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.     

Potential of Beauveria bassiana (Hyphomycetes: Moniliales) for controlling the white pine weevil, Pissodes strobi (Col., Curculionidae)

Abstract:  Studies were conducted under laboratory conditions to document the potential of Beauveria bassiana conidia applications for controlling the white pine weevil, Pissodes strobi (Peck). A screening test including six B. bassiana isolates allowed us to demonstrate that CFL (Centre de Foresterie des Laurentides) and IP-CPB (Île Perrault-charançon du pin blanc) were the most virulent isolates among the ones tested, with percentages of mortality after 3 weeks of 73% and 85.5%, respectively. These two B. basiana isolates were applied either onto soil or branch sections to compare the effectiveness of these potential control strategies. Greater than 75% mortality was observed within 3 weeks for both modes of application using suspensions of B. bassiana at a concentration of 1.0 × 10⁸ (soil application) and 1.0 × 10⁹ conidia/ml (branch application). The results demonstrate for the first time that B. bassiana is an effective entomopathogen against P. strobi .     

粗放经营茶园土壤昆虫病原真菌种群多样性及时间生态位

本文系统调查了粗放经营状态下黄山毛峰茶园根际土壤昆虫病原真菌的种群多样性及其时间生态位水平.于2012年8月至2013年6月,在安徽省黄山市汤口镇选择粗放经营茶园采集土壤样品140份,经稀释平板法分离共获得昆虫病原真菌1041株.结果表明： 昆虫病原真菌种群多样性较为丰富,经鉴定分为6属13种.其中,淡紫紫孢菌、球孢白僵菌和金龟子绿僵菌为优势种群,其菌株数分别为309、255和101株,占总数的29.7%、24.5%和9.7%.茶园土壤中淡紫紫孢菌的时间生态位宽度最大,球孢白僵菌最小.昆虫病原真菌之间以球孢白僵菌和布氏白僵菌的时间生态位重叠值最大,为1.965;而爪哇棒束孢和球孢白僵菌的时间生态位重叠值最小,仅为0.374.     

Genetic diversity among Brazilian isolates of Beauveria bassiana: comparisons with non-Brazilian isolates and other Beauveria species

Aims:  The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp.
Methods and Results:  The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp.
Conclusion:  Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil.
Significance and Impact of the Study:  This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu . The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil.     

Characterization and virulence of Beauveria spp. recovered from emerald ash borer in southwestern Ontario, Canada

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is an invasive wood boring beetle that is decimating North America's ash trees (Fraxinus spp.). To find effective and safe indigenous biocontrol agents to manage EAB, we conducted a survey in 2008-2009 of entomopathogenic fungi (EPF) infecting EAB in five outbreak sites in southwestern Ontario, Canada. A total of 78 Beauveria spp. isolates were retrieved from dead and mycosed EAB cadavers residing in the phloem tissues of dead ash barks, larval frass extracted from feeding galleries under the bark of dead trees. Molecular characterization using sequences of the ITS, 5' end of EF1-α and intergenic Bloc region fragments revealed that Beauveria bassiana and Beauveria pseudobassiana were commonly associated with EAB in the sampled sites. Based on phylogenetic analysis inferred from ITS sequences, 17 of these isolates clustered with B. bassiana, which further grouped into three different sub-clades. However, the combined EF1-α and Bloc sequences detected five genotypes among the three sub-clades. The remaining 61 isolates clustered with B. pseudobassiana, which had identical ITS sequences but were further subdivided into two genotypes by variation in the EF1-α and Bloc regions. Initial virulence screening against EAB adults of 23 isolates representing the different clades yielded 8 that produced more than 90% mortality in a single concentration assay. These isolates differed in virulence based on LC(50) values estimated from multiple concentration bioassay and based on mean survival times at a conidia concentration of 2×10(6) conidia/ml. B. bassiana isolate L49-1AA was significantly more virulent and produced more conidia on EAB cadavers compared to the other indigenous isolates and the commercial strain B. bassiana GHA suggesting that L49-1AA may have potential as a microbiological control agent against EAB.     

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

Numerical taxonomic study of thermophilic Bacillus isolated from three geographically separated deep-sea hydrothermal vents

Abstract: A numerical taxonomic study has been carried out with 80 strains, newly isolated, from three geographically separated deep-sea hydrothermal vents (Mid-Atlantic Ridge, Guaymas Basin and Lau Basin) and eleven thermophilic reference strains representing 11 Bacillus species. The deep-sea isolates were all halotolerant spore-forming rods and grew aerobically above 65°C. Results from unweighted average linkage cluster analysis of a similarity matrix derived from the simple matching coefficient, showed formation of nine major phena, which were defined at the 83% similarity level or above. Seven phena were composed exclusively of strains isolated from the same site (4 from Mid-Atlantic Ridge, 1 from Guaymas Basin and 2 from Lau Basin). The majority of the Lau Basin isolates clustered with 6 of the reference strains in one phenon, while isolates from Mid-Atlantic Ridge and Guaymas Basin were found separated from this phenon at the 69% similarity level. The other reference strains showed less than 69% similarity with the deep-sea isolates.     

马尾松林中球孢白僵菌寄主转移和专化性的SSR标记分析

球孢白僵菌是一种最常见的虫生真菌,已被广泛应用于害虫生物防治。利用SSR简单序列重复(simple sequence repeat,SSR)分子标记对来自安徽省麻姑山马尾松林的102株球孢白僵菌进行基因分型、寄主转移和寄主专化性研究。9对微卫星引物将102株白僵菌分成31个微卫星基因型。在这31个微卫星基因型中,有5种基因型株系为相对优势菌株,这5种基因型株系通过侵染不同寄主昆虫即寄主转移延续自身在马尾松林生态系统中的传播和流行,从而证实寄主转移是白僵菌群体中的普遍现象。同时,这些相对优势基因型并不是在各个月份均匀分布,在大部分月份中,存在1–2种优势度高的基因型。相同基因型株系可侵染不同寄主的结果揭示出白僵菌不仅在种的水平,而且在菌株水平上寄主专化性也较弱。正是白僵菌较弱的寄主专化性特征促使部分基因型株系通过寄主转移,在马尾松林生态系统中得以宿存和延续。     

Avoidance of a generalist entomopathogenic fungus by the ladybird, Coccinella septempunctata

Fungal entomopathogens are ubiquitous within the environment and susceptible insects are predicted to be under strong selection pressure to detect and avoid virulent isolates. Beauveria bassiana is an entomopathogenic fungus with a wide host range including coccinellids. Seven-spot ladybirds, Coccinella septempunctata, overwinter predominantly in leaf litter and B. bassiana is one of their major mortality factors during winter in temperate regions. Behavioural assays were conducted to assess the ability of adult C. septempunctata to avoid lethal densities of B. bassiana conidia in soil or on leaves. Further assays considered avoidance by C. septempunctata of mycosed (B. bassiana) C. septempunctata cadavers compared with uninfected C. septempunctata cadavers or in vitro B. bassiana. Treatments in any bioassays entirely avoided by C. septempunctata were regarded as censored data, to overcome the difficulties associated with zeros in log-ratio analyses. Male and female C. septempunctata avoided contact with leaf surfaces and soil inoculated with B. bassiana and mycosed cadavers. The ability of C. septempunctata to detect and avoid B. bassiana conidia is an adaptation that undoubtedly increases survival and ultimately fitness. We predict that such behavioural responses are widespread and driven by the high cost of fungal infection to a host.     

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

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.     

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection.     

苎麻疫霉群体的RAPD分析

利用从126个RAPD(RandomAmplifiedPolymorphicDNAs)随机引物中筛选到的可扩增出清晰条带、主带明显、稳定的8条引物,对采集自江苏、安徽和江西不同寄主的45个Phytophthoraboehmeriae菌株进行全基因组DNARAPD标记遗传多样性分析。选用引物共标出DNA指纹图带68条,其中多态性条带20条,多态性检测率为29.4%,表明该种内不同地区和寄主来源的菌株间变异较小。利用Popgene软件计算供试菌株间的遗传距离并绘制聚类树状图,供试菌株被划分为2个遗传聚类组。菌株间的遗传相似性与菌株的寄主来源有一定的相关性,来自江苏、江西和安徽棉花上的27个菌株被划分在同一遗传聚类组内,而分离自构树、枫杨和苎麻的18个菌株被划分在另一个遗传聚类组。结果还表明菌株间遗传相似性与其地区来源无直接相关性。     

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (=Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2h of weighted UV-B irradiance at 978mWm(-2) (providing a total dose of 7.04kJm(-2)) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0 degrees to 22 degrees latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22 degrees , however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.     

苎麻疫霉群体的RAPD分析*

利用从126个RAPD(RandomAmplifiedPolymorphicDNAs)随机引物中筛选到的可扩增出清晰条带、主带明显、稳定的8条引物,对采集自江苏、安徽和江西不同寄主的45个Phytophthoraboehmeriae菌株进行全基因组DNARAPD标记遗传多样性分析。选用引物共标出DNA指纹图带68条,其中多态性条带20条,多态性检测率为29.4%,表明该种内不同地区和寄主来源的菌株间变异较小。利用Popgene软件计算供试菌株间的遗传距离并绘制聚类树状图,供试菌株被划分为2个遗传聚类组。菌株间的遗传相似性与菌株的寄主来源有一定的相关性,来自江苏、江西和安徽棉花上的27个菌株被划分在同一遗传聚类组内,而分离自构树、枫杨和苎麻的18个菌株被划分在另一个遗传聚类组。结果还表明菌株间遗传相似性与其地区来源无直接相关性。     

瓜类保护地土壤镰孢菌种群及UP-PCR多样性分析

对采自辽宁省部分地区瓜类保护地的36份土壤样品进行镰孢菌（Fusarium）分离培养,共获得112株镰孢菌,采用传统形态学分类和现代分子生物学方法,确定属于11个种.对其中25株镰孢菌及3株对照镰孢菌进行了通用引物PCR（UP-PCR）多样性分析.结果表明：6条引物扩增出73条带,其中多态性条带66条,占总条带数的90.4%.对供试菌株进行UP-PCR聚类图谱分析,当相似系数为0.736时,可将其划分为8个类群,其中14株尖孢镰孢菌聚为一类.UP-PCR分析体现了镰孢菌菌株间的亲缘关系及差异性,可以作为镰孢菌分类的辅助方法.     

Antifungal Susceptibility, Enzymatic Activity, PCR-Fingerprinting and ITS Sequencing of Environmental Cryptococcus laurentii Isolates from Uberaba, Minas Gerais, Brazil

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human infection have been already reported. This study aimed to evaluate the phospholipase, proteinase and hemolysins activity, the antifungal susceptibility profile, the genetic variability by M13 and (GACA)(4) fingerprinting and the internal transcribe spacer (ITS) sequencing of 38 C. laurentii isolates recovered from captive bird droppings and surrounding hospital areas. All of them exhibited phospholipase activity, while the hemolytic activity was evidenced in 34 (89.4%) isolates. None of them exhibited proteinase activity. Twenty-seven isolates (71.1%) presented susceptibility dose dependent to fluconazole. Most isolates (94.7%) were susceptible to voriconazole, while one (2.65%) was resistant to this drug. Twenty-one (55.3%) isolates showed reduced susceptibility to itraconazole while nine (23.7%) were resistant. Three (7.9%) and five (13.1%) isolates exhibited resistance to ketoconazole and amphotericin B, respectively. Most C. laurentii fingerprinting obtained with M13 and (GACA)(4) showed high heterogeneity. By using the two primers, seven (18.4%) isolates grouped as A (CL2, CL7, and CL8), B (CL35, CL38) and C (CL29, CL30) with 100% similarity. Different from most variable surrounding hospital isolates, all but one of the pet shops strains clustered with the two primers, although they had been recovered from different neighborhoods. All isolates were identified as C. laurentii phylogenetic group I by ITS sequencing. Thus, the presence of virulence factors, a decreased antifungal susceptibility and a heterogeneous molecular pattern of the C. laurentii isolates here described suggests this species can be a potential pathogen in the context of the immunocompromised population.     

中国与欧洲高卢蜜环菌的遗传多样性

高卢蜜环菌(Armillaria gallica)为北半球广布种,不同大陆间的菌株遗传相似性和多样性水平能反映出该种在洲际大陆尺度上的地理遗传变异关系。作者用ISSR(inter-simple sequence repeat)分子标记技术,对从中国和欧洲收集到的高卢蜜环菌79个菌株进行了遗传多样性分析。用6个ISSR引物扩增得到210个位点,其中多态性位点(频率<0.95)为202个,占96.2%,平均每个引物多态位点多达33.6个,表明ISSR标记在蜜环菌中存在较高的多态性。根据非加权类平均法(UPGMA)聚类分析,中国53个菌株中的49个在0.773的相似性水平上聚成了中国类群(China group);而欧洲26个菌株遗传分化较大,分别在0.775和0.763的相似性水平上聚成了欧洲类群A(Europe group A)和B(Europe group B);2个欧洲类群间的相似性水平仅为0.738,而欧洲类群A与中国类群间的相似性却达到了0.770;两个大陆均有少数菌株表现出较为明显的遗传分化,个别菌株的种内遗传相似性甚至低于蜜环菌种间的遗传相似性。结果表明,中欧两个大陆间的A.gallica菌株因地理隔离已经表现出明显的遗传分化,处于异域物种形成过程中;欧洲大陆的菌株遗传分化更为明显,可能是两个大陆A.gallica菌株的起源地。