Effects of different culture media on mycelial growth and sporulation of Fusarium oxysporum f. sp. Lycopersici

Introduction

Tomato (Lycopersicon esculentum Mill.) is one of the most popular, highly nutritious, commercial vegetable crop grown all over the world. It is a Solanaceous family vegetable and origin in Tropical America (Thompson and Kelly, 1957) [1] . It is grown extensively and marketed throughout the world including tropical and temperate regions. Fusarium wilt is one of the most serious diseases affecting tomato plants throughout the world, especially in upland areas. Fusarium oxysporum F. sp. lycopersici is a devastating pathogen of tomato [11] . Management of Fusarium wilt can be done by many ways. Crop rotation with nonsolanaceous crops (non-host) for four to six years is usually recommended to reduce the inoculum level of FOL in the field [2] . The crop should be rotated with cereals crops wherever possible [3] . Physical methods are quite effective but an important hindrance that limits their use is low efficiency and high labour intensive. Synthetic chemicals are commonly used for the management of FOL in tomatoes. Treatment with chemical fungicides especially prochloraz, carbendazim, propiconazole, thiabendazole, benomyl, thiophanate, fuberidazole and benzimidazoles considerably reduce the wilt incidence in tomatoes [4,5] . Khan and Khan [6] reported that the root-dip treatment of carbendazim on tomato seedlings infected with FOL led to a 24% increase in yield. However, the use of fungicides is a bit expensive as well as environmentally undesirable due to damaging effects on the agroecosystem, human beings and animals [7] . Under this situation, all hopes are set on the exploration of biological management of plant diseases. Biological control is one of the best and safest methods of disease management and is devoid of residual toxic effects [12] . This method is based on the idea that pathogen's populations can be decreased by infuencing naturally occurring living microbial species, altering the environment, or introducing antagonists. Several antagonists of FOL are known and their utilization resulted in a substantial decrease in the disease severity and correspondingly increase in the crop yield. Trichoderma spp. are well known, Frequently used biocontrol agents [8][9][10] .

Origin, Taxonomy and Morphology of Fusarium

Fusarium oxysporum f. sp. lycopersici comprise that are widely distributed worldwide in soils. Taxonomically Fusarium oxysporum f. sp. lycopersici belongs to the Kingdom-Fungi, Phylum-Ascomycota, Subphylum-Pezizomycota, Class -Sordariomycetes, Subclass -Hypocromycetidae, Order-Hypocreales, Family-Nectriaceae and Genus -Fusarium. It produces three types of asexual spores: Microconidia, Macroconidia and Chlamydospores.

Macroconidia

Macroconidia were also different shape and size like fusiform, sickle shaped hyaline with mostly 2-5 septate and measured 20.7-28.5 x 2.8-6.09 µm in size.

Microconidia

The microconidia were vary from round to cylindrical shaped with hyaline, single celled (0-2 septate), ovoid to ellipsoidal in shape, straight to slightly curved and measured 5.7-12.7 x 2.8-4.3 µm in size.

Chlamydospores

The chlamydospores produced in 2-3 week old culture were typically intercalary and produced in individual or in a chain. They were globose to sub-globose, aseptate thick walled and smooth surfaced. The pathogen enters through the epidermis of root, later spreads through the vascular tissue and inhabits the plant xylem vessels, resulting in vessel clogging, and severe water stress as a result wilt like symptoms appear (Singh et al., 2017) [13] . The dormant chlamydospore of Fusarium oxysporum f. sp. lycopersici in infested soil can survive indefinitely in the absence of host (Khan et al., 2017 and Cha et al., 2016) [14] .

Materials and Methods Isolation, Purification and characterization Fusarium oxysporum F. spp Lycopersici

To isolate of the indigenous isolate of test fungus Fusarium oxysporum f. sp. lycopersici (FOL), soil samples were collected from agricultural field in and around, Aligarh. The fields from where the samples collected was under tomato cultivation. Soil was collected from the site having continuously good moisture contents. The samples were collected from a depth of 2-4 cm and 10-15cm after removing skin of soil from the rhizosphere of the tomato field. The fungus was isolated using serial dilution technique. Association of FOL with the soil samples was confirmed on the basis of its colony and morphological characteristics. First 10 g soil (properly dried) were taken in sterilized flask with 90 ml water and then stirred with a magnetic stirrer for 20-30 minutes. A commercial blender was used for blending the samples. While the suspension is in motion, withdraw 10 ml and add to 90 ml sterile water in a screw cap flask. Shake for 1 minute and transfer 10 ml of the suspension to another 90 ml sterile water blank. Repeat the process until the desired dilution of 10 5 was obtained. The final 1 ml of diluted samples were spread on a solidified PDA (Potato dextrose agar, HiMediaTM, India) in a Petriplate under a laminar flow using sterile pipette. To avoid the growth of bacteria Streptomycin were added to PDA media. Petri plates were incubated at 25±1 ℃ for 7 days. After incubation the fungal colonies were observed under a microscope and matched with FOL characteristics. Isolates were further purified on another petri dish containing PDA and identified by morphological characters. Relative growth of Fusarium oxysporum f. sp. lycopersici on different media The cultural characters of F. oxysporum f.sp. lycopersici were conducted on the following six synthetic and semi-synthetic media viz., King B medium, Czapek dox agar, rose bengal agar, Richard synthetic agar, Czapek yeast extract agar and potato dextrose agar. To prepare the solid media, Agar-agar was melted in 500 ml distilled water. The other ingredients were dissolved in the remaining 500 ml of distilled water. The two solutions were mixed thoroughly and the volume was made up to one litre. The medium was sterilized at 15 PSI /lbs for 20 minutes at 121℃ in an autoclave. Twenty ml of each solid media was poured into 90 mm diameter Petri plates and three replicates were maintained. Inoculation was made by transferring ½ cm disc of mycelium taken from the periphery seven days old pure. Culture of FOL. The plates were incubated at 25℃ for seven days. The cultural characteristics and the colony diameter (mm) on each of the mediums were recorded and results were analyzed statically.

Acknowledgement

AcknowledgementThe authors are thankful to the Department of Plant Protection, Faculty of Agricultural Science, Aligarh Muslim University for providing all the required facilities to carry out this experiment.

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