球孢白僵菌继代培养中菌落局变现象及环境影响因素的研究

球孢白僵菌野生型多孢标以及从其上分离的单孢株在不同培养基,光照,温度和湿度条件下继代培养。结果显示：无论是多孢株还是单孢株。在传代中均发生显著的菌落局变,并产生形态各异的分离子,多表现为菌苔瘠薄或气生菌丝徒长,肉眼可见产孢量减少,尤其是单孢株菌落局变产生的分离子往往伴随着棕黄色或红棕色色素的分泌。培养基、温度、湿度和光照等环境因素对菌落局变产生具明显诱导作用,以ＳＤＡＹ培养基、偏低的温湿度和全光照     

球孢白僵菌继代培养中的菌落局变现象

通过球孢白僵菌两个野生型菌株及其单孢株在不同培养基、温度、湿度和光照条件下继代培养,结果产生3种不同类型的菌落局变.局变产生的分离子多表现为菌落瘩薄或气生菌丝陡长、产孢量下降、生长速率增加等现象.培养基、温度、湿度和光照等因素对菌落局变有明显诱导作用.湿度为15℃,相对湿度较低及全光照条件下于SDAY培养基上培养,局变发生频率最低.在实验室继代培养中,野生型菌株在4-14代内即可发生局变而被局变分离子所取代.引人注意的是,单孢株局变产生的分离子同时伴随着棕黄色或红棕色素的分泌,仅以菌落形态和颜色等培养特征作为球孢白僵菌种的划分依据是不可靠的.文中提出了菌落局变发生的遗传变异可能机制.     

地生弯颈霉的培养与生长条件

从长白山北坡森林土壤中分离纯化得到1株地生弯颈霉(Tolypocladium geodes),对其培养及生长条件(如培养基、光照、温度、碳源、氮源和pH等)进行了试验。结果表明,地生弯颈霉在麦芽浸膏琼脂培养基(MEA)上菌落生长直径最大,马铃薯葡萄糖琼脂培养基(PDA)上产孢量最高;5~30℃温度范围内均能生长,25℃时生长和产孢量最佳;最适碳源和氮源分别为葡萄糖和柠檬酸铵;光暗交替条件下,最利于其生长和产生孢子;pH4~9范围内均能生长和产孢,pH=8时菌落直径最大,pH=6时产孢量最高。     

球孢白僵菌单孢子分离株在继代培养过程中菌落局变的遗传分析

研究了球孢白僵菌Beauveria bassiana单孢株继代过程中的菌落局变现象,并通过AFLP分析了角变子与原菌株间在分子水平上的差异。结果表明,野生型出发菌株Bb13的单孢分离株13S5和13S8在继代培养过程中均发生生长速率加快和产孢量下降现象。13S5在前5代,13S8在前6代产孢量有所下降但不显著,至第10代时产孢量比第1代分别下降了81.7%和69.0%。菌株角变现象在继代培养5-6代后表现明显,而13S8角变子出现的频率更高。AFLP指纹图谱分析表明,用20个引物组扩增出的98个位点中,两个角变子与野生型菌株间有12条差异条带,变异率达12.2%。由此证明单孢子分离株在继代培养中发生菌落局变后遗传物质已产生了变异。     

一株球孢白僵菌转基因工程菌在继代培养中的变异

基因工程是解决昆虫病原真菌致死慢、致死率低的有效途径,但在传代培养过程中,被转入的基因是否会丢失以及转基因工程株是否会像野生型菌株那样频繁地发生变异,引起产孢减少和毒力减退等生产性状的退化？针对这些问题,研究一株球孢白僵菌Beauveria bassiana转外源蝎毒基因工程菌株在继代培养过程中的的变异现象,发现工程株也和野生型菌种一样会发生菌落局变,但所插入的蝎毒基因并未丢失。与野生型出发菌株及其单孢分离株相比,工程株的角变率居高不下而产孢量及对松毛虫的毒力显著降低。然而,从该工程株筛选出的一株单孢分离     

球孢白僵菌野生及转基因菌株的退化

球孢白僵菌在人工培养基上继代培养容易发生退化变异,表现为菌丝徒长以及产孢量减少和毒力的逐渐下降,给真菌杀虫剂的生产和使用造成较大的损失.没有丢失被插入的外源毒力基因的工程株同样出现退化.培养基成分和含水量以及培养温度和光照等环境因子皆可不同程度地诱发退化的发生,但退化主要由其遗传机制决定,包括线粒体基因突变、异核现象、准性生殖、核基因突变以及有性重组等;菌落局变实质上也是菌种老化的征兆.真菌杀虫剂生产中防止退化的对策除减少传代次数外,还要根据具体菌株确定适宜的环境控制细则.     

响应面法优化蝉棒束孢SLGY-2产孢条件

棒束孢菌Cordyceps sp.是一类重要的昆虫病原真菌,具有开发潜能。前期(通过生物毒力测定)筛选一株对斜纹夜蛾具有较高毒力蝉棒束孢SLGY-2菌株,为进一步提高其产孢活力,分别利用单因素方差法和中心组合设计配合响应面分析两种方法开展了本次研究。本研究以产孢量为指标,选择培养基营养成分含量、pH值、温度以及光照条件等进行单因素试验,通过响应曲面法优化最佳产孢条件。结果表明:SLGY-2菌株在实际和模拟较为吻合,模型中最佳产孢条件为培养基营养成分含量为38.3 g、pH 6.55、温度25.21℃、光照18.04 h。在该培养条件下,产孢量达到5.70×10~8个/mL,可用于孢子批量生产。     

蛹虫草(Cordyceps militaris)无性型的多型现象*

在萨氏琼脂和PDA上,蛹虫草Cordycepsmilitaris（Vuill）Fr.无性型,蛹草拟青霉Paecilomycesmilitaris(Kob）Br.＆Sm．的一些单孢子株可自发产生突变,基于多型现象及其它形态特征可分三种类型：（1）具野生型菌株特征;产孢结构拟青霉型（Paecilomyces－type）,稳定,菌落通常不自发产生角变,大多数单孢子株属此类群。（2）属此类群的单孢子株,在PDA上可形成亮褐色至橙黄色的角变,它们典型的产孢结构为瓶梗轮生,分生孢子常聚集成头状的轮枝孢型（Verticillium－type）。在查氏培养基上则相反,拟青霉型的产孢结构占优势。单孢子株Cm-71是这一类群的代表。（3）单孢子株Cm－42是在所观察单孢子株中形态特征独特的菌株。在PDA上菌丝生长纤细,蛛网状,产孢结构轮枝孢型。蛹草拟青霉对氯酸钾（KClO3）不敏感,在浓度为6％（W／V）的KMM培养基上,23～24℃14天菌落的直径仅1～2cm,无气生菌丝生长。各单孢子株形成的不利用硝酸盐突变株（nit突变株）的途径和形态特征,也可分成类似于粉被虫草CordycepspruinosaPeteh不利用硝酸盐突变株（nit突变株）的三种类型。     

蛹虫草(Cordycepsmilitaris)无性型的多型现象

在萨氏琼脂和PDA上,蛹虫草Cordycepsmilitaris（Vuill）Fr.无性型,蛹草拟青霉Paecilomycesmilitaris(Kob）Br.＆Sm．的一些单孢子株可自发产生突变,基于多型现象及其它形态特征可分三种类型：（1）具野生型菌株特征;产孢结构拟青霉型（Paecilomyces－type）,稳定,菌落通常不自发产生角变,大多数单孢子株属此类群。（2）属此类群的单孢子株,在PDA上可形成亮褐色至橙黄色的角变,它们典型的产孢结构为瓶梗轮生,分生孢子常聚集成头状的轮枝孢型（Verticillium－type）。在查氏培养基上则相反,拟青霉型的产孢结构占优势。单孢子株Cm-71是这一类群的代表。（3）单孢子株Cm－42是在所观察单孢子株中形态特征独特的菌株。在PDA上菌丝生长纤细,蛛网状,产孢结构轮枝孢型。蛹草拟青霉对氯酸钾（KClO3）不敏感,在浓度为6％（W／V）的KMM培养基上,23～24℃14天菌落的直径仅1～2cm,无气生菌丝生长。各单孢子株形成的不利用硝酸盐突变株（nit突变株）的途径和形态特征,也可分成类似于粉被虫草CordycepspruinosaPeteh不利用硝酸盐突变株（nit突变株）的三种类型。     

蛹虫草表型多态性对子实体产生及虫草菌素的影响

为选育高产子实体和虫草菌素的蛹虫草菌株,从野生蛹虫草中分离单孢子并进行分型,之后进行产子实体实验;同时对蛹虫草子实体进行了产孢结构的观察,并用高效液相色谱仪检测了子实体和培养基中的虫草菌素。结果表明,菌落背面颜色为橙铬色的菌株容易产生子实体;出发(原代)菌株所产子实体在形态上更接近野生蛹虫草,而角变株的子实体畸形;出发菌株产子实体能力要比该菌株未发生角变部分分离株和角变部分分离株都强。在虫草菌素的产量上也以出发菌株为高,表明表型多态性对蛹虫草产子实体和虫草菌素有很大的影响。这对于优势菌种的筛选和生产实践有借鉴意义。     

不同寄主来源的串珠镰孢生物学性状及其在无性后代的遗传与变异

自安徽、山东、湖北、江苏等地多点采集棉花红腐病、水稻恶苗病、玉米穗腐病的病组织,经分离、鉴定和纯化,获得107个串珠镰孢（Fusarium monilifoFine）菌株。对上述来源于棉花、水稻、玉米的串珠镰孢的菌落形态、生长速率和产孢量等生物学性状及其在无性后代的遗传与变异进行了研究。结果表明,不同寄主来源的串珠镰孢菌株的菌丝生长温度范围和最适温度大致相同,但在菌落形态特别是色素方面存在明显差异,生长速率和产孢量也存在显著差异。棉花菌株的平均生长速率最大,玉米菌株生长速率最小,水稻菌株生长速率居中,相同群体的不同菌株间生长速率有极显著差异;玉米菌株产孢量最大,棉花菌株产孢量最小,水稻菌株产孢量居中。方差分析显示,不同寄主菌株群体间产孢量存在显著差异,而同一寄主群体的不同菌株间产孢量均无显著差异,说明菌株产孢量大小主要与其寄主种类有关,而与地区来源关系不大。遗传测定结果表明,分离自棉花、玉米和水稻的串珠镰孢的菌落形态和生长速率在单分生孢子后代均可稳定遗传;产孢量性状遗传有两种情况：分离自棉花和水稻的串珠镰孢菌株Fm1和Fm31的产孢量性状在单分生孢子后代均可稳定遗传,而分离白玉米的串珠镰孢菌株Fm19的产孢量性状在单分生孢子第一代（CG1）发生变异。     

苎麻疫霉生物学性状遗传与变异研究*

在实验室条件下研究了不同地区和不同寄主来源的苎麻疫霉（PhytophthoraboehmeriaeSawada）的菌落形态、菌丝线性生长速率（以下简称为生长速率）及同宗配合性状的遗传与变异,结果指出苎麻疫霉菌落形态和生长速率的遗传存在3种类型：（1）在单游动孢子后代和自交后代均可稳定遗传;（2）在单游动孢子后代稳定遗传而在自交后代发生变异;（3）在单游动孢子后代和自交后代均发生变异。结果表明,该菌菌落形态和生长速率的遗传具有多样性,上述两性状可以由细胞核杂合基因控制,也可以由细胞核纯合基因控制,还可能由细胞质因子控制。试验结果还指出,苎麻疫霉的同宗配合性状在单游动孢子和单卵孢后代均可稳定遗传,表明在供试的苎麻疫霉菌株中控制该性状的遗传因子是纯合的。     

苎麻疫霉生物学性状遗传与变异研究

在实验室条件下研究了不同地区和不同寄主来源的苎麻疫霉（PhytophthoraboehmeriaeSawada）的菌落形态、菌丝线性生长速率（以下简称为生长速率）及同宗配合性状的遗传与变异,结果指出苎麻疫霉菌落形态和生长速率的遗传存在3种类型：（1）在单游动孢子后代和自交后代均可稳定遗传;（2）在单游动孢子后代稳定遗传而在自交后代发生变异;（3）在单游动孢子后代和自交后代均发生变异。结果表明,该菌菌落形态和生长速率的遗传具有多样性,上述两性状可以由细胞核杂合基因控制,也可以由细胞核纯合基因控制,还可能由细胞质因子控制。试验结果还指出,苎麻疫霉的同宗配合性状在单游动孢子和单卵孢后代均可稳定遗传,表明在供试的苎麻疫霉菌株中控制该性状的遗传因子是纯合的。     

Cold,pH and salt tolerant Penicillium spp. inhabit the high altitude soils in Himalaya,India

Twenty five fungal cultures (Penicillium spp.), isolated from soil samples from the high altitudes in the Indian Himalayan region, have been characterized following polyphasic approach. Colony morphology performed on five different media gave varying results; potato dextrose agar being the best for the vegetative growth and sporulation as well. Microscopic observations revealed 18 isolates to be biverticillate and 7 monoverticillate. Based on the phenotypic characters (colony morphology and microscopy), all the isolates were designated to the genus Penicillium. Exposure to low temperature resulted in enhanced sporulation in 23 isolates, while it ceased in case of two. The fungal isolates produced watery exudates in varying amount that in many cases increased at low temperature. All the isolates could grow between 4 and 37 °C, (optimum 24 °C), hence considered psychrotolerant. While all the isolates could tolerate pH from 2 to 14 (optimum 5–9), 7 isolates tolerated pH 1.5 as well. While all the fungal isolates tolerated salt concentration above 10 %; 10 isolates showed tolerance above 20 %. Based on ITS region (ITS1-5.8S-ITS2) analysis the fungal isolates belonged to 25 different species of Penicillium (showing similarity between 95 and 100 %). Characters like tolerance for low temperature, wide range of pH, and high salt concentration, and enhancement in sporulation and production of secondary metabolites such as watery exudates at low temperature can be attributed to the ecological resilience possessed by these fungi for survival under low temperature environment of mountain ecosystem.     

Isolation, Growth Characteristics, and Long-Term Storage of Fungi Cultivated by Attine Ants

Seven pure-culture strains of fungi cultivated by attine ants (ant-garden fungi) were isolated from locally maintained leaf-cutting ant colonies. An ant-garden fungus strain obtained from an Atta cephalotes colony, when offered to ants of the colony from which the fungus was isolated, was accepted as their own. Young fungus cultures were harvested and incorporated into the fungus garden, and cultures of intermediate age were used to begin a new fungus garden; old cultures were simply harvested. To facilitate further research on this fungus, growth characteristics of the different isolates were studied under a variety of conditions. They grew better at 24°C than at 30°C, and growth did not occur at an incubation temperature of 37°C. In a broth culture medium, growth was enhanced by aeration of the culture and by addition of yeast extract, olive oil, sesame oil, peanut oil, soybean oil, corn oil, sunflower oil, cottonseed oil, walnut oil, safflower oil, or mineral oil. Glycerol did not noticeably affect growth, but Tween 80 inhibited growth. These fungi were extremely sensitive to cycloheximide, growth being totally inhibited at cycloheximide concentrations ranging from 0.4 to 4.0 μg/ml. To date, the ant-garden fungus isolates have remained viable in long-term mineral oil-overlay storage cultures for up to 4 years.     

Genetic and Phenotypic Diversity among Botrytis cinerea Isolates in Iran

Forty-four Botrytis cinerea isolates from different hosts and geographical regions were studied for colony morphology, mycelial growth rate at different temperatures, pathogenicity and molecular diversity. Botrytis cinerea isolates had temperature optima of 20–25°C and isolates showed variation in growth rate at different temperatures. Two morphological types were identified among tested isolates: mycelial and sclerotial. The pathogenicity of isolates was tested on grapevine leaves, and it was revealed that nine of 44 isolates were non-pathogenic and among them seven were of mycelial type. There was no correlation between mycelium growth rate and pathogenicity. Genetic diversity was investigated using nine arbitrary decaprimers. No relationship was found between molecular clusters and geographical region or sampling time; whereas isolates from the same plant host tended to cluster with each other. Seven of nine non-pathogenic isolates were separated from pathogenic ones.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

Germination and growth of single-pustule isolates of Erysiphe graminis tolerant and sensitive to tridemorph and ethirimol

The germination and colony growth of single-pustule Erysiphe graminis isolates tolerant and sensitive to tridemorph and ethirimol was studied on glass slides and on leaf segments sprayed with tridemorph or left untreated. In the absence of the fungicide, neither tridemorph- nor ethirimol-tolerant isolates differed from the sensitive in germination or colony growth. Germination percentages of all isolates were depressed by high temperature and low humidity, although some isolates grew well in less than optimum conditions. Differences between ethirimol- and tridemorph-tolerant and sensitive isolates for colony size on untreated plants of various cultivars reflected differences in virulence to the host rather than fungicide tolerance. The presence of tridemorph retarded the growth rate of colonies of tolerant and sensitive isolates, but the germination of tridemorph-tolerant isolates was greatly stimulated by low concentrations of tridemorph (50 mg/litre) and was less depressed than that of the sensitive isolates by high concentrations (200 mg/litre).     

MORPHOGENESIS IN VERTICILLIUM: A SELF-INDUCED,NON-HEREDITARY VARIATION IN COLONY FORM

Some homokaryotic isolates of Verticillium albo-atrum (microsclerotial form) vary radically in colony appearance even when temperature, lighting, humidity and medium content are held constant. This variation is so great that it may easily be mistaken for hereditary variation. The environmental factors which control the variation are differences in shape of medium on which the colonies grow and presence or absence of other Verticillium colonies. The cause of the variation appears to be a diffusible substance which Verticillium itself produces.