Posted on 2006-05-31, sourse: Phd Thesis
Molecular and physiological investigations of biocontrol by the genus Hypocrea/Trichoderma. Selected chapters of the Phd Thesis
The genus Hypocrea/Trichoderma
The anamorphic fungal genus Trichoderma (Hypocreales, Ascomycota) is cosmopolitan in soils and on decaying wood and other forms of plant organic matter (Samuels 1996; Klein and Eveleigh 1998). Trichoderma species are among the most widely distributed and common fungi in nature and exist in climates ranging from the tundra to the tropics. This may be attributable to their diverse metabolic capability and aggressively competitive nature (Samuels 1996; Klein and Eveleigh 1998).
Rapid growth rates in culture and the production of numerous spores (conidia) that are mostly varying shades of green characterize fungi in this genus. A growing number of teleomorphs in Hypocrea have been linked to commonly occurring Trichoderma anamorphs, but most strains of Trichoderma are classified as imperfect fungi because they have not been associated with a sexual state (Gams and Bissett 1998). The taxonomy of Hypocrea/Trichoderma is rather difficult and complex due to the plasticity of characters if classical approaches, based on morphological criteria, are applied. The use of molecular phylogenetic markers has refined Hypocrea/Trichoderma taxonomy significantly and phylogenetic analysis of the large number of Hypocrea/Trichoderma spp. is still a field of active ongoing research (Druzhinina and Kubicek 2005).
Hypocrea/Trichoderma spp. have a number of remarkable mechanisms for survival and proliferation including physical attack of other fungi and degradation and utilization of complex carbohydrates. For the most part they are beneficial to man?s economic interests and are used for commercial applications. Hypocrea jecorina (= Trichoderma reesei) is an economically important producer of cellulases and hemicellulases and is used for heterologous protein expression (Kubicek and Penttilń 1998). H. lixii (= T. harzianum),H. atroviridis (= T. atroviride) and T. asperellum are applied as biocontrol agents against plant pathogenic fungi (Hjeljord and Tronsmo 1998) for a wide variety of crops and climates. However, there are also negative effects of Trichderma: due to their high cellulolytic potential they degrade cotton fabrics, strains of T. aggressivum are pathogenic on commercial mushrooms like Agaricus and Pleurotus (Seaby 1998), and more recently T. longibrachiatum was reported to be an opportunistic pathogen of immunocompromised mammals including humans (Kredics et al. 2003).
The reason for plant disease epidemics is that modern agriculture is an ecologically unbalanced system, which is based on growing one or a few crop cultivars on large areas. Prevention of such epidemics has so far been mainly achieved through use of chemical fungicides, but farmers are increasingly often confronted with pathogens resistant to available chemical plant protectants. Additionally, consumers are becoming gradually more concerned about chemical pollution of the environment and pesticide residues in food and there is an increasing demand for products coming from sustainable agriculture and eco-farming. Thus both, customers and industry, are highly interested in finding alternative methods of disease control.
Replacement or reduction of chemical applications has been achieved through use of biologically based pesticides, a concept included in the broad definition of biocontrol proposed by Cook and Baker (1983): ?Biological control is the reduction of the amount of inoculum or disease-producing activity of a pathogen accomplished by or through one or more organisms other than man.? This broad definition includes use of less virulent variants of the pathogen, more resistant cultivars of the host and microbial antagonists ?that interfere with the survival or disease producing activities of the pathogen?.
The advantages of biological pest management are the safety of handling, the self perpetuation and therefore a less frequent need of application and a high degree of host specificity. On the other hand biological control agents are, like any other organism, affected by abiotic and biotic factors such as weather, disease pressure and competition from the indigenous microflora. Chemical pesticides are less prone to such influences and thus the inconsistent performance of biocontrol agents is a major disadvantage of biological pest management.
In greenhouse systems environmental conditions such as temperature and relative humidity are tightly controlled. The high economic value of greenhouse crops can absorb higher inputs to control disease. Because of the reduced area and high density of planting, less inoculum is needed than in treating a field. Additionally, the continuous harvesting of many crops, which puts the workers at greater risk of fungicide exposure, makes the application of many commercial fungicides infeasible. Therefore, biological control of plant diseases in greenhouses is a unique niche and attractive alternative to chemical pesticides. Biological control of greenhouse insects is already the predominant method in e.g. the United Kingdom, but the application of biological fungicides is still a developing market (Paulitz and Belanger 2001).
A large area of interest in biocontrol is the reduction of plant diseases caused by soil-borne and foliar plant pathogenic fungi. Roughly 70% of all the major crop diseases are caused by fungi, or the fungus-like Oomycota (Deacon 1997). Notorious examples are species belonging to the genera Rhizoctonia, Botrytis, Phytophthora, Pythium, Sclerotinia and Fusarium. Most of the formulations of commercially available biocontrol products against plant pathogenic fungi contain the bacteria Pseudomonas and Bacillus or fungi belonging to the genus Hypocrea/Trichoderma (Paulitz and Belanger 2001).
Biocontrol by Hypocrea/Trichoderma
The potential of Hypocrea/Trichoderma species as biocontrol agents of plant diseases was first recognized by Weindling in the early 1930s (Weindling 1934) who described the mycoparasitic action of Hypocrea/Trichoderma on Rhizoctonia and Sclerotinia and its beneficial effects for plant disease control. This has stimulated research on this topic and also the commercial use of several Hypocrea/Trichoderma species for the protection and growth enhancement of a number of crops. Commercially available formulations are e.g. RootShield? , BioTrek 22G? , T-22G? , T-22HB? (Bio-Works, USA), Supresivit? (Borregaard BioPlant, Denmark), Binab? (Bio-Innovation, Sweden), Trichopel? , Trichojet? , Trichodowels?, Trichoseal? (Agimm, New Zealand), Trieco? (Ecosense Labs, India), Gliomix? (Verdera Oy, Finland), Trichodex? (Makhteshim, Israel) SoilGuard? (Thermo Trilogy, USA) or Promot? (J.H. Biotech, USA). However, not all of these products are registered as biocontrol agents, but are marketed as plant growth promoters, plant strengtheners, or soil conditioners. These designations have enabled the products to get to the marketplace with less stringent toxicology or efficacy testing than would be required for plant protectants (Paulitz and Belanger 2001).
One of the most interesting aspects of the research field of biological control is the study of the mechanisms employed by biocontrol agents to accomplish disease control. Past research indicates that the mechanisms are many and varied, even within the genus Hypocrea/Trichoderma. To achieve an optimal application of Hypocrea/Trichoderma for the control of plant diseases during cultivation and storage, a detailed understanding of the biocontrol agents? modes of action and their limitations is essential.
Biocontrol mechanisms of Hypocrea/Trichoderma
Biocontrol by Hypocrea/Trichoderma results from different mechanisms acting synergistically to achieve disease control. Those involve the competition for nutrients and living space with plant pathogenic organisms, the direct attack and destruction of the pathogens (antagonism, mycoparasitism) and promotion of plant beneficial processes such as enhancement of plant growth and induction of systemic and localized resistance.
Competition for nutrients and living space:Hypocrea/Trichoderma spp. have a rapid growth rate, persistent conidia and a broad spectrum of substrate utilization which makes them very efficient in the competition for nutrients and living space (Hjeljord and Tronsmo 1998). Furthermore Hypocrea/Trichoderma spp. are able to produce and/or resist metabolites that either impede spore germination (fungistasis), kill the cells (antibiosis) or modify the rhizosphere, e.g. by acidifying the soil, so that pathogens cannot grow (Benitez et al. 2004). Starvation is the most common cause of death for microorganisms and competition has turned out to be especially important for the biocontrol of phytopathogens such as B. cinerea, the main pathogenic agent during pre- and post-harvest in many countries, which is particularly sensitive to the lack of nutrients (Benitez et al. 2004).
Attack and decomposition of the pathogens: The direct interaction between Hypocrea/Trichoderma spp. and the pathogen is called mycoparasitism. The events leading to mycoparasitism are complex and different between various species, but the mycoparasitic attack generally follows the same scheme: Hypocrea/Trichoderma strains detect other fungi and grow straightly towards them; remote sensing is at least partially responsible for the sequential expression of hydrolytic, cell wall-degrading enzymes (Cortes et al. 1998; Zeilinger et al. 1999; Kullnig et al. 2000). Once the fungi come into contact, Hypocrea/Trichoderma spp. attach to the host, coil around the host hyphae and form appressoria on the host surface (Inbar and Chet 1992; Rocha-Ramirez et al. 2002). Then the Hypocrea/Trichoderma spp. produce a range of fungitoxic, hydrolytic enzymes such as chitinases, glucanases and proteases and other toxic compounds and/or peptaibol antibiotics (Schirmbock et al. 1994; Lorito et al. 1996a; Szekeres et al. 2005). Penetration of the host cell wall is achieved by a synergistic action of the hydrolytic enzymes and antibiotics (Schirmbock et al. 1994; Lorito et al. 1996a; Kubicek et al. 2001; Szekeres et al. 2005). The invasive process is locally restricted to the sites of the appressoria, where holes are produced in the host cell wall, and direct entry ofHypocrea/Trichoderma hyphae into the lumen of the target fungus occurs (Inbar and Chet 1992).
Plant beneficial processes:Hypocrea/Trichoderma spp. can also exert positive effects on plants, which cause an increase in plant growth and root development (biofertilization) and stimulate plant-defense mechanisms (Harman et al. 2004). Some Hypocrea/Trichoderma strains (e.g. of the species H. atroviridis and H. lixii) were shown to establish robust and long-lasting colonizations of root surfaces and to penetrate into the epidermis. They are opportunistic, avirulent plant symbionts and produce a variety of compounds that induce plant defense mechanisms. Interestingly,Hypocrea/Trichoderma spp. are even able to induce systemic resistance, which is characterized by the occurrence of disease control in the plant at a site distant from the location of Hypocrea/Trichoderma. They stimulate the production of low-molecular weight compounds that have antimicrobial activity like e.g. phytoalexins which are normally produced by plants in response to an attack by pathogens. Additionally, proteome analysis of H. lixii identified homologues of the avirulence genes Avr4 and Avr9 from Cladosporium fulvum. The protein products of avirulence genes have been identified in a variety of fungal and bacterial plant pathogens. They usually function as race- or pathovar specific elicitors that are capable of inducing hypersensitive responses and other defense-related reactions in plant cultivars that contain the corresponding resistance gene (Harman et al. 2004).
The (hydro) lytic enzyme system ofHypocrea/Trichoderma
Hypocrea/Trichoderma spp. produce a wide range of enzymes for degradation of homo- and heterpolysaccharides, which are designative for their broad spectrum of substrate utilization and their ubiquitous occurrence in nature. Furthermore they possess a wide spectrum of proteases which help them in the defense of their habitats and the competition for nutrients with other microorganisms. Sequencing of the H. jecorina genome allowed a more detailed and extensive analysis of the genes coding for those enzymes and their regulation and revealed and even larger number of hydrolytic enzymes, such as e.g. 12 genes encoding cellulases, than previously suspected. With the currently ongoing genome sequencing of the mycoparasitic species H. atroviridis the study of lytic enzymes, but also other gene products like Avrs, in biocontrol will be more complete and greatly facilitated. However, even without a sequenced genome a wide variety of hydrolytic enzymes were already cloned from various mycoparasitic Hypocrea/Trichoderma spp., their enzymes characterized and their regulation studied.
Chitin, the (1-4)-b-linked homopolymer of N-acetyl-D-glucosamine, is one of the most abundant polymers in the biosphere, and chitinolytic enzymes are found among all kingdoms, e.g., protista, bacteria, fungi, plants, invertebrates and vertebrates, including humans (Cabib 1987; Gooday 1990; Sahai and Manocha 1993). Enzymatic degradation of chitin is generally involved in many biological processes, such as autolysis (Vessey and Pegg 1972), morphogenesis and nutrition (Griffin 1994) and plays in addition to mycoparasitism also a role in relationships between fungi and other organisms such as plant-fungus and insect-fungus interactions (St. Leger et al. 1987; Mauch et al. 1988).
Chitinolytic enzymes can be divided into exo- and endo-acting enzymes based on their reaction end products and catalytic mechanism.
b-N-acetylglucosaminidases (NAGases, EC 18.104.22.168) catalyze the hydrolysis of terminal non reducing N-acetyl-D-glucosamine (GlcNAc) residues. The tolerance of NAGases for the aglycon moiety is generally quite high which enables the detection of NAGases in enzyme assays with chromogenic substrates (Horsch et al. 1997). NAGases have also already been shown to catalyze transglycosilation reactions and are used in polymer chemistry to synthesize regio- and stereo-selective polymers (Kobayashi et al. 1997).
Additionally to the exo-acting NAGases, endo-b-N-acetylglucosaminidases (EC 22.214.171.124) exist, which catalyze the hydrolysis of the N, N?-diacetylchitobiosyl unit in high-mannose glycopeptides and glycoproteins containing the [Man(GlcNAc)2]Asn-structure, with one GlcNAc residue remaining attached to the protein and the rest of the oligosaccharide being released intact (Horsch et al. 1997).
Chitinases (EC 126.96.36.199) catalyze random hydrolysis of N-acetyl-b-glucosaminide 1,4-b-linkages in chitin and chito-oligomers according to an endo-mechanism with (GlcNAc)2 and some (GlcNAc)3 as the only end products. By using those definitions it is important that a NAGase, that effects a processive degradation of chito-oligomers (i.e. (GlcNAc)n (=2-10)) by successively releasing GlcNAc residues from the nonreducing end of the chain must not be referred to as exo-chitinase. Exo-chitinases would follow a processive mechanism of hydrolysis and likewise release N, N?-diacetylchitobiose units, but starting at the non-reducing terminus of the substrate (Horsch et al. 1997).
The characteristics of the extensive chitinolytic enzyme system of Hypocrea/Trichoderma are discussed in more detail in chapters 4 and 5.
Chitinases and b-1, 3-glucanases are considered the main enzymes responsible for the degradation of the host cell walls by Hypocrea/Trichoderma, as chitin and b-1, 3-glucan are their two major cell wall components (Mahadevan and Tatum 1967).
It has been shown that b-1,3 glucanases inhibit spore germination or the growth of pathogens in synergistic cooperation with chitinases and antibiotics (Benitez et al. 2004). Many b-1, 3-glucanases have been isolated, but only a few genes have been cloned, e.g. bgn13.1 (de la Cruz et al. 1995) and lam1.3 (Cohen-Kupiec et al. 1999) from H. lixii, glu78 from H. atroviridis (Donzelli et al. 2001) and Tv-bgn1 and Tv-bgn2 from H. virens (Kim et al. 2002).
However, other enzymes hydrolyzing less abundant, but structurally important components (as b-1, 6-glucan), can also contribute to the efficient disorganization and further degradation of the cell wall by Hypocrea/Trichoderma. Three b-1, 6 glucanases (BGN16.1-3) have been purified from H. lixii. BGN16.1 and BGN16.2 are secreted under conditions where chitin is present as the only carbon source (de la Cruz and Llobell 1999; Delgado-Jarana et al. 2000) and BGN16.3 is specifically secreted in the presence of fungal cell walls (Montero et al. 2005).
a-1,3-Glucanases (EC 188.8.131.52), also named mutanases, are extracellular enzymes able to degrade polymers of glucose bound by a-1,3-glycosidic links and are classified as endo-hydrolytic when two or more residues of glucose are released as reaction products, and exo-hydrolytic when glucose monomers are the final reaction products.
Two exo a-1, 3 glucanases,agn13.1 and agn13.2 have been cloned from H. lixii and T. asperellum, respectively, and the transcript levels of the genes as well as the enzymatic properties of the proteins were characterized and their involvement in mycoparasitism was studied (Fuglsang et al. 2000; Ait-Lahsen et al. 2001; Sanz et al. 2005).
The study of the proteolytic system of Hypocrea/Trichoderma spp. and their contribution to biocontrol has been receiving increasing attention. Elad and coworkers (Kapat et al. 1998; Elad and Kapat 1999) showed that hydrolytic enzymes produced by B. cinerea were partially deactivated by protease activities of H. lixii, and demonstrated that the protease-containing culture liquid of Hypocrea/Trichoderma reduced germination and germ tube length of the pathogen, suggesting the involvement of proteases in biocontrol. Besides deactivation of plant pathogens? enzymes, proteases may be important for the mycoparasitic process by degrading the protein components of the host well wall. The presence of several different extracellular proteases was detected by IEF and gel filtration chromatography methods (Antal et al. 2001; Delgado-Jarana et al. 2002; Williams et al. 2003; Suarez et al. 2004).
Several protease encoding genes have already been cloned from Hypocrea/Trichoderma spp. The subtilisin-like serine protease of H. atroviridis, Prb1 has already been characterized in more detail and was shown to be involved in mycoparasitism of R. solani (Geremia et al. 1993; Flores et al. 1997; Cortes et al. 1998; Olmedo-Monfil et al. 2002). Its orthologues have also already been cloned from H. virens and T. hamatum (Pozo et al. 2004; Steyaert et al. 2004).
pra1, coding for a trypsin-like serine protease, was shown to be induced by fungal cell walls, nitrogen and carbon starvation and influenced by the pH of the media (Suarez et al. 2004) and the transcription of papA coding for an aspartic protease proved to be influenced by the nitrogen source and was upregulated in plate confrontation assays with R. solani and upon plant root attachment (Delgado-Jarana et al. 2002). Additionally, papB, encoding a vacuolar aspartic protease, and recently the extracellular aspartic protease P6281, which is upregulated upon growth on fungal cell wall were already described (Viterbo et al. 2004; Suarez et al. 2005).
Cellulases, Xylanases and other hydrolytic enzymes
Cellulases (b-1, 4-glucanases) comprise exoglucanases (i.e. cellobiohydrolases EC 184.108.40.206), endoglucanases (EC 220.127.116.11) and b -glucosidases (EC 18.104.22.168), which occur in various isozymic forms. Although cellulose is the major cell wall component of plant pathogenic oomycetes like Pythium, cellulases have not been studied in much detail for this purpose. Migheli et al (1998) overexpressed the cellulase Cel7B in T. longibrachiatum and obtained transformants with increased biocontrol activities. However, the cellulolytic system has been studied extensively in H. jecorina, which is industrially used for production of cellulases and heterologous protein expression (Mach and Zeilinger 2003; Schmoll and Kubicek 2003). Ongoing research in this field is focusing on the induction of cellulases by various carbon sources (Seiboth et al. 2004; Seiboth et al. 2005), the influence of light on this process (Schmoll et al. 2005; Schmoll and Kubicek 2005) and the impact of UPR and stress on protein secretion (Saloheimo et al. 1999; Collen et al. 2005; Pakula et al. 2005). It should also be noted that Saloheimo and coworkers (2002) cloned a gene encoding a protein with sequence similarity to plant expansins. These are plant cell wall proteins which are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. The protein, named swollenin, was found to disrupt the structure of cotton fibers without detectable formation of reducing sugars.
▀-1,4-Xylans are heteropolysaccharides that have a backbone of ▀-1,4-linked xylopyranosyl residues, to which side groups such as D-glucuronic acid, L-arabinose, p-coumaric acid, and ferulic acid are attached and which constitute 20 to 35% of the roughly 830 Gt of annually formed renewable plant biomass (Timell 1965). Enzymes capable of degrading the xylan backbone comprise endoxylanases (EC 22.214.171.124) and ▀-xylosidase (EC 126.96.36.199) (Kulkarni et al. 1999). Xylanases of the ascomycete H. jecorina have received strong attention because of their application in the pulp and paper and feed industry (Buchert et al. 1998) but beyond this the physiological relevance of xylanases for other processes in Hypocrea/Trichoderma in nature has not been studied.
Likewise, studies on other hydrolytic enzymes like e.g. galactosidases, pectinases or mannosidases, are so far only conducted with H. jecorina due to its industrial applications. Additionally, the sequence genome of H. jecorina is publicly available since 03/2005 (http://gsphere.lanl.gov/trire1/trire1.home.html) which is a large advantage, especially in research fields like e.g. genomics (DNA microarrays, RaSH) or proteomics (2D-gel electrophoresis/MS/MS, LC/MS/MS) where large scale screening methods are applied.