菌寄生属研究进展

菌寄生属通常寄生于其他真菌子实体上,是子囊菌的重要类群之一,在生物医药和工农业等方面具有经济重要性。简述了菌寄生属在资源与分类、系统发育和寄主专化性等方面的研究现状及进展。已有的分子系统学研究表明,菌寄生属是多谱系类群,形成多个分支,各分支与其无性型特征和寄主种类有关。今后需进一步澄清属种概念,建立稳定的有性阶段-无性阶段对应关系,在全面了解其物种多样性和地理分布特点基础上,深入探讨种间系统发育关系。     

EPG技术在昆虫学研究中的应用及进展

刺吸行为电图(electrical penetration graph, EPG) 技术在植食性刺吸式昆虫,如蚜虫、粉虱、叶蝉和蓟马等研究中的应用日益深入,尤其是对蚜虫和粉虱的研究,已涉及寄主专化性、植物的抗虫机制以及病毒传播等领域。该文从蚜虫和粉虱等典型的刺吸式昆虫入手,介绍EPG技术的研究进展和应用前景。     

玉米小斑病菌C小种毒素理化性质及光谱特征的研究

就玉米小斑病菌Ｃ小种分泌的毒素进行了分离纯化,得到一对Ｃ细胞质有专化侵染的活性有机物质ＴｏｘｉｎＩ,通过对ＴｏｘｉｎＩ的紫外,红外及质子核磁共振等的测定,确定了ＴｏｘｉｎＩ的一些光谱结构特征,推测其为一种新化合物。     

链格孢菌毒素合成相关基因研究进展

链格孢属真菌(Alternaria Nees)是世界上分布最广泛的真菌之一,可产生9种寄主选择性毒素(host-selective toxins,HST)。根据寄主不同,将链格孢属的7个生理小种称为链格孢菌(Alternaria alternata)的7种致病型。每种致病型产生的HST都是低分子质量的次级代谢产物,只对敏感品种有毒,对抗性品种无毒。参与HST生物合成的基因都是多拷贝的,这些共同表达的基因组成基因簇。各致病型的基因簇位于一条小于2.0Mb的conditionally dispensable(CD)染色体上,CD染色体的不稳定性不影响菌丝的生长和孢子的萌发,但与菌株的致病力有关。从链格孢菌6种致病型得到毒素合成基因,日本梨致病型、草莓致病型、橘致病型有共同的EDA结构域,这3种致病型有很多EDA生物合成需要的同源基因。HST的分子遗传学研究为链格孢菌不同致病型的演化提供了新的见解。     

胶孢炭疽菌的菟丝子专化型

本文报道对寄生菟丝子的两个炭疽菌(Colletotrichum Corda)分离系(鲁保一号New-76)的鉴定结果。两分离系按其培养特性、分生孢子和附着孢的形态、大小及产孢细胞、分生孢子盘的特点,应属于胶孢炭疽菌[Colletotrichum gloeosporioides(Penz.)Sacc.],但在对寄主的选择性方面不同于该种的其他分离系,特别是对菟丝子(Cussuta chinensis)具有寄生专化性,故确定为胶孢炭疽菌菟丝子专化型[C.gloeosporioides(Penz.)Sacc.f.sp.cuseutae]。     

棉花型和瓜型棉蚜产生有性世代能力的分化

为了明确寄主专化性是否会影响棉蚜的繁殖策略或生活史对策,采用低温和短光照组合（18℃,L∶D=8∶16）分别对棉花型和瓜型棉蚜进行有性世代的诱导,比较两寄主专化型棉蚜产生有翅蚜、雄性母、雌性母及无翅孤雌蚜的能力。结果表明：两寄主专化型棉蚜在产生有性世代能力上存在显著差异,表现为棉花型棉蚜在诱导后代中产生有翅蚜、雄性母和雌性母的比率显著高于瓜型棉蚜,并且雌、雄性母产生的时间明显早于瓜型棉蚜。瓜型棉蚜在诱导条件下产生无翅孤雌蚜的比率显著高于棉花型棉蚜,并且发现有不产生有性世代的营专性孤雌生殖个体,而在棉花型棉蚜中没有发现。产生性母蚜的棉花型和瓜型棉蚜均可同时产生孤雌胎生后代。寄主型与诱导时间长短对棉蚜有性世代的产生存在交互作用。采集于田间棉花和黄瓜上的棉蚜,也表现为棉花上的易产生有性世代,在诱导的第2代中产生性母蚜的比率显著高于黄瓜上的棉蚜,并且性母蚜比率在诱导的第2代与第3代间无显著差异; 但黄瓜上的棉蚜性母蚜产生比率第3代显著高于第2代。由此推测,棉蚜寄主专化性的形成与生活史特性的分化有关。     

苹果链格孢菌与寄主叶片互作的超微结构研究

以苹果杂交组合(‘华脆’ב金冠’)F1代杂种实生苗群体易感病株系为材料,通过扫描电镜、透射电镜等观察方法,对苹果链格孢菌与寄主叶片互作过程进行了细胞学观察,分析苹果链格孢菌侵染寄主的动态过程和变化规律,以揭示苹果病害发生机制。结果表明,苹果链格孢菌侵染寄主叶片时,多数在表皮分枝扩展,很少通过气孔器或其他方式入侵细胞内部。互作后期,寄主叶细胞内叶绿体和质膜变化显著,多数细胞器降解,表明病原菌已分泌AM毒素作用于寄主。     

利用脂肪酸生物标记对尖镰孢寄主专化型的快速鉴定

尖镰孢寄主范围广、遗传差异大,其种下存在多种寄主专化型。对尖镰孢寄主专化型的快速鉴定可为科学制定植物病害防控策略提供依据。利用Sherlock MIS脂肪酸鉴定系统对分离自番茄、棉花、黄瓜、茄子等4种寄主专化型的18株尖镰孢进行脂肪酸成分测定,共检测到10种脂肪酸。运用SPSS软件中的PCA法对被检测到的脂肪酸进行主成分分析,确定了18:1CIS9（W9）[X1],18:2 CIS 9,12/18:0a[X2]和18:00[X3]等3个脂肪酸为其主成分。利用Bayes逐步判别法建立了尖镰孢4种不同寄主专化型判别模型为Y1=-157.750+2.809X1+3.391X2+8.099X3;Y2=-178.343+0.586X1+7.587X2- 0.214X3;Y3=-129.132+2.749X1+4.163X2+4.476X3;Y4=-201.307+2.016X1+7.345X2+2.400X3。通过对43株未知寄主专化型菌株主成分脂肪酸的测定,利用判别法对尖镰孢进行判定,结果发现有40株与原寄主来源一致,判对率达93%。表明脂肪酸生物标记法可用于尖镰孢寄主专化型的快速鉴定。     

蚜虫寄主专化型及其成因

综述了蚜虫寄主专化型产生的可能原因。蚜虫寄主专化型的形成在生态学水平上主要与蚜虫对寄主的选择识别能力、天敌作用、其他共生（共存）生物作用、抗药性等有关;并且具有一定的遗传基础,主要表现在蚜虫体内酶系的变化、染色体变异、有性繁殖中同型交配行为及种群的遗传分化上。但是对某种寄主专化型蚜虫而言,其具体的成因尚不明确。     

中国月季上两株链格孢属真菌的分离鉴定及TeA毒素的测定

细交链格孢菌酮酸(Te A)是链格孢菌毒素中最为重要的一种,其在适合的浓度对月季没有伤害而对月季长管蚜有趋避作用。从中国月季上分离纯化得到的两株链格孢属真菌,编号为0363和0645,经过培养性状对比、ITS和18S基因序列分析及显微形态观察,鉴定0363为链格孢(Alternaria alternate.Keissler),0645为细极链格孢(Alternaria tenuissima)。用有机溶剂对两株菌株的毒素进行提取,得到的毒素粗提物利用HPLC分析测定,结果表明,两株菌的毒素粗提物中均含有细交链格孢菌酮酸(Te A)。以上研究结果为中国月季病虫害的防治及寄主植物介导的间接病虫互作机理研究提供实验材料和理论依据。     

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.     

Host-specific toxins: effectors of necrotrophic pathogenicity

Host-specific toxins (HSTs) are defined as pathogen effectors that induce toxicity and promote disease only in the host species and only in genotypes of that host expressing a specific and often dominant susceptibility gene. They are a feature of a small but well-studied group of fungal plant pathogens. Classical HST pathogens include species of Cochliobolus , Alternaria and Pyrenophora . Recent studies have shown that Stagonospora nodorum produces at least four separate HSTs that interact with four of the many quantitative resistance loci found in the host, wheat. Rationalization of fungal phylogenetics has placed these pathogens in the Pleosporales order of the class Dothideomycetes. It is possible that all HST pathogens lie in this order. Strong evidence of the recent lateral gene transfer of the ToxA gene from S. nodorum to Pyrenophora tritici-repentis has been obtained. Hallmarks of lateral gene transfer are present for all the studied HST genes although definitive proof is lacking. We therefore suggest that the Pleosporales pathogens may have a conserved propensity to acquire HST genes by lateral transfer.     

Current advances in aptamer‐assisted technologies for detecting bacterial and fungal toxins

Infectious diseases are among the common leading causes of morbidity and mortality worldwide. Associated with the emergence of new infectious diseases, the increasing number of antimicrobial‐resistant isolates presents a serious threat to public health and hospitalized patients. A microbial pathogen may elicit several host responses and use a variety of mechanisms to evade host defences. These methods and mechanisms include capsule, lipopolysaccharides or cell wall components, adhesions and toxins. Toxins inhibit phagocytosis, cause septic shock and host cell damages by binding to host surface receptors and invasion. Bacterial and fungal pathogens are able to apply many different toxin‐dependent mechanisms to disturb signalling pathways and the structural integrity of host cells for establishing and maintaining infections Initial techniques for analysis of bacterial toxins were based on in vivo or in vitro assessments. There is a permanent demand for appropriate detection methods which are affordable, practical, careful, rapid, sensitive, efficient and economical. Aptamers are DNA or RNA oligonucleotides that are selected by systematic evolution of ligands using exponential enrichment (SELEX) methods and can be applied in diagnostic applications. This review provides an overview of aptamer‐based methods as a novel approach for detecting toxins in bacterial and fungal pathogens.     

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.     

Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.     

EVect of Chitosan on Growth and Toxin Production by Alternaria alternata f. sp. lycopersici

The antifungal activity of chitosan, a biopolymer of beta-1-4 glucosamine, against Alternaria alternata f. sp. lycopersici , causal agent of black mold of tomato, was investigated. Chitosan was incorporated into potato-dextrose broth at concentrations of 100-6400 mug ml - 1, and the growth and toxin production by the fungus were assessed after 15 days of incubation. At the higher concentrations, chitosan significantly aVected both fungal growth and toxin production. However, at lower concentrations toxin production was aVected more than growth. The fungus sporulated excessively in the presence of chitosan, but the spores were less viable. Chitosan also induced aggregation, abnormal shape, excessive branching and hyphal contortion of fungal cells, and leakage of proteins. The virulence of the toxin in culture filtrates of the fungus grown on diVerent concentrations of chitosan was assessed by administering toxin on tomato disks. The phospholipid content, electrolyte leakage and activities of xylanase and pectin methylesterase were measured in the tomato tissue administered with culture filtrates containing fungal toxin. Decreased trends in the tendency to cause electrolyte leakage, phospholipid degradation and activation of xylanase and pectin methylesterase in the tomato tissue were observed with increasing concentrations of chitosan. The results showed that toxin produced in the presence of chitosan was less eVective in causing degradation of tomato tissue compared with the control. Thus, chitosan is a potential antifungal agent which can interfere with the pathogenic factors of the fungus.     

A Novel Strategy for the Invasive Toxin: Hijacking Exosome-Mediated Intercellular Trafficking

Toxins penetrate mammalian cells through various means. In this study, we report a unique strategy used by trichosanthin (TCS), a plant toxin with ribosome-inactivating activity, to penetrate host cells. We found that in both JAR and K562 cells, endocytosed TCS is incorporated into intraluminal vesicles of the multivesicular body (MVB) and is then secreted in association with these vesicles upon fusion of the MVB with the plasma membrane. The secreted TCS-loaded vesicles secreted by K562 cells move throughout the intercellular space and target syngeneic and specific allogeneic cells. Subsequent internalization permits delivery of the toxin into the cytosol, resulting in ribosomal inactivation and cell death. Thus, our findings provide a novel mechanism by which foreign proteins pass between and penetrate into mammalian cells.     

Zygocin,a secreted antifungal toxin of the yeast Zygosaccharomyces bailii,and its effect on sensitive fungal cells

Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.     

Bioproduction of [C]deoxyphomenone, A fungistatic metabolite of the hyperparasite Dicyma pulvinata

Deoxyphomenone is a fungistatic sesquiterpene produced by the fungal hyperparasite Dicyma pulvinata. A study of the kinetics of the synthesis of this toxin was achieved by the use of [U-¹⁴C]acetate. Labelled deoxyphomenone was detectable in the culture filtrate one hour after inoculation of the medium with spores of D. pulvinata. Optimum incorporation of radioactivity in the toxin occurred in the early phase of exponential growth. Consequently, maximum availability of deoxyphomenone is found in the young parts of the hyphae in zones where contact between the hyperparasite and parasite occur. This underlines the role which the toxin can play in early phases of hyperparasitism.