Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

Some factors influencing spore germination and penetration of Alternaria longipes

The ranges over which the germination of conidia of Alternaria longipes was > 50% were 10–35 °C on agar and 15–30 °C on tobacco leaf disks. Germination was optimal at 22.5 °C; so was germ-tube growth, reaching c. 300 and 102 μm on agar and leaf disks respectively after 12 h. On average, 27% more conidia germinated and germ-tubes were 62% longer on disks from leaves washed for 5 min under running water than on disks from unwashed leaves. At controlled saturation deficits germination after 8 h at 1.1 and 2.3 mb was 42.3 and 9.3% respectively and the rate of germ-tube growth was < 0.8 μm/h, compared with 94.4% and 8.3 μm/h in standing water. These results, together with some field data, suggests that germination in the field is largely restricted to periods when free moisture is present on leaves. In Malawi, leaf temperatures and the duration of dew at night were adequate to allow germination and penetration in the absence of rain. Pollen, when applied with the inoculum, had little effect on the number of germinated conidia, but caused a c. tenfold increase in the number of successful penetrations.     

Microbial Oxidation of Cephalosporins to Cephalosporin Sulfoxides

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.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidiogenesis.

A cultivation system has been developed for Penicillium urticae which yields 'microcycle' conidiation in submerged culture. Spherical growth of spores was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged spores to a nitrogen-poor medium at 35 degrees C results in synchronous germination and limited outgrowth followed by roughly synchronous conidiation. A study of the conidiation stage showed that a phialide and an immature conidium began to form at the tip of all germ tubes 18 h after the temperature shift. By 24 h additional phialides commonly appeared as a branch near the tip of the germ tube and the more mature conidia exhibited increasing refractility. The earliest ultrastructural signs of conidiation were various round invaginations in the plasma membrane and a thickening and rounding of the new spore wall which appeared as an inner extension of the phialide cell wall. Upon segregation of the conidium from the phialide cell by conidial wall formation, 'trench-like' invaginations gradually appeared in the plasma membrane and a disorganized rodlet pattern was formed on the outer surface of the maturing conidial wall. Continued maturation involved the formation of chains of conidia and phialide senescence which was characterized by a general degradation of intracellular structure. A comparison with standard surface and submerged culture conidiation indicated that 'microcycle' conidiation, while less prolific, was essentially identical.     

Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates

On agar media the maximum conidia production of Exserohilum monoceras occurred on V-8 juice agar (VA) or centrifuged V-8 juice agar, whereas the optimal radial mycelial growth occurred on Czapek-Dox agar. The optimal temperatures for radial mycelial growth and conidia production were 28 and 27°C respectively. Light prohibited E. monoceras conidia production. The best sporulation occurred under continuous dark conditions. Echinochloa leaf decoction significantly increased conidia production on potato dextrose agar (PDA) and VA, and significantly increased germ tube length on PDA, lima bean agar and VA, but did not affect conidia germination. No conidia were produced in liquid media. Of 22 agricultural-based products evaluated as solid substrates, the most abundant sporulation (1.8 × 10⁶ conidia g^-1 of dry weight) occurred on corn leaves. The conidia production of E. monoceras on corn leaves was affected by incubation period, moisture content and substrate quantity. There were no differences in germination rate, germ tube length and virulence of conidia produced on agar media or corn leaves.     

Variability in conidial thermotolerance of Metarhizium anisopliae isolates from different geographic origins

Notable variability in thermotolerance was found among conidia of 16 isolates of the insect-pathogenic fungi Metarhizium anisopliae var. anisopliae and one M. anisopliae var. acridum isolated from latitudes 61 degrees N to 54 degrees S. Conidial suspensions were exposed to 40 or 45 degrees C for 2, 4, 8, and 12 h. Most of the isolates tolerated 40 degrees C very well, with relative germination (germination relative to unheated controls) above 90% after 12 h of exposure. Exceptions were three isolates originating from high latitude, viz., ARSEF 2038 (38 degrees N, South Korea), 4295 (54.4 degrees S, Australia), and 5626 (61.2 degrees N, Finland) that had approximately 80% germination. High variability, however, was observed among isolates at 45 degrees C; viz., after 2 h exposure, relative germination was above 80% for six isolates, between 50 and 70% for three isolates, and between 0 and 30% for eight isolates. After 8 and 12 h at 45 degrees C, only two M. anisopliae isolates pathogenic to grasshoppers, viz., ARSEF 324 (latitude 19 degrees S, Australia) and 3609 (15 degrees N, Thailand), had high relative germination (91.6 and 79.4%, respectively, for 8 h exposures; and 90 and 47.1%, respectively, for 12 h). These isolates also were the most tolerant to UV-B radiation [J. Invertebr. Pathol. 78 (2001) 98-108]. The median lethal dose (LD50) for isolate ARSEF 324 was 49.4 and 47.9 degrees C, for 2 and 4 h of exposures, respectively. Exposure of conidia to wet-heat greatly delayed germination of some isolates. In general, isolates from higher latitudes demonstrated greater heat susceptibility than isolates from nearer the equator. Dry conidia tolerated 50 degrees C better than 45 degrees C in aqueous suspension.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidial germination and outgrowth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of conidia was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged conidia to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiogenesis. An ultrastructural study of the germination stage indicated nuclear migration into the emerging germ tube whose new cell wall was an extension of the parent conidium's innermost cell wall layer. Septal formation at the neck of the germ tube followed. The septal pore was filled with particulate material and the septal membranes possessed unusual linear elements in their median hydrophobic zones. The germ tube, which possessed a smooth-surfaced plasma membrane, continued to elongate with periodic septum formation. The parent conidium and later the proximal germ tube showed progressive vacuolation and the cytoplasm became largely occupied by electron-translucent material. In older cells the septal pore was blocked by Woronin bodies. Compared with normal conidial germination this microcycle' germination is far more synchronous and the resultant germling is morphologically simpler. In ultrastructural terms, however, germination appears to be identical with that obtained at 28 degrees C.     

A novel computerised image analysis method for the measurement of production of conidia from the aphid pathogenic fungus Erynia neoaphidis

A semi-automated method has been developed for the quantification and measurement of conidia discharged by the aphid pathogen Erynia neoaphidis. This was used to compare conidiation by E. neoaphidis-mycosed pea aphid cadavers, mycelial plugs cut from agar plates, mycelial pellets from shake flasks and by mycelial pellets from different phases of liquid batch fermenter culture. Aphid cadavers discharged significantly more and significantly smaller conidia than plugs or pellets. The volume of conidia discharged was stable over the period of discharge (80 h), but more detailed analysis of the size frequency distribution showed that more very small and very large conidia were discharged after 5 h incubation than after 75 h incubation. Biomass harvested at the end of the exponential growth phase in batch fermenter culture produced significantly more conidia than biomass from any other growth phase. The implications of these findings for the development of production and formulation processes for E. neoaphidis as a biological control agent are discussed.     

Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates

Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY. Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from artificial substrates showed that conidia produced on Czapek's and Emerson's YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

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

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

Behavior of Vibrio cholerae in hot foods.

Four food types held hot at 45 to 60 degrees C were deliberately contaminated with O1 and non-O1 Vibrio cholerae strains. These organisms were assayed for survival and recovery from the foods within 1 h of the time the food was kept hot. The results showed no growth of V. cholerae non-O1 on thiosulfate-citrate bile-sucrose agar plates after 24 h of incubation at 37 degrees C for food held hot at 50 to 60 degrees C. Growth was low for V. cholerae O1 and was not achieved in some instances in which foods were held at either 55 or 60 degrees C after 40 or 60 min of from the time the food was kept hot. Both organisms, however, were recovered equally from all food types held at all temperatures after 48 h of incubation. When incubated for an additional 24 h, the organisms grew to unusually small-sized colonies, measuring 0.1 to 0.3 mm in diameter, on the same agar plates that were negative for growth after an initial 24 h of incubation. It was concluded that V. cholerae survived the period of time held at hot temperatures. Although the organisms were not recovered from some foods when held at some of the temperatures and times after 24 h of incubation, they remained viable. An incubation period of 48 h at 37 degrees C was found to be appropriate for the recovery of V. cholerae from hot foods.     

Behavior of Vibrio cholerae in hot foods

Four food types held hot at 45 to 60 degrees C were deliberately contaminated with O1 and non-O1 Vibrio cholerae strains. These organisms were assayed for survival and recovery from the foods within 1 h of the time the food was kept hot. The results showed no growth of V. cholerae non-O1 on thiosulfate-citrate bile-sucrose agar plates after 24 h of incubation at 37 degrees C for food held hot at 50 to 60 degrees C. Growth was low for V. cholerae O1 and was not achieved in some instances in which foods were held at either 55 or 60 degrees C after 40 or 60 min of from the time the food was kept hot. Both organisms, however, were recovered equally from all food types held at all temperatures after 48 h of incubation. When incubated for an additional 24 h, the organisms grew to unusually small-sized colonies, measuring 0.1 to 0.3 mm in diameter, on the same agar plates that were negative for growth after an initial 24 h of incubation. It was concluded that V. cholerae survived the period of time held at hot temperatures. Although the organisms were not recovered from some foods when held at some of the temperatures and times after 24 h of incubation, they remained viable. An incubation period of 48 h at 37 degrees C was found to be appropriate for the recovery of V. cholerae from hot foods.     

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.     

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.     

Sporulation by Entomophthora schizophorae (Zygomycetes: Entomophthorales) from housefly cadavers and the persistence of primary conidia at constant temperatures and relative humidities

The duration of discharge of Entomophthora schizophorae (Zygomycetes: Entomophthorales) conidia from house fly (Musca domestica, Diptera) cadavers was measured at 7, 18, and 25 degrees C. The higher the temperature, the shorter the duration of conidia discharge. Significantly more conidia were produced per cadaver at 7 degrees C over a period of 120 h than at 18 and 25 degrees C. At 25 degrees C, the initial discharge over the first 10 h was much larger than at the other temperatures, and at 7 degrees C, no peak in discharge was observed. The persistence of E. schizophorae primary conidia was measured on fabricated non-host surfaces typically found in stables (straw, wood, plaster, and glass) at 7, 18, and 25 degrees C or constant relative humidities of 45, 65, and 85%. Persistence, as measured by the subsequent ability to infect flies, was usually only a few days and depended on the temperature and type of surface. It was greatest on straw, followed by wood, glass, and plaster, and at 7 degrees C, followed by 18 and 25 degrees C. Limited transmission took place between flies exposed to conidia and previously unexposed mates.     

Variability in thermal responses among Furia gastropachae isolates from different geographic origins

The effect of temperature ranging from 5-30 degrees C on in vitro vegetative growth and conidial germination of isolates of the entomophthoralean fungus Furia gastropachae was investigated. Eleven isolates were used for growth studies; two from Maryland, six from New York, and three from Ontario. A subset of four isolates, one each from Maryland and New York and two from Ontario, were used in conidial germination experiments. Growth and germination were significantly associated with temperature for all isolates, occurring throughout the range 5-30 degrees C, though both processes were inhibited to varying degrees at upper and lower extremes. Temperature optima for growth ranged from 20 to 27 degrees C, and for germination from 20 to 25 degrees C. Although significant variability was observed among isolates in growth at temperatures above 13 degrees C, temperature optima were not significantly different among isolates, and variability did not appear to relate to the geoclimatic origins of the isolates. In contrast, germination responses to temperature did appear to be related to geographic origin. Furia gastropachae isolates from New York and Maryland germinated more slowly at 10 degrees C than did Ontario isolates, although the percentage of conidia ultimately germinating at each temperature was the same for all isolates. The New York and Maryland isolates performed much better at 30 degrees C, with significantly greater overall germination and secondary conidial discharge, than the Ontario isolates. Compared with other isolates at 30 degrees C, Ontario isolates were the least active, often failing to successfully discharge any secondary conidia.     

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.     

A simple method for counting Staphylococcus aureus in swimming pool water

Staphylococcus aureus counts from swimming pool water were determined by the membrane filtration technique. Water samples were passed through a membrane filter and then put on Baird-Parker media. After incubation, the filters were transferred to nutrient agar, and incubated at 37 degrees C, for 3 h. After removal of the filters, the plates were incubated at 60 degrees C for 2 h. An overlay of toluidine blue agar was added and the plates reincubated for 4 h at 37 degrees C. The formation of thermonuclease correlated with the formation of coagulase, and the results indicated that Staphylococcus aureus could be present in swimming pool water without the presence of either coliform or faecal coliform bacteria.