Biocontrol of plant diseases through induction of systemic resistance is an environmental friendly substitute to chemicals in crop protection measures. Different biotic and abiotic elicitors can trigger the plant for induced resistance. Present study was designed to explore the potential of Pseudomonas aeruginosa PM12 in inducing systemic resistance in tomato against Fusarium wilt. Initially the bioactive compound, responsible for ISR, was separated and identified from extracellular filtrate of P. aeruginosa PM12. After that purification and characterization of the bacterial crude extracts was carried out through a series of organic solvents. The fractions exhibiting ISR activity were further divided into sub-fractions through column chromatography. Sub fraction showing maximum ISR activity was subjected to Gas chromatography/mass spectrometry for the identification of compounds. Analytical result showed three compounds in the ISR active sub-fraction viz: 3-hydroxy-5-methoxy benzene methanol (HMB), eugenol and tyrosine. Subsequent bioassays proved that HMB is the potential ISR determinant that significantly ameliorated Fusarium wilt of tomato when applied as soil drench method at the rate of 10 mM. In the next step of this study, GC-MS analysis was performed to detect changes induced in primary and secondary metabolites of tomato plants by the ISR determinant. Plants were treated with HMB and Fusarium oxysporum in different combinations showing intensive re- modulations in defense related pathways. This work concludes that HMB is the potential elicitor involved in dynamic reprogramming of plant pathways which functionally contributes in defense responses. Furthermore the use of biocontrol agents as natural enemies of soil borne pathogens besides enhancing production potential of crop can provide a complementary tactic for sustainable integrated pest management.
Introduction
Tomato (Solanum lycopersicum L.) is a member of family Solanaceae, cultivated worldwide, ranked first among the processing crops and second as a vegetable crop. In Pakistan it is cultivated on about 58.196 thousand hectares with an annual production of 574.052 thousand tons (FAO, 2013). It contains valuable nutrients like vitamin A and C, calcium, phosphorus, potassium and magnesium (United States Department of Agriculture [USDA], 2009). It is also a source of an antioxidant compound named lycopene that has been found helpful against cancer (Miller et al., 2002). Tomato wilt caused by the fungus Fusarium oxysporum Schlecht. f. sp. lycopersici (Sacc.) W.C. Snyder et H.N. Hansen (FOL) is an alarming disease, causing yield losses up to 25% (Fravel et al., 2005; Park et al., 2013). Disease control strategies include use of resistant tomato varieties with cultural, chemical and biological control (Agrios, 2005; Pottorf, 2006). Cultural control lost its effectiveness as pathogen has a wide host range. Use of resistant varieties is futile due to chances of mutation in Fusarium spp. Chemical control is now losing its ground due to adverse effects of chemicals on environment and soil microbiota that calls for alternative inputs with lower dependency on chemicals for sustainable agriculture (Lucas, 2011). Recently used biological control employed induced systemic resistance (ISR) mechanism using rhizospheric plant growth-promoting bacteria against fungal pathogens (Pieterse et al., 2014).
Plants are devoid of an immune system which is the chief characteristic of mammals. To deal with different types of pathogens, plants have weakly inducible or constitutive defense systems including plant cell walls, cuticles, phytoanticipins (Underwood, 2012; Newman et al., 2013) and inducible defenses in which plants activate their immune system by the stimulus of signal molecules known as elicitors (Henry et al., 2012; Maffei et al., 2012; Newman et al., 2013). Elicitors can be derived from natural/living organisms like plants and microbes or generated synthetically (Walters et al., 2013).
Plant pathogens stimulate the host plant to activate defense responses against the invaders but this weak defense reaction will not limit the spread of the pathogen into the host plant (Thordal-Christensen, 2003). However, the defense responses can be enhanced by triggering the plant before pathogen attack. Interaction of some rhizobacteria with the plant roots has proven to increase plant resistance against some pathogenic bacteria, fungi and viruses. This phenomenon is termed as ISR (Lugtenberg and Kamilova, 2009). Application of microorganisms to control diseases, which is a form of biological control, is an environment-friendly approach (Lugtenberg and Kamilova, 2009). The direct mechanism of PGPR in biocontrol involves antagonism to soil borne pathogens (Supplementary Data Sheet 1) and indirect mechanism depends on induction of systemic resistance (Choudhary, 2011; Glick, 2012). Generally competition for nutrients, niche exclusion, ISR and antifungal metabolites production are some of the chief modes of biocontrol activity in PGPR (Lugtenberg and Kamilova, 2009). Many rhizobacteria have been reported to produce antifungal metabolites like hydrogen cyanide (HCN), phenazines, pyrrolnitrin, 2, 4-diacetylphloroglucinol, pyoluteorin, viscosinamide, and tensin (Bhattacharyya and Jha, 2012). In 2009, De Vleesschauwer and Höfte (2009) coined the term ISR for resistance induced by PGPR which was found to be independent of the pathway involved.
In case of microbially induced resistance the best option is the use of plant growth promoting rhizobacteria (PGPR) as potential elicitors of plant defense mechanisms (Liu et al., 1995). On the onset of plant colonization by the rhizobacteria, metabolic changes may occur in the host, i.e., production of phytoalexins (Van Peer et al., 1991), accumulation of pathogenesis related (PR) Proteins (Zdor and Anderson, 1992) or deposition of structural barriers, etc. (Benhamou et al., 1996a,b).
Origin of elicitor compounds may be biological (plant or microbe derived) or synthetic like beta-amino-butyric acid (BABA), cis-jasmone and acibenzolar-S-methyl (ASM) (Walters et al., 2013). Plants generally recognize three types of chemical elicitors viz; microbe-associated molecular patterns (MAMPs) derived from beneficial microbes, pathogen-associated molecular patterns (PAMPs) released by pathogenic microbes and damage-associated molecular patterns (DAMPs) produced by plants on injury by insects or herbivores or even during microbial degradation (Henry et al., 2012; Newman et al., 2013). These aforementioned molecules are called “patterns that elicit immunity” (PEIs) which are recognized by plants through transmembrane pattern recognition receptors (PRRs) (Jones and Dangl, 2006; Maffei et al., 2012; Newman et al., 2013). After recognition of MAMPs or DAMPs elicitors pattern-triggered immunity (PTI) is activated in plants. This stimulation of defense reaction restricts the pathogen making plant resistant to additional pathogen attack through the mechanism of induction of systemic resistance (Henry et al., 2012).
Bacteria belonging to the genus Pseudomonas involve pathogen inhibition via competition and/or antagonism (Haas and Défago, 2005) and by developing direct interactions with the host plants through ISR (Bakker et al., 2007). Pseudomonas aeruginosa PM12, used in this study was isolated from healthy tomato roots from vegetable garden of University of the Punjab, Lahore, Pakistan (Fatima and Anjum, 2016). It was characterized on molecular grounds through sequencing of 16S rRNA (900bp) and alignment at GeneBank (NCBI, MaryLand), allotted accession number KT966743 (Fatima and Anjum, 2017).
Induced systemic resistance is involved in synthesis of enhanced levels of various secondary metabolites engaged in plant defense mechanisms. These include phytoalexins that increase plant resistance through their toxic action against pathogens. Phytoalexins, i.e., sesquiterpenoids are synthesized by the members of Solanaceae and isoflavonoids are produced in individuals of the Papilionaceae. They inhibit germination of fungal spores and retard fungal growth. Plant tissues exhibiting ISR show increased activity of phenylalanine ammonia lyase (PAL) that leads to resistance response in plants (Verhagen et al., 2010).
Metabolomics is one of the most rapidly growing areas of contemporary science. This technology is now being used to route out reprograming and metabolic fluctuations in plant pathways (De Vos et al., 2007; Zhao et al., 2015). Current investigation revealed that compatible host pathogen interactions are characterized by a lower level of certain defense-related mechanisms compared with host pathogen interactions in the presence of bacterial ISR elicitor that leads to a more dynamic metabolic response over the course of colonization. Furthermore, our results demonstrate that elicitor (HMB) from P. aeruginosa (PM12) induces production of secondary metabolites involved in defense pathways of tested plant.
Materials and Methods
Fungal and Bacterial Strains
Virulent strain of F. oxysporum isolated from diseased tomato plants growing in vegetable garden of University of the Punjab, Lahore was cultured on potato dextrose agar (PDA difco) for 10 days. Conidia were harvested by gentle scraping in sterile water and pathogen inoculum was prepared by adjusting the concentration to 105conidia/ml using haemocytometer. P. aeruginosa (PM12) was grown on LB broth medium (100 ml) for 24 h at 35°C. For collecting extracellular metabolites culture was pelleted by centrifugation at 4000 g for 15 min, and the supernatant obtained was processed for ISR assay. Intracellular metabolites were extracted using sonication. Bacterial cell lysis was performed six times through sonication at resonance amplitude for 15 s at 4°C.
Preliminary Screening of ISR Determinants from P. aeruginosa PM12
This study was performed to identify the involvement of intracellular metabolites and cell-free culture filtrates (CFCF) of P. aeruginosa for inducing systemic resistance against fungal wilt in tomato. Two-weeks old seedlings of Fusarium wilt susceptible tomato variety “Rio-Grande” seeded in sterilized pot media. In total, 50 ml of both extracts (intracellular and extracellular) were supplied to the allocated plastic pots containing 0.5 kg sterilized soil and after a period of 3 days these pots were inoculated with 50 ml of pathogen @ 105conidia/ml. Positive control consisted of P. aeruginosa PM12 suspension made in water by adjusting the concentration to 104 cfu/ml. Fifty milli liter of sterile distilled water was provided to the pots designated as untreated control. All the pots were incubated for 14 days under greenhouse conditions. There were ten replicates against each treatment and experiment was conducted twice. To determine the disease index (DI), wilting was scored based on the criteria developed by Epp (1987) (0 = no wilt symptoms; 1 = less than 25% of the plant turned yellow; 2 = yellowing and browning covered nearly 50% of plant; 3 = whole plant turned brown and died). The equation described by Cachinero et al., 2002 was used to calculate the DI.
