Quantifying the Effects of a G137S Substitution in the Cytochrome bc1 of Venturia effusa on Azoxystrobin Sensitivity Using a Detached Leaf Assay.

The quinone outside inhibitor (QoI) fungicides are known for their inherently high resistance risk owing to substitutions in amino acid residues 129, 137, or 143 of the cytochrome b gene of phytopathogens. In Venturia effusa, cause of pecan scab, an intron adjacent to position 143 likely reduces this risk; however, the effects of a recently discovered substitution at position 137 (G137S) are unknown. Traditional in vitro assays are not useful for determining sensitivity of isolates of V. effusa to the QoI fungicides, owing to the fungitoxic effects of required alternative oxidase inhibitors. A detached leaf assay was developed to quantify the sensitivity of 59 isolates to azoxystrobin: 45 wild-type isolates and 14 carrying G137S. Isolate EC50 values ranged from <0.0001 to 0.3047 µg/ml; EC50 values for wild-type isolates ranged from <0.0001 to 0.2007 µg/ml (median 0.0023 µg/ml), whereas EC50 values for G137S isolates ranged from 0.0033 to 0.3047 µg/ml (median 0.0178 µg/ml). The median EC50 value for G137S isolates was significantly greater than that of the wild-type isolates; however, there was overlap between the two groups. This is the first report of sensitivity of V. effusa isolates to a QoI fungicide and evidence of G137S as a potential mechanism of partial resistance. However, although a complete control failure is unlikely, the impact of this substitution on QoI efficacy in Georgia pecan orchards remains to be determined.

Effect of Planting Date and Peanut Cultivar on Epidemics of Late Leaf Spot in Georgia.

Field trials were conducted in 2015 and 2016 in Tifton, GA to determine the effects of planting dates (24 and 27 April, 4, 11, 19, and 26 May 2015; and 11, 18, and 25 April and 2, 9, and 16 May 2016), peanut (Arachis hypogaea) cultivar (Georgia-06G and Georgia-12Y), and seed treatment (nontreated and treated with azoxystrobin, fludioxonil, and mefenoxam) on epidemics of late leaf spot (Nothopassalora personata), plant populations, and peanut yield. Final severity and AUDPC of late leaf spot increased with later planting dates in both years. For most planting dates in 2015 and the final planting date in 2016, final leaf spot severity and AUDPC were lower for Georgia-12Y than for Georgia-06G. Seed treatment increased plant populations for the 27 April and 4 May planting dates in 2015 and across all other treatments in 2016. Yields were higher for Georgia-12Y than for Georgia-06G in both years. In 2015, yields of both cultivars decreased according to linear functions of day of year of planting date, but there was no effect of planting date on yield in 2016. The combination of early planting with Georgia-12Y shows potential utility for management of leaf spot in situations such as organic production where fungicide use is minimal.

Assessing Fitness Costs and Phenotypic Instability of Fentin Hydroxide and Tebuconazole Resistance in Venturia effusa.

Sensitivity monitoring of Venturia effusa, cause of pecan scab, has revealed insensitivity to fentin hydroxide and tebuconazole, but recent research indicates that the insensitivity to fentin hydroxide is not stable. A study was undertaken to determine if a fitness cost may be responsible for this instability. In this study, experiments were conducted to evaluate fitness components and phenotypic stability of insensitivity of V. effusa to fentin hydroxide and tebuconazole. Conidial production, conidial germination, microcolony growth, sensitivity to osmotic stress, and sensitivity to oxidative stress in the absence of fungicide were compared for isolates with differing sensitivities to both fungicides. Percent conidial germination decreased linearly with increasing fentin hydroxide insensitivity, and microcolony growth on 1.0 mM H2O2 decreased linearly with increasing tebuconazole insensitivity. Stability of resistance was assessed on concentrations of 1.0, 3.0, and 10 µg/ml of both fungicides prior to and after five transfers on non-fungicide-amended medium. Tebuconazole insensitivity was stable after transfers, but fentin hydroxide insensitivity on 1.0 and 3.0 µg/ml decreased significantly after transfers, indicating instability. Here we provide evidence that in V. effusa tebuconazole insensitivity is stable and fentin hydroxide insensitivity is not. These results suggest that fentin-hydroxide-resistant V. effusa isolates have reduced conidial viability compared with sensitive isolates, which may allow the population to regain sensitivity in the absence of this frequently used fungicide.

Relating Peanut Rx Risk Factors to Epidemics of Early and Late Leaf Spot of Peanut.

Previous research has demonstrated the efficacy of prescription fungicide programs, based upon Peanut Rx, to reduce combined effects of early leaf spot (ELS), caused by Passalora arachidicola (Cercospora arachidicola), and late leaf spot (LLS), caused by Nothopassalora personata (syn. Cercosporidium personatum), but the potential of Peanut Rx to predict each disease has never been formally evaluated. From 2010 to 2016, non-fungicide-treated peanut plots in Georgia and Florida were sampled to monitor the development of ELS and LLS. This resulted in 168 cases (unique combinations of Peanut Rx risk factors) with associated total leaf spot risk points ranging from 40 to 100. Defoliation ranged from 13.9 to 100%, and increased significantly with increasing total risk points (conditional R2 = 0.56; P < 0.001). Leaf spot onset (time in days after planting [DAP] when either leaf spot reached 1% lesion incidence), ELS onset, and LLS onset ranged from 29 to 140, 29 to 142, and 50 to 143 DAP, respectively, and decreased significantly with increasing risk points. Standardized AUDPC of ELS was significantly affected by risk points (conditional R2 = 0.53, P < 0.001), but not for LLS. After removing redundant Peanut Rx factors, planting date, rotation, historical leaf spot prevalence, cultivar, and field history were used as fixed effects in mixed effect regression models to evaluate their contribution to leaf spot, ELS or LLS prediction. Results from mixed effects regression confirmed that the selected Peanut Rx risk factors contributed to the variability of at least one measurement of development of combined or separate epidemics of ELS and LLS, but not all factors affected ELS and LLS equally. Historical leaf spot prevalence, a new potential preplant risk factor, was a consistent predictor of the dominant disease(s) observed in the field. Results presented here demonstrate that Peanut Rx is a very effective tool for predicting leaf spot onset regardless of which leaf spot is predominant, but also suggest that associated risk does not reflect the same development for each disease. These data will be useful for refining thresholds for differentiating high, moderate, and low risk fields, and reevaluating the timing of fungicide applications in reduced input programs with respect to disease onset.

Fine-Scale Population Genetic Structure and Within-Tree Distribution of Mating Types of Venturia effusa, Cause of Pecan Scab in the United States.

Scab (caused by Venturia effusa) is the major disease of pecan in the southeastern United States. There is no information available on the fine-scale population genetic diversity or the occurrence of clonal types at small spatial scales that provides insight into inoculum sources and dispersal mechanisms, and potential opportunity for sexual reproduction. To investigate fine-scale genetic diversity, four trees of cultivar Wichita (populations) were sampled hierarchically: within each tree canopy, four approximately evenly spaced terminals (subpopulations) were selected and up to six leaflets (sub-subpopulations) were sampled from different compound leaves on each terminal. All lesions (n = 1 to 8) on each leaflet were sampled. The isolates were screened against a panel of 29 informative microsatellite markers and the resulting multilocus genotypes (MLG) subject to analysis. Mating type was also determined for each isolate. Of 335 isolates, there were 165 MLG (clonal fraction 49.3%). Nei's unbiased measure of genetic diversity for the clone-corrected data were moderate to high (0.507). An analysis of molecular variance demonstrated differentiation (P = 0.001) between populations on leaflets within individual terminals and between terminals within trees in the tree canopies, with 93.8% of variance explained among isolates within leaflet populations. Other analyses (minimum-spanning network, Bayesian, and discriminant analysis of principal components) all indicated little affinity of isolate for source population. Of the 335 isolates, most unique MLG were found at the stratum of the individual leaflets (n = 242), with similar total numbers of unique MLG observed at the strata of the terminal (n = 170), tree (n = 166), and orchard (n = 165). Thus, the vast majority of shared clones existed on individual leaflets on a terminal at the scale of 10s of centimeters or less, indicating a notable component of short-distance dispersal. There was significant linkage disequilibrium (P < 0.001), and an analysis of Psex showed that where there were multiple encounters of an MLG, they were most probably the result of asexual reproduction (P < 0.05) but there was no evidence that asexual reproduction was involved in single or first encounters of an MLG (P > 0.05). Overall, the MAT1-1-1 and MAT1-2-1 idiomorphs were at equilibrium (73:92) and in most populations, subpopulations, and sub-subpopulations. Both mating types were frequently observed on the same leaflet. The results provide novel information on the characteristics of populations of V. effusa at fine spatial scales, and provide insights into the dispersal of the organism within and between trees. The proximity of both mating idiomorphs on single leaflets is further evidence of opportunity for development of the sexual stage in the field.

Isolation of azole-resistant Aspergillus fumigatus from the environment in the south-eastern USA.

Background: Azole resistance in isolates of the fungus Aspergillus fumigatus has been associated with agricultural use of azole fungicides. Environmental isolation of resistant isolates has been reported in Asia, Africa, Europe and South America. Objectives: To determine whether A. fumigatus isolates containing TR34/L98H or TR46/Y121F/T289A can be found in fields in the USA treated with agricultural azoles. Methods: Crop debris was collected and screened for A. fumigatus. All A. fumigatus isolates were screened for azole resistance. The CYP51A gene of azole-resistant isolates was sequenced. The population structure of a subset of isolates was determined using microsatellite typing. Results: This article identifies azole-resistant A. fumigatus isolates containing the TR34/L98H mutation in an experimental peanut field that had been treated with azole fungicides. Conclusions: These findings suggest the development of resistance to azole antifungals in A. fumigatus may be present where agricultural azoles are used in the USA.

Population Genetic Structure of Venturia effusa, Cause of Pecan Scab, in the Southeastern United States.

Venturia effusa is the most important pathogen of pecan in the southeastern United States. Little information exists on the population biology and genetic diversity of the pathogen. A hierarchical sampling of 784 isolates from 63 trees in 11 pecan orchards in the southeastern United States were screened against a set of 30 previously characterized microsatellite markers. Populations were collected from Georgia (n = 2), Florida (n = 1), Alabama (n = 2), Mississippi (n = 1), Louisiana (n = 1), Illinois (n = 1), Oklahoma (n = 1), Texas (n = 1), and Kansas (n = 1). Clonality was low in all orchard populations (≤10.1% of isolates), and there were consistently high levels of genotypic diversity (Shannon-Weiner's index = 3.49 to 4.59) and gene diversity (Nei's measure = 0.513 to 0.713). Analysis of molecular variance showed that, although 81% of genetic diversity occurred at the scale of the individual tree, 16% occurred between orchards and only 3% between trees within orchards. All populations could be differentiated from each other (P = 0.01), and various cluster analyses indicated that some populations were more closely related compared with other pairs of populations. This is indicative of some limited population differentiation in V. effusa in the southeastern United States. Bayesian and nearest-neighbor methods suggested eight clusters, with orchards from Georgia and Florida being grouped together. A minimum spanning tree of all 784 isolates also indicated some isolate identification with source population. Linkage disequilibrium was detected in all but one population (Kansas), although 8 of the 11 populations had <20% of loci at disequilibrium. A Mantel test demonstrated a relationship between physical and genetic distance between populations (Z = 11.9, r = 0.559, P = 0.001). None of the populations were at mutation-drift equilibrium. All but 3 of the 11 populations had a deficiency of gene diversity compared with that expected at mutation-drift equilibrium (indicating population expansion); the remaining populations had an excess of gene diversity compared with that expected at mutation-drift equilibrium (indicating a recent bottleneck). These observations are consistent with the known history of pecan and pecan scab, which is that V. effusa became an issue on cultivated pecan in the last approximately 120 years (recent population expansion). Recently reported mating type genes and the sexual stage of this fungus may help explain the observed population characteristics, which bear a strong resemblance to those of other well-characterized sexually reproducing ascomycete pathogens.

Sensitivity of Fusarium oxysporum f. sp. niveum to Prothioconazole and Thiophanate-Methyl and Gene Mutation Conferring Resistance to Thiophanate-Methyl.

Fusarium wilt, incited by the fungus Fusarium oxysporum f. sp. niveum, is a soilborne disease that affects watermelon production worldwide. Approaches for effective management of Fusarium wilt in watermelon are limited. Studies conducted in recent years indicated that prothioconazole and thiophanate-methyl reduced the disease significantly under field conditions. However, effects of the fungicides on different life stages of F. oxysporum f. sp. niveum and potential existence of fungicide resistance in F. oxysporum f. sp. niveum populations are unknown. In the present study, effects of prothioconazole and thiophanate-methyl on mycelium growth and spore germination of F. oxysporum f. sp. niveum isolates collected in watermelon fields in Georgia were determined. In vitro mycelium growth studies indicated that all 100 isolates evaluated were sensitive to prothioconazole; the effective concentration that suppressed mycelium growth by 50% ranged from 0.75 to 5.69 µg/ml (averaged 1.62 µg/ml). In contrast, 33 and 4% of the isolates were resistant to thiophanate-methyl at 10 and 100 µg/ml, respectively. Microconidial germination assays showed that 36 and 64% of the isolates tested were sensitive or intermediately sensitive to prothioconazole at 100 µg/ml but the fungicide did not inhibit spore germination at 10 µg/ml. Sequencing a portion of the ß-tubulin gene of eight isolates resistant or sensitive to thiophanate-methyl indicated that fungicide resistance was associated with a point mutation at nucleotide position 200, resulting in a substitution of phenylalanine by tyrosine. This is the first report of isolates of F. oxysporum resistant to thiophanate-methyl. Results of the research suggest that prothioconazole may be a viable option for management of Fusarium wilt of watermelon whereas thiophanate-methyl should be used judiciously due to the existence of isolates resistant to the fungicide.

Severity of Scab and its Effects on Fruit Weight in Mechanically Hedge-Pruned and Topped Pecan Trees.

Scab is the most damaging disease of pecan in the southeastern United States. Pecan trees can attain 44 m in height, so managing disease in the upper canopy is a problem. Fungicide is ordinarily applied using ground-based air-blast sprayers. Although mechanical hedge-pruning and topping of pecan is done for several reasons, improved management of scab is an important reason in the humid, wet Southeast. Resulting shoot growth on cut limbs of susceptible cultivars could lead to more severe scab. In three experiments over three years, we explored the effect of hedge-pruning trees to ∼12 to 14 m compared with non-hedge-pruned trees. All trees received fungicide treatments (air-blast sprays and ≤3 aerial applications). Hedge-pruning either had no effect, or increased or decreased scab severity only slightly on leaflets, immature, or mature fruit (a -9.95 to +14.63% difference in scab severity compared with the control). However, height in the canopy invariably had a large and significant effect on scab severity, and amounted to a 0.05 to 73.77% difference in severity between the lowest and highest sample in the canopy. Fruit weight depended on sample height, with fruit most often weighing less when collected at greater sample heights. A robust relationship between fruit weight and scab severity was found at the highest sample heights where scab was also most often severe (R2 = 0.21 to 0.67, P < 0.0001). Hedge-pruning and topping pecan tree canopies to manage tree size will enable better fungicide coverage, reducing risk of a scab epidemic as more of the canopy is assured efficacious fungicide spray coverage.

Differences in Sensitivity to a Triazole Fungicide Among Stagonosporopsis Species Causing Gummy Stem Blight of Cucurbits.

Gummy stem blight (GSB) is a destructive disease of cucurbits caused by three closely related Stagonosporopsis species. In the southeastern United States, GSB management relies heavily on triazole fungicides. Our objectives were to determine if resistance to triazoles has developed in populations of GSB fungi in the southeastern United States, and if so, to investigate the molecular basis of resistance. A tebuconazole sensitivity assay was conducted on 303 Stagonosporopsis citrulli and 19 S. caricae isolates collected from the southeastern United States in 2013 and 2014, as well as three S. citrulli, three S. cucurbitacearum, and six S. caricae isolates from other regions or years. Tebuconazole resistance was detected for all 19 S. caricae isolates from the southeastern United States and one S. caricae isolate from Brazil. All S. citrulli and S. cucurbitacearum isolates were sensitive to tebuconazole. For resistant and sensitive isolates of S. caricae, coding and promoter regions of the target gene Cyp51 were sequenced and expression levels of Cyp51 and ScAtrG (an ATP-binding cassette transporter) were measured. Tebuconazole resistance was not associated with mutations within Cyp51, multiple copies of Cyp51, changes in the promoter region, or increased expression of Cyp51 or ScAtrG. Tebuconazole resistance may explain the increase in frequency of S. caricae isolates recovered from GSB-infected cucurbits in Georgia.

Allele-Specific PCR for the Detection of Azoxystrobin Resistance in Didymella bryoniae.

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is considered the most widespread and destructive disease of watermelon in the southeastern United States. The quinone outside-inhibiting (QoI) fungicide azoxystrobin (AZO), which inhibits mitochondrial respiration by binding to the outer, quinone-oxidizing pocket of the cytochrome bc1 (cyt b) enzyme complex, was initially very effective in controlling GSB. However, resistance to AZO has been observed in D. bryoniae in many watermelon-producing regions. In this study, the DNA sequences of partial cyt b genes of four AZO-resistant (AZO-R) and four AZO-sensitive (AZO-S) isolates of D. bryoniae confirmed the amino acid substitution of glycine by alanine at the 143 codon (G143A) in the AZO-R isolates tested. Allele-specific primers were designed to detect the resistant or sensitive allele at codon 143 of the cyt b gene, which amplified a 165-bp polymerase chain reaction (PCR) product from genomic DNA of nine AZO-R and nine AZO-S isolates of D. bryoniae, respectively. The primer pairs did not amplify DNA from other pathogens tested in the study. The results indicated that the PCR assays developed in the study were specific in differentiating AZO-R and AZO-S isolates and could facilitate AZO resistance detection in D. bryoniae.

Induction of Systemic Acquired Resistance by Rotylenchulus reniformis and Meloidogyne incognita in Cotton Following Separate and Concomitant Inoculations.

Systemic acquired resistance (SAR) can be elicited by virulent and avirulent pathogenic strains and SAR against plant-parasitic nematodes has been documented. Our objective was to determine whether co-infection of cotton by Meloidogyne incognita and Rotylenchulus reniformis affects the population level of either nematode compared to infection by each species individually. Split-root trials were conducted in which plants were inoculated with i) R. reniformis only, ii) M. incognita only, iii) both R. reniformis and M. incognita, or iv) no nematodes. Half of the root system was inoculated with R. reniformis or M. incognita on day 0 and the other half with M. incognita or R. reniformis on day 0 or day 14 depending on the experiment. Experiments were conducted on cotton cultivar DP 0935 B2RF (susceptible to both nematodes), LONREN-1 (germplasm line resistant to R. reniformis), and M-120 RNR (germplasm line resistant to M. incognita), and tests were terminated 8 wk after the last inoculation. Both soil (vermiform) and roots (egg) extracted from each half of the root system to determine the total nematode population levels, and root galling was rated on a 0 to 10 scale. Mixed models analysis and comparison of least squares means indicated no differences in root galling (except on LONREN-1) or population levels when the two nematode species were introduced on the same day. When M. incognita was introduced 14 d after R. reniformis, reduction in galling (36% on DP 0935 and 33% on LONREN-1) and M. incognita population levels (35% on DP 0935 and 45% on LONREN-1) were significant (P ≤ 0.05). When R. reniformis was inoculated 14 d after M. incognita, reduction in R. reniformis population levels (18% on DP 0935 and 26% on M-120) were significant. This study documents for the first time that infection of cotton by a nematode can elicit SAR to another nematode species.

Influence of infection of cotton by rotylenchulus reniformis and meloidogyne incognita on the production of enzymes involved in systemic acquired resistance.

Systemic acquired resistance (SAR), which results in enhanced defense mechanisms in plants, can be elicited by virulent and avirulent strains of pathogens including nematodes. Recent studies of nematode reproduction strongly suggest that Meloidogyne incognita and Rotylenchulus reniformis induce SAR in cotton, but biochemical evidence of SAR was lacking. Our objective was to determine whether infection of cotton by M. incognita and R. reniformis increases the levels of P-peroxidase, G-peroxidase, and catalase enzymes which are involved in induced resistance. A series of greenhouse trials was conducted; each trial included six replications of four treatments applied to one of three cotton genotypes in a randomized complete block design. The four treatments were cotton plants inoculated with i) R. reniformis, ii) M. incognita, iii) BTH (Actigard), and iv) a nontreated control. Experiments were conducted on cotton genotypes DP 0935 B2RF (susceptible to both nematodes), LONREN-1 (resistant to R. reniformis), and M-120 RNR (resistant to M. incognita), and the level of P-peroxidase, G-peroxidase, and catalase activity was measured before and 2, 4, 6, 10, and 14 d after treatment application. In all cotton genotypes, activities of all three enzymes were higher (P ≤ 0.05) in leaves of plants infected with M. incognita and R. reniformis than in the leaves of control plants, except that M. incognita did not increase catalase activity on LONREN-1. Increased enzyme activity was usually apparent 6 d after treatment. This study documents that infection of cotton by M. incognita or R. reniformis increases the activity of the enzymes involved in systemic acquired resistance; thereby providing biochemical evidence to substantiate previous reports of nematode-induced SAR in cotton.

Differences in fungicide resistance profiles and multiple resistance to a quinone-outside inhibitor (QoI), two succinate dehydrogenase inhibitors (SDHI), and a demethylation inhibitor (DMI) for two Stagonosporopsis species causing gummy stem blight of cucurbits.

BACKGROUND: Gummy stem blight (GSB) is a devastating disease of cucurbits that has been effectively managed with fungicide applications. However, the Stagonosporopsis spp. that cause GSB have rapidly evolved resistance to multiple classes of fungicides. To better understand the evolution and persistence of fungicide resistance in field populations, resistance profiles of unique and clonal genotypes of 113 Stagonosporopsis citrulli and 19 S. caricae isolates to four different fungicides were determined based on in vitro mycelial growth assays and molecular markers based on genes encoding fungicide targets. RESULTS: All 19 S. caricae isolates screened were resistant to tebuconazole and azoxystrobin, and sensitive to boscalid and fluopyram. All 113 S. citrulli isolates were sensitive to tebuconazole and sensitive to fluopyram, with one exception that was fluopyram-resistant. All isolates of S. citrulli except two were resistant to azoxystrobin. Phenotypic differences in response to boscalid were detected among S. citrulli isolates, but the phenotypes were not associated with multilocus genotypes (MLG) determined by 16 microsatellite loci. Additionally, isolates sharing the same MLG varied by SdhB genotype. A unique mutation of I229V in SdhB, a target of succinate dehydrogenase inhibitor fungicides, was detected for the fluopyram-resistant isolate of S. citrulli. CONCLUSION: Both the lack of association of fungicide resistance profiles with genetic similarity of isolates based on microsatellite loci and the finding that widely distributed MLG varied in fungicide resistance profiles suggest that independent evolutionary events for resistance to boscalid have likely occurred. Frequent genetic recombination within populations may be responsible for resistance to multiple fungicides. This study provides useful information for effectively managing both species of GSB fungi present in the southeastern USA and understanding the evolution of fungicide resistance within populations of plant-pathogenic fungi. © 2019 Society of Chemical Industry.

A Pictorial Disease Severity Key and the Relationship Between Severity and Incidence for Black Root Rot of Pansy Caused by Thielaviopsis basicola.

A pictorial key was developed and the relationship between disease severity (S) and incidence (I) was examined to aid in the assessment of black root rot of pansy caused by Thielaviopsis basicola. The key consisted of photographs of root segments that represented nine disease severity levels ranging from 1 to 91%. Pansies that had received different fertility treatments, as part of seven separate experiments, were inoculated with T. basicola. Four weeks after inoculation, roots were washed, and incidence and severity of black root rot were visually assessed using a grid-line-intersect method. Disease incidence ranged from 1.3 to 100%, and severity ranged from 0.1 to 21.4% per plant. Four different mathematical models were compared to quantitatively describe the I-S relationship for the combined data from all seven experiments. Although all models provided an adequate fit, the model that is analogous to the Kono-Sugino equation provided the most reliable estimate of severity over the entire range of disease incidence values. The predictive ability and accuracy of this model across data sets was verified by jackknife and cross-validation techniques. We concluded that incidence of black root rot in pansy can be assessed more objectively and with greater precision than disease severity and can be used to provide reliable estimates of disease severity based on derived regression equations that quantify the I-S relationship for black root rot.

Draft genome sequence of Fusicladium effusum, cause of pecan scab.

Pecan scab, caused by the plant pathogenic fungus Fusicladium effusum, is the most destructive disease of pecan, an important specialty crop cultivated in several regions of the world. Only a few members of the family Venturiaceae (in which the pathogen resides) have been reported sequenced. We report the first draft genome sequence (40.6 Mb) of an isolate F. effusum collected from a pecan tree (cv. Desirable) in central Georgia, in the US. The genome sequence described will be a useful resource for research of the biology and ecology of the pathogen, coevolution with the pecan host, characterization of genes of interest, and development of markers for studies of genetic diversity, genotyping and phylogenetic analysis. The annotation of the genome is described and a phylogenetic analysis is presented.

Sensitivity of Sclerotinia homoeocarpa Isolates to Propiconazole and Impact on Control of Dollar Spot.

In response to reports of reduced efficacy of propiconazole for control of dollar spot, isolates of Sclerotinia homoeocarpa were collected from several locations in Georgia and tested for sensitivity to propiconazole and other demethylation-inhibiting (DMI) fungicides. Two discriminatory concentrations of propiconazole (0.02 and 0.2 µg ml-1) were used to detect lower in vitro sensitivity in two populations that had been exposed repeatedly to propiconazole than in four nonexposed populations. Mean 50% effective concentration (EC50) values for a nonexposed population (baseline) and a DMI-exposed population were 0.0049 and 0.0283 µg ml-1, respectively. Positive correlations were significant among log10 EC50 values for propiconazole, fenarimol, and myclobutanil but not between triadimefon and any of the other three fungicides, indicating cross-resistance relationships in this pathogen may not be universal among the DMIs. In greenhouse experiments, propiconazole-treated bentgrass was inoculated with seven isolates of S. homoeocarpa differing in sensitivity to propiconazole. Incubation period decreased and relative area under the disease progress curve and disease severity 28 days after inoculation increased linearly with increasing log10 EC50 value of the isolate. Results of this study confirm a significant relationship between in vitro sensitivity of S. homoeocarpa and in planta control efficacy of propiconazole and provide evidence of field resistance to propiconazole in S. homoeocarpa in Georgia.

Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram.

BACKGROUND: Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B(VHR) ) and highly resistant (B(HR) ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B(VHR) isolates were only highly resistant to penthiopyrad (B(VHR) -P(HR) ), while the B(HR) isolates appeared sensitive to penthiopyrad (B(HR) -P(S) ). In this study, the molecular mechanism of resistance in these two phenotypes (B(VHR) -P(HR) and B(HR) -P(S) ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS: A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B(S) -P(S) ), two B(VHR) -P(HR) and one B(HR) -P(S) isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. CONCLUSION: The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management.