Sung-Chan Lee·Hyo-Rim Lee,2·Dong-Soo Kim·Jun-Hyeong Kwon·Min-Jung Huh·Il-Kwon Park,2

Abstract In this study,we investigated the preventive effects of emamectin benzoate 9.7% SL,which was newly developed to reduce the injection volume and number of injection holes required to protect against pine wood nematode.None of the Pinus thunbergii trees injected with emamectin benzoate 9.7% SL at 0.3 mL/cm diameter at breast height(DBH)died within 2 years of inoculation with pine wood nematodes.Emamectin benzoate 9.7% SL injected at 0.6 mL/cm DBH resulted in no tree mortality for 3 years.Mean residue of emamectin benzoate 9.7% SL in pine twigs injected with 0.3 mL/cm DBH was 0.490 μg/g at 1 year after injection and 0.303 μg/g after 2 years.These residues values are greater than 0.031 μg/g,previously determined IC95 value for emamectin benzoate against the pine wood nematode.Our field experiment and residue analysis showed that emamectin benzoate 9.7% SL could be a substitute agent for emamectin benzoate 2.15% EC,which is widely used to prevent pine wood nematode in the field and that injection volume and number of injection holes can be greatly educed using this new formulation,which will reduce injury to the cambium,interruption of water movement,and infection of inoculation wounds by wood-decay or blue stain fungi.

Keywords Emamectin benzoate 9.7% SL·Emamectin benzoate 2.15% EC·Trunk injection·Pine wood nematode·Residue

Introduction

Pine wilt disease caused by pine wood nematode,Bursaphelenchus xylophilus, is the most serious threat to coniferous forests in Korea(Shin 2008).Since pine wilt disease was first reported in 1988 at Mt.Geumjeong,Busan,Republic of Korea(Choi and Moon 1989),it has spread rapidly to several areas on the Korean Peninsula.Because major coniferous tree species such as Pinus densiflora,Pinus thunbergii,and Pinus koraiensis,are highly susceptible to pine wood nematode,pine wilt disease has caused extensive ecological and economic damage to forest ecosystems in Korea(Lee et al.2017).

The Korea Forest Service and the National Institute of Forest Science have developed several management methods to prevent the spread of pine wilt disease(Shin 2008), including small-scale clear-cutting of seriously infected areas(Kwon et al.2011),cutting and fumigation of infected trees with metam sodium 25% SC to kill the larvae of insect vectors and pine wood nematodes,cutting and fragmentation of dead trees,aerial sprays of thiacloprid 10% SC to manage adult insect vectors,and trunk injection of nematicides such as abamectin 1.8% EC and emamectin benzoate 2.15% EC for preventing pine wilt disease(Shin 2008).

Although trunk injection of nematicides is considered an effective method of preventing the spread of pine wood nematodes,it caused physical damage to pine trees due to inoculation wounds.Injection holes could make pine trees vulnerable to windbreak(Lee et al.2009a).Other side effects of trunk injection include injury to the cambium,interruption of water movement,and wond infection by wood-decay or blue stain fungi.Injection bottles containing nematicides have been developed to reduce the number of injection holes required for treatment. However, this method has limited applicability in the field due to high cost,as well as the difficulty of recovering these injection bottles.In this study,residue analysis and the preventive effect of emamectin benzoate 9.7% SL against pine wood nematode were evaluated for field application since this new formulation was designed to reduce injection volume.

Materials and methods

Chemicals

A new formulation of emamectin benzoate 9.7% (w/w)SL,emamectin benzoate 2.15% EC,and a standard compound of emamectin benzoate(mixture of 91.20% B1a and 3.08% B1b)were supplied by Syngenta Korea(Seoul,Republic of Korea). Acetonitrile (purity ≥99.8% ), dichloromethane(purity ≥99.8% ),water,and n-hexane(purity 95% )were purchased from Avantor Performance Materials(Allentown,PA,USA).Formic acid(purity ≥95% )was purchased from Sigma-Aldrich(St.Louis,MO,USA).Na2SO4(purity ≥99% )and NaCl(purity ≥99.5% )were obtained from Junsei Chemical(Tokyo,Japan).

Rearing of pine wood nematode

Dr.Hye-Rim Han,Gene Bank of Pine Wood Nematode,National Institute of Forest Sciences,Seoul,Republic of Korea,supplied the pine wood nematode strain used in this study.We reared pine wood nematodes on a lawn of Botrytis cinerea cultured on potato dextrose agar(PDA)in the dark at 28°C for 2 months.We extracted pine wood nematodes by using the Baermann funnel method(Chawla and Prasad 1975)1 day before we inoculated pine trees.

Field experiment

The preventive effect of emamectin benzoate 9.7% SL against pine wood nematode was tested in a P.thunbergii forest stand (N35°12′54′′,E128°10′15.8′′) in the Wora Experimental Forest on January 16,2014 in Jinju City,Gyeongsangnam-do,Republic of Korea.We drilled injection holes in the trunk of pine trees 1 m above the ground using a drill bit(? 10 mm,100 mm long).We injected emamectin benzoate 9.7% SL at 0.3 mL/cm DBH(diameter at breast height)or 0.6 mL/cm DBH into different individual trees.P.thunbergii trees injected with emamectin benzoate 2.15% EC at 1 mL/cm DBH were used as a positive control. Untreated P. densiflora trees were observed as a negative control.Ten pine trees were used for each treatment. Table 1 shows the minimum and maximum cm DBH of P.thunbergii trees,injection volume,and number of injection holes.On January 16,2014,emamectin benzoate 9.7% SL was injected at 1.2 mL/cm DBH into three healthy pine trees(16-17 cm DBH)to evaluate the phytotoxicity of the new formulation to pine trees.Phytotoxicity was determined by observing discoloration of the leaves and injury to the trunk near the injection hole on May 27,2014,January 27,2015,January 12,2016,and January 15,2017.To determine the preventive effect of emamectin benzoate,we inoculated treated and untreated P.thunbergii trees with pine wood nematode on May 27,2014,May 15,2015,and June 10,2016.We drilled two holes with a drill bit(? 6 mm,170 mm long)on opposite sides of the trunks,1 m above the ground,and inoculated each tree with about 20,000 pine wood nematodes.The cumulative mortality of treated and untreated pine trees was assessed on January 27,2015,January 12,2016,and January 15,2017.We collected wood chips from dead pine trees,extracted pine wood nematodes with the Baermann funnel method,and identified pine wood nematodes using a microscope.

Calibration curve

For the calibration curve for the residue analysis,we made a stock solution of authentic emamectin benzoate(mixture of 91.20% B1a and 23.08% B1b)at 1000 mg/L acetonitrile,and diluted the stock to 0.01,0.05,0.1,0.25,0.5,1.0,2.5,and 5.0 mg/L with acetonitrile.We created the calibration curve based on the peak areas in the chromatograms obtained by injecting 2 μL standard solutions into the Agilent 6460 Triple Quad LC/MS/MS (Santa Clara,CA,USA).The column was Capcellpak C18MG column (150 mm×2.0 mm i.d., particle size=5 μm;Shiseido Co,Tokyo,Japan)using acetonitrile-water-formic acid(50:50:0.1,v/v/v)at a flow rate of 0.3 mL/min.The column oven temperature was 30°C.The ion source of mass spectrometry was ESI(electrospray ionization),and the flow rate of the drying gas was 10 mL/min.The nebulization pressure was 45 psi,and the capillary voltage was 4000 V.The retention times of emamectin benzoate B1a and B1b were 4.5 min and 3.4 min,respectively.

Table 1 Cumulative mortality of Pinus thunbergii at 1,2,and 3 years after injection with emamectin benzoate

Analysis of emamectin benzoate residue

To analyze the residue of emamectin benzoate,we collected twigs five times between 2014 and 2017 from P.thunbergii that were injected with emamectin benzoate.Pine twigs from both sides of the trunk of four trees per treatment at a height of 8-10 m were cut using a tree pruner.Twigs of untreated pine trees were used as the control.Collection dates were June 17,2014,January 27,2015,June 20,2015,January 12,2016,and January 15,2017.Once collected,the P.thunbergii twigs were transferred to a laboratory.Pine twigs(10 g)were powdered with a tissue homogenizer, extracted with acetonitrile(100 mL),and filtered.The filtrate was concentrated at 40°C with a rotary evaporator.The concentrate was added to NaCl(50 g)and distilled water(100 mL)and partitioned twice with dichloromethane(100 mL).Water was removed by drying the dichloromethane fraction over sodium sulfate and concentrated at 40°C with the rotary vacuum evaporator. The concentrate was dissolved in dichloromethane(10 mL),and 1 mL of solution was loaded on an SPE cartridge(NH2,Agilent)and eluted with dichloromethane-acetone(80:20,v/v).The effluent was concentrated with nitrogen gas.The final extract was dissolved with 1 mL of acetonitrile,and 2 μL of solution was injected into the LC/MS/MS.The target peak area was measured,and the concentration of emamectin benzoate was calculated based on the calibration curve.

Results and discussion

Preventive effect of emamectin benzoate 9.7% SL against pine wood nematode

The preventive effects of emamectin benzoate 9.7% SL injected at 0.3 mL/cm DBH and 0.6 mL/cm DBH are shown in Table 1.No mortality was observed for 2 years,but one pine tree injected with 0.3 mL/cm DBH was dead 3 years after injection.This result indicated that injection of emamectin benzoate 9.7% SL at the volume of 0.3 mL/cm DBH could guarantee protection from pine wood nematode for 2 years.Emamectin benzoate 2.15% EC,a formulation widely used in the field to prevent pine wood nematode, showed similar results. No mortality was observed for 3 years in pine trees treated at 0.6 mL/cm DBH.No phytotoxicity of emamectin benzoate 9.7% SL was observed in pine trees injected at 1.2 mL/cm DBH.The preventive effect of emamection benzoate against pine wood nematode has been reported in previous studies(Takai et al.2000,2003a;Lee et al.2009a).Due to the strong anti-nematodal activity of emamection benzoate in a laboratory study(Takai et al.2000),Takai et al.(2003a)further evaluated the preventive effect of a liquid formulation of emamectin benzoate against pine wilt disease.They reported that injection of Short Wan Liquid Formulation(SWAF)into pine trees at 10 g/m3emamectin benzoate had a preventive effect against pine wilt disease for 3 years.Lee et al.(2009a)reported that emamectin benzoate 2.15% EC at 1 mL/cm DBH,had a preventive effect of ＞90% for 2 years against pine wood nematode.Our study showed that the new formulation of emamection benzoate 9.7% SL could be applied in the field to prevent pine wilt disease for 2 or 3 years depending on the injection volume.

Emamectin benzoate residue in pine trees

Figure 1 shows the mean residues of emamectin benzoate 9.7% SL and 2.15% EC in pine twigs collected at various times after injection.For emamectin benzoate 9.7% SL injected at 0.6 mL/cm DBH,residue was highest at 1 year after injection,followed by 0.5 years,1.5 years,2 years,and 3 years after injection.The residue of emamectin benzoate 9.7% SL injected at 0.6 mL/cm DBH was 1.078 μg/g after 1 year and 0.093 μg/g after 3 years.The residues of emamectin benzoate 9.7% SL injected at 0.3 mL/cm DBH showed a similar pattern to those of emamectin benzoate 9.7% SL injected at 0.6 mL/cm DBH.The highest residue of emamectin benzoate 9.7% SL injected at 0.3 mL/cm DBH was 0.490 μg/g,1 year after injection,and the lowest residue was 0.048 μg/g after 3 years.For comparison with emamectin benzoate 9.7% SL,the residue of emamectin benzoate 2.15% EC was 0.555 μg/g 6 months after injection,and 0.040 μg/g after 3 years.Residues of emamectin benzoate 2.15% EC were higher 0.5 years,1.5 years,and 2 years after injection than those of emamectin benzoate 9.7% SL injected at 0.3 mL/cm DBH.However,residues of emamectin benzoate 2.15% EC were lower at 1 year and 3 years after injection than those of emamectin benzoate 9.7% SL at 0.3 mL/cm DBH.Residue of emamectin benzoate decreased dramatically 3 years after all treatments. This result indicates that emamection benzoate might be more rapidly degraded after a certain period of time by biological or environmental factors.Determining the concentration of the nematicides necessary to inhibit pine wood nematode propagation in current year shoots is very important for field applications.Takai et al.(2003b)reported that the IC95value of emamectin benzoate against pine wood nematode was 0.031 μg/g,which also can prevent reproduction.Takai et al.(2003b)injected a liquid formulation of emamectin benzoate at 10 g/m3,and 27 months later,the residue in pine twigs was still greater than 0.031 μg/g.Although it is difficult to compare injection doses of emamectin benzoate among studies,here we found that the mean residue of emamectin benzoate 9.7% SL and 2.15% EC was greater than 0.031 μg/g 3 years after injection. However, the residue in two of four sampled pine trees injected with emamectin benzoate 2.15% EC at 1.0 mL/cm DBH and in one of four sampled pine trees injected with emamectin benzoate 9.7% SL at 0.3 mL/cm DBH,were less than 0.031 μg/g 3 years after injection.This result was closely related to the mortality of pine trees that were inoculated with pine wood nematodes(Table 1).One of 10 pine trees injected with emamectin benzoate 9.7% SL at 0.3 mL/cm DBH and emamectin benzoate 2.15% EC at 1.0 mL/cm DBH was dead 3 years after injection.In the report of Lee et al.(2009b)on the persistence and distribution of abamectin 1.8% EC injected into trunks at 1.0 mL/cm DBH in Pinus thunbergii,the mean residue in branches on the middle and upper parts of the tree crown was,respectively,0.173 μg/g and ＜0.05 μg/g 180 days after injection.The mean residue in the middle and upper parts of the trunk was,respectively,＜0.05 and ＜0.05 μg/g 180 days after injection.However,it is difficult to compare the study by Lee et al.(2009b)to our study,due to their short study period(180 days)and high detection limit(0.05 μg/g).

Fig.1 Residue of emamectin benzoate 9.7% SL and 2.15% EC in Pinus thunbergii twigs collected at various times after injection

Based on our field experiment and residue analysis,we have suggested various injection volumes of emamectin benzoate 9.7% SL for different DBH measurements,which will be useful for field application(Table 2).Injection volumes of emamectin benzoate 2.15% EC applied in the field were described for comparison.To guarantee the preventive effect of emamectin benzoate 9.7% SL for 2 years,we recommend two injection holes and an injection volume of 8 mL for pine trees with DBH of 14-24 cm.In contrast,more than twice as many injection holes are necessary to guarantee a preventive effect for emamectin benzoate 2.15% EC.

Table 2 Suggested injection volume of emamectin benzoate 9.7% SL for various ranges of DBH(cm)

Conclusions

In this study,we showed that the new formulation of emamectin benzoate 9.7% SL can be substituted for emamectin benzoate 2.15% EC,which is widely used to prevent pine wood nematode in the field.The lower injection volume and fewer injection holes should decrease the susceptibility of trunk-injected pine trees to windbreak,injury of the cambium,interruption of water movement,and infection from wood-decay or blue stain fungi.

AcknowledgementsThis work was supported by a grant from Syngenta Korea to IK Park,entitled‘‘Control and prevention effect of emamectin benzoate 9.7% SL against sycamore lace bug(Corythucha ciliata)and pine wood nematode(Bursaphelenchus xylophilus)’’.