J. Bio.Env. Sci.

2018

Journal of Biodiversity and Environmental Sciences (JBES)

ISSN: 2220-6663 (Print) 2222-3045 (Online)
Vol. 12, No. 5, p. 25-31, 2018
http://www.innspub.net

RESEARCH PAPER OPEN ACCESS

Exploration and mass propagation of nematodes

entomopathogen Steinernema spp. wetlands on mustard
(Brassica juncea L.)

Antar Sofyan and Akhmad Rizali*

Department of Agroecotechnology, Faculty of Agriculture, Lambung Mangkurat University,

Banjarbaru, Indonesia.
Article published on May 13, 2018

Key words: Entomopathogen, Mustard, Steinernema spp., Tenebrio molitor.

Abstract
The use of chemical pesticides in the context of pest control in mustard (Brassica juncea L.)plants is very
dangerous. The used of biological agents of Steinernema spp. in the control of important pests is biological
pesticide to overcome the problem of vegetable pest problems is e new hope foe us all so that farmers no longer us
chemical pesticides that can interfere with human health. The utilization of biological agent in the form of
election of entomopatogen nematode exploration on wetland from district of Barito Kuala, South Kalimantan.
Test of biological agent formulation that is capable not only inhibits the development of plant pests
(biopesticides). Experiment used a completely randomized design with each treatment ranging from control
(without Steinernema spp.), 20, 40, 60, 80 and 100 tail population per unit, experiment with 3 repetitions, with
40 tail caterpillar as feed in mass propagation). The result of the study is that the population of
entomopathogenic nematodes develops well in all treatment of Steinernema spp. DMRT test results showed that
there were differences between treatments tested, only T1 and T2 treatment were not different in effect, because
different initial population. Population with an initial population number of entomopathogen nematodes 80 and
100 tail tended to differ significantly and were stable in the number of late population development observations.
In general that the use of caterpillar T. molitor is suitable in mass breeding entomopathogen nematodes.
*Corresponding Author: Akhmad Rizali  arizali25@yahoo.com

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 J. Bio.Env. Sci. 2018

Introduction pests in various types of crops, whether food crops,

Continuous planting to meet the growing vegetable plantations or horticulture (Grewal et al., 2005).
needs resulted in pest problems also increased due to Indeed, when compared with the use of chemical
pest host always available at all times. To overcome insecticides, the use of nematodes in general is more
these farmers are highly dependent pesticides so that expensive (Arthurs et al., 2004), has a short life cycle,
the spraying of toxic materials is scheduled. While the low stability, and for its application the user must
cultivation of healthy plants can not rely solely on the have sufficient knowledge about nematodes ranging
use of certified healthy seeds and only the use of from knowledge to care until the storage of
synthetic pesticides, because pests are always present nematodes (Umamaheswari et al., 2006).
in the land so that it always threatens the freedom of
plants against pest problems (Bedding et al., However, with the rules and restrictions of the Food
1993)Increasing awareness of quality food, the Quality Protection Act of 1996 on the need for
requirements of pest control measures according to restricting the use of chemical insecticides in order
the Directorate General of Horticulture (2007), must not to poison humans (Anonymous, 1996) and the
meet the ecological aspects (not disturbing human pressure and awareness of the public to reduce the
health, natural enemy life and non-target organisms, use of chemical insecticides will encourage the
environment and not cause harmful residues in the development of safe biological controls, especially the
crops), social aspects (easy to implement, socially use of the pathogenic nematodes Steinernema spp.
acceptable, and motivating community self-reliance) (Koppenhöfer and Fuzy, 2009).
and technical aspects (combining harmonious,
harmonious and balanced ways of controlling, The study of the use of entomopathogenic nematodes
prioritizing cultivation, physical / mechanical, on pests is important in view of the fact that there is a
biological, genetic, and pesticide use if necessary). tendency for migration to organic farming,
commercial market developments, attention to food
The role of horticultural crop commodities in the safety, and the increasing number of pesticides
present and future in life and the national economy withdrawn from circulation, leading to an
becomes very important. Behind its very important increasingly important role of biological control. With
role, this commodity has constraints in cultivation this research is expected nanntinya can be an
and its management. The obstacle is the attack of alternative in plant pests control, especially in
plant pest organism (OPT), one of the pest is a pest mustard plants.
insects are very detrimental to the farmers (Azharie,
2005). The disturbance will be able to decrease the The challenge to meet the public demands for the
quality and the crop production. Important plant availability of healthy and healthy quality foods leads
pests such as Plutella spp Lamprosema spp to the use of synthetic pesticides to overcome pest
Spodoptera spp and Aphis spp, Lyriomysa spp and as diseases when planted or in storage needs to be
well as the presence of many diseases affecting reduced even avoided.
vegetables especially the mustard greens is Fusarium
sp, Sclerotium sp., and Rhizoctonia solani is a land- Therefore, an effective, efficient and environmentally
borne pathogens proven to always exist together in friendly control alternative is needed. This research
soil and often infect vegetable crops on young becomes important to be carried out considering the
mustard and old crops on farmer's land. superior local biomedical exploitation as biopesticide
as a biological agent in mustard plants has never been
The use of insect pathogenic nematodes has been comprehensively studied to obtain appropriate
developed for the biological control of several insect technology.

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 J. Bio.Env. Sci. 2018

Entomopathogenic nematodes are able to inhibit the Propagation Tenebrio mollitor

development of important pests. But unfortunately The sample soil that has been taken from the land is
the existence of that potential has not been explored labeled according to the location of the pickup.
comprehensively, especially in wetlands. Thus, this Tenebrio mollitor feed larvae bought in the bird
research tries to utilize the collaboration between market, larvae are then wrapped with 10 gauze per
entomopathogenic nematodes and is an alternative pack. Sample Land Placement and Ground Feed
that has a good future and representative especially in larvae and Tenebrio mollitor feed larvae are placed
wetlands as biopestisida for the main pest of mustard inside polybags or glass jam jars labeled sample soil
plant in order to fulfill requirement of mustard used. Volume of sample soil when using polybag as
oragnik in the future. much as 500 gram and when using 200-300 gram of
jams. Place Tenebrio mollitor feed larvae randomly.
The ultimate goal of this study is to obtain Tenenbrio mollitor baits are placed as many as 10
appropriate technology for the exploration and mass grams/500 grams of soil if in polybags and if in the
management of organic mustard plants using bottle of jam as much as 4- 5 tails/300 grams of soil.
entomopatogen nematodes Steinerneme spp so as to Then given the aquadest as much as 100 ml so the
affect the high productivity of vegetable crops, ground remains moist and entomopatogen
especially mustard greens. nematodes can live. Day two and so on also remain in
add aquadest as much as 100 ml. Tenebrio mollitor
It is expected that proper entomopathogenic feed duration in polybag or glass jelly bottle for 4-5
nematodes then the management of plants, especially days.
mustard greens can be profitable in a sustainable
manner for the development of organic agriculture. The feed larvae are taken on the 4th or 5th day after
the trapping is done. Pick up the larvae using a pair of
Research sites tweezers first cleaned with 95% alcohol. Prepare the
The material used in this research is the sample soil petri dish and then give the aqua glass cup as high as
taken from the horticultural cultivation land in the 5-6 mm. on top of aqua glass chunks placed in a petri
wetlands Balitra Garden of South Kalimantan dish with a strimine cloth and filter paper. The feed
experiment especially. Survey of the location and lever is placed on a filter paper, then a petri dish filled
sampling is done on the land planting. Taking with a sterile aquadest until it touches the body of the
sampling of existing land vegetable crops. 10 Soil feed larvae. The duration of the feed is in the 3-4 day
samples were taken around the root of the plant petri dish.
(rhizozfer) with a depth of 0-20 cm using a ground
picker at a depth of 15 cm, a 45º angle of 500 grams. Symptom observation
The soil is then put into a clear plastic size of 1 kg and Dead bait larvae are observed for symptoms that
then wrapped with aluminum foil so that the appear on the morphology of the body. If there are
temperature and soil moisture can be maintained. symptoms of discoloration, the body becomes soft
and if in the surgery the inner tissue becomes liquid
The equipment used in this research is microscope, but not foul then it can be concluded that the larvae
glass slide, glass cover, petri dish, erlenmeyer flask, have been attacked by entomopatogen nematodes
dropper dropper, tweezers, cetok, filter paper, plastic (Azharie, 2005). According to Nurcholisah (2005),
bag, jar, strimin cloth, gauze, glass jam jar, hand the infected feed larvae of Steinernema spp., will
sprayer. This research was carried out in the field of change color to beige, sometimes slightly greenish
experiment of Balittra and Laboratory of and the body softens, while the feed larvae turns
Agroteknologi. reddish brown and the body remains hard then

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 J. Bio.Env. Sci. 2018

infected with Heterorhabditis spp. If there are totaling 18 experimental units. Each of the 6
symptoms of feed larvae after 3 days of isolation, the treatments used were: a) T1 100 tail, b) T2 80 tail, c)
liquid in the petri dish is taken as much as 0.5-1 ml T3 60 tail, d) T4 40 Tail, e) T5 tail 20 and f) tail
and placed on a glass slide and covered with a glass Control. Observations were made 2 times after 1
cover. If the nematodes are visible, it is necessary to month 1 and second month. According Sulistyanto
harvest as soon as possible. The liquid in the petri (2001) 24-48 hours of host insects will die from
dish is put into the erlenmeyer flask and then diluted. infection by entomopathogenic nematodes. The last
After that the suspension is taken as much as 0.5-1 ml observation was performed for 1 month after
and seen under a microscope. If there are still many treatment using magnifying glass and surgery in host
nematodes, it needs to be diluted again until they are body.
seen in the microscope only slightly and can be
counted. Counting is done 5 times from the same The dead pest insects were reexamined with the Koch
suspension. postulat method to ascertain the consequences of
insect mortality. Variety analysis according to RBD
Testing entomopatogen nematodes with Koch design format used.
postulat test
This test is performed to test whether the symptoms The experimental design used was a randomized
that occur on the specimen are symptoms caused by block design with 5 replicates. The variance analysis
entomopathogenic nematodes. This kochpostulat test was performed on the difference between treatments
uses only five Tenebrio mollitor feedlots suspected to tested according to Duncan's multiple-range test
be infected with entomopathogenic nematodes, to be (DMRT).
reproduced in new and sterile feeds. If the infected
symptom is the same as the previous one, then the Results and Discussion
infected feed larvae are entomopathogenic The results of the average analysis of the development
nematodes. Feed Testing in Mass Breeding: of Steinernema spp. on Tenebrio molitor media is
very suitable only that affect is the cloud population
The feed test is done by using hongkong caterpillar will affect the final population after 2 months of the
Tenebrio molitor with various population level. The pangamatan. Population with jumkah 100 tail and 80
initial populations tested were 20, 40, 60, 80, 100 tail that is 100 treatment of tail and 80 head per unit
and without population. The tested feed. Then the of experiment is ideal population for development of
data is made report. nematode at host body. 1media using its host insect
larvae is hongkong caterpillar Tenebrio molitors as
Experimental design much as the development of Steinernema spp. the
The experimental design used was a randomized lowest is in population 0 or without treatment where
block design (RBD) using entomopathogenic the caterpillar Does not die until 0 days of
nematodes given 6 treatments and 3 replications, observation (Table 1.).

Table 1. Results of propagation analysis Nematoda Steinernema spp on several treatments.

Source of variation df SS MS F-Calculate F-Table
0,05 0,01
Treatment 4 60228362.252 15057090 18.56 ⃰ 3.06 4.89
Error 15 12169999.772 81333.318
Total 19 72398362.02
⃰ Influential very real.

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 J. Bio.Env. Sci. 2018

DMRT results showed that there were differences 15 days no additional population of Steinernema spp.
between treatments tested, only T1 and T2 treatment because the nematodes are still in the stage of
were not different in effect, because different initial infection to the hongkong caterpillar body.
population (Table 2.). Nematodes Steinernema spp. is quick to find a
suitable host Steinernema spp., entering the insect
The experimental results show the best nematode through natural holes (mouth, anus, or spiracles)
development is in the initial population of 00 and 80 feeding nematodes and developing in the host's body
heads per unit of experiment. Its own insects are with the bacteria inside the gastrointestinal tract
hongkong worms (Tenebrio molitor). During the first (Untung, 2001).

Table 2. DMRT results on the average rate of propagation rate of Nematodes Steinernema spp.
Treatment The average propagation of Steinernema spp.
T1 (Populasi 100 ekor per sat perconaan) 5424.80 a
T2 populasi 80ekor per satuan per percobaan) 5370.00 a
T3 (Populasi 60 ekorSteinernema) 2334.20 b
T4 (Piopulasi 40 dan 20 ekor) 2324.920 c
T5 20 ekor persatuan percobaan 12,00 d
Description: The number followed by the same letter behind the numbers shows a very real effect based on
DMRT 5%.

After the 30th day of observation, the number of growth and development because Tenebrio molitor
nematodes of Steinernema spp. greatly increased as has nutrient content of 48% crude protein, ash
much as 2443.13 because in the body of host insects content 3%, moisture content reaches 57%, crude fat
in the digestive system of insects through hemokoel, 40%. The nutrient content found in Tenebrio molitor
solve the immune system of insects and immediately is good as a feed source for Steinernema spp. Some
release the symbiont bacteria brought by the sources say that the content of fat present is higher
nematodes. Nematodes multiply in one cell of the than protein content (Untung, 2001).
insect's body along with the enzymes produced by
bacterial cells located in the digestive tract of According to (Harahap et al., 2004) the superiority of
nematodes, breaking the body's tissue into nutrients host larvae is easier to breed, cheaper propagation
suitable for nematodes. Nematodes can develop 2-3 costs. The visually generated JI is more uniform in
generations and after depleted nutrients. size and motility, retained in storage and lower
contamination during the propagation process,
The nematodes will re-enter the infective cycle and resulting from extreme environmental survival. In the
migrate from the nutrient-depleted host by carrying host's insect body there is an infective juvenile and all
bacterial cells in the gastrointestinal tract, out of the develop into females and males. JI on the nematodes
body of the insect larvae and can last for months Steinernema spp. can protect from unstable
(Untung, 2001). environmental factors and other microorganisms that
can inhibit the growth of nematode (Poiner, 1979).
On the 30th day the nematodes came out of the body
of a host insect indicated by a change in the insect Effects on drought, high temperature and ultra violet
that is a soft or crushed insect. Nematodes rays even though the nematode is in water.
Steinernema spp. Tenebrio molitor is thought to be Nematodes require high moisture that is over 80%.
quantitatively derived from insect larvae suitable for (Sulistyanto, 2001).

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 J. Bio.Env. Sci. 2018

Nematodes Steinernema spp. also require oxygen, the five treatments for media using embroidered insect
higher the oxygen the faster nematode growth larvae or can be tested with in vivo the best is
(Untung, 2001). The occurrence of population decline Tenebrio molitor.
on days 60 to day 75 due to the available nutrients
has been reduced suspected due to ingestion or Conclusion
evaporation in the media so that nematodes The use of Steinernema spp. nematodes is the best in
Steinernema spp. inactive and off. initial population of 100 and 80 tails in the effective
jouvenil development of insect larvae on the Tenebrio
Utilization can be used for nematode breeding, but molitor caterpillar host. Initial population growth
artificial media containing only high protein from below 60 tails per experiment unit. And Tenebrio
animals and is all the elements needed for growth of molitor is particularly suitable for mass culture of
nematodes. However, this medium can cause a entomopathogenic nematodes.
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