Evidence for Resistance-inducing Compounds in Conidia of Erysiphe graminis f.sp. hordei

Preparations of Erysiphe graminis f.sp. hordei conidia were spray-applied to the first leaf of barley plants which were subsequently challenge inoculated with virulent powdery mildew. The powdery mildew reducing effect of the preparations was assessed by scoring the outcome of the challenge inoculation. Homogenates of ungerminated conidia, germinated conidia, and methanol-water extracts of germinated conidia reduced the number of powdery mildew colonies. Cell wall fragments from ungerminated conidia, germinated conidia, and conidial germination fluid obtained from conidia germinated in aqueous suspension did not reduce the number of powdery mildew colonies. Microsconical analysis of the infection course following challenge inoculation indicated that the powdery mildew reducing effect is due partly to induced resistance.     

Autoregulation of Conidium Germination in Micromycetes of the genus Trichoderma

The amount of germinated conidia of micromycetes belonging to the genus Trichoderma considerably decreased with an increase in the population density. Strains exhibited different ecological strategies. The maximum number of germinated conidia (30–70%) was recorded when the average distance between conidia was 50 m.     

Taka-amylase A in the conidia of Aspergillus oryzae RIB40

A study of Taka-amylase A of conidia from Aspergillus oryzae RIB40 was done. During the research, proteins from conidia and germinated conidia were analyzed using SDS-PAGE, 2-D gel electrophoresis, Western blot analysis, MALDI-TOF Mass spectrometry, and native-PAGE combined with activity staining of TAA. The results showed that TAA exists not only in germinated conidia but also in conidia. Some bands representing degraded products of TAA were detected. Conidia, which formed on starch (SCYA), glucose (DCYA), and glycerol (GCYA) plates, contained mature TAA. Only one active band of TAA was detected after native-PAGE activity staining. In addition, TAA activity was detected in cell extracts of conidia using 0.5 M acetate buffer, pH 5.2, as extraction buffer, but was not detected in whole conidia or cell debris. The results indicate that TAA exists in conidia in active form even when starch, glucose, or glycerol is used as carbon source. TAA might belong to a set of basal proteins inside conidia, which helps in imbibition and germination of conidia.     

Hydrolytic Enzymes of Ungerminated and Germinated Conidia of Erysiphe graminis DC f. sp. hordei Marchal

Activities of hydrolytic enzymes (esterase, lipase, phosphatase, protease, 1,3-β-glucanase, chitinase and amylase) were demonstrated in washings and extracts of ungerminated and germinated conidia of Erysiphe graminis f. sp. hordei , Marchal. No cellulase and only very little xylanase activity was detected. Considerable amounts, 65% of total conidia proteins can be extracted by shaking the conidia when the extraction medium contained non-ionic detergent only. Different extraction procedures were used to determine enzyme distribution between exo- and endoconidial compartments and to evaluate the enzyme extraction efficiency from conidia.
The possible physiological roles of the hydrolytic enzymes in the ungerminated as well as in the germinated conidia were discussed.     

Fungicidal and binding properties of three plant peptides

The fungicidal properties of plant seed peptides from Heuchera sanginea (Hs-AFP₁), Raphanus sativus (EA-AFP₂), and Impatiens balsamina (Ib-AMP₃) were determined for the nongerminated and germinated conidia of Aspergillus flavus and Fusarium moniliforme . These peptides were weakly lethal for germinated but not for nongerminated conidia of A. flavus . Both nongerminated and germinated conidia of F. moniliforme were susceptible to these peptides. Overall, F. moniliforme was more susceptible than A. flavus to the peptides. The peptides bound strongly to chitin, mannan, galactocerebrosides, and sphingomyelin. Binding results varied for ergosterol, cholesterol, and β1,3-glucan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Taka-Amylase A in the Conidia of Aspergillus oryzae RIB40

A study of Taka-amylase A of conidia from Aspergillus oryzae RIB40 was done. During the research, proteins from conidia and germinated conidia were analyzed using SDS–PAGE, 2-D gel electrophoresis, Western blot analysis, MALDI-TOF Mass spectrometry, and native-PAGE combined with activity staining of TAA. The results showed that TAA exists not only in germinated conidia but also in conidia. Some bands representing degraded products of TAA were detected. Conidia, which formed on starch (SCYA), glucose (DCYA), and glycerol (GCYA) plates, contained mature TAA. Only one active band of TAA was detected after native-PAGE activity staining. In addition, TAA activity was detected in cell extracts of conidia using 0.5 M acetate buffer, pH 5.2, as extraction buffer, but was not detected in whole conidia or cell debris. The results indicate that TAA exists in conidia in active form even when starch, glucose, or glycerol is used as carbon source. TAA might belong to a set of basal proteins inside conidia, which helps in imbibition and germination of conidia.     

Mycosporine-alanine: A self-inhibitor of germination from the conidial mucilage ofColletotrichum graminicola

On soft substrata conidia ofColletotrichum graminicola germinated by the formation of germ tubes, whereas on hard, hydrophobic substrata they germinated by the formation of appressoria. Regardless of the substratum, conidia did not germinate at high conidium concentrations, suggesting the presence of an endogenous self-inhibitor. Bioassays to test for the inhibition of germination demonstrated the presence of a low-molecular-weight self-inhibitor in the mucilage which surrounds conidia as they are produced in acervuli. The inhibitory material exhibited an absorbance maximum of 310 nm, indicating that it is a member of the mycosporine family of compounds. The inhibitor was purified to homogeneity and shown by mass spectrometric analysis to have a molecular mass consistent with the structure corresponding to mycosporine-alanine.     

Guanine uptake and metabolism in Neurospora crassa

Guanine is transported into germinated conidia of Neurospora crassa by the general purine base transport system. Guanine uptake is inhibited by adenine and hypoxanthine but not xanthine. Guanine phosphoribosyltransferase (GPRTase) activity was demonstrated in cell extracts of wild-type germinated conidia. The Km for guanine ranged from 29 to 69 micro M in GPRTase assays; the Ki for hypoxanthine was between 50 and 75 micro M. The kinetics of guanine transport differ considerably from the kinetics of GPRTase, strongly suggesting that the rate-limiting step in guanine accumulation in conidia is not that catalyzed by GPRTase. Efflux of guanine or its metabolites appears to have little importance in the regulation of pools of guanine or guanine nucleotides since very small amounts of 14C label were excreted from wild-type conidia preloaded with [8-14C]guanine. In contrast, excretion of purine bases, hypoxanthine, xanthine, and uric acid appears to be a mechanism for regulation of adenine nucleotide pools (Sabina et al., Mol. Gen. Genet. 173:31-38, 1979). No label from exogenous [8-14C]guanine was ever found in any adenine nucleotides, nucleosides, or the base, adenine, upon high-performance liquid chromatography analysis of acid extracts from germinated conidia of wild-type of xdh-l strains. The 14C label from exogenous [8-14C]guanine was found in GMP, GDP, GTP, and the GDP sugars as well as in XMP. Xanthine and uric acid were also labeled in wild-type extracts. Similar results were obtained with xdh-l extracts except that uric acid was not present. The labeled xanthine and XMP strongly suggest the presence of guanase and xanthine phosphoribosyltransferase in germinated conidia.     

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Germination of Aspergillus fumigatus conidia in the lungs of normal and cortisone-treated mice.

Inhaled conidia of Aspergillus fumigatus germinated in the lungs of mice at a low rate but both germinated and ungerminated spores were cleared. Spores germinated at a high rate in the lungs of cortisone-treated mice.     

Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle

The lepidopteran-specific fungal pathogen Entomophaga maimaiga is highly virulent against Lymantria dispar (gypsy moth) larvae, and other members of the family Lymantriidae. Numerous species in the subfamily Cuculliinae (Family Noctuidae) are not susceptible to E. maimaiga due to the inability of this fungus to penetrate the larval cuticle. Conidial attachment and germination were compared among five cuculliine species and L. dispar using bioassays and scanning electron microscopy. Although conidia were showered evenly across larvae during bioassays, on L. dispar conidia were most abundant on segments, where they adhered well to the cuticle and germinated at high percentages. Conidia on cuculliine cuticles were predominantly found in large, loose aggregations in intersegmental areas. Few conidia on cuculliine cuticle germinated and scanning electron microscopy revealed a thick film of mucous enveloping conidia. We hypothesize that the conidia on cuculliines become coated by this film and were only loosely attached to the larval cuticle. No such film was seen on L. dispar larvae where individual conidia appeared well attached. On L. dispar larvae many conidia also adhered to setae. To determine if hydrophobicity affected the ability of E. maimaiga conidia to attach and germinate on a substrate, a goniometer was used to determine relative hydrophobicity of larval cuticles. L. dispar cuticle was more hydrophobic than cuculliine cuticle, suggesting that a high level of hydrophobicity could be a required characteristic for hosts. Cuticles from four cuculliine species and L. dispar were sequentially extracted using hexane, chloroform, and methanol. Conidia were showered onto glass slides coated with the different extracts and germination was quantified. Methanol extracts of cuculliine cuticle consistently decreased germination, compared to all extracts of L. dispar cuticle. For all L. dispar extracts, the majority of conidia produced germ tubes, which is a normal prerequisite for cuticular penetration. For the cuculliines, conidia exposed to hexane and chloroform extracts produced secondary conidia as did all controls, but the conidia exposed to cuculliine methanol extracts that germinated produced germ tubes. These studies demonstrated that a range of factors act in concert to prevent E. maimaiga infection of the cuculliine species investigated.     

Inactivation of Neurospora crassa conidia by singlet molecular oxygen generated by a photosensitized reaction.

Photodynamic damage of Neurospora crassa conidia was studied in the presence of the photosensitizing dye, toluidine blue O. Conidia which germinated to form colonies decreased in number as irradiation time became longer. The photoinactivation of conidia was suppressed by azide, bovine serum albumin, and histidine, and was stimulated in deuterium oxide. Wild-type conidia were less sensitive to the irradiation than albino conidia. In the wild-type, carotenoid-enriched conidia were more resistant against the lethal damage than the conidia which contained small amounts of carotenoids. These results suggest that singlet molecular oxygen causes photodynamic lethal damage to N. crassa conidia and that singlet molecular oxygen is quenched by endogenous carotenoids.     

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。     

Recovery of germinating fungal conidia from the nasal cavity after environmental exposure

Background The expression of fungal allergens is increased by the germination of conidia. We assessed the state of germination of fungal conidia recovered by nasal lavage after environmental exposure. Methods Nasal lavage was performed on twenty adults at three stages: the start of the experiment, after 1 h indoors, and after 1 h outdoors. One half of the lavage liquid was immediately treated to prevent in-vitro germination and stained with periodic acid Schiff (PAS) to enable identification of germinated and ungerminated conidia. The untreated half of the lavage liquid was cultured on nutrient agar plates to enumerate and identify viable fungi. Results PAS staining showed that both ungerminated and germinated conidia, and hyphal fragments, were present in the nasal cavity. The most prevalent fungi recovered were Aspergillus, Alternaria, Cladosporium, Epicoccum, Penicillium, and Yeast species. The number of viable fungi recovered after 1 h indoors was significantly less than after 1 h outdoors (P < 0.01). Conclusions Viable fungi and germinating conidia, in addition to ungerminated conidia and hyphal fragments, were present in the nasal cavity after both indoor and outdoor exposure. This provides novel insight into the pathogenicity of exposure to fungal aeroallergens.     

努利虫疠霉感染桃蚜过程的扫描电镜观察

利用扫描电镜观察了努利虫疠霉Pandora nouryi (Remaudière & Hennebert) Humber初生分生孢子接种桃蚜Myzus persicae (Sulzer)后孢子萌发、入侵以及菌体突破虫体的整个侵染过程。结果表明：附着于虫体表面的初生分生孢子在3～5 h后即有60%以上的萌发率,萌发的孢子形成芽管或产生球形或棍棒状的附着胞;12 h后大部分孢子均已萌发,并成功入侵寄主虫体;接种60 h后,呈掌状分枝的假根首先从桃蚜胸部的腹面突破虫体长出体外,明显区别于新蚜虫疠霉Pandora neoaphidis (Remaudière & Hennebert) Humber假根突破虫体的位置;假囊状体不多见,且仅分布于蚜虫身体两侧,这可在一定程度上解释努利虫疠霉产孢对湿度条件要求较高的生物学现象。     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

Germination of sclerotia of Botrytis tulipae, the cause of tulip fire

Sclerotia of Botrytis tulipae (Lib) Lind. buried in field soil germinated mainly in winter and early spring to produce conidiophores and conidia and then decayed. Except when buried very late in the year sclerotia germinated and decayed in the season following burial. In the laboratory, sclerotia from pure culture germinated in two ways. On sterile water-agar they formed colourless mycelium but no conidiophores; the rate of germination was greatest at 25 d?C. On unsterile filter paper and on soil they produced mainly conidiophores and conidia; the rate of germination was greatest at 5 d?C and was enhanced by previous cold treatment. It was unaffected by soil-moisture or pH. Primary infections of tulips arose from sclerotia placed in the soil but only when they were close to the shoot tip or developing stem. It is suggested that, because of their limited survival, soil-borne sclerotia are of importance only when tulips are planted in annual succession.     

Effects of exogenous nutrients on conidial germination and virulence against the silverleaf whitefly for two hyphomycetes

Exogenous protein and sugar sources were tested for their impact on conidial germination of two silverleaf whitefly (Bemisia argentifolii) pathogens: Beauveria bassiana and Paecilomyces fumosoroseus. In liquid culture, sugars stimulated only 5-27% germination of B. bassiana and < or =11% germination of P. fumosoroseus, whereas, yeast extract or peptone stimulated 95-100% germination. In the absence of additional nutrients, agar alone stimulated approximately 50% germination. Storing spores for different periods of time did not alter their general response to exogenous nutrients. When spores were germinated before being applied to third instar B. argentifolii, mortality was as much as 2.45 times greater and occurred more rapidly than that for fresh spores. For ungerminated conidia, the mean time to death from infection was 5.45 (SE = 0.16) and 4.74 (SE = 0.08) days for application rates of 37 and 144 conidia x mm(-2), respectively. When conidia were germinated before application, infection times dropped to 4.58 (SE = 0.16) and 4.45 (SE = 0.10) days, respectively. A likely explanation for the greater pathogenicity and virulence of germinated over ungerminated B. bassiana conidia is that only a fraction of the spores applied to whitefly nymphs actually germinate on the cuticle. For some specialized applications, such as greenhouse production systems, it may be beneficial to germinate spores immediately prior to application.