Identification of Three Elicitins and a Galactan-Based Complex Polysaccharide from a Concentrated Culture Filtrate of Phytophthora infestans Efficient against Pectobacterium atrosepticum

Guillaume Saubeau,Florence Val,Vincent Ferrières,Fanny Gaillard,Caroline Nugier-Chauvin,Laurent Legentil,Didier Andrivon

doi:10.3390/molecules191015374

Abstract

The induction of plant immunity by Pathogen Associated Molecular Patterns (PAMPs) constitutes a powerful strategy for crop protection. PAMPs indeed induce general defense responses in plants and thus increase plant resistance to pathogens. Phytophthora infestans culture filtrates (CCFs) are known to induce defense responses and decrease the severity of soft rot due to Pectobacterium atrosepticum in potato tubers. The aim of this study was to identify and characterize the active compounds from P. infestans filtrate. The filtrate was fractionated by gel filtration, and the protection effects against P. atrosepticum and the ability to induce PAL activity were tested for each fraction. The fraction active in protection (F1) also induced PAL activity, as did the whole filtrate. Three elicitins (INF1, INF4 and INF5) were identified in F1b, subfraction of F1, by MALDI-TOF-MS and MS/MS analyses. However, deproteinized F1b still showed biological activity against the bacterium, revealing the presence of an additional active compound. GC-MS analyses of the deproteinized fraction highlighted the presence of a galactan-based complex polysaccharide. These experiments demonstrate that the biological activity of the CCF against P. atrosepticum results from a combined action of three elicitins and a complex polysaccharide, probably through the activation of general defense responses.

Highlights

After fractionation of concentrated culture filtrate (CCF) on gel filtration column and analyses of compounds by MALDI TOF and MS/MS, we demonstrated the implication of three elicitins and a galactan-based polysaccharide in the biological activity of culture filtrate
CCF at 400 μg/slice, but not pure pea broth used for growing the pathogen, significantly reduced (p-value = 0.0001) the soft rot severity due to P. atrosepticum compared to the water control
(1) The CCF is fractionated by gel filtration into four fractions (chromatogram (A) F1, F2, F3, and F4) and the induction of phenylalanine ammonia-lyase (PAL) activity is evaluated on F1 and F2 fractions

Summary

Introduction

Pectobacterium atrosepticum, formerly named Erwinia carotovora subsp. atroseptica [1], is a causal agent of bacterial soft rot and black leg, affecting both potato yield and tuber quality [2,3].P. atrosepticum, a pectinolytic Gram negative bacterium, produces extracellular enzymes such as pectate-lyases, pectinases, cellulases and proteases, resulting in tissue maceration and rot symptoms [4]affecting postharvest storage [1] and causing significant economic losses ($20–100 million in multiple crops worldwide every year) [3,5,6].There are currently no efficient curative methods to protect potato against Pectobacterium spp. [7], biocontrol strategies have been tested to control soft rot development. There are currently no efficient curative methods to protect potato against Pectobacterium spp. [7], biocontrol strategies have been tested to control soft rot development. The application of biocontrol agents that are safe for the environment has become an important research area in pest management, and some fluorescent Pseudomonas strains can act as biological control agents against Pectobacterium spp. on melon [8] and on potato [9]. Rhodococcus bacteria can prevent disease due to P. atrosepticum by disrupting the quorum sensing-based communication of P. atrosepticum [11]. These methods are not always efficient enough, and new strategies exploiting plant immunity constitute major opportunities for improved crop protection.

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