Entomopatogen Nematode Exploration and Virulency Against Spodoptera Frugiperda J.E Smith

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Entomopatogen nematode exploration and virulency against Spodoptera frugiperda J.E Smith

Winda Septi Hade1, Djamilah1*, Priyatiningsih 1 Departement of Plant Protection, Faculty of Agriculture, Universitas Bengkulu, Kandang Limun, Bengkulu, Indonesia

*Corresponding author: [email protected]

ABSTRACT : Corn (Zea mays L) is a very important food and animal feed after rice. One of the important pests that attack corn plants is Spodoptera frugiperda J.E Smith. This pest is a new pest and is a major pest in corn. In pest control, farmers generally use synthetic chemicals. In an effort to reduce these negative impacts synthetic chemical, entomopathogenic nematode (NEP) was chosen as one of the more environmentally friendly control techniques. This study aims to obtain NEP isolates, population density and virulence against S. frugiperda. This research was conducted at the Plant Protection Laboratory, Bengkulu University from December 2019 to April 2020. This study used a Factorial Completely Randomized Design (CRD) with 4 treatments and 5 replications. The treatments consisted of NEP concentrations : 0 Jl / ml; 200 Jl / 1ml; 400 Jl / 2ml; 600 Jl / 3ml originating from Bengkulu and Kepahiang isolates. Each experiment consisted of 3 S. frugiperda larvae. The results showed that the NEP obtained from each region, namely the genus Steinermatidae. Population density of 9,005 NEP / 5ml in the Kepahiang area and 6,837 NEP / 5ml in the Bengkulu region. NEP virulence test against S. frugiperda larvae showed that 200 Jl / ml concentration was able to control S. frugiperda in maize , and can kill pests within 4 days. With an LC50 value of 163.5 Jl / ml in Kepahiang isolates and 186.5 Jl / ml in Bengkulu isolates.

Key words: Steinermatidae sp (NEP), S. frugiperda

Reference to this paper should be made as follows : Hade, W. S., Djamilah, and Priyatiningsih. 2020. Entomopatogen nematode exploration and virulency against Spodoptera frugiperda J.E Smith. Agritropica: Journal of Agricultural Science. 3(2): 70-81. Doi: https://doi.org/10.31186/Jagritropica.3.2.70-81

population is still small it will be INTRODUCTION difficult to detect. Imago is a strong flier Cornworm Spodoptera frugiperda J. E. and has a high cruising range (CABI, Smith is an invasive insect that has 2019). Spodoptera frugiperda is become a pest on maize (Zea mays) in polyphagous, some of the main hosts are Indonesia. This insect originates from food plants from the Graminae group America and has spread in various such as maize, rice, wheat, sorghum, and countries. In early 2019, this pest was sugarcane so that the existence and found in maize plants in the Sumatra development of the population is very area (Kementerian Pertanian, 2019). S. concerned. The losses incurred due to frugiperda is also found in maize plants this pest attack on maize crops in in Bengkulu (Nadrawati et al., 2019). African and European countries are These pests attack plant growing between 8.3 and 20.6 million tons per points which can result in failure of year with an economic loss of between shoot formation. S. frugiperda larvae have US $ 2.5-6.2 billion per year (FAO & a high ability to eat. Larvae will enter the CABI, 2019). The control of S. frugiperda plant and actively eat there, so that if the

generally uses synthetic insecticides. The NEP have been widely studied for their use of insecticides by farmers is effectiveness in controlling horticultural, generally very intensive, using either food and plantation pests that can cause single or mixed insecticides, high doses, high mortality (Sulistiyanto, 2008). scheduled spraying, and short-term NEP is a parasite that specifically spraying intervals, which are 2-3 days attacks insect larvae so it is safe for (Koster, 1990; Buurma and Nurmalinda, animals and plants. The use of NEP has 1994). Intensive use of insecticides is a great opportunity to control insect irrational, inefficient, and has the pests. The advantage of NEP over other potential to cause negative impacts on biological agents is that it is easily found the environment and the emergence of in all soils in many areas, in addition to pest resistance to insecticides (Djunaedi, the ease with which NEPs adapt and 2009). Therefore, an effective and survive. Thus, gradually, efforts to use efficient control technique is needed in synthetic chemical pesticides have controlling S. frugiperda. In addition, decreased and have begun to shift to control must also be environmentally controlling the biological agent NEP friendly and not cause resistance, which is safe for the environment (Ria, namely by using biological control. One 2014). of the most effective biological agents is This study aims to obtain the genus the use of entomopathogenic nematodes. NEP, population density and effective Nematodes are worm-like organisms density to kill S. frugiperda. with a size of 700-1200 microns and are in the soil. Among the nematodes found RESEARCH METHOD in the soil, some are free-living (non- The research was carried out from parasitic), plant parasitic nematodes and December 2019 to April 2020 at the insect parasitic nematodes, known as Laboratory, Plant Protection, Faculty of entomopathogenic nematodes (NEP) Agriculture, Bengkulu University. (Nugrohorini, 2010). The tools used are a small shovel, There are two NEP families that act tape measure or ruler, binocular stereo as biological control agents, namely microscope, 9 cm diameter petri dishes Steinernematidae and Heterorhabditidae. and 14 cm petri dishes, nematode NEP infects its host insects by symbiosis counter plates, hand counters, tweezers, with bacteria in their digestive tract. The basins, rubber bands, plastic jars, nematodes of the Steinernematidae family nematode fishing rods, pipettes, 10 ml have symbiosis with the bacteria injection, glass object, label paper, plastic Xenorhabdus sp and the nematodes of the bag, scale, scissors. Heterorhabditidae family have a symbiosis The materials used include gauze, with the genus Photorabdus sp (Smart, filter paper, 3rd instar S. frugiperda 1995). larvae, Hong Kong caterpillar (Tenebrio The entomopathogenic nematode molitor), soil samples, distilled water, which has high pathogenicity to its host 70% alcohol, formal glyserin fixative is Steinernema spp. and Heterorhabditis (Formalin 40% 10 ml, 6 ml glycerol and spp. Both types of NEP have the distilled water 84 ml), glycerol, potential to control insect pests of the methylene blue, and polish. order Lepidoptera, Coleoptera and This research method used Diptera (Chaerani, 1995). Both types of exploratory methods and virulence test

for entomopathogenic nematodes. The Soil is taken from around the exploratory method was carried out in 2 roots of the corn plant. Each location is different areas, namely Kepahiang assigned 5 places randomly. Soil (highlands) and Bengkulu (lowlands) sampling was carried out to a depth of using a sampling technique where each 20 cm. Soil samples were taken using a area was taken 5 points for sampling. shovel. Then the soil samples are stored Virulence test for entomopathogenic in plastic bags with holes for ventilation nematodes against S. frugiperda used a (Baliadi, 2011). completely randomized design (CRD) Entomopathogenic nematodes were with 2 factors, namely: isolated using larvae of Hong Kong Factor 1: the origin of the isolate (W1 = caterpillar (Tenebrio molitor). Soil samples Kepahiang) taken from 10 location points from 2 (W2 = Bengkulu) regions were put into different basins, Factor 2: concentration (K0 = each basin was given 20 T. molitor larvae Control) every day sprayed with distilled water (K1 = 200 Jl / ml) to maintain humidity. After 7 days, the (K2 = 400 Jl / ml) dead larvae were removed and rinsed (K3 = 600 Jl / ml) with distilled water. The Each treatment consisted of 5 entomopathogenic nematode trapping replications and each experiment was then continued using the White trap consisted of 3 S. frugiperda larvae so that extraction method. 40 experimental units were obtained. The dead larvae showing brown or This research was conducted in red symptoms are taken and arranged in several stages. These stages include large petri containing small petri which multiplication of S. frugiperda larvae, soil is turned over and given filter paper that sampling, isolation of entomopathogenic sticks out to the bottom of a large petri nematodes, white trap method, filled with water to half the height of the identification of entomopathogenic small petri. The dead larvae are nematodes, calculation of population incubated for 7-14 days at 25o C and the density and nematode application. nematodes will come out down into the Larvae of S. frugiperda instar 2, 3 water. The 30μ sieve is used to separate and 4 were collected from maize fields the insect tissue from the nematodes by according to the number of needs. Then leaving the filter underneath propped up they are kept in plastic cups and given for a few minutes in a large petri. Storing fresh baby corn as food so they don't get the nematodes in a specimen container / stressed and die. The larvae were bottle at 4 oC for 5 days for application maintained until they became pupae and (Sulistiyanto, 2011). immago, then the immago was transferred to 5L plastic jars that had Observation on host insects serves been given 10% honey, fresh corn leaves to see symptoms of attack by insect and white oil paper as the media for parasitic nematodes on the cuticle area laying eggs. The eggs are reared until which is indicated by a change in color. they hatch and become larvae, larvae are If the insect's body is brownish black / maintained up to instar 3, because larvae caramel, it means that the insect is in instar 3 are the most active larvae in infected with Steinernematidae, and reddish in color if it is infected with destroying corn plants. Heterorhabditidae. This is caused by the

reaction of the symbiont bacteria, percentage of mortality is calculated Xenorhabdus spp. or Photorhabdus spp. using the following formula: which is released by the nematode while inside the host insect. ∑ 푑푒푎푑 푙푎푟푣푎푒 %푀표푟푡푎푙푖푡푦 = × 100% NEP identification is carried out ∑ 푡푒푠푡 푙푎푟푣푎푒 morphologically, namely by observing 1. Observations were made every 24 the morphology of the nematodes using hours for 7 days. The variables a binocular microscope, including observed were: Symptoms and signs observing the nematode's body size, of infected S. frugiperda larvae. head shape, hooks on the head and Observations were made one day longitudinal striations on the nematode's after application (HSA). body. 2. The feeding power of larvae. The feeding power of larvae will be Calculation of the population calculated by weighing 3 grams of density of entomopathogenic nematodes baby corn first, then weighing it again using a binocular microscope, count every 24 hours. Observation of larval plates and hand counters. Then feeding capacity was carried out one calculated using the formula: day after application (HSA). 3. Percentage of S. frugiperda larvae P = P1+P2+P3+P4+P5 푥 X mortality was carried out one day n after application (HSA). The percentage of larval mortality is Information : calculated using a formula P = Population of nematodes per… .ml a 푃 = × 100% n = number of repetitions P1,… P5 a + b Information: X =volume of suspension / volume of P = percentage of test insect mortality sub samples a = number of test insects that died Virulence test was carried out b = number of test insects that are still with filter paper placed on a petri dish. alive Before being used, the plates are 4. The time of death for S. frugiperda sterilized in an autoclave with a pressure larvae after application was calculated of 120 atm. Place 1 larvae of 3rd instar S. from one day after application to the frugiperda into a petri dish. Then the 7th day after application. The time of entomopathogenic nematode isolates death for larvae is calculated using with a concentration of 200 Jl / 1 ml, 400 the formula: ∑( Larvae died on nth day × nth day of observation ) Jl / 2 ml and 600 JI / 3 ml distilled water ∑( 푊= ) 푊 = were inoculated into the cup. In control, Total dead larvae larvae were applied with sterile distilled Information: water. Larvae were given fresh baby W = mean time to death of larvae. corn feed and maintained at room The research data will be analyzed temperature. Observations of the statistically using factorial RAL analyzed number of dead S. frugiperda and with ANOVA level of 5%, Probit movement of sick S. frugiperda and analysis LC50% and LC75%. Then if the changes in appearance were carried out results are significantly different, a every 24 hours for 7 days. The

further test will be carried out with the nematodes and bacteria that produce LSD test at the 5% level. exotoxins. The activity of S. frugiperda larvae RESULTS AND DISCUSSION infected with entomopathogenic Symptoms and Signs of S. frugiperda nematodes will reduce their activity and Larvae Infected with NEP will die due to the toxins released by the Symptoms in larvae infected with symbiont bacteria that live in the NEP show a change in the color of the digestive tract of the nematodes. cuticle of the larvae from green to According to Gaugler and Kaya (1990), blackish brown and the movement of the the nematodes of the Steinermatidae larvae, which initially moved actively, family have a symbiosis with the genus but gradually became slow. When Xenorabdus bacteria and the nematodes infected larvae die, their bodies become of the Heterorhabditidae family have flaccid (Figure 1). If the body of the larva symbiosis with the genus Photorhabdus is pressed by hand, it will easily crumble bacteria. These bacteria play a role in and release a foul-smelling liquid. killing host insects quickly, within 2-3 The symptoms of these changes days. The death of many host insects is have been reported by Djamilah et al., caused by toxins released by bacteria. (2010) on Spodoptera litura, that S. litura Bacteria will grow rapidly in the body of infected with Steinernema shows several the dead host insect and use it as a symptoms, namely the movement of the nutrient. In principle, nematodes feed on larvae becomes inactive or moves these bacteria. slowly. When touched, the larvae show a The results of NEP isolation from different response from healthy larvae, maize fields in the Kapahiang and the larvae become limp and over time Bengkulu regions showed that the the larva's body will become more characteristics of T. molitor larvae that flaccid and change color, the longer the were parasitized by NEP had decreased larvae blacken all over its body. When movement activity, tended to remain pressed, the larva's body will break stationary, and eventually the larvae easily and release a foul-smelling, would die with blackish brown skin yellowish white liquid. Color changes (Figure 2). that occur in insects are caused by a symbiotic mutualism between

a b

Figure 1. Frugiperda larvae infected with entomopathogenic nematodes (a) seen through a microscope (b)

Figure 2. Tenebrio molitor larvae infected with entomopathogenic nematodes

These symptoms are consistent with the culticula and non-ciliated, a complete results of research by Nugrohorini (2007) digestive tract in the form of a straight that S. litura larvae infected with NEP line anterior and posterior anus and a Steinernematidae are immobile and stiff ring of nerves that surrounds the and have discolored cuticles. Another esophagus is the nervous system (Figure symptom that can be observed is the 3). larvae of T. molitor which at first are light The identification results were brown then turn into caramel brown, the adjusted based on the description of the structure of the infected larva's body entomopathogenic nematodes of the tissue becomes soft, although the larva's Steinernematidae family according to body shape remains intact and does not Tanada and Kaya (1993) and Gaugler smell bad. (2001). Entomopathogenic nematodes of the Steinernematidae family have Entomopathogenic Nematode common and distinctive morphological Identification features. The head is smooth and The results of observations based unhooked, the tail is short pointed, has a on morphological characteristics showed vulva that protrudes outward and is that the nematodes found were located in the middle, the nerve ring is entomopathogenic nematodes of the located anteriorly. Steinernematidae family. Steinernematidae nematodes have a long body covered by

Figure 3. Entomopathogenic nematodes of the Steinernematidae family, tail (posterior) (a) head (anterior) (b), magnification 400x The results of the identification of The high nematode population in entomopathogenic nematodes that infect the Kepahiang area is thought to be T. molitor larvae showed blackish brown because the soil type in the Kepahiang color in the larval cuticle. In this study, area is Andosol. Broadly speaking, only one type of NEP was found, namely Andosol soil has quite good physical the genus Steinernema, in accordance and chemical properties, so that the soil with Anggraeni's (2003) opinion that two productivity is medium to high. This different types of NEP were never found land is widely used for horticultural or in one test insect. This is presumably due vegetable crops, flowers, plantations and to the specific relationship between the forestry. In general, Andosol soil is host insect and NEP and the symbiont blackish in color with a crumbly texture bacteria of each type of NEP. that looks looser and has high organic matter content. The substance contained Entomopathogenic Nematode in it is mostly volcanic ash from volcanic Population Density From the calculation eruptions (Barchia 2009). This was also of the average population density of reported by Nugrohorini (2010), that NEP in each region, namely in the nematodes cannot live in clayey clay Kepahiang area there were 15 T. molitor types, because in this type of soil there is larvae attacked by NEP with an average no cavity so that oxygen cannot population density of 9005 NEP / 5ml penetrate the soil optimally. and in the Bengkulu region there were 11 T. molitor larvae attacked by NEP. Results of Analysis of Variants with an average population density of The results of the analysis of 6,837 NEP / 5ml. The population density variance showed that the interaction is higher in the Kepahiang area (Figure between entomopathogenic nematodes 4). and isolates had no significant effect on all the observed variables. Concentrations showed very significant 10.000 9.000 differences for all the observed variables 8.000 (Table 1). 7.000 6.000 Feeding Larvae 5.000 4.000 The results of the analysis of 3.000 variance on observations of larval 2.000 feeding power showed significantly 1.000 0 different results (Table 1). At the time of

Bengkulu Kepahiang observation, the feeding power of larvae ORIGIN OF THE ISOLATE ISOLATE OF ORIGIN THE (NEP/ML) ORIGIN OF THE ISOLATE was calculated by weighing 3 grams of baby corn and weighing it again after 24 hours. Figure 4. Average entomopathogenic nematode population density

Table 1. The summary of the results of the F-count analysis of variance at the 5% level in each observed variable f-count Observationall variables Isolate concentration interaction KK %

Larval feeding power 7.2 ns 6.1 ** 0.3 ns 38.7 Percentage of larval mortality 0.6 ns 8.0 ** 1.9 ns 29.1 The time of death of the larva 0.9 ns 0.3 ** 2.7 ns 29.8 Information: ns = not significantly different, * = significantly different, ** = very significantly different at the 5% level

Table 2. Average feeding power of S. Data on the average feeding frugiperda larvae at various power of larvae (Figure 5) shows that on concentrations and day 1, larvae eat as much as 1.5 grams of entomopathogenic nematode baby corn, but on day 2 to day 4 larvae isolates. feeding power decreases. This is because Feeding Larvae the infected larvae move slowly and Consentration Kepahiang Bengkulu even tend to stay still, so the larvae are Isolate Isolate not active in foraging. On the 5th day the 200 Jl/1ml 7.1 a 6.2 a average feeding power increased by 1.2 400 Jl/2ml 5.5 a 5.6 a g, this is presumably because the larvae 600 Jl/3ml 3.1 b 3.9 a began to move foraging to increase immunity in defense against NEP Based on the average test with a attacks, in accordance with the statement 5% BNT level, it shows that the average of Ehlers (1996) that the killing power of feeding power of larvae in Kepahiang NEP against The host insect is isolates with a concentration of 400Jl / 2 determined not only from the NEP- ml is significantly different from the bacterial symbiont complex, but also the feeding power of larvae at a level of immunity of the host insect. concentration of 600Jl / 3ml, while in However, on the 6th and 7th day, the Bengkulu isolates the average feeding average feeding power of the larvae power is not significantly different at decreased again, from the 3 grams of every concentration. baby corn given, the larvae only ate 0.7 grams. 2 The decreased feeding activity of 1,5 S. frugiperda larvae is caused by disruption of the insect body tissue by 1 the symbiont bacteria produced from NEP as a toxin, causing damage to the power(g) 0,5 digestive tract and destroying the 0

Larvaeaverage feeding immune system of S. frugiperda larvae. 1 2 3 4 5 6 7 Day The percentage of mortality and time of death of S. frugiperda larvae at various Figure 5. Average feeding power of concentrations larvae Observation of the number of S. frugiperda that died was carried out

every 24 hours for 7 days and was where 16 S. frugiperda larvae died in calculated using the percentage Kepahiang isolates and 10 S. frugiperda mortality formula. Based on calculations larvae in Bengkulu isolates. On the 5th to using the percentage mortality formula, the 7th day, the average number of the concentration of NEP 600 Jl / 3 ml larvae that died was 2 S. frugiperda produced the highest percentage of larvae in Kepahiang isolates, while in mortality, namely 100% with Kepahiang Bengkulu isolates larvae mortality only isolates and 93.3% with Bengkulu occurred until the 6th day (Figure 6) isolates. Then followed by the 18 concentration of NEP 400 Jl /2 ml, 16 namely 80% with Kepahiang isolates and 14 73.3% with Bengkulu isolates. This 12 10 shows that the greater the concentration 8 of NEP given to S. frugiperda larvae will 6 spur the mortality percentage of S. 4 frugiperda to increase (Table 3). 2 0

1 2 3 4 5 6 7 Larvae Larvae frugiperda S. mortality Table 3. Average percentage of mortality Day of S. frugiperda larvae at various Kepahiang Bengkulu

concentrations and origins of Figure 6. Graph of S. frugiperda larvae entomopathogenic nematode mortality isolates The pathogenicity mechanism of Mortalitas (%) Steinernema sp. begins with the Concentration Kepahiang Bengkulu penetration of Steinernema sp. Isolate Isolate nematodes. into the body of S. 200 Jl/1ml 60 b 46,6 b frugiperda., presumably through natural 400 Jl/2ml 80 ab 73,3 ab openings such as the spiracle, mouth, 600 Jl/3ml 100 a 93,3 a and anus, then ending with the death of S. frugiperda. Similar research results were also The high pathogenicity of reported in S. exigua larvae that the high Steinernema sp nematodes is thought to concentration of NEP affected the be due to Steinernema sp. has a higher mortality percentage of S. exigua resistance to desiccation (drought), as (Baharuddin, 2014). Wiludjeng and Sri well as the nematode symbiont bacteria (2014), reported that with an increase in Steinernema sp. produce more effective mortality of Spodoptera sp. Larvae. with enzymes and toxins. Likewise, the each increase in the dose of Steinerma higher resistance to the sp. so it can be said that the dose of entomopathogenic nematode Steinerma sp. which was applied had a Steinernema sp. This is supported by the positive effect on the percentage of research report of Surrey and Wharton mortality of larvae of Spodoptera sp. (1995), that several types of The calculation of the mortality of entomopathogenic nematodes, namely S. frugiperda larvae showed that there Steinernema sp. Certain isolates have was an increase in mortality from 1 day higher resistance to desiccation than after application. The highest mortality other types of entomopathogenic occurred on the 4th day after application nematodes, so that this type of nematode

is more resistant to life and attacks the death, then the nematodes leave the host. The presumption is that host's body and look for a new host. Steinernema sp. contains enzymes and toxins that are more effective than other Table 5. Table LC50 and LC75 Spodoptera types of nematodes, it has been reported frugiperda against by Kaya and Koppenhofer (1996) that Entomopathogenic Nematodes Steinernema spp. certain can kill the host Isolate LC50% LC75% insect because the symbiont bacteria Kepahiang 163.5 Jl/ml 319.5 Jl/ml have enzymes and toxins that are very Bengkulu 186.5 Jl/ml 398.7Jl/ml effective. Determination of the values of Table 4. Average time of death of S. LC50 and LC75 is the determination of frugiperda larvae at various the optimal concentration, intended for entomopathogenic nematode Steinermatidae spp. effective for concentrations controlling S. frugiperda larvae. The Time of death (days) results of the NEP petogenicity test on S. Consentration Kepahiang Bengkulu frugiperda larvae showed that there was Isolate Isolate an increase in mortality at each 200 Jl/1ml 3,9 a 4,9 a treatment concentration. The estimated 400 Jl/2ml 3,7 a 3,7 a value of LC50 from probit analysis was 600 Jl/3ml 3.1 a 3,4 a 163.873 Jl / ml in Kepahiang isolates and Note: The numbers followed by different letters in the same column show a significant difference 186.582 Jl / ml in Bengkulu isolates, and in the BNT follow-up test at the 5% level the magnitude of the LC75 estimated Based on the average test with the values from probit analysis was 319.5 Jl BNT level of 5%, the mortality rate at the / ml in Kepahiang isolates and 398.7 Jl / concentration of 200 Jl / ml, 400 Jl / ml ml in Bengkulu isolates. and 600Jl / ml was not significantly CONCLUSION different, even though the NEP concentration was added and NEP The results of the isolation of isolates came from different areas but entomopathogenic nematodes from the results were not different. real, but Bengkulu and Kepahiang regions using the concentration of NEP 200 Jl / ml was Tenebrio molitor larvae obtained able to kill the larvae within 4 days. The entomopathogenic nematodes genus relationship between the NEP Steinernema with an average population concentration and the time of death of S. sdensity of entomopathogenic frugiperda larvae shows that the higher the NEP concentration at the time of nematodes of 9,005 NEP / 5ml in the application, the higher the mortality of S. Kepahiang region and 6,837 NEP / 5ml frugiperda. It is thought that the more in the Bengkulu region. The results of NEP in S. frugiperda's body, the more the entomopathogenic nematode severe the damage to S. frugiperda's body virulence test against S. frugiperda larvae tissue due to NEP attacks. After S. show that the NEP 200 Jl / ml frugiperda dies, the nematodes will come concentration can control S. frugiperda in out and look for new hosts. As reported maize (Zea mays), and can kill pests by Sulistiyanto (1998), Steinerma sp. stay within 4 days. With an LC50 value of in the host's body for 10-14 days or until

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