不同培养基质上球孢白僵菌孢子与菌丝多糖含量的变化

球孢白僵菌Beauveria bassiana(Balsamo)Vuellemin是重要的害虫生防真菌(Feng et al.,1994),其制剂在常温下长期储存是有效应用的技术瓶颈。由于菌体内源营养(如糖类)的积累是一个可控指标, 通过工艺调节内源营养积累可改善孢子的生理状态,有助于增强孢子贮存稳定性(冯明光和应盛华, 2002)。有关生防真菌孢子中糖类的研究多集中于单糖、双糖以及糖醇(Hallsworth&Magan,1994),多糖的报道仅见于芽生孢子内的糖原及其代谢的检测(Lane&Trinci,1991),不同基质对培养物多糖总量的影响还未见诸报道。本文简要报道在不同培养基质上培养的球孢白僵菌菌丝和孢子多糖的测定结果, 并就三种多糖提取方法进行了比较。     

Three new species of <Emphasis Type="Italic">Hyphodontia</Emphasis> s.l. (Basidiomycota) with poroid or raduloid hymenophore

Three species of Hyphodontia (Schizoporaceae) with poroid or raduloid hymenial surface are described as new to science. Hyphodontia chinensis sp. nov. was found in southwest and northeast China, having raduloid hymenophore, ventricose to subulate cystidia, and slightly thickened-walled, ellipsoid basidiospores; H. reticulata sp. nov. was found in central Japan and northern Taiwan, having poroid hymenophore, projecting peg-like structures, and encrusted cystidia; H. subtropica sp. nov. was collected from China (Yunnan province) and northern Vietnam, having poroid hymenophore, slightly apically swollen cystidioles, and pseudodimitic hyphal system. Both maximum parsimony and Bayesian methods were used to construct the phylogenetic trees based on ITS (internal transcribed spacer) sequences. The independent status of each proposed new species is based on morphological study, further confirmed by ITS-based phylogenetic analysis. The recently presented H. dimitica is found to be conspecific with H. nongravis, based on morphological and molecular evidences.     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

Distinctive role of fluG in the adaptation of Beauveria bassiana to insect-pathogenic lifecycle and environmental stresses

The upstream developmental activation (UDA) pathway comprises three fluG-cored cascades (fluG-flbA, fluG-flbE/B/D and fluG-flbC) that activate the key gene brlA of central developmental pathway (CDP) to initiate conidiation in aspergilli. However, the core role of fluG remains poorly understood in other fungi. Here, we report distinctive role of fluG in the insect-pathogenic lifecycle of Beauveria bassiana. Disruption of fluG resulted in limited conidiation defect, which was mitigated with incubation time and associated with time-course up-regulation/down-regulation of all flb and CDP genes and another fluG-like gene (BBA_06309). In ΔfluG, increased sensitivities to various stresses correlated with repression of corresponding stress-responsive genes. Its virulence through normal cuticle infection was attenuated greatly due to blocked secretion of cuticle-degrading enzymes and delayed formation of hyphal bodies (blastospores) to accelerate proliferation in vivo and host death. In submerged ΔfluG cultures mimicking insect haemolymph, largely increased blastospore production concurred with drastic up-regulation of the CDP genes brlA and abaA, which was associated with earlier up-regulation of most flb genes in the cultures. Our results unveil an essentiality of fluG for fungal adaptation to insect-pathogenic lifecycle and suggest the other fluG-like gene to act as an alternative player in the UDA pathway of B. bassiana.     

Oxaloacetate hydrolase gene links the cytoplasmic route of oxalate formation to differentiation and virulence of entomopathogenic fungus Beauveria bassiana

Oxalic acid is used as a functional molecule by most filamentous fungi, and produced via cytoplasmic pathway and mitochondrial pathway. The cytoplasmic pathway of oxalate production from oxaloacetate is a one-step reaction catalyzed by oxaloacetate hydrolase. The entomopathogenic fungus Beauveria bassiana contains a unique oxaloacetate hydrolase gene (BbOAH). The role of cytoplasmic pathway of oxalate production in B. bassiana development and virulence was studied via construction of a targeted gene disruption mutant of BbOAH. Disruption of BbOAH resulted in a slight decrease (~ 30%) in oxalate production, but has no significant influence on fungal growth. The mutant strain displayed a significant delay at early stage of conidial development, and a significant defect in dimorphic transition. Additionally, bioassay using the greater waxmoth as host indicated a slight (~ 20%) decrease in mortality caused by the gene disruption strain. The phenotypic defects of the ΔBbOAH strain could be restored by ectopic integration of BbOAH. Our findings indicate that BbOAH gene connects the cytoplasmic route of oxalate production to fungal development and virulence, although the cytoplasmic route is not indispensable for oxalate production in B. bassiana.     

中国灵芝学名之管见

灵芝是最重要的药用真菌之一,在我国已有2,000多年的记载和利用历史。虽然早在一百多年前法国真菌学家Patouillard就有给中国的灵芝冠上Ganoderma lucidum这一学名,并沿用至今,但随着分子生物学技术的不断发展,人们认识到过去外国人的定名并不正确。实际上,G.lucidum是1871年由William Curtis根据采自英国的标本描述的新物种。最近的研究表明,我国广泛分布和栽培的灵芝与产于欧洲的G.lucidum不同,是一个独立的种,其合法的拉丁学名应为G.lingzhi。鉴于"灵芝"这一名称在中国已使用2,000余年,故建议"G.lingzhi"的汉语学名为"灵芝"(俗称赤芝),而灵芝属的模式种G.lucidum的汉语学名改为"亮盖灵芝"(俗称白肉灵芝或白灵芝)。灵芝Ganoderma lingzhi广泛分布于东亚暖温带和亚热带,其主要形态特征是孔口表面新鲜时浅黄色至硫磺色、成熟时菌肉中有黑褐色区带、管口壁厚度为80–120μm。亮盖灵芝G.lucidum主要分布于欧洲和亚洲,在我国分布于东北、华北、华中和西南海拔较高地区,其孔口表面新鲜时白色至奶油色、成熟时菌肉中无黑褐色区带、管口壁厚度为40–80μm。四川灵芝G.sichuanense尽管其担孢子与灵芝G.lingzhi相似,但基于其模式标本ITS序列的系统发育研究表明,该种与灵芝不同,是个独立的种,且在广东也有发现。     

In vitro and in vivo responses of fungal biocontrol agents to gradient doses of UV-B and UV-A irradiation

Conidia of Beauveria bassiana and Metarhizium spp. smeared on glass slides were assayed for their responses to irradiation with weighted 312-nm UV-B and 365-nm UV-A at gradient doses of 0.005–1.1 and 1.0–18.0 J cm⁻², respectively. All inverted, sigmoid dose–survival trends showed good fit to a survival model (r ² ≥ 0.97), yielding respective UV-B LD₅₀s of 0.23–0.59 and 0.05–0.65 J cm⁻² for 24 B. bassiana and 36 Metarhizium isolates, and UV-A LD₅₀s of 2.78–10.46 J cm⁻² for 24 Metarhizium isolates. Myzus persicae apterae on detached leaves were sprayed with a concentrated spore suspension of B. bassiana or M. anisopliae, followed by exposure to the UV-B doses to cause 10–90% viability losses. These doses caused aphid mortality reductions as expected but affected neither spray-to-death period nor fungal growth on cadavers. The results highlight the merits of using UV-tolerant candidates and photoprotection measures in fungal formulations for pest control.     

抗脂多糖纳米抗体的筛选及其五聚体的制备

目的：筛选抗脂多糖（LPS）纳米单域抗体,并制备抗LPS纳米抗体五聚体。方法：以LPS为抗原,从驼源天然单域重链抗体库中筛选抗LPS纳米抗体,利用分子克隆技术将抗LPS单域抗体基因组装入志贺杆菌样毒素B亚基蛋白结构域（VTB）的五聚体特异性表达载体中进行可溶性表达,并用ELISA法鉴定所获抗体的抗原结合活性和特异性。结果：获得抗LPS纳米单域抗体及LPS纳米抗体五聚体;经鉴定,LPS纳米抗体五聚体的抗原结合活性优于抗LPS单域抗体。结论：利用驼源天然单域重链抗体库制备了抗LPS纳米单域抗体及抗LPS纳米抗体五聚体,为脓毒血症的分子诊断、预后判断及寻找生物治疗新靶点奠定了基础。