Fungicide Sensitivity Research Articles

Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

Fusarium Species and Mycotoxins Associated with Sorghum Grains in Uruguay.

Grain mold and stalk rot are among the fungal diseases that cause significant losses in sorghum worldwide and are caused by different Fusarium spp. The presence of Fusarium species in sorghum grains causes yield losses and mycotoxin contamination, which represents a risk to consumers. In this study, Fusarium graminearum species complex (FGSC) had a high incidence, followed by Fusarium fujikuroi species complex (FFSC) and F. incarnatum-equiseti species complex. Within FFSC, F. proliferatum, F. andiyazi, F. fujikuroi, F. thapsinum, F. verticillioides and F. subglutinans were identified, and this was the first report of F. fujikuroi in sorghum. The most frequent toxins found in sorghum samples were deoxynivalenol (DON) and zearalenone (ZEN). The presence of fumonisins and nivalenol (NIV) was detected at low levels. This study adds new knowledge about the occurrence of Fusarium species and mycotoxins in sorghum grains. Furthermore, this is the first report in Uruguay on fungicide sensitivity for Fusarium isolates from sorghum, which constitutes an important starting point for defining management practices to minimize fungal infection and mycotoxin contamination.

Effect of environmental factors, fungicide sensitivity, and pathogenicity of Fusarium spp. associated with fruit rot of melon

AbstractFusarium rot is one of the main postharvest diseases of melons, directly interfering with the quality and commercial use of the fruit. The purpose of this study was to assess the effect of environmental factors (temperature, pH and salinity) and the pathogenicity of four Fusarium species (F. falciforme, F. kalimantanense, F. pernambucanum and F. sulawesiense) belonging to three different species complexes on disease development on melon, as well as the in vitro and in vivo sensitivity of these species to the fungicides azoxystrobin + fludioxonil, imazalil and thiabendazole. The results showed optimum fungal growth temperatures from 24.5 to 27.2°C, and optimum pH from 6.10 to 8.37 for all Fusarium species. NaCl concentrations (250–1000 mM) significantly reduced in vitro mycelial growth for all isolates. All species were pathogenic to melon plants and fruit, with an isolate of F. falciforme being the most aggressive, causing the highest disease severity in plants and fruit (43.3% and 62.5%, respectively). The isolate of F. sulawesiense tested showed high in vitro sensitivity to two fungicides (azoxystrobin + fludioxonil and imazalil), with EC50 values below 1 mg/L of a.i. Fruit inoculated with the selected isolates of F. falciforme and F. pernambucanum showed a reduction in the disease severity on the peduncle when treated with the fungicide thiabendazole (9.0% and 4.0%, respectively). Azoxystrobin + fludioxonil was responsible for the lowest disease severity in the epicarp caused by the same isolates (0.0% and 2.5%, respectively). These results are relevant to improve management strategies for diseases of melon caused by Fusarium spp.

First Confirmed Report of Rhizoctonia solani AG-7 Causing Potato Stem Canker in Mexico.

Rhizoctonia solani, is the causal agent of black scurf and stem canker of potatoes (Solanum tuberosum L.) throughout the world. In November 2021, stem canker symptoms were observed in two potato fields located in Ahome, Sinaloa, Mexico. The disease incidence was estimated up to 15%. For fungal isolation, fragments of symptomatic stems were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed with sterilized distilled water, and blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25°C in darkness for 4 days. Rhizoctonia-like colonies were consistently obtained and 12 isolates were purified by the hyphal-tip method. Fungal colonies on PDA were white initially and then turned brown, raised, and with entire or undulate edges. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Microscopic examination by staining with 1% safranin O and 3% KOH solution showed multinucleate cells. The morphological features of the isolates resembled those of Rhizoctonia solani (Sneh et al. 1991). Four representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa) under accession nos. CCLF267, CCLF274, CCLF277, and CCLF279. For molecular identification, genomic DNA from each of the four isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP784258 to OP784261). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the four isolates within the R. solani AG-7 clade with high bootstrap support (100%). For pathogenicity tests, certified pathogen-free potato mini-tuber (cv. Fianna) were placed in a polystyrene pot (1 L) filled with a 5 cm layer of a sterile substrate composed of soil and peat moss (2:1 w/w). One rice grain (20 mg) colonized with each isolate was placed 10 mm above the uppermost sprout tip and covered with the sterile substrate (Inokuti et al. 2019). Control plants were inoculated with sterile rice grains. All pots were transferred to a greenhouse where the temperature ranged from 20 to 32°C. Stem necrosis symptoms were observed on all inoculated plants 25 days after emergence, whereas control plants remained symptomless. Pathogenicity test was performed twice with similar results. Fungi were reisolated from the infected stems and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. The AG-7 has been previously reported to cause potato diseases in South Africa (Truter and Wehner 2004). In Mexico, Carling et al. (1998) reported the presence of an isolate of R. solani AG-7 obtained from a potato tuber-borne sclerotium in Toluca; however, there is no information about the methodology used for the characterization of that isolate. To our knowledge, this is the first confirmed report of R. solani AG-7 causing potato stem canker in Mexico. Our findings improve knowledge about R. solani AGs occurring in potato fields in Mexico. So, further studies should be conducted to investigate the diversity, prevalence, and fungicide sensitivity of AGs distributed in the main potato-producing states in Mexico.

MORPHOTYPIC COMPOSITION AND DIFENOCONAZOLE SENSITIVITY OF APPLE SCAB PATHOGEN POPULATIONS THAT DIFFER IN THE HISTORY OF FUNGICIDE APPLICATION

Scab is one of the most harmful diseases of the world's main fruit crop, the apple tree.
 Purpose. To evaluate the fungicide influence on the population structure of Venturia inaequalis, by its sensitivity to difenoconazole and morphotypic composition.
 Materials and methods. The study was conducted on 88 monospore isolates isolated from two populations of V. inaequalis. The baseline population, which was not treated with fungicides, was selected from natural populations of Malus orientalis. A population of commercial apple orchards was treated with difenoconazole. The sensitivity of isolates was evaluated under laboratory conditions by mycelial growth at different fungicide concentrations and expressed as effective 50% concentration (EC50). Morphotypes were distinguished by comparing photo-images of isolates with morphotype images in a database.
 Results. The populations differed in their sensitivity to difenoconazole at a high level of significance (p≤0.003), with median EC50 values of 0.5 and 2.5 mg a.i./l. The resistance factor of the orchard population had a value of 22. Morphological analysis of the isolates also showed an uneven distribution of morphotypes in the populations. Of the 15 morphotypes isolated, 73 % were unique to a particular population. A medium positive correlation between the traits "morphotype" and "fungicide sensitivity" was found.
 Conclusion. For the first time, a change in the population structure of Venturia inaequalis under the influence of a fungicide was shown not only for a trait under selection pressure (sensitivity), but also for an evolutionarily neutral trait, morphotypic composition.

Research to Fungicide Sensitivity of Colletotrichum spp. Isolated from Apple Fruits in Cheongsong, Korea

Apple grower in the Cheongsong region, Korea has reported the increased loss of apple yield due to severe bitter rot incidence. We noticed that this indience is because the <i>Colletotrichum</i> population has developed resistance to commonly used fungicides. We isolated 39 <i>Colletotrichum</i> isolates from 13 orchards in Cheongsong, and all the isolated <i>Colletotrichum</i> species were identified as <i>C. siamense</i> or <i>C. fructicola</i>. These 39 strains were tested for mycelial growth and conidial germination against 12 fungicides. trifloxystrobin (30–55% in recommended concentrations) was shown lower inhibitory effect on mycelial growth. However, the inhibition of conidial germination was shown higher than mycelial growth (62–100%). Kresoxim-methyl was shown lower inhibitory effect on mycelial growth (29–55%). conidial germination inhibitory effect was shown 51% to 96%. dithianon was shown diversity response to inhibition of mycelial growth (43–100%). tebuconazole was shown high inhibitory effect on mycelial growth (84–100%) and conidial germination inhibitory effect was shown to be 64 to 100%. metconazole has been found to display with high inhibitory effect on mycelial growth (79–100%) and conidial germination (70–80%). fluazinam was shown to possess high inhibitory effect on mycelial growth (87–100%) and conidial germination (94–100%). This study provides basic information for the effective management of apple bitter rot.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Sensitivity of Clarireedia spp. to benzimidazoles and dimethyl inhibitors fungicides and efficacy of biofungicides on dollar spot of warm season turfgrass.

Dollar spot caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa) is an economically destructive fungal disease of turfgrass that can significantly compromise turf quality, playability, and aesthetic value. Fungicides are frequently used to manage the disease but are costly and potentially unfavorable to the environment. Repeated use of some active ingredients has resulted in reduced efficacy on C. jacksonii causing dollar spot in cool-season turfgrasses in the US. Experiments were conducted to study fungicide sensitivity of Clarireedia spp. as well as to develop alternatives to fungicides against dollar spot on warm-season turfgrass in Georgia. First, 79 isolates of Clarireedia spp. collected across the state were tested on fungicide-amended agar plates for their sensitivity to thiophanate-methyl (benzimidazole) and propiconazole (dimethyl inhibitor). Seventy-seven isolates (97.5%) were sensitive (0.001 to 0.654 μg/mL) and two isolates (2.5%) were found resistant (>1000 μg/mL) to thiophanate-methyl. However, in the case of propiconazole, 27 isolates (34.2%) were sensitive (0.005 to 0.098 μg/mL) while 52 isolates (65.8%) were resistant (0.101 to 3.820 μg/mL). Next, the efficacy of three bio- and six synthetic fungicides and ten different combinations were tested in vitro against C. monteithiana. Seven bio- and synthetic fungicide spray programs comprising Bacillus subtilis QST713 and propiconazole were further tested, either alone or in a tank mix in a reduced rate, on dollar spot infected bermudagrass 'TifTuf' in growth chamber and field environments. These fungicides were selected as they were found to significantly reduce pathogen growth up to 100% on in vitro assays. The most effective spray program in growth chamber assays was 100% B. subtilis QST713 in rotation with 75% B. subtilis QST713 + 25% propiconazole tank mix applied every 14 days. However, the stand-alone application of the biofungicide B. subtilis QST713 every seven days was an effective alternative and equally efficacious as propiconazole, suppressing dollar spot severity and AUDPC up to 75%, while resulting in acceptable turf quality (>7.0) in field experiments. Our study suggests that increased resistance of Clarireedia spp. to benzimidazoles and dimethyl inhibitors warrants continuous surveillance and that biofungicides hold promise to complement synthetic fungicides in an efficacious and environmentally friendly disease management program.

Identification and Characterization of Diplodia parva and Diplodia crataegicola Causing Black Rot of Chinese Quince.

Fungal isolates from infected Chinese quince trees were found to cause black rot in Yeongcheon, Gyeongsangbuk Province, Korea. The quince leaves withered and turned reddish-brown and fruits underwent black mummification. To elucidate the cause of these symptoms, the pathogen was isolated from infected leaf and fruit tissues on potato dextrose agar and Levan media. Several fungal colonies forming a fluffy white or dark gray mycelium and two types of fungi forming an aerial white mycelium, growing widely at the edges, were isolated. Microscopic observations, investigation of fungal growth characteristics on various media, and molecular identification using an internal transcribed spacer, β-tubulin, and translation elongation factor 1-α genes were performed. The fungal pathogens were identified as Diplodia parva and Diplodia crataegicola. Pathogenicity tests revealed that the pathogen-inoculated fruits exhibited a layered pattern, turning brown rotting; leaves showed circular brown necrotic lesions. The developed symptoms were similar to those observed in the field. Fungal pathogens were reisolated to fulfill Koch's postulates. Apples were inoculated with fungal pathogens to investigate the host range. Strong pathogenicity was evident in the fruits, with browning and rotting symptoms 3 days after inoculation. To determine pathogen control, a fungicidal sensitivity test was conducted using four registered fungicides. Thiophanate-methyl, propineb, and tebuconazole inhibited the mycelial growth of pathogens. To the best of our knowledge, this is the first report on the isolation and identification of the fungal pathogens D. parva and D. crataegicola from infected fruits and leaves of Chinese quince, causing black rot disease in Korea.

Virulence, Aggressiveness, and Fungicide Sensitivity of Phytophthora spp. Associated with Soybean in Illinois.

Phytophthora root and stem rot (PRR), caused by Phytophthora sojae, is one of the most devastating oomycete diseases of soybean in Illinois. Single resistant genes (Rps) are used to manage this pathogen but P. sojae has adapted to Rps, causing failure of resistance in many regions. In addition to P. sojae, recent reports indicate that P. sansomeana could also cause root rot in soybean. Soil samples and symptomatic plants were collected across 40 Illinois counties between 2016 and 2018. P. sojae (77%) was more abundant than P. sansomeana (23%) across Illinois fields. Both species were characterized by virulence, aggressiveness, and fungicide sensitivity. Virulence of all P. sojae isolates was evaluated using the hypocotyl inoculation technique in 13 soybean differentials. Aggressiveness was evaluated in the greenhouse by inoculating a susceptible cultivar and measuring root and shoot dry weight. On average, P. sojae isolates were able to cause disease on six soybean differentials. P. sojae was more aggressive than P. sansomeana. All isolates were sensitive to azoxystrobin, ethaboxam, mefenoxam, and metalaxyl. The characterization of the population of species associated with PRR will inform management decisions for this disease in Illinois.

Species identification and fungicide sensitivity of fungi causing Alternaria Leaf Blight and Head Rot in cole crops in the Eastern U.S.

Alternaria Leaf Blight and Head Rot is an important disease of broccoli and other cole crops. With no resistant host varieties, fungicides are utilized to manage this disease. However, anecdotal evidence suggests that in Southeastern broccoli-producing states in the U.S., there is a loss of disease control through the use of QoI fungicides. To understand why there was a reduced sensitivity to QoI fungicides in these states, we isolated Alternaria species from symptomatic lesions on cole crops from Georgia and Virginia-two states with observations of loss of fungicide sensitivity, as well as New York-a state with no observations of loss of fungicide sensitivity. Using multi-locus sequencing and phylogenetic analysis, we identified two species, A. brassicicola and A. japonica. Whereas A. brassicicola was isolated in all states, A. japonica was only isolated in Georgia. Next, we wanted to determine the sensitivity of these isolates to azoxystrobin-an active ingredient in some QoI fungicides-by estimating the effective concentration at which only 50% of spores germinate (EC50). The EC50 of A. brassicicola ranged from 0.01 to 0.17 ppm, while the EC50 of A. japonica ranged from 7.9 to 27.1 ppm. None of the known target-site mutations that confer resistance to QoI fungicides were identified during screening of either species. Alternaria japonica was first reported on the East Coast of the U.S. in 2020 in South Carolina. The substantially higher EC50 value suggests that its emergence in the Southeastern U.S. may play at least a part in the observed loss of disease control. However, further in planta and field studies are needed to thoroughly test this hypothesis.

Colletotrichum acutatum: Causal Agent of Olive Anthracnose Isolation, Characterization, and Fungicide Susceptibility Screening in Punjab, Pakistan.

Anthracnose of olive fruit caused by Colletotrichum acutatum was a severe epidemic disease in Pakistan that occurred in September 2020. The estimated disease incident was recorded as 59%. Anthracnose causes a significant reduction in yield and quality traits. Anthracnose has been found in several orchards. Agricultural practices, environmental factors, and disease aggressiveness vary between orchards. Therefore, we looked at spore size, cultural traits, morphological variation, growth pattern, and pathogenicity of different strains of C. acutatum from various orchards. Molecular and phylogenetic analysis confirmed the isolated strains as C. acutatum. In all, 15 C. acutatum isolates from olive orchards were tested for susceptibility to four commercial fungicides (P < 0.001). The examined isolates' in vitro fungicide sensitivity varied with fungicide concentration. The concentration at which conidial germination was hindered by 50% compared with the control values was observed for difenoconazole, tebuconazole, carbendazim, and cyprodinil, ranging from 0.12 to 2.69 g ml-1. Based on the findings of the fungal growth inhibition studies, carbendazim has been found to be the only fungicide that effectively reduces (P < 0.001) anthracnose caused by C. acutatum strains. Additionally, results revealed that preharvest site treatments of different fungicides greatly decreased anthracnose infections on olive fruit (70 to 90%), and postharvest site applications significantly reduced disease prevalence and severity (75 to 95%). The fungicide carbendazim significantly decreased pre- and postharvest anthracnose infection on olive cultivars. This study suggests that the latter compound might be used to control olive anthracnose in Pakistan while lowering environmental impact and fungicide resistance.

Identification of Cyprodinil + Fludioxonil to Manage Soybean Red Crown Rot Using the Microplate-Based High-Throughput Screening and Pot Assay.

Red crown rot (RCR), caused by the soilborne fungus Calonectria ilicicola, is an emerging soybean disease in Taiwan, and fungicide screening is desired to identify effective management for C. ilicicola. This study screened 11 fungicides, including azoxystrobin, boscalid, cyprodinil, cyprodinil + fludioxonil, difenoconazole, fluopyram, flutolanil, mancozeb, prochloraz, pyraclostrobin, and tebuconazole, for their inhibitory effects on the mycelial growth of 10 C. ilicicola field isolates. Subsequently, a microplate-based high-throughput screening (MHTS) method was established to measure the fungicide sensitivity in a population composed of 80 C. ilicicola isolates to three effective fungicides, cyprodinil + fludioxonil, fluopyram, and tebuconazole. The MHTS was optimized for multiple factors, including the optical scanning pattern, absorption wavelength, conidial concentration, and measurement timing based on the quality controls of Z' factor and the log-phase growth curve. The population mean EC50 estimated by MHTS were 0.14, 2.34, and 2.46 ppm to cyprodinil + fludioxonil, fluopyram, and tebuconazole, respectively. In addition to the in vitro assessment, fungicide efficacy was evaluated by coating cyprodinil + fludioxonil, fluopyram, or tebuconazole on soybean seeds in the pot assay. The results showed that cyprodinil + fludioxonil significantly reduced both postemergence damping-off and disease severity, while fluopyram and tebuconazole reduced only the postemergence damping-off but not disease severity. Based on the MHTS and the pot assay results, this study demonstrated cyprodinil + fludioxonil to be a potential fungicide to manage soybean RCR.

Within-Season Shift in Fungicide Sensitivity Profiles of Pseudoperonospora cubensis Populations in Response to Chemical Control.

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is an important disease affecting cucurbits worldwide. Chemical control is an effective method for disease control but P. cubensis has a high risk for developing resistance to fungicides. Alternating fungicides with different modes of action is recommended to avoid an increase of resistant subpopulations. Thus, this study was conducted to establish shifts in the sensitivity profiles of P. cubensis isolates during the growing season, wherein chlorothalonil was applied in alternation with either cymoxanil, fluopicolide, or propamocarb in field experiments conducted from 2018 to 2020 at Rocky Mount, NC and in 2018 and 2020 at Charleston, SC. The sensitivity of baseline isolates sampled early in the season or exposed isolates sampled late in the season to these single-site fungicides was determined using a detached-leaf assay, where tested isolates were classified as sensitive or resistant based on the relative disease severity. Based on the Kruskal-Wallis test, the distribution profile of relative disease severity among baseline and exposed isolates was significantly different where chlorothalonil was alternated with fluopicolide (χ2 = 10.82; P = 0.001) but not with cymoxanil (χ2 = 1.39; P = 0.238) or propamocarb (χ2 = 2.37; P = 0.412). Although there was a directional selection toward resistance for isolates sampled from plots that were treated with fluopicolide or propamocarb alternated with chlorothalonil during a growing season, a significant shift in fungicide sensitivity distribution based on combined data were observed for fluopicolide (χ2 = 8.25; P = 0.004) but not propamocarb (χ2 = 1.05; P = 0.461). Baseline and exposed isolates sampled from the cymoxanil-treated plots were all resistant to this fungicide and there was no significant shift in their fungicide sensitivity profile during a growing season (χ2 = 0.06; P = 1.000). These results indicate that a shift toward reduced sensitivity in P. cubensis can occur during a growing season and the efficacy of fluopicolide is likely to decrease as the frequency of the less sensitive subpopulations increases during a production season. The resultant effect on disease severity and selection of an insensitive subpopulation may accelerate the development of resistance to propamocarb in the southeastern United States.

Identification and Fungicide Sensitivity of Colletotrichum spp. from Apple Flowers and Fruitlets in Brazil.

Glomerella leaf spot (GLS) and bitter rot (BR), caused by Colletotrichum spp., are major diseases on apple in southern Brazil. Among integrated pest management tools for disease management in commercial orchards, fungicides remain an important component. This study aimed to identify Colletotrichum spp. from cultivar Eva in Paraná state orchards; evaluate their in vitro sensitivity to cyprodinil, tebuconazole, iprodione, and fluazinam; and determine the baseline in vitro sensitivity of these isolates to benzovindiflupyr and natamycin. Most isolates belonged to Colletotrichum melonis and C. nymphaeae of the C. acutatum species complex. The two species varied in sensitivity to fluazinam and tebuconazole, but no variability was found for any other fungicide. The lowest 50% effective concentration (EC50) values of Colletotrichum spp. were observed for cyprodinil (mean EC50 < 0.02) and benzovindiflupyr (mean EC50 < 0.05); EC50 values were intermediate for fluazinam (mean EC50 < 0.33) and tebuconazole (mean EC50 < 0.14), and they were highest for natamycin (mean EC50 < 5.56) and iprodione (mean EC50 > 12). Cyprodinil and fluazinam are registered for use in Brazil for apple disease management but not specifically for GLS and BR. Tebuconazole is one of the few products registered for Colletotrichum spp. control in apples. In conclusion, flowers and fruitlets can serve as sources of inoculum for GLS and BR disease; C. acutatum was the predominant species complex in these tissues; cyprodinil and fluazinam applications may suppress GLS and BR; and benzovindiflupyr and natamycin warrant further investigation for GLS and BR disease control of apple due to comparably high in vitro sensitivity.

Determining the Distribution of QoI Fungicide-Resistant Cercospora sojina on Soybean from Indiana.

Frogeye leaf spot (FLS) is a foliar disease of soybean (Glycine max) caused by Cercospora sojina. Application of fungicide products that contain quinone outside inhibitor (QoI) active ingredients has been one of the major tools used in the management of this disease, but, since 2010, QoI-resistant C. sojina isolates have been confirmed in over 20 states in the United States, including Indiana. In summer 2019 and 2020, 406 isolates of C. sojina were collected from 32 counties across Indiana and screened for QoI resistance using a PCR-restriction fragment length polymorphism (RFLP) method. An in vitro fungicide sensitivity test was also performed on a subset of isolates to evaluate their sensitivity to three QoI fungicides: azoxystrobin, pyraclostrobin, and picoxystrobin. A discriminatory dose of picoxystrobin was established as 1 µg/ml by testing five concentrations (0.001, 0.01, 0.1, 1, and 10 µg/ml). QoI-resistant isolates were found in 29 counties, and 251 of the 406 isolates (61.8%) were confirmed to be resistant to QoI fungicides based on PCR-RFLP results. Partial nucleotide sequences of the cytochrome b gene from four resistant and four sensitive isolates corroborated the presence and absence, respectively, of the G143A mutation. Results from the sensitivity assays with discriminatory doses of azoxystrobin (1 µg/ml) and pyraclostrobin (0.1 µg/ml) also supported the findings from the PCR-RFLP assay, because all QoI-resistant isolates were inhibited less than 50% relative to a no-fungicide control when exposed to these doses. Resistant isolates harboring the G143A mutation also exhibited resistance to picoxystrobin. The effective concentrations to inhibit mycelial growth by 50% relative to the nonamended control (EC50) in QoI-sensitive isolates ranged from 0.087 to 0.243 µg/ml, with an overall mean of 0.152 µg/ml, while EC50 values in QoI-resistant isolates were established as >10 µg/ml for picoxystrobin. Results from this study indicated that QoI-resistant C. sojina isolates are spread throughout Indiana and exhibit cross-resistance to QoI fungicides.

A multivariate analysis of phenotypic traits of strains of Fusarium graminearum and F. meridionale supports structure by species

AbstractFusarium head blight (FHB) of wheat and Gibberella ear and stalk rot (GER and GSR) of maize are diseases of worldwide importance. Several phylogenetic species belonging to the Fusarium graminearum species complex (FGSC) cause FHB and GER/GSR in Brazil, but the dominant species varies depending on host. Fusarium graminearum is the most frequent cause of FHB, while F. meridionale is more often associated with GER/GSR. These two species are distinguished based on fixed sequence variations at a few genetic loci; however, relevance of these differences to reproduction, pathogenicity and toxigenicity has not been fully explored. A range of phenotypic traits were evaluated for a group of strains of F. graminearum and F. meridionale from wheat and maize. These included radial growth on agar plates, sexual and asexual reproduction, fungicide sensitivity, toxigenicity and pathogenicity on wheat and maize. Although intraspecies variation was observed for most traits, a multivariate analysis showed the strains were strongly structured by species, regardless of host origin. Increased sexual or asexual reproduction, deoxynivalenol production and aggressiveness on wheat heads were most important for distinguishing strains of F. graminearum, while faster mycelial growth in culture, nivalenol production and reduced colonization on detached maize silks were most important for distinguishing strains of F. meridionale. Thus, the phylogenetic distinction between F. graminearum and F. meridionale is supported by phenotypic differences that are relevant for pathogenicity (aggressiveness and mycotoxin production on wheat heads and maize silks, mycelial growth and sexual and asexual reproduction) and disease management (tebuconazole sensitivity).

Clinical Aureobasidium Isolates Are More Fungicide Sensitive than Many Agricultural Isolates.

Fungicide applications in agriculture and medicine can promote the evolution of resistant, pathogenic fungi, which is a growing problem for disease management in both settings. Nonpathogenic mycobiota are also exposed to fungicides, may become tolerant, and could turn into agricultural or medical problems, for example, due to climate change or in immunocompromised individuals. However, quantitative data about fungicide sensitivity of environmental fungi is mostly lacking. Aureobasidium species are widely distributed and frequently isolated yeast-like fungi. One species, A. pullulans, is used as a biocontrol agent, but is also encountered in clinical samples, regularly. Here, we compared 16 clinical and 30 agricultural Aureobasidium isolates based on whole-genome data and by sensitivity testing with the 3 fungicides captan, cyprodinil, and difenoconazole. Our phylogenetic analyses determined that 7 of the 16 clinical isolates did not belong to the species A. pullulans. These isolates clustered with other Aureobasidium species, including A. melanogenum, a recently separated species that expresses virulence traits that are mostly lacking in A. pullulans. Interestingly, the clinical Aureobasidium isolates were significantly more fungicide sensitive than many isolates from agricultural samples, which implies selection for fungicide tolerance of non-target fungi in agricultural ecosystems. IMPORTANCE Environmental microbiota are regularly found in clinical samples and can cause disease, in particular, in immunocompromised individuals. Organisms of the genus Aureobasidium belonging to this group are highly abundant, and some species are even described as pathogens. Many A. pullulans isolates from agricultural samples are tolerant to different fungicides, and it seems inevitable that such strains will eventually appear in the clinics. Selection for fungicide tolerance would be particularly worrisome for species A. melanogenum, which is also found in the environment and exhibits virulence traits. Based on our observation and the strains tested here, clinical Aureobasidium isolates are still fungicide sensitive. We, therefore, suggest monitoring fungicide sensitivity in species, such as A. pullulans and A. melanogenum, and to consider the development of fungicide tolerance in the evaluation process of fungicides.

Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms

This study evaluates the capacity of commercial formulations of synthetic fungicides to inhibit grapevine bacterial growth when sprayed on vineyards to control diseases, such as downy mildew, powdery mildew and secondary rots. Fungicide sensitivity plate assays were carried out on bacteria isolated from vineyards that were also identified and characterized for their plant growth-promoting (PGP) traits and antifungal activity. The high taxonomic variability of bacteria screened with different chemical classes of fungicides is one new finding of this study. Seven out of 11 fungicides were able to inhibit the growth of bacteria at a concentration corresponding to the maximum dose allowed by law in spray treatments of vineyards. Bacterial sensitivity to each fungicide varied greatly. Many sensitive isolates displayed PGP traits and/or antagonistic activity. This study shows the potential impact of fungicidal treatments on grapevine bacterial microbiota. The involvement of bacteria beneficial to the growth and health of plants underlines the importance of this investigation. Our data reveal that the control of a certain disease may be possible using fungicides that have no or low impact on natural non-target microbiota. Understanding the action mechanisms of the active ingredients in these products is a priority for the development of new eco-friendly pesticides.

Diversity in virulence and metalaxyl-m sensitivity of Phytophthora rubi isolates has implications for raspberry root rot and wilting complex management

Raspberry root rot and wilting complex (RRWC), primarily caused by Phytophthora rubi, is the most important disease for raspberry producers in Canada. There is currently little to no information on phenotypic diversity or sensitivity to metalaxyl-m of P. rubi isolates affecting raspberries in Canada. This study was conducted to understand the diversity in virulence and sensitivity to metalaxyl-m of P. rubi populations collected from diverse raspberry cultivars, fields, and locations during 2018–2020. All isolates of P. rubi (n = 24) were pathogenic on ‘Chemainus’ raspberry and showed significant variation (P < 0.05) in days to first foliar symptom development (DFSD) and progress in wilting index, as indicated by relative areas under disease progress curves (rAUDPC). Principal component analyses identified that root rot index, rAUDPC, DFSD, and percentage reduction in root length and weight were important variables in assessing pathogen virulence. Metalaxyl-m sensitivity was assessed using a radial growth assay and effective concentrations that inhibited 50% of mycelial growth (EC50) were determined for 44 isolates. The EC50 values ranged from 0.035–4.71 µg mL−1 with a mean value of 0.793 µg mL−1. About 61% of the isolates were very sensitive (EC50 values <0.20 µg mL−1), 25% of isolates were considered sensitive (EC50 values of 0.21–1.18 µg mL−1), and 14% of isolates showed reduced sensitivity to metalaxyl-m (EC50 value range of 1.19–4.71 µg mL−1). Knowledge of pathogen phenotypic diversity will be useful for raspberry breeders in screening genotypes against RRWC and the results of the fungicide sensitivity study will be useful in developing management strategies for RRWC.