*3.1.2 Molecular identification*

*Organic Agriculture*

human health.

**2. Biopesticides market**

biotechnological products.

and growth promotion in plants [8, 9].

cation of the species which needs work is necessary.

For example, the apple scab caused by *Venturia inaequalis* (Cook) Wint. (Anamorph: *Spilocaea did Fr*.) is the most important disease of this fruit at a worldwide level, which can cause significant economic losses until 100% of the production, affecting the commercial quality of fruits [1, 2]. Generally, its control is based on the use of agrochemicals. In vegetables, wilting of chili pepper and tomato crops is one of the main biological limitations in the production of these crops and can be caused by *Phytophthora capsici*, *Rhizoctonia solani*, and *Fusarium oxysporum* [3]; this disease is reported throughout Mexico, estimating losses of up to 80% due to root rot by invading the vascular system of plants. Likewise, chemical control is the most used method for disease management and is common to reduce the inoculum by disinfecting the soil with metam sodium, 2-thiocyanomethyl benzothiozole (TCMTB), metalaxyl, azoxystrobin, and propanocarp fungicide applications to control *P. capsici* [4]. *R. solani* and *Fusarium* spp. are controlled with tebuconazole, carbendazim, thiabendazole, and methyl thiophanate [5]. The use of this control method significantly increases the production costs and the negative impact it causes on the environment and to human health and induces resistance of the pathogens toward the active ingredients. An alternative is the use of biological control by microorganisms antagonistic to fungi and stramenopiles from the soil, which has little or no effect on the environment and

The worldwide market of biopesticides was of 1213 million dollars in 2010 and 3222 million dollars in 2017; the annual rate increases to 15.8% since 2012 besides 2017. Within this market, bioinsecticides represented 46% in 2011, and biofungicides were of 600.5 million dollars, reaching 1447 million in 2017. The annual rate from 2012 to 2017 grows up at 16.1%. Given that there currently exists a market demand for free products of pesticide waste, huge agrochemical companies are in the market of bioproducts, acquiring biocontrol companies and developing new

**3. Isolation and identification of** *Bacillus* **spp. and** *Trichoderma* **spp.**

*Trichoderma* and *Bacillus* are essential genera of antagonistic microorganisms for control of a large number of phytopathogens. *Trichoderma* is a cosmopolitan soil fungus, which is frequently on soil from the plant root system. This fungus is attractive for organic management of diseases because present different action modes against phytopathogens as competition for nutrients, mycoparasitism, and antibiosis by hydrolytic enzymes and metabolites also produce substances that promote plant growth [6, 7]. On the other hand, *Bacillus* spp. is a large and heterogeneous group of Gram-positive, rod-shaped, aerobic and facultative anaerobic, and endospore-forming bacteria; same as *Trichoderma*, *Bacillus* is an alternative of biological control of plant diseases due to its capability to inhibit phytopathogens

Due to the abovementioned and because there is a large number of species from both microorganisms, their isolation and identification for their possible commercial use are necessary; some of the species of *Trichoderma* are *T. virens*, *T. harzianum*, and *T. viride* and of *Bacillus* spp. are reported as antagonists *B. amyloliquefaciens*, *B. licheniformis*, *B. subtilis*, and *B. pumilus* [10, 11]. Thus a correct identifi-

**98**

This kind of identification has gained acceptance because it presents more precision and reliability among several strains of *Trichoderma.* The phylogeny of this genus has been based in the sequence analysis of the internal transcribed spacers of ribosomal DNA using the universal primers ITS1 and ITS4 with a subsequent sequencing and analysis through databases [15] but also can be identified through specific primers which are a powerful tool that allows to identify a specific species of *Trichoderma* [16] (**Table 1**). In Mexico, diverse species of *Trichoderma* have been isolated and identified [6]; they identified *T. atroviride*, *T. asperellum*, *T. citrinoviride*, *T. ghanense*, *T. harzianum*, *T. inhamatum*, *T. longibrachiatum*, and *T. yunnanense* (**Figure 1**) from samples taken from several agricultural regions. In a similar research, Osorio et al. [23] identified the species as *T. asperellum*, *T. rossicum*, and *T. hamatum* from different localities of Mexican Northeast region.


#### **Table 1.**

*Examples of species-specific primers for* Trichoderma *spp.*

#### **Figure 1.**

*Morphologic characteristics of different* Trichoderma *spp. isolated from samples of different agricultural systems of Mexico.*

#### **3.2 Isolation and identification of** *Bacillus* **spp.**

*Bacillus* spp. is a genus present in the soil of a considerable amount of crops and naturally is on the rhizosphere; due to this, the traditional tools for determining the soil bacterial community and diversity are used [24]. The first step is to make the collecting of the rhizosphere soil, take 10 g of soil with a sterile spoon, and store the sample at 4°C. Heat or pasteurization treatments are the most commonly used techniques to select spores due to this, the sample is diluted in 90 mL of sterile normal saline and heated at 80°C for 10 min to eliminate vegetative cells; once heated, the sample is serially diluted (10<sup>−</sup><sup>1</sup> –10<sup>−</sup><sup>4</sup> ) and placed on 1 mL nutrient agar (NA) medium with cycloheximide (100 mg mL<sup>−</sup><sup>1</sup> ) to prevent fungal growth or carboxymethylcellulose (CMC) agar, and it is incubated at 37°C for 24 h [25, 26].

However, the treatment with heat can be different depending on the species because endospores of some strains are more resistant to heat than others [24]. Due to this, the drying treatment is considered more gentle; this method consists of placing the samples on a dryer at 70°C for 1 h [25]. The considerable variety of physiology of *Bacillus* spp. requires elaborate biochemical and morphological tests for species identification; as colony growth in artificial media, form cell unit, presence, number and orientation of flagella, Gram stain, spore form-position and specific environmental conditions of growth and finally the specific use of carbon sources gave its metabolic diversity [27].

#### *3.2.1 Molecular identification of Bacillus spp.*

Several molecular approaches are currently utilized for the identification of microorganisms; in this sense, the use of polymerase chain reaction (PCR) in combination with 16S rRNA is a tool frequently used for identification of *Bacillus* spp. from various environments including soil. Using the 16S rRNA sequence, five groups within biological control of root pathogens by plant growth-promoting *Bacillus* spp.*,* the genus *Bacillus* spp., where group 1 comprises species *B. amyloliquefaciens*, *B. subtilis*, *B. pumilus*, and *B. licheniformis*, have been identified [24, 25].

*Bacillus* spp. can identify through specific primers (**Table 2**). Such as *Trichoderma* spp., the Mexican agricultural systems are an excellent source to obtain *Bacillus* spp., such as mentioned by Guillén-Cruz et al. [9] and Hernandez-Castillo

**101**

*Biological Efficacy of* Trichoderma *spp. and* Bacillus *spp. in the Management of Plant Diseases*

p-gyrA-r: CAAGGTAATGCTCCAGGCATTGCT

trpE(G) R: ACATACATTTCGGGGGATGA

L1401(R): GCGTGTGTACAAGACCC

BL8AR: CGTGTCCGAGTGTGCGTTATAT

**size (bp)**

741 [28]

78 [29]

490 [30]

247 [31]

**References**

**Species specificity Primer sequences (5′–3′) Product** 

*B. subtilis* p-gyrA-f: CAGTCAGGAAATGCGTACGTCCTT

*B. amyloliquefaciens* trpE(G) F: TTTGAATCCGAGCCCTTATG

*B. pumilus* GC-U968(F): GCAACGCGAAGAACCTTAC

*B. licheniformis* BL8AF: TCACAACCCGTTGACGACAA

et al*.* [32] whom identified several *Bacillus* spp. species as *B. amyloliquefaciens*, *B. pumilus*, *B. licheniformis*, and *B. subtilis* from samples coming from several

**4. Antifungal activity in vitro of** *Trichoderma* **spp. and** *Bacillus* **spp.**

The antifungal activity of *Trichoderma* species has been evaluated in in vitro studies against soilborne and foliar fungi, and there have been acceptable results. The antifungal activity can be determined such a direct manner as indirect manner. In the case of a direct manner, the most used technique is the dual culture where the inhibition percentage, Bell scale, and the days to contact are evaluated to determine the antagonistic activity of *Trichoderma* species. Dual culture consists of Petri dishes with PDA where a disk (5 mm in diameter) with mycelium of the plant pathogen is placed and, on the other side of the Petri dish equidistantly, a disk of mycelium of the same diameter of *Trichoderma* strains under study is placed. The plates inoculated are incubated at 27 ± 1°C until the growth of control treatment (with only plant pathogen disk) covered the Petri dish. The effect of *Trichoderma* strains on plant pathogens is determined by the percentage of mycelial growth inhibition. The days of contact between plant pathogen antagonistic and antagonistic ability of *Trichoderma* isolates according to the methodology proposed by Bell et al. [33] are also determined. Bell et al. [33] classified the antagonism produced by *Trichoderma* as follows: Class I, *Trichoderma* overgrows completely to pathogen and covers the whole surface of the medium; Class II, *Trichoderma* overgrows two-thirds of the surface of the medium; Class III, *Trichoderma* and pathogen colonized each half of the surface, and nobody seems to dominate the other; Class IV, the pathogen colonizes the two-third parts of the media surface and resists invasion by *Trichoderma*; and Class V, the plant pathogen overgrows completely to *Trichoderma* and covers an area total culture media [6]. In case of the desire to determine the antifungal activity of an indirect manner, the volatile compounds are an option; this method is realized as follows. In the center of a Petri dish having only PDA medium, a disk of 5 mm in diameter with active mycelia of the plant pathogen is placed, and the top of the dish is replaced with another Petri dish in which a disk with mycelia of *Trichoderma* strain is placed; in this case, the lid is pierced with a punch (10 mm in diameter), and the Petri dishes are joined and sealed with parafilm paper and incubated at 26 ± 1°C until each pathogen control covered the Petri dish. The effect of volatile compounds is measured considering the diameter of pathogen colonies and expressed as percentage of mycelial growth inhibition [6]. Several research were carried out to determine the antifungal activity of *Trichoderma* spp. strains, due to its potential as biocontroller of plant pathogens,

*DOI: http://dx.doi.org/10.5772/intechopen.91043*

regions of the center and northern of Mexico.

*Examples of species-specific primers for* Bacillus *spp.*

**Table 2.**

*Biological Efficacy of* Trichoderma *spp. and* Bacillus *spp. in the Management of Plant Diseases DOI: http://dx.doi.org/10.5772/intechopen.91043*


**Table 2.**

*Organic Agriculture*

**3.2 Isolation and identification of** *Bacillus* **spp.**

cells; once heated, the sample is serially diluted (10<sup>−</sup><sup>1</sup>

sources gave its metabolic diversity [27].

*3.2.1 Molecular identification of Bacillus spp.*

nutrient agar (NA) medium with cycloheximide (100 mg mL<sup>−</sup><sup>1</sup>

*Bacillus* spp. is a genus present in the soil of a considerable amount of crops and naturally is on the rhizosphere; due to this, the traditional tools for determining the soil bacterial community and diversity are used [24]. The first step is to make the collecting of the rhizosphere soil, take 10 g of soil with a sterile spoon, and store the sample at 4°C. Heat or pasteurization treatments are the most commonly used techniques to select spores due to this, the sample is diluted in 90 mL of sterile normal saline and heated at 80°C for 10 min to eliminate vegetative

*Morphologic characteristics of different* Trichoderma *spp. isolated from samples of different agricultural* 

growth or carboxymethylcellulose (CMC) agar, and it is incubated at 37°C for

However, the treatment with heat can be different depending on the species because endospores of some strains are more resistant to heat than others [24]. Due to this, the drying treatment is considered more gentle; this method consists of placing the samples on a dryer at 70°C for 1 h [25]. The considerable variety of physiology of *Bacillus* spp. requires elaborate biochemical and morphological tests for species identification; as colony growth in artificial media, form cell unit, presence, number and orientation of flagella, Gram stain, spore form-position and specific environmental conditions of growth and finally the specific use of carbon

Several molecular approaches are currently utilized for the identification of microorganisms; in this sense, the use of polymerase chain reaction (PCR) in combination with 16S rRNA is a tool frequently used for identification of *Bacillus* spp. from various environments including soil. Using the 16S rRNA sequence, five groups within biological control of root pathogens by plant growth-promoting *Bacillus* spp.*,* the genus *Bacillus* spp., where group 1 comprises species *B. amyloliquefaciens*, *B. subtilis*, *B. pumilus*, and *B. licheniformis*, have been identified [24, 25]. *Bacillus* spp. can identify through specific primers (**Table 2**). Such as

*Trichoderma* spp., the Mexican agricultural systems are an excellent source to obtain *Bacillus* spp., such as mentioned by Guillén-Cruz et al. [9] and Hernandez-Castillo

–10<sup>−</sup><sup>4</sup>

) and placed on 1 mL

) to prevent fungal

**100**

24 h [25, 26].

**Figure 1.**

*systems of Mexico.*

*Examples of species-specific primers for* Bacillus *spp.*

et al*.* [32] whom identified several *Bacillus* spp. species as *B. amyloliquefaciens*, *B. pumilus*, *B. licheniformis*, and *B. subtilis* from samples coming from several regions of the center and northern of Mexico.
