Co-infection by Soil-Borne Fungal Pathogens Alters Disease Responses Among Diverse Alfalfa Varieties

Abstract

Fusarium oxysporum f. sp. medicaginis (Fom) and Rhizoctonia solani (Rs) are the major soil-borne fungal pathogens that pose severe threats to commercial alfalfa production in China. However, the effects of Fom and Rs co-infection on alfalfa and whether co-infection alters disease resistance responses among diverse varieties remain unknown. A collection of 80 alfalfa varieties (Medicago sativa) originated from seven countries were used to study the effects of Fom and Rs co-infection on alfalfa and host resistance responses. The co-infection resulted in more severe disease and reductions in growth and biomass allocation across varieties in comparison with either single infection by Fom or Rs; in addition, root morphology was much more strongly altered by the co-infection. Principal component analysis based on all plant traits showed that varieties under the co-infection were related to the single infection by Rs, being separated from Fom, and hierarchical clustering found differential response patterns among varieties upon co-infection compared with either single infection, with most varieties being highly susceptible to the co-infection. Furthermore, varieties that were most resistant to either single infection were not effective to co-infection, and there was no individual variety with resistance to both pathogens singly and co-infected. This study reveals for the first time that the co-infection by Fom and Rs alters disease resistance responses among diverse alfalfa varieties and provides useful information for developing alfalfa varieties with resistance to the co-occurrence of different soil-borne pathogens.

Keywords: Fusarium oxysporum; Rhizoctonia solani; alfalfa; co-infection; host disease resistance; root rot; single infection; wilt.

FIGURE 1

Variations in disease severity among 80 alfalfa varieties single or co-inoculated with Fom and Rs. DIS and DIR, disease index of plant shoot and root, respectively. Fo, single inoculation with Fom; Rs, single inoculation with Rs; Fom:Rs, co-inoculation with Fom and Rs. Plants of the control treatment for companions showed no diseases and data were not included. Boxplots show the medians and 25th and 75th percentiles, with whiskers extending to 1.5 times of the interquartile range, and data presented beyond whiskers represent outliers. Different letters above the bars in the same column indicate significant differences (p < 0.001) among treatments according to Fisher’s protected least significant difference (LSD) test. Asterisks between the bars under the same treatment indicate a significant difference (p < 0.001) between traits by Student’s t-test. ns, not significant between traits.

FIGURE 2

Pathogen effects on plant growth among 80 alfalfa varieties single or co-inoculated with Fom and Rs. (A) Variations in plant height (PH) and root length (RL), (B) effect sizes of PH and RL, (C) variations in dry weight of shoot (DWS) and root (DWR), and (D) effect sizes of DWS and DWR. Fom, single inoculation with Fom; Rs, single inoculation with Rs; Fom:Rs, co-inoculation with Fom and Rs. Boxplots show the medians and 25th and 75th percentiles, with whiskers extending to 1.5 times of the interquartile range, and data presented beyond whiskers represent outliers. Different letters above the bars in the same column indicate significant differences (p < 0.001) among treatments according to Fisher’s protected LSD test. Asterisks between the bars under the same treatment indicate a significant difference (p < 0.001) between traits by Student’s t-test. ns, not significant between traits. Three values of DWS larger than 100 (115.1, 145.9, and 119.6 mg/plant for Mvv16, Msv17, and Msv27, respectively) under the control treatment were defined as 100 to maximize resolution. Positive effects (effect size < 0) of pathogen treatment on some traits under Fo (Supplementary Figure 2) were defined as zero to maximize resolution.

FIGURE 3

Pathogen effects on biomass allocations among 80 alfalfa varieties single or co-inoculated with Fom and Rs. (A) Variations in shoot biomass ratio (ST) and root biomass ratio (RT) and (B) effect sizes of ST and RT. Fom, single inoculation with Fom; Rs, single inoculation with Rs; Fom:Rs, co-inoculation with Fom and Rs. Boxplots show the medians and 25th and 75th percentiles, with whiskers extending to 1.5 times of the interquartile range, and data presented beyond whiskers represent outliers. Different letters above the bars in the same column indicate significant differences (p < 0.001) among treatments according to Fisher’s protected LSD test. Asterisks between the bars under the same treatment indicate a significant difference (p < 0.001) between traits by Student’s t-test. ns, not significant between traits.

FIGURE 4

Pathogen effects on root morphology among 12 alfalfa varieties single or co-inoculated with Fom and Rs. Effect sizes of (A) root diameter (RD), (B) total root length (TRL), (C) root surface area (RSA), and (D) root volume (RV). Fom, single inoculation with Fom; Rs, single inoculation with Rs; Fom:Rs, co-inoculation with Fom and Rs. Boxplots show the medians and 25th and 75th percentiles, with whiskers extending to 1.5 times of the interquartile range, and data presented beyond whiskers represent outliers. Different letters above the bars indicate significant differences (p < 0.001) among treatments according to Fisher’s protected LSD test at p = 0.05.

FIGURE 5

Principal component analysis (PCA) on all plant traits and pathogen effect sizes for 80 alfalfa varieties single or co-inoculated with Fom and Rs. (A) PCA of all plant traits under the control and three pathogen treatments. PCA on pathogen effect sizes of plant traits under (B) single inoculation with Fom (Fom), (C) single inoculation with Rs (Rs), and (D) co-inoculation with Fom and Rs (Fom:Rs). Biplot vectors are trait factor loadings, whereas the position of each variety is shown. Kaiser–Meyer–Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity are shown. The study includes 75 M. sativa subsp. sativa (Ms) varieties consisting of 24 domestic bred (BV, circles in black), 4 local varieties (LV, triangles) in China, and 47 introduced varieties overseas (IV, circles in gray), and 5 M. sativa subsp. varia domestic bred varieties (Mv, squares). Numbers of varieties shown in panels (B–D) corresponds to those shown in Supplementary Table 1.

FIGURE 6

Hierarchical clustering of 80 alfalfa varieties based on variations in pathogen effect sizes of all plant traits single and co-inoculated with Fom and Rs. (A) Single inoculation with Fom (Fom), (B) single inoculation with Rs (Rs), and (C) co-inoculation with Fom and Rs (Fom:Rs). Labels on the dendrogram (I, II, III, and IV) represent the main groups. The heat map shows variations in pathogen effect sizes of different traits. The color gradient range (0–100%) is shown on the top right corner. Positive effects (effect size < 0) of pathogen treatment on some traits (Supplementary Figures 1–4) were defined as zero to maximize resolution. DIS and DIR, disease index of plant shoot and root, respectively; PH, plant height; RL, root length; DWS and DWR, dry weight of shoot and root, respectively; ST, shoot biomass ratio; RT, root biomass ratio.