Nematicidal effectivity of neem oil, castor oil, and pyrethroids toward foliar nematode (Aphelenchoides fragariae) mortality

The nematicidal activity of neem oil, castor oil, and pyrethroids (deltamethrin and λ-cyhalothrin) was assessed in triplicate toward A. fragariae mortality in vitro. Carbamate carbosulfan was used as a comparative chemical. All chemicals tested have a contact poison activity for nematodes. About 25 to 50 nematode vermiforms consisting of larvae and adults were soaked in 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% of botanical oils and pyrethroids solutions for 1 hour, and the dead nematode was then counted under 100x magnification of a compound microscope. The results showed that the mortality rate of A. fragariae that was exposed to neem oil, castor oil, deltamethrin, λ-cyhalothrin, and carbosulfan solution with several concentration levels was significantly higher when compared to the mortality rate in the water (P < 0.05). On 1.0% concentration, neem oil was more effective in killing the nematode (84.85% mortality) than castor oil (36.52% mortality), and deltamethrin was more effective (74.27% mortality) than λ-cyhalothrin (54.19% mortality. LC50 of neem oil was 0,680 μlml−1, deltamethrin was 0.517 μlml−1, and carbosulfan was 0.545 μlml−1. The regression equation for neem oil is y = 60.295x with R2 = 0.9551, while for deltamethrin is y = 42.499x + 21.066 with R2= 0.8709.


Introduction
Foliar nematode Aphelenchoides fragariae (Ritzema-Bos, 1891) Christie, 1932 has become one of the important parasitic nematodes that need to be aware of in the horticultural and ornamental agroindustry in Indonesia.This nematode has been reported to be associated with symptoms of bitter king leaf blight, shallot yellowing and wilting leaves, and strawberry necrotic leaves in Java (Banten, DKI Jakarta, West Java, Central Java, East Java) [1,2].
Once A. fragariae is established in the nursery and the field, it is always very hard to eliminate them.Apart from preventive measures such as using healthy stock and resistant plants, foliar nematode control is carried out through the pesticide/ nematicides application.Nematicides were applied in situations of urgency to hinder the spread of nematodes and suppress nematode populations in heavily infested fields.Some chemicals from the group of organophospate (thionazin, demeton, fenamiphos, parathion, mevinphos), carbamate (carbamoyl, oxamyl, aldicarb, methomyl, carbofuran), have been tested and effectively reduced the symptom of A. fragariae infection.However, due to their negative safety impact, some of those chemicals have had their use restricted or prohibited in various countries [3].
Discovering alternative pest control methods that are equally effective as synthetic pesticides, safer for farmers, consumers, and the environment, and reasonably accessible has become a crucial concern.Since the last decade, researchs have been done to discover alternative products for the control of foliar nematodes that impact ornamental and horticultural crops [3].Some of possible alternatives are the use of pesticides from plant origin, biocontrol agents, and modern safer 1297 (2024) 012058 IOP Publishing doi:10.1088/1755-1315/1297/1/012058 2 chemicals.Neem seed powder and its water extract, and CNSL (cashew nut shell liquid) have been tested and effectively suppressed A. fragariae population in bitter king plant leaves [4].Among the several groups of synthetic pesticides (organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids) are used in crop protection, pyrethroids continue to be the least hazardous [5,6].According to Zaim et al., the risk of any form of toxicity is low if pyrethroids are frequently exposed at low doses [5].Pyrethroids that enter the systemic circulation quickly break down into metabolites that are considerably less harmful.In terms of toxicology, pyrethroids have the beneficial feature of causing skin paraesthesia, which serves as an early warning indicator of exposure.This reversible symptom of exposure is not a hazardous impact and is caused by temporary activation of peripheral sensory nerves [5].Among the commercial plantbased pesticide products, neem oil is one of the least toxic to humans and environment; therefore, it is very promising for safer management of many pests [7].Most of the available investigations on neem oil, castor oil, and pyrethroids against nematodes were mostly targeted at root-knot nematodes Meloidogyne spp.[8,9,10] and a few studies on A. fragariae.
This experiment described the results of the efficacy test of the neem oil, castor oil, and pyrethroid chemicals (deltamethrin and λ-cyhalothrin) toward foliar nematode mortality and their effective chemical's lethal concentration required for 50 and 95 percent mortality at exposure time in the laboratory.

Preparation of chemicals and the botanical oil
Neem oil (certified-100% pure neem oil) was purchased from "Happy Green Garden-Jakarta"; and castor oil (100% pure castor oil) was purchased from "Mitra Jaya 889".Pyrethroid chemicals (deltamethrin and lambda (λ)-cyhalothrin) and carbamate (carbosulfan) were purhcased from the agro-chemical shop "Sarana Tani" in Bogor, Indonesia.The botanical pesticide formula were prepared simply by mixing botanical oil with tween 80 (synthesis grade) as an emulsifier.

Preparation of laboratory test foliar nematode
Laboratory test foliar nematode was obtained by extraction of A. fragariae vermiform from the nematode infested bitter-king plant leaves.The extracted nematodes were then made into a suspension by adding 100 ml of sterile water.Extraction of nematodes from plant leaves was carried out using a modified Baermann funnel method [11].The extracted fresh foliar nematodes were directly used for chemical suspension exposures treatment.

Experimental designs
The experiment was conducted in triplicate in the laboratory of Genomic, BRIN.The experiment was designed as randomized block design.The treatment designs were two factors with 5 types of pesticides for the main plot and 4 concentration levels (0.5%, 1.0%, 1.5%, and 2.0%) of each type of pesticides for the co-plot (Table 1).About 25 to 50 foliar nematode vermiforms were used in each replication (Table 2).The testing was carried out using the Bunt method [12], the nematodes vermiforms were soaked to concentrated chemicals suspension for 1 hour/60 minutes exposure time.One (1) ml of fresh water containing 25 to 50 nematode were introduced in to 9 ml of ajusted chemical solution concentration (0.5%, 1.0%, 1.5%, and 2.0%) in glass tubes.After about 1 hour, the dead nematode number was observed and counted under 100 magnification compound microscope, and was expressed in mortality percentage, with assuming that nematodes are considered dead when they do not move in solution and do not react when touched with the tip of a nematode fishing needle.
Where, M = the nematode mortality percentage (%), n = the dead nematode number, N = total nematode number observed.
Data on mortality percentage of A. fragariae were subjected to probit analysis [13] to estimate the values of lethal concentration of botanical oil and pyrethroids dose required for 50 and 95 percent mortality at 1 hour exposure time.Using Abbott's formula [14], the death rates of the nematodes in the exposure groups (PO) were corrected for mortality in the water controls (PC): Where, Pr = the corrected nematode mortality, Po = the death rates of the nematodes in the exposure groups, Pc = the nematode mortality in the water controls.
Correct for mortality in the control treatment using Abbott's formula (% test mortality -% control mortality/ 100 -control mortality x 100).The corrected mortality (P T ) was plotted against the pesticide concentration and fitted using Epa Probit Analysis Program Version 1.5 to determine the LC 50 and LC 95 values.The control data were included as a concentration that was 100 times lower than the lowest test compound concentration since the log scale was used to plot the data.

Statistical data analysis
Data from mortality tests were subjected to analysis of variance (ANOVA) and means compared by Least Significant Difference's Test (P < 0.05 and P < 0.01).Meanwhile, data for toxicological assays 4 were analysed by Epa Probit Analysis Program Version 1.5 to obtain the Median Lethal Concentration (LC 50 and LC 95 ).Data for regression analysis was Microsoft Excell.

Results and discussions
The nematicidal effectivity of botanical oils and pyrethroids toward foliar nematode mortality was estimated through the values of A. fragariae mortality rates, LC 50 and LC 95 , and a linear regression model.The dead nematodes after exposure to tested chemicals were mostly in straight and banana shapes (Figure 1).Based on the dead nematode number counting, the results showed that the mortality rates of A. fragariae after 1 hour of exposure to neem oil, castor oil, carbosulfan, deltamethrin, and λ-cyhalothrin solution with several concentration levels (0.5%, 1.0%, 1.5%, and 2.0%) were significantly higher when compared to the mortality rate to water (control) (Figure 2).Comparative effectivity among chemicals tested was analyzed at 1.0% concentration since the recommended concentration of synthetic chemicals is mostly at 1.0% concentration.On a 1.0% chemical concentration, after 1 hour of exposure to chemicals, it was revealed that neem oil had the highest effectivity toward nematode mortality (84.85%), and the effectivity was similar to pyrethroid deltamethrin (74.27%) and carbamate carbosulfan as a comparative chemical (72.23%) (Table 2).The chemical with the least effectivity was castor oil (36.52%).
From leaf-disc assays of neem oil on A. fragariae by An et al., demonstrated that neem oil caused 36.22% and 91.08% mortality in a 20 and 2-fold dilution (in 4.76% and 33.33% concentration, respectively) within 24 -72 hours of exposure time [15].In a similar in vitro test on M. incognita J2, the use of neem oil in vitro was effective only in the immobility of M. incognita at concentrations of 1% in the control of active J2, with a percentage of 11% immobility that was different statistically from the control with 84.8% and smaller than the other treatments [8].Neem oil is a type of vegetable oil that is made from the neem (Azadirachta indica) seed kernels.Neem oil contains at least 100 compounds that have physiological effects.Their primary components are triterpenes known as limonoids, the most important of which is azadirachtin, which are considered to be responsible for 90% of the effects on most pests.Meliantriol, nimbin, nimbidin, nimbinin, nimbolides, fatty acids (oleic, stearic, and palmitic), and salannin are also included [16].Active components of neem oil, such as triterpenoid (nimbin, nimbicidin) and terpenoid (azadirachtin, nimbin, salannin), have biological activity as nematicidal/insecticidal and antifeedant/growth inhibitor, respectively [15,17,18].Those active component might be responsible for the death of the nematode.

Figure 1. The live body shape (left) and dead body shape (centre and right) of Aphelenchoides. fragariae after chemical treatment
In this in vitro study, castor oil treatment at 1.0% concentration demonstrated significantly lower effectiveness than neem oil toward A. fragariae mortality (Table 2).Castor oil is a vegetable oil obtained from castor seed and has characteristic properties and many industrial uses.It is basically a triglyceride, which contains approximately 90 percent fatty acid chains of ricinoleate; and other components are oleate and linoleate [19].Commonly, products like castor bean meal and crude castor bean extract are frequently used in the nematode control experiment.The main component of castor bean meal and crude castor bean extract was ricin, which was nematotoxic and harmful to plant parasite nematodes [20,21].In vitro and on potted-tomato plants, the crude castor bean extract was nematotoxic to root-knot nematodes [21].All parts of the castor bean plant are poisonous to nematodes, fungi and insects because the bioactive content of ricin is 80-90% and the rest is castor oil [22].From the experiment with botanical oil as coating material for urea amendment, castor oil at 2.0% concentration demonstrated significantly lower effectivity than neem oil at 2.0% concentration in suppressing the number of galls per root system, egg masses, and adult females of Meloidogyne spp.infection [9].Those informations above might be used to justify why castor bean oil was less toxic than neem oil to A. fragariae in this in vitro study, even though the comparative study between A. fragariae and Meloidogyne spp. was not carried out in this in vitro study.Chemical pyrethroid deltamethrin is a contact and stomach poison insecticide used to control plant pests [23], whereas pyrethroid λ-cyhalothrin is a non-systemic insecticide with contact and stomach action as well as repellent qualities, providing quick knockdown and long-lasting residual activity [6].The synthetic pyrethroids deltamethrin, analogs of chrysanthemum-derived terpenoid pyrethrins [24].They are designed to be more stable in the natural environment [25]; low in toxicity to mammals and birds and dissolve very poorly in water [24].From a field investigation by Djouaka et al. on λ-cyhalothrin residues in lettuce and cabbage from farms and markets in Benin, they are within the MRL (maximum residue limit) and hence are relatively safe for consumption [6].Carbamate carbosulfan is systemic insecticides and acaricides, contact and gastric poisons to control pests on plants [23].The in vitro toxicity test by Wiratno et al. on carbosulfan and deltamethrin (40 µl of the synthetic pesticide stocks to 460 µl of fresh tap water) in Meloidogyne sp.J2 resulted in about 73% mortality and 40% mortality, respectively [10].A similar result was showed on carbosulfan effectivity towards A. fragariae in this study (72.23% mortality).However, a different result was showed for deltamethrin in A. fragariae (74.27% mortality) which was higher than result for Meloidogyne J2 (40% mortality).It was confirmed from some experiments that not all pesticides in the same group are equally toxic, or equally effective the same pests [26].
Data on mortality percentage of A. fragariae were subjected to probit analysis [13] to estimate the values of lethal concentration of botanical oil and pyrethroids dose required for 50 and 95percent mortality at 1 hr exposure time.Among all chemicals tested, from the lowest LC 50 to the highest LC 50 value were deltamethrin (0,517 μlml -1 ), then carbosulfan as comparative chemical IOP Publishing doi:10.1088/1755-1315/1297/1/0120586 (0,545 μlml -1 ), neem oil (0,680 μlml -1 ), λ-cyhalothrin (0,712 μlml -1 ), and the highest LC 50 was castor oil (1,179 μlml -1 ) (Table 2).These results indicated that neem oil has higher potential than castor oil to cause mortality of A. fragariae in-vitrolly with LC 50 0,680 μlml -1 ; and pyrethroid deltamethrin has higher potential than pyrethroid λ-cyhalothrin with LC 50 0,517 μlml -1 .The smaller the LC 50 value indicates the greater the influence of the chemical concentration applied.This character is possessed by neem oil from botanical oils and deltamethrin from pyrethroid pesticides tested.Chemicals used in this in vitro study (botanical oils, pyrethroids, and carbamate carbosulfan) were chosen because they have contact nematicidal or insecticidal activity [6,23,27].The oil is considered a contact insecticide, presenting systemic and translaminar activity [25].In vitro testing is a valid method to screen contact poison chemicals against nematodes by exposing the nematode to concentrated solutions for a certain time length, so that the results obtained from this in vitro test can be close to the application performance in the field.Based on the in vitro mortality rate value [10], neem oil was classified as highly toxic (84.85% mortality) to A. fragariae, while pyrethroid deltamethrin and carbosulfan were toxic (74.27% and 72.23% mortality, respectively); pyrethroid λ-cyhalothrin was quite toxic (54.19% mortality), and castor oil was slightly toxic (36.52% mortality) (Table 2).However, λ-cyhalothrin showed increased nematicidal activity with increased concentration and gave similar effectivity as deltamethrin at 1.5% concentration to cause 75.0%mortality in A. fragariae (Tabel 2).This concentration range was similar to the application recommendation dose on the commercial product.
Linear regression analysis was also used to relate mortality rates to chemical concentration level data to investigate the descriptive value of mortality rates.Relationship between mortality rate of A. fragariae and applied botanical oil and chemical concentration level is demonstrated in Figure 3.This mortality rates were used as explanatory concentration level variables (x), and logit transformed data of the efficacy/mortality rate as dependent variables (y).The equations for regression lines that were calculated from the experimental points resulted as follows: y = 60.295x for neem oil, y = 42.50x+ 21.066 for deltamethrin, and y = 47.108x+ 5.6106 for λ-cyhalothrin.These linear regression models provided accurate descriptions of mortality rates variables by neem oil, deltamethrin, and λ-cyhalothrin concentration variables (with determination coefficients of R 2 = 95, R 2 = 87, and R 2 = 99, respectively) (Figure 3).Pyrethroids deltamethrin and λ-cyhalothrin, and carbamate carbosulfan are registered as insecticides for use in Indonesia in control pest Spodoptera exigua on some horticultural crops [23].Their efficient application in the field might be used to simultaneously control leaf pests and foliar nematodes.Insecticide residues of carbamates and pyrethroids found in food commodities in Indonesia are generally still below the maximum residue limit (MRL), which refers to national standards, namely SNI 7313:2008 [28].As they have nematicidal efficacious toward A. fragariae mortality in this in vitro study, there is a need to evaluate those pyrethroids (deltamethrin and λcyhalothrin) and carbamate carbosulfan as alternative approaches for safer and or effective chemicals management of foliar nematode.

Conclusions
Based on the values of mortality rate, LC 50 and the determinant coefficient of the linear regression models indicated that neem oil and pyrethroid deltamethrin have potential as effective and safer nematicide for management of foliar nematode A. fragariae, while carbamate carbosulfan has potential option for effective chemical nematicide.Further studies need to be conducted is to evaluate the efficacy of neem oil, deltamethrin and carbosulfan to control the foliar nematode in field conditions.

Figure 2 .
Figure 2. Mortality rate of Aphelenchoides fragariae after 1 hour of exposure to several concentrations of neem oil, castor oil, carbosulfan, deltamethrin, and λ-cyhalothrin solution (Error bars represent standard deviation of mean, n= 75).

Figure 3 .
Figure 3.The trend curve was plotted between mortality rates of Aphelenchoides fragariae and the various concentrations of neem oil, castor oil, carbosulfan, and pyrethroids (deltamethrin and λ-cyhalothrin) at 1 hour of exposure time.

Table 1 .
Treatment design 2.4.Response designsFor determining the effectivity of chemicals tested in this experiment, parameters observed were the nematode mortality rate, and toxicity level of chemicals (botanical oils and pyrethroids).To calculate the nematode mortality rate, the following formula is used:

Tabel 2 .
Mortality rate of Aphelenchoides fragariae and lethal concentrations (LC 50 and LC 95 ) after 1 hour of exposure to several botanical oils and pyrethroid chemical solution concentrations (n= 75) ) Averages in the same column followed by the same letter are not significantly different by DMRT at α 0.05 *