Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii

doi:10.21203/rs.3.rs-3802508/v1

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Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii

https://doi.org/10.21203/rs.3.rs-3802508/v1

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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 EC₅₀ 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 EC₅₀ 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.

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.

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 EC₅₀ (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 1×10⁷ 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 EC₅₀ 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 EC₅₀ 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\(\times\)10⁷ 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 EC₅₀ 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). EC₅₀ 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 EC₅₀ values (and control efficacy) for coumoxystrobin with and without SHAM.

Toxicity of SHAM to P. litchii. The EC₅₀ values of P. litchii to SHAM on mycelial growth (Fig. 1A) and sporangial germination (Fig. 1B) 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 EC₅₀ values of 0.4531 and 0.0741 µg/mL, respectively. When SHAM (at 50 µg/mL) was potentiated, the EC₅₀ 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 EC₅₀ 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 (EC₅₀) values of coumoxystrobin for *P. litchii* zoospore release
Fungicide	Toxicity regression equation	r	EC₅₀ (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 EC₅₀ values of fungicides to EC₅₀ 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 EC₅₀ 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 (EC₅₀) values of coumoxystrobin for *P. litchii* cystospore germination
Fungicide	Toxicity regression equation	r	EC₅₀ (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 EC₅₀ values of fungicides to EC₅₀ values of fungicides with SHAM.

Respiration inhibition by coumoxystrobin and azoxystrobin with or without SHAM. Respiration inhibition was tested during coumoxystrobin (Fig. 4A) 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. 4B) 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. 5A) against P. litchii was determined on leaves. For all tested concentrations, coumoxystrobin exhibited lower control effects of P. litchii than azoxystrobin (Fig. 5B), 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.

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.

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).

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19 Apr, 2024
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Effect of SHAM on the activity of coumoxystrobin against Phytophthora litchii

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