One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y.

BACKGROUND: Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. RESULTS: We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. CONCLUSION: This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

PCR-denaturing Gradient Gel Electrophoresis as a Simple Identification Tool of Arbuscular Mycorrhizal Fungal Isolates.

Due to their obligate symbiotic nature and lack of long-term storage methods, the strain collection of arbuscular mycorrhizal (AM) fungi requires periodic proliferation using a pot culture with host plants. Therefore, a method to evaluate the purity of proliferated AM fungal cultures is critical for the quality control of their collection. In a simple evaluation of the purity and identity of a proliferated AM fungal culture, DNA extracted from the culture was amplified using AM fungi-specific PCR followed by an analysis with denaturing gradient gel electrophoresis (PCR-DGGE). The present results showed that the DGGE band patterns of AM fungal strains differed according to their phylogenetic positions, allowing for the rapid and easy identification of the proliferated AM fungal strains. When a culture was contaminated with another AM fungal strain, the DGGE pattern became a mixture of those strains. A contaminant strain was detectable even when its ratio was 1/9 of the main strain. It was also possible to confirm the purity of the culture by comparing whether the DGGE band pattern of the proliferated culture was identical to that obtained from single spores isolated from the culture. Therefore, PCR-DGGE is useful as a quality control tool for maintaining culture collections of AM fungi.

Isolation of Native Arbuscular Mycorrhizal Fungi within Young Thalli of the Liverwort Marchantia paleacea.

Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with most land plant species. "Root trap culture" generally has been used for isolating a single regenerated spore in order to establish a monospecific, native AMF line. Roots may be co-colonized with multiple AMF species; however, only a small portion of AMF within roots sporulate, and do so only under certain conditions. In this study, we tested whether young thalli (<2 mm) of the liverwort Marchantia paleacea harbour monospecific AMF, and can be used as a vegetative inoculant line. When M. paleacea gemmae were co-cultivated with roots obtained from the field, the young thalli were infected by AMF via rhizoids and formed arbuscules after 18 days post-sowing. Ribosomal DNA sequencing of the AMF-colonized thalli (mycothalli) revealed that they harboured phylogenetically diverse AMF; however, new gemmae sown around transplanted mycothalli showed evidence of colonization from phylogenetically uniform Rhizophagus species. Of note, mycothalli can also be used as an inoculum. These results suggest that the young thalli of M. paleacea can potentially isolate monospecific AMF from field soil in a spore-independent manner.

A Reduced Phosphorus Application Rate Using a Mycorrhizal Plant as the Preceding Crop Maintains Soybean Seeds' Nutritional Quality.

We tested whether introducing an arbuscular mycorrhizal fungi (AMF)-host plant with a reduced P application rate could maintain soybean seeds' nutrient quality. The dynamic variation of 14 nutrients was analyzed in source and sink organs during the seed-filling stage. The AMF-host and non-AMF-host plants, sunflower and mustard, were grown as preceding crops (PCs). Soybeans, the succeeding crops, were planted with three different phosphorus levels, namely, 0, 50, and 150 kg P2O5 ha-1. The results showed that the AMF-host PC with a reduced P application rate maintained the seed's yield and nutrients quality. During the seed-filling stage, the AMF-host PC with a reduced P application rate increased the uptake of most nutrients compared to the non-AMF-host PC, and improved the remobilization efficiency of all nutrients except Mn, Fe, and Se, compared to the optimal P application rate. These results could help improve the utilization efficiency of P fertilizers and protect soybeans' nutritional value.

Infection Unit Density as an Index of Infection Potential of Arbuscular Mycorrhizal Fungi.

The effective use of arbuscular mycorrhizal (AM) fungal function to promote host plant phosphate uptake in agricultural practice requires the accurate quantitative evaluation of AM fungal infection potential in field soil or AM fungal inoculation material. The number of infection units (IUs), intraradical fungal structures derived from single root entries formed after a short cultivation period, may reflect the number of propagules in soil when pot soil is completely permeated by the host root. However, the original IU method, in which all AM propagules in a pot are counted, requires the fine tuning of plant growing conditions and is considered to be laborious. The objective of the present study was to test whether IU density, not the total count of IU, but the number of IUs per unit root length, reflects the density of AM fungal propagules in soil. IU density assessed after 12 d of host plant cultivation and 3,3'-diaminobenzidine (DAB) staining showed a stronger linear correlation with propagule density than the mean infection percentage (MIP). In addition, IU density was affected less by the host plant species than MIP. We suggest that IU density provides a more rapid and reliable quantitation of the propagule density of AM fungi than MIP or the original IU method. Thus, IU density may be a more robust index of AM fungal infection potential for research and practical applications.

Differential Responses of Soybean and Sorghum Growth, Nitrogen Uptake, and Microbial Metabolism in the Rhizosphere to Cattle Manure Application: A Rhizobox Study.

In this study, we determined the capacity of soybean (Glycine max L. Merr. cv. Hoyoharuka) and sorghum (Sorghum bicolor L. Moench. cv. Hybrid Sorgo) to utilize different forms of nitrogen (N) in a rhizobox system. Seedlings were grown for 35 days without N or with 130 mg N kg-1 soil as ammonium sulfate or farmyard cattle manure. The soil fractions at different distances from the root were sliced millimeter by millimeter in the rhizobox system. We assessed the distribution of different forms of N and microbial metabolism in different soil fractions in the rhizosphere. There are no treatment-dependent changes in biomass production in the roots and shoots of soybeans, however, the ammonium and manure treatment yielded 1.30 and 1.40 times higher shoot biomass of sorghum than the control. Moreover, the depletion of inorganic N and total amino acids (TAA) in the rhizosphere was largely undetectable at various distances from the soybean roots regardless of the treatments employed. The addition of ammonium sulfate resulted in a decrease in the nitrate concentration gradient as the distance decreased from the sorghum roots. The addition of manure to the soil increased the N content in the sorghum shoots, 1.57 times higher than the control; this increase was negatively correlated with the concentrations of TAA in the soil of the root compartment. In addition, the application of manure simultaneously induced TAA depletion (i.e., the TAA concentration in root compartment was 1.48 times higher than that in bulk soil) and greater microbial activity and diversity in the sorghum rhizosphere, where higher microbial consumption of asparagine, glutamic acid, and phenylalanine were also observed near the roots. Our results are first to present the evidence that sorghum may possess a high capacity for taking up amino acids as a consequence of organic matter application, and microbial metabolism.

Ionome of soybean seed affected by previous cropping with mycorrhizal plant and manure application.

Two field experiments were conducted to investigate the effects of previous cultivation of an arbuscular mycorrhizal (AM) host plant and manure application on the concentration of 19 mineral elements in soybean ( Glycine max L. Merr. cv. Tsurumusume) seeds. Each experiment ran for two years (experiment 1 took place in 2007-2008, and experiment 2 took place in 2008-2009) with a split plot design. Soybeans were cultivated after growing either an AM host plant (maize, Zea mays L. cv. New dental) or a non-AM host plant (buckwheat, Fagopyrum esculentum Moench. cv. Kitawase-soba) in the first year in the main plots, with manure application (0 and 20 t/ha) during the soybean season in split plots from both main plots. On the basis of the two experiments, manure application significantly increased the available potassium (K) and decreased the available iron (Fe) and cesium (Cs) in the soil. However, higher concentrations of cadmium (Cd) and barium (Ba) and lower concentrations of Cs in the seed were induced by the application of manure. Cd levels in the seed were decreased by prior cultivation with the AM host plant. The present study showed that the identity of the prior crop and manure application changed the mineral contents of the soybean seed and suggests a connection between environmental factors and food safety.

Cellulase applied to the leaves of sweet pepper (Capsicum annuum L. var. grossum) upregulates the production of salicylic and azelaic acids.

Treating the leaves of sweet pepper (Capsicum annuum L. var. grossum) with an aqueous solution of cellulase resulted in a four-fold increase in the salicylic acid level compared to a control plant. The level of endogenous azelaic acid was also elevated by the cellulase treatment. Azelaic acid has recently been reported to act as a mobile "priming" agent to arm plants against pathogenic attack. Our results are consistent with this and that the cellulase treatment enhanced the ability of sweet pepper to withstand viral attack.

Differences in the metabolite profiles of spinach (Spinacia oleracea L.) leaf in different concentrations of nitrate in the culture solution.

The nitrogen (N) status of a plant determines the composition of its major components (amino acids, proteins, carbohydrates and organic acids) and, directly or indirectly, affects the quality of agricultural products in terms of their calorific value and taste. Although these effects are guided by changes in metabolic pathways, no overall metabolic analysis has previously been conducted to demonstrate such effects. Here, metabolite profiling using gas chromatography-mass spectrometry (GC-MS) was used to evaluate the effect of N levels on spinach tissue, comparing two cultivars that differed in their ability to use N. Wide variation in N content was observed without any distinct inhibition of growth in either cultivar. Principal component analysis (PCA) and self-organizing mapping (SOM) were undertaken to describe changes in the metabolites of mature spinach leaves. In PCA, the first component accounted for 44.5% of the total variance, the scores of which was positively correlated with the plant's N content, and a close relationship between metabolite profiles and N status was observed. Both PCA and SOM revealed that metabolites could be broadly divided into two types, correlating either positively or negatively with plant N content. The simple and co-coordinated metabolic stream, containing both general and spinach-specific aspects of plant N content, will be useful in future research on such topics as the detection of environmental effects on spinach through comprehensive metabolic profiling.