Laboratory bioassays were conducted to assess the virulence of two entomopathogenic fungi, Beauveria bassiana, and Metarhizium anisopliae to 5th instars of the banded sunflower moth, Cochylis hospes,(Lepidoptera: Cochylidae). Temperature conditions of 20 and 25°C and high humidity, (near saturation) were nearly optimal for development of both fungi. Concentrations of 10(7) to 10(8) conidia/ml produced 100% mortality in 10 days or less and 106 conidia/ml produced 90% mortality at 21 to 26 days. Median lethal concentrations of conidia (LC50) from M. anisopliae were 3.6 x 103 at 25°C and 4.1 x 103 at 20°C. The LC50 for B. bassiana was 14.9 x 104 conidia/ml at 20°C and 6.7 x 103 conidia/ml at 25°C. Although B. bassiana tended to be less virulent at 20°C, these differences were not significant. The high humidities required for germination and growth may reduce the usefulness of these fungi as con­trol agents of C. hospes in the northern Great Plains. Further studies and field evaluations are needed to determine if there are microhabitats in the soil or on the sunflower head where the humidity is high enough for germination and growth of B. bassiana or M. anisopliae. Targeting of C. hospes stages in the soil to avoid contaminating the seed or oil with saprophytic fungal spores may be preferred to targeting the sunflower plant for reasons of preserving seed quality, marketing, and consumption

Barker, John F

English

Valparaiso University

The Great Lakes Entomologist Volume 32 Number 3 - Fall 1999 Number 3 - Fall 1999 Article 1 October 1999 Laboratory Evaluation of the Pathogenicity of Beauveria Bassiana and Metarhizium Anisopliae to Larvae of the Banded Sunflower Moth, Cochylis Hospes (Lepidoptera: Cochylidae). John F. Barker USDA Follow this and additional works at: https://scholar.valpo.edu/tgle Part of the Entomology Commons Recommended Citation Barker, John F. 1999. "Laboratory Evaluation of the Pathogenicity of Beauveria Bassiana and Metarhizium Anisopliae to Larvae of the Banded Sunflower Moth, Cochylis Hospes (Lepidoptera: Cochylidae).," The Great Lakes Entomologist, vol 32 (2) Available at: https://scholar.valpo.edu/tgle/vol32/iss2/1 This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 1999 101THE GREAT LAKES ENTOMOLOGIST LABORATORY EVALUATION OF THE PATHOGENICITY OF BEAUVER/A BASS/ANA AND METARH/Z/UM AN/SOPLIAE TO LARVAE OF THE BANDED SUNFLOWER MOTH, COCHYLIS HOSPES (LEPIDOPTERA: COCHYLlDAE). john F. Barker1 ABSTRACT Laboratory bioassays were conducted to assess the virulence of two ento­mopathogenic fungi, Beauveria bassiana, and Metarhizium anisopliae to 5th instars of the banded sunflower moth, Cochylis hospes,(Lepidoptera: Cochyli­dae). Temperature conditions of 20 and 5°C and high humidity, (near satu­ration) were nearly optimal for development of both fungi. Concentrations of 107 to 108 conidia/ml produced 100% mortality in 10 days or less and 106 conidia/ml produced 90% mortality at 21 to 26 days. Median lethal concen­trations of conidia (LC 50) from M. anisopliae were 3.6 x 103 at 25°C and 4.1 x 103 at 20°C. The LC50 for B. bassiana was 14.9 x 104 conidia/ml at 20°C and 6.7 x 103 conidia/ml at 25°C. Although B. bassiana tended to be less virulent at 20°C, these differences were not significant. The high humidities required for germination and growth may reduce the usefulness of th e fungi as con­trol agents of C. hospes in the northern Great Plains. Further studies and field evaluations are needed to de ermine if there are microhabitats in the soil or on the sunflower h ad where the hum dity is high enough for germi­nation and growth of B. bassiana or M. anisopliae. Targeting of C. hospes stages in the soil to avoid contaminating the seed or oil with saprophytic fun­gal spores may be preferred to targeting the sunflower plant for reasons of preserving seed quality, marketing, and consumption. Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotinia: Hyphomycetes) are entomopatho­genic fungi that occur in the soil, have a wide insect host range, and are dis­tributed worldwide (Tanada and Kaya 1993). Virulence to insects varies with the fungal variety or strain, dose, host age, and environmental conditions, particularly emperature and humidity (Miller et al. 1983, Tanada and Kaya 1993). Infection usually takes p ace through chitinolytic penetration of the in­tegument by fungal hyphae and both genera produce mycotoxins CTanada n Kaya 1993). Beauveria bassiana and M. anisopliae are are being studied as alternatives to chemical pesticides for the control of a variety of insect species (Tanada d Kaya 1993, Miller et al. 1983) but neither has been significantly explored for microbial control of the banded sunflower moth Cochylis hospes, or other sunflower insect pests. The life cycle of C. hospes begins about the third week in July when eggs are laid on the bracts of sunflower. The larval 1 USDA, ARS, Biosciences Research Laboratory, P. O. Box 5674, State University Station, Fargo, ND 58105. 1Barker: Laboratory Evaluation of the Pathogenicity of Beauveria BassiaPublished by ValpoScholar, 1999102 THE GREAT LAKES ENTOMOLOGIST Vol. 32, No.3 stages feed on the florets and seeds of the sunflower head. The fifth and ulti­mate instar leaves the sunflower head in late August and early September to ovenvinter in the and pupates in late spring (Westdal 1949, Charlet and Gross 1990, Charlet al. 1997, Beregovoy and Riemann 1989). The adults emerge about mid-July and begin a new generation. The objective of this study was to assess, in controlled laboratory conditions, the virulence of thes fungi to fifth instars and pupae that overwinter in the soil. The latter stages were targeted for control in this study, because application to the sunflower head of large numbers of conidia from saprophytic fungi to control the earlier ins ars, could reduce the quality or acceptability of the seed or oil by con­sumers and the sunflower industry. The high humidity needed for fungal growth is more probable in the soil than on the sunflower capitulum where high humidity is incompatible with maintaining seed quality. MATERIALS AND METHODS Insects and rearing conditions. Cochylis hospes is routinely reared on insect diet in laboratory environmental chambers maintained at 28 ± 1°C, 50% relative humidity, and a 15:9 L:D cycle (Barker 1988). Fifth instars that had ceased feeding were selected primarily because this stage completes its development in the soil where it was suggested that control be targeted and, secondarily, it circumvented complications that would arise from diet spoilage in the presence of saprophytic fungi. In addition, ingestion of coni­dia was not required since infection of insects with fungi is primaril throu h the integument (Tanada and Kaya 1993). Cessation of feeding e 5th-in­star of C. hospes is marked by a change in color about 2 days the molt to the fifth instal'. The insect diet contains Benomy1 (Bonide Products I c. Yorkville, N.YJ which will inhib t but will not prevent fungal growth, how­ever, contamination of the diet is controlled primarily with the use of a ster­ile hood, materials, utensils, and control of excess moisture (Barker 1988). Fungi. M. anisopliae (ATCC # 22099) was obtained from the American Type Culture Collection, Rockville, MD. A strain of B. bassiana, formulated into a commercial microbial insecticide Naturalis-L®, was obtained from Troy Biosciences, (Phoenix AZ). Naturalis-L and ATCC 22099 were stock prepara­tions which were sub-cultured on potato dextose agar plates to produce coni­dia for bioassay. Bioassay. Conidia from sub-cultures of Naturalis-L or ATCC 22099 stock were suspended in 10mM phosphoric acid buffered saline, pH 7.0, prepared with 0.5 % Tween 80 to reduce clumping of the conidia and counted with a hemocytometer. Serial dilutions were prepared with the following concentra­tions of conidia/ml: 108, 107, 106, 105, 104, 103, 102, or 0 (controls). This method of assay has a limitation in that it does not define precisely what dosage each larva but it does define the concentration of conidia to which each larva was exposed. A 1 and a lOplsample of each concentration was streaked on potato dextrose agar plates to check for viability. Thirty, 5th­instal' larvae were transferred to each of the tubes containing conidia and gently shaken for 5-10 sec. by inverting the tube. Longer periods of immer­sion were avoided because of detrimental effects on larval development and survival that were not related to infection. The 30 larvae were confined indi­vidually in 1 ml vented microfuge tubes with a piece of moist p tt ng foam provided for pupation. Moisture was added to the pot ing foam and replen­ished as necessary to facilitate survival of pupae and their successful emer­gence as adults and to facilitate growth of the fungi. Tubes were vented by punching a hole (diameter 4mm) into the cap of the tube and covered with 2The Great Lakes Entomologist, Vol. 32, No. 2 [1999], Art. 1https://scholar.valpo.edu/tgle/vol32/iss2/11999 	 103THE GREAT LAKES ENTOMOLOGIST Nitex (Tetko, Briarcliff Manor, NY) 2-ply polyester (70 threads per cm). The larvae were held at 20±1°C or 25±1cC in environmental chambers main­tained on a 15:9 L:D cycle and 60-70% relative humidity. The humidity within the microfuge tubes, with the moist potting foam, was near satura­tion. Observations for mort lity began on the second day after treatment and every other day thereafter for 21 or 26 d at 25 and 20cC, respectively, until the controls and treated insects that survived had pupated and emerged as adults. The LCso of the treated larv e wa determined at 21d (25°C) and 26 d (20°C). adavers that did not show obvious mycelial growth were macerated with a forceps and the debris streaked on potato dextrose agar and observed for mycelial growth. The bioassay was replicated eight times and an overall average response for each concentration was obtained by averagi g the data from the eight replicates. Statistical methods. POLO-PC (LeOra Software 1987, Berkeley, CA) was used to estimate the L50s and determine the Chi square goodness of fit. Jandel SigmaPlot (version 5, 1992 and version 2, 1995, San Rafael, CA) and Finney (1947) were used to plot probit regression equations. RESULTS AND DISCUSSION Mortality in relation to concentration of conidia and tempera­ture. Mortality that resulted from treatment of C. hospes 5th instars with conidia from B. bassiana and M. anisopliae was 100% in 10 d or less after treatment with 108 and 107 conidia/ml at both temperatures. Fifth instars begin to pupate about 13 days after becoming fifth i stars at 25°C (Barker 1994) but none of the fifths treated with 108 or 7 conidia survived to the pupal stage at 20 or 25°C. Mortality after treatment with 106 conidia of both fungi was approximately 90% at both temperatures over a period f 21 to 26 d (Table 1) while the controls and survivors completed development to the adult stage. The majority of fifths treated v;rith 106 conidia per ml died as fifth instars because development of infected larvae to the pupal stage was retarded relative to the controls, although some pupated and then died. Dia­pause was not induced in the fifths held at 20°C in this study because the Table 1. Mortality of C. hospes larvae after infection with conidia of B. bassiana or M. anisopliae at 20 and 25Q C. 20°C 25°C % mortality % mortality [Cone) 26d 	 21 d M. anisopliae 106 95.8 ± 2.5 92.0 ± 8.5 105 93.3 ± 1.9 83.3 ± 7.7 104 68.0 ± 9.0 63.3 ± 18.4 103 30.0 ± 8.7 37.5 ± 8.8 102 11.1 ± 6.2 16.7 ± 10.9 0 	 6.7 ± 2.2 3.3 ± 2.7 B. bassiana 	 106 88.7 5.9 91.7 3.3 105 68.4 ± 8.4 73.3 ± 16.3 104 48.9 ± 8.8 54.0 ± 12.7 103 32.2 ± 5.1 35.3 ± 4.8 102 10.7 ± 0.7 13.3 ± 6.9 0 	 5.6 ± 4.3 1.3 ± 1.0 3Barker: Laboratory Evaluation of the Pathogenicity of Beauveria BassiaPublished by ValpoScholar, 1999104 THE GREAT LAKES ENTOMOLOGIST Vol. 32, No.3 Table 2. Estimated median lethal concentrations of conid a for C. hospes 5th instars. LC50 Lower limit Upper limit Treatment Temp. Conidialml 0.95 0.95 ± S. E. M. anisopliae 20°C 4,122 2,531 6,269 1.042 ± 0.114 M. anisopliae 3,651 2,111 6,184 0.662 ± 0.071 B. bassiana 14,956 8,122 26,389 0.621 ± 0.060 B. bassiana 8,160 5,268 12,486 0.592 ± 0.051 larvae were reared to the fifth instar under non-diapause inducing conditions (Barker 1994) and then placed at 20°C as control or test insects. Mortality trended lower for both fungi, at both temperatures, in response to exposure to lower concentrations of conidia. The estimated LC50 values of M. aniso­pliae conidia at 20 and 25°C were 4.1 x 103 and 3.6 x 103 conidia/ml at 26 d and 21 d, respectively (Table 2). The estimated LC50 values for larvae treated with B. bassiana at 20 and 25°C were 14.9 x 104 and 8.1 x 103 conidia/ml re­spectively (Table 2). LCso values and slopes in relation to temperature. Optimum tem­peratures for development, pathogenicity, and survival of fungi generally fall between 20 to 30°C (McCoy et al. 1988). The optimal growth temperatures for Beauueria are 23-25°C and 27-28°C for Metarhizium (Ferron 1981). Gen­erally, there is a decline in virulence of entomopathogenic fungi with declin­ing temperature (Deacon 1983, Rath et al. 1995) outside the optimal temper­ature range. The LC50 values at 20 and 25°C overlapped for both fungi indicating that the change of temperature from 25 to 20°C did not signifi­cantly affect mortality. The overlapping regression lines for M. ani opliae did not clearly demonstrate a trend toward lower virulence at 20°C. B. bassiana showed a trend toward reduced virulence at the lower temperatur (Fig.1). The slopes of the regression lines for B. bassiana at 20°C and 25°C are not significantly different since the lines are nearly parallel but the lines reflect a lower virulence at 20°C than at 25°C. Chi-square heterogenity < 1 indi­cated a good fit of the regression equations to the data in each case and P::::;0.05 in the Chi-square goodness of fit test. The results of this study show that B. bassiana and M. anisopliae are pathogenic to C. hospes larvae in laboratory conditions where the tempera­ture and humidity are controlled. The humidity level (around 90%) required by these fungi for optimal germination and infection (Deacon 1983) may limit their use as control agents to periods of high humidity. Studies of microhabi­tats in the sunflower head or the s il may indicate that in those environ­ments the humidity levels are high enough for the development of these fungi. High humidity needed for fungal growth is more probable in the soil than on the sunflower capitulum where it is undesirable because it is incom­patible with maintaining seed quality. Application to the su fl wer head of large numbers of conidia from saprophytic fungi should be avoided since it could reduce the quality or acceptability of the seed or oil. Humidity levels in the soil are probably 90% under some climatic conditions, but targeting con­trol to C. hospes stages in the soil can only be directed to the 5th instar which enters the soil to overwinter, or to the pupal stage which develops in the soil. Application of conidia to the soil in the fall or in the spring and early summer to control overwintering larval and pupal stages of the next generation of C. hospes is a possibility that circumvents objections to treatment of he sun­flower capitulum. Infectivity of the fungus in the soil, however, could be slowed due to suboptimal temperatures that prevail in the fall and spring months. The total mortality from infection with these fungi may be unaf­4The Great Lakes Entomologist, Vol. 32, No. 2 [1999], Art. 1https://scholar.valpo.edu/tgle/vol32/iss2/11999 105THE GREAT LAKES ENTOMOLOGIST 7 ~ 6 -1 t=: c:::: 0 :2 5 III Y = 1.325 + 1.042X I­eo 20°C 0 •c:::: Y = 2.601 + 0.662Xa. 4· 2SoC 3 1 2 3 4 5 6 7 LOG DOSE (M. AN/SOPLIAE. CONIDIA I ML) 7 f; 6 -1 t=: c:::: o 5:2 II1II Y = 2.425 + 0.621XI­eo 20°Co c:::: 0... 4 Y = 2.736 + 0.S92X• 2SoC 3 ~----~--~----~----~----~--~ 1 234 5 6 7 LOG DOSE (B. BASS/ANA CONIDIA I ML) Figure 1. Probit regression lines for the mortality of C. hospes fifth instars treated with conidia from M. anisopliae and B. bassiana at 20 and 25°C. 5Barker: Laboratory Evaluation of the Pathogenicity of Beauveria BassiaPublished by ValpoScholar, 1999-106 THE GREAT LAKES ENTOMOLOGIST Vol. 32, No.3 fected at suboptimal temperatures except that it occurs over x ended peri­ods of time (Ferron 1978, Rath et al. 1995). Isolation or development of fun­strains that germinate and develop at lower temperature and humidity is another alternative. ACKNOWLEDGMENTS The author thanks Sharon Grugel for insect rearing and diet prepara­tion. This article reports the results of research only. Mention of a propri­etary product does not constitute an endorsement or recommendation by the USDA for its use. LITERATURE CITED Barker J. 1988. Laboratory rearing of the banded sunflower moth, Cochylis hospes,(Lepidoptera: Cochylidae). J. Kansas Entomol. Soc. 61:350-352. Barker J. 1994 Development of laboratory reared banded sunflower moth, Cochylis hospes Walsingham (Lepidoptera: Cochylidae), in relation to temperature. J. Kansas Entomo!. Soc. 66:420-426. Beregovoy, V. H. and J. G. Riemann. 1987. Infestation phenology of sunflower by the banded sunflower moth, Cochylis hospes (Cochylidae: Lepidoptera) in the northern plains. J. Kansas Entomol. Soc. 60:517-527. Charlet, L. D., G. J. Brewer and B. Franzmann. 1997. Insect pests, pp. 183-261. In: A. A. Schneiter (ed.), Sunflower technology and production. Agron. Ser. 35. Am. Soc. Agron., Madison, WI. 834 pp. Charlet, L. D. and T. A. Gross. 1990. Bionomics and seasonal abunda ce of the banded sunflower moth (Lepidoptera: Cochylidae) on cultivated sunflower in the northern Great Plains. J. Econ. Entomo!. 83:135-141. Deacon, J. W. 1983 . .Microbial control of plant pests and diseases. Am. Soc. Microbiol., Wash. DC. 88 pp. P. 1981. Pest control by the fungi, Beauveria and Metarhiziurn, pp. 465-476. In: D. Burges (ed.), Vol. 2, Microbial control of pests and plant diseases. Academic Press, New York. 949 pp. Ferron, P. 1978. Biological control of insect pests by entomogenous fungi. Annu. Rev. Ent mo!. 23: 409-442. Finney, D. J. 1947. Probit analysis, a statistical treatment of the sigm id response curve. Cambridge University Pr s , UK. 256 pp. Jandel SigmaPlot software. 1992. SigmaPlot scientific graphing software. User's man­ual. San Rafael, CA. LeOra Software. 1987. POLO-PC a user's guide to Probit Or Logit analysis. Berkeley, CA . .McCoy, C. W., Hill, A. J. and R. F. Kanavel. 1988. Entomogenous fungi, pp. 151-236. In: C. M. Ignoffo (ed.), CRC handbook of natural pesticides. Microbial Insecticides, Part A. Entogenous Protozoa and Fungi, vo!' 5. CRC Press, Boca Raton, FL. Miller, L. K., A. J. and L. A. Bulla, Jr. 1983. Bacterial, viral, and fungal insecti­cides. Science 219: Rath, A. C., G. C. Anderson, D. Worledge and T. B. Koen. 1995. The effecto low tem­peratures on the virulence of Metarhizium anisopliae (Dat F-001) to the subter­ranean scarab, Adoryphorus coulon!. J. Invert. Pathol. 65: 186-192. Tanada, Y. and H. K. Kaya. 1993. Insect pathology. Academic Press, San Diego. 666 pp. Westdal, P. H. 1949. A preliminary report on the biology of Phalonia hospes (Walsing­ham) (Lepidoptera: Phaloniidae), a new pest of sunflowers i Manitoba. 80th Annu. Rep. EntomoL Soc. Ontario, Canada, pp.1-3. 6The Great Lakes Entomologist, Vol. 32, No. 2 [1999], Art. 1https://scholar.valpo.edu/tgle/vol32/iss2/1

Laboratory Evaluation of the Pathogenicity of \u3ci\u3eBeauveria Bassiana\u3c/i\u3e and \u3ci\u3eMetarhizium Anisopliae\u3c/i\u3e to Larvae of the Banded Sunflower Moth, \u3ci\u3eCochylis Hospes\u3c/i\u3e (Lepidoptera: Cochylidae).

John F Barker

Crossref

 (Lepidoptera: Cochylidae).

ValpoScholar

The Great Lakes Entomologist 
Volume 32 
Number 3 - Fall 1999 Number 3 - Fall 1999 Article 1 
October 1999 
Laboratory Evaluation of the Pathogenicity of Beauveria Bassiana 
and Metarhizium Anisopliae to Larvae of the Banded Sunflower 
Moth, Cochylis Hospes (Lepidoptera: Cochylidae). 
John F. Barker 
USDA 
Follow this and additional works at: https://scholar.valpo.edu/tgle 
 Part of the Entomology Commons 
Recommended Citation 
Barker, John F. 1999. "Laboratory Evaluation of the Pathogenicity of Beauveria Bassiana and Metarhizium 
Anisopliae to Larvae of the Banded Sunflower Moth, Cochylis Hospes (Lepidoptera: Cochylidae).," The 
Great Lakes Entomologist, vol 32 (2) 
Available at: https://scholar.valpo.edu/tgle/vol32/iss2/1 
This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. 
It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. 
For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 
1999 101THE GREAT LAKES ENTOMOLOGIST 
LABORATORY EVALUATION OF THE PATHOGENICITY OF 

BEAUVER/A BASS/ANA AND METARH/Z/UM AN/SOPLIAE 

TO LARVAE OF THE BANDED SUNFLOWER MOTH, 

COCHYLIS HOSPES (LEPIDOPTERA: COCHYLlDAE). 

john F. Barker1 
ABSTRACT 
Laboratory bioassays were conducted to assess the virulence of two ento­
mopathogenic fungi, Beauveria bassiana, and Metarhizium anisopliae to 5th 
instars of the banded 
sunflower moth, 
Cochylis hospes,(Lepidoptera: Cochyli­
dae). Temperature conditions of 20 and 5°C and high humidity, (near satu­
ration) were nearly optimal for development of both fungi. Concentrations of 
107 to 108 conidia/ml produced 100% mortality in 10 days or less and 106 
conidia/ml produced 90% mortality at 21 to 26 days. Median lethal concen­
trations of 
conidia (LC 50
) from M. anisopliae were 3.6 x 103 at 25°C and 4.1 x 
103 at 20°C. The LC50 for B. bassiana was 14.9 x 104 conidia/ml at 20°C and 
6.7 x 103 conidia/ml at 25°C. Although B. bassiana tended to be less virulent 
at 
20°C, 
these differences were not significant. The high humidities required 
for germination and growth may reduce the usefulness of th e fungi as con­
trol agents of C. hospes in the northern Great Plains. Further studies and 
field evaluations are needed to de ermine if there are microhabitats in the 
soil or on the sunflower h ad where the hum dity is high enough for germi­
nation and growth 
of 
B. bassiana or M. anisopliae. Targeting of C. hospes 
stages in the soil to avoid contaminating the seed or oil with saprophytic fun­
gal spores may be preferred to targeting the sunflower plant for reasons of 
preserving seed quality, marketing, and consumption. 
Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae 
(Metschnikoff) Sorokin (Deuteromycotinia: Hyphomycetes) are entomopatho­
genic fungi that occur in the soil, have a wide insect host range, and are dis­
tributed 
worldwide (Tanada 
and Kaya 1993). Virulence to insects varies with 
the 
fungal 
variety or strain, dose, host age, and environmental conditions, 
particularly emperature and humidity (Miller et al. 1983, Tanada and Kaya 
1993). Infection usually takes p ace through chitinolytic penetration of the in­
tegument 
by fungal 
hyphae and both genera produce mycotoxins CTanada n 
Kaya 
1993). 
Beauveria bassiana and M. anisopliae are are being studied as 
alternatives 
to chemical pesticides for 
the control of a variety of insect species 
(Tanada d Kaya 1993, Miller et al. 1983) but neither has been significantly 
explored for microbial control of the banded sunflower moth Cochylis hospes, 
or other sunflower insect pests. The life cycle of C. hospes begins about the 
third 
week 
in July when eggs are laid on the bracts of sunflower. The larval 
1 USDA, ARS, Biosciences Research Laboratory, P. O. Box 5674, State University 
Station, Fargo, ND 58105. 
1
Barker: Laboratory Evaluation of the Pathogenicity of Beauveria Bassia
Published by ValpoScholar, 1999
102 THE GREAT LAKES ENTOMOLOGIST Vol. 32, No.3 
stages feed on the florets and seeds of the sunflower head. The fifth and ulti­
mate instar 
leaves 
the sunflower head in late August and early September to 
ovenvinter in the and pupates in late spring 
(Westdal 1949, 
Charlet and 
Gross 1990, Charlet al. 1997, Beregovoy and Riemann 1989). The adults 
emerge about mid-July and begin 
a 
new generation. The objective of this 
study 
was to assess, 
in controlled laboratory conditions, the virulence of thes 
fungi to fifth instars and pupae that overwinter in the soil. The latter stages 
were targeted 
for control 
in this study, because application to the sunflower 
head of large numbers of 
conidia from saprophytic fungi to control 
the earlier 
ins ars
, could reduce 
the quality or acceptability of the seed or oil by con­
sumers and the sunflower 
industry. 
The high humidity needed for fungal 
growth is more probable in the soil than on the sunflower capitulum where 
high humidity 
is incompatible 
with maintaining seed quality. 
MATERIALS AND METHODS 
Insects and rearing conditions. Cochylis hospes is routinely reared on 
insect 
diet 
in laboratory environmental chambers maintained at 28 ± 1°C, 
50% relative humidity, and a 15:9 L:D cycle (Barker 1988). Fifth instars that 
had 
ceased feeding were selected 
primarily because this stage completes its 
development in the soil where it was suggested that control be targeted and, 
secondarily, it circumvented complications that would arise from diet 
spoilage in the presence of saprophytic fungi. In addition, ingestion of coni­
dia was not required 
since infection 
of insects with fungi is primaril throu h 
the integument (Tanada and Kaya 
1993). 
Cessation of feeding e 5th-in­
star of C. hospes is marked by a change in color about 2 days the molt 
to the fifth instal'. The insect diet contains Benomy1 (Bonide Products I c. 
Yorkville, N.YJ which will inhib t but will not prevent fungal growth, how­
ever, contamination of the diet is controlled primarily with the use of a ster­
ile hood, materials, utensils, and control of excess moisture (Barker 1988). 
Fungi. M. anisopliae (ATCC # 22099) was obtained from the American 
Type Culture Collection, Rockville, MD. A strain of B. bassiana, formulated 
into a commercial microbial insecticide Naturalis-L®, was obtained from Troy 
Biosciences, (Phoenix AZ). Naturalis-L and ATCC 22099 were stock prepara­
tions which were sub-cultured on potato dextose agar plates to produce coni­
dia 
for bioassay. Bioassay. 
Conidia from 
sub-cultures of Naturalis-L or ATCC 22099 stock 
were suspended in 
10mM phosphoric acid buffered saline, 
pH 7.0, prepared 
with 
0.5 % Tween 
80 to reduce clumping of the conidia and counted with a 
hemocytometer. Serial dilutions were prepared with the following concentra­
tions of 
conidia/ml: 10
8
, 107, 106, 105, 104, 103, 102, or 0 (controls). This 
method of assay has 
a 
limitation in that it does not define precisely what 
dosage each larva but it does define the concentration of conidia to 
which each larva was exposed. A 1 and a lOplsample of each concentration 
was streaked 
on potato dextrose 
agar plates to check for viability. Thirty, 5th­
instal' larvae were transferred 
to 
each of the tubes containing conidia and 
gently shaken for 5-10 sec. by inverting the tube. Longer periods of immer­
sion were avoided because of detrimental effects on larval development and 
survival that were not related 
to infection. 
The 30 larvae were confined indi­
vidually in 
1 ml 
vented microfuge tubes with a piece of moist p tt ng foam 
provided for pupation. Moisture was added to the pot ing foam and replen­
ished as necessary 
to facilitate 
survival of pupae and their successful emer­
gence as adults and to facilitate growth of the fungi. Tubes were vented by 
punching 
a hole 
(diameter 4mm) into the cap of the tube and covered with 
2
The Great Lakes Entomologist, Vol. 32, No. 2 [1999], Art. 1
https://scholar.valpo.edu/tgle/vol32/iss2/1
1999 	 103THE GREAT LAKES ENTOMOLOGIST 
Nitex (Tetko, Briarcliff Manor, NY) 2-ply polyester (70 threads per cm). The 
larvae were held at 
20±1°C 
or 25±1cC in environmental chambers main­
tained on 
a 15:9 L:D cycle 
and 60-70% relative humidity. The humidity 
within the 
microfuge 
tubes, with the moist potting foam, was near satura­
tion. Observations for mort lity began on the second day after treatment and 
every other day thereafter for 21 or 26 d at 25 and 20cC, respectively, until 
the 
controls 
and treated insects that survived had pupated and emerged as 
adults. The LCso of the treated larv e wa determined at 21d (25°C) and 26 d (20°C). adavers that did not show obvious mycelial growth were macerated 
with a forceps and the debris streaked on potato dextrose agar and observed 
for mycelial growth. The bioassay was replicated eight times and an overall 
average 
response for 
each concentration was obtained by averagi g the data 
from the eight replicates. 
Statistical methods. 
POLO-PC 
(LeOra Software 1987, Berkeley, CA) 
was used 
to 
estimate the L50s and determine the Chi square goodness of fit. 
Jandel SigmaPlot 
(version 5, 1992 
and version 2, 1995, San Rafael, CA) and 
Finney 
(1947) were 
used to plot probit regression equations. 
RESULTS AND DISCUSSION 
Mortality in relation to concentration of conidia and tempera­
ture. Mortality that resulted 
from 
treatment of C. hospes 5th instars with 
conidia from B. bassiana and M. anisopliae was 100% in 10 d or less after 
treatment with 
10
8 and 107 conidia/ml at both temperatures. Fifth instars 
begin to pupate about 13 days after becoming fifth i stars at 25°C (Barker 
1994) but none of the fifths treated with 108 or 7 conidia survived to the 
pupal stage at 
20 or 25°C. Mortality 
after treatment with 106 conidia of both 
fungi was approximately 90% at both temperatures over a period f 21 to 26 
d (Table 1) while the controls and survivors completed development to the 
adult 
stage. 
The majority of fifths treated v;rith 106 conidia per ml died as 
fifth instars because development of infected larvae to the pupal stage was 
retarded relative 
to 
the controls, although some pupated and then died. Dia­
pause was not 
induced 
in the fifths held at 20°C in this study because the 
Table 1. Mortality of C. hospes larvae after infection with conidia of B. bassiana or M. 
anisopliae at 20 and 25Q C. 
20°C 25°C 
% mortality % mortality 
[Cone) 26d 	 21 d 
M. anisopliae 106 95.8 ± 2.5 92.0 ± 8.5 
105 93.3 ± 1.9 83.3 ± 7.7 
104 68.0 ± 9.0 63.3 ± 18.4 
103 30.0 ± 8.7 37.5 ± 8.8 
102 11.1 ± 6.2 16.7 ± 10.9 
0 	 6.7 ± 2.2 3.3 ± 2.7 
B. bassiana 	 106 88.7 5.9 91.7 3.3 
105 68.4 ± 8.4 73.3 ± 16.3 
104 48.9 ± 8.8 54.0 ± 12.7 
103 32.2 ± 5.1 35.3 ± 4.8 
102 10.7 ± 0.7 13.3 ± 6.9 
0 	 5.6 ± 4.3 1.3 ± 1.0 
3
Barker: Laboratory Evaluation of the Pathogenicity of Beauveria Bassia
Published by ValpoScholar, 1999
104 THE GREAT LAKES ENTOMOLOGIST Vol. 32, No.3 
Table 2. Estimated median lethal concentrations of conid a for C. hospes 5th instars. 
LC50 Lower limit Upper limit 
Treatment 
Temp. Conidialml 0.95 
0.95 ± S. E. 
M. 
anisopliae 
20°C 4,122 2,531 6,269 1.042 ± 0.114 
M. anisopliae 3,651 2,111 6,184 0.662 ± 0.071 
B. bassiana 14,956 8,122 26,389 0.621 ± 0.060 
B. bassiana 8,160 5,268 12,486 0.592 ± 0.051 
larvae were reared to the fifth instar under non-diapause inducing conditions 
(Barker 
1994) 
and then placed at 20°C as control or test insects. Mortality 
trended 
lower for 
both fungi, at both temperatures, in response to exposure 
to lower concentrations of conidia. The estimated LC50 values of M. aniso­
pliae conidia at 20 and 25°C were 4.1 x 103 and 3.6 x 103 conidia/ml at 26 d 
and 
21 d, respectively (Table 
2). The estimated LC50 values for larvae treated 
with B. bassiana at 20 and 25°C were 14.9 x 104 and 8.1 x 103 conidia/ml re­
spectively (Table 2). 
LCso values and slopes in relation to temperature. Optimum 
tem­peratures 
for development, pathogenicity, 
and survival of fungi generally fall 
between 20 to 30°C (McCoy et al. 1988). The optimal growth temperatures 
for Beauueria are 23-25°C and 27-28°C for Metarhizium (Ferron 1981). Gen­
erally, there is a decline in virulence of entomopathogenic fungi with declin­
ing temperature 
(Deacon 1983, 
Rath et al. 1995) outside the optimal temper­
ature range. The 
LC
50 values at 20 and 25°C overlapped for both fungi 
indicating that the change of temperature from 25 to 20°C did not signifi­
cantly 
affect mortality. 
The overlapping regression lines for M. ani opliae did 
not clearly demonstrate a trend toward lower virulence at 20°C. B. bassiana 
showed a trend toward reduced virulence at the lower temperatur (Fig.1). 
The slopes of the regression lines for B. bassiana at 20°C and 25°C are not 
significantly different since the lines are nearly parallel but the lines reflect 
a lower virulence at 20°C than at 25°C. Chi-square heterogenity < 1 indi­
cated 
a good 
fit of the regression equations to the data in each case and 
P::::;0.05 
in 
the Chi-square goodness of fit test. 
The results 
of 
this study show that B. bassiana and M. anisopliae are 
pathogenic to C. hospes larvae in laboratory conditions where the tempera­
ture and humidity are 
controlled. The 
humidity level (around 90%) required 
by these fungi for optimal germination and infection (Deacon 1983) may limit 
their use as 
control 
agents to periods of high humidity. Studies of microhabi­
tats in the 
sunflower 
head or the s il may indicate that in those environ­
ments the humidity 
levels 
are high enough for the development of these 
fungi. High humidity needed for fungal growth is more probable in the soil 
than 
on 
the sunflower capitulum where it is undesirable because it is incom­
patible with maintaining seed quality. Application to the su fl wer head of 
large numbers of 
conidia from saprophytic fungi 
should be avoided since it 
could reduce the quality or acceptability of the seed or oil. Humidity levels in 
the 
soil 
are probably 90% under some climatic conditions, but targeting con­
trol to C. hospes stages in the soil can only be directed to the 5th instar which 
enters the 
soil 
to overwinter, or to the pupal stage which develops in the soil. 
Application of conidia to the soil in the fall or in the spring and early summer 
to control overwintering larval and pupal stages of the next generation of C. 
hospes is a possibility that circumvents objections to treatment of he sun­
flower capitulum. Infectivity of the fungus in the soil, however, could be 
slowed due to suboptimal temperatures that prevail in the fall and spring 
months. The total mortality from infection with these fungi may be unaf­
4
The Great Lakes Entomologist, Vol. 32, No. 2 [1999], Art. 1
https://scholar.valpo.edu/tgle/vol32/iss2/1
1999 105THE GREAT LAKES ENTOMOLOGIST 
7 
~ 6 
-1 
t=: 
c:::: 
0 
:2 5 III Y = 1.325 + 1.042X 
I­
eo 
20°C 
0 
•
c:::: Y = 2.601 + 0.662Xa. 4· 
2SoC 
3 

1 2 3 4 5 6 7 

LOG DOSE (M. AN/SOPLIAE. CONIDIA I ML) 
7 
f; 6 
-1 
t=: 
c:::: 
o 5:2 II1II Y = 2.425 + 0.621XI­
eo 
20°C
o 
c:::: 
0... 4 Y = 2.736 + 0.S92X• 
2SoC 
3 ~----~--~----~----~----~--~ 
1 234 5 6 7 
LOG DOSE (B. BASS/ANA CONIDIA I ML) 
Figure 1. Probit regression lines for the mortality of C. hospes fifth instars 
treated with 
conidia from 
M. anisopliae and B. bassiana at 20 and 25°C. 
5
Barker: Laboratory Evaluation of the Pathogenicity of Beauveria Bassia
Published by ValpoScholar, 1999
-106 THE GREAT LAKES ENTOMOLOGIST Vol. 32, No.3 
fected at suboptimal temperatures except that it occurs over x ended peri­
ods of time (Ferron 1978, Rath et al. 1995). Isolation or development of fun­
strains that germinate and 
develop 
at lower temperature and humidity is 
another alternative. 
ACKNOWLEDGMENTS The author thanks Sharon 
Grugel for 
insect rearing and diet prepara­
tion. This article reports the results of research only. Mention of a propri­
etary product 
does 
not constitute an endorsement or recommendation by the 
USDA for its use. 
LITERATURE CITED 
Barker J. 1988. Laboratory rearing of the banded sunflower moth, Cochylis 
hospes,(Lepidoptera: Cochylidae). J. Kansas Entomol. Soc. 61:350-352. 
Barker J. 
1994 Development 
of laboratory reared banded sunflower moth, Cochylis 
hospes Walsingham (Lepidoptera: Cochylidae), in relation to temperature. J. Kansas 
Entomo!. Soc. 66:420-426. 
Beregovoy, V. H. and J. G. Riemann. 1987. Infestation phenology of sunflower by the 
banded 
sunflower moth, 
Cochylis hospes (Cochylidae: Lepidoptera) in the northern 
plains. J. Kansas Entomol. Soc. 60:517-527. 
Charlet, L. D., G. J. Brewer and B. Franzmann. 1997. Insect pests, pp. 183-261. In: A. 
A. Schneiter (ed.), Sunflower technology and production. Agron. Ser. 35. Am. Soc. 
Agron., Madison, WI. 834 pp. 
Charlet, L. D. and T. A. Gross. 1990. Bionomics and seasonal abunda ce of the banded 
sunflower moth (Lepidoptera: Cochylidae) on cultivated sunflower in the northern 
Great 
Plains. 
J. Econ. Entomo!. 83:135-141. 
Deacon, J. W. 1983 . .Microbial control of plant pests and diseases. Am. Soc. Microbiol., 
Wash. DC. 88 pp. 
P. 
1981. 
Pest control by the fungi, Beauveria and Metarhiziurn, pp. 465-476. 
In: D. Burges (ed.), Vol. 2, Microbial control of pests and plant diseases. Academic 
Press, 
New York. 949 pp. Ferron, P. 
1978. Biological control 
of insect pests by entomogenous fungi. Annu. Rev. 
Ent mo!. 23: 409-442. 
Finney, D. J. 1947. Probit analysis, a statistical treatment of the sigm id response 
curve. Cambridge University Pr s , UK. 256 pp. 
Jandel 
SigmaPlot software. 1992. SigmaPlot scientific 
graphing software. User's man­
ual. San Rafael, CA. 
LeOra 
Software. 1987. POLO-PC a 
user's guide to Probit Or Logit analysis. Berkeley, 
CA . 
.McCoy, C. W., 
Hill, 
A. J. and R. F. Kanavel. 1988. Entomogenous fungi, pp. 151-236. 
In: C. M. Ignoffo (ed.), CRC handbook of natural pesticides. Microbial Insecticides, 
Part A. Entogenous Protozoa and Fungi, vo!' 5. CRC Press, Boca Raton, FL. 
Miller, L. K., A. J. and L. A. Bulla, Jr. 1983. Bacterial, viral, and fungal insecti­
cides. Science 219: 
Rath, A. 
C., 
G. C. Anderson, D. Worledge and T. B. Koen. 1995. The effecto low tem­
peratures 
on 
the virulence of Metarhizium anisopliae (Dat F-001) to the subter­
ranean 
scarab, 
Adoryphorus coulon!. J. Invert. Pathol. 65: 186-192. 
Tanada, Y. and 
H. 
K. Kaya. 1993. Insect pathology. Academic Press, San Diego. 666 pp. 
Westdal, P. H. 1949. A preliminary report on the biology of Phalonia hospes (Walsing­
ham) 
(Lepidoptera: Phaloniidae), a new 
pest of sunflowers i Manitoba. 80th Annu. 
Rep. EntomoL Soc. Ontario, Canada, pp.1-3. 
6
The Great Lakes Entomologist, Vol. 32, No. 2 [1999], Art. 1
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https://scholar.valpo.edu/cgi/viewcontent.cgi?article=1984&amp;context=tgle

Laboratory Evaluation of the Pathogenicity of \u3ci\u3eBeauveria Bassiana\u3c/i\u3e and \u3ci\u3eMetarhizium Anisopliae\u3c/i\u3e to Larvae of the Banded Sunflower Moth, \u3ci\u3eCochylis Hospes\u3c/i\u3e (Lepidoptera: Cochylidae).

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