**3. Fungicidal potential of actinobacteria against phytopathogen in crop plants**

Fungal phytopathogens pose serious problems worldwide and cause a number of plants and animal diseases such as ringworm, athlete's foot, and several more serious diseases. Plant diseases caused by fungi include rusts, smuts, rots, and may cause severe damage to crops. Fungi are some of the world's largest and possibly oldest individuals.

Agrochemical treatment may result in environmental impact and pose a threat to humans and animals. As a result, there has been an increase in research on potential Biocontrol agents, aimed at finding a definitive solution or at least reducing pesticide use in the treatment of phytopathogenic diseases.

Actinobacteria have been considered as potential Biocontrol agents of plant diseases. Several investigators have described the *in vitro* and *in vivo* activities of the actinobacteria. Their modes of action includes parasitism of hyphae (El-Tarabily and Sivasithamparam, 2006), oospores or fungal sclerotia (Crawford *et al.,* 1993) competition with pathogens (Kunoh, 2002), antibiotic production (Igarashi, 2004), siderophores (Khamna *et al.,* 2009), as herbicides (Hasegawa *et al.,* 2006), and via enzymes such as cellulases, hemicellulases, chitinases, amylases, and glucanases (Yuan and Crawford, 1995).

In addition, actinobacteria may affect plant growth (Igarashi, 2004). According to Kunoh (2002), endophytic *Streptomyces* may play an important role in the development and health of plants, because it affects plant growth due to its assimilation of nutrients and production of secondary metabolites. The tomato (*Lycopersicon esculentum*) is highly susceptible to phytopathogen attack, and tomato crops are most intensively treated with agrochemicals.

All the clinical pathogens were prepared in 0.85% saline corresponding to No. 0.5 McFarland turbidity standard. All cultures were incubated on a shaker at 37°C for 18 h and then diluted to 1/10 the concentration to yield a culture density of approximately 108CFU/ml. The pathogens used in the study such as *Candida albicans, Cryptococcus* 

Mueller Hinton agar (Beef extract 0.2 g, Peptone 1.75g, Starch 0.15g, Agar 2.0g, Distilled water 100 ml, pH 7.5) prepared with lawn culture using desired test organisms. The inoculated plates were kept aside for few minutes. The discs with fungicidal compound were placed over the medium. After diffusion, the plates were incubated at 28°C for 48hours for antifungal analysis. After incubation, the zone of inhibition was analyzed and recorded.

To measure the MIC value, two-fold serial dilutions of 50, 25, 12.5, 6.25, 3.125, 1.562 and 0.781 mg ml-1 of the fungicidal compound was prepared in solvent and assayed by well diffusion method. The MIC was defined as the lowest concentration able to inhibit any

Fungal phytopathogens pose serious problems worldwide and cause a number of plants and animal diseases such as ringworm, athlete's foot, and several more serious diseases. Plant diseases caused by fungi include rusts, smuts, rots, and may cause severe damage to

Agrochemical treatment may result in environmental impact and pose a threat to humans and animals. As a result, there has been an increase in research on potential Biocontrol agents, aimed at finding a definitive solution or at least reducing pesticide use in the

Actinobacteria have been considered as potential Biocontrol agents of plant diseases. Several investigators have described the *in vitro* and *in vivo* activities of the actinobacteria. Their modes of action includes parasitism of hyphae (El-Tarabily and Sivasithamparam, 2006), oospores or fungal sclerotia (Crawford *et al.,* 1993) competition with pathogens (Kunoh, 2002), antibiotic production (Igarashi, 2004), siderophores (Khamna *et al.,* 2009), as herbicides (Hasegawa *et al.,* 2006), and via enzymes such as cellulases, hemicellulases,

In addition, actinobacteria may affect plant growth (Igarashi, 2004). According to Kunoh (2002), endophytic *Streptomyces* may play an important role in the development and health of plants, because it affects plant growth due to its assimilation of nutrients and production of secondary metabolites. The tomato (*Lycopersicon esculentum*) is highly susceptible to phytopathogen attack, and tomato crops are most intensively treated with agrochemicals.

**3. Fungicidal potential of actinobacteria against phytopathogen in crop** 

crops. Fungi are some of the world's largest and possibly oldest individuals.

chitinases, amylases, and glucanases (Yuan and Crawford, 1995).

**2.7.2 Inoculum preparation** 

**2.7.3 Antifungal assay** 

visible fungal growth.

treatment of phytopathogenic diseases.

**plants** 

*neoformans* and *Aspergillus flavus* for sensitivity assay.

**2.8 Determination of Minimum Inhibitory Concentrations (MIC)** 

Among the many fungal pathogens that attack tomatoes are *Phytophthora infestans, Alternaria solani, Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium oxysporum*, Most of these pathogens not only spread disease in tomato plants, but also affect other crops. According to Cao *et al*. (2004a), *R. solani* can develop in both farmed and unfarmed soils, spreading disease in many crops, including rice. *F. oxysporum* attacks banana plants, causing a disease known as fusarium wilt (Cao *et al.,* 2004b) and also infects wheat (Taechowisan *et al.,* 2003). *R. solanacearum* is an important soil pathogen causing bacterial wilt in more than 200 plant species, including the potato, tomato, pea, tobacco, banana and others (Tan *et al.,* 2006).

Marten *et al*. (2001) reported that RhizovitR from *Streptomyces rimosus* is used in the control of a wide range of fungi such as *Pythium* spp., *Phytophthora* spp., *Rhizoctonia solani*, *Alternaria brassicola*, and *Botrytis* sp. Liu *et al*. (2004a) also reported that *S. rimosus* showed a high antagonism activity against *Fusarium solani*, *F. oxysporium* f sp. *cucumarinum*, *Verticillium dahliae*, *R. solani*, *Fulvia fulva*, *Botrytis cinerea*, *A. alternata*, *Sclerotinia sclerotiorum* and *Bipolaris maydis*. The antifungal antibiotic, which is produced by *S. rimosus*, was purified by silica gel column chromatography. Its ultraviolet (UV) spectrum was consistent with that of polyene macrolide, which had the same absorption peaks at 291, 305, and 318 nm. Antifungal activity can be kept for 20 months at room temperature (12–30C, pH 5.4) (Liu *et al.,* 2004b). So *S. rimosus* will be employed as a target to search for new biocontrol agents or drugs to satisfy public demands, and much interests will be generated (Table1).


Applications of Actinobacterial Fungicides in Agriculture and Medicine 39

Saadoun *et al*. (2000b) identified several *Streptomyces* isolates from soils in northern Jordan which were bioassayed for their antifungal activity against several food-associated fungi and moulds isolated from olive-mill residue. Dhanasekaran et al. (2008) reported the antifungal compound 4' phenyl-1-napthyl-phenyl acetamide from *Streptomyces* sp. DPTB16*.* It showed significant antifungal activity against *Candida albicans* followed by *Aspergillus niger, A. fumigatus, A. flavus* and minimum inhibitory activity was observed with *Mucor* sp*.* and *Penicillium* sp*.* Kumar and Kannabiran (2010b) reported the antifungal activity of *Streptomyces* VITSVK5 spp.

Dermatophyte infections are one of the earliest known fungal infections of mankind and are very common throughout the world. There are three genera of dermatophytes, such as *Trichophyton, Microsporum* and *Epidermophyton*. Dermatophytoses are world wide in distribution with high prevalence in tropical and sub-tropical countries due to the hot and humid climate which favours their growth. As the dermatophytes have developed resistance to antimycotic drugs and due to a lack of safe and effective antifungal antibiotics, there is an urgent need for nontoxic, safe and cost effective antifungal antibiotics. Deepika *et al*. (2009) reported the actinobacteria exhibiting antidermatophytic activity against *Trichophyton rubrum*  were identified among 100 isolates by cross streak method. Among them only two actinobacterial isolates DKD 6 and DKD 7 exhibiting potential antidermatophytic activity and

against drug resistant *Aspergillus* clinical isolates from pulmonary tuberculosis patients.

further characterized and identified as *Streptomyces* sp (Table 2; Plate 3).

Table 2. Fungicidal activity of actinobacterial isolates against human pathogens



#### **4. Fungicidal potential of actinobacteria against human fungal pathogens**

Some species of fungi produce mycotoxins that are very toxic to humans. For example, the fungus *Claviceps purpurea* causes the ergot poisoning. An individual infected with the mycotoxin experiences hallucination, gangrene, and blood flow restrictions in limbs. Humans usually get infected with the fungus after eating cereal grains contaminated with *C. purpurea*.

The incidence of opportunistic mycoses and the number of different fungal pathogens are increasing dramatically. During the 1980s, the frequency of nosocomial candidemia increased as much as 500% over the decade (Mitchell 1998). High mortality and increasing antifungal drug resistance are also major concerns. These trends will continue unless better preventive or treatment measures are developed. The traditional approach is to increase the screening programmes that are still being initiated in various countries for the isolation of antibiotic producing microorganisms from the environment, especially marine and terrestrial soil, which provide a rich source for these organisms, particularly the actinobacteria (Labeda & Shearer 1990). It has been estimated that approximately two-thirds of naturally occurring antibiotics have been isolated from actinobacteria (Takizawa *et al*. 1993). Of these antibiotics, the majority were isolated from the genus *Streptomyces* (Goodfellow & O'Donnell 1989).

Table 1. Fungicidal activity of actinobacterial isolates against plant pathogens

were isolated from the genus *Streptomyces* (Goodfellow & O'Donnell 1989).

**4. Fungicidal potential of actinobacteria against human fungal pathogens** 

Some species of fungi produce mycotoxins that are very toxic to humans. For example, the fungus *Claviceps purpurea* causes the ergot poisoning. An individual infected with the mycotoxin experiences hallucination, gangrene, and blood flow restrictions in limbs. Humans usually get infected with the fungus after eating cereal grains contaminated with *C. purpurea*. The incidence of opportunistic mycoses and the number of different fungal pathogens are increasing dramatically. During the 1980s, the frequency of nosocomial candidemia increased as much as 500% over the decade (Mitchell 1998). High mortality and increasing antifungal drug resistance are also major concerns. These trends will continue unless better preventive or treatment measures are developed. The traditional approach is to increase the screening programmes that are still being initiated in various countries for the isolation of antibiotic producing microorganisms from the environment, especially marine and terrestrial soil, which provide a rich source for these organisms, particularly the actinobacteria (Labeda & Shearer 1990). It has been estimated that approximately two-thirds of naturally occurring antibiotics have been isolated from actinobacteria (Takizawa *et al*. 1993). Of these antibiotics, the majority Saadoun *et al*. (2000b) identified several *Streptomyces* isolates from soils in northern Jordan which were bioassayed for their antifungal activity against several food-associated fungi and moulds isolated from olive-mill residue. Dhanasekaran et al. (2008) reported the antifungal compound 4' phenyl-1-napthyl-phenyl acetamide from *Streptomyces* sp. DPTB16*.* It showed significant antifungal activity against *Candida albicans* followed by *Aspergillus niger, A. fumigatus, A. flavus* and minimum inhibitory activity was observed with *Mucor* sp*.* and *Penicillium* sp*.* Kumar and Kannabiran (2010b) reported the antifungal activity of *Streptomyces* VITSVK5 spp. against drug resistant *Aspergillus* clinical isolates from pulmonary tuberculosis patients.

Dermatophyte infections are one of the earliest known fungal infections of mankind and are very common throughout the world. There are three genera of dermatophytes, such as *Trichophyton, Microsporum* and *Epidermophyton*. Dermatophytoses are world wide in distribution with high prevalence in tropical and sub-tropical countries due to the hot and humid climate which favours their growth. As the dermatophytes have developed resistance to antimycotic drugs and due to a lack of safe and effective antifungal antibiotics, there is an urgent need for nontoxic, safe and cost effective antifungal antibiotics. Deepika *et al*. (2009) reported the actinobacteria exhibiting antidermatophytic activity against *Trichophyton rubrum*  were identified among 100 isolates by cross streak method. Among them only two actinobacterial isolates DKD 6 and DKD 7 exhibiting potential antidermatophytic activity and further characterized and identified as *Streptomyces* sp (Table 2; Plate 3).


Table 2. Fungicidal activity of actinobacterial isolates against human pathogens

Applications of Actinobacterial Fungicides in Agriculture and Medicine 41

Oki *et al.* (1989) studied cispentacin, a new antifungal antibiotic and its *in vitro* and *in vivo* antifungal activities. Novel antifungal antibiotics, Maniwamycins A and B I and II and their

Konishi *et al.* (1989) studied the production, isolation, physico-chemical properties and its structure of cispentacin, a new antifungal antibiotic. A novel hepatoprotective -lactone, MH-031 was discovered and their physico-chemical properties and structure have been reported (Itoh *et al.,* 1991). Nishio *et al.* (1989) reported Karnamicin, a complex of new antifungal antibiotic from *Streptomyces* sp. and its taxonomy, fermentation, physico-chemical

Sawada *et al.* (1990) reported new antifungal antibiotics, pradimicins D and E glycine analogs of pradimicins A and C. Water-soluble pradimicin derivatives synthesis and antifungal evaluation of N, N-dimethyl pradimicins derived from *Actinomadura hibisca* was

Kakushima *et al.* (1990) studied the effect of stereochemistry at the C-17 position on the antifungal pradimicin A. Stephan *et al*. (1996) was observed that Kanchanamycins, new polyol macrolide antibiotics produced by *S. olivaceus*. The structures of the Kanchanamycins were determined by eletrospray MS and modern 2D NMR techniques. A Manumycin type antibiotic (SW-B) was isolated from a solid agar culture of *S. flavus* strain A-11. The structure

Harindran *et al*. (1999) isolated a new antifungal antibiotic, HA-1-92 from the biomass of *Streptomyces* CDRIL-312. The antibiotic is presumed to be an oxehexaene macrolide and showed promising antifungal activity against yeasts and filamentous fungi. The structure elucidation and antifungal activity of plants an anthracycline antibiotic, daunomycin, isolated from *Actinomadura roseola* against *Phytophthora* blight in pepper have been reported (Kim *et al.,* 2000). A new tetraene polyene macrolide antibiotic was isolated from the culture broth of *S. arenae* var*. ukrainiana* and its structure was determined on the basis of spectral data such as UV, IR, 1H, 13C NMR and Mass spectroscopy (Gupte *et al.,* 2000). The isolation and structure elucidation of a new antifungal and antibacterial antibiotic produced by

Hwang *et al.,* (2001) isolated the antifungal substances SH-1 and SH-2 from *Streptomyces humidus* strains SE 55 cultures by various purification procedures and identified as phenyl acetic acid and sodium phenyl acetate respectively based on the nuclear magnetic resonance, electron ionization mass spectral analysis and inductively coupled plasma mass spectral data SH-1 and SH-2. The two compounds were as effective as the fungicide metalaxyl in inhibiting spore germination and hyphal growth. Raytapadar and Paul (2001) found a broad-spectrum antifungal *Streptomyces* isolate IDA-28 from Indian soil, which was characterized and identified as *Streptomyces aburaviensis* var*. ablastmyceticus* (MTCC 2469). Frandberg *et al*. (2000) observed antifungal compounds on solid substrate that inhibit radial growth of fungi among Ascomycetes, Basidomycetes, Deuteromycetes, Oomycetes and Zygomycetes and strongly affected hyphal branching and morphology of the fungus such as

Ellis (2002) reported that Amphotericin B is a polyene macrolide antibiotic derived from the actinomycete, *Streptomyces nodosus*. Amphotericin B has a relatively broad spectrum of

was determined by MS and by 1 and 2D NMR spectroscopy (Kook *et al.,* 1996).

*Aspergillus niger*, *Mucor hiemalis*, *Penicillium roqueforti* and *Paecilomyces variotii*.

*Streptomyces* sp. have been reported (Bordoloi *et al.,* 2001).

structure were studied and reported (Nakayama *et al.,* 1989; Takahashi *et al.,* 1989).

and biological properties.

studied and reported (Oki *et al.,* 1990a,b).

Plate 3. Antifungal activity 4' phenyl-1-napthyl-phenyl acetamide and methyl substituted -lactum compounds of *Streptomyces* isolates DPTB16 and DPTD21
