Efficacy of Metarhizium Anisopliae and Bacillus Thuringiensis against Tomato Leafminer Tuta Absoluta Meyrick ( Lepidoptera : Gelechiidae )

Susceptibility of Tuta absoluta Meyrick (Lepidoptera : Gelechiidae) populations to Metarhizium anisopliae and Bacillus thuringiensis (Bt) were evaluated under laboratory conditions. T. absoluta larvae were treated either individually or in combination with a single dose rate of B. thuringiensis (0.5 μL / L) and three conidial suspensions viz. 1 x 104, 1 x 106 and 1 x 108 spores / mL of M. anisopliae. Larval mortality, pupation, adult emergence, mycosis and sporulation varied depending on the application of different quantities of M. anisopliae alone and in combination with B. thuringiensis. Maximum mortality (100%) was achieved in 2nd instar larvae when M. anisopliae (1 x 108 spores/mL) and B. thuringiensis (0.5μL / L) were applied synergistically, while 4th instar larvae recorded a 95.45% mortality. Compared with the untreated checks, mortality, pupation and adult emergence of both 2nd and 4th instar larvae were significantly reduced with the combined application of M. anisopliae (1 x 108 spores / mL) and B. thuringiensis (0.5μL / L). Μycosis was most prevalent on 2nd and 4th instar larvae, exceeding 88 and 80% respectively, after exposure to 1 × 104 conidia / mL. The results indicate that the entomopathogenic fungi and the insecticidal protein produced by B. thuringiensis can be used in combination as biocontrol agents for the management of T. absoluta. Current Agriculture Research Journal www.agriculturejournal.org ISSN: 2347-4688, Vol. 7, No.(1) 2019, pg. 37-45 CONTACT Spyridon Mantzoukas mantzoukas@upatras.gr Department of Agricultural Technology, Technological Institute of Western Greece, Amaliada, Greece. © 2018 The Author(s). Published by Enviro Research Publishers. This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY). Doi: http://dx.doi.org/10.12944/CARJ.7.1.05 Article History Received: 06 February 2019 Accepted: 15 April 2019

cause severe hazardous effects. 9,19,20Several studies revealed that in the case of lepidopterous pests, commercially available B. thuringiensis enhanced the efficacy of entomopathogenic fungi when used in an integrated fashion. 21,22The enhanced synergistic effect between the two agents could be due to larval starvation, as it is likely that the bacteria arrest the nutrition of insects, thus enabling the fungus to kill the weakened worms more quickly. 23eping in view the significance of these promising alternatives, the current experiment was planned to evaluate the single and combined effects of M. anisopliae and B. thuringiensis on mortality, pupation and adult emergence of T. absoluta.
M. anisopliae was collected from different regions in Western Greece and different hosts/sources.The specific strain was selected for its high entomopathogenicity, as resulting from preliminary assessment (unpublished data).In order to prepare the appropriate suspensions for the experiments, the isolates were grown in 9-cm Ø Petri dishes with Sabouraud Dextrose Agar and kept in the dark for 15 days at 25 o C ± 1.For bioassays, fresh conidia were collected from 15 day-cultures.Conidial suspensions were prepared by scraping the surface of the Petri dish using a sterile loop and transferring the conidia into a 500 mL glass beaker containing 50 mL sterile distilled water plus 0,05% Tergitol® NP9.The conidial suspension was panned across several layers of sterile cloth and mixed with a magnetic stirrer for 5 min. 3,24Subsequently, a Neubauer hemocytometer was used to determine the conidial concentration under a phase contrast microscope at 400 x magnification.

Introduction
Tomato (Solanum lycopersicum L.) is one of the most popular and nutritious vegetable crops in Greece.Application of synthetic insecticides has increased and remains the most common pest control strategy.However, chemical control is difficult due to the rapid development of pest resistance to insecticides as well as due to the negative impact on natural enemies, the environment and human health.[3] Among the possible methods for the management of Tuta absoluta, [4][5][6][7] which is a key pest of tomato, the use of entomopathogenic microorganisms provides an alternative to chemical insecticides with increased environmental safety and pest selectivity; hence they can be used either alone or in combination with other pest control tactics.Moreover, microbial agents facilitate the survival of beneficial fauna due to their high target specificity.The entomopathogenic fungus Metarhizium anisopliae and the bacterium Bacillus thuringiensis have an important role in crop protection, and may represent effective and ecologically sound solutions to pest problems. 9,10 anisopliae mainly uses propagules such as conidia, blastospores or hyphae to infect the host by direct contact; however, secondary infections took place by horizontal transmission of spores from mycosed cadavers.12 Unlike other microbial agents which need to be ingested to manifest their action, the mycospores adhere to and infiltrate the cuticle upon contact, thus growing internally and producing different toxins that kill the insect pests.10,13 B. thuringiensis is an endospore forming bacterium that produces parasporal crystalline proteinaceous inclusions (Cry and Cyt toxins) during its sporulation and stationary growth phases.14,15 The primary effect of Cry toxins is the cessation of feeding due to paralysis of the mouthparts and gut, leading to the formation of pores in the apical microvilli membrane of the target cells, which results in the lysis of midgut epithelial cells and septicemia.15,16 The specific and narrow spectrum of action of these toxic crystal proteins against lepidopteran, dipteran and coleopteran insects 17,18 renders their use in food and other sensitive crops safer than chemicals which may For the bacterial treatments, we used Bactospeine®, a microbial insecticide from Bacillus thuringiensis sub kurstaki (Hellafram A.E, Greece), formulated as granules and wettable powder (WP) and with 32.000 IU / mg potency.Aqueous suspensions of each dose were prepared at the desired concentrations.The powder was mixed with water in a sterilized Erlenmeyer flask (100 mL), using a sterilized spatula. Thn, aqueous suspensions were prepared by mixing the solution with a magnetic stirrer for 3 min.

Bioassay
The mortality of 2 nd and 4 th instar larvae, pupation and adult emergence was determined by treating T. asboluta larvae with a single dose rate of B. thuringiensis (0.5 μL / L) and three different concentrations of M. anisopliae (Ma1: 1 x 10 4 , Ma2: 1 x 10 6 , Ma3: 1 x 10 8 conidia / mL), both individually and in their respective combinations (Bt + Ma1, Bt + Ma2 and Bt + Ma3).B. thuringiensis was applied by dipping tomato leaf discs (3 cm each) from fourweek old seedlings into the bacterial suspension at a dose rate of 0.5 μL / L, for 3 minutes, in a petri dish.Treated tomato leaves were offered to the larvae in a sterilized petri dish for 48 hours.Larvae were then offered the fresh untreated maize leaves until they pupated or died.Before the treatment with B. thuringiensis, tomato leaves were washed in a solution of commercial bleach (3% sodium hypochlorite) for 2-3 minutes to remove any kind of debris or disease-causing agent, as well as in dd water for one minute, and they were then allowed to dry.However, M. anisopliae was applied following the larval immersion method. 25To check the synergistic effect of M. anisopliae and B. thuringiensis, larvae were exposed to B. thuringiensis by feeding them with treated tomato leaves for 48 hours, and they were then dipped in the fungal suspension for 10 seconds. 26The larvae were then allowed to feed on fresh untreated tomato leaves until they pupated or died.Larval mortality, pupation rate, adult emergence, mycosis and sporulation were recorded.The experiment was carried out in a completely randomized design using 15 larvae of both 2 nd and 4 th instars per replicate, and the bioassay was repeated three times.

Mycosis and Sporulation
For mycosis and sporulation, dead T. absoluta individuals from each treatment were collected, and they were counted and refrigerated at 4 o C in plastic vials.Prior to three washings with distilled water, surface sterilization of mycosed cadavers was done with sodium hypochlorite solution (0.05%) for 2-3 min.The cadavers were placed on Sabouraud Dextrose Agar plates and incubated at 25 ± 2 o C and 75% RH for 7 days.The insects showing external fungal growth were determined under microscope.Sporulation data were determined by immersing mycosed cadavers from each replication in 20 mL distilled water with a drop of Tween-80. 27The treatments were replicated independently, three times.The solution was then thoroughly stirred, and the total number of conidia / mL was counted with the help of a haemocytometer, under microscope.

Statistical Analysis
Corrected percent mortality was calculated using Abbott's formula 28  instar larvae were found to be more death susceptible than 4 th instar larvae, as mortality was found to be linearly related to their developmental stage in all tested treatments (Table 1).

Pupation and Adult Emergence
A significantly lower pupation and adult emergence was recorded in tested larval instars of T. absoluta in those treatments where the mortality rate was high (Table 1).In the control, 91.61% and 93.03% of 2 nd and 4 th instar larvae successfully transformed into pupae, followed by 75.00% and 79.16% pupation when 2 nd and 4 th instar larvae were treated with M. anisopliae at a concentration of 1 × 10 4 conidia/mL (Ma1).Conversely, minimum pupation was observed in the case of both 2 nd (0%) and 4 th (4.19%) instars when the highest concentrations of M. anisopliae (1 × 10 8 conidia/mL) and B. thuringiensis were applied simultaneously (Ma3 x Bt) (Table 1).
Similarly, besides the control, maximum adult emergence was observed in larvae of 2 nd (65.38%) and 4 th instars (70.83%) when treated with M. anisopliae (1 × 10 4 conidia / mL); however, no adult emergence was recorded in both 2 nd and 4 th instars when they were treated with a combination of the highest dose rate of M. anisopliae (1 × 10 8 conidia/ mL) and B. thuringiensis (0.75 μL / L) (Table 1).

Discussion
The aim of the present study was to assess the effect of M. anisopliae and B. thuringiensis against 2 nd and 4 th larval instars of T. absoluta both individually and synergistically.Both larval stages showed varied mortality responses to various concentrations of fungi, alone and in combination with B. thuringiensis as used in this study.Individual applications of entomopathogenic fungi have a great potential to suppress lepidopterous pests. 29This was corroborated by our findings in which M. anisopliae showed significant mortality efficiency (> 40%) at the highest dose rate, in both 2 nd and 4 th instar larvae, especially in the 2 nd instar larvae whose mortality exceeded 50%.Mortality of older instars occurs via infection from mycosed cadavers inside the stem. 30imilar effectiveness of M. anisopliae was recorded by Nguyen et al. 31 in their laboratory bioassays, against the various larval instars of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae).In the current experiment, a dose-dependent upsurge in mortality is corroboratory to the study of Sasidharan and Varma, 32 who reported higher larval mortality of Indarbela quadrinotata Walker (Lepidoptera: Cossidae)-up to 100% -when treated with higher doses of Beauveria bassiana (Bals.-Criv.)Vuill., compared to 66.7 % of mortality when treated with lower fungal concentrations.
The declining trend regarding mortality from 2 nd to 4 th instar larvae was recorded for each treatment application in the current study.Similar outcomes were recorded by Inglis et al., 33 who observed that the virulence of entomopathogenic fungi varies with the different developmental stages of insects.This can be attributed to the reduced fungal germ tube penetration inside the insect body because of the enhanced melanin contents in the mid gut and cuticle of the insect. 34Similar findings were also presented by Hafez et al., 35 where B. bassiana was more virulent to neonate larvae than older instars of the potato tuber moth Phthorimaea operculella (Z.) (Lepidoptera: Gelechiidae).Vandenberg et al. 36 on the other hand, found that the 2 nd instar larvae of Plutella xylostella (L.) (Lepidoptera: Plutellidae) were more resistant to entomopathogenic fungi than the 3 rd and 4 th instars.
In  39 reported that the control of the Colorado potato beetle ranged from good to excellent with the application of low to high concentrations of B. thuringiensis respectively.Enzymatic activity is responsible for differences in mortality between different larval instars.It has been stated that the action of detoxification enzymes changes significantly within and among different developmental stages.This action is minor in the egg stage, increases with each nymphal or larval stage and reduces again to zero at the pupal stage. 40,41ese results indicate clearly that a significantly higher larval mortality was observed in both 2 nd and 4 th instar larvae when M. anisopliae was synergized with B. thuringiensis.These outcomes are in accordance with the findings of Lacey et al. 39 who also reported that the highest larval mortality of the Colorado potato beetle occurred in the plots treated with the combined use of B. thuringiensis and entomopathogenic fungi, while the lowest mortality was recorded in untreated checks.Similarly, the combined application of B. bassiana and B. thuringiensis caused significant larval mortality of the Colorado potato beetle, which was not the case when they were individually applied. 42Lewis et al. 43 concluded that the integrated use of B. thuringiensis and B. bassiana enhanced the larval mortality of the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae).The synergistic action of entomopathogenic fungi and bacteria was further supported by Gao et al. 21who reported that the starvation stress triggered by B. thuringiensis intoxication may cause negative effects on host immunity and physiology.The starvation stress also enhanced the inter-molt period, which could be the possible cause for the improved vulnerability of the L. decemlineata larvae. 44Additionally, Lawo et al. 45 found that sub lethal Cry2Aa intoxication of B. thuringiensis in H. armigera increases the efficacy of M. anisopliae.Similarly, the combined application of both biocontrol agents showed a significant effect on the following developmental stages of both instar larvae with no or little pupation and adult emergence.
In the current study, mycosis and sporulation on cadavers was higher at the low concentrations of conidia.However, mycosis and sporulation on cadavers depend on the method of exposure, the conidial concentration and temperature.At high concentrations, numerous larvae died quickly, and the fungal sporulation was seen only in a few cadavers exhibiting small numbers of conidial production.A self-inhibiting mechanism at high concentrations of conidia was observed, which was also demonstrated by Tefera and Pringle 26 in their experiments.The same mechanism has been also reported by Garraway and Evans 46 for other species of fungi against various insect pests.
Our study suggests that M. anisopliae and B.
thuringiensis can be used as potential biocontrol agents, especially when applied synergistically, for the management of the tomato leafminer.Moreover, pest activity will require intensive scouting to determine the correct application timings of control agents.Different T. absoluta instar larvae responded differently to the applied biocontrol agents, exhibiting various behavioral and physiological responses; more research is required to gain insight into the diversity of responses caused by these agents.However, the mortality obtained in the current laboratory bioassays may not predict the correct field mortality.Therefore, extensive field research is important to be conducted in order to examine the combined efficacy of M. anisopliae and B. thuringienis to develop and corroborate successful integrated pest management solutions against T. absoluta.