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Background Red clover (Trifolium pratense), a crop constrained by weak persistence, is the number one forage legume in Northern Europe. Whether plant loss is a sign of an increasing number of known or new pathogens, a decreasing occurrence of beneficial soil microorganisms or other factors is unclear. Here, we collected red clover root samples from Swedish conventional and organic farming sites located between latitudes 55°N and 66°N and analyzed their prokaryotic and eukaryotic communities using amplicon sequencing. We compared the organism diversity, differential abundance, hub taxa and impact of Rhizobium on fungal pathogens in relation to geographic location and farming practices. Results We found that microbial biodiversity was lowest in samples from southern Sweden and on conventional farms. Rhizobium was the most prevalent bacterial genus, followed by Sphingomonas, Mucilaginibacter, Flavobacterium and the unclassified Chloroflexi group KD4-96. The Leptodontidium, Cladosporium, Clonostachys and Tetracladium fungal genera known for endophytic, ectomycorrhizal, saprotrophic and mycoparasitic lifestyles were also frequently observed in all samples. Sixty-two potential pathogenic fungi were identified with a bias toward grass pathogens and a higher abundance in samples from conventional farms. To gain insights into the cooccurrence patterns of red clover root-associated communities, intrakingdom and interkingdom networks were constructed. Bacterial networks were dominated by known beneficial groups of bacterial species. The interaction between Rhizobium leguminosarum bv. trifolii and Aeromicrobium sp. was strongly positive in the north but negative in the south. Different co-occurrence responses were observed when fungal and bacterial data were combined, as exemplified by the interactions with Clonostachys rosea, a beneficial fungus that fluctuated in our dataset and was positively associated with species in Enterobacterales and Aeromicrobium in the north but negatively with species in the south. Conclusions We show that the microbial community is mainly shaped by geographic location and management procedures. R. leguminosarum bv. trifolii is a keystone species in samples from the northern region and Swedish organic farms. This Rhizobium species is negatively associated with all fungal pathogenic species recognized in this study. A number of previously unknown fungal species are identified, particularly those known to produce mycotoxins, which are of major concern for crop productivity and feed safety.

Endophytes, both of bacterial and fungal origin, are ubiquitously present in all plants. While their origin and evolution are enigmatic, there is burgeoning literature on their role in promoting growth and stress responses in their hosts. We demonstrate that a salt-tolerant endophyte isolated from salt-adapted Pokkali rice, a Fusarium sp., colonizes the salt-sensitive rice variety IR-64, promotes its growth under salt stress and confers salinity stress tolerance to its host. Physiological parameters, such as assimilation rate and chlorophyll stability index were higher in the colonized plants. Comparative transcriptome analysis revealed 1348 up-regulated and 1078 down-regulated genes in plants colonized by the endophyte. Analysis of the regulated genes by MapMan and interaction network programs showed that they are involved in both abiotic and biotic stress tolerance, and code for proteins involved in signal perception (leucine-rich repeat proteins, receptor-like kinases) and transduction (Ca2+ and calmodulin-binding proteins), transcription factors, secondary metabolism and oxidative stress scavenging. For nine genes, the data were validated by qPCR analysis in both roots and shoots. Taken together, these results show that salt-adapted Pokkali rice varieties are powerful sources for the identification of novel endophytes, which can be used to confer salinity tolerance to agriculturally important, but salt-sensitive rice varieties.

A total of 507 endophytic fungal isolates belonging to 82 operational taxonomic units (OTUs) were isolated from fifteen plant species naturally occurring in the Thar Desert, Rajasthan, India. Aspergillus, Alternaria, Chaetomium, Penicillium and Nigrospora were among the dominant fungal genera. Thermotolerance of the isolates was evaluated by culturing the fungi at 40 °C and 45 °C in shake flasks. Growth of nine OTUs (ACJ-2, ACJ-5, ACL-2, ACT-2, ACT-3, LAS-4, LAS-6, SAP-3 and SAP-6) was unaffected at 40 °C. Of these, six endophytes, namely, ACJ-2, ACJ-5 (Aspergillus flavus), SAP-3 (Aspergillus sp.), SAP-6, LAS-4 (Aspergillus sp.) and LAS-6 (Chaetomium sp.), were tolerant to as high as 45 °C. Rest of the OTUs did not survive culture temperatures beyond 35 °C. We evaluated the ability of one thermotolerant endophyte, LAS-6 (Chaetomium sp.) to confer high temperature tolerance and three OTUs, namely, LAS-4 (Aspergillus sp.), SAP-3 (Aspergillus sp.) and SAP-6 to confer drought tolerance to a rice cultivar, IR-64, at early seedling stage. Seedlings treated with LAS-6 (Chaetomium sp.) showed a higher survival percentage as well as maintained a significantly higher shoot and root growth under high temperature stress compared to seedlings not treated with the fungus. Under drought stress, endophyte treated seedlings maintained a significantly higher root growth compared to untreated seedlings. The identified thermotolerant fungal sources could be potentially useful in alleviating abiotic stress in agriculturally important crops.

Strawberry powdery mildew, caused by Podosphaeraaphanis is a major fungal disease that affects strawberry yield and quality. In the model plant species Arabidopsis and the crop plants barley, tomato and pea, the Mildew resistance locus O (MLO) proteins have been found to be required for powdery mildew susceptibility. The present study, based on the sequence of a wild plum (Prunus americana) MLO protein, identified 16 MLO genes within the genome of woodland strawberry, Fragaria vesca and examined their expression pattern in response to powdery mildew infection in three diploid strawberry cultivars. Phylogenetic analysis showed that the FvMLO genes can be classified into six clades. Four FvMLO genes were grouped into clade III, which comprises MLO genes from Arabidopsis, tomato and grapevine that mediate powdery mildew susceptibility. A RNA-seq analysis of two diploid strawberry cultivars, F. vescassp. vesca accession Hawaii 4 (HW) and F. vesca f. semperflorens line “Yellow Wonder 5AF7” (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection showed the expression of 12 out of the 16 FvMLO genes. The comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) detected by RNA-seq and expression values of qRT-PCR for FvMLO genes showed substantial agreement. The FvMLO3 gene, which was grouped in clade III and orthologous to the Arabidopsis,tomato and grapevine genes, was highly expressed in YW compared to other FvMLO genes across varieties. The results showed that FvMLO genes can be used as potential candidates to engineer powdery mildew resistance in strawberry based on MLO suppression or genome editing.

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry