Effect of chemical elicitors on the differential expression pattern of PR genes in susceptible and resistant cultivars of tomato against bacterial wilt disease caused by Ralstonia solanacearum

Highlights

• The growth of R. solanacearum was significantly (P < 0.05) inhibited by BABA and SA at all the concentrations (50 μM, 100 μM, 150 μM) under in vitro conditions.

• The pathogenesis-related genes relative expression of PR1a; PR1b and PR5 genes showed significantly (P < 0.05) 1.25–6.44 fold high level of up-regulation than other the treatments with SA and SA + R. solanacearum, as comparative to control plants.

• PR1a, PR1b, and PR2 significantly (P < 0.05) showed up-regulation in Pusa Ruby whereas, down-regulation in Hawaii7996 cultivar of a tomato plant.

• The minimal production of defence-related reactive oxygen species (ROS) was observed in stems and leaves of Pusa Ruby than the Hawaii7996.

• The maximum population of R. solanacearum (6.76 log value/g of plant tissue) after 96h of inoculation and wilt incidence (63.08 %) after 28 was recorded in cv. Pusa Ruby.

• Minimum 9.14% wilt incidence was recorded in SA (100 μM) followed by 15.96% in BABA (50 μM) treated Hawaii7996 cultivar after 28 days of inoculation in combination with pathogen R. solanacearum.

Abstract

Phytopathogens induced pathogenesis-related (PR) proteins as well as defense-related signaling moleculesare imperative part of an innate immune system in plant especially for systemic acquired resistance. The present study was undertaken to find out the role of chemical elicitors on expression of pathogenicity related genes in susceptible and resistant cultivars of tomato (Solanum lycopersicum L) against bacterial wilt and alsoon reduction of disease incidence. Two chemical elicitors i. e. Salicylic acid (SA) and β-amino butyric acid (BABA) were used in vitro, at 50, 100 and 150 μM concentrations to observe their effect on the growth of Ralstonia solanacearum (Smith) (Yabuuchi et al.1995). The growth of R. solanacearum was significantly inhibited by BABA and SA at 50 μM under in vitro conditions significantly. Furthermore, the relative differential expression of PR gene (PR1a, PR1b, PR2, and PR5 genes) transcripts were analyzed in both resistant (cv. Hawaii 7996) and susceptible (cv. Pusa Ruby) tomato cultivars when inoculated with R. solanacearum UTT-25 (RS)eitheralone or in combination with 50 μM concentration of BABA and SA separately at 6, 24, 48 & 96 hpi using RT-PCR. The maximum 5.61 fold up-regulation of PR1a gene was observed with SA (50 μM) + RS while 6.44 fold upregulation of PR1b gene was noticed with SA (50 μM) alone at 48hpi in cv. Pusa Ruby. Whereas, resistant nature of cv. Hawaii7996 does not allow R. solanacearum UTT-25 to multiply, so it does not show any upregulation except with R. solanacearum alone at 96hpi of the treatment. The histochemical detection has also confirmed the minimal production of defence-related reactive oxygen species (ROS) in stems and leaves of cv. Pusa Ruby than cv. Hawaii7996 in the presence of chemical elicitors. The wilt incidences on both the cultivars at varied concentrations of the elicitors used were decreased or delayed as compared to control. In cv. Pusa Ruby with Beta-butyric acid (50 μm) + R. solanacearum, minimum wilt incidence of 9.34 % were noticed on 16th day. However, in Hawaii7996 only 6.79 % wilt incidences were observed with Salicylic acid (50 μm) + R. solanacearumon 24th day as compared to control. Thus, our findings suggest that chemical elicitors SA (50 μM) had inducedresistance in susceptible cultivar to reduce wilt incidence significantly under glasshouse conditions.

Introduction

Tomato (Solanum lycopersicum L) is a protective food containing vitamins, minerals, anthocyanin, phenols, and amide proteins. The crop has the second position after potato in vegetable crops with the estimated total world production of 182,301,395 metric tonnes, and grown in 4.6 million hectares annually in the world. In India, however, tomato has been ranked 3rd after potato and onion in priority while ranked second as per area of cultivation with 21 million MT production [1].

Tomato crop suffers from >50 diseases of varied etiology like fungi, bacteria, phytoplasma, and viruses. Amongst all pathogens, Ralstonia solanacearum [2] is well-known soil-borne phytopathogenic bacterium causes lethal wilt disease in over 450 plant species and 54 botanical families [3]. The losses caused by bacterial wilt in tomato crops are varied from 2.0 to 95.0% [[4], [5], [6], [7]] over the globe depending on the cultivars, climatic conditions as well as virulence of the pathogen. The pathogen is considered to have the potential to developas a bioterrorissm weapon [8]. The strains of R. solanacearum species complex has been grouped in 5 races, 6 biovars, 4 phylotypes and 20 sequevars [9].

The management of tomato bacterial wilt is very challenging due to soil-borne nature of the pathogen, its longer survival periodeven in absenceof the host and its variable nature. Moreover, R. solanacearum is stressed by low temperatures [10] and commence a resistance stage which is called a viable but non-culturable state (VBNC). The bacterial pathogen is readily prone to disseminate through flood irrigation or soils infested with the pathogen. Use of the resistant cultivars to control the wilt disease is well adapted and most effective method [11] but it is not absolute solution for disease control due to lack of resistant gene in the host except tomato cultivar ‘Hawaii7996' which has stable resistance to R. pseudosolanacearum [12]. Recently, some alternative management strategies have been recommended for bacterial wilt such as microbes [11,13] and induced resistance in plants using chemical elicitor [14,15]. Plants have evolved two mechanisms to fend off infection caused by pathogen i.e. innate resistance and acquired or induced resistance [16]. In the second strategy, pre-treatment with/chemicals or bio-inducers confer increased resistance to tolerate/fight subsequent pathogen challenges. The plant disease-inducing agents have been used to stimulate the plant's defence mechanism by producing disease resistance substances without shows direct inhibitory effects on the pathogen [17]. The plant activates many types of induced resistance during infection caused by different pathogens and chemical inducers like salicylic acid (SA) [17], benzothiadiazole, methyl jasmonate, β-aminobutyric acid [18], DL-3- aminobutyric acid, silicon. Kurabachew et al. [19] reported two types of induced resistance in plants viz., ISR (induced systemic resistance) and SAR (systemic acquired resistance).

Numerous physiological and biochemical changes were observed when the plants get infected by the pathogens to express resistance or susceptible reaction to the disease. As far as the biotic stress concerned, PR (pathogenesis related) proteins play a significant role in the plants [20]. Many previous studies had explained significant increase in expression of PR1 gene at transcription and translation level [[21], [22], [23], [24]] in plants when treated with chemical elicitors to induce resistance against pathogen infection. On the basis of structural and functional similarities, the PR-proteins are classified into 17 PR families like PR1, PR2 (endo-1, 3-β-d- glucanase) and PR5 (Thaumatin-like) which are associated with the SA-mediated defence response, while the rest PR-protein families like PR4 (herein-like protein), PR6 (proteinase inhibitor) and PR9 (peroxidase) are very closely associated with the JA-ET pathway [25,26]. The toxicity of PR's may be commonly reported for their functions like hydrolytic, proteinase-inhibitory and differential membrane-permeability activities. Many pathogens may cross the first defence barrier therefore plant should possess an alternate defence mechanism to counter the pathogen further, such defence mechanisms are termed as pathogen induced defence response (PIDR). The pathogen inducible defence responses include hypersensitive response followed by the generation of ROS (Reactive oxygen species), cross-linking of cell wall, and production of phytoalexins and PR proteins. ROS likes H₂O₂, hydrogen radicals (HO), and superoxide anion radicals (O₂⁻) have been considered as subsidiary products of cell metabolism. Production of ROS is a kind of reaction of the plant against stress generated due to the disease. The excessive accumulation of ROS in plant damages protein structure, induces fragmentation of protein and also enhances peroxidation of membrane lipid, which causes irreversible damage and death of the cell [27]. Cohen et al. [18] stated that in accordant to the plant defensive response, the Salicylic acid (SA) plays a vital role as a signaling molecule against pathogens. While β-amino butyric acid (BABA) mediates the conditioning of induced plant defence mechanisms effectively to enhance protection against pathogen. The pre-treatment with BABA or other elicitors can be applied to the plant against biotic stress [28,29]. There are some findings which stated that the stress establishment is a comparatively uncommon case to reset at the time of stress recovery [[30], [31], [32]].

In the present study, an in-vitro evaluation of two elicitor's i.e. Salicylic acid and β-amino butyric acidwas conducted against R. solanacearum infection under controlled environmental conditions. Furthermore, the effect of these elicitors assessed on the relative expression pattern of PR genes, histochemical analysis, colonization/bacterial population and wilt incidence in susceptible and resistant cultivars of tomato under Phytotron conditions.