Diversity in species composition and fungicide resistance profiles in Colletotrichum isolates from apples.

Outbreaks of bitter rot were observed in three commercial apple orchards in Illinois despite best management efforts during the 2018 production season. Three isolates from symptomatic fruit from these orchards and two isolates from an orchard in South Carolina were identified to the species level using morphological tools and calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences. The isolates from Illinois were identified as Colletotrichum siamense of the Colletotrichum gloeosporioides species complex and the ones from South Carolina as Colletotrichum fioriniae and Colletotrichum fructicola of the Colletotrichum acutatum and the C. gloeosporioides species complex, respectively. Two of the three C. siamense isolates from Illinois were resistant to azoxystrobin and thiophanate-methyl as determined in mycelial growth tests in vitro. EC50 values were >100 µg/ml for both fungicides. One isolate was only resistant to azoxystrobin. None of the isolates from South Carolina was resistant to either of the two compounds. All five isolates were sensitive to fludioxonil (EC50 values <0.1 µg/ml), propiconazole (EC50 values ranged from 0.15 to 0.36 µg/ml), and benzovindiflupyr (EC50 values ranged from <0.1 to 0.33 µg/ml). Resistance in C. siamense to azoxystrobin and thiophanate-methyl was confirmed in detached fruit studies using apples treated with label rates of registered product. Resistance to thiophanate-methyl in C. siamense was based on E198A mutation in b-tubulin gene, whereas resistance to azoxystrobin was based on G143A in cytochrome b (CYTB). One isolate resistant to azoxystrobin possessed no amino acid variation in CYTB. This study shows that quinone outside inhibitor fungicide resistance in Colletotrichum from apple has emerged and is being selected for in Illinois apple orchards by current spray strategies. Resistance monitoring may alert growers to potential threats, but the employment of molecular tools based on current knowledge of resistance mechanisms will provide incomplete results.

Infrequent Occurrence of Peach Skin Streaking and the Role of Rainwater Attributes on Symptom Development.

Streaks lacking pigmentation have impacted red blush cultivars of peaches in many East Coast production areas, but the underlying cause is still unclear. Some evidence suggests that streaking may be caused by reactive agents in rainwater. Peach skin streaking was monitored over two consecutive years at a commercial farm with a history of streaking problems located near Ridge Spring, SC. Six cultivars (two early season, two midseason, and two late season) were evaluated, each in two locations (LocA and LocB). Among those 12 experimental block cultivars, streaking occurred only in 2017 in cv. Scarletprince of LocA with an incidence of 6%. That same year two nearby nonexperimental blocks with 'Scarletprince' revealed 11 and 25% streaking. Streaking was also monitored at the Musser Fruit Research Center (MFRC) in Seneca, SC. At that location, a high incidence of streaking was observed, with 50 and 64% in 'Julyprince' (2017) and 'Carored' (2018), respectively. Rainwater pH taken from each of the 12 experimental blocks ranged from 3.03 to 7.4, ozone (O3) levels ranged from <0.02 to 0.37 mg/liter, and chlorine (Cl2) and chlorine dioxide (ClO2) levels were either just above or under the detection limit of 0.01 mg/liter and 0.02 mg/liter, respectively. Although the electrical conductivity (EC) was below 100 µS/cm on average, we did measure EC values as high as 1,500 µS/cm. For all samples, the oxidation-reduction potential (ORP) ranged from 90 to 302 mV, indicating oxidizing conditions. Fruit harvested 1 or 2 weeks prior to commercial maturity and treated with solutions of high (10) or low (3) pH, ozone >0.37 mg/liter, and EC values of up to 3,000 µS/cm did not produce symptoms. However, streaking was reproduced with collected rainwater, but the remaining sample volume did not allow further analyses. Using 0.05% ClO2 to induce streaking, we show that fruit of different cultivars varied in susceptibility when treated 1 week prior to commercial maturity, with 'Juneflame' being the most susceptible and 'August Lady' being the least susceptible. Our study shows that multiple factors determine the occurrence of streaking in peach orchards, including cultivar susceptibility, ripening stage, and the presence of rainwater with sufficient amounts of a yet unknown reactive agent or agent combination.

Characterization of Botrytis cinerea From Commercial Cut Flower Roses.

Botrytis cinerea Pers. infects cut flower roses (Rosa × hybrida L.) during greenhouse production and gray mold symptoms are often expressed in the postharvest environment, resulting in significant economic losses. Disease management is based on cultural practices and preventative chemical treatments; however, gray mold outbreaks continue to occur. Rose tissues from six commercial shipments from two greenhouses in Colombia were evaluated to determine the Botrytis species composition as well as identify other pathogens present, gray mold incidence and severity, and fungicide resistance profiles. Botrytis isolates (49 total) were grouped into six morphological phenotypes, and all were identified to be B. cinerea sensu stricto. Disease incidence was higher in the petals than in the stem, stamen, ovary, sepal, or leaf tissues. Other fungi were isolated infrequently and included Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Penicillium citrinum, Aspergillus brasiliensis, and Diplodia sp. Fungicide resistance profiles were determined using previously established discriminatory doses. Isolates resistant to thiophanate-methyl, iprodione, boscalid, and cyprodinil were found frequently in all shipments and in both greenhouses. The frequency of resistance to penthiopyrad, fenhexamid, fluopyram, isofetamid, and fludioxonil varied between shipments and greenhouses. No resistance to pydiflumetofen was observed at the discriminatory doses tested. Isolates with resistance to multiple chemical classes were commonly found. These results indicate that fungicide resistance management practices may improve preharvest and postharvest gray mold control of cut flower roses.

Fitness and Competitive Ability of Botrytis cinerea Isolates with Resistance to Multiple Chemical Classes of Fungicides.

Resistance to multiple chemical classes of fungicides in Botrytis cinerea isolates from eastern United States strawberry fields is common and strategies to control them are needed. In this study, we compared fitness and competitive ability of eight sensitive isolates (S), eight isolates resistant to five or six chemical classes of fungicides but not to phenylpyrroles (5CCR), and eight isolates resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The latter included the MDR1h phenotype due to overexpression of atrB based on Δ497V/L in mrr1. The 6CCR/MDR1h isolates grew more slowly at 4°C on potato dextrose agar, and both 5CCR and 6CCR/MDR1h isolates were hypersensitive to osmotic stress compared with S isolates. In contrast, no differences were found in oxidative sensitivity, aggressiveness, and spore production in vivo, and sclerotia production and viability in vitro. In competition experiments, the 5CCR and 6CCR/MDR1h isolates were both outcompeted by S isolates and 6CCR/MDR1h isolates were outcompeted by 5CCR isolates in the absence of fungicide pressure. Under selective pressure of a fludioxonil/pyraclostrobin rotation, the 6CCR/MDR1h isolates dominated after coinoculation with 5CCR and S isolates. The competitive disadvantage of 5CCR and especially 6CCR/MDR1h isolates suggest that, in the absence of fungicide selection pressure, S isolates may reduce inoculum potential of multifungicide-resistant isolates under field conditions.

Sensitivity of Colletotrichum Species, Including C. fioriniae and C. nymphaeae, from Peach to Demethylation Inhibitor Fungicides.

Few fungicides are effective against anthracnose, caused by Colletotrichum spp., and emerging resistance makes the search for chemical alternatives more relevant. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and ß-tubulin gene sequences separated the isolates into C. nymphaeae and C. fioriniae. The sensitivity of these and three other previously reported Colletotrichum spp. from peach, including C. fructicola, C. siamense, and C. truncatum, to demethylation inhibitor (DMI) fungicides difenoconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole was determined based upon mycelial growth inhibition. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole and C. nymphaeae was resistant to flutriafol and fenbuconazole based on 50% effective concentration (EC50) values >100 µg/ml. C. fructicola and C. siamense were sensitive to all DMI fungicides (EC50 values of 0.2 to 13.1 µg/ml). C. fioriniae subgroup 2 isolates were less sensitive to DMI fungicides (EC50 values of 0.5 to 16.2 µg/ml) compared with C. fioriniae subgroup 1 (EC50 values of 0.03 to 2.1 µg/ml). Difenoconazole and propiconazole provided the best control efficacy in vitro to all five species, with EC50 values of 0.2 to 2.7 µg/ml. Tebuconazole and metconazole were effective against all Colletotrichum spp., except for C. truncatum. The strong in vitro activity of some DMI fungicides against Colletotrichum spp. may be exploited for improved anthracnose disease management of peach.

Resistance in Alternaria alternata to SDHI Fungicides Causes Rare Disease Outbreak in Peach Orchards.

Alternaria rot, caused by Alternaria alternata, was observed in commercial peach orchards in South Carolina. Single-spore isolates formed two phenotypically different culture morphologies on two artificial media. Isolates highly resistant (HR) to boscalid were also HR to penthiopyrad. Among isolates HR and medium resistant (MR) to boscalid and penthiopyrad, we detected isolates that were MR to fluopyram and HR to fluxapyroxad. Sequence analysis of succinate dehydrogenase (sdh) genes sdhB, sdhC, and sdhD revealed that resistant phenotypes were associated with point mutations leading to amino acid substitutions. In particular, H277Y/R in the SDHB and H134R in SDHC were consistently associated with the boscalid HR phenotype. The highest effective concentration that inhibits growth by 50% (EC50) values to penthiopyrad were conferred by H134R and D123E, whereas H134R conferred low resistance and MR to fluxapyroxad. A previously undescribed mutation, G79R, was identified in our collection conferring HR to both boscalid and penthiopyrad. The point mutations associated with highest EC50 values to all four FRAC 7 fungicides were H277L and H134R. The outbreak of Alternaria rot demonstrates that fungicide programs containing "medium to high-risk fungicides" may promote disease outbreaks by secondary pathogens that typically are outcompeted or controlled effectively, while still controlling the primary target disease.

Fungicide Sensitivity of Sclerotinia sclerotiorum: A Thorough Assessment Using Discriminatory Dose, EC50, High-Resolution Melting analysis, and Description of New Point Mutation Associated with Thiophanate-Methyl Resistance.

Thiophanate-methyl (TM), fluazinam, and procymidone are fungicides extensively used for white mold control of common bean in Brazil. We assessed the sensitivity of Brazilian isolates of Sclerotinia sclerotiorum to these three fungicides using discriminatory doses and concentration that results in 50% mycelial growth inhibition (EC50) values. In total, 282 isolates from the most important production areas were screened and none was resistant to fluazinam or procymidone. The EC50 values varied from 0.003 to 0.007 and from 0.11 to 0.72 µg/ml for fluazinam and procymidone, respectively. One isolate was resistant to TM. The EC50 of the TM-resistant isolate was greater than 100 µg/ml, whereas the EC50 of the sensitive isolates varied from 0.38 to 2.23 µg/ml. The TM-resistant isolate had a L240F mutation in the ß-tubulin gene. This is the first report of mutation at codon 240 causing resistance to a benzimidazole fungicide in S. sclerotiorum. The high-resolution melting analysis allowed the distinction of TM-sensitive and -resistant isolates by specific melting peaks and curves. The TM-resistant isolate had mycelial growth, sclerotia production, and aggressiveness comparable with that of the sensitive isolates, indicating that this genotype will likely compete well against sensitive isolates in the field. This study demonstrates that resistance to TM, fluazinam, and procymidone is nonexistent or rare. Resistance management practices should be implemented, however, to delay the spread of TM-resistant genotypes.

First Report of Anthracnose on Peach Fruit Caused by Colletotrichum truncatum in South Carolina.

In July 2013, two diseased peach fruit (Prunus persica (L.) Stokes) of the cv. Sweet Dream were collected from a commercial orchard in Ridge Springs, South Carolina. Affected peaches were at or near maturity and symptoms resembled anthracnose disease caused by Colletotrichum spp. with circular sunken tan to brown lesions that were firm in touch, and had wrinkled concentric rings. The center of the lesion was covered with black acervuli containing setae. To isolate the causal agent, the two symptomatic fruit were surface-sterilized in 10% bleach for 2 min and rinsed with sterile distilled water. Lesions were cut in half, and necrotic tissue from the inside of the fruit was placed on acidified potato dextrose agar (APDA). Flat colonies covered with olive-gray to iron-gray acervuli developed on APDA incubated at 22°C with a 12-h cycle of fluorescent light and darkness. Morphology of acervuli, setae (avg. 90 to 160 µm), conidiophores (up to 90 um long), and conidia (avg. 22 × 3.8 µm) of single spore isolates were consistent with descriptions of Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore (3), a causal agent of anthracnose disease. Genomic DNA was extracted from isolate Ct_RR13_1 using the MasterPure Yeast DNA Purification Kit (Epicentre, Madison, WI). The ribosomal ITS1-5.8S-ITS2 region and a partial sequence of the actin gene were amplified with primer pair ITS1 and ITS4 (4), and primer pair ACT-512F and ACT-783A (2), respectively. A multilocus sequence identification in Q-bank Fungi revealed a 100% similarity with C. truncatum (1). The C. truncatum sequences from the peach isolate were submitted to GenBank (accessions KF906258 and KF906259). Pathogenicity of isolate Ct_RR13_1 was confirmed by inoculating five mature but still firm peach fruits with a conidial suspension of C. truncatum. Peaches were washed with soap and water, surface-disinfected for 2 min with 10% bleach, rinsed with sterile distilled water, and air dried. Dried fruit were stabbed at three equidistant points, each about 2 cm apart, to a depth of 9.5 mm using a sterile 26G3/8 beveled needle (Becton Dickinson & Co., Rutherford, NJ). For inoculation, a 30-µl droplet of conidia suspension prepared in distilled, sterile water (1 to 2 × 104 spores/ml) was placed on each wound; control fruit received sterile water without conidia. Fruit were incubated at 22°C for 2 days at 100% humidity and another 12 days at 70% humidity. Inoculated fruit developed anthracnose symptoms with sporulating areas as described above and the fungus was re-isolated. All control fruit remained healthy. C. truncatum has a wide host range, including legumes and solanaceous plants of the tropics, and is especially common in the Fabaceae family. Its occurrence in a commercial peach orchard is worrisome because control measures may need to be developed that are different from those developed for endemic species, i.e. C. acutatum and C. gloeoporioides, due to differences in disease cycle or fungicide sensitivity. To our knowledge, this is the first report of C. truncatum causing anthracnose on a member of the genus Prunus. References: (1) P. Bonants et al. EPPO Bull. 43:211, 2013. (2) I. Carbone et al. Mycologia 91:553, 1999. (3) U. Damm et al. Fungal Divers. 39:45, 2009. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Application. Academic Press, NY, 1993.

First Report of Pilidium concavum Causing Tan-brown Rot on Strawberry Nursery Stock in South Carolina.

Pilidium concavum (Desm.) Höhn. [synanamorph: Hainesia lythri (Desm.) Höhn.] is an opportunistic pathogen that causes leaf spots and stem necrosis in a wide range of hosts, including strawberry (Fragaria ananassa) (1,2). In October 2013, 24 strawberry plug plants (cv. Chandler) with brown to dark brown necrotic lesions on stolons were obtained from a nursery in Easley, SC. The lesions were oval shaped and varied in length from 2 to 8 mm. The tips of stolons with larger spots had died. To isolate the causal agent, 3 to 5 cm of necrotic stolon tissue was surface disinfected for 1 min with 10% bleach, rinsed with sterile distilled water, air dried, and placed on potato dextrose agar (PDA). After 7 days of incubation at 22°C, pink-orange masses of spores emerged. Single spore colonies on PDA produced a gray to brown colony with whitish aerial mycelium. Numerous discoid to hemisphaerical conidiomata (0.3 to 2.2 mm in diameter) developed with a dark base and exuded a pink, slimy mass that contained many conidia. Conidiophores (10.2 to 47.8 × 0.8 to 2.0 µm) were hyaline, unicellular, cylindrical, and filiform. Conidia (3.0 to 8.5 × 1.0 to 2.9 µm) were aseptate, fusiform, hyaline, and canoe-shaped to allantoid. On the basis of morphology, the pathogen was identified as P. concavum (3). The internal transcribed spacer region ITS1-5.8S-ITS2 was amplified by PCR and sequenced with primers ITS1 and ITS4 (4). The sequence was submitted to GenBank (Accession No. KF911079) and showed 100% homology with sequences of P. concavum. Pathogenicity was examined on strawberry fruit and leaves. Our previous efforts to achieve infection without wounding failed, which is consistent with experiences of other scientists (not cited). Thus, 24 strawberry fruit were wounded (1 cm deep) with a needle once, and submerged for 3 min in a conidial suspension (2 × 106 conidia ml-1). Controls were wounded and submerged in sterile water. After 4 days of incubation at 22°C, characteristic symptoms were observed at the wound site only on inoculated fruit. Detached leaves (about 6 cm in diameter) from 3- to 4-week-old strawberry plants cv. Chandler were surface sterilized and placed right side up in petri dishes (one leaf per dish) containing water agar. Leaves were inoculated at one site with a 50 µl conidial suspension (2 × 106 conidia ml-1) after inflicting a scraping-type injury with a needle to the surface at the point of inoculation. Control leaves received just water. After 7 days of incubation at 22°C, only the inoculated leaves showed symptoms similar to those observed on strawberry stolons. The fungus was re-isolated from symptomatic fruit and leaf lesions and identity was confirmed based on morphological features. The experiments were repeated. To our knowledge, this is the first report of P. concavum causing tan-brown rot on strawberry tissue in South Carolina. Prior to this study, the pathogen has been described from different hosts and countries including Belgium, Brazil, China, France, Iran, Poland, and the United States. Contamination of strawberry nursery stock by P. concavum could become a plant health management issue in the United States, especially if the pathogen is transferred to strawberry production areas. Further information on in-field occurrence of P. concacum is needed. References: (1) J. Debode et al. Plant Dis. 95:1029, 2011. (2) W. L. Gen et al. Plant Dis. 96:1377, 2012. (3) A. Y. Rossman et al. Mycol. Prog. 3:275, 2004. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Sensitivity of Monilinia fructicola from Peach Farms in China to Four Fungicides and Characterization of Isolates Resistant to Carbendazim and Azoxystrobin.

Brown rot of peach caused by Monilinia fructicola can cause considerable preharvest and postharvest losses in China. Fungicides are increasingly utilized to minimize such losses. Eighty isolates of M. fructicola were collected from commercial peach orchards located in five provinces in China and the sensitivity to carbendazim, azoxystrobin, tebuconazole, and boscalid was determined. Resistance to carbendazim was detected only in the Yunnan province in 15 of 16 isolates. Characterization of carbendazim-resistant isolates revealed stable resistance, no fitness penalty, and negative cross resistance to diethofencarb. Resistant isolates produced disease symptoms on detached fruit sprayed with label rates of formulated carbendazim and possessed the amino acid mutation E198A in ß-tubulin. Resistance to azoxystrobin was detected in 3 of 10 isolates from Fujian. In contrast to carbendazim resistance, however, azoxystrobin resistance was unstable, associated with a fitness penalty, and not associated with mutations in the target gene cytochrome b. The concentration at which mycelial growth is inhibited 50% (EC50) values of the azoxystrobin-sensitive isolates were 0.02 to 1.94 µg/ml, with a mean value of 0.54 µg/ml. All isolates were sensitive to tebuconazole, with a mean EC50 value of 0.03 µg/ml. The EC50 values for boscalid were 0.01 to 3.85 µg/ml, with a mean value of 1.02 µg/ml. Our results indicate that methyl benzimidazole carbamates (MBCs), quionon outside inhibitors, demethylation inhibitor fungicides, and succinate dehydrogenase inhibitors are likely to be very effective in controlling brown rot in many peach production areas in China, but that resistance to MBCs is emerging.

First Report of Fludioxonil Resistance in Botrytis cinerea, the Causal Agent of Gray Mold, from Strawberry Fields in Maryland and South Carolina.

Botrytis cinerea Pers. is the causal agent of gray mold and one of the most economically important plant-pathogenic fungi affecting strawberry (Fragaria × ananassa). Control of gray mold mainly depends on the use of site-specific fungicides, including the phenylpyrrole fludioxonil. This fungicide is currently registered in combination with cyprodinil in form of Switch 62.5WG (Syngenta Crop Protection, Greensboro, NC) for gray mold control of small fruits in the United States. In June 2013, strawberries affected with symptoms resembling gray mold were observed despite the application of Switch in one field located in Federalsburg, MD, and one located near Chesnee, SC. Ten single-spore isolates, each from a different fruit, were obtained from each location and confirmed to be B. cinerea using cultural and molecular tools as described previously (3). In vitro sensitivity to fludioxonil (Scholar SC, 20.4% [v/v] active ingredient, Syngenta Crop Protection, Greensboro, NC) was determined using a conidial germination assay as previously described (4). Eight of the 20 isolates (six from Maryland and two from South Carolina) were moderately resistant to fludioxonil, i.e., they grew on medium amended with 0.1 µg/ml fludixonil and showed residual growth at 10 µg/ml (4). The in vitro assay was repeated obtaining the same results. To assess in vivo sensitivity on fungicide-treated fruit, commercially grown strawberries were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of conidia suspension (106 spores/ml) of either two sensitive or four resistant isolates (two isolates from Maryland and two isolates from South Carolina). Each isolate/treatment combination consisted of 24 mature but still firm strawberry fruit with three 8-fruit replicates. The fruit were kept at 22°C and lesion diameters were measured after 4 days of inoculation. The sensitive isolates developed gray mold symptoms on nontreated (2.5 cm lesion diameter) but not on Scholar SC-treated fruit. The resistant isolates developed gray mold on both, the water-treated control (2.3 cm lesion diameter), and the fungicide-treated fruit (1.8 cm lesion diameter). The experiment was performed twice. To our knowledge this is the first report of fludioxonil resistance in B. cinerea from strawberry fields in Maryland and South Carolina. Resistance to fludioxonil is still rare in the United States and has only been reported in B. cinerea isolates from a Virginia strawberry field (1). The increase in occurrence of resistance to fludioxonil may be a result of increased use of Switch following reports of resistance to other chemical classes in this pathogen in southern strawberry fields (2). References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 96:1198, 2012. (3) D. Fernández-Ortuño et al. Plant Dis. 95:1482, 2011. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

First Report of Fludioxonil Resistance in Botrytis cinerea from a Blackberry Field in Georgia.

Botrytis cinerea Pers. is an important plant-pathogenic fungi responsible for gray mold on more than 230 plant species worldwide, including blackberry (Rubus). One of the main strategies to control the disease involves the application of different classes of fungicides. The phenylpyrrole fludioxonil is currently marketed in combination with the anilinopyrimidine cyprodinil as Switch 62.5WG (Syngenta Crop Protection Inc., Greensboro, NC) for gray mold control. In August 2013, blackberries affected with symptoms resembling gray mold were collected from a field located in Berrien County (Georgia), where Switch 62.5WG had been used extensively over the last 5 years. Three single-spore isolates, each from a different fruit, were obtained and identified as B. cinerea on the basis of morphology and confirmed by a 238-bp PCR amplification product obtained with primer set G3PDH-F1 (5'-GGACCCGAGCTAATTTATGTCACGT-3'), G3PDH-F2 (5'-GGGTGTCAACAACGAGACCTACACT-3'), and G3PDH-R (5'-ACCGGTGCTCGATGGGATGAT-3'). In vitro sensitivity to fludioxonil (Scholar SC, Syngenta) was determined on 1% malt extract agar (MEA) using a conidial germination assay as previously described (4). One isolate was moderately resistant due to growth on medium amended with the discriminatory dose of 0.1 µg/ml fludioxonil and residual growth at 10 µg/ml (4). To assess performance of fludioxonil in detached fruit assays, commercially grown strawberries (24 in total for each isolate and treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water (control fruit) or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Scholar SC was used because fludioxonil was the sole active ingredient in this product and strawberries were used because latent infections in fresh blackberry fruit interfered with inoculation experiments. This dose reflects the rate recommended for postharvest gray mold control according to the Scholar label. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of conidia suspension (106 spores/ml) of the two sensitive or the resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolates developed gray mold on non-treated (2.7 cm lesion diameter) but not on Scholar SC-treated fruit (0.0 cm lesion diameter). The resistant isolate developed gray mold disease on the water-treated control fruit (2.5 cm lesion diameter) and the fungicide-treated fruit (1.8 cm lesion diameter). EC50 values were determined in microtiter assays as described previously (3) using the concentrations of 0.01, 0.04, 0.12, 0.37, 1.1, 3.3, and 10 µg/ml fludioxonil. Values were 0.02 and 0.05 µg/ml for the two sensitive isolates and 3.15 µg/ml for the resistant isolate. All experiments were performed twice. This is the first report of fludioxonil resistance in B. cinerea from blackberry in Georgia. Prior to this study, resistance to fludioxonil in B. cinerea was reported in France, Germany, and only a few states in the United States including Maryland, South Carolina, Virginia, and Washington (1,2). The emergence of resistance to fludioxonil emphasizes the importance of resistance management strategies. References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 98:692, 2013. (3) M. Kretschmer et al. PLOS Pathog. 5:e1000696, 2009. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

First Report of Brown Rot Caused by Monilinia fructicola in Sweet Cherry in Maryland.

Monilinia fructicola (G. Wint.) Honey is the most important causal agent of brown rot of stone fruits in North America. In July 2010, 20 sweet cherry fruit (Prunus avium) of unknown variety with symptoms resembling brown rot were collected from one commercial orchard in Maryland. Each cherry fruit came from a different tree. Symptoms included necrotic areas up to 10 mm in diameter with brown conidia and conidiophores developing from the infection center. Spores from nine symptomatic fruit collected each from different trees of a single orchard were suspended in sterile water, spread onto the surface of 1% agar plates, and incubated at 22°C. After 12 h, single, germinated spores were transferred onto 9-cm petri dishes with potato dextrose agar (PDA). Nine fungal colonies, each from a different fruit, were investigated in three replicates for cultural characteristics on separate petri dishes containing PDA. They were very similar in morphology and grew 12.4 mm per day on average at 22°C, forming branched, monilioid chains of grayish colonies with concentric rings and little sporulation. Rich sporulation was observed on tomato sauce medium (250 ml tomato sauce and 20 g agar in 750 ml water). The lemon-shaped spores had an average size of 15 × 10 µm, which is consistent with M. fructicola. Two colonies were randomly selected to identify the pathogen to the species level using a PCR technique based on cytochrome b sequence amplifications (2). Resulting gel electrophoresis patterns were consistent with M. fructicola. Koch's postulates were fulfilled by inoculating 15 mature sweet cherry fruits of cv. Bing with a conidial suspension (105 spores/ml) of one of the single-spore isolates from cherry. Fruit were stab-inoculated at a point to a depth of 2 mm using a sterile needle. A 10-µl droplet was placed on each wound; control fruit received sterile water without conidia. After 3 days of incubation at room temperature in airtight plastic bags, the inoculated fruit developed typical brown rot symptoms with lesions that were 20.6 mm in diameter. The developing spores on inoculated fruit were confirmed to be M. fructicola. All control fruit remained healthy. The entire detached fruit experiment was repeated 1 week later. M. fructicola is assumed to be the main causal agent of brown rot of sweet cherry in the northeastern United States, but recent studies show that M. laxa is also causing the disease on sweet cherry in many northeastern states (1). For this reason, it is important to delineate species for accurate disease assessments. This study confirms assumptions that M. fructicola is a causal agent of sweet cherry in Maryland. References: (1) K. D. Cox et al. Plant Dis. 12:1584. 2011. (2) J.-M. Hily et al. Pest Manag. Sci. 67:385, 2011.

Field Strains of Monilinia fructicola Resistant to Both MBC and DMI Fungicides Isolated from Stone Fruit Orchards in the Eastern United States.

In 2012, significant brown rot disease was observed on stone fruit in Pennsylvania, Maryland, and South Carolina despite preharvest application of methyl benzimidazole carbamate (MBC) and demethylase inhibitor (DMI) fungicides. In total, 140 Monilinia fructicola isolates were collected from diseased orchards and examined for fungicide sensitivity. In addition to isolates resistant to either the DMI propiconazole or the MBC thiophanate-methyl, 22 isolates were discovered that were resistant to both fungicides, including 4 isolates from peach in South Carolina, 12 isolates from peach and sweet cherry in Maryland, and 6 isolates from sweet cherry in Pennsylvania. Analysis of MBC resistance revealed that dual-resistant isolates from South Carolina carried the ß-tubulin E198A mutation, whereas isolates from Maryland and Pennsylvania carried E198 mutations not previously described in the Monilinia genus, E198Q or F200Y. The genetic element Mona, associated with DMI fungicide resistance in M. fructicola, was detected in the dual-resistant isolates from South Carolina but not in the isolates from the two more northern states. An investigation into the molecular mechanism of DMI resistance in the latter isolates revealed that resistance was not based on increased expression or mutation of MfCYP51, which encodes the target of DMI fungicides. Label rates of formulated propiconazole or thiophanate-methyl were unable to control dual-resistant isolates on detached peach fruit, confirming field relevance of dual resistance. The same isolates were not affected by fitness penalties based on mycelial growth rate, ability to sporulate, and virulence on detached peach fruit. The emergence of M. fructicola strains resistant to both DMI and MBC fungicides in multiple states and multiple stone fruit crops is a significant development and needs to be considered when designing resistance management strategies in stone fruit orchards.

Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid.

In 2011 and 2012, significant brown rot disease caused by Monilinia fructicola was observed in a peach orchard in Spartanburg County, SC, despite preharvest fungicide applications of demethylation inhibitor (DMI), quinone outside inhibitor (QoI), and succinate dehydrogenase inhibitor (SDHI) fungicides. All 22 isolates obtained in 2011 from this orchard were sensitive to the QoI fungicide, azoxystrobin, and the methyl benzimidazole carbamate (MBC) fungicide, thiophanate-methyl. Five were resistant to the DMI fungicide, propiconazole, and were selected, together with five propiconazole-sensitive isolates, for further investigations. One of the 10 isolates was resistant to propiconazole but sensitive to the SDHI fungicide, boscalid (EC50 = 0.42 µg/ml), 3 were resistant to propiconazole with intermediate sensitivity to boscalid (EC50 0.72 to 2.1 µg/ml); 2 were sensitive to propiconazole with intermediate sensitivity to boscalid; 3 were sensitive to propiconazole but resistant to boscalid (EC50 ≥ 2.1 µg/ml); and 1 (isolate MD22) was resistant to both propiconazole and boscalid. Disease incidence on detached fruit treated with formulated propiconazole or boscalid was significantly higher for MD22 compared to a sensitive control isolate. Continued monitoring of fungicide resistance in the same orchard in 2012 revealed an increase of isolates resistant to propiconazole from 22.7% in 2011 to 34.7%, and an increase of isolates resistant to both propiconazole and boscalid from 4.5% in 2011 to 18.4%. Propiconazole resistance was always associated with the presence of the 'Mona' mobile element located upstream of the sterol 14α-demethylase (MfCYP51) gene. To investigate whether mutations in the subunits of the succinate dehydrogenase enzyme were involved in boscalid resistance, significant portions of the M. fructicola SdhA, SdhB, SdhC, and SdhD genes were cloned and analyzed for 2 sensitive, 2 boscalid-resistant, and 6 dual-resistant isolates. Although sequence variation was found among the isolates, no single change correlated with resistance. Interestingly, analysis of isolates collected from orchards in 2001 and 2002, prior to the registration of boscalid, revealed a range of sensitivities to boscalid (EC50 0.03 to 3.46 µg/ml) including boscalid-resistant isolates. The presence of boscalid-resistant isolates in the baseline population was unexpected and requires further investigation.

First Report of Fludioxonil Resistance in Botrytis cinerea from a Strawberry Field in Virginia.

Gray mold caused by Botrytis cinerea Pers.:Fr. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. Control of gray mold mainly depends on fungicides, including the phenylpyrrole fludioxonil, which is currently marketed in combination with cyprodinil as Switch 62.5WG (Syngenta Crop Protection, Research Triangle Park, Raleigh, NC). In 2012, 790 strains of B. cinerea were collected from 76 strawberry fields in eight states, including Arkansas, Florida, Georgia, Kansas, Maryland, North Carolina, South Carolina, and Virginia. Strains were collected from sporulating flowers and fruit and sensitivity to fludioxonil was determined using a conidial germination assay as previously described (2). Only one isolate from a farm located in Westmoreland County, Virginia, grew on medium amended with the discriminatory dose of 0.1 µg/ml fludioxonil and was therefore considered low resistant. The isolate did not grow on 10 µg/ml. All other 789 isolates did not grow at either of the two doses. This assay was repeated twice with a single-spore culture of the same strain. In both cases, residual growth was observed on the fludioxonil-amended medium of 0.1 µg/ml. The single spore isolate was confirmed to be B. cinerea Pers. using cultural and molecular tools as described previously (1). To assess resistance in vivo, commercially grown ripe strawberry fruit were rinsed with sterile water, dried, placed into plastic boxes (eight strawberries per box for each of the three replicates per treatment), and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of fludioxonil (Scholar SC, Syngenta) to runoff using a hand mister. This dose reflects the rate recommended for gray mold control according to the Scholar label. Each fruit was stabbed at three equidistant points, each about 1 cm apart and 1 cm deep using a syringe tip. Wounds were injected with a 30-µl droplet of conidia suspension (106 spores/ml) of either 5 sensitive or the resistant isolate. Control fruit were inoculated with water. After inoculation, the fruit were kept at 22°C for 4 days. In two independent experiments, sensitive and low resistant isolates were indistinguishable in pathogenicity on detached, unsprayed fruit. The low resistant isolate developed gray mold disease on all treated and untreated fruit (100% disease incidence) as determined by the absence or presence of gray mold symptoms. The sensitive isolates only developed disease on untreated fruit. The EC50 values, determined in microtiter assays with concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µg/ml fludioxonil, were 0.01 µg/ml for the sensitive isolates and 0.26 µg/ml for the resistant isolate. To our knowledge, this is the first report of fludioxonil resistance in B. cinerea from strawberry in North America. Our monitoring results indicate that resistance is emerging 10 years after the introduction of fludioxonil and stress the importance of chemical rotation for gray mold control. References: (1) X. P. Li et al. Plant Dis. 96:1634, 2012. (2) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

First Report of Thiophanate-Methyl Resistance in Botrytis cinerea on Strawberry from South Carolina.

Botrytis cinerea Pers.:Fr. is the causal agent of gray mold disease and one of the most important plant-pathogenic fungi affecting strawberry (Fragaria× ananassa). Control of gray mold mainly depends on fungicides, including the methyl benzimidazole carbamate (MBC) thiophanate-methyl. In 2011, strawberries with gray mold symptoms were collected from commercial fields near Chesnee, Florence, Lexington, McBee, Monetta, and North Augusta, all in South Carolina. MBC fungicides were used in most of these fields for gray mold control during the last 3 years. A total of 124 single spore B. cinerea isolates were obtained, each from a different fruit. Resistance to thiophanate-methyl (Topsin M 70WP, Cerexagri-Nisso LLC, King of Prussia, PA) was determined using a conidial germination assay as described previously (1). The majority of isolates (81.4%) were resistant; the rest were sensitive. Resistant isolates were found in all locations with some populations (Chesnee, McBee, and Lexington) revealing no sensitive isolates. Genomic DNA from 35 resistant isolates (representing all locations) and 10 sensitive isolates (from Chesnee, Monetta, and North Augusta, SC) was extracted, and the molecular basis of MBC fungicide resistance was determined as described previously (2). All MBC-resistant isolates possessed the E198A mutation known to confer high levels of MBC fungicide resistance in many fungi, including B. cinerea (2,3). Disease was assessed using a detached strawberry fruit assay. Commercially grown strawberry fruit (24 in total for each isolate and fungicide treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.4 g/liter of Topsin M to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of a conidial suspension (106 spores/ml) of either a sensitive or resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolate developed gray mold disease in untreated but not Topsin M-treated fruit. The resistant isolate developed gray mold disease of equal severity in both, the control and fungicide-treated fruit. This experiment was repeated once. The results of the study show that resistance to MBC fungicides is common and widespread in B. cinerea from strawberry in South Carolina. Prior to this study, resistance to MBCs has only been reported in B. cinerea from ornamental crops grown in greenhouses in South Carolina (4). References: (1) J. E. Luck and M. R. Gillings. Mycol. Res. 99:1483, 1995. (2) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011. (3) O. Yarden and T. Katan. Phytopathology 83:1478, 1993. (4) L. F. Yourman and S. Jeffers. Plant Dis. 83:569, 1999.

First Report of Fusarium Wilt of Strawberry Caused by Fusarium oxysporum in South Carolina.

During October 2011, wilted and dead strawberry (Fragaria × ananassa cv. Albion) plants from two commercial fields in South Carolina were sent to the Clemson University Plant Problem Clinic in Pendleton, SC. Symptoms consisted of wilting and chlorosis of foliage, scorch and dieback of older leaves, and stunting of plants. Internal vascular and cortical tissues of plant crowns showed a distinct reddish brown discoloration. To isolate the causal agent, necrotic crown tissue selected from two symptomatic plants from one location and four symptomatic plants from the other were placed on acidified potato dextrose agar (APDA) and on quarter strength acidified PDA (QPDA). Colonies with light purple mycelia and beige or orange reverse colony colors developed on APDA after 5 days of incubation at 25°C. Colonies on QPDA were light purple. Morphology, growth, and development of macroconidia and microconida were consistent with descriptions of Fusarium oxysporum Schlechtend emend. Snyder & Hansen (3). Genomic DNA from 3 isolates (11-1246A, 11-1247A, and 11-1247B) was extracted and purified according to Chi et al. (1). The internal transcribed spacer region comprising ITS1, ITS2, and 5.8S rRNA was amplified by primers ITS1 and ITS4 (4). The sequence comparison revealed a 100% match with F. oxysporum sequences in GenBank. To confirm the pathogenicity of the fungus, roots of 15 strawberry plants (cv. Albion) were cut and then five plants were soaked for 10 min in either 500 ml of conidial suspension (104 conidia/ml) of one of the two isolates or in sterile distilled water. All were then potted in 15-cm pots with artificial peat-based soil mix and maintained at 25°C in the greenhouse. After 6 weeks, all plants inoculated with isolates 1247A and B were stunted and developed wilt symptoms similar to those observed in the field, while the control plants remained healthy. Support roots on all affected plants were soft and flaccid and new feeder roots had brown lesions. Crowns of three plants inoculated with isolate 1247A and four plants inoculated with 1247B showed vascular discoloration. To reisolate, crowns were plated as above and roots were surface sterilized in 10% bleach for 1 min and rinsed in sterile distilled water prior to plating on QPDA. F. oxysporum was isolated at frequencies of 70 and 100% from crowns and 100% from roots of all inoculated plants. To our knowledge, this is the first report of the occurrence of Fusarium wilt caused by F. oxysporum on strawberry plants in South Carolina. The presence of Fusarium wilt in South Carolina should alert growers, county agents, and specialists to properly identify Fusarium wilt symptoms, which may be confused with Anthracnose or Phytophthora crown rot of strawberry. The disease has been reported previously in other countries including the United States (2). References: (1) M. H. Chi et al. Plant Pathol. J. 25:108, 2009. (2) S. T. Koike et al. Plant Dis. 93:1077, 2009. (3) W. C. Snyder and H. N. Hansen. Am. J. Bot. 27:64, 1940. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Application. Academic Press, NY, 1993.

First Report of Gray Mold of Strawberry Caused by Botrytis caroliniana in North Carolina.

Gray mold caused by Botrytis spp. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. In June 2011, strawberry fruit that was symptomatic of gray mold disease was collected from High Point county in North Carolina. Fruit had brown lesions that enlarged quickly and were covered with green-gray masses of spores followed by a soft rot. To isolate the causal agent, conidia were scraped off the fruit, suspended in 1% Tween 20, spread on water agar amended with 0.1% lactic acid, and emerging colonies were then transferred onto potato dextrose agar (PDA) medium. All but one single-spore colony (designated HP33) were at first colorless and later became gray to brown when the conidiphores and conidia developed on PDA. Isolate HP33 was white to pale gray with short, tufted aerial mycelium, black sclerotia in concentric rings, and did not produce conidia on PDA. Conidia were subhyaline to light brown, smooth, ellipsoid, ovoid or obovoid, and were on average 11.7 × 8.6 µm. The conidiophores were erect, septate, and brown to subhyaline from the base to apex, with swollen basal cell and multiple inflated conidiogenous cells. These morphological features were consistent with Botrytis caroliniana X. P. Li & G. Schnabel sp. nov., a new species isolated recently from blackberry fruit in South Carolina (2). All other single-spore isolates were confirmed to be B. cinerea as described previously (1). To confirm the identity of isolate HP33 to the species level, the necrosis and ethylene-inducing protein 1 (NEP1) was PCR amplified and sequenced by primer pairs NEP1for/NEP1rev as described previously (3). The nucleotide sequence matched the ones published for B. caroliniana (GenBank Accession Nos. JF811593, JF811594, and JF811595). Pathogenicity tests were conducted by inoculating 10 surface-sterilized strawberry fruit with single agar plugs (6 mm in diameter) containing actively growing mycelium; 10 control fruit received agar plugs without mycelium. The inoculated fruit were incubated for 3 days at room temperature in airtight plastic bags and after that developed typical gray mold symptoms. Koch's postulates were fulfilled by the reisolation of B. caroliniana from symptomatic fruit. Control fruit remained healthy. To our knowledge, this is the first report of B. caroliniana on strawberry. It is uncertain whether the new species requires management strategies different from those that control gray mold caused by B. cinerea. References: (1) D. Fernández-Ortuño et al. Plant Dis. 95:1482, 2011. (2) X. P. Li et al. Mycologia 2012, doi:10.3852/11-218. (3) M. Staats et al. Fungal Genet. Biol. 44:52, 2007.

Aboveground Root Collar Excavation of Peach Trees for Armillaria Root Rot Management.

Root collar excavation (RCE) has been applied to established citrus trees and grapevines for Armillaria root rot (ARR) control but, despite its demonstrated effectiveness, this cultural management system is not routinely used for ARR protection in disease-infested replant sites. One major drawback is the difficulty of excavating the belowground root collar, the potential of excavated roots to be covered again with surrounding soil, and the associated labor cost. In this study, a new cultural method was investigated that resulted in trees with aboveground excavated root collars, potentially eliminating many of the drawbacks. Experimental peach trees were planted in two commercial orchards (designated Landrum and Monetta) in South Carolina; each tree replaced one that had declined from ARR disease the year before. Trees were planted approximately 40 cm higher than normal in open-bottom Smart Pots and root collars were excavated above ground level 8 months later. Five years after planting, 30 and 70% of all control trees (planted according to grower standard) had declined from ARR disease in Landrum and Monetta, respectively, whereas only 0 and 10%, respectively, of trees in the aboveground root collar excavation (AGRCE) treatment had declined. The difference in disease pressure between the two locations could not be attributed to differences in nematode pressure. Nonexcavated trees in Smart Pots revealed significantly less tree decline (P ≤ 0.05) compared with the control but tree decline in both locations was greater compared with the AG-RCE treatment (P ≤ 0.05). Trees in the AG-RCE treatment were as vigorous as the controls but produced more root suckers. In this 'prototype' study, we demonstrate the potential of aboveground root collar excavation for ARR management. Its potential for commercial use is discussed.