Differential deoxyribonucleic acid synthesis during microcycle conidiation in Neurospora crassa

In conidia of Neurospora crassa germinating at 25°C, DNA synthesis measured by incorporation of tritiated adenosine reaches a maximum soon after the outgrowth of the germ tube (6–7h after inoculation). In conidia heat-treated at 46°C (for 15h), a maximum of incorporation of the DNA precursor occurs already 1h after inoculation, then the incorporation progressively declines until the end of the heat-shock. When such conidia are shifted to 25°C, a maximum of DNA synthesis occurs during the development of the presumptive conidiophore as at the outgrowth of normal germ tubes. This wave of DNA synthesis is followed by a second maximum of DNA synthesis, occurring only in the microcyclized cultures, when the premature differentiation of proconidia takes place. Prevention of this second wave of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine respectively reduces or fully inhibits such induced conidial differentiation.     

黑星病菌在苹果叶片上的发育过程研究

采用电子显微镜技术,研究了苹果黑星病菌在苹果叶片上发育过程。扫描电子显微镜观察结果表明,接种后12 h 分生孢子即可萌发并形成附着胞,统计结果显示其孢子萌发率在6 h和12 h分别为83% 和95%,附着胞形成率在12 h和24 h 分别为93% 和95%。透射电子显微镜观察结果表明,黑星病菌侵入以后在寄主角质层下和表皮细胞之间扩展、定殖并可形成子座。接种后12d,病菌开始从子座上产生分生孢子梗和分生孢子,分生孢子梗顶端每产生一个单生的分生孢子就形成一个环痕并延伸其长度。分生孢子梗和分生孢子主要沿叶脉形成,在叶片上呈网状扩展,此时叶片表现明显的病害症状。     

Influence of mechanical stress and surface interaction on the aggregation of Aspergillus niger conidia

Productivity of fungal cultures is closely linked with their morphologic development. Morphogenesis of coagulating filamentous fungi, like Aspergillus niger, starts with aggregation of conidia, also denominated as spores. Several parameters are presumed to control this event, but little is known about their mode of action. Rational process optimization requires models that mirror the underlying reaction mechanisms. An approach in this regard is suggested and supported by experimental data. Aggregation kinetics was examined for the first 15 h of cultivation under different cultivation conditions. Mechanical stress was considered as well as pH-dependent surface interaction. Deliberations were based on a two-step aggregation mechanism. The first aggregation step is only affected by the pH-value, not by the fluid dynamic conditions in the bioreactor. The second aggregation step, in contrast, depends on the pH-value as well as on agitation and aeration induced power input. For the given experimental set-up, agitation had much more influence than aeration. In addition, hyphal growth rate was determined to be the driving force for the second aggregation step.     

Development of Powdery Mildew Fungi on Leaves Submerged Under Water

Effects of submerging inoculated leaves under water on the development of Erysiphe graminis f. sp. hordei on barley, E. pisi f. sp. pisi on pea and Sphaerotheca pannosa on rose were investigated. Submerging leaves immediately after inoculation removed 27–40% of conidia from leaves. Additionally, conidia either failed to germinate or germinated abnormally resulting in reduced elongating secondary hyphae (ESH) production. Submerging leaves 24 h after inoculation had, little effect on the numbers of ESH. Conidiophore production from mycelia occurred under water, although mildew growth was slower. Sporulating colonies treated with water produced conidia which had the ability to produce ESH. Experiments with E. aquilegiae on Aquilegia vulgaris, E. depressa on Arctium lappa, E. verbasci on Verbascum thapsus and Microsphaera alphitoides on Quercus rober suggest that the wettability of leaves and the presence of trichomes on leaf, surfaces play an important role in determining germination behaviour and subsequent ESH production on leaves.     

Collection of highly germinative pseudochain conidia of Oidium neolycopersici from conidiophores by electrostatic attraction

A population of simultaneously germinating conidia is an ideal inoculum of the powdery mildew pathogen, Oidium neolycopersici. In conditions of no or low wind velocity, O. neolycopersici successively stacks mature conidia on conidiophores in a chain formation (pseudochain), without releasing the precedent mature conidia. These pseudochain conidia represent a perfect inoculum, in which all conidia used for inoculation germinate simultaneously. However, we found that conidia must be collected before they fall to the leaf surface, because the germination rate was lower among conidia deposited on the leaf surface. We used an electrostatic spore collector to collect the pseudochain conidia, and their high germination rate was not affected by this treatment. The spore collector consisted of an electrified insulator probe, which created an electrostatic field around its pointed tip, and attracted conidia within its electric field. The attractive force created by the probe tip was directly proportional to voltage, and was inversely proportional to the distance between the tip and a target colony on a leaf. Pseudochain conidia were successfully collected by bringing the electrified probe tip close to target colonies on leaves. In this way, conidia were collected from colonies at 3-d intervals. This effectively collected all conidia from conidiophores before they dropped to the leaf surface. A high germination rate was observed among conidia attracted to the probe tip (95.5 ± 0.6 %). Conidia were easily suspended in water with added surfactant, and retained their germination ability. These conidia were infective and produced conidia in pseudochains on conidiophores after inoculation. The electrostatic spore collection method can be used to collect conidia as they form on conidiophores, thus obtaining an inoculum population in which all of the conidia germinate simultaneously.     

Effects of hydrophilic and hydrophobic kaolin-based particle films on pea aphid (Homoptera: Aphididae) and its entomopathogen Pandora neoaphidis (Entomophthorales: Entomophthoraceae)

Hydrophobic and hydrophilic kaolin-based particle films are effective for control of insect pests in certain agricultural crops. How these products interact with potential biological control agents is not well documented. This study was conducted to evaluate the effects of the hydrophobic (M96-018) and hydrophilic (Surround WP) kaolin-based particle films (Engelhard Corporation, Iselin, NJ) on pea aphid, Acyrthosiphon pisum (Harris), on peas (Pisum spp.), and on the fungal aphid pathogen Pandora neoaphidis (Remaudière and Hennebert) Humber. Over two field seasons (2001 and 2002) in northern Idaho, applications of M96-018 significantly reduced the rate of pea aphid increase on pea, but Surround WP, tested only in 2001, did not reduce aphid population growth rate. Neither particle film treatment was as effective as a standard application of esfenvalerate (DuPont Asana). In the laboratory, particle films suppressed pea aphid populations by up to 30%. M96-018 seemed to have some repellent activity based on aphid distributions after treating plants. When applied along with P. neoaphidis conidia, M96-018 but not Surround WP caused higher percentage of infection mortality of pea aphids by P. neoaphidis than occurred on controls treated only with P. neoaphidis conidia. P. neoaphidis conidia deposited on glass slides coated with M96-018, produced more germ tubes and secondary conidia than those deposited on untreated glass slides or slides treated with Surround WP. This result suggests that greater infection of pea aphids on plants treated with M96-018 is in part a result of a direct enhancement of fungal germination by the particle film.     

Production of an Extracellular Matrix as an Isotropic Growth Phase of Penicillium rubens on Gypsum

Indoor mold represents an important environmental concern, but a fundamental knowledge of fungal growth stages is needed to limit indoor fungal proliferation on finishing materials used in buildings. The present study focused on the succession of germination stages of the common indoor fungus Penicillium rubens on a gypsum substrate. This substrate is used as a model system representing porous materials that are widely used in indoor environments. Imaging with cryo-scanning electron microscopy showed that the formation of an extracellular matrix (ECM) is a phase of the isotropic growth of P. rubens that is uniquely related to germinating conidia. Furthermore, the ECM is observed only when a dry-state inoculation of the surface is applied, i.e., applying conidia directly from a 7-day-old colony, mimicking airborne contamination of the surface. When inoculation is done by spraying an aqueous conidial suspension, no ECM is observed. Moreover, it is concluded that the formation of an ECM requires active processes in the fungal cell. The porosity of the substrate proved that the ECM substance has high-viscosity characteristics. The present results stress that studies of indoor fungal growth should consider the method of inoculation, knowing that the common aqueous suspension may obscure specific stages in the initial phases of germination.     

新蚜虫疠霉连续液体培养的菌丝生物量、产孢量及杀蚜毒力

继代培养常被疑为虫霉菌种毒力下降或某些生物学性状发生改变的原因之一。从研究新蚜虫疠霉 (Pandoraneoaphidis)固体平板菌落的液体培养获得的初始菌液出发 ,连续 6次继代液培 ,测定了其在萨氏营养液中继代培养生产的菌丝生物量、产孢量及其对桃蚜 (Myzuspersicae)的毒力。在初始菌液的生物量 8 8mg mL和产孢量 7 2× 1 0 5孢子 mg的条件下 ,以三种转接比 (种液 营养液 ,v v)连续 6次继代培养 ,在 1 2 0转接比下的菌丝生物量和产孢量分别为 6 4～ 1 0 0mg mL和 7 3× 1 0 5～ 1 0 8× 1 0 5孢子 mg,在 2 2 0下为 5 7～ 8 5mg mL和 1 0 0× 1 0 5～ 1 2 1× 1 0 5孢子 mg ,在 4 2 0下为 5 5～ 1 0 9mg mL和 6 4× 1 0 5～ 1 0 9× 1 0 5孢子 mg。方差分析表明 ,继代培养对生物量和产孢量均无显著影响 (P <0 0 5)。用初始菌液和 1 2 0转接比下继代培养的菌液制备而成的接种体对桃蚜进行两组毒力测定 ,每一组测定的接种…     

Characterization of Spanish strains of Verticillium lecanii

We have characterized biologically and physiologically eight Verticillium lecanii strains from several origins including insect pests. Of all the temperatures tested, 25 degrees C was the best for growth and at 40 degrees C none of the strains could grow. At 4 and 7 degrees C, growth was reduced in comparison to warmer temperatures. The strains had better development at pH close to 7 (F = 27.64, P < 0,01) than at pH 3. Self-inhibition of germination of strain 50 was found when more than 0.78 conidia/cm(2) were plated on corn meal agar (CMA). Germination of conidia was close to 100% for all strains except one, three days after inoculation. Among extracellular enzymatic activities studied the fungal strains showed strongest proteolytic activities followed by lipolytic and chitinolytic activities. Some strains showed significant differences (P < 0.05) in conidia production. Most of the fungicides tested (especially benomyl) inhibited radial growth of strain 50 on CMA. Pathogenicity (as median lethal time, LT50) of V. lecanii strains on larvae of Galleria mellonella varied from 2.66 -/+ 0.33 to 4.27 -/+ 0.25 days. We conclude that in vitro tests per se are not sufficient to select the best biocontrol strains of entomopathogenic fungi. Pathogenicity is a complex process in which the presence, timing and regulation of many factors including those covered in this paper, as well as their interactions, are probably involved.     

Histopathology of Nomuraea rileyi in Plathypena scabra larvae

Green cloverworm larvae. Plathypena scabra, were inoculated with Nomuraea rileyi by “tumbling” larvae in a vial of conidia. The ontogeny of the pathogen was followed by using standard histological techniques. N. rileyi conidia germinated on green cloverworm integument within 12 hr after inoculation. Germ tubes penetrated larval cuticle 36 hr after inoculation, then grew parallel to endocuticular laminae. After hyphal penetration of the epidermis ca. 4.5 days after inoculation, hyphal bodies were produced and were transported throughout the hemocoel. Hyphal bodies and hemocytes cohabited the hemocoel, but gut epithelial and muscle tissues were not invaded by Day 5. Hemocytes lysed and mycelia completely ramified throughout all larval tissues by 7 days after inoculation. Death of larvae was followed by conidiogenesis ca. 7.5 days after inoculation.     

Conidiation ofTrichoderma atroviride isolate during submerged cultivation in a laboratory stirred-tank fermenter

Conditions for conidiation of a natural isolate of Trichoderma atroviride during submerged cultivation in Erlenmeyer flasks and in a laboratory stirred-tank fermenter were optimized. From the simple sugars tested, cellobiose was the best substrate for conidia production while cellulose fines from paper mill waste proved to be a suitable cheap complex carbon source. Optimum temperature for conidiation was 24-26 degrees C, and the required dissolved oxygen level was > 40% saturation. After initial slight decrease during the 1st d after inoculation, the pH of the culture medium constantly increased throughout the sporulation period. Attempts to regulate the pH during fermentation did not improve the spore yields. The most intense formation of conidia took place between 2nd and 3rd d of growth and the overall volumetric productivity of conidia was 4.1-8.2 x 10(9) conidia per L/h.     

昆虫病原真菌长孢蜡蚧菌TF-2菌株对豌豆蚜的侵染过程和致病力

【目的】豌豆蚜Acyrthosiphon pisum是世界性的重要农业害虫,给豆类作物造成巨大的经济损失。本研究在前期筛选已获得豌豆蚜致病菌长孢蜡蚧菌Lecanicillium longisporum TF-2菌株的基础上,检测了该致病菌分生孢子对豌豆蚜的毒力效用及侵染方式,以期为利用昆虫病原真菌防治豌豆蚜提供理论基础。【方法】采用离体叶片饲养法检测在不同浓度长孢蜡蚧菌TF-2菌株孢子悬浮液(1.0×10~3～1.0×10~7孢子/mL)中浸渍后对豌豆蚜成虫的致病力;应用扫描电镜和体视显微镜观察豌豆蚜成虫接种TF-2菌株(1.0×10~7孢子/mL)后长孢蜡蚧菌TF-2菌株侵染症状和侵染过程。【结果】不同浓度长孢蜡蚧菌TF-2菌株分生孢子浸染豌豆蚜成虫致病力随分生孢子浓度升高而逐渐增强,侵染后6 d对豌豆蚜成虫的半致死浓度(LC_(50))为3.26×10~4孢子/mL,最高浓度分生孢子(1.0×10~7孢子/mL)对豌豆蚜成虫的半致死时间(LT_(50))为2.92 d。扫描电镜观察结果表明,接种后24 h,TF-2菌株分生孢子在豌豆蚜成虫体表皮萌发形成芽管和附着胞;接种后48 h,芽管伸长在复眼、触角基部、足基节、腹末生殖节等部位形成网络结构;接种后96 h,菌丝布满整个虫体,新的分生孢子产生。【结论】长孢蜡蚧菌TF-2菌株对豌豆蚜成虫具有较强的致病力,扫描电镜观察揭示了其分生孢子在其虫体上的侵染过程,结果为进一步应用昆虫病原真菌进行生物防治提供理论基础和参考依据。     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

Effects of catalase on the accumulation of H2O2 in rice cells inoculated with rice blast fungus, Magnaporthe oryzae

Roles of H₂O₂ in the infection process of Magnaporthe oryzae on rice were investigated. In a leaf sheath assay for up to 48 h post-inoculation, the absence or presence of catalase in the conidia suspension was correlated with the level of accumulated H₂O₂ in infected leaf cells, as observed by staining with 3',3-diaminobenzidine tetrahydrochloride. In the incompatible interaction, the appearance of autofluorescence or frequency of cell death characterized by granulation (symptoms characteristic of hypersensitive responses) was not significantly affected by the presence of catalase in the conidia suspension. In the leaf blade assay, inoculation of compatible conidia in the presence of catalase produced more severe symptoms than that of conidia in the absence of catalase at 6 days post-inoculation. These results suggest that, in this host–parasite interaction, the primary role of host-produced H₂O₂ is in limiting hyphal growth after penetration through toxic action. Furthermore, in incompatible interactions, H₂O₂ is implied not to be a major mediator of hypersensitive cell death.     

Relationship between proteolytic activity of Aspergillus fumigatus and the fungus' invasiveness of mouse brain

This experiment was carried out to determine whether proteolytic activity of Aspergillus fumigatus was enhanced in the mouse brain during advance in growth of hypae, and if any relationship might be demonstrated between the proteolytic activity of the fungus and its invasive ability for the mouse brain.The K-2 strain of A. fumigatus and male mice of ddy line weighing 22±1 g were used in this experiment. Each mouse was inoculated intravenously with 5×10⁶ conidia of the strain suspended in 0.2 ml. phosphate buffer solution supplemented with 0.01 per cent Tween 80. Mice were sacrified at 4 hours' intervals up to 40 hours after inoculation and at an hour's intervals later. Two mice were assigned for each time. Each brain obtained was fixed in 10 per cent formalin and was cut into three sections from which histopathological specimens were prepared.The brains over 40 hours after inoculation were markedly swollen and abundant hypae were observed in the specimens. The extent of the fungal growth in the specimens from the mouse brain was 1.60, 7.71 and 10.84 per cent at 32,45 and 49 hours after inoculation, respectively.For assay of the proteolytic activity in the mouse brain, three groups of fifteen mice each were tested with the K-2 strain. These groups of mice were inoculated with 5×10⁶conidia of the strain and sacrificed 0,32 and 45 hours after inoculation, respectively. Then the brains pooled in each group and four volumes of phosphate buffer solution was added. The material was homogenized and used as test samples (enzyme solution). The proteolytic activity was measured quantitatively by a modification of Anson's method.The proteolytic activity in the mouse brain sacrificed 0,32 and 45 hours after inoculation was 0.00, 0.08 and 0.13, respectively, as expressed by the optical density.In conclusion, it was possible to confirm that proteolytic activity increased in the mouse brain in proportion to the growth of hyphae in it.     

Germination of phytopathogenic fungi conidia

The autoregulation of conidium germination in phytopathogenic micromycetes of the genera Fusarium, Botrytis, and Bipolaris was studied. It was shown that Trichoderma longibrachiatum was less competitive than Fusarium oxysporum after their simultaneous inoculation but inhibited the phytopathogen growth in the case of earlier introduction. In the latter case, no autoinhibition of the germination of F. oxysporum conidia occurred; moreover, cooperative effect was observed, i.e., the number of germinated F. oxysporum conidia increased with an increase in their density.     

Role of the Mf1-1 pheromone precursor gene of the filamentous ascomycete Cryphonectria parasitica

Site-directed recombination was used to obtain a Cryphonectria parasitica strain carrying deletions at the Mf1-1 gene locus. Macroscopic features such as growth rate and conidia production were unaffected by Mf1-1 deletions, but, when a strain containing a complete deletion of Mf1-1 was used as spermatia it was male sterile. The same strain was fully competent as a female parent. Deletion of three of the seven putative pheromone peptide repeats within the gene had no effect on mating. Male fertility of the complete deletion strain was restored when an ectopic copy of the Mf1-1 gene was returned by transformation. Expression of the mating type specific pheromone precursor gene Mf1-1 was stimulated by growth in nutritionally poor liquid media. It was found that age and source of inoculum of liquid cultures influences pheromone precusor gene expression, i.e., conidia did not express Mf1-1 and cultures derived from conidia were significantly delayed in expression of this gene, as were cultures derived from young mycelium. Cultures inoculated with older hyphae, however, expressed Mf1-1 within 1 day after inoculation.     

Freckle disease of banana in Hawaii caused by Phyllostictina musarum (Cke.) Petr

‘Freckle’ (‘black-spot’ disease) of bananas is common on leaves and fruit of Dwarf Cavendish and other varieties in Hawaii, especially after rainy periods. On fruit, symptoms may appear 2–4 weeks after the bunch has opened, and become more severe as maturity is approached. The disease is usually confined to older leaves on affected plants. Freckled tissue contains numerous pycnidia of Phyllostictina musarum and disease was experimentally induced by inoculating leaves and fruit with conidia of this fungus. This appears to be the first record of successful inoculation with P. musarum. Conidia of P. musarum germinate after 3–6 h in a film of water on banana peel, appressoria being formed after 18–30 h. Penetration of the epidermis occurs 24–96 h after inoculation, and is brought about by an infection hypha which grows from the appressorium. The progressive increase in severity of freckle as fruit matures is due to repeated infection by further conidia of P. musarum, rather than to enlargement of original infections. Some banana clones, including Gros Michel, appear to be resistant to the fungus Dispersal of P. musarum conidia immediately after discharge from the pycnidium is chiefly by rainwater and dew. Secondary infections contribute greatly to the total number of infections. Conidium dispersal by water often results in the development of characteristic patterns of spotting, chiefly in the form of streaks or circular areas, coinciding with the directions of movement of rainwater and dew. Large numbers of conidia of P. musarum are washed on to fruit in rainwater and dew running from diseased, overhead leaves.