Reevaluation of Sensitivity of Monilinia fructicola Isolates to the DMI Fungicide Propiconazole in the Southeastern United States and Investigation of the Genetic Element Mona.

Sterol demethylation inhibitor (DMI) fungicides continue to be essential components for the control of brown rot of peach caused by Monilinia fructicola in the United States and worldwide. In the southeastern United States, resistance to DMIs had been associated with overexpression of the cytochrome P450 14α-demethylase gene MfCYP51 as well as the genetic element Mona, a 65 bp in length nucleotide sequence located upstream of MfCYP51 in resistant isolates. About 20 years after the first survey, we reevaluated sensitivity of M. fructicola from South Carolina and Georgia to propiconazole and also evaluated isolates from Alabama for the first time. A total of 238 M. fructicola isolates were collected from various commercial and two experimental orchards, and sensitivity to propiconazole was determined based on a discriminatory dose of 0.3 µg/ml. Results indicated 16.2, 89.2, and 72.4% of isolates from Alabama, Georgia, and South Carolina, respectively, were resistant to propiconazole. The detection of resistance in Alabama is the first report for the state. All resistant isolates contained Mona, but it was absent from most sensitive isolates. It was unclear if the resistance frequency had increased in South Carolina and Georgia. However, the resistance levels (as assessed by the isolate frequency in discriminatory dose-based relative growth categories) did not change notably, and no evidence of other resistance genotypes was found. Analysis of the upstream MfCYP51 gene region in the resistant isolate CF010 revealed an insertion sequence described for the first time in this report. Our study suggests that current fungicide spray programs have been effective against increasing resistance levels in populations of M. fructicola and suppressing development of new resistant genotypes of the pathogen.

Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards.

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 µg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 µg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the ß-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.

Identification of Fungicide Combinations for Overcoming Plasmopara viticola and Botrytis cinerea Fungicide Resistance.

Fungal diseases, including downy mildew (caused by Plasmopara viticola) and gray mold (caused by Botrytis cinerea), significantly impact the marketable yield of grapes produced worldwide. Cytochrome b of the mitochondrial respiratory chain of these two fungi is a key target for Quinone outside inhibitor (QoI)-based fungicide development. Since the mode of action (MOA) of QoI fungicides is restricted to a single site, the extensive usage of these fungicides has resulted in fungicide resistance. The use of fungicide combinations with multiple targets is an effective way to counter and slow down the development of fungicide resistance. Due to the high cost of in planta trials, in silico techniques can be used for the rapid screening of potential fungicides. In this study, a combination of in silico simulations that include Schrödinger Glide docking, molecular dynamics, and Molecular Mechanism-Generalized Born Surface Area calculation were used to screen the most potent QoI and non-QoI-based fungicide combinations to wild-type, G143A-mutated, F129L-mutated, and double-mutated versions that had both G143A and F129L mutations of fungal cytochrome b. In silico docking studies indicated that mandestrobin, famoxadone, captan, and thiram have a high affinity toward WT cytochrome b of Botrytis cinerea. Although the QoIs mandestrobin and famoxadone were effective for WT based on in vitro results, they were not broadly effective against G143A-mutated isolates. Famoxadone was only effective against one isolate with G143A-mutated cytochrome b. The non-QoI fungicides thiram and captan were effective against both WT and isolates with G143A-mutated cytochrome b. Follow-up in silico docking and molecular dynamics studies suggested that fungicide combinations consisting of famoxadone, mandestrobin, fenamidone, and thiram should be considered in field testing targeting Plasmopara viticola and Botrytis cinerea fungicide resistance.

Efficacy of calcium propionate against fungicide-resistant fungal plant pathogens and suppression of botrytis blight of ornamental flowers.

Conventional fungicides are used in IPM programs to manage fungal plant pathogens, but there are concerns about resistance development in target organisms, environmental contamination, and human health risks. This study explored the potential of calcium propionate (CaP), a common food preservative generally recognized as safe (GRAS) to control fungicide-resistant plant pathogens, mainly Botrytis cinerea, and botrytis blight in ornamentals. In-vitro experiments using mycelium growth inhibition indicated a mean EC50 value for CaP (pH 6.0) of 527 mg/L for six isolates of Botrytis cinerea as well as 618, 1354, and 1310 mg/L for six isolates each of Monilinia fructicola, Alternaria alternata, and Colletotrichum acutatum. In vitro efficacy tests indicated CaP equally inhibited mycelium growth of fungal isolates sensitive and resistant to FRAC codes 1, 2, 3, 7, 9, 11, 12, and 17 fungicides. CaP at 0.1% (pH 6.0-6.5) reduced infection cushion (IC) formation in vitro, botrytis blight on petunia flowers, and botrytis blight of cut flower roses with little to no visible phytotoxicity. Although higher concentrations strongly inhibited infection cushion formation, they did not improve efficacy and exhibited phytotoxicity. We hypothesize that high concentrations may create tissue damage that facilitates direct fungal penetration without the need for infection cushion and subsequent appressoria formation. This study indicates the potential usefulness of CaP for blossom blight disease management in ornamentals if applied at concentrations low enough to avoid phytotoxicity.

Development of a Molecular Tool for Identification of a New Neopestalotiopsis sp. Associated with Disease Outbreaks on Strawberry.

A new Neopestalotiopsis sp. was recently reported causing outbreaks of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best, and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida; isolates of N. rosae, N. honoluluana, N. ellipsopora, N. saprophytica, N. samarangensis, and P. rhododendri; and isolates from South Carolina suspected to be the new Neopestalotiopsis sp. were included in this study. This method is based on PCR amplification of a ß-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice, yielding fragments of 290 base pairs (bp) and 130 and 20 bp in size, whereas fragments from other species are only cut once, yielding fragments of 420 and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry.

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.

Characterization of High Fludioxonil Resistance in Botrytis cinerea Isolates from Calibrachoa Flowers.

The fungicide fludioxonil is one of the most effective single-site fungicides available for managing flower blight caused by Botrytis cinerea on fruit and ornamental crops. Although low and moderate levels of resistance to fludioxonil have been reported in the pathogen across the United States and Europe, high resistance has been reported only from greenhouses in China. In this study, two B. cinerea isolates with high resistance (half maximal effective concentration >100 µg/ml) to fludioxonil were detected on ornamental calibrachoa flowers grown in a greenhouse. These isolates exhibited stable resistance for >20 generations, produced symptoms on calibrachoa flowers sprayed with label rates of fludioxonil, and displayed in vitro fitness penalties with decreased mycelial growth (P < 0.0001) and sporulation (P < 0.0001) compared with sensitive isolates. Highly resistant isolates were identified as MDR1h, containing the ΔL/V497 deletion in mrr1. However, resistance levels and in vitro fitness parameter characteristics were not consistent with this phenotype. One isolate contained the mutation L267V between HAMP domains 1 and 2 of the Bos-1 gene, and both isolates exhibited high osmotic sensitivity and reduced glycerol accumulation in the presence of fludioxonil, indicating that high resistance of these isolates may be associated with the high-osmolarity glycerol mitogen-activated protein kinase pathway.

Occurrence of Plasmopara destructor causing Downy Mildew on Impatiens walleriana in Brazil.

Impatiens walleriana (Balsaminaceae), popularly known as Impatiens, is an African succulent and a popular ornamental plant worldwide (GBIF, 2019). In Brazil it is broadly grown indoors and outdoors, including in public parks of Curitiba, State of Paraná (Viezzer et al. 2018). In September 2018, I. walleriana plants showing typical downy mildew symptoms were observed in wastelands and gardens in Curitiba. The symptoms included adaxial chlorotic leaf spots with abundant white sporulation on abaxial side (Supplementary figure 1). The disease led to severe defoliation of the plants and the incidence of the plant disease varied from 20 to 80% of plants in an area ranging from 400 to 40,000 m2. A representative sample was deposited in herbarium of the Museu Botânico Municipal de Curitiba (MBM 331601). The following morphology was observed: Sporangiophores (n = 30), hyaline, thin walled, emerging through stomata, 407.3 to 551.1 µm long, slightly swollen base, first branch at 165.8 to 324.7 µm from base, end branches 5.1 to 13.1 µm long, sporangia (n = 50) hyaline, thin-walled subglobose to ovoid, from 12.8 to 21.9 µm x 12.5 to 17.9 µm, slightly papillate. Due to morphological and genetic variations within the species Plasmopara obducens, Görg et al. (2017) proposed the new species P. velutina and P. destructor. The morphology of the Curitiba specimen was equivalent to that described for P. destructor (Görg et al. 2017). DNA was extracted from LEMIDPRTf-19-02 isolate and the ITS1 and cox2 regions were PCR amplified as described in Görg et al. (2017). The resulting sequences were deposited in GenBank (ITS1, MT680628; cox2, MT952335). A BLASTn analysis of the sequences revealed 100% homology with ITS (MF372742) and cox2 (MF372728) sequences of type strain of P. destructor (GLM-F107554). A Bayesian phylogenetic analysis was performed to compare the sequences from this study with reference sequences for P. obducens, P. destructor and P. velutina (Görg et al. 2017; Salgado-Salazar et al. 2018). The oomycete from Curitiba grouped in a reliable clade with P. destructor (Supplementary figure 2). Pathogenicity was carried out by ex vivo and in vivo tests. For ex vivo, stems with approximately four healthy leaves of I. walleriana (n = 10) were embedded in aluminum grid inside of gerbox with the stem bases immersed in distilled water. The inoculation of five stems was carried out by spraying a suspension with 6 x 104 sporangia mL-1 on the abaxial side of the leaves. Five stems with leaves inoculated with sterile water were used as controls. They were incubated in a growth chamber in the dark for 48 h at 20 °C and another 12 days in a 12 h light photoperiod. The confirmation of pathogenicity in plants (in vivo) was obtained with the inoculation of I. walleriana seedlings (one-month old) grown in 2 dm3 aluminum pots. The inoculation methodology and number of plants were the same as the stems test. After the inoculation, plants were incubated in a growth chamber for 48 h in the dark at 20 °C with 100% RH with nebulization, and another 10 days at a photoperiod of 12 hours of light. For both tests, abundant sporulation was observedwith morphology equivalent to Plasmopara destructor described by Görg et al. (2017). No disease developed on control plants. To our knowledge, this is the first report of P. destructor on I. walleriana in Brazil (Farr and Rossman 2019, Silva et al. 2019) representing a potential loss to flower production and a reduction in flowering period in public gardens and parks.