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Bacterial spot is one of the most serious diseases of peach caused by the pathogen Xanthomonas arboricola pv. pruni (XAP), leading to early defoliation and unmarketable fruit. The pathogen can overwinter in peach twigs and form spring cankers, which are considered the primary inoculum source for early season leaf and fruitlet infection. The amount of overwintering bacterial inoculum plays a critical role for the bacterial spot development, but no reliable quantification method is available. Thus, we developed a long-amplicon propidium monoazide (PMA)-quantitative PCR (qPCR) assay for specific detection of viable XAP cells. The optimized PMA-qPCR assay used 20 μM of PMAxx for pure bacterial suspensions and 100 μM for peach twig tissues. The Qiagen Plant Pro Kit with an additional lysozyme digestion step was the DNA extraction protocol that yielded the best detection sensitivity with the bacteria-spiked peach twig extracts. The PMA-qPCR assay was tested with different mixtures of viable and heat-killed XAP cells in pure bacterial suspensions and bacteria-spiked peach twig tissues. The results showed that this assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 10 3 CFU/ml with the presence of up to 10 7 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. For mixtures of viable and dead cells, the PMA-qPCR results were linearly correlated with the predicted concentrations of viable XAP ( R 2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees., Competing Interests: The author(s) declare no conflict of interest.

Biological control agent Bacillus subtilis formulated as Theia is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (emulsifiable concentrate [EC] and wettable powder) and metconazole (EC and water-dispersible granule) at 0, 10, 50, 100, and 150 µg/ml of active ingredient. After cocultivation, the optical density (OD 600 ) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the wettable powder or water-dispersible granule formulations at 10 or 50 µg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia and each formulated DMI at 50 µg/ml of active ingredient resulted in a significant reduction of Monilinia fructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia with EC formulations showed weaker disease reduction due to antagonism. Only Theia plus non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared with the respective solo treatments and anthracnose disease incidence of cherry compared with the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia with a lower-than-label-rate concentration (50 µg/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects, especially when non-EC formulations were used., Competing Interests: The author(s) declare no conflict of interest.

Xylella fastidiosa causes bacterial leaf scorch in southern highbush ( Vaccinium corymbosum interspecific hybrids) and is also associated with a distinct disease phenotype in rabbiteye blueberry ( V. virgatum ) cultivars in the southeastern United States. Both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex have been reported to cause problems in southern highbush blueberry, but so far only X. fastidiosa subsp. multiplex has been reported in rabbiteye cultivars in Louisiana. In this study, we report detection of X. fastidiosa in rabbiteye blueberry plants in association with symptoms of foliar reddening and shoot dieback. High throughput sequencing of an X. fastidiosa -positive plant sample and comparative analyses identified the strain in one of these plants as being X. fastidiosa subsp. fastidiosa . We briefly discuss the implications of these findings, which may spur research into blueberry as a potential inoculum source that could enable spread to other susceptible fruit crops in South Carolina., Competing Interests: The author(s) declare no conflict of interest.

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni , causes yield loss every year in southeastern U.S. peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in X . arboricola pv. pruni in the United States, adaptation of populations from frequent use is a concern. We collected X . arboricola pv. pruni from shoot cankers, leaves, and fruit of cultivar O'Henry over 2 years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. X . arboricola pv. pruni was isolated from four canker types (bud canker, tip canker, nonconcentric canker, and concentric canker) in early spring (bud break), as well as from leaf and fruit tissues later in the season at the phenological stages of pit hardening and final swell. X . arboricola pv. pruni was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. X . arboricola pv. pruni isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 μg/ml or 51 μg/ml of Cu 2+ . Two phenotypes of copper tolerance in X . arboricola pv. pruni were discovered: low copper tolerance (LCT; growth up to 38 μg/ml Cu 2+ ) and high copper tolerance (HCT; growth up to 51 μg/ml Cu 2+ ). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at the stages of delayed dormancy (high rate; 2,397 μg/ml Cu 2+ ), shuck split (medium rate; 599 μg/ml Cu 2+ ), and during summer cover sprays (low rate; 120 μg/ml Cu 2+ ), and subsequently inoculated with sensitive, LCT, and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of X . arboricola pv. pruni tolerance to copper in commercial peach orchards in the southeastern United States, and suggests its contribution to bacterial spot development under current management practices.

Monilinia fructicola is a destructive pathogen causing brown rot on stone fruits worldwide. Though it is best known as a fruit rot pathogen, M. fructicola also causes blossom blight and, subsequently, twig cankers in the spring. Orchard management strategies often overlook cankers as an inoculum source, though they are an inoculum source of both blossom and fruit infections. In this study, we analyzed the role of cankers as storage structures for diverse genotypes of M. fructicola, examining whether multiple genotypes can be transmitted from blossom to canker. Fungal spores from blossoms, and 2 months later from their corresponding cankers, were collected from a conventional and an unsprayed orchard in 2015 and 2016. Simple sequence repeat markers were used to genotype 10 to 20 single spores from each of four blossom/canker pairs per orchard. Individual blossoms and cankers were detected containing up to four and five genotypes, respectively. The average number of genotypes in blossoms and corresponding cankers were not significantly different (P = 0.690) across both years and farms, showing that a bottleneck for genetic diversity was not generated during the transition from blossom to canker. The average number of genotypes unique to blossom or canker was not significantly different (P = 0.569) and no significant effect of farm (P = 0.961) or year (P = 0.520) was observed, although blossoms had a numerically greater number of unique genotypes in both cases. In conclusion, a single blossom may be infected by one or more genotypes of M. fructicola, and this diversity is being preserved in the corresponding canker. This information implicates M. fructicola cankers as diversity storehouses, and may also apply to other Monilinia spp. and fungal diseases that initiate in reproductive tissue.

Anthracnose disease, caused by Colletotrichum truncatum, affects marketable yield during preharvest production and postharvest storage of fruits and vegetables worldwide. Demethylation inhibitor (DMI) fungicides are among the very few chemical classes of single-site mode of action fungicides that are effective in controlling anthracnose disease. However, some species are inherently resistant to DMIs and more information is needed to understand this phenomenon. Isolates of C. truncatum were collected from the United States and China from peach, soybean, citrus, and begonia and sensitivity to six DMIs (difenoconazole, propiconazole, metconazole, tebuconazole, flutriafol, and fenbuconazole) was determined. Compared with DMI sensitive isolates of C. fructicola, C. siamense, and C. fioriniae (EC 50 value ranging from 0.03 to 16.2 µg/ml to six DMIs), C. truncatum and C. nymphaeae were resistant to flutriafol and fenbuconazole (with EC 50 values of more 50 µg/ml). Moreover, C. truncatum was resistant to tebuconazole and metconazole (with resistance factors of 27.4 and 96.0) and displayed reduced sensitivity to difenoconazole and propiconazole (with resistance factors of 5.1 and 5.2). Analysis of the Colletotrichum spp. genome revealed two potential DMI targets, CYP51A and CYP51B, that putatively encode P450 sterol 14α-demethylases. Both genes were identified and sequenced from C. truncatum and other species and no correlation between CYP51 gene expression levels and fungicide sensitivity was found. Four amino acid variations L208Y, H238R, S302A, and I366L in CYP51A, and three variations H373 N, M376L, and S511T in CYP51B correlated with the DMI resistance phenotype. CYP51A structure model analysis suggested the four alterations may reduce azole affinity. Likewise, CYP51B structure analysis suggested the H373 N and M376L variants may change the conformation of the DMI binding pocket, thereby causing differential sensitivity to DMI fungicides in C. truncatum.

Fungi in the genus Monilinia cause brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: M. fructicola, M. fructigena, M. polystroma, and M. laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.

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.

Strawberry production in Florida and South Carolina is affected by two major diseases, anthracnose fruit rot (AFR) and Botrytis fruit rot (BFR), caused by Colletotrichum acutatum and Botrytis cinerea, respectively. The effective management of both diseases traditionally relied on weekly fungicide applications. However, to improve timing and reduce the number of fungicide sprays, many growers follow the Strawberry Advisory System (StAS), a decision support system for forecasting fungicide applications based on environmental conditions and previously developed models. The objective of this study was to perform a meta-analysis to determine the effectiveness of the StAS for AFR and BFR management compared with a calendar-based spray program. Thirty-nine trials were conducted from 2009 to 2014 in Florida and South Carolina commercial strawberry fields. Meta-analysis was conducted to quantify the treatment effects on four effect sizes, all based on the difference in response variables for StAS and the calendar-based treatments in each trial. The mean difference in BFR incidence, AFR incidence, yield, and number of marketable fruit between the two treatments was not significantly different from 0 (P < 0.05). However, the number of fungicide applications per season was reduced by a median of seven when using the StAS, a 50% reduction in sprays compared with the calendar-based approach. Effect sizes were not influenced by location or the favorability of the environment for disease development. These findings indicate that use of StAS in commercial fields is effective in controlling fruit rot diseases with no reduction in yield while substantially reducing fungicide applications.

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.

Previous research has shown that Botrytis cinerea isolates with resistance to multiple chemical classes of fungicides exist in eastern strawberry fields. In this study, the fungicide resistance profiles of 2,130 isolates from flowers of commercial strawberry fields located in multiple states was determined over four consecutive strawberry production seasons. Producers were asked to alternate single-site fungicides that were considered low risk in their specific location based on resistance monitoring results in their fields. This recommendation led to an increase of chemical class diversity used in the spray programs. Results indicated that simultaneous resistance in individual isolates to two, three, four, five, six, and seven classes of fungicides increased over time. The increase in chemical class resistances within isolates was likely due to a process we termed "selection by association", where fungicide resistance traits were often linked to the trait being selected rather than the selectable trait itself. Data analysis also indicated that the odds were highest for isolates resistant to one chemical class (1CCR) to be resistant to thiophanate-methyl; for 2CCR isolates to be resistant to thiophanate-methyl and pyraclostrobin; and for 3CCR isolates to be resistant to thiophanate-methyl, pyraclostrobin, and either cyprodinil or fenhexamid. We hypothesize that the more chemical classes are used in a spray program, the faster isolates will be selected with increasing numbers of chemical class resistances by virtue of selection by association if such isolates preexist in the population.

Rice false smut disease is an increasing threat to rice production in the world. Despite of best efforts, research for the infection of the fungus has yielded equivocal and conflicting results about where and how the infection is initiated and developed. Here we show a stepwise infection pattern and sophisticated regulation during this process. Initial infection occurred on the filaments, which prevented the production of mature pollen thus blocked the pollination. In the following days, the pathogen invaded the stigmas and styles, occasionally the ovaries. Expression analysis indicated that the fungus mimicked a successful fertilization process and enabled the continuous supply of nutrients for fungus to produce false smut balls. The stepwise infection of flower organs and mimicry of ovary fertilization unveiled in this study guided the rice plant into supplying nutrients for false smut ball development and represents a new and unique biological process of host pathogen interactions., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Leaf wetness duration (LWD) plays a key role in disease development and is often used as an input in disease-warning systems. LWD is often estimated using mathematical models, since measurement by sensors is rarely available and/or reliable. A strawberry disease-warning system called "Strawberry Advisory System" (SAS) is used by growers in Florida, USA, in deciding when to spray their strawberry fields to control anthracnose and Botrytis fruit rot. Currently, SAS is implemented at six locations, where reliable LWD sensors are deployed. A robust LWD model would facilitate SAS expansion from Florida to other regions where reliable LW sensors are not available. The objective of this study was to evaluate the use of mathematical models to estimate LWD and time of spray recommendations in comparison to on site LWD measurements. Specific objectives were to (i) compare model estimated and observed LWD and resulting differences in timing and number of fungicide spray recommendations, (ii) evaluate the effects of weather station sensors precision on LWD models performance, and (iii) compare LWD models performance across four states in the USA. The LWD models evaluated were the classification and regression tree (CART), dew point depression (DPD), number of hours with relative humidity equal or greater than 90 % (NHRH ≥90 %), and Penman-Monteith (P-M). P-M model was expected to have the lowest errors, since it is a physically based and thus portable model. Indeed, the P-M model estimated LWD most accurately (MAE <2 h) at a weather station with high precision sensors but was the least accurate when lower precision sensors of relative humidity and estimated net radiation (based on solar radiation and temperature) were used (MAE = 3.7 h). The CART model was the most robust for estimating LWD and for advising growers on fungicide-spray timing for anthracnose and Botrytis fruit rot control and is therefore the model we recommend for expanding the strawberry disease warning beyond Florida, to other locations where weather stations may be deployed with lower precision sensors, and net radiation observations are not available.

Repeated applications of fungicides with a single mode of action are believed to select for pre-existing resistant strains in a pathogen population, while the impact of sub-lethal doses of such fungicides on sensitive members of the population is unknown. In this study, in vitro evidence is presented that continuous exposure of Monilinia fructicola mycelium to some fungicides can induce genetic change in form of transposon transposition. Three fungicide-sensitive M. fructicola isolates were exposed in 12 weekly transfers of mycelia to a dose gradient of demethylation inhibitor fungicide (DMI) SYP-Z048 and quinone outside inhibitor fungicide (QoI) azoxystrobin in solo or mixture treatments. Evidence of mutagenesis was assessed by monitoring Mftc1, a multicopy transposable element of M. fructicola, by PCR and Southern blot analysis. Movement of Mftc1 was observed following azoxystrobin and azoxystrobin plus SYP-Z048 treatments in two of the three isolates, but not in the non-fungicide-treated controls. Interestingly, the upstream promoter region of MfCYP51 was a prime target for Mftc1 transposition in these isolates. Transposition of Mftc1 was verified by Southern blot in two of three isolates from another, similar experiment following prolonged, sublethal azoxystrobin exposure, although in these isolates movement of Mftc1 in the upstream MfCYP51 promoter region was not observed. More research is warranted to determine whether fungicide-induced mutagenesis may also happen under field conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of small fruit crops and control is largely dependent on the application of fungicides. As part of a region-wide resistance-monitoring program that investigated 1,890 B. cinerea isolates from 189 fields in 10 states of the United States, we identified seven isolates (0.4%) from five locations in four different states with unprecedented resistance to all seven Fungicide Resistance Action Committee (FRAC) codes with single-site modes of action including FRAC 1, 2, 7, 9, 11, 12, and 17 registered in the United States for gray mold control. Resistance to thiophanate-methyl, iprodione, boscalid, pyraclostrobin, and fenhexamid was based on target gene mutations that conferred E198A and F200Y in β-tubulin, I365N/S in Bos1, H272R/Y in SdhB, G143A in Cytb, and T63I and F412S in Erg27. Isolates were grouped into MDR1 and MDR1h phenotypes based on sensitivity to fludioxonil and variations in transcription factor mrr1. MDR1h isolates had a previously described 3-bp deletion at position 497 in mrr1. Expression of ABC transporter atrB was increased in MDR1 isolates but highest in MDR1h isolates. None of the isolates with seven single resistances (SR) had identical nucleotide variations in target genes, indicating that they emerged independently. Multifungicide resistance phenotypes did not exhibit significant fitness penalties for the parameters used in this study, but MDR1h isolates produced more sclerotia at low temperatures and exhibited increased sensitivity to salt stress. In this study we show that current resistance management strategies have not been able to prevent the geographically independent development of resistance to all seven site-specific fungicides currently registered for gray mold control in the United States and document the presence of MDR1h in North America.

Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was ≈200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of strawberry. Control of the disease in commercial fields is largely dependent on the application of fungicides, including the dicarboximide iprodione. Single-spore isolates were collected from strawberry fields in Florida, North Carolina, and South Carolina and subjected to an assay using conidial germination that distinguished sensitive (S) isolates from isolates with various levels of resistance to iprodione. Of the 245 isolates, 1 was highly resistant (HR), 5 were moderately resistant (MR), and 43 had low resistance (LR) to iprodione. LR and MR strains were found in the Florida population and in 9 of 11 locations from North Carolina and South Carolina, indicating that resistance was widespread but accounted for only a relatively small percentage of the B. cinerea population. Sequence analysis of the target gene bos1, which codes for a class III histidine kinase, revealed that the MR phenotype was associated with Q369P and N373S mutations and that the LR phenotype was associated with either a I365S or a I365N mutation. The I365S and I365N mutations were also present in five additionally included HR isolates from North Carolina and South Carolina blackberry fields and one HR isolate from a Virginia strawberry field but no mutation or mutation combinations in bos1 were uniquely associated with the HR phenotype. Expression analysis of bos1 in S and HR isolates did not reveal convincing evidence of the gene's involvement in HR resistance either. The six HR isolates had three different phenotypes with respect to their sensitivity to fludioxonil; two were S, two were LR, and two were MR. The fludioxonil LR and MR isolates were also resistant to tolnaftate, an indication of multidrug efflux pump activity. These data suggest that, in addition to point mutations in bos1, drug efflux pump activity and potentially a third mechanism of resistance may be contributing to the iprodione HR phenotype. Detached fruit studies showed that field rates of Rovral 4 Flowable (iprodione) did not control iprodione MR and HR isolates.

Blackberry fruits symptomatic for gray mold were collected from three commercial blackberry fields in northwestern South Carolina. Single-spore isolates were generated and two distinct phenotypes were discovered in each location; one sporulated on PDA and one did not. One isolate of each phenotype and location (six isolates total) were selected for in depth molecular and morphological characterization. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) coding sequence alignment revealed Botrytis cinerea as the sporulating phenotype and a new yet undescribed species as the non-sporulating phenotype. The new Botrytis sp., described herein as Botrytis caroliniana, was most closely related genetically to B. fabiopsis and B. galanthina, the causal agents of gray mold disease of broad bean and snowdrop, respectively. It produces smaller conidia than either B. fabiopsis or B. galanthina, and sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) also indicated that the Botrytis isolates represent a separate and distinct species. The new species is pathogenic on blackberry fruits and broad bean leaves, which distinguishes it further from B. galanthina. The new species formed white to pale gray colonies with short, tufted aerial mycelium and produced black sclerotia on PDA at 20 C. To our knowledge this is only the third Botrytis species discovered to cause disease on blackberry in the United States.

Gray mold and anthracnose fruit rot are two of the most important diseases threatening strawberry production in the United States. Managing these diseases is critical, but difficult without understanding how the fungus causing each disease operates. Improvements in design technology and computer processing power have enabled us to create a new generation of informational resources that explain disease development in a new and exciting way. High-definition photos and time-lapse videos help us recognize and diagnose diseases from their first appearance in the field. Through illustrations and animations, concepts, such as disease cycles, can be explained more effectively. Free availability of these materials on websites and social media makes them accessible to growers, agents, and specialists. Through these materials we can clarify and justify current management practices so that growers can more effectively combat diseases in the field. Using gray mold and anthracnose fruit rot as an example, we created photo galleries and updated, visually stimulating disease cycles for continued and improved grower training. [ABSTRACT FROM AUTHOR]

BACKGROUND Colletotrichum species cause anthracnose diseases on many plants and crops. A new generation of succinate dehydrogenase inhibitors (SDHIs) was developed recently. The inhibitory activity of the five SDHI fungicides against Colletotrichum species was determined in this study. RESULTS Isolates of C. gloeosporioides, C. acutatum, C. cereale and C. orbiculare were insensitive (naturally resistant) to boscalid, fluxapyroxad and fluopyram on YBA agar medium. In contrast, these isolates were relatively sensitive to penthiopyrad, except for C. orbiculare. Most interestingly, benzovindiflupyr showed highest inhibitory activity against all of these four species. Benzovindiflupyr was effective against C. gloeosporioides and C. acutatum on apple and peach fruit, as well as on cucumber plants inoculated with C. orbiculare. The sdhB, sdhC and sdhD genes encoding the subunits of fungicide-targeted succinate dehydrogenase were sequenced, but, despite high polymorphisms, no apparent resistance mutations were found in Colletotrichum species. CONCLUSIONS This is the first report on the activity of benzovindiflupyr against Colletotrichum species. The broad-spectrum efficacy of benzovindiflupyr within the Colletotrichum genus might be exploited when designing disease management strategies against various pathogens on a wide range of crops. Other mechanism(s) than fungicide target-site modification may be responsible for differential sensitivity of Colletotrichum species to SDHI fungicides. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: Biofumigation of peaches in shipping cartons with pads containing culture of the volatile-producing fungus Muscodor albus was investigated for the control of brown rot caused by Monilinia fructicola. Biofumigation during cold storage (1–2°C) with 50 or 200g of M. albus per plastic bag and enclosed carton containing 14kg of fruit reduced the incidence of brown rot significantly (P <0.05) with ‘Coronet’ and ‘Red Globe’ peaches and disease incidence was comparable or lower (P <0.05) than decay in fungicide-treated peaches. Biofumigation was not as effective in non-bagged cartons, although significant brown rot control was observed in ‘Red Globe’ peaches with the 200-g treatment. Containment of volatile compounds in the cartons is thus important for the efficacy of this treatment in cold storage. [Copyright &y& Elsevier]

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 = 4 1 ), 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peach skin streaking is a previously undescribed skin discoloration affecting red-blush peach cultivars in Georgia and South Carolina. Streaked peach fruit have been observed in the field close to harvest. The cause of streaking is still unknown but one hypothesis is that atmospheric pollutants may be involved. The goal of this study was to establish proof of concept that commonly found air pollutants can produce streaks on peach skin similar to those observed in commercial orchards and investigate the susceptibility of peach fruit during maturation. Common reactive byproducts of atmospheric pollutants, including sulfuric acid (H2SO4), nitric acid (HNO3), and hypochlorite acid (HCl), at concentrations up to 10 µg/ml did not produce streaking under field conditions when applied at week 3, 2, and 1 prior to commercial harvest. However, sodium hypochlorite (NaCIO) in the form of Clorox solution and chlorine dioxide (CIO2) at 100 µg/ml generated from the Aquamira water treatment solution produced streaking symptoms on detached peach fruit under controlled conditions and in the field. Peach fruit were most susceptible to streaking closest to harvest, suggesting that NaCIO and CIO2 interfere with pigment formation. [ABSTRACT FROM AUTHOR]

Colletotrichum species cause diseases on many plants and are among the 'top 10' fungal plant pathogens. Species of the C. gloeosporioides and C. acutatum complexes are particularly important because they infect temperate fruit crops, but their control relies largely on chemical fungicides. In this study, differences in intrinsic fungicide sensitivity were determined in vitro using isolates of the C. gloeosporioides sp. complex (C. fructicola , C. siamense , and C. tropicale) and the C. acutatum sp. complex (C. fioriniae and C. nymphaeae), which had never been exposed to fungicides. Mycelial growth of all isolates was sensitive to the QoI azoxystrobin, the SDHI benzovindiflupyr, and the new DMI fungicide mefentrifluconazole. The isolates of C. nymphaeae were highly sensitive to the phenylpyrrole fungicide fludioxonil. The isolates of C. gloeosporioides sp. complex were sensitive to the bis-guanidine fungicide iminoctadine-albesilate, whereas those of C. acutatum sp. complex were inherently insensitive. These results are valuable when sensitivity of field populations is monitored in resistance management. Although SDHI fungicides are largely not effective against diseases caused by Colletotrichum species, benzovindiflupyr controlled anthracnose disease of various crops such as kidney bean, garland chrysanthemum, and strawberry, caused by C. lindemuthianum , C. chrysanthemi , and C. siamense , respectively, demonstrating this fungicide to be unique among SDHIs and having a broad control spectrum against anthracnose. To help understanding the reason for differential activity of benzovindiflupyr and boscalid, sdhB gene sequences were analyzed but those of C. lindemuthianum , C. chrysanthemi , and C. scovillei revealed no known mutations reported to be responsible for SDHI resistance in other fungi, indicating that other mechanism(s) than target-site modification may be involved in differential sensitivity to benzovindiflupyr and boscalid, found in Colletotrichum species. [Display omitted] • Azoxystrobin sensitivity was high in baseline isolates of Colletotrichum gloeosporioides and C. acutatum sp. complexes • Mefentrifluconazole, with favorable toxicity profile, was effective against Colletotrichum isolates regardless of the species • Mefentriconazole and benzovindiflupyr will become an effective tool to manage anthracnose on multiple crops [ABSTRACT FROM AUTHOR]

Gray mold disease of blackberry and strawberry is caused by Botrytis cinerea and B. caroliniana in the southeastern United States. In this study, methods to distinguish both species were established and their prevalence was determined in commercial blackberry and strawberry fields. Using DNA from B. cinerea and B. caroliniana reference strains, a species-differentiating polymerase chain reaction (PCR) amplification was developed that amplified G3PDH gene fragments of two different sizes depending on the species. The PCR is performed with three primers (two species-differentiating forward primers and one universal reverse primer) and amplified a 238-bp product from B. cinerea and a 536-bp fragment from B. caroliniana reference isolates. A total of 400 Botrytis isolates were collected from 6 commercial blackberry and 11 strawberry fields of the Carolinas and identified to the species level by the new PCR method. Both Botrytis spp. were identified in blackberry and strawberry fields, but B. caroliniana was less common than B. cinerea. Only 33 of 202 isolates from blackberry fields were identified as B. caroliniana, and the majority of these iso-lates came from two fields in South Carolina. Only 1 of 198 isolates from strawberries was identified as B. caroliniana, and this isolate was found in central North Carolina. B. cinerea but not B. caroliniana iso-lates sporulated on potato dextrose agar and Kings medium B. Our results show that B. cinerea and B. caroliniana coexist in at least some commercial blackberry and strawberry fields of the Carolinas, with B. cinerea being the more prevalent species. [ABSTRACT FROM AUTHOR]

Sterol demethylation inhibitor (DMI) fungicide resistance in isolates of Monilinia fructicola from Georgia has been linked to overexpression of the MfCYP51 gene and a corresponding 65-bp genetic element 'Mona' inserted in the upstream region of MfCYP51. In this study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to detect the Mona element. Fourteen DMI fungicide-resistant (DMI-R) and six DMI fungicide-sensitive (DMI-S) isolates from Georgia, six DMI-R and 11 DMI-S isolates from South Carolina, seven DMI-R and nine DMI-S isolates from New York, and two DMI-R and three DMI-S isolates from Ohio were used in this study. The isolates from the southeastern United States and Ohio were collected from peach, whereas isolates from New York were collected from cherry. A 376-bp fragment containing the Mona element was consistently amplified with primer pair INS65-F and INS65-R from DMI-R isolates, and either a 311 -bp or 1,81 5-bp fragment was amplified from DMI-S isolates. The primer pair did not amplify DNA fragments of similar sizes from isolates of five other common fruit rot pathogens of peach, including Alternaria alternata, Colletotrichum acutatum, Gilbertella persicaria, Penicillium expansum, and Rhizopus stolonifer. Gel electrophoresis of the PCR amplicon can distinguish between DMI-R and DMI-S isolates based on the 65-bp size difference of the amplicon; however, the restriction digestion assay can verify questionable results, especially in the absence of a positive control. Only the 376-bp fragment containing the Mona element was digestable with endonuclease BsrBI, resulting in two restriction fragments of 236 and 140 bp in size. In this study, a protocol for Mona detection from aerial fungal structures was developed that can yield results within a few hours of sampling. This study confirms that the Mona element is strongly linked to the DMI-resistance phenotype and reveals that overexpression of the MfCYP51 gene is a common DMI fungicide resistance mechanism in M. fructicola, not only in Georgia but throughout the eastern United States. [ABSTRACT FROM AUTHOR]