Academic Journal of Entomology 6 (3): 127-132, 2013 ISSN 1995-8994 © IDOSI Publications, 2013 DOI: 10.5829/idosi.aje.2013.6.3.7686

Infectivity of Five anisopliae (Deuteromycota: Hyphomycetales) Strains on Whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) Infesting Brinjal, Solanum melongena (Solanaceae)

1L. Norhelina, 21 A.S. Sajap, S.A. Mansour and 1 A.B. Idris

1School of Environmental and Natural Resource Sciences, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 2Department of Forestry, Faculty of Forestry, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Abstract: Theinfectivityof five strains of the , Metarhizium anisopliae (Gennadius) (PR1, GT2, TFFH3, GJ4 and HSAH5) against whitefly, Bemisia tabaci, infesting brinjal, Solanum melongena was studied. Dosage response assays on nymphal mortality rates were conducted with four concentrations of the most virulent strain (10753 , 10 , 10 and 10). Strain GJ4 was found to be the most virulent strain of M. anisopliae against B. tabaci with

the lowest LT50 (2.16 days, 4.48 slope). The second nymphal instar of B. tabaciwas significantly more sensitive 41 5 1 to GJ4 infection (LD50 6.62 x 10 conidia ml ) than was the fourth nymphal instar (LD50 7.87 x 10 conidia ml ). This finding was supported by the results of the dosage response assay which found GJ4 to be12 times more infectiveon the second nymphal instar than on the fourth nymphal instar of B. tabaci. The time needed to cause 71 50% mortality (LT50 ) decreased with increasing dosage of the conidia. A dosage 10 ml on second instar had 7 the shortest LT50 of 2.25 days, indicating both that the 10 dosage was the most infectiveoneamongst the four tested and that the second nymphal instar was more susceptible to this strain than the fourth. M. anisopliae strain GJ4 was found to havepotential for use in control of whiteflies in the field.

Key words: Entomopathogenic Fungi Infectivity Malaysia Biopesticide Dosage-response

INTRODUCTION in insecticidal hazards and pollution of the environment [5]. Toxic chemical residue in the environment and Whitefly, Bemisia tabaci (Gennadius), a common induced resistance in their insect host drive the serious sap-sucking insect from the family Aleyrodidae of the interest in the use of microbial insecticides for biological order Homoptera, has become one of the world’s rather than chemical control of insect pests [6]. important pests [1],causing serious crop losses Public awareness and concern for environmental amounting to hundreds of millions of US dollars quality has led to more focused attention on research annually [2]. It is economic importance as one of the major aiming at developing and finding biological control agents insect pests of a number of crops including brinjal [3]. [7]. A promising strategy with good potential to control Its feeding behavior of piercing the tissues of plants and insect pests while minimizing the adverse effects of sucking sap directly from the vascular bundle reduces chemical insecticides is the use of entomopathogenic host vigor and growth and may induce plant fungi and other microbial agents [8]. Entomopathogenic physiological disorders [4]. B. tabaci (Genn.) has fungi are more promising natural enemies of whiteflies that developed high levels of resistance to insecticides in infect their hosts by direct penetration of the body wall cotton, brinjal and okra, whetherwith higher dosages of than pathogens such as bacteria and viruses that are the same pesticide or with the application of several normally transmitted via host feeding on contaminated pesticides [5]. Pest resistance to insecticides is known not foliage [9]. Most reports of n atural infections of only to increase the cost of pest controlbutalso to result Bemisia spp. have noted such infections byspecies of

Corresponding Author: A.B. Idris, School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, National University Malaysia. 127 Acad. J. Entomol., 6 (3): 127-132, 2013

Hyphomycetes, especially Paecilomyces, Verticillum and Table 1: Metarhiziumanisopliaestrains from the Faculty of Forestry, Aschersonia spp [10]. On the other hand, most study of Universiti Putra Malaysia stock culture Fungus Code of strains Location/State of origin infection with Metarhizium anisopliaehas focused Metarhiziumanisopliae PR 1 PantaiRemis (Isolate 1) primarily on the control of Coleoptera, Lepidoptera GT 2 GunungTahan (Isolate 2) and Orthoptera [11]. Only recently was the efficacy of TFFH 3 Tranum Forest Fraser Hill M. anisopliae against whitefly discovered. Malsam et al. (Isolate 3) [12] first described the efficacy of M. anisopliaeagainst GJ 4 GunungJerai (Isolate 4) HSAH 5 HutanSimpan Ayer Hitam greenhouse whitefly,Trioleurodes vaporariorum.A (Isolate 5) review by Faria and Wraight [10] has reported that isolates of M. anisopliae are highly virulent toward Using a dosage response assay, the superior fungal nymphs of B. tabaci. The present study was designed isolates from five selected isolates that caused the highest to determine the most virulent strain of M. anisopliae mortality on whitefly were chosen in this test. Conidia for the control of whitefly B. tabaci and to determine were prepared in 0.05% Tween 80 solution of distilled dosage-response of the most virulent strains of water. A Neubauer haemocytometer was used to estimate M. anisopliae. the concentration and then a dilution series of 1x107 , 1x1053 , 1x10 and 1x10 conidia ml 1 were prepared. Distilled MATERIALS AND METHODS water containing 0.05% Tween 80 was used as control. Unlike the screening test, which used 34-mm diameter Mature entomopathogenic fungi, M. anisopliae were discs, this part of the study used a topical test, in acquired from the Entomology Laboratory, Faculty of which68-mm discs were punched out of brinjal leaves which were infested with second instar or fourth instar Forestry, Universiti Putra Malaysia, Serdang, Malaysia whitefly. At least 60 individuals of second or fourth (UPM). Five strains of M. anisopliae previously obtained instars whiteflies were counted on each leaf disc. The leaf from Pantai Remis, Hutan Simpan Ayer Hitam, Gunung discs were placed on water agar (1%) in Petri dishes and Jerai, Gunung Tahan and the Tranum Forest of Fraser Hill sprayed with 2ml of a spore suspension (10753 , 10 , 10 and were chosen randomly for this study (Table 1). The fungi 10conidia ml 1) using a hand sprayer. were cultured on Sabouraud Dextrose Agar (SDA). The The experiment was carried out in folowing a cultured plates were kept for 14 days to obtain fungal Complete Randomized Design (CRD) and replicated four spores and were maintained in the Pathology Laboratory times for each treatment. Abbot’s formula was used to of Forest Faculty, UPM. calculate the mortality actually due to fungus in all The experimental technique on leaf discs was bioassays (Abbot, 1925).The mortality data were pooled modified from that of Malsam et al. [13]. In this and calculated using probit analysis in Minitab version experiment, 34-mm diameter discs were punched out of 14 to calculate LD50 and LT50 values; the 95% confidence brinjal leaves infested with second instar and fourth instar limits mean was used to find significant differencesin LD50 whitefly. At least 30 individuals of second instar or fourth or LT50 among treatments [14]. Probit analysis tests the instar whiteflies were counted on each disc. The leaf discs linearity of dose-mortality responses and provides the were placed on water agar (1%) in Petri dishes and slope, median lethal dose (LD50 ) and 95% confidence sprayed with a 2 ml spore suspension (1071 conidia ml ) limits (CL) of the LD50 , for all mortality lines [15]. using a hand sprayer. After drying, the Petri dishes were RESULTS AND DISCUSSION placed upside down on covers and incubated at 25 ± 2°C in the incubator. Screening Test: Strain GJ4 had the lowest LT (2.16 Screening was performed on second instar nymphs, 50 days, 4.48 slope) on second instar, indicating that it was 30 individuals per replicate, four replicates per strain the most virulent strain of the five strains tested for this and six strains per assay including control. The total of instar (Table 2). The GT2 and HSAH5 strains were 720 individual second instar nymphs including the considered to be the least virulent and infective strains as control were screeneddaily for 7 days post-treatment. they took a longer time (2.16 and 3.18 respectively) to kill A similar screening was done on fourth instar whitefly 50% of the B. tabaci tested. The PR1(2.77 days, 4.13 nymphs, giving an overall total of 1440 individuals of slope) and TFFH3 (2.76 days, 4.32 slope) were somewhat B. tabaciscreened. more infective than these two but much less than GJ4.

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71 Table 2: Median lethal times (LT50 ) for the 10 conidia ml dosageof M. anisopliaestrains on nymphal stages of B. tabaci 7 days post-treatment)

Strains a (Intercept) b ± SE (Slope) LT50 (days) 95% Confidence Limit Second Instar PR1 3.174 4.128 ± 0.214 2.77 2.61 ± 2.94 GT2 3.201 3.616 ± 0.200 3.14 2.95 ± 3.35 TFFH3 3.100 4.317 ± 0.223 2.76 2.60 ± 2.92 GJ4 3.503 4.476 ± 0.224 2.16* 2.04 ± 2.29 HSAH5 2.979 4.027 ± 0.220 3.18 3.00 ± 3.37 Fourth Instar PR1 2.603 4.352 ± 0.252 3.55 3.37 ± 3.75 GT2 2.985 3.665 ± 0.217 3.55 3.34 ± 3.77 TFFH3 2.491 4.570 ± 0.266 3.54 3.36 ± 3.73 GJ4 3.042 4.720 ± 0.244 2.60* 2.46 ± 2.75 HSAH5 2.549 4.507 ± 0.257 3.50 3.32 ± 3.69 Control treatment = zero mortality

14 This variation in LT50 was also noted by Altre et al. ml ) with a 95% confidencelimit between 3.12 x 10 – 1.48 [16] who proposed that different strains of the same x 105 was significantly lower than that of GJ4 on fourth species may not have equal potential for control of a instar (7.87 x 1051 conidia ml ) with a 95% confidencelimit given arthropod species. A study by Adane et al. [17] (CL) between 2.90 x 1056 – 2.30 x 10 . These results also demonstrated that,while B. bassiana isolates showed suggest that M. anisopliae GJ4 was more infective on pathogenicity to weevil adults, there were highly second instar nymphs of B. tabaci, being 12 times significant differences among the isolates with respect to more pathogenic against second instar than against virulence. fourth instar nymphs (Table 3). With similar results,

Strain GJ4 also had the lowest LT50 (2.60 days, 4.72 Fransen et al. [20] proposed that the inability of the slope) on fourth instar, indicating that it was the most fungus Aschersonia aleyrodis to penetrate the cuticle virulent strainin this study. The other strains (PR1, GT2, of whitefly in older instar stages maybe due to

TFFH3 and HSAH5) were less infective as their LT50 failure of the fungus to produce enzymes or to ranged from 3.50 to 3.55 days of the B. tabaci tested. inhibition of the germination and penetration processes

The LT50 for second and fourth instars (2.04 ± 2.29; by antifungal substances present in the cuticle of 2.46 ± 2.75) differed significantly as indicated by non- whitefly. overlapping of a 95% confident limit (Table 2) and the The pathogenicity of M. anisopliae GJ4, as shown by second instar was found to be more sensitive to the LT50 values, generally decreased over time with increasing 7 infection of this fungus. dosage. Dosage 10 on second instar had the lowest LT50

The small variance of LT50 across strains could be (2.25 days, 4.50 slopes) indicating it was the most virulent attributable to a decrease in pathogenicity of a strain or a dosage of the four dosages tested (Table 4). In contrast, variable response in replication. A study by Shah et al. dosages 10 and 103 treatments were the least infective [18] suspected that a decrease in pathogen virulence was strains to both instars. Not only did they takelonger than caused by ingredients of the culture media like 7 days to kill 50% but the LT50 was not computable (NA) carbohydrates in the mass production of Metarhizium sp. because mortality was less than 50% at sevendays post- Also, Arzumanov et al. [19] have proposed that the treatment while treatments with dosages 105 and 7 pathogenicity of M. anisopliae was influenced by the 10 reached LT50 within six days. 5 medium of the inocula. Inoculum was reported to be more The LT50 for dose 10 on both second and fourth effective and more virulent on naturally infected insects instars were 5.11 to 5.53 days, respectively, which was than spores harvested from culture media [19]. Overall, somewhat more infective than doses 103 and 10 but much strain GJ4 was proven to be the most virulent strain of longer than at 1077 (2.25 to 2.87 days). Dosage 10 on

M. anisopliae to control B. tabaci. fourth instar also had the lowest LT50 (2.87 days, 4.20 slope), indicating that dosage 107 was the most virulent

Dosage Response Assay: Probit analysis of the dosage dosage of the four dosages tested. LT50 for second and response assay indicated a significant relationship fourth instars for dose 107 differed significantly withthe

(P < 0.05) between log10 dosage and probit mortality. 95% CL not overlapped (2.16 ± 2.35; 2.76 ± 2.99),

Estimates of the median lethal dose (LD50 ) computed for indicating the second instar more sensitive than the M. anisopliae GJ4 on second instar (6.62 x 104 conidia fourth instar to Nosema infection.

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Table 3: Median lethal dose (LD50 ) for different dosages of GJ4 on nymphal stages of B. tabaci

Stage a (Intercept) b ± SE (Slope) LD50 (Dosage) 95% Confident Limit Second instar 2.845 3.154 ± 0.176 6.62 x 104 3.12 x 1045 ± 1.48 x 10 Fourth instar 2.498 3.247 ± 0.214 7.87 x 105 2.90 x 1056 ± 2.30 x 10 Control treatment = zero mortality

a Table 4: Median lethal time (LT50 ) for different dosages of GJ4 on nymphal stages of B. tabaci (7 days post-treatment; P < 0.05; Control = zero mortality)

b1 Dosage (conidia ml ) a (Intercept) b ± SE (Slope) LT50 (days) 95% Confidence Limit Second Instar 1 x 107 3.42 4.50 ± 0.16 2.25 2.16 ± 2.35 1 x 105 2.27 3.85 ± 0.20 5.11 4.89 ± 5.34 1 x 103 NA NA NA NA 1 x 10 NA NA NA NA Fourth Instar 1 x 107 3.08 4.20 ± 0.16 2.87 2.76 ± 2.99 1 x 105 2.14 3.85 ± 0.22 5.53 5.27 ± 5.80 1 x 103 NA NA NA NA 1 x 10 NA NA NA NA

NA = LT50 is not available for mean percent mortality below 50%; Control treatment = zero mortality

LT50 for the second instar nymphs indicated a shorter inhibit penetration of the germinated conidia or spore time to mortality (2.23 days, 4.50 slope) than did the fourth [27, 28]. instar nymphs (2.87 days, 4.20 slope). LT50 values indicate Most of the nymphs were observed to become that second instar nymphs of B. tabaciwere more moribund two days post-treatment and were gradually susceptible than fourth instar nymphs because a shorter covered by mycelia, which appeared pale yellowish white time was needed to reach median lethal time when treated slowly turning light green, then gradually darker. The with the same dosage. This result is in agreement with same phenomena have been described by Sajap and Kaur that of Vicentini et al.[21] who found that the young [29] of colonization of the termite, Coptotermes instars of B. tabaci were more susceptible than the curvignathus by mycelia of this fungus, which appeared older instars to fungal infections ofBeauveria bassiana. pale yellowish white initially, subsequently turning olive Lacey et al. [22] also reported different degrees of green. Similarly, Krutmuang and Mekchay [30] have noted susceptibility between the stages of eggs and second that when the white mycelia of M. anisopliaecovering instar nymphs of whitefly B. argentifolli after infection C. curvignathuschanged to light green four to seven days with Paecilomyces fumosoroseus. Similarly, Aziz & Saleem after death and gradually darkened with time. A study by [23] have reported different degrees of susceptibility at Nugroho and Ibrahim [31] reported that sporulation of different developmental stages of mealy bug when M. anisopliae on mite cadavers appeared greenish infected with M. anisopliae. whileP. fumosoroseus appeared pinkish upon sporulation. However, unlike these studies, Wraight [24] found no direct relationship between instars of B. tabaciand CONCLUSION different dosages of B. bassiana. Meade and Byrne [25] also found no difference in susceptibility among instars Strain GJ4 of M. anisopliae was the most virulent of of B. tabaci treated with Verticillium lecanii. According the five strains studied to both second and fourth to Cuthbertson et al. [26] information on both the biology whitefly nymphalinstars. Interestingly, the GJ4 was and feeding activity of the insect targeted for control and somewhat 12 times more pathogenic to the second than to the most susceptible stage of the pest species is the fourth instars of B. tabaci. Generally, there was important for the successful introduction of a fungal adecreasing in lethal time of the insect with increasing biocontrol agent. The increase in resistance to fungal dosage of GJ4 strain. We propose that the findings of this infection as the species develops from young to older study provide a good impetus for continuing into the field instar or insect is probably due to the increase in the the investigation of the effectiveness of this thickness of the cuticle and its properties that delay and potentialbiopesticide.

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