Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

Effects of Simulated Hail Events and Subsequent Fungicide Applications on Cranberry Fruit Rot Incidence and Yield.

Storms containing hail are a common occurrence in Wisconsin, with a few or many cranberry (Vaccinium macrocarpon) growers being affected every year. Growers usually apply fungicides immediately following hail events to prevent fruit rot, despite a lack of research to support this practice. We conducted field trials in 2010 and 2011 to address the question of whether applying fungicides to injured fruit reduces fruit rot incidence (% rotten fruit). Hail damage was simulated by forcibly projecting pea gravel into cranberry beds using a mist-blower sprayer modified for this purpose, and the fungicides azoxystrobin or copper hydroxide were applied to fruit immediately after applying gravel. Fruit rot incidence and yield were evaluated within 2 weeks prior to commercial harvest in late September and early October. Fruit rot incidence was greater (P ≤ 0.05) and yield was lower (P < 0.05) in plots treated with gravel than in the nontreated control plots in six of seven trials. Fungicides did not reduce fruit rot incidence (P ≥ 0.05) in gravel-treated plots compared to the nontreated control in six of seven trials. However, in a trial conducted on relatively immature berries, fruit rot incidence in gravel-treated plots treated with azoxystrobin was less (P = 0.0103) than fruit rot incidence in gravel-treated plots receiving no fungicide treatment. In that same trial, fruit rot incidence was not reduced (P = 0.1243) in gravel-treated plots treated with copper hydroxide compared to gravel-treated plots that were not treated with fungicide. Results suggest that under most circumstances, if cranberries are damaged by hail, it is unlikely that an application of fungicide will reduce the amount of fruit rot at the time of harvest.

Effects of cherry leaf spot on photosynthesis in tart cherry 'Montmorency' foliage.

Results described here span a total of three field seasons and quantitatively depict the effects of an economically important fungal pathogen (Blumeriella jaapii) on tart cherry (Prunus cerasus 'Montmorency') leaf physiology. For the first time, leaf photosynthesis, stomatal conductance (g(s)), maximum ribulose-1,5-bisphosphate carboxylation rate (V(cmax)), and maximum electron transport (J(max)) were measured as functions of visible cherry leaf spot disease (CLS) severity. Defined as the proportion of chlorotic and necrotic tissue per leaf, CLS severity was estimated from leaves of mature 'Montmorency' trees in 2007, 2008, and 2009. Briefly, as visible disease severity increased, all of the leaf-level physiological parameters decreased significantly (P < 0.01) and disproportionately. Thus, the effects of visible symptoms on leaf photosynthetic metabolic function encroached upon asymptomatic tissue as well. Impairment of photosynthetic metabolism in 'Montmorency' tart cherry leaves due to CLS appears to be mediated through disproportionately large perturbations in g(s), V(cmax), and J(max). These findings offer a new perspective on the amount of damage that this serious disease can inflict.

Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere.

AIMS: Bacterial communities in the apple phyllosphere were examined quantitatively and qualitatively by applying culture-dependent and culture-independent methods. METHODS AND RESULTS: Populations estimated by viewing cells stained with 4',6-diamidino-2-phenylindole generally were at least 100-1000 times greater than populations estimated by culturing on tryptic soy agar (TSA). Of the 44 operational taxonomic units (OTUs; cut-off threshold of 97%) detected in total, five bacterial orders containing 23 OTUs were identified by culturing on TSA, whereas nine orders containing 33 OTUs were identified by 16S rRNA gene cloning of DNA extracted from apple leaf surfaces. Twelve of the 44 OTUs were shared between cultured isolates and 16S rRNA gene clones and included the orders Burkholderiales, Pseudomonadales, Rhizobiales and Sphingomonadales. Three OTUs within the genus Sphingomonas accounted for 40% of isolates and 68% of clones. The Actinomycetales were found only among isolates, whereas the Bacteroidales, Enterobacteriales, Myxococales and Sphingobacteriales were represented in the 16S rRNA gene clone libraries but were absent among isolates. CONCLUSIONS: Culture-independent methods revealed greater numbers and greater richness of bacteria on apple leaves than found by culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to directly compare culture-dependent and independent approaches for assessing bacterial communities in the phyllosphere. The biases introduced by different methods will have a significant impact on studies related to phyllosphere ecology, biological control of plant diseases, reservoirs of antibiotic resistance genes and food safety.

Effects of Copper-Based Fungicides on Foliar Gas Exchange in Tart Cherry.

In the Great Lakes region of the United States, cherry growers are poised to re-adopt copper-based fungicides to manage cherry leaf spot disease (CLS), caused by Blumeriella jaapii. However, application of copper is often associated with leaf bronzing. In growth chamber experiments, bronzing was observed on foliage of tart cherry (Prunus cerasus 'Montmorency') seedlings 1 week following application of a copper-based fungicide, only when leaves were also exposed to nightly dew. In potted, 1-year-old trees outdoors, light-saturated rates of net CO2 assimilation (A) and stomatal conductance (gs) were not affected by treatment with copper sulfate, chlorothalonil, tebuconazole, or trifloxystrobin compared to a nonsprayed control. In 2005 and 2006, A and gs were measured during late summer on leaves of mature trees in an orchard subjected to the following fungicide programs: synthetic fungicides only; synthetic fungicides integrated with copper-based fungicides; or not sprayed. Bronzing symptoms were observed on trees sprayed with copper. Regression analysis revealed that neither A nor gs decreased as leaf surface area affected by bronzing increased (R2 = 0.004, P = 0.80 and R2 = 0.006, P = 0.74, respectively). Leaf bronzing associated with application of copper-based fungicides may therefore be inconsequential to foliar gas exchange in tart cherry during late summer.

Adaptation of an Apple Sooty Blotch and Flyspeck Warning System for the Upper Midwest United States.

A warning system for sooty blotch and flyspeck (SBFS) of apple, developed in the southeastern United States, uses cumulative hours of leaf wetness duration (LWD) to predict the timing of the first appearance of signs. In the Upper Midwest United States, however, this warning system has resulted in sporadic disease control failures. The purpose of the present study was to determine whether the warning system's algorithm could be modified to provide more reliable assessment of SBFS risk. Hourly LWD, rainfall, relative humidity (RH), and temperature data were collected from orchards in Iowa, North Carolina, and Wisconsin in 2005 and 2006. Timing of the first appearance of SBFS signs was determined by weekly scouting. Preliminary analysis using scatterplots and boxplots suggested that cumulative hours of RH ≥ 97% could be a useful predictor of SBFS appearance. Receiver operating characteristic curve analysis was used to compare the predictive performance of cumulative LWD and cumulative hours of RH ≥ 97%. Cumulative hours of RH ≥ 97% was a more conservative and accurate predictor than cumulative LWD for 15 site years in the Upper Midwest, but not for four site years in North Carolina. Performance of the SBFS warning system in the Upper Midwest and climatically similar regions may be improved if cumulative hours of RH ≥ 97% were substituted for cumulative LWD to predict the first appearance of SBFS.

Evaluation of Sampling Strategies for Determining Incidence of Cranberry Fruit Rot and Fruit Rot Fungi.

Two sampling strategies were compared and sources of variability in the sampling protocols analyzed to optimize sampling methods for studies of cranberry fruit rot that occurs in the field (i.e., field rot). For the first method, fruit were dry picked by hand from randomly assigned quadrats; for the second method, fruit were scooped from harvest floodwaters. Rot incidence, which ranged from 1.8 to 9.7%, did not differ significantly between upland and lowland sites or, in general, between dry-picked and wet-harvested samples. There were no consistent differences between upland and lowland sites in the frequency of isolation of any fungus from either rotten or sound fruit. The incidence of certain saprophytic and soilborne fungi was greater in wet-harvested compared with dry-picked fruit. In general, rot incidence and incidence of various fungal taxa isolated from fruit varied more among samples within sites than among sites. Site type (i.e., upland or lowland) was never a major source of variability. These findings suggest that if the goal were to assess the occurrence of cranberry fruit rot within a region, intensive within-site sampling would be necessary, but site type would not be an important consideration, at least in Wisconsin, where this study was conducted.

Effects of Calcium Salts on the Cranberry Fruit Rot Disease Complex.

Calcium salts were applied during the growing season to fresh-fruit cranberry beds to test their effects on cranberry fruit rot incidence and the incidence of specific fungi isolated from rotten and sound cranberry fruit at the time of harvest and after storage. Calcium salts did not affect fruit rot incidence, nor did they affect the recovery of specific fungi from berries. The field treatments did not result in higher calcium content in mature berries, nor did they affect the force required to penetrate the berry epidermis. Calcium propionate inhibited growth in vitro of Allantophomopsis cytisporea, A. lycopodina, Coleophoma empetri, Fusicoccum putrefaciens, and Physalospora vaccinii. Calcium chloride and calcium nitrate inhibited growth of Coleophoma empetri and Fusicoccum putrefaciens, but these salts enhanced growth of Physalospora vaccinii. P. vaccinii was the fungus most frequently isolated from rotten berries at the time of harvest. The fungi most frequently isolated from rotten berries after several weeks in storage varied among sites. P. vaccinii, which was common in sound fruit at harvest, persisted in sound fruit in storage but was also isolated frequently from rotten berries after storage. A. lycopodina and F. putrefaciens, which were isolated infrequently from sound berries at the time of harvest, were isolated frequently from rotten berries after storage. In two of four trials, no fungi were isolated from a large proportion of fruit that decayed in storage.

Outbreak of Leucostoma Canker Caused by Leucostoma cincta on McIntosh Apple Trees in Wisconsin.

In fall 1996, cankers were observed on branches of 7- to 9-year-old apple trees (Malus domestica cvs. Empire and McIntosh, strains Marshall and Redmax) in a research orchard in Sturgeon Bay, WI. By summer 1997, cankers had developed on scaffold limbs or the central leaders of 48 of 48 Marshall McIntosh trees and 26 of 40 Redmax McIntosh trees, but they rarely were found in an adjacent block of 40 Empire trees. In 1998, new cankers were not observed, but existing cankers expanded. By 1999, the Marshall McIntosh block was so severely affected it was removed; 50% of the Redmax trees and 13% of the Empire trees also were removed. Initially, cankers appeared as orange discolored areas on bark and frequently, but not always, were associated with pruning wounds. Canker expansion was greater during spring and fall than during summer. At later stages of canker development, bark cracked and peeled, and leaves distal to cankers died. These features were consistent with descriptions of Leucostoma canker of apple (2,3), a disease previously reported only in Michigan, Germany, and Iran (1-3). All of 10 cankers sampled in 1997 and several more cankers sampled in 1998 yielded fungal colonies on potato-dextrose agar resembling Leucocytospora spp., which are anamorphs of Leucostoma spp. (2). Colonies initially were white to buff and later turned brown. Conidium morphology was consistent with descriptions of Leucocytospora spp. (2). Alternaria spp. also were isolated frequently; other fungi, which were not identified, were isolated rarely. The presence of abundant pycnidia of Leuco-cytospora, but not perithecia of Leucostoma, on cankers was different from the signs described for Leucostoma canker caused by Leucostoma cincta in Michigan (2,3). However, simple matching coefficient analysis of randomly amplified polymorphic DNA data indicated 74% similarity between a typical isolate from apple in Wisconsin (97-82) and two isolates of L. cincta from apple in Michigan (3; ATCC 64878 and 64879) but only 49 to 54% similarity to L. cincta and L. persoonii isolated from stone fruit trees (Prunus spp.). Inoculation of wounded branches of mature apple trees (cvs. Golden Delicious and Cortland) with isolate 97-82 in July 1998 and 1999 resulted in cankers that resembled young cankers observed on trees in the research orchard. Eight weeks after inoculation, the pathogen was reisolated from these cankers, which fulfilled Koch's Postulates. Therefore, we conclude that Leucostoma canker was responsible for the serious damage to apple trees of different cultivars at the research station, with strains of McIntosh affected most severely. We speculate that low-temperature injury during the unusually cold winter of 1995-1996 might have created infection sites and predisposed trees to disease development. This is the first report of Leucostoma canker of apple in Wisconsin. It is rare that this disease causes such significant losses (2). References: (1) M. Ashkan. Iranian J. Plant Pathol. 30:33, 1994. (2) A. L. Jones and H. S. Aldwinckle, eds. 1991. Pages 40-41 in: Compendium of Apple and Pear Diseases. American Phytopathological Society, St. Paul, MN. (3) T. J. Proffer and A. L. Jones. Plant Dis. 73:508, 1989.

Infection of Cranberry Flowers by Monilinia oxycocci and Evaluation of Cultivars for Resistance to Cottonball.

ABSTRACT Infection of cranberry flowers by conidia of Monilinia oxycocci, the cottonball pathogen, was investigated using a squash-mount histological method. Conidia germinated on anthers, nectaries, petals, and stigmata, but not styles. The stigma was the only flower part penetrated by the fungus, but no specialized infection structures were noted. Both fungal and pollen germ tubes grew through the stylar canal and made contact with ovules and nucellar tissue by 72 h after inoculation and pollination. Cottonball incidence was greatest when stigmata were inoculated; the low level of cottonball that resulted from inoculation of other flower parts and in noninoculated flowers was attributed to contamination of stigmata. In greenhouse tests, cottonball incidence was 25, 28, 31, and 38% for cvs. Searles, Pilgrim, Ben Lear, and Stevens, respectively, and was greater for M. oxycocci isolate 593 than isolate 591. We conclude that the stigma is the sole floral infection court for conidia of M. oxycocci and that the most popular cranberry cultivars in Wisconsin do not differ in inherent resistance to cottonball. The relevance of these findings to the long-term management of cottonball is discussed.

Sensitivity of Monilinia oxycocci to Fenbuconazole and Propiconazole in vitro and Control of Cranberry Cottonball in the Field.

The efficacy of fungicides in controlling cottonball disease of cranberry was tested during 1996 to 1998 at three locations in Wisconsin. For some fungicides, the efficacy of four applications, two each during shoot elongation and bloom, was compared with two applications during bloom only. Spraying twice during bloom was as effective in controlling secondary infection as spraying twice during shoot elongation plus twice during bloom. Azoxystrobin, cyprodinil, and propiconazole were equally effective. None of the treatments affected yield, fruit retention, or berry weight compared with the controls. Sensitivity of M. oxycocci, the cottonball pathogen, to fenbuconazole and propiconazole was tested in vitro by comparing the distributions of ED50 values of populations collected from three sites that differed in previous exposure to fungicides. Median ED50 values for fenbuconazole were significantly greater at sites where sterol demethylation inhibitor fungicides had been used compared with a site where fungicides had never been used, but median ED50 values for propiconazole did not differ among sites. There was no correlation between the sensitivities to fenbuconazole and propiconazole. The data will form the basis of recommendations aimed at delaying the onset of fungicide resistance and will provide a baseline for monitoring resistance to fenbuconazole and propiconazole in populations of M. oxycocci in the future.

First Report of Early Rot of Cranberry Caused by Phyllosticta vaccinii in Wisconsin.

Phyllosticta vaccinii Earle causes early rot of cranberry (Vaccinium macrocarpon Aiton) and previously was reported in fruit and leaves from Massachusetts and New Jersey, but not Wisconsin or Washington (2). This fungus previously was introduced into Wisconsin, apparently on planting stock, but did not persist in the field (2). In the present study, rotted fruit were collected in central Wisconsin in September 1997 from research plots adjacent to a commercial planting that had been started from field cuttings from New Jersey. P. vaccinii was isolated from 12 of 31 symptomatic berries, and its identity was verified by cultural and morphological characteristics (3). P. vaccinii was not isolated from rotted fruit from five other sites in central and northern Wisconsin. In three separate experiments, 10 to 25 cv. Stevens or Searles cranberry fruit were punctured with a needle, inoculated with 2 to 4 × 105 conidia from sporulating cultures of P. vaccinii, and incubated at 28°C in a moist chamber. After 5 to 14 days, soft, watery spots developed at the inoculation point on 8 to 22% of the fruit in different experiments, and P. vaccinii was reisolated from the lesions. Fruit that were punctured but not inoculated neither developed symptoms nor yielded P. vaccinii. Previous attempts at fulfilling Koch's postulates by inoculating mature fruit were unsuccessful (1). P. vaccinii is one of approximately 15 species of fungi involved in the cranberry fruit rot complex in the eastern U.S. where fungicides are applied to greater than 95% of cranberry acreage, usually three times per year, primarily to control preharvest fruit rots. In Wisconsin, however, preharvest fruit rots are insignificant; less than 25% of the acreage is treated with fungicides. The occurrence of early rot in Wisconsin and the threat of introducing pathogens on cranberry cuttings are troublesome in light of the Food Quality Protection Act of 1996, which threatens registration of fungicides used to control cranberry fruit rots. References: (1) D. M. Boone. Pages 35-36 in: Compendium of Blueberry and Cranberry Diseases. F. L. Caruso and D. C. Ramsdell, eds. American Phytopathological Society, 1995. (2) G. J. Weidemann and D. M. Boone. Plant Dis. 67:1090, 1983. (3) G. J. Weidemann et al. Mycologia 74:59, 1982.