The New Nematicide Cyclobutrifluram Targets the Mitochondrial Succinate Dehydrogenase Complex in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is a dangerous quarantine pest that causes extensive damage to pine ecosystems worldwide. Cyclobutrifluram, a succinate dehydrogenase inhibitor (SDHI), is a novel nematicide introduced by Syngenta in 2013. However, the nematocidal effect of cyclobutrifluram against plant-parasitic nematodes remains underexplored. Therefore, here, we aim to address this knowledge gap by evaluating the toxicity, effects, and mode of action of cyclobutrifluram on B. xylophilus. The result shows that cyclobutrifluram is the most effective agent, with an LC50 value of 0.1078 mg·L-1. At an LC20 dose, it significantly reduced the population size to 10.40 × 103 ± 737.56-approximately 1/23 that of the control group. This notable impact may stem from the agent's ability to diminish egg-laying and hatching rates, as well as to impede the nematodes' development. In addition, it has also performed well in the prevention of pine wilt disease, significantly reducing the incidence in greenhouses and in the field. SDH consists of a transmembrane assembly composed of four protein subunits (SDHA to SDHD). Four sdh genes were characterized and proved by RNAi to regulate the spawning capacity, locomotion ability, and body size of B. xylophilus. The mortality of nematodes treated with sdhc-dsRNA significantly decreased upon cyclobutrifluram application. Molecular docking further confirmed that SDHC, a cytochrome-binding protein, is the target. In conclusion, cyclobutrifluram has a good potential for trunk injection against B. xylophilus. This study provides valuable information for the screening and application of effective agents in controlling and preventing PWD in forests.

Trunk injection to control Xylosandrus germanus (Coleoptera: Curculionidae) in topworked apple trees.

Xylosandrus germanus (Blandford) is an invasive species of ambrosia beetle known to attack apple trees in North America. Xylosandrus germanus are attracted to ethanol produced by stressed and injured trees and can be a serious problem when grafting a new cultivar onto established fruit trees (topworking). The objective of this study was to evaluate the efficacy of 2 insecticides (emamectin benzoate and azadirachtin) and injection timing (fall and spring) on their ability to control X. germanus colonization in apple trees with simulated topworking. Our study shows evidence that both emamectin benzoate and azadirachtin injections can reduce X. germanus infestations; however, our results were inconsistent. The timing of injections influenced X. germanus, with spring injected azadirachtin being more effective than fall injections. Residue analyses of emamectin benzoate and azadirachtin showed the presence of residues in woody tissue comparable to those found in leaves.

The Potency of Abamectin Formulations against the Pine Wood Nematode, Bursaphelenchus xylophilus.

Abamectin offers great protection against Bursaphelenchus xylophilus, a well-known devastating pathogen of pine tree stands. Trunk injection of nematicides is currently the most preferred method of control. This study aimed to evaluate the potency of the commonly used formulations of abamectin against B. xylophilus. Twenty-one formulations of abamectin were evaluated by comparing their sublethal toxicities and reproduction inhibition potentials against B. xylophilus. Nematodes were treated with diluted formulation concentrations in multi-well culture plates. And, populations pre-exposed to pre-determined concentrations of the formulations were inoculated onto Botrytis cinerea culture, and in pine twig cuttings. Potency was contrastingly different among formulations, with LC95 of 0.00285 and 0.39462 mg/ml for the most, and the least potent formulation, respectively. Paralysis generally occurred at an application dose of 0.06 µg/ml or higher, and formulations with high sublethal toxicities caused significant paralysis levels at the tested doses, albeit the variations. Nematode reproduction was evident at lower doses of 0.00053-0.0006 µg/ml both on Botrytis cinerea and pine twigs, with significant variations among formulations. Thus, the study highlighted the inconsistencies in the potency of similar product formulations with the same active ingredient concentration against the target organism, and the need to analyze the potential antagonistic effects of the additives used in formulations.

Spatiotemporal dynamics of fluopyram trunk-injection in Pinus massoniana and its efficacy against pine wilt disease.

BACKGROUND: Pine wilt disease (PWD) is a destructive disease of pine trees caused by the pinewood nematode, Bursaphelenchus xylophilus. Fluopyram, a novel nematicide compound with systemic activity, is a prospective trunk-injection agent against pinewood nematodes. The disadvantage of current trunk-injection agents is that they were not evenly distributed in tree tissues and were poor in the persistence of effect and efficiency. Therefore, we investigated the spatiotemporal transport pattern and residue behavior of fluopyram following its injection into the trunk of Pinus massoniana. RESULTS: Fluopyram transport in the trunk occurred through radial diffusion and vertical uptake within 1 week of the injection, reaching all tissues of P. massoniana, including apical branches and needles. Three years after the field test, the infection of PWD declined substantially with treatment using the fluopyram trunk-injection agent, which demonstrated 100% efficacy in both the mild and moderate occurrence areas, and 71.1% efficacy in the severe occurrence area. Fluopyram as trunk-injection agent exerted substantial control over PWD, with its efficacy being influenced by the infection time of PWD. The half-life of 10% fluopyram in treated pine trees was 346.6 days with 3-year persistence. CONCLUSION: The advantages of overall distribution and long persistence of fluopyram in the tree after injection help explain its evident efficacy against PWN. Overall, fluopyram trunk-injection has potential to prevent PWD. © 2023 Society of Chemical Industry.

Comparative Bioactivity of Emamectin Benzoate Formulations against the Pine Wood Nematode, Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus is a well-known devastating pathogen of economic importance in the Republic of Korea and other countries. In the Republic of Korea, trunk injection of nematicides is the preferred method of control. In this study, the efficacy of 16 locally produced formulations of emamectin benzoate against the PWN are compared through determining their sublethal toxicities and reproduction inhibition potentials. Nematodes were treated with varying concentrations of the tested chemicals in multi-well culture plates, and rates of paralysis and mortality were determined after 24 h. Reproduction inhibition potential was tested by inoculating pre-treated nematodes onto Botrytis cinerea, and in pine twig cuttings. Despite the uniformity in the concentration of the active ingredient, efficacy was contrastingly different among formulations. The formulations evidently conformed to three distinct groups based on similarities in sublethal activity (group 1: LC95 of 0.00768-0.01443 mg/ml; group 2: LC95 of 0.03202-0.07236 mg/ml, and group 3: LC95 of as high as 0.30643-0.40811 mg/ml). Nematode paralysis generally occurred at the application dose of 0.0134-0.1075 µg/ml, and there were significant differences in nematode paralysis rates among the products. Nematode reproduction was only evident at lower doses both on B. cinerea and pine twigs, albeit the variations among formulations. Group 1 formulations significantly reduced nematode reproduction even at a lower dose of 0.001075 µg/ml. The variations in efficacy might be attributed to differences in inert ingredients. Therefore, there is need to analyze the potential antagonistic effects of the large number of additives used in formulations.

Essential Oil Trunk Injection Into Orchard Trees: Consequences on the Performance and Preference of Hemipteran Pests.

Apples and pears are among the most widely cultivated fruit species in the world. Pesticides are commonly applied using ground sprayers in conventional orchards; however, most of it will not reach the target plant, increasing the contamination of nontarget organisms such as natural predators, pollinators, and decomposers. Trunk injection is an alternative method of pesticide application that could reduce risks to beneficials and workers. Essential oils represent a 'green' alternative to pesticides due to their reported insecticidal, antimicrobial, antiviral, nematicidal, and antifungal properties. The aim of this study was, therefore, to evaluate the impact that the injection of a cinnamon essential oil solution into the trunk of apple and pear trees could have on their respective pests, Dysaphis plantaginea and Cacopsylla pyri, respectively. The feeding behavior (preference), the life history traits (performance), and the timing of this effect were measured. The injection of an essential oil emulsion in trees impacted hemipteran host-plant colonization, as for both species a modification of their preference and of their performance was observed. The feeding behavior of D. plantaginea was altered as a significantly lower proportion of aphids ingested phloem sap on injected trees, suggesting that the aphids starved to death. On the contrary, the feeding behavior of the psyllids was little changed compared to the control condition, implying that the observed mortality was due to intoxication. The results presented here could theoretically be used to control these two orchard hemipteran pests, although the effectiveness in real conditions still has to be demonstrated.

Effects of trunk injection with emamectin benzoate on arthropod diversity.

BACKGROUND: Pine wood nematode is a major plant quarantine object in the world. Trunk injection is an effective method for controlling pests that cause disease. To evaluate the ecological safety of trunk injection with emamectin benzoates in forests of Pinus massoniana, the community diversity and community composition of soil arthropods and flying insects (Hymenoptera) were studied at different stages of trunk injection. RESULTS: The dominant taxonomic groups of soil arthropods were Collembola (30.80%), Insecta (26.42%), and Arachnida (23.84%). The taxonomic groups of flying insects (Hymenoptera) were Ichneumonidae (48.94%), Formicidae (14.10%), and Braconidae (8.44%). Trunk injection with emamectin benzoate has no significant effect on the community diversity indices of total soil arthropods and flying insects (Hymenoptera). However, it has a significant effect on the community diversity indices of detritivores for soil arthropods. It changed the community composition of soil arthropods but did not impact the community composition of flying insects (Hymenoptera). Redundancy analysis of arthropod community structure and environmental variables showed that total potassium, residual of green leaf, and residual of litter leaf have a significant impact on the community structure of soil arthropods, and total phosphorus, total nitrogen, water content, organic matter, and total potassium have a significant impact on the community structure of flying insects (Hymenoptera). CONCLUSION: Trunk injection with emamectin benzoate is safe for the ecological environment. This study provides a new insight into the field for the prevention and control of pine wood nematode disease, which is of great significance to forest management and pest control. © 2022 Society of Chemical Industry.

Preliminary Studies on Endotherapy Based Application of Ozonated Water to Bobal Grapevines: Effect on Wine Quality.

The application of ozonated water in the vineyard is an increasingly popular tool for disease management, but the quality of grapes and resulting wines is likely to be affected. Endotherapy, or trunk injection, is a particularly useful method to apply phytosanitary products since many fungal pathogens colonize the grapevine woody tissues. Thus, the present study aimed to evaluate the effect on wine quality of the ozonated water applied to Bobal grapevines, one of the most cultivated red varieties in Spain, through endotherapy (E) or its combination with spraying (E + S). Endotherapy was carried out four times before harvest for both E and E + S treatments, and spraying was performed 2 days before and after each endotherapy application. Grapes were harvested, vinified, and the quality of the finished wines was evaluated through several enological parameters and the phenolic and volatile composition. Both treatments resulted in less alcoholic and more acidic wines. The E treatment, although it reduced the content of phenolic acids, stilbenes and flavanols, significantly increased anthocyanins, whereas E + S decreased the overall amount of phenolics, which had different implications for wine colour. In terms of aroma, both treatments, but E to a greater extent, reduced the content of glycosylated precursors and differentially affected free volatiles, both varietal and fermentative. Thus, the dose of ozonated water, frequency and/or method of application are determining factors in the effect of these treatments on wine quality and must be carefully considered by winegrowers to establish the optimum treatment conditions so as not to impair the quality of wines.

Biopesticide Trunk Injection Into Apple Trees: A Proof of Concept for the Systemic Movement of Mint and Cinnamon Essential Oils.

The use of conventional pesticides is debated because of their multiple potential adverse effects on non-target organisms, human health, pest resistance development and environmental contaminations. In this setting, this study focused on developing alternatives, such as trunk-injected essential oil (EO)-based biopesticides. We analysed the ecophysiology of apple trees (Malus domestica) following the injection of Cinnamomum cassia and Mentha spicata nanoemulsions in the tree's vascular system. Targeted and untargeted volatile organic compounds (VOCs) analyses were performed on leaf-contained and leaf-emitted VOCs and analysed through dynamic headspace-gas chromatography-mass spectrometry (DHS-GC-MS) and thermal desorption unit (TDU)-GC-MS. Our results showed that carvone, as a major constituent of the M. spicata EO, was contained in the leaves (mean concentrations ranging from 3.39 to 19.7 ng gDW -1) and emitted at a constant rate of approximately 0.2 ng gDW -1 h-1. Trans-cinnamaldehyde, C. cassia's major component, accumulated in the leaves (mean concentrations of 83.46 and 350.54 ng gDW -1) without being emitted. Furthermore, our results highlighted the increase in various VOCs following EO injection, both in terms of leaf-contained VOCs, such as methyl salicylate, and in terms of leaf-emitted VOCs, such as caryophyllene. Principal component analysis (PCA) highlighted differences in terms of VOC profiles. In addition, an analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA) revealed that the VOC profiles were significantly impacted by the treatment. Maximum yields of photosystem II (Fv/Fm) were within the range of 0.80-0.85, indicating that the trees remained healthy throughout the experiment. Our targeted analysis demonstrated the systemic translocation of EOs through the plant's vascular system. The untargeted analysis, on the other hand, highlighted the potential systemic acquired resistance (SAR) induction by these EOs. Lastly, C. cassia and M. spicata EOs did not appear phytotoxic to the treated trees, as demonstrated through chlorophyll fluorescence measurements. Hence, this work can be seen as a proof of concept for the use of trunk-injected EOs given the systemic translocation, increased production and release of biogenic VOCs (BVOCs) and absence of phytotoxicity. Further works should focus on the ecological impact of such treatments in orchards, as well as apple quality and production yields.

Organic Control of Pear Psylla in Pear with Trunk Injection.

Organic production of pears is challenging in part because OMRI (Organic Materials Review Institute) approved biopesticides are short lived when applied as foliar sprays. Trunk injection is an alternative method of insecticide delivery that may enhance the performance of biopesticides for control of pear psylla. The objective of this study is to compare the efficacy of azadirachtin and abamectin in the control of pear psylla using two different application methods, airblast sprayer and trunk injection. Trunk injections of azadirachtin and abamectin were compared to airblast applications of equal labeled rates on 33-year-old Bartlett Pear trees (Pyrus communis L., var "Bartlett"). The azadirachtin and abamectin trunk injected treatments performed equally or better than the two airblast applications in the control of the pear psylla. The trunk injected trees from the first season provided a moderate level of control into the second season, one year after the injections. This study suggests that trunk injection is a superior delivery system for biopesticides used in organic pear production.

Transcriptomic profiling of effects of emamectin benzoate on the pine wood nematode Bursaphelenchus xylophilus.

BACKGROUND: Emamectin benzoate (EB) has recently been successfully applied as a trunk injection for preventative control of the pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Here, a whole-organism transcriptomic analysis provides comprehensive insights into the adverse effects of EB on B. xylophilus. RESULTS: A large set of differentially expressed genes (DEGs) were found, demonstrating the antagonistic effects of EB on B. xylophilus embryonic and larval development, reproduction, nervous and motor systems, and pathogenesis. In toxicity assays with EB, the number of eggs laid, hatching rate, thrashing frequency, and developmental rate of B. xylophilus were significantly suppressed at low concentrations (0.1 µg mL-1 ). Moreover, the transcriptional changes validated by real-time quantitative PCR showed downregulated transcript levels of the genes encoding pectate lyases, ß-1,4-endoglucanases, and upregulated the genes encoding glutamate-gated chloride channel, γ-aminobutyric acid type ß receptor, uridine 5'-diphospho-glucuronosyl transferase, ATP-binding cassette transporter. The potential responses of B. xylophilus to EB included the upregulation of several genes putatively contributing to oocyte protection, stem cell renewal, and xenobiotic degradation, implying the potential for drug resistance to develop. CONCLUSION: Our findings further our understanding of the effects of EB for managing the PWD and may help to improve the pesticide-use strategies for controlling B. xylophilus. © 2019 Society of Chemical Industry.

Insecticide dose and seasonal timing of trunk injection in apples influence efficacy and residues in nectar and plant parts.

BACKGROUND: Trunk injection is an established method for delivering pesticides in ornamental and shade trees, but further research is needed to determine efficacy and pollinator safety in tree fruit crops. Apple trees were injected in 2013 and 2014 with the insecticides emamectin benzoate, imidacloprid, dinotefuran, spinosad, chlorantraniliprole, or abamectin. Additional emamectin benzoate and imidacloprid injections were performed in the spring and fall of 2015. Nectar and pollen were sampled in the following spring to compare the effects of application timings on insecticide loading into flowers. RESULTS: Neonicotinoids reduced Empoasca fabae density in the field. Emamectin benzoate, chlorantraniliprole, and abamectin resulted in moderate to high mortality and reduced Choristoneura rosaceana feeding in bioassays. Imidacloprid was not detected in nectar or pollen when injected in the spring, and was detected at 0.39 ng g-1 in pollen when injected the previous fall. Emamectin benzoate was not detected in nectar or pollen when injected the previous fall, and was detected at 7.36 ng g-1 (nectar) and 1.15 ng g-1 (pollen) when injected in the spring. CONCLUSIONS: This study identified a broader list of possible trunk-injectable pesticides for apple trees. This study also shows that managing the seasonal timing of injection can reduce the risk of insecticide exposure to pollinators. © 2018 Society of Chemical Industry.

Evaluation of the effectiveness of insecticide trunk injections for control of Latoia lepida (Cramer) in the sweet olive tree Osmanthus fragrans.

The screening of suitable insecticides is a key factor in successfully applying trunk injection technology to ornamental plants. In this study, six chemical pesticides were selected and injected into the trunks of Osmanthus fragrans to control the nettle caterpillar, Latoia lepida (Lepidoptera: Limacodidae), using a no-pressure injection system. The absorption rate of the insecticides, the leaf loss due to insect damage, and the mortality and frass amount of L. lepida larvae were evaluated after 77 and 429 days. The results showed that 4% imidacloprid + carbosulfan and 21% abamectin + imidacloprid + omethoate had the fastest conductivity and were completely absorbed into the trunkswithin14 days; however, the efficiencies of these insecticides in controlling L. lepidawere extremely low. Additionally, the treatment 10% emamectin benzoate + clothianidin and 2.5% emamectin benzoate was almost completely absorbed within 30 days and exhibited a longer duration of insecticide efficiency (>80% mortality) in the upper and lower leaves of the canopy. Treatment with these insecticides also resulted in significantly lower leaf loss and frass amounts. We conclude that emamectin benzoate and emamectin benzoate + clothianidin have a rapid uptake into O. fragrans, and are effective as insecticides over long durations. Hence, they may be a suitable control option for L. lepida in O. fragrans plants.

Control of fire blight (Erwinia amylovora) on apple trees with trunk-injected plant resistance inducers and antibiotics and assessment of induction of pathogenesis-related protein genes.

Management of fire blight is complicated by limitations on use of antibiotics in agriculture, antibiotic resistance development, and limited efficacy of alternative control agents. Even though successful in control, preventive antibiotic sprays also affect non-target bacteria, aiding the selection for resistance which could ultimately be transferred to the pathogen Erwinia amylovora. Trunk injection is a target-precise pesticide delivery method that utilizes tree xylem to distribute injected compounds. Trunk injection could decrease antibiotic usage in the open environment and increase the effectiveness of compounds in fire blight control. In field experiments, after 1-2 apple tree injections of either streptomycin, potassium phosphites (PH), or acibenzolar-S-methyl (ASM), significant reduction of blossom and shoot blight symptoms was observed compared to water injected control trees. Overall disease suppression with streptomycin was lower than typically observed following spray applications to flowers. Trunk injection of oxytetracycline resulted in excellent control of shoot blight severity, suggesting that injection is a superior delivery method for this antibiotic. Injection of both ASM and PH resulted in the significant induction of PR-1, PR-2, and PR-8 protein genes in apple leaves indicating induction of systemic acquired resistance (SAR) under field conditions. The time separating SAR induction and fire blight symptom suppression indicated that various defensive compounds within the SAR response were synthesized and accumulated in the canopy. ASM and PH suppressed fire blight even after cessation of induced gene expression. With the development of injectable formulations and optimization of doses and injection schedules, the injection of protective compounds could serve as an effective option for fire blight control.

Spatial and temporal distribution of trunk-injected imidacloprid in apple tree canopies.

BACKGROUND: Pesticide use in orchards creates drift-driven pesticide losses which contaminate the environment. Trunk injection of pesticides as a target-precise delivery system could greatly reduce pesticide losses. However, pesticide efficiency after trunk injection is associated with the underinvestigated spatial and temporal distribution of the pesticide within the tree crown. This study quantified the spatial and temporal distribution of trunk-injected imidacloprid within apple crowns after trunk injection using one, two, four or eight injection ports per tree. RESULTS: The spatial uniformity of imidacloprid distribution in apple crowns significantly increased with more injection ports. Four ports allowed uniform spatial distribution of imidacloprid in the crown. Uniform and non-uniform spatial distributions were established early and lasted throughout the experiment. The temporal distribution of imidacloprid was significantly non-uniform. Upper and lower crown positions did not significantly differ in compound concentration. Crown concentration patterns indicated that imidacloprid transport in the trunk occurred through radial diffusion and vertical uptake with a spiral pattern. CONCLUSION: By showing where and when a trunk-injected compound is distributed in the apple tree canopy, this study addresses a key knowledge gap in terms of explaining the efficiency of the compound in the crown. These findings allow the improvement of target-precise pesticide delivery for more sustainable tree-based agriculture.

Stem injection of a systemic insecticide to control Uraba lugens on urban Lophostemon confertus trees.

BACKGROUND: Uraba lugens Walker (Lepidoptera: Nolidae) has the potential to become a serious pest of susceptible forestry and urban trees in New Zealand. Stem injection of insecticides provides a method for the control of U. lugens in public locations where foliar applications are not suitable. Field and laboratory studies were conducted in 2009 and 2010 to determine a dose response profile of U. lugens larvae to acephate injected at 0.0, 0.25, 0.50 and 1.00 g cm(-1) tree diameter measured at breast height (dbh). The concentration of the injected solution varied across years. RESULTS: The highest mortality (85-100%) of U. lugens larvae occurred at 1.00 g acephate injected cm(-1) dbh. The lower rates (0.25 and 0.50 g acephate cm(-1) ) were more effective in 2010 than in 2009, a factor attributed to the change in volume of injected solution. CONCLUSION: Systemic injections of acephate at rates above 0.50 g cm(-1) dbh provide a tool for the management of U. lugens in public places. It is recommended that stem injection operations for control of U. lugens larvae be timed to coincide with the emergence of first instars so that larvae are exposed to lethal insecticide doses.