A novel α-1, 3-glucan elicits plant defense responses in potato and induces protection against Rhizoctonia solani AG-3 and Fusarium solani f. sp. eumartii

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Abstract

All glucan elicitors from fungal cell walls have been described as ß-glucans. In a previous work, we have isolated and characterized an α-1, 3-glucan from a non-pathogenic Rhizoctonia isolate which induced glucanase activity in potato sprouts. In this work we showed that, in addition to eliciting a wide array of defense reactions, the α-1, 3-glucan induces protection against Rhizoctonia canker and dry rot in plants and tubers, respectively. The α-1, 3-glucan strongly induced ß-1, 3-glucanase and chitinase activities. Immunological analysis showed that the level of the pathogenesis-related proteins increased. Histological studies showed an increase in cell wall deposition of callose and lignin, and ultrastructural analysis showed changes in the cytoplasm and the accumulation of electron-dense bodies in vacuoles. Protection assays showed that pre-treatments of potato sprouts and potato tubers with the α-1, 3-glucan results in a 40% of protection against Rhizoctonia canker and 60% protection against dry rot, respectively.

Introduction

In the course of evolution, any plant/pathogen interaction has developed a complex array of recognition, attack and defense reactions at the plant/microbe interface. Pathogens have developed offensive strategies and, in turn, plants have developed a wide range of sophisticated defense mechanisms to resist the colonization by microbial pathogens and parasites. Pathogen recognition by the host triggers a variety of early defense responses, such as modification of the ion influxes across the plasma membrane, phosphorylation and dephosphorylation of signaling proteins, and production of reactive oxygen species [1]. Later, these events are followed by the induction of a broad spectrum of defense reactions that confer local resistance against pathogens. These include: (i) cell wall reinforcement through lignin synthesis, callose deposition and cross-linking of macromolecular components, (ii) production of signaling secondary metabolites from the phenylpropanoid and octadecanoid pathways and (iii) accumulation of antimicrobial compounds such as phytoalexins, and synthesis of proteins with hydrolytic or inhibitory activity against microbes such as pathogenesis-related (PR) proteins [2]. These proteins have well-described antimicrobial activity against different pathogens [3], [4]. ß-1, 3-glucanase (PR-2 group) hydrolyzes ß-glucans and chitinase (PR-3 group) hydrolyzes chitin, which are major components of fungal cell walls. Hydrolysis of these fungal cell-wall leads to the inhibition of the growth of several fungi [5] and they might also play an important role in the amplification of defense reactions through the release of ß-1, 3-glucans [6] and chitin oligosaccharides [7]. Thaumatin-like proteins (PR-5 group) have showed antifungal activity, particularly against oomycetes [8].

Signal molecules from the pathogen or from the host that are able to trigger defense responses are known as elicitors. Many of these can be surface components released from the cell wall of the microbe or the host. Various types of elicitors have been characterized, including oligosaccharides, lipids, proteins and glycoproteins. ß-glucans, oligogalacturonides and chitin-derived oligomers are well-known elicitors [9], [10], [11], [12].

Due to their activity as plant protectants, by triggering a large array of defense responses, elicitors have been considered as alternative tools for disease control in agronomic crops [11], [12], [13], [14], [15].

All glucans with elicitor activity characterized so far are formed by ß-linked glucose units [16], [17], [18]. However, we recently reported the isolation and characterization of an α-1,3-glucan from the cell wall of a non-pathogenic binucleate Rhizoctonia (BNR) isolate which induces ß-1,3-glucanase activity in potato sprouts [19]. BNR isolates have been characterized as effective biocontrol agents against several plant diseases caused by the pathogenic fungus Rhizoctonia solani Kühn [20], [21], [22], including in potato against Rhizoctonia canker, the disease caused by R. solani AG-3. Symptoms and signs of Rhizoctonia canker include canker on stems, sprouts and stolons, death of pre-emerging sprouts, malformation of tubers and dark brown-black sclerotia formed on the surface of mature tubers.

In this work, we characterized at biochemical level the response elicited by the α-1, 3-glucan in potato sprouts, the primary site of infection by R. solani AG-3, and we tested the ability of the α-1, 3-glucan to protect potato plants against R. solani AG-3 and Fusarium solani f. sp. eumartii. We report here the analysis of the accumulation of PR-proteins, the reinforcement of the cell wall by cytological studies, the ultrastructural changes in the elicited tissue and the results of biological assays of protection.

Section snippets

Plant, fungus and growth conditions

Potato tubers (Solanum tuberosum cv. Kennebec and Solanum tuberosum cv. Pampeana) were obtained from the Experimental Station of the INTA Balcarce, Argentina. Kennebec cultivar was selected because it is moderately susceptible to Rhizoctonia disease and Pampeana cultivar was selected because it is susceptible to dry rot disease caused by F. solani f. sp. eumartii. Potato tubers from Kennebec cultivar were incubated at 18 °C, on the dark, to allow sprouting.

All fungal isolates were obtained from

Kinetics of induction of ß-1, 3-glucanase activity by the α-1, 3-glucan and dose–response assays

In order to test the ability of the α-1, 3-glucan to induce ß-1, 3-glucanase activity, potato sprouts were injected with 250 μg of the glucan or water (control). ß-1, 3-glucanase activity was determined at 0, 4, 6 and 8 days after treatment (Fig. 1). Glucanase activity began to increase significantly at day 4 and maximal increase of ß-1, 3-glucanase activity was detected after 6 days, being 4-fold higher than controls (Fig. 1).

To compare the elicitor activity induced by the α-1, 3-glucan with

Discussion

Non-pathogenic Rhizoctonia isolates have the ability to protect several plants (potato, bean seedlings, poinsettia) against further infection by virulent isolates of Rhizoctonia and it has been proposed that such a protection is due to induced resistance [20], [22], [35]. Induced resistance can also be triggered by elicitors [12], [13], [15], [36] and the elicitation of defense mechanisms is assumed to be a powerful approach for the management of plant diseases and to be an alternative to

Acknowledgments

We are indebted to Dr. Kombrink and Dr. Fritig for kindly providing the antibodies of a basic ß-1, 3-glucanase and PR-proteins, respectively.

This research was supported by the National University of Mar del Plata, the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica and Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC). Daleo, GR is an established researcher of CIC. Andreu AB and Maldonado S are

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