Visual Assessment of Phony Peach Disease: Evaluating Rater Accuracy and Reliability.

Phony peach disease (PPD), found predominantly in central and southern Georgia, is a re-emerging disease caused by Xylella fastidiosa (Xf) subsp. multiplex. Accurate detection and rapid removal of symptomatic trees are crucial to effective disease management. Currently, peach producers rely solely on visual identification of symptoms to confirm PPD, which can be ambiguous if early in development. We compared visual assessment to quantitative PCR (qPCR) for detecting Xf in 'Julyprince' in 2019 and 2020 (JP2019 and JP2020) and in 'Scarletprince' in 2020 (SP2020). With no prior knowledge of qPCR results, all trees in each orchard were assessed by a cohort of five experienced and five inexperienced raters in the morning and afternoon. Visual identification accuracy of PPD was variable, but experienced raters were more accurate when identifying PPD trees. In JP2019, the mean rater accuracy for experienced and inexperienced raters was 0.882 and 0.805, respectively. For JP2020, the mean rater accuracy for experienced and inexperienced raters was 0.914 and 0.816, respectively. For SP2020, the mean rater accuracy for experienced and inexperienced raters was 0.898 and 0.807, respectively. All raters had false positive (FP) and false negative (FN) observations, but experienced raters had significantly lower FN rates compared with the inexperienced group. Almost all raters overestimated the incidence of PPD in the orchards. Reliability of visual assessments was demonstrated as moderate to good, regardless of experience. Further research is needed to develop accurate and reliable methods of detection to aid management of PPD as both FPs and FNs are costly to peach production.

First report of the new Neopestalotiopsis species causing strawberry leaf spot and fruit rot in Georgia.

In April 2023, symptomatic strawberry (Fragaria × ananassa) plants (cv. 'Camarosa' and 'Florida Brilliance') were observed at a commercial farm in Worth County, GA (USA). Symptoms included foliar, irregularly distributed, and different-sized spots (dark brown with light brown centers) and dark brown V-shaped necrotic areas starting at the leaf edge. By the time of sample collection, ~50% incidence was reported in the field. Leaf samples were collected and shipped overnight to the laboratory. Black acervuli were observed readily on old necrotic foliar lesions. Conidial morphology was consistent with that observed with Neopestalotiopsis species (Maharachchikumbura et al. 2014). Conidia were ellipsoid to fusiform, five-celled, with three light brown median cells and one hyaline apical and basal cell. Apical cells had two-to-four flexuous appendages, and the basal cell had one non-flexuous appendage (Fig 1). The average (n = 20) conidia length, not including the appendages, was 26.6 µm (SD: 2.8), and width was 6.3 µm (SD: 0.94). Fungal isolation was conducted on acidified PDA and incubated at 25°C for 6 days. Dense, white mycelia were observed on the upper plate surface, while a pale pink/orange coloration was observed on the underside (Fig 1). Black acervuli formed on the surface of the white mycelial mat. Six isolates were purified and selected to confirm the species identity. DNA was extracted from 6-day-old cultures and PCR was conducted following Kaur et al. (2023). Amplified DNA was digested with the restriction enzyme BsaWI and two bands were clearly visualized (~130 and ~290 bp), along with a faint band of 20-bp (Fig 2). Four of the six isolates were selected for sequencing of the ß-tubulin gene. BLAST queries using the consensus sequence showed that all isolates had 100% identity to strain N21002 from Florida (FL), characterized as Neopestalotiopsis sp. (Kaur et al. 2023). One representative isolate (AJ07-2023) was deposited in GenBank (accession No. PP316103). Pathogenicity tests were performed on 27-day-old transplants of Sensation 'Florida127' provided by Natalia Peres from the UF. Plants were grown in 10.5 cm pots in the greenhouse. Isolate AJ07-2023 was grown on PDA for 30 days at 25°C, and the spore suspension was adjusted at 106 spore/ml. Five strawberry plants were sprayed with 5 ml of inoculum using a Preval sprayer with a CO2 canister, and 5 plants were sprayed with sterile distilled water. Plants were placed in a growth chamber for 6 days and covered with plastic bags after the sixth day to maintain ~85% relative humidity and 25°C. Foliar symptoms, including dark-brown circular lesions occurring towards the edge of leaves with light-brown center and light-yellow halo, developed 13 days after inoculation. No symptoms were observed on control plants. Neopestalotiopsis sp. was reisolated from inoculated plants as described above. Colony, conidial morphology, and PCR results were consistent with the original isolates. Neopestalotiopsis disease has been reported on strawberry in FL (Baggio et al. 2021), OH (Rotondo et al. 2022) and IN (Guan et al. 2023). Although the disease has been observed sporadically in GA since 2020 (Brannen, personal communication), to our knowledge, this is the first official report of the new Neopestalotiopsis sp. in GA. It has been reported that this new strain is more aggressive on fruits and leaves than other Neopestalotiopsis spp. (Baggio et al. 2021), therefore, accurate identification is critical for proper management.

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.

Fungicide Use Patterns in Select United States Wine Grape Production Regions.

Wine grape production (Vitis sp.) in the United States requires fungicide inputs for disease control. Currently, there is limited data available on vineyard fungicide use patterns. This information is important in developing tailored recommendations for disease management and fungicide stewardship. In this paper, we summarize the wine grape vineyard fungicide use patterns from four major regions: Napa and Sonoma valleys (California), Willamette Valley (Oregon), Columbia Valley (Washington), and several smaller regions east of the Mississippi River in years 2009 to 2020. We learned that the average in-season total fungicide applications ranged regionally from 5.6 to 8. The most commonly applied Fungicide Resistance Action Committee (FRAC) codes in spray programs were FRAC 3, 13, and M02 across all regions, with some variation to the top four groups in each region. Most applications were made on 14-day intervals; however, shorter intervals (7-day) were favored early season, and longer intervals (21-day) were favored late season. Tank-mixing multiple active ingredients was common east of the Mississippi River during all stages of grape development; this action was typically favored during the bloom period in other regions. In a subset of records that participated in FRAC 11 fungicide resistance testing, the average number of FRAC 11 applications after testing was reduced to either no applications or one application in Napa and Sonoma valleys. This survey provides regionally specific data related to fungicide stewardship practices that could be a focus for future stewardship messaging and fungicide resistance selection training, including total product use (selection events), spray intervals (selection pressure), and tank mixing (selection management).[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Colletotrichum species causing Glomerella leaf spot and apple bitter rot in the southeastern United States exhibit disparities in relative frequency, morphological phenotype, and QoI sensitivity.

Glomerella leaf spot (GLS), Glomerella fruit rot (GFR) and apple bitter rot (ABR), caused by Colletotrichum spp. are amongst the most devastating apple diseases in the southeastern United States. While several species have been identified as causal pathogens of GLS, GFR, and ABR, their relative frequency and fungicide sensitivity status in the southeastern U.S. is unknown. In total, 381 Colletotrichum isolates were obtained from symptomatic leaves and fruit from 18 conventionally managed apple orchards and two baseline populations in western North Carolina and Georgia in 2016 and 2017. Multilocus DNA sequence analysis revealed that C. chrysophilum was the predominant cause of GLS and GFR, and C. fioriniae was the causal agent of ABR. Baseline and commercial populations of Colletotrichum spp. were evaluated for sensitivity to pyraclostrobin and trifloxystrobin and no statistical differences in sensitivity between the two species were observed for conidial germination. However, EC50 values were significantly lower for C. fioriniae compared to C. chrysophilum for both fungicides regarding mycelial inhibition. Isolates recovered from commercial orchards revealed that 5 populations of C. chrysophilum and 1 population of C. fioriniae had reduced sensitivity to trifloxystrobin, and 1 C. fioriniae population had reduced sensitivity to pyraclostrobin via conidial germination assays. The cytb gene for 27 isolates of C. fioriniae, C. chrysophilum, and C. fructicola with different QoI sensitivities revealed the G143A mutation in a single isolate of C. chrysophilum with insensitivity to both fungicides. Results of these studies suggest that two Colletotrichum spp. predominantly cause GLS and ABR in the southeastern U.S. and that a reduction in sensitivity to some QoI fungicides may be responsible for control failures. This study also provides basis for monitoring shifts in QoI sensitivity in Colletotrichum spp. causing disease on apple in the southeastern U.S.

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.

Prevalence and Distribution of Phony Peach Disease (Caused by Xylella fastidiosa) in the United States.

Peach is an important specialty fruit crop in the United States, and phony peach disease (PPD), caused by Xylella fastidiosa subsp. multiplex, has been a major cause of yield loss since it was first observed in 1885. Under a federal eradication program, surveys of PPD were conducted from 1929 to 1972, when the program was terminated. No surveys have been conducted in approximately 50 years; therefore, the current prevalence of PPD in the United States is unknown, especially in the Southeast, where damage was previously most severe. To ascertain the status of PPD, we surveyed orchards in Alabama, Florida, Georgia, and South Carolina from June to August 2020 and, except for South Carolina and northern Georgia, PPD was prevalent. Trees in 17 orchards were subjected to confirmation of X. fastidiosa using the AmplifyRP XRT+ for X. fastidiosa to corroborate our visual assessments; based on these tests, PPD incidence in the orchards ranged from 0 to 30.5%. Ancillary written surveys of relative PPD presence and prevalence were sent to fruit pathologists from universities in 20 states where PPD was historically reported. Only 35.0% of respondents reported that PPD either currently or recently occurred in their state and, of these, three reported PPD to be of significant concern. The results of the physical and written surveys indicate that PPD remains prevalent mainly in the southeastern region of the United States but, in other states where previously reported, it is either not present or has very low prevalence when compared with historical accounts of the disease.

Distribution, Diversity, and Soil Associations of Wine Grape Plant-Parasitic Nematodes in Georgia, U.S.A., Vineyards.

Wine grape (Vitis vinifera and V. vinifera hybrids) production in Georgia occurs in three distinct regions (North, West, and South) which can be characterized by sandy, sandy-loam, or sandy clay-loam soils. We studied plant-parasitic nematode (PPN) communities in 15 wine grape vineyards from the three primary growing regions to understand which nematodes are a concern and what soil characteristics are associated with their occurrence and relative abundance. Twelve genera of PPNs were detected throughout the state: Belonolaimus, Helicotylenchus, Hemicycliophora, Heterodera, Hoplolaimus, Meloidogyne, Mesocriconema, Paratrichodorus, Paratylenchus, Pratylenchus, Tylenchorhynchus, and Xiphinema. Nonmetric multidimensional scaling ordination and multirank permutation procedure identified PPN community differences and soil characteristics that were associated by region. Indicator species analysis identified Helicotylenchus, Mesocriconema, Tylenchorhynchus, and Xiphinema as statistically associated with the West while Meloidogyne and Paratrichodorus were associated with the South. Our analyses further suggested that soil texture (percent sand, percent clay, and percent silt) and the lime buffer capacity at equilibrium (LBCEQ) were associated with PPN community structure while pH was not. When focused on a single vineyard in the North, multiple logistic regression analysis suggested a statistically significant association between Meloidogyne spp. and soil characteristics, including percentages of sand, pH, and LBCEQ. Our study supports the association between soil characteristics and specific nematode genera, as well as the emergence of LBCEQ, the soil measurement with the strongest statistical association with nematode community structure and Meloidogyne presence.

Mating Type Idiomorphs, Heterothallism, and High Genetic Diversity in Venturia carpophila, Cause of Peach Scab.

Scab (caused by Venturia carpophila) is a major disease affecting peach in the eastern United States. The aims of the study were to characterize the mating-type loci in V. carpophila, determine whether they are in equilibrium, and assess the population genetic diversity and structure of the pathogen. The mating-type gene MAT1-1-1 was identified in isolate JP3-5 in an available genome sequence, and the MAT1-2-1 gene was PCR amplified from isolate PS1-1, thus indicating a heterothallic structure. Mating-type loci structures were consistent with those of other Venturia spp. (V. effusa and V. inaequalis): the mating-type gene is positioned between APN2 encoding a DNA lyase and a gene encoding a Pleckstrin homology domain. Primers designed to each of the mating-type genes and a reference gene TUB2 were used as a multiplex PCR to screen a population (n = 81) of V. carpophila from various locations in the eastern United States. Mating types in five of the nine populations studied were in equilibrium. Among the 81 isolates, there were 69 multilocus genotypes. A population genetic analysis of the populations with >10 individuals (four populations) showed them to be genetically diverse. Linkage disequilibrium was found in five of nine populations with ≥4 isolates. A discriminant analysis of principal components indicated three genetic clusters, although extensive admixture was observed. Mating-type identification in V. carpophila provides a basis for understanding reproductive methods of the pathogen and can be a basis for further studies of the genetics of the peach scab pathogen.

Yield Response to Orange Cane Blotch of Blackberry Grown in the Georgia Coastal Plain.

Orange cane blotch (OCB), an algal disease on commercial blackberry plants in the southeastern United States, has been an increasing concern among producers. The causal agent, Cephaleuros virescens, produces brightly colored green to orange lesions on blackberry stems, but proof of actual damage and impact on crop yield has not been documented. Naturally infected stem sections were viewed using transmission and scanning electron microscopy to evaluate cane damage. Surface abrasions, intercellular growth, and occasional intracellular growth were observed on the surface and epidermal layers. Field studies at four commercial sites over 2 years were conducted to assess the impact of OCB on yield in 'Ouachita' blackberry plants not treated with algicidal chemicals. Neither cane diameter nor berry size was impacted by severity of OCB; however, berry number decreased with increasing OCB intensity in a nonlinear manner, thereby resulting in reduced yields.

Novel Primers and Sampling for PCR Detection of Xylella fastidiosa in Peach.

Epidemics of phony peach disease (PPD), caused by Xylella fastidiosa, are of increasing concern to peach (Prunus persica) producers in the southeastern United States. Primers suitable for both conventional PCR (cPCR) and quantitative PCR (qPCR), along with optimal tissue and sampling time, are needed for comparative and reliable detection of X. fastidiosa. In this study, we developed and assessed novel primers for X. fastidiosa and for peach and compared detection of X. fastidiosa in four peach tissue types sampled at three time points using both cPCR and qPCR. Primer C06Xf-bamA was extensively tested for reliable detection of X. fastidiosa due to the more consistent intensity of the cPCR products and the marginally lower average quantification cycle (Cq) values of the qPCR products, compared with the other primers screened. Among the four peach tissue types tested, only root samples demonstrated reliable and consistent detection of X. fastidiosa; stem, petiole, and leaf samples, regardless of source trees, primers used, sampling times, or PCR methods (cPCR or qPCR), were unreliable for detection, due to insufficient quantity of DNA of X. fastidiosa in these samples based on the relative quantification assay. The Cq means and ratios were compared and statistically analyzed, to ascertain effects of source tree, tissue type, sampling time, and primer. Differences in detection sensitivity and the Cq means among sampled trees, sampling times, tested primers, and tissues (except root) were not significant or were inconsistent precluding further exploitation. In summary, these novel primers are a useful resource for detecting X. fastidiosa, and based on our results, root is the only tissue type reliable for year-round detection of X. fastidiosa in peach. Further research on potential utilization of above-ground tissues for PCR detection of X. fastidiosa are discussed.

Effect of spirotetramat and fluensulfone on population densities of Mesocriconema xenoplax and Meloidogyne incognita on peach.

Management of plant-parasitic nematodes (PPNs) on peach is needed for a longer period of time than is typically afforded by pre-plant fumigant nematicides. Two post-plant nematicides, spirotetramat and fluensulfone, were evaluated for control of Meloidogyne incognita and Mesocriconema xenoplax under laboratory and greenhouse conditions. In vitro assays were conducted to test the effect of spirotetramat at 0.017 and 0.026 kg a.i./ha and fluensulfone at 3.92 kg a.i./ha on the mobility of both M. incognita and M. xenoplax in 24-well plates for 24, 48, and 72 hr, compared to a water control. Both fluensulfone and spirotetramat reduced mobility of M. xenoplax, but only fluensulfone reduced the mobility of M. incognita, compared to the untreated control. In peach greenhouse trials, both spirotetramat at 0.017 kg a.i./ha and fluensulfone at 3.92 kg a.i./ha reduced M. incognita numbers by 62 and 77% at 40 d after inoculation (DAI), respectively; neither chemical reduced populations at 70 DAI. Fluensulfone reduced M. xenoplax numbers by 84, 94, and 96% at 30, 60, and 90 DAI, respectively. No effects were observed for spirotetramat on M. xenoplax. At 40 DAI, dual applications of spirotetramat 30 d apart reduced M. incognita numbers by 58 and 54% for both 0.017 and 0.026 kg a.i./ha rates, respectively; no reductions were observed at 70 DAI. No effect was seen for a dual application of spirotetramat on M. xenoplax. These post-plant nematicides may provide additional options for management of PPNs on peach.Management of plant-parasitic nematodes (PPNs) on peach is needed for a longer period of time than is typically afforded by pre-plant fumigant nematicides. Two post-plant nematicides, spirotetramat and fluensulfone, were evaluated for control of Meloidogyne incognita and Mesocriconema xenoplax under laboratory and greenhouse conditions. In vitro assays were conducted to test the effect of spirotetramat at 0.017 and 0.026 kg a.i./ha and fluensulfone at 3.92 kg a.i./ha on the mobility of both M. incognita and M. xenoplax in 24-well plates for 24, 48, and 72 hr, compared to a water control. Both fluensulfone and spirotetramat reduced mobility of M. xenoplax, but only fluensulfone reduced the mobility of M. incognita, compared to the untreated control. In peach greenhouse trials, both spirotetramat at 0.017 kg a.i./ha and fluensulfone at 3.92 kg a.i./ha reduced M. incognita numbers by 62 and 77% at 40 d after inoculation (DAI), respectively; neither chemical reduced populations at 70 DAI. Fluensulfone reduced M. xenoplax numbers by 84, 94, and 96% at 30, 60, and 90 DAI, respectively. No effects were observed for spirotetramat on M. xenoplax. At 40 DAI, dual applications of spirotetramat 30 d apart reduced M. incognita numbers by 58 and 54% for both 0.017 and 0.026 kg a.i./ha rates, respectively; no reductions were observed at 70 DAI. No effect was seen for a dual application of spirotetramat on M. xenoplax. These post-plant nematicides may provide additional options for management of PPNs on peach.

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.

Blueberry necrotic ring blotch virus in Southern Highbush Blueberry: Insights into In Planta and In-Field Movement.

Blueberry necrotic ring blotch virus (BNRBV) causes an emerging disease of southern highbush blueberry (SHB) in the southeastern United States. Disease incidence and severity vary considerably from year to year within the same planting. Experiments were conducted to determine how the virus spreads in the field. Leaf tissue from symptomatic field plants tested positive for BNRBV in 2011, whereas the same plants were asymptomatic in 2012 and tested negative for the virus. Symptomatic and asymptomatic leaves from individual shoots were tested for the presence of the virus, and symptomatic leaves tested positive (100%), whereas 65.4% of the asymptomatic leaves from the same shoots tested negative. Leaves were selected in which half the leaf blade was symptomatic and the other half was not; symptomatic leaf halves tested positive (100%), whereas 76.0% of the asymptomatic halves from the same leaf tested negative for the virus. When virus-free, potted trap plants were interspersed in the field among established plants that had shown disease symptoms the previous year, disease onset in trap plants was observed 2 to 3 weeks after disease onset in field plants. In a separate experiment, asymptomatic softwood cuttings were collected from mother plants symptomatic for BNRBV, rooted, and monitored for symptom development for a period of 12 to 27 months. No BNRBV symptoms were observed in the progeny, whereas disease incidence was high for cuttings taken at the same time from plants infected with Blueberry red ringspot virus used as a control. Collectively, these studies suggest that BNRBV does not infect SHB plants systemically and is not transmitted through vegetative propagation, and that the virus likely does not persist in plants after natural defoliation in the fall.

Exobasidium maculosum, a new species causing leaf and fruit spots on blueberry in the southeastern USA and its relationship with other Exobasidium spp. parasitic to blueberry and cranberry.

Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species.

Comparative analysis of different molecular and serological methods for detection of Xylella fastidiosa in blueberry.

Bacterial leaf scorch, caused by Xylella fastidiosa, is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x105 cfu/mL of Xylella fastidiosa. Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn't require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X. fastidiosa on blueberry.

A Novel Population of Fusarium fujikuroi Isolated from Southeastern U.S. Winegrapes Reveals the Need to Re-Evaluate the Species' Fumonisin Production.

Mycotoxins pose a challenge to a safe food supply worldwide, and their threat is expected to worsen with our changing climate. The need for diligence is exemplified by the discovery of fumonisin B2 in wine, which joins ochratoxin A as a mycotoxin of concern in the grape-wine chain. To elucidate the mycotoxin risk in southeastern American wine, grape samples were collected from vineyards during harvest in 2013 and potentially mycotoxigenic fungi (Fusarium and Aspergillus) were isolated from the samples. Numerous Fusarium isolates were recovered and identified to the species level by comparison of translation elongation factor 1-α gene sequences to verified strains. Fusarium fujikuroi was the most abundant species recovered (239 isolates), followed by F. proliferatum (52), F. incarnatum-equiseti (14), F. oxysporum (7), F. concentricum (1), and F. solani (1). In vitro assays quantified fumonisin production for representative isolates via liquid chromatography-tandem mass spectrometry. Surprisingly, nearly all F. fujikuroi isolates produced fumonisins B1, B2, and B3 at levels comparable to both the F. proliferatum isolates and the positive control, Fusarium verticillioides. Such capacity for fumonisin production refutes the generally accepted notion that F. fujikuroi produces undetectable or low levels of fumonisins and provides evidence to reconsider this species as a mycotoxigenic threat to economically significant crops.

Elevated Genetic Diversity in the Emerging Blueberry Pathogen Exobasidium maculosum.

Emerging diseases caused by fungi are increasing at an alarming rate. Exobasidium leaf and fruit spot of blueberry, caused by the fungus Exobasidium maculosum, is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA, severely reducing fruit quality in some plantings. The objectives of this study were to determine the genetic diversity of E. maculosum in the southeastern USA to elucidate the basis of disease emergence and to investigate if populations of E. maculosum are structured by geography, host species, or tissue type. We sequenced three conserved loci from 82 isolates collected from leaves and fruit of rabbiteye blueberry (Vaccinium virgatum), highbush blueberry (V. corymbosum), and southern highbush blueberry (V. corymbosum hybrids) from commercial fields in Georgia and North Carolina, USA, and 6 isolates from lowbush blueberry (V. angustifolium) from Maine, USA, and Nova Scotia, Canada. Populations of E. maculosum from the southeastern USA and from lowbush blueberry in Maine and Nova Scotia are distinct, but do not represent unique species. No difference in genetic structure was detected between different host tissues or among different host species within the southeastern USA; however, differentiation was detected between populations in Georgia and North Carolina. Overall, E. maculosum showed extreme genetic diversity within the conserved loci with 286 segregating sites among the 1,775 sequenced nucleotides and each isolate representing a unique multilocus haplotype. However, 94% of the nucleotide substitutions were silent, so despite the high number of mutations, selective constraints have limited changes to the amino acid sequences of the housekeeping genes. Overall, these results suggest that the emergence of Exobasidium leaf and fruit spot is not due to a recent introduction or host shift, or the recent evolution of aggressive genotypes of E. maculosum, but more likely as a result of an increasing host population or an environmental change.

Reduced Sensitivity in Monilinia fructicola to Propiconazole in Georgia and Implications for Disease Management.

Single-spore isolates of Monilinia fructicola were collected from commercial orchards in South Carolina and Georgia with prolonged past exposure to demethylation inhibitor (DMI) fungicides and from an orchard with no DMI history (baseline population). Sensitivity to propiconazole was determined using the concentration in agar media required to suppress radial growth of mycelium by 50% (EC50. Mean EC50 values from six South Carolina populations were not different from the baseline population (P < 0.05). Two of five populations from Georgia revealed (significantly higher mean EC50 values compared with the baseline population (P < 0.05). Isolates with high (AP5 and AP6) and low (DL71 and DL72) EC50 values were selected to determine disease incidence on peach fruit after protective or curative applications of propiconazole at 0.15 or 0.3 liter/ha (half and full label rate, respectively). Disease incidence was significantly greater on peaches inoculated with AP5 and AP6 after curative treatment with propiconazole at 0.15 liter/ha (P < 0.05). Following protective or curative treatments at 0.3 liter/ha, disease incidence was significantly greater for AP6 but not for AP5. These results suggest that a shift toward reduced sensitivity has developed in some M. fructicola populations from Georgia, and that isolates with reduced sensitivity to propiconazole are more difficult to control in the field. Field testing of DMI fungicides, captan, QoI fungicides, and fenhexamid in experimental orchards) indicated that the DMI fungicides are still among the most efficacious products for brown rot (control, and that new products containing QoI fungicides may be viable disease control alternatives or rotation partners.