Phillip M. Brannen

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.

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.

The Bacterial Pathogen Xylella fastidiosa Affects the Leaf Ionome of Plant Hosts during Infection

Xylella fastidiosa is a plant pathogenic bacterium that lives inside the host xylem vessels, where it forms biofilm believed to be responsible for disrupting the passage of water and nutrients. Here, Nicotiana tabacum was infected with X. fastidiosa, and the spatial and temporal changes in the whole-leaf ionome (i.e. the mineral and trace element composition) were measured as the host plant transitioned from healthy to diseased physiological status. The elemental composition of leaves was used as an indicator of the physiological changes in the host at a specific time and relative position during plant development. Bacterial infection was found to cause significant increases in concentrations of calcium prior to the appearance of symptoms and decreases in concentrations of phosphorous after symptoms appeared. Field-collected leaves from multiple varieties of grape, blueberry, and pecan plants grown in different locations over a four-year period in the Southeastern US showed the same alterations in Ca and P. This descriptive ionomics approach characterizes the existence of a mineral element-based response to X. fastidiosa using a model system suitable for further manipulation to uncover additional details of the role of mineral elements during plant-pathogen interactions. This is the first report on the dynamics of changes in the ionome of the host plant throughout the process of infection by a pathogen.

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.

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.

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.

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.

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.

Assessment of Mycotoxins in Vitis vinifera Wines of the Southeastern United States

Mycotoxins pose a serious worldwide threat to the safety of numerous food commodities. Red wine is prone to contamination from ochratoxin A, produced by black-spored Aspergillus spp.; it was recently discovered that some of these species can also produce the mycotoxin fumonisin B2. Although wine surveys in most world regions have determined that mean ochratoxin A levels are below the European Union’s recommended guideline maximum (2 μg/L), monitoring toxin levels in poor vintages and in emerging regions is imperative to ensure safety. The hot, humid environment of the southeastern United States proves challenging to winegrapes because of opportunistic fungal growth and high disease pressure, yet 550+ wineries exist in this expanding region. To determine the mycotoxin potential in southeastern vineyards, 30 cluster samples (representing 10 grape varieties, eight vineyards, and three states) were collected during the 2013 harvest and analyzed for ochratoxin A and fumonisins. Additionally, nearly 200 bottles of 100% southeastern U.S. red vinifera wines were tested for mycotoxins via liquid chromatography–tandem mass spectrometry (LC-MS/MS). These wines represent 18 grape varieties grown across six states during vintages between 2001 and 2013. Only four out of 157 (2.5%) wine samples tested positive for ochratoxin A, with the highest toxin quantity being at a safe level of 0.48 μg/L. Most wines (25/27, 92.6%) contained total fumonisins (B1, B2, and B3) at extremely low concentrations (<15 μg/L), far below a health-risk level. Only one grape must sample was positive for mycotoxins (20.6 μg/L fumonisin B1) out of 30 tested. Based on these results, the southeastern U.S. Vitis vinifera wine industry has a low occurrence of the mycotoxin ochratoxin A and insignificant amounts of fumonisins in red wines.

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.