Factors affecting sporulation and germination of Pandora nouryi (Entomophthorales: Entomophthoraecae), a pathogen of Myzus persicae (Homoptera: Aphididae)

Pandora nouryi discharged large numbers of primary conidia between 8 and 25°C from cadavers on the surface of water-agar. At 8°C conidial discharge lasted for 120 h, but most conidia were produced within 48 h when temperature was >15°C. Saturated humidity alone was not enough to allow for sporulation to occur freely and where RH?<?95%, no conidia were discharged. Light did not affect the pattern of conidial production nor the total number of conidia. Germination percentages of conidia on the surface of water-agar were 40 and 66% at 8 and 30°C, respectively, and were significantly lower than that at 15–25°C where germination was >95%. Conidia on leaves germinated well when RH?>?74%, while no germination occurred when RH?<?100% on cover slips. All eight insecticides tested entirely inhibited conidial germination at recommended doses (R), in particular, both the organophosphorus pesticides Lorsben (chlorpyrifos) and the organochlorine pesticides Thiodan (endosulfan) completely inhibited conidial germination even at 0.2R dose.     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Factors Affecting Germination of Oospores of Phytophthora infestans

When oospores from the pairing between A¹ and A² mating types of Phytophthora infestans were treated with 0.25 % KMnO₄ solution for 15 min and incubated at 19 °C under light on a modified S+L medium, germination commenced within 4 days and reached about 70 % after 20 days. Under these conditions, more than 25 % of oospores obtained from a 4-day-old culture germinated. To obtain a high germination rate of P. infestans oospores, light was essential during germination but not during growth and oospore formation. The optimum time for activation of oospores with 0.25 % KMnO₄ was 15 to 30 min and a suitable concentration of KMnO₄ for 15 min activation was 0.25 to 0.45 %.     

Effect of Flavin Inhibitors on Photoactivation of Oospores of Phytophthora cactorum

When dark-grown mature oospores of Phytophthora cactorum were activated to germinate by exposure to 5 uW cm^-2nm^-1 of fluorescent light at 20–22°C in the presence of certain flavin inhibitors such as KI, salicylhydroxamic acid and phenylaceric acid at 40. 1. and 0.1 mM respectively, photoactivation and hence subsequent germination of oospores were inhibited without appreciable irreversible effect on oospore viability. Likewise, when applied during the light period, NaN₃ and KCN at 1 mM reduced photoactivation but had a minimal effect on dark reactions. Diphenylamine, an inhibitor of certain carotenoids, had no effect on photoactivation of oospores. The data suggest that the photoreceptor pigment for activation of oospore germination is a flavin.     

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.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

Agarose Medium for Germination of Oospores of Phytophthora cactorum

When oospores of Phytophthora caetorum from 30-day-old culture were treated with 0.25% KMnO₄ for 20 min and incubated at 24°C under light for 10 days, 65–75% germinated on water agar and water agarose but only 1–21% germinated on V-8 agar and S+L agar. Water agarose was selected because germinated oospores formed restrieted colonies on this medium that could be isolated easily. KMnO₄ treatment killed sporangia, chlamydospores and mycelial fragments present in oospore suspensions. Under the above conditions, approximately 44% of oospores from 10-day-old culture germinated and the optimum germination rate of about 75% was obtained when oospores reached about 20 days old.     

Optimising inoculum yield and shelf life of Plectosphaerella cucumerina,a potential bioherbicide for Cirsium arvense

Plectosphaerella cucumerina was identified as a potential bioherbicide for controlling Cirsium arvense in Canada and New Zealand. The current study evaluated production conditions using two isolates (one from each country) to determine whether the yield and shelf life of inoculum are suitable for mass production. Mycelial growth and sporulation in culture both increased from 15°C to 25°C and declined at higher temperatures with no mycelial growth at 37°C. The Canadian isolate produced fewer conidia than a New Zealand isolate. Potato dextrose-based liquid media with moderate to high concentrations of carbohydrates (25%, 50%, and 100%) maximised conidia production and these base media produced conidia with the highest germination rate (>80%) both at harvest and after 4 weeks stored at 4°C in 2.5% glycerol, 40% milk glycerol or after air drying. However, after 10-week storage, the conidia failed to germinate. Sporulation occurred during growth on all solid substrates tested (rice, rolled barley, and triticale), but conidial germination was highest on rice and barley, both before and after air drying. By contrast to conidia, 90% of mycelia-infested barley grains were viable after 3 years of storage at room temperature, although viability was lost by this time on the other substrates. This study has shown that the nutritional base is an important determinant of sporulation and shelf life for P. cucumerina. Although the yield of conidia in liquid medium was adequate to justify further development of P. cucumerina as a bioherbicide, improvement in its shelf life, or alternate formulation types that extend the shelf life, must be made for commercial efficiency.     

Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana

The formulation of mycopesticides may require a physical separation of conidia from the substrate and subsequent drying. In the present study, Beauveria bassiana conidia produced by solid-state fermentation were harvested either through a dry or washing protocol. Washed conidia were used to design a water-dispersible granule (WG) formulation, whereas sieved conidia were mixed with an emulsifiable oil to achieve an oil-based formulation (OD). Potential harmful effects caused by the formulation type on the storage stability and insecticidal activity against Hypothenemus hampei were assessed. As expected, the time for initial conidial germination to drop 50% (GT₅₀) in all treatments was deeply influenced by storage temperatures, which varied from over 180 days at 4 °C to less than 90 days at 35°C. In all four tested temperatures, GT₅₀s for unformulated dry conidia were significantly higher than for those formulated as WG, and the latter was similar to conidia formulated as OD in the two highest temperatures. Residual water content in the OD formulation (1,600 vs. 340?ppm) had a negative influence on conidial survival under storage, whereas WG granules immediately dried after the washing protocol showed conidial germination similar to granules exposed to a slower dehydration regime. Mortality of H. hampei adults exposed to different concentrations of B. bassiana formulated as WG was slightly lower (10–15%) than either the OD or the unformulated conidia. In brief, we have demonstrated that formulation type and their moisture level can affect the storage stability and insecticidal activity of B. bassiana conidia toward the coffee berry borer. Of particular importance, we have shown that drying oils prior to formulation could improve the storage of mycopesticides, an approach that may find industrial applications.     

Production of thermotolerant entomopathogenic Isaria fumosorosea SFP-198 conidia in corn-corn oil mixture

Low thermotolerance of entomopathogenic fungi is a major impediment to long-term storage and effective application of these biopesticides under seasonal high temperatures. The effects of high temperatures on the viability of an entomopathogenic fungus, Isaria fumosorosea SFP-198 (KCTC 0499BP), produced on different substrates amended with various additives were explored. Ground corn was found to be superior in producing the most thermotolerant conidia compared to yellow soybean, red kidney bean, and rice in a polyethylene bag production system. Using ground corn mixed with corn oil as a substrate resulted in only 7% reduction in germination compared to ground corn alone (67% reduction) after exposure of conidia to 50°C for 2 h. Corn oil as an additive for ground corn was followed by inorganic salts (KCl and NaCl), carbohydrates (sucrose and dextrin), a sugar alcohol (sorbitol), and plant oils (soybean oil and cotton seed oil) in ability to improve conidial thermotolerance. Unsaturated fatty acids, such as linoleic acid and oleic acid, the main components of corn oil, served as effective additives for conidial thermotolerance in a dosage-dependant manner, possibly explaining the improvement by corn oil. This finding suggests that the corn-corn oil mixture can be used to produce highly thermotolerant SFP-198 conidia and provides the relation of unsaturated fatty acids as substrates with conidial thermotolerance.     

Downstream processing of Beauveria bassiana and Metarhizium anisopliae-based fungal biopesticides against Riptortus pedestris: solid culture and delivery of conidia

We established a fungal production platform by focusing on substrates of solid culture for conidial productivity and thermotolerance, and focusing on surfactants to effectively deliver fungal conidia to the Riptortus pedestris cuticles. First, to produce thermotolerant fungal conidia, 2 of each Beauveria bassiana and Metarhizium anisopliae isolates were cultured on 13 cereal substrates for 10 days. Overall, five substrates (millet, non-glutinous Italian foxtail millet, barley, glutinous Italian foxtail millet, and brown rice) produced greatest number of conidia with thermotolerant conidia. When the selected substrates were mixed with minerals, zeolite, perlite or vermiculite to reduce the amount of cereal grains, vermiculite combination showed relatively high conidial production compared to the other mineral combinations. Next, to efficiently deliver the fungal conidia to the cuticles of R. pedestris, six surfactants, CO-2.5, CO-12, LE-7, PE-61, TED-3, and siloxane were each combined with the fungal conidia. The 0.01% combination showed significantly increased insect mortality, which varied depending on isolate. Virulence tests against R. pedestris were performed with conidial suspensions of isolates to assess their virulence. As a result, isolates showed the highest virulence when a virulence test was conducted at 25°C, rather than 20°C, 30°C and 35°C. This work suggests that the combination of cereal grain substrates and vermiculite could be considered for economic conidial production with high thermotolerance, and the CO-12 surfactant is the most suitable for effective delivery to target insects, followed by the information on optimal temperature for virulence against R. pedestris.     

Isolation of Oospores of Sunflower Downy Mildew, Plasmopara halstedii, and Microscopical Studies on Oospore Germination

A method was elaborated to isolate oospores of Plasmopara halstedii from tissue of its host, Helianthus annuus . Isolated oospores were studied microscopically and germination was documented with respect to the time course and the mode of germination. Formation of primary sporangia was similarly observed in oospores, harvested from 4- to 6-week-old systemically infected sunflower plants, grown under constant conditions at 16°C, as well as from field plants, harvested late in the season. Pretreatment of oospores with cold temperatures, previously assumed to stimulate the rate and to accelerate the speed of oospore germination, did not result in such effects. Germination usually occurred within 10–30 days of incubation at a highly variable rate of about 1 to 17% (average 6.7%) in deionized water.     

Effect of sunscreens,irradiance and resting periods on the germination ofMetarhizium flavoviride conidia

Chemical sunscreens were incorporated into oil formulations of conidia of two isolates of the entomopathogenic fungusMetarhizium spp. and the formulations exposed to simulated solar radiation. After 2 h exposure several sunscreens gave protection as demonstrated by conidial germination after 24 h incubation on gelatin plates at 26°C but only the formulations with Eusolex 8021 showed higher germination than the unprotected control after 48 h incubation. During 5h of exposure, Eusolex 8021 failed to offer significant protection as demonstrated by conidial germination after 48 h incubation. Conidial damage was proportional to the duration of radiation received. Allowing periods of darkness between exposures did not result in decreased loss of viability. Storing conidia, after exposure to simulated radiation, for 24 h prior to germination reduced their viability.     

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.     

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.     

Role of Respiration in the Germination Process of the Pathogenic Mold <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Inhalation of resting conidia is usually the first step of a systemic infection caused by the opportunistic fungal pathogen Aspergillus fumigatus. In the lung, the inhaled spores encounter an environment that permits germination. However, the relative importance of certain environmental conditions for conidial activation and subsequent hyphae formation has so far not been analyzed in detail. In this study, we studied the role of oxygen during germination. We found that inhibitors of the respiratory chain were nearly as efficient in blocking germination as cycloheximide, an inhibitor of protein synthesis, which is already known to prevent germination of Aspergillus nidulans. We also found that A. fumigatus is unable to grow or germinate under anaerobic conditions, and using the fluorescent mitotracker dye we detected active mitochondria already at the stage of swollen conidia, which indicates that respiration is an early event during germination. In line with these data, we found that significant oxygen consumption was detectable early during germination, whereas no oxygen consumption was measurable in suspensions of resting conidia. In summary, the present study provides evidence that respiration is absolutely required for the germination of A. fumigatus conidia. Anela Taubitz and Bettina Bauer contributed equally.     

Factors influencing pathogenicity of Fusarium tumidum on gorse (Ulex europaeus)

Factors promoting pathogenicity of Fusarium tumidum on gorse (Ulex europaeus) were determined to develop a novel strategy for delivering this potential mycoherbicide using insects as vectors of inoculum. Fusarium tumidum sprayed as a suspension of 1×10⁶ conidia mL^?1 on at least 50% of a gorse plant reduced shoot dry weight by 45% (P<0.05). A minimum of 910 viable conidia were required to cause a lesion on leaves. The leaves and flowers were more susceptible to infection than stems, spines and pods. Generally, wounding of gorse leaves and stem increased F. tumidum infection, most likely through releasing nutrients that enhanced conidial germination and hyphal growth. We showed in a separate experiment that conidial germination (93%) and germ tube length (407 µm) were greater when incubated in 0.2% gorse extract solution for 24 h than in water (62% germination, germ tube length 42 µm). Inoculation of gorse with a F. tumidum conidial suspension supplemented with 0.2% gorse extract resulted in a shoot dry weight reduction (P=0.012) equivalent to that of plants that were wounded and inoculated. It is concluded that wounding of older tissues (which mimics insect damage) is required to facilitate F. tumidum infection of mature gorse plants.     

CYTOLOGICAL INVESTIGATIONS OF KABATIELLA CAULIVORA

Cole , Herbert , Jr ., and Houston B. Couch . (Penn. State U., University Park.) Cytological investigations of Kabatiella caulivora. Amer. Jour. Bot. 46(1) : 12-16. Illus. 1959.—Initial growth of K. caulivora on artificial media is characterized by budding, yeast-like, conidia, exclusive of mycelia. After 14 days growth, at 20°C., mycelial growth becomes macroscopically evident. A study of the germination behavior of a total of 5500 conidia from the initial yeast-like growth stage showed 59 to germinate by the production of mycelia, while the balance germinated by budding. Five thousand conidia of the mycelial growth type were studied in a successive, single-spore transfer series, and, in all cases, conidial germination continued to be by means of germ tubes. Conidia of both growth forms were studied by means of bright field and phase contrast microscopy, and found to be multinucleate—possessing variable nuclear numbers, ranging from 1-8 per cell. Mean nuclear number for both the mycelial and conidial types was 2.8. All nuclei of both growth types appeared to contain the same chromosome complement. The cultural variability exhibited by K. caulivora cannot be reconciled with the concept of dual phenomenon. It is suggested, rather, that the mycelial homotype probably arises as the result of a unidirectional mutation within the conidial growth form.     

Survival, Germinability and Infectivity of Oospores of Peronospora viciae f.sp. fabae

A series of experiments was conducted to germinate oospores of Peronospora viciae f.sp. fabae. With rare exceptions, dry-stored oospores did not germinate in water nor did they infect faba bean seedlings in soil. Long-term storage, pre-treatment with KMnO₄ or addition of nutrients to the medium did not induce germination. Survival and infectivity of dry-stored oospores were compared to those of oospores incorporated in a silt loam and a loamy sand soil in the field during 21–22 months. Under dry conditions, the percentage of living oospores did not change as determined by the vital stain tetrazolium bromide. In soil, less than 2% of the oospores had survived after 21 months. Infectivity of oospores was determined by a bioassay 17 and 21 months after oospores had been incorporated in soil. Diseased seedlings were obtained after inoculation of faba bean seeds with oospores extracted from the soil but not with the drystored ones. Soil samples from two field plots naturally infested with oospores 2 and 3 years before the bioassay were infective. Oospores collected with diseased plant material on one of these plots and subsequently stored dry for 3 years were not infective. The results suggested that oospores need a period of natural weathering to become germinable and infective.