红火蚁病原真菌AUGM47早期发育超微结构研究

在前期筛选已获得对红火蚁Solenopsis invicta Buren高效致病真菌罗伯茨绿僵菌Metarhizium robertsii AUGM47的基础上,为进一步明确病原真菌对寄主昆虫的侵染机制。本试验在室内条件下,以红火蚁工蚁为侵染对象,利用荧光显微镜和透射电镜观察了罗伯茨绿僵菌AUGM47侵染单元分生孢子在体表附着萌发、穿透和体内增殖的早期发育过程。结果表明,菌株AUGM47分生孢子在红火蚁体表可萌发并形成附着胞侵入,接种后12 h观察到萌发,在36 h内普遍出现穿透结构穿透体壁。接种后48 h为菌体在血腔内的增殖阶段。菌丝体在穿透表皮和体腔内增殖过程中伴随着机械压力和酶的活动。接种后96 h,观察到自噬现象,菌体通过自噬降解并回收细胞器,为从体内穿出的晚期发育过程提供物质基础。本研究对罗伯茨绿僵菌AUGM47分生孢子在红火蚁体外至体内的发育进程研究证实了菌株的高致病性,为红火蚁生防真菌菌种改良和后续开发利用奠定理论基础。     

飞虱虫疠霉分生孢子在桃蚜体壁上的附着与入侵*

用飞虱虫疠霉(Pandora delphacis)初级分生孢子接种桃蚜(Myzus persicae),24 h内定时取样,在扫描电镜下观察孢子的萌发及其对寄主体壁的入侵。结果表明,附着到蚜体表面的孢子在4 h内有30~40%已萌发产生芽管,其中多数为侵染性芽管,少数是呈叉状分枝的营养生长型芽管。具侵染性芽管的孢子部分陷入体壁蜡质层中,显示孢子有分泌物产生并作用于寄主体壁。接种10 h内,侵染性芽管通过顶端膨大的附着胞或直接穿透侵入寄主体壁。到24 h时,产生侵染性芽管的孢子全部侵入寄主体内,寄主体表仅留下少数未萌发的孢子或营养生长型芽管。初级分生孢子在蚜体表面很少产生次级分生孢子,说明桃蚜是适合该菌侵染的寄主。陷入寄主体壁的孢子不因若蚜蜕皮而被去掉,表明该菌对成蚜和若蚜都具有侵染力。     

飞虱虫疠霉分生孢子在桃蚜体壁上的附着与入侵

用飞虱虫疠霉（Pandora delphacis)初级分生孢子接种桃蚜（Myzus persicae),24h内定时取样,在扫描电镜下观察孢子的萌发及其对寄主体壁的入侵。结果表明,附着到蚜体表面的孢子在4h内有30－40％已萌发产生芽管,少数是呈叉状分枝的营养生长型芽管。且侵染性芽管的孢子部分孢入体壁蜡质层中,显示孢子有分泌物产生并作用于寄主体壁。接种10h内,侵染性芽管通过顶端膨大的附着胞或直接穿透入寄主体壁。到24h时,产生侵染性芽管的孢子全部侵入寄主体内,寄主体表仅留下少数未萌发的孢子或营养生长型芽管。初级分生孢子在蚜体表面很少产生次级分生孢子,说明桃蚜是适合该菌侵染的寄主。陷入寄主体壁的孢子不因若蚜蜕皮而被去掉,表明该菌对成蚜和若蚜都具有侵染力。     

金龟子绿僵菌附着胞分化及其与环腺苷酸cAMP的关联性研究

寄主识别与附着胞分化是虫生真菌启动侵染过程的首要步骤。本文利用先前获得的金龟子绿僵菌基因缺失突变株与其野生型一起进行附着胞分化研究。接种后不同时间下的观察表明,绿僵菌突变株或野生型的附着胞既可以在萌发不久的芽管顶端形成,也可以在伸长菌丝分支的顶端形成。与野生型不同的是,突变株附着胞的分化频率显著下降,附着胞周围也缺乏粘液层的产生。研究表明,绿僵菌的类枯草杆菌类体壁降解酶对于附着胞分化不产生影响,对体壁降解也非完全必需的。与突变株附着胞分化频率显著降低相对应,其胞内环腺苷酸cAMP水平显著下降,而添加外源cAMP能够显著增加其附着胞分化频率,说明绿僵菌cAMP信号途径对于调控附着胞分化起着重要的作用。     

球孢白僵菌在红火蚁体表侵染的扫描电镜观察

利用扫描电镜观察了球孢白僵菌Beauveria bassiana Bb04菌株分生孢子对红火蚁Beauveria bassiana 工蚁体壁的侵染过程。结果表明: 分生孢子多分布在红火蚁工蚁节间膜、胸部的褶皱、气门、体壁的凹陷部位、刚毛窝附近, 以及着生较密刚毛的足上。萌发的分生孢子在节间膜以及体表缝隙、刚毛窝及刚毛稀少的凹陷部位、胸部褶皱和足胫节处入侵。分生孢子在附着12 h后开始萌发, 接种后18 h附着在节间膜处的孢子首先侵入成功, 接种后24 h刚毛窝附近孢子萌发入侵, 接种后60 h胸、腹和足等部位的孢子均成功穿透侵入表皮。分生孢子可以直接以芽管侵入表皮, 也可以产生附着胞再侵入。     

尖角突脐孢菌在稗草和水稻上的萌发和侵染行为比较研究

本试验结合曲利苯兰和荧光素钠两种染色方法的优点,从显微角度研究了尖角突脐孢菌(Exserohilum monoceras)两个菌株X-27和HN-14在稗草和水稻上萌发和侵染行为的差异。结果表明,在寄主稗草上,接种4h后尖角突脐孢菌孢子开始从一端或两端萌发形成初生芽管;10h后芽管顶端膨大形成附着胞,附着在寄主表面,两端萌发的孢子约90%一端败育,仅一端形成成熟附着胞;在接种后24h内成熟附着胞形成率与接种时间成正相关,24h后趋于稳定;16h后在成熟附着胞下方受侵染的细胞内指状吸器开始形成,随后发育为掌状吸器;接种36h后菌丝在组织表面扩展形成网状,同时稗草叶片上显现叶斑病症状,部分菌丝能在细胞间蔓延扩展。在非寄主植物水稻上,同样观察到孢子萌发产生芽管,但是萌发起始时间滞后大约4h,初生菌丝分枝明显减少,且未能观察到附着胞和吸器的产生。     

应用荧光染色法检测寄主昆虫体表病原真菌孢子及其附着孢

采用蝗虫翅膀作为侵染组织,探讨了荧光染色剂Calcofluor White M2R在观测寄主体表绿僵菌孢子及其附着孢形成中的应用。结果表明,在荧光显微镜下,清晰可见蝗虫翅膀上发蓝色荧光的绿僵菌孢子、芽管及附着孢,而蝗虫翅膀未被染色,避免了干扰观察目标物。该方法可以准确观察病原真菌孢子在昆虫体表组织的萌发及附着孢形成。     

绿僵菌侵染小菜蛾幼虫过程的透射电镜观察

利用透射电镜观察了绿僵菌Metarhizium anisopliae对小菜蛾Plutella xylostella幼虫的侵染过程。结果表明：接种后22 h最早观察到绿僵菌穿透到寄主表皮层中,在穿透表皮过程中伴随着机械力和酶的活动。菌体进入寄主血腔后,以菌丝出芽生殖、菌丝分隔及菌丝段分隔3种方式大量增殖,主要形成颗粒状的菌丝段在寄主体腔内扩散,也形成少量的丝状菌丝。     

落叶松-杨栅锈菌在感病杨树寄主上发育过程的组织学研究

采用荧光染色技术、光学显微镜和电子显微镜技术,系统研究了落叶松-杨栅锈菌在感病杨树叶片上的发育过程。结果表明,在侵染前期(接种12h以内),锈菌夏孢子在杨树叶片上萌发,利用芽管或附着胞穿透叶表气孔后形成气孔下囊,进而在胞间产生侵染菌丝。进入活体营养生长阶段(接种后24–96h),锈菌不断产生大量吸器来满足营养需求的同时,侵染菌丝在叶肉细胞间隙蔓延分枝生长至形成菌落结构。最终在产孢阶段(接种120h之后)产孢菌丝分化形成的夏孢子在表皮下聚集成堆,待成熟后突破表皮显露出来。     

几种虫生真菌附着胞的荧光显微及扫描电镜观察

本研究通过对细脚拟青霉、蝉拟青霉、玫烟色拟青霉、金龟子绿僵菌和莱氏野村菌在疏水表面产生附着胞的荧光显微镜和扫描电镜电镜观察,明确五种虫生真菌均可产生附着胞,细脚拟青霉和金龟子绿僵菌产生单附着胞和复合附着胞两种形态,均呈椭圆至长椭圆形,如遇到不合适侵染的部位,则重新产生芽管向前延伸直至找到适合入侵的部位,蝉拟青霉分生孢子多在顶端恨芽成人字形,末端椭圆形附着胞,该拟青霉再生附着胞能力强。金龟子绿僵菌     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Development of Infection Structures by the Direct-Penetrating Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) on Artificial Membranes

The development of infection structures by the directly infecting soybean rust fungus of different artificial membranes was followed by light and scanning electron microscopy. On water agar uredospores developed germ tubes without appressoria. On dialysis membranes more than 80% of the uredospores formed appressoria. With low frequencies (1–7%) also primary hyphae and/or penetration hyphae were present. When cellulose nitrate membrane filters with pore diameters ≤ 0.2 μm were used, uredospores germinated but showed a strongly reduced appressoria formation. Membranes with pores ≥ 0.1 μm allowed a development of infection structures similar to that on dialysis membranes. In experiments with paraffin oil incorporated into collodion membranes more than 90% of the uredospores formed appressoria, about 50% of the appressoria developed hyphae. Ungerminated spores and germ tubes always contained 2 nuclei. In fully developed appressoria 4 nuclei were present. Compared with stomata entering rust fungi appressoria formation by Phakopsora pachyrhizi occurred more frequently and seemed to be less dependent on specific stimuli. Moreover, in most cases only few of the appressoria formed penetration or primary hyphae. The induction of these structures seemed to be dependent on further unknown stimuli.     

Symptoms and Prepenetration Events Associated with the Infection of Common Bean by the Anamorph and Teleomorph of Glomerella cingulata f.sp. phaseoli

Glomerella cingulata f.sp. phaseoli and Colletotrichum lindemuthianum are the teleomorph and anamorph, respectively, of the pathogen causing anthracnose in common bean. The mechanisms relating to the sexual reproduction of this plant pathogen are still unclear, as are the infection structures involved and the symptoms produced. In the present study, bean plants were inoculated with ascospores and conidia, and the events taking place within the following 120 h were investigated using light microscopy and scanning electron microscopy. The symptoms exhibited by plants inoculated with the ascospores were milder than in those inoculated with conidia. Microscopy revealed that most of ascospores produced germ tubes and appressoria at an early stage (24 h after inoculation). From 48 h onwards, the formation of hyphae and the production of germ tubes and appressoria were great. In contrast, infections originating from conidia developed more slowly, and at 24 and 48 h, many non‐germinated conidia were present, whereas only few conidia developed germ tubes and appressoria. Ascospore germination and appressorium formation were similar on both resistant and susceptible cultivars. Hence, the symptoms and the temporal sequence of events associated with the infection of bean plants by the two fungal forms differed, although the structures produced were similar. This is the fist report comparing symptoms and prepenetration events between anamorph and teleomorph of G. cingulata f.sp. phaseoli in common bean.     

A Method for Staining Infection Hyphae in Pine Leaves

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: conidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

Percutaneous infection of Hylobius pales by Metarrhizium anisopliae

The manner in which Metarrhizium anisopliae infects larval and adult Hylobius pales was investigated histologically and by scanning electron microscopy. In the presence of fungal and bacterial contaminants on beetles, none or few conidia of M. anisopliae germinated. Antibiosis is suggested, since the inhibition could be eliminated by surface sterilization. On larvae, the contaminants were scarce, and spore germination was greater. With germination, the germ tubes typically grew extensively over the procuticle or sclerites before producing appressoria of various shapes and sizes. These appressoria consistently produced a light-transmissible mucoid substance. Conidia, germ tubes, and appressoria were frequently fused into infection cushions of random arrangement and size. Sclerites of larvae and adults were not penetrated by the fungus, whereas procuticle and metawings were.     

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (M_r 133,000 and 146,000). Reductions in the M_r of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM extracellular matrix - BPA Bauhinia purpurea agglutinin - BSA bovine serum albumin - DIC differential interference contrast - FITC fluorescein isothiocyanate - GNL Galanthus nivalis lectin - GSI-B₄ Griffonia simplicifolia isolectin B₄ - HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - IIF indirect immunofluorescence - IPC isopycnic centrifugation - MAb monoclonal antibody - PEG polyethylene glycol - PBS phosphate buffered saline - PNGase peptideN-glycosidase - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCS tissue culture supernatant - TEM transmission electron microscopy - TFMS trifluoromethane sulphonic acid     

A method for staining infection hyphae in pine leaves.

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: condidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

Penetration of Phytophthora cinnamomi into disease tolerant and susceptible eucalypts

The mechanisms of penetration of Phytophthora cinnamomi Rands into seedling eucalypt roots were studied by light and electron microscopy. Culture grown seedlings of root-rot tolerant Eucalyptus st johnii and root-rot susceptible Eucalyptus obliqua were inoculated with both zoospores and mycelium. Zoospores encysted on roots of both species and the germ tubes penetrated without the formation of appressoria. Swellings, previously described as appressoria, were formed when the germ tube was slow to enter the host by intracellular penetration. Vegetative hyphae penetrated both inter- and intracellularly into the zones of root elongation and differentiation, often through root hairs. Evidence of hydrolysis of the host cell-wall at the point of penetration was observed in electron micrographs. Several hours after the germ tube penetrated the epidermis, a thick plug of amorphous material formed in the germ tube slightly below the level of the outer walls of the epidermal cells, sealing off the hypha within the root. Behaviour of zoospores and germ tubes and the mechanism of penetration were similar on both hosts. Micrographs do not suggest any kind of a hypersensitive reaction by the host cells during the early stages of infection.     

Germ-tube formation by atypical strains of Candida albicans

Atypical isolates of Candida albicans which failed to produce germ tubes in routine diagnostic procedures were examined for their ability to produce germ tubes in various media. Bovine serum was more effective than defined media for induction of germ tubes in the majority of isolates. A few strains formed appreciable germ tubes only in bovine serum with added thioglycollate or cysteine. One strain did not produce germ tubes in any medium. Germ-tube maturation appeared to be dependent upon mitochondrial RNA polymerase activity. The failure by an isolate to produce germ tubes, particularly in tests without strictly controlled conditions, does not preclude the possibility that the organism is C. albicans.