禾谷镰孢菌对苯醚甲环唑的敏感性基线及与其他杀菌剂敏感性的相关性分析

付刘元, 陈金鹏, 王 栓, 姜 佳, 车志平,田月娥, 陈根强, 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

In recent years,Fusariumhead blight (FHB) has widely occurred in countries and regions such as Asia, Africa, Europe, Australia, and the United States[1-2], and has long been a noteworthy question in the agricultural field. As a cosmopolitan disease, it reduces the quality and yield of cereals including wheat, maize, oat and so on[3-5]. Studies have reported thatFusarium graminearumcan also be isolated from weeds, soybeans and other non-grain hosts[6-7]. When the disease occurs, the ascospores and conidia produced byF. graminearumare spread by airflow and rainwater to infect crops[8]. In the case of wheat,FHB is capable of infecting all parts of it, with ear rot being the most serious, resulting in 10%-20%reduction in wheat yield, even 40%, or even no harvest in pandemic years. In addition to losing the seed and industrial value, contaminated wheat kernels can also produce a variety of trichothecene derivatives including deoxynivalenol (DON), nivalenol (NIV)and certain secondary metabolites[9-12]. Once consumed by humans and animals, it easily causes poisoning, which seriously threatens health, safety and even life[13-15]. Along with the change of global climate and the influence of tillage system such as increasing the area of straw returning to the field, the microclimate of field planting has also changed,resulting in a substantial increase in the frequency and scope of the disease. FHB is primarily relevant to theF. graminearumspecies complex (FGSC), such asF.asiaticum,F. graminearum,F. verticillioides,F.equiseti,F. meridionale,F. vorosii,F. boothii, andF.brasilicum[10-12,16-18].

In China, the occurrence of FHB was first reported in 1936. It has spread from the north to the west since 2010. it has spread from the north to the west[19]. The incidence of diseases in the northwestern wheat region has also increased significantly. The areas with frequent occurrence of wheat head blight are mainly concentrated in the winter wheat areas of the Huanghuai River Basin, the Yangtze River Basin and South China[11,20]. Such diseases also occur in the spring wheat areas of Northeast China. Worse still,the toxin content of wheat scab in some areas even exceeds the present national limit.F. graminearumandF. asiaticumare the most available pathogens in China and the former is the dominant species in Henan Province[21-22]. Henan Province is located in east-central China, the middle and lower reaches of the Yellow River, with a humid to semi-humid, warm temperate to subtropical monsoon climate. Known as the "granary of China", Henan's annual wheat output accounts for more than a quarter of the country's total,making it the largest wheat producing province in China[20]. Therefore, it is particularly crucial to prevent and control wheat diseases.

Epidemiological studies have shown that it is extremely important to control wheat diseases during the initial infection, and this requires the control ofF.graminearumbefore its occurrence. For the time being, due to the lack of wheat disease-resistant varieties, chemical control is still the most effective method in the treatment of FHB. Spraying fungicides during the flowering period of wheat is used for field control[23-24]. Examples include carbendazim (benzimidazoles), tebuconazole (demethylation inhibitors,DMIs) and pydiflumetofen (succinate dehydrogenase inhibitors, SDHIs). However, some fungicides such as carbendazim and tebuconazole have been used to control FHB for many years. With the increasing number and amount of application, a growing body of research has shown that long-term and single use of fungicides possibly give rise to a series of serious problems, such as increasedF. graminearumresistance resulting in an obvious decrease in its effectiveness against target diseases, chemical residues that are difficult to degrade, and environmental pollution which is a threat to biological health[25]. Therefore, we demand to seek new fungicides for controlling efficiently wheat head blight. In the meantime, the neo-fungicides were needed to compare with the fungicides in common use to determine the sensitivity relationship between them.

Difenoconazole, an excellent triazole fungicide developed by Syngenta, is capable of destroying the structure and function of cell membranes by inhibiting the biosynthesis of cellular ergosterol, thereby preventing the growth of pathogenic fungi. It is a highefficiency and low-residue sterol DMI fungicide[26].Moreover, since difenoconazole was launched in 1989, it has shown strong systemic properties and a broad range of antifungal spectrum like other DMI fungicides[27]. It is effective against a variety of fungal diseases including Ascomycotina, Basidiomycotina,Deuteromycotina in apple, wheat, tobacco and other plants, which exhibit high protective and therapeutic effects[28-31]. With regard to the prevention and control of wheat diseases, as of 2020, the most commonly registered control targets of single-agent difenoconazole wereGaeumannomyces graminisandUstilago tritici(http://www.chinapesticide.org.cn). Nevertheless, the single-agent registration ofF. graminearumhas not yet been discovered.

As a consequence, to evaluate the sensitivity ofF. graminearumfield isolates to difenoconazole and clarify the sensitivity correlation between difenoconazole and other fungicides, this study aimed to: (a)determine the EC50values ofF. graminearumfield isolates collected in different wheat regions of Henan Province and establish the baseline sensitivity to difenoconazole. (b) evaluate the sensitivity correlation relationship between difenconazole and each of the fungicides (epoxiconazole, carbendazim, phenamacril,pydiflumetofen, tebuconazole, prothioconazole, and metconazole).

1 Materials and methods

1.1 Isolate collections

When the disease characteristics of wheat ears were remarkable in late April, 107 field isolates were collected from 11 prefecture-level cities in Henan Province from 2016 to 2017 (Kaifeng, Luohe,Nanyang, Pingdingshan, Shangqiu, Xinxiang,Xinyang, Xuchang, Zhengzhou, Zhoukou, and Zhumadian). A few infected ears were sampled randomly from each wheat field and each field was more than 10 kilometers apart. Under a sterile environment, individual wheat grains with obvious FHB symptoms were washed twice in sterile water and then transferred to 0.1% (V/V) sodium hypochlorite solution for 30 s sterilization in the first place.Subsequently, sterilized kernels were rinsed 2-3 times with sterile water. In the end, after being dried with sterile filter paper, all samples were inoculated on potato sucrose agar (PSA, 200 g fresh potato, 20 g sucrose and 15 g agar, boiled and filtered, then diluted to 1 L distilled water) plate and grown under dark conditions for 2-3 days at 25 °C. In order to obtain pure strains, the single-spore isolation technique was performed on the cultures. Using a 5 mm diameter puncher, ten mycelial disks on the edge of the colony grown for 3 days, were added to 100 mL mung bean soup medium (30 g mung beans were boiled in 1 L distilled water for 20 min and residue was removed)and cultured at 25 °C, 120 r/min, dark and light alternately shaken for 7 days. Thereafter, we used double-layer sterile lens cleaning paper to filter, and evenly spread the appropriately diluted filtrate on a water agar (20 g agar per litre of distilled water) glass slide, (it was advisable to observe a single spore at high power), and transferred the single spore to the PSA plate at 25 °C in the dark for 3 days. The isolates ofF. graminearumwere cultured in PSA slants and stored at 4 °C until subsequent experiments.

1.2 Fungicides

All the dosage forms of fungicides in this study were technical-grade original fungicides. All tested fungicides were dissolved to obtain 10 mg/mL stock solution and stored at 4 °C. The information about these fungicides are shown in Table 1.

Table 1 Information of tested fungicides in the study

1.3 Sensitivity to difenoconazole

In this study, the sensitivity of 107F. graminearumisolates to difenoconazole was determined by the mycelial growth rate method[32]. The 50% effective concentration (EC50) required to inhibit mycelial growth was calculated, and it was conducive to analyze the frequency distribution range of EC50value. In this study, 5 mm mycelial disks from the edge of the colonies pre-cultured for 3 days were inoculated on PSA plates containing gradient concentration difenoconazole of 0, 0.1, 0.2, 0.4, 0.6 and 0.8 mg/L and the corresponding concentration of methanol for control treatment (the solvent effect was insignificant and negligible, data not shown). After being cultured at 25 °C for 3 days, the mean diameter of the fungal colonies was measured by criss-cross.The inhibition rate of difenoconazole on mycelial growth was calculated by the formula as previously described[17]. Next, the logarithm of the concentration was taken as the value on the X axis, and the probability value of the inhibition rate was taken as the value on the Y axis, and then the linear regression equation and EC50value ofF. graminearumto difenoconazole were calculated. Three replicates were set for each treatment and the experiment was repeated three times.

1.4 Sensitivity correlation analysis

A total of 20 isolates were selected from all theF.graminearumisolates used above for sensitivity tests as previously described, to demonstrate the sensitivity relationship between difenoconazole and the other seven fungicides (epoxiconazole, carbendazim,phenamacril, pydiflumetofen, tebuconazole,prothioconazole, and metconazole). Four cities were selected from northern Henan to southern Henan and 4-7 isolates were randomly selected from each city among all the isolates from 2016 to 2017. The isolates used in the experiment were numbered as follows:KF1721, KF1723, KF1725, KF1729, XC1710,XC1716, XC1718, KF1618, KF1628, KF1630,NY1604, NY1613, NY1614, NY1615, NY1620,XC1629, ZMD1620, ZMD1623, ZMD1625 and ZMD1634. The sensitivities of theF. graminearumisolates to the test fungicides were determined based on mycelial growth inhibition, as described above(Table 2). The solvent controls were treated likewise(the solvent effect was insignificant and negligible,data not shown). The sensitivity correlation between difenoconazole and the other seven fungicides were analyzed by Spearman's rank correlation. Spearman’s rho (ρ) for the log10of the EC50values of 20 isolates ofF. graminearumbetween difenoconazole and tested fungicides were analysed[18,33]. Three replicates were set for each treatment and the experiment was repeated three times.

Table 2 Concentrations of tested fungicides in the study

1.5 Statistical analysis

All data were analyzed using IBM SPSS Statistics v20 (SPSS Inc., Chicago). The mean value of EC50was tested by Fisher’s least significant difference(LSD,P= 0.01). The log10EC50value of difenoconazole is on the X-axis and the log10EC50values of the other seven fungicides are on the Y-axis, respectively.

2 Results and analysis

2.1 Sensitivity to difenoconazole

The sensitivity of 107F. graminearumisolates in Henan Province to difenoconazole was determined based on mycelial growth. The results indicated that the EC50value range of difenoconazole in 2016-2017 was 0.012 8-0.607 9 mg/L, and the mean EC50± SD value was (0.223 9 ± 0.119 2) mg/L (Table 3).

Table 3 Sensitivity of Fusarium graminearum to difenoconazole in Henan Province from 2016 to 2017

By analyzing the frequency distribution range of the EC50value, we found that the sensitivity curve of difenoconazole appeared in a unimodal form, and the distribution range was relatively narrow and mainly concentrated in the range of 0.1-0.3 mg/L (Fig.1).

After analyzing the geographical distribution region of the EC50value, it was obvious that the EC50value in the southern part of Henan Province was slightly overtopped compared to other areas (Fig.2),particularly the mean EC50value (0.393 3 ± 0.168 1)mg/L of Xinyang, but there was no significant difference compared with the overall. From 2016 to 2017, the strains ofF. graminearumin Henan Province were sensitive to difenoconazole, and no resistant isolates were found.

2.2 Sensitivity correlation analysis

To determine whether there was sensitivity correlation between difenoconazole, epoxiconazole,tebuconazole, prothioconazole, metconazole,carbendazim, phenamacril and pydiflumetofen, 20 randomly selected isolates were tested for sensitivity among allF. graminearumisolates from 2016 to 2017. The results suggested that the mean EC50values of difenoconazole, epoxiconazole, tebuconazole,prothioconazole, metconazole, carbendazim,phenamacril and pydiflumetofen were 0.227 6,0.210 9, 0.083 9, 0.800 9, 0.032 0, 0.476 0, 0.096 0 and 0.069 6 mg/L, respectively (Fig.3).

The sensitivity correlation between difenoconazole and the other seven fungicides were analyzed by Spearman's rank correlation. The Spearman’sρfor the log10of the EC50values of 20 isolates ofF.graminearumbetween difenoconazole and epoxiconazole, carbendazim, phenamacril, pydiflumetofen,tebuconazole, prothioconazole and metconazole were−0.05 (P> 0.05), 0.424 (P> 0.05), 0.269 (P> 0.05),−0.132 (P> 0.05), 0.366 (P> 0.05), 0.027 (P> 0.05)and 0.495 (P≤ 0.05), respectively (Fig.4). The results showed that was low level correlation between difenoconazole and metconazole, but no correlation with other tested fungicides.

3 Conclusion and discussion

As one of the food crops humans mostly consume,wheat disease is regarded as a cosmopolitan food security problem[34]. FHB not only affects the yield and quality of wheat, but reduces the germination rate and flour output of wheat kernels[35]. Hitherto, wheat varieties with high resistance and suitable for largescale planting have not been obtained[36-37]. Thus,chemical control is mainly adapted in production,combined with the reasonable disposal of crop stubble, suitable rotation cultivation and other control measures to reduce the occurrence of FHB to a certain extent[38]. At the moment, the commonly used fungicides are benzimidazoles, SDHIs, DMIs,cyanoacrylate esters, etc[39-40]. Unfortunately,carbendazim-resistant and tebuconazole-resistant strains have been isolated from the field in China[41].Studies have revealed that difenoconazole is used to control pepper anthracnose, pear speck spot, tomato gray mold and other diseases[42-44], but there are few reports on the establishment of wheat head blight sensitivity baseline in China. No resistant strains were found. Therefore, determining the sensitivity ofF.graminearumto difenoconazole plays an essential role in the identification and monitoring of resistance in Henan Province and even China.

In the present study, we detected the sensitivity of 107 wheat scab field isolates collected from different areas of Henan Province in response to difenoconazole. The range of EC50values and mean were 0.012 8-0.607 9 mg/L and (0.223 9 ± 0.119 2) mg/L,respectively. It was significantly lower than the values in Rekanović’s research[45]. We suspected that the utilization of such fungicide was probably continual in Serbia[45]. Our data demonstrated that, even thoughF. graminearumin Henan Province showed sensitivity to difenoconazole in overall tendency, the EC50value in Xinyang was the highest, indicating thatF.graminearumin this region was less sensitive to difenoconazole. The likely explanations might be that Xinyang is located in the southernmost point of Henan Province, south of the Huaihe River, with abundant precipitation and suitable for the propagation of FHB. On the whole, FHB in Henan Province was sensitive to difenoconazole and no resistant isolates were found. The sensitivity basically conformed to the normal distribution and the EC50value was about 0.1-0.3 mg/L. As previously reported,difenoconazole plays a significant control effect onF.graminearum[46].

For the sake of interpreting whether there was a sensitivity correlation relationship between difenoconazole and other fungicides, we selected epoxiconazole, tebuconazole, prothioconazole,metconazole, which belonged to the DMIs, and carbendazim, phenamacril, pydiflumetofen not in the same category as the fungicides with excellent efficacy[17,40,47]. In previous studies, the results showed that there were significant correlations between different DMI fungicides to rice false smut[48].However, in our study, we found that there was low level correlation between difenoconlazole and metconazole, but not with other DMI fungicides. It might be on the score of the fact that the sensitivity correlation is related to different kinds of pathogens.It may also be that DMI fungicides target different CYP51 proteins, including CYP51A, CYP51B, and CYP51C inF. graminearum[49]. Moreover, Beck's research had confirmed there was obvious divergence of the topology with triazolinthion-head of prothioconazole to heme, resulting in the binding mode of prothioconazole and CYP51 distinguished from the classic triazole fungicides[50]. Whether the diverse target binding sites lead to otherness in sensitivity correlation because of different structures for difenoconazole, further verifications should be considered. In the present study, we found that there was no correlation relationship between difenoconazole and other tested fungicides. We speculated that this finding was induced by the diversity in mechanism of action of the different fungicides. The mechanism of action of DMI fungicides is effected by inhibiting the biosynthesis of cellular ergosterol. The mechanism of action of benzimidazole fungicides (carbendazim) is achieved by inhibiting microtubule assembly by binding toβ-tubulin. The mechanism of action of succinate dehydrogenase inhibitor fungicides (SDHIs,pydiflumetofen) is effected by interfering with the respiratory chain complex Ⅱ. Although the mechanism of cyanoacrylate fungicides (phenamacril)is undefined, it is likely related to type I myosin5[51].

Given all that, in order to deal with the risk of FHB, the application of fungicides with highefficiency and low-toxicity is the most effective means of control at present. As far as we know,difenoconazole has a significant inhibitory effect onF. graminearum, and no similar reports have been found in China till now. Thus, we recommend that the baseline sensitivity of difenoconazole in this study should be regarded as a basis and the fungicide used in field production should be publicized. Moreover, it is practicable to reduce the frequency of usage to delay the occurrence of fungicide resistance like applying difenoconazole in combination with other fungicides or in rotation.