Baseline sensitivity and resistance risk assessment of Valsa mali to pyraclostrobin

Pyraclostrobin, a quinone outside inhibitor (QoI) fungicide, has been registered to control apple tree Valsa canker (AVC) caused by Valsa mali in China. However, there is no data available regarding the resistance risk of V. mali to pyraclostrobin. In this study, the sensitivities of 120 V. mali isolates to pyraclostrobin were detected. The isolates were collected from apple orchards with no application of pyraclostrob at six provinces in China during 2013–2015, and showed similar sensitivity to pyraclostrobin. The EC50 values of these 120 V. mali isolates to pyraclostrobin ranged from 0.0014 to 0.0240 μg/mL, indicating an excellent inhibitory efficacy of pyraclostrobin to the pathogen. The EC50 values were distributed as a unimodal curve with a mean value of 0.0091 μg/mL, and the mean EC50 displayed correlation with geographic location. Meanwhile, three pyraclostrobin-resistant mutants (PR mutants) of V. mali were obtained using fungicide adaption method, with a resistance factor (RF) of 41.0, 56.8 and 22.0, respectively. The mutants showed a stable resistance to pyraclostrobin after 10 transfers on pyraclostrobin-free medium. Comparing with the corresponding parental isolates, the hyphal growth, mycelial dry weight and pathogenicity of PR mutants were significantly reduced, but the number of propagules showed no significant difference. More importantly, no cross-resistance of PR mutants to pyraclostrobin, tebuconazole, difenoconazole, imazalil and thiophanate-methyl was detected. In conclusion, V. mali showed a moderate risk to pyraclostrobin, and pyraclostrobin could be used as an alternative fungicide to control AVC in the field in China.


Background
The apple tree Valsa canker (AVC), caused by Valsa mali, is the most serious disease of apple tree in Asia, especially in China (Abe et al. 2007;Suzaki 2008;Wang et al. 2011Wang et al. , 2014Li et al. 2013). Shaving diseased bark tissues and spraying fungicide on the wound were commonly used to control AVC. However, it is very difficult to cure the canker, because the pathogen could penetrate deep into the xylem (Ke et al. 2013). Meanwhile, this method also leads to the weakening of trees, making the tree more susceptible to the disease. Thus, how to inhibit the penetration of pathogen spores is the key for disease prevention. In 2013, annual dissemination of the pathogen in the field was studied systematically, and the peak period of pathogen dissemination was found to be from bud stage to young fruit stage of apple tree . Once pathogen spores colonize on the bark of apple tree, they then penetrate into bark tissues through lenticels, cracks, injuries, etc. (Ke et al. 2013). There is a latent period from pathogen colonizing in superficial tissues to entering into interior tissues, and the length of this latent period depends on the tree vigor (Meng et al. 2019). According to the annual dissemination and main penetration pathways of the pathogen, a prevention technology was developed and confirmed to be effective to prevent AVC by spraying fungicides or biological agents onto the bark surface for two times during June to August (Li et al. 2016). Thus, it is very important to select the appropriate and effective agents. Based on the information from China Pesticide Information Network (http://www.chinapesticide.org.cn/ywb/ index.jhtml), more than 20 kinds of chemical agents were registered to control AVC. Among them, the benzimidazole fungicide thiophanate-methyl was widely used in the past few years, which was effective in preventing infection of V. mali but showed little capacity in inhibiting the expansion of lesions (Tamura and Saito 1982). In recent years, the demethylation inhibitors, tebuconazole and difenoconazole, have been demonstrated to be very effective against V. mali and have become alternative fungicides to control AVC (Chen et al. 2009;Ma et al. 2012;Guo et al. 2015;Gao et al. 2017). Long-term use of single fungicide increased the risk of fungicide resistance. Although no fungicide-resistant strain of V. mali was found, the sensitivity of V. mali to difenoconazole has declined (Liu et al. 2019). Thus, it is urgent to find more fungicides with high efficiency in the control of AVC.
In early 2000s, a new fungicide, pyraclostrobin, belonging to quinone outside inhibitor (QoI) class, was developed by BASF Corporation, which was found to be preventive, curative, and eradicative against many plant diseases by inhibiting spore germination, mycelial growth, and sporulation of the target pathogenic fungi (Bartlett et al. 2002;Liang et al. 2015). However, QoIs are classified as high risk fungicides for triggering fungal resistance by Fungicide Resistance Action Committee (FRAC). In the past years, some populations of Botrytis cinerea have been found to be resistant to pyraclostrobin (Kim and Xiao 2011;Fernández-Ortuño et al. 2012). Meanwhile, isolates of Alternaria alternata have also showed resistance to pyraclostrobin (Avenot and Michailides 2015;Fan et al. 2015).
Pyraclostrobin has been confirmed to be effective against V. mali in laboratory test (Ma et al. 2012;Guo et al. 2015). However, the resistance risk of V. mali to pyraclostrobin is still largely unknown. In this study, the baseline sensitivity to pyraclostrobin of 120 V. mali stains from China was determined. Furthermore, pyraclostrobin-resistant mutants (PR mutants) of V. mali were obtained by fungicide adaption. Fitness of these resistant mutants in vitro and their cross-resistance to pyraclostrobin and other fungicides were subsequently investigated. The results will provide important guidance for scientific and rational use of pyraclostrobin in AVC control.

Results
Baseline sensitivity of V. mali to pyraclostrobin SHAM was often used to inhibit the alternative oxidase (AOX) in determination of the sensitivity of a pathogen to QoI fungicide. Thus, we firstly evaluated its effects on the mycelial growth of eight randomly selected isolates with different genetic background. The results showed that SHAM at 100 μg/mL had no effect on the EC 50 value of V. mali to pyraclostrobinin on AEA medium, whereas had significant inhibitory activity against the mycelial growth of V. mali (Additional file 1: Table S1). Thus, AEA medium without exogenous SHAM was used to detect the baseline sensitivity of V. mali to pyraclostrobin. The EC 50 values of the corresponding isolates to pyraclostrobin ranged from 0.0014 to 0.0240 μg/mL, with a mean value of 0.0091 μg/ mL, and the ratio of the maximum to the minimum EC 50 values was 17.5 (Additional file 2: Table S2). The frequency distribution of the EC 50 values of these V. mali isolates showed a unimodal curve with a positive skew (Fig. 1). Therefore, no resistance was observed in the field isolates of V. mali that we collected. However, the mean EC 50 values of V. mali strains collected from different geographic locations were significantly different. For example, the mean EC 50 values of strains from Liaoning were highest (0.0128 ± 0.0061 μg/mL), while from Xinjiang were lowest (0.0040 ± 0.0015 μg/ mL) ( Table 1).
Generation of pyraclostrobin-resistant (PR) mutants of V. mali and the resistance stability of mutants Two PR mutants (XJVM001R1 and XJVM001R2) were obtained from the parental isolate XJVM001; one PR mutant (972R) was obtained from the parental isolate 972. These three resistant mutants exhibited resistance factor (RF) values of 41.0, 56.8 and 22.0, respectively (Table 2). After 10 transfers on pyraclostrobin-free PDA, XJVM001R1 and XJVM001R2 still kept a moderate resistance to pyraclostrobin, with RF values of 33.9 and 52.5, respectively. However, the RF value of 972R declined from 22.0 to 11.7. Thus, the resistances of XJVM001R1 and XJVM001R2 to pyraclostrobin were more stable than that of 972R.

Characterization of PR mutants and their parental isolates
Compared to their parental isolates, all PR mutants had a smaller colony diameter and a reduced mycelial dry weight. There were no significant differences in propagule number between PR mutants and their parental isolates. However, the virulence of PR mutants was lower than that of their parental isolates ( Fig. 2 and Table 3). The EC 50 values of PR mutants to pyraclostrobin were significantly higher than that of their parental isolates. More importantly, the sensitivity of PR mutants and their parental isolates to pyraclostrobin was not correlated with the sensitivity to tebuconazole, difenoconazole, imazalil, and thiophanate-methyl ( Fig. 3 and Table 4). In other words, no cross-resistance in PR mutants to pyraclostrobin, tebuconazole, difenoconazole, imazalil and thiophanate-methyl was detected.

Discussion
It is of great importance to establish baseline sensitivity data of a phytopathogenic fungus to a fungicide, as such data are useful to evaluate the risk of resistance development in sensitive populations of the fungi, and meanwhile provide evidence to suggest future methods for control of fungicide-resistant mutants . In this study, the baseline sensitivity and resistance risk of V. mali populations in China to pyraclostrobin were reported.
The alternative oxidase (AOX) provides an alternative route for respiration and it will usually be activated after QoIs inhibit mitochondrial respiration in fungi by binding to the Qo site of the cytochrome bc1 complex and blocking electron transfer (Piccirillo et al. 2018). SHAM, an AOX inhibitor, is commonly added into culture media to suppress alternative respiration (Mizutani et al. 1995). In previous study, SHAM was confirmed to possess different effects on the sensitivity of pathogens to QoI fungicide (Di et al. 2016). Thus, it is necessary to firstly detect the effect of SHAM to the mycelial growth of V. mali in this study. According to the results we obtained, SHAM (100 μg/mL) has significant inhibitory activity against the mycelial growth of V. mali, but does not affect the EC 50 value of V. mali to pyraclostrobin. So, we determined the baseline sensitivity of V. mali populations to pyraclostrobin by not amending the medium with SHAM. The results showed that no pyraclostrobin-resistant V. mali isolate was detected. This sensitivity baseline could be used for monitoring any future sensitivity shifts to pyraclostrobin in the field populations of V. mali. Meanwhile, the mean EC 50 values of V. mali isolates to pyraclostrobin were different. Similar result was also observed when the sensitivity of Rhizoctonia cerealis isolates collected from different geographic location to trifluzamide was detected (Qi et al. 2014). It may be related to the natural differences of the strains in different regions, the physiological differences in the strains themselves, as well as the population structure of the strains of V. mali.
Fungal pathogens may develop resistance to different fungicides under certain selection pressures or under conditions of adversity. Resistance to QoI fungicides has been reported in a broad range of plant pathogenic fungi such as Plasmopara viticola (Wong and Wilcox 2000), Venturia inaequalis (Steinfeld et al. 2001) and Magnaporthe grisea (Avila-Adame and Koller 2003). In this study, 14 wild parental isolates of V. mali were randomly selected to produce PR mutants by repeated exposure to pyraclostrobin, and three resistant mutants were obtained. The resistance levels of two PR mutants were still stable over ten generations. The failure in using other parental isolates to generate stable resistant mutants might be related to the genetic variation among different strains (Pang et al. 2013).
Changes in biological traits could affect the competitiveness of resistant mutants and sensitive strains in  nature (Ziogas et al. 2003). Thus, biological fitness parameters are considered to be important factors that affect the formation of a fungicide-resistant population of fungal pathogens in the field (Zhang et al. 2014). The pyraclostrobin-resistant strains of B. cinerea showed decreased sporulation rate, spore germination rate and sclerotial production (Markoglou et al. 2006). Meanwhile, the resistance change of A. alternate to pyraclostrobin did not affect the mycelial growth, conidial germination, and conidial production, but the resistant strains showed similar or increased virulence compared to that of sensitive strains (Vega and Dewdney 2014). In this study, the mycelial growth, mycelial dry weight and pathogenicity of resistant mutants were reduced compared with those of their sensitive parental strains, but there was no significant difference in the propagules number. The reduced pathogenicity of resistant mutants might be related to their decreased growth. Clarifying cross-resistance of a pathogen to different fungicides will help to provide a theoretical basis for scientific application of fungicides on the control of the pathogen. Based on our results, no cross-resistance was detected among pyraclostrobin, tebuconazole, difenoconazole, imazalil and thiophanate-methyl in PR mutants. Due to the low fitness of PR mutants and lack of crossresistance with other fungicides, we concluded that the resistant risk of V. mali to pyraclostrobin was at a lowto-moderate level. To avoid the generation of pyraclostrobin resistance in V. mali, we suggest that fungicides with different mechanisms should be used alternately. Otherwise, applying pyraclostrobin in mixtures with multisite or non-cross-resistant fungicides may be adopted for control of AVC and fungicide resistance management of V. mali.

Conclusions
In this study, baseline sensitivity of V. mali to pyraclostrobin was established and the efficiency of pyraclostrobin against V. mali was subsequently evaluated. All the 120 V. mali isolates showed similar and high sensitivity to pyraclostrobin. Meanwhile, the resistance risk of these V. mali isolates to pyraclostrobin was evaluated, and no cross-resistance between pyraclostrobin and other fungicides including tebuconazole, difenoconazole, imazalil and thiophanate-methyl was detected in PR mutants. Thus, pyraclostrobin could be used as an alternative fungicide to control AVC in China. Meanwhile, V. mali showed a moderated risk to pyraclostrobin, and the corresponding resistance management strategies should also be considered. These results will lay a foundation for the scientific application of pyraclostrobin in the field.

Pathogen isolates, culture media and fungicides
A total of 120 isolates of V. mali were tested in this study (Additional file 3: Table S3), which were collected  Fig. 3 Cross-resistance assays between pyraclostrobin and other four fungicides in Valsa mali. a Cross-resistance assay between pyraclostrobin and tebuconazole. b Cross-resistance assay between pyraclostrobin and difenoconazole. c Cross-resistance assay between pyraclostrobin and imazalil. d cross-resistance between pyraclostrobin and thiophanate-methyl from infected apple trees in orchards with no application of pyraclostrobin at different geographic regions of China (Liaoning, Shandong, Gansu, Shanxi, Shaanxi, and Xinjiang) during 2013-2015. All the isolates were maintained on potato dextrose agar (PDA) medium at 4°C. Alkyl ester agar (AEA) medium was used to detect the sensitivity of V. mali to pyraclostrobin. PDA medium was used to detect the sensitivity of the pathogen to other fungicides. PDB medium was used to culture strains of V. mali. All fungicides used were technical-grade products, including pyraclostrobin (97.5% a.i.; Xi'an Hytech Agrochemicals Co., Ltd., China), tebuconazole (97% a.i.; Jiangsu Flag Chemical Industry Co., Ltd., China), difenoconazole (95% a.i.; Xi'an Hytech Agrochemicals Co., Ltd.), imazalil (98% a.i.; Anhui Guangxin Agrochemical Co., Ltd., China), and thiophanate-methyl (97% a.i.; Anhui Huaxing Chemical Industry Co., Ltd., China). Pyraclostrobin, tebuconazole, difenoconazole and imazalil were respectively dissolved in acetone to produce a stock solution containing 10 mg/mL of the active ingredient. Tiophanate-methyl was dissolved in acetone and then diluted with 0.1% Tween-20 to produce a stock solution containing 3.2 mg/ mL of the active ingredient. Fungicide stock solution was then stored in the dark at 4°C until used.
Baseline sensitivity of V. mali isolates to pyraclostrobin The sensitivities of 120 isolates of V. mali to pyraclostrobin were determined by measuring mycelial growth on agar plates. Mycelial plugs (5 mm in diameter) from the edge of a 3-day-old colony were transferred to a series of AEA plates amended with increasing concentrations of pyraclostrobin (0, 0.000625, 0.0025, 0.01, 0.025, 0.04, 0.16, 0.64, and 2.56 μg/mL), and the final concentration of acetone in the medium was standardized at 0.1% (v/ v). Plates without pyraclostrobin were used as control. The plates were incubated at 25°C for three days in a growth chamber. For each plate, the EC 50 value was calculated by measuring the average colony diameter and regressing the percentage growth inhibition against the log of fungicide concentration. The experiment was repeated twice with three replicates for each treatment.

Generation of PR mutants of V. mali
Fourteen isolates (3)(4)(5)(6)(7)(8)XJVM001,XJVM006,878,881,889,891,893,902,910,972,980,982 and 985) of V. mali were randomly selected to generate PR mutants. Fresh mycelial plugs from colony margins were transferred to AEA plates (seven plugs per plate) containing pyraclostrobin of 80 μg/mL. After incubation at 25°C for two weeks, any fast-growing sectors from the otherwise restricted colonies were transferred to a series of AEA plates amended with increasing concentrations of pyraclostrobin (100, 200 and 500 μg/mL) .

Resistance level and stability of PR mutants
To determine the resistance level of PR mutants to pyraclostrobin, mycelial plugs of the mutants were placed on AEA plates amended with 0, 0.002, 0.02, 0.2, 2, 20, and 200 μg/ mL of pyraclostrobin. The EC 50 values were calculated by regressing the percentage growth inhibition against the log of fungicide concentration. The level of fungicide resistance, termed as resistance factor (RF), was equal to the EC 50 value  . To evaluate the stability of the resistant phenotypes, the PR mutants were subjected to ten successive transfers on new fungicide-free PDA plates. The RFs at the 1st and 10th generations were determined as described by Wang et al. (2015).
Hyphal growth, dry weight, propagule number, and pathogenicity of the PR mutants and their parental isolates Hyphal growth and propagule number of PR mutants and their parental isolates were determined on fungicide-free PDA plates. Colony diameters were measured perpendicularly after incubation at 25°C for 60 h. The number of propagules was subjectively evaluated when the mutants were cultured for 25 d under natural light at room temperature. Moreover, five mycelial plugs taken from the colony edge of each PR mutant and parental isolate were transferred to a flask containing 100 mL of PDB. After 3 d cultivation at 25°C with shaking at 100 rpm, the mycelia were collected, dried (56°C for 12 h), and weighed. There were three replicate plates and three replicate flasks for each isolate, and the experiment was repeated three times. The virulence of PR mutants and parental isolates was determined by a scald wounding method, which was carried out by inoculating detached twigs of Malus domestica Borkh. cv. 'Fuji' with a 5 mm mycelial plug derived from the margin of a young colony (Zang et al. 2007;Wei et al. 2010). After 5 d incubation at 25°C, the length of each necrotic lesion was measured. Three twigs for each isolate were inoculated, and the experiment was performed three times.

Cross-resistance analysis
Pyraclostrobin-resistant and pyraclostrobin-sensitive isolates were used to assess their sensitivity to five fungicides, including pyraclostrobin, tebuconazole, difenoconazole, imazalil, and thiophanate-methyl. It was proceeded by using the same method of mycelial growth inhibition described above with fungicides at various final concentrations (Table 5). The individual EC 50 values for these fungicides in all isolates were calculated. The experiment was repeated three times.

Statistical analysis
The data from different repetitions were used for statistical analysis. All statistical analysis was performed using SPSS 16.0 (Statistical Package for the Social Science, SPSS Inc., Chicago, IL). A regression equation was derived by correlating the log10 of inhibitor concentration of pyraclostrobin with the probit of inhibition percentage of average radial mycelial growth of V. mali, and effective concentration for 50% inhibition (EC 50 ) of V. mali was calculated from the regression equation. The ANOVA procedure of SPSS (P < 0.05) was used to determine significant differences among the EC 50 values.