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

#### **2.1 Crowded plate technique**

32 Fungicides for Plant and Animal Diseases

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

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 potent isolate for fungicidal compound production.

### **2.2 Agar streak method**

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

#### **2.3 Agar disk method**

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

Applications of Actinobacterial Fungicides in Agriculture and Medicine 35

Crowded plate technique Agar streak method Agar streak method

Dual culture technique Dual culture technique Disc diffusion method

Agar overlay method Agar diffusion method Agar streak method

Food poisoning technique MIC

Plate 2. *In vitro* screening methods for Fungicidal substances produced by actinobacteria

SCA medium. Plates were incubated at 24°C for 4-6 days and fungicidal activity was evaluated by measuring the diameter of inhibition zones (mm).

#### **2.4 Dual culture method**

Antifungal activity of actinobacterial isolates were tested by dual culture technique using PDA medium. A mycelial disc of the fungal pathogen (5mm dia.) was placed at one end of the Petri plate. The actinobacterial antagonists were streaked 1 cm away from the periphery of the Petri plate just opposite to the mycelial disc of the pathogen. Visual observation on the inhibition of pathogenic fungal growth was recorded after 96 hours of incubation in comparison with the PDA plate simultaneously inoculated with fungal pathogen only as control.

Percent of test pathogen inhibition by the actinobacterial isolate was evaluated by dual culture technique. The radial growth of mycelium in mm was measured and percent inhibition (PI) was calculated.

$$\text{PI} = \frac{\text{C-T}}{\text{C}} \times 100$$

Where, C is the growth of test pathogen (mm) in the absence of the antagonistic isolate; T is the growth of test pathogen (mm) in the presence of the antagonistic isolate.

#### **2.5 Agar overlay method**

To evaluate the fungicidal activity of the actinobacteria, phytopathogenic filamentous fungi were used as test microorganisms. The actinobacteria were spot inoculated onto SCA medium and incubated at 28°C for 14 days. After this period, the antagonism between actinobacteria and the test fungal pathogen was evaluated using the agar over lay method. For this procedure, 10 ml of Sabouraud soft agar medium was added and inoculated with 106 spores/ml of filamentous fungi. All plates were incubated at 28°C and incubation time of 7-10 days for fungi.

#### **2.6 Well diffusion method**

The sterilized Sabouraud's dextrose agar medium (Dextrose 4.0 g, Mycological peptone 1.0 g, Agar 2.0 g, pH 5.0, Distilled Water 100 ml,) was poured to a petridish in a uniform thickness and kept aside for solidification. Using sterilized swabs, even distribution of lawn culture was prepared using desired fungi such as *A. niger, P. notatum*, *C. albicans* in SDA plates. Using sterile well cutter two wells were made in plates at required distance. 20l of different solvents treated test fungicidal compound was added in to one well and another well was loaded with corresponding control (solvent without compound). The plates were incubated at room temperature for 48 hours. After incubation, the zone of inhibition was analyzed and recorded.

#### **2.7 Paper disc assay**

#### **2.7.1 Preparation of disc**

The filter paper disc were impregnated with 5μg/μl antifungal compound + 25μl distilled water, similar procedures were used to prepare the other concentrations of the disc such as 10μg/μl + 20μl distilled water, 15μg/μl + 15μl distilled water, 20μg/μl + 10μl distilled water, 30μg/μl + 1μl distilled water.

SCA medium. Plates were incubated at 24°C for 4-6 days and fungicidal activity was

Antifungal activity of actinobacterial isolates were tested by dual culture technique using PDA medium. A mycelial disc of the fungal pathogen (5mm dia.) was placed at one end of the Petri plate. The actinobacterial antagonists were streaked 1 cm away from the periphery of the Petri plate just opposite to the mycelial disc of the pathogen. Visual observation on the inhibition of pathogenic fungal growth was recorded after 96 hours of incubation in comparison with the

Percent of test pathogen inhibition by the actinobacterial isolate was evaluated by dual culture technique. The radial growth of mycelium in mm was measured and percent

C-T PI= ×100 C Where, C is the growth of test pathogen (mm) in the absence of the antagonistic isolate; T is

To evaluate the fungicidal activity of the actinobacteria, phytopathogenic filamentous fungi were used as test microorganisms. The actinobacteria were spot inoculated onto SCA medium and incubated at 28°C for 14 days. After this period, the antagonism between actinobacteria and the test fungal pathogen was evaluated using the agar over lay method. For this procedure, 10 ml of Sabouraud soft agar medium was added and inoculated with 106 spores/ml of filamentous fungi. All plates were incubated at 28°C and incubation time

The sterilized Sabouraud's dextrose agar medium (Dextrose 4.0 g, Mycological peptone 1.0 g, Agar 2.0 g, pH 5.0, Distilled Water 100 ml,) was poured to a petridish in a uniform thickness and kept aside for solidification. Using sterilized swabs, even distribution of lawn culture was prepared using desired fungi such as *A. niger, P. notatum*, *C. albicans* in SDA plates. Using sterile well cutter two wells were made in plates at required distance. 20l of different solvents treated test fungicidal compound was added in to one well and another well was loaded with corresponding control (solvent without compound). The plates were incubated at room temperature for 48 hours. After incubation, the zone of inhibition was analyzed and recorded.

The filter paper disc were impregnated with 5μg/μl antifungal compound + 25μl distilled water, similar procedures were used to prepare the other concentrations of the disc such as 10μg/μl + 20μl distilled water, 15μg/μl + 15μl distilled water, 20μg/μl + 10μl distilled

evaluated by measuring the diameter of inhibition zones (mm).

PDA plate simultaneously inoculated with fungal pathogen only as control.

the growth of test pathogen (mm) in the presence of the antagonistic isolate.

**2.4 Dual culture method** 

inhibition (PI) was calculated.

**2.5 Agar overlay method** 

of 7-10 days for fungi.

**2.7 Paper disc assay 2.7.1 Preparation of disc** 

water, 30μg/μl + 1μl distilled water.

**2.6 Well diffusion method** 

Crowded plate technique Agar streak method Agar streak method

Dual culture technique Dual culture technique Disc diffusion method

Agar overlay method Agar diffusion method Agar streak method

Food poisoning technique MIC

Plate 2. *In vitro* screening methods for Fungicidal substances produced by actinobacteria

Applications of Actinobacterial Fungicides in Agriculture and Medicine 37

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).

#### **2.7.2 Inoculum preparation**

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 neoformans* and *Aspergillus flavus* for sensitivity assay.

#### **2.7.3 Antifungal assay**

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.
