A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing.

Spider mites (Acari: Tetranychidae) are polyphagous pests of economic importance in agriculture, among which the two-spotted spider mite Tetranychus urticae Koch has spread widely worldwide as an invasive species, posing a serious threat to fruit tree production in China, including Beijing. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is also a worldwide pest of fruit trees and woody ornamental plants. The cassava mite, Tetranychus truncatus Ehara, is mainly found in Asian countries, including China, Korea and Japan, and mainly affects fruit trees and agricultural crops. These three species of spider mites are widespread and serious fruit tree pests in Beijing. Rapid and accurate identification of spider mites is essential for effective pest and plant quarantine in Beijing orchard fields. The identification of spider mite species is difficult due to their limited morphological characteristics. Although the identification of insect and mite species based on PCR and real-time polymerase chain reaction TaqMan is becoming increasingly common, DNA extraction is difficult, expensive and time-consuming due to the minute size of spider mites. Therefore, the objective of this study was to establish a direct multiplex PCR method for the simultaneous identification of three common species of spider mites in orchards, A. viennensis, T. truncatus and T. urticae, to provide technical support for the differentiation of spider mite species and phytosanitary measures in orchards in Beijing. Based on the mitochondrial cytochrome c oxidase subunit I (COI) of the two-spotted spider mite and the cassava mite and the 18S gene sequence of the hawthorn spider mite as the amplification target, three pairs of specific primers were designed, and the primer concentrations were optimized to establish a direct multiplex PCR system for the rapid and accurate discrimination of the three spider mites without the need for DNA extraction and purification. The method showed a high sensitivity of 0.047 ng for T. truncatus and T. urticae DNA and 0.0002 ng for A. viennensis. This method eliminates the DNA extraction and sequencing procedures of spider mite samples, offers a possibility for rapid monitoring of multiple spider mites in an integrated microarray laboratory system, reducing the time and cost of leaf mite identification and quarantine monitoring in the field.

Acaricidal Activities of Dioscorea japonica Thunb. (Dioscoreales: Dioscoreaceae) Extract and its Constituents Against the Two-spotted Spider Mite, Tetranychus urticae Koch (Trombidiformes: Tetranychidae).

The acaricidal activities of 86 plant extracts were investigated under laboratory conditions. The ethanol extract of Dioscorea japonica Thunb. root showed the strongest acaricidal activity, with 89.3% mortality against two-spotted spider mite, Tetranychus urticae Koch adults at a 2 mg/ml concentration. Bioassay-guided isolation of D. japonica root extract using silica gel open column chromatography, gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS) identified palmitic acid as the primary active compound. The acaricidal activities of palmitic acid against T. urticae were 91.2% and 69.7% at concentrations of 1 and 0.5 mg/ml, respectively. Among nine saturated fatty acids with carbon chains ranging from C8 to C26, the most vigorous acaricidal activity was observed with octanoic acid, followed by palmitic acid, and decanoic acid at a 1 mg/ml concentration. The acaricidal activity of the other fatty acids was less than 40% mortality at a 1 mg/ml concentration. These results indicate that a suitable carbon length is essential for fatty acids to exhibit acaricidal activity. The acaricidal efficacy of Eungjinssag (EJSG), an organic agricultural material authorized for the management of mites in the Republic of Korea, was compared to D. japonica root extract. At concentrations above 1 mg/ml, the acaricidal activity of D. japonica root extract was stronger than that of EJSG. The results of this study show that D. japonica root extract and palmitic acid are promising candidates as new environmentally-friendly control agents against two-spotted spider mite, which is one of the most severely damaging agricultural arthropod pests.

Overexpression of leucoanthocyanidin reductase or anthocyanidin reductase elevates tannins content and confers cassava resistance to two-spotted spider mite.

The two-spotted spider mite (TSSM) is a destructive cassava pest. Intensive demonstration of resistance mechanism greatly facilitates the creation of TSSM-resistant cassava germplasm. Gene to metabolite network plays a crucial role in modulating plant resistance, but little is known about the genes and related metabolites which are responsible for cassava resistance to TSSM. Here, a highly resistant (HR) and a highly susceptible (HS) cassava cultivar were used, integrative and comparative transcriptomic and metabolomic analyses between these two cultivars after TSSM infestation revealed that several genes and metabolites were closely related and significantly different in abundance. In particular, the expression of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes showed a high positive correlation with most of the metabolites in the tannin biosynthesis pathway. Furthermore, transgenic cassava lines overexpressing either of the genes elevated tannin concentrations and conferred cassava resistance to TSSM. Additionally, different forms of tannins possessed distinct bioactivity on TSSM, of which total condensed tannins (LC50 = 375.68 mg/l) showed maximum lethal effects followed by procyanidin B1 (LC50 = 3537.10 mg/l). This study accurately targets LAR, ANR and specific tannin compounds as critical genes and metabolites in shaping cassava resistance to TSSM, which could be considered as biomarkers for evaluation and creation of pest-resistant cassava germplasm.

Chitin deacetylase 2 is essential for molting and survival of Tetranychus urticae.

Chitin metabolism has long been considered promising targets for development of biorational pesticides. Considering the increasing challenges of controlling the twospotted spider mite, Tetranychus urticae Koch, the roles of chitin deacetylases (CDAs) during molting process and mite development are explored. TuCDA1 and TuCDA2 differ in expression patterns during the development process. Feeding of double-strand RNA (dsRNA) against TuCDA1 or TuCDA2 has lethal effects on the mites. Especially TuCDA2 displays a much stronger phenotype than TuCDA1 (p = 0.0003). The treated mites fail to shed the old cuticle and are trapped within exuviate until they die. The aberrant cuticle structure observed by scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM) may be responsible for the lethal phenotype of TuCDA1 and TuCDA2 knocked down mites. However, treatment with both dsRNA-CDA1 and dsRNA-CDA2 cannot significantly enhance the lethal effects of dsRNA-CDA2, which indicates partially redundant function of TuCDA1 and TuCDA2. TuCDA2 may play a key role during the molting and development process. Chitin-modifying enzyme such as TuCDA2 is potential target of RNA interference through feeding.

Surface properties of Tetranychus urticae Koch (Acari: Tetranychidae) and the effect of their infestation on the surface properties of kidney bean (Phaseolus vulgaris L.) hosts.

BACKGROUND: The wettability of the target surfaces affects the wetting and deposition of pesticides on them. The properties of leaf surfaces change after infestation by Tetranychus urticae Koch. Studying the surface wettability of T. urticae and the changes in leaf wettability after infestation is important to guide the use of acaricides. RESULTS: The body surface of T. urticae is an ellipsoidal crown covered with dense cuticle striations and hairs arranged in different directions, which makes the surface of T. urticae rough and hydrophobic. The abaxial surfaces of the leaves are rougher and more hydrophobic than the adaxial surfaces. After infestation by T. urticae, the faded spots were sunken on the adaxial surface and raised on the abaxial surface, where they had formed new wide peaks and valleys. The adaxial surface became obviously rougher and more hydrophobic, while the roughness of the abaxial surface became slightly larger, and the change in hydrophobicity was not obvious. The contact angles of the studied commercial acaricide on these surfaces were greater than 65° and were affected by the infestation. Reducing the surface tension can allow for better wetting of these surfaces and eliminate changes in leaf wettability. CONCLUSION: The surfaces of kidney bean leaves became more hydrophobic after infestation by T. urticae with hydrophobic surface. The wettability of the acaricide solution should be adjusted according to the changes in leaf wettability. This study has important theoretical guiding significance for improving effective deposition of acaricide.

Tomato Cultivars Resistant or Susceptible to Spider Mites Differ in Their Biosynthesis and Metabolic Profile of the Monoterpenoid Pathway.

The two-spotted spider mite (TSSM; Tetranychus urticae) is a ubiquitous polyphagous arthropod pest that has a major economic impact on the tomato (Solanum lycopersicum) industry. Tomato plants have evolved broad defense mechanisms regulated by the expression of defense genes, phytohormones, and secondary metabolites present constitutively and/or induced upon infestation. Although tomato defense mechanisms have been studied for more than three decades, only a few studies have compared domesticated cultivars' natural mite resistance at the molecular level. The main goal of our research was to reveal the molecular differences between two tomato cultivars with similar physical (trichome morphology and density) and agronomic traits (fruit size, shape, color, cluster architecture), but with contrasting TSSM susceptibility. A net house experiment indicated a mite-resistance difference between the cultivars, and a climate-controlled performance and oviposition bioassay supported these findings. A transcriptome analysis of the two cultivars after 3 days of TSSM infestation, revealed changes in the genes associated with primary and secondary metabolism, including salicylic acid and volatile biosynthesis (volatile benzenoid ester and monoterpenes). The Terpene synthase genes, TPS5, TPS7, and TPS19/20, encoding enzymes that synthesize the monoterpenes linalool, ß-myrcene, limonene, and ß-phellandrene were highly expressed in the resistant cultivar. The volatile profile of these cultivars upon mite infestation for 1, 3, 5, and 7 days, revealed substantial differences in monoterpenoid and phenylpropanoid volatiles, results consistent with the transcriptomic data. Comparing the metabolic changes that occurred in each cultivar and upon mite-infestation indicated that monoterpenes are the main metabolites that differ between cultivars (constitutive levels), while only minor changes occurred upon TSSM attack. To test the effect of these volatile variations on mites, we subjected both the TSSM and its corresponding predator, Phytoseiulus persimilis, to an olfactory choice bioassay. The predator mites were only significantly attracted to the TSSM pre-infested resistant cultivar and not to the susceptible cultivar, while the TSSM itself showed no preference. Overall, our findings revealed the contribution of constitutive and inducible levels of volatiles on mite performance. This study highlights monoterpenoids' function in plant resistance to pests and may inform the development of new resistant tomato cultivars.

Effect of broflanilide on the phytophagous mite Tetranychus urticae and the predatory mite Typhlodromips swirskii.

BACKGROUND: Two-spotted spider mite (TSSM), Tetranychus urticae Koch, is one of the most serious pests of agricultural crops. Broflanilide exhibits high lethality against various pests and has been marketed worldwide under the Vedira and Tenebenal brands in 2020. Nevertheless, little information has been reported about its effects on agricultural mites. RESULTS: Broflanilide displayed higher toxicity to TSSM eggs (24 h LC50 , 1.015 mg L-1 ) and adult females (24 h LC50 , 2.062 mg L-1 ) than commercial acaricides, including cyflumetofen, bifenazate, and profenofos. In contrast, the adverse effects of broflanilide on the predatory mite, Typhlodromips swirskii Athias-Henriot, was lower than those of fenpyroximate and abamectin. In the sublethal effect study, while adult females were treated with broflanilide, the number of eggs and longevity were reduced in LC10 and LC30 treatments; when eggs were treated with broflanilide, the egg duration and deutonymph duration were prolonged in LC30 treatment. A significant decrease in the total life span and duration and fecundity of adult females was observed in LC10 and LC30 treatments. Furthermore, the number of eggs per adult female was significantly reduced from 103.48 ± 3.69 in the control group to 69.42 ± 2.22 and 48.33 ± 1.75 in LC10 and LC30 treatments, respectively. In the greenhouse bioassay, broflanilide 5% suspension concentrate (MCI-8007) showed excellent acaricidal activity to TSSM, with 99.22% corrected control, compared with the MCI-8007 untreated group. CONCLUSION: Our results indicated that broflanilide has a high acaricidal activity to TSSM and significant inhibition to fecundity of adult female, and could be considered as a potential alternative for TSSM management. © 2021 Society of Chemical Industry.

Laboratory selection, resistance risk assessment, multi-resistance, and management of Tetranychus urticae Koch to bifenthrin, bifenazate and cyflumetofen on cowpea.

BACKGROUND: Tetranychus urticae (T. urticae) Koch is an important pest of vegetable crops worldwide. In this study, bioassays were carried out to analyze the resistance risk, multi-resistance and management of T. urticae Koch to bifenthrin, bifenazate and cyflumetofen on cowpea. RESULTS: The resistance ratios of the adult T. urticae population to bifenthrin (G16), bifenazate (G12) and cyflumetofen (G12) were 31.29, 9.38 and 5.81, respectively. Realized heritability (h 2 ) analysis showed that, under a selection pressure of 50-90% mortality, the generations needed to increase 10-fold LC50 values of bifenthrin, bifenazate and cyflumetofen were 3.64-8.05, 5.75-12.71, and 10.93-24.15, respectively. No obvious multi-resistance among these three acaricides was observed. Synergist bioassay results showed that microsomal multifunctional oxidase (MFO) was involved in bifenthrin resistance of T. urticae, with a synergistic ratio of 22.38. However, MFO and GSTs were not the main factors conferring the resistance to bifenazate. MFO, glutathione S-transferases(GSTs), together with esterase contributed to the development of the resistance to cyflumetofen. Additionally, the toxicity selection index test showed that bifenazate was safe to the natural enemy Neoseiulus barkeri (N. barkeri) with a toxicity selection index (TSI) >484.85, while bifenthrin was the least safe (TSI = 0.92). CONCLUSIONS: These results demonstrated the T. urticae developed higher resistance risk to bifenthrin compared to bifenazate and cyflumetofen and no obvious multi-resistance among these three acaricides, providing guidance for designing appropriate strategies for the effective application of bifenthrin, bifenazate and cyflumetofen in the field and delaying the development of insecticide resistance. © 2019 Society of Chemical Industry.