Marcos Faria

Biological control of Bemisia tabaci with fungi

Recent advances in production, formulation, and application of insect pathogenic fungi have resulted in improvements in long-standing whitefly mycoinsecticide products based on Verticillium lecanii, and development and registration of several new products based on Paecilomyces fumosoroseus and Beauveria bassiana. These products have the capacity to suppress and, in some instances, provide good control of whiteflies in both greenhouse and field crops. However, numerous factors continue to impede the commercial development of fungi as whitefly biological control agents. These include slow action, poor adulticidal activity, potentially negative interactions with commonly used fungicides, relatively high cost, limited shelf life, and dependence on favorable environmental conditions. Development of methods and strategies for overcoming these limitations has progressed, however, and various practices that enhance mycoinsecticide efficacy have been identified. Principal recommendations include: (1) initiating treatments against the early stages of the pest to prevent population buildup, (2) targeting pest populations developing under moderate environmental conditions (e.g., during spring or fall growing seasons), (3) selecting crops amenable to multiple, highly efficient spray applications, and (4) applying fungi asynchronously with incompatible fungicides. Commercial markets for these products have been slow to develop and remain unstable in the face of strong competition from less costly, highly efficacious chemical insecticides. Nevertheless, continuing problems with chemical insecticide resistance and environmental and food contamination support continued development of fungi as relevant tools in the whitefly biological control arsenal.

Biological control of insects in Brazil and China: history, current programs and reasons for their successes using entomopathogenic fungi

Abstract Brazil and China have been successful in the use of microbial control methods to manage several agricultural and forest insects. In both countries, entomopathogenic fungi (EF) have been used for pest management since the 1970s. However, EF production and commercialization have not been constant in either country. Several companies and cooperatives suspended their activities or shut down from the 1970s to the 1990s. This was due to loss of confidence in available mycoinsecticides by Brazilian farmers or due to reduced involvement and government subsidies for biological control in China; and, consequently, mycoinsecticides were largely replaced by inexpensive chemical insecticides. Starting in the 1990s and continuing until today, however, new Brazilian and Chinese private companies have arisen. In Brazil, the area treated with M. anisopliae for spittlebug control alone is estimated to be approximately one million hectares in 2008, 75% of which was for control of spittlebugs in sugarcane plantations and the remainder for spittlebugs in pasture grass (primarily Brachiaria spp.) and other smaller programs. In China, the fungus Beauveria bassiana was used annually in 0.8–1.3 million ha until the 1980s. Several factors were important for the success of these programs, such as: governmental support (at least during the initial steps of biocontrol programs); availability of indigenous virulent fungal isolates; low-cost substrates for mass production; retail prices of mycoinsecticides lower than their chemical counterparts; and sale by contract which allows the products to be immediately available for use, rather than stored. In this report, we discuss the current biocontrol programs using insect fungi in these two developing countries, as well as the future and main challenges they must face to further encourage the adoption of mycoinsecticides.

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a(w), temperature, and O(2) concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 degrees C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O(2) concentrations and stored at 50 degrees C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O(2) (100% N(2)) to as high as 63-93% when treated with 21-22% O(2). For conidia stored over a broad range of moderate to high temperatures in the absence of O(2), SR-FR differences were <or= 9% at 25-40 degrees C but 30% at 50 degrees C. Germination of stressed Bb and Ma conidia increased substantially when incubation time on the germination substrate was increased from 24 to 72 h, whereas germination of non-stressed conidia showed little change. Conidia debilitated by stress were characterized by hypersensitivity to lethal imbibitional damage (damage that is mitigated by slow rehydration) and slow germination. Viability protocols that may provide more reliable assessments of overall mycopesticide quality are discussed.

Screening of Beauveria bassiana (Deuteromycotina: Hyphomycetes) isolates against nymphs of Bemisia tabaci (Genn.) biotype B (Hemiptera: Aleyrodidae) with description of a new bioassay method

A bioassay method that uses melon leaves as substrate for eggs and nymphs of the biotype B of Bemisia tabaci (Genn.) was established. Root formation of petioles immersed in tap water guaranteed the turgidity and normal coloration of leaves for a period of 20 to 25 days, enabling the execution of tests with entomopathogenic fungi. The virulence of 50 isolates of Beauveria bassiana (Balsamo) Vuillemin was assessed, as an initial action of a research project that aims the development of a bioinsecticide for controlling Bemisia tabaci nymphs in melon crops. Melon leaves were infested with 18 couples of biotype B adults for 26 hours. First-instar nymphs were selected through microscopic observation approximately 4-5 days after adults removal. Application of fungal isolates was performed with a spray tower. Average number (± SEM) of infective propagules deposited on leaf surface was 1.2x104 ± 9.12x102 conidia per cm2. For the control it was applied a Tween 80 solution at 0.1%. Three or four replicates were performed per treatment. Incubation was in chamber regulated to 27±1oC, 70±5% RH and 12 hours photophase. Assessments of dead nymphs were performed at seven and 14 days following spraying. Average mortality in the control treatment was 1.2% at day 14, and 94.4% of the nymphs became adults. Average nymphal mortality at day seven post-spray reached a maximum value of 25.7%. Average mortality at day 14 post-spray varied from 6.1% to 92.3%.

Short-term assessment of bt maize on non-target arthropods in Brazil

Although not yet available for cultivation in Brazil, the effect of Bt maize hybrids on natural enemies and soil dwelling arthropods should be assessed prior to its release to growers. Trials were carried out during one growing season in two different locations with the genetically modified maize hybrids 7590-Bt11 and Avant-ICP4, comparing with their respective non-Bt isogenic hybrids. Arthropods were evaluated through direct observation on plants and pitfall traps. In general, no differences were observed between populations of earwig (Dermaptera: Forficulidae), lady beetles (Coleptera: Coccinellidae), minute pirate bug (Coleoptera: Anthocoridae), ground beetles (Carabidae), tiger beetles (Cicindelidae), and spiders (Araneae). There was no difference in egg parasitism of Helicoverpa zea (Boddie) by Trichogramma sp. (Hymenoptera: Trichogrammatidae). Thus, Bt maize hybrids expressing insecticide proteins Cry1A(b) and VIP 3A do not cause reduction of the main maize dweeling predators and parasitoids.

DOUBLE-STRANDED RNA IN THE ENTOMOPATHOGENIC FUNGUS METARHIZIUM FLAVOVIRIDE

Abstract Bands of dsRNA were detected in five out of seven isolates of the entomopathogenic fungus Metarhizium flavoviride. The identity of these bands was proven by RNase and S1-nuclease treatments. The transference of dsRNA between isolates (from CG291 to CG442) was successfully carried out through forced heterokaryons. Isogenic strains, with or without dsRNA, were submitted to virulence tests against the grasshopper Rhammatocerus schistocercoides. In contrast to what has been found in some phytopathogenic fungi, these dsRNA fragments did not cause hypovirulence to M. flavoviride.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5′ strains by sequencing of the 59′ end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10–12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Influence of some parameters on the germination assessment of mycopesticides.

The substantial negative impact of some parameters on the germination of low-quality conidia (high proportion of slow-germinating propagules) was demonstrated, whereas for high-quality batches their effect was small or even absent. Germination was increased as the initial hydration status of conidia immediately prior to suspension preparation was increased, being ca. 33% and 80% for dehydrated Metarhizium anisopliae propagules (water activity ≤0.314) from low- or high-quality batches after an 18 h incubation period, respectively, and 63% and 95% for hydrated propagules (water activity = 0.933). Germination of low-quality propagules also increased as the time dry conidia were kept in aqueous suspension prior to inoculation onto culture media (15 min, 3 or 24 h) or the incubation time at 25°C before counts (18, 48 or 72 h) was increased. Depending on treatment conditions, average germination of low-quality conidia varied from 53% to 98%. On the other hand, germination for high-quality conidia was always ≥94%. Regarding the relative humidity (RH) of the incubation atmosphere, the average germination rates for low-quality conidia on Potato Dextrose Agar (PDA) in Petri plates was 49%, while germination of these conidia on PDA blocks kept under lower RH inside plastic boxes was ≤23%. Use of lactophenol-staining and/or use of coverslips had a negative effect when germination assessment was performed for low-quality conidia, resulting in distorted counts or increased standard deviations compared to high-quality conidial batches. The occurrence of dislodged conidia (ungerminated conidia outside the inoculation zone due to hydraulic pressure exercised by addition of stains and/or coverslips added to the substrate by the time germination is assessed) was common place, whereas dislodged conidia were not seen in treatments with high-quality batches. This work underscores the importance of a number of parameters that anyone working with low-quality fungi needs to be cognizant of in their research.

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

Abstract 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.

Food consumption of Rhammatocerus schistocercoides Rehn (Orthoptera: Acrididae) infected by the fungus Metarhizium flavoviride Gams & Rozsypal

Foliage consumption by nymphs and adults of the grasshopper Rhammatocerus schistocercoides Rehn infected with the fungus Metarhizium flavoviride Gams & Rozsypal was evaluated. Sixth and eighth instar nymphs and female adults consumed on average (±SEM) 6.9 ± 0.36 cm2 (60.7 ± 2.92 mg of dry matter), 11.0 ± 1.35 cm2 (74.9 ± 9.24 mg d.m.), and 16.0 ± 0.50 cm2 (126.5 ± 6.35 mg d.m.) of sugarcane leaves per day, equivalent to 85.4, 35.9 and 41.9% of the insect body weights, respectively. Eighth instar nymphs and female adults inoculated with M. flavoviride (5,000 conidia / insect) ingested less food from day three onward and showed at day 10 an average consumption 74.5 and 45.6% lower than control insects, respectively. Adult females topically treated with a sub-lethal dosage (3 ml of a 8 ppm suspension / insect) of the chemical insecticide diflubenzuron showed consumption rates comparable to untreated insects (P=0.065). The combination fungus + chemical did not show results different from those obtained with the fungus alone (P=0.405). A strong correlation (r2=0.998) between production of fecal pellets by R. schistocercoides and food consumption was observed. These results confirmed the high voracity of R. schistocercoides and showed a remarkable adverse effect of the fungus on food intake by infected nymphs and adults.