MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 93(6) Nov/Dez 1998
PAGES: 839-846 DOI: Full paper
Selection of Beauveria bassiana and Metarhizium anisopliae Isolates to Control Triatoma infestans

Christian Luz +, Myrian S Tigano*, Ionizete G Silva, Celia MT Cordeiro*, Salah M Aljanabi*

Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Caixa Postal 131, 74001-970 Goiânia, GO, Brasil
*Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia, SAIN Parque Rural, W5 Norte, Caixa Postal 02372, 70849-970 Brasília, DF, Brasil

Abstract

Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%), conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50) varied from 7.1x105 to 4.3x106conidia/ml, for a B. bassiana isolate (CG 14) and a M. anisopliae isolate (CG 491) respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30°C, compared to 15 and 20°C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity).

Entomopathogenic fungi are promising candidates for microbiological control of Triatominae (Hemiptera, Reduviidae) because they invade their hosts through the integument. However, relative humidity (RH) and temperature are known to be limiting environmental factors for fungal development on insects (Glare & Milner 1991, Ferron et al. 1991). High rates of infection and a rapid kill of triatomine bugs by the hyphomycete fungi Beauveria bassiana and Metarhizium anisopliae were obtained at humidities close to saturation (Silva & Messias 1985, Romaña & Fargues 1987, Luz 1990, Romaña 1992, Luz et al. 1994). Infection of bugs diminished with B. bassiana at RH below 97% (Luz 1994). Optimal temperatures for fungal development on the insect host range from 16 to 30°C for B. bassiana and M. anisopliae with a faster development at the higher temperatures (Ferron et al. 1991).

Microclimatic conditions in the natural insect habitat may be unfavorable for fungal infection. Humidity in domestic microhabitats of triatomine bugs may be distinctly lower as shown by Luz (1994) in Rhodnius prolixus. To select isolates for biocontrol, intraspecific differences of fungal behaviour related to abiotic conditions in target insect habitats should be considered. In this study, isolates of B. bassiana and M. anisopliae were screened against Triatoma infestans at RH close to saturation and at 50% RH. The effect of conidial concentrations and temperatures were analyzed for some isolates at the lower RH. Polymorphism among some B. bassiana isolates, with different levels of virulence, was investigated by using random amplification of polymorphic DNA (RAPD) analysis.

 

MATERIALS AND METHODS

Insect rearing - T. infestans was mass-reared in the laboratory. Insects were allowed to feed on chickens every two weeks, and maintained at 25±0.5°C, 75±5% RH with a photophase of 12 hr. The T. infestans colony was originally from the State of Paraná, Brazil, and has been maintained in laboratory since 1981.

Fungal cultures - Most of the 23 B. bassiana and 13 M. anisopliae isolates selected for this study were originally obtained from hemipteran insects in Brazil (Table I). Conidia were obtained from cultures grown on complete media (0.001 g FeSO4, 0.5 g KCl, 1.5 g KH2PO4, 0.5 g MgSO4.7 H2O, 6 g NaNO3, 0.001 g ZnSO4, 1.5 g hydrolysed caseine, 0.5 g yeast extract, 10 g glucose, 2 g peptone, 20 g agar and 1 l distilled water) at 27°C for 15 days. Mycelium used for RAPD analysis was obtained from a submersed culture of conidia in complete medium shaking at 150 rpm at 25°C for three days. Mycelium was harvested by filtration through filter paper (Whatman No. 1), lyophilized and stored at -80°C.

Bioassays - Conidia were harvested from plate cultures and suspended in sterile distilled water with 0.1% Tween 80 (Sigma, St. Louis, MO, USA). Newly emerged and unfed third instar T. infestans nymphs were used in the assays. Tests on pathogenicity at a RH close to saturation and 25°C were done by immersion of ten insects in a conidial suspension (108 conidia/ml) for approximately 6 sec. For all other assays the same method of application was used with four replicates of ten insects. A suspension of 107 conidia/ml was used to evaluate virulence of isolates at 50% RH and 25°C. Maintaining this lower RH, six selected isolates (CG 14, CG 24, CG 144, CG 306, CG 474 and CG 491) were tested at four temperatures (15, 20, 25 and 30°C) and seven conidial concentrations (105, 3x105, 106, 3x106, 107, 3x107 and 108 conidia/ml). Control insects were treated as described above but without conidia. After treatment, insects were transferred to gauze-covered transparent cups (55 mm diameter x 75 mm) and kept in a chamber with regulated temperature and humidity (50±5% RH and 25±1°C). For tests at different temperatures, insects were held in desiccators (53% RH), which were kept in incubators. Humidity inside desiccators was maintained by using a saturated aqueous solution of MgNO3.6H2O (Winston & Bates 1960). For all assays, mortality of insects was recorded daily during 15 days after treatment.

RAPD analysis - Genomic DNA of ten B. bassiana isolates (CG 14, CG 16, CG 19, CG 21, CG 24, CG 136, CG 261, CG 306, CG 474 and CG 516) was obtained using a universal rapid salt extraction method (Aljanabi & Martinez 1997). PCR reactions were performed in 50-ml volumes, with 15 ng of each template, using the PTC-100 programmable thermal controller (MJ Research), and a temperature profile described by Tigano-Milani et al. (1995). Amplifications were done using the following reaction mix: 2 units of Taq polymerase (Cenbiotec), 5 ml of 10x Taq polymerase reaction buffer, 200 mM of each deoxynucleotides triphosphate (Pharmacia Biotec), and 0.4 mM of 10-mer primer (Operon Technologies, Alameda CA.). Ten primers were selected for the analysis: OPE-01, OPE-02, OPE- 03, OPE-04, OPE-07, OPE-14, OPE-15, OPE-16, OPE-19 and OPE-20. Amplified products were separated by electroforesis in 2% LE agarose gel dissolved in 0.5x Tris-borate-EDTA (TBE) buffer. After electrophoresis, gels were stained with ethidium bromide (Sambrook et al. 1989) and photographed under UV light. DNA fingerprints were scored directly from the photographs.

Data analysis - Angular transformed cumulative mortalities were analyzed by ANOVA (analysis of variance) and means compared by cluster analysis (Scott & Knott 1974)Estimates of 50% survival time were calculated (Lee 1980), curves of survival analyzed by log-rank-test and compared by Z-statistics (Fox 1993). Lethal concentrations to kill 50% and 90% (LC50 and LC90) were calculated by probit analysis (SAS Institute Inc. 1989). RAPD characters were analyzed using NTSYS-pc V1.8. A similarity matrix was created using the Jaccard similarity coefficient (Sneath & Sokal 1973). Clustering was done using the unweighted mean pair group arithmetic mean method (UPGMA).

 

RESULTS

Effect of humidity on mortality - Most B. bassiana and M. anisopliae isolates tested at RH nearing saturation and 25°C, induced cumulative mortalities between 90 and 100% in third instar nymphs of T. infestans (Table II). At 50% RH, the virulence of most isolates was reduced, but four B. bassiana isolates (CG 21, CG 306, CG 474 and CG 516) and two isolates of M. anisopliae (CG 144 and CG 491) caused mortalities of 90% or higher. Four other B. bassiana isolates (CG 14, CG 19, CG 24 and CG 261) and one isolate of M. anisopliae (CG 50) caused mortalities over 85% at 50% RH. ANOVA of mortalities showed a significant difference between B. bassiana isolates (F = 4.8, p < 0.0001), but not between isolates of M. anisopliae (F = 1.4, p = 0.1928). Comparison of means resulted in three groups (a = 0.05) for B. bassianaisolates. Estimates of 50% survival time of insects varied from six days, for CG 550, to eleven days for CG 19, CG 42 and CG 125. A significant difference between survival curves of B. bassiana isolates (c2 = 42.9, p < 0.0001) and M. anisopliae isolates (c2 = 37.9, p < 0.0001) was detected.

Effect of temperature and conidial concentration on mortality - The effect of temperature and conidial concentration on fungal virulence were analyzed at 50% RH. Four isolates of B. bassiana (CG 14, CG 24, CG 306, CG 474) and two isolates of M. anisopliae (CG 144, CG 491) were selected for this study. Progress of mortality at different temperatures is demonstrated in Fig. 1. There was a significant effect of the isolate (F = 15.7, p < 0.0001) and temperature (F = 14.8, p < 0.0001) on insect cumulative mortality, 15 days after treatment. Within all temperatures tested, mortality due to the B. bassiana isolates was higher than in the M. anisopliae isolates (7.2 %, I.C. 95 % at 3.9-11.3 %). Isolates showed different patterns of mortality at increasing temperatures (F = 2.6, p < 0.004) with a significant linear effect in CG 14, CG 144 and CG 491, a significant linear and quadratic effect in CG 24 and CG 306. No significant differences between mortalities at different temperatures were observed for CG 474. The values of LC50, 15 days after fungal application, varied from 7.1x105 to 4.3x106 conidia/ml fora B. bassiana isolate (CG 14) and a M. anisopliae isolate(CG 491)respectively (Table III). Values of LC90 varied between 4.6x106 (CG 474) and 1.4x108 (CG 491). Confidence intervals indicate similarity in virulence among B. bassiana isolates, but not in M. anisopliae isolates.

RAPD analysis - The ten primers used for the B. bassiana isolates produced 114 scorable bands, and the average genetic similarity among these isolates was 79%. Cluster analysis of the RAPD data did not produce well defined phenetic groups (Fig. 2). The isolates analyzed presented high similarity (> 73%), although they had been selected for presenting different virulence towards T. infestans (Table II).

 

DISCUSSION

All B. bassiana and M. anisopliae isolates tested proved to be pathogenic to T. infestans at a RH nearing saturation. However, several isolates of B. bassiana and M. anisopliae were also virulent against T. infestans at 50% RH. High mortalities due to infection with B. bassianaindependent of RH or at low RH was reported for other insect pests (Ferron 1977, Doberski 1981, Marcandier & Khachatourians 1987) and has also been observed for other fungal species (Hsiao et al. 1992, Fargueset al. 1997). Exposure of fungus-treated triatomine bugs to undefined humidities resulted in low rates of insect mortality (Dias & Leão 1967, Romaña & Romaña 1981, Sherlock & Guitton 1982). Only Luz (1990) and Romaña (1992) reported a somewhat superior susceptibility of R. prolixus to B. bassiana at 40% RH, compared to higher humidities. Two B. bassiana isolates, CG 449 and CG 550, which were reported to be highly virulent to T. infestans and R. prolixus respectively at RH close to saturation (Romaña & Fargues 1987, Romaña 1992), were distinctly less virulent in the present study when tested at 50% RH and 25°C.

The four B. bassiana isolates, CG 14, CG 24, CG 306 and CG 474 and M. anisopliae isolate CG 144 showed no difference in effectiveness against T. infestans according to concentration of conidia applied. Only M. anisopliae isolate CG 491 was distinctly less virulent. Effectiveness of most isolates tested, particularly of the M. anisopliae isolates (CG 144 and CG 491), was reduced at 50% RH and temperatures of 15 or 20°C. The most virulent isolate at temperatures of 20 and 25°C, temperatures found in domestic habitats of T. infestans, and 50% RH was CG 306, which showed also an elevated virulence at 15 and 30°C. However, isolates CG 14 and CG 24 were the most active at 25 and 30°C. Virulence of the isolate CG 474 to T. infestans proved to be independent of the temperatures tested but was generally reduced compared to the other B. bassiana isolates.

Mietkiewski et al. (1994) found maximal mortality in Galleria melonella treated with M. anisopliae at 30°C. However, the two M. anisopliae isolates tested against T. infestans were not more virulent at 30°C compared to lower temperatures. The intraspecific optimum of temperature for fungal development can vary notably as shown by Moorhouse et al. (1994) who reported a M. anisopliae isolate with highest virulence against the vine weevil, Otiorhynchus sulcatus, at 10°C and another isolate with an optimum at 25°C. Recently Vidal et al. (1997) showed the correlation of geographic origin of Paecilomyces fumo-soroseus isolates and their temperature ranges in terms of vegetative growth on artificial media.

RAPD analysis indicated that the B. bassiana isolates were quite homogeneous, despite their differences in virulence against T. infestans. The virulent isolates were even more homogenous and could not be distinguished by these molecular markers. The analysis of isoenzymes of B. bassiana isolates has shown that it is not possible to correlate molecular polymorphism with virulence (Lecuona et al. 1996) This high similarity could be related to the original host of the strains analyzed. With the exception of CG 16, all others were isolated from heteropteran insects. Maurer et al. (1997) have shown, by RFLP and RAPD analysis, clear relationships between the population structure of B. bassiana and some defined host species. However, a larger sample number of isolates from different origins should be used to allow the identification of related groups.

 

ACKNOWLEDGMENTS

To Raquel A Mello for technical assistance; Jacques Fargues and Roberto Lecuona for providing fungal isolates; Eugenia M Bettiol for bibliographic searches; Peter Inglis for the English review and Bonifácio Magalhães for critical review of the manuscript.

 

REFERENCES

Aljanabi SM, Martinez I 1997. Universal and rapid salt-extraction of high genomic DNA for PCR-based techniques. Nucleic Acid Res 25: 4692-4693.

Dias JCP, Leão AEA 1967. Parasitismo de fungos (Beauveria bassiana) sobre triatomíneos brasileiros criados em laboratório. Atas Soc Biol 2: 85-87.

Doberski JW 1981. Comparative laboratory studies on three fungal pathogens of the elm bark beetle, Scolytus scolytus: effect of temperature and humidity on infection by Beauveria bassianaMetarhizium anisopliae and Paecilomyces farinosus. J Invertebr Pathol 37: 195-200.

Fargues J, Ouedraogo A, Goettel MS, Lomer CJ 1997. Effects of temperature, humidity and inoculation method on susceptibility of Schistocerca gregaria to Metarhizium flavoviride. Biocontrol Science Technol 7: 345-356.

Ferron P 1977. Influence of relative humidity on the development of fungal infection caused by Beauveria bassiana (Fungi imperfecti) in imagines of Acanthoscelides obtectus. Entomophaga 22: 393-396.

Ferron P, Fargues J, Riba G 1991. Fungi as microbial insecticides against pests, p. 665-705. In DK Arora, L Ajello, KG Mukerji (eds), Handbook of Applied MycologyVol. 2, Humans, Animals and Insects, Marcel Dekker Inc., New York.

Fox GA 1993. Failure time analysis: emergence, flowering, survivorship and other waiting times, p. 253-289. In SM Schreiner, J Gurevitch (eds), Design and Analysis of Ecological Experiments, Chapman and Hall, New York.

Glare TR, Milner RJ 1991. Ecology of entomopathogenic fungi, p. 547-612. In DK Arora, L Ajello, KG Mukerji (eds), Handbook of Applied MycologyVol. 2, Humans, Animals and Insects, Marcel Dekker Inc., New York.

Hsiao WF, Bidochka MJ, Khachatourians GG 1992. Effect of temperature and relative humidity on the virulence of the entomopathogenic fungus Verticillium lecanii, toward the oat-bird aphid, Rhopa-losiphum padi (Hom., Aphididae). J Appl Ent 114: 484-490.

Lecuona RE, Tigano MS, Diaz BM 1996. Characterization and pathogenicity of Beauveria bassiana against Diatraea saccharalis (F.) (Lepidoptera: Pyralidae) in Argentina. An Soc Entomol Brasil 25: 299-307.

Lee ET 1980. Statistical Methods for Survival Data Analysis, Wadsworth Inc., Lifetime Learning Publications, Belmont, California, 557 pp.

Luz C 1990. Zur Pathogenität von Beauveria bassiana (Fungi imperfecti) gegenüber mehreren Raub-wanzenarten (Reduviidae, Triatominae) und Einfluss der relativen Luftfeuchtigkeit auf die Infektion von Rhodnius prolixusMitt Dtsch Ges Allg Angew Ent 7: 510-511.

Luz C 1994. Biologische Bekämpfung der Überträger der Chagaskrankheit (Triatominae). Einfluss von Temperatur und Luftfeuchtigkeit auf die larvale Entwicklung von Rhodnius prolixus sowie die Infektion mit Beauveria bassiana (Deuteromycetes) und Sporulation des Pilzes auf den Kadavern, PhD Thesis, Tuebingen, 175 pp.

Luz C, Fargues J, Romaña CA, Moreno J, Goujet R, Rougier M, Grunewald J 1994. Potential of entomopathogenic hyphomycetes for the control of the triatomine vectors of Chagas' disease. Proc 6 Int Coll Invertebr Path Microbiol Control 1: 272-276.

Marcandier S, Khachatourians GG 1987. Susceptibility of the migratory grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera, Acrididae), to Beauveria bassiana (Bals.) Vuillemin (Hypho-mycete): Influence of relative humidity. Can Entomologist 119: 901-907.

Maurer P, Couteaudier Y, Girard PA, Bridge PD, Riba G 1997. Genetic diversity of Beauveria bassiana and relatedness to host insect range. Mycol Res 101: 159- 164.

Mietkiewski R, Tkaczuk C, Zurek M, Geest LPS Van der 1994. Temperature requirement of four entomopathogenic fungi. Acta Mycologica 29: 109-120.

Moorhouse ER, Gillespie AT, Charnley AK 1994. The influence of temperatures on the susceptibility of vine weevil, Otiorhynchus sulcatus (Fabricius) (Coleoptera: Curculionidae), larvae to Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). An Appl Biol 124: 185-193.

Romaña CA 1992. Recherches sur les Potentialités des Hyphomycetes Entomopathogènes (Fungi imperfecti) dans la Lutte Contre les Triatominae (Heteroptera), Thèse de Doctorat, Montpellier, 129 pp.

Romaña CA, Fargues J 1987. Sensibilité des larves de l'hémiptère hématophage Rhodnius prolixus (Triatominae) aux hyphomycètes entomopathogènes. Entomophaga 32: 167-179.

Romaña C, Romaña C 1981. Experimental infection of Triatoma infestans with the fungus Beauveria tenella, p. 215-217. In EV Canning, Parasitological Topics. A Presentation Volume to PCC Garnham FRF on the Occasion of his 80th Birthday, Allen Press Inc., Kansas, USA.

SAS Institute Inc. 1989. SAS/STAT User's Guide, Version 6; Fourth Edition, Volume 1, Cary, NC: SAS Institute Inc., 943 pp.

Sambrook J, Fritsch EF, Maniatis T 1989. Molecular Cloning: A Laboratory Manual, 2nd, ed., Cold Spring Harbor Laboratory Press, NY.

Scott AJ, Knott M 1974. A cluster analysis method for grouping means in the analysis of variance. Biometrics 30: 507-512.

Sherlock IA, Guitton N 1982. Observações sobre a ação do fungo Metarhizium anisopliae Metsch. sobre algumas espécies de Triatominae (Hemiptera, Reduviidae). Rev Inst Med Trop 24: 234-239.

Silva JC, Messias CL 1985. Virulência de Metarhizium var. anisopliae a Rhodnius prolixusCienc Cult 7: 37-40.

Sneath PHA, Sokal RR 1973. Numerical Taxonomy, Freeman, San Francisco, 573 pp.

Tigano-Milani MS, Honeycutt RJ, Lacey LA, Assis R, McClelland M, Sobral BWS 1995. Genetic variability of Paecilomyces fumosoroseus isolates revealed by molecular markers. J Invertebr Pathol 65: 274-282.

Vidal C, Fargues J, Lacey LA 1997. Intraspecific variability of Paecilomyces fumosoroseus: Effect of temperature on vegetative growth. J Invertebr Pathol 70: 18-26.

Winston PW, Bates DH 1960. Saturated solutions for the control of humidity in biological research. Ecology 41: 232-237.

+Corresponding author. Fax: +55-62-202.3066. E-mail:  This e-mail address is being protected from spambots. You need JavaScript enabled to view it.  
Received 20 February 1998
Accepted 27 July 1998

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