Tianpei YANG，Jigang FU，Guangjin ZOU，Tingqing WANG，Jun TANG，Youhua LONG，Ji ZHANG*.Qiandongnan Tobacco Company，Kaili 556000，China；2.College of Agriculture，Guizhou University，Guiyang 550025，China；3.Guizhou Key Laboratory for Tobacco Quality，Guiyang 550025，China

A Preliminary Study on the Resistance to Different Fungicides in Phytophthora parasitica var.nicotianae

Tianpei YANG1，Jigang FU1，Guangjin ZOU1，Tingqing WANG1，Jun TANG1，Youhua LONG2，3，Ji ZHANG1*
1.Qiandongnan Tobacco Company，Kaili 556000，China；
2.College of Agriculture，Guizhou University，Guiyang 550025，China；
3.Guizhou Key Laboratory for Tobacco Quality，Guiyang 550025，China

［Objective］This study aimed to investigate the resistance to different fungicides in Phytophthora parasitica var.nicotianae. ［Method］Under indoor incubation conditions，the resistance to dimethomorph，metalaxyl·mancozeb，propamocarb and ovraclostrobin·dimethomorph in P.parasitica strain isolated from Zhenyuan County in Qiandongnan State was analyzed with colony growth measurement method.［Result］P.parasitica exhibited different levels of sensitivity to four fungicides.To be specific，P.parasitica exhibited the highest resistance to dimethomorph，and EC50reached 1.19 μg/ml.［Conclusion］In Zhenyuan tobacco-growing area，long-term single use of dimethomorph possesses certain resistance risk in prevention and control of black shank disease.

Qiandongnan State；Phytophthora parasitica var.nicotianae；Drug resistance；Fungicide

A s the sole pathogen causing tobacco black shank，Phytophthora parasitica var.nicotianae （Breda de Haan）Tucker has become of the major oomycete diseases seriously endangering tobacco production in China［1］.In 2011，Ding et al.［2］found that tobacco black shank disease caused huge losses to tobacco production in Guizhou Province and occurred frequently with tobacco bacterial wilt disease.Generally，the incidence rate of tobacco black shank disease in tobacco-planting plots is approximately 20%.In serious cases，tobacco black shank disease can lead to total crop failure，thus greatly affecting the income of farmers.

At present，resistance breeding，plant quarantine，chemical control，a gricultural control，physical control and biological control are main methods for prevention and control of tobacco black shank［3-5］.Specifically，chemical control is the most effective method for integrated control of tobacco black shank［6］.Currently，mancozeb，metalaxyl and mixed formulations are commonly used chemicals in China［6］.However，due to long-term large-scale continuous uncontrolled application of mancozeb and metalaxyl，P.parasitica has developed varying degrees of resistance in the field［2，7］.Zhenyuan County is a main tobacco-producing area in Qiandongnan State with serious occurrence of tobacco black shank，where dimethomorph，metalaxyl and propamocarb are commonly used.In this study，the resistance to dimethomorph，metalaxyl，propamocarb and ovraclostrobin·dimethomorph in P.parasitica was analyzed，aiming at providing a theoretical basis for screening fungicides to control tobacco black shank disease in Zhenyuan tobaccogrowing area.

Materials and Methods

Materials

Experimental strain In June 2012，Phytophthora parasitica var.nicotianae （Breda de Haan）Tucker was isolated from typical infected plants in Zhenyuan tobacco-growing area as the experimental strain.The experiment was carried out in Guizhou Key Laboratory for Tobacco Quality.

Fungicides Dimethomorph 50% WP， purchased from Dongguan Ruidefeng Biotechnology Co.，Ltd.；metalaxyl·mancozeb 58%WP，purchased from Dongguan Ruidefeng Biotechnology Co.，Ltd.；propamocarb 72.2%AS，purchased from Dongguan Ruidefeng Biotechnology Co.，Ltd.；ovraclostrobin·dimethomorph 18.7% WG，purchased from BASF SE.

Chemical reagents Experimental reagents were all of analytical grade，including ammonium sulfate，dimethyl hydrogen phosphate，magnesium sulfate，potassium chloride，ethanol，agar，distilled water（made in Guizhou Key Laboratory for Tobacco Quality）.

Instruments SW-CJ-1F double-sided clean bench，produced by Suzhou Purification Equipment Co.，Ltd.；light incubator，produced by Changzhou Aohua Instrument Co.，Ltd.；micro pipettes，produced by Shanghai Qiujing Biochemical Reagent&Instrument Co.，Ltd.；SZ-93 automatic dual water rectifier，produced by Shanghai Yarong Biochemical Instrument Factory；JJ200 electronic balance，produced by Changshu Shuangjie Testing Instrument Factory；portable pressure steam sterilizer，produced by Shanghai Medical Nuclear Instrument Factory.

Methods

Collection of infected tobacco plants Infected tobacco plants with typical symptoms of black shank were collected.Stems and roots （from diseased roots to 5 cm above the junction of diseased part and healthy part）were cut off，placed into double polyethylene film bags and brought back to the laboratory.

Isolation and purification of P.parasitica With tissue isolation method，diseased stems were washed clean，air-dried and sterilized using a cotton ball moistened with 70%alcohol；5 mm×5 mm tissue blocks with fresh typical lesions were collected from the junction of diseased part and healthy part with a sterile scalpel，sterilized in 70%alcohol for 2 min，placed in 0.1% mercuric chloride solution for 1 min，rinsed three times with sterile water for more than 1min，dried with sterile filter paper and placed on corn media plates；3-4 tissue blocks were evenly placed on each plate，placed into a 28℃ incubator and incubated under the light for 2-4 d.After the formation of colonies，5 mm×5 mm mycelial pellets were cut off from the edge of colonies with a sterile scalpel，transferred to corn media plates （3-4 mycelial pellets per plate）and incubated at 28℃in an incubator for 3 d；subsequently，5 mm×5 mm mycelial pellets were cut off from the edge of colonies with a sterile scalpel，transferred to corn slant media，incubated at 28℃in an incubator for 7 d and preserved in a refrigerator.

Isolation and purification of P.parasitica isolates The purified isolates were incubated on corn media for a week to observe the shape of mycelia. Mycelial pellets of each P.parasitica isolate were soaked in 0.1%KNO3solution and incubated at 28℃ in the dark for 48 h to promote the generation of sporangia.The morphological characteristics of sporangia were observed.Subsequently，sporangiumproducing mycelia were preserved at 4℃ for 15-30 min and placed at room temperature for a moment to obtain a large amount of swarm spores.Thus，the isolates were preliminarily identified according to the culture traits，morphological characteristics of hyphae and sporangia and formation of swarm spores.

Preservation of P.parasitica isolates After isolation and purification，mycelial pellets with good growth and no pollutants on corn media were collected using a punching bear with a diameter of 5 mm，transferred to corn slant media，and incubated at 28℃in an incubator.Subsequently，mycelial pellets with good growth were preserved in a refrigerator at 10℃to terminate the growth.

Preparation of medium①Preparation of corn media.Corn media was prepared with 30 g of corn flour，1 g of K2HPO4，0.1 g of MnSO4，3 g of（NH3）2SO4，0.5 g of KCl，0.5 g of Mg-SO4，20 g of agar and 1 L of water.The media was boiled，filtered through four layers of gauze，packed，and sterilized at 121℃for 30 min.②Preparation of fungicide-containing corn medium. Firstly，experimental fungicides were prepared into certain concentrations of stock solutions.According to the amount of corn media（9 ml），a certain amount of fungicide stock solution（1 ml）was collected with a pipette，added into 60℃media and shaken evenly to prepare different fungicide-containing corn medium，including corn medium containing 0.25，0.5，1，2，4 μg/ml dimethomorph 50%WP，corn medium containing 0.17，0.2，0.25，0.33，0.5 μg/ml metalaxyl·mancozeb 58%WP，corn medium containing 0.25，0.5，1，2，4 μg/ml propamocarb 72.2%AS，corn medium containing 0.07，0.08，0.1，0.13，0.17 μg/ml ovraclostrobin· dimethomorph 18.7%WG.

In vitro determination of the resistance to different fungicides in P.parasitica After incubation on corn media at 26℃under the light for 3-4 d，mycelial pellets with consistent growth were collected using a punching bear with a diameter of 5 mm，transferred to corn medium containing five concentrations of dimethomorph，metalaxyl·mancozeb，propamocarb and ovraclostrobin·dimethomorph under sterile conditions，and incubated at 26℃ in an incubator for 72 h.The colony diameter was measured twice with crossing method to determine the inhibitory rate of different fungicides against colony growth.With fungicidefree sterile water as a control，each treatment（each concentration of each fungicide）was repeated three times. Five days later，EC50 of each fungicide against P.parasitica was analyzed with colony growth measurement method［8］.

Data processing Experimental data were analyzed using Microsoft Excel 2003 and SPSS statistical software.

Results and Analysis

As shown in Table 1，P.parasitica exhibited different levels of sensitivity to dimethomorph，metalaxyl·mancozeb，propamocarb and ovraclostrobin· dimethomorph，and EC50reached 1.19，0.36，1.10 and 0.09 μg/ml，respectively.Therefore，P.parasitica exhibited the highest resistance to dimetho-morph，followed by propamocarb；P. parasitica exhibited the lowest resistance to ovraclostrobin·dimethomorph.

Table 1 Resistance to different fungicides in P.parasitica

Conclusions

So far，a large number studies have been conducted on the resistance in P.parasitica against dimethomorph.Based on the method proposed by Wang et al.［9］，P.parasitica strains can be divided into resistant strains （EC50＞10 μg/ml），moderate strains （0.1 μg/ml≤EC50≤10 μg/ml）and sensitive strains（EC50＜0.1 μg/ml）. In this study，Phytophthora parasitica var.nicotianae exhibited moderate resistance against dimethomorph，indicating that P.parasitica strains have developed varying degrees of resistance to dimethomorph，which will lead to a certain risk in case of long-term single use.This conclusion is consistent with the researches of Hu et al.［10］and Su et al.［11］.At present，little information is available on the resistance to metalaxyl·mancozeb，propamocarb and ovraclostrobin·dimethomorph in P.parasitica.

Determination results of the resistance to different fungicides in P.parasitica showed that P.parasitica exhibited the highest resistance to dimethomorph，followed by propamocarb；P. parasitica exhibited the lowest resistance to ovraclostrobin·dimethomorph.Therefore，in Zhenyuan tobacco-growing area，the application of dimethomorph should be reduced in tobacco production，while metalaxyl· mancozeb and propamocarb can be used interchangeably to prevent and control tobacco black shank disease. In addition，P.parasitica is highly sensitive to ovraclostrobin·dimethomorph，but the applicability of ovraclostrobin· dimethomorph should be verified by field efficacy trials.

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Responsible editor:Xiaohui FAN

Responsible proofreader:Xiaoyan WU

煙草黑脛病菌對不同殺菌劑的抗藥性初探

楊天沛1，付繼剛1，鄒光進1，王廷清1，唐軍1，龍友華2，3，張繼1*（1.黔東南州煙草公司，貴州凱里 556000；2.貴州大學農(nóng)學院，貴州貴陽550025；3.貴州省煙草品質(zhì)研究重點實驗室，貴州貴陽 550025）

為了解煙草黑脛病菌對不同殺菌劑產(chǎn)生的抗藥性，在室內(nèi)培養(yǎng)條件下，采用菌落生長測定法，對從黔東南州鎮(zhèn)遠縣分離的煙草黑脛病菌進行了抗性測定。結(jié)果表明：煙草黑脛病菌對不同殺菌劑均存在不同程度的抗藥性，其中對烯酰嗎啉的抗藥性最強，EC50值為1.19 μg/mL。因此，在鎮(zhèn)遠煙區(qū)，長期單一使用烯酰嗎啉防治黑脛病存在抗性風險。

黔東南州；煙草黑脛病菌；抗藥性；殺菌劑

楊天沛（1962-），男，貴州天柱人，農(nóng)藝師，長期從事煙葉生產(chǎn)、科研及管理工作，E-mail：yangtp8888@163.com。*通訊作者，E-mail：zhangj198601@163.com。

2014-12-17

2015-00-00

.E-mail:zhangj198601@163.com

Decembert 17，2014Accepted:zhangj198601@163.com