Dairy manure as a potential source of crop nutrients and environmental contaminants.

Although animal manure is applied to agricultural fields for its nutrient value, it may also contain potential contaminants. To determine the variability in such contaminants as well as in valuable nutrients, nine uncomposted manure samples from Idaho dairies collected during 2.5 years were analyzed for macro- and micro-nutrients, hormones, phytoestrogens, antibiotics, veterinary drugs, antibiotic resistance genes, and genetic elements involved in the spread of antibiotic resistance. Total N ranged from 6.8 to 30.7 (C:N of 10 to 21), P from 2.4 to 9.0, and K from 10.2 to 47.7 g/kg manure. Zn (103 - 348 mg/kg) was more abundant than Cu (56 - 127 mg/kg) in all samples. Phytoestrogens were the most prevalent contaminants detected, with concentrations fluctuating over time, reflecting animal diets. This is the first study to document the presence of flunixin, a non-steroidal anti-inflammatory drug, in solid stacked manure from regular dairy operations. Monensin was the most frequently detected antibiotic. Progesterones and sulfonamides were regularly detected. We also investigated the relative abundance of several types of plasmids involved in the spread of antibiotic resistance in clinical settings. Plasmids belonging to the IncI, IncP, and IncQ1 incompatibility groups were found in almost all manure samples. IncQ1 plasmids, class 1 integrons, and sulfonamide resistance genes were the most widespread and abundant genetic element surveyed, emphasizing their potential role in the spread of antibiotic resistance. The benefits associated with amending agricultural soils with dairy manure must be carefully weighed against the potential negative consequences of any manure contaminants.

Real-time PCR quantification of Fusarium avenaceum in soil and seeds.

The pathogenic fungus Fusarium avenaceum infects a broad range of plant hosts across the globe. While primarily soilborne, F. avenaceum can colonize all plant tissues, including buds, seeds, fruits, stems, crowns, and roots, resulting in significant crop yield reductions and economic losses for growers. In addition to its impact on crop productivity, F. avenaceum produces toxic metabolites that can be transferred to humans and livestock through consumption of infected foods. The ability of F. avenaceum to cause seed decay may be utilized to deplete the weed seedbank in soil, an important integrated weed management strategy. We developed a SYBR Green I-based real-time polymerase chain reaction (qPCR) assay to efficiently detect and quantify F. avenaceum in soil, wild oat (Avena fatua L.) seed caryopses, and wild oat seed hulls. The primer pair was designed from the translation elongation factor 1-alpha (TEF1) gene. In silico and wet lab testing were done to assess the ability of the primers to bind TEF1 sequences from Fusarium spp. and common soil fungi. The findings indicated that the primers were specific to F. avenaceum, and also recognized GenBank TEF1 accessions annotated as F. arthrosporioides, which has been listed as a foliar pathogen of wheat in Oregon, and conspecific with F. avenaceum. Standard curves of F. avenaceum DNA diluted with soil, caryopsis, or hull extracts indicated primer amplification efficiency was not significantly affected by PCR inhibitors. This real-time PCR assay effectively assesses the presence and abundance of F. avenaceum and its close relative F. arthrosporioides, if present, in soil and seed tissues. The assay can be used for endpoint PCR as well.

Defense Enzyme Responses in Dormant Wild Oat and Wheat Caryopses Challenged with a Seed Decay Pathogen.

Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a.1 was relatively rapid in wild oat (Avena fatua L.) isoline "M73," with >50% decay after 8 days with almost no decay in wheat (Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a.1 for 2-3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a.1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a.1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a.1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to wheat. Results are consistent with the hypotheses that (1) dormant seeds are capable of mounting complex responses to pathogens, (2) a diversity of defense enzymes are involved in responses in multiple plant species, and (3) it is possible to identify fungi capable of selective decay of weed seeds without damaging crop seeds, a concept that may be applicable to weed management in the field. While earlier work on seed defenses demonstrated the presence of passive defenses, this work shows that dormant seeds are also quite responsive and capable of activating and releasing defense enzymes in response to a pathogen.