Effects of the timing of applications on the incompatibility of three fungicides and one isolate of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina)

Abstract: The effect of timing of application on the incompatibility of three fungicides (metalaxyl, mancozeb, copper oxide) and isolate MK2001 of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was assessed in vitro . Isolate MK 2001 at 1 × 10⁷ conidia/ml concentration was highly pathogenous to adults of Lygus lineolaris (Hemiptera) (84% mortality at day 4 post-treatment). The fungicides at the manufacturer recommended rate (1X) were strongly fungistatic and inhibited the fungal isolate radial growth on Sabouraud dextrose agar (SDA). Mancozeb, metalaxyl and copper oxide exhibited insecticidal activity of 24, 40 and 48% respectively, on L. lineolaris adults. It is clear that application of the fungal isolate MK2001 2–4 days before applying fungicides metalaxyl, mancozeb or copper oxide synergized the insecticidal effect of the isolate. On the contrary, application of metalaxyl, mancozeb, copper oxide 2–4 days before applying isolate MK2001 antagonized the insecticidal effect. The simultaneous use of each fungicide (metalaxyl, mancozeb or copper oxide) and the isolate gave lesser insect mortality. This study revealed that although some fungicides are incompatible for use alongside fungal isolates, the proper evaluation of their time of use could be beneficial in biological control or IPM programmes. Furthermore, the application of B. bassiana isolate MK2001 followed by fungicide application could synergize insecticidal activity.     

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).      

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Entomopathogenic fungi in predatory beetles (Col.: Carabidae andStaphylinidae) from agricultural fields

Prevalence of entomopathogenic fungi was studied in overwintering ground beetles (Col.: Carabidae) and rove beetles (Col.: Staphylinidae) collected from fields of lucerne, white cabbage and white cabbage undersown with white clover. In general infection levels in adult ground beetles and rove beetles were low (Carabidae: max. 7.6%,Staphylinidae: max. 7.0%). In comparison, prevalence of entomopathogenic fungi in carabid larvae was high (19–50%). At one study site an epizootic ofBeauveria bassiana was observed, infecting 67% of the staphylinidAnotylus rugosus and 37% of the staphylinidGyrohypnus angustatus. Beauveria bassiana was the predominant fungus isolated from ground beetles and rove beetles from all studied sites. Other fungal species included the hyphomycetesMetarhizium anisopliae, Paecilomyces farinosus andVerticillium lecanii as well asZoophthora radicans andZoophthora philonthi (Zygomycetes: Entomophthorales). Two individuals ofAnotylus rugosus were found to have a dual infection ofZoophthora philonthi andBeauveria bassiana.

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Résumé La présence de champignons entomopathogènes a été recherchée chez des carabes (Col.: Carabidae) et des staphylins (Col.: Staphylinidae) récoltés dans des champs de luzerne, de chou et d'une culture mélangée chou-trèfle blanc. Chez les adultes, le taux d'infection dans les deux groupes d'insectes est en général très faible (Carabidae: max. 7,6%, Staphylinidae: max. 7%). Chez les larves de carabes par contre, le taux d'infection par les champignons est élevé (19–50%). Sur l'un des sites de l'étude, une épizootie àBeauveria bassiana a été observée, l'infection portant sur 67% des individus récoltés appartenant à l'espèceAnotylus rugosus et 37% desGyrohypnus angustatus. B. bassiana est le champignon prédominant isolé à partir des coléoptères de tous les sites étudiés. D'autres espèces fongiques ont été relevées: ce sont les hyphomycètesMetarhizium anisopliae, Paecilomyces farinosus etVerticillium lecanii ainsi queZoophthora radicans etZoophthora philonthi. Deux individus d'A. rugosus ont été retrouvés infectés à la fois parErynia etB. bassiana.

     

Natural levels of fungal infections in grasshoppers in Northern Benin

The infection of grasshoppers by naturally occurring, entomopathogenic fungi was monitored at two sites in Malanville, northern Benin, Africa. Grasshoppers were collected and recorded from the sites between June and December 1992 and all of them, barring the first instars, were incubated in ventilated cages. At the first site, 1343 individuals of 35 grasshopper species were incubated, and at the second site, 857 individuals of 36 grasshopper species were incubated. Three hyphomycete fungi (Deuteromycotina: Hyphomycetes), Metarhizium flavoviride Gams and Rozsypal, Beauveria bassiana (Bals.) Vuillemin and Sorosporella sp. were found infecting grasshoppers. The average incidence of M. flavoviride infection was 2.9% and 1.8% at the two sites for all host species. M. flavoviride sporulated on most grasshopper cadavers within 10 days of collection. B. bassiana and Sorosporella sp. were only collected from one and five grasshopper individuals respectively. A significant difference was noted in the time to death of small grasshopper species infected with M. flavoviride compared to larger species. At one site, M. flavoviride infection was positively correlated with rainfall during the 10‐day period in which samples were taken.     

Effects in vitro of nine fungicides on growth of entomopathogenic fungi

The effects of nine fungicides were evaluated in vitro on the entomopathogenic fungi Beauveria bassiana, Conidiobolus coronatus, C. thromboides, Metarhizium anisopliae, Paecilomyces farinosus, P. fumosoroseus, Scopulariopsis brevicaulis and Verticillium lecanii. The susceptibility of the fungi to the fungicides varied. The dithiocarbamate derivations zineb + copper oxychloride, and mancozeb completely inhibited germination of C. coronatus, C. thromboides, B. bassiana, P. farinosus, M. anisopliae and V. lecanii. The fungicides triadimefon, copper oxychloride, metalaxyl, sulfur, sulfur + nitrothal‐isopropyl and hymexazol exhibited various effects on the fungi. Usually, fungistasis was more pronounced at 15°C than at 25°C and the inhibitory effects were in direct proportion to the dosage of active ingredient (recommended field rate, 10‐fold higher and 10‐fold lower). In a few combinations, fungi partially overcame or even recovered from the initial inhibition of growth which might have resulted from delayed germination. In other cases, inhibition of growth occurred only after a delay and its intensity increased with time. Stimulation of fungal growth when it occurred was rarely permanent. Generally, adverse effects were much greater against the entomophthoraleans C. coronatus and C. thromboides than against the Hyphomycetes. Extrapolation of the results to practice and to the field situation is difficult. Nevertheless, the data presented give an idea of possible side‐effects in nature.     

Studies on Prolonged Storage of Beauveria bassiana Conidia: Effects of Temperature and Relative Humidity on Conidial Viability and Virulence against Chickpea Borer,Helicoverpa armigera

Unformulated conidia of Beauveria bassiana were stored at five different temperatures (0°, 10°, 20°, 30° and 40°C) at six different relative humidities (RH) (0, 33, 53, 75, 85 and 98%). Conidial viabilities and virulence against third instar larvae of Helicoverpa armigera were determined over a 24‐month period. Conidia survived longest at lower temperatures (0–20°C) and lower RH levels (0–53% RH). At higher temperatures (30–40°C) conidia did not survive. When the temperature was decreased from 30°C to 0°C, at nearly all RH levels the longevity of conidia increased. Conidia remained virulent for third instar larvae of H. armigera under favourable storage conditions for 24 months.     

28s rDNA group-I introns: a powerful tool for identifying strains of Beauveria brongniartii

The nuclear ribosomal DNA of the entomopathogenic fungus Beauveria brongniartii is polymorphic in terms of both restriction site and length. Insertions of 350–450 bp long, identified as group-I introns, were detected in the 28 s rDNA. A panel of 47 strains of B. brongniartii , two B. bassiana and one Metarhizium anisopliae of various geographical and biological origins were found to contain 14 variant forms of intron differing in size and restriction pattern, at four different positions. Twelve types of ribosomal large subunit were defined on the basis of variant distribution and compared with strain clustering based on internal transcribed spacers analysis. There was a correlation between the characteristic introns and isolates collected from the sugar cane pest Hoplochelus marginalis . Primers for polymerase chain reaction amplification were chosen from these variants, and used to develop a specific method for detecting strains pathogenic towards Hoplochelus .     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.