Hanna Friberg

New primers to amplify the fungal ITS2 region – evaluation by 454-sequencing of artificial and natural communities

With recent methodological advances, molecular markers are increasingly used for semi-quantitative analyses of fungal communities. The aim to preserve quantitative relationships between genotypes through PCR places new demands on primers to accurately match target sites and provide short amplicons. The internal transcribed spacer (ITS) region of the ribosome encoding genes is a commonly used marker for many fungal groups. Here, we describe three new primers - fITS7, gITS7 and fITS9, which may be used to amplify the fungal ITS2 region by targeting sites in the 5.8S encoding gene. We evaluated the primers and compared their performance with the commonly used ITS1f primer by 454-sequencing of both artificially assembled templates and field samples. When the entire ITS region was amplified using the ITS1f/ITS4 primer combination, we found strong bias against species with longer amplicons. This problem could be overcome by using the new primers, which produce shorter amplicons and better preserve the quantitative composition of the template. In addition, the new primers yielded more diverse amplicon communities than the ITS1f primer.

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Influence of soil fauna on fungal plant pathogens in agricultural and horticultural systems

Abstract An understanding of the ecology of plant pathogens is crucial for the development of agricultural and horticultural growing systems that enhance plant health. One important group of organisms that influences plant pathogen survival and dispersal is the soil fauna. This review deals with known and possible interactions between soil animals and different groups of fungal plant pathogens. It is suggested that facultative saprophytes may be affected considerably by mycelial grazing by soil animals, while obligate pathogens may be more influenced by animals that ingest spores and other types of propagules. Both types of pathogens can also be influenced by indirect interactions with the soil fauna. Manipulating the soil environment for enhancement of beneficial soil animals that consume pathogens seems to be a realistic possibility for sustainable agriculture and horticulture.

Germination of Plasmodiophora brassicae resting spores stimulated by a non-host plant

Plant-induced germination of Plasmodiophora brassicae resting spores was studied in a laboratory experiment. Spore reaction was analysed in nutrient solution with exudates from growing roots of different plant species – one host plant (Brassica rapa var. pekinensis) and four non-host plants (Lolium perenne, Allium porrum, Secale cereale and Trifolium pratense) – and in controls with distilled water and nutrient solution. It was found that root exudates from L. perenne stimulated spore germination more than exudates from the other plants, including those from the host plant. The effect could not be explained by differences in the nutritional composition of the solutions due to differential uptake of the plant species, or by differences in root activity, measured as exudation of soluble sugars. This is the first time such a separation of factors has been done in analysing the influence of plants on P. brassicae germination. Although stimulation of P. brassicae resting spore germination is not restricted to the presence of host plants, it seems to vary depending on the plant species.

Fungicide effects on fungal community composition in the wheat phyllosphere.

The fungicides used to control diseases in cereal production can have adverse effects on non-target fungi, with possible consequences for plant health and productivity. This study examined fungicide effects on fungal communities on winter wheat leaves in two areas of Sweden. High-throughput 454 sequencing of the fungal ITS2 region yielded 235 operational taxonomic units (OTUs) at the species level from the 18 fields studied. It was found that commonly used fungicides had moderate but significant effect on fungal community composition in the wheat phyllosphere. The relative abundance of several saprotrophs was altered by fungicide use, while the effect on common wheat pathogens was mixed. The fungal community on wheat leaves consisted mainly of basidiomycete yeasts, saprotrophic ascomycetes and plant pathogens. A core set of six fungal OTUs representing saprotrophic species was identified. These were present across all fields, although overall the difference in OTU richness was large between the two areas studied.

Agricultural factors affecting Fusarium communities in wheat kernels

Fusarium head blight (FHB) is a devastating disease of cereals caused by Fusarium fungi. The disease is of great economic importance especially owing to reduced grain quality due to contamination by a range of mycotoxins produced by Fusarium. Disease control and prediction is difficult because of the many Fusarium species associated with FHB. Different species may respond differently to control methods and can have both competitive and synergistic interactions. Therefore, it is important to understand how agricultural practices affect Fusarium at the community level. Lower levels of Fusarium mycotoxin contamination of organically produced cereals compared with conventionally produced have been reported, but the causes of these differences are not well understood. The aim of our study was to investigate the effect of agricultural factors on Fusarium abundance and community composition in different cropping systems. Winter wheat kernels were collected from 18 organically and conventionally cultivated fields in Sweden, paired based on their geographical distance and the wheat cultivar grown. We characterised the Fusarium community in harvested wheat kernels using 454 sequencing of translation elongation factor 1-α amplicons. In addition, we quantified Fusarium spp. using real-time PCR to reveal differences in biomass between fields. We identified 12 Fusarium operational taxonomic units (OTUs) with a median of 4.5 OTUs per field. Fusarium graminearum was the most abundant species, while F. avenaceum had the highest occurrence. The abundance of Fusarium spp. ranged two orders of magnitude between fields. Two pairs of Fusarium species co-occurred between fields: F. poae with F. tricinctum and F. culmorum with F. sporotrichoides. We could not detect any difference in Fusarium communities between the organic and conventional systems. However, agricultural intensity, measured as the number of pesticide applications and the amount of nitrogen fertiliser applied, had an impact on Fusarium communities, specifically increasing the abundance of F. tricinctum. There were geographical differences in the Fusarium community composition where F. graminearum was more abundant in the western part of Sweden. The application of amplicon sequencing provided a comprehensive view of the Fusarium community in cereals. This gives us better opportunities to understand the ecology of Fusarium spp., which is important in order to limit FHB and mycotoxin contamination in cereals.

Genus-Specific Primers for Study of Fusarium Communities in Field Samples.

ABSTRACT Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

Organic farming increases richness of fungal taxa in the wheat phyllosphere

Organic farming is often advocated as an approach to mitigate biodiversity loss on agricultural land. The phyllosphere provides a habitat for diverse fungal communities that are important for plant health and productivity. However, it is still unknown how organic farming affects the diversity of phyllosphere fungi in major crops. We sampled wheat leaves from 22 organically and conventionally cultivated fields in Sweden, paired based on their geographical location and wheat cultivar. Fungal communities were described using amplicon sequencing and real‐time PCR. Species richness was higher on wheat leaves from organically managed fields, with a mean of 54 operational taxonomic units (OTUs) compared with 40 OTUs for conventionally managed fields. The main components of the fungal community were similar throughout the 350‐km‐long sampling area, and seven OTUs were present in all fields: Zymoseptoria, Dioszegia fristingensis, Cladosporium, Dioszegia hungarica, Cryptococcus, Ascochyta and Dioszegia. Fungal abundance was highly variable between fields, 103–105 internal transcribed spacer copies per ng wheat DNA, but did not differ between cropping systems. Further analyses showed that weed biomass was the strongest explanatory variable for fungal community composition and OTU richness. These findings help provide a more comprehensive understanding of the effect of organic farming on the diversity of organism groups in different habitats within the agroecosystem.

Roots of symptom-free leguminous cover crop and living mulch species harbor diverse Fusarium communities that show highly variable aggressiveness on pea (Pisum sativum)

Leguminous cover crop and living mulch species show not only great potential for providing multiple beneficial services to agro-ecosystems, but may also present pathological risks for other crops in rotations through shared pathogens, especially those of the genus Fusarium. Disease severity on roots of subterranean clover, white clover, winter and summer vetch grown as cover crop and living mulch species across five European sites as well as the frequency, distribution and aggressiveness to pea of Fusarium spp. recovered from the roots were assessed in 2013 and 2014. Disease symptoms were very low at all sites. Nevertheless, out of 1480 asymptomatic roots, 670 isolates of 14 Fusarium spp. were recovered. The most frequently isolated species in both years from all hosts were F. oxysporum and F. avenaceum accounting for 69% of total isolation percentage. They were common at the Swiss, Italian and German sites, whereas at the Swedish site F. oxysporum dominated and F. avenaceum occurred only rarely. The agressiveness and effect on pea biomass were tested in greenhouse assays for 72 isolates of six Fusarium species. Isolates of F. avenaceum caused severe root rot symptoms with mean severity index (DI) of 82 and 74% mean biomass reduction compared to the non-inoculated control. Fusarium oxysporum and F. solani isolates were higly variable in agressiveness and their impact on pea biomass. DI varied between 15 and 50 and biomass changes relative to the non-inoculated control -40% to +10%. Isolates of F. tricinctum, F. acuminatum and F. equiseti were non to weakly agressive often enhancing pea biomass. This study shows that some of the major pea pathogens are characterized by high ecological plasticity and have the ability to endophytically colonize the hosts studied that thus may serve as inoculum reservoir for susceptible main legume grain crops such as pea.