N. Rosenzweig

Microbial Communities Associated with Potato Common Scab-Suppressive Soil Determined by Pyrosequencing Analyses

Potato common scab, caused by Streptomyces spp., is an annual production problem for potato growers, and not effectively controlled by current methods. A field with naturally occurring common scab suppression has been identified in Michigan, and confirmed to have a biological basis for this disease suppression. This field and an adjacent scab nursery conducive to disease were studied using pyrosequencing to compare the two microbial communities. Total DNA was extracted from both the disease-conducive and -suppressive soils. A phylogenetically taxon-informative region of the 16S rRNA gene was used to establish operational taxonomic units (OTUs) to characterize bacterial community richness and diversity. In total, 1,124 OTUs were detected and 565 OTUs (10% dissimilarity) were identified in disease-conducive soil and 859 in disease-suppressive soil, including 300 shared both between sites. Common phyla based on relative sequence abundance were Acidobacteria, Proteobacteria, and Firmicutes. Sequences of Lysobacter were found in significantly higher numbers in the disease-suppressive soil, as were sequences of group 4 and group 6 Acidobacteria. The relative abundance of sequences identified as the genus Bacillus was significantly higher by an order of magnitude in the disease-conducive soil.

Culture-Based Assessment of Microbial Communities in Soil Suppressive to Potato Common Scab

A field in East Lansing, MI, showed a decline of potato common scab compared with an adjacent potato field. To confirm that the decline was due to biological factors, the soil was assayed. In the greenhouse, putative common-scab-suppressive soil (SS) was either treated with various temperatures or mixed with autoclaved SS at various ratios. Pathogenic Streptomyces scabies was incorporated into the treated soil at 106 CFU/cm3 of soil, followed by planting of either potato or radish. Disease severity was negatively correlated with the percentage of SS in the mixture and positively correlated with temperature above 60°C. The soil was screened for four groups of potential antagonists (general bacteria, streptomycetes, fluorescent pseudomonads, and bacilli) pairing in culture with S. scabies. The frequency of antagonistic bacteria in SS was higher than common-scab-conducive soil (CS) in all four groups but only pseudomonads and streptomycetes were significantly higher. The population of pathogenic Streptomyces spp. in the rhizosphere of CS was significantly higher than SS. Dilution plating of CS and SS samples showed no clear trends or differences in populations of total fungi, total bacteria, streptomycetes, fluorescent pseudomonads, and bacilli but terminal restriction fragment polymorphism analysis revealed two distinct microbial communities were present in SS and CS.

Evaluation of QoI fungicide application strategies for managing fungicide resistance and potato early blight epidemics in Wisconsin

Potato early blight (Alternaria solani) is a yield-limiting disease and control depends primarily on multiple fungicide applications. Azoxystrobin, registered in the United States in 1999, initially provided outstanding early blight control. Within 3 years, approximately 80% of the total potato acreage was being treated with azoxystrobin and other quinone outside inhibitor (QoI), fungicides registered subsequently. Alternaria solani isolates with decreased in vitro sensitivity to azoxystrobin were detected in Wisconsin during 2001. Field experiments were conducted in 2001 to 2003 to evaluate season-long fungicide programs and test fungicide resistance management strategies. The fungicide program recommended to growers at that time, which consisted of three applications of azoxystrobin for weeks 1, 3, and 5 alternated with applications of chlorothalonil at label recommended rates, was effective in controlling early blight when conditions were conducive to disease development. Mean sensitivity in vitro of A. solani isolates from fungicide efficacy field experiments in 2001 to 2003 was numerically highest for isolates from the untreated control plots, chlorothalonil-alone plots, or plots treated with three applications of azoxystrobin alternated with chlorothalonil compared with other treatments tested. Three single-nucleotide polymorphisms (SNPs) can cause the F129L substitution (TTC to TTA, CTC, or TTG) that results in decreased sensitivity to azoxystrobin of A. solani. The TTA mutant was the most frequently recovered mutant type in the field experiments. The frequency of recovery of wild-type isolates in experiments was 22% in 2001, 4% in 2002, and 22% in 2003.

Monitoring and Tracking Changes in Sensitivity to Azoxystrobin Fungicide in Alternaria solani in Wisconsin

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

Rhizosphere bacterial communities associated with long-lived perennial prairie plants vary in diversity, composition, and structure.

The goal of this research was to investigate the variation in rhizosphere microbial community composition, diversity, and structure among individual Andropogon gerardii Vitman (big bluestem) and Lespedeza capitata Michx. (bush clover). Bacterial communities from the rhizosphere of 10 plants of each species (n = 20 plants total) were explored using a culture-independent pipeline. Microbial communities associated with both host plants had high bacterial diversity within individual plant rhizosphere and taxa unique to individual rhizospheres. Bacterial communities associated with the rhizosphere of A. gerardii were consistently more diverse than those associated with L. capitata, and there were significant differences between plant species in rhizosphere bacterial community composition. Differences included microbial taxa with no known functional relationship with their preferred host species, including sulfide-methylating obligate anaerobes (Holophaga), complete denitrifiers (Rhodoplanes), sludge inhabitants (Ktedonobacter), and nitrate oxidizers (Nitrospira). These results suggest the potential for plant species to have significant impacts on a broad array of ecosystem functions (e.g., cycling of carbon, nitrogen sulfurs, metals, and trace elements) via their selective impacts on soil microbes. However, sequence-based community analysis and the corresponding lack of intact microbial cultures limits understanding of the potential influences of enriched microbial taxa on plant hosts and their roles in ecosystem functioning.

Use of PCR-RFLP Analysis to Monitor Fungicide Resistance in Cercospora beticola Populations from Sugarbeet (Beta vulgaris) in Michigan, United States

Genetic resistance to Quinone outside inhibitor (QoI) and benzimidazole fungicides may be responsible for a recent decline in efficacy of chemical control management strategies for Cercospora leaf spot (CLS) caused by Cercospora beticola in Michigan sugarbeet (Beta vulgaris) fields. The target genes and fungicide resistance mutations are known for these two fungicides. Based on this, two standard polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays were developed to detect the G143A and E198A point mutations in the fungal mitochondrial cytochrome b and the β-tubulin genes, respectively. These mutations confer a high level of resistance to either QoI or benzimidazole fungicides. The presence of the G143A and E198A mutations was monitored within C. beticola populations recovered from Michigan sugarbeet production fields collected in 2012. Both the QoI-resistant cytochrome b allele and the benzimidazole-resistant β-tubulin allele were detected directly from leaf tissue following a PCR-RFLP assay. Using either detection assay, the G143A and E198A mutations were detected in over 90% of the 118 field samples originating from Michigan sugarbeet production under fungicide management programs for CLS control. Monitoring of the G143A and E198A mutations in fields located in 9 counties and 58 townships indicated that the mutations were widespread in Michigan sugarbeet production areas. The PCR-based assays used and developed in this study were effective in detecting the presence of the G143A and E198A mutations in C. beticola field populations from Michigan.

The Importance and Application of Bacterial Diversity in Sustainable Agricultural Crop Production Ecosystems

Soil inhabiting bacteria are integral to global biogeochemical cycles and influence nutrient cycling and mineral solubilization important to soil health, and crop productivity. Soil bacteria directly impact plant fitness as pathogens, beneficial mutualists, and indirectly as decomposers, or through antagonistic activity against plant pathogens. Moreover, such beneficial bacteria have the capacity to produce plant hormones and induce systemic disease resistance responses in plants. This chapter discusses bacterial diversity and its role in sustainable agriculture in crop ecosystems. The chapter also addresses the potential role of plant growth promoting rhizobacteria (PGPR), biological control agents (BCAs) and the significance of bacterial community diversity associated with soil borne plant disease suppression in sustainable agricultural crop production. The influence of herbicide resistant crop rotation systems on soil bacterial diversity is discussed. Current culture-independent approaches to study bacterial diversity, and directions for future applied research in agricultural production systems are also discussed.

First report of QoI insensitive Cercospora beticola on sugar beet in Ontario, Canada

Cercospora beticola Sacc. causes Cercospora leaf spot (CLS) of sugar beet (Beta vulgaris L.) and is the most destructive foliar disease of sugar beet worldwide (1). The QoI fungicide pyraclostrobin has been an important management tool for CLS in Canada since 2003. Beginning in 2010, some growers reported poor disease control after applying pyraclostrobin. Leaf disk samples with CLS lesions were collected in September 2012 from 16 commercial fields located in Kent and Lambton Counties, Ontario, Canada. These counties (ca. 300,000 ha) encompass the major commercial sugar beet production area in Ontario (ca. 3,925 ha). CLS severity ranged from low to severe among the sampling sites. Leaf discs with a single leaf spot were cut from leaves using a hole punch. Spots were up to 5 mm in diameter with tan, light brown, or sometimes gray centers. DNA was extracted from leaf discs using a Qiagen DNeasy Plant Mini Kit (Germantown, MD) according to the manufacturers instructions. PCR was used to amplify a fragment of the C. beticola cytochrome b (CYTB) gene (4). Pure cultures were obtained by placing plant tissue in a moist chamber and transferring single spores to V8 juice agar. PCR products were sequenced for 32 samples at the Genomics Technology Support Facility (Michigan State University, East Lansing, MI) and 25 were confirmed to have 100% identity with the sequence of QoI-resistant C. beticola from Michigan (2) and to QoI-resistant isolates from GenBank (Accession Nos. JQ619933 and JQ360628). The remaining seven had 100% identity with a sensitive isolate (EF176921.1). Each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A in the cytochrome b gene. This G143A mutation has been associated with QoI resistance in a number of fungi (3). To confirm the result, a conidium germination bioassay was carried out using nine isolates with the G143A mutation on sugar beet leaf agar covered with water agar amended with pyraclostrobin at concentrations ranging from 0 to 54.3 μg/ml and distributed on a spiral gradient using an Eddyjet II spiral plater. The medium was supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway. Following incubation at 25°C for 2 days, the distance between the center of the plate at which conidial germination was 50% of the maximum observed growth (EC) and the point at which conidial germination terminated were measured (TEC). The EC50 values were determined from the SGE software for each isolate by entering the EC and TEC values, respectively. The estimated EC50 for a representative wild type (sensitive) isolate was 0.03 μg/ml, while the value for the resistant isolate could not be calculated because it was greater than the highest concentration tested (54.3 μg/ml). Additionally, in the controls with no SHAM or fungicide, the resistant isolate showed a consistent reduced germination rate compared to the sensitive isolate (30.0% and 93.5% germination, respectively). Confirmation of fungicide insensitivity will require a re-evaluation of current management practices in Ontario to minimize economic losses due to CLS. References: (1) B. J. Jacobsen and G. D. Franc. Compendium of Beet Diseases and Pests, 2nd ed, APS Press, St. Paul, MN, 2009. (2) W. Kirk et al. New Dis. Rep. 26:3, 2012. (3) Z. Ma and T. J. Michailides. Crop Prot. 24:853, 2005. (4) A. Malandrakis et al. Pestic. Biochem. Physiol. 100:87092, 2011.

A Geostatistical Approach to Visualize the Diversity of Soil Inhabiting Bacteria and Edaphic Qualities in Potato ( Solanum tuberosum) Production Systems

A study was conducted in Michigan (MI) to assess spatial patterns of soil biological and physiochemical factors related to yield in potato production. The project developed an approach to integrate techniques including: high-throughput DNA sequencing; GIS; geostatistics; traditional soil analyses; and yield data. Twenty soil samples were taken and GPS marked in the fall of 2012 from a grower’s field scheduled to be in potato production, and total genomic DNA was extracted. Parallel sequencing targeting the 16S rRNA gene was used to assess bacterial diversity. The total number of taxa identified by sequence analysis was 21, 81, 140, 300 and 814 at the level of phyla, class, order, family and genus respectively. Sequencing results and information gathered on yield at each point was used to generate multi-layer GIS-based maps.ResumenSe condujo un estudio en Michigan (MI) para evaluar los patrones espaciales de factores biológicos y fisioquímicos del suelo relacionados al rendimiento en la producción de papa. El proyecto desarrolló un planteamiento para integrar técnicas, incluyendo: secuenciación de ADN de alto rendimiento; GIS, geoestadística, análisis tradicional de suelo; y datos de rendimiento. Se tomaron 20 muestras y se marcaron con GPS en otoño de 2012 en un campo de un productor programado para ser de producción de papa, y se extrajo el ADN total genómico. Se usó secuenciación paralela del gen 16S del ARNr para evaluar la diversidad bacteriana. El número total de taxa identificado por el análisis de secuencia fue 21, 81, 140, 300 y 814 al nivel de phyla, clase, orden, familia y género respectivamente. Los resultados de la secuenciación y la información recopilada en rendimiento en cada punto, se usaron para generar mapas de multicapas basados en GIS.

First report of DMI-insensitive Cercospora beticola on sugar beet in Ontario, Canada

Cercospora leaf spot, caused by the fungal pathogen Cercospora beticola, is an economically important foliar disease of sugar beet in Ontario, Canada. The first demethylation inhibitor (DMI) fungicide registered for sugar beet in Canada …