Keywords

1 Introduction

Like animals, plants do have to fight with a plethora of problems for their very survival. Both biotic and abiotic factors affect plant survival. The major biotic stresses are caused by a wide variety of pathogens that include bacteria, viruses, fungi, nematodes, insects and many herbivorous animals. Being sessile, plants have developed an array of sophisticated innate defense system against the microbial invaders in consequence of the long process of host-pathogen co-evolution (Dodds and Rathjen 2010).

The multiple layers of defense mechanisms evolved by plants ranges from passive-mechanical or preformed chemical barriers, which provide nonspecific/non-host protection (Tyler 2001; Yun and Loake 2002), to active responses that provide host- or cultivar-specific resistance (Jackson and Taylor 1996; Hammond-Kosack and Parker 2003). Passive-mechanical barriers include the various structural features that are characteristic of different plants like thorns, prickles, spines or trichomes, many of these may sometimes also contain irritants or poisons. In addition to these structures, other mechanical barriers to microbial invasion include the waxy outer layer called cuticle, the secondary protective tissue namely the periderm in addition to wax, resins, latex (Agrawal and Konno 2009), lignins etc. that prevent pathogen ingress (Fig. 1). Many plants have non-protoplasmic inclusions primarily in epidermal cells like calcium oxalate crystals (Goldblatt et al. 1984; Rudall and Caddick 1994), starch grains, mucilage (slime) and silica bodies (10–30 μ), which serve as variety of purposes including structural rigidity (Kaufman et al. 1979; Christina et al. 2003), lodging resistance (Ma et al. 2006), mechanical resistance to invading organisms (Namaganda et al. 2009), tolerance to pathogens (Djamin and Pathak 1967) and also to overcome various abiotic stresses like drought, radiation, nutrient imbalance, temperature fluctuations and osmotic imbalances (Hodson et al. 2005; Ma and Yamaji 2006). Pathogen can overcome these physical barriers by way of force penetration using specialized structures, entry through stomata or wounded regions or by production of degradative enzymes (Walton 1994). Since plants produce a wide range of secondary metabolites and many of which posseses antimicrobial activities, the pathogen will be encountered by this second line of defense systems (Table 1) (Edreva et al. 2008). These defensive secondary metabolites in plants can be classified as terpenoids, phenolics and nitrogen compounds (Harborne 1999). The chemical barrier has evolved as a consequence of natural selection and plays decisive role in plant resistance against the invading microbes (Ingham 1973; Osbourn 1996). However, pathogens also evolve themselves to overcome this chemical arsenal by acquiring various types of abilities (Morrisey and Osbourn 1999; Papadopoulou et al. 1999). Once the pathogen breaches the preformed mechanical barriers it comes under the robust surveillance of host resistance/active defense which finally results in resistance or susceptibility of host to the invading pathogen (Jones and Jones 1997; Hammond-Kosack and Jones 1997; Moffett et al. 2002; Morita-Yamamuro et al. 2005).

Fig. 1
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Different types of defense mechanisms in plants. (a) Anatomical barriers; (b) Physical barriers; (c) Sequential defense mechanism

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Table 1 Various allelochemicals, their plant source and target pest against which they show their activity
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In resistant plants, pathogen avirulence (Avr) gene encoded elicitors (Dangl and Holub 1997; Nimchuk et al. 2001) are perceived by the plant R-gene encoded receptors that “guard” the host proteins (Dangl and Jones 2001; Jones 2001; Rathjen and Moffet 2003). This molecular recognition originally proposed by Flor (1956, 1971) and elaborated in the “gene-for-gene model” underlies the molecular basis of defense response and subsequently triggers a hierarchy of defensive molecular events in the plant. After Flor’s classic work, it became evident that the outcome of many host-parasite interactions is governed by matching gene pairs [resistance (R) and avirulence (Avr) genes, respectively] (Crute and Pink 1996). R-proteins localized in the plasma membrane or cytoplasm impart resistance to the host plant either by direct detection of elicitors or indirectly through detection of any pathogen mediated alterations to the “guarded” host proteins (Dangl and Jones 2001). Following the R-Avr gene product interaction, a signaling cascade is activated that includes generation of reactive oxygen intermediates (ROIs), transient ion fluxes, intracellular pH changes, cell wall strengthening, release of secondary signaling molecules like nitric oxide (NO), salicylic acid, jasmonic acid and ethylene and activation of MAPK (Mitogen activated protein kinase) cascades (Martin 1999; McDowell and Dangl 2000; Heath 2000). All these molecular events eventually result in the transcriptional activation of batteries of plant defense genes, such as pathogenesis-related (PR) genes. The cumulative effect of all these cellular events arrests microbial proliferation through collapse of challenged plant cells by a localized programmed cell death (PCD), termed the ‘hypersensitive response’ (Keen et al. 1993) and shares analogy to animal apoptosis (Lam et al. 2001). Hypersensitive response results in establishment of systemic acquired resistance (Ross 1961) that immunizes the entire plant against secondary attack by a broad spectrum of pathogens (Ryals et al. 1996). Systemic acquired resistance is characterized by an increase in endogenously synthesized signaling molecules like salicylic acid (Malamy et al. 1990; Metraux et al. 1990), concomitant activation of PR genes (Bol et al. 1990; Ward et al. 1991) and heightened plant resistance (Staskawicz et al. 1995). Other signaling molecules like jasmonic acid and ethylene have also been observed to induce expression of defense genes (Wasternack and Parthier 1997; van Wees et al. 2000; Hammond-Kosack and Parker 2003) that are not activated by salicylic acid (Penninckx et al. 1996). Apparently it has been proven that jasmonic acid/ethylene act antagonistically to salicylic acid pathway but global gene expression profiling supports existence of substantial cross-talk between the salicylic acid, jasmonic acid and ethylene signaling pathways (Glazebrook et al. 2003; Schenk et al. 2000; Thomma et al. 2001). The defense genes activated by these secondary signaling molecules include proteases (Pechan et al. 2000), protease inhibitors (Azarkan et al. 2004; Kehr 2006; Walz et al. 2004), chitinases (Howard and Glazer 1969; Azarkan et al. 2004; Kim et al. 2003), oxidases like polyphenol oxidase (PPO), peroxidase (Saby et al. 2003) and lipoxygenases (Walz et al. 2004) in addition to phosphatase (Lynn and Clevette-Radford 1987) and lipases (Gandhi and Mukherjee 2000).

2 Chemical Defense in Plants

Among the many different mechanisms employed by plants for combating diseases, the ability to synthesize an arsenal of low-molecular weight volatile and non-volatile chemicals helps them to interact with the ever-changing physical environment (Firn and Jones 2009). These defensive organic compounds called secondary metabolites have been a major counter defense tactics evolved by plants to withstand pathogen attack (Boller 1995). Unlike primary metabolites which are found in every dividing cell, secondary metabolite biosynthesis is regulated by environmental factors, genotype of the plant as well as age (Kossel 1991). There is also difference in distribution with particular metabolites being confined to phylogenetically related taxa while others having much broader distribution (Futuymaa and Agrawal 2009; Dixon 2001). Even in any particular plant the distribution of secondary metabolites may not be uniform but it will be constitutive with an induction seen in response to pathogen attack (Zong and Wang 2006). Constitutive/basal distribution of secondary metabolites, which is central to the optimal defense theory of plant-pathogen interactions (McKey 1979; Rhoades 1979; Strauss et al. 2002; Holland et al. 2009), is of significance considering the crucial role of speed of the host response in determining resistance to the invading pathogen.

Plants mainly produce three kinds of secondary metabolites that are nitrogen compounds, terpenoids, and phenolics (Harborne 1999). Table 2 represents different physical and chemical barriers which are present as barriers against certain pests, herbivory and other harmful effects. Current review emphasizes on chemical defence mechanism of plants against various target pests. The nitrogen compounds include alkaloids, cyanogenic glycosides and glucosinolates. Alkaloids widespread among different plant taxa are derived from various amino acids and are basic organic compounds with heterocyclic nitrogen, the position of which varies according to the plant families (Harborne 1993; Wink 2004). More than 12,000 different types of alkaloids have been discovered from over 300 plant families (Zwenger and Basu 2008). The defensive effects of alkaloids are manifested by their ability to affect cell membrane/cytoskeletal structure, as inhibitors of glycosidases and sugar-metabolizing enzymes (Asano et al. 2000) or by inhibition of protein/DNA synthesis (Schmeller et al. 1997). Cyanogenic glycosides composed of an alpha-hydroxynitrile type aglycone and sugar moiety (mostly D-glucose) are stored in inactive forms in plant vacuoles among a wide range of plant taxa (Vetter 2000). Upon enzymic hydrolysis, cyanogenic glycosides release cyanohydric acid (HCN) or prussic acid (Harborne 1993) which is extremely toxic to a wide spectrum of organism due to its ability to chelate metal ions functioning as co-factors to many key enzymes involved in metabolic processes (McMahon et al. 1995; Francisco and Pinotti 2004). In the unaffected plant the enzyme and cyanogenic glycosides remain separate and are brought in contact upon pathogen attack (Gruhnert et al. 1994). Sulfur containing glucosinolates or thioglucosides produced widely by plants belonging to the genera Cruciferae have well-documented antimicrobial activity (Fenwick et al. 1983). Hydrolysis of glucosinolates liberates D-glucose, sulphate ion and a series of compounds such as isothiocyanate, thiocyanate and nitrile that posseses fungitoxic activity (Mithen 1992; Wink 2004).

Table 2 Summary of the various mechanical and chemical defense systems as barriers in plants
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Most numerous and structurally diverse group of organic secondary metabolites are terpenoids (isoprenoids) derived from five carbon isoprene units and having diverse physiological, metabolic and structural functions in addition to their significant role in plant defense (Rees and Harborne 1985; Sessa et al. 2000; Dussourd 2003). All terpenoids are derived by isomerization of isopentenyl diphosphate to dimethylallyl diphosphate by isopentenyl diphosphate isomerase. Isopentenyl diphosphate and dimethylallyl diphosphate originate from either the plastidial methyl-erythritol 4-phosphate pathway or the cytosolic mevalonate pathway (Chappell 1995; Bochar et al. 1999). Dimethylallyl diphosphate condenses with one, two or three units of isopentenyl diphosphate catalyzed by prenyltransferases to give geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate respectively. These three acyclic prenyl diphosphates are converted by a very large group of enzymes called the terpene (terpenoid) synthases to corresponding monoterpenoid (C10), sesquiterpenoid (C15), and diterpenoids (C20). Subsequent hydroxylation and oxidation of terpenes catalyzed by cytochrome P450 enzymes further diversifies the terpenes (Zulak and Bohlmann 2010). Gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes are primarily responsible for the terpenoid diversity in response to different physiological conditions (Keeling et al. 2008; Trapp and Croteau 2001).

Phenolics with a wide distribution in the plant kingdom are aromatic compounds with hydroxyl groups. Phenolics range from simple plant phenols like hydroxy-benzoic acid to phenylpropanoids and complex phenyl propanoids like flavonoids that give pigmentation to plants and constitute a complex group called tannins (Harborne 1993). Flavanoids are further sub-divided as flavanols, anthocyanidins and chalcones and perform a wide range of biological activities that includes their role as attractants or feeding deterrents (Snook 1994), biotic and abiotic stress protective agents (Tattini et al. 2004; Moore et al. 2005; Schlösser 1994; Inderjit and Gross 2002). Antimicrobial activities of flavanoids are mediated by their ability to inhibit microbial cell wall degrading enzymes and chelation of metals necessary for enzyme activity (Skadhauge et al. 1997). Phenolics generally have antioxidant properties and are thus of therapeutic significance (Dai and Mumper 2010). Systemic induction and accumulation of low molecular weight phenolics and phenolic polymers like lignin and suberin is observed in response to various diseases (Bonello et al. 2006; Wallis et al. 2008; Cvikrova et al. 2006). Inhibition of pathogen colonization by phenolics is mediated via protein precipitation and iron depletion (Scalbert 1991).

3 Molecular Evolution of Allelochemical Diversity

Secondary metabolite chemistry of plants is dynamic and subject to changes depending on the type of environmental stress (Kliebenstein 2004; Hammerschmidt 2005; Mary Ann Lila 2006) with upregulation of key genes observed in response to pathogen attack (Baldwin 1998; Sirvent and Gibson 2002). Pathogen attacks impose natural selection on plants to evolve defensive strategies (Agrawal 2007) that in turn depends on the plants life history, genetic attributes and mating pattern (Futuymaa and Agrawal 2009; Johnson et al. 2009). Reduced recombination and allelic segregation as a consequence of prolonged asexual reproductive strategies negatively affects defense gene evolution as proposed in the Recombination-Mating hypothesis (Levin 1975). It is hypothesized that asexual reproduction not only results in erosion of genetic variation but also is susceptible to Muller’s Ratchet according to which sexual reproduction purges the deleterious mutations that might affect primary or secondary metabolic processes (Muller 1964; Paland and Lynch 2006). As per the Red Queen hypothesis, maintenance of sexual reproduction is of prime significance in the host-pathogen co-evolutionary arms race (Jaenike 1977), thereby equipping plants to synthesize an enormous reservoir of natural chemical diversity (Agrawal 2007). Natural selection pressures will select for alleles that enhance or retain secondary metabolite diversity taking into consideration the fitness cost on the plant (Agrawal and Fishbein 2008). Phylogenetic analysis has revealed the role of recombination, mutations, gene duplications and reshuffling in maintaining allelic diversity in the secondary metabolite as well defense genetic pool (Wagner 1998; Gierl and Frey 2001; Kondrashov et al. 2002). Molecular studies in genes associated with secondary metabolite biosynthesis like polyketide synthases (PKS), genes associated with phenyl propanoid (Milkowski and Strack 2004; Stehle et al. 2006) and alkaloid (Ober and Hartmann 1999) biosynthetic pathway, cytochrome P450 (Scott and Wen 2001) and terpene synthases (Bohlmann et al. 1998; Rohdich et al. 2005) have revealed that environmental selection pressures must have favored duplication followed by functional divergence in generating/maintaining a rich diversity of metabolites that are distinguishable from their biogenetic starter units. Studies have indicated that terpenoid phytochemical complexity is contributed by a multitude of phylogenetically diverse terpene synthases and cytochrome P450 that have been subject to repeated duplication and divergence (Zulak and Bohlmann 2010). Even single amino acid changes have been observed to alter substrate specificity, product profiles and kinetic efficiency of these enzymes (Jez et al. 2002; Keeling and Bohlmann 2006). Nucleotide sequence diversity analysis of such functional genes from resistant and susceptible sources have thus provided a framework for understanding the variations in deployment of metabolites and defensive proteins in plant-pathogen responses (Shonle and Bergelson 2000; Bishop et al. 2000).

Secondary metabolite biosynthetic genes can also evolve by divergent evolution and by domain swapping (O’Brien and Herschlag 1999; Schmidt et al. 2003; Katoh et al. 2004). Simultaneous analysis of diversity at both neutral and defense-related functional loci has helped in elucidating the evolutionary forces shaping resistance evolution (Burdon and Thrall 1999; de Meaux et al. 2003). Adaptive evolution at key residues involved in detection of variable pathogen ligands and recombination events generating paralogues have been suggested as primary mechanisms responsible for generation of diversity at these loci (Ehrlich and Raven 1964; Michelmore and Meyers 1998; Futuymaa and Agrawal 2009). Besides sequence diversity analysis have also revealed the role of balancing selection in maintaining high nucleotide diversity levels at key resistance loci reflecting “trench warfare” (Stahl et al. 1999) or “recycling polymorphism” (Holub 2001) between host-pathogen genotypes (Bergelson et al. 2001).

Unfortunately, domestication and breeding procedures have eliminated many of the genes responsible for the biosynthesis of secondary metabolite in crop plants (Lebot et al. 2005; Olsen and Gross 2008). As a result, the dependence on chemical pesticides/insecticides increased considerably (Holdren and Ehrlich 1974; Nash et al. 2010). This increased susceptibility in cultivars attributed to “domestication bottleneck” eventually reduces overall genetic diversity in the cultivars as against their resistant wild counterparts (Tang and Knapp 2003). Hence to gain a comprehensive understanding of the molecular processes governing host-pathogen interactions, diversity should also be sampled in genes involved in secondary metabolite biosynthesis of both cultivars as well in the relatively undisturbed wild relatives of crop plants (Hawkes 1977). The adverse impact of pesticides on environment and biodiversity is compounded by the susceptibility of cultivars of many crop plants. It has sparked an interest in understanding the mechanisms underlying the evolution and nature of this chemical diversity in developing biologically and ecologically friendly sustainable resistance strategies by making use of the plants own innate immune system.

4 Allelochemicals as Biocontrol Agents for Plant Diseases

Growing global awareness about safe food and increasing public consciousness on the adverse impact of pesticides both on human health and environment has witnessed a boom in organic farming and has fuelled the search for greener alternatives in the agricultural sector (Fravel 2005; Slusarenko et al. 2008). Plant derived allelochemicals or secondary metabolites provide better alternatives than pesticides because of low toxicity, biodegradability and hence reduced risk to environment and human health (Tesar and Marble 1988; Jespers and de Waard 1993; Tripathi and Dubey 2004) and thus serve as leads for new agrochemicals. Allelochemicals produced from a plant source show leaching in the soil, due to precipitation and other factors. There they interact with different soil components and affect the quality of soil in many ways. One among these is the biocontrol of various plant diseases. Leached out allelochemicals and their exudates then enter the groundwater and may be transpored to distant areas where they affect the growth and physiology of receiver plants (Fig. 2). In this way, phytotoxic activity of an allelochemical is influenced by soil factors and plant factors of donor and receiver plants both. While plant factors and soil factors are ultimately affected by meteorological factors.

Fig. 2
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Leaching of allelochemicals and its effects on receiver plant

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Plant derived antimicrobial agents that include essential oils, alkaloids, phenols, flavanoids, tannins and sterols (Burt 2004; Halama and van Haluwin 2004) can contribute to disease control because of their enormous diversity. Many allelochemicals have been identified and characterized so far and includes Heliannuols and guaianolides from H. annuus (Macias et al. 1999); 14,15[beta]-Epoxy-prieurianin from bark of Guarea guidona (Lukacova et al. 1982); Valtrate from Valeriana capense (Fuzzati et al. 1996); insecticidal pyrethrin I and pyrethrin II from Tanacetum cinerariifolium (Staudinger and Ruzicka 1924); Nicotine from Nicotiana tabacum (Elliott et al. 1974), Azadirachtin from Azadirachta indica (Butterworth and Morgan 1968; Abbasi et al. 2005; Rani et al. 2006); rotenone from Derris urucu (Geoffrey 1985), glucosinolates like glucoraphenine isothiocyanate and allyl-isothiocyanate from mustard and horseradish (Ishiki et al. 1992; Delaquis and Mazza 1995) to name a few.

Even though performance of these allelochemicals is considered inferior to non-natural chemical pesticides, the environment friendly and low toxic nature of the allelochemicals outweighs the harmful effects of chemical pesticides, thereby making it one of the most promising and safe alternative to the chemical pesticides and fungicides.

5 Application of Biotechnology in Extraction and Purification of Allelochemicals

Biotechnological approaches employing tissue culture, DNA recombination technology, microbial fermentation, etc. have facilitated easy manipulation of sources and extraction of large quantities of pure allelochemical compounds. Plant tissue culture systems like cell culture and micro propagation etc., aid in the synthesis of large quantities of secondary metabolites without much interference from climatic factors (Junaid et al. 2010). The hairy root cultures, for example, are seen to be one of the most efficient systems for the production of secondary metabolites that are normally biosynthesized in roots (Hu and Du 2006). DNA recombination technology has also facilitated manipulating the metabolic pathways to enhance production of secondary metabolites (Capell and Christou 2004; Park et al. 2002). There have been many attempts to modulate biosynthetic pathway to enhance production of secondary metabolites (Park et al. 2002; Verpoorte and Memelink 2002).

Integration of molecular and genetic approaches may aid in the allelochemicals production in plants. Incidences of multi-gene engineering to introduce completely novel pathways and thus to produce completely new products have also been reported (Tattersall et al. 2001). Identification of individual genes and analysis of their expression pattern is a powerful tool to focus rapidly on genes that can improve complex traits. If operational genetic transformation systems are available for crops, transgenics have a great potential for the test and verification of gene functions identified by classical breeding and marker assisted selection. Areas in the genome of importance for trait variation, i.e., quantitative trait loci (QTL) for complex traits, such as water and nutrient use efficiency, have been identified in mapping populations of major agricultural crops and potential energy crops (Hirel et al. 2001; Rönnberg-Wästljung et al. 2005; Manneh et al. 2007).

Thus, the new tools of molecular biology and plant biotechnology provide much better opportunities to enhance the production and extract large quantities of high quality allelochemical for their use in pest and disease management.

6 Conclusion

Although application of weed and pest controlling chemical agents have steadily increased, yet a number of pesticides have well-documented negative consequences on the environment and on human health. Therefore biological control offers a number of alternative approaches for pest, disease and weed control in agriculture (Jordan 1993; Bond and Grundy 2001; Mason and Spaner 2006), but the application of biological weed control has often been proved difficult in practice (Müller-Schärer et al. 2000). Allelopathy is a promising component of biological control measures (Lovett 1991) which may have direct or indirect effect of one plant (or microorganism) on another mediated through the production of chemical compounds that escape into the environment (Rice 1974; Macías et al. 2007). A comprehensive understanding of the chemical complexities seen in different taxa can thus be utilized towards developing measures for crop protection. Encouraging results have been documented and reported regarding the crucial role played by these antimicrobial secondary metabolites in plant-pathogen interactions. Development of these non-chemical/synthetic plant based formulations on a commercial scale is gaining considerable momentum considering the merits that includes negligible risk to human health and environment as against the chemical pesticides. As many of these secondary metabolites have proven biological activities, scale-up production of active principles/preparations of secondary metabolites, from resistant plants can be optimized and exploited for development of environment friendly biopesticides/microbicides. Alternatively, strategies can be developed through metabolic engineering to mediate the synthesis of these defensive secondary metabolites in susceptible cultivars to combat the invading pathogens.