A cosmopolitan fungal pathogen of dicots adopts an endophytic lifestyle on cereal crops and protects them from major fungal diseases

Abstract

Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to help safeguard such resources for increasing human populations on a global scale. Here, we find that Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, rice, barley, maize, and oat, providing protection against Fusarium head blight, stripe rust, and rice blast. Protection is also provided by disabled S. sclerotiorum strains harboring a hypovirulence virus. The disabled strain DT-8 promoted wheat yields by 4-18% in the field and consistently reduced Fusarium disease by 40-60% across multiple field trials. We term the host-dependent trophism of S. sclerotiorum, destructively pathogenic or mutualistically endophytic, as schizotrophism. As a biotroph, S. sclerotiorum modified the expression of wheat genes involved in disease resistance and photosynthesis and increased the level of IAA. Our study shows that a broad-spectrum pathogen of one group of plants may be employed as a biocontrol agent in a different group of plants where they can be utilized as beneficial microorganisms while avoiding the risk of in-field release of pathogens. Our study also raises provocative questions about the potential role of schizotrophic endophytes in natural ecosystems.

Fig. 1. S. sclerotiorum growing endophytically in wheat roots.

a Visualized by confocal microscopy, hyphae of strain DT-8VF^RFP in a segment of a rootlet stained with wheat germ agglutinin (WGA). a2 Z-stack images to further show the hyphae inside the rootlet of a1; Scale bars for a1 and a2 are 200 and 50 µm. b Visualized by colloidal gold immunoelectron microscopy, hyphae of strain DT-8VF^RFP in the intercellular spaces of roots probed with the anti-mCherry antibody (b1), (b2), and (b3) are enlargements of the boxed regions (i and ii) in b1. c Visualized by scanning electron microscopy, hyphae of strains DT-8VF and DT-8 in the intercellular and intracellular spaces of roots (indicated by arrows). c1 Transverse section of a rootlet of DT-8VF-treated plant; c2–c4 enlargement of the regions in dotted boxes (i–iii) in c1; c2, c4 a single hypha in a root cell; c5 transverse section of a rootlet of strain DT-8-treated plant, showing hyphae in the intercellular and intracellular spaces of roots; and c6 enlargement of the regions in a dotted box (iv) of c5. No hyphae were observed in root of noninoculated wheat (CK), its SEM pictures were presented in Supplementary Fig. 3. Seedlings germinated from surface-sterilized seeds were inoculated with strains DT-8VF and DT-8 and allowed to grow for an additional 15 days on MS medium in a sterile culture bottle before sampling.

Fig. 2. S. sclerotiorum promotes wheat growth.

a Representative image of wheat plants treated with strains DT-8VF and DT-8; seedlings were grown in a greenhouse for 50 days. b Wheat seedling height 50 days after planting (t-test, p < 0.01) (n = 28). c Wheat plant height at the anthesis stage (t-test, p < 0.01) (n = 60). d Flag leaf length at the anthesis stage (t-test, p > 0.05) (n = 60). e Wheat spike length at the anthesis stage (t-test, p < 0.01) (n = 60). f The 1000-grain weight (t-test, p > 0.05) (n = 4). g Representative image of DT-8-treated and nontreated wheat plant at the anthesis stage in field. The length (h), width (i), and thickness (j) of flag leaves, the height of plant (k), and length of spike (l) of wheat plant at the anthesis stage in field (t-test, p < 0.01) (n = 120). m The weight of 1000-grains of DT-8-treated and nontreated wheat plant in field (t-test, p < 0.05) (n = 5). In b–f and h–m, error bars indicate standard deviation and different letters indicate significant differences.

Fig. 3. S. sclerotiorum enhances wheat resistance against Fusarium head blight and stripe rust.

a, b Spikes of wheat plants inoculated with F. graminearum in a greenhouse and under field conditions in 2017; photographs taken at 14 days post inoculation (dpi). Wheat seeds of cultivar Zheng 9023 were inoculated with S. sclerotiorum strains DT-8 and DT-8VF or water (mock) before sowing. c Percent of infected spikelets on F. graminearum-inoculated spikes under greenhouse conditions in 2017, 14 dpi (12 spikes for each treatment) (t-test, p < 0.05) (c; n = 12). d Percent of infected spikelets on F. graminearum-inoculated spikes under natural field conditions in 2017 and 2018. Ten plants from each plot were measured from a total of 30 plants in each treated group, (t-test between the two groups, p < 0.05) (d; n = 3). e DON content in spikelets collected from F. graminearum point-inoculated spikelets 14 days after inoculation. Values on black bars followed by the same letter are not significantly different at p < 0.05 (e; n = 6). f, g Disease index and percent of infected spikelets on spikes from three locations in Hubei province under natural conditions a week before harvest in 2018; 500 spikes were collected from each plot (a total of 1500 spikes from DT-8-treated plots and 1500 spikes from control plots) in each field and then examined (t-test between the two groups, p < 0.01) (f; n = 3) and (g; n = 3). h, i Strain DT-8-treated wheat revealed strong resistance against stripe rust. h Representative images at the anthesis stage of wheat (EZhou Experimental field); i disease index of stripe rust naturally occurred in wheat fields located at EZhou, Hubei province (winter wheat) and Tianzhu, Gansu Province (spring wheat) in 2017. In c–g and i, error bars indicate standard deviation and different letters indicate significant differences.

Fig. 4. S. sclerotiorum grows in and enhances the resistance of barley and rice against blast fungi.

Hyphae of strain DT-8 in the roots of rice (a) and barley (b), located in the intercellular and intracellular spaces. hy indicates hyphae, arrows indicate hyphae in root cells. Images were visualized by SEM. c, d DT-8-treated rice and barley, respectively, showing strong resistance against the rice blast fungus, M. oryzae; representative images of barley and rice inoculated with M. oryzae in a chamber. Barley and rice seeds were treated with DT-8 or water (mock) before sowing; photographs were taken at 5 dpi for barley and 7 dpi for rice.

Fig. 5. Disease resistance-associated processes in wheat spikes harboring endophytic S. sclerotiorum.

a Schematic diagram of wheat disease resistance processes enriched in the Gene Ontology (GO) analysis. The number in the top bracket in each box is the p value, and the lower bracket in the same box represents the number of enriched genes and the total genes in the pathway. The p values in the dark-colored boxes were lower than those in light-colored boxes. b Heat maps of the transcriptional profiles of 60 wheat DEGs associated with the wheat defense response in wheat spikes from DT-8-treated and control plants grown under natural conditions (|log2FC| > 1, FDR < 0.01). Each gene is represented by a colored bar. Significantly upregulated genes are highlighted in red and downregulated genes in green. All genesʼ gene_id were listed in Supplementary Table 4, genes labeled with “*” do not have common name. c Pattern-triggered immunity (PTI) pathway-related genes enriched following the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. d, e Abscisic acid (ABA) and jasmonic acid (JA) metabolism-related genes enriched following the KEGG analysis. Determination of ABA (f; n = 6) and JA (g; n = 6) content in wheat spikes (t-test, p < 0.05). Error bars indicate standard deviation and different letters indicate significant differences.