Polyamines stimulate hyphal branching and infection in the early stage of <Emphasis Type="Italic">Glomus etunicatum</Emphasis> colonization

Polyamines are known to strongly stimulate hyphal growth in arbuscular mycorrhizal fungi. The effect of the polyamines putrescine, spermidine and spermine on spore germination, hyphal elongation and branching by the AM fungus Glomus etunicatum was investigated in this study. The effect of spermine on infection and the development of the host and of daughter spores was further investigated using the dual monoaxenic culture system comprised of Gl. etunicatum fungal cultures in Ri T-DNA transformed carrot hairy roots. Spermidine and spermine showed positive effects on germination and all three polyamines significantly promoted hyphal growth. Hyphal branching was also strongly stimulated by treatment with polyamines, such as an increase in the number of branches. Infection during the early stages of the in vitro co-culture life cycle was enhanced in the presence of spermine, and daughter spores appeared at earlier timepoints compared to the control. Our results demonstrate that polyamines stimulate germination and hyphal branching in the early stage of AM fungal colonization. Moreover, results from the investigations conducted in the fungus-root co-culture suggest that polyamines may be involved in establishing the symbiotic relationship between root and fungus.     

Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insects.     

Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis

Mycelial morphology is a critically important process property in industrial fermentations of filamentous micro-organisms, as particular phenotypes are associated with maximum productivity. However, the accurate quantification of complex morphologies still represents a significant challenge in elucidating this relationship. A system has been developed for high-resolution characterisation of filamentous fungal growth on a solid substrate, using membrane immobilization and fully-automatic plug-ins developed for the public domain, Java-based, image-processing software, ImageJ. The system has been used to quantify the microscopic development of Aspergillus oryzae on malt agar, by measuring spore projected area and circularity, the total length of a hyphal element, the number of tips per element, and the hyphal growth unit. Two different stages of growth are described, from the swelling of a population of conidiospores up to fully developed, branched hyphae 24 h after inoculation. Spore swelling expressed as an increase in mean equivalent spore diameter was found to be approximately linear with time. Widespread germination of spores was observed by 8 h after inoculation. From approximately 12 h, the number of tips was found to increase exponentially. The specific growth rate of a population of hyphae was calculated as approximately 0.24–0.27 h⁻¹. A wide variation in growth kinetics was found within the population. The robustness of the image-analysis system was verified by testing the effect of small variations in the input data.     

Density-dependent insect-mold interactions: effects on fungal growth and spore production

Larvae of saprophagous insects often have been suspected of being competitors of filamentous fungi on decaying organic matter, which negatively influence mold development. Of interest, the role of insects in determining fungal growth and the onset of sporulation largely has been ignored. I used Aspergillus niger and the vinegar fly Drosophila melanogaster as an ecological model system to analyze the influence of insect larvae on daily fungal growth and the start of conidiospore production. I used an artificial substrate to test whether the effect of larval density (one, five and 10 larvae) and inoculation date of the mold (2 and 3 d ahead of the addition of larvae) significantly altered fungal growth. Fungal growth (area covered by hyphal tissue of the artificial patch) was affected negatively by the number of larvae and by the time that elapsed between inoculation with fungal spores and transfer of larvae to the patches. Whereas one larva had only a minor effect on fungal growth, five or 10 larvae strongly hampered mold development. As time between inoculation with spores and introduction of fly larvae increased, mold increased, indicating a priority effect for the fungus. When 10 larvae were transferred at the same time as the patches were inoculated with spores, almost no mold was visible within the period of observation (after 12 d). In comparison with control treatment (no insect larvae), an increase in larval density caused an increasing delay of several days in the start of spore production. Thus only minor changes in the density of insect larvae and the time that larvae entered the patches after inoculation with spores had an enormous effect on fungal growth and spore production. Therefore insects co-occurring with mold on ephemeral resources might constitute an important biotic factor driving local fungal population dynamics. The mechanisms leading to the suppression of fungal growth and the evolutionary implications of insect-mold interactions are discussed.     

Vesicular-arbuscular mycorrhizas and soil salinity

This review discusses the growth and activity of vesicular-arbuscular (VA) mycorrhizal fungi in saline conditions. The review includes examination of the effects of high concentrations of salts on the occurrence of VA mycorrhizal fungi in field soils, and on spore germination, growth of hyphae, establishment of the symbiosis and production of spores in controlled conditions. Information on the growth and reproduction of VA mycorrhizal fungi under saline conditions is scarce and is often circumstantial. There is clear evidence that germination of spores and subsequent hyphal growth of some VA mycorrhizal fungi are reduced by increasing concentration of salts. However, in plant growth experiments, experimental designs and methodologies have generally not allowed the direct effects of salinity on fungal growth to be separated from plant-mediated effects. There is a need for controlled studies to investigate the responses of VA mycorrhizal fungi to soil salinity. Research is required which distinguishes between effects on different phases of the fungus lifecycle and which includes in its design the ability to separate direct effects from plant-mediated influences on fungal growth and reproduction.     

A pattern recognition tool for quantitative analysis of in planta hyphal growth of powdery mildew fungi

The development of fungal pathogens can be quantified easily at the level of spore germination or penetration. However, the exact quantification of hyphal growth rates after initial, successful host invasion is much more difficult. Here, we report on the development of a new pattern recognition software (HyphArea) for automated quantitative analysis of hyphal growth rates of powdery mildew fungi on plant surfaces that usually represent highly irregular and noisy image backgrounds. By using HyphArea, we measured growth rates of colonies of the barley powdery mildew, Blumeria graminis f. sp. hordei, on susceptible and induced-resistant host plants. Hyphal growth was not influenced by the resistance state of the plants up to 48 h postinoculation. At later time points, growth rate increased on susceptible plants, whereas it remained restricted on induced-resistant plants. This difference in hyphal growth rate was accompanied by lack of secondary haustoria formation on induced-resistant plants, suggesting that induced resistance in barley against Blumeria graminis is caused mainly by reduced penetration rates of primary as well as secondary appressoria leading, finally, to fewer and less-developed fungal colonies. No evidence was found for reduced nutrient-uptake efficiency of the primary haustoria in induced-resistant leaves, which would be expected to have resulted in reduced hyphal growth rates during the first 48 h of the interaction.     

青霉TS67菌株活性产物的抗真菌作用

本文初步探讨了青霉TS67(Penicillum sp.)的发酵活性产物对植物病原真菌的抑制作用机理,实验结果表明,用其50%的发酵液分别处理玉蜀黍平脐蠕孢菌(Bipolarismaydis)和大豆尖孢镰刀菌(Fusarium oxysporum)120 h后,菌丝生长的抑制率分别为77.78%和70.30%,对孢子产生的抑制率分别达58.8%和73.5%:同时发现用50%发酵液处理病原茵的无性繁殖孢子12 h后,孢子萌发抑制率分别达78.3%和62.O%.经显微镜观察抗菌活性物质处理后的菌丝体,发现菌丝体表面瘤状畸形、菌丝生长顶端不规则膨胀、内部发生原生质浓缩,初步推测青霉TS67主要通过影响植物病原真菌的细胞壁而实现抑制作用.     

三株球孢白僵菌侵染烟粉虱的比较生长动力学及其毒力

同一种虫生真菌的不同菌株对于特定昆虫宿主的毒力可存在显著差异,真菌在昆虫体内的生长能力不同可能是引起这种差异的原因之一。为了探讨真菌在宿主体内的生长动力学与其毒力的关系,本研究用生测法测定了3株球孢白僵菌（GZGY-1-3、SCWJ-2、WLMQ-20）在高剂量（1×108孢子/mL）和低剂量（1×106孢子/mL）下对烟粉虱4龄若虫的毒力,用实时荧光定量PCR技术对真菌在宿主体内的拷贝数进行了定量,用荧光显微方法观察了白僵菌侵染烟粉虱的过程。生物测定实验结果显示：不论在高剂量还是低剂量下,菌株GZGY-1-3杀死烟粉虱所需的时间最短（在高剂量和低剂量下LT50分别为2.29d和6.10d）,其次是菌株SCWJ-2（LT50分别为3.03d和7.38d）,菌株WLMQ-20所需时间最长（LT50分别为4.13d和9.39d）。对于同一菌株,其高剂量下的毒力显著高于低剂量下的毒力。实时荧光定量PCR实验显示,接触高剂量孢子后烟粉虱获得的孢子数远远高于接触低剂量时的孢子数。接种后,每一菌株和每一剂量下的真菌生长都表现出一个相似的模式。在接种24h后,真菌细胞数量显著下降,在接种后24–48h之间是一个简短的恢复阶段,接种48–72h后是真菌的细胞数略有净增长的稳定时期,在宿主死亡前后的阶段,真菌数量急剧增加,与接种后最初24h相比高了近1 000倍。然而,尽管它们的生长模式相似,但是却存在着量上的差异,由致病性强、剂量高的菌株侵染的昆虫体内的最终真菌菌体数高。荧光显微技术观察到的白僵菌对烟粉虱的侵染过程证实了定量PCR的结果。这些结果说明菌株间毒力的差异在一定程度上是由真菌生长动力学的量化差异所决定的。     

Germination of surface-disinfected resting spores ofPlasmodiophora brassicae and their root hair infection in turnip hairy roots

Germination of surface-disinfected resting spores ofPlasmodiophora brassicae and its infection of turnip hairy root hairs were studied. Surface-disinfected resting spores showed higher germination than non-disinfected resting spores. Root hair infection was most frequent in the section of root formed 1 d before inoculation. Root hair infection began 4 d after inoculation, increased up to 6 d, and continued to increase more slowly until 10 to 12 d after inoculation. Growth ofP. brassicae in the root hair of hairy roots was observed serially. Most primary plasmodia differentiated to mature zoosporangia 8–10 d after inoculation. The secondary zoospores were initially released 6 d after inoculation.     

Differences in susceptibility to peroral inoculation with Bacillus piliformis spores in rats and mice

The median liver lesion producing doses of peroral inoculation with the spores of Tyzzer's organism RJ strain were 10(4. 3) in rats and 10(2. 7) in rats receiving prednisolone treatment for the provocation of Tyzzer's disease. In contrast to rats, liver lesions were detected in few mice inoculated perorally with 10(7) spores. In mice inoculated perorally with 10(7) spores, excretion of infective spores in the feces was detected only on day 1 postinoculation. On the other hand, no difference in susceptibility between rats and mice was detected upon intravenous inoculation with vegetative cells of the RJ strain. These results suggest that germination of the spores in the intestinal tract causes the difference in the susceptibility in rats and mice.     

Status of nuclear division in arbuscular mycorrhizal fungi during in vitro development

Summary The number of nuclei in spores and along hyphae of an arbuscular mycorrhizal fungiGigaspora margarita was measured in digital images of fluorescence arising from mithramycin stained cultures. Typical dormant spores (250 m diameter) contained 2000 nuclei. Eight hundred nuclei were mobilized during the first 3 days of germination. The number of nuclei in the spores nearly returned to the initial number after 22 days of hyphal growth. The average relative DNA content in the nuclei of dormant spores and in the nuclei of spores incubated for 22 days was comparable, as judged from fluorescence intensity. Hyphal elongation occurred with 460 nuclei per cm under a special set of in vitro conditions that promote extensive hyphal growth of arbuscular mycorrhizal fungi. We found an average total of 26000 hyphal nuclei per germinating spore after 22 days. The specific DNA polymerase inhibitor aphidicolin did not inhibit spore germination but it rapidly reduced the rate of hyphal growth and arrested growth after 4 days. No nuclei were produced de novo during this time. These results demonstrate thatG. margarita replicates nuclear DNA and undergoes nuclear division when grown in vitro even in the absence of a plant host.     

A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited approximately 65% survival compared to approximately 10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.     

Trichoderma harzianum antagonistic activity and competition for seed colonization against seedborne pathogenic fungi of sunflower

This study investigated the antagonistic effects of Trichoderma harzianum isolate (TRIC8) on mycelial growth, hyphal alteration, conidial germination, germ tube length and seed colonization by the seedborne fungal pathogens Alternaria alternata, Bipolaris cynodontis, Fusarium culmorum and F. oxysporum, the causes of seedling rot in over 30% of sunflowers. The antagonistic effect of TRIC8 on mycelial growth of pathogens was evaluated on dual culture that included two inoculation assays: inoculation of antagonist at 48 h before pathogen (deferred inoculation) and inoculation at the same time with pathogen (simultaneous inoculation). TRIC8 inhibited mycelial growth of the fungal pathogens between 70·67 and 76·87% with the strongest inhibition seen with deferred inoculation. Alterations in hyphae were observed in all pathogens. Conidial germination of F. culmorum was inhibited by most of the fungal pathogens (38·28%) by TRIC8. Inhibition of germ tube length by the antagonist varied from 31·83 to 37·67%. In seed colonization experiments, TRIC8 was applied in combination with each pathogen to seeds of a sunflower genotype that is highly tolerant to downy mildew. Seed death was inhibited by TRIC8 and the antagonist did not allow growth of A. alternata, B. cynodontis and F. culmorum on seeds and inhibited the growth of F. oxysporum at the rate of 58·32%.     

Effects of ethylene on the susceptibility to Botrytis cinerea infection of different tomato genotypes

Ethylene at 10 and 100 μl/litre stimulated germ-tube elongation of Botrytis cinerea spores incubated within normal and non-ripening nor tomato fruits, but had little influence on the total percent of germination. Values of germ-tube length within the mature-green normal fruits and the mature-green or mature nor fruits were similar to those recorded within the normal mature fruits when held in air. Exposure of the normal and the mutant fruits to 100 μl/litre ethylene immediately after inoculation with B. cinerea insignificantly increased lesion development, but resulted in increased sporulation. When tomato fruits were exposed to ethylene for 3 days before inoculation a marked stimulatory effect on rot development was exhibited on the mature-green normal fruits but not on the nor mutant fruits. The results indicate that exogenous ethylene may directly stimulate germ tube growth of B. cinerea in both normal and mutant fruit, but that it may affect subsequent fungal growth indirectly, via stimulation of the ripening process, only in preclimacteric normal tomato fruit.     

Toxicity of citric and succinic acids for the pycnidiospores ofBotryodiplodia theobromae

The toxic effect of citric and succinic acids on the germination of the pycnidiospores ofBotryodiplodia theobromae, mycelial growth and the killing rate of theB. theobromae spores was investigated. The percentage inhibition of germination of viable fungal spores by 0.01% succinic or citric acid ranged between 51.6 and 58.1%, respectively.B. theobromae was found to grow in 2% malt extract broth at 28°C at the rate of 0.13 CFU/h. Citric acid exhibited a higher killing rate of 0.26 CFU/h and was more effective against the germination of the fungal spores. At concentrations of 0.3% and above, citric acid could be used as pre- and post-infectional fungicide.     

pH值对Gigaspora margarita孢子萌发、菌丝生长与聚磷酸盐含量的影响

土壤pH值是影响AM真菌的生理与生态过程的重要因子之一,本试验在培养基上接种Gigasporamargarita的孢子,研究了pH值分别为5.2、6.0和6.8时孢子萌发率、菌丝生长和菌丝中聚磷酸盐(polyP)的含量。结果表明,不同pH条件下的孢子萌发率没有明显差异,培养12d后的萌发率为70%左右;随着pH的升高,菌丝的长度逐渐增加,表明低pH对菌丝的生长有一定的抑制效应;培养12d后,孢子中polyP含量低于菌丝中polyP含量,pH6.0和pH6.8的条件下菌丝中polyP含量明显高于pH5.2的含量,表明低pH也能降低菌丝中的聚磷酸盐含量。认为低pH对菌丝生长和polyP含量的抑制可能是其限制AM真菌功能发挥的重要机制之一。     

Metabolic products of microorganisms

1. The total nitrogen content of spores of Penicillium notatum increased during swelling and reached a maximum before germ-tubes were formed. It subsequently decreased during germ-tube formation until a constant value in hyphae was reached.
2. An increase in the protein content of the spores was found during swelling, followed by a decrease when germ-tubes were formed. After germination, the protein content increased again to a constant level in hyphae.
3. The content of total lipids steadily decreased during swelling of spores. It reached a minimum value in germinating spores, followed by a net accumulation during hyphal growth. Similar changes occurred in free lipids, phospholipids and the acetone-soluble lipids but not in bound lipids.
4. After an initial decrease during swelling, no further change took place in the neutral carbohydrates content of the spores at the time of germ-tube formation. An accumulation of neutral carbohydrates occurred during late hyphal extension.
5. The nucleic acid content increased sharply during swelling, and reached the highest value in swollen spores just prior to the initiation of germ-tube formation. Afterwards, its content steadily decreased during germ-tube formation and hyphae elongation.

     

A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.     

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

Symbiotic interaction of endophytic bacteria with arbuscular mycorrhizal fungi and its antagonistic effect on Ganoderma boninense

Endophytic bacteria (Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UMPB3), isolated from within roots of oil palm (Elaeis guineensis Jacq.) were tested for their presymbiotic effects on two arbuscular mcorrhizal fungi, Glomus intraradices UT126 and Glomus clarum BR152B). These endophytic bacteria were also tested for antagonistic effects on Ganoderma boninense PER 71, a white wood rot fungal pathogen that causes a serious disease in oil palm. Spore germination and hyphal length of each arbuscular mycorrhizal fungal (AMF) pairing with endophytic bacteria was found to be significantly higher than spores plated in the absence of bacteria. Scanning electron microscopy (SEM) showed that the endophytic bacteria were scattered, resting or embedded on the surface hyaline layer or on the degraded walls of AMF spores, possibly feeding on the outer hyaline spore wall. The antagonistic effect of the endophytic bacteria was expressed as severe morphological abnormalities in the hyphal structures of G. boninense PER 71. The effects of the endophytic bacteria on G. boninense PER 71 hyphal structures were observed clearly under SEM. Severe inter-twisting, distortion, lysis and shriveling of the hyphal structures were observed. This study found that the effect of endophytic bacteria on G. intraradices UT126 and G. clarum BR152B resembled that of a mycorrhiza helper bacteria (MHB) association because the association significantly promoted AMF spore germination and hyphal length. However, the endophytic bacteria were extremely damaging to G. boninense PER 71.