Introductory Chapter: Identification and Isolation of *Trichoderma* spp. - Their Significance in Agriculture, Human Health, Industrial and Environmental Application

*Mohammad Manjur Shah and Hamisu Afiya*

### **1. Introduction**

The genus *Trichoderma* is a diverse group of free-living fungi in the family *Hypocreaceae*, commonly present in all soils [1–6]. These ascomycetes fungi are opportunistic, avirulent plant symbionts inhabiting root ecosystems [3, 7] and parasites on other groups of fungi [2]. They reproduce by chlamydospores and ascospores and proliferate better at mesophilic temperatures (25–35°C) and wide range of pH. Several findings supported this such as [8] who observed no visible growth of conidia at 15°C, but retain growth at 25°C and best results at 30°C [9], evaluated the growth of *Trichoderma* isolates at different temperatures and pH ranges, and reported the highest mycelial growth of *T. hamatum T612*, *T. harzianum T447*, *T. harzianum T969*, and *T. hamatum T614* at 25°C and *T. virens T523* and *Trichoderma* sp. at 30°C. For pH requirement, mycelial growth of *T. hamatum T612*, *T. harzianum T447*, and *T. virens T523* grew best at pH 5 and *T. harzianum T969* and *Trichoderma* sp. at pH 7, while *T. hamatum T614* has best mycelial growth at pH 8. A recent study by [10] reported 25–35°C. Similarly, a pH range of 5.5–8.5 was congenial for *T. harzianum* and *T. hamatum.*

*Trichoderma* colonizes several ecological niches where they play a vital role; they have been earlier recognized as effective biocontrol agents of plant-pathogenic fungi, producers of secondary metabolites of medical importance [3, 11, 12], and agents of bioremediation. Similarly, their ability to degrade lignocellulosic biomass to produce second-generation biofuels and other value-added products has been widely accepted [3, 12].

### **2. Identification of** *Trichoderma* **isolates**

Conventional methods for identification of *Trichoderma* spp. using morphological and cultural approach have earlier been used. These include arrangement of conidiophores, phialides, and conidia, while cultural features include linear growth, colony color, growth pattern, and pigmentation of hyphae. The fungus has revealed different morphologies on various cultivation media due to genetic factors and

environmental and nutritional factors. Green colony pigmentation after incubation for 7 days at 28°C on potato dextrose agar (PDA) was observed in *Trichoderma* cultures isolated from soil samples in Kliran [5]. Rhizospheric isolates revealed pale or yellowish color of reverse colonies at 25 and 30°C with rapid growth, loosely arranged conidia, and effused conidiation [13]. An ellipsoidal, obovoid, and bowling pin phialides were observed in *Trichoderma* spp. [14]; 10 isolates from groundnut rhizosphere revealed different morphological and microscopic features as shown in **Table 1**.


**5**

overlap it [22].

**Figure 1.**

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance…*

Isolates GRT-1, GRT-6, and GRT-9 were confirmed as *Trichoderma viride*; GRT-2, GRT-5, and GRT-8 as *Trichoderma koningii*; *GRT-4* as *Trichoderma reesei*; GRT-7 as *Trichoderma harizanum*; and GRT-10 as *Trichoderma aureoviride*, while GRT-3 could not be identified up to species level (**Figure 1**) [15]. However, conventional methods for the identification of *Trichoderma* spp. using morphological and cultural methods are prone to error and poor documentation of isolates at the culture collection since isolates are not "adequately differentiated" [16]. The use of molecular phylogenetic features coupled with the conventional methods is a better approach for verification of isolates and identification of novel strains [8] than morphological features alone, as anamorph and teleomorph used for defining species have reached their limits [17]. Several literatures have reported various techniques of molecular characterization to confirm *Trichoderma* isolates, such as "amplifying and analyzing the sequences of internal transcribed spacer gene (ITS) 1 and 2 and translation elongation factor 1-alpha (tef1) encoding gene" and BLAST interface in TrichOKEY and TrichoBLAST [16]; "restriction fragment length polymorphism (RFLP) and DNA sequencing" [18–20]; random amplified polymorphic DNAs (RAPD) and rDNA sequencing [14, 21]; sequence-

*Trichoderma isolates after 5 days incubation period [15]. (a) Colony color. (b) Colony reverse color.*

The growth of *Trichoderma* has been screened on different culture media for various studies using available, relatively cheaper supporting media such as corn meal agar, oat meal agar, potato dextrose agar, Czapek's Dox agar, special nutrient media, carrot agar, rose Bengal agar, selective media, etc. However, selective media favor growth of *Trichoderma* strains over other fungi and hence preferred for easy identification of *Trichoderma* isolates over rapidly growing fungi that may

*Trichoderma* selective medium (TSM) is recognized for quantitative isolation of *Trichoderma* spp. from soil. It is composed of low glucose level for rapid growth and sporulation of the fungus. Chloramphenicol is used to inhibit the growth of

related amplified polymorphism (SRAP) marker [7]; etc.

**3. Isolation media for** *Trichoderma*

**3.1** *Trichoderma* **selective media (TSM)**

*DOI: http://dx.doi.org/10.5772/intechopen.83528*

### **Table 1.**

*Morphological characteristics of some Trichoderma isolates.*

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance… DOI: http://dx.doi.org/10.5772/intechopen.83528*

### **Figure 1.**

*Trichoderma - The Most Widely Used Fungicide*

as shown in **Table 1**.

**S/N Isolate Colony** 

2 GRT-2 Dull green

4 GRT-4 Scattered

5 GRT-5 Dell green

6 GRT-6 Dark bluish

7 GRT-7 Dark green

8 GRT-8 Dull green

9 GRT-9 Dark bluish

10 GRT-10 Compute

**color**

to bluish green

in minute tufts and pale yellow green

to bluish green

green

producing tufts or pustules fringed by sterile mycelium

to bluish green

green

dull, green tufts or pustules

*Morphological characteristics of some Trichoderma isolates.*

1 GRT-1 Dark green Amber Long

**Colony reverse color**

Pale yellowish

Pale yellowish

Dull yellowish

Pale yellowish

Colorless Broad,

environmental and nutritional factors. Green colony pigmentation after incubation for 7 days at 28°C on potato dextrose agar (PDA) was observed in *Trichoderma* cultures isolated from soil samples in Kliran [5]. Rhizospheric isolates revealed pale or yellowish color of reverse colonies at 25 and 30°C with rapid growth, loosely arranged conidia, and effused conidiation [13]. An ellipsoidal, obovoid, and bowling pin phialides were observed in *Trichoderma* spp. [14]; 10 isolates from groundnut rhizosphere revealed different morphological and microscopic features

> **Conidiophore character**

> infrequently branching and verticillate

verticillate, and frequently branching

3 GRT-3 White White — — No conidia Abundant,

Rarely branched and verticillate

Broad frequently branching and verticillate

branching and verticillate

Frequently branching and verticillate

Narrow verticillate and frequently branching

branching and verticillate

branching and pyramidal structure

Uncolored Infrequently

Discolored Frequently

Uncolored Infrequently

**Phialide character**

Frequently paired, lageniform, and divergent

Lageniform, divergent, terminal philaid more elongated

Cylindrical or slightly inflated and divergent

Ampulliform and divergent

Lageniform and convergent

Ampulliform and convergent

Ampulliform and divergent

Lageniform and convergent

Lageniform and divergent

**Conidia shape**

Globose to ellipsoidal

Sub cylindrical to narrow ellipsoidal

Sub cylindrical

Sub globose to obovoid

Sub cylindrical to narrow ellipsoidal

Globose to ellipsoidal

Globose to ellipsoidal

**Chlamydospore formation**

Infrequent, terminal, and intercalary

Frequent, intercalary, and terminal

terminal, and intercalary

intercalary and terminal

Infrequent, intercalary, and terminal

Frequently intercalary and terminal

Infrequent, internally and terminally

Infrequent, intercalary, and terminal

Infrequent, intercalary, and terminally

intercalary, and terminal

Obovoid Frequently,

Ellipsoidal Frequently

**4**

*Source: [15].*

**Table 1.**

*Trichoderma isolates after 5 days incubation period [15]. (a) Colony color. (b) Colony reverse color.*

Isolates GRT-1, GRT-6, and GRT-9 were confirmed as *Trichoderma viride*; GRT-2, GRT-5, and GRT-8 as *Trichoderma koningii*; *GRT-4* as *Trichoderma reesei*; GRT-7 as *Trichoderma harizanum*; and GRT-10 as *Trichoderma aureoviride*, while GRT-3 could not be identified up to species level (**Figure 1**) [15]. However, conventional methods for the identification of *Trichoderma* spp. using morphological and cultural methods are prone to error and poor documentation of isolates at the culture collection since isolates are not "adequately differentiated" [16]. The use of molecular phylogenetic features coupled with the conventional methods is a better approach for verification of isolates and identification of novel strains [8] than morphological features alone, as anamorph and teleomorph used for defining species have reached their limits [17]. Several literatures have reported various techniques of molecular characterization to confirm *Trichoderma* isolates, such as "amplifying and analyzing the sequences of internal transcribed spacer gene (ITS) 1 and 2 and translation elongation factor 1-alpha (tef1) encoding gene" and BLAST interface in TrichOKEY and TrichoBLAST [16]; "restriction fragment length polymorphism (RFLP) and DNA sequencing" [18–20]; random amplified polymorphic DNAs (RAPD) and rDNA sequencing [14, 21]; sequencerelated amplified polymorphism (SRAP) marker [7]; etc.

### **3. Isolation media for** *Trichoderma*

The growth of *Trichoderma* has been screened on different culture media for various studies using available, relatively cheaper supporting media such as corn meal agar, oat meal agar, potato dextrose agar, Czapek's Dox agar, special nutrient media, carrot agar, rose Bengal agar, selective media, etc. However, selective media favor growth of *Trichoderma* strains over other fungi and hence preferred for easy identification of *Trichoderma* isolates over rapidly growing fungi that may overlap it [22].

### **3.1** *Trichoderma* **selective media (TSM)**

*Trichoderma* selective medium (TSM) is recognized for quantitative isolation of *Trichoderma* spp. from soil. It is composed of low glucose level for rapid growth and sporulation of the fungus. Chloramphenicol is used to inhibit the growth of

bacteria, while pentachloronitrobenzene, p-dimethylaminobenzenediazo sodium sulfonate, and rose bengal are used as selective fungal inhibitors [22].

*3.1.1 TSM recipe*


10.0.2 g of pentachloronitrobenzene.

Recipe is dissolved in 1000 ml distilled water and autoclaved at 121°C, 1.4 kg cm<sup>−</sup><sup>1</sup> for 15 min. Then add 0.25 g chloramphenicol and 0.2 g pentachloronitrobenzene into the solution. Keep/store media at 45°C to prevent solidification.

### **3.2** *Trichoderma harzianum* **selective medium (THSM)**

Selection of THSM enables comparison between aggressive and non-aggressive *Trichoderma* groups. The antimicrobials chloramphenicol, streptomycin, quintozene, and propamocarb are added to the medium to highly select *T. harzianum* in compact colonies without visible contamination [23].

**7**

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance…*

phenicol, 9.0 mL of streptomycin, 1.2 mL of propamocarb, and 0.2 g of quintozene

RBA is a nonselective medium for isolation of *Trichoderma* which is developed by Jarvis in 1973, for enumeration of molds and yeasts from food. The medium is suitable with protein foods and tolerates high temperatures. Chloramphenicol or

Add the ingredients to the distilled water and boil to dissolve completely. Add 10 mL of chloramphenicol or chlortetracycline; shake and autoclave at 121°C for

Several methods are available for the isolation of *Trichoderma*; however, one of the commonest methods reported in literature is the serial dilution of samples [22, 25–28]. This technique is simple, cost-effective, and appropriate to handle

Soil samples are collected, air dried, and ground into powder. Stock solution of sample is prepared by dissolving 10 g of powdered soil sample into 90 mL of

One milliliter of each of the prepared dilution is spread evenly on a suitable medium

Continuous use of chemical pesticides to manage fungal pathogens (which are known to cause major diseases in agriculture) has led to destruction of soil structure, soil infertility, and accumulation of toxic compounds on crops. Moreover, "chemical fungicides have less influence on pathogens due to their diversity,

, 10<sup>−</sup><sup>2</sup>

…10<sup>−</sup><sup>5</sup> .

distilled water. Next, serial dilution of samples were prepared as 10<sup>−</sup><sup>1</sup>

**5. Agricultural significance of** *Trichoderma* **spp.**

chlortetracycline is added to suppress the growth of bacteria [24].

, for 15 min, and 0.25 g of chloram-

*DOI: http://dx.doi.org/10.5772/intechopen.83528*

**3.3 Rose Bengal agar (RBA)**

*3.3.1 Recipe for rose Bengal agar (RBA)*

1.Mycological peptone 5.0 g

2.Rose bengal 0.05 g

4.Chloramphenicol 0.1 g

5.Dipotassium phosphate 1.0 g

15 min. Store in the dark at 4°C for further use.

**4. Method for isolation of** *Trichoderma*

on a petri dish at 28 ± 1°C for 7 days.

3.Glucose 10.0 g

6.Agar 15.0 g

large samples.

7.MgSO4.7 H20 0.5 g

8.Distilled 1000 mL

are added.

Media is autoclaved at 121°C, 1.4 kg cm<sup>−</sup><sup>1</sup>

*3.2.1 Recipe for THSM*


*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance… DOI: http://dx.doi.org/10.5772/intechopen.83528*

Media is autoclaved at 121°C, 1.4 kg cm<sup>−</sup><sup>1</sup> , for 15 min, and 0.25 g of chloramphenicol, 9.0 mL of streptomycin, 1.2 mL of propamocarb, and 0.2 g of quintozene are added.

### **3.3 Rose Bengal agar (RBA)**

*Trichoderma - The Most Widely Used Fungicide*

7H2O.

*3.1.1 TSM recipe*

1.0.2 g of MgSO4<sup>∙</sup>

2.0.9 g of K2HPO4.

4. 1.0 g of NH4NO3.

5. 3.0 g of glucose.

7. 20 g of agar.

1.4 kg cm<sup>−</sup><sup>1</sup>

*3.2.1 Recipe for THSM*

1.0.2 g of MgSO4∙7H2O.

2.0.9 g of K2HPO4.

3. 1.0 g of NH4NO3.

5.0.15 g of rose bengal.

8.950 mL of distilled water.

4.0.15 g of KCl.

6. 3 g of glucose.

7. 20 g of agar.

6.0.15 g of rose bengal.

8.0.25 g of chloramphenicol.

10.0.2 g of pentachloronitrobenzene.

colonies without visible contamination [23].

3.0.15 g of KCl.

bacteria, while pentachloronitrobenzene, p-dimethylaminobenzenediazo sodium

sulfonate, and rose bengal are used as selective fungal inhibitors [22].

9.0.3 g of p-dimethylaminobenzenediazo sodium sulfonate.

**3.2** *Trichoderma harzianum* **selective medium (THSM)**

Recipe is dissolved in 1000 ml distilled water and autoclaved at 121°C,

trobenzene into the solution. Keep/store media at 45°C to prevent solidification.

Selection of THSM enables comparison between aggressive and non-aggressive *Trichoderma* groups. The antimicrobials chloramphenicol, streptomycin, quintozene, and propamocarb are added to the medium to highly select *T. harzianum* in compact

for 15 min. Then add 0.25 g chloramphenicol and 0.2 g pentachloroni-

**6**

RBA is a nonselective medium for isolation of *Trichoderma* which is developed by Jarvis in 1973, for enumeration of molds and yeasts from food. The medium is suitable with protein foods and tolerates high temperatures. Chloramphenicol or chlortetracycline is added to suppress the growth of bacteria [24].

### *3.3.1 Recipe for rose Bengal agar (RBA)*


Add the ingredients to the distilled water and boil to dissolve completely. Add 10 mL of chloramphenicol or chlortetracycline; shake and autoclave at 121°C for 15 min. Store in the dark at 4°C for further use.

### **4. Method for isolation of** *Trichoderma*

Several methods are available for the isolation of *Trichoderma*; however, one of the commonest methods reported in literature is the serial dilution of samples [22, 25–28]. This technique is simple, cost-effective, and appropriate to handle large samples.

Soil samples are collected, air dried, and ground into powder. Stock solution of sample is prepared by dissolving 10 g of powdered soil sample into 90 mL of distilled water. Next, serial dilution of samples were prepared as 10<sup>−</sup><sup>1</sup> , 10<sup>−</sup><sup>2</sup> …10<sup>−</sup><sup>5</sup> . One milliliter of each of the prepared dilution is spread evenly on a suitable medium on a petri dish at 28 ± 1°C for 7 days.

### **5. Agricultural significance of** *Trichoderma* **spp.**

Continuous use of chemical pesticides to manage fungal pathogens (which are known to cause major diseases in agriculture) has led to destruction of soil structure, soil infertility, and accumulation of toxic compounds on crops. Moreover, "chemical fungicides have less influence on pathogens due to their diversity,

adaptability and increasing resistance" [4]. Various microbial biocontrol agents serve a solution for management of the aforementioned to attain a sustainable agriculture for future generations [29].

Knowledge about biocontrol potential of the fungus *Trichoderma* spp. has been recognized as early as 1920 [30, 31], although it received researcher's interest with advances in genetic engineering [12]. This technology has made it easy to isolate, characterize, clone, sequence, and express the roles of specific genes in the biocontrol mechanism. The genes encoding the enzymes play vital roles in biotic and abiotic stress tolerance, growth of hyphae, degradation of cell wall, and antagonistic activity against plant pathogens [29]. *Trichoderma harzianum* (Th. Azad) and *Trichoderma viride* (01PP) are used as biopesticides and biofertilizers [32, 33], growth promoters, and inducers of disease resistance in plants [12, 33]. The former is the main antagonist utilized in management of plant diseases in agriculture [34, 35] due to its cost-effectiveness and minimal effects on the ecological balance [34].

*Trichoderma* is efficient in improving vegetative growth of plants and nutrient content of soil through decomposition and biodegradation [33]. Active substance such as fungal spores is applied as foliar sprays and pre- and post-planting treatments, during watering and transplanting. *Trichoderma*-based products are marketed worldwide and applied in fields, nurseries, and horticulture for management of fungal soil-borne pathogens such as *Pythium* and *Rhizoctonia* [33, 35]. It is a safe and environmentally friendly method to reduce the detrimental effects of chemical pesticides [36].

Various articles reported on the role of *Trichoderma* spp. as antagonist to plant pathogens such as *T. harzianum*, *T. asperellum*, and *T. virens* against *Phytophthora capsici* in red pepper [16]; *Trichoderma* isolates against *Sclerotium rolfsii*, *Colletotrichum gloeosporioides*, *C. capsici* [37], *S. minor* and *S. sclerotiorum* in the in vitro experiments [38]; *T. atroviride SY3A* and *T. harzianum SYN* were effective biological control agents of *R. solani* damping-off of cucumber [39]; *Trichoderma* isolates were antagonist to soil-borne phytopathogenic fungi (*Fusarium graminearum*, *Rhizoctonia solani*, *Macrophomina phaseoli*, and *Phytophtora cactorum*) [9]; *Trichoderma* species was antagonist to anthracnose of strawberry [5]; *Trichoderma* isolates inhibit and control the growth of *Fusarium oxysporum* with *Trichoderma harzianum* being the most effective [40]; *T. viride*, *T. polysporum*, and *T. harzianum* inhibit more than 60% growth of *C. paradoxa* [19]; *T. hamatum LU593* and *T. virens LU556* delayed aphids manifestation on cabbage [41]; *Trichoderma* isolates against *Sclerotium rolfsii* [6]; *Trichoderma* isolates against *Fusarium sambucinum* [42]; and *Trichoderma* spp. and *Bacillus* spp. in seed treatment against root knot nematode *Meloidogyne javanica* [43].

### **6. Mechanism of biological control**

Several researches have revealed the mechanism of biocontrol in *Trichoderma* via mycoparasitism (by coiling around the host, formation of appressoria and breakdown of the host cell wall), antibiosis, and competition for resources (space and nutrients). A peptaibol from *Trichoderma* may induce apoptosis in plantpathogenic fungi through complex mechanisms. Trichokonins, a type of peptaibol from *Trichoderma pseudokoningii SMF2*, produced a molecular biocontrol mechanism which efficiently induces apoptosis in fungal cells. Apoptotic hallmarks such as the deposition of cytoplasmic vacuoles, presence of reactive oxygen species, breakage of DNA molecule, and exposure of phosphatidylserine were observed in *Fusarium oxysporum* cells treated with Trichokonins [44]. Mycoparasitism has been reported in *Trichoderma* species antagonist to Anthracnose disease of strawberry.

**9**

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance…*

Microscopic examination revealed that the hyphae of *Trichoderma* "grew alongside and coiled compactly around the hyphae of the fungal pathogen isolates" [5]. Recent development has further shown the significance of *Trichoderma* to induce systemic/localized resistance in plant by colonizing the root epidermis and subsequent release of bioactive metabolites, to transform the transcriptome and proteome of resultant plants [11, 30]. It further revealed that metabolites/ enzymes produced in *Trichoderma* such as chitinase and β-1,3 glucanase as recorded in *T. viride* and *T. harzianum*, respectively [37]; cellulases and hemicellulases in *Trichoderma* spp. [12, 45]; and proteases and lipases [46] are responsible for breaking down the component of the fungal cell wall to reduce the integrity of the pathogen cell. Relevant biocontrol processes such as production of hydrolytic enzymes and antifungal metabolites and the formation of infection structures are controlled by heterotrimeric G proteins and mitogen-activated protein (MAP) kinases. Similarly, induction of plant systemic resistance in *Trichoderma virens* and hyperosmotic stress response in *Trichoderma harzianum* are related to MAPK signaling [47], while biocontrol associated with coiling and chitinase production in

"*Trichoderma* species are possible source of important antimicrobial agents against gram negative, positive, fungi and yeast" [48]. Earlier in 1995, isolated peptides from *Trichoderma* strains showed antibacterial activity against *S. aureus* [49]; *T. harzianum* produced *44.06* μg/mL of the well-known antifungal drug, cyclosporine [50]. Similarly, 'Trichodermanins C–E (1–3), new diterpenes with a rare fused 6-5-6-6ring system, have been isolated from a fungus *Trichoderma harzianum*" detached from a marine sponge *H. okadai*. Cytotoxicity assay using three cancer cell lines showed significant activity in 1 [51]. Broth extracts of *Trichoderma* species (*Trichoderma harzianum*, *Trichoderma longibrachiatum*, and *T. koningii*) showed antifungal and antibacterial activity against *Paecilomyces variotii*, *Penicillium notatum*, *Nematospora coryli*, *Mucor miehei*, *Bacillus brevis*, *Bacillus subtilis*, *Enterobacter* 

*dissolvens*, and *Sarcina lutea* using agar disk diffusion method [48].

**8. Industrial and environmental applications of** *Trichoderma* **spp.**

*Trichoderma* are utilized in the production of low-cost enzymes for applications in food, pulp, and paper and textile industries to generate biofuel. The biotechnological workhorse of *Trichoderma* is the production of cellulases [52], in addition to the extracellular laccase [53]. Together with cellulases, endoglucanases (EG1, EG2, EG3, and EG5), and β-glucosidase, these enzymes catalyze the breakdown of ligninolytic biomass to produce an important industrial enzyme for the production of second-generation biofuels and other value-added products such as fermentable sugars, organic acids, solvents, drink softeners, etc. [54]. The production of proteins such as heterogenous proteins from cellobiohydrolase I (cbhI), a strong and inducible promoter of the gene encoding the major cellulose [55], and hydrophobins HFBI and HFBII [56–58] for industrial applications has been reported. Research efforts have focused on increasing enzyme yield and other valuable products from *Trichoderma* spp. through genome sequencing [45]. The highest yield of 1.4 g hydrophobins HFBI and 0.24 g hydrophobins HFBII per liter was obtained from the fungus through genetic manipulation on glucose-containing culture medium [59]; up to 40 mg/l of chymosin was produced from a transformed strain [55]; newly

*DOI: http://dx.doi.org/10.5772/intechopen.83528*

*Trichoderma* is regulated by internal cAMP level.

**7.** *Trichoderma* **in human health**

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance… DOI: http://dx.doi.org/10.5772/intechopen.83528*

Microscopic examination revealed that the hyphae of *Trichoderma* "grew alongside and coiled compactly around the hyphae of the fungal pathogen isolates" [5].

Recent development has further shown the significance of *Trichoderma* to induce systemic/localized resistance in plant by colonizing the root epidermis and subsequent release of bioactive metabolites, to transform the transcriptome and proteome of resultant plants [11, 30]. It further revealed that metabolites/ enzymes produced in *Trichoderma* such as chitinase and β-1,3 glucanase as recorded in *T. viride* and *T. harzianum*, respectively [37]; cellulases and hemicellulases in *Trichoderma* spp. [12, 45]; and proteases and lipases [46] are responsible for breaking down the component of the fungal cell wall to reduce the integrity of the pathogen cell. Relevant biocontrol processes such as production of hydrolytic enzymes and antifungal metabolites and the formation of infection structures are controlled by heterotrimeric G proteins and mitogen-activated protein (MAP) kinases. Similarly, induction of plant systemic resistance in *Trichoderma virens* and hyperosmotic stress response in *Trichoderma harzianum* are related to MAPK signaling [47], while biocontrol associated with coiling and chitinase production in *Trichoderma* is regulated by internal cAMP level.

### **7.** *Trichoderma* **in human health**

*Trichoderma - The Most Widely Used Fungicide*

agriculture for future generations [29].

knot nematode *Meloidogyne javanica* [43].

**6. Mechanism of biological control**

pesticides [36].

adaptability and increasing resistance" [4]. Various microbial biocontrol agents serve a solution for management of the aforementioned to attain a sustainable

Knowledge about biocontrol potential of the fungus *Trichoderma* spp. has been recognized as early as 1920 [30, 31], although it received researcher's interest with advances in genetic engineering [12]. This technology has made it easy to isolate, characterize, clone, sequence, and express the roles of specific genes in the biocontrol mechanism. The genes encoding the enzymes play vital roles in biotic and abiotic stress tolerance, growth of hyphae, degradation of cell wall, and antagonistic activity against plant pathogens [29]. *Trichoderma harzianum* (Th. Azad) and *Trichoderma viride* (01PP) are used as biopesticides and biofertilizers [32, 33], growth promoters, and inducers of disease resistance in plants [12, 33]. The former is the main antagonist utilized in management of plant diseases in agriculture [34, 35] due to its cost-effectiveness and minimal effects on the ecological balance [34]. *Trichoderma* is efficient in improving vegetative growth of plants and nutrient content of soil through decomposition and biodegradation [33]. Active substance such as fungal spores is applied as foliar sprays and pre- and post-planting treatments, during watering and transplanting. *Trichoderma*-based products are marketed worldwide and applied in fields, nurseries, and horticulture for management of fungal soil-borne pathogens such as *Pythium* and *Rhizoctonia* [33, 35]. It is a safe and environmentally friendly method to reduce the detrimental effects of chemical

Various articles reported on the role of *Trichoderma* spp. as antagonist to plant pathogens such as *T. harzianum*, *T. asperellum*, and *T. virens* against *Phytophthora capsici* in red pepper [16]; *Trichoderma* isolates against *Sclerotium rolfsii*, *Colletotrichum gloeosporioides*, *C. capsici* [37], *S. minor* and *S. sclerotiorum* in the in vitro experiments [38]; *T. atroviride SY3A* and *T. harzianum SYN* were effective biological control agents of *R. solani* damping-off of cucumber [39]; *Trichoderma* isolates were antagonist to soil-borne phytopathogenic fungi (*Fusarium graminearum*, *Rhizoctonia solani*, *Macrophomina phaseoli*, and *Phytophtora cactorum*) [9]; *Trichoderma* species was antagonist to anthracnose of strawberry [5]; *Trichoderma* isolates inhibit and control the growth of *Fusarium oxysporum* with *Trichoderma harzianum* being the most effective [40]; *T. viride*, *T. polysporum*, and *T. harzianum* inhibit more than 60% growth of *C. paradoxa* [19]; *T. hamatum LU593* and *T. virens LU556* delayed aphids manifestation on cabbage [41]; *Trichoderma* isolates against *Sclerotium rolfsii* [6]; *Trichoderma* isolates against *Fusarium sambucinum* [42]; and *Trichoderma* spp. and *Bacillus* spp. in seed treatment against root

Several researches have revealed the mechanism of biocontrol in *Trichoderma* via mycoparasitism (by coiling around the host, formation of appressoria and breakdown of the host cell wall), antibiosis, and competition for resources (space and nutrients). A peptaibol from *Trichoderma* may induce apoptosis in plantpathogenic fungi through complex mechanisms. Trichokonins, a type of peptaibol from *Trichoderma pseudokoningii SMF2*, produced a molecular biocontrol mechanism which efficiently induces apoptosis in fungal cells. Apoptotic hallmarks such as the deposition of cytoplasmic vacuoles, presence of reactive oxygen species, breakage of DNA molecule, and exposure of phosphatidylserine were observed in *Fusarium oxysporum* cells treated with Trichokonins [44]. Mycoparasitism has been reported in *Trichoderma* species antagonist to Anthracnose disease of strawberry.

**8**

"*Trichoderma* species are possible source of important antimicrobial agents against gram negative, positive, fungi and yeast" [48]. Earlier in 1995, isolated peptides from *Trichoderma* strains showed antibacterial activity against *S. aureus* [49]; *T. harzianum* produced *44.06* μg/mL of the well-known antifungal drug, cyclosporine [50]. Similarly, 'Trichodermanins C–E (1–3), new diterpenes with a rare fused 6-5-6-6ring system, have been isolated from a fungus *Trichoderma harzianum*" detached from a marine sponge *H. okadai*. Cytotoxicity assay using three cancer cell lines showed significant activity in 1 [51]. Broth extracts of *Trichoderma* species (*Trichoderma harzianum*, *Trichoderma longibrachiatum*, and *T. koningii*) showed antifungal and antibacterial activity against *Paecilomyces variotii*, *Penicillium notatum*, *Nematospora coryli*, *Mucor miehei*, *Bacillus brevis*, *Bacillus subtilis*, *Enterobacter dissolvens*, and *Sarcina lutea* using agar disk diffusion method [48].

### **8. Industrial and environmental applications of** *Trichoderma* **spp.**

*Trichoderma* are utilized in the production of low-cost enzymes for applications in food, pulp, and paper and textile industries to generate biofuel. The biotechnological workhorse of *Trichoderma* is the production of cellulases [52], in addition to the extracellular laccase [53]. Together with cellulases, endoglucanases (EG1, EG2, EG3, and EG5), and β-glucosidase, these enzymes catalyze the breakdown of ligninolytic biomass to produce an important industrial enzyme for the production of second-generation biofuels and other value-added products such as fermentable sugars, organic acids, solvents, drink softeners, etc. [54]. The production of proteins such as heterogenous proteins from cellobiohydrolase I (cbhI), a strong and inducible promoter of the gene encoding the major cellulose [55], and hydrophobins HFBI and HFBII [56–58] for industrial applications has been reported. Research efforts have focused on increasing enzyme yield and other valuable products from *Trichoderma* spp. through genome sequencing [45]. The highest yield of 1.4 g hydrophobins HFBI and 0.24 g hydrophobins HFBII per liter was obtained from the fungus through genetic manipulation on glucose-containing culture medium [59]; up to 40 mg/l of chymosin was produced from a transformed strain [55]; newly

constructed *Trichoderma* strains designed for specific industrial applications such as biofinishing and biostoning of cotton increased the cellulose CBHI by 1.5-fold and CBHII by fourfold as to the main strain. These were further increased to 1.6-fold CBHI and 3.4-fold CBHII by transformation as compared to the host strain. In addition, CBHII proteins were produced by the gene promoter (*Trichoderma* cellulases).

### **9. Conclusion**

*Trichoderma* spp. is one of the frequently isolated fungal genera from soil and plant roots that have been extensively studied for their vast metabolites with various applications (agricultural, industrial, health, etc.).

In the field of agriculture, *Trichoderma* are suitable antimicrobial agents against pathogenic bacteria, fungi, and yeast. Similarly, they play a vital role in improving the vegetative growth of plants and nutrient content of soil through decomposition and biodegradation. It is a safe, cost-effective, and environmentally benign technology to attain a sustainable agriculture.

In the field of medicine, different metabolites of medical importance have been reported from *Trichoderma*. Earlier in 1995, isolated peptides from *Trichoderma* strains showed antibacterial activity against *S. aureus* [49]. *T. harzianum* produced 44.06 μg/mL of the well-known antifungal drug, cyclosporine [50].

Cellulases, an important industrial enzyme from *Trichoderma*, are essential in the breakdown of biomass to produce second-generation biofuels and other valueadded products such as fermentable sugars, organic acids, solvents, drink softeners, [54] etc., in addition to the laccase production for textile industries. With advances in genetic engineering, efforts are focused on designing new strains of *Trichoderma* spp. through genome sequencing for production of novel metabolites of various applications.

### **Author details**

Mohammad Manjur Shah\* and Hamisu Afiya Department of Biological Sciences, Yusuf Maitama Sule University, Kano, Nigeria

\*Address all correspondence to: mmanjurshah@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**11**

2017

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance…*

Scientific and Industrial Research.

[9] Hajieghrari B, Torabi-giglou M, Mohammadi MR, Davari M. Biological potential of some Iranian *Trichoderma* isolates in the control of soil borne plant pathogenic fungi. African Journal of Biotechnology. 2008;**7**(8):967-972

[10] Shravan kashid V. Studies on Compatibility of *Trichoderma* spp. with Agrochemicals. Parbhani: Vasantrao Naik Marathwada Krishi Vidyapeeth;

[11] Mukherjee PK, Horwitz BA, Herrera-Estrella A, Schmoll M, Kenerley CM. *Trichoderma* research in the genome era. Annual Review of

105-129. Available from: http://www. annualreviews.org/doi/10.1146/ annurev-phyto-082712-102353

[12] Błaszczyk L, Siwulski M, Sobieralski K, Lisiecka J, Jędryczka M. *Trichoderma* spp.—Application and prospects for use in organic farming and industry. Journal of Plant Protection Research.

[13] Sharma KK, Singh US. Cultural and morphological characterization of rhizospheric isolates of fungal antagonist *Trichoderma*. Journal of Applied and Natural Science.

[14] Choi I-Y, Hong S-B, Yadav MC. Molecular and morphological characterization of green mold, *Trichoderma* spp. isolated from oyster mushrooms. Mycobiology. 2003;**31**(2):74-80. Available from: https://www.mycobiology.or.kr/ Synapse/Data/PDFData/0184MB/

[15] Sekhar YC, Ahammed SK, Prasad TNVKV. Identification of *Trichoderma*

Phytopathology. 2013;**51**(1):

2014;**54**(4):309-317

2014;**6**(2):451-456

mb-31-74.pdf

2011;**2**(8):294-306

2017

*DOI: http://dx.doi.org/10.5772/intechopen.83528*

[2] Harman GE, Howell CR, Viterbo A,

[3] Schuster A, Schmoll M. Computerassisted corneal topography. Highresolution graphic presentation and analysis of keratoscopy. Applied Microbiology and Biotechnology.

[4] Matei S, Matei G-M, Cornea P, Popa G. Characterization of soil *Trichoderma* isolates for potential biocontrol of plant pathogens. Factori şi procese pedogenetice din zona

[5] Mutia D, Prilya F. Exploration of *Trichoderma* spp. and fungal pathogen that causes a strawberry anthracnose and examination of in vitro antagonistic activity. Biotika. 2017;**5**(18):58-68

[6] Mohan PN. Studies on antagonistic potential of *Trichoderma* spp. from saline soils [Internet]. Agricultural University; 2017. Available from: http://krishikosh.egranth.ac.in/

[7] Digamber PS. Geographical diversity analysis of *Trichoderma* spp*.* isolates based on sequence related amplified polymorphism (SRAP) marker. Latur: College of Agricultural Biotechnology;

temperată. 2011;**10**:29-37

handle/1/5810030745

[8] Soesanto L, Sri-Utami D, Rahayuniati RF. Morphological characteristics of four *Trichoderma* isolates and two endophytic *Fusarium*

isolates. Canadian Journal on

[1] Samuels GJ. *Trichoderma*: A review of biology and systematics of the genus. Mycological Research. 1996;**100**(8):923-935. DOI: 10.1016/

S0953-7562(96)80043-8

2010;**87**:787-799

Chet I, Lorito M. *Trichoderma* spp.—Opportunistic, avirulent plant symbionts. Nature Reviews. Microbiology. 2004;**2**(1):43-56

**References**

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance… DOI: http://dx.doi.org/10.5772/intechopen.83528*

### **References**

*Trichoderma - The Most Widely Used Fungicide*

applications (agricultural, industrial, health, etc.).

ogy to attain a sustainable agriculture.

**9. Conclusion**

applications.

**Author details**

constructed *Trichoderma* strains designed for specific industrial applications such as biofinishing and biostoning of cotton increased the cellulose CBHI by 1.5-fold and CBHII by fourfold as to the main strain. These were further increased to 1.6-fold CBHI and 3.4-fold CBHII by transformation as compared to the host strain. In addition, CBHII proteins were produced by the gene promoter (*Trichoderma* cellulases).

*Trichoderma* spp. is one of the frequently isolated fungal genera from soil and plant roots that have been extensively studied for their vast metabolites with various

In the field of agriculture, *Trichoderma* are suitable antimicrobial agents against pathogenic bacteria, fungi, and yeast. Similarly, they play a vital role in improving the vegetative growth of plants and nutrient content of soil through decomposition and biodegradation. It is a safe, cost-effective, and environmentally benign technol-

In the field of medicine, different metabolites of medical importance have been reported from *Trichoderma*. Earlier in 1995, isolated peptides from *Trichoderma* strains showed antibacterial activity against *S. aureus* [49]. *T. harzianum* produced

Cellulases, an important industrial enzyme from *Trichoderma*, are essential in the breakdown of biomass to produce second-generation biofuels and other valueadded products such as fermentable sugars, organic acids, solvents, drink softeners, [54] etc., in addition to the laccase production for textile industries. With advances in genetic engineering, efforts are focused on designing new strains of *Trichoderma* spp. through genome sequencing for production of novel metabolites of various

44.06 μg/mL of the well-known antifungal drug, cyclosporine [50].

**10**

provided the original work is properly cited.

Mohammad Manjur Shah\* and Hamisu Afiya

\*Address all correspondence to: mmanjurshah@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Biological Sciences, Yusuf Maitama Sule University, Kano, Nigeria

[1] Samuels GJ. *Trichoderma*: A review of biology and systematics of the genus. Mycological Research. 1996;**100**(8):923-935. DOI: 10.1016/ S0953-7562(96)80043-8

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[3] Schuster A, Schmoll M. Computerassisted corneal topography. Highresolution graphic presentation and analysis of keratoscopy. Applied Microbiology and Biotechnology. 2010;**87**:787-799

[4] Matei S, Matei G-M, Cornea P, Popa G. Characterization of soil *Trichoderma* isolates for potential biocontrol of plant pathogens. Factori şi procese pedogenetice din zona temperată. 2011;**10**:29-37

[5] Mutia D, Prilya F. Exploration of *Trichoderma* spp. and fungal pathogen that causes a strawberry anthracnose and examination of in vitro antagonistic activity. Biotika. 2017;**5**(18):58-68

[6] Mohan PN. Studies on antagonistic potential of *Trichoderma* spp. from saline soils [Internet]. Agricultural University; 2017. Available from: http://krishikosh.egranth.ac.in/ handle/1/5810030745

[7] Digamber PS. Geographical diversity analysis of *Trichoderma* spp*.* isolates based on sequence related amplified polymorphism (SRAP) marker. Latur: College of Agricultural Biotechnology; 2017

[8] Soesanto L, Sri-Utami D, Rahayuniati RF. Morphological characteristics of four *Trichoderma* isolates and two endophytic *Fusarium* isolates. Canadian Journal on

Scientific and Industrial Research. 2011;**2**(8):294-306

[9] Hajieghrari B, Torabi-giglou M, Mohammadi MR, Davari M. Biological potential of some Iranian *Trichoderma* isolates in the control of soil borne plant pathogenic fungi. African Journal of Biotechnology. 2008;**7**(8):967-972

[10] Shravan kashid V. Studies on Compatibility of *Trichoderma* spp. with Agrochemicals. Parbhani: Vasantrao Naik Marathwada Krishi Vidyapeeth; 2017

[11] Mukherjee PK, Horwitz BA, Herrera-Estrella A, Schmoll M, Kenerley CM. *Trichoderma* research in the genome era. Annual Review of Phytopathology. 2013;**51**(1): 105-129. Available from: http://www. annualreviews.org/doi/10.1146/ annurev-phyto-082712-102353

[12] Błaszczyk L, Siwulski M, Sobieralski K, Lisiecka J, Jędryczka M. *Trichoderma* spp.—Application and prospects for use in organic farming and industry. Journal of Plant Protection Research. 2014;**54**(4):309-317

[13] Sharma KK, Singh US. Cultural and morphological characterization of rhizospheric isolates of fungal antagonist *Trichoderma*. Journal of Applied and Natural Science. 2014;**6**(2):451-456

[14] Choi I-Y, Hong S-B, Yadav MC. Molecular and morphological characterization of green mold, *Trichoderma* spp. isolated from oyster mushrooms. Mycobiology. 2003;**31**(2):74-80. Available from: https://www.mycobiology.or.kr/ Synapse/Data/PDFData/0184MB/ mb-31-74.pdf

[15] Sekhar YC, Ahammed SK, Prasad TNVKV. Identification of *Trichoderma* species based on morphological characters isolated from rhizosphere of groundnut (*Arachis hypogaea* L). International Journal of Science, Environment and Technology. 2017;**6**(3):2056-2063

[16] Savitha MJ, Sriram S. Morphological and molecular identification of *Trichoderma* isolates with biocontrol potential against *Phytophthora blight* in red pepper. Pest Management In Horticultural Ecosystems. 2015;**21**(2):194-202

[17] Samuels GJ. *Trichoderma*: Systematics, the sexual state, and ecology. The Nature and Application of Biocontrol Microbes II: Trichoderma spp. 2006;**96**(2):1-12

[18] Bourguignon E. Ecology and diversity of indigenous *Trichoderma* species in vegetable cropping systems. Lincoln University; 2008

[19] Kannangara S, Dharmarathna R, Jayarathna DL. Isolation, identification and characterization of *Trichoderma* species as a potential biocontrol agent against *Ceratocystis paradoxa*. The Journal of Agricultural Science. 2016;**12**(1):51- 62. Available from: http://jas.sljol.info/ article/10.4038/jas.v12i1.8206/

[20] Hatvani L. Mushroom pathogenic *Trichoderma* species: Occurrence, biodiversity, diagnosis and extracellular enzyme production. University of Szeged; 2008

[21] Narayan K, Kotasthane A. Genetic relatedness among *Trichoderma* isolates inhibiting a pathogenic fungi *Rhizoctonia solani*. Journal of Biotechnology. 2006;**5**:580-584

[22] Elad Y, Chet I, Henis Y. A selective medium for improving quantitative isolation of *Trichoderma* spp. from soil. Phytoparasitica. 1981;**9**(1):59-67

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quantitative reisolation of *Trichoderma harzianum* from *Agaricus bisporus* compost. Applied and Environmental Microbiology. 2003;**69**(7):4190-4191

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[26] Khandelwal M, Datta S, Mehta J, Naruka R, Makhijani K, Sharma G, et al. Isolation, characterization & biomass production of *Trichoderma viride* using various agro products—A biocontrol agent. Advances in Applied Science Research. 2012;**3**(6):3950-3955

[27] Roy MK, Hembram S, Debnath A. Effect of different media and pH on growth and sporulation of different native *Trichoderma* spp. An International Quarterly Journal of Life Sciences. 2015;**10**(4):1833-1837

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[29] Sharma P, Vignesh KP, Ramesh R, Saravanan K, Deep S, Sharma M, et al. Biocontrol genes from *Trichoderma* spp.: A review. African Journal of Biotechnology. 2011;**10**(86):19898-19907

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**13**

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PHYTO-96-0190

php?aid=30417

*Introductory Chapter: Identification and Isolation of Trichoderma spp. - Their Significance… DOI: http://dx.doi.org/10.5772/intechopen.83528*

[31] Harman GE. Overview of mechanisms and uses of *Trichoderma* spp. Phytopathology. 2006;**96**(2): 190-194. Available from: http:// apsjournals.apsnet.org/doi/10.1094/ PHYTO-96-0190

*Trichoderma - The Most Widely Used Fungicide*

quantitative reisolation of *Trichoderma harzianum* from *Agaricus bisporus* compost. Applied and Environmental Microbiology. 2003;**69**(7):4190-4191

[24] Rose bengal chloramphenicol (RBC) agar. International Journal of Food Microbiology. Elsevier;

[25] Mustafa A, Khan AM, Inam-ul-Haq M, Pervez AM, Umar U-D. Usefulness of different culture media for in-vitro evaluation of *Trichoderma* spp. against seed-borne fungi of economic importance. Pakistan Journal of Phytopathology. 2009;**21**(1):83-88

[26] Khandelwal M, Datta S, Mehta J, Naruka R, Makhijani K, Sharma G, et al. Isolation, characterization & biomass production of *Trichoderma viride* using various agro products—A biocontrol agent. Advances in Applied Science Research. 2012;**3**(6):3950-3955

[27] Roy MK, Hembram S, Debnath A. Effect of different media and pH on growth and sporulation of different native *Trichoderma* spp. An International Quarterly Journal of Life

Sciences. 2015;**10**(4):1833-1837

2017;**5**(3):1640-1646

2011;**10**(86):19898-19907

2006;**96**(2):190-194

[30] Harman G. Overview of

mechanisms and uses of *Trichoderma* spp. In: The Nature and Application of Biocontrol Microbes II: *Trichoderma* spp. Overview. Phytopathology.

[28] Iqbal S, Ashfaq M, Malik AH, Khan KS, Mathew P. Isolation,

[29] Sharma P, Vignesh KP, Ramesh R, Saravanan K, Deep S, Sharma M, et al. Biocontrol genes from *Trichoderma* spp.: A review. African Journal of Biotechnology.

preservation and revival of *Trichoderma viride* in culture media. Journal of Entomology and Zoology Studies.

1987;**5**(3):261-262

[16] Savitha MJ, Sriram S. Morphological

species based on morphological characters isolated from rhizosphere of groundnut (*Arachis hypogaea* L). International Journal of Science, Environment and Technology.

and molecular identification of *Trichoderma* isolates with biocontrol potential against *Phytophthora blight* in red pepper. Pest Management In Horticultural Ecosystems.

[17] Samuels GJ. *Trichoderma*: Systematics, the sexual state, and ecology. The Nature and Application of Biocontrol Microbes II: Trichoderma

[18] Bourguignon E. Ecology and diversity of indigenous *Trichoderma* species in vegetable cropping systems.

[19] Kannangara S, Dharmarathna R, Jayarathna DL. Isolation, identification and characterization of *Trichoderma* species as a potential biocontrol agent against *Ceratocystis paradoxa*. The Journal of Agricultural Science. 2016;**12**(1):51- 62. Available from: http://jas.sljol.info/

article/10.4038/jas.v12i1.8206/

Szeged; 2008

[20] Hatvani L. Mushroom pathogenic *Trichoderma* species: Occurrence, biodiversity, diagnosis and extracellular enzyme production. University of

[21] Narayan K, Kotasthane A. Genetic relatedness among *Trichoderma* isolates inhibiting a pathogenic fungi *Rhizoctonia solani*. Journal of Biotechnology. 2006;**5**:580-584

[22] Elad Y, Chet I, Henis Y. A selective medium for improving quantitative isolation of *Trichoderma* spp. from soil. Phytoparasitica. 1981;**9**(1):59-67

[23] Williams J, Clarkson JM, Mills PR, Cooper RM. A selective medium for

2017;**6**(3):2056-2063

2015;**21**(2):194-202

spp. 2006;**96**(2):1-12

Lincoln University; 2008

**12**

[32] Kumar V, Shahid M, Srivastava M, Sonika P, Singh A, Sharma A. Role of secondary metabolites produced by commercial *Trichoderma* spp. and their effect against soil borne pathogens. Biosensors. 2014;**03**(01):1-5. Available from: http://omicsonline.com/openaccess/role-of-secondary-metabolitesproduced-by-commercial-trichodermaspecies-and-their-effect-against-soilborne-pathogens-2090-4967-3-108. php?aid=30417

[33] Woo SL, Ruocco M, Vinale F, Nigro M, Marra R, Lombardi N, et al. *Trichoderma*-based products and their widespread use in agriculture. The Open Mycology Journal. 2014;**8**(1):71-126. Available from: http://benthamopen. com/ABSTRACT/TOMYCJ-8-71

[34] Monte EL. *Trichoderma* in organic agriculture. In: Proceedings V World Avocado Congress. 2003. pp. 725-733

[35] Kumar S, Thakur M, Rani A. *Trichoderma*: Mass production, formulation, quality control, delivery and its scope in commercialization in India for the management of plant diseases. African Journal of Agricultural Research. 2014;**9**(53):3838- 3852. Available from: http://www. academicjournals.org/AJAR

[36] Hermosa R, Botella L, Montero-Barrientos M, Alonso-Ramírez A, Arbona V, Gómez-Cadenas A, et al. Biotechnological applications of the gene transfer from the beneficial fungus *Trichoderma harzianum* to plants. Plant Signaling & Behavior. 2011;**6**(8):1235-1236

[37] Kumar K, Amaresan N, Bhagat S, Madhuri K, Srivastava RC. Isolation and characterization of *Trichoderma* spp. for antagonistic activity against root rot and foliar pathogens. Indian Journal of Microbiology. 2012;**52**(2):137-144

[38] Elias LM, Domingues MVPF, De Moura KE, Harakava R, Patricio RA. Selection of *Trichoderma* isolates for biological control of *Sclerotinia minor* and *S. sclerotiorum* in lettuce. Summa Phytopathologica. 2016;**42**(23):216-221

[39] Yobo KS. Biological control and plant growth promotion by selected *Trichoderma* and *Bacillus* species. KwaZulu-Natal University; 2005

[40] Shankar GK. Induction of Systemic Resistance in Tomato by using *Trichoderma*. India: PhD Thesis submitted to PhD Thesis submitted to Mahatma Phule Krishi Vidyapeeth; 2015. 219 pp. Available from: http://krishikosh.egranth.ac.in/ bitstream/1/95336/1/GHUTUDKAR.pdf

[41] Pus W. Plant-Mediated Effects of *Trichoderma* spp. and *Beauveria bassiana* Isolates on Insect and Pathogen Resistance. New Zealand: Lincoln University; 2017

[42] Ru Z, Di W. *Trichoderma* spp. from rhizosphere soil and their antagonism against *Fusarium sambucinum*. African Journal of Biotechnology. 2012;**11**(18):4180-4186. Available from: http://www.academicjournals.org/AJb/ abstracts/abs2012/1Mar/Ru and Di.htm

[43] Chinheya CC. Use of *Trichoderma* and *Bacillus* isolates as seed treatments against the root knot nematode *Meloidogyne Javanica* (Chitwood). University of KwaZulu-Natal; 2015

[44] Shi M, Chen L, Wang X, Zhang T, Zhao P, Song X, et al. Antimicrobial peptaibols from *Trichoderma pseudokoningii* induce programmed cell death in plant fungal pathogens. Microbiology. 2012;**158**:166-175

[45] Seidl V, Seiboth B. *Trichoderma reesei*: Genetic approaches to improving strain efficiency. Biofuels. 2010;**1**(2):343-354

[46] Jorge L. *Trichoderma* strains as biocontrol agents. Advancements in Genetic Engineering. 2014;**03**(01):4172. Available from: http://www.omicsgroup. org/journals/trichoderma-strains-asbiocontrol-agents-2169-0111.1000e110. php?aid=25241

[47] Zeilinger S, Omann M. *Trichoderma* biocontrol: Signal transduction pathways involved in host sensing and mycoparasitism. Gene Regulation and Systems Biology. 2007;**1**:227-234

[48] Tarus PK, Chhabra SC, Langat-Thoruwa C, Wanyonyi AW. Fermentation and antimicrobial activities of extracts from different spp. of *Trichoderma*. African Journal of Health Sciences. 2004;**11**:33-42

[49] Goulard C, Hlimi S, Rebuffat S, Bodo B. Trichorzins HA and MA, antibiotic peptides from *Trichoderma harzianum* I. Fermentation, isolation and biological properties. Journal of Antibiotics (Tokyo). 1995;**48**(11):1248-1253

[50] Azam A, Anjum T, Irun W. *Trichoderma harzianum*: A new fungal source for the production of cyclosporin A. Bangladesh Journal of Pharmacology. 2012;**7**:33-35

[51] Yamada T, Suzue M, Arai T, Kikuchi T, Tanaka R. Trichodermanins C–E, new diterpenes with a fused 6-5-6-6 ring system produced by a marine sponge-derived fungus. Marine Drugs. 2017;**15**:169

[52] Schuster A, Schmoll M. Biology and biotechnology of *Trichoderma*. Applied Microbiology and Biotechnology. 2010;**87**:787-799

[53] Cázares-García S, Vázquez-Garcidueñas M, Vázquez-Marrufo G. Structural and phylogenetic analysis of laccases from *Trichoderma*: A bioinformatic approach. PLoS One. 2013;**8**(1):e55295

[54] Kumar R, Singh S, Singh O. Bioconversion of lignocellulosic biomass: Biochemical and molecular perspectives. Journal of Industrial Microbiology & Biotechnology. 2008;**35**(5):377-391

[55] Uusitalo AH, Penttila BM, Knowles J, Novel Fungal A. Expression system: Secretion of active calf chymosin from the filamentous fungus *Trichoderma reesei*. Biotechnology. 1989;**7**:596-603

[56] Linder M, Selber K, Nakari-Setälä T, Qiao M, Kula MR, Penttilä M. The hydrophobins HFBI and HFBII from *Trichoderma reesei* showing efficient interactions with nonionic surfactants in aqueous two-phase systems. Biomacromolecules. 2001;**2**(2):511-517

[57] Askolin S, Nakari-Setälä T, Tenkanen M. Overproduction, purification, and characterization of the *Trichoderma reesei* hydrophobin HFBI. Applied Microbiology and Biotechnology. 2001;**57**(1-2):124-130

[58] Mustalahti E, Saloheimo M, Joensuu JJ. Intracellular protein production in *Trichoderma reesei (Hypocrea jecorina)* with hydrophobin fusion technology. New Biotechnology. 2013;**30**(2):262- 268. DOI: 10.1016/j.nbt.2011.09.006

[59] Askolin S. Characterization of the *Trichoderma reesei* hydrophobins HFBI and HFBII. VTT Publications. Helsinki University of Technology; 2006

**15**

**Chapter 2**

**Abstract**

**1. Introduction**

*Snježana Topolovec-Pintarić*

*Trichoderma*: Invisible Partner for

Species of genus *Trichoderma* may benefit as plant pathogen control agent (mycofungicide) and plant growth promoter (biofertilizer) and their application may lower the production costs and environmental impact. Direct effects of these fungi on plant growth and development are crucially important for agricultural uses and for understanding the roles of *Trichoderma* in natural and managed ecosystems. The *Trichoderma* potential as bioagent is utilized through the commercial production of *Trichoderma*-based product. Commercial products of *Trichoderma*-based biofungicides account for about 60% of the biofungicide market, while the availability and dispersion of *Trichoderma*-based biofertilizers are more widespread than commonly known with a tendency to expand due to the easier registrations. Limiting factors for availability of commercial products are expensiveness of registration requirements as they must be registered as pesticides, especially patenting, efficacy testing, toxicological, and biosafety testing. This chapter intends to give insight into agricultural importance of *Trichoderma* and current status of implementation of *Trichoderma* products in developing and in the developed countries.

**Keywords:** biocontrol, biotechnological patent, microbial products, pesticide

The credo of fabulous Spanish architect Antoni Gaudi that *Anything created by human beings is already in the great book of nature* is true for the exploitation of *Trichoderma* benefits. Since the early 1930s, when Weindling reported that *T. lignorum* produces and excretes a "lethal principle" in the surrounding, the scientists become involved in investigation of antifungal ability of various *Trichoderma* species, although *T. harzianum* arisen as the most prominent species of the genus. Today, their agricultural importance is good antagonistic abilities against soil born plant pathogenic fungi, thanks to different mechanisms of antagonism: the production of antifungal metabolites (antibiosis), competition for space and nutrients, induction of defense responses in plant and mycoparasitism. Along with revelation of diverse antifungal mechanisms of *Trichoderma*, the ability to promote plant growth and to increase plant height, leaf area and dry weight were perceived. First, this ability was treated as side effect of suppression of plant pathogenic fungi which leading to stronger root growth and nutrient uptake. Also, positive influence of *Trichoderma* to a faster germination and increase in percentage of emergency were perceived. Nowadays, *Trichoderma* species are considered as opportunistic plant symbionts because they colonize root surface and even penetrate into the epidermis of root tissue and a few cell layers below this level establishing pseudomycorrhizal

Visible Impact on Agriculture

### **Chapter 2**

*Trichoderma - The Most Widely Used Fungicide*

[53] Cázares-García S, Vázquez-Garcidueñas M, Vázquez-Marrufo G. Structural and phylogenetic analysis of laccases from *Trichoderma*: A bioinformatic approach. PLoS One.

[54] Kumar R, Singh S, Singh O. Bioconversion of lignocellulosic biomass: Biochemical and molecular perspectives. Journal of Industrial Microbiology & Biotechnology.

[55] Uusitalo AH, Penttila BM, Knowles J, Novel Fungal A. Expression system: Secretion of active calf chymosin from the filamentous fungus *Trichoderma reesei*. Biotechnology. 1989;**7**:596-603

[56] Linder M, Selber K, Nakari-Setälä T, Qiao M, Kula MR, Penttilä M. The hydrophobins HFBI and HFBII from *Trichoderma reesei* showing efficient interactions with nonionic surfactants

in aqueous two-phase systems. Biomacromolecules. 2001;**2**(2):511-517

[57] Askolin S, Nakari-Setälä T, Tenkanen M. Overproduction, purification, and characterization of the *Trichoderma reesei* hydrophobin HFBI. Applied Microbiology and Biotechnology. 2001;**57**(1-2):124-130

[58] Mustalahti E, Saloheimo M, Joensuu JJ. Intracellular protein production in *Trichoderma reesei (Hypocrea jecorina)* with hydrophobin fusion technology. New Biotechnology. 2013;**30**(2):262- 268. DOI: 10.1016/j.nbt.2011.09.006

[59] Askolin S. Characterization of the *Trichoderma reesei* hydrophobins HFBI and HFBII. VTT Publications. Helsinki

University of Technology; 2006

2013;**8**(1):e55295

2008;**35**(5):377-391

[45] Seidl V, Seiboth B. *Trichoderma reesei*: Genetic approaches to

improving strain efficiency. Biofuels.

[46] Jorge L. *Trichoderma* strains as biocontrol agents. Advancements in Genetic Engineering. 2014;**03**(01):4172. Available from: http://www.omicsgroup. org/journals/trichoderma-strains-asbiocontrol-agents-2169-0111.1000e110.

[47] Zeilinger S, Omann M. *Trichoderma*

biocontrol: Signal transduction pathways involved in host sensing and mycoparasitism. Gene Regulation and Systems Biology. 2007;**1**:227-234

[48] Tarus PK, Chhabra SC, Langat-Thoruwa C, Wanyonyi AW. Fermentation and antimicrobial activities of extracts from different spp. of *Trichoderma*. African Journal of

Health Sciences. 2004;**11**:33-42

*harzianum* I. Fermentation, isolation and biological properties. Journal of Antibiotics (Tokyo).

[50] Azam A, Anjum T, Irun W. *Trichoderma harzianum*: A new fungal source for the production of cyclosporin A. Bangladesh Journal of Pharmacology.

[51] Yamada T, Suzue M, Arai T, Kikuchi T, Tanaka R. Trichodermanins C–E, new diterpenes with a fused 6-5-6-6 ring system produced by a marine sponge-derived fungus. Marine Drugs.

[52] Schuster A, Schmoll M. Biology and biotechnology of *Trichoderma*. Applied Microbiology and Biotechnology.

1995;**48**(11):1248-1253

2012;**7**:33-35

2017;**15**:169

2010;**87**:787-799

[49] Goulard C, Hlimi S, Rebuffat S, Bodo B. Trichorzins HA and MA, antibiotic peptides from *Trichoderma* 

2010;**1**(2):343-354

php?aid=25241

**14**

## *Trichoderma*: Invisible Partner for Visible Impact on Agriculture

*Snježana Topolovec-Pintarić*

### **Abstract**

Species of genus *Trichoderma* may benefit as plant pathogen control agent (mycofungicide) and plant growth promoter (biofertilizer) and their application may lower the production costs and environmental impact. Direct effects of these fungi on plant growth and development are crucially important for agricultural uses and for understanding the roles of *Trichoderma* in natural and managed ecosystems. The *Trichoderma* potential as bioagent is utilized through the commercial production of *Trichoderma*-based product. Commercial products of *Trichoderma*-based biofungicides account for about 60% of the biofungicide market, while the availability and dispersion of *Trichoderma*-based biofertilizers are more widespread than commonly known with a tendency to expand due to the easier registrations. Limiting factors for availability of commercial products are expensiveness of registration requirements as they must be registered as pesticides, especially patenting, efficacy testing, toxicological, and biosafety testing. This chapter intends to give insight into agricultural importance of *Trichoderma* and current status of implementation of *Trichoderma* products in developing and in the developed countries.

**Keywords:** biocontrol, biotechnological patent, microbial products, pesticide

### **1. Introduction**

The credo of fabulous Spanish architect Antoni Gaudi that *Anything created by human beings is already in the great book of nature* is true for the exploitation of *Trichoderma* benefits. Since the early 1930s, when Weindling reported that *T. lignorum* produces and excretes a "lethal principle" in the surrounding, the scientists become involved in investigation of antifungal ability of various *Trichoderma* species, although *T. harzianum* arisen as the most prominent species of the genus. Today, their agricultural importance is good antagonistic abilities against soil born plant pathogenic fungi, thanks to different mechanisms of antagonism: the production of antifungal metabolites (antibiosis), competition for space and nutrients, induction of defense responses in plant and mycoparasitism. Along with revelation of diverse antifungal mechanisms of *Trichoderma*, the ability to promote plant growth and to increase plant height, leaf area and dry weight were perceived. First, this ability was treated as side effect of suppression of plant pathogenic fungi which leading to stronger root growth and nutrient uptake. Also, positive influence of *Trichoderma* to a faster germination and increase in percentage of emergency were perceived. Nowadays, *Trichoderma* species are considered as opportunistic plant symbionts because they colonize root surface and even penetrate into the epidermis of root tissue and a few cell layers below this level establishing pseudomycorrhizal

relationship with plant host. This intimate relationship is what induces localized and systemic resistance plant responses to pathogen attack. For the *Trichoderma*, abundant healthy roots are environment where it grows and proliferates best owing to the main carbohydrates secreted by plant roots. Furthermore, roots are resort of plant pathogenic fungi and nematodes, the target for *Trichoderma* as mycoparasite and nematophagous. The plants also benefit from this relationship through increased root and shoot growth and increased macro- and micronutrient uptake. Therefore, *Trichoderma* may be benefit as growth promotant (biofertilizer) as well as pathogen control agent (mycofungicide), and their application may lower the production costs and environmental impact. Recently, it is recognized that *Trichoderma* positive effect on plant growth is independent ability and equally remarkable and significant as its antifungal ability because growth enhancement has been observed in the absence of any detectable disease and in sterile soil. Therefore, today is considered that the direct effects of these fungi on plant growth and development are crucially important for agricultural uses and for understanding the roles of *Trichoderma* in natural and managed ecosystems.

To exploit *Trichoderma* benefits, it must be isolated from soil, studied, and encapsulated in formulation which will allow application into soil. But, reintroduction to soil, even the most strongly rhizosphere competent such as *Trichoderma* can be difficult. *Trichoderma* reintroduced into soil must compete with spectrum of rhizosphere microbes while trying to colonize available sites along the plant roots. Therefore, it needs to be applied in low cost but high density inocula engineered to maintain fungal propagule viable during the transport, storage, and application. To accomplished mentioned goals and effective dispersal of fungal inocula, it is necessity to choose the fungal inoculum carrier and the type of formulation. The *Trichoderma* potential as bioagent is utilized through the commercial production of *Trichoderma*-based biofungicides, which account for about 60% of the biofungicide market. The availability and dispersion of *Trichoderma*based biofertilizers are more widespread than commonly known with a tendency to expand due to the easier registrations because they are not registered as pesticides. Limiting factors for availability of commercial products are expensiveness of registration requirements, especially patenting, efficacy testing, toxicological, and biosafety testing.

### **2. Historical and commercial background**

In 1794 the mycologist C. H. Persoon proposed and named the genus *Trichoderma* after mycelial appearing, like hairy (Gr. thrix, genitive trikhos) cowering on decaying wood surface (Gr. derma). Association with teleomorphs in *Hypocrea* was done by Tulasne brothers in 1865. The genus came in focus after Weindling's article about *T. lingorum* as parasite of soil fungi, followed by another article in 1934 about parasitism on *Rhizoctonia solani* helped by some kind of a toxic compound [1, 2]. In that article, Weindling gave definition of antibiosis as suppressive mechanism based on production on secondary metabolites with antimicrobial effect and also named this *T. lingorum* lethal metabolite, gliotoxin. Further Weindling's papers defined biocontrol of plant pathogens through the *Trichoderma* strains and their following unique mechanisms: mycoparasitism, competition for area and nutrients in the rhizosphere and antibiosis, and production of antibiotics [3–5]. Existence of volatile organic compounds produced by *Trichoderma* that can inhibit the growth of fungi responsible for wood decay was published by Dennis and Webster in 1970 [6]. Since then, species of genus *Trichoderma* become among the most commonly studied biocontrol microbes and are presently marketed as

**17**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

active ingredients of biopesticides, biofertilizers, plant growth enhancers, and

The *Trichoderma* potential as biocontrol agent is utilized through the commercial production of biofungicides and always registers for use as microbial fungicides. *Trichoderma* based biofungicides are today presented with more than 250 products available worldwide and account for about 60% of the international biofungicide market. The leading country in terms of enormous use of *Trichoderma* products is India which comprises 90% of Asian market [7]. Following is Brazil with the greatest production on the South and Central America. For example, in Venezuela and Cuba, the development and use of *Trichoderma*-based products are government supported and officially recommended [8]. Prevalent species in majority of *Trichoderma*-based products is *T. harzianum* (83%), of which 55% of these are combined with *T. viride* and 28% are with *T. koningii* [7]. Widely used *T. harzianum* and *T. viride* are mostly applicate as soil treatment in around 87 various crops against 70 soil-borne and 18 foliar-borne pathogens, mostly fungi [9].

The first *Trichoderma* biofungicide commercialized worldwide was Trichodex produced by Makhteshim Agan Industries (Beersheba, Israel). Trichodex is based on *T. harzianum* isolate T39 studied since 1986 by its creator Dr. Yigal Elad of the Volcani Center, Israel where research and development were carried out [10]. Worked as a biopesticide researcher, Dr. Elad had primarily studying the biological control of economically significant plant pathogens primary gray mold causal, ascomycete fungus *Botrytis cinerea*. Early contact with industry company and sign of agreement with the aim of developing a biocontrol preparation for the control of gray mold were crucial, and Dr. Elad's collaboration with Makhteshim-Agan Industries led to the development and launch of the biopesticide product, Trichodex. Around the world, Makhteshim carried out efficacy trials of Trichodex in controlling of gray mold in vineyards in more than 130 experiments on 34 varieties, under diverse commercial conditions around the world [11]. In 1993, Trichodex has been registered in countries such as Argentina, Australia, Bulgaria, California, Chile, Colombia, Croatia, Cyprus, Greece, Guatemala, Hungary, Israel, Italy, Morocco, Paraguay, Romania, Turkey, Slovenia, South Africa, and the USA. Trichodex was in Croatia registered as contact antibiotic fungicide with enzymatic activity against *B. cinerea* on grapevine and strawberries. In Croatia, microplots with Trichodex were carried out until 1999 in vineyards of famous winegrowing region Kutjevo, situated in the continental part of Croatia were the gray mold disease inflicts damages of 50–60%. Trichodex shown efficacy in interval of 13–55% depending on weather conditions in year which was satisfactory control and occasionally equally efficient as synthetic fungicide Kidan (a.i. iprodione, Bayer, Germany) registered in Croatia at that time [12, 13]. In 1990s, beside Volcani center (Israel) Research institute for Plant Pathology, investigations of *Trichoderma* biocontrol efficacy were conducted at INRA (Paris, France), Institute for Biological Control (Darmstadt, Germany) and in USA at

Another most famous and useful *T. harzianum* strain was T22 (also known as 1295-22, KRL-AG2, and ATCC 20847). It was produced by Dr. Harman in 1980s and was licensed from Cornell University by the Eastman Kodak Company, which developed the toxicity package and environmental studies and make registration possible. In about 1990, Kodak decided to abandon the agricultural pesticide market and gifted the registration of T22 and other data they had generated to Dr. Harman and his colleagues at the Cornell Research Foundation who founded a company, now BioWorks Inc. This company was founded by Dr. Harman and two of his colleagues previously under name TGT Inc. as their efforts were to develop biocontrol systems for commercial agriculture and to translate biocontrol research into biocontrol

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

Cornel University (Geneva, NY) Dr. Harman.

stimulants of natural resistance.

### *Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

*Trichoderma - The Most Widely Used Fungicide*

relationship with plant host. This intimate relationship is what induces localized and systemic resistance plant responses to pathogen attack. For the *Trichoderma*, abundant healthy roots are environment where it grows and proliferates best owing to the main carbohydrates secreted by plant roots. Furthermore, roots are resort of plant pathogenic fungi and nematodes, the target for *Trichoderma* as mycoparasite and nematophagous. The plants also benefit from this relationship through increased root and shoot growth and increased macro- and micronutrient uptake. Therefore, *Trichoderma* may be benefit as growth promotant (biofertilizer) as well as pathogen control agent (mycofungicide), and their application may lower the production costs and environmental impact. Recently, it is recognized that *Trichoderma* positive effect on plant growth is independent ability and equally remarkable and significant as its antifungal ability because growth enhancement has been observed in the absence of any detectable disease and in sterile soil. Therefore, today is considered that the direct effects of these fungi on plant growth and development are crucially important for agricultural uses and for understand-

ing the roles of *Trichoderma* in natural and managed ecosystems.

To exploit *Trichoderma* benefits, it must be isolated from soil, studied, and encapsulated in formulation which will allow application into soil. But, reintroduction to soil, even the most strongly rhizosphere competent such as *Trichoderma* can be difficult. *Trichoderma* reintroduced into soil must compete with spectrum of rhizosphere microbes while trying to colonize available sites along the plant roots. Therefore, it needs to be applied in low cost but high density inocula engineered to maintain fungal propagule viable during the transport, storage, and application. To accomplished mentioned goals and effective dispersal of fungal inocula, it is necessity to choose the fungal inoculum carrier and the type of formulation. The *Trichoderma* potential as bioagent is utilized through the commercial production of *Trichoderma*-based biofungicides, which account for about 60% of the biofungicide market. The availability and dispersion of *Trichoderma*based biofertilizers are more widespread than commonly known with a tendency to expand due to the easier registrations because they are not registered as pesticides. Limiting factors for availability of commercial products are expensiveness of registration requirements, especially patenting, efficacy testing, toxicological,

**16**

and biosafety testing.

**2. Historical and commercial background**

In 1794 the mycologist C. H. Persoon proposed and named the genus *Trichoderma* after mycelial appearing, like hairy (Gr. thrix, genitive trikhos) cowering on decaying wood surface (Gr. derma). Association with teleomorphs in *Hypocrea* was done by Tulasne brothers in 1865. The genus came in focus after Weindling's article about *T. lingorum* as parasite of soil fungi, followed by another article in 1934 about parasitism on *Rhizoctonia solani* helped by some kind of a toxic compound [1, 2]. In that article, Weindling gave definition of antibiosis as suppressive mechanism based on production on secondary metabolites with antimicrobial effect and also named this *T. lingorum* lethal metabolite, gliotoxin. Further Weindling's papers defined biocontrol of plant pathogens through the *Trichoderma* strains and their following unique mechanisms: mycoparasitism, competition for area and nutrients in the rhizosphere and antibiosis, and production of antibiotics [3–5]. Existence of volatile organic compounds produced by *Trichoderma* that can inhibit the growth of fungi responsible for wood decay was published by Dennis and Webster in 1970 [6]. Since then, species of genus *Trichoderma* become among the most commonly studied biocontrol microbes and are presently marketed as

active ingredients of biopesticides, biofertilizers, plant growth enhancers, and stimulants of natural resistance.

The *Trichoderma* potential as biocontrol agent is utilized through the commercial production of biofungicides and always registers for use as microbial fungicides. *Trichoderma* based biofungicides are today presented with more than 250 products available worldwide and account for about 60% of the international biofungicide market. The leading country in terms of enormous use of *Trichoderma* products is India which comprises 90% of Asian market [7]. Following is Brazil with the greatest production on the South and Central America. For example, in Venezuela and Cuba, the development and use of *Trichoderma*-based products are government supported and officially recommended [8]. Prevalent species in majority of *Trichoderma*-based products is *T. harzianum* (83%), of which 55% of these are combined with *T. viride* and 28% are with *T. koningii* [7]. Widely used *T. harzianum* and *T. viride* are mostly applicate as soil treatment in around 87 various crops against 70 soil-borne and 18 foliar-borne pathogens, mostly fungi [9].

The first *Trichoderma* biofungicide commercialized worldwide was Trichodex produced by Makhteshim Agan Industries (Beersheba, Israel). Trichodex is based on *T. harzianum* isolate T39 studied since 1986 by its creator Dr. Yigal Elad of the Volcani Center, Israel where research and development were carried out [10]. Worked as a biopesticide researcher, Dr. Elad had primarily studying the biological control of economically significant plant pathogens primary gray mold causal, ascomycete fungus *Botrytis cinerea*. Early contact with industry company and sign of agreement with the aim of developing a biocontrol preparation for the control of gray mold were crucial, and Dr. Elad's collaboration with Makhteshim-Agan Industries led to the development and launch of the biopesticide product, Trichodex. Around the world, Makhteshim carried out efficacy trials of Trichodex in controlling of gray mold in vineyards in more than 130 experiments on 34 varieties, under diverse commercial conditions around the world [11]. In 1993, Trichodex has been registered in countries such as Argentina, Australia, Bulgaria, California, Chile, Colombia, Croatia, Cyprus, Greece, Guatemala, Hungary, Israel, Italy, Morocco, Paraguay, Romania, Turkey, Slovenia, South Africa, and the USA. Trichodex was in Croatia registered as contact antibiotic fungicide with enzymatic activity against *B. cinerea* on grapevine and strawberries. In Croatia, microplots with Trichodex were carried out until 1999 in vineyards of famous winegrowing region Kutjevo, situated in the continental part of Croatia were the gray mold disease inflicts damages of 50–60%. Trichodex shown efficacy in interval of 13–55% depending on weather conditions in year which was satisfactory control and occasionally equally efficient as synthetic fungicide Kidan (a.i. iprodione, Bayer, Germany) registered in Croatia at that time [12, 13]. In 1990s, beside Volcani center (Israel) Research institute for Plant Pathology, investigations of *Trichoderma* biocontrol efficacy were conducted at INRA (Paris, France), Institute for Biological Control (Darmstadt, Germany) and in USA at Cornel University (Geneva, NY) Dr. Harman.

Another most famous and useful *T. harzianum* strain was T22 (also known as 1295-22, KRL-AG2, and ATCC 20847). It was produced by Dr. Harman in 1980s and was licensed from Cornell University by the Eastman Kodak Company, which developed the toxicity package and environmental studies and make registration possible. In about 1990, Kodak decided to abandon the agricultural pesticide market and gifted the registration of T22 and other data they had generated to Dr. Harman and his colleagues at the Cornell Research Foundation who founded a company, now BioWorks Inc. This company was founded by Dr. Harman and two of his colleagues previously under name TGT Inc. as their efforts were to develop biocontrol systems for commercial agriculture and to translate biocontrol research into biocontrol

reality [14]. They encapsulated T22 in commercial products RootShield and T22 Planter Box and marketed under new company BioWorks Inc. (Geneva, NY, USA). Sales of those products began in 1993, and in 1998, sales have increased for 20% per year where it was marketing internationally with special consideration for its limited shelf life. Strain T22 was generally promoter of plant growth as well as mycofungicide against soil-borne plant pathogens. In plant, strain T22 enhances expression of proteins involved in photosynthesis and starch accumulation, and supposing its effects are due to increased photosynthetic rates in infected plants [8]. Interestingly, this strain was produced using protoplast fusion in order to be highly rhizosphere competent that also possess substantial ability to compete with spermosphere bacteria. Strains that were fused were *T. harzianum* T-95 and T-12 because first was a rhizosphere competent mutant produced from a strain isolated from a *Rhizoctonia*suppressive Colombian soil, and second was more capable of competing with spermosphere bacteria than T-95 under iron-limiting conditions [14, 15]. The novel generation of RootShield is based on two *Trichoderma* species and contains strain *T. virens* G-41 together with strain *T. harzianum* T-22. Commercial name of product is RootShield PLUS and some with instruction that should be applied to disease-free plants previously chemically treated with fungicide, because the aggressively growing strains T-22 and G-41 are growing on the outside of roots and do not enter the plant tissue.

The availability and dispersion of *Trichoderma*-based biofertilizers are more widespread then commonly known with the tendency to expand due to the easier registrations. Mostly permitted for use in organic farming in Europe is: RootShield, Plant Box and Bio Trek (northern Europe, USA), Binap (Switzerland, Sweden, UK, USA), Bio fungus (Belgium), Supersivit (Czech Republic), Trichodex (Italy), Trifender (Hungary), and Trianum (Avantagro, Spain) [16]. Novel *Trichoderma* formulations are not based on single culture of one species but come as consortia or mix of at least two or three species or different strains of same species. Compatible consortia of compatible strains with different mechanisms, disease suppressive, or plant growth promoting, which complementary each other were found to be more effective than the application of individual organisms. Mixtures for biocontrol have a broadened range of pathogens against which are effective. Mixtures in plant growth promoters are based on insight that metabolic products of various *Trichoderma* strains are not identical and they have selective character to different plant species and even a variety [17, 18]. Seems this may be due to better interaction of some *Trichoderma* species or some strains with certain plant species because root exudates may induce or inhibit their mycelial growth [19]. In development are other types of consortia, mixtures of *Trichoderma* strains with other organisms, fungi or bacteria that are known as bioagent also. Because knowing of action mechanisms allows combining of strains with different modes of action in order to anticipate efficacy of final product, investigations of effectiveness differences between species and biotypes of same species are in progress.

### **3.** *Trichoderma* **as opportunistic plant symbiont**

Mycoparasitic and nematophagous *Trichoderma* species found their prey in rhizosphere where roots are resort of plant pathogenic fungi and nematodes. Therefore, it becomes usual to define species of genus *Trichoderma* as free living rhizosphere organisms which colonize plant root surface as opportunistic plant symbionts. Biocontrol of plant pathogens by *Trichoderma* was from the first Weindling report in 1932 [1] considered as the direct ability of these fungi to interact with soil pathogens. Along with revelation of diverse antifungal mechanisms of

**19**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

total more than a billion of nitrogen kilos annually.

uptake and disease protection as well.

emergency were perceived also [17, 18, 29].

*Trichoderma*, the ability to promote plant growth and to increase plant height, leaf area, and dry weight were perceived. Positive influence of *Trichoderma* to a faster germination and increase in percentage of emergency were perceived also. Abundant healthy roots are from environment where *Trichoderma* grows and proliferates best. The sucrose that leaks from roots stimulates growth of *Trichoderma* mycelium and leads to interaction with plant. In order to stimulate plant to provide more sucrose, *Trichoderma* has evolved numerous mechanisms for better routing. With its enzyme arsenal, *Trichoderma* enhances solubility of soil nutrients which will be otherwise unavailable to plant. Further, *Trichoderma* enhances nutrient uptake by plant and better plant nourishment that will result in stronger routing which are frequently associated with increase in yield [20]. Special benefit is induction of increased nitrogen use efficiency in plants. This mechanism is not enlightened yet but probably is connected to *Trichoderma* stimulation of deeper rooting, and thereby increasing the volume of soil colonized by plant roots. Plants approximately take up only 33% of the amount of applied nitrogen fertilizer while some field trial data on *Trichoderma* treatments in several different crops indicate possibility of reduction nitrogen application rates by 30–50% with no reduction in yield [21, 22]. Calculation says that if this reduction was applied to the 30 million hectares of wheat in the USA, the savings in nitrogen application would

Sucrose metabolism increased by *Trichoderma* stimulates the resistance response in the leaves that leads to increased photosynthesis and respiration because growth induced by *Trichoderma* plant requires energy, and sunlight energy utilized in increased photosynthesis will be the energy source needed for plant growth enhancement by *Trichoderma* mechanisms. Of course, better photosynthesis enables that more sucrose is translocating to the roots and metabolic circle continues. Increased leaf mass enables increased photosynthesis but the *Trichoderma* has the abilities to increase photosynthetic efficiency [21, 23]. It was demonstrated that electron flow was substantially increased by root colonization and electron transport strongly enhanced [24]. In trial with barley exposed to water deficiency *T. harzianum* substantially increases water deficit tolerance and consequently reduces effects on photosynthetic systems even when plants were at or approaching the permanent wilting point after 2 weeks of withholding irrigation. Therefore, plants benefit from relationship with *Trichoderma* through increased root, shoot, and leaf growth and increased macro- and micronutrient

First scientific papers about effects of *Trichoderma* on plant growth promotion, mostly of horticultural crops and conifers, began to appear in 1980s and continuing in 1990s [18, 25–28]. There is ample documentation for the *Trichoderma* influence on plants which are colonized with effective *Trichoderma* strain and that they are substantially different from an uncolonize plants in quality and quantity of yield, withstand to adverse environmental conditions and pathogen attack. Positive influence of *Trichoderma* to a faster germination and increase in percentage of

In Croatia, investigations with autochthon *Trichoderma* strains and their influence to growth of fiber flax, lettuce, tomato, cabbage, and red beet were conducted [16, 30–32]. Significant increase of some lettuce quality characteristics was gained with *T. viride* strain TPS applied in the form of alginate-pellets in two frequently used commercial potting compost mixture: Klasmann-Deilmann P 002 (Germany) and Stender A240 (Germany). Except dry weight, TPS enhanced the formation of leaves: at Stender difference against control varies for 1–2 leaves more, at Klasmann for 1 leaf more. Leaf length was longer for 2 cm at Stender and Klasmann amended with TPS-pellets than at control, while leaf width was wider for 3.15 cm at Stender

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

### *Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

*Trichoderma - The Most Widely Used Fungicide*

reality [14]. They encapsulated T22 in commercial products RootShield and T22 Planter Box and marketed under new company BioWorks Inc. (Geneva, NY, USA). Sales of those products began in 1993, and in 1998, sales have increased for 20% per year where it was marketing internationally with special consideration for its limited shelf life. Strain T22 was generally promoter of plant growth as well as mycofungicide against soil-borne plant pathogens. In plant, strain T22 enhances expression of proteins involved in photosynthesis and starch accumulation, and supposing its effects are due to increased photosynthetic rates in infected plants [8]. Interestingly, this strain was produced using protoplast fusion in order to be highly rhizosphere competent that also possess substantial ability to compete with spermosphere bacteria. Strains that were fused were *T. harzianum* T-95 and T-12 because first was a rhizosphere competent mutant produced from a strain isolated from a *Rhizoctonia*suppressive Colombian soil, and second was more capable of competing with spermosphere bacteria than T-95 under iron-limiting conditions [14, 15]. The novel generation of RootShield is based on two *Trichoderma* species and contains strain *T. virens* G-41 together with strain *T. harzianum* T-22. Commercial name of product is RootShield PLUS and some with instruction that should be applied to disease-free plants previously chemically treated with fungicide, because the aggressively growing strains T-22 and G-41 are growing on the outside of roots and do not enter the

The availability and dispersion of *Trichoderma*-based biofertilizers are more

widespread then commonly known with the tendency to expand due to the easier registrations. Mostly permitted for use in organic farming in Europe is: RootShield, Plant Box and Bio Trek (northern Europe, USA), Binap (Switzerland, Sweden, UK, USA), Bio fungus (Belgium), Supersivit (Czech Republic), Trichodex

(Italy), Trifender (Hungary), and Trianum (Avantagro, Spain) [16]. Novel *Trichoderma* formulations are not based on single culture of one species but come as consortia or mix of at least two or three species or different strains of same species. Compatible consortia of compatible strains with different mechanisms, disease suppressive, or plant growth promoting, which complementary each other were found to be more effective than the application of individual organisms. Mixtures for biocontrol have a broadened range of pathogens against which are effective. Mixtures in plant growth promoters are based on insight that metabolic products of various *Trichoderma* strains are not identical and they have selective character to different plant species and even a variety [17, 18]. Seems this may be due to better interaction of some *Trichoderma* species or some strains with certain plant species because root exudates may induce or inhibit their mycelial growth [19]. In development are other types of consortia, mixtures of *Trichoderma* strains with other organisms, fungi or bacteria that are known as bioagent also. Because knowing of action mechanisms allows combining of strains with different modes of action in order to anticipate efficacy of final product, investigations of effectiveness differences between species and biotypes of same species are in progress.

**3.** *Trichoderma* **as opportunistic plant symbiont**

Mycoparasitic and nematophagous *Trichoderma* species found their prey in rhizosphere where roots are resort of plant pathogenic fungi and nematodes. Therefore, it becomes usual to define species of genus *Trichoderma* as free living rhizosphere organisms which colonize plant root surface as opportunistic plant symbionts. Biocontrol of plant pathogens by *Trichoderma* was from the first Weindling report in 1932 [1] considered as the direct ability of these fungi to interact with soil pathogens. Along with revelation of diverse antifungal mechanisms of

**18**

plant tissue.

*Trichoderma*, the ability to promote plant growth and to increase plant height, leaf area, and dry weight were perceived. Positive influence of *Trichoderma* to a faster germination and increase in percentage of emergency were perceived also.

Abundant healthy roots are from environment where *Trichoderma* grows and proliferates best. The sucrose that leaks from roots stimulates growth of *Trichoderma* mycelium and leads to interaction with plant. In order to stimulate plant to provide more sucrose, *Trichoderma* has evolved numerous mechanisms for better routing. With its enzyme arsenal, *Trichoderma* enhances solubility of soil nutrients which will be otherwise unavailable to plant. Further, *Trichoderma* enhances nutrient uptake by plant and better plant nourishment that will result in stronger routing which are frequently associated with increase in yield [20]. Special benefit is induction of increased nitrogen use efficiency in plants. This mechanism is not enlightened yet but probably is connected to *Trichoderma* stimulation of deeper rooting, and thereby increasing the volume of soil colonized by plant roots. Plants approximately take up only 33% of the amount of applied nitrogen fertilizer while some field trial data on *Trichoderma* treatments in several different crops indicate possibility of reduction nitrogen application rates by 30–50% with no reduction in yield [21, 22]. Calculation says that if this reduction was applied to the 30 million hectares of wheat in the USA, the savings in nitrogen application would total more than a billion of nitrogen kilos annually.

Sucrose metabolism increased by *Trichoderma* stimulates the resistance response in the leaves that leads to increased photosynthesis and respiration because growth induced by *Trichoderma* plant requires energy, and sunlight energy utilized in increased photosynthesis will be the energy source needed for plant growth enhancement by *Trichoderma* mechanisms. Of course, better photosynthesis enables that more sucrose is translocating to the roots and metabolic circle continues. Increased leaf mass enables increased photosynthesis but the *Trichoderma* has the abilities to increase photosynthetic efficiency [21, 23]. It was demonstrated that electron flow was substantially increased by root colonization and electron transport strongly enhanced [24]. In trial with barley exposed to water deficiency *T. harzianum* substantially increases water deficit tolerance and consequently reduces effects on photosynthetic systems even when plants were at or approaching the permanent wilting point after 2 weeks of withholding irrigation. Therefore, plants benefit from relationship with *Trichoderma* through increased root, shoot, and leaf growth and increased macro- and micronutrient uptake and disease protection as well.

First scientific papers about effects of *Trichoderma* on plant growth promotion, mostly of horticultural crops and conifers, began to appear in 1980s and continuing in 1990s [18, 25–28]. There is ample documentation for the *Trichoderma* influence on plants which are colonized with effective *Trichoderma* strain and that they are substantially different from an uncolonize plants in quality and quantity of yield, withstand to adverse environmental conditions and pathogen attack. Positive influence of *Trichoderma* to a faster germination and increase in percentage of emergency were perceived also [17, 18, 29].

In Croatia, investigations with autochthon *Trichoderma* strains and their influence to growth of fiber flax, lettuce, tomato, cabbage, and red beet were conducted [16, 30–32]. Significant increase of some lettuce quality characteristics was gained with *T. viride* strain TPS applied in the form of alginate-pellets in two frequently used commercial potting compost mixture: Klasmann-Deilmann P 002 (Germany) and Stender A240 (Germany). Except dry weight, TPS enhanced the formation of leaves: at Stender difference against control varies for 1–2 leaves more, at Klasmann for 1 leaf more. Leaf length was longer for 2 cm at Stender and Klasmann amended with TPS-pellets than at control, while leaf width was wider for 3.15 cm at Stender

and for 4.27 cm at Klasmann. Fresh weight was greater for 5.36 g at Stender and for 4.68 g at Klasmann against control. Dry weight was only characteristic on which TPS pellets did not have significant influence perhaps due to the similar nutrient content of Stedman and Klasmann substrates. These substrates are characterized by the use of fine peat with the addition of nutrient specially designed to meet the needs of young plants, so they similar in nutrient content (N 150–260 mg l<sup>−</sup><sup>1</sup> , P 180–280 mg l<sup>−</sup><sup>1</sup> , K 200–350 mg l<sup>−</sup><sup>1</sup> , and Mg 80–150 mg l<sup>−</sup><sup>1</sup> ). *Trichoderma* is able to solubilize nutrients but only the ones present in substrate, and as Klasmann and Stender are enriched with the similar nutrients, there were no significant differences among them as trial variants. The differences were bespeaking when the TPS pellets were added against control. In Croatian trial with cabbage and red beet, the indigenous *T. viride* isolates STP16 and STP8 enhanced plant growth in only one trial vegetation season, and results confirm the hypothesis that biotypes of same species differ in their abilities to induce plant growth, so that growth promotion of *Trichoderma* is not species dependent as well as that biotypes of same species differ in their abilities for inducing plant growth. Influence of strains STP16 and STP8 on cabbage growth was estimated by weighing the heads. Fresh weight was greater at STP16 treatment (FW = 1666.5 g) than at STP8 treatment (FW = 1372.5 g) but not statistically different although in comparison to control (FW = 1291 g) significantly increased. Dry weight was slightly but statistically significantly increased at STP16 treatment (DW = 8.2 g) against STP8 treatment (DW = 7.2 g) which was statistically equal to control (DW = 6.3 g). Growth promotion index showed that STP16 treatment promotes fresh weight for 29% while STP8 treatment only for 6.3% and dry weight for 30.16% while STP8 for 14.29%. Influence of those strains on red beet growth was estimated by weighing the root. Fresh weight was increased by both isolates, STP16 (FW = 725 g) and STP8 (FW = 607.5 g). There was no statistically significant difference between isolate influences although STP16 significantly increased fresh weight in comparison to control (FW = 569 g), while STP8 did not. Dry weight was greater at STP16 treatment (DW = 13.1 g) and statistically significant in comparison to STP8 treatment (DW = 12.2 g) and control (DW = 11.6 g). Growth promotion index showed that STP16 treatment increased root fresh weight for 27.42%, while STP8 treatment for only 6.44%. Index calculated for dry weight showed that STP16 increased dry weight for 12.93% and STP8 for 5.17%. In trial with fiber flax indigenous *T. harzianum* strain STP, applied in the form of alginate-pellets and through pelleted seeds, positively influenced germination, seedling emergence and plant growth. Seedling emergence from pelleted seeds were delayed, and on 7th day, after seeding, 16% were emerged, while at STP-pellet treatment, 66% emerged and at control 53%. In the presence of strain STP, plants grow higher (66 cm) than on control (62 cm) and the highest where plants from pelleted seeds (72 cm).

First, this ability was treated as side effect of suppression of plant pathogenic fungi [25, 33–35]. Other possible explanations of this phenomenon include: control of minor pathogens leading to stronger root growth and nutrient uptake [26], secretion of plant growth regulatory factors such as phytohormones [33, 34, 36, 37], and release of soil nutrients and minerals by increased saprophytic activity of *Trichoderma* in the soil. Removing of a toxic and inhibitory material to plant growth from the soil was also presented as interesting explanation of *T. harzianum* plant growth promotion [25].

Today, *Trichoderma* positive effect on plant growth is considered as independent ability and equally remarkable and significant as its antifungal ability because growth enhancement has been observed in the absence of any detectable disease and in sterile soil [17, 28, 31]. Novel genetically analysis shown that the most of *Trichoderma* biocontrol activity is through their abilities to induce plant defense mechanism described as systemic disease resistance [21]. For example, antibiosis of *T. virens* against

**21**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

benefits to plants for at least the life of an annual crop [8].

*Rhizoctonia solani* on cotton seedlings and mycoparasitism *T. harzianum* on *Pythium* were found to be due solely to induced resistance [22, 38]. Therefore, the intimate *Trichoderma*-plant relationship is what induces localized and systemic resistance plant responses to pathogen attack, promoting plant growth and support/encourage biocontrol. From now on, the meaning of biological control must be expanded to include induction of plant defense mechanism up to the disease resistance and plant growth promotion along with classical antagonism like antibiosis and mycoparasitism. Symbiotic *Trichoderma*-plant relationship results with effects that extend beyond biocontrol because *Trichoderma* directly influence plant physiology. That is why it is considered that they establish pseudomycorrhizal relationship with plant host. Species of *Trichoderma* are actually strong plant invaders and can colonize plant internally in endophytic manner with the ability to grow with plants. These allow much longer periods of efficacy than nonendophytic organisms and provide

*Trichoderma* species penetrate into the root cortex, epidermis, and a few cell layers below this level, based upon evidence with *Trichoderma* mutant strains that produce green fluorescent protein and electron microscopy and therefore are similar to endomycorrhizal fungi. Small protein from hydrophobin group is produced on the surface of *Trichoderma* hyphae and facilitates their attachment to root. For penetrations, *Trichoderma* uses appressoria which coil about root hairs and are similar to those observed in mycoparasitism. The *Trichoderma* enables further its entry with help of small protein swolenin TasSwo that recognize cellulose and modify plant cell structure. Entering the cells they have access to plant nutrients,

Penetration of *Trichoderma* hyphae into plant tissue is infection similar to one of other fungal plant pathogens but does not incite parasitism even though they have enzyme systems fully capable of macerating plant tissue. Although rarely phytopathogenic, one case of *T. virens* pathogenic on cotton seedlings was described and enlightened. It happened because protein responsible for antibiotic production, a single 18 kDa, that induces resistance was not expressed, so resistance was not induced [39, 40]. This nonpathogenic yet plant beneficial life style is a successful strategy for the fungus because it provides *Trichoderma* with more sucrose from enlarged roots due to better plant nourishment and also prey for mycoparasitic and nematophagous strains. Recently, endophytic *Trichoderma* strains are known which colonize vascular systems of certain plants [39] like it is known for other root colonizing biocontrol fungi such as binucleate *Rhizoctonia* and nonpathogenic *Fusarium* species [41, 42]. It was discovered that cocoa permits *Trichoderma* strain ramification throughout their structure. Mostly, plants will not permit that so the same strain will function only as root colonists applied to other plants [43].

After penetration into the root tissue, *Trichoderma* establishes chemical communication with the plant and interact at molecular level. Inside the root cells, *Trichoderma* can modify plant's gene expression to activate its immune system. These result initially in an induction of resistance mechanisms, so plant form thickened cell walls and produce phenolic depositions that intercept the *Trichoderma* to the area of infection and prevent further plant colonization [39, 44]. This is the type of plant localized resistance to *Trichoderma*. Therefore, disease development does not occur and this is asymptomatic infection but plant defense system is triggered. Plants have two immune systems: basal disease resistance is systemic acquired resistance (SAR) and the other is induced systemic resistance (ISR). SAR is induced

by pathogens and follows the salicylic acid pathway to reduce the severity of pathogenicity, while ISR follows jasmonic acid pathway. The first plant response to *Trichoderma* infection was found to increase in salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity. Infected plant cells recognize

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

which allows them to proliferate.

### *Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

*Trichoderma - The Most Widely Used Fungicide*

, K 200–350 mg l<sup>−</sup><sup>1</sup>

highest where plants from pelleted seeds (72 cm).

First, this ability was treated as side effect of suppression of plant pathogenic fungi [25, 33–35]. Other possible explanations of this phenomenon include: control of minor pathogens leading to stronger root growth and nutrient uptake [26], secretion of plant growth regulatory factors such as phytohormones [33, 34, 36, 37], and release of soil nutrients and minerals by increased saprophytic activity of *Trichoderma* in the soil. Removing of a toxic and inhibitory material to plant growth from the soil was also presented as interesting explanation of *T. harzianum* plant growth promotion [25]. Today, *Trichoderma* positive effect on plant growth is considered as independent ability and equally remarkable and significant as its antifungal ability because growth enhancement has been observed in the absence of any detectable disease and in sterile soil [17, 28, 31]. Novel genetically analysis shown that the most of *Trichoderma* biocontrol activity is through their abilities to induce plant defense mechanism described

as systemic disease resistance [21]. For example, antibiosis of *T. virens* against

P 180–280 mg l<sup>−</sup><sup>1</sup>

and for 4.27 cm at Klasmann. Fresh weight was greater for 5.36 g at Stender and for 4.68 g at Klasmann against control. Dry weight was only characteristic on which TPS pellets did not have significant influence perhaps due to the similar nutrient content of Stedman and Klasmann substrates. These substrates are characterized by the use of fine peat with the addition of nutrient specially designed to meet the needs of young plants, so they similar in nutrient content (N 150–260 mg l<sup>−</sup><sup>1</sup>

to solubilize nutrients but only the ones present in substrate, and as Klasmann and Stender are enriched with the similar nutrients, there were no significant differences among them as trial variants. The differences were bespeaking when the TPS pellets were added against control. In Croatian trial with cabbage and red beet, the indigenous *T. viride* isolates STP16 and STP8 enhanced plant growth in only one trial vegetation season, and results confirm the hypothesis that biotypes of same species differ in their abilities to induce plant growth, so that growth promotion of *Trichoderma* is not species dependent as well as that biotypes of same species differ in their abilities for inducing plant growth. Influence of strains STP16 and STP8 on cabbage growth was estimated by weighing the heads. Fresh weight was greater at STP16 treatment (FW = 1666.5 g) than at STP8 treatment (FW = 1372.5 g) but not statistically different although in comparison to control (FW = 1291 g) significantly increased. Dry weight was slightly but statistically significantly increased at STP16 treatment (DW = 8.2 g) against STP8 treatment (DW = 7.2 g) which was statistically equal to control (DW = 6.3 g). Growth promotion index showed that STP16 treatment promotes fresh weight for 29% while STP8 treatment only for 6.3% and dry weight for 30.16% while STP8 for 14.29%. Influence of those strains on red beet growth was estimated by weighing the root. Fresh weight was increased by both isolates, STP16 (FW = 725 g) and STP8 (FW = 607.5 g). There was no statistically significant difference between isolate influences although STP16 significantly increased fresh weight in comparison to control (FW = 569 g), while STP8 did not. Dry weight was greater at STP16 treatment (DW = 13.1 g) and statistically significant in comparison to STP8 treatment (DW = 12.2 g) and control (DW = 11.6 g). Growth promotion index showed that STP16 treatment increased root fresh weight for 27.42%, while STP8 treatment for only 6.44%. Index calculated for dry weight showed that STP16 increased dry weight for 12.93% and STP8 for 5.17%. In trial with fiber flax indigenous *T. harzianum* strain STP, applied in the form of alginate-pellets and through pelleted seeds, positively influenced germination, seedling emergence and plant growth. Seedling emergence from pelleted seeds were delayed, and on 7th day, after seeding, 16% were emerged, while at STP-pellet treatment, 66% emerged and at control 53%. In the presence of strain STP, plants grow higher (66 cm) than on control (62 cm) and the

, and Mg 80–150 mg l<sup>−</sup><sup>1</sup>

,

). *Trichoderma* is able

**20**

*Rhizoctonia solani* on cotton seedlings and mycoparasitism *T. harzianum* on *Pythium* were found to be due solely to induced resistance [22, 38]. Therefore, the intimate *Trichoderma*-plant relationship is what induces localized and systemic resistance plant responses to pathogen attack, promoting plant growth and support/encourage biocontrol. From now on, the meaning of biological control must be expanded to include induction of plant defense mechanism up to the disease resistance and plant growth promotion along with classical antagonism like antibiosis and mycoparasitism.

Symbiotic *Trichoderma*-plant relationship results with effects that extend beyond biocontrol because *Trichoderma* directly influence plant physiology. That is why it is considered that they establish pseudomycorrhizal relationship with plant host. Species of *Trichoderma* are actually strong plant invaders and can colonize plant internally in endophytic manner with the ability to grow with plants. These allow much longer periods of efficacy than nonendophytic organisms and provide benefits to plants for at least the life of an annual crop [8].

*Trichoderma* species penetrate into the root cortex, epidermis, and a few cell layers below this level, based upon evidence with *Trichoderma* mutant strains that produce green fluorescent protein and electron microscopy and therefore are similar to endomycorrhizal fungi. Small protein from hydrophobin group is produced on the surface of *Trichoderma* hyphae and facilitates their attachment to root. For penetrations, *Trichoderma* uses appressoria which coil about root hairs and are similar to those observed in mycoparasitism. The *Trichoderma* enables further its entry with help of small protein swolenin TasSwo that recognize cellulose and modify plant cell structure. Entering the cells they have access to plant nutrients, which allows them to proliferate.

Penetration of *Trichoderma* hyphae into plant tissue is infection similar to one of other fungal plant pathogens but does not incite parasitism even though they have enzyme systems fully capable of macerating plant tissue. Although rarely phytopathogenic, one case of *T. virens* pathogenic on cotton seedlings was described and enlightened. It happened because protein responsible for antibiotic production, a single 18 kDa, that induces resistance was not expressed, so resistance was not induced [39, 40]. This nonpathogenic yet plant beneficial life style is a successful strategy for the fungus because it provides *Trichoderma* with more sucrose from enlarged roots due to better plant nourishment and also prey for mycoparasitic and nematophagous strains. Recently, endophytic *Trichoderma* strains are known which colonize vascular systems of certain plants [39] like it is known for other root colonizing biocontrol fungi such as binucleate *Rhizoctonia* and nonpathogenic *Fusarium* species [41, 42]. It was discovered that cocoa permits *Trichoderma* strain ramification throughout their structure. Mostly, plants will not permit that so the same strain will function only as root colonists applied to other plants [43].

After penetration into the root tissue, *Trichoderma* establishes chemical communication with the plant and interact at molecular level. Inside the root cells, *Trichoderma* can modify plant's gene expression to activate its immune system. These result initially in an induction of resistance mechanisms, so plant form thickened cell walls and produce phenolic depositions that intercept the *Trichoderma* to the area of infection and prevent further plant colonization [39, 44]. This is the type of plant localized resistance to *Trichoderma*. Therefore, disease development does not occur and this is asymptomatic infection but plant defense system is triggered.

Plants have two immune systems: basal disease resistance is systemic acquired resistance (SAR) and the other is induced systemic resistance (ISR). SAR is induced by pathogens and follows the salicylic acid pathway to reduce the severity of pathogenicity, while ISR follows jasmonic acid pathway. The first plant response to *Trichoderma* infection was found to increase in salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity. Infected plant cells recognize

that they are under pathogen attack by detecting pathogen-associated molecular patterns (PAMPs), also called microbe-associated molecular patterns (MAMPs) which is more suitable to use for *Trichoderma.* This molecular pattern is essential for the pathogen life which is binding to pattern recognition receptors (PPT) on cell surface, and this triggers basic immunity system SAR. PAMPs are not found in plant, so thus the plant's receptor recognized them as being potentially dangerous and this triggers the plant SAR [45]. Simultaneously, infected plant cell recognizes pathogen through toxins considered being effectors, and this triggers plant's effector-triggered immunity (ETI). ISR is triggered by infection of beneficial microorganisms such as *Trichoderma*, recognized by chitin presence and induced by jasmonic acid and ethylene by wound signals, which they transmit from roots to other plant's part. When *Trichoderma* penetrates root, the ISR is induced by pattern recognition receptors localized in the plasma membrane of plant cell. First response is hypersensitive reaction resulting from production of antimicrobial compounds intent to restrict/restrain the potential pathogen. Plant can identify *Trichoderma* by the following PAMPs: cellulases, chitinases, endopolygalacturonase, peptaibols, and 6-pentyl-α-pyrone [46]. More *Trichoderma* biocontrol compounds that are able to induce plant defenses and are connected with plant-beneficial effects are different proteins, ceratoplatanins, polygalacturonase, cellulose-binding-domain proteins, and nonactive xylanase, and secondary metabolites, peptaibiotics, pyrones, pyridines, and butenolides, The first discovered chemical communicator of *Trichoderma* was a 22 kDa xylanase, protein which induces localized resistance in plants [39]. The peptides or proteins that are effective have masses of 6.5, 18, 20, 32, and 42 kDa. Finding that many of them retained their activity as denatured proteins was lead to premise that particular amino acid sequences are the important factor in their activity rather than enzymatic function.

Once when this *Trichoderma*-root biochemical cross-talk begins, *Trichoderma* may influence plant response to other pathogens attack by increasing SAR and ETI [47]. Effective biocontrol strains of *Trichoderma* changes in the amplitude of plant ETI by using the zig-zag model proposed by Jones and Dangle [48]. *Trichoderma* strains that activate both ISR and SAR or only SAR were also known. In investigation of cucumber root colonization by *Trichoderma* applied at high concentrations, 28 proteins whose expression was affected were identified in cotyledons [49]. All were regulated by *Trichoderma*, and among them, 17 were found to be upregulated, while 11 were downregulated. Proteins differentially regulated by *Trichoderma* were involved in isoprenoid and ethylene biosynthesis, and in metabolism of photosynthesis, photorespiration, and carbohydrate. Important finding is that *Trichoderma* can influence plant's oxidizing, reactive chemical species containing oxygen (ROS). They are a natural by-product of the normal metabolism of oxygen which is important in cell signaling and homeostasis. ROS are influenced by plant responses to stress, mostly to abiotic environmental conditions, so their levels can drastically increase under stress. They have high energy and are unstable so easily react with other species, like biomolecules such as DNA, and oxidizing them. This is known as oxidative stress and may result in significant damage to cell structures. Involvement of *Trichoderma* in ROS scavenging enlightens why *Trichoderma* treated plants are better coping the stress by drought or salinity. One of the pathways is the glutathione-ascorbate cycle, and *Trichoderma* enhancement of enzymes in it will recycle antioxidants more rapidly and thereby reduce stresses effects. In trial with barley exposed to water deficiency, *T. harzianum* substantially increases water deficit tolerance and consequently reduces effects on photosynthetic systems even when plants were at or approaching the permanent wilting point after 2 weeks of withholding irrigation. That shows possible *Trichoderma* influence on photosynthesis. Plants enhanced by *Trichoderma* have increased leaf mass that enables increased

**23**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

photosynthesis, but *Trichoderma* also have the abilities to increase photosynthetic efficiency [21, 23]. It was demonstrated that electron flow was substantially increased by root colonization and electron transport strongly enhanced [43]. Some *Trichoderma* strains can counteract pathogen toxins (effectors) in two ways: inhibiting pathogenicity factors and influencing pathogen dispersal and nutrition, whereby the ETI is induced as mentioned earlier. *Trichoderma* can improve ETI by releasing compounds that plant receptors will recognize as pathogen effectors or causing faster response. Other strains can induce stronger immunologic plant response than pathogen, meaning they induce ISR more than pathogen induces SAR, by producing a variety of MAMPs such as hydrophobins, expansin-like proteins, secondary metabolites and enzymes having direct antimicrobial activity. There is interesting example of involvement of *T. arundinacea* in induction of plant defense. Its trichothecene toxin harzianum A has been recognized as MAMPs in tomato seedlings and activated both ISR and SAR and primed them against *B. cinerea* and *R. solani* [50]. Trichothecenes are produced by a number of fungal genera like *Fusarium*, *Stachybotrys*, and *Myrothecium*. So far, *Trichoderma* is the only trichothecene producer in which the tri5 gene is not located in the main tri cluster. *Trichoderma* species produces two trichothecene toxins: harzianum A

Evidently, *Trichoderma* antifungal activity against phytopathogenic fungi and nematodes, as well as competition for nutrients conferring a nutritional advantage, are carried out by production of extracellular hydrolytic enzymes and/or the production of secondary metabolites with antifungal activity. It is postulated that the *Trichoderma* ability to act as soil colonizer, pseudomycorrhizal relationship with plant host with well being for plant health and fitness, mycoparasite and nematofag are to be provided by *Trichoderma* genome. Genome coevolution has been demonstrated in many plant-pathogen interactions, so it can be considered in the case of some plant and *Trichoderma* species. Strong genetic components to the responses of at last maize to *T. harzianum* T22 were confirmed in trials with a series of inbred lines which were preceded by large trial conducted in U.S. corn-belt with 160 maize hybrids and T22 [44]. Today, several hundred separate plant genes or proteins are known whose expression is altered by *Trichoderma* root colonization although the expression consequences are more pronounce in the shoots than in the roots [22]. Considering all described, it is evident that proteomics study is need to give an understanding of how *Trichoderma*-treated plants become more resistant to

Scientific articles describing excellent antifungal *Trichoderma* efficacy against some phytopathogenic fungi or enhancement of plant growth and yield are published worldwide each year. Many originate from countries having economics primarily based on agriculture and describe mostly experimental results achieved by native strain or created product based on it and may not be applicable on largescale crop production. Furthermore, novel articles summarize biocontrol programs, models of commercialization, and registration requirements. Dominantly is emphasizing that full-scale production, marketing, and registration requirements are unfavorable for products of biocontrol agents and simply too expensive, especially for the agricultural-based countries in which they are needed the most. All those articles clearly show that isolation, experimenting to evaluate biocontrol activity of strain and encapsulating it in formulations for low-scale trial needs are achievable part of developing biocontrol product. To be able to overcome issues on product

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

and trichothecene.

pathogen attacks.

**4. Applying** *Trichoderma* **in agriculture**

### *Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

*Trichoderma - The Most Widely Used Fungicide*

their activity rather than enzymatic function.

that they are under pathogen attack by detecting pathogen-associated molecular patterns (PAMPs), also called microbe-associated molecular patterns (MAMPs) which is more suitable to use for *Trichoderma.* This molecular pattern is essential for the pathogen life which is binding to pattern recognition receptors (PPT) on cell surface, and this triggers basic immunity system SAR. PAMPs are not found in plant, so thus the plant's receptor recognized them as being potentially dangerous and this triggers the plant SAR [45]. Simultaneously, infected plant cell recognizes pathogen through toxins considered being effectors, and this triggers plant's effector-triggered immunity (ETI). ISR is triggered by infection of beneficial microorganisms such as *Trichoderma*, recognized by chitin presence and induced by jasmonic acid and ethylene by wound signals, which they transmit from roots to other plant's part. When *Trichoderma* penetrates root, the ISR is induced by pattern recognition receptors localized in the plasma membrane of plant cell. First response is hypersensitive reaction resulting from production of antimicrobial compounds intent to restrict/restrain the potential pathogen. Plant can identify *Trichoderma* by the following PAMPs: cellulases, chitinases, endopolygalacturonase, peptaibols, and 6-pentyl-α-pyrone [46]. More *Trichoderma* biocontrol compounds that are able to induce plant defenses and are connected with plant-beneficial effects are different proteins, ceratoplatanins, polygalacturonase, cellulose-binding-domain proteins, and nonactive xylanase, and secondary metabolites, peptaibiotics, pyrones, pyridines, and butenolides, The first discovered chemical communicator of *Trichoderma* was a 22 kDa xylanase, protein which induces localized resistance in plants [39]. The peptides or proteins that are effective have masses of 6.5, 18, 20, 32, and 42 kDa. Finding that many of them retained their activity as denatured proteins was lead to premise that particular amino acid sequences are the important factor in

Once when this *Trichoderma*-root biochemical cross-talk begins, *Trichoderma* may influence plant response to other pathogens attack by increasing SAR and ETI [47]. Effective biocontrol strains of *Trichoderma* changes in the amplitude of plant ETI by using the zig-zag model proposed by Jones and Dangle [48]. *Trichoderma* strains that activate both ISR and SAR or only SAR were also known. In investigation of cucumber root colonization by *Trichoderma* applied at high concentrations, 28 proteins whose expression was affected were identified in cotyledons [49]. All were regulated by *Trichoderma*, and among them, 17 were found to be upregulated, while 11 were downregulated. Proteins differentially regulated by *Trichoderma* were involved in isoprenoid and ethylene biosynthesis, and in metabolism of photosynthesis, photorespiration, and carbohydrate. Important finding is that *Trichoderma* can influence plant's oxidizing, reactive chemical species containing oxygen (ROS). They are a natural by-product of the normal metabolism of oxygen which is important in cell signaling and homeostasis. ROS are influenced by plant responses to stress, mostly to abiotic environmental conditions, so their levels can drastically increase under stress. They have high energy and are unstable so easily react with other species, like biomolecules such as DNA, and oxidizing them. This is known as oxidative stress and may result in significant damage to cell structures. Involvement of *Trichoderma* in ROS scavenging enlightens why *Trichoderma* treated plants are better coping the stress by drought or salinity. One of the pathways is the glutathione-ascorbate cycle, and *Trichoderma* enhancement of enzymes in it will recycle antioxidants more rapidly and thereby reduce stresses effects. In trial with barley exposed to water deficiency, *T. harzianum* substantially increases water deficit tolerance and consequently reduces effects on photosynthetic systems even when plants were at or approaching the permanent wilting point after 2 weeks of withholding irrigation. That shows possible *Trichoderma* influence on photosynthesis. Plants enhanced by *Trichoderma* have increased leaf mass that enables increased

**22**

photosynthesis, but *Trichoderma* also have the abilities to increase photosynthetic efficiency [21, 23]. It was demonstrated that electron flow was substantially increased by root colonization and electron transport strongly enhanced [43].

Some *Trichoderma* strains can counteract pathogen toxins (effectors) in two ways: inhibiting pathogenicity factors and influencing pathogen dispersal and nutrition, whereby the ETI is induced as mentioned earlier. *Trichoderma* can improve ETI by releasing compounds that plant receptors will recognize as pathogen effectors or causing faster response. Other strains can induce stronger immunologic plant response than pathogen, meaning they induce ISR more than pathogen induces SAR, by producing a variety of MAMPs such as hydrophobins, expansin-like proteins, secondary metabolites and enzymes having direct antimicrobial activity. There is interesting example of involvement of *T. arundinacea* in induction of plant defense. Its trichothecene toxin harzianum A has been recognized as MAMPs in tomato seedlings and activated both ISR and SAR and primed them against *B. cinerea* and *R. solani* [50]. Trichothecenes are produced by a number of fungal genera like *Fusarium*, *Stachybotrys*, and *Myrothecium*. So far, *Trichoderma* is the only trichothecene producer in which the tri5 gene is not located in the main tri cluster. *Trichoderma* species produces two trichothecene toxins: harzianum A and trichothecene.

Evidently, *Trichoderma* antifungal activity against phytopathogenic fungi and nematodes, as well as competition for nutrients conferring a nutritional advantage, are carried out by production of extracellular hydrolytic enzymes and/or the production of secondary metabolites with antifungal activity. It is postulated that the *Trichoderma* ability to act as soil colonizer, pseudomycorrhizal relationship with plant host with well being for plant health and fitness, mycoparasite and nematofag are to be provided by *Trichoderma* genome. Genome coevolution has been demonstrated in many plant-pathogen interactions, so it can be considered in the case of some plant and *Trichoderma* species. Strong genetic components to the responses of at last maize to *T. harzianum* T22 were confirmed in trials with a series of inbred lines which were preceded by large trial conducted in U.S. corn-belt with 160 maize hybrids and T22 [44]. Today, several hundred separate plant genes or proteins are known whose expression is altered by *Trichoderma* root colonization although the expression consequences are more pronounce in the shoots than in the roots [22]. Considering all described, it is evident that proteomics study is need to give an understanding of how *Trichoderma*-treated plants become more resistant to pathogen attacks.

### **4. Applying** *Trichoderma* **in agriculture**

Scientific articles describing excellent antifungal *Trichoderma* efficacy against some phytopathogenic fungi or enhancement of plant growth and yield are published worldwide each year. Many originate from countries having economics primarily based on agriculture and describe mostly experimental results achieved by native strain or created product based on it and may not be applicable on largescale crop production. Furthermore, novel articles summarize biocontrol programs, models of commercialization, and registration requirements. Dominantly is emphasizing that full-scale production, marketing, and registration requirements are unfavorable for products of biocontrol agents and simply too expensive, especially for the agricultural-based countries in which they are needed the most. All those articles clearly show that isolation, experimenting to evaluate biocontrol activity of strain and encapsulating it in formulations for low-scale trial needs are achievable part of developing biocontrol product. To be able to overcome issues on product

industrialization and commercial and registration requirements, *Trichoderma* research community depends on stakeholders for investments in that part of developing process and industrial linkages. That's why the use of *Trichoderma*-products in developing countries falls under local production model for using microbial agents. This model is one of the four economic models for using microbial agents proposed by Hartman et al. [8].

First model is the microbial pesticide model which implies full registration of microbial product as pesticide and marketing worldwide. It is established in developed countries in USA, Canada, and EU; although in USA, it is substantially different. Registration of microbial products as pesticides is based on the interpretation of the term "pesticide." It does not necessarily refer to killing (e.g., fungicide) or inhibiting (e.g., fungistatic) pest only that it is controlled. Therefore, *Trichoderma*products fall into the scope of pesticide although registration requirements will be substantially less than those for synthetic chemicals, but remain difficult. Good example for this model is T22-product, and data that approximately \$12 million were required for registration, development of product facility, formulation, and marketing system before its sales began to grow. In USA, using the microbial pesticide model requires a minimum of \$8 million and 3–6 years before highly effective product is established in the market. In Canada in EU, registration regulatory also requires efficacy evaluation with toxicological and environmental testing, while in USA, they are required over time. Efficacy tests are required for almost every crop-pathogen combination and for *Trichoderma*-based products with broad capabilities on many crops and pathogens; this limits or even precludes registration and even makes it almost impossible from a financial standpoint. On the other hand, marketing them as plant-growth enhancements and strengthening agents gives them a market advantage because registration requirements like time and efficacy tests on pathogens are excluded. Although, in the USA, mycorrhizal fungi and rhizobia are not subject to regulatory approval for use while in EU and Canada are needed. Need to overcome the expensive efficacy evaluation of *Trichoderma*products led to creation of model named *Inoculants, plant strengthening agents and biofertilizers*. This model is used in various agricultural systems where *Trichoderma* products are marketed as plant inoculants for improvement of plant performance, but the pesticidial claims are not made although their diseases control benefits and well known. This gives those product marketplace advantages because many necessities for registration are avoided and therefore takes less time to reach the marketplace. Sales of *Trichoderma*-products may be larger than the sales of registered products. Great example is the product based on strain T22 which was registered as biofungicide in USA but in EU was just the beginning registration process in 2010. Until then, it was sold as plant strengthening agent named Triannum although its biofungicidal activities were known. As reason for delayed EU registration product, authors instigate economic because that the return on investment for full European registration was unlike to occur. In comparison to microbial pesticide model, the plant inoculants model leak two steps required for registration. Both models have followed steps: identification of good agent; development of production and formulation system; patenting of strain and/or process; building large-scale production and nationwide or international marketing, but steps: toxicology and other testing and registration are required only in microbial pesticide model. Local production model has only one step—discovery of good strain. As name tells, the production of autochthon strain is local. Strains are grown and multiplied in order with wellknown methods for semi-solid cultivation on wheat or corn bran, rice or similar substrate or in liquid fermentation. Cultivation and growing the required amount of inoculum is timed to be delivered directly before the application, date can be ordered by the grower, what eliminates extensive production and formulating. This

**25**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

model describes production of plan growth promotion rhizobacteria (PGPR) and *Trichoderma*-formulation at Faculty of Agriculture University of Zagreb in Croatia. The last one is in Croatia produced only by this chapter author. Although it is regulated by the government, local production model is different from model named *Governmental monopolies or state-supported production*. This model is based on Cuban rapidly shift from conventional agriculture to semi-organic farming. After collapse of trade relations in 1989, Cuba sets in economic and food crisis. As the pesticide and fertilizer imports were reduced to more than 80%, they established alternative agricultural technology and urban agriculture (composed of about 8000 gardens nationwide) with biological pest control practices supported with production on biopesticides and biofertilizers on a large scale. During 1996, Havana's urban farms provided the city's urban population with 8500 tons of agricultural produce, 4 million dozens of flowers, 7.5 million eggs, and 3650 tons of meat. This averted the crisis as it was said in Ref. [8, 51, 52] "It thus helps refute the most common argument—that we couldn't "feed the hungry" without pesticides—against taking the "ethical" position in real-world pest management policy debates." In Venezuela, a complete on large-scale implementation of biopesticide/biofertilizer development program has been funded and run through the Instituto Nacional de Investigaciones Agricola. In India, government is promoting all steps connected to adoption of biopesticides through various types of legislative, like the National Farmer Policy.

Members of genus *Trichoderma* are among the most prevalent cultivable fungi in soils, based upon the frequency of isolation on suitable media. They are present in all types of temperate and tropical soils and constituted up to 3% of the total fungal propagules (mycobiota) in forest and 1.5% in pasture soils, and their populations

isolated from soil, dominantly forest soil, and organic substrates form rhizosphere: alive, dead, healthy, or diseased plant tissue (root, green parts), common on wood decaying and fungal structures (fruiting bodies, sclerotia, and mycelial mats) [53]. Mentioned sclerotia and mycelial mats can be used as bates in search for antagonist for specific fungal pathogen. For that purpose, those mycelial structures are burying in natural soil sample. In looking for strain for biocontrol, it is recommended to identify the problem, the target pathogen (soil borne or aerial, source of inoculum, biology, and epidemiology) and its niche, host crop, and environment conditions. The most effective strains are sought in the geographic center of plant origin because as pathogens are coevolving with the host, their antagonist coevolving also. Strain can be isolated from stromata of teleomorph *Hypocrea* which are often found on wood and less frequently on some Basidiomycetous fungi (sedges, bracket). Isolating process is not inexpensive, requires time, and needs labor. When isolating from various sources in nature, one must have in mind that it is easy to obtain mixed cultures of *Trichoderma* species, as well as teleomorphic *Hypocrea* state, because they usually intermingle. To proceed investigation with pure culture, it is necessary to grow culture from single spore, conidia, chlamydospores, or better ascospores if it is possible and even hyphal tips. This can be achieved using dilution series of soils, root, and other plant tissue macerates suspensions of fungal structures which can be then plated on potato dextrose agar (PDA), corn meal agar (CMA) or *Trichoderma* medium E (TME) or other selective methods have been devised by numbered authors. For example, there is selective media developed to distinguish "P" strains of *T. virens* that produce the antibiotic gliovirin and are effective against *Pythium*, than "Q" strains that produce gliotoxin and are effective against *Rhizoctonia*. Cultivation of strain and multiplication for further tests is not difficult, and the methods

per gram of soil. Therefore, *Trichoderma* strains are mostly

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

**4.1 Assay for biological activity**

to 103

range from 102

*Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

*Trichoderma - The Most Widely Used Fungicide*

proposed by Hartman et al. [8].

industrialization and commercial and registration requirements, *Trichoderma* research community depends on stakeholders for investments in that part of developing process and industrial linkages. That's why the use of *Trichoderma*-products in developing countries falls under local production model for using microbial agents. This model is one of the four economic models for using microbial agents

First model is the microbial pesticide model which implies full registration of microbial product as pesticide and marketing worldwide. It is established in developed countries in USA, Canada, and EU; although in USA, it is substantially different. Registration of microbial products as pesticides is based on the interpretation of the term "pesticide." It does not necessarily refer to killing (e.g., fungicide) or inhibiting (e.g., fungistatic) pest only that it is controlled. Therefore, *Trichoderma*products fall into the scope of pesticide although registration requirements will be substantially less than those for synthetic chemicals, but remain difficult. Good example for this model is T22-product, and data that approximately \$12 million were required for registration, development of product facility, formulation, and marketing system before its sales began to grow. In USA, using the microbial pesticide model requires a minimum of \$8 million and 3–6 years before highly effective product is established in the market. In Canada in EU, registration regulatory also requires efficacy evaluation with toxicological and environmental testing, while in USA, they are required over time. Efficacy tests are required for almost every crop-pathogen combination and for *Trichoderma*-based products with broad capabilities on many crops and pathogens; this limits or even precludes registration and even makes it almost impossible from a financial standpoint. On the other hand, marketing them as plant-growth enhancements and strengthening agents gives them a market advantage because registration requirements like time and efficacy tests on pathogens are excluded. Although, in the USA, mycorrhizal fungi and rhizobia are not subject to regulatory approval for use while in EU and Canada are needed. Need to overcome the expensive efficacy evaluation of *Trichoderma*products led to creation of model named *Inoculants, plant strengthening agents and biofertilizers*. This model is used in various agricultural systems where *Trichoderma* products are marketed as plant inoculants for improvement of plant performance, but the pesticidial claims are not made although their diseases control benefits and well known. This gives those product marketplace advantages because many necessities for registration are avoided and therefore takes less time to reach the marketplace. Sales of *Trichoderma*-products may be larger than the sales of registered products. Great example is the product based on strain T22 which was registered as biofungicide in USA but in EU was just the beginning registration process in 2010. Until then, it was sold as plant strengthening agent named Triannum although its biofungicidal activities were known. As reason for delayed EU registration product, authors instigate economic because that the return on investment for full European registration was unlike to occur. In comparison to microbial pesticide model, the plant inoculants model leak two steps required for registration. Both models have followed steps: identification of good agent; development of production and formulation system; patenting of strain and/or process; building large-scale production and nationwide or international marketing, but steps: toxicology and other testing and registration are required only in microbial pesticide model. Local production model has only one step—discovery of good strain. As name tells, the production of autochthon strain is local. Strains are grown and multiplied in order with wellknown methods for semi-solid cultivation on wheat or corn bran, rice or similar substrate or in liquid fermentation. Cultivation and growing the required amount of inoculum is timed to be delivered directly before the application, date can be ordered by the grower, what eliminates extensive production and formulating. This

**24**

model describes production of plan growth promotion rhizobacteria (PGPR) and *Trichoderma*-formulation at Faculty of Agriculture University of Zagreb in Croatia. The last one is in Croatia produced only by this chapter author. Although it is regulated by the government, local production model is different from model named *Governmental monopolies or state-supported production*. This model is based on Cuban rapidly shift from conventional agriculture to semi-organic farming. After collapse of trade relations in 1989, Cuba sets in economic and food crisis. As the pesticide and fertilizer imports were reduced to more than 80%, they established alternative agricultural technology and urban agriculture (composed of about 8000 gardens nationwide) with biological pest control practices supported with production on biopesticides and biofertilizers on a large scale. During 1996, Havana's urban farms provided the city's urban population with 8500 tons of agricultural produce, 4 million dozens of flowers, 7.5 million eggs, and 3650 tons of meat. This averted the crisis as it was said in Ref. [8, 51, 52] "It thus helps refute the most common argument—that we couldn't "feed the hungry" without pesticides—against taking the "ethical" position in real-world pest management policy debates." In Venezuela, a complete on large-scale implementation of biopesticide/biofertilizer development program has been funded and run through the Instituto Nacional de Investigaciones Agricola. In India, government is promoting all steps connected to adoption of biopesticides through various types of legislative, like the National Farmer Policy.

### **4.1 Assay for biological activity**

Members of genus *Trichoderma* are among the most prevalent cultivable fungi in soils, based upon the frequency of isolation on suitable media. They are present in all types of temperate and tropical soils and constituted up to 3% of the total fungal propagules (mycobiota) in forest and 1.5% in pasture soils, and their populations range from 102 to 103 per gram of soil. Therefore, *Trichoderma* strains are mostly isolated from soil, dominantly forest soil, and organic substrates form rhizosphere: alive, dead, healthy, or diseased plant tissue (root, green parts), common on wood decaying and fungal structures (fruiting bodies, sclerotia, and mycelial mats) [53]. Mentioned sclerotia and mycelial mats can be used as bates in search for antagonist for specific fungal pathogen. For that purpose, those mycelial structures are burying in natural soil sample. In looking for strain for biocontrol, it is recommended to identify the problem, the target pathogen (soil borne or aerial, source of inoculum, biology, and epidemiology) and its niche, host crop, and environment conditions. The most effective strains are sought in the geographic center of plant origin because as pathogens are coevolving with the host, their antagonist coevolving also. Strain can be isolated from stromata of teleomorph *Hypocrea* which are often found on wood and less frequently on some Basidiomycetous fungi (sedges, bracket). Isolating process is not inexpensive, requires time, and needs labor. When isolating from various sources in nature, one must have in mind that it is easy to obtain mixed cultures of *Trichoderma* species, as well as teleomorphic *Hypocrea* state, because they usually intermingle. To proceed investigation with pure culture, it is necessary to grow culture from single spore, conidia, chlamydospores, or better ascospores if it is possible and even hyphal tips. This can be achieved using dilution series of soils, root, and other plant tissue macerates suspensions of fungal structures which can be then plated on potato dextrose agar (PDA), corn meal agar (CMA) or *Trichoderma* medium E (TME) or other selective methods have been devised by numbered authors. For example, there is selective media developed to distinguish "P" strains of *T. virens* that produce the antibiotic gliovirin and are effective against *Pythium*, than "Q" strains that produce gliotoxin and are effective against *Rhizoctonia*. Cultivation of strain and multiplication for further tests is not difficult, and the methods

described by Rifai (1969) are generally still followed. Recommended are inoculation on oatmeal, cornmeal or malt extract agars, and incubation under daylight for 5–7 days at a temperature of 20–25°C as they allow observation of stable morphological features [54–58]. Less expensive isolation is achieved when strain occurs naturally and is isolated from an area where they will be used after semi-solid cultivation, so it is connected to local production model. Hence, just isolation is not expensive but finding the useful strains in evaluating process is.

After isolation, bioassay for biological activity will be required to determine their nature, whether it is suppressing pathogens or enhancing plant growth, and if it is performed satisfactory. Moreover, the molecular identification of species is required so that the harmful (mushroom pathogens) and dangerous (trichothecene producers) ones can be avoided. Serious mushroom diseases can cause *T. aggressivum*, *T. pleurotum*, and *T. pleuroticola*. This species are genetically distinct from well known biocontrol strains [8, 59, 60]. Trichothecene production and their role in induction of plant defense were discussed earlier but it needs to be emphasizing that for registration and marketing strain must be nontoxic as microbial pesticides model requires toxicity testing. But, as most strains will be marketed under other production model, the potential for harm cannot be avoided entirely. Toxicity information is available for five *Trichoderma* species. Some testing reported acute oral toxicity as being >500 to <2.000 mg/kg, so at highest level, no effects were seen, and further, there have been no reported reactions after many years of extensive use. The species in *T. brevicompactum* complex are trichothecene producers, and they are as well not related to *Trichoderma* strains that are registered as bioagent. Immunosuppressive mycotoxin gliotoxin is produced by *T. virens* "Q" strains earlier mentioned, but they were no reported mycotoxicosis attributed to *T. virens* like it were for *Aspergillus* spp. Antibiotic important for biocontrol is volatile lactone and pyrone as it inhibits spore germination of *Phytophthora*, *Botrytis*, and other fungi. It is produced by *T. viride*/*H. rufa* clade only. Pyrone has pleasant coconut odor and is present in fruits, and it is used as flavoring agent, and therefore not considered as high hazard. In connection to plant defense system, peptaibols were mentioned earlier also. As they can lyses red blood cells that can be potentially harmful but it was reported that the syntheses are induced only by fungal cell walls or other elicitors, so again are connected with *Trichoderma* antifungal activity.

Considering all, finding a biologically useful and perspective strain for encapsulation into successful formulation which will be commercially viable is hard. *Trichoderma* has advantages because it can be easily isolated, grown, and tested for selection of efficient strains, manipulated and encapsulated in various formulations as they have good shelf life, which aids commercialization. Most strains used in bioapplications were local in origin and were used locally or regionally. Only smaller number of strains will show to be useful in various locations and environmental conditions, and therefore they become widely adopted and commercially available, like famous T22 and T39. So, what are the characteristics of perspective *Trichoderma* strain? One of the most important traits of beneficial culture is rhizosphere competence, the ability to survive in the environment, mostly rhizosphere. Rhizosphere competence or competitive saprobic ability is culture ability to compete other microbes in colonizing cellulose-rich substrates or the intercellular spaces of the surface layers of host roots. Therefore, *Trichoderma* culture would require cellulolytic enzymes to occupy this region and interact with the plant at a molecular level, either for plant growth promotion or inducing defense mechanism and inhibiting pathogenicity [57, 61]. Rhizosphere competence is always associated to cellulose production, and this is used for assessment of culture ability to metabolize cellulose. Method is simple as some cellulose materials (straw, cellophane discs) are buried into filed soil, then inoculate with a known quantity of fungal propagule

**27**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

produced is directly related to the culture rhizosphere competence.

(conidia, chlamydospores) and after incubation reisolate. The amount of cellulose

After isolation of strain with high rhizosphere and competitive saprophytic competence which is easily artificially mass multiplicative and safe for the environment, it must be perspective by its broad spectrum of activity. It can provide excellent and reliable control against a more of pathogens or/and can enhance growth of different plant species. The presentation must be of low effective doses. Classical method *in vitro* for assessment of biocontrol efficiency is "dual-culture" method. Probably the oldest one dual culture technique was described in 1955 by Morton and Strouble [62]. In one Petri plate, usually 10 mm diameter one, pathogen and *Trichoderma* are confronted as antagonist in the form of mycelial disc cut from the margin of the 7 days-old culture with a circular cutter. The mycelial disc is 5 mm in diameter but there are variations, so it can be 10 cm in diameter [63]. Both discs are placed on the substrate on the opposite of the plate, usually PDA, in Petri plate in the manner that the mycelium side facing the substrate and gently pressed in. The interspace between them amounted to 5 cm and interspace between micellar discs and edge of plate 2 cm. In control plate, *Trichoderma* disc is replaced with sterile agar disc. Inoculated plates are incubated at 25 ± 1°C for 7 days usually, although it could be 10–14 days depending on the pathogen species. Recommendable is to do readings on 2, 4, and 6 days after the incubation period by measuring radial growth of pathogen. The inhibition is quantified with index of inhibition (I) which represents inhibition percent of average pathogen radial growth (T) in the presence of *Trichoderma* and is calculated in relation to growth of the controls (C) as follows: I(%) = [(C − T)/C] × 100 [64]. Basic mechanisms can be determined: competition for substrate and nutrients as antagonist grow faster and colonize substrate; mycoparasitism if antagonist colonize pathogen mycelial mat, sclerotia or fruiting body and diminish and decay of those structure can be monitored; antibiosis when inhibition zone is formed because toxins spread through substrate and inhibited growth of pathogen. There is possibility to misinterpret mycoparasitic capability of the antagonist if neglected that it can absorb nutrients from the agar. Antibiosis by production of volatile metabolites can be tested using slight modification of dual culture technique [6]. The mycelial discs are placed centrally, but the *Trichoderma* disc is placed in the lid while pathogen disc on the bottom of the plate sealed

Furthermore, perspective culture needs to be compatible with other bioagents or tolerant to pesticides commonly used in agriculture which facilitates integrated control. For instance, it is recommended by commercial seed treatment systems to apply a chemical pesticide with the effective *Trichoderma* strains. While chemical will provide short term protection, the *Trichoderma* provides season-long benefits to plants as colonizes roots. Culture should even tolerate oxidizing agent, UV radiations, desiccation, heat, draught, etc. [21, 57, 65, 66]. Because these characteristics are strain depended, fewer than 1% of screened cultures will meet the expectations. Therefore, screening for bioactivity is the critical step when hundreds or thousands of cultures are tested. First, efficacy bioassays are performed in small-scale laboratory trials, *in vitro* and *in vivo,* which are usually random. Recommended is so called three-partner model, a system with potential *Trichoderma* agent, pathogen, and plant. Screening process is obviously time and money consuming as well as requires labor of more than one analyst. Molecular techniques of assisted selection using phenotypic or/and genotypic markers perhaps can improve screening productivity and shorten it but will raise the costs. Moreover, only in few cases, connection with bioactivity and molecular markers was proved. Few perspective cultures from lab-trials are proceeding to greenhouse and small pilot trials at open. At this stage, testing is conducted in different environmental conditions and crops and several

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

together with adhesive paraffin tape.

### *Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

*Trichoderma - The Most Widely Used Fungicide*

finding the useful strains in evaluating process is.

tors, so again are connected with *Trichoderma* antifungal activity.

Considering all, finding a biologically useful and perspective strain for encapsulation into successful formulation which will be commercially viable is hard. *Trichoderma* has advantages because it can be easily isolated, grown, and tested for selection of efficient strains, manipulated and encapsulated in various formulations as they have good shelf life, which aids commercialization. Most strains used in bioapplications were local in origin and were used locally or regionally. Only smaller number of strains will show to be useful in various locations and environmental conditions, and therefore they become widely adopted and commercially available, like famous T22 and T39. So, what are the characteristics of perspective *Trichoderma* strain? One of the most important traits of beneficial culture is rhizosphere competence, the ability to survive in the environment, mostly rhizosphere. Rhizosphere competence or competitive saprobic ability is culture ability to compete other microbes in colonizing cellulose-rich substrates or the intercellular spaces of the surface layers of host roots. Therefore, *Trichoderma* culture would require cellulolytic enzymes to occupy this region and interact with the plant at a molecular level, either for plant growth promotion or inducing defense mechanism and inhibiting pathogenicity [57, 61]. Rhizosphere competence is always associated to cellulose production, and this is used for assessment of culture ability to metabolize cellulose. Method is simple as some cellulose materials (straw, cellophane discs) are buried into filed soil, then inoculate with a known quantity of fungal propagule

described by Rifai (1969) are generally still followed. Recommended are inoculation on oatmeal, cornmeal or malt extract agars, and incubation under daylight for 5–7 days at a temperature of 20–25°C as they allow observation of stable morphological features [54–58]. Less expensive isolation is achieved when strain occurs naturally and is isolated from an area where they will be used after semi-solid cultivation, so it is connected to local production model. Hence, just isolation is not expensive but

After isolation, bioassay for biological activity will be required to determine their nature, whether it is suppressing pathogens or enhancing plant growth, and if it is performed satisfactory. Moreover, the molecular identification of species is required so that the harmful (mushroom pathogens) and dangerous (trichothecene producers) ones can be avoided. Serious mushroom diseases can cause *T. aggressivum*, *T. pleurotum*, and *T. pleuroticola*. This species are genetically distinct from well known biocontrol strains [8, 59, 60]. Trichothecene production and their role in induction of plant defense were discussed earlier but it needs to be emphasizing that for registration and marketing strain must be nontoxic as microbial pesticides model requires toxicity testing. But, as most strains will be marketed under other production model, the potential for harm cannot be avoided entirely. Toxicity information is available for five *Trichoderma* species. Some testing reported acute oral toxicity as being >500 to <2.000 mg/kg, so at highest level, no effects were seen, and further, there have been no reported reactions after many years of extensive use. The species in *T. brevicompactum* complex are trichothecene producers, and they are as well not related to *Trichoderma* strains that are registered as bioagent. Immunosuppressive mycotoxin gliotoxin is produced by *T. virens* "Q" strains earlier mentioned, but they were no reported mycotoxicosis attributed to *T. virens* like it were for *Aspergillus* spp. Antibiotic important for biocontrol is volatile lactone and pyrone as it inhibits spore germination of *Phytophthora*, *Botrytis*, and other fungi. It is produced by *T. viride*/*H. rufa* clade only. Pyrone has pleasant coconut odor and is present in fruits, and it is used as flavoring agent, and therefore not considered as high hazard. In connection to plant defense system, peptaibols were mentioned earlier also. As they can lyses red blood cells that can be potentially harmful but it was reported that the syntheses are induced only by fungal cell walls or other elici-

**26**

(conidia, chlamydospores) and after incubation reisolate. The amount of cellulose produced is directly related to the culture rhizosphere competence.

After isolation of strain with high rhizosphere and competitive saprophytic competence which is easily artificially mass multiplicative and safe for the environment, it must be perspective by its broad spectrum of activity. It can provide excellent and reliable control against a more of pathogens or/and can enhance growth of different plant species. The presentation must be of low effective doses. Classical method *in vitro* for assessment of biocontrol efficiency is "dual-culture" method. Probably the oldest one dual culture technique was described in 1955 by Morton and Strouble [62]. In one Petri plate, usually 10 mm diameter one, pathogen and *Trichoderma* are confronted as antagonist in the form of mycelial disc cut from the margin of the 7 days-old culture with a circular cutter. The mycelial disc is 5 mm in diameter but there are variations, so it can be 10 cm in diameter [63]. Both discs are placed on the substrate on the opposite of the plate, usually PDA, in Petri plate in the manner that the mycelium side facing the substrate and gently pressed in. The interspace between them amounted to 5 cm and interspace between micellar discs and edge of plate 2 cm. In control plate, *Trichoderma* disc is replaced with sterile agar disc. Inoculated plates are incubated at 25 ± 1°C for 7 days usually, although it could be 10–14 days depending on the pathogen species. Recommendable is to do readings on 2, 4, and 6 days after the incubation period by measuring radial growth of pathogen. The inhibition is quantified with index of inhibition (I) which represents inhibition percent of average pathogen radial growth (T) in the presence of *Trichoderma* and is calculated in relation to growth of the controls (C) as follows: I(%) = [(C − T)/C] × 100 [64]. Basic mechanisms can be determined: competition for substrate and nutrients as antagonist grow faster and colonize substrate; mycoparasitism if antagonist colonize pathogen mycelial mat, sclerotia or fruiting body and diminish and decay of those structure can be monitored; antibiosis when inhibition zone is formed because toxins spread through substrate and inhibited growth of pathogen. There is possibility to misinterpret mycoparasitic capability of the antagonist if neglected that it can absorb nutrients from the agar. Antibiosis by production of volatile metabolites can be tested using slight modification of dual culture technique [6]. The mycelial discs are placed centrally, but the *Trichoderma* disc is placed in the lid while pathogen disc on the bottom of the plate sealed together with adhesive paraffin tape.

Furthermore, perspective culture needs to be compatible with other bioagents or tolerant to pesticides commonly used in agriculture which facilitates integrated control. For instance, it is recommended by commercial seed treatment systems to apply a chemical pesticide with the effective *Trichoderma* strains. While chemical will provide short term protection, the *Trichoderma* provides season-long benefits to plants as colonizes roots. Culture should even tolerate oxidizing agent, UV radiations, desiccation, heat, draught, etc. [21, 57, 65, 66]. Because these characteristics are strain depended, fewer than 1% of screened cultures will meet the expectations. Therefore, screening for bioactivity is the critical step when hundreds or thousands of cultures are tested. First, efficacy bioassays are performed in small-scale laboratory trials, *in vitro* and *in vivo,* which are usually random. Recommended is so called three-partner model, a system with potential *Trichoderma* agent, pathogen, and plant. Screening process is obviously time and money consuming as well as requires labor of more than one analyst. Molecular techniques of assisted selection using phenotypic or/and genotypic markers perhaps can improve screening productivity and shorten it but will raise the costs. Moreover, only in few cases, connection with bioactivity and molecular markers was proved. Few perspective cultures from lab-trials are proceeding to greenhouse and small pilot trials at open. At this stage, testing is conducted in different environmental conditions and crops and several

pathogens in the case of biocontrol activity. Satisfaction of these very limiting conditions is important for future possible wide range of applicability of bioagent which is the most important property needed for registration and marketing. For commercialization, the bioagent should be produced on industrial scale.

### **4.2 Methods of formulation**

*Trichoderma* is mostly fermented in solid state with the aim to achieve highest yield with lower cost of culture medium. Obtained fungal biomass needs to be immobilized in certain carriers in low cost but high density inocula and encapsulated into formulation engineered to maintain fungal propagule viable during the transport, storage, and application. *Trichoderma* has advantages because it can be easily manipulated for encapsulation which means mixing wet or dry fungal biomass with a matrix forming material, such as gelatinized polysaccharide or an oil emulsion. Matrix is serving as carrier of fungal inoculums. Most of the examples on different types of *Trichoderma* inoculum for biocontrol include peat, granular vermiculite or clay mixtures, grains, and alginate pellets. Encapsulation in formulation of alginate pellets has been studied and positively evaluated by various authors as found to be successful for the *Trichoderma* delivery [30–32, 67–72]. In the receipt of chapter's author for small trial purpose, culture is grown on Petri dishes 10 cm in diameter containing 20 ml of PDA and incubated in humid chamber at 25°C for 7 days until conidiation. After incubation, the substrate altogether with hyphal biomass and conidia from two Petri dishes are upraised with spatula and transferred into glass with 50 ml sterile DI water. These are mixed by common blender at low speed for 3–5 min in order to make a suspension. The final concentration to be used contained 4 × 106 spores ml<sup>−</sup><sup>1</sup> . The suspension is mixed with 100 g l<sup>−</sup><sup>1</sup> talcum and 10 g l<sup>−</sup><sup>1</sup> sodium alginate. The formed matrix is placed in a separator funnel modified in order to allow suspension to drip into a 0.1 M suspension of calcium gluconate under stirring on magnetic agitator. Drops of alginate matrix dripped into calcium gluconate suspension transformed to gelatinized spherules or pellets. Pellets were removed from suspension within 10 min, rinsed with distilled water, and allowed to dry on waxed paper under a sterile vertical flow for 12–24 h [16, 30, 31]. In India is quite popular a talc-based formulation of *Trichoderma* developed at Tamil Nadu Agricultural University [64]. There are also oil-based formulations prepared with a combination of vegetable/mineral oils and they are suitable for foliar spraying under dry weather. Some formulations use organic wastes like coffee husk from coffee industry and press mud, byproduct of sugar factory.

Carriers of *Trichoderma* inoculum must be cheap, should dissolve well in water, and preserve fungal viability to insure formulation shelf-life. Formulation good for the commercialization should have increased shelf-life to fulfill requirements for storage and transport, and this is one of the most limiting factors. Further, it should deliver viable propagules in adequate concentration through adequate application. In the case of endophytic strains that grow with plant roots, only small amounts of inoculum are needed to be provided for long-term benefits. The minimal propagule number should not be less than 2 × 106 per milliliter or gram of formulation. Commercial preparations are mostly high concentrated with more than of 1010 propagules per gram. Thus, commercial preparations need to be applied in dose of 500 mg per hectare or for addition to greenhouse potting soils only 104 –105 per cm3 [21].

The most important act before commercialization is that formulation, or even strain/culture should be legally protected by means of patent. It is patented as biotechnological invention because it is based on microbe and its mechanisms or its metabolic products. The strain pure culture needs to be deposited in an officially recognized microbial collection by Budapest treaty signed by all countries

**29**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

98/44CEE on the patentability of biotechnological inventions.

pertaining to the World Intellectual Property Organization. It should be emphasized that patent is not authorization for commercial use, and it does not connote registration for agricultural use. All patents are regulated by legal treaties. There are series of international and national treaties: Union of Paris of 1883, Patent Cooperation Treaty of 1970, and European Patent Agreement of 1973. In the USA is United States Patents while in EU the legal framework is regulated by Directive

Present pest control intend to kill, even eradicate organisms but without choosing between harmful and benefit ones. Biocontrol could help in providing low-cost and environmentally safe technologies to farmers especially today when food security and rural livelihood are a key priority. Original paper updates on basic and applied research in all aspects of biological control of invertebrate, vertebrate and weed pests, and plant diseases can be found in the BioControl, the official journal of the International Organization for Biological Control (IOBC). For developing world, biopesticides and biofertilizer are considered extremely important as perceived by Association of Asian Pacific Agricultural Research Institution (AAPARI).

Thus are in progress biopesticide researches of the agriculturally important microorganisms led by the *Trichoderma* that can be encapsulated in bioproducts. Although the chemical control of plant diseases differs tremendously from biocontrol by microbial-based biopreparations, registration regulations remain the same or similar depending on country. In USA, the Environmental Protection Agency (EPA) registers pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). In EU, registration is defined by Regulatives 91/414/CEE, 2092/91, 1488/97, etc. In Croatia, it is defined by Plant Protection Law, which is harmonized with EU Regulatives. In general, registration of any product making a pesticidal claim is obligation defined by law in order to prevent unreasonable, adverse effects on consumer health, or the environment. Registration requires time, expenses, and efforts for conducting toxicological, environmental, and efficiency testing. Especially is difficult to enlighten excessive specificity of bioagent as success of control by any bioagent depends on three living system: pathogen, plant, and bioagent. In this new era, the meaning of biological control must be expanded to include plant growth promotion and disease resistance along with classical antibiosis and mycoparasitism because *Trichoderma* biocontrol and plant performances-enhancing activities overlap. Even better, *Trichoderma* research community emphasizes that considering biocontrol as primary ability of *Trichoderma* may influence biocontrol system in development because it means optimizing conditions for wrong mechanism. It must be taking into account that *Trichoderma*, as pseudomycorrhizal plant partner, has effects that extend beyond biocontrol. These fungi produce changes in plant metabolism, which have direct effects on plant physiology, like increasing growth and enhancing resistance, and even reprogramming of plant gene expression owing to coevolution with plants. Thus is especially pronounced that the control of plan response to abiotic and biotic stresses, such as diseases, is only a subset of their activities and benefits to plant. In future can be expected momentum of proteome study in order to help giving an understanding of how *Trichoderma* treated plants become more resistant to pathogen attacks. Also, needed is development of easy and inexpensive screening methods whose test conditions approach as much as possible the real system where biocontrol has to be. Registration requirements need to be revised and include fact that *Trichoderma*-fungicides are entering market as plant growth promoters. There are opinions that future of biopesticides

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

**5. Concluding remarks**

pertaining to the World Intellectual Property Organization. It should be emphasized that patent is not authorization for commercial use, and it does not connote registration for agricultural use. All patents are regulated by legal treaties. There are series of international and national treaties: Union of Paris of 1883, Patent Cooperation Treaty of 1970, and European Patent Agreement of 1973. In the USA is United States Patents while in EU the legal framework is regulated by Directive 98/44CEE on the patentability of biotechnological inventions.

### **5. Concluding remarks**

*Trichoderma - The Most Widely Used Fungicide*

**4.2 Methods of formulation**

contained 4 × 106

10 g l<sup>−</sup><sup>1</sup>

spores ml<sup>−</sup><sup>1</sup>

coffee industry and press mud, byproduct of sugar factory.

per hectare or for addition to greenhouse potting soils only 104

gule number should not be less than 2 × 106

pathogens in the case of biocontrol activity. Satisfaction of these very limiting conditions is important for future possible wide range of applicability of bioagent which is the most important property needed for registration and marketing. For

*Trichoderma* is mostly fermented in solid state with the aim to achieve highest yield with lower cost of culture medium. Obtained fungal biomass needs to be immobilized in certain carriers in low cost but high density inocula and encapsulated into formulation engineered to maintain fungal propagule viable during the transport, storage, and application. *Trichoderma* has advantages because it can be easily manipulated for encapsulation which means mixing wet or dry fungal biomass with a matrix forming material, such as gelatinized polysaccharide or an oil emulsion. Matrix is serving as carrier of fungal inoculums. Most of the examples on different types of *Trichoderma* inoculum for biocontrol include peat, granular vermiculite or clay mixtures, grains, and alginate pellets. Encapsulation in formulation of alginate pellets has been studied and positively evaluated by various authors as found to be successful for the *Trichoderma* delivery [30–32, 67–72]. In the receipt of chapter's author for small trial purpose, culture is grown on Petri dishes 10 cm in diameter containing 20 ml of PDA and incubated in humid chamber at 25°C for 7 days until conidiation. After incubation, the substrate altogether with hyphal biomass and conidia from two Petri dishes are upraised with spatula and transferred into glass with 50 ml sterile DI water. These are mixed by common blender at low speed for 3–5 min in order to make a suspension. The final concentration to be used

. The suspension is mixed with 100 g l<sup>−</sup><sup>1</sup>

per milliliter or gram of formulation.

–105

per cm3

[21].

sodium alginate. The formed matrix is placed in a separator funnel modified

in order to allow suspension to drip into a 0.1 M suspension of calcium gluconate under stirring on magnetic agitator. Drops of alginate matrix dripped into calcium gluconate suspension transformed to gelatinized spherules or pellets. Pellets were removed from suspension within 10 min, rinsed with distilled water, and allowed to dry on waxed paper under a sterile vertical flow for 12–24 h [16, 30, 31]. In India is quite popular a talc-based formulation of *Trichoderma* developed at Tamil Nadu Agricultural University [64]. There are also oil-based formulations prepared with a combination of vegetable/mineral oils and they are suitable for foliar spraying under dry weather. Some formulations use organic wastes like coffee husk from

Carriers of *Trichoderma* inoculum must be cheap, should dissolve well in water, and preserve fungal viability to insure formulation shelf-life. Formulation good for the commercialization should have increased shelf-life to fulfill requirements for storage and transport, and this is one of the most limiting factors. Further, it should deliver viable propagules in adequate concentration through adequate application. In the case of endophytic strains that grow with plant roots, only small amounts of inoculum are needed to be provided for long-term benefits. The minimal propa-

Commercial preparations are mostly high concentrated with more than of 1010 propagules per gram. Thus, commercial preparations need to be applied in dose of 500 mg

The most important act before commercialization is that formulation, or even strain/culture should be legally protected by means of patent. It is patented as biotechnological invention because it is based on microbe and its mechanisms or its metabolic products. The strain pure culture needs to be deposited in an officially recognized microbial collection by Budapest treaty signed by all countries

talcum and

commercialization, the bioagent should be produced on industrial scale.

**28**

Present pest control intend to kill, even eradicate organisms but without choosing between harmful and benefit ones. Biocontrol could help in providing low-cost and environmentally safe technologies to farmers especially today when food security and rural livelihood are a key priority. Original paper updates on basic and applied research in all aspects of biological control of invertebrate, vertebrate and weed pests, and plant diseases can be found in the BioControl, the official journal of the International Organization for Biological Control (IOBC). For developing world, biopesticides and biofertilizer are considered extremely important as perceived by Association of Asian Pacific Agricultural Research Institution (AAPARI). Thus are in progress biopesticide researches of the agriculturally important microorganisms led by the *Trichoderma* that can be encapsulated in bioproducts. Although the chemical control of plant diseases differs tremendously from biocontrol by microbial-based biopreparations, registration regulations remain the same or similar depending on country. In USA, the Environmental Protection Agency (EPA) registers pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). In EU, registration is defined by Regulatives 91/414/CEE, 2092/91, 1488/97, etc. In Croatia, it is defined by Plant Protection Law, which is harmonized with EU Regulatives. In general, registration of any product making a pesticidal claim is obligation defined by law in order to prevent unreasonable, adverse effects on consumer health, or the environment. Registration requires time, expenses, and efforts for conducting toxicological, environmental, and efficiency testing. Especially is difficult to enlighten excessive specificity of bioagent as success of control by any bioagent depends on three living system: pathogen, plant, and bioagent.

In this new era, the meaning of biological control must be expanded to include plant growth promotion and disease resistance along with classical antibiosis and mycoparasitism because *Trichoderma* biocontrol and plant performances-enhancing activities overlap. Even better, *Trichoderma* research community emphasizes that considering biocontrol as primary ability of *Trichoderma* may influence biocontrol system in development because it means optimizing conditions for wrong mechanism. It must be taking into account that *Trichoderma*, as pseudomycorrhizal plant partner, has effects that extend beyond biocontrol. These fungi produce changes in plant metabolism, which have direct effects on plant physiology, like increasing growth and enhancing resistance, and even reprogramming of plant gene expression owing to coevolution with plants. Thus is especially pronounced that the control of plan response to abiotic and biotic stresses, such as diseases, is only a subset of their activities and benefits to plant. In future can be expected momentum of proteome study in order to help giving an understanding of how *Trichoderma* treated plants become more resistant to pathogen attacks. Also, needed is development of easy and inexpensive screening methods whose test conditions approach as much as possible the real system where biocontrol has to be. Registration requirements need to be revised and include fact that *Trichoderma*-fungicides are entering market as plant growth promoters. There are opinions that future of biopesticides

lies in plant-protecting pesticides or self-protecting plant, a high-value crop plant with embedded genes from bioagent. Yet, are not the same concerns influencing transgenic plant and biopesticide in the light of biosafety? Nontarget effects, toxicity, and possible pathogenicity for plant, animals and humans, allergenicity or horizontal gene transfer to nontarget organisms are exactly limiting issues for both transgenic plant and biopesticide. Further, for both groups are present consumer concerns about living microorganisms in connection with bioterrorism and foodbone diseases. Noticeable is that all that is needed is socially receptive environment and that should be developed and promoted by *Trichoderma* research community.

### **Author details**

Snježana Topolovec-Pintarić Department of Plant Pathology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia

\*Address all correspondence to: tpintaric@agr.hr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**31**

*Trichoderma: Invisible Partner for Visible Impact on Agriculture*

research in India: A review. Indian Phytopathology. 2014;**67**:1-119

[11] O'Neill TM, Elad Y, Shtienberg D, Cohen A. Control of grapevine grey mould with *Trichoderma harzianum*

[12] Topolovec-Pintarić S, Cvjetković

Research. 1999;**10**(1):33-41. DOI: 10.1080/09571269908718156

[13] Topolovec-Pintarić S, Cvjetković B. The sensitivity of *Botrytis cinerea* Pers:Fr. to pyrimethanil in Croatia. Journal of Plant Diseases and Protection. 2002;**109**(1):74-79. DOI:

[14] Harman GE. Myths and dogmas

[15] Ahmad JS, Baker R. Rhizosphere competence of *Trichoderma harzianum*. Phytopathology. 1987;**77**:182-189. DOI:

[16] Topolovec-Pintarić S, Žutić I, Đemić E. Enhanced growth of cabbage and red beet by *Trichoderma viride*. Acta Agriculturae Slovenica. 2013;**101**(1):87-92.

of biocontrol. Plant Disease. 2000;**84**(4):377-393. DOI: 10.1094/

10.1007/BF03356289

PDIS.2000.84.4.377

10.1094/Phyto-7-182

DOI: 10.2478/acas-2013-0010

T39. Biocontrol Science and Technology. 1996;**6**:139-146. DOI: 10.1080/09583159650039340

B, Jurjević Z. Experience in integrated chemical-biological control of grey mould (*Botrytis cinerea*) on grapevines in Croatia. Journal of Wine

[10] Elad Y, Gessler C, Pertot I. Integrated pest management—Italian Israeli cooperation in research and development. In: Proceedings of the Italian-Israeli Workshop on Agriculture, Research and Cooperation: 2003; Israel. Business Conference. 14-17 December 2005; Hong Kong. New York: IEEE;

2006. pp. 866-870

*DOI: http://dx.doi.org/10.5772/intechopen.83363*

[1] Weindling R. *Trichoderma lignorum* as a parasite of other soil fungi. Phytopathology. 1932;**22**:8372-8451

**References**

[2] Weindling R. Studies on a lethal principle effective in the parasitic action of *Trichoderma lingorum* on *Rhizoctonia solani* and other soil fungi. Phytopathology. 1934;**24**:1153-1179

[3] Dennis C, Webster J. Antagonistic properties of species groups of

[4] Weindling R. Studies on a lethal principle effective in the parasitic action of *Trichoderma lignorum* on *Rhizoctonia solani* and other soil fungi. Phytopathology. 1934;**24**:1153-1179

[5] Weindling R. Experimental consideration of the mold toxins of *Gliocladium* and *Trichoderma*. Phytopathology. 1941;**31**:991-1003

[6] Dennis C, Webster J. Antagonistic properties of species-groups of *Trichoderma*: II. Production of volatile antibiotics. Transactions of the British Mycological Society. 1971;**57**:41-48. DOI: 10.1016/S0007-1536(71)80078-5

[7] Woo SL, Ruocco M, Vinale F, Nigro M, Marra R, Lombardi N, et al. *Trichoderma*-based products and their widespread use in agriculture. The Open Mycology Journal. 2014;**8**:71-126. DOI:

10.1007/s00253-016-7792-1

PDIS-94-8-0928

[8] Harman GE, Ma O, Samules GJ, Lorto M. Changing models for commercialization and implementation of biocontrol in the developing and the developed world. Plant Disease. 2010;**94**(8):928-939. DOI: 10.1094/

[9] Sharma P, Sharma M, Raja M, Shanmugam V. Status of *Trichoderma*

*Trichoderma* I, production of non-volatile antibiotics. Transactions of the British Mycological Society. 1971;**57**:25-39

*Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

### **References**

*Trichoderma - The Most Widely Used Fungicide*

lies in plant-protecting pesticides or self-protecting plant, a high-value crop plant with embedded genes from bioagent. Yet, are not the same concerns influencing transgenic plant and biopesticide in the light of biosafety? Nontarget effects, toxicity, and possible pathogenicity for plant, animals and humans, allergenicity or horizontal gene transfer to nontarget organisms are exactly limiting issues for both transgenic plant and biopesticide. Further, for both groups are present consumer concerns about living microorganisms in connection with bioterrorism and foodbone diseases. Noticeable is that all that is needed is socially receptive environment and that should be developed and promoted by *Trichoderma* research community.

**30**

**Author details**

Zagreb, Croatia

Snježana Topolovec-Pintarić

provided the original work is properly cited.

\*Address all correspondence to: tpintaric@agr.hr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Plant Pathology, University of Zagreb Faculty of Agriculture,

[1] Weindling R. *Trichoderma lignorum* as a parasite of other soil fungi. Phytopathology. 1932;**22**:8372-8451

[2] Weindling R. Studies on a lethal principle effective in the parasitic action of *Trichoderma lingorum* on *Rhizoctonia solani* and other soil fungi. Phytopathology. 1934;**24**:1153-1179

[3] Dennis C, Webster J. Antagonistic properties of species groups of *Trichoderma* I, production of non-volatile antibiotics. Transactions of the British Mycological Society. 1971;**57**:25-39

[4] Weindling R. Studies on a lethal principle effective in the parasitic action of *Trichoderma lignorum* on *Rhizoctonia solani* and other soil fungi. Phytopathology. 1934;**24**:1153-1179

[5] Weindling R. Experimental consideration of the mold toxins of *Gliocladium* and *Trichoderma*. Phytopathology. 1941;**31**:991-1003

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[32] Topolovec-Pintarić S, Vinceković M, Jalšenjak N, Martinko K, Žutić I, Đermić E. Prototype of tomato biofertilizer: *Trichoderma viride* and calcium based

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AEM.68.8.4044-4060.2002

S1049-9644(02)00185-8

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[39] Harman GE, Shoresh M. The mechanisms and applications of opportunistic plant symbionts. In: Vurro M, Gressel J, editors. Novel Biotechnologies for Biocontrol Agent

*Trichoderma* inocula on flowering and shoot growth of bedding plants. Scientia Horticulturae. 1994;**59**:147-155. DOI: 10.1016/0304-4238(94)90081-7

10.1094/PD-70-145

BF01876444

10.1094/Phyto-76-518

PHYTO-96-0178

*Trichoderma: Invisible Partner for Visible Impact on Agriculture DOI: http://dx.doi.org/10.5772/intechopen.83363*

microcapsules. In: Proceedings of the 52nd Croatian and 12th International Symposium on Agriculture; 12-17 February 2017; Dubrovnik. Osijek: Faculty of Agriculture University of J.J. Strossmayer; 2017. pp. 100-107

*Trichoderma - The Most Widely Used Fungicide*

Biosystems. 2008;**142**:191-198. DOI:

[25] Ousley MA, Lynch JM, Whipps JM. Potential of *Trichoderma* spp. as consistent plant growth stimulators. Biology and Fertility of Soils. 1994;**17**:85-90. DOI: 10.1007/

[26] Ousley MA, Lynch JM, Whipps JM. Effect of *Trichoderma* on plant growth: A balance between inhibition and growth promotion. Microbial Ecology. 1993;**26**:277-285. DOI: 10.1007/

[27] Lynch JM, Wilson KL, Ousley MA, Whipps JM. Response of lettuce to *Trichoderma* treatment. Letters in Applied Mitrobiology. 1991;**12**:59-61. DOI: 10.1111/j.1472-765X.1991.

[28] Kleifeld O, Chet I. *Trichoderma harzianum*—Interaction with plants and effect on growth response. Plant and Soil. 1992;**144**:267-272. DOI: 10.1007/

[29] Koch E. Effect of biocontrol agents on plant growth in the absence of pathogens. IOBC/WPRS Bulletin.

[30] Topolovec-Pintarić S. Influence of *Trichoderma harzianum* Rifai on the fiber flax germination and growth. Növénytermelés. 2010;**59**(4):421-424. DOI: 10.1556/Novenyterm.59.2010.

[31] Topolovec-Pintarić S, Žutić I, Lončarić I. Enhancing plant growth by *Trichoderma viride* based pellets. Növénytermelés. 2011;**60**(S1):177-180. DOI: 10.1556/Novenyterm.60.2011.

[32] Topolovec-Pintarić S, Vinceković M, Jalšenjak N, Martinko K, Žutić I, Đermić E. Prototype of tomato biofertilizer: *Trichoderma viride* and calcium based

10.1080/11263500802150225

BF00337738

BF00176959

tb00503.x

BF00012884

2001;**24**(1):81-89

Suppl.4

Suppl.1

[17] Celar F, Valic N. Effects of *Trichoderma* spp. and *Gliocladium roseum* culture filtrates on seed germination of vegetables and maize. Journal of Plant Diseases and Protection. 2005;**112**:343-350

[18] Gupta O, Sharma ND. Effect of fungal metabolites on seed germination

(*Phaseolus mungo* L.). Legume Research.

[20] Altomare C, Norvell WA, Björkman

and root length of black gram.

[19] Bal U, Altinatas S. Effects of *Trichoderma harzianum* on lettuce in protected cultivation. Journal of Central European Agriculture. 2008;**9**(1):63-70

T, Harman GE. Solubilization of phosphates and micronutrients by the plant—Growth—Promoting and biocontrol fungus *Trichoderma harzianum* Rifai. Applied and Environmental Microbiology.

[21] Harman GE. *Trichoderma*— Not just for biocontrol anymore. Phytoparasitica. 2011;**39**:103-108. DOI:

[22] Shoresh M, Harman GE, Mastouri F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annual Review of Phytopathology. 2010;**48**:21-23. DOI: 10.1146/ annurev-phyto-073009-114450

[23] Vargas WA, Mandawe JC, Kenerley CM. Plant-derived sucrose is a key element in the symbiotic association between *Trichoderma virens* and maize plants. Plant Physiology. 2009;**151**:792-808. DOI: 10.1104/

[24] Rai MK, Shende S, Strasser RJ. JIP test for fast fluorescence transients as a rapid and sensitive technique in assessing the effectiveness of arbuscular mycorrhizal fungi in *Zea mays*: Analysis of chlorophyll a fluorescence. Plant

10.1007/s12600-011-0151-y

1995;**18**:64-66

1999;**65**:2926-2933

**32**

pp.109.141291

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[34] Chang Y-C, Chang Y-C, Baker R, Kleifeld O, Chet I. Increased growth of plants in presence of the biological control agent *Trichoderma harzianum*. Plant Disease. 1986;**70**:145-148. DOI: 10.1094/PD-70-145

[35] Inbar J, Abramsky M, Cohen D, Chet I. Plant growth enhancement and disease control by *Trichoderma harzianum* in vegetable seedlings growth under commercial conditions. European Journal of Plant Pathology. 1994;**100**:337-346. DOI: 10.1007/ BF01876444

[36] Windham MT, Elad Y, Baker R. A mechanism for increased plant growth induced by *Trichoderma* spp. Phytopathology. 1986;**6**:518-521. DOI: 10.1094/Phyto-76-518

[37] Ousley MA, Lynch JM, Whipps JM. The effects of addition of *Trichoderma* inocula on flowering and shoot growth of bedding plants. Scientia Horticulturae. 1994;**59**:147-155. DOI: 10.1016/0304-4238(94)90081-7

[38] Howell CR. Understanding the mechanisms employed by *Trichoderma virens* to effect biological control of cotton diseases. Phytopathology. 2006;**96**:178-180. DOI: 10.1094/ PHYTO-96-0178

[39] Harman GE, Shoresh M. The mechanisms and applications of opportunistic plant symbionts. In: Vurro M, Gressel J, editors. Novel Biotechnologies for Biocontrol Agent Enhancement and Management. NATO Security Through Science Series. Dordrecht: Springer; 2007. pp. 131-155. DOI: 10.1007/978-1-4020-5799-1\_7

[40] Howell CR. Mechanisms employed by *Trichoderma* species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease. 2003;**87**:4-10. DOI: 10.1094/ PDIS.2003.87.1.4

[41] Benhamou N, Garand C, Goulet A. Ability of nonpathogenic *Fusarium oxysporum* strain Fo47 to induce resistance against *Pythium ultimum* infection in cucumber. Applied and Environmental Microbiology. 2002;**68**:4044-4060. DOI: 10.1128/ AEM.68.8.4044-4060.2002

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[43] Bae H, Roberts DP, Lim HS, Strem M, Park SC, Ryu CM, et al. Endophytic *Trichoderma* isolates from tropical environments delay disease and induce resistance against *Phytophthora capsici* in hot pepper using multiple mechanisms. Molecular Plant-Microbe Interactions. 2011;**24**:336-351. DOI: 10.1094/ MPMI-09-10-0221

[44] Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. *Trichoderma* species—Opportunistic, avirulent plant symbionts. Nature Reviews. Microbiology. 2004;**2**(1):43-56. DOI: 10.1038/nrmicro797

[45] Bittel P, Robatzek S. Microbeassociated molecular patterns (MAMPs) probe plant immunity. Current Opinion in Plant Biology. 2007;**10**:335-341. DOI: 10.1016/j. pbi.2007.04.021

[46] Hermosa R, Viterbo A, Chet I, Monte E. Plant-beneficial effect of *Trichoderma* and its genes. Microbiology. 2012;**158**:17-25. DOI: 10.1099/mic.0.052274-0

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Society; 2015. p. 196. ISBN-13:

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1969. p. 56. ISBN: 0851990002

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[62] Morton DT, Stroube NH.

9780890544846

9780851990002

1998. p. 278. ISBN: 0748405720

[46] Hermosa R, Viterbo A, Chet I, Monte E. Plant-beneficial effect of *Trichoderma* and its genes. Microbiology. 2012;**158**:17-25. DOI:

[47] Lorito M, Woo SL, Harman GE, Monte E. *Trichoderma*: From 'Omics to the field. Annual Review of Phytopathology. 2010;**48**:395-417. DOI: 10.1146/ annurev-phyto-073009-114314

[48] Jones J, Dangl J. The plant immune system. Nature. 2006;**444**:323-329. DOI:

[49] Seggara G, Casanova E, Bellido D, Odena MA, Oliveira E, Trillas I. Proteome, salicylic acid, and

[50] Malmierca MG, Cardoza RE, Alexander NJ, McCormic SP, Hermosa

R, Monte E, et al. Involvement of *Trichoderma* trichotecens in biocontrol activity and induction of plant defense-related genes. Applied and Environmental Microbiology. 2012;**78**:4856-4868. DOI: 10.1128/

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[52] Rosset PM. Cuba: Ethics, biological control, and crisis. Agriculture and Human Values. 1997;**14**(3):291-302. DOI: 10.1023/a:1007433501248

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Section 2

Trichoderma as Biological

Control Agent

## Section 2
