Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii

Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii Suyue Jing, Fadi Zhu, Xiaodong Wen, Jing Zhang, Gang Feng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3802508/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Litchi downy blight, caused by Phytophthora litchii , presents significant challenges to litchi production, storage, and transportation. Previous studies have shown that coumoxystrobin exhibits effective inhibitory activity against P . litchii . Salicylhydroxamic acid (SHAM), an alternative respiratory pathway inhibitor, is commonly used to evaluate the efficacy of cytochrome respiratory pathway inhibitor like coumoxystrobin against fungal phytopathogens in vitro. In this study, the toxicity of SHAM on various developmental stages of P . litchii , including mycelial growth, sporangial germination, zoospore release, and cystospore germination, was assessed. The EC 50 values for SHAM were determined as 166.72, 150.69, 333.97, and 240.91 μg/mL, respectively. Subsequently, the activity of coumoxystrobin against P . litchii was assessed in the presence of SHAM at a concentration of 50 μg/mL, which showed slight inhibition below 20% for all four developmental stages. The addition of SHAM significantly improved the inhibitory activity of coumoxystrobin against P . litchii at different stages, with reductions in EC 50 values ranging from 7.55- to 122.92-fold. Moreover, respiration assays revealed that a concentration of 5 μg/mL coumoxystrobin inhibited P . litchii mycelial respiration to a lesser extent compared to the combined effect of coumoxystrobin and SHAM. SHAM also enhanced the control efficacy of coumoxystrobin against phytophthora blight development on litchi leaves. Previously, we reported that coumoxystrobin effectively controls postharvest downy mildew on litchi fruit. Consequently, coumoxystrobin holds promise as an agent for litchi downy blight control in the field and after harvest. Furthermore, similar to previous studies, SHAM, an alternative oxidase (AOX) inhibitor, was found to significantly enhance the activity of the two aforementioned QoI fungicides against P . litchii , both in vitro and in vivo. This suggests that further exploration of AOX inhibitors and the role of AOX in plant diseases could contribute to the rational use of QoI fungicides and improve control efficiency for plant diseases. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Litchi, a subtropical to tropical fruit, is popular in consumer markets and cultivated in more than 20 countries due to its attractive fruity aroma, delicious taste, and high nutritional value (Xu et al. 2018). Litchi downy blight caused by Phytophthora litchii is one of the most important oomycete diseases that occurs during litchi production, storage and transportation (Zhang et al. 2021a). The disease not only affects tender leaves, twigs, and flowers at unripe stages but also causes damage to mature litchi fruit, leading to enormous yield losses and severe commercial losses of more than 60% in southern China (Zheng a et al. 2019; Guo et al. 2021 ). Currently, chemical fungicides remain the main and most effective strategy to control litchi downy blight (Zheng b et al. 2021). ‘Traditional’ fungicides, such as the multisite inhibitor mancozeb, target site-specific phenylamide fungicide metalaxyl, and carboxyl acid amide (CAA) fungicide dimethomorph, were used for the control of P. litchii in the 1990s (Wang et al. 2009 ). Additionally, quinone outside inhibitor (QoI) fungicides, such as azoxystrobin and pyraclostrobin, have been registered for the control of litchi downy blight according to data from the China Pesticide Information Network (Wang et al. 2010 ). Coumoxystrobin((E)-methyl-2-{2-[(3-n-butyl-4-methylcoumarin-7-yloxy) methyl] phenyl}-3-methoxyacrylate), as a new QoI fungicide, has recently been registered for the control of several crop diseases in China, such as cucumber downy mildew, apple Valsa canker, and citrus scab ( http://www.icama.org.cn/hysj/index.jhtml ). In our previous studies, coumoxystrobin exhibited a strong inhibitory effect on both P. litchii at different developmental stages in vitro and downy blight in detached litchi fruit in the laboratory. (Zhang et al. 2021b), which indicated a potential for coumoxystrobin application in the control of litchi downy blight caused by P. litchii . To better understand the use of coumoxystrobin to control litchi downy blight, it is important to further study the effect of coumoxystrobin against P. litchii . Alternative oxidase (AOX) can affect fungal sensitivity to some QoI fungicides by bypassing the cytochrome pathway when normal electron transport pathways are inhibited (Bradley and Pedersen. 2011; Duan et al. 2012 ; Chaulagain et al. 2019 ; Barsottini et al. 2019 ). To more accurately evaluate the in vitro activity of QoI fungicides against plant pathogens, salicylhydroxamic acid (SHAM), an AOX inhibitor, has been suggested as a supplement in QoI activity tests to suppress alternative respiration (Ziogas et al. 1997 ; Walker, et al. 2009 ; Liang et al. 2015 ). The addition of 50 or 100 µg/mL SHAM under normal circumstances will increase the inhibition activity of QoI fungicides against the pathogen (Jin et al. 2009 ; Piccirillo et al. 2018 ). However, SHAM does not always enhance the inhibitory effect of QoI fungicides toward pathogens due to the differences among pathogens and chemical features (Walker et al. 2009 ). Due to the toxic effects of these AOX inhibitors on pathogens such as Fusicladium effusum , Sclerotinia sclerotiorum and Botrytis cinerea in vitro , inhibition activity for QoIs should be performed without the addition of these inhibitors (Seyran et al. 2010 ; Liang et al. 2019 ). To date, there is no report about the in vitro activity of SHAM and its effect on the coumoxystrobin activity against P. litchii . Therefore, the aims of the current research were to (i) explore the toxicity of SHAM on P. litchii , (ii) determine the activity of coumoxystrobin combined with SHAM against P. litchii at different developmental stages, (iii) examined the rate of mycelial respiration of P. litchii when treated with coumoxystrobin alone or mixed with SHAM, and (iv) assess the control efficacy of coumoxystrobin and SHAM against phytophthora downy blight in vivo . Materials and Methods Fungal isolates. P. litchii isolated from diseased fruit of litchi in Hainan (Sun, et al., 2017 ) was provided by the Postharvest Pathology and Preservation Laboratory, Environment and Plant Protection Institute of the Chinese Academy of Tropical Agricultural Science in Hainan Province, China. Fungicides and SHAM. Technical grade coumoxystrobin (96% active ingredient [a.i.], Shenyang Research Institute of Chemical Industry) and azoxystrobin (95% a.i., Shenyang Research Institute of Chemical Industry) were dissolved in acetone to make 10 g/L stock solutions. SHAM (99% a.i., Sigma-Aldrich Shanghai Trading Co. Ltd.) was dissolved in methanol to make a stock solution with a concentration of 40 g/L. All stock solutions were stored at 4°C in the dark until further use. Effect of SHAM on in vitro mycelial growth. The inhibition activity of SHAM against P. litchii was determined by the mycelial growth inhibition method (Zhou et al. 2016 ). Mycelial plugs (with a diameter of 5 mm) were picked from 72-h colonies of the isolate and transferred to white kidney bean ( Phaseolus lunatus Linn.) agar (WKBA) plates amended with SHAM at 25, 50, 100, 200, and 400 µg/mL. WKBA plates with less than 1% solvent served as controls. All plates were incubated for 4 days in 60 mm Petri dishes at 28°C on WKBA medium in the dark. Colony diameters were measured at perpendicular angles, and the average diameter (minus the original diameter of the inoculation plug) were used to calculated mycelial growth inhibition. The EC 50 (median effective concentration) were calculated by regressing percentage growth inhibition against the log of fungicide concentration. Three replicates were used per concentration. The experiment was performed in triplicate. Effect of SHAM on in vitro sporangial germination. Sporangia were obtained from 7-day-old plates of P. litchii incubated in WKBA medium, and the sporangia were suspended in sterile distilled water until a concentration of 1×10 7 sporangia/mL was obtained. The inhibition activitiy of SHAM against P. litchii was investigated according to Zhou et al. ( 2016 ), with minor adjustments. In brief, 50 µL of the sporangial suspension was spread evenly on WA (9 g of agar, 1000 mL of distilled water) plates amended with 50, 60, 100, 160 and 240 µg/mL SHAM and without SHAM as the control group. For each concentration, three replicate plates were prepared. After incubating for 10 h at 28°C in the dark, germination was quantified by counting 100 sporangia per plate under a microscope. If the germ tube length was greater than the length of the sporangia, the sporangia were considered to be germinated, and one hundred germinated sporangia on each plate were counted under the microscope. The sporangial germination inhibition rate was calculated. The EC 50 were calculated by regressing germination inhibition rate against the log of fungicide concentration. The experiment was performed three times. Effect of fungicides with and without SHAM on mycelial growth. The activity of fungicides with and without SHAM on the mycelial growth of P. litchii was determined according to the assay described by Zhou et al. ( 2016 ), with minor modifications. Briefly, mycelial plugs from the actively growing colony margin were transferred to WKBA plates supplemented with 0.078, 0.156, 0.3125, 0.625 and 1.25 µg/mL coumoxystrobin and with 0.039, 0.078, 0.156, 0.3125, and 0.625 µg/mL azoxystrobin. When 50 µg/mL SHAM was present, the concentrated fungicides were diluted 10-fold. WKBA medium without fungicides was used as a control. The final amount of solvent never exceeded 1%(v/v) in our plates. For each fungicide and fungicide concentration, three replications were prepared. After 4 days at 28°C in the dark, colony diameters were measured and EC 50 values were calculated as previously described. This experiment was repeated three times. Effect of fungicides with and without SHAM on sporangial germination. Sporangia suspensions preparation and sporangial germination were conducted as described above. Coumoxystrobin concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µg/mL and azoxystrobin concentrations of 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 µg/mL were tested according to Zhou et al. ( 2016 ), with minor modifications. When SHAM was present, the fungicide concentrations were 0.001, 0.004, 0.016, 0.032, 0.064, and 0.128 µg/mL. Effect of fungicides with and without SHAM on zoospore release. A sporangial suspension (1 \(\times\) 10 7 sporangia/mL) of P. litchii was prepared, and the empty sporangia were counted microscopically according to Zhou et al. ( 2016 ), with minor modifications. For activity tests, the sporangial suspension was added to a 2 mL microcentrifuge tube (1:1, vol/vol) with coumoxystrobin at final concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µg/mL, azoxystrobin at final concentrations of 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 µg/mL, and without fungicides (in the control group). When SHAM was present, the fungicide concentrations were 0.001, 0.004, 0.016, 0.032, 0.064, and 0.128 µg/mL. Then, microcentrifuge tubes were incubated for 4 h under intense light at 15°C. The 100 sporangia were observed under a microscope, and the percent inhibition of zoospore release was calculated. Three replicates were included per treatment. The experiment was repeated three times. Effect of fungicides with and without SHAM on cystospore germination. The cystospore germination assay preparation and the assays of fungicide toxicity with and without SHAM for cystospore germination of P. litchii were conducted according to Zhou et al. ( 2016 ). The final coumoxystrobin concentrations were 0.01, 0.02, 0.04, 0.08, 0.16, and 0.64 µg/mL, and the final azoxystrobin concentrations were 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 µg/mL. To determine EC 50 values in the absence of SHAM, 50 µL zoospore suspensions were spread evenly over WA plates amended with fungicides at final concentrations of 0.001, 0.004, 0.008, 0.016, 0.032, 0.064 and 0.128 µg/mL, and the WA plates were transferred to an incubator for 6 h at 20°C in the dark. If the germ tube length was greater than the cystospore length, the cystospore was regarded as germinated, and the number of germinated cystospores was counted under a microscope. Three replicates were included per treatment. The experiment was repeated three times. Effect of fungicides with and without SHAM on respiratory rates. Oxygen consumption rate of P. litchii mycelia treated by fungicides with and without SHAM was measured according to a previously described method (Zhang et al. 2021). Briefly, mycelia were collected, filtered, and washed three times with fresh PBS (pH = 7.2 ~ 7.4). Then, stock solutions of coumoxystrobin and azoxystrobin, with or without SHAM, were diluted with sterile distilled water to 0.5, 2.5, 5, 10, and 20 µg/mL, respectively. At room temperature, fresh mycelia were added to the different solutions, and the oxygen concentration was measured after 2 minutes. The oxygen consumption inhibition rate was calculated as follows: oxygen consumption inhibition rate (%) = (oxygen consumption of control - oxygen consumption of each treatment) \(\times\) 100/oxygen consumption of control. Three replicates were included per treatment. The experiment was repeated three times. Effect of SHAM on the control efficacy of fungicides in vitro . The control efficacy of the fungicides on litchi leaves in the presence or absence of SHAM was measured by the detached leaf assay (Chaulagain et al. 2019 ). The healthy, tender leaves of ‘Yutan mili’ were cleaned with distilled water, and each leaf was surface disinfected with 75% ethyl alcohol and then washed four times with sterile water and allowed to dry. A small wound was made on each leaf by aseptic puncture. Then, the leaves were immersed in fungicide dilutions containing coumoxystrobin or azoxystrobin at 4, 20, and 100 µg/mL for 5 s with and without SHAM. Leaves treated with sterile water containing 0.1% methanol served as controls. All leaves were air-dried. A 6 mm mycelial plug was transferred onto leaves wounded by a hole puncher. The inoculated samples were stored in an incubator for 3 days at 28°C and 80% relative humidity, and lesion diameters were measured twice at right angles. Mycelial plugs from WKBA medium were used as a negative control. Six leaves were included in each treatment. The experiment was repeated twice. Statistical analysis. All data were analyzed by data processing software (SPSS Science, version 26). EC 50 values were estimated from the fitted regression line of the log-transformed percentage inhibition plotted against the log-transformed fungicide concentration. Control efficacies were calculated by the following formula (the diameters of the mycelial plugs were subtracted from the lesion diameters before calculations): Control efficacy (%) = [(lesion diameter of the control – lesion diameter of the treated sample)]/[lesion diameter of the control] * 100. Student’s paired t test was employed to determine significant differences in the mean EC 50 values (and control efficacy) for coumoxystrobin with and without SHAM. Results Toxicity of SHAM to P. litchii. The EC 50 values of P. litchii to SHAM on mycelial growth (Fig. 1 A) and sporangial germination (Fig. 1 B) were 166.72 and 150.69 µg/mL, respectively. SHAM at 50 µg/mL weakly inhibited mycelial growth and sporangial germination in P. litchii , with both of inhibition rates below 20%, respectively, while at 100 µg/mL, the inhibition rates were increased to 38.31% and 38.54%, respectively. Thus, SHAM at 50 µg/mL was used to study the effect of SHAM on the activity of coumoxystrobin against P. litchii . In vitro toxicities of fungicides with and without SHAM on mycelial growth. P. litchii was grown on WKBA medium to determine the mycelial growth inhibition activity of coumoxystrobin and azoxystrobin against mycelial growth in the presence and absence of SHAM (Fig. 2 ). Without adding SHAM, the fungicides coumoxystrobin and azoxystrobin exhibited good inhibitory effect on the mycelial growth of P. litchii in vitro , with EC 50 values of 0.4531 and 0.0741 µg/mL, respectively. When SHAM (at 50 µg/mL) was potentiated, the EC 50 values of coumoxystrobin and azoxystrobin for the mycelial growth of P. litchii were reduced 12.11-fold ( P < 0.05) and 8.13-fold ( P < 0.05), respectively. The results showed that the activity of coumoxystrobin against P. litchii in the presence of SHAM was significantly ( P < 0.05) improved compared with that using fungicides alone according to the paired t test. In vitro toxicities of fungicides with and without SHAM against sporangial germination. Coumoxystrobin was found to have excellent inhibitory effect against sporangial germination (Fig. 3 ) of P. litchii . The inhibition rates increased significantly (P < 0.05) with the fungicide concentrations increasing, as shown clearly by the dose-reaction curves. Low concentrations (from 0.1 to 3.2 µg/mL) of coumoxystrobin exhibited promising inhibition effect on sporangial germination in P. litchii , with mycelial tube germination inhibition values of 31.56–95.09%. SHAM at 50 µg/mL, with inhibition rate below 10% against sporangial germination, significantly ( P < 0.05) increased the activity of coumoxystrobin and azoxystrobin against P. litchii . In vitro toxicities of fungicides with and without SHAM against zoospore release. Results of the toxicity test of the fungicides coumoxystrobin and azoxystrobin against zoospore release (Table 1 ) of P. litchii showed that coumoxystrobin and azoxystrobin had inhibited zoospore release of P. litchii in a dose-dependent manner, with EC 50 values of 0.4425 and 0.0553 µg/mL, respectively. Significant ( P < 0.05) differences were observed between the presence and absence of SHAM. When SHAM was added at 50 µg/mL, the inhibition activity of coumoxystrobin and azoxystrobin were increased by 122.92 and 8.38-fold, respectively, compared to those with fungicides alone. Table 1 Effect of SHAM on the median effective concentration (EC 50 ) values of coumoxystrobin for P. litchii zoospore release Fungicide Toxicity regression equation r EC 50 (µg/mL) PF a Coumoxystrobin y = 1.3394x + 5.4742 0.9348 0.4425 Azoxystrobin y = 1.2795x + 6.6090 0.9918 0.0553 SHAM y = 3.7272x-4.4065 0.9857 333.97 Coumoxystrobin + SHAM y = 0.6619x + 6.6168 0.9858 0.0036 122.92 Azoxystrobin + SHAM y = 1.3707x + 7.9858 0.9742 0.0066 8.39 a PF, potentiation factor, the ratio of EC 50 values of fungicides to EC 50 values of fungicides with SHAM. In vitro toxicities of fungicides with and without SHAM on cystospore germination. Compared with other developmental stages, coumoxystrobin showed greater inhibition of cystospore (Table 2 ) germination in P. litchii , with EC 50 values of only 0.0830 µg/mL, and it exhibited a significant ( P < 0.05) difference from azoxystrobin. Furthermore, when SHAM was added, coumoxystrobin was more effective against cystospore germination than the single fungicides, with a potentiation factor of 7.55, and similar results were found for azoxystrobin. Table 2 Effect of SHAM on the median effective concentration (EC 50 ) values of coumoxystrobin for P. litchii cystospore germination Fungicide Toxicity regression equation r EC 50 (µg/mL) PF a Coumoxystrobin y = 1.3631x + 6.4731 0.9860 0.0830 Azoxystrobin y = 2.0496x + 8.1522 0.9855 0.0290 SHAM y = 2.8959x-1.8975 0.9957 240.9100 Coumoxystrobin + SHAM y = 1.8172x + 8.5595 0.9602 0.0110 7.55 Azoxystrobin + SHAM y = 0.8942x + 6.8906 0.9693 0.0077 3.77 a PF, potentiation factor, the ratio of EC 50 values of fungicides to EC 50 values of fungicides with SHAM. Respiration inhibition by coumoxystrobin and azoxystrobin with or without SHAM. Respiration inhibition was tested during coumoxystrobin (Fig. 4 A) treatment at different concentrations. The results showed that coumoxystrobin at 2.5 µg/mL could inhibit the mycelial respiration of P. litchii , and the inhibition rate was 52.54%. For all tested concentrations, the oxygen consumption inhibition rate of coumoxystrobin in the presence of SHAM was significantly ( P < 0.05) improved compared to that with the use of fungicides alone ( P < 0.05), except at 0.5 µg/mL. Coumoxystrobin and azoxystrobin (Fig. 4 B) had similar response trends in inhibiting the mycelial respiration of P. litchii . Effect of SHAM on the control efficacy of coumoxystrobin against P. litchii. The control efficacy of coumoxystrobin (Fig. 5 A) against P. litchii was determined on leaves. For all tested concentrations, coumoxystrobin exhibited lower control effects of P. litchii than azoxystrobin (Fig. 5 B), except at 100 µg/ml, however, no significant difference was observed ( P > 0.05). Coumoxystrobin at 100 µg/ml provided 100% control efficacy. When SHAM was added at 100 µg/mL, the control efficacy of coumoxystrobin at 4 µg/mL was significantly ( P < 0.05) increased. The control efficacy of coumoxystrobin at 20 µg/mL combined with 100 µg/mL SHAM was increased potentiated by 20.16%. Similar results were found for the reference QoI fungicide azoxystrobin. Discussion SHAM was previously recommended to be added to the culture medium to inhibit AOX in the alternative respiratory pathway to reasonably evaluate the activity of QoIs fungicides, but many studies have shown that this approach may not be reasonable due to the toxicity of SHAM against different plant pathogens (Seyran, et al., 2010 ; Ma, et al., 2018 ; Liang, et al., 2019 ). In current study, SHAM exhibited an inhibitory effect on the mycelial growth, sporangial germination of P. litchii . Thus, we suggested not to add SHAM in coumoxystrobin and azoxystrobin activity assay against P. litchii . In addition, a potentiated effect between SHAM and coumoxystrobin against P. litchii was observed in our research. This consistent potentiation has also been reported in other plant pathogens (Liang, et al., 2019 ). Nalumpang reviewed that SHAM could not only inhibit the activity of AOX, but also suppress the activity of other essential enzymes of fungi (Nalumpang, et al., 2021 ). In current research, the potentiation effect tends to be stronger with a lower concentration of coumoxystrobin in the control efficacy test. However, the activity of AOX and other essential enzymes were not directly measured in this study. Thus, the potentiation mechanism needs to be further explored. QoI fungicides are safe, broad spectrum, efficient and environmentally friendly, but plant pathogens easily develop resistance against these fungicides, which restricts their application in plant disease control (Zhang et al., 2019 ). A study on the mechanism of resistance to QoI fungicides found that the AOX in the alternative oxidation pathway of many plant pathogens affects the activity of QoI fungicides, and overexpression of the AOX gene is one of the mechanisms of resistance of many plant pathogens. Studies have shown that the resistance of plant pathogenic fungi such as Septoria tritici (Ziogas et al., 1997 ), Mycosphaerella fijensis (Sierotzki et al., 2000 )d oryzae (Zhang et al., 2019 ) to QoI fungicides is related to AOX. Similar results were also found for the pathogenic oomycete Plasmopara viticola (Fontaine et al., 2019 ). Many studies have found that the addition of the AOX inhibitor SHAM can increase the sensitivity of resistant pathogens to QoI fungicides (Ziogas et al., 1997 ; Sierotzki et al., 2000 ; Seyran et al., 2010 ; Liang, et al., 2019 ; Zhang et al., 2019 ) and even make the pathogens resistant to azoxystrobin sensitive again (Ziogas et al., 1997 ). In this study, adding a lower dose of SHAM (50 µg/mL) significantly improved the activity of coumoxystrobin against P. litchii . The test of azoxystrobin also showed a similar trend, which is consistent with previous research results (Seyran et al., 2010 ; Liang, et al., 2019 ). Further respiratory inhibition tests showed that when the concentration was greater than 5 mg/L, the inhibitory effects of coumoxystrobin on oxygen consumption by P. litchii were no longer dose dependent and remained unchanged. However, the inhibition rate of the treatment with SHAM increased by more than 20%, indicating that P. litchii may activate the alternative oxidation pathway under the stress of these two fungicides. Using two or more fungicides with different modes of action in controlling the same pathogen is an important approach to delaying resistance and is recommended by the FRAC. The abovementioned similar research results regarding the activities and synergistic effect of AOX inhibitors suggest a potential new strategy for the rational use of and resistance control for QoI fungicides, namely, using AOX as an auxiliary fungicide target. In recent years, scientists from Brazil have begun to pay attention to the toxicity of AOX inhibitors to plant pathogens, their synergistic effect with fungicides and exploring novel AOX inhibitors for plant pathogens (Seyran et al., 2010 ; Liang, et al., 2019 ; Barsottini et al., 2019 ; Shi et al. 2020 ). Godwin and Tian also published similar ideas about AOX as a potential target (Ebiloma et al., 2019 ; Tian, et al., 2020 ). However, based on some current researches (Juárez, et al., 2006 ; Thomazella, et al., 2012 ; Ca´rdenas-Monroy, et al., 2017 ; Lin, et al. 2019 ), AOX is believed not to be essential in most fungi, yet AOX is important in their stress response and contributes to their pathogenicity and virulence. We believe that further study on the functions and inhibitors of AOX in plant pathogenic fungi or oomycetes will be helpful for the rational use of QoI fungicides, resistance management and more efficient plant disease control. In conclusion, coumoxystrobin exhibited strong inhibitory activity at different stages of the P. litchii life cycle. SHAM could enhance the antifungal activity of the QoI fungicides coumoxystrobin and azoxystrobin against P. litchii in vitro and in vivo . This enhanced activity from using coumoxystrobin and SHAM together may shed some light on the control of litchi downy blight and the rational use of QoI fungicides. To reasonably evaluate the control effect of coumoxystrobin on downy mildew of litchi caused by P. litchii , more strains from field and field trials are needed for verification. In addition, we suggest further biochemical and molecular studies to help elucidate the active role of AOX in plant pathogen physiology and its potential as an auxiliary target for disease control in plants. Declarations Authors’ contributions Jing SY and Zhu FD contributed equally to this work. Zhang J and Zhu FD designed and supervised the project. Jing SY, Zhu FD and Wen XD performed most of the experiments. Zhu FD and Wen XD analyzed the data and participated in interpretation of the results. Zhu FD and Wen XD prepared the manuscript. Zhang J and Feng G helped to revise the manuscript. All authors have approved the final version of the manuscript. Data availability statement The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. Conflicts of interest No conflict of interest exists for all authors. Acknowledgments This research was financially supported by the National Natural Science Foundation of China (32302413) and Chinese Academy of Tropical Agricultural Sciences for Science and Technology Innovation Team of National Tropical Agricultural Science Center (CATASCXTD202305). References Bradley CA, Pedersen DK (2011) Baseline Sensitivity of Cercospora zeae-maydis to Quinone Outside Inhibitor Fungicides. Plant Dis 95:189–194 Barsottini MR, Pires BA, Vieira ML, Pereira JG, Costa PC, Sanita J, Coradini A, Mello F, Marschalk C, Silva EM, Paschoal D, Figueira A, Rodrigues FH, Cordeiro AT, Miranda PC, Oliveira PS, Sforca ML, Carazzolle MF, Rocco SA, Pereira GA (2019) Synthesis and testing of novel alternative oxidase (AOX) inhibitors with antifungal activity against Moniliophthora perniciosa (Stahel), the causal agent of witches' broom disease of cocoa, and other phytopathogens. Pest Manag Sci 75:1295–1303 Barsottini MR, Copsey A, Young L, Baroni RM, Cordeiro AT, Pereira GA, Moore AL (2020) Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa . Commun Biology 3:263 Ca´rdenas-Monroy CA, Pohlmann T, Piño´n-Za´rate G, Matus-Ortega G, Guerra G, Feldbru¨gge M et al (2017) The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in Ustilago maydis . PLoS ONE 12:e0173389 Chaulagain B, Dufault N, Raid RN, Rott P (2019) Sensitivity of two sugarcane rust fungi to fungicides in urediniospore germination and detached leaf bioassays. Crop Prot 117:86–93 Duan YB, Liu SM, Ge CY, Feng XJ, Chen CJ, Zhou MG (2012) In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystrobin, SHAM, and thiram. Pesticide Biochemistry And Physiology. 103:101–107 Ebiloma GU, Balogun EO, Cueto-Díaz EJ, de Koning HP, Dardonville C (2019) Alternative oxidase inhibitors: mitochondrion‐targeting as a strategy for new drugs against pathogenic parasites and fungi. Med Res Rev 39:1553–1602 Fontaine S, Remuson F, Caddoux L, Barrès B (2019) Investigation of the sensitivity of Plasmopara viticola to amisulbrom and ametoctradin in French vineyards using bioassays and molecular tools. Pest Manag Sci 75:2115–2123 Guo HY, Bao JD, Lin LY, Wang ZX, Shi MY, Huang YL, Wang R, Li B, Liu BJ, P. Q., and, Chen QH (2021) Genome sequence data of Peronophythora litchii , an oomycete pathogen causing litchi downy blight. Mol Plant-Microbe Interact J 34:707–710 Jin LH, Chen Y, Chen CJ, Wang JX, Zhou MG (2009) Activity of azoxystrobin and SHAM to four phytopathogens. Agricultural Sci China 8:835–842 Juárez O, Guerra G, Velázquez I, Flores-Herrera O, Rivera‐Pérez RE, Pardo JP (2006) The physiologic role of alternative oxidase in Ustilago maydis . FEBS J 273:4603–4615 Liang HJ, Di YL, Li JL, You H, Zhu FX (2015) Baseline sensitivity of pyraclostrobin and toxicity of SHAM to Sclerotinia sclerotiorum . Plant Dis 99:267–273 Liang H, Li J, Luo C, Li J, Zhu FX (2019) Effects of SHAM on the sensitivity of Sclerotinia sclerotiorum and Botrytis cinerea to QoI fungicides. Plant Dis 103:1884–1888 Lin ZS, Wu JY, Jamieson PA, Zhang CQ (2019) Alternative oxidase is involved in the pathogenicity, development, and oxygen stress response of Botrytis cinerea . Phytopathology 109:1679–1688 Ma DC, Jiang JG, He LM, Cui KD, Mu W, Liu F (2018) Detection and characterization of Qol-resistant Phytophthora capsici causing pepper phytophthora blight in China. Plant Dis 102:1725–1732 Nalumpang S, Poti T, Akimitsu K (2021) Effect of salicylhydroxamic acid on mycelial growth and baseline sensitivity to azoxystrobin in Phytophthora infestans causing potato late blight in Thailand. Int J Agricultural Technol 17(4):12 Piccirillo G, Carrieri R, Polizzi G, Azzaro A, Lahoz E, Fernández-Ortuno D, Vitale A (2018) In vitro and in vivo activity of QoI fungicides against Colletotrichum gloeosporioide s causing fruit anthracnose in Citrus sinensis . Scientia Horticulturae. 236:90–95 Seyran M, Brenneman TB, Stevenson KL (2010) vitro toxicity of alternative oxidase inhibitors salicylhydroxamic acid and propyl gallate on Fusicladium effusum . J Pest Sci 83:421–427 Shi N, Ruan H, Gan L, Dai YL, Yang XJ, Du YX, Chen FR (2020) Evaluating the sensitivities and efficacies of fungicides with different modes of action against Phomopsis asparagi . Plant Dis 104:448–454 Sierotzki H, Parisi S, Steinfeld U, Tenzer I, Poirey S, Gisi U (2000) Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijensis field isolates. Pest Manag Sci 56:833–841 Sun JH, Gao ZY, Zhang XC, Zou XX, Cao LL, Wang JB (2017) Transcriptome analysis of Phytophthora litchii reveals pathogenicity arsenals and confirms taxonomic status. PLoS ONE 12:e0178245 Thomazella D, Teixeira P, Oliveira HC, Saviani EE, Pereira AG et al (2012) The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development. New Phytol 194:1025–1034 Tian F, Lee SY, Woo SY, Chun HS (2020) Alternative Oxidase: A potential target for controlling aflatoxin contamination and propagation of Aspergillus flavus . Front Microbiol 11:419 Walker AS, Auclair C, Gredt M, Leroux P (2009) First occurrence of resistance to strobilurin fungicides in Microdochium nivale and Microdochium majus from French naturally infected wheat grains. Pest Manag Sci 65:906–915 Wang HC, Sun HY, Ma JX, Stammler G, Zhou MG (2009) Fungicide effectiveness during the various developmental stages of Peronophythora litchii in vitro . J Phytopathol 157:407–412 Wang HC, Sun HY, Stammler G, Ma JX, Zhou MG (2010) Generation and characterization of isolates of Peronophythora litchii resistant to carboxylic acid amide fungicides. Phytopathology 100:522–527 Zhang C, Gao XH, Zhou YX, Liu XL (2019) Research progress on mode action and molecular resistance mechanism of complex Ⅲ inhibitors in cellular respiration chain. Chin J Pesticide Sci 21:747–758 Zhanga ZK (2021) Inhibition of downy blight and enhancement of resistance in litchi fruit by postharvest application of melatonin. Food Chem 347:129009 Zhangb J (2021) I nhibitory activity and action mechanism of coumoxystrobin against Phytophthora litchii , which causes litchi fruit downy blight. Postharvest Biol Technol. 181 Zhenga L (2019) Identification of volatile organic compounds for the biocontrol of postharvest litchi fruit pathogen Peronophythora litchii . Postharvest Biol Technol 155:37–46 Zhengb L (2021) Biocontrol using Bacillus amyloliquefaciens PP19 against litchi downy blight caused by Peronophythora litchii . Front Microbiol 11:1–9 Zhou YX, Yang YB, Zhang Y, Li B, Si NG, Liu CL, Liu XL (2016) Sensitivity of Peronophythora litchii at different developmentstages to four QoI fungicides. Chin J Pesticide Sci 18:57–64 Ziogas BN, Baldwin BC, Young JE (1997) Alternative respiration: a biochemical mechanism of resistance to azoxystrobin (ICIA 5504) in Septoria tritici . Pest Sci 50:28–34 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revisions 19 Apr, 2024 Reviewers agreed at journal 29 Jan, 2024 Reviewers invited by journal 10 Jan, 2024 Editor invited by journal 08 Jan, 2024 Editor assigned by journal 04 Jan, 2024 First submitted to journal 02 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3802508","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266596338,"identity":"645614b8-4f85-4412-8c77-f6f4088ddb59","order_by":0,"name":"Suyue Jing","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Suyue","middleName":"","lastName":"Jing","suffix":""},{"id":266596339,"identity":"8d2f9ebc-a78e-4eaf-8b36-38be0dba77a7","order_by":1,"name":"Fadi Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYDACCQaGDwxsEkgi7I2NDz/g18I4A1ULz+FmYwmc6uFaUETS2wR48OiQn91j2PCjzMJefkbu4Y8/dxxO3D7zYRvQIDs53QbsWhjnnDFs7DknkbjhRl6ahOSZw8YytxPbHhQwJBubHcCuhVkix/wBb5tEgoF0jhmDYdthOQnpxHYDCYYDidtwaGGTyDFs/NsmYS8/O8f4Q2LbYR4JyYNtEjx4tPAAtTQDbWFsuJ1jIHEQZIsEI34tEhJphc0yIL/cf2Mm2Xgm3ViCJxEYyAa4/SI/I3lj45uyOnv5njPGwBCzTpzBfvzhww8VdnK4tKACxoZmKMuAGOUQLXXEKh0Fo2AUjIIRBABSZ1y7GJu4+gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3035-792X","institution":"Chinese Academy of Tropical Agricultural Sciences Environment and Plant Protection Institute","correspondingAuthor":true,"prefix":"","firstName":"Fadi","middleName":"","lastName":"Zhu","suffix":""},{"id":266596340,"identity":"4877e39c-9c91-4954-a987-b01e0338a063","order_by":2,"name":"Xiaodong Wen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Wen","suffix":""},{"id":266596341,"identity":"9007aa63-1e04-4b1d-bdc6-525c787b3aea","order_by":3,"name":"Jing Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":266596342,"identity":"f9a2f19d-4280-483a-84ad-60b27c206974","order_by":4,"name":"Gang Feng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2023-12-25 00:46:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3802508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3802508/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49614159,"identity":"28fe303e-9389-4bcc-b596-16adc14d4815","added_by":"auto","created_at":"2024-01-15 09:45:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":153858,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salicylhydroxamic acid (SHAM) on mycelial growth (A) and sporangial germination (B) of \u003cem\u003eP. litchii\u003c/em\u003e. The data represent the mean of three tests. Error bars denote the standard errors of three tests.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3802508/v1/3fc034acf922dcdac4a15258.jpg"},{"id":49614156,"identity":"3f5f0a9c-02f8-4d8c-865a-a21ea6ba4429","added_by":"auto","created_at":"2024-01-15 09:45:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34105,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salicylhydroxamic acid (SHAM) on the activity of coumoxystrobin against\u003cem\u003e P. litchii\u003c/em\u003e at the mycelial growth stage. The data represent the mean of three tests. Error bars denote the standard errors of three tests. Mean EC50 values followed by asterisks are significantly different based on Student’s paired t test (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3802508/v1/e86b892a658181751ba2a6be.jpg"},{"id":49614157,"identity":"23a2ac43-4dfb-4dec-8965-39650f2233a5","added_by":"auto","created_at":"2024-01-15 09:45:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75114,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salicylhydroxamic acid (SHAM) on the spore germination inhibition activity of coumoxystrobin against\u003cem\u003e P. litchii\u003c/em\u003e. The data represent the mean of three tests. Error bars denote the standard errors of three tests.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3802508/v1/0164935689a7999b49e30c14.jpg"},{"id":49614160,"identity":"6359fd0f-e404-47ed-a5cd-da204290bee3","added_by":"auto","created_at":"2024-01-15 09:45:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":207843,"visible":true,"origin":"","legend":"\u003cp\u003eA and B, Inhibition of \u003cem\u003eP. litchii\u003c/em\u003e respiration by coumoxystrobin and azoxystrobin in the presence or absence of SHAM. Error bars denote the standard errors of three tests. Asterisks represent significant differences based on Student’s paired t test (*P \u0026lt; 0.05, ** P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3802508/v1/0f32e8555885163812e5462d.jpg"},{"id":49614750,"identity":"2478bade-35a6-41ea-a4fc-817ee7b55f87","added_by":"auto","created_at":"2024-01-15 09:53:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":255562,"visible":true,"origin":"","legend":"\u003cp\u003eControl efficacy of coumoxystrobin (A) and azoxystrobin (B) against \u003cem\u003eP. litchii\u003c/em\u003e in the presence or absence of SHAM. Error bars denote the standard errors of three tests. Asterisks represent significant differences based on Student’s paired t test (*P \u0026lt; 0.05, ** P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3802508/v1/75fc23ed107128a990980a69.jpg"},{"id":49615023,"identity":"cfc230c8-256d-4f53-b7bb-0c76e6bdd9c3","added_by":"auto","created_at":"2024-01-15 10:01:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":641940,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3802508/v1/c431d291-d8a0-4349-8fd2-ce59492ea151.pdf"}],"financialInterests":"","formattedTitle":"Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLitchi, a subtropical to tropical fruit, is popular in consumer markets and cultivated in more than 20 countries due to its attractive fruity aroma, delicious taste, and high nutritional value (Xu et al. 2018). Litchi downy blight caused by \u003cem\u003ePhytophthora litchii\u003c/em\u003e is one of the most important oomycete diseases that occurs during litchi production, storage and transportation (Zhang et al. 2021a). The disease not only affects tender leaves, twigs, and flowers at unripe stages but also causes damage to mature litchi fruit, leading to enormous yield losses and severe commercial losses of more than 60% in southern China (Zheng\u003csup\u003ea\u003c/sup\u003e et al. 2019; Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Currently, chemical fungicides remain the main and most effective strategy to control litchi downy blight (Zheng\u003csup\u003eb\u003c/sup\u003e et al. 2021). \u0026lsquo;Traditional\u0026rsquo; fungicides, such as the multisite inhibitor mancozeb, target site-specific phenylamide fungicide metalaxyl, and carboxyl acid amide (CAA) fungicide dimethomorph, were used for the control of \u003cem\u003eP. litchii\u003c/em\u003e in the 1990s (Wang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, quinone outside inhibitor (QoI) fungicides, such as azoxystrobin and pyraclostrobin, have been registered for the control of litchi downy blight according to data from the China Pesticide Information Network (Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCoumoxystrobin((E)-methyl-2-{2-[(3-n-butyl-4-methylcoumarin-7-yloxy) methyl] phenyl}-3-methoxyacrylate), as a new QoI fungicide, has recently been registered for the control of several crop diseases in China, such as cucumber downy mildew, apple Valsa canker, and citrus scab (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.icama.org.cn/hysj/index.jhtml\u003c/span\u003e\u003cspan address=\"http://www.icama.org.cn/hysj/index.jhtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In our previous studies, coumoxystrobin exhibited a strong inhibitory effect on both \u003cem\u003eP. litchii\u003c/em\u003e at different developmental stages \u003cem\u003ein vitro\u003c/em\u003e and downy blight in detached litchi fruit in the laboratory. (Zhang et al. 2021b), which indicated a potential for coumoxystrobin application in the control of litchi downy blight caused by \u003cem\u003eP. litchii\u003c/em\u003e. To better understand the use of coumoxystrobin to control litchi downy blight, it is important to further study the effect of coumoxystrobin against \u003cem\u003eP. litchii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlternative oxidase (AOX) can affect fungal sensitivity to some QoI fungicides by bypassing the cytochrome pathway when normal electron transport pathways are inhibited (Bradley and Pedersen. 2011; Duan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Chaulagain et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barsottini et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To more accurately evaluate the \u003cem\u003ein vitro\u003c/em\u003e activity of QoI fungicides against plant pathogens, salicylhydroxamic acid (SHAM), an AOX inhibitor, has been suggested as a supplement in QoI activity tests to suppress alternative respiration (Ziogas et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Walker, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The addition of 50 or 100 \u0026micro;g/mL SHAM under normal circumstances will increase the inhibition activity of QoI fungicides against the pathogen (Jin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Piccirillo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, SHAM does not always enhance the inhibitory effect of QoI fungicides toward pathogens due to the differences among pathogens and chemical features (Walker et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Due to the toxic effects of these AOX inhibitors on pathogens such as \u003cem\u003eFusicladium effusum\u003c/em\u003e, \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e and \u003cem\u003eBotrytis cinerea in vitro\u003c/em\u003e, inhibition activity for QoIs should be performed without the addition of these inhibitors (Seyran et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To date, there is no report about the \u003cem\u003ein vitro\u003c/em\u003e activity of SHAM and its effect on the coumoxystrobin activity against \u003cem\u003eP. litchii\u003c/em\u003e. Therefore, the aims of the current research were to (i) explore the toxicity of SHAM on \u003cem\u003eP. litchii\u003c/em\u003e, (ii) determine the activity of coumoxystrobin combined with SHAM against \u003cem\u003eP. litchii\u003c/em\u003e at different developmental stages, (iii) examined the rate of mycelial respiration of \u003cem\u003eP. litchii\u003c/em\u003e when treated with coumoxystrobin alone or mixed with SHAM, and (iv) assess the control efficacy of coumoxystrobin and SHAM against phytophthora downy blight \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eFungal isolates.\u003c/b\u003e \u003cem\u003eP. litchii\u003c/em\u003e isolated from diseased fruit of litchi in Hainan (Sun, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was provided by the Postharvest Pathology and Preservation Laboratory, Environment and Plant Protection Institute of the Chinese Academy of Tropical Agricultural Science in Hainan Province, China.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFungicides and SHAM.\u003c/b\u003e Technical grade coumoxystrobin (96% active ingredient [a.i.], Shenyang Research Institute of Chemical Industry) and azoxystrobin (95% a.i., Shenyang Research Institute of Chemical Industry) were dissolved in acetone to make 10 g/L stock solutions. SHAM (99% a.i., Sigma-Aldrich Shanghai Trading Co. Ltd.) was dissolved in methanol to make a stock solution with a concentration of 40 g/L. All stock solutions were stored at 4\u0026deg;C in the dark until further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SHAM on\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003emycelial growth.\u003c/b\u003e The inhibition activity of SHAM against \u003cem\u003eP. litchii\u003c/em\u003e was determined by the mycelial growth inhibition method (Zhou et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Mycelial plugs (with a diameter of 5 mm) were picked from 72-h colonies of the isolate and transferred to white kidney bean (\u003cem\u003ePhaseolus lunatus\u003c/em\u003e Linn.) agar (WKBA) plates amended with SHAM at 25, 50, 100, 200, and 400 \u0026micro;g/mL. WKBA plates with less than 1% solvent served as controls. All plates were incubated for 4 days in 60 mm Petri dishes at 28\u0026deg;C on WKBA medium in the dark. Colony diameters were measured at perpendicular angles, and the average diameter (minus the original diameter of the inoculation plug) were used to calculated mycelial growth inhibition. The EC\u003csub\u003e50\u003c/sub\u003e (median effective concentration) were calculated by regressing percentage growth inhibition against the log of fungicide concentration. Three replicates were used per concentration. The experiment was performed in triplicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SHAM on\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003esporangial germination.\u003c/b\u003e Sporangia were obtained from 7-day-old plates of \u003cem\u003eP. litchii\u003c/em\u003e incubated in WKBA medium, and the sporangia were suspended in sterile distilled water until a concentration of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e sporangia/mL was obtained. The inhibition activitiy of SHAM against \u003cem\u003eP. litchii\u003c/em\u003e was investigated according to Zhou et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with minor adjustments. In brief, 50 \u0026micro;L of the sporangial suspension was spread evenly on WA (9 g of agar, 1000 mL of distilled water) plates amended with 50, 60, 100, 160 and 240 \u0026micro;g/mL SHAM and without SHAM as the control group. For each concentration, three replicate plates were prepared. After incubating for 10 h at 28\u0026deg;C in the dark, germination was quantified by counting 100 sporangia per plate under a microscope. If the germ tube length was greater than the length of the sporangia, the sporangia were considered to be germinated, and one hundred germinated sporangia on each plate were counted under the microscope. The sporangial germination inhibition rate was calculated. The EC\u003csub\u003e50\u003c/sub\u003e were calculated by regressing germination inhibition rate against the log of fungicide concentration. The experiment was performed three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fungicides with and without SHAM on mycelial growth.\u003c/b\u003e The activity of fungicides with and without SHAM on the mycelial growth of \u003cem\u003eP. litchii\u003c/em\u003e was determined according to the assay described by Zhou et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with minor modifications. Briefly, mycelial plugs from the actively growing colony margin were transferred to WKBA plates supplemented with 0.078, 0.156, 0.3125, 0.625 and 1.25 \u0026micro;g/mL coumoxystrobin and with 0.039, 0.078, 0.156, 0.3125, and 0.625 \u0026micro;g/mL azoxystrobin. When 50 \u0026micro;g/mL SHAM was present, the concentrated fungicides were diluted 10-fold. WKBA medium without fungicides was used as a control. The final amount of solvent never exceeded 1%(v/v) in our plates. For each fungicide and fungicide concentration, three replications were prepared. After 4 days at 28\u0026deg;C in the dark, colony diameters were measured and EC\u003csub\u003e50\u003c/sub\u003e values were calculated as previously described. This experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fungicides with and without SHAM on sporangial germination.\u003c/b\u003e Sporangia suspensions preparation and sporangial germination were conducted as described above. Coumoxystrobin concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 \u0026micro;g/mL and azoxystrobin concentrations of 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 \u0026micro;g/mL were tested according to Zhou et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with minor modifications. When SHAM was present, the fungicide concentrations were 0.001, 0.004, 0.016, 0.032, 0.064, and 0.128 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fungicides with and without SHAM on zoospore release.\u003c/b\u003e A sporangial suspension (1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e7\u003c/sup\u003e sporangia/mL) of \u003cem\u003eP. litchii\u003c/em\u003e was prepared, and the empty sporangia were counted microscopically according to Zhou et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with minor modifications. For activity tests, the sporangial suspension was added to a 2 mL microcentrifuge tube (1:1, vol/vol) with coumoxystrobin at final concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 \u0026micro;g/mL, azoxystrobin at final concentrations of 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 \u0026micro;g/mL, and without fungicides (in the control group). When SHAM was present, the fungicide concentrations were 0.001, 0.004, 0.016, 0.032, 0.064, and 0.128 \u0026micro;g/mL. Then, microcentrifuge tubes were incubated for 4 h under intense light at 15\u0026deg;C. The 100 sporangia were observed under a microscope, and the percent inhibition of zoospore release was calculated. Three replicates were included per treatment. The experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fungicides with and without SHAM on cystospore germination.\u003c/b\u003e The cystospore germination assay preparation and the assays of fungicide toxicity with and without SHAM for cystospore germination of \u003cem\u003eP. litchii\u003c/em\u003e were conducted according to Zhou et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The final coumoxystrobin concentrations were 0.01, 0.02, 0.04, 0.08, 0.16, and 0.64 \u0026micro;g/mL, and the final azoxystrobin concentrations were 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 \u0026micro;g/mL. To determine EC\u003csub\u003e50\u003c/sub\u003e values in the absence of SHAM, 50 \u0026micro;L zoospore suspensions were spread evenly over WA plates amended with fungicides at final concentrations of 0.001, 0.004, 0.008, 0.016, 0.032, 0.064 and 0.128 \u0026micro;g/mL, and the WA plates were transferred to an incubator for 6 h at 20\u0026deg;C in the dark. If the germ tube length was greater than the cystospore length, the cystospore was regarded as germinated, and the number of germinated cystospores was counted under a microscope. Three replicates were included per treatment. The experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fungicides with and without SHAM on respiratory rates.\u003c/b\u003e Oxygen consumption rate of \u003cem\u003eP. litchii\u003c/em\u003e mycelia treated by fungicides with and without SHAM was measured according to a previously described method (Zhang et al. 2021). Briefly, mycelia were collected, filtered, and washed three times with fresh PBS (pH\u0026thinsp;=\u0026thinsp;7.2\u0026thinsp;~\u0026thinsp;7.4). Then, stock solutions of coumoxystrobin and azoxystrobin, with or without SHAM, were diluted with sterile distilled water to 0.5, 2.5, 5, 10, and 20 \u0026micro;g/mL, respectively. At room temperature, fresh mycelia were added to the different solutions, and the oxygen concentration was measured after 2 minutes. The oxygen consumption inhibition rate was calculated as follows: oxygen consumption inhibition rate (%) = (oxygen consumption of control - oxygen consumption of each treatment) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e100/oxygen consumption of control. Three replicates were included per treatment. The experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SHAM on the control efficacy of fungicides\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e. The control efficacy of the fungicides on litchi leaves in the presence or absence of SHAM was measured by the detached leaf assay (Chaulagain et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The healthy, tender leaves of \u0026lsquo;Yutan mili\u0026rsquo; were cleaned with distilled water, and each leaf was surface disinfected with 75% ethyl alcohol and then washed four times with sterile water and allowed to dry. A small wound was made on each leaf by aseptic puncture. Then, the leaves were immersed in fungicide dilutions containing coumoxystrobin or azoxystrobin at 4, 20, and 100 \u0026micro;g/mL for 5 s with and without SHAM. Leaves treated with sterile water containing 0.1% methanol served as controls. All leaves were air-dried. A 6 mm mycelial plug was transferred onto leaves wounded by a hole puncher. The inoculated samples were stored in an incubator for 3 days at 28\u0026deg;C and 80% relative humidity, and lesion diameters were measured twice at right angles. Mycelial plugs from WKBA medium were used as a negative control. Six leaves were included in each treatment. The experiment was repeated twice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e All data were analyzed by data processing software (SPSS Science, version 26). EC\u003csub\u003e50\u003c/sub\u003e values were estimated from the fitted regression line of the log-transformed percentage inhibition plotted against the log-transformed fungicide concentration. Control efficacies were calculated by the following formula (the diameters of the mycelial plugs were subtracted from the lesion diameters before calculations): Control efficacy (%) = [(lesion diameter of the control \u0026ndash; lesion diameter of the treated sample)]/[lesion diameter of the control] * 100. Student\u0026rsquo;s paired t test was employed to determine significant differences in the mean EC\u003csub\u003e50\u003c/sub\u003e values (and control efficacy) for coumoxystrobin with and without SHAM.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eToxicity of SHAM to\u003c/b\u003e \u003cb\u003eP. litchii.\u003c/b\u003e The EC\u003csub\u003e50\u003c/sub\u003e values of \u003cem\u003eP. litchii\u003c/em\u003e to SHAM on mycelial growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and sporangial germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) were 166.72 and 150.69 \u0026micro;g/mL, respectively. SHAM at 50 \u0026micro;g/mL weakly inhibited mycelial growth and sporangial germination in \u003cem\u003eP. litchii\u003c/em\u003e, with both of inhibition rates below 20%, respectively, while at 100 \u0026micro;g/mL, the inhibition rates were increased to 38.31% and 38.54%, respectively. Thus, SHAM at 50 \u0026micro;g/mL was used to study the effect of SHAM on the activity of coumoxystrobin against \u003cem\u003eP. litchii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etoxicities of fungicides with and without SHAM on mycelial growth.\u003c/b\u003e \u003cem\u003eP. litchii\u003c/em\u003e was grown on WKBA medium to determine the mycelial growth inhibition activity of coumoxystrobin and azoxystrobin against mycelial growth in the presence and absence of SHAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Without adding SHAM, the fungicides coumoxystrobin and azoxystrobin exhibited good inhibitory effect on the mycelial growth of \u003cem\u003eP. litchii in vitro\u003c/em\u003e, with EC\u003csub\u003e50\u003c/sub\u003e values of 0.4531 and 0.0741 \u0026micro;g/mL, respectively. When SHAM (at 50 \u0026micro;g/mL) was potentiated, the EC\u003csub\u003e50\u003c/sub\u003e values of coumoxystrobin and azoxystrobin for the mycelial growth of \u003cem\u003eP. litchii\u003c/em\u003e were reduced 12.11-fold (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 8.13-fold (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively. The results showed that the activity of coumoxystrobin against \u003cem\u003eP. litchii\u003c/em\u003e in the presence of SHAM was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) improved compared with that using fungicides alone according to the paired t test.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etoxicities of fungicides with and without SHAM against sporangial germination.\u003c/b\u003e Coumoxystrobin was found to have excellent inhibitory effect against sporangial germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) of \u003cem\u003eP. litchii\u003c/em\u003e. The inhibition rates increased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the fungicide concentrations increasing, as shown clearly by the dose-reaction curves. Low concentrations (from 0.1 to 3.2 \u0026micro;g/mL) of coumoxystrobin exhibited promising inhibition effect on sporangial germination in \u003cem\u003eP. litchii\u003c/em\u003e, with mycelial tube germination inhibition values of 31.56\u0026ndash;95.09%. SHAM at 50 \u0026micro;g/mL, with inhibition rate below 10% against sporangial germination, significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased the activity of coumoxystrobin and azoxystrobin against \u003cem\u003eP. litchii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etoxicities of fungicides with and without SHAM against zoospore release.\u003c/b\u003e Results of the toxicity test of the fungicides coumoxystrobin and azoxystrobin against zoospore release (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) of \u003cem\u003eP. litchii\u003c/em\u003e showed that coumoxystrobin and azoxystrobin had inhibited zoospore release of \u003cem\u003eP. litchii\u003c/em\u003e in a dose-dependent manner, with EC\u003csub\u003e50\u003c/sub\u003e values of 0.4425 and 0.0553 \u0026micro;g/mL, respectively. Significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) differences were observed between the presence and absence of SHAM. When SHAM was added at 50 \u0026micro;g/mL, the inhibition activity of coumoxystrobin and azoxystrobin were increased by 122.92 and 8.38-fold, respectively, compared to those with fungicides alone.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of SHAM on the median effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) values of coumoxystrobin for \u003cem\u003eP. litchii\u003c/em\u003e zoospore release\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eToxicity regression equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePF \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumoxystrobin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;1.3394x\u0026thinsp;+\u0026thinsp;5.4742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzoxystrobin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;1.2795x\u0026thinsp;+\u0026thinsp;6.6090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;3.7272x-4.4065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e333.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumoxystrobin\u0026thinsp;+\u0026thinsp;SHAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;0.6619x\u0026thinsp;+\u0026thinsp;6.6168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e122.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzoxystrobin\u0026thinsp;+\u0026thinsp;SHAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;1.3707x\u0026thinsp;+\u0026thinsp;7.9858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea PF, potentiation factor, the ratio of EC\u003csub\u003e50\u003c/sub\u003e values of fungicides to EC\u003csub\u003e50\u003c/sub\u003e values of fungicides with SHAM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etoxicities of fungicides with and without SHAM on cystospore germination.\u003c/b\u003e Compared with other developmental stages, coumoxystrobin showed greater inhibition of cystospore (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) germination in \u003cem\u003eP. litchii\u003c/em\u003e, with EC\u003csub\u003e50\u003c/sub\u003e values of only 0.0830 \u0026micro;g/mL, and it exhibited a significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) difference from azoxystrobin. Furthermore, when SHAM was added, coumoxystrobin was more effective against cystospore germination than the single fungicides, with a potentiation factor of 7.55, and similar results were found for azoxystrobin.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of SHAM on the median effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) values of coumoxystrobin for \u003cem\u003eP. litchii\u003c/em\u003e cystospore germination\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eToxicity regression equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePF \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumoxystrobin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;1.3631x\u0026thinsp;+\u0026thinsp;6.4731\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzoxystrobin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;2.0496x\u0026thinsp;+\u0026thinsp;8.1522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;2.8959x-1.8975\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9957\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e240.9100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumoxystrobin\u0026thinsp;+\u0026thinsp;SHAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;1.8172x\u0026thinsp;+\u0026thinsp;8.5595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzoxystrobin\u0026thinsp;+\u0026thinsp;SHAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;0.8942x\u0026thinsp;+\u0026thinsp;6.8906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea PF, potentiation factor, the ratio of EC\u003csub\u003e50\u003c/sub\u003e values of fungicides to EC\u003csub\u003e50\u003c/sub\u003e values of fungicides with SHAM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRespiration inhibition by coumoxystrobin and azoxystrobin with or without SHAM.\u003c/b\u003e Respiration inhibition was tested during coumoxystrobin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) treatment at different concentrations. The results showed that coumoxystrobin at 2.5 \u0026micro;g/mL could inhibit the mycelial respiration of \u003cem\u003eP. litchii\u003c/em\u003e, and the inhibition rate was 52.54%. For all tested concentrations, the oxygen consumption inhibition rate of coumoxystrobin in the presence of SHAM was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) improved compared to that with the use of fungicides alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), except at 0.5 \u0026micro;g/mL. Coumoxystrobin and azoxystrobin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) had similar response trends in inhibiting the mycelial respiration of \u003cem\u003eP. litchii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SHAM on the control efficacy of coumoxystrobin against\u003c/b\u003e \u003cb\u003eP. litchii.\u003c/b\u003e The control efficacy of coumoxystrobin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) against \u003cem\u003eP. litchii\u003c/em\u003e was determined on leaves. For all tested concentrations, coumoxystrobin exhibited lower control effects of \u003cem\u003eP. litchii\u003c/em\u003e than azoxystrobin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), except at 100 \u0026micro;g/ml, however, no significant difference was observed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Coumoxystrobin at 100 \u0026micro;g/ml provided 100% control efficacy. When SHAM was added at 100 \u0026micro;g/mL, the control efficacy of coumoxystrobin at 4 \u0026micro;g/mL was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased. The control efficacy of coumoxystrobin at 20 \u0026micro;g/mL combined with 100 \u0026micro;g/mL SHAM was increased potentiated by 20.16%. Similar results were found for the reference QoI fungicide azoxystrobin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSHAM was previously recommended to be added to the culture medium to inhibit AOX in the alternative respiratory pathway to reasonably evaluate the activity of QoIs fungicides, but many studies have shown that this approach may not be reasonable due to the toxicity of SHAM against different plant pathogens (Seyran, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ma, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liang, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In current study, SHAM exhibited an inhibitory effect on the mycelial growth, sporangial germination of \u003cem\u003eP. litchii\u003c/em\u003e. Thus, we suggested not to add SHAM in coumoxystrobin and azoxystrobin activity assay against \u003cem\u003eP. litchii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn addition, a potentiated effect between SHAM and coumoxystrobin against \u003cem\u003eP. litchii\u003c/em\u003e was observed in our research. This consistent potentiation has also been reported in other plant pathogens (Liang, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Nalumpang reviewed that SHAM could not only inhibit the activity of AOX, but also suppress the activity of other essential enzymes of fungi (Nalumpang, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In current research, the potentiation effect tends to be stronger with a lower concentration of coumoxystrobin in the control efficacy test. However, the activity of AOX and other essential enzymes were not directly measured in this study. Thus, the potentiation mechanism needs to be further explored.\u003c/p\u003e \u003cp\u003eQoI fungicides are safe, broad spectrum, efficient and environmentally friendly, but plant pathogens easily develop resistance against these fungicides, which restricts their application in plant disease control (Zhang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A study on the mechanism of resistance to QoI fungicides found that the AOX in the alternative oxidation pathway of many plant pathogens affects the activity of QoI fungicides, and overexpression of the AOX gene is one of the mechanisms of resistance of many plant pathogens. Studies have shown that the resistance of plant pathogenic fungi such as \u003cem\u003eSeptoria tritici\u003c/em\u003e (Ziogas et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), \u003cem\u003eMycosphaerella fijensis\u003c/em\u003e (Sierotzki et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e)d \u003cem\u003eoryzae\u003c/em\u003e (Zhang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to QoI fungicides is related to AOX. Similar results were also found for the pathogenic oomycete \u003cem\u003ePlasmopara viticola\u003c/em\u003e (Fontaine et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Many studies have found that the addition of the AOX inhibitor SHAM can increase the sensitivity of resistant pathogens to QoI fungicides (Ziogas et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Sierotzki et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Seyran et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liang, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and even make the pathogens resistant to azoxystrobin sensitive again (Ziogas et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In this study, adding a lower dose of SHAM (50 \u0026micro;g/mL) significantly improved the activity of coumoxystrobin against \u003cem\u003eP. litchii\u003c/em\u003e. The test of azoxystrobin also showed a similar trend, which is consistent with previous research results (Seyran et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liang, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further respiratory inhibition tests showed that when the concentration was greater than 5 mg/L, the inhibitory effects of coumoxystrobin on oxygen consumption by \u003cem\u003eP. litchii\u003c/em\u003e were no longer dose dependent and remained unchanged. However, the inhibition rate of the treatment with SHAM increased by more than 20%, indicating that \u003cem\u003eP. litchii\u003c/em\u003e may activate the alternative oxidation pathway under the stress of these two fungicides.\u003c/p\u003e \u003cp\u003eUsing two or more fungicides with different modes of action in controlling the same pathogen is an important approach to delaying resistance and is recommended by the FRAC. The abovementioned similar research results regarding the activities and synergistic effect of AOX inhibitors suggest a potential new strategy for the rational use of and resistance control for QoI fungicides, namely, using AOX as an auxiliary fungicide target. In recent years, scientists from Brazil have begun to pay attention to the toxicity of AOX inhibitors to plant pathogens, their synergistic effect with fungicides and exploring novel AOX inhibitors for plant pathogens (Seyran et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liang, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barsottini et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Godwin and Tian also published similar ideas about AOX as a potential target (Ebiloma et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tian, et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, based on some current researches (Ju\u0026aacute;rez, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Thomazella, et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ca\u0026acute;rdenas-Monroy, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lin, et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), AOX is believed not to be essential in most fungi, yet AOX is important in their stress response and contributes to their pathogenicity and virulence. We believe that further study on the functions and inhibitors of AOX in plant pathogenic fungi or oomycetes will be helpful for the rational use of QoI fungicides, resistance management and more efficient plant disease control.\u003c/p\u003e \u003cp\u003eIn conclusion, coumoxystrobin exhibited strong inhibitory activity at different stages of the \u003cem\u003eP. litchii\u003c/em\u003e life cycle. SHAM could enhance the antifungal activity of the QoI fungicides coumoxystrobin and azoxystrobin against \u003cem\u003eP. litchii in vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. This enhanced activity from using coumoxystrobin and SHAM together may shed some light on the control of litchi downy blight and the rational use of QoI fungicides. To reasonably evaluate the control effect of coumoxystrobin on downy mildew of litchi caused by \u003cem\u003eP. litchii\u003c/em\u003e, more strains from field and field trials are needed for verification. In addition, we suggest further biochemical and molecular studies to help elucidate the active role of AOX in plant pathogen physiology and its potential as an auxiliary target for disease control in plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing SY and Zhu FD contributed equally to this work. Zhang J and Zhu FD designed and supervised the project. Jing SY, Zhu FD and Wen XD performed most of the experiments. Zhu FD and Wen XD analyzed the data and participated in interpretation of the results. Zhu FD and Wen XD prepared the manuscript. Zhang J and Feng G helped to revise the manuscript. All authors have approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exists for all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the National Natural Science Foundation of China (32302413) and Chinese Academy of Tropical Agricultural Sciences for Science and Technology Innovation Team of National Tropical Agricultural Science Center (CATASCXTD202305).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBradley CA, Pedersen DK (2011) Baseline Sensitivity of \u003cem\u003eCercospora zeae-maydis\u003c/em\u003e to Quinone Outside Inhibitor Fungicides. Plant Dis 95:189\u0026ndash;194\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarsottini MR, Pires BA, Vieira ML, Pereira JG, Costa PC, Sanita J, Coradini A, Mello F, Marschalk C, Silva EM, Paschoal D, Figueira A, Rodrigues FH, Cordeiro AT, Miranda PC, Oliveira PS, Sforca ML, Carazzolle MF, Rocco SA, Pereira GA (2019) Synthesis and testing of novel alternative oxidase (AOX) inhibitors with antifungal activity against \u003cem\u003eMoniliophthora perniciosa\u003c/em\u003e (Stahel), the causal agent of witches' broom disease of cocoa, and other phytopathogens. Pest Manag Sci 75:1295\u0026ndash;1303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarsottini MR, Copsey A, Young L, Baroni RM, Cordeiro AT, Pereira GA, Moore AL (2020) Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen \u003cem\u003eMoniliophthora perniciosa\u003c/em\u003e. Commun Biology 3:263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCa\u0026acute;rdenas-Monroy CA, Pohlmann T, Pi\u0026ntilde;o\u0026acute;n-Za\u0026acute;rate G, Matus-Ortega G, Guerra G, Feldbru\u0026uml;gge M et al (2017) The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in \u003cem\u003eUstilago maydis\u003c/em\u003e. PLoS ONE 12:e0173389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaulagain B, Dufault N, Raid RN, Rott P (2019) Sensitivity of two sugarcane rust fungi to fungicides in urediniospore germination and detached leaf bioassays. Crop Prot 117:86\u0026ndash;93\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan YB, Liu SM, Ge CY, Feng XJ, Chen CJ, Zhou MG (2012) \u003cem\u003eIn vitro\u003c/em\u003e inhibition of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e by mixtures of azoxystrobin, SHAM, and thiram. Pesticide Biochemistry And Physiology. 103:101\u0026ndash;107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbiloma GU, Balogun EO, Cueto-D\u0026iacute;az EJ, de Koning HP, Dardonville C (2019) Alternative oxidase inhibitors: mitochondrion‐targeting as a strategy for new drugs against pathogenic parasites and fungi. Med Res Rev 39:1553\u0026ndash;1602\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFontaine S, Remuson F, Caddoux L, Barr\u0026egrave;s B (2019) Investigation of the sensitivity of \u003cem\u003ePlasmopara viticola\u003c/em\u003e to amisulbrom and ametoctradin in French vineyards using bioassays and molecular tools. Pest Manag Sci 75:2115\u0026ndash;2123\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo HY, Bao JD, Lin LY, Wang ZX, Shi MY, Huang YL, Wang R, Li B, Liu BJ, P. Q., and, Chen QH (2021) Genome sequence data of \u003cem\u003ePeronophythora litchii\u003c/em\u003e, an oomycete pathogen causing litchi downy blight. Mol Plant-Microbe Interact J 34:707\u0026ndash;710\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin LH, Chen Y, Chen CJ, Wang JX, Zhou MG (2009) Activity of azoxystrobin and SHAM to four phytopathogens. Agricultural Sci China 8:835\u0026ndash;842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJu\u0026aacute;rez O, Guerra G, Vel\u0026aacute;zquez I, Flores-Herrera O, Rivera‐P\u0026eacute;rez RE, Pardo JP (2006) The physiologic role of alternative oxidase in \u003cem\u003eUstilago maydis\u003c/em\u003e. FEBS J 273:4603\u0026ndash;4615\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang HJ, Di YL, Li JL, You H, Zhu FX (2015) Baseline sensitivity of pyraclostrobin and toxicity of SHAM to \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. Plant Dis 99:267\u0026ndash;273\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang H, Li J, Luo C, Li J, Zhu FX (2019) Effects of SHAM on the sensitivity of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e and \u003cem\u003eBotrytis cinerea\u003c/em\u003e to QoI fungicides. Plant Dis 103:1884\u0026ndash;1888\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin ZS, Wu JY, Jamieson PA, Zhang CQ (2019) Alternative oxidase is involved in the pathogenicity, development, and oxygen stress response of \u003cem\u003eBotrytis cinerea\u003c/em\u003e. Phytopathology 109:1679\u0026ndash;1688\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa DC, Jiang JG, He LM, Cui KD, Mu W, Liu F (2018) Detection and characterization of Qol-resistant \u003cem\u003ePhytophthora capsici\u003c/em\u003e causing pepper phytophthora blight in China. Plant Dis 102:1725\u0026ndash;1732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNalumpang S, Poti T, Akimitsu K (2021) Effect of salicylhydroxamic acid on mycelial growth and baseline sensitivity to azoxystrobin in \u003cem\u003ePhytophthora infestans\u003c/em\u003e causing potato late blight in Thailand. Int J Agricultural Technol 17(4):12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiccirillo G, Carrieri R, Polizzi G, Azzaro A, Lahoz E, Fern\u0026aacute;ndez-Ortuno D, Vitale A (2018) \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e activity of QoI fungicides against \u003cem\u003eColletotrichum gloeosporioide\u003c/em\u003es causing fruit anthracnose in \u003cem\u003eCitrus sinensis\u003c/em\u003e. Scientia Horticulturae. 236:90\u0026ndash;95\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeyran M, Brenneman TB, Stevenson KL (2010) \u003cem\u003evitro\u003c/em\u003e toxicity of alternative oxidase inhibitors salicylhydroxamic acid and propyl gallate on \u003cem\u003eFusicladium effusum\u003c/em\u003e. J Pest Sci 83:421\u0026ndash;427\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi N, Ruan H, Gan L, Dai YL, Yang XJ, Du YX, Chen FR (2020) Evaluating the sensitivities and efficacies of fungicides with different modes of action against \u003cem\u003ePhomopsis asparagi\u003c/em\u003e. Plant Dis 104:448\u0026ndash;454\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSierotzki H, Parisi S, Steinfeld U, Tenzer I, Poirey S, Gisi U (2000) Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of \u003cem\u003eMycosphaerella fijensis\u003c/em\u003e field isolates. Pest Manag Sci 56:833\u0026ndash;841\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun JH, Gao ZY, Zhang XC, Zou XX, Cao LL, Wang JB (2017) Transcriptome analysis of \u003cem\u003ePhytophthora litchii\u003c/em\u003e reveals pathogenicity arsenals and confirms taxonomic status. PLoS ONE 12:e0178245\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomazella D, Teixeira P, Oliveira HC, Saviani EE, Pereira AG et al (2012) The hemibiotrophic cacao pathogen \u003cem\u003eMoniliophthora perniciosa\u003c/em\u003e depends on a mitochondrial alternative oxidase for biotrophic development. New Phytol 194:1025\u0026ndash;1034\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian F, Lee SY, Woo SY, Chun HS (2020) Alternative Oxidase: A potential target for controlling aflatoxin contamination and propagation of \u003cem\u003eAspergillus flavus\u003c/em\u003e. Front Microbiol 11:419\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker AS, Auclair C, Gredt M, Leroux P (2009) First occurrence of resistance to strobilurin fungicides in \u003cem\u003eMicrodochium nivale\u003c/em\u003e and \u003cem\u003eMicrodochium majus\u003c/em\u003e from French naturally infected wheat grains. Pest Manag Sci 65:906\u0026ndash;915\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang HC, Sun HY, Ma JX, Stammler G, Zhou MG (2009) Fungicide effectiveness during the various developmental stages of \u003cem\u003ePeronophythora litchii in vitro\u003c/em\u003e. J Phytopathol 157:407\u0026ndash;412\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang HC, Sun HY, Stammler G, Ma JX, Zhou MG (2010) Generation and characterization of isolates of \u003cem\u003ePeronophythora litchii\u003c/em\u003e resistant to carboxylic acid amide fungicides. Phytopathology 100:522\u0026ndash;527\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Gao XH, Zhou YX, Liu XL (2019) Research progress on mode action and molecular resistance mechanism of complex Ⅲ inhibitors in cellular respiration chain. Chin J Pesticide Sci 21:747\u0026ndash;758\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhanga ZK (2021) Inhibition of downy blight and enhancement of resistance in litchi fruit by postharvest application of melatonin. Food Chem 347:129009\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhangb J (2021) I nhibitory activity and action mechanism of coumoxystrobin against \u003cem\u003ePhytophthora litchii\u003c/em\u003e, which causes litchi fruit downy blight. Postharvest Biol Technol. 181\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhenga L (2019) Identification of volatile organic compounds for the biocontrol of postharvest litchi fruit pathogen \u003cem\u003ePeronophythora litchii\u003c/em\u003e. Postharvest Biol Technol 155:37\u0026ndash;46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhengb L (2021) Biocontrol using \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e PP19 against litchi downy blight caused by \u003cem\u003ePeronophythora litchii\u003c/em\u003e. Front Microbiol 11:1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou YX, Yang YB, Zhang Y, Li B, Si NG, Liu CL, Liu XL (2016) Sensitivity of \u003cem\u003ePeronophythora litchii\u003c/em\u003e at different developmentstages to four QoI fungicides. Chin J Pesticide Sci 18:57\u0026ndash;64\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiogas BN, Baldwin BC, Young JE (1997) Alternative respiration: a biochemical mechanism of resistance to azoxystrobin (ICIA 5504) in \u003cem\u003eSeptoria tritici\u003c/em\u003e. Pest Sci 50:28\u0026ndash;34\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3802508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3802508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLitchi downy blight, caused by \u003cem\u003ePhytophthora litchii\u003c/em\u003e, presents significant challenges to litchi production, storage, and transportation. Previous studies have shown that coumoxystrobin exhibits effective inhibitory activity against \u003cem\u003eP\u003c/em\u003e. \u003cem\u003elitchii\u003c/em\u003e. Salicylhydroxamic acid (SHAM), an alternative respiratory pathway inhibitor, is commonly used to evaluate the efficacy of cytochrome respiratory pathway inhibitor like coumoxystrobin against fungal phytopathogens in vitro. In this study, the toxicity of SHAM on various developmental stages of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003elitchii\u003c/em\u003e, including mycelial growth, sporangial germination, zoospore release, and cystospore germination, was assessed. The EC\u003csub\u003e50\u003c/sub\u003e values for SHAM were determined as 166.72, 150.69, 333.97, and 240.91 μg/mL, respectively. Subsequently, the activity of coumoxystrobin against \u003cem\u003eP\u003c/em\u003e. \u003cem\u003elitchii\u003c/em\u003e was assessed in the presence of SHAM at a concentration of 50 μg/mL, which showed slight inhibition below 20% for all four developmental stages. The addition of SHAM significantly improved the inhibitory activity of coumoxystrobin against \u003cem\u003eP\u003c/em\u003e. \u003cem\u003elitchii\u003c/em\u003e at different stages, with reductions in EC\u003csub\u003e50\u003c/sub\u003e values ranging from 7.55- to 122.92-fold. Moreover, respiration assays revealed that a concentration of 5 μg/mL coumoxystrobin inhibited \u003cem\u003eP\u003c/em\u003e. \u003cem\u003elitchii\u003c/em\u003e mycelial respiration to a lesser extent compared to the combined effect of coumoxystrobin and SHAM. SHAM also enhanced the control efficacy of coumoxystrobin against phytophthora blight development on litchi leaves. Previously, we reported that coumoxystrobin effectively controls postharvest downy mildew on litchi fruit. Consequently, coumoxystrobin holds promise as an agent for litchi downy blight control in the field and after harvest. Furthermore, similar to previous studies, SHAM, an alternative oxidase (AOX) inhibitor, was found to significantly enhance the activity of the two aforementioned QoI fungicides against \u003cem\u003eP\u003c/em\u003e. \u003cem\u003elitchii\u003c/em\u003e, both in vitro and in vivo. This suggests that further exploration of AOX inhibitors and the role of AOX in plant diseases could contribute to the rational use of QoI fungicides and improve control efficiency for plant diseases.\u003cbr\u003e\n\u003c/p\u003e","manuscriptTitle":"Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-15 09:45:12","doi":"10.21203/rs.3.rs-3802508/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-04-19T10:15:42+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-01-29T06:06:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-11T01:49:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Tropical Plant Pathology","date":"2024-01-08T17:41:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-05T03:01:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Plant Pathology","date":"2024-01-02T21:01:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9dc28216-e9e7-41ed-8bdb-b36428457e88","owner":[],"postedDate":"January 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-02T02:31:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-15 09:45:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3802508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3802508","identity":"rs-3802508","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}