On the Bacteriostatic Activity of Bacillus subtilis and Pyraclostrobin as Well as Their Mixtures to Grape Anthracnose and the Field Disease Control Efficiency

Muxiang Jl，Nini MAO，Guoping Ll，Kebing YAO，Jixiang LlU，Hongzhou CHEN，Xiang WU，Jian GUO，Dongming RUl

Zhenjiang Institute of Agricultural Sciences in Jiangsu Hilly Area，Jurong 212400，China

On the Bacteriostatic Activity of Bacillus subtilis and Pyraclostrobin as Well as Their Mixtures to Grape Anthracnose and the Field Disease Control Efficiency

Muxiang Jl，Nini MAO，Guoping Ll*，Kebing YAO，Jixiang LlU，Hongzhou CHEN，Xiang WU，Jian GUO，Dongming RUl

Zhenjiang Institute of Agricultural Sciences in Jiangsu Hilly Area，Jurong 212400，China

［Objective］To screen out the biological compound bactericides for grape anthracnose，reduce and replace the use of chemical pesticide.［Methods］The determination on the indoor bacteriostatic activity of different proportions of Bacillus subtilisand pyraclostrobin to grape anthracnose was carried out，and mycelial growth rate method was adopted to determine the toxicity of Bacillus subtilis and pyraclostrobin as well as their 5 mixtures to grape anthracnose. ［Results］The EC50of Bacillus subtilis and pyraclostrobin as well as their mixture combinations of 1∶1，1∶2，1∶3，1∶4 and 1∶5 to grape anthracnose were respectively 1.969 8，1.527 4，1.373 2，1.294 8 and 1.247 3 μg/ml；the synergistic coefficients（SR）of the 5 mixture combinations to grape anthracnose were 1.70，1.25，1.13，1.12 and 1.12，respectively，in which the synergistic effect of 1∶1 was the largest.The indoor biological activity of pyraclostrobin（EC50was 1.054 0 μg/ml）was higher than that of Bacillus subtilis（EC50was 15.017 5 μg/ml）.50 d after the agentia（before the harvesting），the investigation results showed that 1 000-fold dilution，1 500-fold dilution and 2 000-fold dilution as well as each single dosage of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder all had better control efficiency to grape anthracnose after fruit setting and before bagging，in which the treatments of high concentration and middle concentration were higher than the treatments of low concentration and two single dosages:the highest control efficiency of high concentration was 90.03%，which was higher than all other treatments；the control efficiency of middle concentration was 87.01%，which was higher than that of low concentration and each single dosage；the control efficiency of low concentration was 84.11%，which was higher than 1 000-fold dilution of 1 000×108cfu/g Bacillus subtili wettable powder（the control efficiency was 64.60%）and 2 000-fold dilution of 250 g/L Bacillus subtili wettable powder（the control efficiency was 81.07%）.In addition，each treatment also had better control efficiency to other cluster diseases，such as white rot，etc.，and the control efficiency was almost the same as that of anthracnose.［Conclusion］It was suggested that the prevention concentration of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder to grape anthracnose after fruit setting and before bagging was 1 000-fold-2 000-fold dilution.

Grape anthracnose；Bacillus subtilis；Pyraclostrobin；Mixture；Bacteriostatic activity；Field control efficiency

G rape anthracnose is one of the main diseases in grape production，it mainly harms nearly-mature fruit，so it is also called“Wan Fu Bing”germs［1-2］.After suffering the anthracnose，the surface of grape fruit will first generate pinheadsized small round brown fleck，then the fleck will gradually enlarge and hollow，and many rotiform black dotswill appear on the surface，these are the acervulus of germs.In wet weather，the eruptive pionnotes with pink gloea are the most obvious character of it，in severe case，the scab may expand to the whole fruit surface.If infected at the later stage，the fruit will be soft and fall off，or gradually lose water and become stiff fruit.If fruit stem and spike-stalk are infected，the obround fuscous hollow fleck will occur，in severe case，the fruit of the whole panicle will be dried-up or fall off.In general，the disease can cause the yield loss of grape by 10%-15%，if in the moist and rainy year，the disease will become pandemic，and the diseased panicle rate will reach 50%-70%［3-4］，especially in summer and autumn，if the rainy weather is continuous and more，the field humidity will be high，then grape anthracnose will be pandemic.

At present，the prevention of the disease has mainly been chemical prevention，and some common bactericides have been used，such as mancozeb，thiophanate methyl，prochloraz and difenoconazole，and so on；if depending on and using lots of organic synthetic chemical pesticide for a long time，the germs will easily have resistance，thereby causing excessive pesticide residues and environmental pollution.With more and more drugresistant phytopathogen emerging，seeking safe and effective pesticides with little toxic and side effect has become the focus issue of agricultural sustainable development.Biopesticide has the characteristics of safety，efficiency and no pollution，etc.，which coincides with the requirements of ecologicalenvironmentalprotection and food safety production［5-6］.Currently，special biopesticide preparations successfully preventing grape anthracnose have been few.Biocontrol bacterium genera（species）which can control diseases using biological control function reported by domestic and foreign literatures were bacterial fungicides（mainly including Bacillus licheniformis，Bacillus subtilis and Pseudomonas sp.，etc.）and mycofugicide（mainly including Trichoderma sp. and Gliocladium，etc.）［7-8］.Because of unstable control efficiency of single biocontrol bacteria，its actual use is less，therefore，the control strategy of grape anthracnose must be adjusted for rationally selecting and using bactericides，at the same time，it is also necessary to screen out more bactericides preventing grape anthracnose.

The bacterial strains of Bacillus subtilis were screened and isolated from the roots of Rhododendron simsii Planch.by Zhenjiang Institute of Agricultural Sciences，and preserved in China General Microbiological Culture Collection Center on July 2 in 2012，the preservation No.was CGMCC No. 6314，the thalli of Bacillus subtilis will generate many active substances during the process of growth，such as subtilin，polymyxin，nystatin and gramicidin，etc.，these active substances have obvious inhibiting effect to pathogenic bacteria or endogenous opportunistic pathogen.Pyraclostrobin is a new broad spectrum fungicide，its functional mechanism is mitochondria respiration inhibitor，namely it prevents electron transfer during the synthesis of cytochrome and has the functions of protection，treatment，blade penetration and conduction，the compound is low toxicity and safe to non-target organisms as well as friendly to users and the environment，it has already been listed as“candidate drugs reducing risk”by U.S.Environmental Protection Agency［9-10］.

In this study，the mixture of Bacillus subtilis and pyraclostrobin was adopted to make up for the deficiency of single antibiological inoculant，improve the using effect of biopesticide and reduce chemical control.Therefore，to screen out new biological compound pesticides，the joint action of Bacillus subtilis and pyraclostrobin as well as their 5 mixtures to the germs was determined，and the field control test was carried out，so as to provide some bases for the development and production of the mixed agentia.

Materials and Methods

The determination of indoor antibacterial activity

Experimental time and site The experiment was carried out at Modern Agriculture Laboratory in Zhenjiang Institute of Agricultural Sciences during April-July in 2012.

Experimental strains Grape anthracnose was collected from Dingzhuang Village，Maoshan Town，Jurong City，Jiangsu Province，its variety was Kyoho，then it was isolated，identified，verified according to Koch’s Rule［11-12］and preserved for using by Modern AgriculturalLaboratory of Zhenjiang Institute of Agricultural Sciences.The bacterialstrains were stored on the bevel of potato sucrose agar（PSA）at 4℃.

Experimental agentia 1 000×108cfu/g Bacillus subtili technical concentrate was provided by Zhenjiang Institute of Agricultural Sciences；97%pyraclostrobin technical was provided by Jiangsu Frey Agrochemicals Co.，Ltd.

lnstrument and equipment Electronic scales（the sensitive quality was 0.1 mg），GZP-300A incubator，petri dish with the diameter of 75 mm，triangularflask，liquid-moving machine，transfer pipette，aurilave，puncher，inoculative knife，and so on.

Experimental methods

The preparation of liquid agentia and the design of concentration

（1）The preparation of mother liquor. 1 000×108cfu/g Bacillus subtili technical concentrate was mixed with sterile water to make 1×104μg/ml，97%pyraclostrobin technical was dissolved by appropriate acetone， then 10% Tween-80 was added，finally 1 000 μg/ml mother liquor was made and put in 4℃refrigerator as the standby.

（2）The concentration design of single dosage of each agentia and the mixture. Bacillus subtilis， Pyraclostrobin and the mixtures of Bacillus subtilis and Pyraclostrobin （1∶1，1∶2，1∶3，1∶4 and 1∶5）were designed as 8 PSA media with different concentration gradients，namely 200.00，100.00，50.00，25.00，12.50，6.25，3.125 and 1.562 5 μg/ml，respectively.Except mother liquor，all other medicine liquids were used in time after they were dispensed.

Experimental methods Mycelium growth rate method［13］was adopted to successively dilute the medicine liquids of each single dosage and mixtures to the required concentrations，then 1 ml medicine liquid was mixed with 9 ml medium on the petri dish to make PSA medium containing series concentrations of reagents，and aquae sterilisata was used as blank control，each treatment repeated 4 times.The reserved grape anthracnose germs were transferred to PSA plate and activated for 72 h under 25℃，then bacterial cake with the diameter of 5 mm was got using puncher from the edge of bacterial colony，and transferred to the medicated PSA plate and blank control；then cultured for 96 h under 25℃ ；when the diameter of bacterial colony grew to about 4/5 of plate diameter，the diameter of bacterial colony was measured using crossing method.

The mean of colony diameter was calculated，and the average inhibition ratio of mycelial growth was calculated according to the following formula: Average inhibition ratio of mycelial growth=｛（Mean of colony diameter of CK-Mean of colony diameter of treatment groups）/（Mean of colony diameter of CK-The diameter of vaccinal bacterial cake）｝×100%，DPS13.0 professional data processing system was adopted to calculate the regression equation，EC50and 95%confidence limit of the inhibition of the agentia to the growth ofgrape anthracnose mycelia.

Based on Wadley method［5］，synergistic coefficients（SR）were calculated.The joint action types of agentia combination were evaluated according to SR，namely SR＜0.5 represented antagonistic action，0.5≤SR≤1.5 represented additive effect，SR＞1.5 represented synergistic effect.

Hereinto，A and B respectively represented single dosage of fungicide，a and b represented the proportion of relevant single dosage in the mixture，EC50（Eth）represented EC50theoretical value of the mixture，and EC50（Eob）represented EC50measured value of the mixture.

The experiment offield disease prevention

The experiment was carried out at the orchard of Huayang Town in Jurong City，the experimental variety was Summer Black，the tree age was four years and the cultivation pattern was trellis.

There were 1 000-fold dilution of 1 000×108cfu/g Bacillus subtili wettable powder（produced by Jiangsu Lvdun Plant Protection Agrochemical Experimental Co.，Ltd.），2 000-fold dilution of 250 g/L pyraclostrobin missible oil（produced by BASF SE），and 1 000-fold dilution，1 500-fold dilution and 2 000-fold dilution（hereafter respectively called high concentration，middle concentration and low concentration）of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder（prepared by Zhenjiang Institute of Agricultural Sciences in Jiangsu Hilly Area），and clean water as the control. When the diameter of the grape fruit was 0.5 cm，soaked treatment was conducted，and commercialpaper bags were used after the airing of the fruit，there were 6 treatments，2 replications，12 plots and randomized block arrangement.

50 d after the agentia and before the harvesting，10 clusters of each plot were chosen，then total fruit grain number，the grain number of anthracnose fruits and other fruit grain diseases were investigated，finally the diseased fruit grain rate and control efficiency were respectively counted.

Results and Analyses

The measured results of indoor bacteriostatic activity

Effects of Bacillus subtilis and pyraclostrobin to the growth of grape anthracnose mycelia From Table 1，the results showed that when the concentrations of Bacillus subtilis and pyraclostrobin were among 1.562 5-200 μg/ml，their antibacterial rates to grape anthracnose were respectively 13.26%-85.08%and20.49%-96.59%.

Effects of the mixtures of Bacillus subtilis and pyraclostrobin to the growth of grape anthracnose mycelia As shown in Table 2，it can be shown that when the concentrations of 5 mixtures of Bacillus subtilis and pyraclostrobin were among 0.156 25-20 μg/ml，the antibacterial rates of 1∶1，1∶2，1∶3，1∶4 and 1∶5 combinations to grape anthracnose were respectively17.26%-96.45%，17.26%-95.94%，18.78%-95.43%，21.32%-94.92%and 20.81%-95.43%.

The toxicity to strawberry blight germs and the joint action types of the mixtures The interaction models of 2 kinds of single dosage and 5 kinds of mixtures to grape anthracnose were seen in Table 3，the correlation coefficients of concentration logarithm and the probability of control efficiency were both≥0.976 3，indicating that the variation of the probability of control efficiency was 97.63%or more from the variation of concentration logarithm，showing thatthe obtained models can express the relationship between concentration logarithm and the probability of control efficiency.

Table 1 The inhibitory effects of Bacillus subtilis DJ-6 and pyraclostrobin to grape anthracnose 72 h

The data of Table 3 showed that the EC50values of Bacillus subtilis and pyraclostrobin to grape anthracnose germs were 15.017 5 and 1.054 0 μg/ml，respectively；the indoor biological activity of pyraclostrobin to grape anthracnose germs was significantly higher than that of Bacillus subtilis.It was also shown from the Table 3 that the synergistic coefficients of the 5 mixtures between Bacillus subtilis and pyraclostrobin were all greater than 1，indicating that the joint action types were all additive effect or synergistic effect；hereinto，the SR with the ratios of 1∶2，1∶3，1∶4 and 1∶5 were 1.25，1.13，1.12 and 1.12，respectively，the SR were among 0.5-1.5，the joint action types were additive effect；when the mixture ratio was 1∶1，the SR was 1.7，which was larger than 1.5，thus the joint action types were synergistic effect.

Table 2 The inhibitory effects of different proportions of Bacillus subtilis and pyraclostrobin to grape anthracnose 72 h

Table 3 The inhibiting effect of Bacillus subtilis and pyraclostrobin as well as their mixtures to grape anthracnose

The effect of field disease prevention

50 d after the agentia and before the harvesting，the survey results in Table 4 showed that for the control efficiency of anthracnose，the diseased fruit grain rate of CK was 6.92%，and the highest control efficiency of highconcentration 20% pyraclostrobin· 200×108cfu/g Bacillus subtili wettable powder was 90.03%，which was higher than all other treatments；the control efficiency of middle concentration was 87.01%，which was higher than that of low concentration and each single dosage；the control efficiency of low concentration was 84.11% ，which was higher than 1 000-fold dilution of 1 000×108cfu/g Bacillus subtili wettable powder （the control efficiency was 64.60%）and 2 000-fold dilution of 250 g/L Bacillus subtili wettable powder （the control efficiency was 81.07%）.In addition，each treatment also had better control efficiency to other cluster diseases，such as white rot，etc.，and the control efficiency was almost the same as that of anthracnose，namely high concentration of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder﹥middle concentration ﹥low concentration﹥2 000-fold dilution of 250 g/L pyraclostrobin missible oil﹥1 000-fold dilution of 1 000×108cfu/g Bacillus subtili wettable powder.Therefore，It was suggested that the prevention concentration of 20%pyraclostrobin· 200×108cfu/g Bacillus subtili wettable powder to grape anthracnose after fruit setting and before bagging was 1 000-fold-2 000-fold dilution.

Conclusions and Discussions

For the lack of anthracnose resistance varieties and the high pressure of chemical prevention pesticide residue and environmental pollution in grape production［14-15］，although single biocontrol agent is safe，environmental and harmless，the efficiency is unstable，and the control efficiency is not ideal，therefore，the study on using the combination between antagonistic bacterium and biochemical resistance against diseases，enhancing the disease resistance of the plants and improving control efficiency is a new topic preventing grape diseases［16-18］. The study used the scientific mixtureof Bacillus subtilis and pyraclostrobin，and took full advantage of the inhibiting effect of beneficial antagonistic organism to grape germs and disease prevention effectofpyraclostrobin. The determination of indoor bacteriostatic activity showed that the indoor biological activity of pyraclostrobin to grape anthracnose was higher than Bacillus subtilis，the synergistic coefficients of 5 proportions between Bacillus subtilis and pyraclostrobin were among 0.5-1.5，the joint action types were additive effect；when the mixture ratio was 1∶1，the SR was 1.7，which was larger than 1.5，thus the joint action types were synergistic effect.1 000-fold dilution，1 500-fold dilution and 2 000-fold dilution as well as each single dosage of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder all had better control efficiency to grape anthracnose after fruit setting and before bagging；50 d after the agentia and before the harvesting，the control efficiency of cluster anthracnose was investigated，and the highest control efficiency was 90.03%，which was higher than all other treatments；the control efficiency of middle concentration was 87.01%，which was higher than that of low concentration and each single dosage；the control efficiency of low concentration was 84.11%，which was higher than 1 000-fold dilution of 1 000×108cfu/g Bacillus subtili wettable powder（the control efficiency was 64.60%）and 2 000-fold dilution of 250 g/L Bacillus subtili wettable powder （the control efficiency was 81.07%）.In addition，each treatment also had better control efficiency to other cluster diseases，such as white rot，etc.，and the control efficiency was almost the same as that of anthracnose，namely high concentration of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder﹥ middle concentration﹥low concentration﹥2 000-fold dilution of 250 g/L pyraclostrobin missible oil﹥1 000-fold dilution of 1 000×108cfu/g Bacillus subtili wettable powder.Therefore，It was suggested that the prevention concentration of 20%pyraclostrobin· 200×108cfu/g Bacillus subtili wettable powder to grape anthracnose after fruit setting and before bagging was 1 000-fold-2 000-fold dilution.

Table 4 The field control efficiency of different concentrations of 20%pyraclostrobin·200×108cfu/g Bacillus subtili wettable powder to grape anthracnose

The rational mixture of Bacillus subtilis and pyraclostrobin is beneficial to overcome or delay the resistance of grape anthracnose to chemical pesticide，improve the safety quality of grape，and reduce chemical pesticide residue and environmental pollution，thereby providing a new thought and a new method for the prevention of grape disease，it is of great application prospect.The effect of the mixture of Bacillus subtilis and pyraclostrobin on Plasmopara uiticola and other crop diseases needs to be further studied.

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Responsible editor：Nanling WANG

Responsible proofreader：Xiaoyan WU

枯草芽孢杆菌与吡唑醚菌酯及其混配对葡萄炭疽病菌的抑菌活性与田间防病效果

吉沐祥，毛妮妮，李国平*，姚克兵，刘吉祥，陈宏州，吴祥，郭建，芮东明 （江苏丘陵地区镇江农业科学研究所，江苏句容212400）

［目的］筛选葡萄炭疽病的增效生物复配杀菌剂，减少与替换化学农药使用。［方法］进行了枯草芽孢杆菌与吡唑醚菌酯不同比例混配对葡萄炭疽病的室内抑菌活性测定，采用菌丝生长速率法测定了枯草芽孢杆菌与吡唑醚菌酯及其5种配比对葡萄炭疽病菌的毒力。［结果］枯草芽孢杆菌与吡唑醚菌酯及1∶1、1∶2、1∶3、1∶4、1∶5混配组合对葡萄炭疽病菌的EC50分别为1.969 8、1.527 4、1.373 2、1.294 8、1.247 3 μg/ml；5种混配组合对葡萄炭疽病菌的增效系数（SR）分别是1.70、1.25、1.13、1.12、1.12，其中以1∶1增效作用最大。吡唑醚菌酯（EC50为1.054 0 μg/mL）的室内生物活性高于枯草芽孢杆菌 （EC50为15.017 5 μg/ml）。药后50 d（采收前）调查结果表明，20%吡唑醚菌酯·200亿cfu/g枯草芽孢杆菌可湿性粉剂1 000倍液、1 500倍液、2 000倍液以及各单剂在葡萄结果期套袋前浸果对炭疽病等均有较好的防治效果，其中高浓度、中浓度处理高于低浓度与两个单剂处理：高浓度防效最高为90.03%，均高于其他各处理，其次为中浓度防效为87.01%，高于低浓度和各单剂，低浓度防效为84.11%，高于1 000亿cfu/g枯草芽孢杆菌可湿性粉剂1 000倍液（防效为64.60%）和250 g/L吡唑醚菌酯乳油2 000倍液（防效81.07%），对其他果穗病害如白腐病等也均有较好防治效果，防治效果与炭疽病防效趋于一致。［结论］20%吡唑醚菌酯·200亿cfu/g枯草芽孢杆菌可湿性粉剂在葡萄套袋前浸果防治葡萄炭疽病等果穗病害建议使用浓度为1 000～2 000倍液。

葡萄炭疽病；枯草芽孢杆菌；吡唑醚菌酯；混配；抑菌活性；田间防治效果

江苏省农业科技自主创新项目［CX（14）2056］；镇江市农业科技支撑项目（NY20-14005）；句容科技创新基金项目（NY2013026）。作者简介 吉沐祥（1963-），男，江苏宝应人，研究员，主要从事植保农药研发和果树病虫害绿色防控技术研究，E-mail：jilvdun2800@163.com。

2015-06-13

Supported by the Independent Innovation Fund Project of Agricultural Science and Technology in JiangsuProvince［CX（14）2056］；Agricultural Science&Technology Supporting Program of Zhenjiang City（NY2014005）；Science and Technology Innovation Items of Jurong City（NY2013026）.

*Corresponding author.E-mail:jrlgp@126.com

Received:June 13，2015 Accepted:November 27，2015

*通讯作者，研究员，主要从事丘陵农业资源开发利用得果树栽培技术研究工作，E-mail：jrlgp@126.com。

修回日期 2015-11-27