International Science Congress Thailand 2017

International Science Congress Thailand 2017 Oct 02-06, 2017 Venue : Asian Institute of Technology Conference Centre, Klong Luang, Pathumthani, Bangkok, Thailand www.iscth2017.wixsite.com/conference | Email : [email protected]

Conference Coordinator : Dr. Chai Ching Tan, Faculty of Management Sciences Mae Fah Luang University, Thailand

Conference Convener : Dr. Worakamol Wisetsri, Faculty of Social Sciences King Mongkut's University of Technology, Thailand

International Advisory Members : Dr. Jagmohan Bajaj, Expert in Bangkok UNESCO, Spl Envoy, Ministry of DT : 02/09/2017 Education & Science, Republic of Macedonia. Dear (Prof/Dr/Mr/Ms) Rungkiat Kawpet, Samaporn Saengyot Prof.Francisco Jose Alcala Torreslanda, Vice President, Kellogg Co, USA. Congratulations! Based on the Recommendations of the Reviewers and the Technical Program Committees, we are pleased to inform you that Prof.Mrinalini B Chavan, your Paper Kirti College, Mumbai, India

Prof.Mora Fernandez, T049A : EFFECT OF ARTIFICIAL AND LOCAL MASS PRODUCTION MEDIA La Casa Mandarina, Mexico. ON GROWTH OF ENDOGENOUS STRAINS OF Beauveria bassiana TOWARDS SUSTAINABLE INSECT CONTROL Prof. Katja Petakova, Jensen Education, Sweden. has been accepted for both publication and presentation (PPT 20 min). Dr. Jeyarani, American College, Madurai, India You are cordially invited to present the paper at International Science Congress Thailand to be held October 02-06, 2017 at Asian Institute of Prof.Lorena Kourousias, La Casa Mandarina, USA Technology Conference Centre, Klong Luang, Pathumthani, Bangkok, Thailand Dr. Suresh Samson, CEC, Chirala, India Looking forward to see you at the Conference.

Dr. Kankeyanathan Kannan , Jaffna University, Sri Lanka.

For ISCTH 2017

Note: All Overseas delegates please come with 6 Months Validity Passport and Return Flight Ticket along with Hotel Accommodation Confirmation.

Proceedings of the International Science Congress Thailand 2017 @ AIT Bangkok, Thailand.

EFFECT OF ARTIFICIAL AND LOCAL MASS PRODUCTION MEDIA ON GROWTH OF ENDOGENOUS STRAINS OF BEAUVERIA BASSIANA TOWARDS SUSTAINABLE INSECT CONTROL

RUNGKIAT KAWPET, SAMAPORN SAENGYOT1

Abstract: The objective of this study is to evaluate the effects of artificial media comprising of Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Agar Supplemented with Yeast Extract (SDAY), Malt Extract Agar (MEA), Nutrient Agar (NA) and Water Agar (WA), and sterilized local mass production media consisting of cooked rice, unmilled rice, sorghum seed and dog feed on growth of entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillenim (Acomycota: Hypocreales) isolate 01 (Bbsoi). The studies showed that NA gave highest growth rate (3.04 ± 0.17 mm. per day) while SDAY induced highest sporulation (8.80 ± 0.33 spore per ml.). The fungus cultured on SDAY was highest at 7 days Percent Cumulative Mortality (PCM) to aphids, Lipaphis erysimi (Hemiptera: Aphididae), flea beetle, PhyUotreta sinuata (Coleoptera: Chrysomelidae) and cutworm, Spodoptera litura (Lepidoptera: Noctuidae) averaging 94.20 ± 4.02, 71.4 ± 3.85 and 79.00 ± 1.58 percent respectively.

Keywords: Beauveria Bassiana, Economic Insect, Entopathogenic Fungi, Mass Production. Introduction: Beauveria bassina fungus (Balsamo) Vuillenim (Ascomycota: Hypocreales) is a disease- generating fungus in insects, which has been one of the fungus most widely studied and utilized for pest control. This is because the said fungus can destroy various types of pest, such as Hemiptera, Lepidoptera, Diptera, Hymenoptera and Coleoptera insects [1]. There have been reports of this fungus being used in the form of microbial insecticide, which works well in controlling pest affecting economically significant crops, such as European corn-borers, Ostrinia nubilalis [2]. Many isolates of this fungus have been developed into a commercial formulation, such as Bbi47 formulation, extracted from o. nubilalis. It was used for pest control of Asiatic Corn borer (o.furnacalis), coffee berry borer, white grubs, bollworm, cutworm and brown planthopper. The commercial names were Bio-power, Beauverin, Boverol, Boveosil, Naturalis-O, Naturalis-T and Back-off [3]. However, the commercial formulations are not quite widespread and does not make up a significant part of the pest control market yet. Furthermore, it is expensive, making it unsuitable for application by low-income farmers. For effective pest control using fungus, particularly in developing countries, the most effective isolate found in local areas will be used. As much culture available in the local area would be used [4H6]. As for Thailand, there have been research efforts on culture method to increase B.bassiana fungus, the most effective local isolate in controlling pest, found in Thailand. The culture can also be found locally [7].

Factors influencing the growth of fungus that can be utilized in the form of an insecticide formulae, specifically in agricultural plots, are such as virulence, or pathogenicity and environmental tolerance, namely lights, temperature and humidity of the fungus. These are mostly directly inherited from genotypes [8]. Additionally, there are reports that artificial media impacts the strengths of B. bassiana and may also impact the fungus’ genetic characters as well [9]. This information leads to the question that, even if a local isolate of pest control fungus is selected to use, due to its capacity to pest in the lab, and that it can create a sufficient amount of spores for certain media, it is possible that the fungus strengths may be reduced as a result of the artificial media or culture used. Molecular level biological indicators are related to the strength level of the fungus, which is the enzyme production [10] connects to the genetic characteristics of the fungus. If the type of the culture impacts the amount of enzyme production and/or the genetic characteristics, it would impact the general usage. However, further impacts may include the investment value in economic terms, if the artificial media used for commercial formulation was not appropriate.

This research is a continuation of the work by the researchers who selected B.bassiana fungus found in local areas for the benefits of pest control. They receive the research funds from National Research Council of Thailand (NRCT). It was discovered that B. bassiana isolate Bbsoi is the most effective pest control for many variety, such as citrus aphid, diamondback moth, and common cutworm. In the Financial year of 2012, a research grant to study the disease causing strengths, and the molecular level genetics of B. Bassiana, a local strain, which was a result from artificial media and growing culture for the benefits of sustainable pets control. In 2012, there has been a selection on the culture type suitable for growing the volume of B. bassiana fungus, isolate Bbsoi. Considerations are made from indicators, both in quantitative term of the fungus external

ISBN 978-93-86435-17-0 I 54 Proceedings of the International Science Congress Thailand 2017 @ AIT Bangkok, Thailand. characters, from the ability to grow, the spore production. There is also a study on the level of relationship between quantitative indicators and the culture, which link to quality indicator. It is also the disease-causing strength, leading to a guideline for maximum utilization of existing natural resource, making the most of the investment. Therefore, finding a clear conclusion on the relationship and the impacts of the culture towards productivity and the fungus strengths need further research. It allows identification of artificial media and culture suitable to bring in local strain focus for usage. Material and Methods: Selecting the Suitable Type of Artificial Media for the Cultivation and Preparation of Leavening Agent: Select the type of artificial agent suitable for the cultivation and preparation of B. Bassiarta leavening agent, by considering from the ability to grow and produce spores on artificial media, which are Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Agar Supplemented with Yeast Extract (SDAY), Malt Extract Agar (MEA), Nutrient Agar (NA) and W ater Agar (WA).

To find the growth rate of a single colony on various type of media using cork borer with a diameter of 0.7 cm., take out a piece of the media with the hyphae of B. Bassiana grew on PDA media for 3-5 days, or until there is no contamination from bacteria or other fungi. Then relocate the fungus onto the PDA media until the fungus is 5 days old. Use cork borer the size of 0.7 cm., take out the piece of media with hyphae, and place it on the plate with artificial media you would like to test, all 6 types. Bring the plate on the shelf with lighting hours: dark hours of 12:12 hours, at 25±3 degree Celsius. Then measure the diameter of the fungus very 24 hours until the media plate has B. bassiana spread throughout the plate (diameter of around 9 cm.). Plan the experiment in the format of CRD, repeat each experiment 3 times.

Find the spore production rate of B. Bassiana fungus by moving pure fungus onto the PDA media for 5 days. Use cork borer the size of 0.7 cm., take out the media with the hyphae on the plate with the media we would like to test. Bring the plate onto the shelf, and set for light hours:dark hours of 12:12 hours, at the temperature of 25+ 3 degree Celsius for 10 days. Afterwards, use the cork borer with the diameter of 0.7 cm to take out the media with the hyphae and spores from the plate. 3 Pieces a plate. Put it in distilled water of 10 ml., and count the spore numbers in the unit of spores per ml (ท=3). For each repetition, the number of spore counts from c Cell equates to 0.02 X 0.02 X 001 cm. = 4.0x106 cubic centimeters. Divide it by (2.5x105) (Goettel and Inglis, 1997, pp. 213-249. Plan out the experiment in a CRD format and test it 5 times each.

Bring the average of the B. bassiana fungus isolate BbsoF 24 hours growth rate on each type of media for analysis to look for the fluctuation, in order to look for statistically significance level of 95 and 99%. If it was found to be statistically significant, do a comparison on the difference between average using DMRT method. Then bring the average number of spores from each B. bassiana, isolate Bbsoi created on the media each type. Analyze for the fluctuations to identify what is statistically significance at the level of 95 and 99%. If there is a significance, compares the difference between averages using DMRT. Selection of Mass Production Media That Could Be Found Locally and Are Suitable For Cultivation and Preparation of Leavening Agent: Prepare the mass production media that can be found in local area, such as cooked rice, unmilled rice, sorghum seed and dog feed. Prepare the cooked rice by using streaming jasmine rice until its 50% cooked. Then spread it out to cool in a bamboo-woven tray to reduce the excess moisture. Prepare the unmilled rice by bringing it to clean, and immerse it in water for 24 hours. Afterwards, boil it for 15 minutes before dry it to dry in the opened air for 30 minutes on a bamboo tray. Sorghum seed should be prepared the same way with unmilled rice. As for the dog feed (Plemo brand for all types of full grown dogs, liver flavor), immerse it in clean water for 1.5 minute, then spread it out to dry on the bamboo tray. Put each type of culture in the heat-tolerant plastic bags, 150 gram each. Use the plastic bottle necks to seal and close up the bottle necks with cotton. Cover it with thick tissues and tie it tightly. Place it for sterilization at the temperature of 121 degree Celsius, for 30 minutes, then bring it to cool at room temperature. Move the pure fungus of 7 days old and put it on PDA media. Use the cork borer with a diameter of 0.7 cm to impale the section of the media with the fungus hyphae. Place it in the bag with prepared culture on the shelf, ensure the light hours: dark hours are at 12:12 hours, at the temperature of 25±3 degree Celcius, until the fungus spread out through any type of media. Prepare and grow fungus on the culture and incubate the fungus according to the method above. Bring the fungus incubated for 240 hours to monitor the volume of spore production on fungus grown on different type of culture. Measure the hyphae growth of the fungus by recording the hours

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the focus hyphae used to grow. Use this information to calculate for the growth percentage of the fungus on different type of fungus, as follows: No. days of fungus uses to grow' X loo Percentage of fungus growth = No. days of fungus grown up first full for a cultivation bag ' Plan for the experiment in the format of CRD by repeating it 5 times each. Then use the average value of B. bassiana growth on each type of culture. Analyze the fluctuation for the statistically significance at the confidence intervals of 95 and 99%. If it พ7aร found to be significant, then compare the difference between the average using DMRT. To find out the amount of fungus spore production on each type of media, bring the food with fungus growing on them and dissolve it in พ7ater at the volume of 500 ml. Bring out 0.05 ml. for count on the hemacytomer and find the spore count in the unit of spores per ml. according to 1), the experiment should be planned in a CRD format, and repeated 5 times each. Afterwards, bring the average number of spore counts of B. Bassiana ร, isolate Bbsoi grown on each type of media. Analyze the fluctuations to identify for statistically significance level of confidence level 95 and 99%. If there are any significance found, compare the difference of average values using DMRT. Results: Artificial Media Suitable for Culture and Preparation of Leavening Agent: Selection of the artificial media type suitable for growing and preparing the Beauveria bassiana leavening agent, isolate Bbsoi. Considerations are made from the capability to grow7 and produce spores on artificial media, such as Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Agar Supplemented with Yeast Extract (SDAY), Malt Extract Agar (MEA), Nutrient Agar (NA) and Water Agar (WA). It พ7aร discovered that the grow7th rate of a single colony of B. bassiana per hour has statistically significant difference. (p=o.oi) (Table 1). The highest and low7est average are 3.21 and 0.53 mm. per 24 hours. Once grown on NA and WA media respectively, the fungus that can expand the peripheral of the colony the fastest on NA media พ7as 3.04 ± 0.17 mm. per 24 hours. There was no statistically significant difference for growths on MEA media. As for measuring the amount of production spore, it appears that the type of artificial media used can impact the spore production of B. bassiana isolate Bbsoi significantly. In the duration of 10 days in laboratory', the fungus can produce as much as 9.35 spores and low7est at 1.57 spores per ml., upon being growrn on SDA and WA media. The amount of spore produced on each type of artificial media for B.bassiana isolate Bbsoi are significantly different. For the grow7th on SDA media, the highest average found was 8.80 ± 0.55 spores per ml. (table 2). Table 1: The Growth Rate of A Single Colony Of Beauveria Bassiana Isolate BbbsOl On Different Types of Artificial Media Artificial media Growth rate (mm. per 24 hour) Potato Dextrose Agar (PDA) 2.36 ± 0.30c Sabouraud Dextrose Agar (SDA) 2.72 ± 0.08b SDA with Yeast Extract (SDAY) 2.70 ± 0.17b Malt Extract Agar (MEA) 2.78 ± 0.19a Nutrient Agar (NA) 3.04 ± 0.17a Water Agar (WA) 0.68 ± 0.i5d

Note: The average ± SD from repetitions, 5 times each, following the different alphabets means there are statistically significant difference at the confidence interval of 99% by comparing the difference in average values with DMRT. Table 2: Amount of spore Production of Beauveria Bassiana Isolate Bbsoi on Different Types of Artificial Media Artificial media A m ount o f Spore Production (spores per ml.) ๐ k> D-

Potato Dextrose Agar (PDA) 6.62 ± 'ง Sabouraud Dextrose Agar (SDA) 8.80 ± 0.55a SDA with Yeast Extract (SDAY) 7.40 + 0.43c Malt Extract Agar (MEA) 8.10 + 0.45b Nutrient Agar (NA) 7.40 ± 0.84c W ater Agar (WA) 2.20 ± ๐-45e

ISBN 978-93-86435-17-0 I 56 Proceedings of the International Science Congress Thailand 20ใ7 @ AIT Bangkok, Thailand.

Mass Production Media That Could Be Found Locally Suitable fo r the Culture and Preparation o f Leavening Agent: The culture whiqh can be found locally comprises of cooked rice, unmilled rice, sorghum seed and dog feed, which have been sterilized with steaming. It appears that the growth percentage of B. bassiarta isolate Bbsoi fungus on 150-g of culture. There is a statistically significant difference (p=o.oi) (Table 3). The highest and lowest value is at 99.89 and 66.49% respectively. The media with hyphae spread to the greatest coverage is sorghum seed. However, there has been no difference between using the cooked rice as media. The average growth percentage of the fungus on the media is 98.80 ± 1.09 and 94.60 ± 3.63 percent respectively. The amount of spore production for fungus in media appears to have significant difference. The highest is at 10.60 spores per ml., when grown on the sorghum seed, it was 7.40 spores per ml. when grown with dog feed. The spore generation is the highest on sorghum seed with the average of 10.60 ± 0.89 spores per ml., and follows by cooked rice is 10.20 ± 1.30 spores per ml. (table 4) which has no statistically difference. Table 3ะ Growth Percentage of Beauveria Bassiana Isolate Bbsoi on Mass Production Media Mass Production Media G row th (%) Cooked rice 94.60 ± 3.65a Unmilled rice 87.60 ± 8.26b # Sorghum seed 98.80 ± 1.09a ± Dog feed 70.40 ______ms______Note: The average + SD from repetitions, 5 times each, following the different alphabets means there are statistically significant difference at the confidence interval of 99% by comparing the difference in average values with DMRT. Table 4: Amount of Spores per Ml. of Beauveria Bassiana Isolate Bbsoi on Mass Production Media Mass Production Media Amount of Spore Production (spores per ml.) Cooked rice 10.20 - j- 1.30a Unmilled rice 8.60 ± 1.34b Sorghum seed 10.60 ± 0.89a Dog feed______7.40 ± 0.55b

Note: The average + SD from repetitions, 5 times each, following the different alphabets means there are statistically significant difference at the confidence interval of 99% by comparing the difference in average values with DMRT.

Discussion: Results of Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Agar Supplemented with Yeast Extract (SDAY), Malt Extract Agar (MEA), Nutrient Agar (NA) and W ater Agar (WA) artificial media and culture, which could be locally sourced (Cooked rice, Unmilled rice, Sorghum seeds and Dog feed) to the growth and spore production of Beauveria bassiana isolate Bbsoi, in consideration to the growth rate of individual colony and production generation of B. bassiana, from Table 1 and 2, there are inconsistencies with the research result. The growth rate of an individual colony for 24 hours, reveals that the fungus can expand its colony the fastest on NA media NA (3.04 ± 0.17). However there is no statistically significant difference on the growth of MEA media (p = 0.01). The spore production per ml. unit of the spore suspended solution in this experiment show's that steamed Sorghum seeds, which are sterile, are suitable for spore production. The spore production of insect disease fungus is crucial to the application of said fungus. It has influences over the disease causing ability in quantitative term [11]. This study shows that the culture on SDA media gives the highest number of spores on average (8.80 ± 0.53 spores per ml.). As for this part, from observation of the growth rate of fungus on the media, it was revealed that even if the fungus can grow' quickly on NA and/or MEA media, the hyphae is fluff and loosely consolidated. It may be this point that cause the fungus grown on aforementioned food to create larger amount of spores, as the hyphae, is the origin of its reproductive part, such as conidiophore and spores. Cooked rice, unmilled rice, sorghum seeds and dog feed

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are culture that can be locally sourced. It can be used to grow B. bassiana appropriately. With scientific evidence to substantiate, it would be great if the B. bassiana is used to control crops7 pests safely in the agricultural practice. After considerations of 2 indicators, which hare the growth percentage of fungus and the amount of spore production on 150 g. culture. This result shows that the culture with fungus hyphae of best coverage is steamed sorghum seeds and/or cooked rice (98.80 ± 1.09 and 94.60 ± 3.65 percentage respectively). In this case, the result IS consistent with spore production in the culture, the fungus has the highest spore production when grown on sorghum seed (ท.49 spores per ml), follows closely by cooked rice (10.20 ± 1.30 spores per ml). There was no statistically significant difference respectively (Table 3 and 4). Therefore, for initial consideration, the said material is suitable to be used for growing B. bassiana fungus. Acknowledgment: This research was financially supported by Suan Dusit University, Project No. 82358 by National Research Council of Thailand. References: 1. Tanada, Y. & Kaya, H.K.. “Insect pathology.77 San Diego, USA: Academic Press. (1993) 2. Lewis, L. c., Berry, E.c. & Bing, K.G. “Aptness of insecticides (Bacillus thuringiensis and carbofuran) with endophytic Beauveria bassiana (Balsamo) Vuillemin for European corn borer control program for immediate and season-long suppression.77 Canadian Entomologist, 123 (1996): 387-393. 3. Copping, L.G. “The manual of biocontrol agents77 (4th ed.). Hampshire, UK: BCPC. (2009) 4. Lopez, L., Carbonell, L.v. & Salinas, J. “Colonization of plant waste substrates by entomopathogenic and mycoparasitic fungi- a SEM study77. Micron, 3. 4 (1999): 325-333. 5. Sahayaraj, K., & Namasivayam, ร. K. R. “Mass production of entomopathogenic fungi using agricultural products and by- products77. African Journal of Biotechnology, 7.12 (2008): 1907-1910. 6. Posada-Florez, F.J. “Production of Beauveria bassiana fungal spores on rice to control the coffee berry borer, Hypothenemus hampei, in Colombia.77 Journal of Insect Science, 8. 41 (2008): 1-13. 7. Saengyot, ร., & Napompeth, B. “Simple technique for mass propagation of Bueaveria bassiana for biological control in Thailand.77 Journal of International Society of Southeast Asian Agricultural Sciences (ISSAAS), supplement, (2007): 1- 7. 8. Butt, T.M., Jackson, C.W., & Magan, N. Fungi as biocontrol agents: progress, problems and potential. London, UK: CABI Publishing. (2001). 9. 9. Rodriguez-Gomez, Loera, D. o. , Saucedo-Castaneda, G., & Viniegra-Gonzalez, G. “Substrate influence on physiology and virulence of Beauveria bassiana acting on larvae and adults of Tenebrio molitor ” Microbiology and Biotechnology 25. 3 (2009): 513-518. 10. Wang, c., M.A. Typas and T.M. Butt. Detection and characterization of pn virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae. FEMS Microbiology Ecology Letters, 213 (2002): 251- 255- 11. Fargues, J., Maniania, N.K. & Delmas, J.c. Infectivity of propagules of Paecilomyces fumosoroseus during in vitro development to Spodoptera frugiperda. Journal of Invertebrate Pathology, 64 (1994): 173-178.

*** Rungkiat Kawpet Faculty of Science and Technology/Suan Dusit University/Bangkok, 10700/Thailand Samaporn Saengyot Department of Plant Protection/Faculty of Agricultural Production Mae Jo University/Chiang Mai, 50290/Thailand

ISBN 978-93-86435-17-0 I 58 ISCTH 2017 International Sciecnce Congress Thailand 2017

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IMRF INTERNATIONAL PUBLISHING HOUSE, # 1-90, Near VTPS Main Gate, Ibrahimpatnam, Krishna Dt, A.P., India Email: [email protected] Tel: +91 9533 42 1234 TEAM ISCTH 2017 Conference Director ะ Dr.Ratnakar D. B, Director (Academics), IMRF

Conference Rector: Dr. M Lellis Thivagar Professor & Head, School of Mathematical Sciences Madurai Kamaraj University, India. Conference Moderator ะ Dr. Jagmohan Bajaj Expert in UNESCO, Spl Envoy, Ministry of Education & Science, Republic of Macedonia. Conference Coordinator ะ Dr. Chai Ching Tan, Faculty of Management Sciences Mae Fah Luang University, Thailand Conference Convener ะ Dr. Worakamol Wisetsri, Faculty of Social Sciences King Mongkut's University of Technology, Thailand Mathematics, Engineering & Physical Sciences Coordinator: Dr. Pankaj Srivastava, Professor & Head, Dept of Mathematics Motilal Nehru National Institute of Technology, India Life Sciences , Agriculture Coordinator ะ Dr. p Bhaskar Reddy, Dept of Microbiology and biotechnology GPG College, Vikram University, Ratlam, Madhya Pradesh, INDIA Business Sciences & Legal Studies Coordinator ะ Prof. Vijaya Vani, Faculty of International Business Studies Acharya Nagarjuna University, India Social Sciences, Women Studies & Education Coordinator ะ Prof. David Sweeney, Chartered Accountant Bournemouth, United Kingdom English Studies Coordinator ะ Prof. Kenichiro Higuchi Faculty of Culture - Information Studies Sugiyama Jogakuen Univeristy, Japan Medical Sciences Coordinator ะ Dr. D Krupa Daniel, Professor, Dept, of Human Anatomy, Southern Medical University, Guangzhou, China

International Advisory Members Dr. Tarni Mandal, Dept of Maths, NIT, Jameshdpur, India Prof.Francisco Jose Alcala Torreslanda, Vice President, Kellogg Co, USA. Prof.Mrinalini B Chavan, Kirti, Mumbai, India Prof.Mora Fernandez, La Casa Mandarina, Mexico. Dr. Shamim Bandey, Poonch, India Prof. Katja Petakova, Jensen Education, Sweden. Dr. Jeyarani, American College, Madurai, India Prof.Lorena Kourousias, La Casa Mandarina, USA Dr. Suresh Samson, CEC, Chirala, India Dr. Kankeyanathan Kannan , Jaffna University, Sri Lanka. Prof. Katrina Ericson, Jensen Education, Sweden. Prof. Jean, Philippines.

And Supporting Team from AIT Bangkok Thailand. CONTENTS

Agriculture & Life Sciences T003A EXPOSURE TO ENVIRONMENTAL ENDOCRINE DISRUPTING 41 CHEMICALS Dr. Reddy, P.B T056A ABUNDANCE OF INDIAN HONEY BEE APIS CERANA CERANA AT 42 DIFFERENT ALTITUDES IN HILLY AREAS OF UTTARAKHAND, INDIA R.M.Srivastava, Pankaj Kumar Toi2A RICKETTSIOSES o f w il d BOAR'S TICKS OF ALGERIA 43 Faygal Zeroual, Hamza Leulmi, Idir Bitam, Ahmed Benakhla, Nassim Didier Raoult, Philippe Parola T033A INFLUENCE OF VARYING PLANT DENSITY ON PER CENT 44 SURVIVABILITY AND MORTALITY OF ‘SABRINA’ STRAWBERRY (. FRAGARIAXANANASSA DUCH.) IN VERTICAL GROWING SYSTEM UNDER NATURALLY VENTILATED POLYHOUSE D. Madaiah, RaghavendraPrasad, M.Dinesh Kumar, B.c. Dhartanjaya T043A • IMPACT OF SPRINKLER IRRIGATION SYSTEM ON MAIZE 47 PRODUCTIVITY AND PROFITABILITY IN NORTHERN KARNATAKA, INDIA-AN ECONOMIC ANALYSIS ShreeshailRudrapur, ร. M. Mundinamani, M. V. Manjurtatha, S.B.Hosamani, V. R Kiresur, ร.ร. Dolli T049A EFFECT OF ARTIFICIAL AND LOCAL MASS PRODUCTION 54 MEDIA ON GROWTH OF ENDOGENOUS STRAINS OF BEAUVERIA BASSIANA TOWARDS SUSTAINABLE INSECT CONTROL Rungkiat Kawpet, Samaporn Saengyot T055A IN-VITRO ANTIOXIDANT ACTIVITY, TOTAL PHENOLICS, TOTAL 59 FLAVANOIDS AND ANTIUNGAL ACTIVITY OF

T065A AGRONOMIC MANAGEMENT OF RABI PIGEONPEA UNDER DIFFERENT 59 PLANT DENSITIES IN NEPZ (NORTH EAST PLAIN ZONE) OF INDIA Akhilesh Mishra, Geeta Rai, Arvind Srivastav T003A APPLICATION OF MULTIPLE FISH BIOMARKERS FOR THE PREDICTION 60 OF AQUATIC POLLUTION

T057A STUDY ON CULTURING METHODS TO INCREASE THE 61 QUANTITIES OF OPHIOCORDYCEPS FOUND IN THE AREA OF DOI INTHANON NATIONAL PARK, CHIANG MAI PROVINCE, THAILAND Rungkiat Kawpet, Samaporn Saengyot T073A IDENTIFICATION OF PLANT HOPPERS AND LEAF HOPPERS 66 ON COCONUT PALMS AFFECTED BY WELIGAMA COCONUT LEAF WILT DISEASE (WCLWD) IN THE MATARA DISTRICT, SRI LANKA Nuwan Rumesh พ eeranansha ISCTH 2017 ISBN 978-93-86435-17-■0

Conference Coordinator : Dr. Chai Ching Tan, Faculty of Management Sciences Mae Fah Luang University, Thailand

Conference Convener : Dr. Worakamol Wisetsri, Faculty of Social Sciences King Mongkut's University of Technology, Thailand

Prof.Mora Fernandez, T057A : STUDY ON CULTURING METHODS TO INCREASE THE QUANTITIES OF La Casa Mandarina, Mexico. OPHIOCORDYCEPS FOUND IN THE AREA OF DOI INTHANON NATIONAL PARK, CHIANG MAI PROVINCE, THAILAND Prof. Katja Petakova, Jensen Education, Sweden. has been accepted for both publication and presentation (PPT 20 min). Dr. Jeyarani, American College, Madurai, India You are cordially invited to present the paper at International Science Congress Thailand to be held October 02-06, 2017 at Asian Institute of Prof.Lorena Kourousias, Technology Conference Centre, Klong Luang, Pathumthani, Bangkok, La Casa Mandarina, USA Thailand Dr. Suresh Samson, CEC, Chirala, India Looking forward to see you at the Conference.

Dr. Kankeyanathan Kannan , Jaffna University, Sri Lanka.

For ISCTH 2017

Note: All Overseas delegates please come with 6 Months Validity Passport and Return Flight Ticket along with Hotel Accommodation Confirmation.

Proceedings of the International Science Congress Thailand 2017 @ AIT Bangkok, Thailand.

STUDY ON CULTURING METHODS TO INCREASE THE QUANTITIES OF OPHIOCORDYCEPS FOUND IN THE AREA OF DOIINTHANON NATIONAL PARK, CHIANG MAI PROVINCE, THAILAND

RUNGKIAT KAWPET, SAMAPORN SAENGYOT

Abstract: The investigation was carried out by surveying and field collecting, the entomopathogenic fungi in the genus Ophiocordyceps (Hypocreales: Ophiocordycipitaceae) in Doi Inthanon National Park, Chiang Mai Province, found that the majority specimens were identified as Ophiocordyceps unilateralis, o. sobolifera and 0. sphecocephala respectively; a few specimens were identified as o. irangiensis and three specimens collected 1. e. Ophiocordyceps sp.1, Ophiocordyceps sp.2 and Ophiocordyceps sp.3 could not be identified. All species were isolated as pure cultures by single spore isolation and tissue culture techniques. Each of the Ophiocordyceps cultures were tested for growth on various media, temperature and pH levels in the dark for 15-60 days. It was found that all of them except o. unilateralis grew well on potato dextrose agar at 25° c, pH 5-6 and pseudostroma were developed.

Keywords: Classification, Entomopathogenic Fungi, Field Surveying, spatial Distribution. Introduction: Thailand is one of the highest sources of various kinds of fungal especially the fungal in Cordycipitaceae, Clavicipitaceae, and Ophiocordycipidae in Division (Phylum) Ascomycota and Hypocreales. In Thailand, there are some reports revealing that the Cordyceps fungal in Cordycipitaceae and Ophiocordyceps in Ophiocordycipidae is the mostly found one [1]. There is also the report on finding three kinds of Cordyceps spp. destroying the cicada larvae (which must be correctly as cicada nymphs) for the first time in the North east of Thailand. One kind was found in Kuchinarai District, Kalasin Province and two kinds were found in Muang District, Maha Sarakham Province. However, the new name was not given or separated [2] which might be Cordyceps cicadae found in Thailand [3]. It was the fungal called cicada flower (Cordyceps sobolifera) in the past or Ophiocordyceps sobolifera (Zhu et al., 2016, pp.619-627) at present.

Several kinds of fungal of Ophiocordyceps spp. could be benefited in agriculture, medical, and pharmaceutical such as being used as microbial insecticides both in formulations and the production with simple local technology for controlling the insects as same as Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces spp, etc. [4]. Regarding medical and pharmaceutical, there is the report on the medical properties of the fungal Ophiocordyceps spp. especially for the fungal Ophiocordyceps sinensis the fungal known well as “chong cao” (Dong-chong-xia-cao or called Chong cao in short) on moth caterpillar in Thitarodes (Lepidoptera: Hepialidae) called “ghost moth”. It was reported that this kind of fungal is used as Chinese herb from the ancient Chinese era. According to Scientific data, it was reported that some kinds of fungal in Ophiocordyceps can produce the bioactive compounds with medical and pharmaceutical activity namely cordycepin, ophicordin, galactomannan, and polysaccharide which are the bioactive compounds detectable in fruiting body of the fungal. After having tested, it was found that these substances had the activity of antitumor, antibacterial, antifungal, etc. [5] especially o. sinensis which was the kind found to destroy the caterpillar in Thitarodes in the highland in the autonomy area of Thibet, China having the report referring to very high marketing value [6]. Moreover, there was the report of finding the essential amino acids, vitamin K, vitamin Bi, vitamin B2, and vitamin B12 in the fungal Ophiocordyceps [7]. Therefore, the fungal in Ophiocordyceps is considered a kind of microorganism beneficially for human in medical and pharmaceutical fields.

There is the report referring the numbers, kinds, and varieties of fungal Ophiocordyceps partly both domestically and internationally [1], [6], [ร}. However, as Thailand is one of the country having varieties in the lands of microorganism highly as well as having the data sources on the classifications of fungal Ophiocordyceps spp. systematically and sufficiently for further studying in order to create the knowledge for benefiting from the existing natural resources, this research project studies the culture methods to increase the quantities of Ophiocordyceps fungal species found in the area of Doi Inthanon National Park, Chiang Mai Province, by selecting the artificial media suitable for cultivating each land of fungal.

Material and Methods: Study on the culture methods to increase the quantities of Ophiocordyceps species found in the area of Doi Inthanon National Park, Chiang Mai Province, by selecting the artificial media suitable for cultivating each kind of fungal.

ISBN 978-93-86435-17-0 61 Proceedings of the International Science Congress Thailand 2017 @ AIT Bangkok, Thailand.

Pure Fungal Separation: Bring the specimens of Ophiocordyceps of each species to be separted to obtain the pure culture by using the methods of single spore isolation and tissue culture.

Single Spore Isolation: Rinse the soil or dirt out of the specimens with clean water. Sterilize the outer surface of the specimens by using the method of triple surface sterilization as follows. Soak in alcohol 70% for 1 minute and soak in sodium hypochlorite 5.25% for 1 minute. Then, soak in alcohol 95% for 30 minutes. Wash the excessive solution out by soaking in the steriled distilled water for 1 minute and then place the specimens on the culture plate with steriled tissue paper to absorb water until getting dry. Cut the specimens on the fertile part horizontally with the razor. Drop 1-2 drops of steriled distilled water on the slide. Use the forceps to pick the specimens and turn upside down on the side with the cut on the drops of distilled water for 2-3 times. The asci will be noticed in the opaque features floating in the distilled water. Touch the asci to be separated with needle. Use Pasteur pipette to suck the spore suspensions on the slide to put into the chloramphenicol 0.01% solution in 2 ml. steriled distilled water for removing the microorganism accompanied by the spores. Incubate at the room temperature (27 ± 2° C) in the darkness for 24 hours! Examine the sprouting of spores under the microscope of combined lens. Use micro pipette to suck the spore or sprouting part-spores to put into the culture plate with potato dextrose agar (PDA) 5, 1 spore or 1 part-spore each. Place at the room temperature (27 ± 2° C) in the darkness for 15-60 days. Notice the growth of mycelia. Confirm the results of pure fungal isolation of the fungal on the culture media which have to contain similar features in all points without the contamination of bacteria or yeast. Store the pure fungal in the test tube with media of potato dextrose agar in the refrigerator (temperature around 10° C) to be used in the experiment further.

Tissue Culture: Rinse the soil or dirt out of the specimens with clean water. Sterilize the outer surface of the specimens by using the method of triple surface sterilization as follows. Soak in alcohol 70% for 1 minute and soak in sodium hypochlorite 5.25% for 1 minute. Then, soak in alcohol 95% for 30 minutes. Wash the excessive solution out by soaking in the steriled distilled water for 1 minute and then place the specimens on the culture plate with steriled tissue paper to absorb water until getting dry. Cut the specimens on the fertile part horizontally with the razor. Use the needles to separate the tissue inside to be placed on 5 points on the culture plate with potato dextrose agar. Incubate at the room temperature (27 ± 2° C) in the darkness for 15-60 days. Notice the growth of fiber out of the tissue. Confirm the results of pure fungal isolation of the fungal on the culture media which have to contain similar features in all points without the contamination of bacteria or yeast. Store the pure fungal in the test tube with media of potato dextrose agar in the refrigerator (temperature around 10° C) to be used in the experiment further.

Study on The Growth of Pure Fungal on the Media: Take the pure culture Ophiocordyceps of each species to be cultured in the media of potato dextrose agar. Incubate at the room temperature (27 ± 2° C) in the darkness for 15-60 days. Use the cork borer of 5 mm diameter to bore the fiber at the colony edge. Put it on the media used in studying the growth of the pure fungal namely corn meal agar(CMA), Czapek’s agar, malt exract agar (MEA), oat meal agar (OA), and potato dextrose agar (PDA). Incubate at the room temperature (27 ± 2° C) in the darkness for 15 days. For some species of pure fungal of Ophiocordyceps slowly grown, the period is extended to 30-60 days. Notice and measure the diameters of the colony. Record the results.

Study on the Growth of Pure Fungal on the Media at Various Tempetures: Take the pure culture of Ophiocordyceps of each species to be cultured in the media of potato dextrose agar. Incubate at the room temperature (27 ± 2° C) in the darkness for 15-60 days. Use the cork borer of 5 mm diameter to bore the mycelia at the colony edge. Put it on the media used in studying the growth of the pure culture of Ophiocordyceps for such species to grow best for 6 sets of experiment. Repeat each set 3 times and bring the media plate of each set to incubate in the temperatures of 20, 25, 30, 37, 45° c the room temperature (27 ± 2° C) repectively in the darkness for 15 days. For some species of pure culture of Ophiocordyceps, the period is extended to 30-60 days. Notice and measure the diameters of the colony. Record the results.

Study on the Growth of Pure Fungal on the Media at Various pH: Take the pure culture of Ophiocordyceps of each species to be cultured in the media of potato dextrose agar. Incubate at the room temperature (27 ± 2° C) in the darkness for 15-60 days. Use the cork borer of 5 mm diameter to bore the mycelia at the colony edge. Put it on the media on which such species of Ophiocordyceps can grow best for 6 sets of experiment. For each set, adjust pH to be 4, 5, 6, 7, and 8 by using the lactic acid or hydrogen peroxide and the set without adjusting pH, respectively. Repeat each set 3 times and bring the media plate of each set to incubate in the temperatures which the pure culture of Ophiocordyceps of such species can grow best in the

ISBN 978-93-86435-17-0 62 Proceedings of the International Science Congress Thailand 2017 @ AIT Bangkok, Thailand. darkness for 15 days. For some species of pure culture of Ophiocordyceps, the period is extended to 30-60 days. Notice and measure the diameters of the colony by using the ruler. Record the results.

Results: The results of study on the methods to increase the quantities of Ophiocordyceps found by selecting the artificial media suitable for culturing each fungal I I Isolation of the Pure Fungal: From bringing the specimens of Ophiocordyceps of each species to be sterilized on the surface by using the method of triple surface sterilization and segregate for the pure fungal using the method of single spore isolation and tissue culture in the media of potato dextrose agar, according to the initial experiment, it is found that the isolation of pure fungal by using the method of single spore isolation gives better results than the method of tissue culture as there is less contamination of bacteria or yeast. This method is selected in segregating the fungal and can segregate the pure culture of Ophiocordyceps of every species.

The Growth of Pure Fungal on Various Media: From bringing the pure culture of Ophiocordyceps of each species to be cultured in 5 media; corn meal agar, Czapeks agar, malt extract agar, oat-meal agar, and potato dextrose agar and leaving at the room temperature (27 ± 2° C) for 13-60 days, it is found that most pure culture of Ophiocordyceps grow well in the media of potato dextrose agar followed by malt extract agar, corn meal agar, oat meal agar, and Czapeks agar, respectively. When comparing the growth period of Ophiocordyceps of each species, it is found that Ophiocordyceps sp.1, Ophiocordyceps sp.2, and Ophiocordyceps sp.3 grow fast in the media whereas o. unilateralis grow quite slowly (Table 1).

Table 1: Diameter of colony of Ophiocordyceps on Various Media at the Temperature Room (27 ± 2° C) for 15-60 days Diameter oi M edia Co ony (m m.) Period Corn Malt Oat Potato Ophiocordyceps Czapek’s (days) meal extract meal dextrose agar agar agar agar agar o.irangiensis 30 15 7-5 19-5 17-5 18 5 O.sobolifera 30 7-4 5-5 10 7-5 16 O.unUateralis 60 8 5-5 15-5 8 18 O.sphecocephala 30 16 6.5 18.5 13 22.5 Ophiocordyceps sp.1 15 47 24.5 51-5 46.5 54 Ophiocordyceps sp.2 15 49-5 26.5 53 49 57 Ophiocordyceps sp.3 15 48 25 52 47 54

The Growth of Pure Fungal On the Media in Various Temperatures: When bringing the pure culture of Ophiocordyceps of each species to be cultured in potato dextrose agar which is the media growing best and incubate at various temperatures for 15-60 days, it is found that most pure culture of Ophiocordyceps grow best at the tempertures of 20, 23, and room temperature (27 ± 2° C). It grows well at 25° c. However from 4 species such as o. iranensis, Ophiocordyceps sp.1, Ophiocordyceps sp.2, and Ophiocordyceps sp.3 grow well at 30° c. From the experiment, no species can grow at 37 and 45° c (Table 2).

Table 2: Diameter of Colony of Ophiocordyceps on the Media of Potato Dextrose Agar Incubated At

Diameter of Media Colony (mm.)* Period Room Ophiocordyceps (days) temperature 20° c 25° c 30° c 37° c 4 5 ° C (2 7 ± 2 ° C) O.irangiensis 30 20.5 19 20 7 - - O.sobolifera 30 17 17-3 19 - - - 0.unilateralis 60 18 21 22.5 - - - O.sphecocephala 30 22.5 23 25 - - - Ophiocordyceps sp.1 15 54 55.6 59 6 - - Ophiocordyceps sp.2 15 57 59-5 63 37 - - Ophiocordyceps sp.3 15 56 57 60 37 - - *- signifies no growth

ISBN 978-93-86435-17-0 63 Proceedings of the International Science Congress Thailand 20ใ7 @ AIT Bangkok, Thailand.

The Growth of Pure Fungal on the Media at Various pH: When bringing the pure culture of Ophiocordyceps of each species to be cultured in potato dextrose agar which is the media growing best. Adjust pH to become 4, 5, 6, 7, and 8 compared to the controlling set. Incubate at 25° c which is the temperature in which the pure fungal of Ophiocordyceps growing best for 15-60 days, it is found that most pure culture of Ophiocordyceps can grow well in the media of potato dextrose agar with pH from 5-6. In the media with pH8, the pure fungal of Ophiocordyceps cannot grow well (Table 3) I

Table 3ะ Diameter of Colony of Ophiocordyceps on the Media of Potato Dextrose Agar at Various pH Incubated at 25° c for 15-60 Days Diameter of media colony (mm.) Period Ophiocordyceps (days) Controlling pH pH pH pH pH set (pH - 5 .6) 4 5 7 o.irangiensis 30 21 18 21 21.5 16.5 15 O.sobolifera 30 18 17 - 18.5 18 14-5 13-5 o.unilateralis 60 23-5 20 22 22.5 13 10.5 22 21 O.sphecocephala 30 25 23-5 . 25 25-5 Ophiocordyceps sp.1 15 59 56 ’ 59 61 54 52-5 Ophiocordyceps sp.2 15 63 59-5 62.5 63 56.5 54-5 Ophiocordyceps sp.3 15 59 57 58 62 55 53

Discussion: The isolation of pure culture of Ophiocordyceps can be done by using the method of single spore isolation and tissue culture in the media potato dextrose agar. According to the initial study, it is found that the isolation by using the method of tissue culture takes long time in culturing from tissue to grow to fiber. In addition, the contamination from bacteria or yeast on the tissue placed on the media often occurs. Therefore, the isolation of pure culture is difficult. For segregating by using the method of single spore isolation, it is found that spore can develop to mycelia and can grow on the media more rapidly. There is also less contamination. Thus, this method may be the method suitable for isolating the pure culture of Ophiocordyceps.

From the experiment in culturing the culture of Ophiocordyceps on the media of corn meal agar, Czapeks agar, malt extract agar, oat meal agar, and potato dextrose agar at the room temperature, it is found that Ophiocordyceps can grow in the media of potato dextrose agar better than other media. It is because this media contains the nutrients suitable for the growth of Ophiocordyceps most compared to other kinds of media (corn meal agar, malt extract agar, and oat meal agar). It is possibly because the media of potato dextrose agar contains the carbon which is the single molecule sugar. The culture of Ophiocordyceps can be used more easily whereas the carbon source in other kinds of media must pass the decomposition process before being applied. For the artificial media (Czapek’s agar), the pure fungal of Ophiocordyceps cannot grow well because this kind of media contains the difficultly-decomposed carbon sources. There is also shortage of vitamin and mineral essential for the growth of mycelia. Ophiocordyceps can grow very little. If the formula of Czapek's agar is adjusted to be suitable for the growth of Ophiocordyceps, this kind of media can be used in testing the demand of the media source of the culture of Ophiocordyceps. This experimental result is correspondent with the experimental result of [9] culturing the pure culture of Ophiocordyceps in 7 species and [10] culturing the pure fungal of Ophiocordyceps in 6 species. It is found to grow well in the media of potato dextrose agar.

When comparing the growth rate in media of potato dextrose agar of the pure culture of Ophiocordyceps and the growth of other kinds of fungi, it is found that Ophiocordyceps can grow quite slowly. The cause is possibly because Ophiocordyceps wants some nutrients from insects in the growth of mycelia. It can be seen from the experimental of [11] experimenting to culture Ophiocordyceps militaris in the media with rice and insect's pupa. It is found that the pure culture of Ophiocordyceps can grow well and creating the reproduction structure in the media.

When using the pure culture of Ophiocordyceps to culture on the media of potato dextrose agar with different temperature of 20, 25, 30, 37 45° c and the room temperature (27 ± 2° C), it is found that Ophiocordyceps of all species can grow well in the temperature range of 20, 25 and the room temperature (27 ± 2° C). It can grow at 250 c. Some species can be grown well at 30° c but they can not grow well in the temperatures of 37 and 45°c.,

ISBN 978-93-86435-17-0 I 64 Proceedings of the International Science Congress Thailand 2017 @ AIT Bangkok, Thailand. no growth of the pure fungal is found. This experimental result is quite similar to the culture of Ophiocordyceps of [12] and [10] reporting that the pure culture of Ophiocordyceps can grow well at 22 and 20° c, respectively.

From culturing the pure culture of Ophiocordyceps on the media of potato dextrose agar, pH is initially adjusted to 4, 5, 6, 7, 8, and not adjust pH (pH 5.6) at 250 c. It is found that the pure culture of Ophiocordyceps can grow well in the media of all experimented pH. It can be said that the width of colony is similar. The media w ith pH 5-6 will be the media with the mycelia of the pure culture growing best. It can be seen that Ophiocordyceps can grow พ,ell in the media with pH both acid and base. It will grow พell in the media with the soft: acidic media.

Acknowledgment: This research W'as financially supported by Suan Dusit University, Project No. 232559 by National Research Council of Thailand. References:

1. Suchada Mongkolsamrit, Jenet Jenifer Lueang Sa-at, Kanoksi Thatsanathai & Som Sak Siwichai. Entomopathogenic fungi in Thailand. National Center for Genetic Engineering and Biotechnology.Pathum Thani. (2010). 2. Srivilai, p., Surapron, ร. & Louchamvoot, p. First report of Cordyceps sp. isolated from cicada in Northeastern Thailand and their characterizations. Journal of Biological Sciences, 13. 7 (2013): 587-595. Doi: 10.3923Zjbs.2013.587.595 3. Hsu, J.H., Jhou, B.Y., Yeh, S.H., Yen-lien Chen, Y.I. and Chin-Chen, c. c. Healthcare Functions of Cordyceps cicadae. Journal of Nutrition & Food Sciences, 5.6 (2015): 432. Doi: 10.4172/2155-9600.1006432 4. Copping, L.G. The manual of biocontrol agents. 4th ed. Hampshire, UK: BCPC, (2009): 702. 5. Jone, K. A brief pharmacologic review of Cordyceps sinensis and c. ophiolglossoides. Armana Research. (1993)- 6. Evan, H. c., & Samson, R.A. Cordyceps species and their anamorphs pathogenic on ants (Formicidae) in tropical forest ecosystems: The Cephalotes (Myrmicinae) complex. Transaction British Mycological Society, 79-3 (1982): 43^ 453- 7. Holliday, J. & Cleaver, M. Medicinal value of the caterpillar fungi species of Genus Cordyceps (Hr.) Ling (Sacomycetes). A review'. International Journal of Medicinal Mushroom, 10.3 (2008): 219-234. 8. Hywel-Jones, N. L. Cordyceps khaoyaiensis and Cordyceps pseudomilitaris, tw70 new7 pathogens of lepidopteran larvae from Thailand. Mycological Research, 98 (1994): 939-942. 9. Wang, Z.N. Cultural characteristics of imported Cordyceps fungi and their pathogenicity to some insects. Report of the Thiw'an Sugar Research Institute, 126 (1989): 13-25. 10. 10. Sung, J.M., and Hyun, K.L. Classification of Cordyceps spp. by morphological characteristics and protein banding pattern. Korean Journal of Mycology, 23 (1995): 92-104 11. Sung, J.M., Kim, C.H., Yang, K.J., Lee, H.K. and Kim, Y.s. Studies on distribution and utilization of Cordyceps militaris an d c. nutans. Korean Journal of Mycology, 21 (1993): 94-105. 12. Hywel-Jones, N. L. Cordyceps khaoyaiensis and Cordyceps pseudomilitaris, tw70 new7 pathogens of lepidopteran larvae from Thailand. Mycological Research, 98 (1994): 939-942.

**★ Rungkiat Kawpet Faculty of Science and Technology/Suan Dusit University/Bangkok- 10700/Thailand Samaporn Saengyot Department of Plant Protection/Faculty of Agricultural Production Mae Jo University/Chiang Mai-50290/Thailand

ISBN 978-93-86435-17-0 I 65 ISCTH 2017 International Sciecnce Congress Thailand 2017

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