Rumakanta Sapkota

An improved high throughput sequencing method for studying oomycete communities

Culture-independent studies using next generation sequencing have revolutionized microbial ecology, however, oomycete ecology in soils is severely lagging behind. The aim of this study was to improve and validate standard techniques for using high throughput sequencing as a tool for studying oomycete communities. The well-known primer sets ITS4, ITS6 and ITS7 were used in the study in a semi-nested PCR approach to target the internal transcribed spacer (ITS) 1 of ribosomal DNA in a next generation sequencing protocol. These primers have been used in similar studies before, but with limited success. We were able to increase the proportion of retrieved oomycete sequences dramatically mainly by increasing the annealing temperature during PCR. The optimized protocol was validated using three mock communities and the method was further evaluated using total DNA from 26 soil samples collected from different agricultural fields in Denmark, and 11 samples from carrot tissue with symptoms of Pythium infection. Sequence data from the Pythium and Phytophthora mock communities showed that our strategy successfully detected all included species. Taxonomic assignments of OTUs from 26 soil sample showed that 95% of the sequences could be assigned to oomycetes including Pythium, Aphanomyces, Peronospora, Saprolegnia and Phytophthora. A high proportion of oomycete reads was consistently present in all 26 soil samples showing the versatility of the strategy. A large diversity of Pythium species including pathogenic and saprophytic species were dominating in cultivated soil. Finally, we analyzed amplicons from carrots with symptoms of cavity spot. This resulted in 94% of the reads belonging to oomycetes with a dominance of species of Pythium that are known to be involved in causing cavity spot, thus demonstrating the usefulness of the method not only in soil DNA but also in a plant DNA background. In conclusion, we demonstrate a successful approach for pyrosequencing of oomycete communities using ITS1 as the barcode sequence with well-known primers for oomycete DNA amplification.

High-throughput sequencing of nematode communities from total soil DNA extractions

BackgroundNematodes are extremely diverse and numbers of species are predicted to be more than a million. Studies on nematode diversity are difficult and laborious using classical methods and therefore high-throughput sequencing is an attractive alternative. Primers that have been used in previous sequence-based studies are not nematode specific but also amplify other groups of organisms such as fungi and plantae, and thus require a nematode enrichment step that may introduce biases.ResultsIn this study an amplification strategy which selectively amplifies a fragment of the SSU from nematodes without the need for enrichment was developed. Using this strategy on DNA templates from a set of 22 agricultural soils, we obtained 64.4% sequences of nematode origin in total, whereas the remaining sequences were almost entirely from other metazoans. The nematode sequences were derived from a broad taxonomic range and most sequences were from nematode taxa that have previously been found to be abundant in soil such as Tylenchida, Rhabditida, Dorylaimida, Triplonchida and Araeolaimida.ConclusionsOur amplification and sequencing strategy for assessing nematode diversity was able to collect a broad diversity without prior nematode enrichment and thus the method will be highly valuable in ecological studies of nematodes.

Fungal Communities Including Plant Pathogens in Near Surface Air Are Similar across Northwestern Europe

Information on the diversity of fungal spores in air is limited, and also the content of airborne spores of fungal plant pathogens is understudied. In the present study, a total of 152 air samples were taken from rooftops at urban settings in Slagelse, DK, Wageningen NL, and Rothamsted, UK together with 41 samples from above oilseed rape fields in Rothamsted. Samples were taken during 10-day periods in spring and autumn, each sample representing 1 day of sampling. The fungal content of samples was analyzed by metabarcoding of the fungal internal transcribed sequence 1 (ITS1) and by qPCR for specific fungi. The metabarcoding results demonstrated that season had significant effects on airborne fungal communities. In contrast, location did not have strong effects on the communities, even though locations were separated by up to 900 km. Also, a number of plant pathogens had strikingly similar patterns of abundance at the three locations. Rooftop samples were more diverse than samples taken above fields, probably reflecting greater mixing of air from a range of microenvironments for the rooftop sites. Pathogens that were known to be present in the crop were also found in air samples taken above the field. This paper is one of the first detailed studies of fungal composition in air with the focus on plant pathogens and shows that it is possible to detect a range of pathogens in rooftop air samplers using metabarcoding.

Effect of Verticillium dahliae Soil Inoculum Levels on Spinach Seed Infection

Verticillium dahliae is a soilborne pathogen and a threat to spinach seed production. The aim of this study was to understand the relation between V. dahliae soil inoculum and infection in harvested seed. Quantitative polymerase chain reaction was used for quantification of the pathogen. Semifield experiments in which spinach was grown in soils with different inoculum levels enabled us to determine a threshold level for V. dahliae DNA of 0.003 ng/g of soil for seed infection to occur. Soils from production fields were sampled in 2013 and 2014 during and before planting, as well as the harvested seed. Seed from plants grown in infested soils were infected with V. dahliae in samples from both the semifield and open-field experiments. Lower levels of pathogen were found in seed from spinach grown in soils with a scattered distribution of V. dahliae (one or two positive of three soil subsamples) than in soils with a uniform distribution of the pathogen (three of three positive soil subsamples). Our results showed that infection of V. dahliae in harvested seed strongly depended on the presence of pathogen inoculum in the soil.

Spatiotemporal Variation and Networks in the Mycobiome of the Wheat Canopy

The phyllosphere is an important habitat for a diverse microbiome and an important entry point for many pathogens. Factors that shape the phyllosphere microbiome and also the co-existence among members and how they affect disease development are largely understudied. In this study we examined the wheat mycobiome by using metabarcoding of the fungal ITS1 region. Leaf samples were taken from four cultivars grown at two locations in Denmark. Samples were taken from the three uppermost leaves and at three growth stages to better understand spatiotemporal variation of the mycobiome. Analysis of read abundances showed that geographical location had a major effect in shaping the mycobiome in the total dataset, but also leaf position, growth stage and cultivar were important drivers of fungal communities. Cultivar was most important in explaining variation in older leaves whereas location better explained the variation in younger leaves, suggesting that communities are shaped over time by the leaf environment. Network analysis revealed negative co-existence between Zymoseptoria tritici and the yeasts Sporobolomyces, Dioszegia, and Cystofilobasidiaceae. The relative abundance of Z. tritici and the yeasts was relatively constant between individual samples, suggesting that fast growing fungi rapidly occupy empty space in the phyllosphere.

Oomycete Communities Associated with Reed Die-Back Syndrome

Phragmites australis (Cav.) Trin. ex Steud. die-back is a widely-studied phenomenon that was first discovered in northern Europe and that, until recently, was almost unknown in the Mediterranean basin. It has been described as a complex syndrome affecting reed populations leading to their retreat and decline, with significant impacts on valuable ecosystem services. Among the factors that cause the decline, soil-living microorganisms can be crucial. The aims of this study were to analyze the diversity of oomycetes communities associated with reed stands, and to understand whether they could play a key role in the decline. Variations in the structure of oomycetes communities were studied by metabarcoding of the internal transcribed spacer (ITS) 1 region of ribosomal DNA, from the sediments of five Italian freshwater ecosystems. They were chosen to cover a large variability in terms of surface area, water depth, microclimate, and presence of documented reed retreat. From 96 samples collected from reed roots, rhizosphere, and bulk soil, we assembled 207661 ITS1 reads into 523 OTUs. We demonstrated that oomycete communities were structured by several factors, among which the most important was die-back occurrence. Our study also indicates that Pythiogeton spp. could be potentially involved in the development of die-back. The role of heavy metals in the soil was also explored, and cadmium concentration was shown to affect oomycetes distribution. This study represents a significant step forward for the characterization of microbial communities associated with reed die-back syndrome and helps to gain knowledge of the complexity of these important wet ecosystems.