Identification and Fungicide Sensitivity of Fusarium spp. Associated with Root Rot of Scutellaria baicalensis in Shanxi Province, China.

Fusarium root rot is usually classified as an extremely destructive soilborne disease. From 2020 to 2021, Fusarium root rot was observed in production areas and seriously affected the yield and quality of Scutellaria baicalensis in Shanxi Province, China. Based on morphological characteristics and combined analysis of the internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-alpha sequences, 68 Fusarium isolates obtained in this work were identified as F. oxysporum (52.94%), F. acuminatum (20.59%), F. solani (16.17%), F. proliferatum (5.88%), F. incarnatum (2.94%), and F. brachygibbosum (1.47%). In the pathogenicity tests, all Fusarium isolates could infect S. baicalensis roots, presenting different pathogenic ability. Among these isolates, F. oxysporum was found to have the highest virulence on S. baicalensis roots, followed by F. acuminatum, F. solani, F. proliferatum, F. brachygibbosum, and F. incarnatum. According to fungicide sensitivity tests, Fusarium isolates were more sensitive to fludioxonil and difenoconazole, followed by carbendazim, thiophanate-methyl, and hymexazol. In brief, this is the first report of Fusarium species (F. oxysporum, F. acuminatum, F. solani, F. proliferatum, F. incarnatum, and F. brachygibbosum) as causal agents of root rot of S. baicalensis in Shanxi Province, China. The fungicide sensitivity results will be helpful for formulating management strategies of S. baicalensis root rot.

A Survey of Phytophthora spp. in Eastern Indian Nurseries and Their Sensitivity to Six Oomycete-Targeted Commercial Fungicides.

A survey of the flori-horticultural nurseries in eastern India found Phytophthora nicotianae to be the most widespread Phytophthora species associated with different foliar symptoms of nursery plants and identified the presence of P. palmivora in eastern Indian nurseries for the first time. The survey also led to the first worldwide finding of P. nicotianae on Dipteracanthus prostratus (Poir.) Nees; Ocimum tenuiflorum L. (syn. Ocimum sanctum L.); Philodendron xanadu Croat, Mayo & J. Boos; and Pyrostegia venusta (Ker-Gawl.) Miers and P. palmivora on Episcia cupreata (Hook.) Hanst., as well as the first report from India of P. nicotianae on Spathiphyllum wallisii Regel; Anthurium andraeanum Linden ex André; and Adenium obesum (Forsk.) Roem. & Schult. Sensitivity to commercial fungicides Glazer 35WS, Rallis India (metalaxyl, FRAC code 4); Ridomil Gold, Syngenta (mefenoxam + mancozeb); Revus, Syngenta (mandipropamid, FRAC code 40); Aliette Bayer (fosetyl-Al, FRAC code 33); Acrobat, BASF (dimethomorph, FRAC code 40); and Amistar, Syngenta (azoxystrobin, FRAC code 11) was analyzed, showing EC50 values ranging from 0.75 to 16.39 ppm, 0.74 to 1.45 ppm, 2.43 to 17.21 ppm, 63.81 to 327.31 ppm, 8.88 to 174.69 ppm, and 0.1 to 1.13 ppm, respectively, with no cross-resistance of the isolates to the fungicides. The baseline information produced about these Phytophthora spp. from ornamental and horticultural host associations could help prevent the pathogens from becoming primary drivers of new disease outbreaks and their large-scale distribution beyond their natural endemic ranges.

Identification, Pathogenicity, and Fungicide Sensitivity of Colletotrichum Species Associated with Anthracnose on Italian Ryegrass in Southwestern China.

Italian ryegrass (Lolium multiflorum L.) is widely cultivated as an important forage worldwide because of its high nutritional value and good palatability. Anthracnose caused by Colletotrichum species was a common and new emerging disease of Italian ryegrass. In this study, 88 Colletotrichum isolates were collected from diseased leaves of Italian ryegrass planting regions in Sichuan, Chongqing and Guizhou provinces of southwestern China between 2019 and 2022. By pure culture technique, 15 representative single-spore isolates were obtained for further study. Multi-locus phylogenetic analysis coupled with morphological features showed that these isolates were finally identified as six new record species: C. cereale of the C. graminicola species complex, C. fioriniae and C. nymphaeae of the C. acutatum species complex, C. boninense and C. citricola of the C. boninense species complex, and C. nageiae. Pathogenicity tests indicated that all species could induce anthracnose symptoms; of these, C. cereale was more invasive than other species, followed by C. fioriniae, C. nageiae, C. citricola and C. boninense; C. nymphaeae was weakest pathogenic to Italian ryegrass plants (P ≤ 0.05). Fungicide sensitivity assays showed that iprodione, propineb and oxime·tebuconazole had strong inhibitory effect on the mycelial growth of six Colletotrichum species; in addition, azoxystrobin and fludioxonil also significantly inhibited the mycelial growth of C. nymphaeae and C. fioriniae, respectively. These results provide the basis for the diagnosis and detection in the field, pathogen identification and management of anthracnose on Italian ryegrass.

Characterization and Fungicide Sensitivity of Colletotrichum spp. from Capsicum peppers in South Korea.

Capsicum peppers, peppers from plants of the genus Capsicum (family Solanaceae), are widely cultivated in South Korea, where annual production was 92,756 tons in 2021, 54.4% higher than that of the previous year. Occurring throughout the production cycle, anthracnose is a major disease limiting commercial Capsicum pepper production worldwide, including in South Korea. This study investigates the diversity and pathogenicity of Colletotrichum species responsible for Capsicum pepper anthracnose in Gyeongbuk, South Korea, focusing on disease incidence and symptomatology in the field and the identification, morphological characteristics, pathogenicity, and fungicide sensitivity of the causative species. Disease incidence ranged from 30 to 50%, with samples categorized into three distinct symptom types, aiding accurate field diagnosis. Phylogenetic analysis classified 41 isolates into six species in the acutatum, gloeosporioides, and truncatum species complexes, revealing significant genetic diversity. Morphological characterization supported these identifications, providing a comprehensive profiling. Pathogenicity tests confirmed that all identified species induced typical anthracnose lesions, with lesion size variations suggesting differential aggressiveness. Temperature significantly influenced mycelial growth, with optimal growth between 20 and 26°C, and C. truncatum demonstrating high-temperature tolerance.In vitro fungicide sensitivity tests showed variable responses, with tebuconazole being generally effective. These findings underscore the need for species-specific fungicide recommendations and highlight the importance of continuous monitoring of Colletotrichum species. Future research should explore the molecular mechanisms of pathogenicity, host specificity, and fungicide resistance, integrating these findings with breeding programs to develop resistant pepper varieties. This study provides critical insights for effective anthracnose management in pepper cultivation and future research directions.

Host Range Characterization of Phytophthora sansomeana Across Corn, Soybean, Wheat, Winter Cereal Rye, Dry Bean, and Oats and an In Vitro Assessment of Seed Treatment Sensitivity.

Formally described in 2009, Phytophthora sansomeana is a pathogen of increasing interest in native, agricultural, and horticulturally important plant species. The objective of this study was to elucidate the symptomatic and asymptomatic host range of P. sansomeana on six agricultural crop species commonly used in field crop rotations in Michigan. In addition, sensitivity to oomicides commonly used in seed treatments, including oxathiapiprolin, mefenoxam, ethaboxam, and pyraclostrobin, was performed to aid in disease management recommendations. Plant biomass, quantity of P. sansomeana DNA in roots, and reisolations were used to assess pathogenicity and virulence of 18 isolates of P. sansomeana on each plant species using an inoculated seedling growth chamber assay. Isolates displayed varying levels of virulence to the hosts tested. Reisolations were completed for each plant species tested, and varying quantities of P. sansomeana DNA were found within all plant species root samples. Corn, wheat, soybean, dry bean, and winter cereal rye plants were symptomatic hosts with significant reduction observed in the total plant biomass. No significant reduction in total plant biomass was observed in oats, and oat roots harbored the least amount of P. sansomeana DNA. No P. sansomeana isolates were insensitive to the oomicide compounds tested with mean absolute inhibition (EC50) values of fungicide required for 50% growth inhibition values of 7.8 × 10-2 µg/ml for mefenoxam, 1.13 × 10-1 µg/ml for ethaboxam, 2.6 × 10-2 µg/ml for oxathiapiprolin, and 3.04 × 10-1 µg/ml for pyraclostrobin. These results suggest that common crop rotations in Michigan may not be a viable option to reduce soilborne inoculum accumulation and oomicide seed treatments could be considered for early-season management of P. sansomeana.

Multistate Sensitivity Monitoring of Fusarium virguliforme to the SDHI Fungicides Fluopyram and Pydiflumetofen in the United States.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean (Glycine max). From 1996 to 2022, cumulative yield losses attributed to SDS in North America totaled over 25 million metric tons, which was valued at over US $7.8 billion. Seed treatments are widely used to manage SDS by reducing early season soybean root infection by F. virguliforme. Fluopyram (succinate dehydrogenase inhibitor [SDHI] - FRAC 7), a fungicide seed treatment for SDS management, has been registered for use on soybean in the United States since 2014. A baseline sensitivity study conducted in 2014 evaluated 130 F. virguliforme isolates collected from five states to fluopyram in a mycelial growth inhibition assay and reported a mean EC50 of 3.35 mg/liter. This baseline study provided the foundation for the objectives of this research: to detect any statistically significant change in fluopyram sensitivity over time and geographical regions within the United States and to investigate sensitivity to the fungicide pydiflumetofen. We repeated fluopyram sensitivity testing on a panel of 80 historical F. virguliforme isolates collected from 2006 to 2013 (76 of which were used in the baseline study) and conducted testing on 123 contemporary isolates collected from 2016 to 2022 from 11 states. This study estimated a mean absolute EC50 of 3.95 mg/liter in isolates collected from 2006 to 2013 and a mean absolute EC50 of 4.19 mg/liter in those collected in 2016 to 2022. There was no significant change in fluopyram sensitivity (P = 0.1) identified between the historical and contemporary isolates. A subset of 23 isolates, tested against pydiflumetofen under the same conditions, estimated an absolute mean EC50 of 0.11 mg/liter. Moderate correlation was detected between fluopyram and pydiflumetofen sensitivity estimates (R = 0.53; P < 0.001). These findings enable future fluopyram and pydiflumetofen resistance monitoring and inform current soybean SDS management strategies in a regional and national context.

Virulence, structure, and triadimefon sensitivity of the Puccinia striiformis f. sp. tritici population in Shaanxi Province, China.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most destructive fungal disease affecting wheat in China, especially in Shaanxi Province, an important epidemiological region connecting the western Pst over-summer regions and the central and eastern spring epidemic regions in the country. In the present study, 291 Pst isolates from Shaanxi Province were studied for their virulence using two sets of wheat differentials, population structure using single-nucleotide polymorphism (SNP) markers, and sensitivity to fungicide. When the isolates were tested on the Chinese differentials of 19 wheat cultivars, 72 races were identified, which belonged to three groups, including the Guinong 22 group (48.45%), Hybrid 46 group (31.62%), and Suwon 11 group (19.93%). The three most predominant races were CYR34 (15.46%), G22-14 (11.68%), and CYR32 (10.65%). When the isolates were tested on the 18 Yr single-gene differentials, 95 races were identified, but none of the isolates were virulent to either Yr5 or Yr15. Cluster analyses of the virulence data based on the two sets of differentials and the SNP marker data consistently separated the Shaanxi Pst population into two clusters in the central part and southern part of the Province. Triadimefon sensitivity testing across different concentrations showed a broad range of half-maximal effective concentration (EC50) values, from 0.03 to 5.99 µg mL-1, with a mean EC50 of 0.46 µg mL-1. The majority of isolates (90.72%) were sensitive to the fungicide. The correlation analyses of the virulence, SNP marker, and the triadimefon sensitivity data showed no significant correlations, except a logarithmic relationship between the EC50 value and the number of avirulence factors. This study is the first to determine the relationship of virulence and SNP markers with triadimefon sensitivity in a regional Pst population.The findings provide valuable insights for breeding resistant wheat cultivars and integrated management of stripe rust.

Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

Fungicide Sensitivity of Fusarium oxysporum f. sp. lentis and Fusarium acuminatum Affecting Lentil in the Northern Great Plains.

Fusarium oxysporum f. sp. lentis and F. acuminatum cause wilting and root rot in pulse crops including lentil. Fungicide seed treatments are widely used, but information about Fusarium spp. sensitivity in lentils is limited. Here, 30 F. oxysporum f. sp. lentis and 30 F. acuminatum isolates from Montana, southern Canada, North Dakota, and Washington were identified, tested for pathogenicity, and assayed for in vitro sensitivity to pyraclostrobin, prothioconazole, ipconazole, and thiophanate-methyl. F. oxysporum f. sp. lentis and F. acuminatum differed in their sensitivity to all fungicides. No resistant isolates were identified, but F. oxysporum f. sp. lentis had lower EC50 values in pyraclostrobin (averaging 0.47 µg a.i./ml) than F. acuminatum (averaging 0.89 µg a.i./ml) for mycelia assays. Both species had lower EC50 values in prothioconazole, averaging EC50 0.23 in F. oxysporum f. sp. lentis and 0.53 µg a.i./ml in F. acuminatum. F. oxysporum f. sp. lentis isolates had the lowest EC50 values on ipconazole compared to F. acuminatum (0.78 and 1.49 µg a.i./ml). The pathogens were least sensitive to thiophanate-methyl (1.74 µg a.i./ml for F. oxysporum f. sp. lentis and 1.91 µg a.i./ml for F. acuminatum). Overall sensitivity to the fungicides was higher in F. oxysporum f. sp. lentis than F. acuminatum. This study provides reference EC50 values while pointing to the possibility of differential fungicide efficacies on Fusarium spp. This will be helpful to monitor shifts in sensitivity of Fusarium spp. and devise robust root rot/wilt management approaches.

Sensitivity of Puccinia triticina f. sp. tritici from China to Triadimefon and Resistance Risk Assessment.

Wheat leaf rust, caused by Puccinia triticina f. sp. tritici (Pt), is distributed widely in wheat-producing areas and results in serious yield losses worldwide. In China, leaf rust has been largely controlled with a demethylation inhibitor (DMI) fungicide, triadimefon. Although high levels of fungicide resistance in pathogens have been reported, no field failure of wheat leaf rust to DMI fungicides has been reported in China. A resistance risk assessment of triadimefon to Pt was investigated in the present study. The sensitivity of 197 Pt isolates across the country to triadimefon was determined, and the density distribution of EC50 values (concentration at which mycelial growth is inhibited by 50%) showed a continuous multimodal curve because of the extensive use of this fungicide in wheat production, with a mean value of 0.46 µg/ml. The majority of the tested Pt isolates were sensitive to triadimefon, whereas 10.2% developed varying degrees of resistance. Characterization of parasitic fitness revealed that the triadimefon-resistant isolates exhibited strong adaptive traits in urediniospore germination rate, latent period, sporulation intensity, and lesion expansion rate. No correlation was observed between triadimefon and tebuconazole and hexaconazole, which have the similar mode of action, or pyraclostrobin and flubeneteram, which have different modes of action. Overexpression of the target gene Cyp51 led to the triadimefon resistance of Pt. The risk of resistance to triadimefon in Pt may be low to moderate. This study provided important data for fungicide resistance risk management against wheat leaf rust.

Molecular Characterization and Fungicide Sensitivity of Jujube Pathogens Colletotrichum gloeosporioides Sensu Stricto and Colletotrichum nymphaeae in South Korea.

Jujube (Ziziphus jujuba) is cultivated across South Korea because of its medicinal and economic value. It is used as a sweetener in jam, tea, and snacks and a garnish in many cuisines. Anthracnose caused by Colletotrichum spp. accounts for huge economic losses for jujube growers. In 2019 and 2020, severe anthracnose was observed in the jujube-growing areas of South Korea. The infected fruit displayed small, water-soaked, sunken, circular spots. Infected fruit were collected from different commercial orchards of Boeungun and Gyeongsan regions of South Korea, and putative causal agents were isolated on potato dextrose agar. Based on the morphological and molecular characteristics, the fungal isolates were identified as Colletotrichum gloeosporioides sensu stricto and C. nymphaeae. The pathogenicity of these isolates was confirmed by inoculating a conidial suspension (1 × 106 conidia ml-1) on healthy fruit. The in vitro sensitivity of the fungal isolates to tebuconazole, carbendazim, and azoxystrobin was also tested. All isolates showed high sensitivity to azoxystrobin in terms of mycelial growth inhibition (half maximal effective concentration value of 0.01 to 0.6 µg/ml). To the best of our knowledge, this is also the first report of jujube anthracnose caused by C. nymphaeae in South Korea.

Identification and Fungicide Control of Bipolaris sorokiniana Causing Leaf Spot and Blight on Common Hop (Humulus lupulus) in Korea.

Hop (Humulus lupulus) is a perennial herbaceous vine belonging to the family Cannabaceae. This crop is commercially grown for the brewing industry for its bitter and aromatic flavor, as well as its antiseptic properties. In June 2021, leaf spot and blight was observed on common hop plants in Buan-gun, Jeollabuk-do, South Korea. The typical symptoms were small to large, dark-brown, necrotic lesions with yellow halos on the leaves. This study aimed to clarify the causal agent of this disease. Two fungal species, Alternaria alternata and Bipolaris sorokiniana, were isolated from the diseased leaf samples and identified by combining morphological observations and phylogenetic analysis using sequence datasets of internal transcribed spacer (ITS), Alt a1, rpb2, endoPG, and OPA10-2; and ITS, gpd, and tef1, respectively. Pathogenicity of the fungal isolates on detached leaves and living plants revealed that B. sorokiniana is the causal pathogen of this disease, while A. alternata is potentially a saprophyte. Fungicide sensitivity of the pathogen B. sorokiniana was further estimated in vitro using three classes of fungicides represented by fluxapyroxad, pyraclostrobin, and hexaconazole. The effective concentrations that inhibited 50% of spore germination (EC50) were 0.72, 1.90, and 0.68 µg ml-1, respectively. Moreover, all of these fungicides were able to control B. sorokiniana on detached common hop leaves at their recommended concentrations. In conclusion, this study reports leaf spot and blight of common hop caused by B. sorokiniana for the first time and proposes potential fungicides for this disease.

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.

Characterization of Colletotrichum Isolates Causing Colletotrichum Dieback of Citrus in California.

Dieback caused by Colletotrichum spp. is an emerging disease in California citrus groves. A large-scale survey with emphasis on seasonal variations of latent infections was conducted throughout citrus orchards in Fresno, Kern, and Tulare counties in 2019 and 2020. Latent infections on citrus leaves and twigs varied markedly between years. Isolates of Colletotrichum spp. were obtained from asymptomatic tissue, and two groups were formed based on colony and spore morphology. The morphological groups were further identified based on multigene sequence analysis using the DNA regions ITS1-5.8S-ITS2, TUB2, and GAPDH. Results revealed that isolates belong to two phylogenetic species, C. gloeosporioides and C. karstii, being C. karstii more frequently isolated. Representative isolates of each species were further selected and characterized based on the response of physiological variables to temperature. Both species had similar optimum growth temperatures but differed in maximum growth rates, with C. gloeosporioides exhibiting a greater growth rate than that of C. karstii on media. Pathogenicity tests on citrus trees demonstrated the ability of C. gloeosporioides and C. karstii to cause lesions on twigs and no differences in aggressiveness. A fungicide screening performed in this study determined that the DMI fungicides were the most effective in reducing the mycelial growth of C. gloeosporioides and C. karstii. The QoI fungicides showed a remarkably inhibitory impact on spore germination of both species. On average, C. karstii was more sensitive to the DMI fungicides than C. gloeosporioides. The findings of this study provide new information to understand the Colletotrichum dieback of citrus.

Fusarium Head Blight of Small Grains in Pennsylvania: Unravelling Species Diversity, Toxin Types, Growth, and Triazole Sensitivity.

Fusarium graminearum is the main causal species of Fusarium head blight (FHB) globally. Recent changes in the trichothecene (toxin) types in the North American FHB pathogens support the need for continued surveillance. In this study, 461 isolates were obtained from symptomatic spikes of wheat, spelt, barley, and rye crops during 2018 and 2019. These were all identified to species and toxin types using molecular-based approaches. An additional set of 77 F. graminearum isolates obtained from overwintering crop residues during winter 2012 were molecularly identified to toxin types. A subset of 31 F. graminearum isolates (15 15-acetyl-deoxynivalenol [15ADON] and 16 3-acetyl-deoxynivalenol [3ADON]) were assessed for mycelial growth, macroconidia, perithecia, and ascospore production, and sensitivity to two triazoles. Ninety percent of isolates obtained from the symptomatic spikes (n = 418) belonged to F. graminearum, with four other species found at a lower frequency (n = 39). The F. graminearum isolates from symptomatic spikes were mainly of the 15ADON (95%), followed by 3ADON (4%), nivalenol (0.7%), and NX-2 (0.3%) toxin types. All F. graminearum isolates obtained from overwintering residue were of the 15ADON type. The toxin types could not be differentiated based on the multivariate analysis of growth and reproduction traits. All isolates were sensitive to tebuconazole and metconazole fungicides in vitro. This study confirms the dominance of F. graminearum and suggests ecological and environmental factors, to be further identified, that lead to similar composition of toxin types in the northern United States. Our results may be useful to assess the sustainability of FHB management practices and provide a baseline for future FHB surveys.

Off-target selection of resistance to azoxystrobin in Aspergillus species associated with grape late season rots.

Due to recent evidence of Aspergillus uvarum pathogenicity on wine grapes and variable fungicide sensitivity to quinone outside inhibitor (QoI) fungicides, the identity and QoI sensitivity of Aspergillus isolates from the Mid-Atlantic United States was investigated. Phylogenic analysis of 31 isolates revealed 26 as A. uvarum and 5 as A. japonicus, both of which have been previously isolated from grape. The A. uvarum isolates had variable sensitivities to the QoI azoxystrobin, and the genomic region that codes for the target of QoIs, cytochrome b, was sequenced. Translation of the cytochrome b coding sequence revealed that the most resistant isolates (termed cytb3) contained three mutations, S108A, F129L, and A194V, and the moderately sensitive isolates (termed cytb2) contained two mutations S108A and A194V. This is the first report of an amino acid variation in cytochrome b at position 108. Cytb3 isolates were significantly less inhibited than the cytb2 and wild-type isolates (cytbWT) in vitro, and were significantly less inhibited than the cytbWT isolates on detached fruit. Molecular docking analysis revealed similar differences, with azoxystrobin binding most securely in the cytbWT variant of cytochrome b than cytb2 and cytb3. As Aspergillus rot has not been a target disease of fungicide sprays in the U.S., the selection of resistant phenotypes is likely resultant from sprays for other diseases. Resistance is of concern due to the pathogenicity of A. uvarum and A. japonicus on wine grapes, and the ability of these species to be mycotoxigenic or pathogenic for humans.

Sensitivity and Resistance Risk Assessment of Puccinia striiformis f. sp. tritici to Triadimefon in China.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat that seriously threatens production safety in wheat-producing areas worldwide. In China, the disease has been largely controlled with the fungicide triadimefon. Although high levels of fungicide resistance in other fungal pathogens have been reported, failure to control Pst with any fungicides has seldomly been reported, and fungicide sensitivity of Pst has not been evaluated in China. The distribution of triadimefon-resistant Pst isolates was investigated in the present study. The baseline sensitivity of 446 Pst isolates across the country to triadimefon was determined, and the concentration for 50% of maximal effect showed a unimodal distribution curve, with a mean value of 0.19 µg ml-1. The results indicated a wide range of sensitivity to triadimefon, with more insensitive isolates collected from Pst winter-increasing areas and northwest oversummering areas, whereas more sensitive isolates were collected from southwest oversummering areas and epidemic areas of Xinjiang and Tibet. The majority of the tested Pst isolates were sensitive to triadimefon; only 6.79% had developed varying degrees of resistance. Characterization of parasitic fitness revealed that the triadimefon-resistant isolates exhibited strong adaptive traits in the urediniospore germination rate, latent period, sporulation intensity, and lesion expansion rate. Positive cross-resistance was observed between triadimefon and tebuconazole or hexaconazole, but not between pyraclostrobin or flubeneteram. The point mutation Y134F in the 14α-demethylase enzyme (CYP51) was detected in triadimefon-resistant isolates. A molecular method (kompetitive allele-specific PCR) was established for the rapid detection of Y134F mutants in the Pst population. Two genotypes with one point mutation Y134F conferred resistance to triadimefon in Pst. The risk of resistance to triadimefon in Pst may be low to moderate. This study provided important data for establishment of high throughput molecular detection methods, fungicide resistance risk management, and the development of new target fungicides.

Response of the mushroom pathogen Cladobotryum mycophilum to prochloraz and metrafenone fungicides and Streptomyces flavovirens actinobacteria.

After an outbreak of cobweb disease of cultivated button mushroom in Serbia in 2003, the isolated fungal pathogen was initially identified as Cladobotryum dendroides (teleomorph Hypomyces rosellus) based on morpho-physiological traits. Molecular analysis indicated re-classification of two strains (isolated in 2004 and 2007) as Cladobotryum mycophilum (teleomorph Hypomyces odoratus). However, subsequent analysis of further five strains (isolated over the period 2003-2010) within the frames of the present study, also confirmed their identification as the exclusive cobweb causal agent C. mycophilum. After artificial inoculation, the symptoms observed on harvested and growing mushrooms were consistent with the appearance of cobweb disease. Pathogen sensitivity to fungicides was estimated by probit analyses. Fungicide susceptibility tests showed that C. mycophilum strains were highly sensitive both to prochloraz (ED50<0.087 µg mL-1) and the newly introduced metrafenone (ED50<0.15 µg mL-1). Furthermore, the growth of all examined strains of C. mycophilum was significantly inhibited by the indigenous actinobacterial strain Streptomyces flavovirens A06. A dual culture assay showed after 72 h that the percentage of radial growth inhibition of the pathogen ranged from 22.38 to 55.73%. Our findings suggest that the antagonistic S. flavovirens A06 might be a potential candidate for controlling the cobweb disease of cultivated button mushroom.

Correlations among population genetic structure, geographic origin, growth rate, and fungicide resistance of Rhizoctonia solani AG 1-IA, the pathogen of rice sheath blight.

Rhizoctonia solani AG1-IA is a necrotrophic fungus that causes rice sheath blight and results in severe yield and quality reductions in rice worldwide. Differences of genetic structure and fungicide sensitivity of the pathogen have significant effects on the severity and control effect of this disease in the field. To determine correlations among population genetic structure, geographic origin, growth rate, and fungicide resistance of the pathogen, 293 strains of R. solani were isolated from diseased rice collected from 13 cities of Jiangsu Province and five regions of China. Simple sequence repeat (SSR) molecular marker technology was used to analyze the genetic diversity of these strains, and a total of 74 bands were amplified by nine pairs of primers. Population genetic structure analysis showed that strains from Central China and northern Jiangsu had the highest Nei's gene diversity index and Shannon diversity index. The vast majority of strains grew fast with colony diameters of more than 60.0 mm cultured at 28 °C for 36 h. The half-maximal effective concentration (EC50) of them to tebuconazole, thifluzamide, and propiconazole varied ∼16.2-, 3.8-, and 7.5-fold. However, the genetic diversity of R. solani had no significant correlation with their geographic origin, growth rate or fungicide sensitivity.

A Putative MAPK Kinase Kinase Gene Ssos4 is Involved in Mycelial Growth, Virulence, Osmotic Adaptation, and Sensitivity to Fludioxonil and is Essential for SsHog1 Phosphorylation in Sclerotinia sclerotiorum.

The high osmolarity glycerol (HOG) pathway, comprising a two-component system and the Hog1 mitogen-activated protein kinase (MAPK) cascade, plays a pivotal role in eukaryotic organisms. Previous studies suggested that the biological functions of some key genes in the HOG pathway varied in filamentous fungi. In this study, we characterized a putative MAPK kinase kinase gene, Ssos4, in Sclerotinia sclerotiorum, which encoded a phosphotransferase in the MAPK cascade. Compared with the wild-type progenitor HA61, the deletion mutant ∆Ssos4-63 exhibited impaired mycelial growth, sclerotia formation, increased hyphal branches, and decreased virulence. The deficiencies of the deletion mutant ∆Ssos4-63 were recovered when the full-length Ssos4 gene was complemented. Deletion of Ssos4 increased the sensitivity to osmotic stresses and cell wall agents and the resistance to fludioxonil and dimethachlon. Intracellular glycerol accumulation was not induced in the deletion mutant ∆Ssos4-63 when treated with fludioxonil and NaCl and the phosphorylation of SsHog1 was also cancelled by the deletion of Ssos4. Consistent with the glycerol accumulation and increased expression levels of SsglpA and Ssfps1, controlling glycerol synthesis and close of glycerol channel under hyperosmotic stress, respectively, were detected in the wild-type strain HA61 but not in the deletion mutant ∆Ssos4-63. Moreover, the relative expression level of Sshog1 significantly decreased, whereas the expression level of Ssos5 increased in the deletion mutant ∆Ssos4-63. These results indicated that Ssos4 played important roles in mycelial growth and differentiation, sclerotia formation, virulence, hyperosmotic adaptation, fungicide sensitivity, and the phosphorylation of SsHog1 in S. sclerotiorum.