Temperature increase modifies susceptibility to Verticillium wilt in Medicago spp and may contribute to the emergence of more aggressive pathogenic strains

Abstract

Global warming is expected to have a direct impact on plant disease patterns in agro-eco-systems. However, few analyses report the effect of moderate temperature increase on disease severity due to soil-borne pathogens. For legumes, modifications of root plant-microbe interactions either mutualistic or pathogenic due to climate change may have dramatic effects. We investigated the effect of increasing temperature on the quantitative disease resistance to Verticillium spp., a major soil-borne fungal pathogen, in the model legume Medicago truncatula and the crop M. sativa. First, twelve pathogenic strains isolated from various geographical origin were characterized with regard to their in vitro growth and pathogenicity at 20°C, 25°C and 28°C. Most of them exhibited 25°C as the optimum temperature for in vitro parameters, and between 20°C and 25°C for pathogenicity. Second, a V. alfalfae strain was adapted to the higher temperature by experimental evolution, i.e. three rounds of UV mutagenesis and selection for pathogenicity at 28°C on a susceptible M. truncatula genotype. Inoculation of monospore isolates of these mutants on resistant and susceptible M. truncatula accessions revealed that at 28°C they were all more aggressive than the wild type strain, and that some had acquired the ability to cause disease on resistant genotype. Third, one mutant strain was selected for further studies of the effect of temperature increase on the response of M. truncatula and M. sativa (cultivated alfalfa). The response of seven contrasted M. truncatula genotypes and three alfalfa varieties to root inoculation was followed using disease severity and plant colonization, at 20°C, 25°C and 28°C. With increasing temperature, some lines switched from resistant (no symptoms, no fungus in the tissues) to tolerant (no symptoms but fungal growth into the tissues) phenotypes, or from partially resistant to susceptible. Further studies in greenhouse evidence the reduction in plant fitness due to disease in susceptible lines. We thus report that root pathogenic interactions are affected by anticipated global warming, with trends towards increased plant susceptibility and larger virulence for hot-adapted strains. New threats due to hot-adapted strains of soil-borne pathogens, with possibly wider host range and increased aggressiveness, might occur.

Keywords: aggressiveness; experimental evolution; global warming; mutation; plant disease; temperature-adapted pathogens; virulence.

Figure 1

Effect of temperature on in vitro growth and sporulation of Verticillium strains. (A) strains were grown on PDA medium in Petri dishes at 20°C, 25°C and 28°C. Radial growth was measured during 14 days and expressed as Area Under Growth Progress Curve (AUGPC). (B) Conidia released into distilled water were counted with a hematocytometer. Each point shows the mean values of three independent experiments, with two Petri dishes each. Bars indicate standard error of observed values.

Figure 2

Aggressiveness of Verticillium strains on M. truncatula at different temperatures, shown by maximum symptom scores. Four M. truncatula lines (A17, DZA315.16, DZA45.5 and F83005.5) were root-inoculated with a spore suspension of the 12 Verticillium strains, and maintained at 20°C, 25°C and 28°C for four weeks. Symptoms were scored regularly on a scale from 0 to 4 and Maximum Symptom Scores (MSS) and Area Under the Disease Progress Curve (AUDPC, not shown) were determined at the end of the experiment. The values are means from three independent experiments, with eight plants each. Bars indicate standard error of observed values.

Figure 3

Aggressiveness of V. alfalfae V31-2 strains before and after successive steps of UV mutagenesis and in planta selection at 28°C. Plants of the susceptible line F83005.5 were root-inoculated and the symptom score after four weeks was determined, on a scale from 0 (healthy plant) to 4 (dead plant). One hundred fifty plants were inoculated for each cycle.

Figure 4

Assessment of Maximum Symptom Score of M. truncatula to V. alfalfae V31-2 and the AS38 derived mutant at different temperatures. Seven M. truncatula lines (A17, PRT180-A, DZA45.5, SA09048, F83005.5, DZA315.16 and SA03780) were root-inoculated with a spore suspension of Verticillium strains V31-2 (A) and AS38 (B) and maintained at 20°C, 25°C and 28°C. Symptoms were scored regularly on a scale from 0 to 4 during four weeks. At the end of the experiment, maximum symptom scores were determined for each plant. The values are from three independent experiments with at least eight plants per combination. Each bubble’s diameter is proportional to the percentage of plants belonging to a symptoms class, with this proportion indicated at the center. Colour code from green (score “0”, no symptoms) to red (score “4”, dead plant). Score classes at the end of the experiments were analyzed using proportional-odds models, used for modeling the dependence of an ordinal response (disease symptom scores) on discrete (pathogen strains and plant lines) or continuous (temperature) covariates. Multiple comparisons of odd-ratio were done using least square means and Tukey HSD. (C) NS/S indicates if a significant difference exists between the response of a M. truncatula accession to AS38 compared to its response to V31-2, for a given temperature.

Figure 5

Assessment of Quantitative Disease Resistance (QDR) of M. truncatula to V. alfalfae V31-2 and AS38 its derived mutant at different temperatures, using Maximum Symptom Score and plant colonization evaluation. Seven M. truncatula lines (A17, PRT180-A, DZA45.5, SA09048, F83005.5, DZA315,16 and SA03780) were root-inoculated with a spore suspension of Verticillium strains V31-2 (A) and AS38 (B) and maintained at 20°C, 25°C and 28°C. Symptoms were scored regularly on a scale from 0 to 4 during four weeks. At the end of the experiment, maximum symptom scores were determined, and stem sections were harvested for fungus re-isolation. The values for MSS and re-isolation percentage are means from three independent experiments, with at least eight plants each. Bars indicate standard error of mean values.

Figure 6

Effect of temperature on total lifetime fitness of M. truncatula inoculated, or not, with Verticillium strain V31-2. The M. truncatula lines A17, PRT180-A, DZA45.5, SA09048, F83005.5, DZA315,16 and SA03780 were mock-inoculated or root-inoculated with a spore suspension of Verticillium strain V31-2 and maintained at 20°C,25°C and 28°C during one month, then transferred to the greenhouse. The greenhouse conditions were identical for all plants. (A) pod number (B) pod weight and (C) weight of dry aerial mass were measured at the end of the plant’s cycle (approx. six months). Bars indicate standard error of the means of two independent experiments, with three blocks each.

Figure 7

Mortality at the end of the experiment for three M. sativa varieties (Prunelle, Magali and Lifeuil) infected with V. alfalfae V31-2 and its derived mutant AS38, each at three temperatures. Data for each combination are from three independent experiments, each with 100 plants per variety. Bars indicate standard error of observed values.