A Review of the Applications of Chitin and Its Derivatives in Agriculture to Modify Plant-Microbial Interactions and Improve Crop Yields
Russell G. Sharp
Moulton College, Moulton, Northamptonshire, NN3 7RR, UK; E-Mail: russell.g.sharp@gmail.com; Tel.: +44-1604-491-131
Received: 9 August 2013; in revised form: 8 September 2013 / Accepted: 9 September 2013 / Published: 21 November 2013
Abstract: In recent decades, a greater knowledge of chitin chemistry, and the increased availability of chitin-containing waste materials from the seafood industry, have led to the testing and development of chitin-containing products for a wide variety of applications in the agriculture industry. A number of modes of action have been proposed for how chitin and its derivatives can improve crop yield. In addition to direct effects on plant nutrition and plant growth stimulation, chitin-derived products have also been shown to be toxic to plant pests and pathogens, induce plant defenses and stimulate the growth and activity of beneficial microbes. A repeating theme of the published studies is that chitin-based treatments augment and amplify the action of beneficial chitinolytic microbes. This article reviews the evidence for claims that chitin-based products can improve crop yields and the current understanding of the modes of action with a focus on plant-microbe interactions.
Keywords: chitin; chitosan; plant growth-promoting rhizobacteria; induced defenses
- Introduction
After cellulose, chitin is the second most abundant polysaccharide on the planet [1]. Chitin is found in, and can be sourced from, a variety of different organisms, with the notable exceptions of higher plants and vertebrate animals. Chitin-rich animal tissues include the exoskeletons of arthropods (including insects, crustaceans and arachnids), the beaks of cephalopods and the eggs and gut linings of nematodes [2]. Various microbes also produce chitin in cell walls, membranes and spores, including fungi [3], and the spines of diatoms [4].
OPEN ACCESS
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Chitin shares a number of biochemical similarities with the cellulose found in plant cell walls. In common with cellulose, it is a long-chained linear, neutrally charged polymeric polysaccharide. Furthermore, like cellulose, chitin is used to construct mechanical and physical barriers that provide structural stability. However, unlike cellulose, chitin has an innate rigidity. Chitin is composed of repeating saccharide monomers of N-acetylglucosamine, which is a modified form of glucose with an amino group substituted at carbon 2 (Figure 1). As is the case with the cellulose in plant cell walls, the chitin polysaccharide is combined with other compounds to produce strengthened tissues. Both polysaccharides form microfibrils which differ in length and construction depending on the species and cellular location [5]. In fungi this involves cross linkages to glucan polymers to create a meshed hyphal wall [6,7]. Due to the involvement of other polymers, such as glucans, the chitin content of fungal cell walls ranges from 22%â40% [8]. In invertebrate tissues the chitin is supplemented with substantial amounts of proteins and calcium minerals [9].
Figure 1. The structural representation of the repeating polymer chains of (A) cellulose, (B) fully acetylated chitin and (C) fully deacetylated chitosan, evidencing their structural similarity. In addition to being deacetylated, chitosan applied in agriculture is also commonly shorter chained. Taken from RamĂrez et al. [10].
1.1. Chitin Biochemistry and Production
The majority of the chitin produced for agricultural purposes is sourced from the exoskeletons of crustaceans farmed/harvested for human consumption, chiefly shrimp, crab, and lobster. In addition to possessing a high chitin content, the use of crustacean exoskeletons provides a way of utilizing a major source of waste in the shrimp farming industry. Accurate data on global crustacean farming do not exist, but the Food and Agriculture Organization of the United Nations estimate that in 2011 global crustacean production was 5.9 Mt [11], with 35%â45% of this amount being discarded waste (head and thorax). This means that the global chitinous waste production from this source is 2.1â2.7 Mt,
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most of which could be productively utilised in agriculture. While attempts have been made to extract chitin from the waste produced from edible fungi cultivation [12,13] or by using fungi to ferment plant material [14], these enterprises are currently conducted on a much smaller scale to crustacean-derived chitin production.
The chitin polysaccharide can be partially depolymerized to produce oligosaccharide derivatives [10]. These oligosaccharides can be produced with varying polymer length or completely depolymerized to N-acetylglucosamine. If the chitin oligomers are deacetylated, the resultant compound produced is called chitosan (Figure 1). The protonation of the amino group resulting from its deacetylation makes chitosan one of the few cationic polymers found in nature [10]. Chitosan is produced commercially by exposing crustacean exoskeletons to high temperatures and alkali conditions which deacetylates the polymer and aids the removal of proteins and calcium minerals. Further purification steps are required if pigments and fats need to be removed, but these contaminations may be acceptable depending upon the final use. Purer forms of chitin and its derivatives are white, odorless and tasteless crystalline solids [10]. Chitosan is soluble in weak acid and so, once the alkali is neutralized, it can be safely applied to plants/soil as a solution or as a dry powder.
As is the case with the production of other natural polysaccharide products, such as fibres [15], considerable research effort is now focused on optimizing methods for enzymatic digestion to replace the use of strong acids/alkali, which are themselves a problematic waste product of chitosan production. Chitinases, chitosanases, chitin deacetylases and proteases [16] from natural sources have been isolated and trialed to develop environmentally friendly chitin and chitosan production [17], as has lactic acid fermentation methods [18].
The cationic properties of the chitosan oligosaccharide imbue it with unique properties that can be exploited by biotechnologists; including applications in the fields of medicine [19â21], material science [7], and crop science. Chitin, chitosan (of various chain lengths), and glucosamine have all been experimentally trialed on crop plants with a range of beneficial agronomical responses recorded. These can be broadly divided into four main areas, each dealt with in a separate section in this review: 1. Direct antibiosis against pests and pathogens of crops; 2. Enhancement of beneficial microbes, both in plant defense and growth; 3. Stimulation of plant defense responses against biotic stress; 4. Up-regulation of plant growth, development, nutrition, and tolerance to abiotic stresses. Positive responses to chitin and its derivatives have been reported in numerous economically important crop species that themselves represent a broad coverage of the plant kingdom, including monocotyledons, eudicotyledons, magnoliids and gymnosperms [22,23]. - Direct Antibiosis of Chitin
Chitosan has been repeatedly found to exhibit potent antimicrobial activity (Reviewed in RamĂrez et al., 2010 [10] and El Hadrami et al., 2010 [24]), which has been attributed to its cationic properties and the disruption of potassium signaling in pathogens [25,26]. However, chitosan could also be acting by creating barrier films, chelating mineral nutrients making them inaccessible to pathogens, and/or preventing the release of mycotoxins from the pathogen [27â29]. The polymeric form of chitin does not show substantial antimicrobial activity and this lack of antimicrobial activity
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has been attributed to chitinâs insolubility and uncharged nature [10]. Thishypothesis is supported by the finding that uncharged chitin oligomers lack antifungal activity [30]. While it is possible to show direct toxicity of pathogens in in vitro cultures, when chitosan is applied to field-grown crops it is less clear if the effects observed are due to direct toxicity of chitosan to the pathogen, the induction of plant defenses, and/or the stimulation of beneficial microbes.
2.1. Effectiveness of Chitin-Based Treatments against Fungal Pathogens
Soil amendment with chitosan has repeatedly been shown to control fungal diseases in numerous crops, especially Fusarium wilts [31â33] and grey mould [34,35]. It is also of note that these studies show chitosan to be fungistatic against both biotrophic and necrotrophic pathogens.
The control of oomycete pathogens has also been achieved with chitosan treatment, with Phytophthora capsici controlled on peppers [26] and Phytophthora infestans in potato [36]. This is despite oomycetes lacking chitinous cell walls, like true-fungi (eumycota). In the study by Xu et al. [26] on Phytophthora capsici in peppers, it was reported that the main effect observed in the pathogen was the disruption of the endomembrane system, especially the integrity of the vacuoles.
2.2. Effectiveness of Chitin-Based Treatments against Bacterial Pathogens
Despite chitin not being a component of bacterial cells [2], chitosan has been shown to possess antibacterial activity [37,38]. The majority of studies have been concerned with the control of human pathogens such as Escherichia coli, Staphylococcus aureus and certain Bacillus species. While in vitro studies show clear antibiotic activity, there is limited evidence for the antibiotic action of chitosan against major bacterial pathogens in planta. Chitosan toxicity has been shown in the major bacterial plant pathogen Pseudomonas syringae [39], but again, this study was conducted in vitro. Chitin in the form of ground shrimp waste was found to control the pathogen Streptomyces scabies, which causes scab disease on potato tubers [40], minimising the infection of the scab susceptible potato cultivar âBentjeâ to 4%, compared to 22% in the control group. However, rather than direct antibiosis, it was concluded by the author that chitin was active by promoting the growth of microbial species with antagonistic action against the pathogen.
2.3. Antiviral Action of Chitin
Chitosan has been shown to control viral diseases in plants [41]. However, it is yet to be shown that viruses are directly inactivated by chitosan, which in itself would appear to be unlikely as viruses are not composed of chitin or related polysaccharides. Therefore, rather than direct toxicity, it has been proposed that chitosan is effective against plant viruses by modifying the plantâs response to infection. It is hypothesised that viral particle transfer is disrupted by chitosan application and its induction of the hypersensitivity response [42â44].
2.4. Effectiveness of Chitin-Based Treatments against Insect Pests Chitosan has been found to show strong insecticidal activity in some plant pests [45]. Rabea et al. [45] found that a chitin derivative (N-(2-chloro-6-fluorobenzyl-chitosan) caused 100%
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mortality of larvae of the cotton leafworm (Spodoptera littoralis) that consumed it when incorporated into an artificial diet at 5 g kgâ1. Despite these positive results and the ubiquitous nature of insect pests, there are still only a limited number of studies on the effects of chitin derivatives on insect pests of plants. Of the reports published in peer-reviewed journals effective control with chitosan has been demonstrated for insect pests in the orders Hemiptera (including aphids) [46] and Lepidoptera (chiefly moth pests) [45,46]. However, there is a notable absence of information on the effects on pests in the orders Coleoptera (beetles), Diptera (true flies), and Hymenoptera (wasps, termites, ants and sawflies), which together represent thousands of economically important plant pests.
Mites are another group of economically important arthropod pests for which there is no information on the effects of chitin-based treatments. Mites, being arachnids, possess a chitinous exoskeleton [47]. There are reports that the chitin synthesis inhibitor nikkomycin disrupts many aspects of the development in the glasshouse mite (Tetranychus urticae); especially cuticular development [48], but there are no published reports of the effects of chitin/chitosan treatments on phytophagous mites on searchable databases.
While chitosan treatments have been found to effective at controlling herbivorous insect pests, it has actually been used successfully as an ingredient in the artificial diet fed to carnivorous insects being reared for use in the biological control of chitinous pests [49]. This finding suggests that chitin-based products could potentially be less harmful to non-target insects than conventional insecticides. However, there is not enough published data on other beneficial insects, such as pollinators, to come to firm conclusions on this matter.
2.5. Effectiveness of Chitin-Based Treatments against Nematodes
From the 1980s onwards a number of studies found that chitin was effective at controlling plant pathogenic nematode populations [50â52]. Chitinous amendments resulted in impressive reductions in the levels of the phytopathogenic nematode species Meloidogyne arenaria [50,51] and Heterodera glycines [52]. The level of control of nematodes by chitin-based products was sufficient for them to be registered and marketed as commercial nematocides (e.g., ClandoSanÂź618) [53]. However, Westerdahl et al. [54] found in an independent study that, although the level of control of nematodes on potatoes and walnuts was good, it was not at the level achieved with the synthetic nematicide 1, 3-dichloropropene. Furthermore, in a study on tomato Belair and Tremblay [55] found that, while plant growth was improved by chitin addition, no nematode control was observed. It has been proposed that chitin controls pathogenic nematodes by acting as a prebiotic promoting the growth of the beneficial chitinolytic microbes that parasitized the eggs of the nematodes [52,56]. However the exact mode of action remains unclear. Both Duncan [57], and Stirling [58] concluded that there was insufficient evidence to back up this mode of action with no detectible parasitism of eggs by chitinolytic fungi when chitin was applied to soil. Therefore, an alternative mode of action, whereby chitin breaks down in the soil to release nematicidal levels of ammonia has been proposed. This would therefore represent a more direct nematicidal action for chitin treatments. This hypothesis is supported by the finding that chitin decomposition in the soil releases significant amounts of ammonia [51]. However, the control of nematode populations by chitin addition has also been found over longer
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periods than would be expected from the short-term release of ammonia gas, which would quickly dissipate [56], thus indicating another control mechanism may be operating in chitin-amended soils. - Enhancement of Beneficial Microbes, both in Plant Defense and Growth
There is now a substantial body of evidence that the addition of chitin alters the environmental conditions in the rhizosphere and phyllosphere to shift the microbial balance in favour of beneficial organisms and to the detriment of plant pathogens. Chitinolytic microbes produce extracellular chitinase enzymes to degrade chitin-rich tissues of other organisms. While many chitinolytic organisms are pathogenic or parasites, many are also saprotrophic/necrotrophic feeding off dead material or are in a mutualistic relationship with plants. As a result, chitinolytic microbes are essential to plant and ecosystem health and nutrition. It is also important to note that chitinases are also used by organisms for reasons other than to utilize chitin as a food source. Firstly, chitin-containing organisms (both beneficial and pathogenic) use chitinases to regulate their growth and development by controlling the synthesis and lysis of cell walls and skeletons. Secondly, chitinases are also produced in organisms that do not produce chitin themselves, such as higher plants, bacteria and vertebrates, as well as viruses where they are used for detecting, consuming, and interacting with chitin-containing organisms [59,60]. Therefore, adding chitin to a growing environment can have a range of effects on the organisms present.
3.1. Stimulation of Antagonistic Biological Control Agents
One of the best-studied responses to chitin addition is the effect on the microbial species that act as antagonists of crop pathogens. Antagonistic microbes employ a number of methods to attack plant pests and pathogens. This includes, but is not limited to, the production of chitinases [61], the production of toxins (e.g., antibiotics and toxins), direct parasitism, competition for nutriment, and the induction of defense responses in the plant. Therefore, adding chitin-based products to the growing environment may aid beneficial antagonists by stimulating the production and activation of chitinases that can then be used to attack pests and pathogens, or be used as a stable nitrogen-rich polysaccharide food source that boosts the population to the level where other mechanisms control the plant pathogens. While the addition of chitin to the soil around cultivated crops may promote the growth of antagonistic microbes, owing to the nature of such a complex system, this is extremely difficult to monitor precisely. As a result, the majority of trials have monitored the effect of chitin addition on isolated and cultured antagonists applied to the same plants. The bacterium Bacillus subtilis is a pathogen of fungi and is one of the most widely used biopesticide in agriculture (product name = Serenade ASO) [62]. B. subtilis is known to secrete chitinases into the medium in which it is growing [63]. Manjula and Podile [64] showed that the addition of chitin to the carrier material improved the multiplication of B. subtilis, and improved the bacteriaâs fungicidal action and improved the control of Fusarium wilt in pigeon pea and crown rot in peanut caused by Aspergillus niger. Chitosan addition also improved the action of B. subtilis against powdery mildew in strawberry [65].
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The beneficial effect of chitin-based treatments to antagonistic bacteria is not restricted to B. subtilis, with both chitin and chitosan improving the control of Fusarium wilt in both tomato [66] and cucumber [67] when applied to the soil with a range of different species of chitinolytic microbes. Kishore et al. [68] found that chitin addition improved the control of Phaeoisariopsis personata, the causal agent of late leaf blight in peanut, by the bacterium Serratia marcescens. In addition to direct antibiosis, the study by Kishore et al. [68] found that these applications also increased the activity of key plant defense enzymes.
A number of soil-borne fungi have been reported to exhibit a chitinolytic activity that surpasses that of bacteria. Strongly chitinolytic species in the Aspergillus and Trichoderma genera are the most commonly studied, but many more are present in the soil [69]. As with chitinolytic bacteria, chitinase levels and activity are raised upon sensing chitin-containing material [70]. Trichoderma species are useful antagonists that utilise chitinases and other hydrolase enzymes against plant pests and pathogens and have now been developed into a number of biopesticide products [62]. The chitinases produced by Trichoderma are now known to be extremely antifungal and work on a wide range of fungal plant pathogens [71].
As a substantial body of evidence has built up to support the premise that incorporating chitin and its derivatives enhances the efficacy of natural biological control agents (both bacterial and fungal) [10,72], a number of commercial products have been developed that supply antagonistic microbe strains supplemented with chitin or encapsulated within a chitinous matrix [72,73]. The use of chitin/chitosan to encapsulate microbes also assists with the practicalities of storing and applying microbes on farms and nurseries, which has been one of the major restriction to the use of biopesticides in recent times [74].
In addition to the control of fungal pathogens, chitinolytic bacteria and fungi have considerable potential for the biological control of animal pests, especially insects, mites and nematodes. Of these, the effects of chitinolytic microbes on insects are the best-studied and have been developed as biopesticides. Entomopathogenic fungi, overcome the physical barrier presented by the insectâs exoskeleton and gut lining by producing multiple extracellular enzymes, including chitinases, which aid cuticular penetration and subsequent infection [75,76]. As a result, a number of chitinase producing entomopathogenic fungi, such as Beauveria bassiana, have been developed into biopesticides that successfully control a range of invertebrate pests [2].
The bacterium Bacillus thurigensis is the most widely used biopesticide worldwide (product name = Dipel DF) [62]. B. thuringiensis produces the insecticidal Cry-protein toxin. When plant tissue treated with the bacterium is consumed by an insect pest, the Cry-protein is activated by the alkaline conditions in its gut [77]. While the primary mode of action of B. thuringiensis is not via chitinase activity, the bacterium can utilize chitin as a source of carbon [78]. In addition, Ortiz-RodrĂguez et al. [79] showed that B. thuringiensis does produce an endochitinase, ChiA74, which when expressed in Escherichia coli growing on a chitin-rich media was able to generate chitin-derived oligosaccharides with antibacterial activity against food-borne human pathogenic bacteria. This indicates that chitin can both be used to stimulate the growth of beneficial bacteria with functions other than just degrading chitinous organisms, and that if chitin can be used to upregulate chitinases in a variety of bacteria used for the control of insects (via Cry-protein toxins), they could also potentially be used to control pathogenic microbes (via chitinase activity). In addition, specific
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strains of B. thuringiensis have been found to produce a chitin-binding protein that both potentiates the insecticidal activity of the Cry-proteins and is directly fungistatic [78].
The caterpillars of the spruce budworm moth (Choristoneura fumiferana) died more rapidly when exposed to a mixture of chitinase and B. thurigensis than when exposed to either the enzyme or bacterium alone [80]. These findings substantiate the previously stated hypothesis that chitinases can assist the penetration of entomopathogenic bacteria which then use other methods of killing their host. As enzymes are relatively expensive to produce and apply as agrochemicals on farms, the organism that they were isolated from in the above study, the entomopathogenic fungus Beauveria bassiana could be used in concert with B. thurigensis to increase the effectiveness of bioinsecticide preparations. This synergistic approach of applying both microbial species has already proved successful experimentally for the control of two major beetle pests; the spotted asparagus beetle (Crioceris quatuordecimpunctata) [81] and the Colarado potato beetle (Leptinotarsa decemlineata) [82].
In addition to promoting bacterial growth, and stimulating the activation of chitinase enzymes, chitin addition has also been shown to have other beneficial effects on rhizobacteria. It was shown by Lo Scrudato and Blokesch [83] that the presence of chitin in the growing media of bacterium Vibro cloerae induced horizontal gene transfer (natural competence) where DNA was absorbed and recombined into the chitinolytic bacterium. Horizontal gene transfer allows for quick adaptation to changes in growing conditions with the bacteria being naturally genetically transformed. Another potential mechanism by which chitin aids the action of beneficial bacteria is by disrupting the formation of biofilms produced by pathogenic microbes [84]. Such biofilms are increasingly being found to be important regulators of pathogenicity and involve quorum sensing of a diverse range of different species [85]. Therefore, if chitin biopolymers disrupt pathogenic film formation and favour the generation of beneficial microbial ones, it could aid plant health.
Entomopathogenic baculoviruses have also been found to utilize chitinases [60] to aid their penetration of their host [86]. It has also been shown that if viruses are transformed with foreign chitinase genes it can increase their virulence [87]. This work holds promise for increasing the effectiveness of baculoviruses when used as biopesticides. The use of baculoviruses is a relatively minor area of pest control in agriculture at present, but is forecast to increase dramatically as insecticides are withdrawn or replaced with viral products which possess greater specificity to pest species [88]. However, unlike the culturing, activation and delivery of chitinolytic microbe biopesticides, viruses cannot be grown on purified chitin and need a living organism for their multiplication.
3.2. Chitin as a Signalling Molecule for Growth-Promoting Microbes
It is well known that a mutualistic symbiotic relationship exists between legume plants and Rhizobium bacteria present in specialised root nodules. However, root nodule formation only occurs after the symbiotic partners exchange specific signalling molecules; flavonoids from the legume stimulating the production of chitin-based âNodâ factors from the bacterium. After successful recognition, there is a series of events that results in nodule formation by the plant and the supply of assimilates to the bacterium, which in turn fix atmospheric nitrogen into a form utilisable by plants.
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The Nod factors produced by Rhizobium bacteria are classified as lipochitooligosaccharides (LCOs), which are composed of an acylated chitin oligomer backbone with various functional group substituted onto the terminal or non-terminal residues. The number of N-acetylglucosamine monomers in a nod factor varies between species; however, generally it is 3 to 5 monomers in length [89]. The exact chemical structure of the Nod factors is thought to vary between bacterial species and strains in order that there is host-symbiont specificity. Staehelin et al. [89] demonstrated that the addition of short-chain acetylated chitin derivatives with structural similarity to Nod factors can induce nodulation in Medicago sativa. However, this study also showed that there needs to be a fair degree of biochemical similarity between the chitin-derivative applied and the Nod factors excreted by Rhizobium sp. This would need to be taken into account when trying to improve nitrogen fixation in legume crops by applying chitin derivatives. Actinorhizal plants, such as alders (Alnus), also possess a symbiotic relationship with a bacterium that fixes nitrogen in their roots. Unlike legume plants, the nitrogen-fixing bacterium associated with actinorhizal plants is not a Rhizobium species, but actinobacteria in the genus Frankia [90]. No Nod factor genes have yet been found in the Frankia genome [91]. However, it is thought that the signaling compounds produced by bacteria are biochemically similar to Rhizobium Nod factors, but they have not yet been confirmed as lipochitooligosaccharides [92]; therefore, the involvement of chitinous compounds in these symbiotic relationships remains uncertain.
3.3. Chitinâs Interaction with Mycorrhizal Fungi
Considering that an estimated 90% of plant species form mycorrhizal connections with fungi [93], there is currently a dearth of published data on the effect of chitin on mycorrhizal fungi. Lowe et al. [65] found that chitosan addition amplified the benefits of mycorrhizal inoculation in strawberry with Glomus sp.; specifically increased growth, fruit yield and a delay of the onset of powdery mildew. Gryndler et al. [94] found that chitin addition to a soil-based growing media promoted the growth of Glomus claroideum mycelium and its colonization of the roots of a number of plant species. However, this is countered by two reports that chitin addition inhibited the growth of mycorrhizae-infected sorghum and broad bean plants [95,96].
Recently it was found that mycorrhizal fungi in the Glomus genus secrete lipochitooligosaccharides which stimulate the formation of root connections in plant species belonging to diverse families [97]. At least three genes in Medicago truncatula that are stimulated by Nod factors are also involved in the formation of an arbuscular mycorrhizal symbiosis [98]. The fact that the genes that are essential to the response to chitinous Nod factors by legumes are also present and functional in non-legumes [99] also suggests that lipochitooligosaccharides could play a wider role in plant-microbe interactions and plant development, and not just nodule formation. This indicates that both rhizobial and mycorrhizal symbioses may share some common mechanisms and hints at the existence of a chitin-based âMyc factorâ.
It is also thought that, in addition to Nod/Myc factors and flavonoids, plant chitinases also play a key role in the recognition and formation of connection with mycorrhizal fungi and nitrogen-fixing bacteria [100]. In Medicago truncatula different sets of chitinase genes are expressed depending upon
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whether it is a mycorrhizal fungus, nodulating bacterium, or a pathogen being interacted with [100]. It could therefore be that chitin-based products make the plant super-sensitized to the presence of fungi.