Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidylinositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and virulence

Beauveria bassiana is a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes β-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putative B. bassiana GPI-anchored β-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized. B. bassiana targeted gene knockouts lacking Bbgas1 were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including β-1,3-glucans, in the ΔBbgas1 strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1 conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1 conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1 knockout showed reduced virulence in Galleria mellonella insect bioassays in both topical and intrahemocoel-injection assays. B. bassiana ΔBbgas1 strains complemented with the complete Bbgas1 open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. The Bbgas1 gene did not complement the phenotype of a Saccharomyces cerevisiae β-1,3-glucanosyltransferase Δgas1 mutant, indicating that this family of enzymes likely possess discrete cellular functions.     

Map kinases in fungal pathogens

MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens.     

A virulence-related lectin traffics into eisosome and contributes to functionality of cytomembrane and cell-wall in the insect-pathogenic fungus Beauveria bassiana

Lectins are characterized of the carbohydrate-binding ability and play comprehensive roles in fungal physiology (e.g., defense response, development and host–pathogen interaction). Beauveria bassiana, a filamentous entomopathogenic fungus, has a lectin-like protein containing a Fruit Body_domain (BbLec1). BbLec1 could bind to chitobiose and chitin in fungal cell wall. BbLec1 proteins interacted with each other to form multimers, and translocated into eisosomes. Further, the interdependence between BbLec1 and the eisosome protein PliA was essential for stabilizing the eisosome architecture. To test the BbLec1 roles in B. bassiana, we constructed the gene disruption and complementation mutants. Notably, the BbLec1 loss resulted in the impaired cell wall in mycelia and conidia as well as conidial formation capacity. In addition, disruption of BbLec1 led to the reduced cytomembrane integrity and the enhanced sensitivity to osmotic stress. Finally, ΔBbLec1 mutant strain displayed the weakened virulence when compared with the wild-type strain. Taken together, BbLec1 traffics into eisosome and links the functionality of eisosome to development and virulence of B. bassiana.     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

Effect of temperature and poultry litter in Beauveria bassiana (bals.) Vuill. and Metarhizium anisopliae (Metsch) virulence against the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate the influence of temperature and poultry litter on germination vegetative growth virulence and conidial production of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates on larvae and adults of the lesser mealworm (Alphitobius diaperinus) (Panzer). The vegetative growth and conidial production were evaluated on culture media. Virulence was studied submerging larvae and adults in a conidial suspension (1 x 10(8) conidia/ml). All the experiments were carried out in growth chamber (26 degrees C and 32 degrees C and 14h photophase). Fungus-killed insects were daily collected and used for microscopic conidial counts. The poultry litter effect was evaluated by submerging the insects in a fungal suspension (10(8) conidia/ml) and then transferring them to cups containing poultry litter (new and used). B. bassiana isolates were more sensitive than M. anisopliae to high temperature because conidia viability, vegetative growth and virulence were negatively affected (P < 0.05). The conidial production was higher to B. bassiana in 26 degrees C (7 to 11 x 10(8) conidia/larval cadaver and 8 x 10(8) conidia/adult cadaver) (P < 0,05). Larval stage was about 10 times more sensitive to M. anisopliae at 26 degrees C than adults stage. Regarding B. bassiana, differences on sensitivity between larval stages and adults were not observed at this temperature. However, at 32 degrees C, larval stage was more sensitive for CB116 and UEL50 isolates. Mortality was higher when larvae and adults (15.7 and 66.7% respectively) were treated by B. bassiana and maintained on new poultry litter at 26 degrees C) (P < 0.05).     

生防真菌分生孢子抗氧化能力与多糖含量的关系

作为重要的丝孢类昆虫病原真菌,球孢白僵菌和玫烟色棒束孢因其易于生产和环境友好等优点而在害虫生防防治中受到广泛青睐。为初步探求孢子耐氧化力及其与孢子多糖含量的关系,球孢白僵菌和玫烟色棒束孢11株菌经胁迫后的残存指数随氧化剂H2O2浓度增加而减小。所有菌株的残存指数均能良好地与Logistic方程拟合,并计算出各菌株在氧化胁迫条件下的半致死浓度。结果显示玫烟色棒束孢孢子的耐氧化力强于球孢白僵菌。两种真菌的分生孢子耐氧力与各自多糖含量呈现良好线性正相关。培养基碳源成分和浓度变化可影响球孢白僵菌孢子耐氧化力,但耐氧化力与多糖含量依旧呈现线性正相关。由此可见,生防真菌分生孢子的耐氧化力的确与多糖积累有关,并在一定程度上受培养条件的调节。研究结果有望为提高生防真菌孢子环境稳定性提供新的策略。     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

MybA,a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA‐sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane‐associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.     

常用化学杀螨剂对两种生防真菌孢子的相容性测定

用营养液萌发法和平板萌发法测定了10种常用化学杀螨剂与球孢白僵菌（Beauveria bassiana）和玫烟色拟青霉（Paecilomyces fumosoroseus）分生孢子的生物相容性.结果表明,营养液萌发法比平板法灵敏.以24 h内的孢子萌发率作为指标,供试杀螨剂的常用浓度及其5和10倍稀释液对活孢率的影响在不同杀螨剂间差异极显著,并在两种真菌间也存在差异.三唑锡、浏阳霉素、三氯杀螨醇和阿维菌素对生防真菌孢子的抑制力很强,不宜混用.速螨酮、克螨特、乐斯本、尼索朗及双甲脒等杀螨剂与真菌孢子的相容性优良,可与菌剂田间混用.将速螨酮、克螨特和乐斯本分别按3种浓度与白僵菌的孢子悬乳剂混配后在4 ℃恒温和室内变温下长期共贮,结果显示,3种杀螨剂对孢子活力的影响迥异,混配制剂在室内变温下的贮存期均未达到12个月,离商品化的要求差距较大.乐斯本的长期相容性相对较优,与菌剂低浓度混配可在常温下贮存6.5个月,中浓度和低浓度混配在4 ℃下可贮存12个月.     

Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana

Knockout and complement mutants of mannitol-1-phosphate dehydrogenase (MPD) and mannitol dehydrogenase (MTD) were constructed to probe the roles of both enzymes in the mannitol metabolism and multi-stress tolerances of entomopathogenic fungus Beauveria bassiana. Compared with wild-type and complement mutants, ΔBbMPD lost 99.5% MPD activity for reducing fructose-6-phosphate to mannitol-1-phosphate while ΔBbMTD lost 78.9% MTD activity for oxidizing mannitol to fructose. Consequently, mannitol contents in mycelia and conidia decreased 68% and 83% for ΔBbMPD, and 16% and 38% for ΔBbMTD, accompanied by greatly enhanced trehalose accumulations due to 81-87% decrease in their neutral trehalase expression. Mannitol as mere carbon source in a nitrate-based minimal medium suppressed the colony growth of ΔBbMTD instead of ΔBbMPD, and delayed more conidial germination of ΔBbMTD than ΔBbMPD. Based on median lethal responses, conidial tolerances to H(2) O(2) oxidation, UV-B irradiation and heat stress at 45°C decreased 38%, 39% and 22% in ΔBbMPD, and 18%, 16% and 11% in ΔBbMTD respectively. Moreover, ΔBbMPD and ΔBbMTD lost 14% and 7% of their virulence against Spodoptera litura larvae respectively. Our findings highlight the primary roles of MPD and MTD in mannitol metabolism and their significant contributions to multi-stress tolerances and virulence influential on the biocontrol potential of B.bassiana.     

Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae)

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 10(7)cells/ml were used. At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC(50) was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.     

Cell wall integrity and high osmolarity glycerol pathways are required for adaptation of Alternaria brassicicola to cell wall stress caused by brassicaceous indolic phytoalexins

Camalexin, the characteristic phytoalexin of Arabidopsis thaliana, inhibits growth of the fungal necrotroph Alternaria brassicicola. This plant metabolite probably exerts its antifungal toxicity by causing cell membrane damage. Here we observed that activation of a cellular response to this damage requires cell wall integrity (CWI) and the high osmolarity glycerol (HOG) pathways. Camalexin was found to activate both AbHog1 and AbSlt2 MAP kinases, and activation of the latter was abrogated in a AbHog1 deficient strain. Mutant strains lacking functional MAP kinases showed hypersensitivity to camalexin and brassinin, a structurally related phytoalexin produced by several cultivated Brassica species. Enhanced susceptibility to the membrane permeabilization activity of camalexin was observed for MAP kinase deficient mutants. These results suggest that the two signalling pathways have a pivotal role in regulating a cellular compensatory response to preserve cell integrity during exposure to camalexin. AbHog1 and AbSlt2 deficient mutants had reduced virulence on host plants that may, at least for the latter mutants, partially result from their inability to cope with defence metabolites such as indolic phytoalexins. This constitutes the first evidence that a phytoalexin activates fungal MAP kinases and that outputs of activated cascades contribute to protecting the fungus against antimicrobial plant metabolites.     

R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular vesicle fusion, which is an essential cellular process of the eukaryotic cells. To investigate the role of SNARE proteins in the rice blast fungus Magnaporthe oryzae, MoSec22, an ortholog of Saccharomyces cerevisiae SNARE protein Sec22, was identified and the MoSEC22 gene disrupted. MoSec22 restored a S. cerevisiae sec22 mutant in resistance to cell wall perturbing agents, and the ΔMosec22 mutant also exhibited defects in mycelial growth, conidial production, and infection of the host plant. Treatment with oxidative stress inducers indicated a breach in cell wall integrity, and staining and quantification assays suggested abnormal chitin deposition on the lateral walls of hyphae of the ΔMosec22 mutant. Furthermore, hypersensitivity to the oxidative stress correlates with the reduced expression of the extracellular enzymes peroxidases and laccases. Our study thus provides new evidence on the conserved function of Sec22 among fungal organisms and indicates that MoSec22 has a role in maintaining cell wall integrity affecting the growth, morphogenesis, and virulence of M. oryzae.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.