Endophytic fungus Acrophialophora jodhpurensis induced resistance against tomato early blight via interplay of reactive oxygen species, iron and antioxidants
Introduction
Root endophytes can protect plants from pathogens by direct and indirect mechanisms. Direct mechanisms include antibiosis, lytic enzyme secretion, phosphate solubilization, competition and production of phytohormones and siderophore. Indirect mechanisms consist of inducing resistance, stimulating plant secondary metabolites, promotion of plant growth and physiology [1]. Indirect mechanisms describe effect of endophytes on the pathogen, which is mediated by the host plant and induction of plant defense against future pathogen attacks [2]. Induced resistance using beneficial fungi has been studied against Alternaria spp. as destructive necrotrophic pathogens on tomato plants by many researchers. For example pre-inoculation with nonpathogenic A. alternata isolate against the pathogenic isolate of A. alternata [3], and application of Trichoderma spp. against A. solani [4] activated plant defense responses, which let to plant protection against the pathogen.
Early defense mechanisms after the pathogen attack in plants include production of reactive oxygen species (ROS), activity of defense-related enzymes, cell wall reinforcement, accumulation of phenolics and lignin [5], which variation in the host response is related to the life style and nutrition strategies of pathogenic fungi [6]. The ROS include free radicals, such as superoxide (O2•−) and hydroxyl radical (OH•), and non-radicals like hydrogen peroxide (H2O2) and singlet oxygen (1O2) [7]. The excessiveness of ROS causes oxidative damage to protein, DNA and lipids, which leads to the changes in cellular function. The ROS are not only known as toxic products of aerobic metabolism, but also as signal molecules and second messengers in plant defense pathways. The ROS control processes such as growth, development and response to biotic and abiotic stress in various plant species. Therefore, they act as secondary messengers in various key physiological phenomena, and also induce oxidative damages under several environmental stress conditions [8].
Various types of ROS can be produced under both normal and stressful conditions in the chloroplasts, mitochondria, peroxisomes, plasma membranes, endoplasmic reticulum and cell wall. The major sources of ROS production are chloroplasts and peroxisomes in presence of light, and the mitochondrion under dark conditions [9]. Asselbergh et al. [10] reported critical role of ROS in defense mechanisms against Botrytis cinerea as a necrotrophic pathogen, which produced as a first line of defense and involved in oxidative cross-linking of cell wall components, induced hypersensitive response, and activated an array of protective genes involved in plant defense signaling.
Changes in the ROS levels cause oxidative stress, therefore ROS scavenging systems are essential for managing the ROS levels both in plants and in pathogens. Destructive, protective, or signaling roles of ROS in the living cells depends on the equilibrium between ROS production and scavenging at appropriate time points and locations. Necrotrophic fungal pathogens kill the host cells to access nutrients via activating ROS production [11], but biotrophic fungi benefit from living cells. Therefore, induced ROS accumulation in the plant tissues might be involved in suppressing the growth of biotrophic pathogens and limit the spread of biotrophics, but not necrotrophic fungi and reported as a helper during the necrotrophics infection process [6].
Production of H2O2 was studied in the interaction of endophytic fungus Piriformospora indica and Rhizoctonia solani as a necrotrophic pathogen in rice [12]. The effect of P. indica in production of H2O2 was investigated against Fusarium pseudograminearum in wheat [13]. Also, H2O2 and O2− accumulations were evaluated in the cucumber plants inoculated with A. alternata and Streptomyces lydicus compared to the leaf discs only inoculated with A. alternata [14]. These investigations revealed that decreased disease progress was associated with decreased levels of ROS in the host plants via application of beneficial microorganisms [[12], [13], [14]].
Iron is an essential component for both plants and pathogenic fungi. In the nature, iron is present in two important forms, which are ferrous (Fe2+) and ferric (Fe3+). Redox cycling between two forms of iron can catalyse the production of dangerous free radicals [15]. Also, excess iron can lead to the formation of harmful hydroxyl radicals. Therefore, the iron balance in living cells must be controlled. Plants use various strategies to reduce pathogen virulence as iron-withholding, and also activate a toxic oxidative burst using locally increased iron levels [16]. Herlihy et al. [17] reported that iron has a critical role in ROS generation during plant immunity, therefore could increase or delay host defense responses, depending on virulence strategy of the pathogen. Systemically high iron levels could make plants more susceptible to necrotrophic pathogens and lead to production of ROS and cell death. But for biotrophics, it could facilitate production of ROS and initiate hypersensitive response (HR) cell death using ferroptosis or other mechanisms, that would be effective in resistance [17].
Plants have developed effective enzymatic and non-enzymatic antioxidant systems, which both work as ROS scavengers. Enzymatic antioxidants, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), guaiacol peroxidase (GPX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), are capable of scavenging ROS and protecting plant cells from oxidative damage [8]. Activation of various antioxidants is known to be related with the ROS homeostasis and induction of resistance in different pathosystems via application of beneficial fungi [12,13].
To our knowledge, induction of resistance using A. jodhpurensis against phytopathogens has not been investigated in any pathosystem, till now. Thus, the aims of this study were to (i) determine the possibility of increasing growth parameters and protecting tomato plants against A. alternata using the endophytic fungus A. jodhpurensis, (ii) examine the involvement of ROS, iron ions and enzymatic antioxidants in the induced defense responses of tomato against the pathogen, and to (iii) determine effect of the beneficial fungus on the levels of phenolics, lignin, relative water content, and cell membrane stability in tomato plants infected with A. alternata.
Section snippets
Fungal isolates
The isolates of Alternaria alternata and Acrophialophora jodhpurensis used in this study were obtained from Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad in Iran, which were previously isolated from infected and healthy tomato plants, respectively. These fungi were kept on potato dextrose agar (PDA) medium and lyophilized filter papers for short-term and long-term storage, respectively.
Inoculum preparation
For preparing A. alternata inoculum, this fungal pathogen was grown
Detection of A. jodhpurensis in tomato roots and percentage of root colonization
Microscopic detection of A. jodhpurensis was carried out at 50 dpi in tomato roots inoculated by spore suspension of the endophytic fungus. Intracellular hyphae of A. jodhpurensis were observed in tomato roots (Fig. 1A). Colonization percentage of tomato roots by the endophytic fungus was 86% at 50 dpi, compared to 50% colonization at 30 dpi (Fig. 1B).
Effect of A. jodhpurensis on protecting tomato plants and the leaf discs against A. alternata
Inoculating tomato roots with A. jodhpurensis led to significant reduction in the disease index of A. alternata on tomato leaves. The beneficial
Discussion
This is the first report on effect of the endophytic fungus A. jodhpurensis (C. jodhpurense Lodha) on defense responses involved in induction of resistance against A. alternata in tomato. The role of A. jodhpurensis on plant defense mechanisms such as accumulations of ROS and iron ions, lignification, total phenolics, antioxidant enzymes (such as POX, CAT, SOD and APX), RWC and MSI in tomato -A. alternata interaction was investigated in the present research. Our results revealed that the
Conclusions
In overall, the endophytic fungus A. jodhpurensis is capable of inducing tomato resistance against A. alternata by increasing activity of antioxidative enzymes (such as POX, CAT, SOD and APX), total phenolics, lignification, RWC, MSI, ROS accumulations and also via reducing iron ions accumulations. Therefore, this beneficial fungus could be suggested as a powerful candidate for plant protection against A. alternata. Future researches seems to be necessary to investigate the effect of A.
CRediT authorship contribution statement
Zoha Daroodi: Methodology, Resources, Writing - original draft, Formal analysis, Software, Visualisation. Parissa Taheri: Conceptualization, Data curation, Project administration, Formal analysis, Funding acquisition, Validation, Writing – review & edition. Saeed Tarighi: Supervision, Writing – review & edition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Ferdowsi University of Mashhad, Iran, for financial support of this research with project number 3/47823 approved on 22/9/2018. The authors have no conflict of interest to declare.
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