Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 375
IDENTIFICATION OF ENTOMOPATHOGENIC ON
Crosidolomia binotalis
[ZELLER] LEPIDOPTERA : PHYRALIDAE USING 16S RNA GENE
Rantje Lilly Worang
1
, Mokosuli Yermia Semuel
1,2
1
Department of Biology, Faculty Mathematics and Natural Science, State University of Manado, Minahasa, Indonesia.
2
Laboratory Bioactivity and Molecular Biology, Faculty Mathematic and Natural Science, State University of Manado, Minahasa , Indonesia
yere_bikipbyahoo.co.id
Abstract The aims of this reserach were to isolating and to identifying bacteria entomopathogenic on caterpillar
pests in cabbage. The study was conducted in three phases. The first stage isolation of bacteria from cabbage caterpillar pests, the second stage of microscopic characterization of bacterial isolates and the
third phase molecular identification of bacteria isolates using 16S RNA gene. The results were obtained five bacterial isolates of caterpillar pests in cabbage which 4 isolates shaped cocus, three
isolates of gram positive and gram negative two isolates. All of isolates were able to degrade D- glucose. Isolates were translucent white color, white yellowish and brownish white. Generally isolates
are not motile dan growth in the media to the surface. Results of DNA extraction and amplification of 16S RNA gene, showing the purity and concentration of DNA is good, confirmed by electrophoresis,
amplicons formed band at 1500 bp according to the characteristics of the 16 S RNA gene.
Keywords: entomopathogenic, cabbage caterpillar, 16S RNA gene
1. Introduction
North Sulawesi has two regional centers of cultivation of horticultural crops namely Tomohon town and District Modoinding South Minahasa District. Vegetable plants of the genus
Brassicaseae such as cabbage, broccoli, cauliflower, mustard greens and turnip are the main vegetable crops are cultivated because it has a high economic value. The vegetable crops in
addition to meeting local demand in North Sulawesi also exported to Singapore among others. This led to the cultivation of vegetables of the genus Brassicasea done by farmers
nevertheless mainly armyworm pest Crocidolomia binotalis and caterpillars Plutella xylostella Plutella become major problems faced by farmers. Both types of pest attack can
damage the plant to 100 when not carried out effective controls Elstein
et. Al
. 2005; Hashim
et. Al
., 2009 . Naturally every existing organisms in an ecosystem strives to maintain the continuity and
existence of species. In agro-ecosystems or organisms that damage the entire crop is classified as pests. Judging from the ecological point of view, every organism has natural enemies
present in the food chain. Natural enemies can be in the form of organisms or microorganisms that can suppress the growth or survival of a species. Insects fitofag in the pre-stage and
mature as armyworm caterpillars that attack the plant genus Plutella Brassicaseae have natural enemies, especially microorganisms which are pathogenic or known as entomopathogenic.
Entomopathogenic microbes can be viruses, bacteria, fungi, protozoa and nematodes the microscopic stage. Entomopathogenic microbes could potentially be used as biological
control agents for specific pests and is more specific because it is isolated directly from the target insect pest control.
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 376
The concept of biological control is currently being developed for chemical control is mostly done over the long term this would lower the carrying capacity of the environment to
agriculture. Chemical insecticides in various research reports damage the soil structure because besides kill insect pests also kill soil microflora act as biofertiliser and break the cycle
of soil nutrient involving microorganisms. Besides, synthetic insecticides also leave residues on agricultural products that can accumulate on the leaves of vegetables, tubers, stems bungga
and later consumed by humans. Accumulation of residues on plant parts which are then consumed by humans will experience the process of bioaccumulation in the long term to
stimulate and initiate various degenerative diseases, especially cancer and mental retardation in children. This led to many developed countries are beginning to use chemical insecticides
to increase production began to shift in the concept of biological control.
By knowing the pests that attack plants, indirectly that it can also appropriate species of entomopathogenic fungi to control measures, because every species of entomopathogenic
fungi have a specific host. Fungus Metarhizium anisopliae, for example, is known to infect several species of insects of the order Coleoptera, Lepidoptera, Homoptera, Hemiptera, and
Isoptera. Sosa-Gomex et. Al., 2003; Butt et. Al., 1994, Rao et al., 2006; Sauche et al. 2000; Nakinga et al. 1996; Strack 2003. But M. anisopliae most effective when used to control
pests of the order Isoptera Strack 2003. This is because the relationship between the behavior of the insect host with the effectiveness of entomopathogenic fungi. The
phenomenon shown by the test results of re-infection. Of the several species of entomopathogenic fungi obtained from armyworm Spodoptera litura and Helicoverpa
armigera, the most effective fungus rileyi Nomuraea controlling S. litura with mortality reaching 100 Prayogo et al. 2005. The fungus is reported is one potential biological agents
to control pests of the order Lepidoptera, though also able to infect insects from other orders Indrayani 2011; Hashim, 2005.
Until now, information utilization entomopathogenic fungi in Indonesia more specifically in North Sulawesi is still very little. Entomopathogenic bacterial identification is required as the
basis for further studies that test the pathogenicity and biopesticide formulations. There are many methods of identification of bacteria but the selected molecular identification method
that has a higher accuracy than the biochemical and morphological identification method only. Bacterial identification methods are widely recommended is the analysis of bacterial
DNA through the reading of the nucleotide sequence of nitrogenous bases making up the gene 16S rDNA fragments bacteria. Judging from several considerations, this method is considered
better than the phenotypic analysis method. The first consideration is the 16S rDNA gene of almost all species of bacteria have been determined sequence of nitrogenous bases that can be
used as guidelines if found new species [12]. The second consideration is the base sequence of 16S rDNA gene nitrogen has a lower intraspecific diversity than other protein-coding
genes, as well as the nature of 16S rDNA fragments were sustainable Thoyibatun and Son, 2012.
Research utilization entomopathogenic microbes is very important in developing specific biological biopesticide control pests on specific crops. Binotalis C. and P. xylostella which is
a major pest Brasicaseae genus has many detrimental to farmers because it can reduce crop production even menggaggalkan. This study aims to: Isolate and characterize the plant genus
isolatmikroba entomopathogenic Brasicaseae cultivated in aquaculture Sentra Hortikulturan Tomohon. Identifying entomopathogenic isolates using molecular techniques 16S rRNA gene.
Getting LC50 lethal concentration or concentrations that can kill 50 percent of the insect population in laboratory tests.
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 377
2. Materials and Methods
