Association of hydrogen peroxide with commercial fungicide formulations in the control of Asian soybean rust

ABSTRACT: In recent years, there have been reductions in the efficacy of the fungicidal control of Phakopsora pachyrhizi, thereby hindering the management of soybean rust and compromising crop yield. This study evaluated the effects of incorporating hydrogen peroxide (H2O2) in commercial fungicide formulations on the control of soybean rust. We conducted two experiments, one of which was performed in a greenhouse environment and the other under field conditions. In both environments, we examined the following four control programs using commercial fungicide formulations: (I) azoxystrobin + cyproconazole (quinone outside inhibitor [QoI] + demethylation inhibitor [DMI]); (II) picoxystrobin + cyproconazole (QoI + DMI); (III) pyraclostrobin + epoxiconazole + fluxapyroxad (QoI + DMI + succinate dehydrogenase inhibitor); and (IV) water (H2O) (program without fungicide application), combined with the incorporation of (i) H2O2; (ii) mancozeb (positive control I); (iii) chlorothalonil (positive control II); or (iv) water (H2O) alone. Analyses of infected leaf area and grain yield revealed that the addition of H2O2 to the formulations of DMI and QoI fungicides led to a reduction in disease severity of between 33% and 44% relative to the effects of these products used alone, as well as an increase in yield and SPAD values. The use of H2O2 and multi-site fungicides alone failed to provide effective control of soybean rust. In addition to enhancing the efficacy of disease control, the use of H2O2 associated with commercial fungicide mixtures was shown to be a potential tool for the management of fungicide resistance and reduction in losses from Asian soybean rust.

Evidence for the agricultural origin of resistance to multiple antimicrobials in Aspergillus fumigatus, a fungal pathogen of humans

Pathogen resistance to clinical antimicrobial agents is an urgent problem. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible humans annually. Azoles, which are widely used in both clinical and agricultural settings, are currently the most effective treatment, but resistance to clinical azoles is emerging worldwide. Here we report the isolation and analysis of azole-sensitive and azole-resistant A. fumigatus from agricultural environments in the southeastern United States (USA) and show that the USA pan-azole-resistant isolates form a clade with pan-azole-resistant isolates from the United Kingdom, the Netherlands, and India. We show that several pan-azole-resistant isolates from agricultural settings in the USA and India also carry alleles with mutations conferring resistance to agricultural fungicides from the benzimidazole (MBC) and quinone outside inhibitor (QoI) classes. We further show that pan-azole-resistant A. fumigatus isolates from patients in clinical settings in the USA, India and the Netherlands also carry alleles conferring resistance to MBC and QoI agricultural fungicides. The presence of markers for resistance to agricultural-use fungicides in clinical A. fumigatus isolates is strong evidence for an agricultural origin of pan-azole resistance in patients. The presence of multiple fungicide-resistance alleles in agricultural and clinical isolates further suggests that the unique genetics of the pan-azole-resistant clade enables the evolution and/or persistence of antimicrobial resistance mutations leading to the establishment of multi-fungicide-resistant isolates.

The G143A Mutation in the Cytochrome b Gene is Associated with Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina from Soybean Fields in Wisconsin

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important management tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from Wisconsin soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in Wisconsin. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in Wisconsin will need to use an integrated approach of cultural practices, genetic resistance, and use fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Resistance to Boscalid, Fluopyram and Fluxapyroxad in Blumeriella jaapii from Michigan (U.S.A.): Molecular Characterization and Assessment of Practical Resistance in Commercial Cherry Orchards

Management of cherry leaf spot disease, caused by the fungus Blumeriella jaapii, with succinate dehydrogenase inhibitor (SDHI) fungicides has been ongoing in Michigan tart cherry orchards for the past 17 years. After boscalid-resistant B. jaapii were first isolated from commercial orchards in 2010, premixes of SDHI fungicides fluopyram or fluxapyroxad with a quinone outside inhibitor were registered in 2012. Here, we report widespread resistance to fluopyram (FluoR), fluxapyroxad (FluxR), and boscalid (BoscR) in commercial orchard populations of B. jaapii in Michigan from surveys conducted between 2016 and 2019. A total of 26% of 1610 isolates from the 2016–2017 surveys exhibited the fully-resistant BoscR FluoR FluxR phenotype and only 7% were sensitive to all three SDHIs. Practical resistance to fluopyram and fluxapyroxad was detected in 29 of 35 and 14 of 35 commercial tart cherry orchards, respectively, in surveys conducted in 2018 and 2019. Sequencing of the SdhB, SdhC, and SdhD target genes from 22 isolates with varying resistance phenotypes showed that BoscS FluoR FluxS isolates harbored either an I262V substitution in SdhB or an S84L substitution in SdhC. BoscR FluoR FluxR isolates harbored an N86S substitution in SdhC, or contained the N86S substitution with the additional I262V substitution in SdhB. One BoscR FluoR FluxR isolate contained both the I262V substitution in SdhB and the S84L substitution in SdhC. These mutational analyses suggest that BoscR FluoR FluxR isolates evolved from fully sensitive BoscS, FluoS, FluxS isolates in the population and not from boscalid-resistant isolates that were prevalent in the 2010–2012 time period.

Distribution of G143A mutations conferring fungicide resistance in Virginia populations of Parastagonospora nodorum infecting wheat

Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum is an important leaf spot disease in the mid-Atlantic U.S. Disease management approaches include use of resistant varieties, cultural control, and foliar fungicides. Frequent use of foliar fungicides can select for fungicide resistance within pathogen populations. Recently, the first report of quinone outside inhibitor (QoI) fungicide resistance in the U.S. was made based on a relatively small collection of P. nodorum isolates from Virginia. The objective of this study was to conduct a state-wide, two-year survey of P. nodorum populations in Virginia wheat and quantify frequencies of the target-site mutation that confers QoI resistance. A total of 318 isolates of P. nodorum were obtained from wheat collected at seven locations distributed throughout the wheat-growing regions of Virginia in 2018 and 2019. A previously designed pyrosequencing assay that detects the G143A substitution in the cytochrome b gene of P. nodorum was used to screen isolates for the presence or absence of the target site mutation. The G143A substitution was detected in all sampled fields. Among locations and years, frequencies of the mutation in P. nodorum populations ranged from 5-32% (mean = 19%). Thus, the QoI-resistance conferring G143A mutation was widespread in P. nodorum populations in Virginia and it occurred at a relatively high frequency. Results suggest that fungicides containing QoI active ingredients may not be effective for controlling SNB in Virginia and the surrounding region, and application of stand-alone QoI fungicides for disease control in wheat is not recommended.

Detection of the G143A Mutation in the Cytochrome b Gene of Corynespora cassiicola Isolates from Soybean in Tennessee

Corynespora cassiicola is the causal pathogen of target spot in soybean and cotton grown in the United States. With target spot increasing in importance, fungicides are becoming an important tool for control of this disease. Unfortunately, there are reports of C. cassiicola isolates in other crops being resistant to some fungicide classes. The objective of this study was to identify if resistance to quinone outside inhibitor (QoI) fungicides is present in Tennessee soybean and cotton isolates of C. cassiicola. Four isolates of C. cassiicola were evaluated at a range of doses for the fungicide pyraclostrobin. Isolates were also sequenced to determine if the G143A mutation was present in the cyt b gene. Two isolates previously reported to be resistant to QoIs were also used as positive checks. Two isolates of C. cassiicola from Tennessee soybean were found to have the G143A mutation. EC50 values for the two isolates ranged from 15.7 to 121 μg/ml. As a result of this study, C. cassiicola isolates have exhibited resistance to QoI fungicides in Tennessee soybean.

Assessing Missouri soybean fields for azoxystrobin resistant cercospora sojina

Successful management of Cercospora sojina, the causal agent of frogeye leaf spot, can be achieved through utilizing resistant varieties and fungicide applications. Fungicides in the quinone outside inhibitor (QoI) class are most effective in controlling C. sojina in the field. Unfortunately, azoxystrobin fungicide-resistant isolates of C. sojina have been recovered in many soybean growing areas of the U.S. Fungicide-resistant isolates of C. sojina were first detected in 2011 and 2012 in two counties in southeast Missouri, but no further assessment was conducted. In this study, 121 isolates were collected from 15 surveyed counties between 2019 and 2020 in an effort to understand the geographical distribution of fungicide-resistant C. sojina. Isolates were collected from fields based on the presence of frogeye leaf spot symptoms. Samples were brought to the laboratory and isolates were recovered from individual lesions. A fungicide sensitivity bioassay was conducted to determine which isolates were resistant to the QoI class of fungicides. The fungicide sensitivity bioassay consisted of full-strength PDA amended with technical grade azoxystrobin at five different concentrations and a no fungicide control. Out of 121 isolates, 81 were fungicide-resistant representing 13 of the 15 Missouri counties included in the survey. Notably, the northwest corner of Missouri had the highest concentration of fungicide-resistant isolates, consistent with the recent recovery of fungicide-resistant C. sojina isolates in Iowa and Nebraska. The widespread recovery of fungicide-resistant C. sojina in multiple counties throughout Missouri provides new insight into disease management in the state.

Investigating the sensitivity of Venturia inaequalis isolates to difenoconazole and pyraclostrobin in apple orchards in China

The resistance level of 90 single lesion conidial isolates of Venturia inaequalis collected from multiple commercial orchards in Shaanxi and Gansu Provinces and Xinjiang Autonomous Region of China to the demethylation inhibitor (DMI) fungicide difenoconazole and quinone outside inhibitor (QoI) fungicide pyraclostrobin was examined. The EC50 values of the 90 isolates to difenoconazole and pyraclostrobin ranged from 0.143 to 6.735 μg/mL and 0.084 to 2.026 μg/mL, respectively. Among the isolates, 19 had resistance, 66 had reduced sensitivity, and five had sensitivity to difenoconazole; eight had resistance, 81 had reduced sensitivity, and one had sensitivity to pyraclostrobin. Although a weak correlation between difenoconazole and pyraclostrobin was detected, four isolates were identified as resistant to difenoconazole and pyraclostrobin. However, isolates with practical resistance were not found widely in our study and were only sporadic in a few places, indicating that at present, difenoconazole and pyraclostrobin are still safe for disease management in the apple-growing areas of Shaanxi, Gansu and Xinjiang. However, the risk of fungicide resistance should be managed with caution, and yearly monitoring of resistance development is necessary.

Detection of a Point Mutation (G143A) in Cyt b of Corynespora cassiicola That Confers Pyraclostrobin Resistance

Point mutation G143A in the cytochrome b (Cyt b) protein commonly confers resistance to quinone outside inhibitor (QoI) fungicides in phytopathogenic fungi, including Corynespora cassiicola, which causes cucumber target spot disease. However, the effect of G143A on the binding between the QoI fungicide and the Cyt b protein, and the use of LAMP (loop-mediated isothermal amplification) to detect this point mutation had not been reported previously in C. cassiicola. In this study, the sensitivity of 131 C. cassiicola isolates—collected from Shandong province, China in 2019 and 2020—to pyraclostrobin was determined. The EC50 values ranged from 1.67 to 8.82 μg/mL, and sequencing results showed that all C. cassiicola isolates contained the G143A mutation. Molecular docking results suggested that G143A significantly alters the affinity of pyraclostrobin to the Cyt b protein. Following development of three LAMP primer pairs, the best reaction condition for LAMP analysis was 65 °C for 60 min, and the detection limit was 0.01 ng/μL of DNA containing the point mutation. In conclusion, the G143A mutation conferring pyraclostrobin resistance is widespread in C. cassiicola from Shandong province, and the LAMP method can be used to monitor QoI resistance in C. cassiicola caused by the G143A mutation in the field.

Sensitivity of C. fructicola and C. siamense of peach in China to multiple classes of fungicides and characterization of pyraclostrobin-resistant isolates

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including C. fructicola and C. siamense, is a devastating disease of peach. The chemical control has been widely used for years and management failures have increased towards commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance and 26 % were low resistant, all of the C. siamense isolates were low resistant. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin, even they are members of the same quinone outside inhibitor (QoI) fungicide group, and between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for mycelial growth rates were detected between highly resistant and low-resistant isolates of C. fructicola. Molecular characterization of Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the current resistance status of C. fructicola and C. siamense to different fungicides and their future perspectives. Demethylation inhibitor (DMI) fungicides are the best option among different chemicals to control peach anthracnose in China.