Patricia S. McManus

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

ABSTRACT 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 β-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional β-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 β-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.

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.

Unexpected Diversity during Community Succession in the Apple Flower Microbiome

ABSTRACT 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. 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 microbiota 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 of 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.

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.

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.

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.

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.

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 systems 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.

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.