Gabriel Moura Mascarin

Liquid culture fermentation for rapid production of desiccation tolerant blastospores of Beauveria bassiana and Isaria fumosorosea strains

A major constraint to the commercial use of fungal biocontrol agents is the availability of low-cost production media and processes. Previous attempts in producing Beauveria blastospores using liquid culture fermentation processes required long fermentation times (6-8days) and produced cells that had poor survival after desiccation and storage. In this study, isolates of Beauveria bassiana and Isaria fumosorosea were evaluated for blastospore yield, desiccation tolerance, storage stability, and biocontrol efficacy using fermentation media containing acid hydrolyzed casein or cottonseed flour as the nitrogen source. Cultures of B. bassiana and I. fumosorosea grown in media containing cottonseed flour produced high blastospore concentrations (>1×10(9)mL(-1)) after 3days which is comparatively less expensive nitrogen source than acid hydrolyzed casein. The resultant air-dried blastospores (<3% moisture) of all fungal isolates survived drying (61-86% viability), irrespective of the nitrogen source tested. Storage stability at 4°C varied with nitrogen source and fungal strain. Air-dried blastospores of B. bassiana strains showed half-lives >14months in contrast to 9.2-13.1months for I. fumosorosea. Blastospores of B. bassiana and I. fumosorosea killed Bemisia tabaci whitefly nymphs faster and required lower concentrations compared with aerial conidia. Our findings support the use of liquid culture fermentation as a cost-effective process to rapidly produce high yields of stable and infective blastospores of either B. bassiana or I. fumosorosea. These results support further evaluation of blastospore sprayable formulations for the control of soft-bodied insects.

The production and uses of Beauveria bassiana as a microbial insecticide

Among invertebrate fungal pathogens, Beauveria bassiana has assumed a key role in management of numerous arthropod agricultural, veterinary and forestry pests. Beauveria is typically deployed in one or more inundative applications of large numbers of aerial conidia in dry or liquid formulations, in a chemical paradigm. Mass production is mainly practiced by solid-state fermentation to yield hydrophobic aerial conidia, which remain the principal active ingredient of mycoinsecticides. More robust and cost-effective fermentation and formulation downstream platforms are imperative for its overall commercialization by industry. Hence, where economics allow, submerged liquid fermentation provides alternative method to produce effective and stable propagules that can be easily formulated as dry stable preparations. Formulation also continues to be a bottleneck in the development of stable and effective commercial Beauveria-mycoinsecticides in many countries, although good commercial formulations do exist. Future research on improving fermentation and formulation technologies coupled with the selection of multi-stress tolerant and virulent strains is needed to catalyze the widespread acceptance and usefulness of this fungus as a cost-effective mycoinsecticide. The role of Beauveria as one tool among many in integrated pest management, rather than a stand-alone management approach, needs to be better developed across the range of crop systems. Here, we provide an overview of mass-production and formulation strategies, updated list of registered commercial products, major biocontrol programs and ecological aspects affecting the use of Beauveria as a mycoinsecticide.

Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani.

Media and culturing protocols were identified that supported the formation of submerged conidia and microsclerotia (MS) by Trichoderma harzianum Rifai strain T-22 using liquid culture fermentation. Liquid media with a higher carbon concentration (36 g L(-1)) promoted MS formation at all C:N ratios tested. Hyphae aggregated to form MS after 2 d growth and after 7 d MS were fully melanized. This is the first report of MS formation by T. harzianum or any species of Trichoderma. Furthermore, submerged conidia formation was induced by liquid culture media, but yields, desiccation tolerance, and storage stability varied with C:N ratio and carbon rate. Air-dried MS granules (<4% moisture) retained excellent shelf life under cool and unrefrigerated storage conditions with no loss in conidial production. A low-cost complex nitrogen source based on cottonseed flour effectively supported high MS yields. Amending potting mix with dried MS formulations reduced or eliminated damping-off of melon seedlings caused by Rhizoctonia solani. Together, the results provide insights into the liquid culture production, stabilization process, and bioefficacy of the hitherto unreported MS of T. harzianum as a potential biofungicide for use in integrated management programs against soilborne diseases.

Compatibility of conventional agrochemicals used in rice crops with the entomopathogenic fungus Metarhizium anisopliae

The toxicological impact of chemical pesticides on fungal entomopathogens and their use in tank-mixing can be directly measured through in vitro compatibility tests. This study reports the in vitro toxicity of eight insecticides, four fungicides and five herbicides in the conidial germination, vegetative growth and conidiation of Metarhizium anisopliae (strain CG 168). A conidial suspension containing the pesticide at recommended field dosage was subjected to constant agitation in a rotary shaker for 3h to simulate a tank mixing. Then, aliquots of each suspension were used to determine conidial germination, vegetative growth and conidiation on potato dextrose agar (PDA). The fungicides difenoconazole (69 mL ha-1), propiconazole (75 mL ha-1), trifloxystrobin (313 g ha-1) and azoxystrobin (56 mL ha-1) were the most harmful products to all biological stages of M. anisopliae and they should not be applied together with this fungus in tank mixing. The insecticides exhibited the least degree of toxicity to this fungal pathogen, whereas the herbicides had the greatest impact on mycelial growth. The agrochemicals compatible with M. anisopliae were the insecticides methyl parathion (240 mL ha-1), thiamethoxam (31 g ha-1), and lambda-cyhalothrin (6.3 mL ha-1) and the herbicides glyphosate (1560 mL ha-1), bentazon (720 mL ha-1), and imazapic+ imazapyr (84 g ha-1). The compatible pesticides could be simultaneously used with this bio-control agent for integrated pest management in rice production systems.

Natural occurrence of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), an invasive pest recently found in Brazil.

Occurrence of Zoophthora radicans infecting nymphs and adults of Thaumastocoris peregrinus Carpintero and Dellape, 2006 is reported in Brazil. This is a new record of host for this fungal species and the first fungal pathogen associated with this pest worldwide. Infection of Z. radicans on T. peregrinus populations on commercial Eucalyptus plantation (Eucalyptus spp.) reached up to 100%, and low insect densities were associated with high levels of fungal infection in three out of seven plots. This pathogen seems to be virulent against T. peregrinus and may play an important role in population regulations of this invasive pest through naturally induced epizootics.

A protocol for determination of conidial viability of the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae from commercial products

Techniques for directly determining conidial viability have widespread use but also have limitations for quality control assessments of formulated mycoinsecticides, especially in emulsifiable oil. This study proposes a new method based on adaptations of already established protocols that use the direct viability method to make it more economical and accurate, thus enabling its use in the evaluation of formulated products. Appropriate parameters and conditions were defined using products based on Beauveria bassiana and Metarhizium anisopliae in the forms of pure conidia, fungus-colonized rice, ground fungus-colonized rice and oil dispersion. The established protocol, named ESALQ, consists of the transfer of 150 μL of a suspension containing about 0.7 and 1 × 10(6) conidia/mL onto Rodac® plates with 5 mL of potato dextrose agar culture medium + 5 mg/L of Pentabiotic® and 10 μL/L of Derosal® (Carbendazim) and subsequent counting of germinated and non-germinated conidia. For the ground fungus-colonized rice and oil dispersion formulations, prior to transferring the fungal suspension to the medium, rice should be decanted and the oil removed, respectively. This method was compared with another direct viability method and with the Colony-forming unit (CFU) and Fluorescence viability methods, comparing the accuracy obtained using the coefficient of variation (CV) of the analysis of each method. The results showed that in addition to the ease of application, the developed method has higher accuracy than the other methods (with a CV up to seven times lower than in the Standard method and up to 32 times lower than CFU). The CFU method underestimated the concentration of viable conidia in most of the tested fungal forms, and in the emulsifiable oil products, these values were 54% lower for B. bassiana and 84% lower for M. anisopliae. The adaptations and standardizations proposed in the ESALQ method showed effective improvements for routine quality assessment of mycoinsecticides.

Effect of heat stress and oil formulation on conidial germination of Metarhizium anisopliae s. s. on tick cuticle and artificial medium

The effect of heat stress (45°C) versus non-heat stress (27°C) on germination of Metarhizium anisopliae sensu stricto (s.s.) isolate IP 119 was examined with conidia formulated (suspended) in pure mineral oil or in water (Tween 80, 0.01%), and then applied onto the cuticle of Rhipicephalus sanguineus sensu lato (s.l.) engorged females or onto culture medium (PDAY). In addition, bioassays were performed to investigate the effect of conidia heated while formulated in oil, then applied to blood-engorged adult R. sanguineus females. Conidia suspended in water then exposed to 45°C, in comparison to conidia formulated in mineral oil and exposed to the same temperature, germinated less and more slowly when incubated on either PDAY medium or tick cuticle. Also, conidial germination on tick cuticle was delayed in comparison to germination on artificial culture medium; for example, germination was 13% on tick cuticle 72h after inoculation, in contrast to 61.5% on PDAY medium. Unheated (27°C) conidia suspended in either water or oil and applied to tick cuticle developed appressoria 36h after treatment; whereas only heat-stressed conidia formulated in oil developed appressoria on tick cuticle. In comparison to conidia heated in mineral oil, there was a strong negative effect of heat on germination of conidia heated in water before being applied to arthropod cuticle. Nevertheless, bioassays [based primarily on egg production (quantity) and egg hatchability] exhibited high percentages of tick control regardless of the type of conidial suspension; i.e., water- or oil-formulated conidia, and whether or not conidia were previously exposed to heat. In comparison to aqueous conidial preparations, however, conidia formulated in oil reduced egg hatchability irrespective of heat or no-heat exposure. In conclusion, mineral-oil formulation protected conidia against heat-induced delay of both germination and appressorium production when applied to the cuticle of R. sanguineus.

Thermal Requirements and Development of Herpetogramma phaeopteralis (Lepidoptera: Crambidae: Spilomelinae)

ABSTRACT The tropical sod webworm, Herpetogramma phaeopteralis Guenée is a major turfgrass pest in the southeastern United States. We evaluated larval development on five artificial diets and at six temperatures (15, 20, 25, 30, 32.5, 35 ± 1°C) on St. Augustinegrass (Stenotaphrum secundatum (Walter) Kuntze). Only larvae fed St. Augustinegrass and soy-wheat germ diets completed their lifecycles. None of the artificial diets tested (corn-based, soy-wheat germ, corn cob-wheat germ, corn cob-soy flour, or pinto bean) were suitable for rearing this species, because of high mortality and slower developmental time. Total developmental time (oviposition to adult) on S. secundatum significantly decreased from 47.8 d at 20°C to 21.1 d at 30°C, and then increased to 32.6 d at 32.5°C. Tropical sod webworm failed to complete larval development at 15 and 35°C. The relationship between temperature and developmental rate was described using linear (common and polynomial) and nonlinear models (Briere-1, Briere-2, and Lactin-2). The estimated lower temperature thresholds using a linear model for eggs, first, second, third, fourth, fifth, and sixth instars, prepupa, pupa, and total development were 10.1, 6.9, 12.3, 10.5, 15.3, 13.9, 9.1, 13.1, 12.0, and 13.1°C, and the thermal constant of these stages were 62.9, 66.2, 38.2, 40.3, 24.9, 32.3, 51.9,106.4,109.9, and 370.4 degree-days, respectively. The Briere-1 model provided the best fit with estimated lower, upper, and optimum thresholds for total development of 14.9, 34.3, and 29.4°C, respectively. The developmental requirements of H. phaeopteralis can be used to help predict the distribution and seasonal phenology of this pest.

Unveiling chemical defense in the rice stalk stink bug against the entomopathogenic fungus Metarhizium anisopliae

Eggs, nymphs (1st-5th instar) and adults of Tibraca limbativentris were challenged by conidial suspensions of its major fungal pathogen Metarhizium anisopliae in order to assess their susceptibility. The role of chemical defensive compounds from exocrine secretions produced by both nymphs and adults were examined for their participation on M. anisopliae infection. Although insect susceptibility to M. anisopliae followed a dose-dependent manner, adults followed by older nymphs displayed the highest resistance. Eggs were highly susceptible showing >96% fungal infection. Crude extracts isolated from metathoracic scent gland and dorsal abdominal glands of adults and nymphs, respectively, showed fungistatic effects by impairing spore germination, vegetative growth and sporulation. Gas chromatography-mass spectrometry analysis of these extracts revealed that the major components were short-chain hydrocarbons (C10-13) and unsaturated aldehydes. In vitro tests with the corresponding synthetic standards indicated compounds with greater antifungal activity including (E)-2-hexenal, (E)-2-octenal, and (E)-2-decenal, with the latter being the most deleterious to fungal fitness. We demonstrated that differential susceptibility of the rice stalk stink bug to M. anisopliae infection is age-specific and partly mediated by fungistatic properties of aldehydes, which are produced by scent glands of both nymphs and adults.

Current status and perspectives of fungal entomopathogens used for microbial control of arthropod pests in Brazil

Entomopathogenic fungi play a central role in Brazils biopesticide market. Approximately 50% of registered microbial biopesticides comprise mycoinsecticides and/or mycoacaricides consisting of hypocrealean fungi, with most based on Metarhizium anisopliae sensu stricto (s. str.) and Beauveria bassiana s. str. These fungi are mainly used to control spittlebugs in sugarcane fields and whiteflies in row crops, respectively, with annual applications surpassing three million hectares. Research also emphasizes the potential of fungal entomopathogens to manage arthropod vectors of human diseases. Most registered fungal formulations comprise wettable powders or technical (non-formulated) products, with relatively few new developments in formulation technology. Despite the large area treated with mycoinsecticides (i.e., approx. 2 million ha of sugarcane treated with M. anisopliae and 1.5 million ha of soybean treated with B. bassiana), their market share remains small compared with the chemical insecticide market. Nevertheless, several major agricultural companies are investing in fungus-based products with the aim at achieving more sustainable IPM programs for major pests in both organic and conventional crops. Government and private research groups are pursuing innovative technologies for mass production, formulation, product stability and quality control, which will support cost-effective commercial mycoinsecticides. Here, we summarize the status of mycoinsecticides currently available in Brazil and discuss future prospects.