Chemical preparation, biological evaluation and 3D-QSAR of ethoxysulfuron derivatives as novel antifungal agents targeting acetohydroxyacid synthase.

Accetohydroxyacid synthase (AHAS) is the first enzyme involved in the biosynthetic pathway of branched-chain amino acids. Earlier gene mutation of Candida albicans in a mouse model suggested that this enzyme is a promising target of antifungals. Recent studies have demonstrated that some commercial AHAS-inhibiting sulfonylurea herbicides exerted desirable antifungal activity. In this study, we have designed and synthesized 68 novel ethoxysulfulron (ES) derivatives and evaluated their inhibition constants (Ki) against C. albicans AHAS and cell based minimum inhibitory concentration (MIC) values. The target compounds 5-1, 5-10, 5-22, 5-31 and 5-37 displayed stronger AHAS inhibitions than ES did. Compound 5-1 had the best Ki of 6.7 nM against fungal AHAS and MIC values of 2.5 mg/L against Candida albicans and Candica parapsilosis after 72 h. A suitable nematode model was established here and the antifungal activity of 5-1 was further evaluated in vivo. A possible binding mode was simulated via molecular docking and a comparative field analysis (CoMFA) model was constructed to understand the structure-activity relationship. The current study has indicated that some ES derivatives should be considered as promising hits to develop antifungal drugs with novel biological target.

Movement patterns in Entomopathogenic nematodes: Continuous vs. temporal.

To exploit resources, animals implement various foraging behaviors to increase their fitness. Entomopathogenic nematodes are obligate parasites of insects in nature. In previous studies, entomopathogenic nematodes were reported to exhibit group movement behavior in the presence and absence of insect hosts. However, it was not determined if group movement is continuous or temporal. For example, nematode movement behavior upon emergence from the host might start out in an independent fashion prior to aggregation, or group movement may be exhibited continuously. In the present study, we explored the propensity for innate group movement behavior of two insect parasitic nematodes in two families and genera: Heterorhabditis indica and Steinernema carpocapsae. We hypothesized the nematode populations would initially move independently from their origin and then come together for group movement. Movement patterns were investigated in sand when nematodes were applied in aqueous suspension (via filter paper) to a specific locus or when the nematodes emerged naturally from infected insect hosts. To compare nematode movement behavior over time and space, nematode dispersal was monitored at three distances (2.5, 4.5 and 8.0 cm) from the center (origin) and at two different time periods, 2 days and 3 days after nematode addition. We discovered that nematode dispersal continuously exhibited an aggregative pattern (independent movement was not observed). Results from both nematode species as well as the host-cadaver and filter paper (aqueous nematode suspension) application methods indicated a continuous aggregative pattern. The discovery of continuous aggregative movement patterns in steinernematid and heterorhabditid nematodes elucidates further the complexity of their foraging behavior and may serve as basis for exploring foraging behavior in other host-parasite systems.

A Comparison of Novel Entomopathogenic Nematode Application Methods for Control of the Chive Gnat, Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga Yang & Zhang (Diptera: Sciaridae) is the most serious pest of Chinese chive, Allium tuberosum Chemical pesticide application has become a necessary step to control B. odoriphaga after each of the four to six harvests during the growing season. We compared the effects of application type (nematode-infected cadaver, aqueous nematode suspension, and mixture of cadaver and aqueous suspension) and nematode species (Heterorhabditis bacteriophora vs. Steinernema carpocapsae) on B. odoriphaga control. Nematode species combinations and the use of the cadaver method has only been tested in a relatively few studies, and has not been tested for this target pest, B. odoriphaga. Furthermore, this is the first report of combining application methods (aqueous and cadaver). Results indicated that the cadaver treatment produced higher mortality of B. odoriphaga than the aqueous treatment, and H. bacteriophora caused higher mortality of B. odoriphaga than S. carpocapsae. The mortality of B. odoriphaga was 96.7% in H. bacteriophora cadaver treatment and only 27.5% in control treatment without nematode addition. Cadaver treatments caused higher biomass of A. tuberosum than that of aqueous treatment. The plant biomass in H. bacteriophora cadaver treatment was 10 folds as much as that in the control treatment. The mixed aqueous suspension of H. bacteriophora and S. carpocapsae did not increase the mortality in pot trial. Our findings indicate that entomopathogenic nematodes applied as cadavers might be a potential alternative biological agent in the integrated management of B. odoriphaga for Chinese chive production.

Desiccation and cold storage of Galleria mellonella cadavers and effects on in vivo production of Steinernema carpocapsae.

BACKGROUND: Direct application of insect cadavers infected with entomopathogenic nematodes (EPN) can successfully control target pest insects. Little is known about the effects of environmental factors (desiccation and temperature) on the production process for infective juveniles (IJ) in insects. RESULTS: We examined the effects of desiccation time and cold storage (6.7 °C) on IJ production of the nematode Steinernema carpocapsae in Galleria mellonella cadavers at 30.8 and 57% humidity. Under desiccation, the IJ yield in cadavers increased gradually and reached a maximum on day 5. IJ yield gradually declined from day 6 onwards and was almost zero by day 15. In general, cold storage at 6.7 °C caused negative effects on IJ production in desiccated cadavers. Approximately 56 h post infection was the time at which nematodes were most sensitive to low temperatures during development in cadavers. Five-day desiccated cadavers generated higher mortality and more rapid death of Galleria mellonella larvae than using newly (day 0) desiccated cadavers. CONCLUSION: This study describe methods of optimizing rearing techniques such as desiccation and cold storage to promote the mass production and application of EPN- infected host cadavers for the field control of insect pests.

Potential antagonistic effects of nine natural fatty acids against Meloidogyne incognita.

Fatty acids, the essential components of life, were widely present in various seed cakes, gutter oil, and other resources. The objective of this study was to evaluate the potential antagonistic effects of nine fatty acids (FAs) against Meloidogyne incognita (root-knot nematodes). The results showed that butyric, caprylic, capric, lauric, myristic, palmitic, and oleic acids significantly reduced M. incognita reproduction, whereas cucumber (Cucumus sativus) biomass was not adversely affected by the tested FAs and was even significantly increased in several fatty acids treatments. All nine tested fatty acids showed apparent inhibitory effects on egg hatching on day 21, especially capric acid with which the hatching rate was reduced to 15.8% as compared to that using sterile distilled water. Caproic, caprylic, capric, lauric, myristic, and palmitic acids caused significantly higher mortality of the second-stage juvenile of M. incognita than the other three FAs, and both caprylic and capric acids resulted in approximately 50% mortality (2000 µmol/L) after a 24 h exposure. In conclusion, fatty acids showed the nematicidal effect differently, among which capric acid showed a strong nematicidal effect and might be a powerful active substance for integrated M. incognita management. Given the general nematicidal properties of FAs, farmers might utilize waste resources, such as oil seed cake, gutter oil, etc., containing various FAs or use pure FAs for effective M. incognita management.

[Effects of root-knot nematodes on cucumber leaf N and P contents, soil pH, and soil enzyme activities].

A pot experiment was conducted to study the effects of inoculation with root-knot nematodes on the cucumber leaf N and P contents, and the rhizospheric and non-rhizospheric soil pH and enzyme activities. The rhizospheric soil pH didn't have a significant decrease until the inoculation rate reached 6000 eggs per plant. With the increase of inoculation rate, the leaf N and P contents, rhizospheric soil peroxidase activity, and rhizospheric and non-rhizospheric soil polyphenol oxidase activity all decreased gradually, rhizospheric soil catalase activity was in adverse, non-rhizospheric soil pH decreased after an initial increase, and non-rhizospheric soil catalase activity had no regular change. After inoculation, rhizospheric soil urease activity decreased significantly, but rhizospheric and non-rhizospheric soil phosphatase activity and non-rhizospheric soil peroxidase activity only had a significant decrease under high inoculation rate. In most cases, there existed significant correlations between rhizospheric soil pH, enzyme activities, and leaf N and P contents; and in some cases, there existed significant correlations between non-rhizospheric soil pH, enzyme activities, and leaf N and P contents.

Combining maxRatio analysis with real-time PCR and its potential application for the prediction of Meloidogyne incognita in field samples.

Diagnosing and quantifying plant-parasitic nematodes is critical for efficient nematode management. Several studies have been performed intending to demonstrate nematode quantification via real-time quantitative PCR. However, most of the studies used dilution of DNA templates to make standard curves, while few studies used samples with different nematode numbers to make the standard curve, resulting in a high standard error. The objective of the present study was to develop a high quality standard curve using samples containing different numbers of the root-knot nematode Meloidogyne incognita and evaluate the results of real time qPCR with maxRatio analysis. The results showed that a high quality standard curve was obtained with different nematode numbers using specific primers and cycle threshold (Ct)-PCR (R²=0.9962, P<0.001, n=9). With the maxRatio analysis, the fractional cycle number (FCN)-PCR cycle curve and adjusted FCN (FCNadj)-PCR cycle curve had similar patterns as those of the Ct-PCR cycle curve. For quantification of nematodes in field soil samples, qPCR estimations with a FCNadj-PCR cycle standard curve was very close to microscope counting of second-stage juveniles (R²=0.9064, P<0.001, n=10), qPCR estimations with a FCN-PCR cycle standard curve was comparably good (R²=0.8509, P<0.001, n=10), and the biases with a Ct-PCR cycle standard curve were large (R²=0.7154, P<0.001, n=10). Moreover, we found that the concentration of Triton X-100 had less of an effect on FCN as compared to Ct, with delta FCN 0.52, and delta Ct 3.94 at 0.8% Triton. The present study suggests, that combined with maxRatio methods, real time qPCR could be a practical approach for quantifying M. incognita in field samples.