/100 Gpi isozyme electromorph. Five different allozyme genotypes of the Pep loci were identified: 92/92, 96/96, 100/100, 92/100, 83/100.     

Isolation of New Aureobasidium Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation

New isolates of Aureobasidium pullulans were obtained from plant leaf surfaces gathered in San Diego County. The new fungal isolates were identified as A. pullulans on the basis of the appearance of polymorphic colonies formed on agar plates, the electrophoretic profiles of repeated genomic DNA sequences, and the production of pullulan in shake flask cultures. The isolates showed different degrees of pigmentation. One of the natural isolates was nonpigmented under mock production conditions in liquid culture, but was still able to synthesize a reduced amount of pigment on agar plates at late times. A mutagenic treatment with ethidium bromide produced derivatives of normally pigmented natural isolates that exhibited an increased tendency toward yeastlike growth and reduced pigmentation. Additionally, some of the new isolates and mutant derivatives accumulated pullulan of relatively high molecular weight in the culture broths.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.