Elsevier

Phytochemistry

Volume 135, March 2017, Pages 169-180
Phytochemistry

Triterpene derivatives from Euphorbia enhance resistance against Verticillium wilt of tomato

https://doi.org/10.1016/j.phytochem.2016.12.017Get rights and content

Highlights

  • Four triterpenes were hemisynthetized from α-euphorbol and 31-norlanostenol.

  • They were able to protect tomato plants against Verticillium wilt disease.

  • They were able to elicit production of H2O2 and to activate plant defense.

Abstract

Oxidation of α-euphorbol and 31-norlanostenol, two triterpenic compounds isolated from the latex of Euphorbia resinifera and Euphorbia officinarum respectively, yielded four products named 3β-tosyloxy-4α,14α-dimethyl-5α-cholesta-7,9-diene; 4α,14α-dimethyl-5α-cholesta-7,9-dien-3β-ol; 24-methylen-elemo-lanosta-8,24-dien-3-one and elemo-lanost-8-en-3,11,24-trione. They were evaluated for protection of tomato plants against Verticillium dahliae in a greenhouse. The four semisynthesized products were phytotoxic at higher concentrations as they completely inhibited tomato germination at 100 and 500 μg/ml. However at lower concentrations (10 and 50 μg/ml) germination and root length were not affected. Disease resistance against Verticillium wilt was assessed in tomato plants derived from seeds that germinated in the presence of 10 and 50 μg/ml of the four products. All of them were able to reduce significantly disease severity, with 10 μg/ml being more effective than 50 μg/ml. Reduction of leaf alteration index and of stunting index ranged from 52 to 68% and from 43 to 67%, respectively, while vessel discoloration was reduced by at least 95%. The compounds were also able to elicit H2O2 accumulation before and after fungal inoculation and to significantly enhance peroxidase and polyphenol oxidase activities. These results suggest that the hemisynthetized triterpenes can be used as elicitors of disease resistance.

Graphical abstract

Four triterpenes deriving from Euphorbia were synthetized and applied to tomato plants. Pre-treated plants were protected against Verticillium wilt disease and exhibited elevated H2O2 content and enhanced defense.

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Introduction

Verticillium wilt, caused by Verticillium dahliae is one of the most important vascular diseases reported on tomato plants. After penetration through root tips or wounds the mycelium colonizes the xylem, leading to systemic infection. Affected plants show stunting and leaf wilting alterations, leading in some instances to death of the whole plant (Fradin and Thomma, 2006, Klosterman et al., 2009). Chemical control is based on the use of fumigants such as methyl bromide and on systemic fungicides such as benzimidazole (Bubici and Cirulli, 2008). However, the high toxicity of these chemicals, environmental concerns, and the emergence of tolerant strains are among the problems with their usage that have fostered the emergence of new strategies that are reliable, efficient and that reduce environmental impacts (Damalas and Eleftherohorinos, 2011, Hollomon, 2015).

The spectacular advances of knowledge regarding the mechanisms involved in induced resistance in plants have favored the development of initiatives to enhance the natural defense strategy for plants to cope with diseases in a context combining the preservation of the environment and intensive agricultural production. This strategy involves using formulations containing synthetic inducers or natural elicitors of defenses as active ingredient (Benhamou and Rey, 2012, Walters et al., 2013). Among them the synthetic analogue of salicylic acid called benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl-ester (benzothiadiazole (BTH); acibenzolar-S- methyl (ASM)), commercialized by Syngenta as Bion or Actigard, is known to induce resistance against a broad spectrum of pathogens in several plant species (Walters et al., 2013). Recently, Zine et al. (2016a) showed that a new derivative 1,3,4-thiadiazole was able to induce resistance against tomato Verticillium wilt. Milsana, a plant extract derived from Reynoutria sacalinensis was marketed as inducer of resistance against powdery mildiou and Botrytis cinerea. Thyme extract, registered as BioZell-2000B was effective in controlling fire blight disease of apple, caused by Erwinia amylovora (Zeller, 2006). This bacterium secretes a Hrp effector, called harpin, that was commercialized by the global company Plant Health care (Buckinghamshire, UK) under the name of Messenger and was revealed to be effective against several tomato diseases including late blight caused by Phytophthora infestans (Fontanilla et al., 2005). Other commercialized elicitors derived from pathogens such as fungal chitosan have been shown to induce defence responses in a wide range of plant species (Zhang et al., 2002).

The Euphorbia genus includes a broad and diverse group of plants, which are characterized by the presence of an irritant latex rich in bioactive compounds such as α-euphol and α-euphorbol triterpenes (Singla and Pathak, 1990). Triterpenoids are among of the largest classes of plant specialized metabolites synthesized via the acetate/mevalonate pathway and 2,3-oxidosqualene cyclization (Crozier et al., 2006), sharing then a common biosynthetic origin with sterol (Abe, 2007). These compounds have been reported to possess interesting pharmacological properties, including anti-inflammatory, antimicrobial and antiplasmodial activities (Dzubak et al., 2006). Nevertheless, there is no report on their use in plant protection or inducers of disease resistance against phytopathogens.

This work aimed to test the action of four semisynthesized triterpene derivatives from the latex of two Moroccan Euphorbiaceae (Euphorbia resinifera Berg. and Euphorbia officinarum L.) on Verticillium wilt of tomato. Greenhouse experiments showed that all triterpenes afforded a good protection against the fungus. In addition, they were able to induce an oxidative burst and to activate two enzymes involved in plant defense; peroxidase and poly phenol oxidase.

Section snippets

Triterpene derivatives are phytotoxic at higher concentrations

Oxidation of α-euphorbol and 31-norlanostenol yielded four previously undescribed products named 3β-tosyloxy-4α,14α-dimethyl-5α-cholesta-7,9-diene (F1); 4α,14α-dimethyl-5α-cholesta-7,9-dien-3β-ol (F2); 24-methylen-elemo-lanosta-8,24-dien-3-one (F3) and elemo-lanost-8-en-3,11,24-trione (F5) (Scheme 1). When tested for their phytotoxicity, all of these previously undescribed compounds inhibited completely germination at concentrations of 500 μg/ml (Fig. 1A) and 100 μg/ml (Fig. 1 B). Germination

Discussion

In this work we showed that tomato seeds germinating in the presence of the four tritepene derivatives prompted resistance to Verticillium wilt caused by V. dahliae. Concentrations as low as 10 μg/ml were effective in reducing external symptoms of Verticillium wilt, revealed by leaf alteration and growth reduction, as well internal symptoms such as vessels discoloration. Despite their extensive pharmacological bioactivities and their therapeutic implications in the field of medicine (Dzubak

General experimental procedures

1H and 13C NMR spectra were recorded on AMX2 Brucker 400 MHz using TMS as an internal standard and CDCl3 as the solvent. Melting points were measured capillaries in a Buchi 510 apparatus. Chemical shifts are given in ppm. Mass spectra were obtained with an Autospect Instrument 70 ev spectrometer. Column chromatography was carried out on silica gel (Merk Art. 15111, 7741) using hexane-EtOAc as eluent. p-Toluenesulfonyl chloride (TsCl), chromium trioxide (CrO3), sodium periodate (NaIO4),

Acknowledgements

This research was supported by the Moroccan ‘Ministère de l'enseignement supérieur et de la recherche’. The authors are grateful to Professor S. El Idrissi for reviewing the English of the manuscript.

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