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Bulgarian Journal of Agricultural Science, 12 (2006), 637-643 National Centre for Agrarian Sciences

Bioassays with Isolates of Entomopathogenic Fungi against Ephestia kuhniella Zell. (: Pyralidae)

S. DRAGANOVA1 and E. MARKOVA2 1 Plant Protection Institute, BG - 2230 Kostinbrod, Bulgaria 2 Biological faculty, Sofia University "St. Kliment Ohridski", BG - 1164 Sofia, Bulgaria

Abstract

DRAGANOVA, S. and E. MARKOVA, 2006. Bioassays with isolates of Entomopathogenic fungi against Ephestia kuhniella Zell. (Lepidoptera: Pyralidae). Bulg. J. Agric. Sci., 12: 637-643

Bioassays with conidial suspensions of isolates of the entomopathogenic fungi - four of Beauveria bassiana (Bals.) Vuill., two of Metarhizium anisopliae (Metsch.) Sorok., one of Verticillium lecanii (Zimm.) Viegas have been proved against larvae of Ephestia kuhniella Zeller (Lepidoptera: Pyralidae). Lethal effect of each fungal isolate was estimated by values of the cumulative mortality (%) in treated populations during 8 days. Virulence of each fungal isolate was estimated by values of the median lethal time (LT50), calculated by probit analysis. It has been established that the isolate 383Bb of B. bassiana caused the highest lethal effect to larvae - 87.78% ± 13.18, following by isolates 399Bb and 382Bb of B. bassiana - 60.00% ± 8.14 and 58.89% ± 8.75, respectively. Isolates 15Ma and 31Ma of M. anisopliae and the isolate 32Vl of V. lecanii showed less lethal effect in comparison with the other tested isolates. It has been established that only one isolate (383Bb of B. bassiana) was with effi- cacy over 70% to larvae of E. kuhniella. The isolate 383Bb of B. bassiana was with the highest virulence. It's values of the median lethal time varied within a narrow confidence interval from 4.811 to 5.234 days (at p-level < 0.05), with the average value of LT50 5.019 days. Key words: entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii, Ephestia kuhniella, virulence

Introduction ous pest species in mills in Bulgaria and its control has been proved by fumi- The Mediterranean flour gation with methyl bromide. Alterna- (Ephestia kuhniella Zeller, Lepi- tive methods to control doptera: Pyralidae) is the most seri- infesting stored products have been [email protected] 638 S. Draganova and E. Markova developed in order to avoid applica- Control, Plant Protection Institute, tion of methyl bromide after its prohi- Bulgaria. Four isolates of B. bassiana bition. (382Bb, 383Bb, 386Bb and 399Bb) According to Scholler (1998) prom- were initially isolated from caterpillars ising combinations represent integra- of Lepidoptera species: 382Bb and tion of biological and non-biological 399Bb - from Cydia pomonella L. methods - beneficials and hygiene, (Tortricidae), 383Bb - from Archips modified atmospheres, Bacillus rosana L. (Tortricidae) and 386Bb - thuringiensis, combination of certain from Yponomeuta malinella Zell. species of beneficials and some natu- (Yponomeutidae). Isolate 32Vl of V. le- ral insecticides. canii was initially isolated from larva of Biological methods for control of Lepi- Trialeurodes vaporariorum Westw. (Ho- doptera pests include application of moptera: Aleurodidae). Isolates 15Ma and bioagents - predatory mites and parasitoids 31Ma of M. anisopliae were obtained (Hansen, 1998; Nielsen, 1998) or from P. Ferron (INRA, France). pathogens - nuclear-polyhedroses-viruses Conidia of the isolates used as infec- (Scholler, 1998), bacterium B. thuringiensis tious units in bioassays were obtained af- (Cox and Wilkin, 1998; Scholler, 1998), ter fifteen-days cultivation on SDA protozoan parasites such as the (Sabouraud dextrose agar) in tubes at 25oC neogregarine species from genus Mattesia following by washing down with sterilized (Gencheva, 1995, Lord, 2003) and water. The concentrations of conidia were entomopathogenic fungi (Bischoff, 1998; determined by counting in haemocy- Scholler, 1998; Lord, 2005; Wakefield et tometer. Aqueous suspensions applied in al., 2005). bioassays were prepared by dilution at Bioassays with isolates of entomopa- concentration of 1x108 conidia/ml. thogens are the first step to select more Larvae of E. kuhniella in last stages virulent ones for the future studies in de- of development used in bioassays veloping biological methods of pest con- were from laboratory population trol. reared on grain of wheat according to The aim of the study was to estimate Gencheva (1995) at 25+2oC, 60+5 % the virulence of isolates of entomo-patho- RH, on 16L: 8D photoperiod. They genic fungi Beauveria bassiana were treated for 24 h by a surface con- (Balsamo) Vuillemin, Metarhizium tact with 1 ml of conidial suspensions anisopliae (Mets-chnikoff) Sorokin placed on filter paper discs (100 mm and Verticillium lecanii (Zimmerman) in diameter) in Petri dishes (Dra- Viegas to larvae of Ephestia kuhniella ganova and Staneva, 1988). Experi- Zeller (Lepidoptera: Pyralidae). ments were carried out in three repli- cates with 30 larvae per replicate at Materials and Methods temperature 25+2 oC and 60 % RH. were fed with semolina 24 h Isolates used in the study were from after the treatment. Larvae in control the Culture Collection of Entomopa- variant were treated with water in- thogenic Fungi maintained in the Depart- stead of conidial suspension. ment of Biological and Integrated Pest Insect mortality was noted daily for 8

Entomopathogenic Fungi against Ephestia kuhniella Zell... 639

   90.00% 

  

 1 day 2 day 3 day

80.00%

     4 day 5 day 6 day

   70.00%  7 day 8 day

  

60.00% 

    

     50.00% 

    

40.00% 

     

       30.00% 

           

20.00% 

                

10.00%                     0.00% 32Vl 15Ma 31Ma 382Bb 383Bb 386Bb 399Bb Control Fig. 1. Cumulative mortality (%) of larvae of Ephestia kuhniella Zeller (Lepidoptera: Pyralidae) in bioassays with entomopathogenic fungal isolates days. Dead larvae were placed into a were with the highest initial effect to humid chamber for fungal pathogen treated larvae of E. kuhniella - 7.78% exhibition expressed as produced hy- ± 2.51 and 6.67% ± 1.36, respectively. phal growth. Results of the bioassays It could be explained with the lethal were evaluated as percentages of cu- effect of produced toxic metabolites by mulative daily mortality due to my- the isolates to the susceptible part of cosis. Efficacy of fungal isolates was the treated insect population. recorded according to Abbott (1925). According to Roberts (1981) spe- Virulence of each fungal isolate cies M. anisopliae releases toxic me- was estimated by values of the median tabolites destruxins as a step of the lethal time (LT50), calculated by probit development of mycosis. Destruxins analysis (Finney, 1971). induce immediate tetanus in most lepi- Statistical analyses performed in the dopterous larvae. study were Descriptive Statistics and Later isolates 15Ma and 31Ma of Probit Analysis. M. anisopliae and the isolate 32Vl of V. lecanii showed less lethal effect in comparison with the other tested iso- Results and Discussion lates. On the 8th day it has been cal- culated the cumulative mortality Results of the bioassays were exhib- 27.78% ± 2.51; 17.78% ± 1.36 and ited in Figure 1, Tables 1 and 2. 23.33% ± 2.80 in variants treated with As it has been shown on Figure 1, iso- conidial suspensions of the isolates lates 15Ma and 31Ma of M. anisopliae 15Ma, 31Ma and 32Vl, respectively 640 S. Draganova and E. Markova

Table 1 Lethal effect and efficacy of isolates of entomopathogenic fungi in bioassays with larvae of Ephestia kuhniella Zeller (Lepidoptera: Pyralidae) Isolates and species of Mean percentage mortality ± SE Efficacy, % entomopathogenic fungi 8 days after the treatment with isolates 382Bb Beauveria bassiana 58.89 ± 8.75 55.58 383Bb Beauveria bassiana 87.78 ± 13.18 84.4 386Bb Beauveria bassiana 15.56 ± 2.17 12.23 399Bb Beauveria bassiana 60.00 ± 8.14 56.69 15Ma Metarhizium anisopliae 27.78 ± 2.51 24.46 31Ma Metarhizium anisopliae 17.78 ± 1.36 14.45 32Vl Verticillium lecanii 23.33 ± 2.80 20.01 Control 3.33 ± 0.29

Table 2 Virulence of isolates of entomopathogenic fungi to larvae of Ephestia kuhniella Zeller (Lepidoptera: Pyralidae) Values of the median lethal Regression Isolate and species of time (LT50), days coefficient entomopathogenic fungus Average values Confidence intervals (b ± S ε b ) of LT50 from to 382Bb Beauveria bassiana 6.386 5.853 6.966 3.695 ± 0.297 383Bb Beauveria bassiana 5.019 4.811 5.234 6.587 ± 0.297 386Bb Beauveria bassiana 11.82 10.16 13.74 5.739 ± 0.692 399Bb Beauveria bassiana 7.408 6.879 7.979 5.390 ± 0.297 15Ma Metarhizium anisopliae 24.86 24.14 25.6 1.227 ± 0.201 31Ma Metarhizium anisopliae 177 29.2 1074 0.807 ± 0.201 32Vl Verticillium lecanii 18.88 14.42 24.73 2.182 ± 0.201 *Confidence intervals at statistical significance level P < 0.05

(Figure 1 and Table. 1). Low lethal ues of the cumulative mortality on the effect could be explained by the de- 8th day were 15.56% ± 2.17. Isolate crease in the virulence of the isolates 383Bb of B. bassiana caused the high- of M. anisopliae as a result of their est lethal effect to larvae - 87.78% ± durable grown for years on SDA. 13.18, following by isolates 399Bb and Isolate 386Bb of B. bassiana caused 382Bb of B. bassiana - 60.00% ± 8.14 low lethal effect to larvae of E. and 58.89% ± 8.75, respectively. kuhniella as well. The calculated val- Isolates of the entomopathogenic Entomopathogenic Fungi against Ephestia kuhniella Zell... 641 fungi M. anisopliae, B. bassiana, from 12 tested isolates of B. bassiana Paecilomyces farinosus (Holm) caused 100% mortality of larvae of E. Brown et Smith and Paecilomyces kuhniella 10 days after treatment with fumosoroseus (Wize) Brown et Smith conidial suspensions 1x108 conidia/ml. have been tested against the stored Comparison between isolates could product infesting Plodia be proved according to the values of interpunctella Hb and E. kuhniella by the median lethal time (LT50) as an Bischoff (1998). According to him the estimation of their virulence. As it has virulence of the isolates and the envi- been shown on Table 2 the isolate ronmental conditions were the mean 383Bb of B. bassiana was with the factors for a successful use of ento- highest virulence. It's values of the mopathogenic fungi. Furthermore, the median lethal time varied within a nutritional state of the fungi, the sus- narrow confidence interval from 4.811 ceptibility of the moths and the den- to 5.234 days (calculated at statisti- sity of inoculums influenced the effect cal significance level P < 0.05), with of fungal treatment. the average value of LT50 - 5.019 days. Efficacy of isolates has been deter- Differences between virulence of the mined on the 8th day (Table 1). It has been isolates 382Bb and 399Bb of B. established that only one isolate (383Bb bassiana couldn't be proved at P-level of B. bassiana) was with efficacy over < 0.05. Their values of LT50 varied 70% to larvae of E. kuhniella and an- within overlapped confidence intervals other two (382Bb and 399Bb of B. from 5.853 to 6.966 days and from bassiana) - with 55.58% and 56.69%, 6.879 to 7.979 days, respectively. The respectively. Efficacy of isolates isolate 386Bb was less virulent in com- 15Ma of M. anisopliae, 32Vl of V. parison with the other isolates of B. lecanii and especially efficacy of iso- bassiana applied in bioassays. Its av- lates 386Bb of B. bassiana and 31Ma erage value of LT50 was 11.820 days of M. anisopliae were very insuffi- and confidence interval - from 10.160 cient. Only the isolate 383Bb of B. to 13.740 days. bassiana could be used for further bio- Regression coefficient showed sharp- assays. ness of the lethal effect of the mycosis Better results were obtained accord- caused by the certain isolate. It's values ing to Wakefield et al. (2005) in the project calculated for the isolate 383Bb of B. "Mycopest". The aim of the project was bassiana were 6.587 ± 0.297. Regres- to identify the most effective naturally sion coefficients were rather low for occurring insect specific fungi for the con- the other isolates, especially for the trol of residual infestations of storage pests isolate 31Ma of M. anisopliae (0.807 in storage structures. The work under- ± 0.201). This isolate caused mycosis taken in the project demonstrated that the with very low lethal effect. Calculated entomopathogenic fungus B. bassiana values of its median lethal time varied is present in UK grain stores and that within an unreal wide confidence in- under laboratory conditions very terval - from 29.200 to 1074.000 days. good control of a range of insect and It is an indicator that only some indi- mite pests could be achieved. Four viduals of the treated population of E. 642 S. Draganova and E. Markova kuhniella have been affected by the kuhniella - 87.78% ± 13.18 in bioas- isolate. says. Isolates 15Ma of M. anisopliae and • Isolates 15Ma and 31Ma of M. 32Vl of V. lecanii were low virulent anisopliae and the isolate 32Vl of V. to larvae of E. kuhniella. It has been lecanii showed less lethal effect in com- established that differences between parison with the other tested isolates. their virulence were not significant (at • It has been established that only

P-level < 0.05). Values of their LT50 one isolate (383Bb of B. bassiana) was varied within overlapped confidence with efficacy over 70% to larvae of intervals - from 24.140 to 25.600 day E. kuhniella. (for 15Ma of M. anisopliae) and from • The strain 383Bb of B. bassiana 14.420 to 24.730 days (for 32Vl of V. was with the highest virulence. It's lecanii). values of the median lethal time var- In our previous studies (Draganova ied within a narrow confidence inter- and Balabanov, 1984; Draganova, val from 4.811 to 5.234 days (at P level 1990) larvae of E. kuhniella were dis- < 0.05), with average value of LT50 tinguished as resistant to fungal infec- 5.019 days. tions, particularly to mycoses caused by M. anisopliae and V. lecanii. Iso- lates of B. bassiana applied in bioas- References says showed relatively low-virulence as well, with values of LT50 over ten Abbott, W. S., 1925. A method of comput- days. ing the effectiveness of an insecticide. J. Results obtained in the current bioas- Econ. Entomol., 18: 265 - 267. says with isolates of entomopathogenic Bischoff, R., 1998. Untersuchungen zur fungi distinguished 383Bb of B. Wirksamkeit Insektenpathogener Pilze bassiana as isolate with moderate viru- gegen die vorratsschadlichen Motten lence against larvae of E. kuhniella. Ephestia kuhniella und Plodia inter- According to Lord (2005) application punctella (Lepidoptera: Pyralidae). Ber- of entomopathogens such as fungi lin, Humboldt - Univ., Diss. who's host range is relatively Cox, P. D. and D. R. Wilkin, 1998. A review of favourable (benign for non-target in- the options for biological control against sects) needs to improve their perfor- invertebrate pests of stored grain in the mance. The most apparent means to UK. In: C. Adler and M. Schoeller (Editors), achieve efficacy improvement is to Integrated Protection of Stored Products, combine fungi with other control mea- IOBC wprs Bull. 21 (3): 27 - 32. sures (for example diatomaceous Draganova, S., 1990. Virulence of strains of earth). entomopathogenic fungi from genus Beauveria Vuill. Biotechnology, 1: 22 - Conclusions 25. (Bg) Draganova, S. and V. Balabanov, 1984. Some • It has been established that the iso- biological characteristics of ento- late 383Bb of B. bassiana caused the mopathogenic Fungi impecfecti. In: B. highest lethal effect to larvae of E. Botev (Editor), Problems of biological pest Entomopathogenic Fungi against Ephestia kuhniella Zell... 643

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