绿僵菌侵染小菜蛾体表过程的显微观察

采用扫描电镜研究了小菜蛾Plutella xylostella体表结构对绿僵菌入侵行为的影响及绿僵菌的侵染过程。结果表明: 绿僵菌孢子在小菜蛾体表萌发后可形成附着胞,寄主体表结构影响形成附着胞的快慢、多少及穿透体壁时芽管长度, 在平缓结构区和刺状结构区比嵴状结构区更易形成附着胞,且芽管较短。在所有结构区,LF68菌株穿透芽管均短于LD65菌株的芽管。接种后7 h,分生孢子在小菜蛾体表开始萌发,LF68与LD65菌株分别于接种后10 h和13 h出现侵染构造穿透体壁。     

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。     

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。     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

红火蚁工蚁对绿僵菌侵染蛹的防御行为

研究了红火蚁工蚁感染绿僵菌后在蛹室的行为变化,以及健康工蚁对侵染蛹的行为保护机制.结果表明: 工蚁被绿僵菌侵染后,在蛹室的活动时间逐渐减少,由第1天的103.4 s降至第3天的38.5 s;而且育幼时间占蛹室活动时间的比例也下降,由第1天的13.6%降至第3天的3.5%.当蛹被绿僵菌侵染后,工蚁对侵染蛹的梳理总时间为对照组的5.3倍,每次梳理的平均持续时间为对照组的5.2倍.梳理行为能显著减少侵染蛹的体表分生孢子数量,在无工蚁、2只工蚁和10只工蚁存在条件下,蛹体表平均孢子数分别为103.1、51.6和31.3个.工蚁的存在能抑制蛹体表孢子的萌发,处理20 h后,无工蚁、2只工蚁和10只工蚁存在条件下,蛹体表孢子萌发率分别为95.1%、80.4%和59.9%.蛹的羽化率随着工蚁数量增加显著升高.红火蚁工蚁通过社会行为防御病原真菌侵染蛹的策略为种群的延续和发展提供了保障.     

玫烟色棒束孢侵染小菜蛾的透射电镜观察

利用透射电镜观察了玫烟色棒束孢Isaria fumosorosea(=Paecilomyces fumosoroseus)对小菜蛾Plutella xylostella(L.)的侵染过程及菌体在虫体内的增殖方式。以浓度为1×107孢子/mL的孢子悬浮液接种小菜蛾4龄幼虫,在透射电镜下对虫体各部位的观察结果表明:接种后1h玫烟色棒束孢菌株EBCL03011的分生孢子开始萌芽,至4h可观察到附着孢的形成和穿透,接种后24h已普遍侵入体腔。玫烟色棒束孢在寄主表皮和体腔内,以菌丝段出芽生殖、菌丝分隔及菌丝段分隔3种方式大量增殖,主要以颗粒状的菌丝段在寄主体腔内扩散,菌丝段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。     

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。     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

黄绿绿僵菌对褐飞虱侵染过程的扫描电镜观察

本实验利用扫描电镜观察了黄绿绿僵菌Metarhizium flavoviride菌株分生孢子对褐飞虱Nilaparvata lugens(St(a)l)的侵染过程.结果表明:分生孢子多分布在褐飞虱节间膜、体表的褶皱凹陷等部位,主要以芽管或产生附着胞入侵,然后在体表长出菌丝和产孢.菌体进入寄主血腔后,利用体腔内营养大量增...     

Invasion of the pathogenic fungus Metarhizium anisopliae through the guts of germfree desert locusts,Schistocerca gregaria

Less than 1% of an ingested inoculum of the pathogenic fungus Metarhizium anisopliae was retained for long enough (ca. 24 h) in the gut of the desert locust, Schistocerca gregaria, for germination and penetration to have occurred. The residual inoculum did not initiate an infection in guts of fed conventional or axenic locusts. However, symptoms of mycosis (hyphal bodies in the haemolymph, fungal penetration of the hindgut intima and epithelium, tetanic paralysis) were consistently observed in axenic but not conventional locusts which were starved post-inoculation.It is concluded that the antifungal toxin produced by the gut bacteria defends the desert locust against gut invasion by Metarhizium anisopliae during periods of starvation when the physical defences, prominent in fed insects, are less apparent.     

The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development

? Premise of the study: The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia. ? Methods: We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed. ? Key results: Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1-2 d). Plants inoculated with M. robertsii ΔMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. ? Conclusions: These results suggest that M. robertsii is not only rhizosphere competent but also displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs.     

A novel Botrytis cinerea-specific gene BcHBF1 enhances virulence of the grey mould fungus via promoting host penetration and invasive hyphal development

Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea-specific pathogenicity-associated factor BcHBF1 (h yphal b ranching-related f actor 1), identified from virulence-attenuated mutant M8008 from a B. cinerea T-DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic- and oxidative-stress adaptation, as well as cell wall integrity of the ∆Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up-regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

New insights into the infection process of Rhynchosporium secalis in barley using GFP

Through the use of a Rhynchosporium secalis isolate transformed with the green fluorescent protein gene and LASER scanning confocal microscopy (LSCM), fungal development during the R. secalis/barley interaction was analysed. Each infection stage was investigated from 0.5h to 14 days post-inoculation (p.i.) with extensive sampling within the first 48 h p.i. Early germination events were observed that had not been previously described. A specific time of germination was noted, with germ tube formation appearing as early as 1h p.i. Conidia were observed within anticlinal grooves of epidermal cells and the formation of bubbles within these pectin-rich regions was observed within 24h p.i. The study reports R. secalis pectinase production and suggests degradation of these pectin-rich regions. Reactive oxygen species were present during early penetration, 3h p.i. and co-localised with fungal development. LSCM allowed the visualisation of fungal growth deep within tissues at the later stage of the infection.     

Insertion of an esterase gene into a specific locust pathogen (Metarhizium acridum) enables it to infect caterpillars

An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene.     

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.     

Infection and colonization of tissues of the aphid Myzus persicae and cassava mealybug Phenacoccus manihoti by the fungus Beauveria bassiana

Histopathogenesis of living insects of Myzus persicae Sulzer (Hemiptera: Aphididae) and Phenacoccus manihoti Matile‐Ferrero (Hemiptera: Pseudococcidae) by Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was monitored from penetration through insect death. Important events in aphids included fungal penetration of the integument of the less-resistant leg intersegmental membrane and invasion of natural openings, formation of hyphal bodies in live aphids by three days post-inoculation (PI), and extensive hyphal colonization of the two leg segments closest to the insect body at death of the aphids. Confocal microscopy of green fluorescent protein-labeled B. bassiana in live mealybugs indicated the fungus penetrated the host through the legs and mouthparts. The fungus was scarce in live mealybugs at 1–5 days PI, formed hyphal bodies by six days PI, and growth was limited to parts of dead hosts at 6–7 days PI. In dead mealybugs, hyphal bodies were near solid tissue. Blastospores were in the hemolymph.