Sara M. Villani

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group-I-like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143-associated intron.

Prevalence of Myclobutanil Resistance and Difenoconazole Insensitivity in Populations of Venturia inaequalis

Demethylation inhibitors (DMIs) are a class of single-site fungicides with high levels of protective and curative efficacy against Venturia inaequalis, the causal agent of apple scab. To determine the prevalence of resistance to the DMI fungicide myclobutanil, 3,987 single-lesion conidial V. inaequalis isolates from 141 commercial, research, and baseline orchard populations were examined throughout New England, the mid-Atlantic, and the Midwest from 2004 to 2013. Of these orchard populations, 63% had practical resistance, 13% had reduced sensitivity, and 24% were sensitive to myclobutanil. A sensitivity baseline for the recently introduced DMI fungicide difenoconazole was established to make comparisons with myclobutanil sensitivity in orchard populations. The mean effective concentration of difenoconazole at which mycelial growth was inhibited by 50% (EC50) was determined to be 0.002 μg ml-1 for 44 baseline isolates of V. inaequalis. From 2010 to 2013, 1,012 isolates of V. inaequalis from 37 of the 141 orchard populations above were screened for sensitivity to difenoconazole. In all, 1 orchard population had reduced sensitivity to difenoconazole, while the remaining 36 orchard populations were sensitive to the fungicide. In field experiments, difenoconazole demonstrated high levels of apple scab control on mature apple fruit, despite the fact that the population of V. inaequalis had practical resistance to difenoconazole. Although our results indicate widespread resistance to myclobutanil but not difenoconazole, due to the propensity for cross-sensitivity among DMI fungicides, growers with myclobutanil resistance should be cautious when using difenoconazole for disease management.

Prevalence and Stability of Qualitative QoI Resistance in Populations of Venturia inaequalis in the Northeastern United States

Quinone-outside-inhibitor (QoI) fungicides are a safe and effective means of managing apple scab caused by Venturia inaequalis. To determine the prevalence of both quantitative (partial) and qualitative (complete) QoI resistance in V. inaequalis in the northeastern United States, we sampled single-lesion conidial isolates (n = 4,481) from 120 commercial and research orchards from 2004 to 2011 with a range of exposure to QoI fungicides from none to several applications a year. In all, 67% of these orchard populations of V. inaequalis were sensitive to QoI fungicides, 28% exhibited QoI practical resistance, and 5% were not sensitive QoI fungicides but had not become practically resistant. Isolates with qualitative QoI resistance, conferred by the G143A cytochrome b gene mutation, were found in 13 of the 34 QoI-resistant orchard populations. To evaluate the stability of the G143A mutation, 27 isolates were selected from different orchard populations to represent the scope of regional populations. These isolates were subcultured continuously in the presence or absence of the QoI fungicide trifloxystrobin. All isolates that initially possessed qualitative resistance maintained the resistant genotype (G143A) for six transfers over 6 months in both the absence and presence of trifloxystrobin. Given the observed QoI resistance in orchard populations of V. inaequalis and the stability of the G143A mutation in individual isolates, apple scab management paradigms must encompass strategies to limit selection of QoI resistance in the sensitive orchard populations remaining in the region.

Heteroplasmy of the cytochrome b Gene in Venturia inaequalis and Its Involvement in Quantitative and Practical Resistance to Trifloxystrobin

Quantitative (partial) and qualitative (complete) resistance responses to quinone outside inhibitor (QoI) fungicides have been documented for the apple scab pathogen Venturia inaequalis. Resistance monitoring efforts have traditionally focused on the detection of qualitative resistance based on a single point mutation, G143A, within the cytochrome b (cyt b) gene. In order to better understand the role of heteroplasmy of the cyt b gene in the QoI resistance response for isolates and populations of V. inaequalis, an allele-specific quantitative polymerase chain reaction was developed to quantify the relative abundance of the A143 (resistant) allele in 45 isolates of V. inaequalis with differing in vitro resistance responses to the QoI fungicide trifloxystrobin. Although a high relative abundance of the A143 allele (>62%) was associated with isolates with high resistance responses (50 to 100% relative growth on trifloxystrobin-amended medium), heteroplasmy of the cyt b gene was not the primary factor involved in isolates with moderate resistance responses (29 to 49% relative growth). The relative abundance of the A143 allele in isolates with moderate resistance to trifloxystrobin rarely exceeded 8%, suggesting that other resistance mechanisms are involved in moderate resistance and, therefore, that the Qol resistance response is polygenic. In research orchards where QoI fungicides failed to control apple scab (practical resistance), field trials were conducted to demonstrate the link between practical resistance and the abundance of the A143 allele. Relative abundance of the A143 allele in these orchard populations exceeded 20% in 2011 and 2012. Similarly, of the eight additional commercial orchards screened in 2011, the relative abundance of the A143 allele always exceeded 20% in those with QoI practical resistance. Although heteroplasmy of the cyt b gene did not entirely explain the response of isolates with moderate resistance to QoIs, the relative abundance of A143 in orchard populations of V. inaequalis helps to explain the point of emergence for practical resistance to trifloxystrobin across several orchard populations with differing production histories.

Confirmation of European Brown Rot Caused by Monilinia laxa on Tart Cherry, Prunus cerasus, in Western New York

Monilinia fructicola (G. Wint.) Honey and M. laxa (Aderh. & Ruhl.) Honey are two pathogens causing brown rot in the United States. While the presence of M. fructicola has been confirmed in all major stone-fruit-production regions in the United States, M. laxa has yet to be detected in much of the eastern production regions. In July 2008, a planting of tart cherries cv. Surefire in Appleton, NY developed severe shoot blight. Blighted shoots (>15% of first-year shoots) were wilted and light brown with the blight encompassing the distal end and often extending into second-year tissue with a distinct sunken margin. Leaves on symptomatic shoots had flushed, but were blighted. Blossom spurs were either blighted at bloom or bore fruit, which were subsequently blighted. Gummosis was commonly observed from cankers at the base of spurs. Both mature and immature mummified fruit in addition to spurs and shoot tissue were sporulating in a manner characteristic of Monilinia (2). Eleven branches displaying symptoms were removed for isolation. Sections of symptomatic shoots (5 cm long) were surface sterilized in 0.6% NaOCl for 1 min and rinsed in sterile dH2O. Cross sections of shoot tissue (3 mm thick), in addition to spores from fruit and spurs, were placed on potato dextrose agar amended with 50 μg/ml of streptomycin sulfate. Following incubation at 24°C for 5 days, 24 colonies exhibiting morphology consistent with that of M. fructicola (uniform colony margin) were obtained, along with nine colonies exhibiting lobed colony margins, commonly associated with M. laxa (3). All colonies resembling M. fructicola were isolated from fruit, whereas those resembling M. laxa were isolated from spurs and shoots. Conidia from both colony morphotypes were lemon-shaped, but those from putative M. laxa isolates were smaller on average (10.75 × 12.0 μm) compared with those from putative M. fructicola isolates (15.75 × 18.25 μm). Confirmation of M. laxa was also accomplished by inoculation of mature green pear (2). Pears inoculated with 104 putative M. laxa conidia per ml produced a region of white-buff colored mycelium but no spores within the inoculated area, while M. fructicola-inoculated pears sporulated abundantly. Identity was further confirmed by PCR amplification of the β-tubulin gene using M. laxa specific primers as previously described (1). Pathogenicity was proven by inoculating flowering shoots of tart cherry trees (cv. Montmorency) in spring 2009. Twenty shoots were spray inoculated with either 104 M. laxa conidia per ml or sterile dH2O and covered with plastic bags for 24 h. Shoots were monitored for symptom development on a weekly basis. Shoots inoculated with M. laxa developed characteristic shoot blight symptoms, while those inoculated with water remained healthy. M. laxa was reisolated from symptomatic shoots and spurs, but not water-inoculated tissues. The presence of M. laxa is reported for the Great Lakes region, which includes New York, but to our knowledge, this report is the first confirmed instance of economically devastating brown rot caused by M. laxa in New York. In the coming seasons, tart cherry growers must consider revising chemical management programs to protect against European brown rot infection during bloom. References: (1) Z. Ma et al. Pest Manag. Sci. 61:449, 2005. (2) J. M. Ogawa et al. Compendium of Stone Fruit Diseases. The American Phytopathological Society. St. Paul, MN, 1995. (3) G. C. M. van Leeuwen and H. A. van Kesteren. Can. J. Bot. 76:2042, 1998.

Impact of White Pine Blister Rust on Resistant Cultivated Ribes and Neighboring Eastern White Pine in New Hampshire

To determine the impact of white pine blister rust (WPBR) following the recent breakdown of the Cr resistance dominant gene in cultivated Ribes spp., 255 plants of 19 Ribes cultivars and 445 neighboring eastern white pine (Pinus strobus) from 42 sites across New Hampshire were evaluated. Of the 19 Ribes cultivars evaluated, 15 were WPBR resistant, and 4 of these were labeled as black currant (Ribes nigrum) with the Cr gene (Cr Ribes cultivars). Incidence of WPBR ranged from 0 to 88% for WPBR-resistant Ribes cultivars. Mean WPBR severity was 14 and <6% of leaf area for Cr Ribes and partially resistant cultivars, respectively. The presence of Cronartium ribicola was confirmed on 17 of the 19 Ribes cultivars screened with polymerase chain reaction analysis and DNA sequencing. Reference accessions of Cr Ribes cultivars from the Canadian Clonal Genebank were successfully infected with C. ribicola inoculum collected in New Hampshire from Cr Ribes cultivars and P. strobus, confirming that the vCr race of C. ribicola that has overcome the Cr resistance dominant gene in cultivated Ribes spp. is present in New Hampshire. The probability of finding pine trees with WPBR was greater for trees neighboring infected Cr Ribes cultivars (0.18) than trees neighboring WPBR-free Ribes cultivars (0.02). Results from this study suggest that the breakdown of Cr-based resistance in Ribes spp. poses a threat to the white pine resource and to cultivated Ribes production.

First Report of White Pine Blister Rust Caused by Cronartium ribicola on Immune Black Currant Ribes nigrum Cv. Titania in Preston, Connecticut

Since the relaxation of federal sanctions on the planting of Ribes crops because of the development of white pine blister rust (WPBR) immune Ribes cultivars (3), a small industry for the production of Ribes fruit for fresh and processing markets was established in New York and surrounding New England states. The majority of Ribes acreage in the region is planted to a WPBR immune black currant R. nigrum cv. Titania. From 2008 to June 2011, symptoms resembling those caused by WPBR pathogen Cronartium ribicola were observed at a large (>12 ha) R. nigrum cv. Titania planting in Preston, CT. In 2008, infection was restricted to a single field (100% incidence), but in 2009, all fields suffered from premature defoliation by late July. In 2010 and 2011, there was considerable incidence (>85%), but premature defoliation was kept in check by chemical management. Symptoms began as chlorotic lesions (0.5 to 4.3 mm in diameter) on both sides of the leaf. These chlorotic lesions had margins delineated by leaf veins and many developed necrotic (0.3 to 0.9 mm in diameter) centers on the upper surface of leaves within 2 to 3 weeks. The undersides of lesions developed blisters containing orange uredinia (0.1 to 0.33 mm in diameter) with smooth peridia that broke with the production of yellow-orange urediniospores (30 × 15 to 25 μm). Symptoms and signs were consistent with published descriptions of C. ribicola (1) and with WPBR infections on highly susceptible R. nigrum cv. Ben Alder planted at the New York State Agricultural Experiment Station in Geneva. Additional confirmation was provided by sequencing the two internal transcribed spacer (ITS) regions and the 5.8S gene (GenBank Accession No. JN587805; 98% identity with No. DQ533975) in the nuclear ribosomal repeat using primers ITS1-F and ITS4 as described previously (2,4). Furthermore, an attempt was made to confirm pathogenicity in the greenhouse by inoculating shoots of potted nursery stock of R. nigrum cv. Titania. Shoots were inoculated by rubbing leaves with either an uninfected currant leaf or a currant leaf from Preston, CT sporulating with urediniospores. Plants were subsequently misted with dH20 and covered with plastic bags for 24 h. Plants were watered biweekly and kept in a greenhouse with 39 to 65% relative humidity at 21 to 26°C. Shoots were monitored for symptom development on a weekly basis. After 3 weeks, 2 of the 10 plants inoculated with infected leaves developed chlorotic lesions and uredinia identical to those on leaves from Preston, CT, while all others remained healthy. Although not easily reproducible in a greenhouse, the breakdown of immunity in R. nigrum cv. Titania was observed for the last 4 years in Connecticut. Given that WPBR immunity was one of the conditions for legalized planting of Ribes, the breakdown of immunity has potentially deleterious implications particularly for nearby states like Massachusetts and New York, in which the Ribes industries are expanding. Moreover, Ribes growers may need to rely on chemical management programs to manage WPBR in the future. References: (1) G. B. Cummins. Illustrated Genera of Rust Fungi. Burgess Publishing Company, Minneapolis, MN, 1959. (2) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (3) S. McKay. Hortic. Technol. 10:562, 2000. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Molecular Characterization of the sdhB Gene and Baseline Sensitivity to Penthiopyrad, Fluopyram, and Benzovindiflupyr in Venturia inaequalis

The succinate dehydrogenase inhibiting (SDHI) fungicides are a class of single-site fungicides that are increasingly important in the management of Venturia inaequalis. In this study, the baseline sensitivity of V. inaequalis to penthiopyrad, fluopyram, and benzovindiflupyr was investigated. In all, 35 to 70 isolates with no prior exposure to single-site fungicides were used to determine the effective concentration at which growth was inhibited by 50% (EC50). Mean EC50 values for the conidial germ tube growth stage for penthiopyrad, fluopyram, and benzovindiflupyr were 0.086, 0.176, and 0.0016 μg ml-1, respectively. Linear correlation analysis revealed a significant and positive correlation between fluopyram and penthiopyrad (P ≤ 0.0001, r = 0.66) and fluopyram and benzovindiflupyr (P = 0.0014, r = 0.52). Baseline sensitivities of V. inaequalis during the mycelial growth stage were also determined for fluopyram and benzovindiflupyr. EC50 values were higher for fluopyram and benzovindiflupyr during this stage compared with the conidial germ tube growth stage, with means of 0.043 and 2.02 μg ml-1, respectively. In addition, the sdhB gene was characterized for three isolates of V. inaequalis collected from a research, baseline, and commercial orchard population. No common mutation sites associated with SDHI resistance in other phytopathogenic fungi were discovered in these isolates or isolates that were recovered following field applications of SDHI fungicides. The results of this study suggest that SDHI fungicides have a high level of activity during the conidial germ tube elongation stage in V. inaequalis and provide a basis for phenotypic and genotypic monitoring of shifts toward resistance of V. inaequalis populations to the SDHI fungicide class.

Xylosandrus germanus (Coleoptera: Curculionidae: Scolytinae) Occurrence, Fungal Associations, and Management Trials in New York Apple Orchards

Abstract Xylosandrus germanus (Blandford) has caused increasing damage in high-density New York apple orchards since 2013, resulting in tree decline and death. We documented their occurrence and timing in > 50 orchards using ethanol-baited traps from 2014 to 2016. First captures ranged from 48 to 83 degree days (base 10 °C) from 1 January. Captures were numerically higher at the orchard–woods interface than within the orchard interior, but differences were not significant in locations with lower populations. Control using insecticide trunk sprays was tested in potted, waterlogged apple trees placed in orchards and nurseries, and inside wooded areas adjacent to orchards. A verbenone repellent was used in combination with trunk sprays to improve control. Overall, insecticide sprays were inconsistent and marginal in preventing new infestations. Chlorpyrifos significantly reduced infestations versus lambda-cyhalothrin and untreated trees at one location in the 2015 orchard trials, and versus untreated trees at one location in the 2016 nursery trials, but otherwise performed no better than other treatments. The addition of verbenone to either the check or permethrin treatments resulted in significantly fewer attack sites containing brood at one orchard site in 2016. Chlorpyrifos, lambda-cyhalothrin, and permethrin significantly reduced the number of attack sites containing adults compared with untreated trees at one nursery trial location in 2016, but were otherwise ineffective in reducing numbers of trees in other locations and infestation categories. We found several fungal and bacterial species associated with X. germanus and its infestation of apples. These microbes likely play a minimal role in apple decline.

The Effect of Delayed-Dormant Chemical Treatments on Demethylation Inhibitor (DMI) Sensitivity in a DMI-resistant Population of Venturia inaequalis

Demethylation inhibitor (DMI) fungicides are an effective means to manage apple scab caused by Venturia inaequalis. Unfortunately, practical resistance to DMI fungicide chemistries is prevalent in populations in New York and the New England states. Management practices that delay the development of DMI resistance in V. inaequalis populations are highly desired by regional apple producers. Trials were conducted in a New York apple orchard during the 2011 and 2012 growing seasons to determine the impact of delayed-dormant (after bud break, but prior to green tissue) chemical treatments on the DMI sensitivity of a V. inaequalis population with stable resistance to DMI fungicides. Delayed-dormant treatment programs consisted of either an application of a copper fungicide, a manganese sanitation product, a DMI fungicide (myclobutanil), or no fungicide. Sensitivity to the DMI fungicide myclobutanil was evaluated for a minimum of 25 V. inaequalis single lesion conidial isolates from each of four replicated treatment blocks. In both years, mean percent relative growth on myclobutanil amended media for V. inaequalis isolates from the copper treatment program were significantly (P < 0.05) lower than isolates from blocks did not receive a delayed dormant fungicide treatment. The effect of the manganese treatment was inconsistent between years. V. inaequalis isolates collected from the myclobutanil treatment program were not significantly (P > 0.05) different in myclobutanil sensitivity from isolates collected from the blocks that did not receive a delayed dormant fungicide treatment. Overall, the results suggest that delayed dormant treatments of copper may favorably impact the myclobutanil sensitivity for a population of V. inaequalis with resistance to DMI fungicides, and should be considered as a standard management practice in apple production.