3.1.1 Trichoderma Species Identification
The genus Trichoderma contains species that occur in soils throughout the world. Most species are fast-growing saprophytes with the ability to survive under a range of environmental conditions by utilizing different substrates for growth (Hjeljord and Tronsmo 1998; Samuels 1996). The most common biological control agents in the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride (Hermosa et al. 2000). Characterization of 16 biocontrol strains, identified previously as Trichoderma harzianum Rifai and one biocontrol strain recognized as T. viride, has been carried out using several molecular techniques. A certain degree of polymorphism was detected among isolates in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) of ribosomal DNA revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum—T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride—T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride—T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum that cause mushroom green mold. A similar study by Dodd et al. (2000) utilized ITS1 and ITS2 sequence data to group 50 isolates of Trichoderma species with biocontrol potential, while ITS1
Table 1 Examples of fungal biological control agents that prevent seed rots, damping-off, and root, crown, and vascular diseases caused by pathogenic fungi on vegetable
Target pathogen and host
T. harzianum, T. hamatum T. harzianum T. harzianum T. hamatum, T. harzianum,
T. viride, T. virens T. harzianum T. longibrachiatum G. virens G. virens G. virens GL-3
G. virens GL-3, GL-21 G. catenulatum J1446 P. oxalicum Nonpathogenic
F. oxysporum Nonpathogenic F. oxysporum, F. solani Nonpathogenic
F. oxysporum Nonpathogenic R. solani
P. oligandrum P. oligandrum T. flavus T. flavus T. flavus C. minitans S. sclerotivorum C. foecundissimum
F. oxysporum f. sp. radicis-lycopersici on tomato F. oxysporum f. sp. lycopersici on tomato P. capsici on pepper P. ultimum and R. solani on bean R. solani on eggplant
S. sclerotiorum on pea
P. ultimum on cucumber
F. oxysporum f. sp. lycopersici on tomato
P. ultimum on cucumber; R. solani on peas
R. solani, P. ultimum, S. rolfsii, and F. oxysporum on tomato and pepper S. rolfsii on carrot Pythium on cucumber F. oxysporum f. sp. lycopersici on tomato F. oxysporum f. sp. lycopersici on tomato
F. oxysporum f. sp. lycopersici on tomato
F. oxysporum f. sp. cucumerinum on cucumber
R. solani and Pythium on cucumber and pepper
R. solani on bean, cabbage
Pythium spp. on cress V. dahliae on pepper S. rolfsii on bean V. dahliae on eggplant S. rolfsii on bean S. sclerotiorum on lettuce S. minor on lettuce
P. ultimum and R. solani on eggplant and pepper
Datnoff et al. (1995), Nemec et al. (1996), and Sivan and Chet (1993)
Larkin and Fravel (1998)
Knudsen and Eschen (1991) Migheli et al. (1998) Larkin and Fravel (1998) Koch (1999) Mao et al. (1998)
Ristaino et al. (1994)
Niemi and Lahdenpera (2000) and Punja and Yip (unpublished) De Cal et al. (1999)
Alabouvette et al. (1993), Duijff et al. (1998), and Fuchs et al. (1999) Larkin and Fravel (1998)
Mandeel and Baker (1991)
Cubeta and Echandi (1991), Harris and Adkins (1999), and
Villajuan-Abgona et al. (1996) Jabaji-Hare et al. (1999) and Ross et al. (1998)
McQuilken et al. (1992) Al-Rawahi and Hancock (1998) Madi et al. (1997)
Budge and Whipps (2001)
Adams and Fravel (1990)
Lewis and Larkin (1998)
sequence data and RFLP analysis were used to distinguish amongst isolates of T. harzianum (Gams and Meyer 1998). These studies demonstrated the utility of molecular methods to resolve the identity of strains of Trichoderma with potential biocontrol activity that were overlapping in morphological features. This approach could also be used to develop strain-specific markers for a desired biocontrol strain. Molecular markers were developed and used to detect and trace a strain of T. hamatum in potting mix (Abbasi et al. 1999).
Several studies have shown that T. harzianum can control diseases caused by many root-infecting pathogens, including Fusarium, Rhizoctonia, and Pythium (Table 1). T. harzianum strain KRL-AG2, commercially formulated as F-Stop, when added to a potting soil mix prior to seeding with tomatoes, reduced the incidence and severity of Fusarium root and crown rot caused by Fusarium oxysporum f. sp. radicis-lycopersici (Datnoff et al. 1995). T. harzianum strain T-22 also provided control of Fusarium crown rot of tomato (Nemec et al. 1996), and the fungus could be recovered from the roots of treated plants 26 days after application, suggesting it had colonized the roots. T. hamatum reduced the incidence of Fusarium wilt of tomato, caused by F. oxysporum f. sp. lycopersici, when added to potting medium prior to seeding (Larkin and Fravel 1998). A commercial formulation of T. harzianum (Rootshield strain T-22) was also evaluated against this disease and was found to significantly reduce it when incorporated into potting mix at 0.2% (Larkin and Fravel 1998). Strain T-22 of T. harzianum was generated by fusing a mutant strain capable of colonizing plant roots with a strain able to compete with bacteria under iron-limiting conditions using protoplast fusion techniques (Harman 2000). This new strain had the enhanced ability to colonize the root system of host plants, resulting in greater efficacy as a biological control agent for long-term root protection (Harman 2000; Harman and Bjorkman 1998; Sivan and Harman 1991).
A number of commercial formulations are available that contain strains of T. harzianum for use against different diseases on a wide range of crops. These products are registered for use against a number of soilborne pathogens. Among these products, RootShield™, T-22G, and T-22 Planter Box™ contain T. harzianum strain T-22 that is able to survive well in the rhizosphere of plants (Fravel 2000).
Trichoderma species can confer biological control against soilborne diseases through a number of mechanisms, including antibiosis, parasitism, competition, and the induction of host plant resistance (Hjeljord and Tronsmo 1998). Trichoderma species are known to produce a range of volatile and nonvolatile secondary metabolites, some of which inhibit other microorganisms and are considered to be antibiotics. These fungi can also penetrate and infect pathogen structures, such as hyphae, causing them to be degraded through the production of cell wall degrading enzymes, such as chitinases, glucanases, cellulases, and proteinases (Geremia et al. 1993; Schirmbock et al. 1994; Thrane et al. 1997; Zeilinger et al. 1999). Trichoderma species can compete with pathogens for nutrients, rapidly colonize a substrate and exclude pathogens from infection sites, and colonize senescing tissues and wounds to reduce pathogen colonization (Hjeljord and Tronsmo 1998). Some strains of Trichoderma are good root colonizers (rhizosphere competent). It has also been reported that T. harzianum (strain T-22) has the ability to directly enhance root growth and plant development in the absence of pathogens (Harman 2000), and it has been suggested that this was due to the production of a growth-regulating factor by the fungus (Windham et al. 1986). Altomare et al. (1999) proposed that the ability of T. harzianum to increase plant growth was partially due to the organism's ability to solubilize nutrients, thus making them more available to host plants. These observations indicate the versatility through which Trichoderma species can manifest biological control activity. Finally, it has been reported that a strain of T. harzianum was able to trigger host defense mechanisms in cucumber plants through enhanced cell wall depositions and induction of defense enzymes, suggesting an indirect effect in the host plant by the biocontrol agent (Yedidia et al. 1999).
Techniques in biotechnology have been applied to elucidate the role of hydrolytic enzymes, such as chitinases and glucanases, in mycoparasitism by Trichoderma that could lead to biological control activity. Transformants of T. longibrachiatum expressing extra copies of the ß-1,4-endoglucanase gene egl1 were found to be better at suppressing Pythium development on cucumber compared to wild-type strains (Migheli et al. 1998). In addition, transformants of T. harzianum overproducing the proteinase gene prb1 had up to a five-fold increase in ability to protect cotton seedlings from R. solani (Flores et al. 1996). Transformants of T. harzianum overexpressing an endochi-tinase gene chit33 were more effective in inhibiting growth of the pathogen R. solani in vitro compared with wild-type strains (Limon et al. 1999). A mutant of T. harzianum that was selected for its enhanced ability to hydrolyze pustulan, a polymer of ß-1,6-glucan, had 2-4 times more chitinase, ß-1,3 and ß-1,6 glucanase activity compared to the wild-type, produced three times more extracellular proteins and other compounds, and showed greater inhibition of B. cinerea in vitro (Rey et al. 2001).These studies reaffirm the roles played by fungal enzymes in biocontrol of plant pathogens and also highlight the successes in manipulating biocontrol strains to genetically engineer them to enhance efficacy. Specificity in the activity of the hydrolytic enzymes was suggested in a study by Woo et al. (1999), in which the endochitinase ech42 gene encoding for the secreted 42 Kda endochitinase
(CHIT 42) was silenced by targeted disruption; it was found that the endochitinase-deficient mutant had similar activity as the wild-type strain against Pythium ultimum, but had enhanced activity against R. solani, and reduced activity against B. cinerea.
A genetically marked strain of T. harzianum was developed by transformation with the ^-glucuronidase (uid A) gene and the hygromycin B (hygB) gene for use in population dynamics studies (Thrane et al. 1995). Techniques utilizing protoplast transformation as well as particle bombardment of conidia have been described for Tricho-derma (Lorito et al. 1993; Thrane et al. 1995). Population densities of the transformed strain could be monitored in a potting mix (Green and Jensen 1995), and the presence of the biocontrol agent around wounded tissues was reported.
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