Medicago truncatula ecotypes A17 and R108 show variations in jasmonic acid/ethylene induced resistance to Macrophomina phaseolina

Abstract

The soilborne necrotrophic fungal pathogen Macrophomina phaseolina (Tassi) Goid causes charcoal rot disease in many economically important crops. There are no effective control methods for this disease and no completely disease-resistant cultivar has been identified in crop species against M. phaseolina, including alfalfa, soybean and sorghum. In our previous study, we have established a pathosystem using the model legume Medicago truncatula to study the molecular interactions between the pathogen and its host. In this study, we investigated the possible variability in host response to M. phaseolina infection among different Medicago ecotypes. We found that disease progression in different ecotypes had little variation, and no ecotype was identified that showed resistance. We have previously found that methyl jasmonate (MJ) and ethylene (ET) could induce partial resistance in Medicago ecotype A17 against M. phaseolina. Here, we compared the disease progression after MJ or ET treatment between A17 and R108, another commonly used ecotype. Unlike A17, our results showed that R108 plants treated with MJ and ET did not show any induced resistance to M. phaseolina. The possible molecular mechanisms that led to these variations are further discussed.

Résumé

L'agent pathogène fongique nécrotrophique terricole Macrophomina phaseolina (Tassi) Goidanich cause la pourriture noire chez de nombreuses plantes agricoles de grande importance économique. Il n'existe pas de méthode de lutte efficace contre cette maladie, pas plus qu'il y a de cultivars de variétés agricoles entièrement résistants à M. phaseolina, y compris chez la luzerne, le soja et le sorgho. Lors de notre étude précédente, afin d'examiner les interactions moléculaires entre l'agent pathogène et son hôte, nous avons établi un pathosystème basé sur la légumineuse Medicago trunculata. Dans celle-ci, nous avons examiné la variabilité possible de la réponse de l'hôte à l'infection par M. phaseolina chez différents écotypes de Medicago. Nous avons trouvé que, chez les différents écotypes, la progression de la maladie affichait peu de variations et aucun écotype n'affichait de résistance. Nous avions déjà constaté que le jasmonate de méthyle (JM) et l'éthylène (ET) pouvaient induire une résistance partielle chez l'écotype A17 de Medicago infecté par M. phaseolina. Ici, nous avons comparé la progression de la maladie après traitement au MJ ou à l'ET chez A17 et R108, un autre écotype utilisé fréquemment. Contrairement à A17, nos résultats montrent que les plants de R108 traités avec MJ et ET n'ont pas affiché de résistance induite à M. phaseolina. Les mécanismes moléculaires probables qui ont mené à ces variations sont discutés plus en détail.

Keywords:

• charcoal rot
• disease resistance
• ethylene
• jasmonic acid
• Macrophomina phaseolina
• Medicago truncatula

Mots clés:

• acide jasmonique
• éthylène
• Macrophomina phaseolina
• Medicago trunculata
• pourriture noire
• résistance à la maladie

Introduction

Charcoal rot is a plant disease caused by the soil fungus Macrophomina phaseolina (Tassi) Goid. This fungus has a wide range of plant hosts, readily invading over 500 different monocotyledonous and dicotyledonous species, including various important crops such as soybean, sorghum, maize and alfalfa (Wyllie, 1989). Plants infected with M. phaseolina develop symptoms ranging from leaf yellowing, wilting to plant death, and the severity of the disease usually increases in hot and dry conditions (Smith & Wyllie, 1999). Charcoal rot can result in severe losses due to reduced yield and low seed quality. For instance, the US soybean yield reduction due to charcoal rot was estimated to be around 1.9 million tons in 2003 (Wrather & Koenning, 2006). The loss could be more severe in years with hot and dry summers which could become more frequent as a consequence of global warming. Charcoal rot has a wide geographical distribution, and the disease has been reported in southern and north-central regions of the United States (Mihail et al., 1990; Bradley & del Rio, 2003; Yang & Navi, 2005), and in tropical and subtropical regions of the world (Karadimos et al., 2002; Gaetan et al., 2006; Khangura & Aberra, 2009; Mahmoud & Budak, 2011).

M. phaseolina exists in soil as compact masses of hardened mycelium called sclerotia, which can remain dormant for many years. Under appropriate conditions, hyphae germinated from the sclerotia infect the roots of plant hosts (Ammon et al., 1974). After decay of infected plants, sclerotia are released into the soil and the infection cycle can start again in the following years. The pathogen can also be transmitted through infected seeds (Abawi & Pastorcorrales, 1990). The common management strategies that are currently applied to control the disease include crop rotation (Mueller et al., 1985; Francl et al., 1988), adjusting planting dates (Todd, 1993), irrigation (Wyllie, 1989), and cover crops (Rothrock et al., 1995). Other management approaches including the application of fungicides, plant-derived natural products as well as biological control using competing soil microbes have also been investigated (Ghaffar et al., 1969; Abawi & Pastorcorrales, 1990; Siddiqui & Mahmood, 1993; Brooker et al., 2000; Haas & Défago, 2005). However, none of these approaches are very effective. Host resistance may be the only feasible approach to manage the disease; however, the search for naturally existing resistant cultivars has not yielded success (Smith & Carvil, 1997). Moreover, genetic engineering of resistant plants is not feasible currently due to the lack of knowledge on the molecular processes that occur during M. phaseolina–host interactions.

In order to better understand the host responses to M. phaseolina infection at the molecular level, we have established a model pathosystem for charcoal rot using Medicago truncatula (M. truncatula) in our previous study (Gaige et al., 2010). M. truncatula is the primary model legume species for genomic and functional genomic research because of its relatively small genome size and available genomic resources (Thoquet et al., 2002; Choi et al., 2004). Our inoculation protocol was established using the commonly used M. truncatula ecotype Jemalong A17. This ecotype was used in the Medicago genome sequence project to generate the reference genome (Town, 2006). Plants of this ecotype infected with M. phaseolina developed disease symptoms such as wilting and leaf yellowing at 1 day-post-inoculation (dpi), and most of the plants died by 4 dpi. Roots from infected plants showed browning and necrotic lesions (Gaige et al., 2010). The disease phenotypes were consistent with what has been observed in the field (Wyllie, 1989). Using this ecotype, we have found that A17 plants treated with methyl jasmonate (MJ) and/or ethylene (ET) gained partial resistance to M. phaseolina (Gaige et al., 2010). Jasmonic acid (JA) and ET are two plant hormones that are involved in both plant development and defence responses, especially against several necrotrophic pathogens in different plant species (McDowell & Dangl, 2000; Glazebrook, 2001). For example, Arabidopsis mutants that have defects in JA biosynthesis or signalling were more susceptible to Pythium mastophorum Drechsler, a soil-borne pathogen causing root rot disease (Vijayan et al., 1998) and to the necrotrophic fungus Botryotinia cinerea (De Bary) Whetzel (Thomma et al., 1998). Arabidopsis plants carrying mutations in EIN2, the key ethylene signalling gene, were more susceptible to B. cinerea (Thomma et al., 1999). Similarly, Medicago sickle mutants that are defective in EIN2 ortholog were less tolerant to the necrotrophic pathogens Rhizoctonia solani J.G. Kühn and Phytophthora medicaginis E.M. Hansen & D.P. Maxwell (Penmetsa et al., 2008).

To explore naturally existing genetic variations in M. truncatula against M. phaseolina, we first examined the severity of disease symptoms in 323 different M. truncatula ecotypes to evaluate the possible host resistance in this species. These ecotypes include 322 accessions obtained from the USDA National Plant Germplasm System (NPGS) collection and one commonly used ecotype R108. The NPGS collection includes 322 ecotypes collected from different areas around the globe (http://www.ars-grin.gov/cgi-bin/npgs/html/tax_acc.pl) (Bauchan & Greene, 2002). Since R108 has been frequently used in genetic studies and it is distinct from A17, we decided to test if the genetic variations existing in these ecotypes led to differences in JA and ET-induced resistance against M. phaseolina. Ecotypes A17 and R108 are phenotypically and genotypically distinct. A17 was originally isolated from Australia, whereas R108 was a derivative of ecotype 108-1 through in vitro regeneration (Hoffmann et al., 1997).

Materials and methods

Plant materials

Seeds for 322 M. truncatula accessions were obtained from the USDA National Plant Germplasm System (NPGS) collection (provided by Dr Stephanie Greene). Seeds for Jemalong A17 and R108 were obtained from the Samuel Roberts Nobel Foundation, Ardmore, OK (provided by Dr Srinivasa Rao Uppalapati). M. truncatula seeds were treated with concentrated sulphuric acid for 8 min, rinsed five times with distilled water, followed by surface sterilization with freshly prepared 20% bleach (Chlorox). After sterilization, the seeds were rinsed three times with sterilized distilled water and germinated on half-strength Murashige and Skoog (MS) salt (Murashige & Skoog, 1962) (Sigma-Aldrich, St. Louis, MO) with 1% agar at room temperature in the dark for 3 days. The seedlings were then transplanted to plastic pots filled with wet sterilized soil (Sun Gro Horticulture, Bellevue, WA) and grown in the growth chamber (27 °C, 12-h photoperiod, 44% relative humidity [RH], photon flux density 150–200 μmol m⁻² s⁻¹).

Fungal inoculum

M. phaseolina isolate and inoculum preparation were described previously (Gaige et al., 2010). Briefly, the isolate was cultured on potato dextrose agar (PDA) at 27 °C for 3 days. Then, agar pieces were cut out and transferred to potato dextrose broth (PDB). After incubating at 27 °C for 14 days, the fungal mat from PDB was collected using a sterile spatula and air-dried overnight at room temperature. The dried material containing mostly sclerotia was ground to fine powder and used to prepare the inoculum.

Inoculations

Four-week-old M. truncatula plants were removed from the pots, and rinsed with running tap water to remove potting mix from the roots. The roots then were submerged in M. phaseolina inoculum suspension (1 g of dried sclerotia in 10 mL 0.015% agarose) for 30 s, then re-potted into sterilized soil. Control plants were dipped in 0.015% agarose only (Gaige et al., 2010). The low percentage of agarose helped to evenly suspend the sclerotia and also kept the sclerotia from sticking onto plant roots. Treated plants were maintained as described above. Disease symptoms were monitored daily from 1 dpi to 7 dpi using digital imaging with a Nikon CoolPix digital camera under ambient light condition without flash. The disease symptoms were also assessed based on a scoring matrix on a scale of 0–6 (0 = no detectable symptom; 1 = 1–10% chlorotic or 1–5% necrotic; 2 = 10–20% chlorotic or 5–10% necrotic; 3 = 20–40% chlorotic or 10–20% necrotic; 4 = 40–60% chlorotic or 20–40% necrotic; 5 = 60–80% chlorotic or 40–60% necrotic; 6 = > 80% necrotic or dead plant). For the ecotype screen, A17 was used as a reference for every inoculation. For each accession, at least 3 plants were mock-treated, and at least 6 plants were treated with the fungal inoculum. Experiments were repeated for ecotypes that showed altered susceptibility.

Chemical preparation and application

Four-week-old plants were sprayed with either 0.1% MJ (Sigma-Aldrich, St. Louis, MO), or 3.0 mM ethephon (2-chloroethyl phosphonic acid) (Sigma-Aldrich), or the combination of both 3 days before inoculation. Ethephon is a direct ethylene source when applied to plants, and is much easier to handle than ethylene gas (Cooke & Randall, 1968; Khan, 2004). MJ solution was prepared fresh by diluting the stock solution in water containing 0.01% of Tween-20. Ethephon was freshly prepared by dissolving in water containing 0.01% Tween-20 to a final concentration of 3.0 mM. Mock treatment was water with 0.01% Tween-20. Plants sprayed with the chemicals were covered with a plastic dome and kept in a separate area from untreated plants. For large-scale experiments, 120 4-week-old plants were divided into four groups; each group was treated with either mock control, MJ, ET or MJ plus ET 3 days prior to inoculation. The experiments were performed twice.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA). The statistical significance of ecotype variations in hormone responses was based on two-way ANOVA with P < 0.05 or P< 0.01.

Results and discussion

Using our established model pathosystem for charcoal rot, we plan to identify genetic factors that result in changes in host susceptibility to M. phaseolina using a forward genetics approach. With this in mind, we first conducted the screen with the available 323 accessions to see if there were any naturally existing variations in M. truncatula against M. phaseolina. Any accession that showed different disease symptoms when compared with A17 (either died more quickly or showed partial resistance) was screened again to confirm the results. After completing the screen for 320 accessions (PI-577600, PI-577602 and PI-577608 could not be screened due to low germination rates), we did not observe any major differences in disease progression among different ecotypes (data not shown). Compared with A17 plants, all other ecotypes developed disease symptoms at a similar pace. The inoculated plants started to show leaf yellowing and wilting at 1 day-post-inoculation (dpi). The symptoms became more severe at 2 dpi and 3 dpi, and the plants died at 4 dpi. The roots of the dead plants were completely colonized by M. phaseolina sclerotia (data not shown).

This result is not surprising, considering that previous efforts to identify naturally existing host resistance to M. phaseolina have not been successful (Smith & Wyllie, 1999).

In our previous study, we found that the expression of genes in several disease response pathways was affected by M. phaseolina infection, and treating A17 plants with methyl jasmonate (MJ) and/or ethylene (ET) prior to inoculation induced partial resistance against M. phaseolina. Here, we tested the effects of these two hormones on R108 plants. Ecotype A17 plants treated with MJ or ET developed disease symptoms more slowly, and the infected plants survived up to 7 dpi. In addition, A17 plants treated with both hormones showed an increase in resistance compared with the ones treated with either one of the hormones alone. To our surprise, R108 plants did not show a similar response to the hormone treatments. Ecotype R108 plants treated with MJ or ET developed disease symptoms similar to the untreated R108 plants. To test whether the different responses we observed between A17 and R108 were statistically significant, we increased the number of plants in each treatment group and performed the large-scale experiment twice. After hormone treatment and inoculation, the disease symptoms were scored daily for each plant based on a scale of 0 to 6. The numbers of plants with severe symptoms (scored 5 and 6) at each time point were compared between treatment groups (Fig. 1). The impact of hormone treatments on disease progression was significantly different between A17 and R108 from 3 dpi and later (Fig. 1). Mock-treated, infected A17 and R108 plants all died around 4 to 5 dpi, and the same was observed for R108 plants treated with either MJ or ET alone or with both hormones. However, disease progression in A17 plants treated with MJ or ET was slower. A17 plants treated with both hormones showed more resistance to the pathogen, and the difference in disease progression was significant compared with plants treated either MJ or ET alone (Fig. 1B and 1C).

Fig. 1. Effect of MJ and ET on disease caused by M. phaseolina on Medicago ecotypes A17 and R108. Each treatment was done on 30 plants, and the experiment was performed twice. The percentage of plants that developed severe symptoms (scored 5–6) after M. phaseolina infection in each treatment group was compared at (A) 3 dpi, (B) 4 dpi and (C) 5 dpi. The data were analyzed using two-way ANOVA. * and ** refer to significant differences at P < 0.05 and 0.01, respectively.

The finding that R108 did not show an induced level of resistance by MJ and ET was intriguing. Ecotypes A17 and R108 have genetic variations that can result in phenotypic differences ranging from salt stress responses to symbiotic interactions with rhizobia (Salzer et al., 2004; de Lorenzo et al., 2007). It was reported that class IV chitinase gene was inducible by symbiotic rhizobia in A17, but not in R108 (Salzer et al., 2004). Several chitinases are JA or ET-inducible in Arabidopsis (van Loon et al., 2006). Although the inducible response of chitinases to JA and ET is not well characterized in M. truncatula, it is possible that these genes could be involved in the differences in MJ- and ET-induced partial resistance between A17 and R108. We are also exploring other possible genetic variations between these two ecotypes that could lead to the different hormonal responses.

Acknowledgements

This study was supported in part by a grant to BS from the Kansas Soybean Commission. We thank Dr Srinivasa Rao Uppalapati at the Samuel Roberts Noble Foundation (Ardmore, OK, USA) and Dr Stephanie Greene at the USDA (USDA, Washington State University, Pullman, WA) for providing plant materials for our study.