Genetic diversity, reproductive biology, and speciation in the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin.

Beauveria bassiana, a mitosporic fungus used for the biological control of many insect species, is recognized as a "species complex" comprising genetically diverse lineages. Being predominantly asexual, mating tests cannot be applied to delimit species in this species complex. Genetic tests offer an indirect means of identifying species among isolates. To this end, molecular genetic analysis of a sample of B. bassiana isolates with 2 subsamples, 1 representing a worldwide collection and another from a localized epizootic population was carried out. DNA markers generated through AFLPs (amplified fragment length polymorphisms) and SSCPs (single-strand conformation poly morphisms) and nucleotide sequence data of different allelic forms of 3 genes (large and small subunits of rRNA and beta-tubulin) were evaluated. The B. bassiana isolates from the worldwide sample showed 11% overall similarity and no closely clustered groups. Phylogenetic trees generated from the AFLP and SSCP data of this sample resolved the different isolates into distinct phylogenetic lineages. In the epizootic B. bassiana population, prevalence of recombination was evident from random association of alleles in multilocus tests and lack of phylogenetic concordance among 3 gene genealogies. Thus, the worldwide sample of B. bassiana exhibits a predominantly clonal structure, hinting at species divergence leading to cryptic speciation with recombination being customary among isolates sharing a close ecological niche.     

Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants

We have examined the global population genetic structure of Haemonchus contortus. The genetic variability was studied using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. To examine the performance and information content of the two different marker systems, comparative assessment of population genetic diversity was undertaken in 19 isolates of H. contortus, a parasitic nematode of small ruminants. A total of 150 individual adult worms representing 14 countries from all inhabited continents were analysed. Altogether 1,429 informative AFLP markers were generated using four different primer combinations. Also, the genetic variation was high, which agrees with results from previous AFLP studies of nematode parasites of livestock. The genetic structure was high, indicating limited gene flow between the different isolates and populations from each continent mostly formed monophyletic groups in the phylogenetic analysis. However, for isolates representing Australia, Greece and one laboratory strain that originated from South Africa (WRS), there was no clear genetic relationship between the isolates and the distance between their geographical origins. Basically the same pattern was observed for the mitochondrial marker, although the phylogenetic analysis was less resolved than for AFLP. In contrast with previous findings on the population genetic structure of H. contortus, the calculation of population structure gave high values (Nst=0.59). The strong structure was present also for the four Swedish isolates (Nst=0.16) representing a small geographical area.     

AFLP genotyping of Candida metapsilosis clinical isolates: Evidence for recombination

In a collection of 395 independent clinical isolates classified as Candida parapsilosis on a biochemical profile basis, 20 Candida metapsilosis strains were identified by molecular tests with an isolation frequency of 5%. Isolates were screened for their susceptibility to conventionally used antifungals and for virulence determinants, such as biofilm formation and protease production. Molecular characterization of C. metapsilosis independent isolates by amplified fragment length polymorphism (AFLP) revealed a high percentage of polymorphic bands. Statistical analysis of the pairwise genetic distances and bootstrapping revealed that recombination occurs and significantly contributes to C. metapsilosis genetic population variability. No association between specific AFLP markers and drug resistance or other phenotypes was observed.     

Parasexual recombination in fungi

Parasexual recombination is a valuable tool in the laboratory, particularly for asexual fungi, and a number of developments in methodology are outlined. In biotechnology, the parasexual cycle has proved less useful than at one time predicted, but it retains a function in analysis of the products of genetic manipulation, and as a convenient detection system for environmental chemicals that may disturb mitosis. In nature, recent evidence suggests that parasexual recombination is rare, in part at least because of the prevalence of heterokaryon incompatibility of many wild fungi.     

Development of multiple genetic markers for studies of genetic variation in arbuscular mycorrhizal fungi using AFLP™

Intraspecific and interspecific genetic variation was studied among arbuscular mycorrhizal fungi. DNA was extracted from single spores and variation was analysed by AFLP (Amplified Fragment Length Polymorphism). The patterns of amplified fragments revealed substantial genetic variation between spores from the same isolates. Possible recombination in the fungi was studied by comparing the obtained data with data generated by artificial recombination of the data sets. No evidence for recombination was found by the analysis, suggesting that the fungi reproduce clonally. The AFLP technique generated a large number of fragments, and the potential for using the technique in population genetic studies of these important unculturable biotrophic fungi is discussed.     

In vitro spore formation and completion of the asexual life cycle of Neozygites parvispora, an obligate biotrophic pathogen of thrips

Capilliconidia, the asexual secondary spores of Neozygites parvispora (Zygomycetes, Entomophthorales) were produced in vitro either by entrapment of vegetative cells (hyphal bodies) in alginate pellets or after plating them onto water agar. Cultivation of the fungus for 3 days in a medium lacking hemolymph increased spore production 30 to 40-fold, and about 10% of the cells produced capilliconidia. The in vitro produced capilliconidia were infectious to Thrips tabaci and the fungus was reisolated from infected insects, thus completing its asexual life cycle under laboratory conditions. A decrease in capilliconidia production and a modification of the number of nuclei per spore were observed for isolates cultivated in vitro for more than 2 months, but subsequent host passages restored and increased sporulation efficiency without influencing the number of nuclei. Fungal cultures were stored at —80 °C for up to 7 months, and the capability to sporulate and infect T. tabaci was preserved. A bioassay procedure for infecting T. tabaci with N. parvispora is described, the first mycosed insects dying usually after 8 d of incubation.     

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.     

Studies on the diversity of the distinct phylogenetic lineage encompassing Glomus claroideum and Glomus etunicatum

Morphological and molecular characters were analysed to investigate diversity within isolates of the Glomus claroideum/Glomus etunicatum species group in the genus Glomus. The inter- and intra-isolate sequence diversity of the large subunit (LSU) rRNA gene D2 region of eight isolates of G. claroideum and G. etunicatum was studied using PCR-single strand conformational polymorphism (SSCP)-sequencing. In addition, two isolates recently obtained from Southern China were included in the analysis to allow for a wider geographic screening. Single spore DNA isolation confirmed the magnitude of gene diversity found in multispore DNA extractions. An apparent overlap of spore morphological characters was found between G. claroideum and G. etunicatum in some isolates. Analysis of the sequence frequencies in all G. etunicatum and G. claroideum isolates (ten) showed that four LSU D2 sequences, representing 32.1% of the clones analysed for multispore extraction (564) were found to be common to both species, and those sequences were the most abundant in four of the ten isolates analysed. The frequency of these sequences ranged between 23.2% and 87.5% of the clones analysed in each isolate. The implications for the use of phenotypic characters to define species in arbuscular mycorrhizal fungi are discussed. The current position of G. claroideum/G.etunicatum in the taxonomy of the Glomeromycota is also discussed.     

The survival ofZoophthora radicans (Zygomycetes: entomophthorales) isolates as hyphal bodies in mummified larvae ofPlutella xylostella (Lep.: Yponomeutidae)

The survival of three isolates ofZoophthora radicans (NW 250, NW 253 & NW 182) as hyphal bodies in dried larvae ofPlutella xylostella stored at 4, 10 and 20°C and 20% R.H was determined. After storage at 20°C, the production of conidia by all isolates was unaffected after 2 weeks but diminished increasingly after 4 and 8 weeks and was entirely lost after 16 weeks. By comparison conidium production at 10°C was unaffected after 16 weeks (isolates NW 250 and NW 182) and, 24 weeks (NW 253) of storage though it declined rapidly in all isolates thereafter. At 4°C many conidia were produced by all isolates even after 34 weeks of storage. These results are consistent with work on other entomophthoralean fungi in dried cadavers suggesting that this may be a common survival strategy in these fungi. NW 250, 253 and 182 were isolated fromP. xylostella in Malaysia and Taiwan, where conditions allow the host to remain active throughout the year. None produced resting sporesin vivo orin vitro but as hosts are always available the ability to survive short dry periods is probably more important than long-term survival for which resting spores are most adapted.      

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.     

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.     

Genetic variation in Armillaria mellea subsp. nipponica estimated using IGS-RFLP and AFLP analyses

In this study, genetic variation of Armillaria mellea subsp. nipponica was estimated using intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) and amplified fragment length polymorphism (AFLP) analyses. Four IGS-RFLP phenotypes were produced, of which two have never been reported. AFLP analysis suggested that the 11 isolates used could be divided into five subgroups, and the isolates within the same subgroup were distributed throughout a relatively large area in Japan. A parental isolate and its offspring (single-spore isolates) showed an almost identical AFLP profile to each other. These results suggest that the large distribution of the isolates within the same subgroup were established via the basidiospore from a common parental strain. Contribution no. 378 of The Tottori Mycological Institute     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

Phytophthora infestans isolates from Northern China show high virulence diversity but low genotypic diversity

Phenotypic and genotypic characteristics of 48 Phytophthora infestans isolates , collected in five provinces in Northern China between 1997 and 2003, were determined and compared with reference isolates. Characterisation included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphisms (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from SSR genotypes found in the reference isolates, including those representing the 'old' US-1 lineage that dominated the P. infestans population worldwide prior to 1980. In contrast, the virulence spectra were highly variable and virulence to all resistance genes present in the standard differential set ( R1 to R11 ) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this rather uniform population is discussed in the framework of recent insights into the molecular determinants of avirulence in potato– P. infestans 'gene-for-gene' interactions.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

In vitro soil receptivity assays to egg-parasitic nematophagous fungi

Soil application of nematophagous fungi for the biological control of plant-parasitic nematodes often fails, and in many cases it has been difficult to reisolate the agent delivered to the soil. A reason for these results could be the inability of the fungi to proliferate in soil. We used a soil–membrane technique to study the capacity of several isolates of the nematophagous fungi Pochonia chlamydosporia and Paecilomyces lilacinus to grow and establish in sterilized and nonsterilized sandy soils from SE Spain and Western Australia. Growth of all fungi tested was inhibited in nonsterilized soil, although there was intraspecific variability in sensitivity among isolates of the same species. With respect to hyphal density, P. chlamydosporia isolate 5 (from Italy) was the least inhibited in nonsterilized soil from both sites. Relative growth analyses confirmed this result for soil from SE Spain, while with this method, P. chlamydosporia isolate 4624 (from Australia) appeared to be least inhibited in the Australian soil. The results indicate that a soil can be more receptive to its indigenous isolates than to nonindigenous isolates. Apparently, soil microbiota can determine the ability of nematophagous fungi to proliferate in soil.     

A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae)

We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 10(8)conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis.     

Isolation of a homothallic mutant in <Emphasis Type="Italic">Lentinula edodes</Emphasis>

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny. Contribution no. 385 from the Tottori Mycological Institute     

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。     

丝核菌的分类系统：现状及存在问题

以重要植物病原菌为特征的丝核菌是一类在土壤中广泛分布的丝状真菌,通常不产孢,以菌丝或菌核的形式存在,多样性非常丰富。本文基于国内外最新研究进展,对依据菌丝体的细胞核数目、菌丝融合、有性生殖和系统进化等方面的基本特征展开的丝核菌分类体系及分类现状进行了综述。基于菌丝的细胞核数目,丝核菌被分为单核、双核和多核丝核菌三大类群。自然界中单核丝核菌数量极少,多核和双核丝核菌在全球分布广泛,占丝核菌的绝大多数。基于菌丝融合试验的结果,目前多核丝核菌被分为13个菌丝融合群,双核丝核菌被分为18个菌丝融合群。部分融合群内又根据一些稳定的特征分了亚群,但亚群的建立标准并不统一。目前的分子系统学研究结果基本支持丝核菌的菌丝融合群及亚群的分类。基于部分有性世代被发现的菌株的形态特征,多核和双核丝核菌分别被鉴定为亡革菌属和角担菌属。此外,目前已有分属重要植物病原菌和兰科菌根菌类群的至少9个融合群或亚群的17个菌株完成了基因组测序,比较基因组学和线粒体组学开始在丝核菌分类和进化研究中发挥作用。丝核菌分类系统特殊且复杂,作者在文末提出了目前丝核菌分类学研究面临的问题和今后研究的趋势,期待更多的学者参与到这个重要菌...