A robust hydroponic-based system for screening red clover (Trifolium pratense) for Fusarium avenaceum

Red clover (Trifolium pratense) is an important forage legume crop that suffers likemost perennial crops from attacks by soil-borne pathogens.Fusariumroot rot is oneof the most serious diseases and at the same time problematic to identify resistancebecause of its hidden life in the soil. Current screening methods are laborious andhampered by limited reproducibility. To remedy this situation, we aimed to establisha simple and reliable hydroponics-based screening system to facilitate studies of redclover–Fusarium avenaceuminteractions. First, the fungal spore concentrations werebalanced toward the development of red clover plants grown hydroponically. Wefound that the optimum concentration was 30,000 spores in 2 L of hydroponicmedium to ensure infection during the plant growth period in this system. The proce-dure was scaled-up to screen plants from 25 populations to identify red clover indi-viduals with the improved resistance toF. avenaceum. Susceptible plants hadapproximately two-fold higher amounts of fungal DNA than resistant plants, demon-strating a correlation between the disease readings of the plants and pathogen DNA.We foresee this screening procedure meeting the needs of both applied breedingwork and in-depth molecular studies of responses between this pathogen and itshost plant. This method could be applied for the screening of other plant species forresistance toFusariumspp. or to other root microbes

Identification and expression analysis of Fragaria vesca MLO genes involved in interaction with powdery mildew (Podosphaera aphanis)

Strawberry powdery mildew, caused by Podosphaera aphanis 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. vesca ssp. 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

Global transcriptome analysis and identification of differentially expressed genes after infection of Fragaria vesca with powdery mildew (Podosphaera aphanis)

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 strawberr

Red clover root-associated microbiota is shaped by geographic location and choice of farming system

Aims: This study evaluated the red clover (Trifolium pratense) root-associated microbiota to clarify the presence of pathogenic and beneficial microorganisms in 89 Swedish field sites.Methods and results: 16S rRNA and ITS amplicon sequencing analysis were performed on DNA extracted from the red clover root samples collected to determine the composition of the prokaryotic and eukaryotic root-associated microbe communities. Alpha and beta diversities were calculated and relative abundance of various microbial taxa and their co-occurrence were analyzed. 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, 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.Conclusions: We showed that the microbial community was mainly shaped by geographic location and management procedures. Co-occurrence networks revealed that the Rhizobiumleguminosarum bv. trifolii was negatively associated with all fungal pathogenic taxa recognized in this study

Molecular studies of plant-pathogen interactions in strawberry

Powdery mildew is an important disease of crop plants. In strawberry, the disease is caused by the obligate biotrophic fungus Podosphoera ophonis (syn: Sphoeratheco mocu/oris f. sp. Fragorioe) and causes significant economic loss in the cultivated strawberry. This study used the diploid species Fragorio vesco as a model to study plant pathogen interactions. Initial studies involved in the identification of mildew resistance locus 0 (MLO) genes in the diploid species, F. vesco f. vesco and identified 17 FvMLO genes. Real-time quantitative PCR (qRT-PCR) results showed differential expression of 12 FvMLO genes in four strawberry varieties, F. vesco f. vesco (Fv), F. vesco ssp. vesco accession Hawaii 4 (HW), F. vesco f. semperf/orens line "Yellow Wonder 5AF7" (YW) and Eluica (octoploid strawberry). The FvML03 gene which is orthologous to the Arabidopsis AtML02, AtML06 and AtML012 and tomato S/ML01 genes required for powdery mildew susceptibility was highly expressed (164 fold) 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. To investigate the molecular mechanism underlying strawberry-powdery mildew interaction, RNA-seq was employed to generate a large transcriptome dataset in HW and YW at 1 d and 8 d after powdery mildew infection. We identified about 999 million (92%) reads mapped to the F. vesco genome. Transcripts were identified from a total of 23,470 and 23,464 genes in HW and YW, respectively at all three stages (control, 1DAI and 8DAI). Differential gene expression analysis identified 1,567, 1,846 and 1,145 upregulated genes between control and 1DAI, control and 8DAI, and 1DAI and ! lDAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were upregulated in YW. Also 646, 1,098 and 624 down regulated genes were identified in HW, while 571, 754 and 627 genes were downregulated in YW between all three stages, respectively. The study also investigated differentially expressed genes (log2 fold changes <:5) between control and 1DAI in both HW and YW. A large number of genes related to secondary metabolism, signal transduction, transcriptional regulation and disease resistance were expressed predominantly either in HW or YW. These included flavonoid 3' -monooxygenase, peroxidase 15, glucan endo-1, 3-beta-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This first application of RNA-seq to any pathogen interaction in strawberry provides wealth of genomic information for future studies in understanding molecular and cellular processes of the strawberry defence response to powdery mildew. It was also shown that metabolic lSN hydroponic isotope labelling of entire plants (HILEP) can be achieved in strawberry for quantitative proteomics analysis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A robust hydroponic‐based system for screening red clover (Trifolium pratense) for Fusarium avenaceum

Abstract Red clover (Trifolium pratense) is an important forage legume crop that suffers like most perennial crops from attacks by soil‐borne pathogens. Fusarium root rot is one of the most serious diseases and at the same time problematic to identify resistance because of its hidden life in the soil. Current screening methods are laborious and hampered by limited reproducibility. To remedy this situation, we aimed to establish a simple and reliable hydroponics‐based screening system to facilitate studies of red clover–Fusarium avenaceum interactions. First, the fungal spore concentrations were balanced toward the development of red clover plants grown hydroponically. We found that the optimum concentration was 30,000 spores in 2 L of hydroponic medium to ensure infection during the plant growth period in this system. The procedure was scaled‐up to screen plants from 25 populations to identify red clover individuals with the improved resistance to F. avenaceum. Susceptible plants had approximately two‐fold higher amounts of fungal DNA than resistant plants, demonstrating a correlation between the disease readings of the plants and pathogen DNA. We foresee this screening procedure meeting the needs of both applied breeding work and in‐depth molecular studies of responses between this pathogen and its host plant. This method could be applied for the screening of other plant species for resistance to Fusarium spp. or to other root microbes

Thermotolerance of fungal endophytes isolated from plants adapted to the Thar Desert, India

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

Role of endophytes in early seedling growth of plants: a test using systemic fungicide seed treatment

Systemic fungicide seed treatments are routinely used in conventional agriculture to control soil and seedborne diseases, but little is known about their unintended adverse effects on non-target beneficial fungal endophytes that are known to be involved in plant growth and development. This study evaluated the seed treatment effect of a broad spectrum systemic fungicide, carbendazim (bavistin) on symbiotic association of fungal endophytes in rice and on early seedling growth of rice, green gram, soybean, and cowpea. Seeds were surface sterilized with sodium hypochlorite followed by 0.2% bavistin treatment. Growth of fungal endophytes was significantly affected by the seed treatment with fungicide in rice seedlings, while shoot and root growth was suppressed in all the crops. Quantitative real time PCR showed that the level of expression of two basal transcriptional regulator genes, OsBTF3 and OsNFYC1 that are required for seed germination and seedling growth significantly decreased in bavistin treated rice seedlings. Re-inoculation of consortia of fungal endophytes onto bavistin treated rice seedlings significantly recovered seedling growth and development. These results suggest that fungicide treatment of seeds affects early seedling growth and has negative impact on beneficial fungal endophytes that are involved in plant growth and development. This study provides information on possible ill effects of fungicide on beneficial fungal endophytes that play key roles in early seedling growth of plants and also open up the prospect to additional research on different crops in vitro and field conditions to determine the consequences of fungicide effects and optimise fungicide application strategies to develop sustainable disease control methods