International Journal of Scientific & Engineering Research, Volume 7, Issue 9, September-2016 - 727 - ISSN 2229-5518 Impact of Entomopathogenic Fungi and fumosorosea on Cruciferous Brevicoryne Brassicae L.

1Ahmed A.A. Saleh, 2Gamal M.A. Lashin and 1Ahmed A.M. Ali 1Plant Protection Research Institute, Zagazig Branch, Egypt. 2Department of botany, Faculty of science, Zagazig University, Zagazig, Egypt.

ABSTRACT: An experiment was conducted to evaluate the role of two entomopathogenic fungi: Beauveria bassiana (Bals.) and Isaria fumosorosea (Wize) against the adults of cruciferous aphid (cabbage aphid), Brevicoryne brassicae L. under laboratory 5 6 7 conditions at plant protection institute, Sharkia Branch. Three tested concentrations (10P P, 10P P and 10P P spores/ml) of B. bassiana formulations were used against aphid B. brassicae. Mortality percentage after 7 days of application showed 35.55, 46.66 and 6 9 64.44%, respectively. The obtained results revealed that LCR50R was 1.1×10P P spores/ml and LCR90R was 3.4×10P P spores/ml. While, 6 7 8 Three tested concentrations (10P P, 10P P and 10P P spores/ml) of I. fumosorosea formulation were used against the same aphid. Mortality percentage after 7 days of application showed 37.77, 60.0 and 73.33%, respectively. The obtained results revealed 6 9 that LCR50R was 3.9×10P P spores/ml and LCR90R was 2.1×10P P spores/ml.

Keywords: Biological control, Entomopathogenic fungi, Beauveria bassiana, Isaria fumosorosea, Brevicoryne brassicae

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conditions 25±1°C, 65±5 RH% and 12 hr INTRODUCTION photoperiod. The cabbage leaves that were used for laboratory evaluation contained a midrib in order The aphid Brevicoryne brassicae L. is a to survive for a long period of time. were major pest on cruciferous plants, in several parts of taken by a fine brush and located on cabbage the world and also in EgyptIJSER especially on cabbage leaves. The leaves were incubated under the same cauliflower (Horn 1989 and Saleh 2004 and 2014). laboratory conditions 25±1°C, 65±5 RH% and 12 Entomopathogenic fungi are natural enemies of hr, photoperiod. After 48 hr, three leaves were insects and arachnids and they contribute in the picked up and put in a plastic Petri plates on filter regulation of their host population. In agriculture, papers that saturated daily with water. they have been observed to cause mortality in pest's population (El-husseini et al., 2010). Several 2. Fungal inocula species of entomopathogenic fungi can be used as Spores of fungal isolate were harvested by natural enemies against many pests, including rinsing with sterilized water containing 0.005% Onidiobolus obscures, Erynid neophidis, Tween80 from 7days old culture (Dox medium Verticillium lecanii, various species of Beauveria, grown at 25±1°C for B. bassiana and I. and Isaria fumosorosea (Wize) (Roberts and fumosorosea isolates). The suspensions were Yendol 1971, Samson et al. 1988 and Hayden et filtered through cheesecloth to reduce mycelium al. 1992). clumping. The spores were counted in the suspensions using a haemocytometer. The 5 6 7 MATERIALS AND METHODS concentrations were adjusted to 10P ,P 10P P and 10P P 6 7 8 for B. bassiana and 10P ,P 10P P and 10P P for I. 1. Culture of B. brassicae fumosorosea. The cabbage's aphid B. brassicae, was reared on cabbage leaves under laboratory 3. Experimental work

IJSER © 2016 http://www.ijser.org International Journal of Scientific & Engineering Research, Volume 7, Issue 9, September-2016 - 728 - ISSN 2229-5518 Studies regarding the effect of the and 2, after the treatment, the mortality then on the infected leaves of cabbage were applied on dramatically increased from day 7 to 9. The three replicates each consists of fifteen individuals mortality in infected aphids with fungal isolates of B. brassicae on cabbage leaves. Leaves were increased with increase in spore concentration of sprayed with two ml of spore suspension and the conidial suspensions and exposure time. The control was treated with two ml of sterilized water susceptibility of target insect to fungal infection is containing 0.005 % tween80 only. The treatments dose dependant (Liu and Chen, 2002; Wright et al., and control were incubated for 7 days under 2005). laboratory conditions (25±1°C, 65±5 RH% and 12 Meanwhile, Akmal et al. (2013) showed hr photoperiod). Larval mortality was observed that the maximum mortality 100% of B. bassiana

after 1, 3, 5and 7 days. LCR50 Rand LCR90R and slop on B. brassicae was obtained at 7th day post 8 values were calculated after 5 and 7 days according treatment at a concentration of 1×10P ,P while to Finny (1971). The presented results are means minimum mortality of 99.2% with treatment of 6 of each treatment. 1×10P .P In contrast to this no mortality was recorded 5 The obtained results were statistically in control. The value of LCR50R 6.28×10P P showed analyzed by using Costat (2005) computer that the 50% mortality was obtained at 3rd day of program. treatment. On the other hand, Akbari et al. (2014) in Iran RESULTS AND DISSCUTION showed that the adult aphids B. brassicae were 3 treated with fungal concentrations of 1 × 10P P to 1 × 7 1. Laboratory evaluation of the efficiency of 10P P spores/ml. The lowest LTR50R was obtained at entomopathogenic fungi on B. brassicae 7.67 days for Iran 429C (B. bassiana) isolate at 7 1.1. The entomopathogenic fungi B. bassiana: concentration 1 × 10P P spores/ml. Laboratory evaluation: Data given in Table (1) shows the efficacy of B. bassiana spores 1.2. The I. suspension on nymph instars of cabbage aphid B. fumosorosea: brassicae after application with different Laboratory evaluation: Data given in Table (2) concentrations of B. bassiana spores under shows the efficacy of I. fumosorosea spores laboratory conditions of (25±1°C, R.H. 65±5%). suspension on nymph instars of cabbage aphid B. 5 6 The concentrations were adjusted to 10P ,10P P P and brassicae after application with different 7 10P P spores/ml. MortalityIJSER percentages after 5 days concentrations of I. fumosorosea spores under of application showed 22.22%, 28.88% and laboratory conditions of (25±1°C,R.H. 65±5%). 6 7 42.22% and after 7 days showed 35.55%, 46.66% The concentrations were adjusted to 10P ,10P P P and 8 and 64.44%, respectively. 10P P spores/ml. Mortality percentage values after 5

Data given in Table (3) clarified the LCR50R days of application showed 24.44, 40 and 51.11%,

and LCR90R values of B. bassiana spores/ml after 5 and after 7 days showed 37.77,60 and 73.33%, and 7 days of application on nymph instars of respectively.

cabbage aphid, B. brassicae. The obtained results Data given in Table (4) clarified the LCR50R 7 revealed that after 5 days LCR50R: 5.8×10P P spores/ml and LCR90R values of I. fumosorosea spores/ml after 12 and LCR90R: 1.7×10P P spores/ml (Fig. 1) and after 7 5 and 7 days of application on nymph instars of 6 9 days LCR50R: 1.1×10P P spores/ml and LCR90R: 3.4×10P P cabbage aphid, B. brassicae. The obtained results 7 spores/ml (Fig. 2). revealed that after 5 days LCR50R: 7.1 × 10P P 11 Entomopathogenic fungi have been spores/ml and LCR90R: 2.7 × 10P P spores/ml (Fig. 3) 6 observed to cause mortality in pest population and and after 7 days LCR50R: 3.9 × 10P P spores/ml and 9 thus, investigated for their potential as biological LCR90R: 2.1 × 10P spores/mlP (Fig. 4). control agents (Hesketh et al., 2008) or Asi et al. (2009b) showed that the successfully developed as biocontrol agent for a concentration of the entomopathogenic fungus I. number of different pests, including aphids (Shah fumosorosea affected the mortality of cabbage and Pell, 2003; De Faria and Wraight, 2007). aphids B. brassicae differently. The LCR50R of this Time-dose dependent mortality response fungus against the adults of aphids 7 days after 6 experiments were designed as a measure of conidial treatment was 2.22 × 10P P conidia/ ml. this st mortality of different fungal isolates against fungus resulted in mortality of 5.83% on the 1P P day aphids. The mortality observed was low on day 1 of treatment. The mortality then increased on days IJSER © 2016 http://www.ijser.org International Journal of Scientific & Engineering Research, Volume 7, Issue 9, September-2016 - 729 - ISSN 2229-5518 5-7. The cumulative mortality caused by the Egyptian Journal of Biological Pest fungus after 7 days of treatment was 18.14- Control, 20(2): 179-183, 2010. 90.82%. The mortality increased with the increase D.W. Roberts and W.G. Yendol: Use of fungi for in the spores concentration and exposure time. microbial control of insects PP.125-149. In Also, Asi et al. (2009a) showed that the H.D. Burges and N.W. Hussey (eds.). 6 concentration (1 × 10P P conidia/ml) of the Microbial Control of insects and mites, entomopathogenic fungus I. fumosorosea resulted Academic, New York, 1971. in 20.17% mortality of adults of cabbage aphids B. R.A. Samson, H.C. Evans and J.P. Latge: Atlas of brassicae three days after conidial treatment. The entomopathogenic Fungi. Springer, New mortality percentage of B. brassicae was 48.40% York. 1988. and 68.57% after 5 and 7 days of conidial T.P. Hayden, M.J. Bidochka and G.G. treatment. However, virulence potential of Khachatourians: Entomopathogenicity of entomopathogenic fungi against target aphid varies several fungi toward the English grain aphid from isolate to isolate and from strain to strain. The (Homoptera: Aphididae) and enhancement of susceptibility of the same aphid species may vary virulence with host passage of to different fungal strains. Even biotypes or farinosus. J. Agric., Res., 83(3):1249-1291, different colons of the same aphid species may 1992. rd have varying susceptibility to fungal infection D.I. Finny: Probit Analysis. 3P P Ed., Cambridge (Ferrari et al., 2001 and Blanford et al., 2003). V. University Press, London, 1971. lecanii, M. anisopliae and P. fumosoroseus can Costat Statistical Software: Microcomputer effectively control aphids (Asi et al., 2009a). program analysis version, 6. 311. CoHort Software, Monterey, California, 2005. CONCLUSION H. Hesketh, P.G. Alderson, B.J. Pye and J.K. Pell: The development and multiple uses of The two entomopathogenic fungi: B. a standardized bioassay method to select bassiana and I. fumosorosea have efficiency in hypocrealean fungi for biological control of controlling cabbage aphid B. brassicae and can be aphids. Biological Control, 46: 242-255, used as biological control agents against B. 2008. brassicae. P.A. Shah and J.K. Pell: Entomopathogenic fungi as biological agents. Appl. Microbiol REFERENCESIJSER Biotechnol., 61: 413-423, 2003. M.R. De Faria and S.P. Wraight: Mycoinsecticides D.J. Horn: Secondary parasitism and population and mycoacaricides: a comprehensive list with dynamics of aphid parasitoids worldwide coverage and international (Hymenoptera: Aphidiidae). J. of the classification types. Biol. Control. 43:387-400, Kansas Entomol. Soci., 62(2): 203-210, 2007. 1989. X.Z. Liu and S.Y. Chen: Nutritional requirements A.A.A. Saleh: Mass production and field of the nematophagous fungus Hirsutella application of some aphid natural enemies. rhossiliensis. Biocontrol Science and Ph.D. Thesis, Fac. of Agric., Mansoura Univ., Technology, 12(3): 381- 393, 2002. pp 161, 2004. M.S. Wright, A.K. Raina and A.R. Lax: A strain of A.A.A. Saleh: Efficacy of the aphid parasitoid the fungus for Diaeretiella rapae (M'Intosh) to control controlling subterranean termites. J. Econ. Brevicoryne Brassicae L., Aphis craccivora Entomol., 98: 1451-1458, 2005. (Koch) and Aphis neriiboyer at Sharkia M. Akmal, S. Freed, M.N. Malik and H.T Gul: governorate, Egypt. J. Agric. Res., 92(1): 21- Efficacy of Beuaveria bassiana 31, 2014. (Deuteromycotina: Hypomycetes) against M.M. El-Husseini, E.A. Agamy, H.K. Bekheit different aphid species under laboratory and S.S. Ali: Virulence of Destruxins from conditions. Pakistan J. zool., vol. 45(1), pp. two Metarhizium anisopliae (Metch.) 71-78, 2013. Isolates versus larvae of the sugar beet S. Akbari, S.A. Safavi and Y. Ghosta: Efficacy of worm Spodoptera exigua (Hiibner). Beauveria bassiana (Blas.) Vuill. against cabbage aphid Brevicoryne brassicae L. IJSER © 2016 http://www.ijser.org International Journal of Scientific & Engineering Research, Volume 7, Issue 9, September-2016 - 730 - ISSN 2229-5518 (Hem.: Aphidiidae) in laboratory condition. aphid, Brevicoryne brassicae L. Pak. Entomol. Archives of Phytopathology and Plant Vol. 31: 43-46, 2009a. Protection, 47(12): 1454-1458, 2014. V. Ferrari, C.B. Moller, A.R. Kraaijeveld and M.R. Asi, M.H. Bashir, M. Afzal and S. Imran: H.C.J. Godfray: Clonal variation and Effect of conidial concentration of covariation in aphid resistance to parasitoids entomopathogenic fungi on mortality of and a pathogen. Evolution, 55(9): 1805-1814, cabbage aphid, Brevicoryne brassicae L. Pak. 2001. J. Life Soc. Sci., (2): 175-180, 2009b. S. Blanford, B. Thomas, C. Pugh and J.K. Pell: M.R. Asi, M.H. Bashir, J.H. Mirza, M. Afzal and Temperature checks the Red Queen resistance S. Imran: In vitro efficacy of and virulence in a fluctuating environment. entomopathogenic fungi against cabbage Ecology Letters, 6(1): 2-5, 2003.

Table (1): Mortality percentages of nymph instars of B. brassicae after application with different concentrations of B. bassiana spores suspension under laboratory conditions (25±1°C, R.H. 65±5%) during winter season of 2012/2013. After 1 day After 3 days After 5 days After 7 days

Concentration Mortality Mortality Mortality Mortality Life Dead Life Dead Life Dead Life Dead % % % %

5 1×10P P spores/ml 44 1 2.22 39 6 13.33 35 10 22.22 29 16 35.55

6 1×10P P spores/ml 42 3 6.66 36 9 20 32 13 28.88 24 21 46.66

7 1×10P P spores/ml 41 4 8.88 34 11 24.44 26 19 42.22 16 29 64.44

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Table (2): Mortality percentage values of nymph instars of B. brassicae after application with different concentrations of I. fumosorosea spores suspension under laboratory conditions (25±1°C, R.H. 65±5%) during winter season of 2012-2013. After 1 day After 3 days After 5 days After 7 days

Concentration Mortality Mortality Mortality Mortality Life Dead Life Dead Life Dead Life Dead % % % %

6 1×10P spores/mlP 44 1 2.22 40 5 11.11 34 11 24.44 28 17 37.77

7 1×10P P spores/ml 43 2 4.44 37 8 17.77 27 18 40.00 18 27 60.00

8 1×10P P spores/ml 41 4 8.88 33 12 26.66 22 23 51.11 12 33 73.33

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Table (3): Lethal concentration (LC25-99) of B. bassiana spores/ml after 5 and 7 days of application against nymph instar of B. brassicae under laboratory conditions (25 ± 1°C, R.H. 65± 5%) during winter season of 2012/2013. Concentration of B. bassiana spores/ml Lethal Slope Concentration Lower limit Upper limit concentration After 5 days After 7 days After 5 days After 7 days After 5 days After 7 days After 5 days After 7 days

LC25 258760 18062.3 8837 353.66 874830 81558.6 7 6 6 5 10 6 LC50 5.8 × 10P 1.1 × 10P 9.5 × 10P 4.5 × 10P 9.1 × 10P 3.5 × 10P 10 7 8 7 17 9 LCR75 1.3 × 10P 7.9 × 10P 2.9 × 10P 1.5 × 10P 3.2 × 10P 5.4 × 10P 12 9 9 8 23 12 0.287 0.37 LCR90 1.7 × 10P 4.3 × 10P 6 × 10P 2.2 × 10P 2.8 × 10P 7.2 × 10P 13 10 10 9 27 14 LCR95 3.1 × 10P 3.3 × 10P 3.6 × 10P 1 × 10P 1 × 10P 5.5 × 10P 19 12 12 10 33 18 LCR99 7.4 × 10P 2.2 × 10P 1 × 10P 1.8 × 10P 5.1 × 10P 1.9 × 10P

Table (4): Lethal concentration (LCR25-99R) of I. fumosorosea spores/ml after 5 and 7 days of application against nymph instar of B. brassicae under laboratory conditions (25 ± 1°C, R.H. 65± 5%) during winter season of 2012-2013. Concentration of I. fumosorosea spores/ml Lethal Slope Concentration Lower limit Upper limit concentration After 5 days After 7 days After 5 days After 7 days After 5 days After 7 days After 5 days After 7 days IJSER6 5 LCR25 921610 143230 51058.9 8777.84 2.9 × 10P 5.3 × 10P 7 6 7 6 8 6 LCR50 7.1 × 10P 3.9 × 10P 2.5 × 10P 1.4 × 10P 7.1 × 10P 8.1 × 10P 9 8 8 7 12 8 LCR75 5.4 × 10P 1 × 10P 5.9 × 10P 4.2 × 10P 3.6 × 10P 6.6 × 10P 11 9 9 8 15 10 0.357 0.469 LCR90 2.7 × 10P 2.1 × 10P 8.4 × 10P 4.1 × 10P 9.6 × 10P 7.7 × 10P 12 10 10 9 18 12 LCR95 2.8 × 10P 1.2 × 10P 4 × 10P 1.5 × 10P 1 × 10P 1.4 × 10P 14 11 11 10 21 14 LCR99 2.3 × 10P 3.6 × 10P 7.6 × 10P 1.7 × 10P 7.8 × 10P 3.2 × 10P

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Fig. (1): Concentration mortality probit line of B. Fig. (2): Concentration mortality probit line of B. bassiana spores/ml on nymph instars of B. bassiana spores/ml on nymph instars of B. brassicae under laboratory conditions (25 ± 1°C, brassicae under laboratory conditions (25 ± 1°C, 65± R.H. 5%) after 5 days. 65± R.H. 5%) after 7 days.

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Fig. (3): Concentration mortality probit line of Fig. (4): Concentration mortality probit line of I. fumosorosea spores/ml on nymph instars of I. fumosorosea spores/ml on nymph instars of B. B. brassicae under laboratory conditions (25 ± brassicae under laboratory conditions (25 ± 1°C, 65± R.H. 5%) after 5 days. 1°C, 65± R.H. 5%) after 7 days.

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