Protection of entomopathogenic conidia against chemical fungicides afforded by an oil-based formulation

We evaluated the protection afforded by an oil formulation against non-compatible fungicides in mixtures with conidia of the entomopathogenic fungi Metarhizium anisopliae (Ma) and Beauveria bassiana (Bb). Under laboratory conditions, viability of unformulated (aqueous suspensions) Ma conidia was harmed by recommended label doses of carbendazim (not tested for Bb), and both Ma and Bb conidia were affected by triadimefon. On the other hand, effect of fungicides was usually nil or minimal on conidia formulated as oil-containing suspensions (emulsifiable oil + water). Germination rates for unformulated and oil-formulated Ma conidia subjected to carbendazim were reduced by 77.3 and 12.1%, respectively, compared to their fungicide-free counterparts. Germination rates at 16 h post-inoculation for unformulated and oil-formulated Bb conidia subjected to triadimefon were reduced by 20.5 and 5.5%, respectively, compared to their fungicide-free counterparts. No differences were observed at 20 h post inoculation, indicating a fungistatic action of this compound on Bb conidia. Virulence of unformulated conidia amended with fungicides against third instar Diatraea saccharalis larvae was negatively affected compared to their formulated counterparts. These results suggest that oil-formulated conidia can be effectively protected from damage caused by chemicals, which could have applications in tank mixing or alternate applications with shared spraying equipment, being especially relevant for IPM programs in which mycopesticides and chemicals are simultaneously sprayed.     

Evaluation of Isaria fumosorosea (Hypocreales: Cordycipitaceae) for control of the Asian citrus psyllid,Diaphorina citri (Hemiptera: Psyllidae)

A laboratory bioassay was developed to evaluate strains of Isaria fumosorosea Wize, against Diaphorina citri. Up to 100% of adult psyllids were killed at concentrations between 10⁶ and 10⁷ blastospores/ml after 12 days, with derived LC₅₀ values (at 7 days post treatment) between 1.4 × 10⁵ and 2.0 × 10⁶ blastospores/ml for strains ARSEF 3581, FE 9901 and Apopka-97. A significantly higher value (1.5 × 10⁷) was obtained with a conidial formulation of Apopka-97. Average survival times were dosage dependent, i.e. between 10.2 days at 10³ blastospores/ml and 3.5 days at 10⁸ blastospores/ml. Rates of mycosis were also dosage dependent, with up to 100% sporulation on cadavers at 10⁸ blastospores/ml but declining at lower concentrations. The Apopka-97 strain (commercially available as PFR-97) was tested against established D. citri infestations in potted citrus in greenhouse cages. Treatments at label rates reduced psyllid populations by approximately 50% over 3 weeks. The combination of PFR-97 with emulsifiable oils (0.25% v/v) did not increase psyllid mortality compared with either agent alone. Imidacloprid applied as a drench killed 100% of psyllids within 3 weeks. Subsequent greenhouse tests during humid conditions were hampered by natural dissemination of I. fumosorosea to untreated psyllids, suggesting that this fungus is spread by air movement and may be highly effective under very humid conditions. In later tests, a Cladosporium sp. rapidly colonised psyllid cadavers and leaf surfaces, but was not pathogenic in laboratory tests. Our studies confirm the potential of I. fumosorosea to be used in IPM strategies for D. citri that rely on other tactics, such as insecticidal oils and native or introduced biological control agents.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum, Part 1: Comparison of cell wall characteristics and drying stability among three spore types

Metarhizium anisopliae var. acridum (IMI 330189) can produce at least three spore types in vitro; blastospores, submerged conidia, and aerial conidia, as defined by culturing conditions, sporogenesis, and spore morphology. This study compares morphological characteristics (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), and performance (germination rate and drying stability) among these three spore types. Submerged conidia and aerial conidia both possessed thick, double-layered cell walls, with hydrophobic regions on their surfaces. However, in contrast to aerial conidia, submerged conidia have: (1) a greater affinity for the lectin concanavalin-A; (2) more anionic net surface charge; and (3) a less distinct outer rodlet layer. Blastospores were longer and more variable in length than both submerged conidia and aerial conidia, and had thinner single-layered cell walls that lacked an outer rodlet layer. Also, blastospores had a greater affinity than either conidia type for the lectin, wheat germ agglutinin. Blastaspores lacked hydrophobic regions on their surface, and had a lower anionic net surface charge than submerged conidia. In culture, blastospores germinated the fastest followed by submerged conidia, and then aerial conidia. Survival of submerged conidia and aerial conidia were similar after drying on silica gel, and was greater than that for blastospores. We provide corroborating information for differentiating spore types previously based on method of production, sporogenesis, and appearance of spores. These physical characteristics may have practical application for predicting spore-performance characteristics relevant to production and efficacy of mycoinsecticides.     

Fungal pathogens for arthropod pest control in orchard systems: mycoinsecticidal approach for pear psylla control

This paper reviews the research on entomopathogenic fungi in orchard systems and presents research on a mycoinsecticidal approach to an important pest of pear, the pear psylla. The review identifies the host-pathogen relationships that have been examined to date, the microbial formulation and application strategies that have been used, and the results that were obtained. The mycoinsecticides used in the pear psylla research were based on conidia of Beauveria bassiana (ARSEF #2860) and Paecilomyces fumosoroseus (ARSEF #2658). These were formulated into sprayable solutions containing water, 0.1% or 0.5% Ultrafine Sunspray oil (paraffinic oil) in water, or 0.1% acrylic polymer (Stressguard^TM) in water. Final spray solutions that contained 6 × 10⁶ conidiospores/ml were applied to psylla nymph infested trees at a rate of 5.39 × 10¹³ conidiospores/ha during the 1993--1995 seasons. In addition, two commercial formulations of B. bassiana, GHA from Mycotech and Naturalis L from Fermone Corp., were mixed in water at the same conidiospore application rates as the other fungal isolates. Single applications of the ARSEF fungal strain/formulation combinations produced psylla nymphal mortalities that ranged from 18.2--37.1%, but the results varied with formulation. Conidia formulated with acrylic polymers in water caused significantly higher mortalities several days earlier than either the water or water and oil combination in 1993. However, no significant differences among pathogen/formulation combinations occurred in 1994 or 1995. The performance of Naturalis L was comparable to the ARSEF fungal strain/formulation combinations with peak nymphal mortalities of 34.1%, while GHA produced a significantly lower peak mortality of only 10.8%. However, because of the low conidiospore concentrations in the Naturalis L formulation, final spray solutions contained nearly 25% of the oil-like carrier. Thus, psylla mortalities may not have been entirely attributed to mycosis. Based on the results from the ARSEF fungal formulations, a mycoinsecticidal approach to pear psylla management could be a useful component in an integrated pest management program for pear.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

Coating Beauveria bassiana with lignin for protection from solar radiation and effects on pathogenicity to Lygus lineolaris (Heteroptera: Miridae)

The entomopathogenic fungus, Beauveria bassiana, is highly susceptible to the damaging effects of solar radiation. This study demonstrates protection from simulated solar radiation by coating B. bassiana (GHA) spores with lignin and effects on pathogenicity to Lygus lineolaris (Palisot de Beauvois) adults in direct spray and contact uptake bioassays. Spores were coated with either lignin or cross-linked lignin by spray drying to produce spore coatings of high and low water solubility, respectively. Non-coated spores and the two spore coating treatments were suspended in either water (0.04% Silwet L77) or oil (Orchex 692) to produce six formulations. Rates of loss in spore viability under simulated solar radiation were approximately ten times lower for the three formulations in which spores remained coated in suspension (cross-linked lignin in water, cross-linked lignin in oil, and lignin in oil). However, these three formulations were the least pathogenic. Estimates of LC₅₀ and LC₇₅ values for the most pathogenic formulation (non-coated in oil) were 5 and 12 times lower, respectively, than the least pathogenic formulation (cross-linked lignin in oil), but these differences were not significant. The three lignin-coated formulations that provided the greatest UV protection were also the least pathogenic based on LT values, which were significant. Overall, L. lineolaris mortality was approximately 80 times less when exposed to treated broccoli rather than sprayed directly, based on LC₅₀ values. If the contribution of spore uptake from plant surfaces to L. lineolaris mortality is similarly low under field conditions, then improving persistence may be less important for improving efficacy. However, under control conditions where solar radiation significantly impacts mycoinsecticide efficacy, the improved persistence of lignin coating formulations may outweigh negative effects on pathogenicity.     

Development of Beauveria bassiana (Ascomycota: Hypocreales) as a mycoinsecticide to control green peach aphid,Myzus persicae (Homoptera: Aphididae) and investigation of its biocontrol potential

Long-term and intensive use of synthetic insecticides to control the green peach aphid, Myzus persicae (Homoptera: Aphididae) in agricultural production in the world has resulted in pest resistance and environmental pollution. The aim of the study was to develop an environmentally-friendly and effective mycoinsecticide from a local fungal isolate against M. persicae. According to the results of the screening experiments using 15 isolates (6 × Metarhizium, 5 × Beauveria, 2 × Isaria, 2 × Lecanicillium) at 1 × 10⁷ conidia ml^?1 concentrations and the dose–response experiments at 1 × 10⁵ –1 × 10⁹ conidia ml^?1 concentrations against M. persicae nymphs, Beauveria bassiana (KTU-24) was identified as the most promising isolate. The strain KTU-24 is also had tolerance to ecological conditions such as temperature, and UV-B. KTU-24 with advantageous and superior properties was used to develop a test mycoinsecticide. Mass production of spores by KTU-24 was conducted out by liquid-state fermentation using liquid medium. Spores harvested from the sporulated biomass were used to develop an oil-based mycoinsecticide, and the product was designated as AFIDISIDAL-OD Bbas-TR61. The product had lethal effect on M. persicae nymphs at a concentration of 1 × 10⁸ conidia ml^?1 in leaf-disc (82.5%) and pot (84.33%) experiments in a climate chamber. The oil-based mycoinsecticide developed in this study could be profitable when used in aphid-IPM prgrams by reducing crop loss and synthetic pesticides use.     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

The infectivity of Metarhizium anisopliae to two insect pests of coconuts

Papua New Guinea isolates of Metarhizium anisopliae from the rhinoceros beetles, Scapanes australis and Oryctes rhinoceros; the black palm weevil, Rhynchophorus bilineatus (Coleoptera); and three other insect orders (Hemiptera, Lepidoptera, and Dermaptera) were all short-spored (M. anisopliae var. anisopliae). Isolates from Scapanes were also pathogenic to Rhynchophorus and Oryctes. When a Scapanes isolate was grown on brown rice and released into the frond axils of young palms, 32% of dead Scapanes adults collected during the following 48 weeks were infected compared to 1% in the control nonrelease palms. Some infection of Rhynchophorus also occurred. Infection in Scapanes but not Rhynchophorus also increased in a plot of palms adjacent to the release zone. The rice inoculum could be recovered and the fungus reisolated up to 84 days after release, but infection of Scapanes continued to occur after all trace of the inoculum had disappeared.