**Applications of Actinobacterial Fungicides in Agriculture and Medicine**

D. Dhanasekaran1, N. Thajuddin1 and A. Panneerselvam2 *1Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamilnadu, 2P.G. & Research Department of Botany & Microbiology, A.V.V.M. Sri Pushpam College, (Autonomous), Poondi, Tamil Nadu, India* 

## **1. Introduction**

28 Fungicides for Plant and Animal Diseases

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Actinobacteria are found in virtually every natural substrate, such as soils and compost, freshwater basins, foodstuffs and the atmosphere. Deep seas, however, do not offer a favorable habitat. These organisms live and multiply most abundantly in various depths of soil and compost, in cold and in tropical regions. Alkaline and neutral soils are more favorable habitats than acid soils and neutral peats are more favorable than acid peats.

The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active antifungal compounds are obtained from the actinobacteria. A number of these isolates were capable of suppressing the fungal pathogens *Rhizoctonia solani*, *Pythium* sp. and *Gaeumannomyces graminis* var. *tritici*, both *in vitro* and in plants indicating the potential of the actinobacteria to be used as biocontrol agents.

The principal reason behind the actinobacteria having such important roles in the soil and in plant relationships comes from the ability of the actinobacteria to produce a large number of secondary metabolites, many of which possess antibacterial activity. Actinobacteria produce approximately two-thirds of the known antibiotics produced by all mircoorganisms. The genus *Streptomyces* produces nearly 80% of the actinobacterial antibiotics, with the genus *Micromonospora* producing one-tenth as many as the *Streptomyces*. In addition to the production of antibiotics the actinobacteria produce many secondary metabolites with a wide range of activities. Activities of the secondary metabolites include antifungal agents

Applications of Actinobacterial Fungicides in Agriculture and Medicine 31

that degrade cell walls and inhibit the synthesis of mannan and β-glucan enzymes, antiparasitic agents and insecticidal agents.

Actinobacteria produce a number of plant growth regulatory compounds, some of which have been used commercially as herbicides. Not all secondary metabolites are antimicrobial. Others are enzyme inhibitors, immunomodulators and antihypertensives. The actinobacteria produce over 60% of secondary metabolites produced by microorganisms, with *Streptomyces* accounting for over 80%.

In some cases actinobacteria form a pathogenic relationship with plants. *Streptomyces scabies*  is a soil-borne actinobacterium that is the principal causal agent of scab diseases, which affect a variety of underground tuberous vegetables such as potato. *S. scabies* produces thaxtomin, a family of phytotoxins, that induce the development of necrotic lesions in potato. There is a 100% correlation between pathogenicity and the ability to produce thaxtomin. Scab suppressive soils have been identified and it has been found that the lenticels on these tubers are colonised by *Streptomyces* (Schottel *et al.,* 2001). Suppressive strains of *Streptomyces* isolated from a naturally scab suppressive soil produced antibiotics that inhibited *S. scabies in vitro* (Neeno-Eckwall and Schottel, 1999).

*Streptomyces* species have also been implicated in the biological control of a number of other pathogens. *S. ambofaciens* inhibited *Pythium* damping-off in tomato plants and *Fusarium* wilt in cotton plants. *S. hygroscopius* var. *geldanus* was able to control *Rhizoctonia* root rot in pea plants and the inhibition was due to the production of the antibiotic geldanamycin. *Streptomyces lydicus* WYEC108 inhibited *Pythium ultimum* and *R. solani in vitro* by the production of antifungal metabolites (Yuan and Crawford, 1995). A number of other actinobacteria that are used in inhibiting the human and animal pathogens such as *Aspergillus niger, Penicillium* sp., *Mucor* sp., *Rhizopus* sp. *Candida albicans*, *Cryptococcus neoformans.* This chapter describes the potential applications of fungicidal substances from actinobacterial origin, screening methods, mode of action of fungicides against plant and animal fungal pathogens.

#### **1.1 Antagonistic actinobacteria**

The actinobacteria first recognized as potential destroyers of fungi and bacteria by Gasperini (1890). Tims (1932) studied an actinobacteria antagonistic to *Pythium* of sugarcane. Waksman (1937) made a detailed survey of actinobacteria possessing antagonistic effect upon the activity of other microorganisms in their studies on decomposition.

Dhanasekaran *et al.,* (2009a) screened 78 *Streptomyces* isolates for their antimicrobial activity against pathogenic fungi by agar overlay assay method. Among the 78 isolates, 18 isolates showed antifungal activity. The maximum percentage of the isolates of *Streptomyces*, which showed antifungal antagonistic activity, was found in sea shore soil (13/27 isolates, 48.14 %) followed by salt pan soil (4/9 isolates, 44.44 %), estuarine soil (3/12 isolates, 25 %) and agricultural field soil (5/30 isolates, 16.6 %). Among the 18 isolates tested, all the isolates showed extracellular antifungal activity including 8 isolates having both extra and intracellular antifungal activity (Fig.1; Plate 1). They also studied the antifungal actinobacteria in marine soil of Tamilnadu against *Candida albicans, Aspergillus niger* using agar overlay, diffusion assay method (Dhanasekaran *et al.,* (2005b) and estuarine *Streptomyces* against the *Candida albicans* (Dhanasekaran *et al.,* (2009b)

that degrade cell walls and inhibit the synthesis of mannan and β-glucan enzymes,

Actinobacteria produce a number of plant growth regulatory compounds, some of which have been used commercially as herbicides. Not all secondary metabolites are antimicrobial. Others are enzyme inhibitors, immunomodulators and antihypertensives. The actinobacteria produce over 60% of secondary metabolites produced by microorganisms,

In some cases actinobacteria form a pathogenic relationship with plants. *Streptomyces scabies*  is a soil-borne actinobacterium that is the principal causal agent of scab diseases, which affect a variety of underground tuberous vegetables such as potato. *S. scabies* produces thaxtomin, a family of phytotoxins, that induce the development of necrotic lesions in potato. There is a 100% correlation between pathogenicity and the ability to produce thaxtomin. Scab suppressive soils have been identified and it has been found that the lenticels on these tubers are colonised by *Streptomyces* (Schottel *et al.,* 2001). Suppressive strains of *Streptomyces* isolated from a naturally scab suppressive soil produced antibiotics

*Streptomyces* species have also been implicated in the biological control of a number of other pathogens. *S. ambofaciens* inhibited *Pythium* damping-off in tomato plants and *Fusarium* wilt in cotton plants. *S. hygroscopius* var. *geldanus* was able to control *Rhizoctonia* root rot in pea plants and the inhibition was due to the production of the antibiotic geldanamycin. *Streptomyces lydicus* WYEC108 inhibited *Pythium ultimum* and *R. solani in vitro* by the production of antifungal metabolites (Yuan and Crawford, 1995). A number of other actinobacteria that are used in inhibiting the human and animal pathogens such as *Aspergillus niger, Penicillium* sp., *Mucor* sp., *Rhizopus* sp. *Candida albicans*, *Cryptococcus neoformans.* This chapter describes the potential applications of fungicidal substances from actinobacterial origin, screening methods, mode of action of fungicides against plant and

The actinobacteria first recognized as potential destroyers of fungi and bacteria by Gasperini (1890). Tims (1932) studied an actinobacteria antagonistic to *Pythium* of sugarcane. Waksman (1937) made a detailed survey of actinobacteria possessing antagonistic effect

Dhanasekaran *et al.,* (2009a) screened 78 *Streptomyces* isolates for their antimicrobial activity against pathogenic fungi by agar overlay assay method. Among the 78 isolates, 18 isolates showed antifungal activity. The maximum percentage of the isolates of *Streptomyces*, which showed antifungal antagonistic activity, was found in sea shore soil (13/27 isolates, 48.14 %) followed by salt pan soil (4/9 isolates, 44.44 %), estuarine soil (3/12 isolates, 25 %) and agricultural field soil (5/30 isolates, 16.6 %). Among the 18 isolates tested, all the isolates showed extracellular antifungal activity including 8 isolates having both extra and intracellular antifungal activity (Fig.1; Plate 1). They also studied the antifungal actinobacteria in marine soil of Tamilnadu against *Candida albicans, Aspergillus niger* using agar overlay, diffusion assay method (Dhanasekaran *et al.,* (2005b) and estuarine

upon the activity of other microorganisms in their studies on decomposition.

*Streptomyces* against the *Candida albicans* (Dhanasekaran *et al.,* (2009b)

antiparasitic agents and insecticidal agents.

with *Streptomyces* accounting for over 80%.

animal fungal pathogens.

**1.1 Antagonistic actinobacteria** 

that inhibited *S. scabies in vitro* (Neeno-Eckwall and Schottel, 1999).

Applications of Actinobacterial Fungicides in Agriculture and Medicine 33

Fig. 1. Antifungal activity of Extra and Intracelluar compounds of *Streptomyces* isolates

A series of test tubes containing 9 ml of sterile water was taken. From the stock culture, 1 ml suspension was transferred aseptically to the 1st tube (10-1) and mixed well. Further serial dilutions were made to produce 10-5 suspensions. Suspension (0.1 ml) from each test tube was spread on sterile soyabean-casein digest medium (SBCD), actinobacteria isolation agar (AIA) medium and starch-casein agar medium plates aseptically in a laminar air flow cabinet. The plates were incubated at 27 ± 2°C for 84 h. The plates were observed intermittently during incubation. After 72 h, whitish pin-point colonies, characteristic of actinobacteria and with clear zone of inhibition around them were observed. The pinpoint colonies with inhibitory or clear zone of inhibition were selected and purified and used as a

The Fungicidal activity of the soil actinobacterial isolates were analyzed by agar streak method. Each of the isolate was streaked as a straight line on Starch casein agar (SCA) medium and incubated at 27°C for 6 days. After the 6th day, different fungal pathogens were streaked at right angle, but not touching each other, and then incubated at 28°C for 48 h. If the organism is susceptible to the antibiotic produced by actinobacteria, then it will not grow near the

The *Streptomyces* isolate was smeared on SCA medium as a single streak and incubated at 28°C for 4-6 days, from well grown streaks 6 mm agar disks of *Streptomyces* colony mass was prepared by using sterile cork borers. Disks were then aseptically transferred to PDA plates having fresh lawn cultures of *Aspergillus* isolate. Controls included using plain disks from

actinobacteria. The zone of inhibition against each test fungal pathogen was noted.

**2.** *In vitro* **screening methods of fungicidal substances produced by** 

**actinobacteria** 

**2.1 Crowded plate technique** 

**2.2 Agar streak method** 

**2.3 Agar disk method** 

potent isolate for fungicidal compound production.

Plate 1. Cultural and microscopic view of *Streptomyces* isolates aerial mycelium with spores

Fig. 1. Antifungal activity of Extra and Intracelluar compounds of *Streptomyces* isolates
