Role of Yeasts in the Cranberry Fruit Rot Disease Complex.

Cranberry fruit rot (CFR) is an economically important disease caused by at least 10 species of filamentous fungi. Despite the application of fungicides, incidence of CFR is sometimes high, raising the possibility of a role for microbes other than fungi in the CFR complex. Isolation of microbes from rotten berries on culture media that favor either bacteria or yeasts resulted in mucoid colonies from <15% of dry-harvested rotten berries but up to 60% of wet-harvested berries. The mucoid colonies were identified as yeasts, primarily in the family Saccharomycetaceae. Inoculation of sound berries with three yeasts, Hanseniaspora uvarum, Pichia fermentans, and Pichia terricola, resulted in significantly higher incidence and severity of rot symptoms compared with mock-inoculated controls; these yeasts were recovered from inoculated berries, providing evidence of their pathogenicity. The minimum concentrations of azoxystrobin, chlorothalonil, and prothioconazole that resulted in 80% inhibition of growth compared with untreated controls (MIC80) were determined for a subset of yeasts. In general, MIC80s were higher for azoxystrobin and prothioconazole (usually >64 µg/ml) than for chlorothalonil (usually ≤1 µg/ml). To complement culture-dependent studies, DNA was isolated from wet- and dry-harvested rotten berries, and fungi were identified to the level of family by high-throughput sequencing of the fungal internal transcribed spacer region. There were no fungal families consistently detected among samples by one method (culturing or high-throughput sequencing) and missed by the other that have not previously been reported in cranberry; however, some fungal families were found to be more abundant by one method versus the other. Harvest method (wet or dry) had a significant effect on the composition of fungal communities of rotten berries (P < 0.001), and operational taxonomic units representing the Saccharomycetaceae were more abundant in wet- than dry-harvested berries. Taken together, the results suggest that some yeasts are pathogenic to cranberry and may be especially relevant in wet-harvested berries.

Characterization of Blueberry shock virus, an Emerging Ilarvirus in Cranberry.

Blueberry shock virus (BlShV), an Ilarvirus sp. reported only on blueberry, was associated with scarring, disfigurement, and premature reddening of cranberry fruit. BlShV was detected by triple-antibody sandwich enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction, and isometric virions of 25 to 28 nm were observed in cranberry sap. The virus was systemic, although unevenly distributed in plants. The coat protein of BlShV from cranberry shared 90% identity compared with BlShV accessions from blueberry on GenBank. Phylogenetic analysis of isolates of BlShV from cranberry collected from Wisconsin and Massachusetts did not indicate grouping by state. BlShV was detected in cranberry pollen, and seed transmission of up to 91% was observed. Artificial inoculation of cranberry flowers by pollination did not cause virus transmission. In some Nicotiana spp., rub inoculation of leaves with homogenized BlShV-positive cranberry flowers resulted in systemic infection. Cranberry plants recovered from symptoms the year after berry scarring occurred but continued to test positive for BlShV. The virus caused significant reduction in the average number of marketable fruit and average berry weight in symptomatic cranberry plants but recovered plants yielded comparably with healthy plants. Although recovery may limit the immediate economic consequences of BlShV, long-term implications of single- or mixed-virus infection in cranberry is unknown.

Susceptibility of Cold-Climate Wine Grape Cultivars to Downy Mildew, Powdery Mildew, and Black Rot.

Lack of knowledge regarding the susceptibility of cold-climate hybrid wine grape cultivars may be leading to the overuse of fungicides and underutilization of plant host resistance to combat disease in the northern United States. To provide new insights on diseases of cold-climate cultivars and to update management recommendations, disease was evaluated in three vineyards containing eight cultivars that were not sprayed with fungicides in 2015 and 2016. Disease severity or incidence of downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator), and black rot (Guignardia bidwellii) were measured from bud break until 2 weeks after harvest. Cold-climate cultivars ranged widely in susceptibility to different diseases and, although several cultivars were relatively resistant to two diseases, no cultivar was highly resistant to all three diseases. Additionally, a difference between foliar and fruit susceptibility for all three diseases was noted in several cultivars. These data provide a foundation for developing low-spray and certified organic disease management strategies for cold-climate wine grape cultivars based on susceptibility to disease.

Recovery of Cranberry Plants Infected With Tobacco streak virus and Incidence and Distribution of TSV in the Field.

Tobacco streak virus (TSV) has been detected in cranberry plants in Wisconsin, New Jersey, and Massachusetts, and is associated with berry scarring symptoms. In the current study, cv. Mullica Queen plants that produced scarred, symptomatic, TSV-positive fruit in one year produced nonscarred, asymptomatic, TSV-positive fruit in subsequent years, consistent with the "recovery" phenomenon previously documented in other ilarvirus-woody plant interactions. In field trials, fruit set and berry weight were significantly reduced (P < 0.05) in symptomatic, TSV-positive cranberry shoots but not in recovered, TSV-positive shoots compared with healthy, TSV-negative shoots. Likewise, return bloom in the first year following berry scarring was not negatively impacted in recovered shoots. Detection of TSV in various plant parts throughout the growing season was more variable in symptomatic shoots than in recovered shoots. Of all plant parts tested, TSV detection was lowest in berries at the time of harvest in both symptomatic and recovered shoots. Minimal increases in incidence of TSV-infected shoots in cranberry beds from one year to the next, and spatial autocorrelation of TSV-infected shoots, suggest that most new infections result from TSV spreading within a bed, rather than frequent introductions of the virus from external sources.

Does a drop in the bucket make a splash? Assessing the impact of antibiotic use on plants.

Antibiotics are applied to plants to prevent bacterial diseases, although the diversity of antibiotics and total amounts used are dwarfed by antibiotic use in animal agriculture. Nevertheless, the release of antibiotics into the open environment during crop treatment draws scrutiny for its potential impact on the global pool of resistance genes. The main use of antibiotics on plants is application of streptomycin to prevent fire blight, a serious disease of apple and pear trees. A series of recent studies identified and quantified antibiotic resistance genes and profiled bacterial communities in apple orchard plots that were or were not sprayed with streptomycin. While the specific objectives and methods varied, the results of these studies suggest that streptomycin application for fire blight control does not influence bacterial community structure or increase the abundance of resistance genes in orchards.

Streptomycin application has no detectable effect on bacterial community structure in apple orchard soil.

Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 µg/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielou's evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria.

Unexpected diversity during community succession in the apple flower microbiome.

UNLABELLED: Despite its importance to the host, the flower microbiome is poorly understood. We report a culture-independent, community-level assessment of apple flower microbial diversity and dynamics. We collected flowers from six apple trees at five time points, starting before flowers opened and ending at petal fall. We applied streptomycin to half of the trees when flowers opened. Assessment of microbial diversity using tag pyrosequencing of 16S rRNA genes revealed that the apple flower communities were rich and diverse and dominated by members of TM7 and Deinococcus-Thermus, phyla about which relatively little is known. From thousands of taxa, we identified six successional groups with coherent dynamics whose abundances peaked at different times before and after bud opening. We designated the groups Pioneer, Early, Mid, Late, Climax, and Generalist communities. The successional pattern was attributed to a set of prevalent taxa that were persistent and gradually changing in abundance. These taxa had significant associations with other community members, as demonstrated with a cooccurrence network based on local similarity analysis. We also detected a set of less-abundant, transient taxa that contributed to general tree-to-tree variability but not to the successional pattern. Communities on trees sprayed with streptomycin had slightly lower phylogenetic diversity than those on unsprayed trees but did not differ in structure or succession. Our results suggest that changes in apple flower microbial community structure are predictable over the life of the flower, providing a basis for ecological understanding and disease management. IMPORTANCE: Flowering plants (angiosperms) represent a diverse group of an estimated 400,000 species, and their successful cultivation is essential to agriculture. Yet fundamental knowledge of flower-associated microbiotas remains largely unknown. Even less well understood are the changes that flower microbial communities experience through time. Flowers are particularly conducive to comprehensive temporal studies because they are, by nature, ephemeral organs. Here, we present the first culture-independent time series of bacterial and archaeal communities associated with the flowers of apple, an economically important crop. We found unexpected diversity on apple flowers, including a preponderance of taxa affiliated with Deinococcus-Thermus and TM7, phyla that are understudied but thought to be tolerant to an array of environmental stresses. Our results also suggest that changes in microbial community structure on the apple flower may be predictable over the life of the flower, providing the basis for ecological understanding and disease management.

Culturing captures members of the soil rare biosphere.

The ecological significance of rare microorganisms within microbial communities remains an important, unanswered question. Microorganisms of extremely low abundance (the 'rare biosphere') are believed to be largely inaccessible and unknown. To understand the structure of complex environmental microbial communities, including the representation of rare and prevalent community members, we coupled traditional cultivation with pyrosequencing. We compared cultured and uncultured bacterial members of the same agricultural soil, including eight locations within one apple orchard and four time points. Our analysis revealed that soil bacteria captured by culturing were in very low abundance or absent in the culture-independent community, demonstrating unexpected accessibility of the rare biosphere by culturing.

Effect of streptomycin treatment on bacterial community structure in the apple phyllosphere.

We studied the effect of many years of streptomycin use in apple orchards on the proportion of phyllosphere bacteria resistant to streptomycin and bacterial community structure. Leaf samples were collected during early July through early September from four orchards that had been sprayed with streptomycin during spring of most years for at least 10 years and four orchards that had not been sprayed. The percentage of cultured phyllosphere bacteria resistant to streptomycin at non-sprayed orchards (mean of 65%) was greater than at sprayed orchards (mean of 50%) (P = 0.0271). For each orchard, a 16S rRNA gene clone library was constructed from leaf samples. Proteobacteria dominated the bacterial communities at all orchards, accounting for 71 of 104 OTUs (determined at 97% sequence similarity) and 93% of all sequences. The genera Massilia, Methylobacterium, Pantoea, Pseudomonas, and Sphingomonas were shared across all sites. Shannon and Simpson's diversity indices and Pielou's evenness index were similar among orchards regardless of streptomycin use. Analysis of Similarity (ANOSIM) indicated that long-term streptomycin treatment did not account for the observed variability in community structure among orchards (R = -0.104, P = 0.655). Other variables, including time of summer, temperature and time at sampling, and relative distance of the orchards from each other, also had no significant effect on bacterial community structure. We conclude that factors other than streptomycin exposure drive both the proportion of streptomycin-resistant bacteria and phylogenetic makeup of bacterial communities in the apple phyllosphere in middle to late summer.

A New View of Sooty Blotch and Flyspeck.

Sooty blotch and flyspeck (SBFS) fungi colonize the surface wax layer of the fruit of apple, pear, persimmon, banana, orange, papaya, and several other cultivated tree and vine crops. In addition to colonizing cultivated fruit crops, SBFS fungi also grow on the surfaces of stems, twigs, leaves, and fruit of a wide range of wild plants. The disease occurs worldwide in regions with moist growing seasons. SBFS is regarded as a serious disease by fruit growers and plant pathologists because it can cause substantial economic damage. The smudges and stipples of SBFS often result in downgrading of fruit from premium fresh-market grade to processing use. This review describes the major shifts that have occurred during the past decade in understanding the genetic diversity of the SBFS complex, clarifying its biogeography and environmental biology, and developing improved management strategies.

Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins.

To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a beta-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional beta-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a beta-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.

Integration of Copper-Based and Reduced-Risk Fungicides for Control of Blumeriella jaapii on Sour Cherry.

Practical resistance to sterol demethylation inhibitor (DMI) fungicides among populations of Blumeriella jaapii, the cherry leaf spot (CLS) pathogen, was documented in 2005. In the present study, strategies to reduce selection for DMI-resistant strains of B. jaapii and adapt to possible restrictions on the use of chlorothalonil are described. Ten field trials were conducted on the sour cherry cultivars Balaton and Montmorency to test the efficacy of integrating respiration-inhibitor and copper-based fungicides into spray programs. Programs that included up to three sprays of copper-based fungicides were among the most effective for controlling CLS, although leaf phy-totoxicity was sometimes observed. Under high disease pressure, eliminating chlorothalonil compromised CLS control. 'Balaton' and 'Montmorency' did not differ in the percentage of leaves with CLS or defoliation resulting from CLS. The physical modes of action of representative DMI, QoI, and copper-based fungicides were evaluated in a leaf disk assay. Trifloxystrobin, a QoI fungicide, provided the best protection against infection by B. jaapii. All fungicides were more effective than water when applied 46 h postinfection, although differences were not statistically significant in one of two trials. Tebuconazole, a DMI, was the only fungicide that was more effective than water in preventing resporulation from existing lesions in both trials. Isolates of B. jaapii, which varied in DMI-sensitivity, all were sensitive to copper in vitro.

Occurrence, Distribution, and Polymerase Chain Reaction-Based Detection of Resistance to Sterol Demethylation Inhibitor Fungicides in Populations of Blumeriella jaapii in Michigan.

ABSTRACT The intensive use of site-specific fungicides in agricultural production provides a potent selective mechanism for increasing the frequency of fungicide-resistant isolates in pathogen populations. Practical resistance occurs when the frequency and levels of resistance are great enough to limit the effectiveness of disease control in the field. Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii, is a major disease of cherry trees in the Great Lakes region. The site-specific sterol demethylation inhibitor fungicides (DMIs) have been used extensively in the region. In 2002, CLS control failed in a Michigan orchard that had used the DMI fenbuconazole exclusively for 8 years. That control failure and our observations from around the state suggested that practical resistance had developed in B. jaapii. Field trial data covering 1989 to 2005 for the DMIs fenbuconazole and tebuconazole supported observations of reduced efficacy of DMIs for controlling CLS. To verify the occurrence of fungicide-resistant B. jaapii, monoconidial isolates were collected in two surveys and tested using a fungicide-amended medium. In one survey, 137 isolates from sites with different DMI histories (no known history, mixed or alternated with other fungicides, and exclusive use) were tested against 12 concentrations of fenbuconazole, tebuconazole, myclobutanil, and fenarimol. Isolates from sites with no prior DMI use were DMI sensitive (DMI(S) = no colony growth at 0.2 mug/ml a.i.) whereas the isolates from the site with prior exclusive use showed growth at DMI concentrations 3 to >100 times higher, and were rated as DMI resistant (DMI(R)). A second survey examined 1,530 monoconidial isolates, including 1,143 from 62 orchard sites in Michigan, where DMIs had been used to control CLS. Resistance to fenbuconazole was detected in 99.7% of the orchard isolates. All isolates from wild cherry trees were sensitive and isolates from feral and dooryard trees showed a range of sensitivities. A polymerase chain reaction (PCR)-based detection method for identifying B. jaapii and DMI(R) was developed and tested. The species-specific primer pair (Bj-F and Bj-R) based on introns in the CYP51 gene of B. jaapii, and the DMI(R)-specific primer pair (DMI-R-Bj-F and DMI-R-Bj-R) based on an insert found upstream of CYP51 in all DMI(R) isolates, provided an accurate and rapid method for detecting DMI(R) B. jaapii. The PCR-based identification method will facilitate timely decision making and continued monitoring of DMI(R) subpopulations in response to management programs.

Indole-3-acetic Acid-producing bacteria are associated with cranberry stem gall.

ABSTRACT Cranberry stem gall is characterized by tumors that girdle stems, thereby killing all distal leaves, flowers, and fruit. Among bacteria isolated from galls, all 11 isolates that were identified as members of the family Enterobacteriaceae caused galls on 50 to 100% of micropropagated cranberry plants that were inoculated. Four of fifteen isolates identified as Pseudomonas spp. caused galls on 10 to 83% of plants inoculated. Twelve of fifteen isolates identified as either Agrobacterium spp. or Rhizobium spp. caused galls on 10 to 50% of plants inoculated, but the galls were smaller than those caused by members of the family Enterobacteriaceae or Pseudomonas spp. There was a positive correlation between the ability of bacteria to produce IAA in vitro and cause galls. In 2002 and 2003, bacteria were isolated from plant and soil samples collected from beds where stem gall had been observed in the past 2 years and beds where stem gall had never been observed. IAA-producing bacteria were common in all samples, although trends were different across years. The results of this study support the hypothesis that IAA-producing bacteria cause cranberry stem gall and suggest that rather than one bacterial species being the cause, multiple strains of bacteria that produce IAA may be responsible for gall formation.

Anatomy of cranberry stem gall and localization of bacteria in galls.

ABSTRACT Cranberry stem gall is characterized by tumors that girdle stems, thereby killing all distal leaves, flowers, and fruit. Bacteria that produce high levels of the plant growth hormone indole-3-acetic acid (IAA) are associated with and believed to cause cranberry stem gall. The anatomy of naturally occurring galls on woody cranberry plants and galls caused by inoculation of micropropagated cranberry plants with Pantoea agglomerans strain 4/99 was consistent with elevated levels of IAA in plants. Field galls exhibited hypertrophy and hyperplasia of tissue external to the vascular cambium, resulting in extensive stem swelling and splitting of the periderm. Similarly, galls on micropropagated plants contained enlarged cortical parenchyma cells. The current year's xylem vessels in field galls were narrow and dense compared with xylem vessels of healthy stems. Curved xylem elements apparently developed de novo within field galls and galls on inoculated plants. Cavities and fissures in both types of galls contained dense aggregates of bacteria. Treatment of micropropagated plants with synthetic IAA caused hypertrophy of cortical parenchyma and formation of adventitious roots. The results support the hypothesis that IAA-producing bacteria cause cranberry stem gall.

Antibiotic use in plant agriculture.

Antibiotics have been used since the 1950s to control certain bacterial diseases of high-value fruit, vegetable, and ornamental plants. Today, the antibiotics most commonly used on plants are oxytetracycline and streptomycin. In the USA, antibiotics applied to plants account for less than 0.5% of total antibiotic use. Resistance of plant pathogens to oxytetracycline is rare, but the emergence of streptomycin-resistant strains of Erwinia amylovora, Pseudomonas spp., and Xanthomonas campestris has impeded the control of several important diseases. A fraction of streptomycin-resistance genes in plant-associated bacteria are similar to those found in bacteria isolated from humans, animals, and soil, and are associated with transfer-proficient elements. However, the most common vehicles of streptomycin-resistance genes in human and plant pathogens are genetically distinct. Nonetheless, the role of antibiotic use on plants in the antibiotic-resistance crisis in human medicine is the subject of debate.

Virulence of Botryosphaeria dothidea and Botryosphaeria obtusa on Apple and Management of Stem Cankers with Fungicides.

The virulence of isolates of Botryosphaeria dothidea and B. obtusa was compared on apple fruit, trunks of 2-year-old apple trees, and twigs of mature apple trees. In general, B. dothideaisolates were more virulent than B. obtusa isolates. There was no correlation between virulence on fruit and virulence on 2-year-old trees. Several compounds were tested as topical wound treatments to control stem cankers caused by B. dothidea and B. obtusa. Benomyl, kresoxim-methyl, and trifloxystrobin, when applied at rates recommended for foliar application, consistently reduced the incidence and size of cankers compared with the water control. Clove oil, garlic extract, and neem oil did not reduce the incidence and size of cankers compared with the water control. Kresoxim-methyl was also effective when applied with an air-blast sprayer, despite incomplete spray coverage. Although fungicides reduced external canker symptoms, B. dothidea and B. obtusa were frequently isolated from xylem tissue, suggesting that fungicides might not provide long-term control of these pathogens.