河南省油菜菌核病菌对氯氟联苯吡菌胺及其复配剂的敏感性

李路怡, 苗淑斐, 钱 乐, 姜 佳, 刘圣明

(河南科技大学 园艺与植物保护学院 植物保护系，河南 洛阳 471023)

0 Introduction

Sclerotinia sclerotiorumis one of the most devastating fungal pathogens in the world, which is capable of attacking more than 700 plant species[1].Sclerotinia stem rot, a destructive fungal disease caused by the fungusS.sclerotiorum, presents in oilseed rape and many other broadleaf crops[2].It can also damage commercial crops such as crucifers,legumes, compositae, etc.[3].The infection ofS.sclerotiorumbegins when ascospores colonize, bloom and invade the stem[4].S.sclerotioruminfects all aboveground parts of oilseed rape plants, including leaves, stems, flowers and pods.Sclerotinia stem rot is characterized by necrosis of the plant, reduced pod numbers and stunted growth.It may also lead to a decreased yield and quality of oilseed rape[5].

Currently, sclerotinia stem rot is mainly controlled by chemicals[6-7].The main method to control sclerotinia stem rot is to spray benzimidazole fungicides and the mixtures of fungicides at the flowering stage of oilseed rape[8].However, with the long-term use of these fungicides,S.sclerotiorumcaused very obvious resistance to these fungicides such as carbendazim and cyprodinil[9-10].As the distribution of fungicide-resistant isolates expands each year, the control efficiency of fungicides decreases each year, or even becomes ineffective eventually, leading to control failure[11].Therefore,there is an urgent need to screen out fungicides with new modes of action and better effects on sclerotinia stem rot.The succinate dehydrogenase inhibitors(SDHIs) belong to the fungicides of pyrazole carboxamides such as penthiopyrad and bixafen.The SDHIs are characterized by a broad spectrum of fungicidal activity and are also effective against agronomically important Ascomycetes[12-13].

Bixafen, discovered by Bayer Crop Science in 2001, has shown high inhibitory activity against many cereal pathogens in field trials[14-15].Bixafen has a long time of action, various types, high efficiency and strong selectivity.It has excellent internal absorption and conductivity abilities.It has both therapeutic and preventive effects and inhibits the production of spores and the growth of fungal hyphae.As the fungicide is not currently registered on oilseed rape in China, bixafen has not been used inS.sclerotiorum.In a previous report, the sensitivity ofS.sclerotiorumagainst an SDHI fungicide boscalid was determined.The EC50values ofS.sclerotiorumto boscalid ranged from 0.0073 to 0.3880 μg/mL, and the mean EC50value was (0.15 ± 0.09) μg/mL.The frequency distribution was unimodal[16].However, there are few reports on the application of bixafen to prevent and controlS.sclerotiorumin Henan Province and other areas in China.

The aim of this study was to investigate the baseline sensitivity of bixafen againstS.sclerotiorumobtained from oilseed rape fields in Henan, China.To establish the baseline sensitivity ofS.sclerotiorumto bixafen in Henan Province, the sensitivities of 119 isolates ofS.sclerotiorumto bixafen were determined based on the mycelial growth rate method.The mixture of fungicides with different modes of action and structural typesis a common and effective method in production.It improves the control efficiency and delays the development of resistance.In order to determine whether bixafen can be mixed with other fungicides, the fungicides with different modes of action, including the phenylpyrrole fungicide fludioxonil, DMI fungicides prothioconazole and metconazole, benzomezole fungicide carbendazim and dicarboxylic fungicide dimetachlone were chosen.The sensitivity of each fungicide mixture toS.sclerotiorumwas calculated based on the synergistic ratio (SR) values.The results of this study provide important information for the control ofS.sclerotiorumin Henan Province and the monitoring of the sensitivity ofS.sclerotiorumto bixafen.

1 Materials and methods

1.1 Isolate collections

In 2015 and 2016, plots with the most serious sclerotium stem rot were selected from Jiaozuo, Luohe,Nanyang, Pingdingshan, Zhengzhou, Zhoukou and other places in Henan Province and the sclerotia were collected randomly at certain distances from the roots and stems of diseased plants.These plants have not been previously exposed to bixafen and other SDHI fungicides.Three to five sclerotia were obtained from each collected plot, then cut into small pieces.After sterilization in a 0.1% sodium hypochlorite disinfection solution for 30 s and dried with sterile filter paper, sclerotia were transferred to potato dextrose agar (PDA: 200 g of potato, 15 g of agar, and 20 g of dextrose per liter of distilled water) plates for 2 days at 25 ℃ in the dark.The purified and retained single mycelium was transferred to a PDA slant, cultivated for 2 days, and stored at 4 ℃[17].In this study, a total of 119 non-duplicate isolates ofS.sclerotiorumwere collected from these isolates, including Pingdingshan(n= 24), Nanyang (n= 11), Zhengzhou (n= 33),Zhoukou (n= 14), Jiaozuo (n= 15), Luohe (n= 12),and Xuchang (n= 10).

1.2 Fungicides

All fungicides used in this study were technical grade.Bixafen (98% a.i.) was supplied by Shandong Weifang Runfeng Chemical Co., Ltd.(Shandong,China).Carbendazim (98% a.i.) was supplied by Jiangsu Rotam Chemistry Co., Ltd.(Suzhou, China).Fludioxonil (95% a.i.) was supplied by Syngenta Crop Protection Co., Ltd.(Basel, Switzerland).Prothioconazole (97% a.i.) was kindly provided by Shandong Hailier Chemical Co., Ltd.(Shandong,China).Dimetachlone (95% a.i.) was supplied by Jiangxi Heyi Chemical Co., Ltd.(Jiangxi, China).Metconazole (95% a.i.) was provided by the Jiangsu Huifeng Biological Agriculture Co., Ltd.(Yancheng,China).Fungicides were dissolved in the respective solvents, to obtain 10 000 μg/mL stock solutions(Table 1).All stock solutions were stored at 4 ℃.

Table 1 Concentrations of tested fungicides used in the study

1.3 Sensitivity to bixafen

In order to establish the baseline sensitivity to bixafen, the mycelium growth inhibition of 119 isolates ofS.sclerotiorumwas determined[18].All 119 isolates ofS.sclerotiorumwere transferred from the PDA slants to 9-cm PDA plates and cultured at 25 ℃in the dark for 2 days.The 5 mm mycelial plugs were cut from the edge of a 2-day-old colony of eachS.sclerotiorumisolate and transferred to PDA plates containing bixafen at concentrations of 0.00625,0.025, 0.10, 0.40 and 1.60 μg/mL and a blank solvent control without fungicide was set.After 2 days of incubation at 25 ℃ in the dark, the diameter of each colony was measured in two perpendicular directions.The average values obtained from the above measurement were used to calculate the mycelial growth rate according to the following formula (1):

Iis the inhibition rate;Dckis the average of the diameter of the control strain, cm;Dtis the average of the diameter of the treated strain, cm.The EC50value was calculated based on the linear relationship between the log10of each concentration and the probability of inhibition.Based on EC50value, the baseline sensitivity ofS.sclerotiorumto bixafen was established.Fisher’s protected LSD (P= 0.05) was used to compare the mean EC50values[18].For each group of experiments, three groups of repetitions were set and the experiment was repeated three times.

1.4 Synergistic interaction in mixtures.

The sensitivity of the bixafen mixtures with carbendazim, fludioxonil, dimetachlone, prothioconazole, or metconazole againstS.sclerotiorumwas determined by the degree of inhibition onS.sclerotiorumby different concentration gradients and ratios of concentration of the fungicide mixtures above.Isolates of PDS1604 were also carried out according to the above methods.The experiment combined bixafen and carbendazim, fludioxonil, dimetachlone, prothioconazole, metconazole according to (1 : 1, 1 : 3, 3 : 1,1 : 5, 5 : 1,V/V).In this study, 5-mm-diameter plugs were taken from the edge of each colony with a puncher and inoculated on PDA medium containing each concentration (Table 2).After 2 days of incubation at 25 ℃ in the dark, the diameter of each colony was measured in two perpendicular directions.The EC50value was obtained according to the measurement and calculation method described above.The theoretical inhibition concentration of each mixture(EC50(Exp)) was obtained by formula (2)[19].The SR was calculated according to the formula (3).When SR≥ 1.5, the interaction is defined as synergistic.When 1.5 ＞ SR ≥ 0.5, the interaction is defined as additive.When SR ＜ 0.5, the interaction is defined as antagonistic[20].The SR value was used to judge the inhibition of each ratio of fungicides toS.sclerotiorum.Three groups of replicates were set for each group of experiments and the experiment was repeated twice.

A, B are combined fungicides;a,bis the proportion of the fungicide in the mixture; EC50(Exp)is the theoretical suppressed medium concentration;EC50(Obs) is the actual measured medium concentration.

1.5 Statistical analysis

DPS software (version 7.05; Zhejiang University,Hangzhou, China) was used to obtain the regression equation of toxicity, the EC50value, and the 95%confidence limit.The bioassay option in DPS was used to determine the EC50value ofS.sclerotiorum.The resistance ratios were calculated as the EC50value of theS.sclerotiorumisolates[21].The statistical differences between sensitivity of different isolates were analyzed via the least significant difference(LSD) test atP= 0.05.In order to minimize errors,these tests were repeated twice, and the average of both tests was taken as the final result.

2 Results and analysis

2.1 Sensitivity to difenoconazole

The sensitivity of 119S.sclerotiorumisolates to bixafen was determined based on mycelial inhibition.By analyzing the results of these mycelial growth inhibition of isolates, the EC50values of bixafen to the 119 isolates ofS.sclerotiorumranged from 0.0417 to 0.4732 μg/mL and the mean EC50value(mean ± SD) was (0.1968 ± 0.1053) μg/mL (Table 3).The frequency distribution range curve was unimodal with a narrow range (Fig.1).The average EC50value obtained can be used as the baseline sensitivity ofS.sclerotiorumto bixafen in Henan Province.

Fig.1 Distribution of EC50 values for sensitivity to bixafen of 119 isolates of S.sclerotiorum collected from oilseed rape fields in different regions of Henan Province,China, in 2015 and 2016

2.2 Synergistic interaction in mixtures

The results showed that the EC50of the bixafen,carbendazim, fludioxonil, prothioconazole, dimeta-chlone and metconazole againstS.sclerotiorumwas 0.1256, 0.1122, 0.0229, 0.0651, 0.8057, and 0.0278 μg/mL, respectively, indicating that the test fungicides have high inhibitory activity on the growth ofS.sclerotiorum.The mixture combining bixafen with carbendazim, fludioxonil, sclerotium, prothioconazole, or metconazole according to (1 : 1, 1 : 3, 3 : 1, 1 : 5, 5 : 1,V/V) five treatments shows that the SR values against the mycelial growth ofS.sclerotiorumranged from 0.54 to 3.57 (Table 4).Among them, the highest SR value of 3.57 was obtained by the concentration ratio experiment of bixafen and dimetachlone 1 : 3.These mixtures are synergistic : bixafen and carbendazim(1 : 1, 1 : 3, 3 : 1, 5 : 1,V/V), bixafen and fludioxonil(1 : 3, 1 : 5, 3 : 1, 5 : 1,V/V), bixafen and prothioconazole (1 : 1, 5 : 1,V/V), bixafen and dimetachlone (1 : 1, 1 : 3, 1 : 5, 5 : 1,V/V), bixafen and metconazole (1 : 1,V/V).Meanwhile, the SR values of all treatments were greater than 0.5.This indicates that the effect of the mixtures of these fungicides is synergistic or additive, which may be due to the influence of the modes of action of each fungicide.

Table 2 Concentrations of fungicides mixtures used to determine the sensitivity of S.sclerotiorum

Table 4 Effect of different mixtures on the virulence of S.sclerotiorum

3 Conclusion and discussion

The application of a single fungicide induces the resistance ofS.sclerotiorumin oilseed rape to benzimidazole fungicides, dicarboximide fungicides and SDHI fungicides[22].At the same time, the resistance of fungicides has a clear trend of stable inheritance, which reduces the control efficiency of these agents on diseases such as sclerotinia stem rot[23-26].Therefore, the selection of new fungicides with different modes of action as parts of action is currently a key issue[27].

In previous studies, the SDHI fungicides have been used to prevent sclerotinia stem rot.In one study, 120 isolates ofS.sclerotiorumwere collected and isolated from oilseed rape in different areas of Jiangsu Province from 2006 to 2008.The baseline sensitivity of the isolates to boscalid was measured to be 0.028 to 0.398 μg/mL.The average EC50value was(0.17 ± 0.09) μg/mL[28].In another study from Anhui,Hubei, Jiangsu, and Zhejiang provinces, 161 isolates ofS.sclerotiorumwere collected and isolated from oilseed rape from 2001 to 2008.The baseline sensitivity to boscalid was 0.002-0.391 μg/mL, and the average EC50value was 0.042 μg/mL[29].In this experiment, there was no significant difference in the concentration of EC50inhibition of 119 isolates ofS.sclerotiorum, distributed from 0.0417 to 0.4732 μg/mL and the average EC50value of (0.1968 ± 0.1053) μg/mL.Although they were both SDHI fungicides, the geographical origin of the isolates, the number of the tested isolates, and the duration of treatment had a great influence on the sensitivity ofS.sclerotiorum.In addition, it is reported that SDHI-resistant isolates such as boscalid-resistant isolates have appeared in some areas in China[30].

Bixafen is an SDHI, which acts on the nodes of electron transport and oxidative phosphorylation in fungi, leading to the termination of the tricarboxylic acid cycle, affecting its energy metabolism, hindering the growth and the death of plant-pathogenic fungi,and finally achieving the goal of scientific crop disease control.Fludioxonil belongs to phenylpyrrole fungicide, which is a division protein activated kinase and histidine kinase inhibitor of osmotic signal transduction and inhibits the growth of pathogenic fungal mycelium by inhibiting the transfer related to glucose phosphorylation[31-34].Prothioconazole is a DMI fungicide, and its mechanism of action is to inhibit the demethylation of lanosterol or 2,4-methylene dihydrolanosterol 14, a sterol precursor in fungi, which affects the biosynthesis of ergosterol and inhibits the growth of pathogenic mycelia[35].Metconazole, a DMI fungicide,is a C-14 demethylase inhibitor that primarily inhibits ergosterol biosynthesis in plant-pathogenic fungi[36].Carbendazim, a benzomezole fungicide, is widely used to control many fungal diseases during agricultural production.Its mechanism of action is to inhibit microtubule assembly by binding toβ-tubulin orβ2-tubulin genes in pathogenic fungi[37-38].Dimetachlone is a dicarboxylic fungicide whose mechanism of action is binding to two-component histidine kinase[39].The results of the single fungicide sensitivity determination study showed that these five test fungicides had good inhibitory effects on the growth ofS.sclerotiorum.This study showed that when the volume ratio of 1 : 1, 1 : 3, 1 : 5, 3 : 1 and 5 : 1 was used, 15 of the 25 combinations of the mixtures were synergistic.In these ratios, bixafen and dimetachlone (1 : 3,V/V) had the best synergistic effect.Other mixtures with different proportions showed additive effects, indicating that the modes of action of each mixture did not affect each other.

This experiment filled a gap in the study of bixafen's sensitivity toS.sclerotiorumin Henan Province from 2015 to 2016 because no one had conducted a study of bixafen's sensitivity toS.sclerotiorumbefore.It can also be used to make more scientific guidance for the application of bixafen in Henan Province.The results proved thatS.sclerotiorumin the seven regions of Henan Province showed sensitivity to bixafen.In order to avoid resistance, bixafen can be used in combination with other fungicides to reduce the frequency of resistance and improve the control efficiency of sclerotinia stem rot.