Quinone Outside Inhibitor Research Articles

Fungicide resistance in Cercospora species causing cercospora leaf blight and purple seed stain of soybean in Argentina

AbstractCercospora species cause cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean. Because there are few resistant soybean varieties available, CLB/PSS management relies heavily upon fungicide applications. Sensitivity of 62 Argentinian Cercospora isolates to demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) fungicides, and mancozeb was determined in this study. All isolates were sensitive to difenoconazole, epoxiconazole, prothioconazole, tebuconazole, and cyproconazole (EC50 values ranged from 0.006 to 2.4 µg/ml). In contrast, 51% of the tested isolates were sensitive (EC50 values ranged from 0.003 to 0.2 µg/ml), and 49% were highly resistant (EC50 > 100 µg/ml) to carbendazim. Interestingly, all isolates were completely resistant to azoxystrobin, trifloxystrobin, and pyraclostrobin, and insensitive to boscalid, fluxapyroxad, and pydiflumetofen (EC50 > 100 µg/ml). The G143A mutation was detected in 82% (53) of the QoI‐resistant isolates and the E198A mutation in 97% (31) of the carbendazim‐resistant isolates. No apparent resistance mutations were detected in the succinate dehydrogenase genes (subunits sdhB, sdhC, and sdhD). Mancozeb completely inhibited mycelial growth of the isolates evaluated at a concentration of 100 µg/ml. All Argentinian Cercospora isolates were sensitive to the DMI fungicides tested, but we report for the first time resistance to QoI and MBC fungicides. Mechanism(s) other than fungicide target‐site modification may be responsible for resistance of Cercospora to QoI and MBC fungicides. Moreover, based on our results and on the recent introduction of SDHI fungicides on soybean in Argentina, Cercospora species causing CLB/PSS are insensitive (naturally resistant) to SDHI fungicides. Insensitivity must be confirmed under field conditions.

Efficacy of fungicide treatments for Plasmopara viticola control and occurrence of strobilurin field resistance in vineyards in Georgia, USA

Grapevine downy mildew, caused by Plasmopara viticola, is a major disease of cultivated wine grapes in Georgia (USA), where the climate is favorable for rapid epidemic development. Although loss of fungicide sensitivity has been documented for P. viticola populations in other viticultural regions, it had not been confirmed previously in Georgia. Downy mildew samples from three vineyards, two managed commercially and one located at a research station, were subjected to polymerase chain reaction (PCR) assays to identify the presence of known mutations conferring quinone outside inhibitor (QoI) and carboxylic acid amide (CAA) fungicide resistance. PCR assays detected the presence of the G143A mutation which confers resistance to the QoI fungicides in 100% of samples (n = 39) and the absence of the G1105S mutation that confers resistance to CAA fungicides at all three locations. To determine fungicide efficacy, field trials were conducted in these same vineyards with 10 oomycete-active fungicides or combinations. A significant reduction in efficacy of QoI fungicides (azoxystrobin and pyraclostrobin), with disease levels statistically equivalent to those of untreated vines, was observed. Based on the presence of the G143A mutation that confers resistance to QoIs and the associated field disease control failures observed where QoIs were applied, this fungicide class may no longer be effective for downy mildew management in many Georgia vineyards.

Combining penthiopyrad with azoxystrobin is an effective alternative to control seedling damping-off caused by Rhizoctonia solani on sugar beet

The soil-borne fungus Rhizoctonia solani causes damping-off on sugar beet seedlings. Growers rely on fungicides to protect sugar beet in fields affected by R. solani. Quinone outside inhibitor (QoI) fungicides, such as azoxystrobin, have been applied as in-furrow and foliar sprays to manage R. solani, but repeated use of QoI fungicides pose risks in fungicide resistance. Penthiopyrad is a novel fungicide with the succinate dehydrogenase inhibitor (SDHI) mode of action. The objectives of this study were to compare the efficacy of penthiopyrad used as a sole seed treatment versus azoxystrobin as an in-furrow or a post-planting application for controlling R. solani; to determine if a penthiopyrad seed treatment combined with azoxystrobin as a post-planting application can improve control of R. solani over sole penthiopyrad seed treatment, azoxystrobin in-furrow or post-planting spray application. Seedling survival rate and area under disease progress curve (AUDPC) for seedling loss rate were used to measure the efficacy of each treatment. A sole penthiopyrad seed treatment at 14 g a. i. kg−1 of seeds, and penthiopyrad seed treatments at 7 and 14 g a. i. kg−1 of seeds combined with one azoxystrobin in-furrow application 14 days after planting resulted in similar seedling survival rate and AUDPC as achieved with the standard azoxystrobin in-furrow application. However, post-planting foliar spray of azoxystrobin alone failed to control seedling damping-off. Our research suggests that penthiopyrad can be used as a seed treatment to provide early protection to vulnerable seedlings while azoxystrobin can be used as a post-planting application to protect the ensuing adult plants.

Multiple resistance of Plasmopara viticola to QoI and CAA fungicides in Brazil

AbstractGrapevine downy mildew, caused by Plasmopara viticola, is commonly controlled by quinone outside inhibitor (QoI) and carboxylic acid amide (CAA) fungicides. QoI resistance is conferred by a mutation resulting in a G143A alteration in cytochrome b, while CAA resistance is associated with a G1105S mutation in cellulose synthase 3. The aims of this study were to (a) verify the presence of QoI and CAA resistance in P. viticola in Brazil, (b) develop a single nucleotide polymorphism (SNP) detection pyrosequencing assay for quantitative detection of G143A, and (c) determine the geographic prevalence and distribution of G143A and G1105S in Brazilian field populations. P. viticola isolates were sampled from two vineyards reporting failures of QoI and CAA control. Their growth was unaffected by azoxystrobin and mandipropamid, and resistance was associated with the G143A and G1105S mutations, respectively. The SNP detection pyrosequencing assay developed for G143A detection was precise and accurate. Pyrosequencing assays revealed that G143A and G1105S frequencies varied among the 66 field populations and were positively correlated with the number of QoI and CAA applications, respectively. The G143A mutation was observed in 60 populations, 39 of them with frequencies over 90%. G1105S was detected in 20 populations with frequencies ranging from 18.3% to 100%, and which also carried the G143A mutation. Multiple resistance to QoI and CAA in P. viticola was confirmed for the first time in South America. Future resistance monitoring surveys and management strategies must be adopted to prevent further increase in QoI and CAA resistance in Brazil.

Effect of membrane integrity on survival competition of Botrytis cinerea upon QoI fungicide pyraclostrobin

AbstractBotrytis cinerea, the causal agent of the grey mould disease, developed resistance to multiple fungicides. However, the role of cell membrane in survival competition of B. cinerea upon quinone outside inhibitor (QoI) fungicide has not yet been elucidated. In this paper, the enhancement of cystamine, a transglutaminase inhibitor, on membrane integrity of B. cinerea was determined, and the effect of the enhancement on the sensitivity of B. cinerea to pyraclostrobin was investigated. The results showed that pyraclostrobin inhibited mycelial growth with EC50 as 1.122 and 3.042 μg/ml at 24 and 48 hr, respectively. In the treatment of 5 and 50 μg/ml pyraclostrobin, membrane integrity of B. cinerea was broken, causing high permeability, lipid peroxidation, flocculent and malformed surface with vague septum and abundant agglomerates inside and outside the mycelia. Cystamine even at 50 and 200 μg/ml had little inhibitory effect on mycelial growth. However, in presence of 50 or 200 μg/ml cystamine, the mycelia from pyraclostrobin treatment possessed a significantly reduced leakage, lower MDA content, and a revived fibrous and transparent surface. Meanwhile, SEM images showed that membrane integrity of the mycelia was significantly improved and the agglomerates were dramatically disappeared. Synergy assays further revealed that B. cinerea regained less sensitivity to pyraclostrobin inhibition. In conclusion, membrane integrity controls mycelia sensitivity and is required for survival competition of B. cinerea upon pyraclostrobin.

Resistance of Cercospora beticola Sacc isolates to thiophanate methyl (benzimidazole), demethylation inhibitors and quinone outside inhibitors in Morocco

Sugar beet is a major crop in Morocco and Cercospora leaf spot is one of its most important fungal diseases. In Morocco, thiophanate methyl (benzimidazole) and difenoconazole (demethylation inhibitor, DMI) have been used extensively in the management of Cercospora leaf spot. In this study, samples of Cercospora beticola Sacc were collected from four major production areas. The identification of all isolates was confirmed using a PCR test with specific primers. Radial mycelia growth of each isolate in unmodified potato dextrose agar medium was compared to mycelia growth in the same medium modified with thiophanate methyl (1, 5, 10 and 50 ppm) or the DMIs difenoconazole, epoxiconazole and tetraconazole (0.1, 0.5, 1, 5, 10 and 50 ppm) or the quinone outside inhibitors (QOIs) azoxystrobin and trifloxystrobin (1, 5, 10 and 50 ppm). The percentage of inhibition obtained was used for the half maximal effective concentration (EC50) calculation. All the isolates showed resistance to the thiophanate methyl molecule to different degrees. Three groups were identified: low resistance with EC50 less than 100 ppm, moderate resistance with EC50 between 100 and 1000 ppm, and very resistant with EC50 more than 1000 ppm. For difenoconazole, 41% of isolates were sensitive (EC50 < 0.05 ppm) and 59% were resistant, while for tetraconazole and epoxiconazole 6% were sensitive (EC50 < 0.01 ppm), 66.7 % had medium resistance (0.01 < EC50 < 1 ppm) and 27.3% were resistant (EC50 > 1 ppm). For QOIs, azoxystrobin was less effective for mycelial inhibition than trifloxystrobin, with 54.5% of isolates with resistance to azoxystrobin (EC50 > 100 ppm).

Development of novel pesticides in the 21st century.

General trends and strategies for novel pesticides are summarized. Global pesticide sales and pesticide discovery research are also briefly reviewed. At least 105 chemical pesticides have been launched during the past decade or are under development: 43 fungicides, 34 insecticides/acaricides, 6 nematicides, 21 herbicides, and 1 herbicide safener. Most of them are safe to humans and environmentally friendly. The most developed fungicides are SDHI (succinate dehydrogenase inhibitors), DMI (demethylation inhibitors), QoI (quinone outside inhibitors), and QiI (quinone inside inhibitors). Due to the development of resistance to fungicides with existing modes of action, many fungicides possessing various novel modes of action have been launched or are under development. The trend of insecticide development is changing from organophosphorus, carbamate, and synthetic pyrethroids to nicotinic and diamide insecticides. During the past decade, compounds possessing a variety of novel modes of action have also been launched or are under development. Flupyradifurone and flupyrimin, exhibiting extremely low honeybee toxicity, have been developed and subjected to practical use. Herbicides possessing varied modes of action, such as acetolactate synthase, p-hydroxyphenylpyruvate dioxygenase, protoporphyrinogen oxidase, and very-long-chain fatty acid elongase inhibition, have been developed, but no herbicides possessing a novel mode action have commercialized in nearly 30 years. It is of interest that cyclopyrimorate, which was recently launched, and tetflupyrolimet, which is under development, have novel modes action: homogentisate solanesyltransferase (HST) and dihydroorotate dehydrogenase (DHODH) inhibition, respectively. The development of useful acaricides and nematicides is also progressing. Some natural product origin pesticides are getting attention.

Three-dimensional visualization of mitochondria in conidia of Pyricularia oryzae using green fluorescent protein (GFP) fused with citrate synthase (CitA)

The mitochondrion is the target organelle for QoI (quinone outside inhibitor) antifungal drugs, but mitochondrial behavior in Pyricularia oryzae remains unclear. Conidial mitochondria in fungal isolate transformed with citrate synthase (CitA)-GFP fusion gene, were observed in situ using laser-scanning confocal microscopy and computer analysis. After mycelium was harvested at 36 h and aerial mycelia removed, conidia with 1, 2, or 3 cells were observed over time. The 3D visualization of mitochondria in these conidia showed that mitochondria were initially shaped like dots and developed into a network as the conidia aged.

Sensitivity of Colletotrichum acutatum Isolates from Citrus to Carbendazim, Difenoconazole, Tebuconazole, and Trifloxystrobin.

Postbloom fruit drop (PFD) of citrus is caused by the Colletotrichum acutatum and C. gloeosporioides species complexes. The disease is important when frequent rainfall occurs during the flowering period of citrus trees. In Brazil, until 2012, PFD was mainly controlled by preventive applications of the methyl-benzimidazole carbamate (MBC) carbendazim and demethylation-inhibitor (DMI) fungicides such as difenoconazole. Since then, mixtures containing the DMI tebuconazole and the quinone-outside inhibitor (QoI) trifloxystrobin have been commonly used. Fungicides are often applied preventively, sometimes even when conditions are not conducive for PFD development. Excessive fungicide applications may favor the selection of resistant populations of Colletotrichum spp. In this study, we assessed the fungicide sensitivity of C. acutatum isolates collected during the two distinct periods of PFD management in Brazil: before and after the trifloxystrobin and tebuconazole mixture became widely employed. The sensitivity of 254 C. acutatum isolates to carbendazim and difenoconazole and of 164 isolates to tebuconazole and trifloxystrobin was assessed. Mycelial growth inhibition of these isolates was evaluated for all the fungicides using either serial dilution of fungicide rates or the spiral gradient dilution method. In addition, inhibition of conidial germination was also assessed for trifloxystrobin. Analysis of partial β-tub, cytb, and cyp51b gene sequences did not reveal any mutations related to resistance to MBCs, QoIs, and DMIs, respectively. In mycelial growth assays, mean EC50 values were 0.14, 0.11, and 0.21 μg/ml for difenoconazole, tebuconazole, and trifloxystrobin, respectively. The conidial germination inhibition by trifloxystrobin was similar among the tested isolates, and the mean EC50 value was 0.002 μg/ml. All isolates had similar mean mycelial growth inhibition for carbendazim, regardless of the fungicide concentrations. Therefore, based on similar EC50 values and molecular analyses, no shift in the sensitivity of isolates has been observed to the fungicides commonly used in different citrus-producing areas in Brazil.

Rapid Detection of Cercospora beticola in Sugar Beet and Mutations Associated with Fungicide Resistance Using LAMP or Probe-Based qPCR.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection "toolbox" for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in β-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates.

Temporal population monitoring of fungicide sensitivity in Cercospora beticola from sugarbeet (Beta vulgaris) in the Upper Great Lakes

In recent years, there has been an increase in the frequency of Cercospora beticola (Sacc.), the cause of Cercospora leaf spot (CLS) in sugarbeet (Beta vulgaris L.), isolates with demethylase inhibitor (DMI) and quinone outside inhibitor (QoI) fungicide resistance collected from commercial fields in Michigan, USA and Ontario, Canada. This study evaluated sensitivity distributions of C. beticola populations in the Upper Great Lakes sugarbeet production region to DMIs and organotin fungicides and determined shifts in the temporal frequency of fungicide sensitivity phenotypes from 2014 through 2017. The effective concentration (mg L−1) for inhibiting growth by 50% (EC50) was determined for six chemistries from the above fungicide classes. Isolates of C. beticola were categorized phenotypically into five sensitivity categories based on EC50 values: Sensitive (<1); reduced sensitive (1–10); moderately insensitive (10–50); insensitive (50–100) and resistant (>100). Significant temporal shifts towards insensitivity were detected from 2014 to 2017 in the population median and mean EC50 values, and frequencies of sensitivity phenotypes to DMI and organotin fungicides. Individual isolates of C. beticola were recovered with cross resistance to DMI and organotin fungicides. There was a population shift towards insensitivity, and thus an increase in the frequency of recovered isolates of C. beticola with lower sensitivity to five DMI fungicides (difenoconazole, fenbuconazole, flutriafol, prothioconazole and tetraconazole) and fentin hydroxide. Results from sensitivity monitoring agree with other studies that an integrated approach, combining knowledge of fungicide efficacy and pathogen biology is essential in developing fungicide resistance management recommendations and thus effective disease management strategies.

Sensitivity of Colletotrichum Isolates Collected from Strawberries in Georgia to Pyraclostrobin, a Quinone Outside Inhibitor (QoI) Fungicide

Anthracnose fruit rot disease, caused by Colletotrichum spp., is the most significant disease problem of commercial strawberry (Fragaria × ananassa) production in the southeastern United States. The hot, humid weather and continuous rainfall in Georgia make Colletotrichum-induced fruit rot a widespread problem in strawberry production. In order to control this disease, growers mainly rely on preventive fungicide applications from flower bud emergence to harvest. The most commonly used single-site fungicides are quinone outside inhibitors (QoIs); the QoI active ingredients azoxystrobin and pyraclostrobin are utilized to manage anthracnose fruit rot. In 2019, we collected 108 strawberry fruits with visible rot symptoms from seven different strawberry farms in Georgia. These farms had received multiple applications of QoI fungicides during the 2019 growing season, as well as in previous seasons. Sensitivities to pyraclostrobin were assessed on 1% malt extract agar using a mycelial growth inhibition assay. Our results demonstrated that a majority of Colletotrichum isolates collected in 2019 were not inhibited by pyraclostrobin, suggesting a growing resistance issue with the QoI fungicides. A PCR-restriction fragment length polymorphism assay showed the presence of the G143A mutation in all QoI “resistant” C. acutatum isolates and none for isolates labeled “reduced sensitivity” or “sensitive”. These results further prove that C. acutatum isolates with the G143A mutation are highly resistant to the QoI fungicide. These findings suggest that there is a high risk of resistance development associated with using pyraclostrobin (likely all QoIs) for controlling anthracnose fruit rot of strawberry in Georgia.

Resistance to Quinone Outside Inhibitor Fungicides Conferred by the G143A Mutation in Cercospora sojina (Causal Agent of Frogeye Leaf Spot) Isolates from Michigan, Minnesota, and Nebraska Soybean Fields

Frogeye leaf spot, caused by Cercospora sojina, is an important disease of soybean (Glycine max) in the United States. An important tactic to manage frogeye leaf spot is to apply foliar fungicides. Isolates of C. sojina were collected from soybean fields in one county in Michigan, three counties in Minnesota, and 10 counties in Nebraska in 2019, and they were tested for resistance to quinone outside inhibitor (QoI) fungicides using a discriminatory dose assay, a PCR assay, and DNA sequencing. Results of the testing indicated that QoI fungicide-resistant isolates were detected in isolates from all counties. Testing results also indicated that the G143A mutation was responsible for the QoI fungicide resistance. This is the first report of QoI fungicide-resistant C. sojina isolates in Michigan, Minnesota, and Nebraska and expands the geographical distribution of QoI fungicide-resistant C. sojina isolates to 18 states in total.

Fungicide Sensitivity Survey of Plasmopara viticola Populations in Georgia Vineyards

Grapevine downy mildew, caused by Plasmopara viticola, is among the most damaging diseases of grapes globally and in the viticultural regions of Georgia (U.S.A.). Although management of this disease typically involves fungicide applications, resistance development in P. viticola can render chemical management ineffective. The objective of this study was to survey fungicide sensitivities of P. viticola populations in vineyards across three regions in Georgia. Samples were collected from eight vineyards in 2017 and four in 2018 and tested phenotypically for sensitivity to quinone outside inhibitor (QoI), carboxylic acid amide (CAA), and phenylamide (PA) fungicides using leaf disc bioassays. In addition, DNA was extracted from P. viticola samples collected from 2015 through 2018 in these 12 vineyards and six others for a total of 18 vineyards sampled. All available DNA samples were tested for the presence of the known resistance-causing mutations G143A (QoI) and G1105S (CAA) using polymerase chain reaction (PCR). This study documented widespread occurrence of QoI resistance in P. viticola in Georgia, with 83.0% of isolates collected in 2017 and 2018 testing positive for QoI resistance based on bioassay and 82.9% of isolates collected from 2015 to 2018 testing positive for QoI resistance through PCR testing. In total, 94.4% (17/18) of surveyed vineyards had confirmed QoI resistance by the conclusion of the survey period. No reduced sensitivity to CAA or PA fungicides was identified. Due to widespread QoI resistance, these fungicides should not be relied upon for downy mildew management in Georgia vineyards with a long history of their use.

Cercospora nicotianae Isolates from Flue-Cured Tobacco in North Carolina Found with G143A and F129L Mutations in Cytochrome b Gene

Frogeye leaf spot of tobacco caused by Cercospora nicotianae (Ellis &amp; Everhart) is a widespread disease of cultivated tobacco. Recently, flue-cured tobacco producers in North Carolina reported losses due to frogeye leaf spot disease despite the use of strobilurin fungicides. Isolates (n = 4) were obtained in 2018 from affected tobacco leaves from Cumberland, Lenoir, and Nash counties. In 2019, isolates (n = 28) were collected from a field in Wilson county. After sequencing the cytb region of 32 isolates, 30 contained a single point mutation conferring a G143A or F129L amino acid change that resulted in quinone outside inhibitor (QoI) fungicide resistance. Although these resistance mutations have been found in air-cured tobacco in Kentucky, to the best of our knowledge, the present study is the first to report QoI resistance mutations in C. nicotianae populations in flue-cured tobacco and a first report in North Carolina.

Colletotrichum Species Isolated from Massachusetts Cranberries Differ in Response to the Fungicide Azoxystrobin

Fruit rot is the most prevalent disease of cranberry (Vaccinium macrocarpon). Colletotrichum species (C. acutatum and C. gloeosporioides) have been identified as cranberry fruit rot pathogens. Resistance to azoxystrobin by Colletotrichum has been documented in other fruit crops. Azoxystrobin, a quinone-outside inhibitor fungicide, was approved for use in cranberry in 2003, and a single application delivers between 78 and 202 ppm under typical chemigation conditions. Twenty-nine isolates were collected from diseased fruit in commercial Massachusetts cranberry farms. Six isolates were collected prior to registration of azoxystrobin (unexposed), and 22 isolates were from locations where the fungicide had been applied (exposed). This study is the first report of the relative sensitivity of exposed and unexposed field-acquired cultures for the cranberry fruit rot pathogen, Colletotrichum, to azoxystrobin.

Widespread QoI Fungicide Resistance Revealed Among Corynespora cassiicola Tomato Isolates in Florida.

Target spot of tomato caused by Corynespora cassiicola is one of the most economically destructive diseases of tomato in Florida. A collection of 123 isolates from eight counties in Florida were evaluated for sensitivity to azoxystrobin and fenamidone based on mycelial growth inhibition (MGI), spore germination (SG), detached leaflet assays (DLAs), and sequence-based analysis of the cytochrome b gene (cytb). Cleavage of cytb by restriction enzyme (Fnu4HI) revealed the presence of a mutation conferring a glycine (G) to alanine (A) mutation at amino acid position 143 (G143A) in approximately 90% of the population, correlating with quinone outside inhibitor (QoI) resistance based on MGI (<40% at 5 μg/ml), SG (<50% at 1 and 10 μg/ml), and DLA (<10% severity reduction). The mutation conferring a phenylalanine (F) to leucine (L) substitution at position 129 (F129L) was confirmed in moderately resistant isolates (#9, #19, and #74) based on MGI (40 to 50% at 5 μg/ml), SG (<50% at 1 μg/ml and >50% at 10 μg/ml), and DLA (>10% and <43% severity reduction) for both QoI fungicides, whereas sensitive isolates (#1, #4, #7, #28, #29, #46, #61, #74, #75, #76, #91, #95, and #118) based on MGI (>50% at 5 μg/ml), SG (>50% at 1 μg/ml and 10 μg/ml), and DLA (>50% severity reduction) correlated to non-mutation-containing isolates or those with a silent mutation. This study indicates that QoI resistance among C. cassiicola isolates from tomato is widespread in Florida and validates rapid screening methods using MGI or molecular assays to identify resistant isolates in future studies.

Soybean Fungal Endophytes Alternaria and Diaporthe spp. are Differentially Impacted by Fungicide Application.

In field trials in Iowa, we investigated the association of a fungicide applied at early pod set to the diversity and composition of foliar endophytic fungi in presenescent soybeans. The main purpose of our study was to determine whether fungicides affect the microbiome of soybean plants during the pod-fill reproductive stage. In a replicated experiment focused on the impact of a fungicide application including a quinone outside inhibitor (QoI) and a pyrazole-carboxamide spanning two growing seasons, healthy stems and leaves near the tops of soybean were sampled for endophytic fungi. The survey yielded 1,791 isolates belonging to 17 putative species, identified by morphology and sequence analysis of the ribosomal DNA internal transcribed spacer region. Taxa were grouped by genus into operational taxonomic units: Alternaria, Colletotrichum, and Diaporthe were the dominant genera isolated. Plant parts were analyzed separately using a multivariate community analysis of isolate counts per plant. The 14.3% fluxapyroxad and 28.6% pyraclostrobin fungicide spray significantly increased the proportion of Diaporthe isolates over no-spray controls, whereas the inverse occurred for foliar Alternaria isolates. In addition, seed harvested from fields with shorter-season varieties and sprayed with fungicide showed higher percentages of Diaporthe isolates than fields with no fungicide spray. In conclusion, soybean farmers may want to consider that the application of a QoI fungicide in the absence of disease pressure might adversely impact seed quality.

Emergence of Stemphylium Leaf Blight of Onion in New York Associated With Fungicide Resistance.

A complex of foliar diseases affects onion production in New York, including Botrytis leaf blight (Botrytis squamosa), purple blotch (Alternaria porri), Stemphylium leaf blight (SLB; Stemphylium vesicarium), and downy mildew (Peronospora destructor). Surveys were conducted in 2015 and 2016 to evaluate the cause of severe premature foliar dieback in New York onion fields. SLB was the most prevalent disease among fields with the greatest incidence, surpassing downy mildew, purple blotch, and Botrytis leaf blight. Sequencing of the internal transcribed spacer region of ribosomal DNA and the glyceraldedyhe-3-phosphate dehydrogenase and calmodulin genes identified S. vesicarium as the species most commonly associated with SLB. S. vesicarium was typically associated with a broad range of necrotic symptoms but, most commonly, dieback of leaf tips and asymmetric lesions that often extended over the entire leaf. Because of the intensive use of fungicides for foliar disease control in onion crops in New York, the sensitivity of S. vesicarium populations to various fungicides with site-specific modes of action was evaluated. Sensitivity of S. vesicarium isolates collected in 2016 to the quinone outside inhibitor (QoI) fungicide, azoxystrobin, was tested using a conidial germination assay. Isolates representing a broad range of QoI sensitivities were selected for sequencing of the cytochrome b gene to evaluate the presence of point mutations associated with insensitivity to azoxystrobin. The G143A mutation was detected in all 74 S. vesicarium isolates with an azoxystrobin-insensitive phenotype (effective concentrations reducing conidial germination by 50%, EC50 = 0.2 to 46.7 µg of active ingredient [a.i.]/ml) and was not detected in all 31 isolates with an azoxystrobin-sensitive phenotype (EC50 = 0.01 to 0.16 µg a.i./ml). The G143A mutation was also associated with insensitivity to another QoI fungicide, pyraclostrobin. Sensitivity to other selected fungicides commonly used in onion production in New York was evaluated using a mycelial growth assay and identified isolates with insensitivity to boscalid, cyprodinil, and pyrimethanil, but not difenoconazole. The frequency of isolates sensitive to iprodione, fluxapyroxad, and fluopyram was high (93.5 to 93.6%). This article discusses the emergence of SLB as dominant in the foliar disease complex affecting onion in New York and the complexities of management posed by resistance to fungicides with different modes of action.

Rapid quantification of the G143A mutation conferring fungicide resistance in Virginia populations of Cercospora sojina using pyrosequencing

Quinone outside inhibitors (QoI) inhibit a wide spectrum of fungal pathogens, but there is high risk of resistance development with this class of fungicides. QoI resistance in Cercospora sojina, the causal agent of frogeye leaf spot in soybean, has been confirmed from 14 states. An amino acid substitution at position 143 of the cytochrome b (cytb) gene was confirmed as the mechanism of resistance. Cercospora sojina has not been well characterized in the mid-Atlantic U.S. The objectives of this study were to (i) determine if QoI-resistant C. sojina is present in Virginia, (ii) develop a pyrosequencing-based method to quantify G143A frequencies, and (iii) determine frequencies and distributions of QoI-resistant C. sojina. Based on in vitro fungicide sensitivity and PCR detection of the G143A mutation, QoI-resistant C. sojina was confirmed from nine Virginia soybean fields in 2015. A pyrosequencing assay to amplify amino acid position 143 was designed to detect frequencies of the G143A substitution from mixtures of C. sojina DNA. This assay was used to quantify G143A frequencies within fields sampled in a statewide survey from 2015 to 2017. G143A frequencies ranged from 1 to 99% (mean = 27%), with 80% of fields having a mutation frequency ≤50% and 40% with frequencies <10%. Using pyrosequencing, G143A substitution frequencies, and subsequent QoI resistance, was monitored in C. sojina populations across locations and over time. Relatively low frequencies of QoI-resistant C. sojina were detected in Virginia, but fungicide resistance management practices, including monitoring fields for G143A substitution frequencies, should be continued to track the development and spread of resistance.