Resch et al. Instead, the analysis identified two regions that displayed high amino acid diversity in combination with positively selected sites in Streptomyces, but not in Trichoderma (A and B), and one region of high sequence diversity in the fungal orthologs only (C) (Fig. does have an effect on its mycoparasitic abilities and underlines the yet unexplored potential of the chitin machinery in Trichoderma. The analyses show that the numbers of subgroup B chitinases in the mycoparasites T. atroviride and T. virens, but not in the saprotrophic T. reesei, are significantly higher than what would be expected from a random evolutionary process (Fig. Proteases and chitinases were analyzed in order to establish a screening method for identification of high virulent strains (Sun et al., 2013). Mycoparasitism depends on a combination of events that include lysis of the preys' cell wall (Harman etal., 2004; Howell, 2003; Lorito etal., 2010). 5.5). (2011) studied the effect of glycerol, an osmoticant, on the shelf life of T. harzianum. Iqbal, in International Journal of Biological Macromolecules, 2021. Yedidia etal. (2011) identified a plant-like sucrose transporter (TvSut) in the genome of T. virens, suggesting that at least in Trichoderma, there is active sucrose transference from the plant to the fungal cells during their beneficial associations. Dubbed as a multifunctional endophytic plant symbionts (Shoresh etal., 2010; Harman, 2011a,b), some Trichoderma species colonize plants roots and effect multiple benefits to their host plants. Another class of genes of relevance to mycoparasitism are those involved in the formation of secondary metabolites. Biofuels are eco-sustainable and possess high-energy proficiency than that to fossil-based fuels. FIGURE 5.7. (2010) supplemented commercial Trichoderma and powder formulations with NPK which improved growth response but with no reduction in root disease symptoms of specific apple replant disease (Kandula et al., 2010). Many authors report increases in stress and/or defense-related enzyme activities such as phenylalanine ammonia-lyase, peroxidase, lipooxygenase, polyphenoloxidase, -1,3-glucanase, chitinase, and other defense-related genes, as well as induction of specific PRs in leaves of plants of which the roots were colonized by biocontrol Trichoderma strains (Shoresh etal., 2005; Gallou etal., 2009; Shoresh etal., 2010; Tucci etal., 2011; Yoshioka etal., 2012). Shanmugam et al. inoculation to be quantified similar to the case of rhizobacteria (van Loon, 2007). The second ENGase (Eng18B) does not contain a signal peptide and is therefore an intracellular protein, presumably involved in the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins. However, contradictory results have also been obtained: under the same testing conditions Penicillium oxalicum secretes more cellulases than Aspergillus niger and Trichoderma reesei (Glass et al., 2013), but Aspergillus niger secretes more enzymes for xyloglucan conversion than do Penicillium oxalicum and Trichoderma reesei (Gong et al., 2015). Initial genome analysis studies in Trichoderma species was carried out over 20 years ago using electrophoretic karyotyping. ), increases total absorptive surface of the roots, thereby facilitating nutrient uptake resulting to increased plant growth (Contreras-Cornejo etal., 2009; Samolski etal., 2012). (2018) characterized a ~44kDa chitinase from Aspergillus niveus works well at 65C and pH 5.0. Both of these chitinases are lacking CBMs. Interestingly, genes encoding fungal specific Zn(2)Cys(6) transcription factors were also among the most abundant types within the 1273 shared by the two mycoparasites, which may be involved in the regulation of gene expression related to mycoparasitism and/or interaction with the plant. The addition of glycerol in the production medium at 3% and 6% extended shelf-life prolonged the shelf-life of talc formulation to 7 and 12 months, respectively, compared to 45 months in formulations without glycerol. Gene expression of chit36 (chi18-15) is induced by various different stimuli including growth on chitin and fungal cell walls, mycoparasitism and starvation (Viterbo etal., 2002). (2015) found that Aspergillus niger secreted more biomass-degrading enzymes than did Trichoderma reesei under the same testing conditions. The molecular mechanisms that govern the recognition and association between Trichoderma and their hosts are still largely unknown. The depolymerization and elimination of lignin from lignocellulosic biomass is a requisite step before transforming into biofuels by fermentation technology [155]. The successful colonization of Trichoderma strains of its hosts' roots imply a reprogramming of the plant which is often beneficial; improving growth, yield and resistance to pathogens (Mukherjee etal., 2012b). Besides PKS and NRPS, T. atroviride and T. virens have further augmented their antibiotic arsenal with genes encoding cytolytic peptides such as aegerolysins, pore forming cytolysins typically present in bacteria, fungi and plants, yeast-like killer toxins and cyanovirins (Kubicek etal., 2011). (1993) found that T. harzianum and Trichoderma viride had six chromosomal DNA bands varying in size from 2.2Mb to 7.7Mb, with the total genome sizes estimated to range from 31Mb to 39Mb. Theoretically, several synergistically acting endochitinase isozymes may be equally advantageous for degradation of dead fungal biomass as for the mycoparasitic attack. [157] immobilized laccase from Trichoderma asperellum onto Fe3O4@SiO2-chitosan nanocarrier support for lignocellulosic biomass delignification and then exploited for biohydrogen synthesis. Subsequently, MYB72 putatively interacts with the transcription factor EIL3. Even when coinoculated with other beneficial microorganism like the AMF, Trichoderma spp. Bernal-Vicente et al. (2009) demonstrated that an intracellular invertase from Trichoderma virens (TvInv) is responsible for sucrose hydrolysis and normal growth of T. virens in the presence of sucrose. Trichoderma spp. Signaling between plant roots and soil microorganisms like the Trichoderma spp. Trichoderma-induced resistance is generally visually manifested as reduced disease symptom expression. A. phoenicis and Paecilomyces variotii. Saskia CM Van Wees, Corn MJ Pieterse, in Current Opinion in Plant Biology, 2008. The sequenced T. reesei strain shows a saprotrophic lifestyle on decaying wood, so the analysis of newly sequenced Trichoderma genomes (already Trichoderma longibrachiatum, Trichoderma citrinoviride, Trichoderma asperellum and T. harzianum are available at the Mycocosm portal of DOE JGI website) will determine if the selection for high numbers of subgroup B endochitinases is specific for mycoparasitism or if this also applies to the mycotrophic lifestyle. ISR inducibility by Trichoderma was also tested with 36 phytohormone-affected mutants of A. thaliana. It is known that the addition of a CBM increases the adherence of an enzyme to insoluble substrates because the enzyme does not dissociate from the substrate after successful cleavage, which would normally be the case for proteins with shallow substrates binding site as subgroup B endochitinases. The population peaked at 144h and the formulation was used to treat fruits infected with black rot disease caused by Thielaviopsis paradoxa. 5.6), from which two were predicted to influence the catalytic cleft structure of the enzyme (Ihrmark etal., 2010). B. haptosporus NFCCI 1922 hydrolyzed the mycelia of tested fungal isolates as a major carbon source to produce chitinase. Evolutionary analysis of Chit36 and ChiJ orthologs. It is furthermore necessary to determine whether the high sequence diversity between orthologs is the result from low selective constraint, i.e. In cucumber increased chitinase (PR3), -1,3-glucanase (PR2), cellulase, and peroxidase activities were detected locally and systemically in roots and leaves of Trichoderma harzianum T-203-treated plants in comparison to nontreated ones (Yedidia etal., 1999, 2000). By using reverse genetic experiments, Vargas etal. The invert emulsion (water-in-oil type) formulation of T. harzianum prolonged the postharvest shelf life of the fruit (Batta, 2007). Disruption mutants of Eng18B in T. atroviride show defects in vegetative growth, tolerance to abiotic stress, conidiation, chitin utilization and mycoparasitism of Botrytis cinerea (Dubey etal., 2012). We use cookies to help provide and enhance our service and tailor content and ads. showed that signs of accelerated evolution were not directly located in the catalytic cleft (Ubhayasekera and Karlsson, 2012), which suggests that the primary substrate for Chit36 and ChiJ is of similar composition, presumably fungal cell wall material. Comparative genome analysis of T. atroviride, T. virens, and T. reesei, revealed that these three Trichoderma species display a remarkable conservation of gene order (7896%), and a lack of active mobile elements (transposons). Chit36 (Chi18-15) is a chitinase that was acquired through horizontal gene transfer from an actinobacterial origin by an ancestor of the genus Trichoderma and can also be attributed to subgroup B chitinases (Karlsson and Stenlid, 2009). Another type of GH family 18 proteins that are phylogenetically associated with subgroup B is the subgroup B5 ENGases, which have only recently been described in fungi. The secretory strategies of lignocellulose degrading enzymes that are used by various microorganisms, such as those by Ganoderma lucidum (Manavalan et al., 2012) and Fusarium graminearum (Ji et al., 2013), can vary greatly. However, as outlined by Lee (2008), residues important for enzyme properties may be expected to display higher diversity than other positions in closely related orthologs due to selection for modified enzymatic properties between species. (2015) and Gong et al. Adav et al. Canola root growth is regulated by an ACC deaminase (similar to ACCDs of PGPR) encoded by an ACCD gene from T. asperellum T203 (Viterbo etal., 2010). It was shown that Trichoderma induces the octadecanoic pathway and the synthesis of JA immediately after root colonization. An evolutionary analysis of Chit36 and its bacterial ortholog ChiJ from Streptomyces spp. and their gene or gene products are found to induce resistance to a wide range of plant species (Table 35.1), from monocots to dicots and even trees. Attack by pathogens or insects, as depicted on the right side of the figure, activates defense responses in the plant (yellow arrows), which is accelerated in ISR-primed plants (combined blue and yellow arrows). (1999), "Species 2000 & ITIS Catalogue of Life: 2011 Annual Checklist", https://en.wikipedia.org/w/index.php?title=Trichoderma_asperellum&oldid=1049477266, Creative Commons Attribution-ShareAlike License 3.0, This page was last edited on 12 October 2021, at 01:59. lachrymans (Psl) by induction of systemic resistance, since Trichoderma did not antagonize Psl in dual cultures. Irpex lacteus, Phanerochaete chrysosporium secreted more enzymes related to polysaccharides hydrolysis, Pleurotus ostreatus produced more oxidoreductases. As compared to the free enzyme, the immobilized biocatalyst showed high potential for lignin removal in sweet sorghum Stover and recycled in eight repeated cycles. 5.4). is often based on root-derived chemicals. (2016) performed comparative secretome analyses of Penicillium echinulatum wild-type 2HH and its mutant strain S1M29, and found that the latter was more efficient than the former in producing enzymes that participate in cellulose and hemicellulose degradation. Trichoderma spp. Gene expression of ech30 was found to be elevated during growth on fungal cell walls and in mycoparasitism assays (Seidl etal., 2005), which indicates that the function of Ech30 is indeed degradation of fungal cell wall chitin. With respect to these, the three Trichoderma spp. Gains and losses of chitinase genes in Trichoderma. Phylogenetic analysis of the available genome sequence data indicates that the powerful antagonists of other fungi, T. atroviride and T. asperellum, are ancestral species, suggesting that mycoparasitism was the ancestral life style of the genus (Kubicek etal., 2011). In conclusion, the immobilized laccase could be effectively used for lignin valorization in multiple cycles. Recent advances in molecular and genetic techniques afforded us some notion unto the attraction, attachment, penetration, and internal colonization of Trichoderma spp. The respective fungal strains overexpressing the hybrid chitinases also displayed increased mycoparasitic activities in comparison to strains overexpressing the native chitinases (Limon etal., 2004, 2001). (B.) Muhammad Bilal, Hafiz M.N. Once inside the roots, Trichoderma spp. (For color version of this figure, the reader is referred to the online version of this book.). Chitinase synthesizing fungi (Conidiobolus coronatus NFCCI 1235, C. couchii NFCCI 719, C. coronatus NFCCI 718, Basidiobolus haptosporus NFCCI, 1922 and Basidiobolus haptosporus NFCCI, 1923) were isolated from the two different genera of fungus, Conidiobolus and Basidiobolus. Christian Joseph R. Cumagun, in Biotechnology and Biology of Trichoderma, 2014. Amino acid diversity of Trichoderma Ech30 orthologs are estimated using Rate4Site, based on a Clustal X alignment. The application of alginate formulation and paraffin oil in Trichoderma increased its shelf life (Al-Taweil et al., 2010). The reason for improved performance is the sustained colonization of T. harzianum SQR-T037 in the rhizosphere soil (Yang et al., 2011). Biochemical analysis have revealed local or systemic accumulation of phytoalexins, phenolic compounds, terpenoids, superoxides, or lignifications in plants inoculated by Trichoderma prior to subsequent pathogen inoculation (Ahmed etal., 2000; Howell etal., 2000; Koike etal., 2001; Yedidia etal., 2003). Dried conidial pellets of T. harzianum were more effective antagonist formulation than liquid formulation in inhibiting sclerotial germination of Rhizoctonia solani (Cumagun and Ilag, 1998). Hongliang Guo, Duu-Jong Lee, in Bioresource Technology, 2018. The relatively low number of subgroup B chitinases in T. reesei is a consequence of the switch from mycoparasitism to a saprotrophic lifestyle (Kubicek etal., 2011), which is accompanied by a loss of endochitinase genes. With respect to secretion by mutants, Schneider et al. (For color version of this figure, the reader is referred to the online version of this book.). Also, it was stable in many organic and inorganic compounds. Trichoderma spp. This type of coevolutionary interactions may leave an imprint on the selective signature of the participating enzymes, and the previously mentioned Ech30 is one possible example of this. Amino acid diversity of Trichoderma Chit36 and Streptomyces ChiJ orthologs are estimated using Rate4Site, based on a Clustal X alignment. Structural modeling showed that this is due to subtle differences in the substrate binding cleft in comparison to the well characterized plant chitinase hevamine, resulting from small insertions and deletions in loops on the noncatalytic side of the TIM barrel. Copyright 2022 Elsevier B.V. or its licensors or contributors. With respect to the quantity and quality of secretion by a pure culture, Borin et al. But prior to stimulating defense responses in its host plants, Trichoderma spp. Later, the T. atroviride sequenced strain (IMI 206040) was estimated to have a genome size of 39.1Mb (Gmez etal., 1997). Further studies on the induction of induced resistance in cucumber by T. asperellum T-203 extended previous investigations by testing gene expression of more components involved in the different defense response pathways. Systemically, induction of ISR is associated with priming for enhanced expression of a set of JA-responsive and/or ET-responsive genes and increased formation of callose-containing papillae at the site of attempted pathogen entry. (2015). The ISR signal transduction cascade requires NPR1, probably in the systemic tissue. Phanerochaete chrysosporium secretes more enzymes that are related to the hydrolysis of polysaccharides than does Irpex lacteus whereas Pleurotus ostreatus produces more oxidoreductases than did Irpex lacteus (Salvacha et al., 2013). facilitate root colonization of their hosts by the production and regulation of hormonal signals. Colonization of the plant root system by Trichoderma spp. Shotgun sequencing of the T. reesei genome resulted in a slightly larger estimate, indicating a total genome size of approximately 34.1Mb, containing 9129 protein-encoding genes (Martinez etal., 2008). Evolutionary analysis of Ech30 orthologs. Samuels, G.J. Half of the secondary metabolite gene clusters present in T. virens and T. atroviride are not present in T. reesi, but all those present in the latter were also found in the mycoparasites (Kubicek etal., 2011). It reduced the benzyl phenyl ether content from 95.90% to 75.63% after 48h agitation in a shaker at 25C. As the main product of photosynthesis, sucrose acts as an important molecule in carbohydrate-mediated signaling in plants and degraded by plant cells to yield a carbon source for microbes during plantmicrobe associations (Koch, 2004). Sriram et al. T. versicolor secreted high levels of laccase, Highlighted differences of strategies utilizing hydrolytic enzymes to degrade cellulose among two different lignocellulolytic microorganisms, Highlighted specially expressed proteins among lignocellulolytic strains, Penicillium echinulatum wild-type was more efficient in production of enzymes involved in cellulose and hemicellulose degradation. Shoresh etal. The addition of organic fertilizer enhanced the performance of T. harzianum SQR-T037 as compared to conidial suspension alone in controlling Fusarium wilt of cucumbers (Yang et al., 2011). Trichoderma virens exhibited the highest number (50) of PKS, NRPS and PKSNRPS fusion genes, mainly due to the abundance of NRPS genes. Plant lignocellulose degradation by S. commune involves a hydroxyl radical-mediated mechanism for lignocellulose modifcation in parallel with the synergistic system of various polysaccharide-degrading enzymes. Yet, T. virens and T. atroviride contain about 2756 and 2510 genes, respectively, which have no true ortholog in any of the other species. Trichoderma genomes contain two genes encoding ENGases, of which one (Eng18A, Endo T) is secreted and may thus be responsible for postsecretorial modifications of glycan structures on endogenous glycoproteins such as cellulases, as has been reported for T. reesei (Stals etal., 2010). By continuing you agree to the use of cookies. trichotoxins in Trichoderma asperellum, and trichostromaticins in Trichoderma stromaticum. Ecotype Col-0 was found to be more ISR-inducible than Landsberg, Nossen or Wassilewskija. Although priming with BABA and with avirulent bacteria leads to the same resistance phenotype, their impact on the plant's metabolome partially differs [5]. This suggests that T. atroviride and T. virens may contain an as yet undiscovered reservoir of secondary metabolites, which may contribute to their success as mycoparasites (Kubicek etal., 2011). (2013) also identified differences between the strategies of Hypocrea jecorina and Clostridium thermocellum for using hydrolytic enzymes to degrade cellulose. The Trichoderma subgroup B chitinases Ech30 (Chi18-13) and Chit33 have already been biochemically characterized (Boer etal., 2007; Hoell etal., 2005; Lienemann etal., 2009). Phylogenetic relationships suggest a single neofunctionalization event that resulted in evolution of enzymes with ENGase activity from a chitinase ancestor (Karlsson and Stenlid, 2009). GDFS1009 synthesized the chitinase, glucanase, and protease, which can hydrolyzed the fungal cell walls. TABLE 33.1. In comparison, the mycoparasitic species possess genomes that range between 33.48Mb and 40.98Mb (Table 33.1). Microorganisms seem to exhibit no universal secretory strategy for lignocellulolytic enzymes (Table 2). Phylogenetic relationships among fungal species are shown, including estimated divergence dates in millions of years. Microscopic techniques such as confocal, epiflourescence, and transmission or scanning electron microscopy function as supporting tools which allow the monitoring of pathogen development in plant cells, and histochemical studies like the visualization of ROS, callose and other determinants of induced resistance (Yedidia etal., 2000, 2003; Gallou etal., 2009; Salas-Marina etal., 2011; Palmieri etal., 2012). Model for the ISR signaling pathway. Therefore, white-rot fungi and brown-rot fungi should have different strategies for using hydrolytic enzymes to degrade cellulose (Hori et al., 2013). From: Biotechnology and Biology of Trichoderma, 2014, Barbara Reithner, Robert L. Mach, in Biotechnology and Biology of Trichoderma, 2014. However, compared with non-primed plants, T. asperellum-inoculated plants that suffered subsequent infection by Pst showed major differences in defence gene expression patterns [17]. The secretome of Trichoderma asperellum has been identified as containing more diverse hemicellulases and -glucosidases than that of Trichoderma reesei (Marx et al., 2013). As already mentioned, the genome sequence also revealed that 1273 orthologous genes are shared between T. atroviride and T. virens but absent from T. reesei. Analyses of plant genotypes, particularly mutants (disrupted, overexpressing or knock out mutants) and transgenics that either do not accumulate or do not respond to SA, JA, ET and other defense-related pathways help elucidate the signaling molecules essential for basal resistance to varying pathogens (Korolev etal., 2008; Salas-Marina etal., 2011; Malmierca etal., 2012). Ech30 is a small chitinase (30kDa) but has as many as seven subsites for sugar binding. (2015) and Gong et al. must suppress or tolerate plant defense mechanisms in order to facilitate root invasion. (2013) noted the differences among the secretomes of seven polyporales that are grown under identical conditions, including those of white-rot fungi (Bjerkandera adusta, Ganoderma spp, Phlebia brevispora, Dichomitus squalens and Trametes versicolor) and brown-rot fungi (Fomitopsis pinicola and Wolfiporia cocos). Table 2. Artwork: Wouter Boog. FIGURE 5.5. In general, no conclusions can be drawn from the comparative studies stated above on how different strains would select their strategies to secrete lignocellulose-degrading enzymes. Based on the versatility and importance of Trichoderma species in ecosystem health, it is not surprising that recently the JGI decided to get involved in sequencing projects targeting seven of the over 200 species of the genus (Kubicek and Druzhinina, 2013), namely, Trichoderma reesei, Trichoderma virens, Trichoderma atroviride, Trichoderma harzianum, Trichoderma asperellum, Trichoderma longibrachiatum and Trichoderma citrinoviride (http://genome.jgi-psf.org/programs/fungi). Kandula et al. However, enzymes involved in other parasitehost interactions sometimes evolve rapidly in response to a coevolutionary arms race, resulting in continuous selection for adaptive modifications. All subgroup B chitinases have a signal peptide at their N-terminus and are therefore targeted to the secretory pathway. (2011) incubated the biocontrol agent in yeast waste residue medium. The most important molecular characteristics are divergent ITS-1 and 28S sequences and RFLP's of the endochitinase gene. These genes include, solute transporters, and several putative oxidoreductases and monooxygenases that may be involved in AMP-activation of acids and phosphopathetheine attachment, and synthesis of isoquinoline alkaloids. equally utilize a vast array of proteins to facilitate root colonization. FIGURE 5.6. Comparative proteomic analyses for secretomes among functional strains. 5.7). Boxed numbers represent numbers of subgroup A, B (excluding ENGase) and C chitinase genes in extant species and estimated equivalents in ancestral species. Different mutants of the same bacterial strain, such as Trichoderma reesei mutant Rut C30 (Couturier et al., 2012) and mutant CL847 (Jourdier et al., 2013), can also differ greatly. Verena Seidl-Seiboth, Magnus Karlsson, in Biotechnology and Biology of Trichoderma, 2014. Genes from Trichoderma as a Source for Improving Plant Resistance to Fungal Pathogen, Advances in Formulation of Trichoderma for Biocontrol, Genome-Wide Approaches toward Understanding Mycotrophic Trichoderma Species. In addition, the rampant population growth, and food versus fuel discussion, has diverted the emphasis on the synthesis of biofuel from non-edible feedstock's [153,154]. The biocontrol ability in cucumber was associated with the accumulation of phytoalexins. Thangavelu et al. (2012a) reached the same conclusion based on their comparative analysis of secretomes for Trichoderma reesei and its mutant Rut C30. The development of molecularbiological techniques has allowed the reaction of plants to Trichoderma inoculations to be determined at the transcriptional level by analyzing differential gene expression by a variety of techniques (Alfano etal., 2007; Segarra etal., 2007; Shoresh and Harman, 2008a; Bae etal., 2011; Contreras-Cornejo etal., 2011; Mathys etal., 2012; Palmieri etal., 2012). In contrast to the free enzyme, the catalytic products of MnPs-immobilized laccase have a 6-fold increase in 2-styrylphenol, 4-fold in phenol, and 2-phenyloxirane, and 1.5-fold enhancement in other products. While in A. fumigatus a fivefold knockout strain of subgroup B chitinases, including a GPI-anchor containing ortholog of A. nidulans ChiA, did not show any phenotypic defects with respect to germination and hyphal growth (Alcazar-Fuoli etal., 2011), deletion of the Neurospora crassa ortholog chit-1 gene resulted in a reduced growth rate compared with the wild type strain (Tzelepis etal., 2012). Their expression experiments suggest the existence of a sucrose-dependent network in the fungal cells that regulates the symbiotic association between Trichoderma and its host plants. Identifications of lignocellulolytic enzymes secreted by G. lucidum, 87 proteins were identified, 63 were secretory proteins in F. graminearum, Highlighted 51 variable secreted proteins among six wild-type strains with different host range grown in liquid minimal medium, Highlighted lignocellulose degradation mechanism by filamentous fungi, and indicated each of 20 fungi in test could act as a supplement for industrial T. reesei enzymatic cocktail to improve sugar release, Sugar substrate composition strongly influenced composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode, Under same condition, A. niger secreted more enzymes (quantitatively and qualitatively) related to biomass degradation than T. reesei, A. niger secreted more enzymes in degradation of (galacto) mannan and xyloglucan, Secretome of T. asperellum contained high diversity of main and side chain hemicellulases and -glucosidases, and an increased abundance of some of these proteins compared with T. reesei, Highlighted importance of low abundant auxiliary proteins for efficient lignocellulose degradation, and differential expressions of pectin-degrading proteins, peptidase and proteases in different substrates, Highlighted new approach for improving cellulose accessibility in biofuel feedstocks, fresh material and cell wall residues from different plants.