Rogério Biaggioni Lopes

Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae).

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 10(7)cells/ml were used. At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC(50) was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.

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.

External development of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in the subterranean termite Heterotermes tenuis

The subterranean termite Heterotermes tenuis is one of the main pests of sugarcane and eucalyptus in Brazil, and the use of entomopathogenic fungi, alone or associated to chemicals, is an efficient and environmentally favorable method for its control. Studies related to the fungal development on these insects are important due to the effect of insect behavior on entomopathogens. The objective of this work was to describe the external development of Beauveria bassiana and Metarhizium anisopliae on H. tenuis using Scanning Electron Microscopy (SEM), determining the duration of the different phases of fungal infection. Two fixation techniques for preparing SEM samples were also evaluated. Worker specimens of H. tenuis were inoculated with a 1 x 109 conidia mL -1 suspension of the fungi and maintained at 25±1 o C and 70±10% relative humidity. Insects were collected from 0 to 144 hours after inoculation and prepared on SEM stubs for each of the two fixation techniques. The results obtained with the two techniques were compared and duration of the different phases of the infection process were estimated from SEM observations and compared for three fungal isolates. B. bassiana and M. anisopliae have similar development cycles on the termite, but some important differences exist. The penetration, colonization and conidiogenesis phases are relatively faster for M. anisopliae than for B. bassiana, which results in a faster rate of insect mortality. The fixation technique with OsO 4 vapor is suitable for preparation of insects to be used in SEM observation of the developmental stages

Culture media selection for mass production of Isaria fumosorosea and Isaria farinosa

This work investigated the production of the fungi Isaria fumosorosea and Isaria farinosa in biphasic fermentation using agro-industrial products and residues. Combinations of natural liquid substrates, alternative to the complete medium and potato dextrose medium, were evaluated. The best liquid media were sugarcane molasses + rice broth, rice broth + yeast and sugarcane molasses + yeast + rice broth, which resulted in the highest viable propagule concentration. The molasses + rice broth medium was selected for the next phase of the study in which the production of both fungal isolates was evaluated in solid grain substrates. In solid-state fermentation, the best conidia production was achieved with the soybean meal and broken corn for I. farinosa, and whole rice and broken rice for I. fumosorosea. Results demonstrated that the two fungal species could be rapidly produced with higher yield of conidia on agro-industrial resources by using biphasic fermentation techniques.

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.

Genetic Diversity and Pathogenicity of Metarhizium spp. Associated with the White Grub Phyllophaga capillata (Blanchard) (Coleoptera: Melolonthidae) in a Soybean Field

Phyllophaga capillata (Blanchard) was recently described as a soybean pest in Brazil. The occurrence of Metarhizium spp. associated with adults and their pathogenicity were investigated. Natural prevalence of Metarhizium anisopliae was 0.51% in adults collected by light trap. Most Metarhizium isolates were identified as Metarhizium robertsii, although M. anisopliae sensu stricto and a few unidentified isolates were also found. Bioassays with representative isolates selected from different genetic groups resulted in ≤5.6% confirmed mortality against larvae and adults, suggesting low potential of microbial control of this pest by use of indigenous Metarhizium isolates.

Conidial vigor vs. viability as predictors of virulence of entomopathogenic fungi.

We tested the hypothesis that debilitated conidia exhibiting slow-germination (requiring>16h to germinate) are less virulent than vigorous conidia exhibiting fast germination (requiring⩽16h to germinate). Preparations of Beauveria bassiana s.l. strain CG 1027 with variable ratios of vigorous to debilitated conidia were assayed against third-instar larvae of Spodoptera frugiperda. As the proportion of debilitated conidia in test preparations increased, LC50 expressed in terms of total viable conidia increased, while LC50 expressed solely in terms of vigorous conidia remained constant, indicating that vigorous conidia were responsible for nearly all mortality observed in the assays. Larvae treated with conidia from low-quality batches (with high proportions of debilitated conidia) survived consistently longer than those treated with comparable doses of conidia from high-quality batches. These results confirm our previous hypotheses that inclusion of debilitated conidia in viability assessments can lead to overestimation of the quality (potency) of mycoinsecticide preparations and support our recommendation for use of short incubation periods for assessing viability whenever viability is relied upon as an indicator of product quality.

The fungistatic and fungicidal effects of volatiles from metathoracic glands of soybean-attacking stink bugs (Heteroptera: Pentatomidae) on the entomopathogen Beauveria bassiana.

This study was initially designed to evaluate the differential susceptibility of three soybean-attacking pentatomids to the entomopathogenic fungus Beauveria bassiana in standardized bioassays. Euschistus heros (Eh) was shown to be significantly less susceptible than Chinavia ubica (Cu), whereas Dichelops melacanthus (Dm) adults were highly susceptible to fungal infections. A deeper look at the mechanisms involved in the possible role of volatiles from metathoracic glands on fungal infections was undertaken, and gland extracts from Nezara viridula (Nv), a species known for its resilience to fungal infections, were also included in the assays. Atmospheres with volatiles from pentatomids with very low-susceptibility to B. bassiana infections (Eh and Nv) had a significant effect on speed of germination as shown in counts performed up to 22h post-inoculation, by which time 0.1 (control), 0.6 (Dm), 17.9 (Cu), 32.6 (Eh), and 43.4% (Nv) of conidia had not germinated. The fungistatic (inhibitory) and fungicidal (lethal) effects of Eh and Nv volatile-rich atmospheres were subsequently quantified in Petri dishes with either PDA or PDA medium amended with carbendazim, which allowed germination rates to be determined at 18 and 48h post-inoculation, respectively. As opposed to control, Eh volatile-rich atmosphere had a clear fungistatic effect, since germination rate was only 27.4% within 18h, but reached 99.4% at 48h post-inoculation. For Nv volatile-rich atmospheres, only 15.1% of conidia germinated within 18h, and by 48h post-inoculation, approx. 18% of conidia were unviable (neither germ tubes nor intumescence), whereas in the control treatment rates were >99% at both reading times. Therefore, the gaseous phase of defensive secretions from fungus-resilient pentatomids possess a strong inhibitory effect and may display a less pronounced lethal effect on fungal germination, as was the case for Nv.