Probing the Interaction between Isoflucypram Fungicides and Human Serum Albumin: Multiple Spectroscopic and Molecular Modeling Investigations.

To better understand the potential toxicity risks of isoflucypram in humans, The interaction between isoflucypram and HSA (human serum albumin) was studied through molecular docking, molecular dynamics simulations, ultraviolet-visible absorption, fluorescence, synchronous fluorescence, three-dimensional fluorescence, Fourier transform infrared spectroscopies, and circular dichroism spectroscopies. The interaction details were studied using the molecular docking method and molecular dynamics simulation method. The results revealed that the effect of isoflucypram on human serum albumin was mixed (static and dynamic) quenching. Additionally, we were able to obtain important information on the number of binding sites, binding constants, and binding distance. The interaction between isoflucypram and human serum albumin occurred mainly through hydrogen bonds and van der Waals forces. Spectroscopic results showed that isoflucypram caused conformational changes in HSA (human serum albumin), in which the α-helix was transformed into a ß-turn, ß-sheet, and random coil, causing the HSA structure to loosen. By providing new insights into the mechanism of binding between isoflucypram and human serum albumin, our study has important implications for assessing the potential toxicity risks associated with isoflucypram exposure.

Interaction between the flagellum of Candidatus Liberibacter asiaticus and the vitellogenin-like protein of Diaphorina citri significantly influences CLas titer.

Huanglongbing (HLB) is a global devastating citrus disease that is mainly caused by "Candidatus Liberibacter asiaticus" (CLas). It is mostly transmitted by the insect Asian citrus psyllid (ACP, Diaphorina citri) in a persistent and proliferative manner. CLas traverses multiple barriers to complete an infection cycle and is likely involved in multiple interactions with D. citri. However, the protein-protein interactions between CLas and D. citri are largely unknown. Here, we report on a vitellogenin-like protein (Vg_VWD) in D. citri that interacts with a CLas flagellum (flaA) protein. We found that Vg_VWD was upregulated in CLas-infected D. citri. Silencing of Vg_VWD in D. citri via RNAi silencing significantly increased the CLas titer, suggesting that Vg_VWD plays an important role in the CLas-D. citri interaction. Agrobacterium-mediated transient expression assays indicated that Vg_VWD inhibits BAX- and INF1-triggered necrosis and suppresses the callose deposition induced by flaA in Nicotiana benthamiana. These findings provide new insights into the molecular interaction between CLas and D. citri.

Effects of Rhizosphere Microorganisms on the Uptake and Translocation of Organic Compounds in Maize Seedlings.

The plant root is a key pathway to absorb insecticides from soil and is colonized by beneficial and pathogenic microbial communities. Our study demonstrated that colonizing roots by nitrogen-fixing bacterium Pseudomonas stutzeri and pathogenic Fusarium graminearum and Pythium ultimum increased the uptake of insecticides into maize roots from soil. An alteration in the permeability of root cells contributed to this increased uptake. For the subsequent root-to-shoot translocation, the relationship between translocation and log P of the compound satisfied a Gaussian distribution. Relatively beneficial P. stutzeri can promote maize seedling growth and increase translocation, whereas Fusarium and Pythium pathogens can retard the seedling growth and reduce the translocation. Furthermore, the relationship between the concentration difference (difference of an insecticide from inoculation treatment to control) and log P also showed a Gaussian distribution. The maximum concentration difference from the Gaussian equation can be applied to assess the capacity of rhizosphere microorganisms to influence translocation.

Sulfoxaflor adversely influences the biological characteristics of Coccinella septempunctata by suppressing vitellogenin expression and predation activity.

Sulfoxaflor is a widely used sulfoximine insecticide that has been regarded as an important alternative insecticide for IPM strategies, but a comprehensive study of its potential ecological toxicity is still lacking. In the present work, the growth, longevity, predation and reproduction toxicity of Coccinella septempunctata caused by sulfoxaflor were evaluated. In addition, the potential mechanisms of decreased fecundity in C. septempunctata were investigated by analyzing the transcriptional and protein levels of reproduction-related gene vitellogenin (Vg). In a 20-day acute contact toxicity test, decreased survival proportion, pupation rate, adult emergence ratio, and increased hazard quotient (HQ) values were observed. Moreover, sublethal dosages of sulfoxaflor significantly inhibited the predation, longevity, fecundity and net reproduction rate of progeny. In addition, LR30 of sulfoxaflor dramatically down-regulate the mRNA-expression (F0: 65.38-fold, F1: 2.24-fold) and protein content (F0: 1.35-fold, F1: 1.36-fold) of Vg in the F0 and F1 generations. These results suggested that sulfoxaflor could inhibit the gene and protein content of Vg, thereby reducing the fecundity of C. septempunctata. Our study indicated that sulfoxaflor has potential risks to parent and progeny generations of C. septempunctata. These results provide valuable reference for optimal usage of sulfoxaflor in IPM systems.

Seed treatment with chlorantraniliprole and carbaryl mixture for managing fall armyworm on maize: systemic synergism, control efficiency and synergistic mechanism.

BACKGROUND: Fall armyworm (Spodoptera frugiperda) is one of the major invasive pests in China, and has been widely controlled by labor-intensive foliar sprays of agrochemicals in maize (Zea mays L.). RESULTS: Systemic bioassay showed that mixtures of chlorantraniliprole (Chlor) and carbaryl (Carb) had dramatically synergistic effect on toxicity to S. frugiperda. Particularly, a mixture of Chlor with Carb at a mass ratio of 2:1 (MCC) exhibited the highest toxicity to S. frugiperda. Therefore, seed treatment of Chlor mixed with Carb was studied as a simple, accurate, efficient and low-cost control technology. Our results showed that MCC treatment shortened the median lethal time and 90% lethal time to S. frugiperda compared to Chlor- and Carb-alone treatments. Meanwhile, smaller leaf consumption by S. frugiperda was recorded under MCC treatment compared to Chlor- and Carb-alone treatments. In field trial, maize-seed treatment with MCC showed efficacy up to 39 days post-emergence in preventing S. frugiperda foliar damage at a low infestation pressure. Moreover, chemical quantification by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) showed that Chlor residues were more absorbed and concentrated in maize leaves of MCC treatment, compared to that of Chlor-alone treatment. CONCLUSION: These results suggested that seed treatment with MCC can be applied to increase the control efficacy and reduce the cost of Chlor-alone treatment for controlling S. frugiperda. The present study provided evidence of an enhanced translocation and accumulation of Chlor residues in maize leaves under MCC treatment, which likely contributed to a synergistic effect against S. frugiperda. © 2022 Society of Chemical Industry.

First Report of Anthracnose Caused by Colletotrichum kahawae and Colletotrichum horri on Tea-oil Tree in China.

Tea-oil tree (Camellia oleifera Abel) is an important economic woody plant in southern China. The buds, fruits, and leaves of tea oil tree are all susceptible to the disease, causing the wilt or even fall of the plant. Every year, the disease incidence of anthracnose reached 20%-40% in diseased fields. In 2020, leaves with anthracnose were collected from the main producing areas of tea-oil tree in Yunnan Province and Guizhou Province, China. To isolate the pathogen, several fragments of diseased tissues (5×5mm) were disinfected in 75% alcohol for 40 s, and rinsed 3 times in sterilized water. Then, tissues were placed on PDA medium and incubated at 26℃ for 5 days. Fungal isolates with morphology characteristics similar to Colletotrichum spp. were consistently recovered from diseased tissues. Eighteen fungal isolates were obtained. Among them, 3 representative single-spore isolates (C2, gy15, Ch) were picked for further analysis. The isolates C2 and gy15 on the PDA were gray-white in the initial stage, and later became olive green and spread to the edge. Abundant orange-red conidial masses were present in the colony surface. Conidia were cylindrical and with blunt circles at both ends, with a size of 9.9 µm ~ 21.8 µm × 4.0 µm ~ 6.8 µm (n=50). The hyphae of isolate Ch on PDA were thin, cotton-like, gray to gray-black; the center of the back of the colony was brown, and the color of the colony became darker, and concentric rings could be produced. The conidia were cylindrical , with blunt circles at both ends, with a size of 6.3 µm ~15.0 µm × 3.2 µm ~ 7.0µm (n=50). In order to further identify the pathogens, the internal transcribed spacer (ITS) region of ribosomal DNA, actin (ACT), chitin synthase (CHS), ß-tubulin (TUB2), calmodulin (CAL) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified (Weir et al. 2012; Yang et al. 2009). The resulting sequences were deposited under the GenBank accession numbers OK148894, OM397909, OM249943, OL422149, OM184266 and OM718003 for C2,OK148975, OM397910, OM249944, OL422150, OM184267, OM718004 for gy15, OK148976, OM397911, OM249945, OL422151, OM184268 and OM718005 for Ch. A BLAST search showed that the sequences of isolates C2 and gy15 had 99.57% to 100% similarity to the type strain of Colletotrichum kahawae ICMP12952. The sequences of isolate Ch had 99.03% to 100% similarity to the type strain of Colletotrichum horri ICMP 17968. Further, a phylogenetic tree based on the combined ITS, ACT, CHS, TUB, CAL, and GAPDH sequence using the neighbor-joining algorithm revealed that the isolates were C. kahawae and C. horri (Fig. 1). Pathogenicity assays were conducted on healthy leaves collected from 1-year-old tea-oil tree. The experiments were repeated twice. The leaves were surface-sterilized with 75% ethanol. After drying, they were placed in a plastic box pre-laid with sterilized absorbent paper. The leaf surface was slightly pierced with a sterile needle, and each stab wound was inoculated with 10 µL of conidial suspension (1×106 conidia/ml). All inoculated leaves were placed in a moist chamber at 25℃ with 80% relative humidity. After 10 days, inoculated leaves showed similar symptoms as observed in the field, whereas controls remained symptomless. C. kahawae and C. horri were re-isolated from the diseased leaves, and identifed by sequencing. C. kahawae is widespread on coffee in Africa (Waller et al. 1993). C. horri has been associated with fruit and stem diseases of Diospyros kaki from China, Japan, and New Zealand (Weir et al. 2010). To our knowledge, this is the first report of C. kahawa and C. horri causing anthracnose of tea-oil tree.

Occurrence of root rot caused by Fusarium fujikuroi and Fusarium proliferatum on peanut in China.

Root rot of peanut, caused by Fusarium spp., is a devastating disease in most peanut cultivation regions. In this reported outbreak, Fusarium root rot of peanut has been observed in Henan province, China in July 2021. About 20% of peanuts in a field (0.66 ha) were affected. Early symptoms comprised the wilt of the lower leaves, and the darkening of the vascular tissue of roots, which turned brown. Progressively, the whole plant wilted, the roots rotted, and the plant ultimately died. Pathogenic species were isolated from plants showing symptoms of root rot in the field. A total of 206 Fusarium isolates were generated, and 16 isolates were preliminarily identified as Fusarium fujikuroi based on morphological characteristics. Isolates were obtained and grown on PDA plates. Isolates developed floccose white aerial mycelia with reddish-pink coloration in the medium in 2 weeks on the benchtop. Macroconidia were 3-5 septate, measuring 27.5 to 48.8 × 2.6 to 3.8 µm (avg. 36.7 × 3.6 µm, n=50). Microconidia were abundant in chains, mainly asepatate, oval to kidney-shaped, 4.0 to 11.6 × 2.5 to 4.1 µm (avg. 5.8 ×3.1 µm, n=50). DNA was extracted from mycelium and the following genes were amplified and sequenced: the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al.1990) (Genbank assessions MZ831304 to MZ831308), the partial calmodulin gene (CAM, primer CL1/CL2A, O'Donnell.)(Genbank assession MZ856333 to MZ856337) and the partial translation elongation factor (EF-1α) using primer EF1/EF2 (Geiser et al.)(Genbank assession MZ856338 to MZ8564342). FUSARIUM-ID analysis showed 98.18% to 100% similarity with sequences of the F. fujikuroi species complex. The phylogenetic analysis was conducted using a neighbor-joining algorithm based on the ITS, CAM, and EF-1α gene sequences. The isolates were clustered with F. fujikuroi clade (Supplementary Fig.1). Koch's postulates were conducted using a sand-cornmeal-inoculum-layer method (Bilgi et al.). Briefly, 400 ml plastic boxes were filled with 15g of sterilized premium-grade coarse vermiculite, followed by a 15 g of inoculum prepared as sand-cornmeal mixture inoculum. The inoculum for each F. fujikuroi isolates was prepared by infesting a pre-sterilized sand-cornmeal mixture with three 5 mm plugs of cultures. Three F. fujikuroi isolates and PDA plugs were inoculated to serve as positive control and non-inoculated control treatments, respectively. The completed colonization of the sand-cornmeal mixture was finished by incubating at 25 ℃ for 7-10 days. Eight pre-germinated seeds of cv. Luhua No.1 was then covered with another 8 g of vermiculite. Peanuts were grown at 25 °C with 85% relative humidity under a light/dark cycle of 14h/10h. After 14 days of incubation, the inoculated plants showed typical symptoms of root rot similar to those in the field: pre-emergence damping-off, reddish-brown lesions on the tap, and lateral roots. F. fujikuroi was successfully re-isolated from inoculated plants but not from the controls and identified as described above. F. fujikuroi was reported to cause bakanae disease of rice (Amatulli et. al.), and root rot of soybeans (Zhao et. al.). To the best of our knowledge, this is the first record of F. fujikuroi causing root rot of peanut in China.

Comparative Toxicity and Joint Effects of Chlorantraniliprole and Carbaryl Against the Invasive Spodioptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm, Spodoptera frugiperda, is one of the most devastating invasive pests in China. Chlorantraniliprole (CH) is currently the main agent for controlling S. frugiperda. Carbaryl (CA) has been widely used as a foliar treatment to control S. frugiperda, although the pest has become highly resistant to it. This study investigates the comparative toxicity and joint effects of CH and CA on S. frugiperda. Time-toxicity results showed that CH had high toxicity to 1st and 3rd instar larvae, whereas CA had very low toxicity to 1st and 3rd instar larvae. The mixtures of CH and CA at different mass ratios showed strong synergistic effects on toxicity, and the mass ratio of 2:1 exhibited the highest toxicity to S. frugiperda. Furthermore, the synergistic toxicity of CH and CA at the 2:1 mass ratio (CH+CA) was also verified in field populations of S. frugiperda. The life-history parameters showed that CH+CA dramatically decreased the survival rate and fecundity of the parent population (F0) compared with CH treatment at the same concentration. Besides, CH and CH+CA mixture showed induction effect on cytochrome P450s and glutathione-S-transferases (GSTs) activities in S. frugiperda, with cytochrome P450s enzyme responding the fastest. In conclusion, this research found CH+CA provided synergistic effects on the toxicity and the sublethal effect on larvae. The joint effects on the life-history parameters and the detoxifying enzymes in S. frugiperda, may be useful for implementing IPM programs against this Lepidoptera pest.

Primary Mode of Action of the Novel Sulfonamide Fungicide against Botrytis cinerea and Field Control Effect on Tomato Gray Mold.

Botrytis cinerea is considered an important plant pathogen and is responsible for significant crop yield losses. With the frequent application of commercial fungicides, B. cinerea has developed resistance to many frequently used fungicides. Therefore, it is necessary to develop new kinds of fungicides with high activity and new modes of action to solve the increasingly serious problem of resistance. During our screening of fungicide candidates, one novel sulfonamide compound, N-(2-trifluoromethyl-4-chlorphenyl)-2-oxocyclohexyl sulfonamide (L13), has been found to exhibit good fungicidal activity against B. cinerea. In this work, the mode of action of L13 against B. cinerea and the field control effect on tomato gray mold was studied. L13 had good control against B. cinerea resistant to carbendazim, diethofencarb, and iprodione commercial fungicides in the pot culture experiments. SEM and TEM observations revealed that L13 could cause obvious morphological and cytological changes to B. cinerea, including excessive branching, irregular ramification or abnormal configuration, and the decomposition of cell wall and vacuole. L13 induced more significant electrolyte leakage from hyphae than procymidone as a positive control. L13 had only a minor effect on the oxygen consumption of intact mycelia, with 2.15% inhibition at 50 µg/mL. In two locations over 2 years, the field control effect of L13 against tomato gray mold reached 83% at a rate of 450 g ai ha-1, better than the commercial fungicide of iprodione. Moreover, toxicological tests demonstrated the low toxicological effect of L13. This research seeks to provide technical support and theoretical guidance for L13 to become a real commercial fungicide.

Rapid visual detection of benzimidazole resistance in Botrytis cinerea by recombinase polymerase amplification combined with a lateral flow dipstick.

BACKGROUND: Benzimidazole resistance in Botrytis cinerea is related to point mutations in the target ß-tubulin gene (TUB2). Three mutations (E198A, E198K, E198V) at codon 198 account for most of the resistant strains. A rapid on-site diagnostic assay would be useful to detect the presence and monitor further spread of this resistance mechanism. RESULTS: A recombinase polymerase amplification combined with lateral flow detection (RPA-LFD) method was established for the rapid detection of methyl benzimidazole carbamate (MBC) resistance in B. cinerea. Based on the three mutations at TUB2 codon 198, three sets of RPA-LFD primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation; no amplification was detected with other mutated or wild-type DNA. The assay was optimized for specificity and sensitivity and was shown to detect the presence of 2 × 102 copies µl-1 of target DNA per reaction within 10 min. DNA from eight other common fungal species of small fruit did not yield a signal. The system worked well over a wide range of temperatures from 25 to 45°C. Crude DNA obtained from boiled mycelium and conidia of symptomatic fruit could be used as templates, which simplified the assay process. CONCLUSION: This study developed a novel assay based on RPA-LFD for the rapid and equipment-free detection of MBC-resistant isolates. In combination with a simple DNA extraction method, the assay could detect B. cinerea MBC-resistant isolates even without specialized equipment within 30 min. Considering its specificity, stability and simplicity, the RPA-LFD assay could be a promising tool for rapid on-site diagnosis of fungicide-resistant isolates. © 2021 Society of Chemical Industry.

Controlled-release granules for the delivery of pymetrozine to roots of transplanted rice seedlings with decreased phytotoxicity and enhanced control efficacy against paddy planthoppers.

BACKGROUND: Seedling transplanting is widely used in rice cultivation. Systemic insecticides can be delivered to seedling roots by application through rice seedling boxes before transplanting. The most challenging aspect is to provide long-term control of rice pests and overcome transplanting shock. Precise control of the release rate of insecticide can meet these requirements. Pymetrozine is a promising insecticide used for the control of rice planthoppers resistant to neonicotinoid insecticides. RESULTS: In this study, four controlled-release granular formulations of pymetrozine were prepared based on a mixture of cost-effective and biodegradable kaolin and xanthan gum or a mixture of calcined kaolin and xanthan gum. Fluorescence images showed that different 3D networks were formed in the four granular formulations. The four granular formulations showed different water uptake rates and release rates of pymetrozine in water. Pymetrozine release rate was positively correlated with the water uptake capacity, rather than the water uptake rate of granules. Diffusion was the dominant mechanism for the release of pymetrozine from granules. Pymetrozine was found to reduce the survival of transplanted rice seedlings suffering from transplanting shock. Incorporating pymetrozine in controlled-release granules alleviated this phytotoxicity. The survival rate of rice seedlings in granular pymetrozine treatments ranged 68.8-85.0%, whereas the survival rate was <50% for powdered pymetrozine treatments. Additionally, four prepared granule formulations had a significant control effect on rice planthopper with efficacies ranging from 76.7% to 98.0% 40 days after seedling box treatment. CONCLUSIONS: The granule with an intermediate release rate of pymetrozine was shown to be more suitable for seedling box treatment than field application and traditional liquid spraying for the long-term control of paddy planthoppers. © 2021 Society of Chemical Industry.

Broadcasting of tiny granules by drone to mimic liquid spraying for the control of fall armyworm (Spodoptera frugiperda).

BACKGROUND: The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is an invasive pest. Maize is one of its preferred host plants. The traditional application of granules has been effective for the control of FAW. Nevertheless, the challenge is to develop an application method that is less labor-intensive and also efficient. RESULTS: Tiny granules in the size range of 0.38-0.55 mm were prepared by the centrifugal granulation method for drone application. The in-swath distribution patterns of granules deposited on sticky tapes were similar to the distribution of aqueous droplets through drone spraying. In the range of 1.5-3.5 m, a relatively higher aircraft height of flight can provide a more uniform distribution pattern of deposited granules, but resulted in less deposit of tiny granules in the whorl of maize plants. Similarly, the deposit of granules in the whorl of maize plants significantly decreased when the flight speed increased from 4 to 6 m s-1 . Granules gathered in the whorl accounted for 50.8-58.7% of total granules deposited in the maize canopy. The field experiment demonstrates granular formulations (containing 0.25% chlorantraniliprole + 0.15% emamectin benzoate, or higher concentrations) can provide better and longer duration of FAW control than the aqueous spray formulation at the whorl stage of maize plants. CONCLUSION: Broadcasting of tiny granules by drone can provide better FAW control than liquid spray. The architecture of the maize plant and the downward airstream of the drone create favorable conditions for the deposition of granules in whorls. © 2021 Society of Chemical Industry.

Factors Underlying the Prevalence of Pythium Infection of Corn Seeds Following Seed Treatment Application of Tebuconazole.

Microbial communities are essential for soil health, but fungicide application may have significant effects on their structure. It is difficult to predict whether nontarget pathogens of applied fungicides in the soil will cause crop damage. Tebuconazole is a triazole fungicide that can be used as a seed treatment and, thereby, introduced to the soil. However, seed-applied tebuconazole has a potential risk of causing poor emergence of corn (Zea mays) seedlings. Using soil with a history of poor corn seedling emergence, we demonstrate through TA cloning and isolation that the poor emergence of corn seedlings from tebuconazole-coated corn seeds was primarily because of infection by surviving soil pathogens, specifically Pythium species that are not targeted by tebuconazole, rather than the phytotoxic effects of tebuconazole. Bioassay tests on tebuconazole-amended media showed that tebuconazole can suppress soil fungi while allowing Pythium to grow. Pythium species primarily contributing to the corn seed rot were more pathogenic at cooler temperatures. Furthermore, the nontarget biocontrol agent of Trichoderma spp. was strongly inhibited by tebuconazole. Taken together, the nontarget effects of tebuconazole are likely not significant under favorable plant growing conditions but are considerable because of low-temperature stress.

Synergistic Effects of Clonostachys rosea Isolates and Succinate Dehydrogenase Inhibitors Fungicides against Gray Mold on Tomato.

Gray mold caused by Botrytis cinerea is a devastating disease in tomatoes. Site-specific fungicide application is still key to disease management; however, chemical control has many drawbacks. Here, the combined application of a biological agent, Clonostachys rosea, with newly developed succinate dehydrogenase inhibitors (SDHI) fungicides showed stronger synergistic effects than the application of SDHI fungicides alone on tomato gray mold control. C. rosea 67-1 has been reported as an efficient biological control agent (BCA) for B. cinerea. Little information is currently available about the combination of C. rosea and fungicides in the control of gray mold. By testing the sensitivity to fungicides with different action mechanisms, C. rosea isolates showed high tolerance to SDHI fungicides (1000 µg mL-1) on PDA, and the conidial germination rate was almost not affected under 120 µg mL-1 of fluxapyroxad and fluopyram. In greenhouse experiments, the control effect of the combination of C. rosea and fluxapyroxad or fluopyram against tomato gray mold was significantly increased than the application of BCA or SDHI fungicides alone, and the combination allows a two-fold reduction of both the fungicide and BCA dose. Further, the biomass of B. cinerea and C. rosea on tomato plants was determined by qPCR. For B. cinerea, the trend of detection level for different treatments was consistent with that of the pot experiments, and the lowest biomass of B. cinerea was found when treated with C. rosea combined with fluxapyroxad and fluopyram, respectively. For C. rosea, qPCR assay confirmed its colonization on tomato plants when mixed with fluopyram and fluxapyroxad. These results indicated that combining C. rosea 67-1 with the SDHI fungicides could synergistically increase control efficacy against tomato gray mold.

Field-evolved resistance to 11 insecticides and the mechanisms involved in Helicoverpa armigera (Lepidoptera: Noctuidae).

BACKGROUND: To understand the ongoing resistance of cotton bollworm, Helicoverpa armigera, the sensitivity of five field populations to commonly used insecticides, indoxacarb, abamectin, methoxyfenozide, chlorfenapyr, chlorantraniliprole, spinetoram, lambda-cyhalothrin, carbosulfan, metaflumizone, chlorpyrifos, and flufenoxuron, were evaluated. Furthermore, the biochemical and molecular mechanisms of field-evolved resistance in H. armigera were also investigated. RESULTS: Five field populations of H. armigera showed moderate resistance to indoxacarb, chlorantraniliprole, metaflumizone, methoxyfenozide, carbosulfan and lambda-cyhalothrin. The resistance ratio (RR) of indoxacarb was significantly correlated with glutathione-S-transferases (GSTs) activity (r = 0.913, P = 0.011). Methoxyfenozide RR was largely correlated with cytochrome P450s activity (r = 0.860, P = 0.028). Besides, six cytochrome P450s genes of CYP4L5 in AQP, CYP6B7 and CYP9A14 in HDP and BDP, CYP9A17V2 in HDP and YSP, CYP332A1 in HDP, LFP, AQP and YSP, CYP337B1 in YSP, and two GSTs genes of GSTd1 and GSTs1 in HDP were overexpressed (>5-fold). Moreover, indoxacarb RR was positively correlated with the overexpression of GSTs1, GSTd1 and CYP9A14 genes (r = 0.880, 0.98 and 0.86, P = 0.021, 0.001 and 0.028, respectively). The transcript of CYP9A17V2 and CYP337B1 were found to be correlated with metaflumizone RR (r = 0.950, P = 0.004) and carbosulfan RR (r = 0.850, P = 0.033), respectively. CONCLUSION: H. armigera can be effectively controlled using abamectin, chlorfenapyr, chlorpyrifos and spinetoram in Hebei and Shandong provinces. The present study demonstrated that the relative expression level of GSTs1, GSTd1, CYP9A14, CYP9A17V2 and CYP337B1 genes were significantly correlated with the resistance ratio to indoxacarb, metaflumizone and carbosulfan in field H. armigera.

First Report of Pythium torulosum Causing Corn Root Rot in Northeastern China.

In October 2017, we collected five soil samples from each of several fields with a history of severe corn (Zea mays) seedling disease in Heilongjiang province of China. Affected seedlings were wilted with severe root rot, and a high incidence of seedling death was observed in the fields. Corn seeds were seeded in the collected soil samples and grown in a growth chamber for 21 days set at the following incubation temperatures: 21℃/7℃ for 6 days, 10℃/3℃ for 4 days, 16℃/7℃ for 5 days, 20℃/20℃ for 6 days (16 h/8 h, light/dark) (Tang et al. 2019). The corn seedlings in the growth chamber showed the same symptoms observed in the field as mentioned above. Corn root rot samples were collected from several symptomatic plants in the growth chamber to isolate the possible pathogen. Symptomatic roots were washed in 0.5% NaOCl for 2 min, rinsed in sterile water and cut into 1-2 mm segments and then plated on corn meal agar amended with pimaricin (5 µg/ml), ampicillin (250 µg/ml), rifampicin (10 µg/ml), pentachloronitrobenzene (50 µg/ml), and benomyl (10 µg/ml) (PARP+B), which is selective for oomycetes (Jeffers and Martin 1986). After 3 days of incubation in the dark at 25℃, colonies were transferred to 10% V8 juice agar and incubated at 25℃ for 2 weeks. Six isolates were identified as Pythium torulosum based on the morphology of sexual and asexual structures following van der Plaats-Niterink's key (van der Plaats-Niterink 1981). On 10% V8 juice agar, the hypha were aseptate and colonies had filamentous sporangia with a dendroid or globose structure. The oogonia were globose or subglobose, laevis, terminal, rarely intercalary, ranging from 12-19 (average 16) µm. Antheridia were mostly sessile or brachypodous, and each oogonium was supplied by 1-2 antheridia cells. Oospores were globose, plerotic, ranging from 9-16 (average 13) µm. For the molecular identification, two molecular targets, the internal transcribed spacer (ITS) region of ribosomal DNA and cytochrome c oxidase subunit II (CoII), were amplified and sequenced using universal primer sets DC6/ITS4 (Cooke et al. 2000) and FM58/FM66 (Villa et al. 2006), respectively for one isolate, "copt". BLAST analyses of a 971 bp ITS segment amplified from copt (GenBank Accession No. MT830918) showed 99.79% identity with a P. torulosum isolate (GenBank Accession No. AY598624.2). For the COⅡ gene of copt, BLAST analyses of a 553 bp segment (GenBank Accession MT843570) showed 98.37% identity with P. torulosum isolate (GenBank Accession No. AB095065.1). Thus, the isolate, copt, was identified as P. torulosum based on morphological characteristics and molecular analysis. To confirm pathogenicity and Koch's postulates, a pathogenicity test was conducted as described by Zhang et al. (2000). Briefly, a 5 mm culture plug from the P. torulosum isolate, copt, was transferred to a 9-cm petri dish containing 20mL 10% V8 juice agar and incubated in the dark at 25℃ for 7 days. The culture was cut into small pieces and mixed with a sterilized soil mix (40% organic peat substrate, 40% perlite, and 20% soil) at a ratio of one petri dish per 100 g soil mix. Ten corn seeds were planted at a depth of 2 cm in a 500-mL pot containing the inoculated soil mix. The control pots were mock inoculated with plain 10% V8 juice agar. Pots were incubated in a greenhouse at temperatures ranging from 21 to 23℃. There were four replications. After 14 days, corn roots brown and rotted were observed, which was similar to those observed in the field and growth chamber. Control plants remained symptomless and healthy. P. torulosum copt was consistently re-isolated from the symptomatic roots. To our knowledge, this is the first report of P. torulosum causing root rot of corn in Northeastern China. Corn is an important crop in Heilongjiang and the occurrence of root rot caused by this pathogen may be a new threat to corn plants. There is a need to develop management measures to control the disease.

Mechanisms of Increased Indoxacarb Toxicity in Methoxyfenozide-Resistant Cotton Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae).

Indoxacarb is an important insecticide for the selective control of Helicoverpa armigera. It can be bioactivated to the more effective N-decarbomethoxylated indoxacarb (DCJW) by esterases in pests. It was observed that both field and laboratory selected populations of H. armigera showed negative cross-resistance between indoxacarb and methoxyfenozide. The Handan population exhibited moderate resistance to indoxacarb, but was susceptible to methoxyfenozide; the Baoding and Yishui populations exhibited moderate resistance to methoxyfenozide, but they were susceptible to indoxacarb. Moreover, the toxicity of indoxacarb was enhanced 1.83-fold in the laboratory methoxyfenozide-resistant H. armigera, and susceptibility to methoxyfenozide was increased 2.81-fold in the laboratory indoxacarb-resistant H. armigera. In vivo, DCJW concentrations in the susceptible and methoxyfenozide-selected (laboratory methoxyfenozide-resistant) populations were 4.59- and 4.31-fold greater than in the indoxacarb-resistant Handan population 1 h after dosing. After 2 h, the highest concentrations of DCJW and indoxacarb appeared in the methoxyfenozide-selected population. Meanwhile, increased carboxyl esterase (CarE) and decreased glutathione S-transferase (GST) activities were observed in the methoxyfenozide-selected population. However, the indoxacarb-selected (laboratory indoxacarb-resistant) and Handan populations showed a higher disappearance of indoxacarb and DCJW, and the activity of cytochrome P450 mono-oxygenase in these populations were significantly increased. This study showed that the improved toxicity of indoxacarb, as observed in the methoxyfenozide-selected H. armigera, was correlated with increased CarE activity, decreased GST activity, and the in vivo accumulation of indoxacarb and DCJW. The significantly increased cytochrome P450 activity and higher disappearance of indoxacarb and DCJW in indoxacarb-resistant H. armigera resulted in the decreased toxicity of indoxacarb.