BIOTROPIKA Journal of Tropical Biology
https://biotropika.ub.ac.id/
Vol. 10 | No. 2 | 2022 | DOI: 10.21776/ub.biotropika.2022.010.02.01 COMPATIBILITY OF NEEM (Azadirachta indica) AND Beauveria bassiana FOR CONTROL OF Spodoptera exigua AND THE THEORETICAL IMPACT TO THE
AGROECOSYSTEM
KOMPATIBILITAS MIMBA (Azadirachta indica) DAN Beauveria bassiana UNTUK PENGENDALIAN Spodoptera exigua DAN DAMPAK TEORITISNYA TERHADAP
AGROEKOSISTEM
Agung Sih Kurnianto1)*, Santi Prastiwi1), Nilasari Dewi1), Ahmad Ilham Tanzil1), Wildan Muhlison1), Wagiyana2)
ABSTRACT
Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) is the biggest threat to onion farming, especially in the vegetative phase. In severe attacks, this pest can cause yield losses of up to 100%. Compatibility is one way to increase the efficiency and effectiveness of pest control by using plant-based pesticides or biological agents. Neem Leaf Extract (Azadirachta indica, acronym=NLE) has azadirachtin compound, which can inhibit insect growth, reduce appetite, reproduction, and egg hatching. The fungus Beauveria bassiana (acronym=Bb) can secrete chitinase, lipase, and proteinase enzymes that are able to decompose insect cuticles. NLE was obtained through the extraction method of plant- based pesticides and stored at 4ºC until the experiment time. The Bb used was a commercial B. bassiana inoculum in the flour form with a density of 4.5 x 1010 spores/gram (trade name = Natural BVR). To determine the advantages of compatibility, this study was held by a single toxicity test of Bb and NLE, and a combined toxicity test of both. This study used a completely randomized design (CRD) consisting of six treatments with four replications, and each replication used 12 larvae (a total of 48 larvae).
Combination toxicity had higher toxicity than the single use of NLE and Bb. At the LC 95 level, the combination treatment of Bb and NLE was 1.06-1.15 times more toxic than the single treatment of Bb, and 6.87-7.79 times more toxic than the single treatment of NLE.
Therefore, NLE and Bb were considered to have high compatibility (strong synergistic with GI value <0.5). Theoretically, the compatibility of NLE plant-based pesticides and Bb is very promising in replacing chemical pesticides that have long-term adverse effects on agroecosystems.
Keywords: agroecosystem, Azadirachta indica, Beauveria bassiana, compatibility, Spodoptera exigua
ABSTRAK
Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) merupakan ancaman terbesar bagi budidaya bawang merah terutama pada fase vegetatif. Pada serangan berat, hama ini DATat menyebabkan kehilangan hasil hingga 100%. Kompatibilitas merupakan salah satu cara untuk meningkatkan efisiensi dan efektivitas pengendalian hama dengan menggunakan pestisida nabati atau agens hayati. Ekstrak Daun Mimba (Azadirachta indica, akronim = EDM) memiliki senyawa azadirachtin yang dapat menghambat pertumbuhan serangga, mengurangi nafsu makan, reproduksi dan penetasan telur. Jamur Beauveria bassiana (akronim=Bb) dapat mensekresi enzim kitinase, lipase, proteinase yang mampu menguraikan kutikula serangga. NLE diperoleh melalui metode ekstraksi pestisida nabati dan disimpan pada suhu 4ºC sampai waktu percobaan. Bb yang digunakan adalah inokulum B. bassiana komersial dalam bentuk tepung dengan densitas 4,5 x 1010 spora/gram (nama dagang = Natural BVR). Untuk mengetahui keunggulan kompatibilitas, penelitian ini dilakukan dengan uji toksisitas tunggal Bb dan NLE, dan uji toksisitas gabungan keduanya. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) yang terdiri dari 6 perlakuan dengan 4 ulangan, setiap ulangan menggunakan 12 larva (total 48 larva). Toksisitas kombinasi memiliki toksisitas yang lebih tinggi daripada penggunaan tunggal NLE dan Bb. Pada kadar LC 95, perlakuan kombinasi Bb dan NLE 1,06-1,15 kali lebih toksik dibandingkan perlakuan tunggal Bb, dan 6,87-7,79 kali lebih toksik dibandingkan perlakuan tunggal NLE. NLE dan Bb dianggap memiliki kompatibilitas yang tinggi (bersinergi kuat dengan nilai GI<0,5). Secara teoritis, kompatibilitas pestisida nabati NLE dan Bb sangat menjanjikan untuk menggantikan pestisida kimia yang memiliki efek merugikan jangka panjang pada agroekosistem.
Kata kunci: agroekosistem, Azadirachta indica, Beauveria bassiana, kompatibilitas, Spodoptera exigua
Received : March, 22 2022 Accepted : July, 1 2022
Authors affiliation:
1)Agrotechnology Study Program, Faculty of Agriculture, University of Jember.
Kalimantan street, No 37.
Tegalboto, Jember, East Java Province 68121, Indonesia
2)Plant Protection Study Program, Faculty of Agriculture, University of Jember.
Kalimantan street, no 37.
Tegalboto, Jember, East Java Province 68121, Indonesia
Correspondence email:
How to cite:
Kurnianto, AS, S Prastiwi, N Dewi, AI Tanzil, W Muhlison, Wagiyana. 2022. Compatibility of neem (Azadirachta indica) and Beauveria bassiana for control of Spodoptera exigua and the theoritical impact to the agroecosystem. Journal of Tropical Biology 10 (2): 89-96.
INTRODUCTION
Environmental sustainability is crucially affected by agricultural management, especially the use of agrochemicals derived from fertilizers and pesticides. The consumption of pesticides is very large and is a basic need in onion agricultural management [1]. Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) is a major threat to onion farming, especially in the vegetative phase.
In severe attacks, this pest can cause yield losses of up to 100% [2]. Onion farmers use a lot of chemical pesticides to control this pest [3, 4, 5].
However, as studies have been conducted by Aoun [6], Tudi et al. [7], and Trisyono [8], chemical pesticides have long-term adverse effects on agroecosystems, such as pollution, pest resurgence, and death of natural enemies and pollinators. Therefore, control using plant-based pesticides and biological agents is considered promising. However, many authors have studied studies that reveal the ineffectiveness of single- use plant-based pesticides or biological agents [9, 10].
Compatibility is one way to increase the effectiveness of pest control by using plant-based pesticides or biological agents. This effectiveness has an impact on cost savings, reducing the risk of poisoning in non-target organisms, increasing the spectrum of activity, and slowing down pest resistance [11]. Studies related to compatibility have been carried out by several authors, including combining Beauveria bassiana (Bb) with several insecticides and fungicides [12] and applied as an Integrated Pest Management [13].
Neem leaves (Azadirachta indica) were chosen because of the content of azadirachtin compounds that can inhibit insect growth, reduce appetite, reproductive rate, and egg hatching [14]. In addition, B. bassiana could secrete chitinase, lipase, and proteinase enzymes that could break down insect cuticles. Beauveria bassiana also produces the beauvericin toxin and oxalic acid, which can cause an increase in pH, blood clots, damage to the digestive tract, muscles, nervous system, and respiration in insects [15]. Based on research by Afandhi et al. [16], the concentration of neem leaf extract was 0.25%, 0.5%, and 1%
compatible because they did not inhibit the growth of the fungus, as indicated by the compatibility value (T) above 60. This fact shows that Bb and NLE are very promising to be combined and can increase the mortality of S.
exigua larvae. This study aims to examine the compatibility between plant-based pesticides made from neem leaves and biological agents of fungus (B. bassiana) and analyze their theoretical impact on agroecosystems.
METHODS
Study sites and preparation. This research was conducted at the Green House of the Faculty of Agriculture, the University of Jember. Data analysis was carried out at the Laboratory of Agrotechnology, Faculty of Agriculture, in March-September 2021. Five onion seedlings were planted in each 35x30 cm polybag and were filled with soil, manure, and husk (1:1:2). There are 50 polybags used in this study. After seven days, the plants were fertilized with NPK (trademark: Phonska) 1 g/polybag. S. exigua larvae were obtained from Kraksaan, Probolinggo Regency, East Java. The third instar larvae were used and cultured by rearing method (Figure 1) [17]. Neem leaf extract (NLE) was obtained through a plant-based pesticide extraction method [18] and stored at 4ºC until the experiment. The commercial inoculum of B. bassiana experiment in the form of flour with a density of 4.5 x 1010 spores/gram (trademark Natural BVR) was used in this research. This inoculum was produced by PT. Natural Nusantara Yogyakarta and has a recommended concentration of 2 g/l.
A
B
Figure 1. A. S. exigua collection in the field, B.
Rearing process in the laboratory
To determine the compatibility effect of NLE and Bb, this study consisted of two tests, namely the single toxicity test of Bb and NLE and a combination of both. This study used a completely randomized design (CRD) consisting of 6 treatments with four replications, and each replication used 12 larvae (total = 48 larvae).
The feed dye method was used in this study [19]. Three pieces of spring onion were dipped in each suspension (Table 1), then dried. Then, the leaves were given to the larvae that had been fasted for 12 hours and kept in a plastic box. This treatment is carried out for 1-2 days.
In addition, a combination toxicity test was conducted to determine the compatibility effect on S. exigua. Combination insecticide concentration based on LC50 value from single toxicity test results. The combination test was carried out with five levels of concentration (Table 2).
Observation. There were three observation parameters carried out in this study. Observations were made from the first day of treatment until it reached 90% of larval death. Below is the formula for calculating mortality :
𝑃 = 𝑎
𝑎+𝑏× 100%...(1)
Keys: P = Mortality; a = Number of dead larvae; b
= Number of remaining larvae.
If there was larval mortality in control, the Abbott formula was used as shown below [20].
𝑃𝑡 = 𝑃𝑜−𝑃𝑐
100−𝑃𝑐× 100%...(2)
Keys: Pt = Percentage of dead larvae after correction; Po = Percentage of larvae that died during treatment; Pc = Percentage of dead larvae
The toxicity test was carried out by calculating the Lethal Concentration (LCx) and Lethal Time (LTx) values. The toxicity values of Lethal Concentration 50 and Lethal Concentration 95 (LC50 and LC95) are concentrations that can cause 50% and 95% of insect deaths in a given duration.
LT50 toxicity is the time required to kill 50% of the tested insects.
The interaction of Bb and NLE was analyzed by calculating the combination index at the LC50
and LC95 levels. The combined index (IK) at the LCx level is calculated by the formula below [21].
𝐼𝐾 =𝐿𝐶𝑥1(𝑐𝑚)
𝐿𝐶𝑥1 +𝐿𝐶𝑥2(𝑐𝑚)
𝐿𝐶𝑥2 + [𝐿𝐶𝑥1(𝑐𝑚)
𝐿𝐶𝑥1 +𝐿𝐶𝑥2(𝑐𝑚) 𝐿𝐶𝑥2 ] Keys: LCx1 and LCx2 = Single concentrations of insecticides 1 and 2 that resulted in x mortality.
LCx1(cm) dan LCx2(cm) = The mixture concentration of insecticides 1 and 2, which resulted in x mortality.
IK<0.5 indicates strong synergism; 0.5 IK 0.77 indicates weak synergism; 0.77< IK 1.43 indicates a combination of additives; CI>1.43 indicates an antagonistic combination.
Data analysis. The toxicity value of each insecticide to S. exigua larvae can be determined by the LC50 value, LC95 value, and LT50 value.
Toxicity values were obtained from insect mortality data, then processed by probit analysis [22] using Microsoft Excel 2010 software.
Table 1. Concentrations of B. bassiana (Bb) and Neem Leaf Extract (NLE) used in the treatment. Keys: a = aquadest, as = agristick solvent (trade name: Agristick Bayer, active compound: alkil aril polyglycol ether 400 ml/l)
Treatments Concentration (%, w/v) Information
Bb Control 100 ml a + 0.05 ml as
0.0125 0.0125 g Bb + 100 ml a + 0.05 ml as 0.025 0.0250 g Bb + 100 ml a + 0.05 ml as
0.05 0.05 g Bb + 100 ml a + 0.05 ml as 0.1 0.1 g Bb + 100 ml a + 0.05 ml as 0.2 0.2 g Bb + 100 ml a + 0.05 ml as
NLE Control 100 ml a + 0.05 ml as
0.3125 0.3125 g NLE +100 ml a + 0.05 ml as 0.625 0.625 g NLE + 100 ml a + 0.05 ml as
1.25 1.25 g NLE + 100 ml a + 0.05 ml as 2.5 2.5 NLE + 100 ml a + 0.05 ml as
5 5 g NLE + 100 ml a + 0.05 ml as
Table 2. Concentration of the combination of Bb and NLE. Keys: a = aquadest, as = agristick solvent
Concentration (%, w/v) Information
Control 100 ml a + 0.05 ml as
0.04125 0.04125 g combination +100 ml a + 0.05 ml as 0.0825 0.0825 g combination + 100 ml a + 0.05 ml as
0.165 0.165 g combination + 100 ml a + 0.05 as ml
0.33 0.33 g combination + 100 ml a + 0.05 ml as
0.66 0.66 g combination + 100 ml a + 0.05 ml as
RESULTS AND DISCUSSION
Larval mortality after insecticide application continued to increase with time of observation.
Single treatment resulted in mortality of 16.67%- 91.67% at 7 Day After Treatment (DAT) and 18.75%-95.83% at 8 DAT (Table 3). Insecticide concentration affected the insect mortality rate.
Increased insecticide concentrations had an impact on higher mortality.
Observations at 7 DAT on Bb treatment with a concentration of 0.0125% could kill larvae up to 16.67%, and at a concentration of 0.025%-0.2%
could kill 20.83%-79.17% (Table 4). NLE concentration 0.3125% could kill larvae up to 27.08% and concentration 0.625%-5% could kill 43.75%-91.67% larvae. Based on the observations of 8 DAT, it was known that the Bb treatment concentration of 0.0125% killed the larvae up to 18.75%, and the concentration of 0.025%-0.2%
killed the larvae between 22.92%-89.58%.
Meanwhile, NLE treatment with a concentration of 0.3125% could kill larvae up to 33.33%, and a concentration of 0.625%-5% can kill larvae between 47.92%-98.83%.
Previous studies have shown that low concentrations of plant-based insecticides act as repellants and antifeedants, so high concentrations were required to be toxic to target insects.
Therefore, combinations of compatible insecticides would require lower concentrations than single applications [23, 24].
Toxicity values of LC50 and LC95 in the single treatment of Bb and NLE were shown by probit analysis (Table 4). The smaller the insecticide toxicity value, the more toxic an insecticide was.
Observation of single Bb treatment at 7 DAT showed LC50 and LC95 values of 0.08% and 0.98%, while the NLE single treatment showed LC50 and LC95 values of 0.70% and 6.32%, respectively. Observation of single Bb treatment at 8 DAT showed LC50 and LC95 toxicity values of 0.06% and 0.47%, respectively, while NLE single treatment showed LC50 and LC95 toxicity values of 0.58% and 4.44%, respectively.
Observation of single Bb treatment at 8 DAT showed that the highest concentration (0.2%) took 5.20 days to kill 50% of larvae, and the 0.1%
concentration took 6.70 days to kill 50% of larvae (Table 5). Then, the single treatment of NLE at
the highest concentration (5%) took 4.68 days to kill 50% of the test larvae, and at a concentration of 1.25%-2.5%, it took 5.42-5.99 days to kill.
50% larvae. In general, NLE with a concentration of 5% killed the test larvae faster than Bb at a concentration of 0.2%.
Observation of combination treatment with a concentration of 0.04125% at 7 DAT could kill up to 41.67% of larvae, and a concentration of 0.825% killed 47.92% of larvae (Table 6).
Observation of 8 DAT with a concentration of 0.4125% could kill 45.83% larvae, and a concentration of 0.0825%-0.66% could kill 64.58%-97.92% larvae.
The results of the toxicity of LC50 and LC95
with the combination treatment of Bb and NLE and a ratio of 1:10 based on the results of probit analysis could be seen in Table 7. The lower the toxicity value of the combination, the more toxic the insecticide. The combination treatment of Bb and NLE observed in 7 DAT showed LC50 and LC95 values of 0.07% and 0.92%, respectively.
Meanwhile, in the observation of 8 DAT, the LC50
and LC95 values were 0.05% and 0.41%, respectively. Then, the combination treatment of Bb and NLE at 7 DAT showed LC50 and LC95
values of 0.07% and 0.92%, respectively.
Meanwhile, in the observation of 8 DAT, it was known that the LC50 and LC95 values were 0.05%
and 0.41%, respectively. At the LC95 level, the combination treatment of B. bassiana and neem leaf extract was 1.06-1.15 times more toxic than the single treatment of B. bassiana and 6.87-7.79 times more toxic than the single treatment of neem leaf extract.
The combination treatment of Bb and NLE was more effective than the single treatment. This was because B. bassiana and neem leaf extract have different active ingredients, penetration methods, targets, and effects in killing insects [25]. This was also confirmed by the research of Sain et al. [26], that combining entomopathogenic fungi with plant-based insecticides was more effective. Combination applications have different active ingredients so that they can complement each other. In addition, combination treatment can delay pest resistance.
The LT50 value indicated that the combination treatment at several concentrations resulted in the
mortality of more than 50% of S. exigua larvae (Table 8). The combination of Bb and NLM with a concentration of 0.0825% took 7.29 days to kill 50% of the larvae. The combination with the highest concentration (0.66%) was able to kill faster, which was 4.65 days to kill 50% of larvae.
The combination treatment had different
characteristics in killing insects, so it became more effective. The influencing factors included the level of pest resistance, host resistance, microenvironmental conditions in the host's body, as well as different compositions of active ingredients [27].
Table 3. Effect of a single treatment of Bb and NLE on mortality of S. exigua larvae. Keys: DAT = days after treatment
Treatments Concentration (%, w/v) Larval death 7 DAT (%) Larval death 8 DAT (%)
Bb
Control 0 0
0.0125 16.67 18.75
0.025 20.83 22.92
0.05 29.17 31.25
0.1 54.17 68.75
0.2 79.17 89.58
NLE
Control 0 0
0.3125 27.08 33.33
0.625 43.75 47.92
1.25 70.83 75.00
2.5 83.33 87.50
5 91.67 95.83
Table 4. Estimating parameters of single toxicity of Bb and NLE to S. exigua larvae. Keys: DAT = days after treatment, a = probit regression intercept, b = probit regression slope, GB = standard error, LC = lethal concentration
Time (DAT) a ± GBb b ± GBb LC50
(SK 95%) (%) b
LC95
(SK 95%) (%) b
Bb 7 0.66±0.09 1.50±0.22 0.08 (0.06-0.10) 0.98(0.74-1.29)
8 0.07±0.09 1.79±0.24 0.06 (0.05-0.07) 0.47 (0.38-0.59)
NLE 7 1.66±0.09 1.73±0.23 0.70 (0.54-0.91) 6,32 (4.89-8.16)
8 2.00±0.09 1.86±0.25 0.58 (0.45-0.75) 4.44 (3.43-5.76)
Table 5. LT50 of single Bb and NLE values for S. exigua. Keys: a = probit regression intercept, b = probit regression slope, GB = standard error, LT = lethal time
Concentration (%) a ± GBb b ± GBb LT50 (days)b
Bb 0.1 1.37±0.08 4.39±0.62 6.70 (6.08-7.38)
0.2 0.75±0.08 5.94±0.67 5.20 (4.88-5.55)
NLE 1.25 0.65±0.08 5.59±0.66 5.99 (5.60-6.42)
2.5 0.21±0.08 6.53±0.68 5.42 (5.11-5.74)
5 0.58±0.09 6.59±0.70 4.68 (4.40-4.98)
Table 6. Effect of combination treatment of Bb and NLE on mortality of S. exigua larvae
Treatments Concentrations (%, w/v) Mortality of larvae at 7 DAT (%)
Mortality of larvae at 8 DAT (%)
BB+NLE (1:10) Control 2.08 2.08
0.04125 41.67 45.83
0.0825 47.92 64.58
0.165 77.08 81.25
0.33 85.42 93.75
0.66 91.67 97.92
Table 7. Estimating the toxicity parameters of the combination of Bb and NLE on S. exigua larvae. Keys: a
= probit regression intercept, b = probit regression slope, GB = standard error, LC = lethal concentration Time (DAT) a ± GBb b ± GBb LC50 (%) b LC95 (%) b BB + NLE (1:10) 7 0.87±0.09 1.46±0.23 0.07 (0.06-0.10) 0.92 (0.62-1.12)
8 0.74±0.09 1.57±0.25 0.05 (0.03-0.06) 0.41 (0.33-0.64)
The interaction properties of the 7 DAT observations at the LC50 and LC95 levels had a combination index of 0.18 and 0.23. While the observation of 8 DAT in the LC50 and LC95 levels had a combination index of 0.17 and 0.18. Based on these data, it could be seen that the combination of Bb and NLE had a strong synergistic interaction. This showed that Bb and NLE could be applied together [28].
There was a greater increase in mortality of S.
exigua larvae in the combination treatment at 8 DAT compared to the insecticide treatment alone (Table 9). The combination of Bb and NLE provides a synergistic or compatible effect. This result implied that both could be applied together and deserved to be tested further in the field. The combination of entomopathogenic fungi with plant-based insecticides was more effective in controlling the target pests when compared to the single treatment of entomopathogenic fungi [29].
Bb and NLE had different ways of penetrating and working systems. Bb was an entomopathogenic fungus that was applied in the form of conidia that could infect insects through the skin, cuticle, mouth, and segments found on the insect's body [15]. Conidia contacted the integument, then germinated and formed hyphae.
Germinated hyphae would secrete beauvericin and oxalic acid. These poisons caused the nervous system to be disturbed and ultimately led to the death of the insect. Neem leaves contained several active ingredients, including azadirachtin, meliantriol, solanine, and nimbin, which were secondary metabolites of the neem plant [30]. The active compound azadirachtin acted as an ecdysone blocker or a substance that could inhibit the process of insect metamorphosis. Insects that were poisoned would be disturbed in their metamorphosis process, from eggs to larvae, and eventually cause the death of insects.
Impact on agroecosystem. The environmental impact of chemical pesticides was enormous and should be minimized as much as possible.
Disturbances caused by chemical pesticides caused a long-term imbalance of agroecosystems
[31, 32, 33]. Compatibility resulted greatly impacts the environment. The effectiveness of the compatibility treatment on a broader and complementary spectrum had a beneficial impact on the environment. Previously, chemical pesticides were widely used as part of compatibility with several biological agents, and many had a significant impact with reduced concentrations of chemical pesticides used [34, 35].
As carried out in this study, the use of a combination of plant-based insecticides was synergistic and could increase application efficiency due to lower concentrations compared to the single application model. In other words, the combination of the two insecticides could also overcome the limitations of plant-based insecticide raw materials. This was because plants as sources of plant-based insecticides were not always abundant in every area. The results of this study were very promising to be used as a reference that the combination of B. bassiana with neem leaf extract could be used as a substitute for chemical insecticides, which were more environmentally friendly and still had an effect on the mortality of S. exigua larvae. Several treatments with positive controls and in the field in the future are expected to be carried out to strengthen expectations on compatibility, and in the future, compatibility can be a hope to escape from dependence on chemical pesticides.
CONCLUSION
The combination of B. bassiana and NLE was synergistic. The combined index was less than 0.5 on both LC50 and LC95 in 7 and 8 DAT. The use of a combination of plant-based insecticides was synergistic and could increase application efficiency due to lower concentrations compared to the single application model. The results were very promising to be used as a reference that the combination of B. bassiana with NLE could be used as a substitute for chemical insecticides, which were more environmentally friendly.
Table 8. LT50 values for the combination of Bb and NLE for S. exigua larvae. Keys: a = probit regression intercept, b = probit regression slope, GB = standard error, LT = lethal time
Concentration (%) a ±GBb b ± GBb LT50 (days)b
BB+ NLE (1:10) 0.0825 0.11±0.09 5.67±0.92 7.29 (6.67-7.97)
0.165 0.57±0.08 5.89±0.65 5.64 (5.29-6.01)
0.33 0.47±0.09 6.29±0.68 5.24 (4.93-5.57)
0.66 0.90±0.09 6.13±0.68 4.65 (4.35-4.97)
Table 9. Interaction of the combination of Bb and NLE on S. exigua larvae Days (DAT) Combination Index Interaction
LC50 LC95 LC50 LC95
BB+ NLE (1:10) 7 0.18 0.23 Strong synergistic Strong synergistic
8 0.17 0.18 Strong synergistic Strong synergistic
ACKNOWLEDGMENT
The authors thank the Faculty of Agriculture, University of Jember, for the research facilities.
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