Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Effect of Secondary Metabolites of Tomato (Solanum lycopersicum) on Chemotaxis of Ralstonia solanacearum, Pathogen of Bacterial Wilt Disease Takuya Hasegawa,† Yusuke Kato,‡ Atsushi Okabe,‡ Chie Itoi,‡ Atsushi Ooshiro,§ Hiroshi Kawaide,# and Masahiro Natsume*,#
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Department of Biological Production Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan ‡ Department of Bioregulation and Biointeraction, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan § Okinawa Prefectural Agricultural Research Center, 820, Makabe, Itoman, Okinawa 901-0336, Japan # Division of Bioregulation and Biointeraction, Institute of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan S Supporting Information *
ABSTRACT: The chemotactic activity of the pathogen of bacterial wilt disease, Ralstonia solanacearum, was tested against 30 aromatic acids and plant hormones infused on filter discs in bioassays on agar plates. 4-Hydroxycinnamic acid (p-coumaric acid) and 4-hydroxybenzoic acid were strong chemoattractants, 3,4-dihydroxybenzoic acid (protocatechuic acid) and jasmonic acid were weak attractants, and 2-hydroxybenzoic acid (salicylic acid) showed both attracting and repelling activity depending on dose. Examination of the dose dependency revealed that the ED50 for 4-hydroxycinnamic acid and 4-hydroxybenzoic acid was 0.08 and 0.39 μmol/disc, respectively. 2-Hydroxybenzoic acid showed chemoattractant activity at 0.33 μmol/disc but chemorepellent activity at 3.3 μmol/disc, and bacterial random motility was activated at 1.0 μmol/disc and bacterial activity was suppressed at 33 μmol/disc. Although water-soluble attractants including amino acids and organic acids have been previously investigated, this is the first report of hydroxylated aromatic acids (HAAs) as chemoattractants of R. solanacearum. KEYWORDS: Solanum lycopersicum, Ralstonia solanacearum, bacterial wilt, aromatic acids, tomato
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rhizobium into plant tissues, and nodulation.7,8 The host plant and symbiotic rhizobium species are strictly interrelated by the specific signaling substances. Besides flavonoids, hydroxylated aromatic acids including protocatechuic acid (3,4-dihydroxybenzoic acid) and p-hydroxybenzoic acid and vanillyl alcohol are also known to be chemoattractants for rhizobia.4 In addition to symbiotic bacteria, phytopathogenic bacteria, antagonistic bacteria, and plant growth-promoting rhizobacteria (PGPR) also respond to plant components; Agrobacterium tumefaciens, a gram-negative bacterium that forms crown gall in plants, shows chemotaxis to a phenolic compound, acetosyringone, that is released by disordered plant tissues and which also induces the Ti plasmid virulence operons in Agrobacterium.9,10 Chemotaxis toward malic acid and citric acid in the exudates was shown to be an important trait for root colonization by PGPR, Pseudomonas fluorescens, in tomato.11 Ralstonia solanacearum is a soil-borne gram-negative bacterium that causes bacterial wilt and is one of the most notorious phytopathogens from three perspectives: (i) It has a
INTRODUCTION Plants produce a remarkably diverse array of secondary metabolites including alkaloids, terpenoids, polyketides, and phenylpropanoids. Secondary metabolites are generally defined as small organic molecules produced by an organism that are not essential for the growth, development, or reproduction of the producer.1,2 However, subsequent research has revealed that secondary metabolites produced by plants are secreted into the rhizosphere where they have been shown to interact in a variety of ways with other organisms.3−5 The phenomenon by which a chemical substance produced by a plant inhibits, promotes, or otherwise influences other organisms is called allelopathy, and the active substance is termed an allelochemical.6 The most well-known mutual interaction between plants and microorganisms is the species-specific symbiotic relationship between leguminous plants and rhizobia. This relationship, known as “a reciprocal molecular conversation”,7 involves an exchange of signaling molecules. That is, a flavonoid secreted from the roots of a leguminous plant acts as a chemoattractant for a rhizobium, and the flavonoid induces gene expression involved in symbiosis in the rhizobium. The gene product, lipochitooligosaccharide, which is called Nod factor, is secreted by the rhizobium and acts on the plant to induce expression of nodulin genes leading to root hair deformation, inclusion of © XXXX American Chemical Society
Received: November 12, 2018 Revised: December 28, 2018 Accepted: January 25, 2019
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DOI: 10.1021/acs.jafc.8b06245 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry wide host range as it infects more than 250 species from 54 monocotyledon and dicotyledon families including economically important crops such as tomato, potato, eggplant, pepper, tobacco, and banana.12 (ii) It has a broad geographic distribution as it is widespread in tropical, subtropical, and some warm temperate regions of the world.13 (iii) It has high endurance as it can survive for long periods in water, soil, and latently infected plants.14 The initiation of bacterial wilt by R. solanacearum in tomato is heavily reliant on the chemotaxis of the bacterium to the substances exuded from the host plant.15,16 Many reports on the chemotaxis of R. solanacearum have been published, but most focus on constituents common to many plants such as amino acids16,17 and organic acids,16−18 while chemotaxis toward the specific secondary metabolites as was observed in chemotaxis of root nodule bacteria to their host plants has not been reported. On the basis of the hypothesis that the specific metabolites of the host plant are involved in host recognition by R. solanacearum, we searched for chemoattractant(s) in the root exudates of tomato using a bioassay.19,20 Several secondary metabolites secreted from the roots of tomatoes have been reported; improvement of growth and fruit yield in hydroponic culture of tomato has been demonstrated by dipping activated carbon in nutrient solutions,21 and several carboxylic acids including benzoic, vanillic, and caffeic acids have been identified as autotoxic substances.22 Tomatoes are also known to contain large amounts of flavonoids and phenolic compounds,23 though it is not clear whether flavonoids are secreted from the roots of tomato. On the basis of the possibility that secondary metabolites produced by tomato act as chemoattractants, chemotactic activity of secondary metabolites produced by tomato and plant hormones was examined.
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Table 1. Chemotactic Activity of Secondary Metabolites of Tomato and Plant Hormones group
chemotactic activity (mm)a
compounds
benzoic acidb caffeic acidb,c chlorogenic acidc,d cinnamic acidc,d p-coumaric acidc ellagic acid ferulic acidb,c gallic acid 4-hydroxybenzoic acidb,c isoferulic acid protocatechuic acidc,d sinapic acidb,c syringic acid vanillic acidb,c flavonoids kaempferolc myricetinc naringeninc quercetinc rutin plant hormones (±)-abscisic acid brassinolide ethephon (+)-gibberellin A3 indole-3-acetic acid (+)-jasmonic acid kinetin phenylacetic acidb salicylic acidc,d synthetic strigolactone, (+)-GR24 other palmitic acidb Positive control L-glutamine
aromatic acids
MATERIALS AND METHODS
Chemicals. Tested aromatic acids and flavonoids listed in Table 1 were selected from among autotoxic substances identified in the hydroponic culture solution of tomato,22 secondary metabolites identified in the root exudates of tomato,23 and flavonoids contained in the fruit of tomato.24 A synthetic analog of strigolacone,25 (+)-GR24,26,27 was provided by Koich Yoneyama and Xiaonan Xie of Utsunomiya University (Utsunomiya, Japan). Other chemicals were reagents marketed by Fujifilm Wako Pure Chemical (Osaka, Japan) or Tokyo Chemical Industry (Tokyo, Japan). Chemotaxis Assay and Evaluation of Activity. Bioassay of chemoattractant or chemorepellent activity was examined by the semisolid agar plate method using R. solanacearum MAFF 730138 (race 1, biovar 3, isolated from tomato; Genebank Project, National Agriculture and Food Research Organization, Tsukuba, Japan) as reported previously.19 Briefly, a 20 μL aliquot of the bacterial suspension was spotted in the center of a semisolid agar plate (in a 6 cm i.d. Petri dish), and two paper discs (8 mm diameter, 1.5 mm thickness), one containing sample and a blank disc (control), were placed on the plate 20 mm from the edge of the bacterial suspension (Figure 1). The maximum distance from the edge of the bacterial suspension to the paper disc was 17 mm. The plates were incubated at 28 °C for 7 days in the dark. The distance between the bacterial halo to the sample disc and to the blank disc (migratory distance) was measured at day 3 and day 7 after the start of incubation. Initial Screening. An initial screening of 30 secondary metabolites and plant hormones (Table 1) was conducted with positive control Lglutamine. Migratory distance for the blank disc at day 7 was subtracted from that of the sample disc and reported as the chemotactic activity. A greater migratory distance toward the sample disc indicates positive chemotactic (chemoattractant) activity. Differ-
× − − × + 9.7 ± 1.0 − − − + 7.0 ± 0.9 − + 2.7 ± 0.1 − − − − − − − − − − − − × + 2.5 ± 0.0 − − − 5.0 ± 1.3 − − + 6.7 ± 0.3
a Chemotactic activity examined at a dose of 2 μmol/disc is the day 7 difference in migratory distance = migratory distance toward the sample disc − migratory distance toward the blank disc. Positive and negative values indicate chemoattractant and repellent activity, respectively. In the case that the value was
