Variations of phyllosphere microorganisms in asymptomatic and tobacco brown spot leaves before and after spraying 12% difenoconazole + fluxapyroxad SC

12%difenoconazole+fluxapyroxad SC(commercial name:Jiangong)was first released by BASF in China in 2016.It has been registered to control many diseases,including pear scab,apple Alternaria leaf spot,tomato early blight,cucumber powdery mildew,etc.This study evaluated the bioactivity of Jiangong against Alternaria alternata and explored variations of phyllosphere microorganisms in both asymptomatic and tobacco brown spot leaves at different persistence periods(0,5,10,and 15 days post-fungicide application)using high-throughput sequencing technology.The results indicated that Jiangong effectively inhibited mycelial growth(average EC_(50) value of 0.51μg/mL),conidia germination(average EC_(50) value of 3.47μg/mL),and the carbon metabolism of A.alternata.Both asymptomatic and symptomatic leaves presented complex microbial communities.Higher fungal diversity was noted in asymptomatic leaves,while higher bacterial diversity was found in symptomatic leaves.After application,the diversity and abundance of microbial community structures in both types of leaves changed over time.Fungal microbiome communities showed greater sensitivity than bacterial groups,with the microbiome communities of asymptomatic leaves being more affected than those of symptomatic leaves.Fungal community diversity decreased for both symptomatic and asymptomatic leaves after 5 days of application,while the diversity of fungal community in symptomatic leaves showed an upward trend after 10 days of application.Meanwhile,bacterial community diversity increased in both symptomatic and asymptomatic leaves after 5 days of application but then declined in asymptomatic leaves after 15 days.The abundance of the dominant function group of phyllosphere bacteria(metabolism,genetic information processing,environmental information processing)was not affected by the application of Jiangong.However,the abundance of the dominant function group of phyllosphere fungi(animal pathogen-endophyte-wood saprotroph,endophyte-plant pathogen,plant pathogen-undefined saprotroph)was significantly affected by the application of Jiangong,and high variation was found in symptomatic leaves than that of asymptomatic leaves.The application of Jiangong-induced alterations in the community structure of the tobacco phyllosphere microbiome provides a basis for future tobacco brown spot control strategies based on phyllospheric microecology.

Degradation Dynamics of Triadimefon and Difenoconazole Residues in Strawberry and Soil

A field experiment was employed to study the degradation of triadimefon and difenoconazole residues in strawberry and soil. The results showed that the degradation of both the two kinds of pesticides was in accordance with the first order kinetic equation. The half-lives of difenoconazole in strawberry fruit, strawberry stem leaf and soil were 4.09, 8.21 and 13.38 d, respectively; and the half-lives of triadimefon in strawberry fruit, strawberry stem and leaf and soil were 3.38, 5.31 and 15.64 d, respectively. The maximum residue limit （MRL） of difenoconazole in strawberry was recommended as 0.5 mg/kg, and 10% difenoconazole WG and 20% triadimefon EC should be applied four times with a safety interval of 7 d according to the amounts of 100 and 60 g a. i./hm2,respectively.

Researches on Methodologies to Verify Reduced Sensitivities of <i>Venturia inaequalis</i>in Field to Difenoconazole and First Indications of a Survey in Italy

Resistance of Venturia inaequalis to the first IBS triazoles occurred in the 80’s in Italy and other countries. Successively number of applications sensibly decreased and problems of resistance to IBS generally disappeared. In 2010 suspects of resistance to the newer IBS difenoconazole, the actually most used IBS, appeared in several countries. In vitro tests the results confirmed the previous researches: the monoconidia from populations which were poorly controlled showed moderate reduced sensitivity respect to well controlled and wild-types. New in vivo tests were realized with preventive and curative applications on seedlings. No different activity among the three types of populations (poorly, well-controlled and wild-types) was found in preventive applications. On the contrary, in curative applications, wild-types and well controlled populations showed a visible and statistically significant activity respect to the poorly controlled. A survey of sensitivity to difenoconazole was carried out in two Italian apple areas in 2013-2015 and showed the presence of populations resistant to difenoconazole. Probably the cause of the resistance spread was the increase of the number of applications of this IBS in the last years. However the problem appears more complex because nowadays resistance regards about all fungicides and with a higher severity respect previous decades. An increase of virulence of V. inaequalis strains appears to be the cause of this situation also for the high presence of cultivars with high or moderate susceptibility to scab. Foreseeable remedies consist in a differentiate and appropriate setting of each chemical groups of fungicides in apple scab control, but in long terms new strategies in apple cultivation should be necessary.

A New Synthesis Process of Highly Efficient and Low Toxic Fungicide Difenoconazole

A new synthesis method of difenoconazole is studied.Difenoconazole is prepared from m-dichlorobenzene by Friedel Crafts reaction,cyclization,bromination,nucleophilic substitution and etherification.The new process improves the selectivity of the reaction and simplified the pu-rification process.The total yield of this process is 84%,which provides a green and economical synthetic route for industrialization.

Bioaccumulation and the expression of hepatic cytochrome P450 genes in marine medaka(Oryzias melastigma)exposed to difenoconazole

This study was conducted to assess the effects of difenoconazole（DFZ）, a triazole fungicide,on the hepatic biotransformation system and its bioaccumulation in marine medaka（Oryzias melastigma）. Fish were exposed to DFZ（1, 10, 100, 1000 ng/L） for 180 days. The results showed that：（1） The m RNA levels of hepatic CYP1A1, CYP1 B, CYP1C1, CYP27 B and CYP3A40 were up-regulated, but those of CYP3A38 and CYP27A1 were down-regulated.（2） The activity of ethoxyresorufin-O-deethylase（EROD） and the content of reduced glutathione（GSH） in the liver were increased in the DFZ-treated groups, and glutathione S-transferase（GST） activity was increased in the 100 and 1000 ng/L groups.（3） DFZ was accumulated in the muscle and the biological concentration factors in the 10, 100, and1000 ng/L groups were respectively 149, 81 and 25. These results suggested that long-term exposure to DFZ at low concentrations would result in a bioaccumulation of this compound and disturb the biotransformation system.

Dissipation pattern of azoxystrobin and difenoconazole in red dragon fruit(Hylocereus polyrhizus)cultivated in Indonesian highland(West Java)and coastal area(D.I.Jogyakarta)and its implication for dietary risk assessment

Background:Cultivation of red dragon fruit(Hylocereus polyrhizus)in Indonesian orchards is hampered by anthracnose and stem canker.A mixture of azoxystrobin 200 g/L+difenoconazole 125 g/L is recommended in supporting the obligated integrated pest management.For the consumer safety,dietary intake of the residues from fresh edible part should be assessed;therefore,information on correct dissipation pattern of the applied pesticide is required.Materials and Methods:Field residue trials in Indonesian highland(West Java)and coastal area(Special Region of Yogyakarta)were done at recommended dose was 0.151 kg ai/ha azoxystrobin and 0.094 kg ai/ha difenoconazole,three times at 10 days interval.Whole fruit samples were collected at 70%-100%ripeness at−1,0,1,3,7,10,and 14 days after last application,processed and determined its residue as soon as possible.Result and Discussion:Photoisomerization of azoxystrobin was observed at Day 0,especially in coastal area;however,it has been dissipated to below lowest validated level in the following day.At Day 0,3%-5%of the deposited azoxystrobin and difenoconazole penetrate into the flesh;however,most residues(95%-97%)were retained in peel,and dissipated in prolonged day.The dissipation pattern was non-linear.The dissipation data were better fit with bi-exponential double-first-order in parallel than single first-order kinetics model.The DT50 of both azoxystrobin and difenoconazole was 3 days.At harvest time,seventh day,only azoxystrobin residue was detected in flesh at 0.006 mg/kg;therefore,the long-term dietary risk was 0 per cent acceptable daily intake.Conclusion:Fresh red dragon fruit is safe to consume.

Exposure to difenoconazole inhibits reproductive ability in male marine medaka(Oryzias melastigma)

Difenoconazole（DFZ） is a triazole fungicide which has been detected in the aquatic environment, including estuaries and embayments. However, few studies addressing the reproductive toxicity and transgenerational effects of DFZ on marine fishes are available.The present study was conducted to investigate the effects of DFZ on male marine medaka（Oryzias melastigma）. After exposure of the embryo to 1, 10, 100 and 1000 ng/L DFZ for180 days, the gonadosomatic index was significantly decreased in the 1000 ng/L treatment.The number of sperm was reduced while the abundances of spermatocytes and spermatogonia in the testes were increased in all the treatments. The m RNA levels of salmontype gnrh（sgnrh）, the luteinizing hormone（lhβ） and the follicle-stimulating hormone（fshβ）genes in the brain all exhibited a significant down-regulation, the expression of androgen receptors（arα and arβ） was decreased and that of estrogen receptor β and cytochrome P450 aromatase（cyp19 B） was increased in the testes. The expression levels of cyp19 A and cyp19 B were increased in the liver. The decrease of ars m RNA levels might be one of the reasons causing the reduction of sperm. The down-regulation of sgnrh, lhβ and fshβ m RNA levels suggested that DFZ might impact the spermatogenesis via the brain–pituitary–gonad pathway. The decrease of the fertilization success, the hatch ability and the swim-up success in the F1 generation indicated that DFZ pollution at environmental levels might cause a decrease of wild fish populations.

Rapid and sensitive determination of difenoconazole in cucumber and pear samples using an immunochromatographic assay

Difenoconazole is triazole fungicide,widely used to prevent the growth and reproduction of fungi and to control fungal diseases in fruits and vegetables.In this work,we prepared a hapten of difenoconazole and produced a highly-sensitive anti difenoconazole-monoclonal antibody(mAb)with an IC50 of 0.64 ng/mL.Using the mAb,an immunochromatographic assay(ICA)was developed to analyze difenoconazole residues in pear and cucumber samples.The ICA exhibited LOD values of 2.60 and 2.30 ng/g for difenoconazole in pear and cucumber samples,respectively.The ICA showed average recoveries of difenoconazole ranging from 94.3%±3.6%–105.4%±1.6%,and CVs of 1.5–4.2%in cucumber and pear samples.When used to measure difenoconazole in samples,the ICA results were compatible with those detected by LC/MSMS.The results of this study support the idea that an ICA is a practical and effective tool for high-throughput and rapid analysis of difenoconazole residues in vegetables and fruits.

Water-Based Environmentally Friendly Pesticide Formulations Based on Cyclodextrin/Pesticide Loading System

Difenoconazole(DIF)is a representative variety of broad-spectrum triazole fungicides and liposoluble pesticides.However,the water solubility of DIF is so poor that its application is limited in plant protection.In addition,the conventional formulations of DIF always contain abundant organic solvents,which may cause pollution of the environment.In this study,two DIF/cyclodextrins(CDs)inclusion complexes(ICs)were successfully prepared,which were DIF/β-CD IC and DIF/hydroxypropyl-β-CD IC(DIF/HP-β-CD IC).The effect of cyclodextrins on the water solubility and the antifungal effect of liposoluble DIF pesticide were investigated.According to the phase solubility test,the molar ratio and apparent stability constant of ICs were obtained.Fourier transform infrared spectroscopy,thermal gravity analysis,X-ray diffraction and scanning electron microscopy were used systematically to characterize the formation and characteristics of ICs.The results noted that DIF successfully entered the cavities of two CDs.In addition,the antifungal effect test proved the better performance of DIF/HP-β-CD IC,which exceeded that of DIF emulsifiable concentrate.Therefore,our study provides informative direction for the intelligent use of liposoluble pesticides with cyclodextrins to develop water-based environmentally friendly formulations.

应用GC-MS测定葡萄中的5种农药残留

酿酒葡萄是河北昌黎的主要水果种植品种,由于受海洋气候的影响,当地空气湿热,葡萄病害十分严重,因而农药使用十分频繁,为了评价其农药残留水平,我们采用丙酮提取,凝胶分离,硅胶小柱净化,GC-MS的选择离子方式检测对采集的葡萄样品进行5种农药(百菌清、精甲霜灵、苯醚甲环唑、嘧菌酯、高效氯氟氰菊酯)的残留分析.……

Control Effect of Four Kinds of Agents for Anthracnose in Autumn Greenhouse Cowpea

In order to mitigate the occurrence and damage of cowpea anthracnose and reduce the chemical pesticide consumption, residue and environmental pollution, the methods of forecasting, randomized block design and statistical analysis were conducted to study the control effect of biopesticide chitosan, biochemical compound Prochloraz-Chitosan and chemical pesticides difenoconazole and thifluzamide by field efficacy trials. The results showed that under serious occurrence of anthracnose in autumn greenhouse cowpea, when the 2% chJtosan AS （2 250 g/hm^2）, 46% Prochloraz-Chitosan EW （450 g/hm^2）, 10% difenoconazole WG （900 g/hm^2, CK） or 24% thifluzamide SC （360 ml/hm^2） were applied three times with an interval of 5-8 d, the control efficiency on day 7, 14 and 20 reached 83%, 78% and 73%, respectively. No significant difference was found in control efficiency among the four kinds of agents. These four kinds of agents, especially chitosan and Prochloraz-Chitosan, could be used as ideal agents for controlling anthracnose, as well as for modern agricultural demonstration zones, pollution-free agricultural products, green agricultural products and organic agricultural products,thereby meeting the develop- ment needs of ＇modern agriculture, green agriculture, organic agriculture and ecological agriculture.