A panel of qPCR assays to detect and quantify soybean soil-borne pathogens.

Fusarium oxysporum,F. graminearum,F. acuminatum,F. equiseti,F. proliferatum,F. solani, and Rhizoctonia solani are soil-borne fungal pathogens that cause substantial yield loss in a widespread list of crops worldwide. The objective of this study was to develop a panel of TaqMan assays for the detection and quantification of these six widespread soil-borne fungal species using real-time polymerase chain reaction (qPCR). The primers and probes were designed based on the intergenic spacer ribosomal RNA and translation elongation factor 1-alpha gene (tef1). These assays, although not multiplexed, can be performed simultaneously as they have similar reaction conditions, allowing more efficiency when targeting multiple pathogens in a sample. The assays presented high efficiency (94.3%-108.9%) and sensitivity, with a limit of detection of 0.05 picograms (50 femtograms) of target DNA. Results from an assay targeting 19 non-target and closely related species confirmed the specificity of the developed assays. The assays were also evaluated to detect the target species in different matrices, such as soil and plant material. This panel of qPCR assays is an additional tool that can be used by plant pathologists, microbiologists, plant breeders, diagnostic clinics, and other researchers interested in these fungal species.

Ecology and diversity of culturable fungal species associated with soybean seedling diseases in the Midwestern United States.

AIMS: To isolate and characterize fungi associated with diseased soybean seedlings in Midwestern soybean production fields and to determine the influence of environmental and edaphic factors on their incidence. METHODS AND RESULTS: Seedlings were collected from fields with seedling disease history in 2012 and 2013 for fungal isolation. Environmental and edaphic data associated with each field was collected. 3036 fungal isolates were obtained and assigned to 76 species. The most abundant genera recovered were Fusarium (73%) and Trichoderma (11.2%). Other genera included Mortierella, Clonostachys, Rhizoctonia, Alternaria, Mucor, Phoma, Macrophomina and Phomopsis. Most recovered species are known soybean pathogens. However, non-pathogenic organisms were also isolated. Crop history, soil density, water source, precipitation and temperature were the main factors influencing the abundance of fungal species. CONCLUSION: Key fungal species associated with soybean seedling diseases occurring in several US production regions were characterized. This work also identified major environment and edaphic factors affecting the abundance and occurrence of these species. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and characterization of the main pathogens associated with seedling diseases across major soybean-producing areas could help manage those pathogens, and devise more effective and sustainable practices to reduce the damage they cause.

Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

Microbial Communities Associated With Long-Term Tillage and Fertility Treatments in a Corn-Soybean Cropping System.

Tillage and fertilization are common practices used to enhance soil fertility and increase yield. Changes in soil edaphic properties associated with different tillage and fertility regimes have been widely examined, yet, the microbially mediated pathways and ecological niches involved in enhancing soil fertility are poorly understood. The effects of long-term conventional tillage and no-till in parallel with three fertility treatments (No fertilization, N-only, and NPK) on soil microbial communities were investigated in a long-term field study that was established in the 1970's. Here, we used high-throughput sequencing of bacterial, fungal and oomycetes markers, followed by community-level functional and ecological assembly to discern principles governing tillage and fertility practices' influence on associated soil microbiomes. Both tillage and fertilizer significantly altered microbial community structure, but the tillage effect was more prominent than the fertilizer effect. Tillage significantly affected bacteria, fungi, fusaria, and oomycete beta-diversity, whereas fertilizer only affected bacteria and fungi beta-diversity. In our study different tillage and fertilizer regimes favored specific networks of metabolic pathways and distinct ecological guilds. No-till selected for beneficial microbes that translocate nutrients and resources and protect the host against pathogens. Notably, ecological guilds featuring arbuscular mycorrhizae, mycoparasites, and nematophagous fungi were favored in no-till soils, while fungal saprotrophs and plant pathogens dominated in tilled soils. Conventional till and fertilizer management shifted the communities toward fast growing competitors. Copiotrophic bacteria and fusarium species were favored under conventional tillage and in the presence of fertilizers. The analysis of the metagenomes revealed a higher abundance of predicted pathways associated with energy metabolism, translation, metabolism of cofactors and vitamins, glycan biosynthesis and nucleotide metabolism in no-till. Furthermore, no specific pathways were found to be enriched under the investigated fertilization regimes. Understanding how tillage and fertilizer management shift microbial diversity, structure and ecological niches, such as presented here, can assist with designing farming systems that can maintain high crop yield, while reducing soil erosion and nutrient losses.

Targeting Aflatoxin Biosynthetic Genes.

Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities are mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requires early detection of the aflatoxin-causing organisms. Detection and quantification of aflatoxin-producing fungi has always been a challenge, especially within species of Aspergillus and Penicillium. Culture-based methods require a high level of expertise and a list of sophisticated equipment. Furthermore, even for a trained taxonomist, species that are identical in morphology, physiology, and nutritional aspects can be challenging to classify. Fungal taxonomy has changed over the past few decades; more species are being reclassified, and new species are being described due to advances in sequencing and genome assembly. These developments make the use of PCR-based approaches practical, rapid, and more reliable for the identification of fungi to the species level. This chapter presents a variety of protocols to detect and quantify aflatoxin-producing fungi using mycotoxin biosynthesis pathway genes.