Evaluation of entomopathogenic fungus ศูนย์นวัตกรรมและการวิจัยกลุ่มมิMetarhiziumRDI anisopliaeตรลลfor controlling MITR PHOL INNOVATIONDorysthenes AND RESEARCHbuqueti CENTERSugarcane Stem Boring Grub (SSBG) Pratchya Swangmaneecharern, Manuwat Tintarasaranaratchaseema, Varinthon Jarnkoon, Peeraya Klomsaard and Klanarong Sriroth MITR PHOL INNOVATION AND RESEARCH CENTER Sugarcane Stem Boring Grub outbreak in northeast of Thailand ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER Without Stem Boring Grub Stem boring grub zone The sugarcane stem boring grub (SSBG), Dorysthenes buqueti Guerin is one of major sugarcane insect pest in northeastern Thailand. SSBG become a serious sugarcane insect pest. It can cause more than 70% yield loss in outbreak area. This insect pest attack sugarcane plant by the larval stage into the ratoon or base of stalk tissue inside which results the whole plant turned brown and die. MITR PHOL INNOVATION AND RESEARCH CENTER Adult tapping control ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล April – May 2019 MITR PHOL INNOVATION AND RESEARCH CENTER Sugarcane Famer solve the problem by adult trapping for make food. But also we need to control insect in larva stage because cause high sugarcane damage. MITR PHOL INNOVATION AND RESEARCH CENTER Insecticide effects concern ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER Allergy and irritation on human Water contamination Fingernail damage Pesticides are used to control insect pest that effect on human health, animal and environment. Lead to mitrphol group concern about pesticide use and turn to biological control. Fugal is an alternative mean to control insect pests. -Md. Wasim AKTAR, 2009, “Impact of pesticides use in agriculture:their benefits and hazards”, Interdisc Toxicol, Vol. 2, 1–12. MITR PHOL INNOVATION AND RESEARCH CENTER -Damalas, Christos A. and Ilias G. Eleftherohorinos.” Pesticide Exposure, Safety Issues, and -Risk Assessment Indicators.” International Journal of Environmental Research and Public Health. 6 May 2011. Web of Science. Biological control ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER Termite Stem Boring Grub Metarhizium anisopliae or longhorn stem borer Dorysthenes buqueti Kingdom: Fungi Division: Ascomycota Class: Sordariomycetes Order: Hypocreales 7-10 um Family: Clavicipitaceae Genus: Metarhizium Species: M. anisopliae White Grub Lepidiota stigma MITR PHOL INNOVATION AND RESEARCH CENTER Stem Boring Grub life cycle ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER Morphology Jun Jul Aug Sep Oct Nov Dem Jan Feb Mar Apr May MITR PHOL INNOVATION AND RESEARCH CENTER Infection process of Metarhizium spp. ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER First step, Metarhiziuum conidia germinate on the host surface and differentiate the infection structure termed appressorium. Then, the appressorium penetrates the cuticle and enters the haemocoel. Then hypal bodies and the potential toxin produced lead to the insect death. 25 MITR PHOL INNOVATION AND RESEARCH CENTER Evaluation of entomopathogenic fungus Metarhizium anisopliae for controlling Dorysthenes buqueti Sugarcane Stem Boring Grub (SSBG) Objectives 1. To screen potential isolateds to control sugarcane stem boring grub ศูนย์นวัตกรรมและการวิจัยกลุ่มมิ2. To develop Mearhizium biopesticide product applyตรลล associated machine (Application improvement) MITR PHOL INNOVATION AND RESEARCH CENTER Timeline and Methodology 1) Fungal isolation and identification 2) Lab test (Fungi pathogenicity test) 3) Green house test 4) Field trial In Ratoon cane MITR PHOL INNOVATION AND RESEARCH CENTER Fungal isolation ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER D C B E A A B C D E MITR PHOL INNOVATION AND RESEARCH CENTER Fungal identification result Samples Nucleotide Nucleotide sequence (5’ to 3’) Similrity% ศูนย์นวัตกรรมและการวิจัยกลุ่มมิregion ตรลล MITR PHOL INNOVATION AND RESEARCH CENTER ACCTGCGGAGGGATCATTACCGAGTTATCCAACTCCCAACCCCTGTGAATTATACCTTTCATTGTTGCTTCGGCGGGACTTCGCGCCCGCCGG GGACCCAAACCTTCTGAATTTTTTAATAAGTATCTTCTGAGTGGTTAAAAAAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGG 99.44% CATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGTCA GTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACGCCCCTCAAGTCCCCTGTGGACTTGGTGTTGGGGATCGGCGAGGCTGGTTTTCCAG CACAGCCGTCCCTTAAATTGATTGGCGGTCTCGCCGTGGCCCTCCTCTGCGCAGTAGTAAAACACTCGCAACAGGAGCCCGGCGCGGTCCAC A TGCCGTAAAAACCCCCAACTTTTTATAGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTTAAGCATATC GACCTGCGGAGGGATCATTACCGAGTTATCCAACTCCCAACCCCTGTGAATTATACCTTTCATTGTTGCTTCGGCGGGACTTCGCGCCCGCCG GGGACCCAAACCTTCTGAATTTTTTAATAAGTATCTTCTGAGTGGTTAAAAAAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG 99.08% GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGT CAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACGCCCCTCAAGTCCCCTGTGGACTTGGTGTTGGGGATCGGCGAGGCTGGTTTTCC AGCACAGCCGTCCCTTAAATTGATTGGCGGTCTCGCCGTGGCCCTCCTCTGCGCAGTAGTAAAACACTCGCAACAGGGAGCCCGGCGCGGT B CCACTGCCGTAAAAAACCCCCAACTTTTTATAGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAA ATGTGAATTATACCTTTCATTGTTGCTTCGGCGGGACCTCGCGCCCGCCGGGGACCCAAACCTTCTGAATTTTTTAATAAGTATATTCTGAGT ITS rDNA GGTTAAAAAAAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTG 99.57% AATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGTCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACGCCC CTCAAGTCCCCTGTGGACTTGGTGTTGGGGATCGGCGAGGCTGGTTTTCCAGCACAGCCGTCCCTTAAATTGATTGGCGGTCTCGCCGCGGC CCTCCTCTGCGCAGTAGTAAAACACTCGCAACAGGAGCCCGGCGCGGTCCACTGCCGTAAAACCCCCCCCCCAACTTTTTATAGTTGACCTCG C AATCAGGTAGGACTACCC GACCTGCGGAGGGATCATTACCGAGTTATCCAACTCCCAACCCCTGTGAATTATACCTTTCATTGTTGCTTCGGCGGGACTTCGCGCCCGCCG GGGACCCAAACCTTCTGAATTTTTTAATAAGTATCTTCTGAGTGGTTAAAAAAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG 99.00% GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGT CAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACGCCCCTCAAGTCCCCTGTGGACTTGGTGTTGGGGATCGGCGAGGCTGGTTTTCC AGCACAGCCGTCCCTTAAATTGATTGGCGGTCTCGCCGTGGCCCTCCTCTGCGCAGTAGTAAAACACTCGCAACAGGAGCCCGGCGCGGTCC D ACTGCCGTAAAAACCCCCAACTTTTTATAGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTTAAGCATATC TTCCGTAGGGTGAACCTGCGGAGGGATCATTACCGAGTTATCCAACTCCCAACCCCTGTGAATTATACCTTTCATTGTTGCTTCGGCGGGACT TCGCGCCCGCCGGGGACCCAAACCTTCTGAATTTTTTAATAAGTATCTTCTGAGTGGTTAAAAAAAAATGAATCAAAACTTTCAACAACGGAT 99.44% CTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCAC ATTGCGCCCGTCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACGCCCCTCAAGTCCCCTGTGGACTTGGTGTTGGGGATCGGCGAG GCTGGTTTTCCAGCACAGCCGTCCCTTAAATTGATTGGCGGTCTCGCCGTGGCCCTCCTCTGCGCAGTAGTAAAACACTCGCAACAGGAGCC E CGGCGCGGTCCACTGCCGTAAAAACCCCCAACTTTTTATAGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTTA Confirmed by PCR. The result showed 99% similar closely related with Metarhizium anisopliae. MITR PHOL INNOVATION AND RESEARCH CENTER Results (Lab test: Fungi pathogenicity test) ศูนย์นวัตกรรมและการวิจัยกลุ่มมิThe mortality of sugarcane on 16 daysตรลล after infected 5 isolated of Metarhizium 120 MITR PHOL100 INNOVATION AND RESEARCH CENTER100 100 100 100 100 100 100 100 90 90 80 80 80 80 80 80 80 80 70 60 60 60 60 60 60 50 50 50 50 40 40 40 40 40 30 30 20 20 20 10 0 0 0 0 0 0 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A B C D E Time (days) Virulence of spore % mortality of sugarcane stem boring grub stem % mortality of sugarcane Samples LT50 (Days) 100% Mortality (Days) Met A 9 c 16 c Met B 6.5 b 10 a Met C 9.5 c 14 b Met D 5.4 a 9 a Met E 6 ab 9 a In an in-vitro experiment, In a laboratory experiment to determining virulence, all five isolates of M. anisopliae were tested for SSBG control with second and fourth instar larvae stage condition. Mortality was evaluated for 16 days causing 100% infection all treatments.From five isolates of M. anisopliae isolated D were found to be high performance pathogenic 100% mortality at nine days and LT50 5.4 days (fast to 50% kill insect ) MITR PHOL INNOVATION AND RESEARCH CENTER Greenhouse test ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER Treatments %Cane damage Treated (Fresh spore) 55% (45% decreased) untreated (No spore) 100% No spore Spore Two months after SBG inoculation & cane 4 month MITR PHOL INNOVATION AND RESEARCH CENTER How to control Sugarcane Stem Boring Grub in field ? ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล MITR PHOL INNOVATION AND RESEARCH CENTER 1 1 1 2 3 4 5 In the field trial, M. anisopliae isolated D was applied in two ratoon sugarcane field (SRT1 and SRT2) located in Phuvieng district, KhonKaen Province, Thailand. One month after inoculation, %cane damage were recorded. In this experiment, the application of bio-pesticide Metarhizium product at four kilograms of M. anisopliae conidia spore mix with chemical fertilizer formula 21-7-18 during ratoon sugarcane was used. MITR PHOL INNOVATION AND RESEARCH CENTER Field trial in ratoon sugarcane at MPV (Zone1 SRT1) ศูนย์นวัตกรรมและการวิจัยกลุ่มมิตรลล 1MITRMPV- YAPHOL-SSBG- HillINNOVATION(Zone1) AND RESEARCH CENTER Treated (Granule conidia spore) Untreated (No spore) 4 kg/Rai Details Treated Untreated %Cane damage (before) 28% 28% %Cane damage (two months after inoculation) 10.66% (45.69%) 23.33% (100%) %Sugarcane protection 12.67 (54.31%) - The result shows that the percentage of cane loss from SSBG after two months of inoculation in SRT 1 field presented 10.66% cane damage in granule conidia spore treatment in comparison to 23.33% cane damage in the treatment of no granule conidia spore. MITR PHOL INNOVATION AND RESEARCH CENTER Field trial in ratoon sugarcane