Madeline Elizabeth Dowling

Resistance in Colletotrichum siamense From Peach and Blueberry to Thiophanate-Methyl and Azoxystrobin

Anthracnose fruit rot was observed in some late-season peach cultivars in South Carolina in the 2012 and 2013 production seasons as well as increased anthracnose leaf spot of blueberry in a commercial operation of the same state in 2012. Single-spore isolates of Colletotrichum siamense were either sensitive or resistant to both thiophanate-methyl and azoxystrobin with the concentration of the fungicide at which fungal development is inhibited by 50% of ≥100 μg/ml. Resistant isolates revealed the E198A mutation in β-tubulin and the G143A mutation in cytochrome b. Nucleotide sequence analysis of the complete CYTB gene from genomic DNA of C. siamense isolates revealed an intronless genotype (CsI) and a genotype revealing two introns (CsII) at amino acid positions 131 and 164. Resistance to thiophanate-methyl or azoxystrobin was not found in isolates of C. fructicola collected from peach fruit. The CYTB gene of isolates of this species was of the CfII genotype or revealed a unique CfIIa genotype. Phylogenetic analysis of C. siamense isolates from different locations and different crops showed that the resistant isolates were genetically closer to each other than to sensitive isolates, suggesting that field resistance to thiophanate-methyl and azoxystrobin fungicides is derived from a common ancestor.

Botrytis fragariae, a New Species Causing Gray Mold on Strawberries, Shows High Frequencies of Specific and Efflux-Based Fungicide Resistance

ABSTRACT Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey of German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed that these strains belong to an undescribed species in Botrytis clade 2, named Botrytisfragariae sp. nov. Isolates of B. fragariae were detected in strawberry fields throughout Germany, sometimes at frequencies similar to those of B. cinerea, and in the southeastern United States. B. fragariae was isolated from overwintering strawberry tissue but not from freshly infected fruit. B. fragariae invaded strawberry tissues with an efficiency similar to or lower than that of B. cinerea but showed poor colonization of inoculated nonhost plant tissues. These data and the exclusive occurrence of this fungus on strawberry plants indicate that B. fragariae is host specific and has a tissue preference different from that of B. cinerea. Various fungicide resistance patterns were observed in B. fragariae populations. Many B. fragariae strains showed resistance to one or several chemical classes of fungicides and an efflux-based multidrug resistance (MDR1) phenotype previously described in B. cinerea. Resistance-related mutations in B. fragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA), azoxystrobin (G143A in cytB), iprodione (G367A+V368F in bos1), and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene and overexpression of the drug efflux transporter gene atrB). The widespread occurrence of B. fragariae indicates that this species is adapted to fungicide-treated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinerea. IMPORTANCE Gray mold is the most important fruit rot on strawberries worldwide and requires fungicide treatments for control. For a long time, it was believed to be caused only by Botrytis cinerea, a ubiquitous pathogen with a broad host range that quickly develops fungicide resistance. We report the discovery and description of a new species, named Botrytisfragariae, that is widely distributed in commercial strawberry fields in Germany and the southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host specific on the basis of its occurrence and artificial infection tests. B. fragariae has also developed resistance to several fungicides that is caused by mutations similar to those known in B. cinerea, including an efflux-based multidrug resistance. Our data indicate that B. fragariae could be of practical importance as a strawberry pathogen in some regions where its abundance is similar to that of B. cinerea.

Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.

Characterization of Botrytis cinerea Isolates from Strawberry with Reduced Sensitivity to Polyoxin D Zinc Salt

Polyoxin D is a Fungicide Resistance Action Committee (FRAC) code 19 fungicide that was recently registered for gray mold control of strawberry in the United States. In this study, we determined the sensitivity to polyoxin D zinc salt (hereafter, polyoxin D) of Botrytis cinerea isolates from 41 commercial strawberry farms in South Carolina, North Carolina, Maryland, Virginia, and Ohio and investigated the fitness of sensitive (S) and reduced sensitive (RS) isolates. Relative mycelial growth ranged between 0 and over 100% on malt extract agar amended with a discriminatory dose of polyoxin D at 5 μg/ml. Isolates that grew more than 70% at that dose were designated RS and were found in three of the five states. The 50% effective dose (EC50) values of three S and three RS isolates ranged from 0.59 to 2.27 and 4.6 to 5.8 μg/ml, respectively. The three RS isolates grew faster on detached tomato fruit treated with Ph-D WDG at recommended label dosage than S isolates (P < 0.008). In all, 25 randomly selected RS isolates exhibited reduced sporulation ability (P < 0.0001) and growth rate (P < 0.0001) but increased production of sclerotia (P < 0.0386) compared with 25 S isolates. Of 10 isolates tested per phenotype, the number of RS isolates producing sporulating lesions on apple, tomato, and strawberry was significantly lower compared with S isolates (P < 0.0001 for each fruit type). The results of this study indicate that resistance management is necessary for fungicides containing polyoxin D. To our knowledge, this is the first study demonstrating reduced sensitivity to FRAC 19 fungicides in B. cinerea isolates from the United States.

Genotypic and Phenotypic Variations in Botrytis spp. Isolates from Single Strawberry Flowers

Gray mold, caused by Botrytis spp., is among the most devastating diseases affecting strawberry worldwide. The great diversity present in the pathogen enhances its ability to survive and adapt in the field. In this study, we explored the genotypic and phenotypic diversity present in single strawberry flowers. In total, 192 isolates were collected from 19 flowers and four farms, and 9 to 12 isolates were collected from each flower. Forty-two haplotypes were found using microsatellite fragment analysis. Multiple haplotypes of two different Botrytis spp. (Botrytis cinerea and B. fragariae) were found in 12 flowers. In the remaining seven flowers, the single-spore isolates examined were of identical haplotypes. In three flowers, the two Botrytis spp. were found to coexist. Isolates were either sensitive (zero chemical class resistance) or resistant to one, two, three, four, or five chemical classes of fungicides. Resistance to multiple fungicides was commonly observed in both species but resistance to boscalid and penthiopyrad was only found in B. cinerea isolates. Resistance to cyprodinil was found in B. fragariae for the first time in the United States. Each haplotype was generally linked to a single resistance profile; however, a single resistance profile often was represented by multiple haplotypes. Isolates from the same flower of multiple haplotypes were largely identical in resistance profiles. This study is a first detailed investigation of genotypic diversity combined with phenotypic analysis of Botrytis spp. at the single-tissue level. It demonstrates that high genotypic and phenotypic diversity is present not only within fields but also in individual blossoms as well. This information is important for understanding the epidemiology of Botrytis and also has implications for fungicide resistance management, particularly related to resistance monitoring practices.

Identification and characterization of Botrytis fragariae isolates on strawberry in the United States

Gray mold is a devastating disease on strawberry, and may be caused by several species of Botrytis. The goal of this study was to better understand and characterize the species of Botrytis with reduced sensitivity to the fungicide Polyoxin D, particularly Botrytis fragariae. In total, 78 Botrytis isolates of unknown species that were sensitive (28 isolates; S), moderately sensitive (22 isolates; MS), or reduced sensitive (28 isolates; RS) to Polyoxin-D were collected from commercial strawberry fields of five states in the United States, identified to the species level, and characterized. The majority (75%) of S isolates were Botrytis cinerea and the majority (79%) of RS isolates were the recently described species B. fragariae, indicating an innate ability of B. fragariae to tolerate Polyoxin-D. B. fragariae produced fluffy, white mycelium and was less likely to sporulate on potato dextrose agar than B. cinerea. Isolates from a commercial field recovered from blossoms in early spring were all B. fragariae, those from leaves of the same plants in late spring were a mixture of B. fragariae and B. cinerea, and those from fruit in early summer were all B. cinerea, indicating that B. fragariae may preferentially colonize blossom tissue. A polymerase chain reaction-based assay was developed based on NEP2 sequence variability to distinguish B. fragariae from other Botrytis spp. that have been reported on strawberry, including B. cinerea, B. mali, B. caroliniana, and B. ricini. None of the isolates collected from Canada, California, or North Carolina nurseries were B. fragariae, indicating that the newly described species may not exist or not be widely distributed in planting stock.

Fungicide Resistance in B. fragariae and Species Prevalence in the Mid-Atlantic United States

Botrytis fragariae was recently described causing gray mold of strawberry in Germany and the United States. The goal of the present study was to determine its prevalence, distribution, and sensitivity to fungicides in strawberry fields of five states. In total, 188 Botrytis isolates were obtained from flowers and fruit collected from the states of Maryland (n = 35), Virginia (n = 38), North Carolina (n = 46), South Carolina (n = 41), and Georgia (n = 28). Only 13 of these were fruit samples and came from South Carolina (n = 5) and Georgia (n = 8). B. fragariae made up 35.1% of the entire collection, and composed close to half of the Botrytis population in North Carolina (43.4%), South Carolina (61.0%), and Georgia (42.9%). One isolate of B. mali was also found, and the rest of the isolates were B. cinerea (sensu lato). B. fragariae and B. cinerea were found coexisting in 11 fields, while other field samples consisted of only B. fragariae (n = 3) or only B. cinerea (n = 10) isolates. B. fragariae isolates with resistance to one or more fungicides were found, and resistance profiles differed from those of B. cinerea, in that no resistance to cyprodinil (FRAC 8) or boscalid and other FRAC 7 botryticides was detected. We detected B. fragariae resistance to the active ingredients thiophanate-methyl, iprodione, fludioxonil, and fenhexamid. We also detected B. fragariae isolates with resistance to up to four chemical classes of fungicides, though most isolates were resistant to one or two chemical classes. In conclusion, isolates of the newly detected species B. fragariae were commonly found on strawberry flowers in the Mid-Atlantic United States, and have developed resistance to many of the most commonly used botryticides. Though the relevance of this species to pre- and postharvest fruit infections is unknown, fludioxonil applications may give this species a competitive advantage over B. cinerea. Controlling this fungus with FRAC 7 fungicides may be an effective way of limiting its spread in strawberry fields.

Novel gene‐sequence markers for isolate tracking within Monilinia fructicola lesions

BACKGROUND Monilinia fructicola is a diverse pathogen of pome and stone fruits that causes severe economic losses each year. However, little is known about inoculum flow within or between orchards and pathogen establishment in an orchard, because few methods exist for detecting diversity or tracking isolates over time. SSR loci are an effective option, but may be confounded by a high degree of mutability and potential sensitivity to abiotic stress. RESULTS Through transcriptome analysis, we identified novel markers mrr1, DHFR and MfCYP01 and validated stability of these markers under fungicide stress in natural infection sites. Nucleotide variation within mrr1, DHFR and MfCYP01 sequences differentiated isolates at all spatial scales: within the same infection site, between trees and between two farms. Sequenced regions were also effective for matching isolates collected from blossoms at the beginning of the season to progeny in cankers obtained at the end of the season. CONCLUSIONS Collectively, results show that mrr1, DHFR and MfCYP01 are able to accurately differentiate M. fructicola isolates at the population level, can be used to track isolates over time, and are more stable than SSRs under external stresses. Either by themselves or combined with SSR markers, these gene-encoding regions are a much-needed tool for better understanding M. fructicola population dynamics.