Hirsutella thompsoniU a fungal pathogen of mites. II. Host-pathogen interactions

The moniliaceous fungus, Hirsutella thompsoniU grown on potato-dextrose agar (PDA) or on sterile wheat bran, was highly pathogenic to the carmine spider mite, Tetranychus cinnabarinus, and, grown on PDA, to the oriental spider mite, Eutetranychus orientalis. The fungus penetrated the mites' integument mainly through the legs and formed hyphal bodies in chains in the haemolymph. Hyphae, on which the spores were produced, began to emerge through the genital and anal apertures and then all over the body. Various mycophagous mites, a predaceous species and the tick Argas persicus were immune. H. thompsonii grew well on cadavers of diverse insects and mites. The fungus killed most mites and quickest, usually by the 2nd day, at 25^o, 27^o and 30 ^oC; least at 13^o and 35 ^oC. It sporulated best on mites at 24^o, 27^o and 30 ^oC (full sporulation at 27 ^oC took place within 12 h after death); it was good, but slower, at 13^o and 35 ^oC. The fungus germinated, penetrated mites and sporulated there very poorly below 100% r.h. When the daily number of hours at 100% r.h. was reduced from 24 to 18 or 6 h, fungus-associated mortality dropped greatly. In the field, use of the fungus for mite control would be suitable particularly in tropical and humid subtropical areas, and its success in Israel would not be assured. In glasshouses, H. thompsonii could induce epizootics in Israel and elsewhere only if humidities were raised to saturation and the daytime temperature kept below 37 ^oC.     

Susceptibility of Brevipalpus phoenicis to entomopathogenic fungi

The pathogenicity of 52 isolates from several fungus species was studied for the false spider mite Brevipalpus phoenicis. In addition, the main stages during the course of infection by Hirsutella thompsonii, by far the most virulent pathogen, were studied by means of light and electron microscopy. Adult mites were confined to arenas prepared with citrus leaves in acrylic dishes containing agar–water. Conidial suspensions containing 10⁸ conidia/ml were applied, except for H. thompsonii, where a concentration of 10⁷ conidia/ml was used. The H. thompsonii isolates caused higher mortality, with indices higher than 90%. Observations under the scanning electron microscope (SEM) were performed at 0, 6, 12, 24, 48, 72, and 120 h after application of a H. thompsonii suspension containing 10⁷ conidia/ml. Twenty-four hours after inoculation, H. thompsonii conidia were observed attached to the mite’s integument. The conidia germinated and penetrated through the base of the setae on the hysterosoma. Colonization occurred after 48 h, as evidenced by mortality. Conidiogenesis occurred after 120 h, with the development of mycelium and conidiophores emerging from the posterior and anterior parts of the mite.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Physiological effects of nanosilver on vegetative mycelium,conidia and the development of the entomopathogenic fungus,Isaria fumosorosea

An assessment of the influence of water nanosilver suspension was made at a concentration of 10 mg·L^?1 on biological material (i.e., vegetative mycelium and conidia of Isaria fumosorosea entomopathogen) on a background of the silver nitrate ionic form used. Conidia of I. fumosorosea treated with silver nitrate for more than 168 h were completely deactivated. The application of nanosilver on Isaria hyphae resulted in a quantitative limitation of mycelium growth and its weaker sporulation after culturing compared to the control. The pathogenicity of a conidial suspension obtained from such culturing towards test insects did not diverge from that observed in the standard culture. No obvious toxic effects of nanosilver were observed on I. fumosorosea conidia. Isaria conidia, after exposure to nanosilver over a period between 1 and 800 h, initially demonstrated weaker vegetative mycelium formation in culture on solid medium and, as a consequence, this mycelium often sporulated in a poorer manner. In one case, there was a significant stimulation of the sporulation process for nanosilver treatment before culture for 168 h. Concurrently, conidial suspensions obtained from the culture after exposure of over 168 h to nanosilver exhibited enhanced pathogenicity towards test insects, which may be considered a beneficial phenomenon in terms of the function played by Isaria in the whole environment. The reaction of conidia with nanosilver indicates the deactivation of conidia cells in suspensions and a possibility of selection in increased pathogenicity.     

Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Efficacy of Exserohilum longirostratum on barnyard grass (Echinochloa crus-galli spp. crusgalli) under field conditions

An isolate of Exserohilum longirostratum obtained from Rottboelia cochinchinensis in Malaysia was highly pathogenic to barnyardgrass (Echinocloa crus-galli,) a serious weed in rice fields in Malaysia. In glasshouse trials, high levels of barnyardgrass control were achieved when E. longirostratum was applied as a conidial concentration at 10⁵ conidia/mL. This conidial concentration may not be sufficient for field use; therefore, a mini plot trial was carried out to assess the bioherbicidal potential of the fungus and the efficacy of different inoculum types under field conditions. Excellent barnyard grass disease as indicated by Area Under Disease Progress Curve (AUDPC) was achieved with mycelium (AUDPC = 583.8 unit²); mycelium + pretichlaclor (AUDPC = 610.4 unit²) and conidia alone (AUDPC = 468.3 unit²) compared to conidia + pretichlaclor (AUDPC = 395.8 unit²). Although conidia caused lower disease severity on barnyard grass compared to mycelium, this inoculum equally reduced the competition from barnyard grass as indicated by lower mean dry weight and fewer barnyard grass tiller numbers. Positive correlations between AUDPC of rice and its tiller number and AUDPC of rice and its dry weight were recorded while negative correlations were observed for the AUDPC of barnyard grass and its tiller number and AUDPC of barnyard grass and its dry weight. These findings confirmed that E. longirostratum applied either in the form of conidia or mycelium was effective against barnyard grass under field conditions.     

Survival of Neozygites cf. floridana (Zygomycetes: Entomophthorales) in mummified cassava green mites and the viability of its primary conidia

The survival of Neozygites cf. floridana (Weiser and Muma) as dry hyphal bodies in mummified cassava green mites, Mononychellus tanajoa (Bondar), at 5.0% RH in the dark was affected by storage temperature. Survival of the fungus in mummies kept at 24±1.0°C could be demonstrated for 6–7 months. When stored at 4°C, the fungus sporulated from 90% of the mummies liberating an average of 186.9 primary conidia per mummy even after a storage period of 16 months, when the experiment was terminated. The temperature, humidity and light condition significantly affected the viability of primary conidia. The percent viability across all factors dropped from 98.4% after 0 h (beginning of the experiment) to 23.4% after a 1 h exposure to the conditions tested. Lower temperatures maintained higher viabilities with 86.3% of the conidia surviving after 18 h at 18°C, whereas almost all conidia died after 12 h at 33°C. Conidia survived less than 1 h when exposed to SDs (saturation deficit) of 2.0 mm Hg or higher at any tested temperature.     

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.     

Stemphylium botryosum f. lactucae- its developmentjn vifro and its pathogenicity to lettuce leaves

Stemphylium botryosum f. lactucae, incitant of a leaf-spot disease of stored lettuce, was found to be relatively restricted in its host range. Cross-inoculations with spore suspension of this fungus failed to induce symptoms in any of the host plants tested, except carrot. Among isolates of S. botryosum from various hosts, only the isolate from carrot induced slight symptoms on lettuce. While mycelial growth of the lettuce isolate was confined to the range 13–37 ^oC spores germinated at more extreme temperatures. The optimum temperature for germination and for radial growth on PDA was found to be between 25 and 30 ^oC. Wet spores were quickly inactivated at 50 ^oC, whereas more than 40 % of dry spores withstood a 24 h exposure to that temperature. Only the outer leaves of lettuce responded readily to inoculation with a spore suspension, the required incubation period being 3 days at 25 ^oC. Symptoms developed less readily on bruised leaves. Relative humidity approaching saturation was necessary for prompt and typical infection, notably during the 24 h following inoculation. Short dry periods (60 % r.h.) interposed at a later stage, while somewhat inhibitory, did not prevent infection.     

The enhanced infectivity of Metarhizium flavoviride in oil formulations to desert locusts at low humidities

Formulation in non-evaporative diluents such as oils is required to exploit the advantages of controlled droplet application methods for biopesticides based on conidial suspensions of entomopathogenic fungi. In laboratory assays on desert locusts (Schistocerca gregaria), formulations of Metarhizium flauoviride conidia in cotton seed oil showed superior performance to water-based suspensions and this was especially pronounced at low humidities (35% r.h.). The LD₅₀s for oil and water suspensions at 5 days were 8900 and > 10⁶ conidia/insect respectively. The dose-mortality regression line for oil was more than four times steeper and median lethal times were typically 76% of those for aqueous suspensions. Formulation in oil improves the efficacy of this fungus and may extend the application of species with lipophilic conidia into less humid agricultural environments.     

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.     

Production of Conidia by Botrytis fabae grown in vitro

Conidiation in Botrytis fabae was stimulated by irradiating 1 to 3 day old, but not 4 to 5 day old mycelium. Three cycles of 12 h irradiation + 12 h darkness stimulated the production of about twice as many spores compared with only 12 h irradiation. At 18°C all the spores had been produced within 3 days but not within 2 days from the start of irradiation. Near-u.v. irradiation at wavelengths of 375–400 nm induced most sporulation. Red light at 600–650 nm also stimulated conidiation but irradiation at other wavelengths from 300 to 700 nm was ineffective. Fewer conidia were produced when the fungus was kept in darkness at 4°C between periods of irradiaton at 18°C compared with continuous 18°C. The optimum osmotic potential of the culture, medium for conidiation was about-27 bar although more mycelium grew at even lower osmotic potentials. Abundant spore production occurred when the fungus was grown in media with a wide range of pH values.     

In vitro-Untersuchungen und mikroskopische Studien zur Entwicklung von Phoma chrysanthemicola Hollos f. sp. chrysanthemicola, dem Erreger einer Wurzel- und Stengelgrundfäule der Chrysantheme

In vitro investigations and microscopic studies about the development of Phoma chrysanthemicola Hollos f. sp. chrysanthemicola, the causal pathogen of a collar and root rot of chrysanthemums Different media were tested for cultivation of Phoma chrysanthemicola. The pathogen grew best on PDA and carrot-Agar at a temperature of 25°C. The three strains used showed many variations, concerning the structure and colour of the mycelium. Fructification has been promoted by irradiation (ultra-violer ray). The morphogenesis, especially the formation of pycnidia, conidia and sclerotial structures has been examined with help of the scanning electrone microscope (SEM), results are presented., So far unknown organs probably belonging to the fungus were observed, their identity is still not clear.     

LABORATORY SUSCEPTIBILITY OF PLUTELLA XYLOSTELLA TO METARHIZIUM ANISOPLIAE AND NOMURAEA RILEYI*

Abstract Five isolates of Metarhizium anisopliae and one isolate of Nomuraea rileyi were bioassayed against larvae of Plutella xylostella. Larvae were treated by exposing cabbage leaf discs previously immersed in conidial suspension, and were then incubated at 25^oC and observed daily. One of M. anisopliae isolates, F11248, originally from Mastotermes darwiniensis was found being most virulent against P. xylostella. The LC₅₀ was estimated as 2.03 times 10⁴conidia/ml with 9 d mortality data, and the LT₅₀ was 4.97 d at concentration of 10⁷conidia/ml. Isolate F163 (N. rileyi) showed virulence to P. xylostella in both tests, but no cadavers sporulated.     

Reaction of <Emphasis Type="Italic">Bipolaris euphorbiae</Emphasis> to environmental factors and its tolerance to solar and ultraviolet radiation

The determination of the ideal cultivation conditions and the knowledge of solar radiation tolerance are important aspects that must be investigated for the use of Bipolaris euphorbiae Muchovej and Carvalho as a biocontrol agent. The present study was done to assess the growth rate, sporulation and viability of the fungus cultivated under different conditions of the initial pH value of the growth medium, temperature and photoperiod. The tolerance of this fungus’ conidia to light from a source simulating solar and ultraviolet radiation was also assessed. B. euphorbiae was affected by different pH values of the growth medium being 6.0 the adequate pH for fungus cultivation. Incubation of the fungus under temperatures of 22, 25 and 28°C enhanced mycelium growth, while sporulation and viability of conidia were better at 22 and 16°C. Different regimens of exposure to light of fungal cultures only affect sporulation. Conidia exposed to solar and ultraviolet radiation for 8 h and 90 min, respectively, presented viabilities higher than 92%, indicating high tolerance levels to radiation exposure.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).      

Time-concentration-mortality response of potato tuber moth pupae to the biocontrol agent Cordyceps tenuipes

ABSTRACT

The potato tuber moth (PTM) Phthorimaea operculella is a critical potato pest. Larvae infest both foliage and tubers and mature larvae pupate in the soil or other safe places. Cordyceps tenuipes, an entomopathogenic fungus, infect lepidopteran pupae. To determine the effectiveness of this fungus as a biocontrol agent for PTM, we evaluated the time-concentration-mortality (TCM) response of PTM pupae to C. tenuipes using the following bioassays: (1) direct immersion in conidial suspensions, (2) incubation in sterilised or (3) unsterilised soilpremixed with conidia, and (4) incubation in unsterilised soil drenched with conidial suspensions to simulate field conditions. Fungal infection caused 100%, 83.3%, 73.3%, and 85.0% mortality of PTM pupae in assays 1–4, respectively. At 10⁸ conidia/mL or conidia/g concentration, assays 1 and 4 had short lethal times (LT₅₀) of 2.2 and 2.6 days compared with 3.7 and 4.8 days for assays 2 and 3, respectively. On day 7 after inoculation, assays 1 and 4 also had low lethal concentrations (LC₅₀) of 1.69 × 10³ conidia/mL and 1.10 × 10⁵ conidia/g compared with those of assays 2 and 3, which showed low virulence, with LC₅₀ of 3.50 × 10⁵ and 3.60 × 10⁶ conidia/mL, respectively. Our results demonstrate that C. tenuipes is a promising candidate for PTM biocontrol at the pupal stage. Drenching the soil surface with conidial suspensions may be the most effective method of field application.     

The Biology of the Acaropathogenic Fungus Hirsutella kirchneri

The acaropathogenic fungus Hirsutella kirchneri (Rostrup) Minter , Brady and Hall grew best and produced most mycelia on a medium containing yeast extract , dextrose and agar; conidial production , however , was maximal or potato dextrose agar (PDA) . The best growth on both media was at 25 C and conidial germination was high within a wide range of temperatures (10 - 35 C) . Colony growth , mycelial mass production and conidial yield were best under alternating dark and light regimes . Maximal germination occurred under dark conditions . When grown in continuous light the fungus produced synnemata (compacted conidiophores) which remained viable for 22 weeks . Of six species of phytophagous mites assayed , three spider mites and a rust mite became infected by the fungus , as did , to a limited degree , a parasitic mite . Another four mites , including two pests , a scavenger and a predator , were unaffected . The fungus grew on and sporulated from heat - killed cadavers of a dead mealybug (Homoptera) . Conidial germination and penetration into live mites , under saturation condi tions , were affected little by temperatures . Intra - host growth was temperature dependent , with mite death beginning on the second or third day post - infection . Maximal sporulation from infected mites took place at 25 C . Mortality was quickest at 25 C when mites were held under saturation conditions , but some death also occurred at lower relative humidities . These data are discussed with a view to using H. kirchneri in the biological control of plant mites .     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

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.