**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, as reported by Hernandez et al. [6] who evaluated several strains of *Trichoderma* spp. against *Sclerotinia sclerotiorum* and *Sclerotium cepivorum* through dual culture and observed rate of inhibition of 45–63.8% and 50.9–81.5 for *S. sclerotiorum* with *T. ghanense* and *T. longibrachiatum*, respectively, and 81.5 and 81.2% of *S. cepivorum* with *T. inhamatum* and *T. asperellum*, respectively. For the Bell scale and contact days for both phytopathogenic fungi, the mean was of 2 days and scale of I, II, and III with all the *Trichoderma* spp. strains. Some research about the inhibition of secondary metabolites, precisely the volatile compounds, present inhibition of *S. sclerotiorum* and *S. cepivorum* against *T. longibrachiatum* with 28.1 and 73.8%, respectively, followed by *T. harzianum* with 12.5 and 62.5%.

Some studies by Osorio et al. [23] mentioned a Trichoderma spp. as controller. They reported the overgrowth of *Trichoderma* spp. strains over *Phytophthora capsici*; in total 13 *Trichoderma* spp. strains showed level 1 according to the Bell scale. This effect can be attributed to enzyme production (β-1, 3-glucanase, chitinase, protease, and cellulose) by these *Trichoderma* spp. strains. As too volatile compounds produced by the *Trichoderma* spp. strains, they reported inhibition of *P. capsici* mycelial growth ranged between 4.3 and 48.8, the major effect observed with the *T. asperellum* and the least with *Trichoderma* sp. strain. Some studies mentioned that *Trichoderma* spp. produces volatile compounds, carbon dioxide, oxygen, ethylene, and 6-pentyl-α-pyrone (6PP) which cause adverse effect in the development of phytopathogenic fungus. Furthermore, *Trichoderma* spp. can inhibit the growth of foliar fungus as *Colletotrichum* spp., such as mentioned by Tucuch et al. [34] who determined the antifungal activity *Trichoderma* spp. strains and reported antagonism level 1 in Bell scale by *T. asperellum*, while for volatile compounds, the species *T. lignorum* affected the fungus inhibiting its developments in 24.02% (**Figure 2**).

Usually, *Trichoderma* spp. inhibit several phytopathogenic fungi due to their capacity to produce enzymes, volatile compounds and compete for nutrients against phytopathogens. Studies realized by Samaniego-Fernández et al. [35] and Kumar et al. [7] showed the antagonistic capacity of *Trichoderma* spp.; in the first case, the species *T*. *harzianum* and *T. viride* are controlled to *S. rolfsii* y *Fusarium* spp.; in the second study, several *Trichoderma* spp. showed mycelial growth inhibition of *S. rolfsii* more than 50%.

Likewise, then *Trichoderma* spp. strains, the antifungal activity of *Bacillus* spp.*,* can be tested by dual culture; nevertheless, the method is different than with *Trichoderma* spp. PDA disk (5 mm) with active mycelium of the phytopathogen is placed in the center of a Petri dish with PDA; on the same plate, at a distance of 1.5 cm in the four cardinal points, a loopful of antagonistic bacterial isolates is placed. Plates inoculated with the pathogen culture serve as controls. In order to quantify the antagonistic potential of bacterial strains, the size of growth inhibition zones measured after 6 days of incubation at 25–28°C and the percent of radial growth inhibition (PICR) are calculated [36]. In this sense, study also showed the capacity of *Bacillus* spp. to inhibit the growth of phytopathogenic fungi. Thereby

#### **Figure 2.**

*Inhibition of* Colletotrichum *spp. by volatile compounds produced by different* Trichoderma *spp. strains (a) control, (b)* T. asperellum*, (c)* T. yunnanense*, and (d)* T. lignorum*.*

**103**

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

Jimenez et al. [36] reported the inhibition of *Venturia inaequalis* mycelia by

*(b)* Fusarium oxysporum*, (c)* Phytophthora capsici, *and (d)* Colletotrichum *spp.*

**5. Obtaining secondary metabolites of** *Trichoderma* **spp. and** *Bacillus* **spp. and their antifungal activity in vitro (PDA methods and** 

petri dish, after that the percentage inhibition is determined [23].

*B. subtilis* and *B. licheniformis* ranged 33.4–41.3%, respectively, and Tucuch et al. [34] observed 50% of inhibition from *B. subtilis* against *Colletotrichum* spp. (**Figure 3**).

*Antagonistic effect of* Bacillus *spp. strains against different phytopathogenic fungus (a)* Rhizoctonia solani*,* 

Generally, the production of the secondary metabolites from biological agents such as *Trichoderma* spp. and *Bacillus* spp. is carried out using liquid media by a fermentation process in a reactor, which can be of different types from a simple bottle to until an automated reactor, where the temperature and shaking rate are the key variables for the emission of secondary metabolites. The liquid medium is integrated by several components such as carbon sources and mineral salts, where the biological microorganism is inoculated [37]. The secondary metabolites obtained from the fermentation of *Bacillus* spp. and *Trichoderma* spp. are filtered with nitrocellulose membrane of 0.22 um; after the recovery of these metabolites, it is necessary to perform a screening to determine their ability to inhibit phytopathogenic microorganisms. There are many methods to determine the antifungal activity from secondary metabolites of antagonistic microorganisms, the most common is the poisoned medium, adding the substance to evaluate in the culture medium before solidification, which consists in adding 200 μl of the secondary metabolites on PDA medium in the center of Petri dish and 5 mm mycelium disk of the phytopathogen, then Petri dishes are incubated at 28 ± 1°C until the control treatment covers the

However, our workgroup has standardized the method in microdilution on plate, which consists in an adaptation of the technique proposed by Masoko et al. [38]; polystyrene microplates of 96 wells are used; in all wells, 100 μl of liquid medium is placed; column 1 is the negative control, column 2 consists of the positive control, and column 3 is a control which consists of the fermentation medium. Starting in column 4, 100 μl of the secondary metabolites from strains is mixed in a pipette with 100 μl of the liquid medium, and then 100 μl of the mixture is transferred to the next column, discarding the last 100 μl from column 12, to get serial microdilutions to 50.00% of the secondary metabolites. Once the microdilutions are carried out, the growth developer 2,3,5-triphenyltetrazolium chloride is added in the whole plate; the concentration of the growth developer is the lowest reported in the literature, as an excess of this indicator can interfere with the growth of the pathogen or react with reagents from the medium; this indicator measures the respiratory activity associated with electron transport chains, and when reduced, it precipitates forming a complex, intense red color; its use is due to its high sensitivity to detect

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

**microplate dilution)**

**Figure 3.**

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

#### **Figure 3.**

*Organic Agriculture*

as reported by Hernandez et al. [6] who evaluated several strains of *Trichoderma* spp. against *Sclerotinia sclerotiorum* and *Sclerotium cepivorum* through dual culture and observed rate of inhibition of 45–63.8% and 50.9–81.5 for *S. sclerotiorum* with *T. ghanense* and *T. longibrachiatum*, respectively, and 81.5 and 81.2% of *S. cepivorum* with *T. inhamatum* and *T. asperellum*, respectively. For the Bell scale and contact days for both phytopathogenic fungi, the mean was of 2 days and scale of I, II, and III with all the *Trichoderma* spp. strains. Some research about the inhibition of secondary metabolites, precisely the volatile compounds, present inhibition of *S. sclerotiorum* and *S. cepivorum* against *T. longibrachiatum* with 28.1 and 73.8%,

Some studies by Osorio et al. [23] mentioned a Trichoderma spp. as controller. They reported the overgrowth of *Trichoderma* spp. strains over *Phytophthora capsici*; in total 13 *Trichoderma* spp. strains showed level 1 according to the Bell scale. This effect can be attributed to enzyme production (β-1, 3-glucanase, chitinase, protease, and cellulose) by these *Trichoderma* spp. strains. As too volatile compounds produced by the *Trichoderma* spp. strains, they reported inhibition of *P. capsici* mycelial growth ranged between 4.3 and 48.8, the major effect observed with the *T. asperellum* and the least with *Trichoderma* sp. strain. Some studies mentioned that *Trichoderma* spp. produces volatile compounds, carbon dioxide, oxygen, ethylene, and 6-pentyl-α-pyrone (6PP) which cause adverse effect in the development of phytopathogenic fungus. Furthermore, *Trichoderma* spp. can inhibit the growth of foliar fungus as *Colletotrichum* spp., such as mentioned by Tucuch et al. [34] who determined the antifungal activity *Trichoderma* spp. strains and reported antagonism level 1 in Bell scale by *T. asperellum*, while for volatile compounds, the species *T. lignorum* affected the fungus inhibiting its developments in 24.02% (**Figure 2**). Usually, *Trichoderma* spp. inhibit several phytopathogenic fungi due to their capacity to produce enzymes, volatile compounds and compete for nutrients against phytopathogens. Studies realized by Samaniego-Fernández et al. [35] and Kumar et al. [7] showed the antagonistic capacity of *Trichoderma* spp.; in the first case, the species *T*. *harzianum* and *T. viride* are controlled to *S. rolfsii* y *Fusarium* spp.; in the second study, several *Trichoderma* spp. showed mycelial growth inhibition of *S. rolfsii* more than 50%. Likewise, then *Trichoderma* spp. strains, the antifungal activity of *Bacillus* spp.*,* can be tested by dual culture; nevertheless, the method is different than with *Trichoderma* spp. PDA disk (5 mm) with active mycelium of the phytopathogen is placed in the center of a Petri dish with PDA; on the same plate, at a distance of 1.5 cm in the four cardinal points, a loopful of antagonistic bacterial isolates is placed. Plates inoculated with the pathogen culture serve as controls. In order to quantify the antagonistic potential of bacterial strains, the size of growth inhibition zones measured after 6 days of incubation at 25–28°C and the percent of radial growth inhibition (PICR) are calculated [36]. In this sense, study also showed the capacity of *Bacillus* spp. to inhibit the growth of phytopathogenic fungi. Thereby

*Inhibition of* Colletotrichum *spp. by volatile compounds produced by different* Trichoderma *spp. strains* 

*(a) control, (b)* T. asperellum*, (c)* T. yunnanense*, and (d)* T. lignorum*.*

respectively, followed by *T. harzianum* with 12.5 and 62.5%.

**102**

**Figure 2.**

*Antagonistic effect of* Bacillus *spp. strains against different phytopathogenic fungus (a)* Rhizoctonia solani*, (b)* Fusarium oxysporum*, (c)* Phytophthora capsici, *and (d)* Colletotrichum *spp.*

Jimenez et al. [36] reported the inhibition of *Venturia inaequalis* mycelia by *B. subtilis* and *B. licheniformis* ranged 33.4–41.3%, respectively, and Tucuch et al. [34] observed 50% of inhibition from *B. subtilis* against *Colletotrichum* spp. (**Figure 3**).

### **5. Obtaining secondary metabolites of** *Trichoderma* **spp. and** *Bacillus* **spp. and their antifungal activity in vitro (PDA methods and microplate dilution)**

Generally, the production of the secondary metabolites from biological agents such as *Trichoderma* spp. and *Bacillus* spp. is carried out using liquid media by a fermentation process in a reactor, which can be of different types from a simple bottle to until an automated reactor, where the temperature and shaking rate are the key variables for the emission of secondary metabolites. The liquid medium is integrated by several components such as carbon sources and mineral salts, where the biological microorganism is inoculated [37]. The secondary metabolites obtained from the fermentation of *Bacillus* spp. and *Trichoderma* spp. are filtered with nitrocellulose membrane of 0.22 um; after the recovery of these metabolites, it is necessary to perform a screening to determine their ability to inhibit phytopathogenic microorganisms. There are many methods to determine the antifungal activity from secondary metabolites of antagonistic microorganisms, the most common is the poisoned medium, adding the substance to evaluate in the culture medium before solidification, which consists in adding 200 μl of the secondary metabolites on PDA medium in the center of Petri dish and 5 mm mycelium disk of the phytopathogen, then Petri dishes are incubated at 28 ± 1°C until the control treatment covers the petri dish, after that the percentage inhibition is determined [23].

However, our workgroup has standardized the method in microdilution on plate, which consists in an adaptation of the technique proposed by Masoko et al. [38]; polystyrene microplates of 96 wells are used; in all wells, 100 μl of liquid medium is placed; column 1 is the negative control, column 2 consists of the positive control, and column 3 is a control which consists of the fermentation medium. Starting in column 4, 100 μl of the secondary metabolites from strains is mixed in a pipette with 100 μl of the liquid medium, and then 100 μl of the mixture is transferred to the next column, discarding the last 100 μl from column 12, to get serial microdilutions to 50.00% of the secondary metabolites. Once the microdilutions are carried out, the growth developer 2,3,5-triphenyltetrazolium chloride is added in the whole plate; the concentration of the growth developer is the lowest reported in the literature, as an excess of this indicator can interfere with the growth of the pathogen or react with reagents from the medium; this indicator measures the respiratory activity associated with electron transport chains, and when reduced, it precipitates forming a complex, intense red color; its use is due to its high sensitivity to detect

inhibition of microorganisms with deficient amounts of antimicrobial products; besides that, the red coloration is a visual indicator of the antimicrobial activity of the treatment. Finally, starting in column 2, 10 μl of a spore solution of the fungus at a concentration of 1 × 108 in all wells is added, keeping all the wells a volume of 150 μl in total; each microplate is considered a replicate. The microplates are incubated in agreement with the necessary conditions of the fungus on absorbance realized at 490 nm in a spectrophotometer. The secondary metabolites from the different strains placed in the rows A to F. To calculate the growth and inhibition percentage, the following formulas used: % Growth = (A − B/C)(100); where A is treatment absorbance, B is negative control absorbance, C is positive control absorbance, and % Inhibition = 100 – % Growth.

In general, the selection principle of strains is the determination of their antagonistic capacity; the method of microdilution in plate is to some extent interesting since it allows determined quickly and efficiently in time and costs its capacity of antifungal inhibition. Several studies demonstrate the effectiveness of secondary metabolites in the control of phytopathogenic fungi with PDA medium; Osorio et al. [23] mentioned that the inhibiting effect by *T. asperellum* and *T. hamatum* against *P. capsici* ranged to 15–20%; this inhibition attributed to the concentration of metabolites like glycotoxins, viridine, trichodermin, furanone, and 6-pentyl-α-pyrone (**Figure 4**).

Likewise, the PDA method or the microdilution in plate method can be an excellent technique to evaluate substance with antifungal activity as the secondary metabolites; in this sense, Jimenez et al. [36] observed that secondary metabolites obtained from *T. yunnanense* and T. *harzianum* at a concentration of 50 and 25% showed an inhibiting total effect of 100% of mycelial growth of *V. inaequalis*, while the metabolites obtained from *T. asperellum* and *T. lignorum* at a concentration of 50% showed an inhibiting effect from 90 to 84%, respectively (**Figure 5**). On the other hand, Jimenez et al. [36] reported that secondary metabolites obtained from *B. licheniformis* at a concentration of 50 and 25% showed an inhibiting effect in 100% against *V. inaequalis*, while the metabolites obtained from *B. subtilis* at a concentration of 50% showed an inhibiting effect near to 78% of the development of this pathogen (**Figure 5**).

In another study, Tucuch-Pérez et al. [39] reported six *Bacillus* spp. strains with antifungal activity against *F. oxysporum*; in this case, the species *B. licheniformis* and *B. subtilis* showed the highest inhibition percentages ranged from 80 to 100%, being the lowest inhibition percentage registered of 50% (**Figure 6**).

The results generated by the microplate dilution method are consistent with the results obtained from indirect test by confrontation or dual test, such as shown in the following results against fungi isolated from crops as melon, pepper, and others, which are produced in different zones from Mexico. In melon crop, the root and stem rot disease is a big problem; in this context, Espinoza-Ahumada et al. [40] studied the in vitro antagonist effects of *Trichoderma* spp. and found that *T. asperellum* have excellent biological activity against *Fusarium* strains, isolated from melon,

#### **Figure 4.**

*Antifungal activity of secondary metabolites from different* Trichoderma *spp. strains against phytopathogenic fungi; (a)* T. asperellum *vs.* Fusarium oxysporum*, (b)* T. yunannense *vs.* Phytophthora capsici*, (c)* T. longibrachiatum *vs.* Rhizoctonia solani*, (d)* T. asperellum *vs.* Sclerotinia sclerotiorum *and (e)* T. asperellum *vs.* Sclerotium cepivorum*.*

**105**

**Table 3.**

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

*Percentage of inhibition of secondary metabolites obtained from* Bacillus *spp. (a) and* Trichoderma *spp.* 

shown in **Table 3**. In general, these authors report that the inhibition of *Fusarium* spp. is higher when *Trichoderma* spp. are used (62.4–54.8%), in contrast when

*(a) Percentage inhibition of microbial extracts from* Bacillus *spp. metabolite dilutions against* F. oxysporum*. (b) Microplate with treatments to several concentrations, and the pathogen elapsed 48 h after incubation. Row A= B-AN1, B = B-AN2, C = B-AN3, D = B-AN4, E = B-AN5, F = B-AN6; column 1 = negative witness, column 2 = positive witness, 3 = growth medium of* Bacillus *spp., 5 = 50%, 5 = 25%, 6 = 12.50%, 7 = 6.25%,* 

In a work carried out by Francisco et al. [41], where the behavior of *Bacillus* spp. against *Fusarium* species was studied, it showed low inhibition values. However, they report that the species *B. pumilus* and *B. liquefaciens* can be used effectively against many *Fusarium* species. On the other hand, higher effectiveness of *Bacillus*

*B. liquefaciens* 46.9A,ab 38.4B,abc 31.4BC,bc 34.8CD,bc 28.2CD,c 41.6DE,ab *B. amyloliquefaciens* 51.4AB,ab 38.9B,c 37.8B,c 42.9BC,bc 41.2BC,bc 55.2C,a *B. subtilis* 45.7B,a 38.6B,a 37.2B,a 42.4B,ca 34.6C,a 46.4D,a *T. asperellum* 57.2A,b 61.5A,ab 64.3A,ab 62.3A,ab 54.9A,b 74.1A,a *T. harzianum* 54.9AB,b 55.4A,b 56.1A,ab 60.5A,ab 57.9A,b 64.7B,a *T. viride* 49.8A,ab 58.3A,a 54.9A,a 51.1A,ab 50.3BC,a 64.7B,a *High letters indicate comparison between columns; low letters indicate comparison between rows. Percentages of* 

**FRR-1 FRG-2 FAF-3 FRE-4 FCA-5 FHA-6**

*Bacillus* spp. (44.5–36.9%) is used (**Figure 7**).

*inhibition with different letters are significantly different (p* ≤ *0.5).*

**Antagonistic agent** *Fusarium* **strain**

*Percentage of antagonism of different biological agents against* Fusarium *spp. strains.*

*8 = 3.13%, 9 = 1.56%, 10 = 0.78%, 11 = 0.39%, and 12 = 0.20%.*

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

**Figure 5.**

**Figure 6.**

*(b) against* Venturia inaequalis.

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

#### **Figure 5.**

*Organic Agriculture*

at a concentration of 1 × 108

of this pathogen (**Figure 5**).

absorbance, and % Inhibition = 100 – % Growth.

inhibition of microorganisms with deficient amounts of antimicrobial products; besides that, the red coloration is a visual indicator of the antimicrobial activity of the treatment. Finally, starting in column 2, 10 μl of a spore solution of the fungus

of 150 μl in total; each microplate is considered a replicate. The microplates are incubated in agreement with the necessary conditions of the fungus on absorbance realized at 490 nm in a spectrophotometer. The secondary metabolites from the different strains placed in the rows A to F. To calculate the growth and inhibition percentage, the following formulas used: % Growth = (A − B/C)(100); where A is treatment absorbance, B is negative control absorbance, C is positive control

In general, the selection principle of strains is the determination of their antagonistic capacity; the method of microdilution in plate is to some extent interesting since it allows determined quickly and efficiently in time and costs its capacity of antifungal inhibition. Several studies demonstrate the effectiveness of secondary metabolites in the control of phytopathogenic fungi with PDA medium; Osorio et al. [23] mentioned that the inhibiting effect by *T. asperellum* and *T. hamatum* against *P. capsici* ranged to 15–20%; this inhibition attributed to the concentration of metabolites like glycotox-

ins, viridine, trichodermin, furanone, and 6-pentyl-α-pyrone (**Figure 4**).

the lowest inhibition percentage registered of 50% (**Figure 6**).

Likewise, the PDA method or the microdilution in plate method can be an excellent technique to evaluate substance with antifungal activity as the secondary metabolites; in this sense, Jimenez et al. [36] observed that secondary metabolites obtained from *T. yunnanense* and T. *harzianum* at a concentration of 50 and 25% showed an inhibiting total effect of 100% of mycelial growth of *V. inaequalis*, while the metabolites obtained from *T. asperellum* and *T. lignorum* at a concentration of 50% showed an inhibiting effect from 90 to 84%, respectively (**Figure 5**). On the other hand, Jimenez et al. [36] reported that secondary metabolites obtained from *B. licheniformis* at a concentration of 50 and 25% showed an inhibiting effect in 100% against *V. inaequalis*, while the metabolites obtained from *B. subtilis* at a concentration of 50% showed an inhibiting effect near to 78% of the development

In another study, Tucuch-Pérez et al. [39] reported six *Bacillus* spp. strains with antifungal activity against *F. oxysporum*; in this case, the species *B. licheniformis* and *B. subtilis* showed the highest inhibition percentages ranged from 80 to 100%, being

The results generated by the microplate dilution method are consistent with the results obtained from indirect test by confrontation or dual test, such as shown in the following results against fungi isolated from crops as melon, pepper, and others, which are produced in different zones from Mexico. In melon crop, the root and stem rot disease is a big problem; in this context, Espinoza-Ahumada et al. [40] studied the in vitro antagonist effects of *Trichoderma* spp. and found that *T. asperellum* have excellent biological activity against *Fusarium* strains, isolated from melon,

*Antifungal activity of secondary metabolites from different* Trichoderma *spp. strains against phytopathogenic fungi; (a)* T. asperellum *vs.* Fusarium oxysporum*, (b)* T. yunannense *vs.* Phytophthora capsici*, (c)* T. longibrachiatum *vs.* Rhizoctonia solani*, (d)* T. asperellum *vs.* Sclerotinia sclerotiorum *and* 

in all wells is added, keeping all the wells a volume

**104**

**Figure 4.**

*(e)* T. asperellum *vs.* Sclerotium cepivorum*.*

*Percentage of inhibition of secondary metabolites obtained from* Bacillus *spp. (a) and* Trichoderma *spp. (b) against* Venturia inaequalis.

#### **Figure 6.**

*(a) Percentage inhibition of microbial extracts from* Bacillus *spp. metabolite dilutions against* F. oxysporum*. (b) Microplate with treatments to several concentrations, and the pathogen elapsed 48 h after incubation. Row A= B-AN1, B = B-AN2, C = B-AN3, D = B-AN4, E = B-AN5, F = B-AN6; column 1 = negative witness, column 2 = positive witness, 3 = growth medium of* Bacillus *spp., 5 = 50%, 5 = 25%, 6 = 12.50%, 7 = 6.25%, 8 = 3.13%, 9 = 1.56%, 10 = 0.78%, 11 = 0.39%, and 12 = 0.20%.*

shown in **Table 3**. In general, these authors report that the inhibition of *Fusarium* spp. is higher when *Trichoderma* spp. are used (62.4–54.8%), in contrast when *Bacillus* spp. (44.5–36.9%) is used (**Figure 7**).

In a work carried out by Francisco et al. [41], where the behavior of *Bacillus* spp. against *Fusarium* species was studied, it showed low inhibition values. However, they report that the species *B. pumilus* and *B. liquefaciens* can be used effectively against many *Fusarium* species. On the other hand, higher effectiveness of *Bacillus*


*High letters indicate comparison between columns; low letters indicate comparison between rows. Percentages of inhibition with different letters are significantly different (p* ≤ *0.5).*

#### **Table 3.**

*Percentage of antagonism of different biological agents against* Fusarium *spp. strains.*

#### **Figure 7.**

*Microbial agents antagonist to* Fusarium oxysporum *(FAF-3, FRE-4, FHA-6) and* Fusarium solani *(FRR-1, FRG-2, FCA-5).*

spp. was observed when applied in the early stage of growth [42], showing that *B. cereus* was most effective against Fusarium dry rot when applied as young cultures (24 h), however *B. thuringiensis* strains was most effective when applied as older cultures (48–72 h). Nevertheless, different studies revealed that *B. pumilus* produced different antifungal compounds as "iturin" which inhibits the growth of *Aspergillus* sp. and their production of aflatoxins [30]. Osorio et al*.* [23] found an inhibition ranged between 4.3 and 48.8% of *P. capsici* mycelial growth induced by the volatile compounds produced by *Trichoderma* spp. strains. The Tukey test indicated that 21 *Trichoderma* spp. strains showed the highest percentage inhibition. *T. asperellum* (T25) strain present the best result for activity inhibition, strain (T9) being the one with the least inhibition activity. It observed that the 31 *Trichoderma* spp. strains were able to produce volatile compounds with inhibitory properties against *P. capsici.*

### **6. Antifungal activity bioassay under greenhouse conditions**

From different research projects under greenhouse conditions, we have found satisfactory results both in the disease suppression and in the promotion of growth and quality in crops. Hernández-Castillo et al. [24], made an experiment under greenhouse conditions using silty clay soil from an experimental batch previously plot with chile crop and were symptoms of wilting incidence were express. The experiment included three bacterial strains of the genus *Bacillus* spp. (B1, B3, and B13), a chemical control (thiabendazole, T), and control (TA) without fungicide. Before the application of the suspension, an initial colony-forming units (CFU) count of the pathogen involved by the dilution method is performed. The application of spores of the bacterial strains is performed at the time of the transplant. The seedlings are immersed in a spore suspension at a concentration of 108 (CFU/mL for 15 min). Subsequently, at 20 and 40 days after transplantation, the same spore suspension was applied to the stem base. In the final evaluation of each treatment (10 adult plants), plant height, fresh fruit weight per cut, root length, dry root weight, and incidence and severity of wilt are measured. The determination of the severity of the disease was with the scale reported by Copes and Stevenson [43]. As a result of this work, a very low wilt incidence found for plants

**107**

**Figure 9.**

**Figure 8.**

*(TA) and chemical control (T = thiabendazole).*

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

is inoculated with biological strains (B13 and B3) with an incidence of less than 10%, while values of 60 and 40% for TA and T, respectively, were observed (**Figure 8A**). Likewise, the wilting showed a reduction in severity in those treatments where three bacterial strains were applied (**Figure 8B**), in contrast to the control treatments where

In **Figure 9**, we can see that the harmful microbiological population rate also reduced with the use of organisms considered as beneficial, according to the final count at the end of the experiment; that could be because antagonistic bacteria are capable of influencing biocontrol mechanisms against phytopathogenic fungi such as antibiosis, siderophores, competition for nutrients, and production of hydrolytic enzymes. Similarly, Ulacio et al. [44] evaluated organic matter and antagonistic microorganisms as management strategies against white rot in garlic cultivation. These authors reported that the fungus *Sclerotium cepivorum* is significantly reduced and there was a lower incidence of the disease in the treatments where the fungus

Some microorganisms posess the ability by several ways to reduce the incidence

*T. harzianum*, the bacteria *B. firmus*, and vermicompost were combined.

and severity of diseases in crops, and also can participate in the stimulation of plant growth, yield, and crop quality. **Figure 10A** and **B** shows the values related to the promotion of root length (A) and its weight (B), where this effect is clearly observed. In **Figure 10C** and **D**, it was observed that *Bacillus* spp. strains increase the height of the plant by 28% compared to treatment T, and 34.5% concerning the TA. These results coincide with previous work where the biological effectiveness

*Incidence (A) and severity (B) in plant traits with* Bacillus *spp. strains (B1, B2, B3) in contrast with control* 

*Colony-forming units from initial (Pi) and final populations of phytopathogenic soil fungi after applying* 

Bacillus *spp. strains (B1, B2, B3) against chemical (T = thiabendazole) and control (TA).*

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

the severity of the damage was more considerable.

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

is inoculated with biological strains (B13 and B3) with an incidence of less than 10%, while values of 60 and 40% for TA and T, respectively, were observed (**Figure 8A**). Likewise, the wilting showed a reduction in severity in those treatments where three bacterial strains were applied (**Figure 8B**), in contrast to the control treatments where the severity of the damage was more considerable.

In **Figure 9**, we can see that the harmful microbiological population rate also reduced with the use of organisms considered as beneficial, according to the final count at the end of the experiment; that could be because antagonistic bacteria are capable of influencing biocontrol mechanisms against phytopathogenic fungi such as antibiosis, siderophores, competition for nutrients, and production of hydrolytic enzymes. Similarly, Ulacio et al. [44] evaluated organic matter and antagonistic microorganisms as management strategies against white rot in garlic cultivation. These authors reported that the fungus *Sclerotium cepivorum* is significantly reduced and there was a lower incidence of the disease in the treatments where the fungus *T. harzianum*, the bacteria *B. firmus*, and vermicompost were combined.

Some microorganisms posess the ability by several ways to reduce the incidence and severity of diseases in crops, and also can participate in the stimulation of plant growth, yield, and crop quality. **Figure 10A** and **B** shows the values related to the promotion of root length (A) and its weight (B), where this effect is clearly observed. In **Figure 10C** and **D**, it was observed that *Bacillus* spp. strains increase the height of the plant by 28% compared to treatment T, and 34.5% concerning the TA. These results coincide with previous work where the biological effectiveness

#### **Figure 8.**

*Organic Agriculture*

spp. was observed when applied in the early stage of growth [42], showing that *B. cereus* was most effective against Fusarium dry rot when applied as young cultures (24 h), however *B. thuringiensis* strains was most effective when applied as older cultures (48–72 h). Nevertheless, different studies revealed that *B. pumilus* produced different antifungal compounds as "iturin" which inhibits the growth of *Aspergillus* sp. and their production of aflatoxins [30]. Osorio et al*.* [23] found an inhibition ranged between 4.3 and 48.8% of *P. capsici* mycelial growth induced by the volatile compounds produced by *Trichoderma* spp. strains. The Tukey test indicated that 21 *Trichoderma* spp. strains showed the highest percentage inhibition. *T. asperellum* (T25) strain present the best result for activity inhibition, strain (T9) being the one with the least inhibition activity. It observed that the 31 *Trichoderma* spp. strains were able to produce volatile compounds with inhibitory properties

*Microbial agents antagonist to* Fusarium oxysporum *(FAF-3, FRE-4, FHA-6) and* Fusarium solani *(FRR-1,* 

**6. Antifungal activity bioassay under greenhouse conditions**

From different research projects under greenhouse conditions, we have found satisfactory results both in the disease suppression and in the promotion of growth and quality in crops. Hernández-Castillo et al. [24], made an experiment under greenhouse conditions using silty clay soil from an experimental batch previously plot with chile crop and were symptoms of wilting incidence were express. The experiment included three bacterial strains of the genus *Bacillus* spp. (B1, B3, and B13), a chemical control (thiabendazole, T), and control (TA) without fungicide. Before the application of the suspension, an initial colony-forming units (CFU) count of the pathogen involved by the dilution method is performed. The application of spores of the bacterial strains is performed at the time of the transplant. The seedlings are immersed in a spore suspen-

after transplantation, the same spore suspension was applied to the stem base. In the final evaluation of each treatment (10 adult plants), plant height, fresh fruit weight per cut, root length, dry root weight, and incidence and severity of wilt are measured. The determination of the severity of the disease was with the scale reported by Copes and Stevenson [43]. As a result of this work, a very low wilt incidence found for plants

(CFU/mL for 15 min). Subsequently, at 20 and 40 days

**106**

against *P. capsici.*

**Figure 7.**

*FRG-2, FCA-5).*

sion at a concentration of 108

*Incidence (A) and severity (B) in plant traits with* Bacillus *spp. strains (B1, B2, B3) in contrast with control (TA) and chemical control (T = thiabendazole).*

#### **Figure 9.**

*Colony-forming units from initial (Pi) and final populations of phytopathogenic soil fungi after applying*  Bacillus *spp. strains (B1, B2, B3) against chemical (T = thiabendazole) and control (TA).*

#### **Figure 10.**

*Root length (A), dry rot weight (B), height (C), fresh fruit weight (D), and increments in chile pepper plant by effect* Bacillus *strains (B1, B3, B13) against chemical (T = thiabendazole) and control (TA). Different letters with bars indicate significant differences among treatments (p* ≤ *0.05)*.

of 57 strains of the genus *Bacillus* spp. isolated from the rhizosphere of commercial sowing chile plants in Northeast Mexico was analyzed, which showed an apparent antagonistic effect against *P. capsici*, *F. oxysporum*, and *R. solani* fungi. The plants inoculated with *Bacillus* spp. strains significantly increased height and dry weight in 191 and 60.2%, respectively [12]. The application of native *Bacillus* spp. strains shows a clear tendency to produce more biomass compared to chemical (T) and control (TA) treatments.

Likewise, del Ángel et al. [45] found a decrease in the incidence and severity of the disease caused by *Rhizoctonia solani* and *Fusarium oxysporum* with formulated endophytic bacteria, which induce a positive effect on the promotion of growth in the bean crop, increasing height and stem diameter in the treatments. Those formulated with bacteria in the absence of the phytopathogen stood out for their stimulating effect on the growth of the plants under study. This stimulating growth effect is observed in

#### **Figure 11.**

*Effect of endophytic bacteria on plant height and stem diameter in bean crop under greenhouse condition.*  Fusarium solani*: height (A), diameter (B), and* Rhizoctonia solani*: height (C), diameter (D). Means with the same letter are not significantly different according to the Tukey test (p* ≤ *0.05). Error bars are a standard error of the mean.*

**109**

**Table 4.**

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

plants treated with those formulated and inoculated at the same time with pathogens. It is essential to mention that the plants grew under no chemical treatment. Therefore,

Jimenez et al. [36] report results obtained on apple fruit and trees under the direct

) to

and for the control

and control, respectively, after 15 days of

was lower in first evaluation (after 15 days of

**15 days 60 days 15 days 60 days**

influence of the application of CFU from *Bacillus* spp., and *Trichoderma* spp., as control agents against the incidence and severity of *Venturia inaequalis* under field conditions in commercial apple cultivar. **Table 4** shows the incidence of fungus *Venturia inaequalis* in fruit, and this incidence varied from 5.6 to 6.25 when biological agents (*Trichoderma* spp. and *Bacillus* spp.) were used in maxima doses (2 L ha<sup>−</sup><sup>1</sup>

19.3% for the control, respectively, after 15 days of a first application. After 60 days from the start of the applications, the incidence is expressed in a range of 42.5–

observed a 91.2%. The range of severity is observed between 1.8 and 2.6 of lesions per

application initiation. After 60 days of treatment application appears first symptoms, so it was evaluated on a range of the number of lesions per fruit (severity) from 5.3 to 14.5 corresponding to *Bacillus* spp., 2 L ha−1, and control, respectively (**Table 4**). The treatment with the best antagonism effect under field conditions was *Bacillus* spp.,

the control, which showed 91.2% incidence and 14.5 lesions per fruit (**Figure 12**). The field experiment is carried out to test biocontrol agents for control *V. inaequalis* in commercial apple cultivar; the statistical analysis showed highly significant differences between treatments (p ≤ 0.5), the incidence in foliage

first application) and until harvest. This treatment expressed 10.6% incidence and two lesions per leaf, in contrast to the control which showed 31.8% and three lesions per leaf (**Table 5**). On the other hand, severity did not show significant differences

Espinoza-Ahumada et al. [40] aimed to find more environmentally friendly alternatives to the wilting of chile pepper; they evaluated the application of

**Treatment Incidence (%) in fruit Severity (lesions) in fruit**

*Bacillus* spp. 1 L ha<sup>−</sup><sup>1</sup> 18.12 ± 2.4a 51.87 ± 5.5b 1.82 ± 0.6ab 8.02 ± 0.7b *Bacillus* spp. 2 L ha<sup>−</sup><sup>1</sup> 6.25 ± 5.2b 42.50 ± 6.5b 1.07 ± 0.8b 5.30 ± 0.5b *Trichoderma* spp. 1 L ha<sup>−</sup><sup>1</sup> 15.00 ± 3.5a 55.00 ± 5.4b 1.77 ± 0.5ab 7.62 ± 0.2b *Trichoderma* spp. 2 L ha<sup>−</sup><sup>1</sup> 5.62 ± 4.7b 45.62 ± 5.2b 1.00 ± 0.0b 6.32 ± 0.7b Control 19.37 ± 4.7a 91.25 ± 4.3a 2.65 ± 0.5a 14.57 ± 0.3a

*Treatments with the same letter are statistically equal to each other (p < 0.05).*

*The incidence in apple fruits by* Venturia inaequalis.

, who expressed 42.5% by incidence and five lesions per fruit in contrast to

they did not receive fertilization by any chemical source (**Figure 11**).

**7. Bioassays of antifungal activity under field conditions**

46.62% for *Bacillus* spp. and *Trichoderma* spp. at doses of 2 L ha<sup>−</sup><sup>1</sup>

fruit by treatment *Trichoderma* spp. 2 L ha<sup>−</sup><sup>1</sup>

treated with *Trichoderma* spp. 2 L ha<sup>−</sup><sup>1</sup>

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

**7.1 Fruits results**

2 L ha<sup>−</sup><sup>1</sup>

among treatments.

**7.2 Vegetable results**

plants treated with those formulated and inoculated at the same time with pathogens. It is essential to mention that the plants grew under no chemical treatment. Therefore, they did not receive fertilization by any chemical source (**Figure 11**).
