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Article

Toxicity of an Emamectin Benzoate Microemulsion against Bursaphelenchus xylophilus and Its Effect on the Prevention of Pine Wilt Disease

by
Wanjun Zhang
1,2,
Xiaoqin Wu
1,2,*,
Jianren Ye
1,2,
Changqiang Li
1,2,
Longjiao Hu
1,2,
Lin Rui
1,2,
Yan Zhang
1,2,
Xiufeng Shi
1,2 and
Lei Wang
1,2
1
Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(7), 1476; https://doi.org/10.3390/f14071476
Submission received: 8 June 2023 / Revised: 2 July 2023 / Accepted: 8 July 2023 / Published: 18 July 2023
(This article belongs to the Section Forest Health)
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Abstract

:
(1) Pine wilt disease (PWD) is a devastating disease of pine forests caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. Control of the disease is a worldwide problem due to the impossibility of using chemical nematicides on a large scale and for long periods. (2) Based on preliminary tests of microemulsion quality and stability, the optimum formulation was selected from 14 formulated microemulsions. The median lethal concentration (LC50) of the selected formulation at 48 h after treatment of B. xylophilus was 31.45 μg/mL of emamectin benzoate. The active ingredient reached the apical branches of Pinus thunbergii within 90 days of injection. (3) P. thunbergii was inoculated with B. xylophilus at 100 days post-injection, and the trees treated with the formulation remained uninfected for 450 days. Trunk injection exerted substantial control over PWD. (4) These results indicate that this formulation has the advantages of good transportability and long persistence in pine trees after injection and that it effectively prevents PWD. Therefore, this emamectin benzoate formulation can effectively reduce PWD occurrence in pine forests.

1. Introduction

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a global plant parasitic nematode that infects pine trees and causes severe ecological damage and economic losses [1,2,3,4]. In 1982, pine wilt disease (PWD) was first discovered in Nanjing, China, and it has now spread to 19 provinces (National Forestry and Grassland Administration Announcement No. 7, 2023). PWD is rapidly expanding throughout the entire country, so control is urgently needed.
Currently, the main control measures include plant quarantine, removing and burning dead wood, breeding resistant pine trees, injecting nematicides into pine trees, and controlling the population of vector beetles [5,6,7]. Chemical control has become a commonly used control method due to its advantages of convenience, ease of operation, and effectiveness. At the phytophagous stage, the nematode migrates to the xylem resin and ray canals and feeds on parenchyma cells, leading to cell death, which makes trunk injection the preferred mode of nematicide application for prevention of the disease [8,9]. It was reported that compounds such as fluopyram, cyhalodiamide, avermectin, and emamectin benzoate have a preventive effect on PWD after trunk injection [10,11,12,13]. However, some pesticide formulations have a large quantity of added organic solvents, and their application easily causes damage to forest trees and other problems, making efficient and environmentally friendly agents a hot spot for scholars worldwide [14].
Water-based pesticide microemulsions are thermodynamically stable liquid solutions with water as the medium, with few or no organic solvents, and with appropriate amounts of surfactant and co-surfactant [15,16,17]. They have the advantages of a small droplet size, strong permeability, and high safety [18,19]. In recent years, microemulsions have continuously been developed and applied as water-based environmentally friendly formulations. In contrast to emulsifiable concentrate, the water content of microemulsions accounts for approximately half of the entire system, significantly reducing the amount of organic solvents added to the formulation and further reducing the development cost of the formulation and the tree damage caused by solvents. As such, microemulsions could provide a new chemical delivery form for the control of PWD.
At present, most of the commercially available trunk injection agents are based on traditional dosage forms of emulsifiable concentrate. Among them, products developed with avermectin and emamectin benzoate as active ingredients are more prominent and have a duration that can reach approximately 3 years [14,20]. Studies showed that the insecticidal activity of emamectin benzoate is one to three orders of magnitude higher than that of avermectin [21]. However, the transport distribution and residual dynamics of the agent in the tree after trunk injection was rarely systematically reported. In this context, there is an urgent need to develop a high-efficiency and low-toxicity trunk injection formulation and to understand its transport and residual period after injection.
In the present study, an emamectin benzoate microemulsion was selected to explore the prevention and control of PWD. After screening, a stable and qualified microemulsion formulation with reference to the relevant quality index of pesticides was used for an indoor toxicity investigation against B. xylophilus. The distribution, residual duration, and persistence period in pine trees after trunk injection were further investigated. These results can provide a basis for explaining the control efficiency of emamectin benzoate against PWD.

2. Materials and Methods

2.1. Description of Biological Materials and Reagents

The highly virulent strain B. xylophilus AMA3 strain used in this study originated from pinewood chips of infested Pinus massoniana in Maanshan, Anhui, China [22]. The nematodes were kept in the Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Nanjing Forestry University, Nanjing, Jiangsu, China. Nematodes were cultured on a mycelial mat of Botrytis cinerea grown on potato dextrose agar (PDA) at 25 °C for 1 week and were extracted by a Baermann funnel [23]. A nematode suspension was prepared with approximately 5000 nematodes per millilitre. Pinus thunbergii was obtained from a large shed at Nanjing Forestry University and cultivated. Healthy P. thunbergii with a ground diameter of 3–4 cm was selected for trunk injection.
Emamectin benzoate (72% active ingredient) was purchased from Xingbai Agricultural (Hebei, China); Song-liang (active ingredient concentration: 2% emamectin benzoate) was supplied by Shijia Technology (commercial agent) (Zhejiang, China); methanol (purity 99.5%), n-butanol (purity 98%), n-pentanol (purity 98%), diethylene glycol monobutyl ether (purity 99%), ethanol (purity 99.7%), dimethyl sulfoxide, dodecylphenol polyoxyethylene ether (10) (OP-10), and nonylphenol polyoxyethylene ether (10) (NP-10) were purchased from Boqiao Biological Technology (Nanjing, China); HPLC-grade methanol (purity 99.9%), acetonitrile (purity 99.9%), and triethylamine (purity 99.5%) were purchased from Macklin Biochemical Technology (Shanghai, China).

2.2. Preparation of Emamectin Benzoate Microemulsions

The 2% emamectin benzoate microemulsion was prepared using the phase inversion method. A certain amount of emamectin benzoate was weighed into a 250 mL brown conical flask; then, compound solvents (mn-butanol:mdiethylene glycol monobutyl ether = 5:3), surfactants (OP-10 or NP-10), co-surfactants (methanol, n-butanol, or n-pentanol), and deionized water were added to bring the mass to 100 g. The relatively stable microemulsion formulations were screened by observing their viscosity and phase separation after treatment at room temperature [(25 ± 2) °C, 24 h], under cold storage [(0 ± 2) °C, 7 days], and under high temperature storage [(54 ± 2) °C, 14 days].

2.3. Quality Index Determination for Emamectin Benzoate Microemulsions

The stability of the selected microemulsions was assessed by referring to the determination method of emulsion stability for pesticides (Standard number: GB/T 1603-2001) [24]. All the relatively stable formulations were diluted 200-fold with standard hard water, placed in a constant-temperature water bath at 30 ± 2 °C and allowed to stand for 1 h. We observed whether the diluted solution showed an oil layer or turbidity. Each experiment was performed at least three times.
The transparent temperature ranges of the selected microemulsions were determined by referring to guidelines on drafting specifications of pesticides (Standard number: HG/T 2467.10-2003) [25]. All the formulations were placed in an adjustable refrigerator, and the temperatures at which freezing (t1) and turbidity (t2) occurred were recorded; the transparent temperature range was t1–t2. Each experiment was performed at least three times.
The pH values of the selected microemulsions were determined by referring to the determination method of pH value for pesticides (Standard number: GB/T 1601-1993) [26]. A total of 1 g of formulation 13 was weighed into a 100 mL beaker, 100 mL of distilled water was added to it, and the mixture was stirred vigorously for 1 min and allowed to stand for 1 min. The pH value was determined with a pH meter. Each experiment was performed at least three times.
The thermal stabilities of the selected microemulsions were determined by referring to the testing method for the storage stability at elevated temperature of pesticides (Standard number: GB/T 19136-2003) [27]. The screened emamectin benzoate microemulsions were placed at 54 ± 2 °C for 14 days, and the concentration and decomposition rate of emamectin benzoate in the screened formulations were determined using high-performance liquid chromatography (HPLC) as described by Jia [14]. Each experiment was performed at least three times.

2.4. Toxicity of the Selected Formulation to B. xylophilus

Preparations of the selected formulation containing 2000, 800, 400, 200, and 100 μg/mL emamectin benzoate were selected to determine the median lethal concentration (LC50) against B. xylophilus. Song-liang, a solution without emamectin benzoate, and sterile water were used as the positive control, solvent control, and blank control, respectively. Individual 1.5 mL centrifuge tubes were filled with 10 μL of formulation at different concentrations and 90 μL of the nematode suspension (50 mixed-stage nematodes). Therefore, nematodes were exposed to the formulation at final concentrations of 200, 80, 40, 20, and 10 μg/mL. Then, the tubes were incubated in the dark at 25 °C. After 48 h of incubation, individuals that did not move and showed “J” or “C” shapes or that had stiff bodies were judged to have died [28]. Live and dead nematodes were counted under a microscope (Leica DM500, Wetzlar, Germany), and the corrected mortality rate was calculated [29]. Then, photographs were taken with a Zeiss fluorescence microscope (Zeiss, Oberkochen, Germany). Each treatment was prepared with three replicates. The corrected mortality was calculated using the following formula:
corrected   mortality   % = mortality   %   of   treatments   −   mortality   %   of   control 1 −   mortality   %   of   control

2.5. Distribution of Emamectin Benzoate within P. thunbergii after Trunk Injection

Trunk injections of five-year-old P. thunbergii (ground diameter of 3–4 cm) were administered in the large shed of the Xiashu Forestry Farm, Jurong City, Jiangsu Province, China, in December 2018, with an indoor temperature of approximately 0 ± 2 °C. Injections were performed by drilling injection ports (2–3 cm depth) into the trunk xylem tissue at a height of 30 cm from the ground at an inclination of 45 degrees. According to the pesticide application standard of 1 mL per 1 cm diameter, each pine was injected with 3 mL of the formulation (the total amount of emamectin benzoate injected was 60 mg). Emamectin benzoate residue tests were conducted on the treated trees at 30, 90, and 540 days post-injection. P. thunbergii injected with Song-liang was used as the control (3 mL/pine; the total amount of emamectin benzoate injected into each pine was 60 mg). Three P. thunbergii trees were injected for each formulation treatment.

2.6. Extraction of Tree Samples Prior to HPLC Analysis

The samples were pre-processed prior to HPLC detection. The extraction method was slightly modified from previous methods [14,30,31]. Branches and trunks were shade-dried and crushed (FW100, Tian Jin, China). Each sample (10 g of sawdust) was homogenized with methanol (80 mL) for 1 h via an ultrasonic cleaner (KQ-500E, Kunshan, China). Subsequently, the homogenate was filtered. The filtrate was then evaporated to dryness using a rotary evaporator (Eyela, Tokyo, Japan) at 40 °C. The sample volume was adjusted to 10 mL using methanol/water (v:v = 7:3) and filtered through a 0.22 μm organic membrane for HPLC analysis.
Trunk samples were collected from 30 cm and 60 cm above the injection site and in the apical branches of P. thunbergii at 30 and 90 days post-injection using the extraction method described above. One P. thunbergii was taken at each of the different sampling times after injection to analyse the distribution and residue of the formulation in the trees. A total of three samples were collected from a single tree. Each sample was analysed three times. P. thunbergii injected with Song-liang was used as the control. The residual concentration of emamectin benzoate in P. thunbergii was determined via HPLC analysis. Waters XBridge C18 chromatography columns (4.6 mm × 250 mm × 5 mm) were used, and the column temperature was maintained at 25 °C. The mobile phase was acetonitrile, methanol, and 0.1% aqueous triethylamine (v:v:v = 50:45:5). The detection wavelength was set to 244 nm. Each sample (volume of 20 μL) was analysed at a flow rate of 1 mL/min [14,30]. The residual emamectin benzoate concentration in each sample was estimated as follows:
C = V · C 0 m
where C is the emamectin benzoate concentration in the sample (μg/g); C0 is the measured emamectin benzoate content of the sample (μg/mL); V is the volume of the sample (mL); and m is the mass of the sample (g).

2.7. Efficacy of the Emamectin Benzoate Formulation against PWD after Trunk Injection

To evaluate the efficacy of emamectin benzoate against PWD in the forest, nematodes were inoculated into P. thunbergii at 100 days post-injection with the selected formulation (3 mL/pine), and the incidence of infection of P. thunbergii was calculated continuously. A sterile blade was used to cut a small wound deep into the xylem of five-year-old P. thunbergii stems, and a cotton ball was placed in the wound. The incision and cotton ball were then covered with a funnel-shaped piece of parafilm. A 100 µL suspension containing approximately 3000 mixed-stage nematodes was transferred into the xylem of each pine tree using a pipette gun. P. thunbergii inoculated with B. xylophilus after injection of Song-liang, P. thunbergii inoculated with B. xylophilus after injection of the solvent control, and P. thunbergii inoculated with B. xylophilus after injection of sterile water were used as the positive control, the negative control, and the blank control, respectively. Four P. thunbergii trees were inoculated in each treatment.
The infection rate and disease severity index (DSI) were used to assess B. xylophilus infections. The DSI of P. thunbergii was classified into five levels: 0, all needles were green; I, a few needles turned yellow; II, approximately half of the needles turned brown; III, most of the needles turned brown, the tree began to wilt, and the terminal shoots of tree were drooping; and IV, all of the needles turned brown, and the entire tree was wilting [32,33]. The infection rate and DSI of P. thunbergii were calculated according to Yu et al. [34]. The calculation formula is as follows:
Infection   rate   % = ∑ Number   of   infected   plants   with   symptoms Total   number   of   plants × 100
Disease   severity   index   DSI = ∑ Number   of   disease   plants   ×   symptom   stage Total   number   of   plants   ×   highest   symptom   stage × 100

2.8. Persistence of Emamectin Benzoate in P. thunbergii

At 540 days post-injection, the residues of emamectin benzoate at 30 cm and 60 cm above the injection site and in the apical branches of P. thunbergii were measured. One P. thunbergii was cut for analysis of the distribution and residues of the formulation in the tree. We collected a total of three samples from a single tree. Each sample was analysed three times. P. thunbergii injected with Song-liang was used as the control. The extraction method was consistent with that described in Section 2.6.

2.9. Statistical Analyses

All data were recorded using Microsoft Excel 2020. The data were subjected to analysis of variance (ANOVA) followed by Duncan’s multiple comparison test with SPSS 21.0 software (IBM Inc., Armonk, NY, USA) or independent samples t-test to determine significant differences (p < 0.05). Graphs were generated using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA). The LC50 value was calculated using DPS 15.10 (DPS data processing software, Ltd., Hangzhou, China).

3. Results

3.1. Screening the Formulations of 2% Emamectin Benzoate Microemulsions

To prepare a relatively stable emamectin benzoate microemulsion, we placed the prepared microemulsions at room temperature [(25 ± 2) °C, 24 h], in cold storage [(0 ± 2) °C, 7 days], and in thermal storage [(54 ± 2) °C, 14 days] to observe the viscosity and phase separation of the microemulsions. Finally, we screened 14 semi-stable formulations of 2% emamectin benzoate microemulsions (Figure 1, Table S1).

3.2. Determining the Optimal Formulation of 2% the Emamectin Benzoate Microemulsion

The diluted microemulsions were placed in a constant-temperature water bath at 30 °C for 1 h. Formulas 1, 2, 3, 7, 8, 9, 13, and 14 were colourless and transparent, while the dilutions of formulas 4, 6, 10, and 11 were milky white, and the dilutions of formulas 5 and 12 were light blue, indicating that the first set of formulations of the former was more stable (Table 1). Considering the formulation costs and the transparent temperature ranges of the microemulsions, formulations 1 and 13 were finally selected for the next step of this study.
The decomposition rate of pesticide active ingredients is one of the important indicators of the stability of pesticide formulations, and the decomposition rate is regulated by the pH value [35,36]. For this reason, we further analysed formulations 1 and 13; the results showed that the pH values of these formulations were 5.69 and 6.27, respectively, and the decomposition rate of emamectin benzoate in formulation 13 was 3.46%, which was significantly lower than that of formulation 1 (7.94%) (Table 1). This indicated that formulation 13 had good stability and could be further evaluated for its virulence effect against B. xylophilus.

3.3. Formulation 13 Possesses Strong Nematicidal Activity

To determine the nematicidal activity of formulation 13 against B. xylophilus, the survival of nematodes was observed after 48 h of treatment. The results showed that 48 h after nematode treatment, the LC50 value of formulation 13 was 31.45 μg/mL, while the LC50 value of Song-liang (positive control) was 29.31 μg/mL, indicating that the nematicidal activity of formulation 13 was close to that of the positive control (Table 2). In addition, microscopy observation revealed a large number of vacuoles in dead nematodes in both the formulation 13 and positive control treatments compared to the solvent control and sterile water treatments (Figure 2). These results suggest that formulation 13 may exhibit promise in applications for the control of PWD.

3.4. Distribution of Formulation 13 in P. thunbergii

To determine whether formulation 13 was transportable in pine, we measured the emamectin benzoate residues in P. thunbergii at 30 and 90 days post-injection. For the transport distribution trial, we collected samples from 30 cm and 60 cm above the injection site and in the apical branches, with one samples from each height in a single tree. As determined from HPLC analysis, the emamectin benzoate residues had a spatially heterogeneous distribution in the three vertically collected samples of P. thunbergii at both 30 and 90 days post-injection (Figure 3). The emamectin benzoate residues in the trunk 30 cm and 60 cm above the injection site and in the apical branches increased significantly at 90 days post-injection compared with samples taken at 30 days post-injection (Figure 3).
In addition, emamectin benzoate was not detected in the apical branches of P. thunbergii at 90 days post-injection of Song-liang (positive control), and the emamectin benzoate residue concentrations in the trunk 30 cm and 60 cm above the injection site were significantly higher at 30 days post-injection than at 90 days post-injection (Figure 3). The rate of upwards transport of formulation 13 in P. thunbergii was lower than that of the positive control at 30 days post-injection. This further indicates that formulation 13 had good transport and stability within the pine trees.

3.5. Formulation 13 Has a Preventive Effect against PWD

We further explored whether formulation 13 could delay the occurrence of PWD by the inoculation of nematodes in P. thunbergii at 100 days post-injection. For the infection assay, each 5-year-old P. thunbergii was inoculated with B. xylophilus, and P. thunbergii individuals were treated with formulation 13, Song-liang, the solvent control, and sterile water. We recorded the symptoms of pine trees inoculated with nematodes at different stages of infection. The results showed that P. thunbergii injected with either formulation 13 or Song-liang remained healthy, while P. thunbergii injected with either the solvent control or sterile water showed signs of infection at 70 days post-inoculation (dpi). Until 110 dpi, all P. thunbergii injected with either the solvent control or sterile water displayed symptoms for the whole pine, but P. thunbergii injected with either formulation 13 or Song-liang had no disease (Figure 4a). Observation continued for 450 dpi. P. thunbergii injected with either formulation 13 or Song-liang remained healthy (Figure 4a). At 70 dpi, the number of nematodes in P. thunbergii injected with sterile water, the solvent control, Song-liang and formulation 13 were 512, 486, 0.22, and 0.15 times higher than the number of nematodes in the original inoculation, respectively (Figure 4b). In addition, the infection rate of P. thunbergii injected with sterile water or the solvent control reached 100% at 110 dpi, and the susceptibility index was more than 68 (Table 3). These results showed that formulation 13 could effectively resist infestation by PWN.

3.6. Residues of Formulation 13 in P. thunbergii

To determine the persistence of formulation 13 in the pine trees, we detected emamectin benzoate residues in the trunk 30 cm and 60 cm above the injection site and in the apical branches in P. thunbergii at 540 days post-injection. P. thunbergii injected with Song-liang was used as a positive control. Emamectin benzoate was detected in the three parts of P. thunbergii, with residual concentrations of 6.10 μg/g, 5.55 μg/g, and 7.15 μg/g in the vertically upwards, respectively. In addition, the residue concentrations at the corresponding sampling sites were significantly higher than those of P. thunbergii injected with Song-liang (4.25 μg/g, 3.59 μg/g, and 4.65 μg/g, respectively) (Figure 5). This indicates that formulation 13 not only has a residual persistence period of at least 18 months but also has good stability.

4. Discussion

PWN can move and propagate in the resin canal of pine trees, and thus, trunk injection of nematicides has become an effective way to control the associated disease [10,37]. However, the issues of high-toxicity, high-cost, and the lack of systematic analysis of the transport distribution and residual kinetics of agents in trees remain to be addressed [13,38]. Emamectin benzoate, a new antibiotic biogenic insecticide, has the advantages of broad-spectrum, high-efficiency, low-toxicity, and low-residue amounts [39,40]. Therefore, based on the characteristics of emamectin benzoate, we developed a high-efficiency and environmentally friendly water-based microemulsion and determined its transport distribution, residual duration, and effectiveness of control of PWD.
Microemulsions are isotropic and thermodynamically stable dispersion systems formed spontaneously by oil, water, surfactants, and co-surfactants [16,17]. The content of each component plays a key role in the formation of microemulsions. Combining multiple solvents compounding can reduce the total amount of organic solvents used in microemulsions [41]. Surfactants and co-surfactants can regulate the hydrophilic–lipophilic balance (HLB) value of microemulsions and improve their flowability, thus making the oil-water interface layer more stable [42,43,44]. In addition, studies have shown that an excellent microemulsion should have a wide transparent temperature range (−5–60 °C) to ensure stability under any conditions [45]. Emamectin benzoate is not easily decomposed in acidic and neutral environments, which indicates that pH plays a key role in the decomposition rate of active ingredients in microemulsions [46,47]. In this study, the most stable formulation was screened based on the basic requirements for microemulsion formulations and assessment of the quality index of pesticide-related compounds. The present findings enrich the variety of agents available for the control of PWD.
Determination of the nematicidal activity of a preparation is the first task before trunk injection can be performed to prevent PWD. To clarify whether formulation 13 had nematicidal activity, we treated nematodes with formulation 13 for 48 h and found that the LC50 value of formulation 13 was 31.45 μg/mL. Jia showed that the LC50 value of emamectin benzoate against B. xylophilus was 0.23 mg/L when the nematodes were treated for 24 h [14]. The LC50 values of formulation 13 against B. xylophilus was higher than those in previous studies, and it was speculated that this might be related to the formulation of the agent, the activity of the nematode itself, and the criteria used to judge nematode mortality.
Second, trunk injections must have good transport ability. In the present study, formulation 13 injected into the trunk exhibited a non-uniform distribution in pine trees. In addition, formulation 13 was transported to all the tissues of P. thunbergii, including the apical branches, within 90 days. Meanwhile, the amount of emamectin benzoate decreased as the distance travelled increased, but the amount of antinematode was retained, which is consistent with the study of Takai et al. [30]. In addition, as the time after trunk injection increased, the emamectin benzoate concentrations in the three vertically collected samples gradually increased in P. thunbergii injected with formulation 13, while the emamectin benzoate concentrations in P. thunbergii injected with Song-liang gradually decreased at 30 cm and 60 cm above the injection point. It was speculated that the fast transport of Song-liang in pine trees and the poor stability of the formulation led to the lack of detection of emamectin benzoate in the apical branches of pine trees at 90 days post-injection. This result provides a reference for the selection of injection time for the prevention of PWD in forests in the future.
A good trunk injection agent should be effective in controlling PWD and be inexpensive to purchase. Residues of a novel emamectin benzoate liquid formulation injected at a dose of 10 g emamectin benzoate m-3 in P. thunbergii tissue still exceeded the IC95 value (0.031 μg/g) after 27 months, and the preventive effect lasted for 3 years [31]. P. thunbergii trees were injected with emamectin benzoate 9.7% soluble liquid at 0.3 mL/cm diameter at breast height (DBH); none died within 2 years of inoculation with B. xylophilus, and the mean residue concentration in the twigs was 0.303 μg/g [48]. However, we found that the nematode population was significantly suppressed in P. thunbergii injected with formulation 13 at 70 dpi, and none of the P. thunbergii showed symptoms of disease for 450 days. In addition, the emamectin benzoate concentrations at the three sampling points above the injection point were 6.10 μg/g, 5.55 μg/g, and 7.15 μg/g at 540 days post-injection of formulation 13. It is difficult to compare the duration of prevention of PWD between studies because of the different injection doses of emamectin benzoate. However, the residue value in this study was greater than the IC95 value (0.031 μg/g) for emamectin benzoate against B. xylophilus reported by Takai et al. [31]. Thus, formulation 13 can be considered an effective control measure for PWD, but further studies are needed to determine whether this formulation can consistently control the occurrence of PWD.
To improve the stability of emamectin benzoate microemulsions, some stabilizers (e.g., butyl hydroxyanisole and tert-butyl hydroquinone) and large amount of organic solvents were added to the formulation, and the addition of these auxiliary agents led to a higher cost of microemulsion preparation [13,49]. Formulation 13 contains only a few low-toxicity organic solvents and low-cost surfactants, and its preparation cost is approximately 50% lower than that of some commercially available formulations. This formulation may prove useful in the selection of agents for the prevention of PWD in the future.

5. Conclusions

This study showed that formulation 13 has nematicidal activity and that it could be effectively transported in P. thunbergii tissues with a long residual period after injection into the trunk. The effectiveness of formulation 13 for PWD was further evaluated, and the results provide theoretical and practical support for PWD control. In conclusion, formulation 13 has the potential to control PWN in pine trees.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14071476/s1, Table S1. 14 formulations of 2% emamectin benzoate microemulsions.

Author Contributions

W.Z., X.W. and J.Y. conceived and designed the research and finalized the manuscript. W.Z. performed the experiments, performed the data analysis, and wrote the draft. C.L., X.S., L.W., L.R. and Y.Z. assisted in the development of the experiment. X.W., J.Y. and L.H. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by grants from the National Key Research and Development Program of China (2021YFD1400903) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Forests 14 01476 g001 550
Figure 1. Component percentages of 14 formulations of 2% benzoate microemulsions. All numbers indicate the percentage contents in the formulation.
Figure 1. Component percentages of 14 formulations of 2% benzoate microemulsions. All numbers indicate the percentage contents in the formulation.
Forests 14 01476 g001
Forests 14 01476 g002 550
Figure 2. Morphology of Bursaphelenchus xylophilus after 48 h of treatment with formulation 13 and the controls.
Figure 2. Morphology of Bursaphelenchus xylophilus after 48 h of treatment with formulation 13 and the controls.
Forests 14 01476 g002
Forests 14 01476 g003 550
Figure 3. Distribution of residues of formulation 13 within Pinus thunbergii. The labels “30 cm” and “60 cm” indicate samples taken from the trunk at 30 cm and 60 cm above the injection site. All values indicate the emamectin benzoate concentrations at different sampling points. Values represent the mean ± SD of three independent biological samples. Different letters over error bars indicate statistically significant differences in pairwise comparisons of the same sampling times between formulation 13 and Song-liang using Duncan’s multiple range test (p < 0.05).
Figure 3. Distribution of residues of formulation 13 within Pinus thunbergii. The labels “30 cm” and “60 cm” indicate samples taken from the trunk at 30 cm and 60 cm above the injection site. All values indicate the emamectin benzoate concentrations at different sampling points. Values represent the mean ± SD of three independent biological samples. Different letters over error bars indicate statistically significant differences in pairwise comparisons of the same sampling times between formulation 13 and Song-liang using Duncan’s multiple range test (p < 0.05).
Forests 14 01476 g003
Forests 14 01476 g004 550
Figure 4. Test on the ability of P. thunbergii to resist B. xylophilus infection after injection with formulation 13. (a) The effect of P. thunbergii against B. xylophilus after injection. (b) The number of nematodes in P. thunbergii at 70 days post-infection. Values represent the mean ± SD of three independent biological samples. Different letters over error bars indicate statistically significant differences determined using Duncan’s multiple range test (p < 0.05).
Figure 4. Test on the ability of P. thunbergii to resist B. xylophilus infection after injection with formulation 13. (a) The effect of P. thunbergii against B. xylophilus after injection. (b) The number of nematodes in P. thunbergii at 70 days post-infection. Values represent the mean ± SD of three independent biological samples. Different letters over error bars indicate statistically significant differences determined using Duncan’s multiple range test (p < 0.05).
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Forests 14 01476 g005 550
Figure 5. Emamectin benzoate residual concentrations in P. thunbergii at 540 days post-injection. Values represent the mean ± SD of three independent biological samples. Different letters over error bars indicate statistically significant differences in a pairwise comparison between formulation 13 and Song-liang using Duncan’s multiple range test (p < 0.05).
Figure 5. Emamectin benzoate residual concentrations in P. thunbergii at 540 days post-injection. Values represent the mean ± SD of three independent biological samples. Different letters over error bars indicate statistically significant differences in a pairwise comparison between formulation 13 and Song-liang using Duncan’s multiple range test (p < 0.05).
Forests 14 01476 g005
Table
Table 1. Determination of the quality and stability of different formulations of 2% emamectin benzoate microemulsions.
Table 1. Determination of the quality and stability of different formulations of 2% emamectin benzoate microemulsions.
FormulationEmulsion StabilityTransparency Temperature Range (t1–t2)/°CpH ± SDDecomposition Rate (%) ± SD
1colourless, transparent−17~615.69 ± 0.11687.91 ± 1.4687
2colourless, transparent−20~63//
3colourless, transparent−18~67//
4transparent, milky white///
5transparent, light blue///
6transparent, milky white///
7colourless, transparent−10~65//
8colourless, transparent−17~65//
9colourless, transparent−11~70//
10transparent, milky white///
11transparent, milky white///
12transparent, light blue///
13colourless, transparent−16~776.27 ± 0.08023.75 ± 1.4036
14colourless, transparent−10~69//
Note: “/” represents no test. Values represent the mean ± SD of three independent biological samples.
Table
Table 2. Toxicity of formulation 13 against Bursaphelenchus xylophilus.
Table 2. Toxicity of formulation 13 against Bursaphelenchus xylophilus.
TreatmentRegressionLC50
(μg/mL)		95% Confidence Limit
(μg/mL)		R2
Formulation 13Y = 0.871 x + 3.69631.4523.7616–41.63170.975
Song-liangY = 0.910 x + 3.66529.3122.7528–37.74750.978
Table
Table 3. Susceptibility of P. thunbergii inoculated with B. xylophilus after injection with formulation 13.
Table 3. Susceptibility of P. thunbergii inoculated with B. xylophilus after injection with formulation 13.
TreatmentInfection Rate (%)DSI
70 days90 days110 days450 days70 days90 days110 days450 days
Sterile water5010010010018.7556.2587.50100
Solvent control5010010010031.2562.5068.75100
Formulation 1300000000
Song-liang00000000
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Zhang, W.; Wu, X.; Ye, J.; Li, C.; Hu, L.; Rui, L.; Zhang, Y.; Shi, X.; Wang, L. Toxicity of an Emamectin Benzoate Microemulsion against Bursaphelenchus xylophilus and Its Effect on the Prevention of Pine Wilt Disease. Forests 2023, 14, 1476. https://doi.org/10.3390/f14071476

AMA Style

Zhang W, Wu X, Ye J, Li C, Hu L, Rui L, Zhang Y, Shi X, Wang L. Toxicity of an Emamectin Benzoate Microemulsion against Bursaphelenchus xylophilus and Its Effect on the Prevention of Pine Wilt Disease. Forests. 2023; 14(7):1476. https://doi.org/10.3390/f14071476

Chicago/Turabian Style

Zhang, Wanjun, Xiaoqin Wu, Jianren Ye, Changqiang Li, Longjiao Hu, Lin Rui, Yan Zhang, Xiufeng Shi, and Lei Wang. 2023. "Toxicity of an Emamectin Benzoate Microemulsion against Bursaphelenchus xylophilus and Its Effect on the Prevention of Pine Wilt Disease" Forests 14, no. 7: 1476. https://doi.org/10.3390/f14071476

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