Conidial Germination Rate Research Articles

In vivo observation of conidial germination at the oxic–anoxic interface and infection of submerged reed roots by Microdochium bolleyi

The underground plant parts of reed ( Phragmites australis) growing in anoxic soil of the littoral zone of lakes are provided with oxygen via an aerenchyma. Some of this oxygen is released into the rhizosphere, which creates a potential microhabitat for aerobic fungi. Although fungal endophytes of reed have been shown to occur also in roots of flooded habitats, it is not known whether or how fungi can infect roots growing in anoxic or hypoxic soil. To study fungal infection of reed roots in the laboratory, we developed an incubation chamber to expose reed roots to conidia of Microdochium bolleyi in an anoxic agar medium and to observe fungal infection in vivo. Germination rates of conidia were high close to living roots, but decreased to zero in anoxic areas of the chamber. Conidial germ tubes located up to 200 μm from the roots grew preferentially towards the living roots. Conidia also germinated close to air-filled Teflon tubes and exhibited germ-tube tropism, but not as distinctly as on living reed roots. Conidia did not germinate in the neighbourhood of dead roots in anoxic agar. However, in the aerated margin of the incubation chamber most conidia germinated and exhibited tropic growth towards dead roots. Penetration of M. bolleyi through several cell layers of living roots was observed in cryo-microtome sections. Penetration was significantly deeper with illuminated plants than with plants kept in the dark; in some cases even the stele was reached. This is the first observation of oxygen released from roots to support growth of an aerobic fungus and of fungal penetration into root tissue in an anoxic environment.

ストロビルリン系薬剤耐性ナスすすかび病菌の発生

Seventy isolates of Mycovellosiella nattrassii, collected from eggplants in Kochi Prefecture, Japan, were tested for sensitivity to strobilurin fungicides in 2000. The frequency distribution of minimum inhibitory concentrations of azoxystrobin against mycelial growth had two distinct peaks at 3.13ppm and over 1600ppm, considered to be sensitive and resistant, respectively. Isolates resistant to azoxystrobin had cross resistance to kresoxim-methyl. The EC50 of azoxystrobin and kresoxim-methyl for mycelial growth and the inhibitory rate of conidial germination by azoxystrobin were also clearly distinguishable between resistant and sensitive strains. Control efficacy of these two fungicides was very low against resistant strains in an inoculation test.

Alternaria alternata에 의한 들깨 잎마름병

최근 들깨잎 주산지인 부산시 강동지역과 경남 밀양지역 하우스내의 들깨잎에 가장자리가 검게 변하고 마르는 증상을 띄는 병해가 발생되고, 심하면 잎 전체가 고사하는 병이 발생되었다. 이런 병반으로부터 병원성이 강하고 분생포자 형성이 많은 SD1 균주를 분리하여 병원균의 형태 및 배양적 특성을 조사한 결과 PDA 평판배지에서 <TEX>$25^{\circ}C$</TEX>, 암조건으로 배양할 경우 균사생장이 가장 왕성하였으며, 분생포자 형성량은 V8A 평판배지에서 가장 많았다. 또한 SD1 균주의 균사생장 적온은 <TEX>$25^{\circ}C$</TEX>, 분생포자 형성 적온은 3<TEX>$0^{\circ}C$</TEX>였으며, 분생포자 현탁액에 첨가한 영양원에 따른 분생포자 발아율과 발아관의 신장은 10% 토마토쥬스에서 가장 양호하였는데 24시간 후 발아율은 거의 100%였으며, 36시간 후 발아관 길이는 535.2<TEX>$\mu\textrm{m}$</TEX>로 PDB보다 1.4배정도 길었다. 또한 SD1 균주의 병원성을 검정한 결과 10% 토마토쥬스를 첨가한 분생포자 현탁액으로 접종할 경우 자연 발병된 병징과 동일하였으며 발병율도 100%로서 PDB 분생포자 현탁액에서는 60%, 살균수에서는 전혀 발병되지 않았다. 본 균주는 균총 형태와 색깔, 광학현미경을 이용한 형태적 특성과 몇 가지 배지 등을 이용한 배양적 특성 및 병원성 검정 결과에 따라 Alternaria alternata로 동정되었으며, 들깨 잎마름병으로 명명하였다. A new leaf blight was found on the perilla leaves at the major perilla-cultivating areas such as Kangdong in Busan and Miryang in Kyungnam province. Symptoms of the disease initially appeared on the edge of perilla leaves showing black necrosis and drying, and the infected leaves were finally fell down. The SD1 isolate showing strong pathogenicity and forming abundant conidial spores on the diseased lesions was isolated. Among the tested media, mycelial growth was abundant on PDA (Potato Dextrose Agar) medium at <TEX>$25^{\circ}C$</TEX> under dark condition, but conidial formation was greater on V8A (V-8 juice A8ar) medium than that on PDA medium. Optimal temperatures for mycelial growth and conidial formation on PDA medium were respectively <TEX>$25^{\circ}C$</TEX> and 3<TEX>$0^{\circ}C$</TEX>. The rate of conidial germination and the elongation of germ tube were more effective in 10% tomato juice than those in PDB (Potato Dextrose Broth) and sterile water. In 10% tomato juice, the rate of conidial germination and the length of germ tube were 100% after incubation for 24k and 535.2<TEX>${\mu}{\textrm}{m}$</TEX> after incubation for 36hr, respectively. According to the result of pathogenicity, it revealed that conidial suspention with 10% tomato juice was the most effective for pathogenicity test showing as 100% of disease incidence, and the symptoms caused by artificial inoculum were same as those of naturally infected perilla. In this study, the SD1 isolate according to the results of morphological characteristics, the incubation characteristics and pathogenicity was firstly identified A. alternata, and named as leaf blight of perilla.

Efeito de fungicidas em Colletotrichum acutatum e controle da antracnose do morangueiro

Onze fungicidas foram analisados in vitro e seus efeitos testados em campo para o controle de Colletotrichum acutatum, agente da flor-preta do morangueiro (Fragaria X ananassa). Os tratamentos (campo) foram (dosagens de i.a/100 l): iprodione (75 ml), benomil (100 g), tebuconazole (50 ml), tiofanato metílico (70 g), prochloraz (100 ml), propiconazole (50 ml), mancozeb (200 g), folpet (270 g), sulfato de cobre (200 g) e chlorotalonil (200 g), em pulverizações semanais (protetores) e quinzenais (sistêmicos). Foram avaliados a produção de frutos e o número de flores com sintoma de queima. Nos testes de fungitoxicidade in vitro, estudou-se o crescimento micelial, a germinação de conídios e a formação de apressórios. Nos testes in vitro foram utilizadas as concentrações de 1 e 10 ppm de i.a. para todos os fungicidas e posteriormente testadas as concentrações de 0,01, 0,1 e 1 ppm para os mais eficientes, e 10, 50 e 100 ppm para os menos eficazes. Os que resultaram em maior inibição micelial em baixas concentrações foram prochloraz e tebuconazole e os que menos inibiram o crescimento micelial foram sulfato de cobre, clorotalonil e folpet. Para inibir a germinação conidial mostraram-se mais eficientes chlorotalonil, tebuconazole, prochloraz e benomil, e para inibir a formação de apressórios, chlorotalonil e benomil. Os resultados de campo diferiram parcialmente dos testes in vitro: prochloraz e sulfato de cobre apresentaram os menores percentuais de flores doentes (53-55%), enquanto benomil apresentou 100% de ataque. Embora prochloraz, seguido de iprodione, folpet e mancozeb tenham resultado em maiores produções, nenhum fungicida controlou a doença satisfatoriamente.

Biological Control ofBotrytis cinereaPers. ex Fr. in Strawberry byPaenibacillus polymyxa(Isolate 18191)

Isolate 18191, obtained from mature strawberry fruit and determined asPaenibacillus polymyxahas shown an antagonistic potential againstBotrytis cinerea, the causal agent of grey mould in strawberries. Germ tube growth of conidia ofB. cinereawas strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density ofB. cinereaby 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 106, 1 × 107, 1 × 108) showed that the lowest density already reduced incidence ofB. cinereaby 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness ofP. polymyxait was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density ofB. cinereaby 53 and 58%, respectively. In 3‐year field trials the effectiveness ofP. polymyxawas in a range of 24–36% as compared to the water control.

The resistance to Oidium lycopersici conferred by ol-2 gene in tomato

The powdery mildew caused by Oidium lycopersici is one of the most destructive diseases in glass-house-grown tomato and is widespreading all over the world. A high level of resistance to O. lycopersici was found in an accession of Lycopersicon esculentum var. cerasiforme at the Department of Biology and Plant Pathology, University of Bari. The genetic analysis of F1, F2 and BC plants indicated that the resistance is conferred by a single recessive gene, designed as ol-2. Studies on the infection process of O. lycopersici on susceptible and ol-2 gene resistant tomatoes were carried out at 24 °C and 90 % relative humidity. Light microscope observations on conidia germination, formation of primary appressoria, elongation of hyphae and sporulation were made on artificially inoculated basal, intermediate and apical leaves. Inoculation was made by shaking mildewed tomato leaves over each test plant. Disease development were assessed by removing the fungal structure from the leaf surface with the ceroidin film technique and by direct observations of stained inoculated leaves. The rate of conidial germination and the appressoria formation was not affected by host genotype. Mycelia growth and sporulation on leaf surface of resistant plant was strongly restricted and influenced by the leaf age. The results indicated that the resistance in ol-2 tomato is postinfectional and is not associated with a hypersensitive response.

Impact of Temperature and Humidity on Host–Pathogen Interactions Between Beauveria bassiana and a Coccinellid

We tested the effect of 2 exposure methods and 2 environmental factors, temperature and humidity, on Beauveria bassiana (Deuteromycotina: Hyphomycetes) mycosis in Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae). We found that the standard EPA bioassay method of exposing nontarget insects by immersion resulted in a per-insect dose that was 5 times greater than that from spray applications. However, this difference did not significantly affect mycosis levels in a simulation of field applications conducted in the greenhouse. In contrast, differences in environmental conditions between the laboratory and greenhouse had a large effect on mycosis. Constant temperatures between 15 and 35°C had a significant effect on both germination rate and vegetative growth of B. bassiana , with the fastest germination occurring at 25-32°C and the fastest growth occurring at 30°C. Although temperature affected the rate of conidial germination, total germination eventually reached 97-100% at all temperatures. Contrary to these results, levels of mycosis in lady beetles decreased as temperatures increased over this same range. Although high levels of humidity are required for conidial germination, we found no well-defined threshold period of high humidity exposure required for mycosis in this insect. In other words, the effects of temperature and humidity on free-living stages of the pathogen are not sufficient to explain the effect of these factors on mycosis. We suggest that temperature and humidity also affect physiological interactions between the host and pathogen.

Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of Coniothyrium minitans

The effects of growth media, temperature, pH and light on the development of four isolates of Coniothyrium minitans (CONIO and CH8 (colony type 3), G4 (colony type 4) and G9 (colony type 5)) were examined. Conidial germination, pycnidial production and hyphal extension rate were initially studied on seven different agar-based growth media at 18–20 °C. Potato-dextrose agar (PDA) and malt extract agar (MEA) consistently gave the greatest conidial germination, pycnidial production and hyphal extension rate for all four isolates. Growth and development on molasses-yeast agar was equivalent to that on PDA and MEA except that hyphal extension rate was slower. Subsequently, the effects of temperature, pH and light on the development of C. minitans were investigated on PDA only. The temperature range of conidial germination and pycnidial production of the four isolates was between 10–25° with the optimum at approximately 20°. Hyphal extension occurred over a greater temperature range, between 4 and 25°, with a maximum extension rate of approximately 3·5 mm d − for all isolates occurring between 20–25°. Conidial germination, pycnidial production and hyphal extension occurred over a pH range between 3–8 with optimum values for all growth assessments occurring between pH 4·5 and 5·6. Increasing light period from continuous dark, to 12 h light 12 h dark or continuous light had no effect on conidial germination or extension growth, but significantly increased pycnidial production. Isolates G4 and G9, previously characterized by sparse production of pycnidia in comparison with CONIO and CH8, consistently exhibited a reduced production of pycnidia on all media, at all temperatures and pH ranges, and all light regimes tested. This demonstrates the stability of this character among these isolates of C. minitans . The significance of these results for improving production of inoculum of this biocontrol agent and in the identification and classification of isolates of C. minitans is discussed.

Improvement of Infectivity of Metarhizium anisopliae(METSCHNIKOFF)to Anomala cuprea HOPE(Coleoptera:Scarabaeidae)by Ultraviolet Irradiation of Protoplasts

Enhancement of infectivity of a wild strain of Metarhizium anisopliae, F1011, against Anomala cuprea was attempted by Ultraviolet irradiation of the protoplasts isolated from mycelia. The protoplasts were regenerated in L-broth containing 2% agar in 0.7 M KCl. Among 38 regenerants obtained, the regenerant No.31 (RNO31), which originated from the protoplasts irradiated by UV for 30 s, was 21 times more virulent than the original strain F1011 against 1st instar larvae of A. cuprea, based on LC50 values. Fewer days were required to achieve a 50% death rate by RNO31 than by F1011. RNO31 also exhibited stronger insecticidal activity against Popillia japonica and Anomala daimiana than did F1011. The number of surviving larvae of A. cuprea in the RNO31 treated pot was lower than that in the F1011 treated pot. Effects of temperatures and agricultural chemicals on the growth of RNO31 were similar to those for the F1011, whereas the effect of pH values was not. Germination rate of conidia from RNO31 was about 2.5 times higher than that from F1011 at 16 h after the inoculation.

Morphology and growth in culture of Microhilum oncoperae, the cause of a mycosis of Oncopera spp. larvae

Microscopic and colony morphology of thirteen isolates of the entomopathogenic fungus Microhilum oncoperae were studied under a range of cultural conditions. Contrary to the original description, several conidia can be produced from a single denticle. All thirteen isolates of M. oncoperae were able to germinate, produce hyphae and sporulate on several different mycological media at temperatures between 5 and 25 °C. None of the isolates could grow above 30°, and at 5° isolates differed in the rate of conidial germination. M. oncoperae conidia germinated more rapidly at 5° than those of two Metarhizium anisopliae and five Beauveria bassiana isolates. Selective agars used for isolating other entomopathogenic fungi from soil were inhibitory to the growth of M. oncoperae .

Anti-conidial germination factors induced in the presence of probenazole in infected host leaves. I. Isolation and properties of four active substances.

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.

Mitochondrial biogenesis in Neurospora crassa. I. An ultrastructural and biochemical investigation of the effects of anaerobiosis and chloramphenicol inhibition.

The isolation of a new class of mutants permitting facultative anaerobiosis in Neurospora crassa is described. Backcross analyses to the obligate aerobe prototroph (An(-)) indicate single nuclear gene inheritance (An(-)/An(+)). An(+) and An(-) are indistinguishable in morphology and growth rates under aerobic conditions. Anaerobic growth requires nutritional supplements that are dispensable for aerobic growth. Conidiogenesis, conidial germination, and vegetative growth rate are suppressed by anaerobiosis. An(+) mutants produce substantial quantities of ethanol under anaerobic conditions. Anaerobiosis and chloramphenicol both affect mitochondrial enzyme activity and morphology. Chloramphenicol inhibition leads to reduction in cytochrome oxidase and swollen mitochondria with few cristae. Anaerobiosis leads to reduction in both cytochrome oxidase and malate dehydrogenase activities, enlarged mitochondria with fewer cristae, enlarged nuclei, and other alterations in cellular morphology. The fine structure of anaerobically grown cells changes with the time of anaerobic growth. We conclude that either inhibition of mitochondrial membrane synthesis or inhibition of respiration might lead to the observed alterations in mitochondria.

Investigations on a disease of Coffea arabica caused by a form of Colletotrichum coffeanum Noack : I. Some factors affecting infection by the pathogen

The berry disease of Coffea arabica, caused by a form of Colletotrichum coffeanum Noack, may have arisen by mutation of the fungus from a mildly parasitic to a virulent form at some time prior to 1922. Conidia from different sources were found to vary in viability and infectivity, those from lesions on ripe berries being superior to those from green, and conidia from pure cultures on potato-dextrose agar being the least satisfactory. Different stages of the crop varied markedly in susceptibility, flowers, very young fruit, and ripe berries being the most susceptible; germination rates of conidia on the surface of flowers were comparable with those on nutrient agar. All parts of the plant can be attacked, and the fungus, which habitually colonizes the maturing bark external to the developing phellogens, can cause a disease of twigs (Elgon die-back) under abnormal conditions. There is a difference in varietal reaction to attack, berries of the Harar variety and its derivatives being the most susceptible known, and Blue Mountain the most resistant. Wounding increased susceptibility in all varieties; addition to the infection drop of extracts from berries of several different varieties increased susceptibility, with Blue Mountain having the greatest stimulatory effect and Harar none. Varietal reaction of a berry to infection cannot, however, be correlated either with the amount of germination on its surface, or with stomatal numbers, but increased germination does increase the invasion rate in individual flowers or berries. Variation between varieties in susceptibility is ascribed to some chemical or physical difference in the cuticle, making penetration relatively more difficult in the resistant variety. At the optimum temperature of around 22° C., and at a suitable humidity, infection can take place in as little as 2 hr.