Resistance in Alternaria alternata to SDHI Fungicides Causes Rare Disease Outbreak in Peach Orchards.

Alternaria rot, caused by Alternaria alternata, was observed in commercial peach orchards in South Carolina. Single-spore isolates formed two phenotypically different culture morphologies on two artificial media. Isolates highly resistant (HR) to boscalid were also HR to penthiopyrad. Among isolates HR and medium resistant (MR) to boscalid and penthiopyrad, we detected isolates that were MR to fluopyram and HR to fluxapyroxad. Sequence analysis of succinate dehydrogenase (sdh) genes sdhB, sdhC, and sdhD revealed that resistant phenotypes were associated with point mutations leading to amino acid substitutions. In particular, H277Y/R in the SDHB and H134R in SDHC were consistently associated with the boscalid HR phenotype. The highest effective concentration that inhibits growth by 50% (EC50) values to penthiopyrad were conferred by H134R and D123E, whereas H134R conferred low resistance and MR to fluxapyroxad. A previously undescribed mutation, G79R, was identified in our collection conferring HR to both boscalid and penthiopyrad. The point mutations associated with highest EC50 values to all four FRAC 7 fungicides were H277L and H134R. The outbreak of Alternaria rot demonstrates that fungicide programs containing "medium to high-risk fungicides" may promote disease outbreaks by secondary pathogens that typically are outcompeted or controlled effectively, while still controlling the primary target disease.

Management of Meloidogyne incognita with Jesup (Max-Q) Tall Fescue Grass Prior to Peach Orchard Establishment.

The effects of two preplant Jesup (Max-Q) tall fescue (Schedonorus arundinaceus) ground cover treatments as alternatives to chemical control of Meloidogyne incognita were investigated from 2006 to 2013. The experiment was initiated in 2006 in a site known to be infested with M. incognita. Treatments included (i) 1 year of peach followed by 1 year of Jesup (Max-Q), (ii) 2 years of continuous Jesup (Max-Q), (iii) 2 years of continuous peach, and (iv) 2 years of continuous peach followed by fumigation with 1,3-dichloropropene (1,3-D). Both Jesup (Max-Q) treatments suppressed (P ≤ 0.05) population densities of M. incognita second-stage juveniles (J2) compared with the 2-year continuous peach treatments (prior to fumigation); no J2 were detected in soil samples taken from 2-year continuous Jesup (Max-Q) plots. Evaluation of the effects of the Jesup (Max-Q) treatments on subsequent peach tree planting was initiated in 2008, when herbicide was applied to the tall fescue treatment plots and half the continuous peach plots were fumigated with 1,3-D (nematicide treatment control). Peach trees were planted into all plots in 2009. By the end of the experiment (48 months after orchard establishment), trunk diameter was greater (P ≤ 0.01) in both of the Jesup (Max-Q) treatments and in the 1,3-D-fumigated treatment than in the nonfumigated treatment. Moreover, trunk diameter did not differ among the Jesup (Max-Q) and the fumigated treatments. Preplant Jesup (Max-Q) tall fescue was as effective as 1,3-D fumigation in increasing tree growth in an M. incognita-infested site.

First Report of Fludioxonil Resistance in Botrytis cinerea from a Blackberry Field in Georgia.

Botrytis cinerea Pers. is an important plant-pathogenic fungi responsible for gray mold on more than 230 plant species worldwide, including blackberry (Rubus). One of the main strategies to control the disease involves the application of different classes of fungicides. The phenylpyrrole fludioxonil is currently marketed in combination with the anilinopyrimidine cyprodinil as Switch 62.5WG (Syngenta Crop Protection Inc., Greensboro, NC) for gray mold control. In August 2013, blackberries affected with symptoms resembling gray mold were collected from a field located in Berrien County (Georgia), where Switch 62.5WG had been used extensively over the last 5 years. Three single-spore isolates, each from a different fruit, were obtained and identified as B. cinerea on the basis of morphology and confirmed by a 238-bp PCR amplification product obtained with primer set G3PDH-F1 (5'-GGACCCGAGCTAATTTATGTCACGT-3'), G3PDH-F2 (5'-GGGTGTCAACAACGAGACCTACACT-3'), and G3PDH-R (5'-ACCGGTGCTCGATGGGATGAT-3'). In vitro sensitivity to fludioxonil (Scholar SC, Syngenta) was determined on 1% malt extract agar (MEA) using a conidial germination assay as previously described (4). One isolate was moderately resistant due to growth on medium amended with the discriminatory dose of 0.1 µg/ml fludioxonil and residual growth at 10 µg/ml (4). To assess performance of fludioxonil in detached fruit assays, commercially grown strawberries (24 in total for each isolate and treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water (control fruit) or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Scholar SC was used because fludioxonil was the sole active ingredient in this product and strawberries were used because latent infections in fresh blackberry fruit interfered with inoculation experiments. This dose reflects the rate recommended for postharvest gray mold control according to the Scholar label. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of conidia suspension (106 spores/ml) of the two sensitive or the resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolates developed gray mold on non-treated (2.7 cm lesion diameter) but not on Scholar SC-treated fruit (0.0 cm lesion diameter). The resistant isolate developed gray mold disease on the water-treated control fruit (2.5 cm lesion diameter) and the fungicide-treated fruit (1.8 cm lesion diameter). EC50 values were determined in microtiter assays as described previously (3) using the concentrations of 0.01, 0.04, 0.12, 0.37, 1.1, 3.3, and 10 µg/ml fludioxonil. Values were 0.02 and 0.05 µg/ml for the two sensitive isolates and 3.15 µg/ml for the resistant isolate. All experiments were performed twice. This is the first report of fludioxonil resistance in B. cinerea from blackberry in Georgia. Prior to this study, resistance to fludioxonil in B. cinerea was reported in France, Germany, and only a few states in the United States including Maryland, South Carolina, Virginia, and Washington (1,2). The emergence of resistance to fludioxonil emphasizes the importance of resistance management strategies. References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 98:692, 2013. (3) M. Kretschmer et al. PLOS Pathog. 5:e1000696, 2009. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

Incidence and Pathogenicity of Plant-Parasitic Nematodes Associated with Blueberry (Vaccinium spp.) Replant Disease in Georgia and North Carolina.

Blueberry replant disease (BRD) is an emerging threat to continued blueberry (Vaccinium spp.) production in Georgia and North Carolina. Since high populations of ring nematode Mesocriconema ornatum were found to be associated with commercially grown blueberries in Georgia, we hypothesized that M. ornatum may be responsible for predisposing blueberry to BRD. We therefore tested the pathogenicity of M. ornatum on 10-wk-old Rabbiteye blueberries (Vaccinium virgatum) by inoculating with initial populations (Pi) of 0 (water control), 10, 100, 1,000. and 10,000 mixed stages of M. ornatum/pot under both greenhouse (25 ± 2°C) and field microplot conditions. Nematode soil population densities and reproduction rates were assessed 75, 150, 225, and 255, and 75, 150, 225, and 375 d after inoculation (DAI) in both the greenhouse and field experiments, respectively. Plant growth parameters were recorded in the greenhouse and field microplot experiments at 255 and 375 DAI, respectively. The highest M. ornatum population density occurred with the highest Pi level, at 75 and 150 DAI under both greenhouse (P < 0.01) and field (P < 0.01) conditions. However, M. ornatum rate of reproduction increased significantly in pots receiving the lowest Pi level of 10 nematodes/plant compared with the pots receiving Pi levels of 100, 1,000, and 10,000 nematodes 75 DAI. Plant-parasitic nematode populations were determined in commercial blueberry replant sites in Georgia and North Carolina during the 2010 growing season. Mesocriconema ornatum and Dolichodorus spp. were the predominant plant-parasitic nematodes in Georgia and North Carolina, respectively, with M. ornatum occurring in nearly half the blueberry fields sampled in Georgia. Other nematode genera detected in both states included Tylenchorhynchus spp., Hoplolaimus spp., Hemicycliophora spp., and Xiphinema spp. Paratrichodorus spp. was also found only in Georgia. In Georgia, our results indicate that blueberry is a host for M. ornatum and its relationship to BRD warrants further investigation.

Reduced Sensitivity in Monilinia fructicola Field Isolates from South Carolina and Georgia to Respiration Inhibitor Fungicides.

Quinone outside inhibitor (QoI) and succinate dehydrogenase inhibitor (SdhI) fungicides are respiration inhibitors (RIs) used for preharvest control of brown rot of stone fruit. Both chemical classes are site-specific and, thus, prone to resistance development. Between 2006 and 2008, 157 isolates of Monilinia fructicola collected from multiple peach and nectarine orchards with or without RI spray history in South Carolina and Georgia were characterized based upon conidial germination and mycelial growth inhibition for their sensitivity to QoI fungicides azoxystrobin and pyraclostrobin, SdhI fungicide boscalid, and a mixture of pyraclostrobin + boscalid. There was no significant difference (P = 0.05) between EC50 values for inhibition of conidial germination versus mycelial growth. The mean EC50 values based upon mycelial growth tests for 25 isolates from an orchard without RI-spray history were 0.15, 0.06, 2.23, and 0.09 µg/ml for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. The respective mean EC50 values for 76 isolates from RI-sprayed orchards in South Carolina were 0.9, 0.1, 10.7, and 0.13 µg/ml and for 56 isolates from RI-sprayed orchards in Georgia were 1.2, 0.1, 8.91, and 0.17 µg/ml. Overall, mean EC50 values of populations from RI-sprayed orchards increased three-, two-, five-, and twofold between 2006 and 2008 for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. A subset of 10 M. fructicola isolates representing low and high EC50 values for azoxystrobin, boscalid, and boscalid + pyraclostrobin was selected for a detached fruit assay to determine disease incidence and severity following protective treatments of formulated RI fungicides at label rates. Brown rot incidence was greater than 50% when fruit were inoculated with isolates having EC50 values of 2, 4, and 0.6 µg/ml for azoxystrobin, boscalid, and pyraclostrobin + boscalid, respectively. Pyraclostrobin failed to control any of the isolates tested in detached fruit assays. Based on minimum inhibitory concentration and brown rot incidence data, we recommend using 3 and 0.75 µg/ml as discriminatory doses to distinguish between sensitive isolates and those with reduced sensitivity to azoxystrobin and pyraclostrobin + boscalid, respectively. Results from our in vitro and in vivo assays indicate a shift toward reduced sensitivity in M. fructicola from the southeastern United States. No cross-resistance was observed between the QoI and the SdhI fungicides, which implies that rotation or tank mixtures of these two chemical classes can be used as a resistance management strategy.

Laboratory Evaluation of Three Rapid, Agar-Based Assays to Assess Fungicide Sensitivity in Monilinia fructicola.

Three rapid, agar-based assays were compared with a traditional petri dish method for assessing the sensitivity of Monilinia fructicola to propiconazole (0.3 and 2.0 µg/ml), thiophanate-methyl (1.0 and 50 µg/ml), and azoxystrobin (1.0 and 35 µg/ml) in the laboratory. The three assays were based on mycelial growth inhibition on agar disks sliced from lipbalm tubes filled with fungicide-amended potato dextrose agar (PDA), on PDA-coated cotton swabs, or in PDA-filled microcentrifuge tubes. Mycelial growth inhibition of eight previously characterized isolates (two resistant to propiconazole, two highly resistant to thiophanate-methyl, two with low levels of resistance to thiophanate-methyl, and two sensitive to all three fungicides) was determined visually 24, 48, and 72 h after inoculation. The 48-h time point was the earliest suitable time to collect data for all methods because insufficient growth was recorded in the petri dish and tube assays after 24 h. With the exception of the swab assay, all methods classified the isolates previously determined to be fungicide sensitive correctly (i.e., no fungal growth was observed for these isolates). For propiconazole-resistant isolates, the lipbalm assay resulted in levels of growth inhibition very similar to the petri dish method, whereas the swab assay and the tube assay overestimated and underestimated, respectively, the level of resistance. Both the lipbalm and the swab assays classified isolates correctly as being thiophanate-methyl resistant, and both were able to discriminate the isolates previously classified as having low versus high levels of resistance when treated with this fungicide at 50 µg/ml, as was the petri dish method. None of the eight isolates which previously were determined to be azoxystrobin sensitive grew on azoxystrobin-amended media, regardless of the assay type. Overall, the average percentage of correct isolate classifications (relative to their previously determined resistance status) on propiconazole- and thiophanate-methyl-amended media after 48 h ranged from 87.5 to 100, 85.3 to 100, 63.2 to 94.5, and 50.5 to 81.0% for the petri dish, lipbalm, swab, and tube assays, respectively. The lipbalm assay provided the most accurate assessments (85.3 to 100%) after only 24 h of incubation, supporting its use as a rapid and simple tool to monitor resistance levels in M. fructicola field populations.

Septoria Leaf Spot Reduces Flower Bud Set and Yield Potential of Rabbiteye and Southern Highbush Blueberries.

In field trials on Premier rabbiteye blueberry, individual shoots were selected and tagged in the fall of 2001, 2002, and 2003 to quantify the effects of Septoria leaf spot severity and disease-induced premature defoliation on flower bud set and return yield. Experiments were carried outsimilarly on Bluecrisp southern highbush blueberry using shoots tagged after fruit harvest in the summer of 2002 and 2003. Leaves on the distal 20-cm segments of these shoots were monitored for disease severity (number of spots per leaf) through the remainder of the growing season; at the same time, defoliation (expressed as the proportion of nodes with missing leaves) was recorded for each of the shoot segments. Flower bud set was assessed subsequently in winter or early spring, and berries were harvested as they matured the following summer to determine return yield. For both cultivars, higher flower bud numbers were more likely to occur on shoots with lower disease levels the previous fall (P ≤ 0.0462 based on a Kolmogorov-Smirnov test). The data further showed that flower bud set potential (i.e., the maximum number of buds on shoots within a given disease severity range) decreased linearly as disease severity increased (r2 ≥ 0.926, P ≤ 0.0005). Based on the slope of this relationship, flower bud set potential decreased by one bud per shoot as disease severity the previous fall increased by 18 and 12 spots per leaf for Premier and Bluecrisp, respectively. Relationships between yield and disease variables were similar to those of flower bud numbers and disease, except that the decrease in yield potential (i.e., the maximum fruit weight per shoot within a given disease severity range) was less gradual than for flower bud set potential. On Premier, yield potential dropped markedly and significantly as disease severity the previous fall exceeded about 50 to 60 spots per leaf on average (P < 0.0001 based on a Kruskal-Wallis test). Evidence for such a threshold effect was weaker on Bluecrisp, presumably because of the lower number of data points for this cultivar combined with lower yields due to poor pollination.

Viability and stability of biological control agents on cotton and snap bean seeds.

Cotton and snap bean were selected for a multi-year, multi-state regional (south-eastern USA) research project to evaluate the efficacy of both commercial and experimental bacterial and fungal biological control agents for the management of damping-off diseases. The goal for this portion of the project was to determine the viability and stability of biological agents after application to seed. The biological seed treatments used included: (1) Bacillaceae bacteria, (2) non-Bacillaceae bacteria, (3) the fungus Trichoderma and (4) the fungus Beauveria bassiana. Seed assays were conducted to evaluate the following application factors: short-term (< or = 3 months) stability after seed treatment; quality (i.e. isolate purity); compatibility with chemical pesticides and other biocontrol agents; application uniformity between years and plant species. For the bacterial treatments, the Bacillaceae genera (Bacillus and Paenibacillus) maintained the greatest population of bacteria per seed, the best viability over time and the best application uniformity across years and seed type. The non-Bacillaceae genera Burkholderia and Pseudomonas had the least viability and uniformity. Although Beauveria bassiana was only evaluated one year, the seed fungal populations were high and uniform. The seed fungal populations and uniformity for the Trichoderma isolates were more variable, except for the commercial product T-22. However, this product was contaminated with a Streptomyces isolate in both the years that it was evaluated. The study demonstrated that Bacillaceae can be mixed with Trichoderma isolates or with numerous pesticides to provide an integrated pest control/growth enhancement package.