FARIA, M.

FRAZÃO, H.

MAGALHÃES, B.

Portuguese

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An. Soc. Entomol. Brasil 26(3) 569Dezembro, 1997Scientific NoteA Technique to Estimate the Conidial Viability of Metarhiziumflavoviride Gams & Rozsypal (Hyphomycetes) Formulatedin Vegetable OilBonifácio Magalhães1, Marcos Faria1 and Heloiza Frazão11Embrapa - Recursos Genéticos e Biotecnologia, Caixa postal 02372,70847-970, Brasília, DF.An. Soc. Entomol. Brasil 26(3): 569-572 (1997)Técnica de Determinação da Viabilidade de Metarhizium flavoviride Gams &Rozsypal (Hyphomycetes) Formulado em Óleo VegetalRESUMO - Foi desenvolvida uma técnica simples para determinação daviabilidade de conídios do fungo patogênico a gafanhotos Metarhiziumflavoviride Gams & Rozsypal (Hyphomycetes), formulado em óleo vegetal. Atécnica consiste em espalhar a suspensão fúngica oleosa sobre meio de cultura ecobrir com uma lamínula. Assim, uma suspensão de 10 μl fúngica oleosa ouaquosa (106 conídios/ml) foi espalhada sobre blocos (1.0 x 1.0 x 0.2 cm) domeio de cultura BDA (batata, dextrose e agar). Em seguida, a suspensão foifirmemente coberta com uma lamínula estéril. O conjunto foi então transferidopara placas de Petri e mantido a 28°C, com observações realizadas 6, 9, 12, 15e 18 h após inoculação. Foram registrados altos níveis de germinação (> 90%)de M. flavoviride formulado em óleo com ou sem querosene, a partir de 15 h.Após 18 h esses níveis e o crescimento vegetativo foram comparáveis àquelesobservados em suspensão aquosa.PALAVRAS-CHAVE: Insecta, gafanhoto, fungos entomopatogênicos,formulação oleosa, viabilidade. The fungus Metarhizium flavovirideGams & Rozsypal is under investigation inBrazil for the biological control of the grass-hoppers Rhammatocerus schistocercoides(Rhen) and Schistocerca pallens (Thunberg)(Magalhães et al. 1996, Moreira et al. 1996,Vicentini & Magalhães 1996), and the walk-ing stick Stiphra robusta Mello-Leitão (S.Vicentini, pers. commun.). The most promis-ing formulation for M. flavoviride againstgrasshoppers is based on oils amended forultra-low volume application (Lomer & Prior1992, Bateman et al. 1993, Magalhães et al.1996).The viability estimate of M. flavovirideconidia formulated in vegetable oils usingaqueous agar media is hindered by the cover-age of conidia with oil. Due to polarity dif-ferences between oil and water, conidia donot have a direct contact with the aqueousmedium when seeded. Attempts to remove theoil from the conidial surface using differentconcentrations (0-10%) of detergents (Tween80, Triton) have led to unconsistent results.Stathers et al. (1993) developed a techniqueto remove oil from conidia involving vacuum570filtration through a cellulose nitrate mem-brane, followed by incubation in gelatin me-dium. Another method to assess fungal viabil-ity is based on the enumeration of colony-forming-units (CFU) (Inglis et al. 1993).However, it requires a selective medium, se-rial dilutions and a fine adjustment for eachsystem added to the problem of high varia-tion of CFU between replicates. In this note,we present a simpler technique to estimate theviability of M. flavoviride, in which conidiaformulated in oil are spread on nutrient me-dium and covered with a coverslip.Conidia of M. flavoviride (CG 423) wereproduced on parboiled rice as described byMagalhães & Frazão (1996), with some modi-fications as follows. After eight days incuba-tion at 30°C, conidia were harvested using a500 μm sieve and formulated in soybean oil,with and without kerosene (5%), and storedat 4°C. Kerosene was used to suspend theconidia more uniformly in the formulation. Toestimate viability, blocks (1.0 x 1.0 x 0.2 cm)of potato dextrose agar medium (potato 170g,dextrose 20g, agar 20g, water 1 liter) wereplaced on sterile glass slides. Ten μl of the oilsuspension (106 conidia/ml) were transferredto the agar blocks, firmly covered with a ster-ile glass coverslip (22 x 22 mm), and thepreparation incubated at 28°C. Germinationand growth of M. flavoviride formulated insoybean oil, soybean oil + kerosene (5%), andwater + Tween 80 (0.1%) were recorded at 6,9, 12, 15 and 18 h. For comparison, conidiaformulated in soybean oil + kerosene and inwater + Tween 80 were also spread on agarFigure 1. Germination of Metarhizium flavoviride (CG 423) at 28°C, during 15 h, usingdifferent techniques for sample preparation.Magalhães  et al.An. Soc. Entomol. Brasil 26(3) 571Dezembro, 1997medium (conventional method, without cov-erslip). A conidium was considered viablewhen the germ tube length was at least equalto its width. The number of viable and non-viable conidia (> 400/slide or Petri plate), andthe germ tube length were assessed under anoptical microscope (400x magnification).Germ tube length data were compared usingthe Student-Newman-Keuls test (P = 0.05).M. flavoviride germinated at similar per-centages when formulated in oil, with or with-out kerosene, plated on nutrient medium andcovered with a coverslip (Fig. 1). At 18 h,air bubbles. The growth rate of M. flavovirideformulated in oil or in oil + kerosene, under acoverslip, was comparable to the growth ratein aqueous suspension with no coverslip (con-ventional method). However, with a coverslipon top of the aqueous formulation the growthrate was significantly higher (P < 0.001) (Fig.2). After 12 h, the germ tube growth in aque-ous suspension, under the coverslip, was soprofuse that measurements were not possible.The use of the technique described hereinprevents the formation of air bubbles and al-lows a more precise determination of the co-germination was very high (> 97%) and com-parable to that estimated by the conventionalaqueous suspension method. However, whenthe oil formulation was plated and no cover-slip used, germination was poor and very dif-ficult to quantify, since non-germinated co-nidia were frequently confounded with smallnidial viability of M. flavoviride. In addition,this method is suitable for viability estima-tion of other species of fungi, such as M.anisopliae (Metsch.) Sorokin and Beauveriabassiana (Balsamo) Vuillemin (B. Magalhães& H. Frazão, unpublished). Its simplicity maymake this technique a very useful tool in pro-Figure 2. Germ tube growth of Metarhizium flavoviride (CG 423) at 28°C, during 15 h,using different techniques for sample preparation.572grams involving mycopesticide applicationsin the field.AcknowledgmentsTo Dr. Peter W. Inglis for kindly review-ing the manuscript. This research was partiallyfunded by the Conselho Nacional de Pesquisae Desenvolvimento Científico e Tecnológico(CNPq), Brazil.References CitedBateman, R.P., M. Carey, D. Moore & C.Prior. 1993. The enhanced infectivity ofMetarhizium flavoviride in oilformulations to desert locusts at lowhumidities. Ann. Appl. Biol. 122: 145-152.Inglis, D.G., M.S. Goettel & D.L. Jonhson.1993. Persistence of the entomo-pathogenic fungus, Beauveria bassiana,on phylloplanes of crested wheatgrass andalfalfa. Biol. Control 3: 258-270.Lomer, C.J. & C. Prior. 1992. Biologicalcontrol of locusts and grasshoppers.Oxon, UK, CAB International, 394p.Magalhães, B.P. & H.S. Frazão. 1996.Effects of temperature, water content andsubstrate on conidial production ofMetarhizium flavoviride. Rev. Microbiol.27: 242-246.Magalhães, B.P., M.R. Faria & W.D.Guerra. 1996. Desenvolvimento debioinseticidas  para controle de gafa-nhotos no Brasil. p. 6-11. In An. EncontroInter. Biol., Comport. Contr. Gafanhoto,1. Cuiabá, MT. Minist. Agric. Abast., 11p.Moreira, M.A., B.P. Magalhães, M.C.C.Valadares & M.C.M. Chagas. 1996.Occurrence of Metarhizium flavovirideGams & Rozsypal (Hyphomycetes) onSchistocerca pallens (Thunberg)(Orthoptera: Acrididae) in Rio Grande doNorte, Brazil. An. Soc. Entomol. Brasil25: 359-361.Stathers, T.E., D. Moore & C. Prior. 1993.The effects of different temperatures onthe viability of Metarhizium flavovirideconidia stored in vegetable and mineraloils. J. Invertebr. Pathol. 62: 111-115.Vicentini, S. & B.P. Magalhães. 1996.Infection of the grasshopper Rham-matocerus schistocercoides Rehn by theentomopathogenic fungus Metarhiziumflavoviride Gams & Rozsypal.  An. Soc.Entomol. Brasil 25: 309-314.Received 04/II/97. Accepted 02/IX/97.Magalhães  et al.

A technique to estimate the conidial viability of Metarhizium flavoviride Gams & Rozsypal (Hyphomycetes) formulated in vegetable oil.

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A technique to estimate the conidial viability of Metarhizium flavoviride Gams & Rozsypal (Hyphomycetes) formulated in vegetable oil.

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