Lili Jiang,Rong Zhang,Xiaojuan Zong,Xiaofang Wang,Chong Wu*

1.Shandong Institute of Pomology/Shandong Technology Innovation Center of Modern Protected Fruit,Taian 271000,China;2.Forestry Protection and development Center of Daiyue District in Taian City,Taian 271000,China

Abstract[Objective]The paper was to isolate Colletotrichum sp.from strawberry and to screen effective fungicides.[Method]The indoor toxicity of 7 fungicides to Colletotrichum sp.was measured by growth rate method.The potted control effect of 3 fungicides on Colletotrichum sp.was evaluated.[Result]Fludioxonil exhibited the highest indoor toxicity to Colletotrichum sp.,with the EC50value of 0.117 mg/L,and the EC50value of prochloraz and difenoconazole were 0.141 and 0.357 mg/L,respectively.The pot experiment showed that the potted control effect of 20% fludioxonil 2 000 times dilution on Colletotrichum sp.was the highest of 92.86%.450 g/L prochloraz EW 4 500 times dilution and 250 g/L difenoconazole EC 2 500 times dilution also received high control effects against Colletotrichum sp.,which were 91.68% and 89.29%,respectively.[Conclusion]Fludioxonil,prochloraz and difenoconazole could be used for the control of strawberry anthracnose.

Keywords Colletotrichum sp.;Indoor toxicity;Pot experiment;Fungicide selection

Strawberry(Fragaria×ananassa Duchesne)is a perennial herb belonging to Fragaria,Rosaceae[1].Strawberry,deeply loved by consumers due to rich nutrition and good taste,is an important cash crop in China.In recent years,with the continuous expansion of cultivation area of facility strawberry,it is difficult to rotate strawberry in large-scale production areas,and the occurrence of diseases is increasingly severe[2].Strawberry anthracnose caused by Colletotrichum sp.is a major disease that damages strawberry and causes yield reduction[3].Anthracnose occurs throughout the whole growth period of strawberry,and is most common in the stage of seedling breeding and early transplant.It mainly damages the root and stolon of strawberry,causing root rot and whole plant wilting and death[4].

At present,agricultural control,biological control and chemical control are the major measures against strawberry anthracnose.Agricultural control measures mainly include breeding disease-resistant varieties and strengthening field management[5].Biological control is mainly in the research stage,and product registration has not been obtained[6].The market has high requirements on the taste and quality of strawberries since it is a fresh fruit.Currently,most of the main domestic strawberry varieties are highly susceptible to anthracnose[7],and chemical control is still the effective means to control anthracnose[8].In this study,7 commonly used fungicides were selected to evaluate their indoor toxicity to Colletotrichum sp.,and highly efficient fungicides were selected for pot experiment,in order to provide the theoretical basis for scientific control of strawberry anthracnose.

1 Materials and Methods

1.1 Agents The fungicides of technical grade used in the test included 98% carbendazim(Shandong Weifang Rainbow Chemical Co.,Ltd.),97% pyraclostrobin(Zhejiang Yulong Biotechnology Co.,Ltd.),97% difenoconazole and 98% trifloxystrobin(Shandong United Pesticide Industry Co.,Ltd.),98% azoxystrobin(Shandong Weifang Shuangxing Pesticide Co.,Ltd.),98% fludioxonil(Hebei Xingbai Agricultural Science and Technology Co.,Ltd.),and 97% prochloraz(Shandong Huayang Pesticide Chemical Group Co.,Ltd.).Carbendazim was dissolved in 3% hydrochloric acid solution,and other raw fungicides were dissolved in acetone.The mother liquors with the concentration of 1×104mg/L were prepared and stored at 4℃for later use.

450 g/L prochloraz EW was produced by Zhejiang Tianfeng Biological Science Co.,Ltd.;20% fludioxonil SC was derived from Qingdao Kaiyuanxiang Chemical Co.,Ltd.;250 g/L difenoconazole EC was obtained from Jiangsu Heben Biochemical Co.,Ltd.

1.2 Isolation of the pathogen The strawberry plants infected by anthracnose were collected from strawberry greenhouses in Jinniushan Experimental Base of Shandong Institute of Pomology.The pathogen was isolated by disease tissue separation[9]:the junction of disease and health tissues(Fig.1)was soaked in 75% ethanol solution for 30 s,and then rinsed with sterilized water for 3 times;the treated diseased tissues were placed in the center of PDA medium and cultured in darkness at 25℃for 5 d;after the formation of colonies,marginal mycelia were picked and further purified and cultured on a new PDA plate.Finally,the mycelia were stored on PDA slant medium at 4℃for later use.

Fig.1 Junction of disease and healthy tissues of anthracnose

According to Koch’s rule[10],several small openings were cut at the stem base of strawberry ‘Akihimei’ with a scalpel,and the Colletotrichum sp.colonies cultured for 7 d were re-inoculated to the incision together with the medium for routine cultivation management.Whether the incidence symptoms of inoculated plants were consistent with field symptoms was observed.

1.3 Indoor toxicity measurement The indoor toxicity of fungicides was determined by mycelial growth rate method[11].On the ultra-clean workbench,the mother liquors of 7 fungicides were diluted by gradient with sterile water.1 mL of fungicides of each concentration was added to 49 mL of PDA medium which cooled to 50℃-60℃.After mixed thoroughly,the plates were prepared.Each concentration was repeated three times.The concentrations of prochloraz and fludioxonil were 0.05,0.10,0.20,0.40 and 1.00 mg/L;the concentrations of difenoconazole were 0.1,0.2,0.4,1.0 and 2.0 mg/L;the concentrations of carbendazim were 1.0,2.5,5.0,10.0 and 25.0 mg/L;and the concentrations of pyraclostrobin,azoxystrobin and trifloxystrobin were 2.5,5.0,10.0,25.0 and 50.0 mg/L,respectively.

The fungal cakes were punched on the edge of activated colony of Colletotrichum sp.with a puncher(diameter 7 mm),and inoculated onto the above of drug-containing PDA plates.Those inoculated with sterile water were used as the control.The plates were cultured in darkness at 25℃.When the colony in control grew to 2/3 of the petri dish,the colony diameter was measured by crossing method[12],and the inhibition rate of each fungicide treatment on mycelial growth of Colletotrichum sp.was calculated.The EC50of each fungicide was calculated by SPSS 18.0 software,and the EC50of the fungicide with the lowest toxicity was used as the reference value to calculate the relative toxicity index of other agents.

Inhibition rate（%）=(Colony diameter in control-Colony diameter in treatment)/(Colony diameter in control-fungal cake diameter)×100.

1.4 Pot efficacy test The pot experiment was conducted according to the recommended dosage of fungicides published by China Pesticide Information Network.Soil was taken from the site where strawberry anthracnose had seriously occurred,and packed into plastic flowerpots(diameter 15 cm).The strawberry seedlings‘Akihime’ at three-leaf and one-heart stage were transplanted to the plastic flowerpots.Four treatments were designed in the test,including 20% fludioxonil SC 2 000 times dilution(20 mL/plant),450 g/L prochloraz EW 4 500 times dilution(20 mL/plant),250 g/L difenoconazole EC 2 500 times dilution(20 mL/plant),and water control.Each treatment was repeated 3 times,10 pots each repeat.

The incidence rate and disease level of strawberry anthracnose were investigated at 14 d after transplanting,and the control effect was calculated.The grading criteria of the disease was as follows[13]:level 0,the plant was health without any symptoms;level 1,there were punctate spots with the length less than 2.0 mm;level 3,the lesions were 2.1-5.0 mm;level 5,the lesions were 5.1-9.0 mm;level 7,the lesions were 9.1-15.0 mm;level 9,the plant were wilted.

Incidence rate（%）=Number of diseased plants/Total number of plants×100;

Disease index=∑ (Disease level×Number of plants at this level)/(The highest level of the disease×Total number of plants)×100;

Control effect（%）=(Disease index in control-Disease index in treatment)/Disease index in control×100.

2 Results and Analysis

2.1 Isolation of the pathogen As shown in Fig.2,the pathogen isolated from diseased strawberry plants was cultured in darkness at 25℃for 5 d,and the colony was thick with grayish aerial villi.After cultured for 10 d,orange viscous sori appeared in the center of the colony,which was consistent with the characteristics of Colletotrichum sp.of strawberry reported by Zhang et al.[14].

Fig.2 Colony morphology of Colletotrichum sp.

When the pathogen was re-inoculated to healthy strawberry plants ‘Akihime’ for 7 d,the stem base of strawberry turned brown,and the plants wilted(Fig.3),which was consistent with the initial symptoms in the field.

Fig.3 Symptoms of Colletotrichum sp.after re-inoculation in strawberry

2.2 Inhibition rates of 7 fungicides against Colletotrichum sp.at different concentrations As shown in Table 1,fludioxonil had the best inhibitory effect on Colletotrichum sp.,and the inhibition rate was 37.73%-75.17% when the concentration was 0.05-1.00 mg/L.When the concentration of prochloraz was 0.05-1.00 mg/L,the inhibition rate was 29.10%-81.52%.When the concentration of difenoconazole was 0.1-2.0 mg/L,the inhibition rate was 33.63%-68.03%,and the antifungal activity was high.When the concentration of carbendazim was 1.0-25.0 mg/L,the inhibition rate was 29.12%-84.02%.The inhibitory effects of pyraclostrobin,azoxystrobin and trifloxystrobin on Colletotrichum sp.were relatively low.When the concentration was 2.5-50.0 mg/L,the inhibition rates of pyraclostrobin,azoxystrobin and trifloxystrobin were 36.18%-78.06%,29.85%-71.49% and 31.08%-69.99%,respectively.

Table 1 Inhibition rates of 7 fungicides against Colletotrichum sp.at different concentrations

2.3 Toxicity of 7 fungicides to Colletotrichum sp. As shown in Table 2,fludioxonil had the highest indoor toxicity to Colletotrichum sp.,with the EC50of 0.117 mg/L,and the relative toxicity index was as high as 104.66 compared to azoxystrobin.Prochloraz and difenoconazole had high indoor toxicity on Colletotrichum sp.,with the EC50of 0.141 and 0.357 mg/L,and their relative toxicity indexes were 86.84 and 34.30,respectively.Azoxystrobin had the lowest indoor toxicity to Colletotrichum sp.,with the EC50of 12.245 mg/L.Carbendazim,pyraclostrobin and trifloxystrobin had relatively low indoor toxicity to Colletotrichum sp.,with the EC50of 3.475,5.703 and 10.345 mg/L,and their relative toxicity indexes were 3.52,2.15 and 1.18,respectively.

Table 2 Toxicity of 7 fungicides to Colletotrichum sp.

2.4 Potted control effect of 3 fungicides against anthracnose As shown in Table 3,the incidence rate of anthracnose in control was as high as 46.67%.By root irrigation,the potted control effect of 20% fludioxonil 2 000 times dilution on anthracnose was the highest of 92.86%.450 g/L prochloraz EW 4 500 times dilution and 250 g/L difenoconazole EC 2 500 times dilution also received high control effects on anthracnose, which were 91.68% and 89.29%,respectively.

Table 3 Potted control effects of 3 fungicides on anthracnose

3 Conclusions and Discussion

Colletotrichum sp.is a complex genus with strong pathogenicity,including multiple pathogenic species,and the sensitivity of the same strain to different fungicides varies greatly[15].Through indoor toxicity determination,it was found that Colletotrichum sp.was the most susceptible to fludioxonil,and the EC50was only 0.117 mg/L.Prochloraz and difenoconazole also had high indoor toxicity on Colletotrichum sp.,with the EC50of 0.141 and 0.357 mg/L,respectively.This is consistent with the results of Liu[16]and Zhang[17].

Fludioxonil belongs to pyrrole nonsystemic fungicide,and has a wide spectrum of disease prevention.Prochloraz is a imidazole demethylation inhibitor with high efficiency,broad spectrum and high permeability,it blocks the synthesis of ergosterol by inhibiting the activity of demethylase,and results in abnormal formation of fungal cell membranes,thereby leading to cell death[18].As a triazole sterol demethylation inhibitor, difenoconazole has strong systemic absorption[19]and has long-lasting protective and therapeutic effects on a variety of diseases[20].By root irrigation,the potted control effect of 20% fludioxonil 2 000 times dilution on anthracnose was the highest of 92.86%.450 g/L prochloraz EW 4 500 times dilution and 250 g/L difenoconazole EC 2 500 times dilution also received high control effects on anthracnose,which were 91.68% and 89.29%,respectively.Therefore,fludioxonil,prochloraz and difenoconazole can be used to control strawberry anthracnose,but their field efficacy needs to be further studied.