Virulence of selected indigenous Metarhizium pingshaense (Ascomycota: Hypocreales) isolates against the rice leaffolder, Cnaphalocrocis medinalis (Guenèe) (Lepidoptera: Pyralidae)

Accepted Manuscript Virulence of selected indigenous Metarhizium pingshaense (Ascomycota: Hypocreales) isolates against the rice leaffolder, Cnaphalocrocis medinalis (Guenèe) (Lepidoptera: Pyralidae) Suyambulingam Arunachalam Kirubakaran, Ahmed Abdel-Megeed, Sengottayan Senthil-Nathan PII:

S0885-5765(17)30157-1

DOI:

10.1016/j.pmpp.2017.06.004

Reference:

YPMPP 1268

To appear in:

Physiological and Molecular Plant Pathology

Received Date: 21 May 2017 Accepted Date: 8 June 2017

Please cite this article as: Kirubakaran SA, Abdel-Megeed A, Senthil-Nathan S, Virulence of selected indigenous Metarhizium pingshaense (Ascomycota: Hypocreales) isolates against the rice leaffolder, Cnaphalocrocis medinalis (Guenèe) (Lepidoptera: Pyralidae), Physiological and Molecular Plant Pathology (2017), doi: 10.1016/j.pmpp.2017.06.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Address Correspondence to:

Prepared for Publication in:

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Dr. S. Senthil-Nathan,

Physiological and Molecular Plant Pathology

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Phone & Fax: + 91 4634 283066; Email - [email protected]

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Title:

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Virulence of selected indigenous Metarhizium pingshaense (Ascomycota: Hypocreales)

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isolates against the rice leaffolder, Cnaphalocrocis medinalis (Guenèe) (Lepidoptera:

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Pyralidae)

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Authors

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Suyambulingam Arunachalam Kirubakarana,b,, Ahmed Abdel-Megeed3 , Sengottayan Senthil-

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Nathan1

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Affiliation

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a

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Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi

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– 627 412, Tirunelveli, Tamil-Nadu, India.

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b

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Science College (Autonomous), Trichy-5, Tamil-Nadu, India.

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c

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P.O.Box.21531, Alexandria- 21526, Egypt

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Funding Source:

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This research was supported by the Department of Biotechnology, Government of India

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(BT/PR13126/GBD/27/194/2009).

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Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for

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Post Graduate and Research Department of Biotechnology, Srimad Andavan Arts and

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Department of Plant Protection, Faculty of Agriculture, Saba Basha, Alexandria University,

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Abstract The aim of this study was to screen and select Metarhizium spp. for the effective management of rice pest Cnaphalocrocis medinalis Guenèe. The virulent screening assay

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with nine Metarhizium soil isolates at the concentration of 1×108 conidia/ml on third-instar

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larvae of C. medinalis have shown that isolates RMT4 and RMT10 caused significant

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mortality (above 90% ) with shorter lethal time than other isolates tested. Molecular

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sequencing of the EFα-1 region of high virulent Metarhizium isolates RMT4 and RMT10

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have confirmed that these isolates belong to the species M. pingshaense. The effect of the two

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virulent M. pingshaense (RMT4 and RMT10) isolates on larvae development, food

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consumption of C. medinalis was studied. Larvae were susceptible to both fungal isolates,

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causing the same level of mortality. In larvae the first instars were the most sensitive with

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shorter lethal times, while the longest lethal time occurred in fourth instars. Estimation of the

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LC50 on third instars, showed a lower LC50 for RMT10 treatment than RMT4. Both isolates

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(RMT4 and RMT10) caused reduced growth and food consumption on third instars.

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Nutritional indices were declined significantly, but the approximate digestibility (AD) of

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treated larvae was significantly higher with increase concentration of the fungal conidia

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(1×105, 1×106 and 1×107). The M. pingshaense isolates are known to cause lethal effects on

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the pupae of C. medinalis. The RMT10 showed lower LC50 value of 7.94×105 conidia/ml on

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pupae than RMT4, which is 2.75×106 conidia/ml. The M. pingshaense RMT10 exhibited

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greater potential as a treatment against C. medinalis, rice leaf folder (RLF)

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Keywords: Rice pest; EPF; elongation factor α-1; Metarhizium pingshaense; Galleria bait

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method; mortality

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1. Introduction The rice leaf folder (RLF), Cnaphalocrocis medinalis (Guenèe) (Lepidoptera:

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Pyralidae) is a migratory destructive pest of the rice crops (Oryza sativa L.) and widely

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distributed in Asia, Oceania and Africa [1-3]. Monoculture of a dominant rice cultivar has

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resulted in lower genetic variability, increased pest pressure, which was exacerbated with

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constant applications of high nitrogen and other fertilizers. These factors have led to

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outbreaks of the pests in tropical and sub-tropical rice growing regions of Asian countries

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[3-5].

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The rice crops were damaged by larval stages of RLF from vegetative to late

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reproductive stages. The RLF damaged leaves show linear pale white stripes due to loss of

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green chlorophyll. The folding and feeding of leaves by RLF larvae reduce the leaf area

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which further leads to the reduction in photosynthesis and increase in transpiration, which

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directly affect the growth and vigour of the plant [8]. The damaged regions of the leaves also

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provide pathways for access of pathogenic fungi and bacteria within plant [2].

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The damage caused by the RLF has led to a dangerous loss in total productivity of

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rice. The yield loss was correlated with the percentage of the leaves damaged by RLF [9].

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The maximum yield loss in rice is reported to be due to the feeding of RLF on the flag leaf

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[10,11]

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Use of chemical insecticides to reduce RLF has also caused a reduction of natural

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enemies in the rice ecosystem and heightened the RLF problem [7, 12]. Furthermore,

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increased applications of insecticides has caused resurgence of secondary pests, the brown

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planthopper, Nilaparvata lugens [13, 14]. Hence there is a great need for alternative control

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strategies to manage this insect pest [15-17].

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Expanding integrated pest management (IPM) strategies depend more on biopesticides and microbial pesticides, which results in exploration of entomopathogens, like

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ACCEPTED MANUSCRIPT fungi, to provide effective sustainable management of pests, while reducing dependence

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solely upon chemical pesticides [16, 18]. Thus, biocontrol of agricultural insect pests with

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entomopathogenic fungi (EPF) can be a promising alternative to chemical pesticides and is an

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important contribution to Integrated Pest Management (IPM) [19, 20]. The use of

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entomopathogenic fungi in pest control holds several benefits including: 1) economic mass

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production, 2) easy to manipulate and formulate, 3) easy integration into agricultural pest

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management systems, 4) safer for the environment, while not causing harm to natural

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enemies with less toxicity to non-target organisms [19, 21, 22]

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The entomopathogenic fungi employed in the present study belongs to Phylum: Ascomycota; Order: Hpyocreales; Genus: Metarhizium, are mitosporic anamorphous fungi

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which have high potency to kill pests from different insect orders. Moreover, they are known

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to cause natural epizootics among insect pest populations in agro and forest ecosystem [22-

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25] .Some species of this genus are cosmopolitan in distribution and are frequently recorded

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in the soil of the agro-ecosystem [26,27].

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The major species of Metarhizium genera have been well studied and developed as

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commercial myco-insecticides in the regulation of major agricultural pests worldwide [28]

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(Faria and Wraight, 2007). The unique infection mechanism, secretion of cuticle degrading

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enzymes and toxic compounds, along with the horizontal transmission of the spores within

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insect populations, production of aerial conidia, with excellent survivability in adverse

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conditions with continuous proliferation where applied make Metarhizium as successful

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biocontrol agents [22, 29]

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Previous reports showed that Metarhizium species were effective against different life

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stages of the lepidopteran pests in vitro, and under field condition [30-35]. However, very

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limited studies have been conducted to evaluate the pathogenicity of Metarhizium species on

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C. medinalis. Therefore, research was conducted towards development of highly virulent

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ACCEPTED MANUSCRIPT indigenous Metarhizium isolates, for suppression of C. medinalis populations on rice.

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Elongation factor alpha-1 (EFα-1) gene sequencing was performed to identify the selected

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Metarhizium isolates. Isolates were evaluated for efficacy on larvae survival and food

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consumption of larvae and pupae of C. medinalis under laboratory conditions.

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2. Materials and methods

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2.1. Cnaphalocrocis medinalis culture

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C. medinalis culture methodology was adopted from the procedure described by

Senthil-Nathan et al. [5]. The culture of C. medinalis was initially raised with larvae collected

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from paddy field grown in and around the Tirunelveli, TN, India. The collected C. medinalis

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larvae were grown in green house at 28±1°C under a 14:10 L:D photoperiod at 85% RH on

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potted rice plants placed in the cage (60×40×70cm3). The resulting pupae in the cage were

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transferred to oviposition cage for adult emergence. In oviposition cage 13 female and 12

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male moths were maintained with one potted rice plant and the moths were fed with 10%

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sucrose solution prepared with a few drops of vitamin mixture (Bluvital-PD, India) to

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increase their oviposition. After 4-7 days the potted plants were removed from the oviposition

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cage. The leaf portions containing the eggs were clipped and placed on moist filter paper in a

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petri dishes (100×15mm) (Hi-media, India). The eggs obtained were used to maintain the

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culture and newly hatched larvae from the eggs were placed on 50-day-old rice plants. The

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insects from the second generation culture were used for the studies. Rice plants of Karnataka

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ponni strain were used for the culture of C. medinalis. The plants were grown in earthenware

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pots, 18 cm tall with a 20 cm diameter top and each pot held 12-15 plants. The pots

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containing rice plants were placed in about 10 cm of water in a metal tray in the greenhouse.

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The rice plants were grown in the control condition without exposure to any chemical

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insecticides.

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2.2. Fungal isolation

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The fungal isolates used in the study were isolated from the soil samples collected

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from rice field habitats of Tirunelveli district, Tamil Nadu state, India. The altitudes and

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location of the sampled soil were recorded using global positioning system (GPS) equipment

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(eTrex summit®, USA) (Table 1). “Galleria bait method” [36] was employed to isolate the Metarhizium species from

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the soil samples. From each soil sample a Metarhizium isolate was recovered for the study. In

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baits, dead Galleria larvae were surface sterilized with the 3% sodium hypochlorite (Hi-

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media, India) (3 min) and washed in sterile distilled water and placed in moist chamber

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(petriplate with wetted filter paper sealed with Parafilm) incubated at 28±1°C in dark and

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observed for the external growth of the fungi and for their aerial conidia production. The

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conidia with typical morphological characters of Metarhizium in Galleria cadavers were

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transferred using sterile inoculation needle to quarter strength of Sabouraud dextrose agar

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(SDA) (Hi-media, India) amended with chloramphenicol (500µg/ml) (Hi-media, India) and

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cycloheximide (250µg/ml) (Hi-media, India) and incubated at 28±10C, 90% RH in B.O.D.

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incubator. The pure isolates of Metarhizium spp. was maintained in colonies with the above

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mentioned media.

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2.3. Morphological confirmation of Metarhizium spp.

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The fungal propugules from pure colony of each isolates were placed in the glass-

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slide using inoculation needle and mounted with lactophenol-cotton blue (Hi-Media, India)

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and observed through microscopic at 40×(Optika-Fluo series B-600TiFL, Italy) and the

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fungal genera were identified and confirmed based on morphological character using

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taxonomic keys [37]. The taxonomic keys such as conidial colour, size and reproductive

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structure of fungi were used for identification of the fungi.

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2.4. Conidial suspension preparation

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ACCEPTED MANUSCRIPT The conidial suspension of each isolate for the bioassay was harvest from 15-20 days-

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old culture of the Metarhizium spp. The conidia were scraped from the medium surface using

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sterile inoculation loop and transfer to 30ml universal glass bottle (Essco Glass Ampoules

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and Vials, India) containing 20ml of sterile 0.05% Triton-X-100 (Hi-media) solution with

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glass beads (3mm) (Hi-media, India). The conidial suspension was vortex for 15 min to break

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the conidial clumps to obtain the homogenous suspension. The concentration of the initial

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stock conidial suspension was determined by counting under microscope (Optika-Fluo series

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B-600TiFL, Italy) at 40× using improved Neubauer haemocytometer chamber. Then it was

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diluted to obtain desired concentration of 1×108 conidia/ml for virulent screening assay.

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2.5. Virulent screening bioassay with third-instar larvae of C. medinalis

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Bioassays were performed with nine isolates of Metarhizium spp. on newly moulted third-instars of C. medinalis. The young rice (Karnataka ponni strain) blade was

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used in the bioassay experiment. The leaves were treated with the fungal inoculums by

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spraying 5ml of conidial suspension using regulator-controlled spray applicator at the single

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concentration of 1×108 conidia/ml and air-dried in the chamber for 10 min to remove excess

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moisture. The control leaves were sprayed with 0.05% Triton-X-100 water solution. The

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treated leaves were placed in bioassay chamber (9×5×4cm3) dampened with wetted cotton

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and tissue paper, to prevent leaves from drying out during the experimental period. The

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healthy third instars were introduced in bioassay chamber containing treated leaves and the

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larvae were allowed to feed on the leaves. The experiment was carried out individually for

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each isolate. Five replications were carried out for each treatment and twenty larvae were

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used per replication. The bioassay chamber was incubated at 28±10C with 90% humidity and

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15:9 L: D photoperiod throughout the experimental period. The mortality of larvae was

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observed from the second day onwards until ninth day after treatment. The dead larvae were

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placed in petridishes (90×15mm) (Hi-media) lined with the filter paper for the observation of

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external growth of the fungi. The mortality of third-instars was recorded and percentage of

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the mortality was calculated and corrected with Abbott’s formula [38]. The lethal time 50

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(LT50) for each isolate were calculated by using Probit analysis [39].

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2.6. DNA Extraction and EFα-1 Sequencing The high virulent Metarhizium isolates (RMT4 and RMT10) were cultured in the 100ml

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conical flask containing 50ml quarter strength SD broth amended with 1% yeast. The culture

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was incubated at 280C for 4-7 days on an incubator shaker (Lark, India) at 200rpm. The

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mycelium was obtained from broth by filtration and weighed 50mg approximately,

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lyophilized and crushed into ground using 1.5 ml microcentrifuge tubes and pestles (Sigma-

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Aldrich, USA) placed in liquid nitrogen. The DNA was extracted from crushed mycelium

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using the GeneiPure™ Plant Genomic DNA Purification Kit (Genei, India) according to

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instruction mentioned by the manufactures.

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The region of the nuclear gene EF-1α was amplified in the thermal cycler (Eppendorf Mastercycler®personal, Germany) under the PCR conditions followed by Rehner and

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Buckley (2005)[40] using the primers: Forward primer EF1T

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5′ATGGGTAAGGARGACAAG AC-3′ and reverse primer PGEF2R

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5′GAACTTGCADGCRATGTGVG-3′. The 50µl PCR reaction mixture contained 5.0µl of

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10×reaction buffer (100 mM Tris– HCl, pH 8.8, 50 mM KCl, 0.1 Triton X-100 and 1.5 mM

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MgCl2), 5.0µl 2mM dNTP Mix; 10 pmol each of the opposing amplification primers; 2.0µl

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DNA template (50ng) and 0.5µl Taq DNA polymerase (5U/µl).

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The obtained PCR product was analysed by electrophoresis in 1.0% agarose gels, the

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gel containing DNA bands were stained with ethidium bromide and illuminated on a UV

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transilluminator captured with gel documentation system (Photostation-Silver plus gel

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documentation system, Lark, India). PCR products were purified using GeneiPure™ Quick

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PCR Purification Kit (Genei, India) and purified products were sequenced (Applied

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Biosystem 370xl DNA analyzer).

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The EF-1α sequence of Metarhizium was analysed and compared with the other Metarhizium isolates in Genbank, NCBI (National Center for Biotechnology Information)

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using Basic Local Alignment Search Tool (BLAST) program. The phylogenetic dendogram

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was constructed with the computer software MEGA6 [41].

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2.7. Dose-dependent mortality studies with M. pingshaense isolates RMT4 and RMT10

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against third-instars of C. medinalis

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Bioassays were performed with the M. pingshaense isolates RMT4 and RMT10 with different concentrations (1×104, 1×105, 1×106, 1×107 and 1×108 conidia/ml) on third-

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instars of C. medinalis to estimate LC50. The leaves of rice were sprayed with the 5ml

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conidial suspension at different concentrations (1×104-1×108 conidia/ml), the control leaves

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were treated with 0.05% Triton-X-100 solution using regulator-controlled spray applicator.

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The newly moulted third instars were placed on treated leaves to feed. Five replications were

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carried out for each treated concentration per isolate and twenty larvae were used per

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replication. The mortality of the larvae was recorded from the second to ninth day post

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treatment. Lethal concentration (LC50) was calculated for each M. pingshaense isolate for

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third-instar C. medinalis, calculated with by Probit analysis [39].

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2.8. Larval age dependent mortality of C. medinalis treated with highly virulent M.

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pingshaense RMT4 and RMT10

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Bioassays were carried out with the virulent M. pingshaense RMT4 and RMT10

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isolates on the first, second, third and fourth instars of C. medinalis. The various stages of

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larvae were treated with the M. pingshaense RMT4 and RMT10 individually at the single

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concentration of 1×108 conidia/ml and LT50 of fungal isolates on each larval stage were

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calculated using Probit analysis [39] (Finney, 1971). The bioassay procedure for larval age

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dependent mortality was carried by above performed bioassay procedure in virulent screening

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assay.

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2.9. Food utilization, consumption and nutritional indices of third-instar larvae of C.

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medinalis treated with M. pingshaense RMT4 and RMT10 The effect of M. pingshaense RMT4 and RMT10 on the food consumption of third

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instars of C. medinalis were studied by treating the diet (rice leaves) at different

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concentration of fungal conidia (1×104, 1×105 and 1×106 conidia/ml). The fresh rice leaves

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were sprayed with 5ml of conidial suspension and control leaf were treated with 0.05%

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Triton-X-100 solution and then air-dried in laminar airflow chamber. The initial weights of

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4h starved third instar were measured and allowed to feed treated and control leaves (10

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larvae per concentration, five replicates) for period of 24h. At the end of 24h, uneaten leaves

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were removed and replaced with fresh untreated leaves. Larvae fresh weight gained, uneaten

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leaves and feces produced were measured using an electronic balance (Shimadzu, Japan).

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Estimated percentages of dry weight of experimental larvae, used sample larvae live weight

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and then post oven-dried for 48h at 60°C dry weight. The uneaten leaves were weighed and

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then oven-dried and re-weighed to estimate diet dry weight. To estimate food ingestion, the

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leaves remaining at the end of the experiment was subtracted from the total dry weight of the

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leaves. Feces were collected, weighed, oven dried and then re-weighed to estimate the dry

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weight of the excreta. Observations were recorded for every 24h and experiments were

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sustained for 5d. Consumption, growth rates, and post-ingestive food utilization efficiencies

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were evaluated according to Waldbauer [42] and Senthil-Nathan et al. [43].

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2.10. Bioassay on C. medinalis pupae with M. pingshaense RMT4 and RMT10

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The pupal assay was performed with M. pingshaense RMT4 and RMT10 at different

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concentrations (1×104-1×108 conidia/ml) on 24h old healthy pupae from the culture of C.

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medinalis. The exposure of conidia to pupae was different from larvae treatments. Ten µl of

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ACCEPTED MANUSCRIPT conidial suspension containing desired concentration (i.e.1×104-1×108 conidia/ml) was

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applied over the individual pupae and spread using fine brush. The control pupae were treated

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with the sterile 0.05% Triton-X-100 solution and the pupae were air-dried in chamber to

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remove excess moisture and then placed in the petridishes (100×15mm) (Hi-media, India)

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lined with the cotton cloth. The entire set was kept in dark incubation at temperature of

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28±10C with (90%) RH. Five replications were carried out for each isolate per concentration

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and twenty pupae were used per replication. The treated pupae were monitored from second

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day of the treatment until 10th day for adult emergence. Lethal concentration (LC50) required

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for M. pingshaense RMT4 and RMT10 to cause 50% of lethal effect on pupae were calculated by Probit regression analysis [39]

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2.11. Statistical analysis

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The mortality and nutritional indices data were expressed as the mean of five replications and normalized by arcsine-square root transformation of percentages. The

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transformed percentages were subjected to analysis of variance (ANOVA). Differences

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between the treatments were determined by Tukey’s multiple range test (p < 0.05) [44].

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Lethal time (LT50) and the lethal concentration (LC50) required by the fungal isolates were

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estimated by Probit analysis [39]. The statistical analysis was performed using Minitab®16

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statistical software package.

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3. Results

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3.1. Virulent screening assay

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In virulent screening assay, nine Metarhizium spp. isolates were tested against third-

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instar larvae of C. medinalis. The results from bioassay experiments showed a significant

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difference in virulence among Metarhizium spp. isolates against third instars of C. medinalis

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(F8,36 =46.40, P<0.0001). Among the tested Metarhizium spp. isolates, the RMT10 caused

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higher mortality, 96%, with a shorter lethal time (LT50) of 3.23 d (2.72−3.68) followed by

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ACCEPTED MANUSCRIPT RMT4 which caused 92% mortality, with LT50 of 3.53 d (3.08−3.96) on third-instar larvae of

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C. medinalis (Fig. 1 and Table 2). The RMT3 was the least virulent isolate comparable to

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other tested isolates, and caused lower percentage of mortality, 51%, and a higher lethal time,

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LT50 of 7.90 d (7.61−8.26) on larvae of C. medinalis (Fig. 1 and Table 2). The isolates RMT4

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and RMT10 showed increased mortality rates, above 90%, with shorter lethal times on third

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instars of C. medinalis. These two competent fungal isolates were selected for further study.

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3.2. Molecular identification of RMT4 and RMT10 Metarhizium isolates

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Sequence analyses of the EFα-1 gene region was amplified and sequenced in order to

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identify the species of each selected highly virulent Metarhizium isolates. Agarose gel analysis of the amplified PCR products showed the EFα-1 gene was nearly 1500 bp in length

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(Fig. 2). The BLASTn analysis of the RMT4 and RMT10 isolates EFα-1 gene sequence

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shows that these isolates belong to the species of M. pingshaense. The isolates RMT4 and

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RMT10 have shown 100% similarity respectively to others M. pingshaense isolates.

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Subsequently the sequence of RMT4 and RMT10 were submitted to NCBI GenBank

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database and accession numbers were obtained (Accession No: KC870067 and KC870068).

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The phlyogenetic analysis of M. pingshaense isolates RMT4 and RMT10 was carried

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out by Neighbour-Joining (NJ) method based on the Kimura 2-parameter model [45] with the

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computer programme MEGA6, using nucleotide sequences data from Genbank (Table 3).

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The phlyogenetics dendogram and nucleotide analyses have shown that the isolates RMT4

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and RMT10 are closely related to other M. pingshaense isolates (Fig. 3).

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3.3. Larval-age dependent mortality of C. medinalis

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First, second, third and fourth instar larvae of C. medinalis were treated with the M.

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pingshaense RMT4 and RMT10 at the concentration of 1×108 conidia/ml. All tested larval

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stages were susceptible to the both M. pingshaense isolates. M. pingshaense infected C.

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medinalis larvae have shown external growth of fungi mycelium and aerial conidia

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ACCEPTED MANUSCRIPT production, when incubated at 280C, 90% relative humidity (Fig. 4). The results also shows

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that there was no significant difference observed in mean percentage mortality among the

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larval stages in treatment with RMT4 (F3,16=3.25, P<0.0001) and RMT10 (F2,12=2.46,

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P<0.0001) (Fig. 5). However there was difference in lethal time (LT50) among larval stages

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were observed in treatment with M. pingshaense RMT4 and RMT10 individually (Table 4).

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The fungal isolates shows shorter lethal time 50 to kill first instar larvae than other tested

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larval instar, which shows the first instar were highly susceptible.

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Shorter LT50 of (2.58 (2.29−2.84) days were recorded in first instar larvae of C.

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medinalis followed by second, third and fourth instar larvae in treatment with M. pingshaense RMT4 (Table 4). Similarly in treatment of all larval stages of C. medinalis with

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M. pingshaense RMT4, shorter LT50 of 2.31 (1.56−2.81) were recorded in the first larval

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instar than other larval stages. The isolates RMT4 and RMT10 has shown longer lethal time

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(LT50) of 4.35 (3.90−4.82) and 3.86 (3.54−4.17) on the fourth instar of C. medinalis (Table

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4). Altogether the above results revealed that fourth instar larvae of C. medinalis were less

15

susceptible to the both fungal isolates than other tested larval stages.

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3.4. LC50 estimation of M. pingshaense RMT4 and RMT10 on third-instar larvae

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The lowest LC50 value of 8.34×104 conidia/ml was recorded in RMT10

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followed by RMT4 with LC50 of 4.45×105 conidia/ml on third-instar larvae of C. medinalis

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(Table 5). The result shows that the mortality of the larvae was and then it was observed that

20

increase in the concentration of the conidia significantly enhances the mortality of the larvae

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in each treatment (P<0.05).

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3.5. Effect of M. pingshaense RMT4 and RMT10 on the food consumption of C. medinalis

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The treatment of third instar larvae with M. pingshaense RMT10 and RMT4 at

24

different concentration (1×104, 1×105 and 1×106 conidia/ml) are known to affect the growth

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and consumption rate of C. medinalis larvae compared to control (Table 6 and 7).

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ACCEPTED MANUSCRIPT M. pingshaense RMT4 at different concentration, significantly reduced ECI (F3,8=

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14.54; P< 0.0001), ECD (F3,8= 47.14; P< 0.0001), RCR (F3,8= 27.87; P< 0.0001) and RGR

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(F3,8= 14.68; P< 0.0001) in larvae of C. medinalis than control (Table 6). Similarly in

4

treatment with RMT10 at various concentration, the isolates significantly reduced ECI (F3,8=

5

31.29; P< 0.0001), ECD (F3,8= 57.58; P< 0.0001), RCR (F3,8= 22.26; P< 0.0001) and RGR

6

(F3,8= 43.36; P< 0.0001) in third-instar larvae of C. medinalis than the control larvae (Table

7

7).

The approximate digestibility of the third-instar larvae of C. medinalis larvae

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significantly increased at the concentration of 1×104, 1×105 and 1×106 of M. pingshaense

10

RMT4 (F3,8= 44.25; P< 0.0001) and RMT10 (F3,8=53.30; P< 0.0001)than control (Table 6

11

and 7). The experiment has clearly indicated that the decrease in food consumption and

12

growth rate was much greater in M. pingshaense RMT10-infected larvae than in RMT4-

13

infected larvae.

The relative consumption rate and the relative growth regression were plotted to

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determine the effects of M. pingshaense (RMT4 and RMT10) isolates on third-instar larvae

16

of C. medinalis and the regression lines represent the reduced growth level of the larvae

17

which fed on the M. pingshaense treated leaves (Fig. 6A and 6B).

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3.6. Pupicidal activity of M. pingshaense RMT4 and RMT10 on C. medinalis

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The pupicidal activity of both M. pingshaense RMT4 and RMT10 isolates were tested

20

on the newly moulted pupae of C. medinalis. The treatment of pupae with the different

21

concentration of conidia has shown that the mortality of the pupae was dose-dependent.

22

The Lethal concentration of M. pingshaense (LC50) on pupae was estimated using

23

Probit regression analysis. The isolates M. pingshaense RMT10 record lower LC50 value of

24

7.94×105 conidia/ml (Fig. 7A) than RMT10 isolate LC50 (2.75×106 conidia/ml) (Fig. 7B).

25

The LC50 value data has revealed that the RMT10 isolate was most potent virulent isolate on

15

ACCEPTED MANUSCRIPT the pupae of C. medinalis than RMT4. The fungal infected pupae have a growth of fungi

2

mycelia and the incubation of the infected pupae in the humid chamber has shown

3

sporulation of green aerial conidia on pupae. This observation was recorded in all fungal

4

(RMT4 and RMT10) infected pupae (Fig. 8).

5

4. Discussion

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Entomopathogenic fungi have been widely used as biological control agents to reduce economically important insect pests of agricultural crops [19, 46]. Biocontrol agents, such as

8

entomopathogenic fungi, especially Metarhizium species to the rice pest are needed. The

9

isolates evaluated in this study were competent on C. medinalis larvae and pupae. From nine Metarhizium isolates collected from soils around the Tirunelveli district,

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Tamil-Nadu, India two isolates were shown to have significant activity to reduce third instars

12

of C. medinalis. Third instars were susceptible to all tested indigenous Metarhizium species.

13

All the isolates show mortality rates above 50% on the larvae at the concentration of 1×108

14

conidia/ml. However there were significant differences in virulence among the tested isolates.

15

Only four of the isolates of Metarhizium cause larval mortality above 70%. Two isolates

16

though (RMT4 and RMT10) cause a significantly higher mortality rate above 90%, with a

17

shorter lethal time (Fig. 1). The variation in the virulence among the isolates is unknown, but

18

may be due to the differences in the production level of cuticle degrading enzymes [47,48] or

19

toxic secondary metabolites [49, 50] The identification and species confirmation of two

20

indigenous highly virulent Metarhizium isolates RMT4 and RMT10 provide new biological

21

control agents with potential for development into commercial products to reduce C.

22

medinalis. Studies using molecular approaches to identify Metarhizium species report using

23

nuclear elongation factor alpha-1 sequence [51,52]. Our phlyogenetic analysis of EF-1α

24

nucleotide sequence from Metarhizium isolates RMT4 and RMT10 show they have

25

significant homology to the species M. pingshaense (Fig. 3).

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The M. pingshaense isolates have the ability to infect and kill larval stages causing significant mortality (Fig. 4). Even so, larvae of C. medinalis show variation in their

3

susceptibility upon treatment with highly virulent M. pingshaense RMT4 and RMT10

4

isolates. The variation in susceptibility of lepidopteran larvae to Metarhizium species has

5

been reported previously [33-35]. In this experiment the first and second instars were highly

6

susceptible to M. pingshaense isolates with shorter lethal times, compared with third and

7

fourth instars. Similarly Kirubakaran et al. [35] reported that second instar of C. medinalis

8

show higher the rice pest are needed. The isolates evaluated in this study were competent on

9

C. medinalis larvae and pupae. Nguyen et al. [33] also reports that third, fourth and fifth

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instars were less susceptible than second instars of Helicoverpa armigera Hüb. upon

11

treatment with M. anisopliae. In contrast, Maniania et al. [34] reported that Lepidopteran pest

12

Busseola fusca Ful. second and third instar were more susceptible than the first instars to M.

13

anisopliae treatments.

The dose-dependent mortality with M. pingshaense isolates on third instars show that,

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RMT10 was more virulent at lower doses producing a lower LC50 compared to RMT4.

16

Increase in the conidial concentration of M. pingshaense produce prominent mortality in

17

larvae of C. medinalis. Similarly, Nguyen et al. [33] reported that species of Metarhizium

18

exhibits its virulent against the lepidopteran pest, H. armigera also in a dose-dependent

19

manner.

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The treatment of the third instars of C. medinalis with M. pinghaense cause effects in

21

the food consumption, growth rate and utilization efficiencies. Tefera and Pringle [31] also

22

report a reduction in the food consumption of second and third larvae of Chilo partellus

23

(Swinhoe) treated with different concentration of M. anisopliae. Moore et al. [53] and

24

Thomas et al. [54] suggested that there is a reduction in the feeding of the desert locust,

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Schistocerca gregaria For., and the variegated grasshopper, Zonocerus variegatus L., upon

2

infection with the fungi M. flavoviride.

3

There were significant reduction of both the RCR and RGR in larvae as the concentration of M. pinghaense increases (Tables 5 and 6). The fungi infected larvae have the

5

ability to maintain AD, but fail to maintain the RGR during larval development. The AD

6

could not be retained due to the continuous decline in RGR (Table 6 and 7). The ECI and

7

ECD are significantly reduced with an increase in concentration of fungal conidia. The ECI

8

is a measure of larvae capacity to utilize the food that it ingests for growth. The ECD was

9

reduced as the quantity of digested food metabolized for energy increases [55].

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The reduction in the food consumption and growth rate in treated larvae was due to

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the toxicity of metabolites produced by the fungi and depletion of the insect’s nutrient

12

reserves, combined with degradation of tissues by the invading fungal hyphae [31, 54].

13

Fungal metabolites affect the insect gut and other tissue physiology, which directly effects the

14

feeding of the insect [56, 57]

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Pupae are dormant and often great challenges as a target with biocontrol agents. Targeting the pupae of insects using entomopathogens has advantages over other control

17

agents, such as the ability to penetrate the cuticle [58].The pupae assay revealed that both

18

isolates have the ability to penetrate the external barrier of the pupae cuticle killing them. The

19

potential use of Metarhizium species against lepidopteran pupae were reported in several

20

studies [33, 58]. The infection of the M. pinghaense isolates to the pupae was also dose-

21

dependent. Similarly Anand et al. [58] revealed the effectiveness of fungal species on pupae

22

of S. litura with LC50 and LC90 values for the entomopathogenic fungi M. anisopliae var.

23

anisopliae, Lecanicillium muscarium and Cordyceps cardinalis. Their study also correlated

24

efficacy and virulence of M. anisopliae var. anisopliae with lower LC50 and LC90 values of

25

1.2×107 conidia/ml and 4.8×108 conidia/ml than other tested fungal species.

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ACCEPTED MANUSCRIPT The results support that M. pinghaense RMT10 and RMT4 isolates are effective

1

entomopathogen against C. medinalis larvae and pupae under laboratory conditions. Based

3

on these results, the more severe isolate, M. pinghaense RMT4 may provide a suitable

4

microbial agent for inclusion in current IPM programs to reduce reliance on conventional

5

chemical pesticides in the control of C. medinalis in rice.

6

Acknowledgements

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This research was supported by the Department of Biotechnology, Government of

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India (BT/ PR13126/GBD/27/194/2009). The authors sincerely thank to Dr Wayne B Hunter

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for his suggestion and comments on an earlier version of the manuscript. Reference

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10 11

AC C

EP

TE D

12

ACCEPTED MANUSCRIPT Table legends 1. Details of Metarhizium spp. isolates used in the studies. 2. Lethal time (LT50) and 95% confidence interval of Metarhizium spp. isolates treated at concentration (1×108conidia/ml) against third -instar larvae of C. medinalis.

RI PT

3. Details of fungal isolates used in the phylogenetic analysis and GenBank accession number of translation elongation factor α-1 gene sequences of the isolates (Bischoff et al., 2009)

SC

4. Lethal time (LT50) and 95% confidence interval (C.I.) of M. pingshaense isolates RMT4 and RMT10 on the different larval stages of C. medinalis.

M AN U

5. Lethal concentration (LC50) estimation of highly virulent M. pingshaense isolates RMT4 and RMT10 on the third larvae of C. medinalis.

6. The Nutritional indices of third-instar larvae of C. medinalis treated with M. pingshaense RMT4

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7. The Nutritional indices of third-instar larvae of C. medinalis treated with M.

AC C

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pingshaense RMT10

ACCEPTED MANUSCRIPT Table 1.

Collection sitea

RMT2

Panakudi

RMT3

Origin of substrateb

Geographic location

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Metarhizium spp. isolates code

Longitude. E

Soil

8.340499

77.569914

Kodumudiyaru

Soil

8.425993

77.536260

RMT4

Kalakadu

Soil

8.527112

77.516278

RMT5

Karisalapatti

Soil

8.623687

77.574285

RMT6

Karukurichi

Soil

8.697190

77.537800

RMT7

Singampatty

Soil

8.664821

77.436436

RMT8

Athanallur

Soil

8.719036

77.471120

RMT9

Papanasam

Soil

8.716174

77.365995

RMT10

Alwarkurichi

Soil

8.777401

77.403764

M AN U

SC

Latitude. N

AC C

EP

TE D

Collection site mentioned in the table belong to Tirunelveli district, Tamil-Nadu, India. b -All the soil substrate was collected from the rice field habitat.

a

-

ACCEPTED MANUSCRIPT Table 2.

LT50 (in days)

Confidence interval Lower

Upper 6.96

6.69

6.45

RMT3

7.90

7.61

RMT4

3.53

3.08

RMT5

4.36

3.97

RMT6

4.90

4.75

RMT7

6.14

RMT8

7.42

RMT9

5.71

3.23

AC C

EP

8.26 3.96 4.80 5.05

5.51

7.18

7.16

7.72

5.15

6.54

2.72

3.68

M AN U

TE D

RMT10

RI PT

RMT2

SC

Metarhizium spp. isolates

ACCEPTED MANUSCRIPT Table 3. -Indicate data not available

Substrate/source

M. pingshaense

CBS:257.90 ARSEF:3210 ARSEF:7929 ARSEF:4342 ARSEF:7501 ARSEF:4739 ARSEF:7450 ARSEF:4303 ARSEF: 6238 ARSEF:7505 ARSEF:1015 ARSEF:4628

Coleoptera Coleoptera Isoptera Coleoptera N/Aa Soil Coleoptera Soil Lepidoptera Coleoptera Lepidoptera Soil

M. roberstii M. anisopliae M. guizhouense M. majus

AC C

EP

TE D

M. lepidiotae

Geographical location China India Australia Solomon Islands Australia Australia Australia Australia China Australia Japan Australia

Accession number EU248850 DQ463995 EU248847 EU248851 EU248849 EU248848 EU248852 EU248859 EU248857 EU248870 EU248866 EU248863

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Isolate

M AN U

Species

SC

a

ACCEPTED MANUSCRIPT Table 4.

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M. pingshaense isolates RMT4 RMT10 LT50 (days) and C.I LT50 (days) and C.I

First

2.58 (2.29 2.84)

2.31 (1.56 2.81)

Second

2.92 (2.28 3.44)

2.55 (2.35 2.73)

Third

3.53 (3.08 3.96)

Fourth

4.35 (3.90 4.82)

SC

Larval instar of C. medinalis

3.23 (2.72 3.68)

AC C

EP

TE D

M AN U

3.86 (3.54 4.17)

ACCEPTED MANUSCRIPT

LC50 (conidia/ml) 4.45×105 8.35×104

Confidence interval

Slope

Intercept

χ2

p

2.24 8.44×105 2.39×104 2.09×105

1.45 1.49

-3.22 -2.37

9.36 12.91

0.002 0.005

AC C

EP

TE D

M AN U

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M. pingshaense isolates RMT4 RMT10

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Table 5.

ACCEPTED MANUSCRIPT Table 6.

RCR (mg/mg/day) 1.23±0.05a

ECI (%) 21.18±0.50a

ECD (%) 45.01±3.53a

AD (%) 45.21±1.05c

RI PT

Treatment RGR** (conidia/ml) (mg/mg/day) Control 4.92±0.11a* 1×104

4.74±0.41ab

0.78±0.36ab

20.02±0.78a

41.88±1.89a

46.54±1.55b

1×105

3.95±0.74bc

0.54±0.27b

18.05±0.62b

38.52±1.73b

47.89±1.57b

1×106

2.67±0.30c

0.36±0.20c

15.13±0.57b

34.43±2.79b

49.56±2.64a

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**Relative consumption rate (RCR); relative growth rate (RGR); approximate digestibility (AD); efficiency of conversion of ingested food (ECI); efficiency of conversion of digested food (ECD)

AC C

EP

TE D

M AN U

* Means (±SE) followed by the same letters within columns of indicate no significant difference (p< 0.05) in a Tukey test.

ACCEPTED MANUSCRIPT Table 7.

RCR (mg/mg/day) 1.19±0.10a

ECI (%) 21.45±0.50a

ECD (%) 45.65±2.67a

AD (%) 45.71±1.05c

RI PT

Treatment RGR** (conidia/ml) (mg/mg/day) Control 4.98±0.26a*

SC

1×104 4.51±0.41a 0.65±0.20b 19.08±0.15ab 40.06±3.24a 46.10±1.55bc 1×105 3.67±0.43b 0.42±0.04b 16.36±0.32bc 35.24±2.89b 48.27±1.57b 1×106 2.25±0.26c 0.28±0.35c 13.74±1.28c 30.10±2.79b 51.92±3.25a **Relative consumption rate (RCR); relative growth rate (RGR); approximate digestibility (AD); efficiency of conversion of ingested food (ECI); efficiency of conversion of digested food (ECD)

AC C

EP

TE D

M AN U

* Means (±SE) followed by the same letters within columns of indicate no significant difference (p< 0.05) in a Tukey test.

ED

M AN U

CE

PT

ED

M AN US

PT ED

M AN US

EP

TE D

M AN US C

TE D

M AN US

AC C

EP

TE D M AN US C

RI

AC C

EP

TE D

M AN US C

RI

ED

M AN U

ACCEPTED MANUSCRIPT

Highlights High virulent isolates (RMT4 and RMT10) were identified as M. pingshaense through comparative gene sequencing, (EFα-1, elongation factor 1-alpha).

•

Treated larvae and pupae were susceptible to the M. pingshaense in dose-dependent manner.

•

M. pingshaense affected food consumption of third-instars of C. medinalis

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M .pinghanese (RMT10) was the more effective isolate against C. medinalis

AC C

EP

TE D

M AN U

SC

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•