General instruments. The 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were obtained on a Bruker Biospin Avance 400 spectrometer with tetramethylsilane (TMS) as the internal standard. Electrospray ionization–mass spectrometry (ESI–MS) was performed on an Agilent 6430 LC/MS/MS and a 1100 LC/MS spectrometer. The high-performance liquid chromatography (HPLC) system comprised a Hitachi Model L-2130 pump, L-2400 UV detector, and YMC J’sphere ODS H-80 column (4 mm, 20 × 150 mm, YMC Co., Ltd).
Chemicals. Highly pure, chemical-grade n-Hexane, chloroform (CHCl3), ethyl acetate (EtOAc), n-buthanol (n-BuOH), and acetone were purchased from Samchun Pure Chemical Co., Ltd., Korea. Dimethyl sulfoxide (DMSO, 99.9%, spectrophotometric grade), trifluoroacetic acid (TFA, for HPLC, ≥ 99.0%), proteinase K, abamectin, propylene glycol monomethyl ether (PGME), propylene glycol (PG),thiobarbituric acid, trichloroacetic acid, and Na2CO3 were obtained from Sigma–Aldrich Chemie GmbH, Steinheim, Germany. MeOH and water for HPLC analysis were purchased from Duksan Pure Chemicals Co., Ltd., Korea. Folin-Ciocalteu’s phenol reagent was obtained from Merck KGaA, Darmstadt, Germany. Tris buffer and gallic acid were purchased from Bioneer Corp and MP Biomedicals, Korea, respectively.
Isolation and molecular identification of Strain AE170020. Strain AE170020 was isolated from root tissues of pine tree samples collected from Jinju (J, 35º12.319ʹ N, 128º10.49ʹ E, altitude, 91 m), Korea. Pine tree samples were obtained with permission from the National Institute of Forest Science. We complied with relevant institutional, national and international guidelines and laws in the study of the use of pine samples.
Plant samples were washed with an ultrasonic step (160 W, 30 min) to remove the surface soils and epiphytes. The washed plants were put into sterilized Petri dishes with aseptic filter paper to remove the surface water. After drying, plants were subjected to a seven-step surface sterilization procedure: immersion in 0.1% Tween 20 for 1 min, followed by a 6-min wash in 5% sodium hypochlorite, a 10-min wash in 2.5% sterilized sodium thiosulfate, three washes in sterile water, a 4-min wash in 70% ethanol, five washes in sterile distilled water, and a final rinse in 5% sodium bicarbonate for 10 min. Samples were then put into 2 ml sterile tubes and grinded using liquid nitrogen and then put onto TWYE media (0.25g Yeast extract, 0.5g K2HPO4, agar, 1L DW). After six weeks of inoculation, the isolate was selected and purified. Genomic DNA was extracted, and the 16S rRNA gene was amplified using polymerase reaction. The resulting 16S rRNA gene sequence was compared with the curated sequences from EzBioCloud (http://www.ezbiocloud.net/) and a phylogenetic tree was built with the neighbor-joining method.
Acquisition of pine wood nematode (PWD). B. xylophilus were supplied by the National Institute of Forest Science, Seoul, Korea. B. xylophilus were reared on a lawn of Botrytis cinerea cultured on potato dextrose agar (Difco, BD) medium in the dark at 25°C for 7 days; nematodes were then extracted using the Baermann funnel method for bioassay22. To obtain bacteria-free PWNs, nematodes were extracted and transferred to Petri dishes containing sterile distilled water to allow egg laying for 3 h. Most eggs adhered to the bottom surface of the Petri dish and were gently washed with distilled water many times to remove the hatched nematodes. Then, 15% H2O2 was added into the Petri dish, which was rinsed for 60 min at 25°C; next, the Petri dish was washed with sterile water three times. Eggs were then resuspended in sterile distilled water and allowed to hatch. The PWN J2S that were almost synchronized after 24 h at 25°C were obtained and used for mortality assays. The J2S were transferred onto a culture of B. cinerea on a PDA plate, and J3S and J4S/adults were collected at 30 h and 78 h after feeding re-initiation, respectively.
Detecting nematicidal activity of strain Streptomyces sp. AE170020. The strain was inoculated into the GSS broth (20 g glucose; 10 g soluble starch; 25 g soybean meal; 1 g beef extract; 4 g yeast extract; 2 g NaCl; 0.25 g K2HPO4; 2 g CaCO3, and 1 L DW) for 36 h. Two percent of the seed was transferred to a 500 mL flask containing 50 mL of GSS broth and incubated on a rotary shaker at 150 rpm at 28°C for 1–10 days. The culture of strain AE170020 was then centrifuged at 13,000 rpm for 10 min to separate the broth and cells. The resultant supernatant was aseptically transferred to sterile tubes and used for a nematicidal bioassay, as described by Gao et al. (2016), with some modification23. Briefly, 50 µL of the supernatant and bacteria-free L2s of B. xylophilus (n = 30–40) specimens were added to each well of a 96-well tissue culture plate. Later, 100 µg mL− 1 streptomycin was also added to each well to inhibit bacterial contamination, and GSS broth was used as a control.
Detecting the stability of nematicidal activity of Streptomyces sp. AE170020 culture. The stability of strain AE170020 was detected following the method of Gao et al. (2016). The supernatant of strain Streptomyces sp. AE170020 was treated with 20 mg mL− 1 of proteinase K at 37°C for 30 min or boiled at 100°C for 5 min. Thereafter, the nematicidal activity of the supernatant was detected. This supernatant was also adjusted to pH values from 1.0 to 11.0 by HCl or NaOH solution and incubated for 2 h; the nematicidal activity of the supernatant was then detected after the pH value was re-adjusted to 7.0.
Fermentation, extraction, and purification of nematicidal substances from Streptomyces sp. AE170020 culture. Strain cultures were prepared as described above and centrifuged at 6,000 rpm for 30 min. The collected supernatant was then sequentially extracted with an equivalent volume of n-hexane, CHCl3, EtOAc, and n-BuOH. The mycelium of strain AE170020 was extracted with acetone, sonicated for 30 min, and kept overnight. All extractions were performed thrice. Different solvent layers were concentrated in vacuo to obtain the dry extracts. The nematicidal activity of the extracts was determined: 2 µL of the solvent extract of Streptomyces sp. AE170020 was dissolved in DMSO and added to the 96-well plate, containing approximately 40–50 L2s nematodes in 98 µL of sterile water. The final concentrations of solvent extracts were 0.0625, 0.1250, 0.2500, 0.500, and 1.000 mg mL− 1, and 2 µL of DMSO was used as a control. Acetone extracts, which showed the highest activity against PWN, were used for further purification.
The fractions extracted by acetone were applied on a silica gel column and eluted with a stepwise n-hexane/acetone gradient of increasing polarity. Fractions were checked on a TLC plate, and those with similar TLC patterns were pooled for nematicidal activity testing. One of the fractions that showed high nematicidal activity was applied on a Sephadex LH-20 column and eluted with MeOH. Nematicidal activity of subfractions was tested and combined as above. Further, active subfractions were purified with preparative TLC plates, and two active compounds were obtained. HPLC with a gradient solvent system of aqueous MeOH, containing 0.04% TFA, as a mobile phase was used to check the purity of the compound.
Different developmental stages of B. xylophilus mortality assay. Two of the most potent purified compounds, alloaueohtin and aureothin, were further explored for their effects on the different stages, reproduction, growth, and behavior of B. xylophilus. Different developmental stages of nematode were prepared as previously described, and L2s were exposed to the two compounds at final concentrations of 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, and 4.00 µg mL− 1; L3s and L4s/adults were exposed to concentrations of 0.25, 0.50, 1.00, 2.50, 5.00, and 10.00 µg mL− 1. The commercial nematicide, abamectin, at the same concentrations and DMSO were used as positive and solvent controls, respectively.
The treated plates were stored in the dark at 25°C, and the mortality of PWNs was recorded after 24-h treatment by visualization under a light microscope (Nikon, SMZ-U). The LC50 values of active compounds at different life stages were estimated. Mortality was defined based on the observation of motility, a visibly moving nematode was marked as alive, and nematodes that failed to respond after several touches were marked as dead. The bioassays were performed three times in triplicate. Mortality was calculated according to the following formula: mortality (%) = dead juveniles/total juveniles × 100.
Effects of compounds on the locomotion behavior of B. xylophilus. In liquids, a single movement of the stylet knob forwards and then backwards to its original position was called “thrashing”24. To assess the impact of alloaureothin and aureothin on B. xylophilus mortality, thrashing of L3s nematode was assayed. Thrashing rate was scored for 1 min at the following time points: 2, 6, and 24 h in the presence of two compounds at the indicated concentrations (1.0, 2.5 and 5.0 µg mL− 1). Control assays were conducted in the presence of DMSO or abamectin, and all assays were conducted at 22°C. Two trials were performed for this experiment, and six numbers of surviving nematode in each treatment were used to test the thrashing behavior.
Nematode population inhibition assay. A population inhibition assay was conducted according to the method described by Cheng et al. (2017) with some modification. Briefly, adult nematodes (~ 500) were initially treated with alloaureothin and aureothin at concentrations of 0.5 and 1.0µg mL− 1, and incubated for 24 h at 22°C. After 24 h, 10 female and male nematodes were randomly selected and placed on a PDA plate fully covered with Botrytis cinerea and allowed to grow. When the B. cinerea has been completely consumed by nematodes in control plates, nematodes were extracted from plates using distilled water. The recovered nematodes were then serially diluted and numbers of nematodes in 100 µL suspensions were counted. The reproduction rates (Pf/Pi, Pf, final nematode population; Pi, initial nematode population) were calculated. The experiment was conducted twice, with three replicates per treatment.
Nematode hatch inhibition assay. Nematode eggs were obtained as above and used for the embryonic lethality tests. Experiments were conducted using established procedures24. Egg suspensions were exposed to alloaureothin and aureothin at final concentrations of 20, 15, 10, 5, 2, 1, 0.5, and 0.1µg mL− 1. Two microliters of DMSO and abamectin at the same concentration were served as negative and positive, respectively. Plates were incubated at 22°C and nematodes of the L2s stage were scored after 48 h. The hatching rate was calculated according to the following formula: Hatching rate (%, HR) = [juveniles / (eggs + juveniles) × 100]. Each treatment was repeated in three wells, and the experiment was repeated three times.
Egg deposition assay. One-day-old female nematodes in the presence of alloaureothin, aureothin or abamectin (5 ug mL− 1) were used in the egg deposition assay according to an established protocol24. B. xylophilus (about 100 numbers) were treated with compound for 24 h in the 96-well plates. Approximately 10 male and 10 female nematodes were then selected randomly and transferred to Petri dishes and allowed to lay eggs for 24 h. The eggs laid by a single female were then recorded and the nematodes inside the female body were also imaged. The experiments were performed two times with six repetitions.
Suppression of pine wilt disease under pot condition. Injection of active substances into pine trees was performed to evaluate the control efficacy in vivo, as previously described25. The pot experiments were conducted in a greenhouse at Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) and Pinus densiflora were transferred to a greenhouse for at least one month prior to the pot test and watered every other day and fertilized if necessary. The average temperature of the greenhouse was 25 ± 5°C. Acetone extracts of strain AE170020 were prepared as described above and dissolved using DPPT solvent (20% DMSO, 20% propylene glycol monomethyl ether, 50% propylene glycol, and 10% Tween-20). The solution of abamectin in DPPT solvent served as a positive control. Holes were made in the trunk of pine trees (average height, 124 cm; average basal diameter, 1.4 cm) by using an electric drill about 5 cm above the ground level. Then, 200 µL of strain extract was injected into holes at different concentrations, and holes were immediately covered with parafilm. The final concentration of the acetone extract was 7.2 mg, 3.6 mg, and 1.8 mg per tree and abamectin was 3.6 mg per tree. Control plants were injected with the DPPT solution. Inoculations of B. xylophilus were performed one week after the injection of chemicals in the trunk. B. xylophilus were reared on B. cinerea and extracted; they were then washed with distilled water at least five times to remove any fungal hyphae present. Final nematode density in the suspension was adjusted to about 20,000 nematodes per mL. A hole in the trunk of each pine tree was made approximately 20 cm above the soil. A 0.1 mL aliquot of the nematode suspension (containing about 2,000 nematodes: a mixture of adults and juveniles) was pipetted into the holes and the holes were covered with parafilm. Control plants were also wounded but distilled water was used instead of the PWN solution. Pine trees were watered three times a week. Three runs with five replicates for each treatment were conducted. External symptoms of pine trees were visually assessed at 15, 30, 45, and 60 days after the inoculation and results were recorded at 60 days.
Sample collection and processing. Sixty days after inoculation, three pine trees from each treatment in one trial were randomly selected and collected. Plants were cut immediately above the ground. Needles and the twigs were removed, and the stems were chopped into small pieces. Stems were immediately weighed and used for nematode quantification. The remaining stems and leaves were stored at − 80°C for chemical analysis.
Nematode quantification in inoculated trees. The nematode population was measured using established procedures26. Five grams of the stem pieces were weighed and transferred into gauze equipped in the upper part of a 50 mL falcon tube. The plant tissues were then immersed in water for 24 h, and after that the gauze and plants were gently removed from tubes and allowed to precipitate for 12 h. Nematodes were mostly located at the bottom of the falcon tube after the precipitation and the upper water was removed to make a final volume of 10 mL. Then, 100 µL of the extracted nematodes was transferred into 96-well tissue plates, and live nematodes were counted under a light microscope. Nematode density was expressed as the number of nematodes in the stem per gram of wood. The data of each treatment were generated from three trees and obtained in triplicate.
Chemical analysis. Chemical analyses were performed on leaf tissues (total chlorophyll, total polyphenolics, and lipid peroxidation) and stem tissues (water content). Leaves collected as previously described were used for chlorophyll quantification. The extraction and determination of the chlorophyll content were performed as per the method described by26. Briefly, needles were homogenized with liquid nitrogen, and samples (about 0.1 g) were extracted with 10 mL of cold acetone/Tris buffer (80:20 vol:vol, pH = 7.8). The samples were incubated at 4°C for 72 h, and the tubes were centrifuged at 13,000 rpm for 5 min. The absorbances were then recorded at 537, 647, and 663 nm in a UV/visible spectrophotometer (Pharmacia Biotech, Ultraspec 3000). The amount of chlorophyll was calculated as follows: total chlorophyll concentration in the extract (mmol mL− 1) = (0.001373A663 – 0.000897A537 – 0.003046A647) + (0.02405A647 – 0.004305A537 – 0.005507A663).
The concentration of total soluble phenolics was determined using the protocol described by Ainsworth et al (2007)27. Needles were ground with liquid nitrogen; about 100 mg of leaves was extracted with 95% methanol and sonicated for 15 min. Samples were then gently shook and allowed to react overnight in the dark at room temperature. Later, 200 µL of 10% (vol/vol) Folin–Ciocalteu’s phenol reagent was added to 100 µL of the methanolic extract. The solution was mixed thoroughly and allowed to stand for 5 min, after which 800 µL of 700 mM Na2CO3 was added, and the tubes were incubated at ambient room temperature for 2 h. Next, 150 µL of each sample solution was loaded on a 96-well microplate and the absorbance was measured at 760 nm with a spectrophotometric microplate reader (VERSAmax™). The total phenolic concentration was calculated from the gallic acid calibration curve. Data were expressed as gallic acid equivalents (GA)/g of extracts averaged from each treatment.
To gain insights about plant oxidative stress and cell damage, lipid peroxidation was measured. The level of lipid peroxidation in tissues was determined in terms of the malondialdehyde (MDA) content by the method of Nunes et al (2015). After homogenization with liquid nitrogen, about 0.1 g of leaf sample was extracted with 10 mL 0.5% thiobarbituric acid in 20% trichloroacetic acid. The sample was then vortexed thoroughly and incubated at 100°C for 30 min. Next, reaction solutions were immediately transferred to ice for 5 min and then centrifuged at 4,000 rpm for 15 min. The absorbance was monitored at 450, 532, and 600 nm in a UV/visible spectrophotometer. Calculation of MDA was based on the following formula: MDA concentration (µmol L− 1) = 6.45(A532 – A600) – 0.56A450.
Specific water content was used as a proxy for water stress, which is usually associated with PWD. For water content determination, each wood sample was oven dried for 72 h, and the relative water content was evaluated through the following formula: relative water content (%) = (fresh weight – dry weight) / fresh weight × 100. All the chemical data were obtained from six trees and three samples per tree.
Statistical analysis. Data analyses were performed using SPSS 18.0 software (SPSS Inc.), and the LC50 values were determined via a probit analysis. Values are expressed as mean ± standard deviation (SD) unless indicated otherwise. The data were compared using analysis of variance (ANOVA) followed by a post-hoc test, as appropriate. Significant differences were determined according to thresholds of *p < 0.05; **p < 0.01, and ***p < 0.001. All charts and figures generated in this study were constructed using GraphPad Prism version 8.0.2.