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

*Francisco Daniel Hernández-Castillo, Francisco Castillo-Reyes, Marco Antonio Tucuch-Pérez and Roberto Arredondo-Valdes*

### **Abstract**

This chapter will cover topics about the microbial antagonists *Trichoderma* spp. and *Bacillus* spp. from the perspective of use as potential biological control agents on plant diseases. Results obtained in the laboratory about from their isolation, microbial strain collections for both genera, taxonomic identification, antifungal activity in in vitro tests, obtained evaluation of the antifungal effect of secondary metabolites from microbial antagonists will be shown. Besides, results obtained from bioassays in the greenhouse and field are used as biopesticides in the control of diseases in fruit trees and vegetables and their effects on the promotion of plant growth and increased crop yield.

**Keywords:** inhibition, disease, plant pathogen, incidence, severity, antagonist microorganisms

### **1. Introduction**

The agricultural production systems are generally based on technological dependence of high-yield varieties and the use of agrochemicals, causing an imbalance in different agroecosystems. The crops had turned to be susceptible to plague organisms to which before they were not and have proliferated because their natural enemies have eliminated the selection pressure has been favored by the monoculture or by the excessive use of plaguicides, what has conducted to the rupture of resistance of host and resistance toward the pesticides.

Those, as mentioned above, do rethink the actual technological management of the crops, researching less aggressive options with low or without environmental impact. This focus allows the searching of microbial alternatives as biological control agents of diseases, as a viable option to reduce their impact, enhancing the yield and quality of the agricultural products. The economic production losses caused by pests and diseases worldwide are estimated to be 36.5% on average, where 14.1% is caused by diseases, 10.2% by insects, and 12.2% by weeds, without considering the 6–12% of agricultural products postharvest losses. Although it estimated that in developing countries, these could reach up to 50% of production losses, considering only the disease, it estimates that annual losses worldwide can reach about 220 billion dollars. Overall, the diseases from plants can destroy crops before and after harvest or yield partial losses and cause loss of quality in the products harvested.

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 human health.

### **2. Biopesticides market**

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 biotechnological products.

### **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 and growth promotion in plants [8, 9].

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 identification of the species which needs work is necessary.

**99**

**Table 1.**

**Species specificity**

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

The first step for correct identification of antagonist microorganisms depends on isolation. In the case of *Trichoderma* spp., they are present in a great variety of agricultural and natural soils. The soil sampling for its isolation is relatively simple; using a shovel at 10–20 cm depth, 500 g of soil is taken and deposited in plastic bags; after, the samples will be moved to a laboratory and placed on storage at 4°C until used. Purification of *Trichoderma* spp. it is essential on investigations and present many ways or techniques; nevertheless, the monosporic culture is suggested by Trichoderma on culture media as potato dextrose agar (PDA) or *Trichoderma* Specific Medium (TSM), and incubated at 28 ± 2°C for 96 h [11–13]. Once the monosporic culture is obtained, the identification of *Trichoderma* species can be realized using taxonomic keys through its morphological features or with molecular biology, extracting DNA and utilizing general or specific primers. In case of the use of taxonomic keys, structures as width and length of phialide, length and width of conidia,

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*,

and *T. hamatum* from different localities of Mexican Northeast region.

HAR-1.6R: GGCTATGACCATGATTACGC

T2AR: CGATAGTGGGGTTGCCGTCAA

TvCTT56r: GAAGAGAGGACATAGGGTCTGG

Q01\_4R: CACGCTGACAATGACCGACAC

Th-R: TGTCACCCGTTCGGATCATCCG

PSrev1: GCGACACAGAGCACGTTGAATC

*T. harzianum* HAR-1.6F: GTACCTCGCGAATGCATCTA

*T. asperellum* T2AF: CTCTGCCGTTGACTGTGAACG

*T. virens* TvCTT56f: CTTGATGACAAGCCAAAAGG

*T. atroviride* Q01\_4F: GCACACCAACTGCTGGAGCTT

*T. aggressivum* Th-F: CGGTGACATCTGAAAAGTCGTG

*T. pleuroti* FPforw1: CACATTCAATTGTGCCCGACGA

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

*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*,

**Primer sequences (5′–3′) Product size (bp) References**

1600 [17]

507 [18]

289 [19]

1017 [20]

444 [21]

218 [22]

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

**3.1 Isolation and identification of** *Trichoderma* **spp.**

and presence of chlamydospores will be observed [7, 14].

*3.1.2 Molecular identification*

*3.1.1 Isolation and morphological identification of Trichoderma spp.*
