Pheromone-Based Mating Disruption of Conogethes punctiferalis (Lepidoptera: Crambidae) in Chestnut Orchards.

Chestnuts (Castanea crenata Siebold and Zucc.) are one of the major agroforestry products in Korea, and Conogethes punctiferalis is a major pest of the chestnut fruit. Pheromone-based mating disruption (MD) has emerged as a promising eco-friendly approach to reduce population levels and ultimately mitigate fruit damage. Field trials were conducted over two years (2022-2023) in two commercial chestnut orchards in Central Korea that were infested with C. punctiferalis. Compared with the control treatment, the MD treatment effectively reduced the number of male C. punctiferalis captured in the MD treatment plots. Male catch inhibition (MCI) rates ranged from 70.5% to 82.7% in 2022 and from 87.8% to 95.1% in 2023. The MD efficacy (%) was calculated based on the total number of chestnut fruits collected and the number of fruits damaged by C. punctiferalis. In 2022, the MD efficacy of the single-dosage treatment (TS, 50 g/ha) was 63.9% in Orchard A and 73.6% in Orchard B. In 2023, the MD efficacies of the double-dosage treatment (TD, 100 g/ha) and the two-application treatment (TT, 50 g/ha in June and August) were 60.2% and 77.9% in Orchard A and 50.9% and 64.8% in Orchard B, respectively. This study confirms the efficacy of pheromone-based MD in reducing the C. punctiferalis numbers in chestnut orchards and damage to chestnut fruits.

Effect of electron beam irradiation on the pine wood nematode, Bursaphelenchus xylophilus.

PURPOSE: Reproduction inhibition of the pine wood nematode (PWN) by electron beam (e-beam) irradiation both in vitro and in vivo was tested to determine if ionizing radiation could control the PWN by reducing survival and preventing reproduction, thus reducing the risk of pine wilt disease (PWD) spread. MATERIALS AND METHODS: E-beam (10 MeV) irradiation treatment at different doses (0-4 kGy) was applied to PWNs in a Petri dish. Treatment of pine wood logs infested with PWNs was performed at 10 kGy. Mortality was determined by comparing the survival rates before and after irradiation treatment. DNA damage by e-beam irradiation (0-10 kGy) in the PWN was determined using the comet assay. RESULTS: E-beam irradiation increased mortality and suppressed reproduction with increasing doses. The lethal dose (LD) values (kGy) were estimated as follows: LD50 = 2.32, LD90 = 5.03, and LD99 = 9.48. E-beam irradiation of pine wood logs significantly suppressed PWN reproduction. Comets of e-beam-irradiated cells showed an increased tail DNA level and moment with an increasing dose. CONCLUSION: This study suggests that e-beam irradiation could be used as an alternative method for the management of pine wood logs infested with PWNs.

Identification and Field Bioassay of Rusicada privata (Lepidoptera: Erebidae) Sex Pheromone.

Rusicada privata (Lepidoptera: Erebidae) is a major pest insect on Hibiscus syriacus L. (Malvaceae: Malvales), which is usually planted in urban landscapes. Insecticidal control of R. privata is not ideal for urban landscaping due to its harmful effects and risk to human health. Therefore, nonchemical and ecofriendly alternatives are needed. To identify the sex pheromone of R. privata, abdominal tip extracts of male and female R. privata were analyzed by gas chromatography-mass spectrometry. The abundance of 7-methylheptadecane (7Me-17Hy) in female R. privata abdominal tip extracts led us to hypothesize that it is the major sex pheromone. The compound was tentatively identified by a mass spectral library and confirmed by matching retention times and mass spectra of the female-produced compound with those of a synthetic standard. The compounds elicited electroantennographic (EAG) activity. In a field trapping experiment, only synthetic lures containing 7Me-17Hy attracted R. privata males. The EAG activity and field trapping results confirmed that 7Me-17Hy is the sex pheromone of female R. privata. The results will aid in developing sex pheromone-based R. privata control techniques, such as mating disruption.

Post-fumigation sub-lethal activities of phosphine and ethyl formate on survivorship, fertility and female sex pheromone production of Callosobruchus chinensis (L.).

Phosphine (PH3) and ethyl formate (EF), the two popular fumigant disinfectants of stored product insect pests, are primarily evaluated for their knock down effects without considering their post-fumigation sub-lethal activities. The sub-lethal activities (adult survivorship, fecundity, sterility and female sex pheromone production) of the fumigants were evaluated on a field-to-storage insect pest adzuki bean beetle, Callosobruchus chinensis (L.). The adults' survivorship and female fecundity, both were dose-dependently affected by sub-lethal PH3 and EF fumigation exposures. Hatchability of the eggs laid by fumigated female adults were also significantly affected. Gas-chromatography mass-spectrometry analysis of solid-phase micro-extraction from virgin fumigated C. cinensis females revealed that the PH3 LC25 (the lethal concentration required to kill the 25% of the population) fumigated female C. chinensis released significantly less amount of the pheromone components. In contrast, EF LC25 exposure did not affect the pheromone release. This study unveils the facts that the EF and PH3 fumigation have detrimental bioactivities against C. chinensis. Notably, this suggests to consider the sub-lethal EF and PH3 fumigation rather than the dose required to instantly kill all the C. chinensis individuals for disinfestation of stored adzuki bean.

Nematicidal Activities of Saccharin and Erythritol Against Pinewood Nematode.

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Bührer), causes pine wilt disease (PWD) resulting in severe environmental damage to pine forest ecosystems worldwide. To develop alternative strategies for managing PWD, the nematicidal activities of two sweeteners, erythritol and saccharin, were investigated. Among these two sweeteners, saccharin induced higher mortality in a dose-dependent manner. The LC50 and LC90 values of saccharin were estimated to be 0.321 M and 0.615 M, respectively. However, erythritol did not exhibit nematicidal activities. The results of our study demonstrated that saccharin is lethal to PWN and shows nematicidal effects in a dose-dependent manner. Although the mechanisms of saccharin toxicity are not yet investigated, saccharin could be used as an effective alternative for the management of PWN.

Fumigant Activity of Ethyl Formate against the Chestnut Weevil, Curculio sikkimensis Heller.

Castanea crenata Siebold & Zucc. (Fagales: Fagaceae), a species of chestnut native to Korea and Japan, is distributed in Korea, Japan, and northeastern China, where chestnuts are a major economic agroforest product. Curculio spp. is among the main known pests of chestnuts around the world. In Korea, only phosphine (PH3) is permitted for the fumigation of C. sikkimensis larva-infested chestnuts. However, it is applied for large-scale fumigation, and its use is restricted. Moreover, it requires a long exposure time and an application device; thus, it cannot be used by small-scale farmers. In this study, the activity of ethyl formate (EF) as a fumigant against Curculio sikkimensis in chestnuts was investigated, and its potential for practical use by farmers was evaluated. The sorption of EF according to the filling ratio (FR) and fumigation time was tested, and the results revealed that 2.5% FR was the most effective. For C. sikkimensis in chestnuts, the mortality rate increased proportionately with the dose of EF. After exposure to 160 g/m3 of EF in a 12 L desiccator, the adult C. sikkimensis showed 100% mortality. According to the time-dose mortality data collected over 12 h of fumigation, the LCT90 and LCT99 values were estimated as 1052.0 and 1952.0 g·h/m3. The results revealed that immersion was not an effective method for controlling C. sikkimensis. According to the LCT values, a dose of 180.0 g/m3 and 12 h of fumigation resulted in 100% mortality on a small scale (2 m3). The results of this experiment indicate that EF could be conveniently used as a fumigant by farmers.

New discovery on the nematode activity of aureothin and alloaureothin isolated from endophytic bacteria Streptomyces sp. AE170020.

Endophytic bacteria, a rich source of bioactive secondary metabolites, are ideal candidates for environmentally benign agents. In this study, an endophytic strain, Streptomyces sp. AE170020, was isolated and selected for the purification of nematicidal substances based on its high nematicidal activity. Two highly active components, aureothin and alloaureothin, were identified, and their chemical structures were determined using spectroscopic analysis. Both compounds suppressed the growth, reproduction, and behavior of Bursaphelenchus xylophilus. In in vivo experiments, the extracts of strain Streptomyces sp. AE170020 effectively suppressed the development of pine wilt disease in 4-year-old plants of Pinus densiflora. The potency of secondary metabolites isolated from endophytic strains suggests applications in controlling Bursaphelenchus xylophilus and opens an avenue for further research on exploring bioactive substances against the pine wood nematode.

Effect of storage conditions on the shelf-life extension of fungus-colonized substrates based on Metarhizium anisopliae using modified atmosphere packaging.

Metarhizium anisopliae is a promising alternative to chemical pesticides against pine wilt disease caused by Bursaphelenchus xylophilus. Herein, we investigated the efficacy of modified atmosphere packaging (MAP) to prolong the shelf-life of the M. anisopliae conidia. The effects of various conditions on its stability were also examined. M. anisopliae-inoculated millet grains were treated in a MAP system with different packaging materials (polypropylene, PP; polyethylene terephthalate, PET; ethylene vinyl alcohol, EVOH), gas compositions (high CO2 atmosphere, ≈ 90%; high O2 atmosphere, > 95%; high N2 atmosphere, > 95%; 30% CO2 + 70% N2; 50% CO2 + 50% N2; 70% CO2 + 30% N2), and storage temperatures (4 and 25 °C). Results revealed EVOH film as the best for the preservation of gases at all concentrations for 28 days. MAP treatment in the high-barrier EVOH film under an atmosphere of 30% CO2 + 70% N2 achieved 80.5% viability of dried conidia (7.4% moisture content), with 44.2-64.9% viability recorded with the other treatments. Cold storage for technical concentrates formulation promoted extension of shelf-life of MAP-treated conidia. These results imply that MAP under optimized conditions could enhance the shelf-life of fungus-based biopesticides in fungus-colonized substrates formulations.

Intraocular lens power calculation using adjusted corneal power in eyes with prior myopic laser vision correction.

PURPOSE: To evaluate the prediction accuracy of the intraocular lens (IOL) power calculation using adjusted corneal power according to the posterior/anterior corneal curvature radii ratio in the Haigis formula (Haigis-E) in patients with a history of prior myopic laser vision correction. METHODS: Seventy eyes from 70 cataract patients who underwent cataract surgery and had a history of myopic laser vision correction were enrolled. The adjusted corneal power obtained with conventional keratometry (K) was calculated using the posterior/anterior corneal curvature radii ratio measured by a single Scheimpflug camera. In eyes longer than 25.0 mm, half of the Wang-Koch (WK) adjustment was applied. The median absolute error (MedAE) and the percentage of eyes that achieved a postoperative refractive prediction error within ± 0.50 diopters (D) based on the Haigis-E method was compared with those in the Shammas, Haigis-L, and Barrett True-K no-history methods. RESULTS: The MedAE predicted using the Haigis-E (0.33 D) was significantly smaller than that obtained using the Shammas (0.44 D), Haigis-L (0.43 D), and Barrett True-K (0.44 D) methods (P < 0.001, P = 0.001, and P = 0.014, respectively). The percentage of eyes within ± 0.50 D of refractive prediction error using the Haigis-E (78.6%) was significantly greater than that produced using the Shammas (57.1%), Haigis-L (58.6%), and Barrett True-K (61.4%) methods (P = 0.025). CONCLUSION: IOL power calculation using the adjusted corneal power according to the posterior/anterior corneal curvature radii ratio and modified WK adjustment in the Haigis formula could improve the refraction prediction accuracy after cataract surgery in eyes with prior myopic laser vision correction.

Diel Rhythmicity of Field Responses to Synthetic Pheromone Lures in the Pine Sawyer Monochamus saltuarius.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes lethal pine wilt disease (PWD) in Asia and Europe and has become a serious threat to global pine forest ecosystems. In Korea, Monochamus saltuarius transmits PWN not only to Pinus densiflora, but also to Pinus koraiensis, which is widely distributed across eastern Asia. The diel rhythmicity of M. saltuarius in response to its aggregation pheromone was studied with the aim of providing reliable data for the prevention of PWD and control of Monochamus spp. Using a spray dispenser controlled with an electronic timer, M. saltuarius pheromone and attractants (PA) were sprayed to determine the diel rhythm of the response to PA. The spraying period was divided into four time periods: 05:00-11:00 (time period A), 11:00-17:00 (time period B), 17:00-23:00 (time period C), and 23:00-05:00 (time period D). The largest number of M. saltuarius was caught in time period B, followed by A, C, and D. It could be concluded that the flight activity of M. saltuarius in response to PA was diurnal. The results of this study improve the understanding of the behavioral biology of M. saltuarius, allowing for the development of pest management strategies to prevent the spread of PWN and control its vector.

Directed evolution of glycosyltransferase for enhanced efficiency of avermectin glucosylation.

Avermectin, produced by Streptomyces avermitilis, is an active compound protective against nematodes, insects, and mites. However, its potential usage is limited by its low aqueous solubility. The uridine diphosphate (UDP)-glycosyltransferase (BLC) from Bacillus licheniformis synthesizes avermectin glycosides with improved water solubility and in vitro antinematodal activity. However, enzymatic glycosylation of avermectin by BLC is limited due to the low conversion rate of this reaction. Thus, improving BLC enzyme activity is necessary for mass production of avermectin glycosides for field application. In this study, the catalytic activity of BLC toward avermectin was enhanced via directed evolution. Three mutants from the BLC mutant library (R57H, V227A, and D252V) had specific glucosylation activity for avermectin 2.0-, 1.8-, and 1.5-fold higher, respectively, than wild-type BLC. Generation of combined mutations via site-directed mutagenesis led to even further enhancement of activity. The triple mutant, R57H/V227A/D252V, had the highest activity, 2.8-fold higher than that of wild-type BLC. The catalytic efficiencies (Kcat/Km) of the best mutant (R57H/V227A/D252V) toward the substrates avermectin and UDP-glucose were improved by 2.71- and 2.29-fold, respectively, compared to those of wild-type BLC. Structural modeling analysis revealed that the free energy of the mutants was - 1.1 to - 7.1 kcal/mol lower than that of wild-type BLC, which was correlated with their improved activity. KEY POINTS: • Directed evolution improved the glucosylation activity of BLC toward avermectin. • Combinatorial site-directed mutagenesis led to further enhanced activity. • The mutants exhibited lower free energy values than wild-type BLC.

Nematicidal Activity of Benzyloxyalkanols against Pine Wood Nematode.

Pine wilt disease (PWD) is caused by the pine wood nematode (PWN; Bursaphelenchus xylophilus) and causes severe environmental damage to global pine forest ecosystems. The current strategies used to control PWN are mainly chemical treatments. However, the continuous use of these reagents could result in the development of pesticide-resistant nematodes. Therefore, the present study was undertaken to find potential alternatives to the currently used PWN control agents abamectin and emamectin. Benzyloxyalkanols (BzOROH; R = C2-C9) were synthesized and the nematicidal activity of the synthetic compounds was investigated. Enzymatic inhibitory assays (acetylcholinesterase (AChE) and glutathione S-transferase (GST)) were performed with BzOC8OH and BzOC9OH to understand their mode of action. The benzyloxyalkanols showed higher nematicidal activity than did benzyl alcohol. Among the tested BzOROHs, BzC8OH and BzC9OH showed the strongest nematicidal activity. The LD50 values of BzC8OH and BzC9OH were 246.1 and 158.0 ppm, respectively. No enzyme inhibitory activity was observed for BzC8OH and BzC9OH. The results suggested that benzyloxyalcohols could be an alternative nematicidal agent.

Response of Pine Rhizosphere Microbiota to Foliar Treatment with Resistance-Inducing Bacteria against Pine Wilt Disease.

In this study, two bacterial strains, IRP7 and IRP8, were selected to induce resistance against pine wilt disease (PWD). Foliar application with these strains to nematode-inoculated pine seedlings significantly reduced PWD severity. The effect of nematode inoculation and bacterial treatment on the rhizosphere bacterial community was investigated. The results indicated that the rhizosphere of nematode-inoculated seedlings contained a lower relative abundance of beneficial microbes such as Paraburkholderia, Bradyrhizobium, Rhizobacter, Lysobacter, and Caballeronia. Bacterial treatment resulted in significant changes in the microbes that were represented in relatively low relative abundance. Treatment with IRP7 resulted in an increase in the relative abundance of Nitrospirillum, Bacillus, and Luteibacter, which might be useful for protection against infection. Treatment with IRP8 resulted in an increase in the relative abundance of obligate bacterial predators of the Bdellovibrio genus that were previously shown to control several bacterial phytopathogens and may have a role in the management of nematode-carried bacteria. The selected bacteria were identified as Pseudomonas koreensis IRP7 and Lysobacter enzymogenes IRP8 and are suggested as a potential treatment for induced resistance against PWD. To our knowledge, this is the first report on the effect of foliar treatment with resistance-inducing bacteria on the rhizosphere microbiota.

Pesticide residue monitoring and risk assessment in the herbal fruits Schisandra chinensis, Lycium chinense, and Cornus officinalis in Korea.

Schisandra chinensis, Lycium chinense, and Cornus officinalis are cultivated in South Korea, China and Japan. Because of their beneficial biological effectiveness, the consumption of these herbs is increasing. The objective of this study was to analyze the pesticide residue levels on these herbal fruits produced in Korea. A short- and long-term risk assessment of the pesticides in herbal fruits was conducted. Pesticides were detected in most samples, and all samples with multiple residues were also noted. The average numbers of pesticides detected in the S. chinensis, L. chinense, and C. officinalis samples were 1.84, 10.28, and 5.87, respectively. The short-term and highest long-term risks, and the cumulative risk (cHI) were 0.633%, 1.14%, and 7.56% (S. chinensis), 1.0 × 10-1%, 1.1 × 10-5%, and 4.0 × 10-5% (L. chinense), and 2.2 × 10-5%, 1.6 × 10-5%, and 8.9 × 10-5% (C. officinalis). No significant health risk for consumers via herbal fruit consumption was detected.

Systemic Acquired Resistance-Mediated Control of Pine Wilt Disease by Foliar Application With Methyl Salicylate.

Pine wilt disease (PWD), caused by the pinewood nematode, is the most destructive disease in pine forest ecosystems worldwide. Extensive research has been done on PWD, but effective disease management is yet to be devised. Generally, plants can resist pathogen attack via a combination of constitutive and inducible defenses. Systemic acquired resistance (SAR) is an inducible defense that occurs by the localized infection of pathogens or treatment with elicitors. To manage PWD by SAR in pine trees, we tested previously known 12 SAR elicitors. Among them, methyl salicylate (MeSA) was found to induce resistance against PWD in Pinus densiflora seedlings. In addition, the foliar applications of the dispersible concentrate-type formulation of MeSA (MeSA 20 DC) and the emulsifiable concentrate-type formulation of MeSA (MeSA 20 EC) resulted in significantly reduced PWD in pine seedlings. In the field test using 10-year-old P. densiflora trees, MeSA 20 DC showed a 60% decrease in the development of PWD. Also, MeSA 20 EC gave the best results when applied at 0.1 mM concentration 2 and 1 weeks before pinewood nematode (PWN) inoculation in pine seedlings. qRT-PCR analysis confirmed that MeSA induced the expression of defense-related genes, indicating that MeSA can inhibit and delay the migration and reproduction of PWN in pine seedlings by modulating gene expression. These results suggest that foliar application of MeSA could reduce PWD incidence by inducing resistance and provide an economically feasible alternative to trunk-injection agents for PWD management.

Nematicidal activity of teleocidin B4 isolated from Streptomyces sp. against pine wood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Pine wilt disease (PWD) is a plant disease that causes serious damage to pine trees. PWD occurs when the host plant is infected with pinewood nematode (PWN), Bursaphelenchus xylophilus. In this study, a compound with nematicidal activity was isolated from actinomycetes and its efficacy was investigated in vitro. RESULT: We screened and selected Streptomyces sp. 680560, which had nematicidal activity against B. xylophilus. Based on 16S rRNA sequence analysis, it showed 99.93% similarity with Streptomyces blastmyceticus NRRB-5480T . Furthermore, the active compound was isolated and identified as teleocidin B4. Teleocidin B4 at concentrations ranging from 6.25 to 100 µM had low nematicidal activity after 24 and 36 h against adult and stage juveniles (J2) of B. xylophilus, but after 48 h nematicidal activity exceeded 95%. The rate of inhibition of egg hatching for Teleocidin B4 6.25, 12.5, 25, 50, and 100 µM was confirmed to be dose-dependently inhibited after 48 h of treatment. Teleocidin B4 is not only toxic to hatched B. xylophilus, but also affects egg hatching. CONCLUSION: This study was carried out to isolate actinomycete metabolites from pine tree endophytes from various natural environments for control of PWD. A compound with nematicidal activity was isolated from a selected strain and its structure was identified as teleocidin B4. The nematicidal effect of the isolated active substance, teleocidin B4, was confirmed. This is the first report of the effect of teleocidin B4 on B. xylophilus, suggesting its possibility as a PWD control agent. © 2020 Society of Chemical Industry.

Multizonal Design Multifocal Intraocular Lens-Induced Astigmatism According to Orientation.

PURPOSE: To evaluate the differences in intraocular lens (IOL)-induced astigmatism according to differences in orientation of a multizonal multifocal IOL, the Precizon Presbyopic NVA IOL (Ophtec BV). METHODS: The clinical study reviewed 80 eyes from 40 patients with cataracts who underwent Precizon Presbyopic IOL implantation. Residual astigmatism, as measured by autorefraction and manifest refraction, was investigated using vector analysis of eyes implanted with vertical (90 ± 30 degrees) and horizontal (180 ± 30 degrees) orientations of the first near segment of the IOL. In the ray-tracing simulation study, pseudophakic eyes with the Precizon Presbyopic IOL were modeled. The modulation transfer function (MTF) of each case was compared with respect to the amount of corneal astigmatism of the model eyes and the orientation of the first near segment. RESULTS: The mean IOL-induced astigmatism measured by autorefraction was 0.68 ± 0.58 diopters (D) at 1 degree in the vertical orientation of the first near segment (n = 52) and 1.05 ± 0.81 D at 96 degrees in the horizontal orientation (n = 28). However, the mean IOL-induced astigmatism measured by manifest refraction was 0.14 ± 0.44 D at 171 degrees and 0.46 ± 0.40 D at 95 degrees. The MTF analysis showed that the highest MTF values were measured in eyes without corneal astigmatism in both the vertically and horizontally implanted IOLs. CONCLUSIONS: Autorefraction measurement indicates induction of with-the-rule astigmatism by the Precizon Presbyopic IOL when implanted vertically with respect to the first near segment, and against-the-rule astigmatism when implanted horizontally. However, this astigmatism is clinically insignificant according to manifest refraction and ray-tracing simulation. [J Refract Surg. 2020;36(11):740-748.].

Mechanisms of Insecticidal Action of Metarhizium anisopliae on Adult Japanese Pine Sawyer Beetles (Monochamus alternatus).

Pine wilt disease, caused by Bursaphelenchus xylophilus (pine wood nematode), leads to severe environmental and economic damage. Here, we report the results of experiments on the biological control of pine wilt disease through termination of the insect vector of the nematode and the mechanism of the insecticidal action of Metarhizium anisopliae JEF-279 against Monochamus alternatus (Japanese pine sawyer). A combined treatment with a fungal conidia suspension and a fungal protease-containing culture filtrate caused 75.8% mortality of the insect vector. Additionally, the presence of destruxins was confirmed in the dead Japanese pine sawyer adults, and half of the 10 protein spots in proteomic analysis were identified as an actin related to muscle contraction. Based on proteomic and microscopic analyses, the infection cycle of the Japanese pine sawyer by M. anisopliae JEF-279 was inferred to proceed in the following sequence: (1) host adhesion and germination, (2) epicuticle degradation, (3) growth as blastospore, (4) killing by various fungal toxins (insecticidal metabolites), (5) immune response as defense mechanism, and (6) hyphal extrusion and conidiation. Consequently, the combined fungal conidia suspension and protease-containing culture filtrate treatment may be applied as an insecticidal agent, and flaccid paralysis is likely a major mechanism underlying the insecticidal action of M. anisopliae JEF-279 on host insects.

A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the devastating diseases affecting pine forests worldwide. Although effective control measurements are still missing, induction of resistance could represent a possible eco-friendly alternative. In this study, induced resistance-based in vitro and in vivo screening tests were carried out for selection of bacteria with the ability to suppress PWD. Out of 504 isolated bacteria, Bacillus thuringiensis JCK-1233 was selected for its ability to boost pathogenesis-related 1 (PR1) gene expression, a marker of systemic acquired resistance. Moreover, treatment of pine seedlings with B. thuringiensis JCK-1233 resulted in increased expression of other defense-related genes, and significantly inhibited PWD development under greenhouse conditions. However, B. thuringiensis JCK-1233 showed no direct nematicidal activity against B. xylophilus. To identify the effective compound responsible for the induction of resistance in B. thuringiensis JCK-1233, several diketopiperazines (DPKs) including cyclo-(D-Pro-L-Val), cyclo-(L-Pro-L-Ile), cyclo-(L-Pro-L-Phe), and cyclo-(L-Leu-L-Val) were isolated and tested. Foliar treatment of pine seedlings with Cyclo-(L-Pro-L-Ile) resulted in suppression of PWD severity and increased the expression of defense-related genes similarly to B. thuringiensis JCK-1233 treatment. Interestingly, treatment with B. thuringiensis JCK-1233 or cyclo-(L-Pro-L-Ile) showed moderately enhanced expression of PR-1, PR-2, PR-3, PR-4, PR-5, and PR-9 genes following inoculation with PWN compared to that in the untreated control, indicating that they mitigated the burst of hypersensitive reaction in susceptible pine seedlings. In contrast, they significantly increased the expression levels of PR-6 and PR-10 before PWN inoculation. In conclusion, foliar spraying with either B. thuringiensis JCK-1233 culture suspension or DPKs could induce resistance in pine seedlings, thereby alleviating the serious damage by PWD. Taken together, this study supports aerial spraying with eco-friendly biotic or abiotic agents as a valuable strategy that may mark an epoch for the control of PWD in pine forests.

Comparative Transcriptome Analysis of Pine Trees Treated with Resistance-Inducing Substances against the Nematode Bursaphelenchus xylophilus.

The pinewood nematode (PWN) Bursaphelenchus xylophilus causes pine wilt disease, which results in substantial economic and environmental losses across pine forests worldwide. Although systemic acquired resistance (SAR) is effective in controlling PWN, the detailed mechanisms underlying the resistance to PWN are unclear. Here, we treated pine samples with two SAR elicitors, acibenzolar-S-methyl (ASM) and methyl salicylic acid (MeSA) and constructed an in vivo transcriptome of PWN-infected pines under SAR conditions. A total of 252 million clean reads were obtained and mapped onto the reference genome. Compared with untreated pines, 1091 and 1139 genes were differentially upregulated following the ASM and MeSA treatments, respectively. Among these, 650 genes showed co-expression patterns in response to both SAR elicitors. Analysis of these patterns indicated a functional linkage among photorespiration, peroxisome, and glycine metabolism, which may play a protective role against PWN infection-induced oxidative stress. Further, the biosynthesis of flavonoids, known to directly control parasitic nematodes, was commonly upregulated under SAR conditions. The ASM- and MeSA-specific expression patterns revealed functional branches for myricetin and quercetin production in flavonol biosynthesis. This study will enhance the understanding of the dynamic interactions between pine hosts and PWN under SAR conditions.