Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt

Abstract

Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

Fig. 1.

Verticillium wilt symptoms in Medicago truncatula. (A–C) Phenotypes of the two most contrasted lines of M. truncatula (Jemalong-A17 and F83005.5) after root inoculation with a conidia suspension of Verticillium albo-atrum V31-2 (10⁶ spores ml^–1) in peat substrate (A), hydroponic system (B), and in vitro culture (C). (D) Symptom scale for disease index scoring. Scores from 0 to 4 correspond to characteristic stages of disease development.

Fig. 2.

Evolution of wilting symptoms and disease evaluation parameters of six Medicago truncatula lines with different levels of quantitative resistance. (A) Fitted logistic curves for disease kinetics for six genotypes (F83005.5, DZA315.16, A20, DZA45.5, TN1.11, Jemalong-A17) in three independent experiments (RB1, RB2, and RB3), following root inoculation in peat substrate with Verticillium albo-atrum V31-2 at 10⁶ spores ml^–1. Wilting symptoms were scored for 20–30 days after inoculation, on a scale from 0 to 4. Raw disease curves were modelled with a non-linear mixed model. Lines are ordered by decreasing susceptibility from left to right. (B) Distribution of the disease evaluation parameters after root inoculation among the six lines. Lines with the same letter belong to the same group of means, defined by Newman-Keuls test. AUDPC, area under disease progress curve. Data were obtained in three independent experiments performed at INP-ENSAT and R2n sites with 8–10 plants per line per experiment.

Fig. 3.

Root colonization of Medicago truncatula F83005.5 (susceptible line) by a GFP-expressing strain of Verticillium albo-atrum, observed by confocal laser scanning microscopy. (A) At 2 hours post inoculation (HPI), Conidia are seen in xylem vessels and accumulate at the end of a tracheid (arrow). (B) Conidia penetrating with its germination hypha through the plaque at the end of a xylem vessel (enlargement of A). (C) At 24 HPI, germinating conidia and hyphae. (D) At 7 days post inoculation (DPI), fungal hyphae in the central cylinder; no hyphae detected in the cortex. (E) At 7 DPI, proliferation of mycelium in xylem vessels and penetration into adjacent vessels (asterisk). (F and G) At 21 DPI, proliferation of mycelium in vascular (F) and cortical (G) tissues. Bars = 50 µm, and 10 µm in B. Longitudinal sections in A, B, C, E, G; cross-sections in D and F. cor = cortex; cw = cell wall; hy = hypha; sp = conidia; xy = xylem elements.

Fig. 4.

Position of quantitative trait loci (QTL) for Verticillium albo-atrum resistance in Medicago truncatula assessed through maximum fitted disease index and AUDPC in LR4 and LR5 populations and proportion of dead plants in LR3, LR4, and LR5 populations. Bars at the left of considered linkage groups indicate 1 logarithm of odd score support for QTL interval.

Fig. 5.

Diversity in the response of Medicago truncatula to Verticillium albo-atrum within the Core Collection 32. (A) Fitted logistic curves for disease kinetics; M. truncatula lines are ordered from top left to bottom right by increasing maximum disease index, from the resistant line Jemalong-A17 (L738) to the most susceptible line L321; similar results were obtained in two independent experiments. (B) Quantitative resistance levels as evaluated through maximum fitted disease index and time to reach 50% of maximum disease index, adjusted over two biological repeats. Each of the four putative M. truncatula sub-populations (Ronfort et al. 2006) is identified by a distinct symbol (1=●, 2=■, 3=◆, 4=▲). DZA45.6 is a sister line of DZA45.5.