Effect of Media Composition on Vegetative and Reproductive Growth of Alternaria brassicicola and Bipolaris sorokiniana

Experiments were conducted to evaluate the effect of some culture media on the vegetative and reproductive growth of Alternaria brassicicola and Bipolaris sorokiniana. Twenty one (21) treatment combinations each with three replications were employed. Potato Dextrose Agar (PDA) was used as the basic growth medium in this study. Supplementation with different plant extracts with this medium produced differential growth and sporulation of the tested fungi. PDA media supplemented with mustard leaf extract showed significantly highest vegetative growth (7.8 cm) and PDA with the combination of mustard leaf, tomato fruit, carrot fruit and cabbage leaf extracts showed the highest sporulation (11×105 spores/ml) of A. brassicicola. In case of B. sorokiniana, the highest vegetative growth (7.4 cm) and highest sporulation (45×104 spores/ ml) were obtained by the supplementation of mustard and wheat leaf extracts respectively. Plant extract supplementation proved better than PDA as growth medium of A. brassicicola and B. sorokiniana. article history Received: 5 October 2017 Accepted: 28 November 2017


introduction
The growth of microorganisms in an artificial medium is influenced by several physical and chemical factors.A nutrient material prepared for the growth of microorganisms in a laboratory is called culture medium and the nutrient composition of a culture medium plays a major role in microbial growth (Toratora et al.) 27 .In laboratory, fungi are isolated on specific and non-specific culture medium for cultivation, preservation, microscopic examination as well as biochemical and physiological characterization (Northolt and Bullerman; Kuhn and Ghannoum; Kumara and Rawal,) 16,13,12 .The most common growth media for bacteria are nutrient broths and for fungi are agar plates; specialized media are sometimes required for microorganism and cell culture growth (Madigan and Martinko) 14 .
In this study, two pathogens were used, Alternaria brassicicola and Bipolaris sorokiniana.Alternaria species are known as major plant pathogen.There are 299 species in the genus (Kirk et al.) 10 , they are ubiquitous in the environment and are a natural part of fungal floral almost everywhere.The spores are airborne and found in the soil and water, as well as indoors and on objects.The club-shaped spores are single or form long chains.They can grow thick colonies which are usually green, black, or gray (Nowickiand Marcin) 17 .Alternaria attacks a wide range of host plants such as cole crops, crucifers, brassica etc. Alternaria cause black-spotting of crucifers: Alternaria brassicae and Alternaria brassicicola.Conidia of Alternaria brassicicolaare similar in length as Alternaria japonica, but have fewer longitudinal septa, are not constricted at the transverse septa, and are produced in longer chains (Tewari and Buchwaldt) 26 .Bipolaris is a genus of fungi belonging to the family Pleosporaceae.It was circumscribed by mycologist Robert A. Shoemaker in 1959 19 .Bipolaris is a large genus of dematiaceous hyphomycetes with more than 100 species, most of them being saprobes in soil and pathogens of plants, while some of the saprobic species are potentially able to infect humans and animals (Sivanesan) 23 .The typical morphological features of Bipolaris species include rapidly growing dark colonies, geniculate conidiophores with sympodial conidiogenesis, and large conidia with transversedistosepta, usually without a protuberant hilum (a basal structure scar indicating the point of attachment in the conidiogenous cell) and with bipolar germination.Morphologically similar anamorphic genera are Drechslera, Curvularia, and Exserohilum (Sivanesan) 23 .
Fungi grow on diverse habitat in nature and are cosmopolitan in distribution requiring several specific elements for growth and reproduction.A wide range of media are used for isolation of different groups of fungi that influence the vegetative growth, colony morphology, pigmentation and sporulation depending upon the composition of specific culture medium, pH, temperature, light, water availability and surrounding atmospheric gas mixture (Northolt and Bullerman) 16 .However, the requirements of fungal growth are generally less stringent than for the sporulation.Physical and chemical factors have a pronounced effect on diagnostic characters of fungi.
Hence, it is often necessary to use several media while attempting to identify a fungus in culture since mycelial growth and sporulation on artificial media are important biological characteristics (St-Germain and Summerbell) 25 .The experiments were performed in the laboratory condition to find out the effect of media composition on vegetative and reproductive growth of Alternaria brassicicola and Bipolaris sorokiniana on different culture media and this is the first attempt in this regard.

Materials and Methods
The experiments were conducted in the Plant Disease Diagnostic Clinic (PDDC), Department of Plant Pathology and Seed Pathology Centre (SPC), Bangladesh Agricultural University, Mymensigh-2202.The experiments were performed during the period from November 2015 to December, 2016.

Collection, isolation and identification of Alternaria brassicicola and Bipolaris sorokiniana
A. brassicicola and B. sorokiniana isolates were isolated from infected mustard and wheat seeds, respectively collected from farmer's own stored seeds.After collection of infected seeds, A. brassicicola and B. sorokiniana were isolated through Standard blotter method (ISTA) 9 .In this blotter method, 25 (1+8+16) mustard and wheat seeds were placed respectively on moist blotter paper (wetted with sterile water) in plastic petridishes.Then the petridishes were incubated at the incubation room 25°C to provide the conditions for the pathogen to sporulate and after 7-10 days pathogenic structures (spores and mycelia) on seeds were observed under stereo binocular microscope.Then the infested seeds were maintained on Potato Dextrose Agar (PDA) medium for 7-10 days in the incubation room and ensured about the presence of concern pathogens by preparing slide and comparing the morphological characters following the key outlined by Barnett 1 and Ellis 6 (Figure 1).The pure culture of both fungi were obtained by culturing fungi on PDA medium and making the fresh culture from "hyphal tip" selected from the periphery of actively growing colony under aseptic conditions.After inoculation of fresh PDA plates with fungal block, the inoculated plates were sealed with parafilm and the fungal isolates were grown under optimum growth conditions (23 ± 1°C) for 10 days.

Experimental Treatments
To study the effect of media composition on vegetative and spore production of A. brassicicola and B. sorokiniana, eleven treatments were used for A. brassicicola and ten treatments for B. sorokiniana.pda Medium/Growth Medium preparation For this preparation 200g of clean peeled slice potato tubers were boiled in 500 ml of water.Water was separated from boiled potato slices.20g Dextrose and 15g Agar was added to separated boiled water and made the final 1000 ml volume of PDA medium with distilled water.Then the medium was autoclaved at 121˚C , 15 PSI for 15 minutes and after proper cooling the medium, the PDA plates were prepared within laminar air flow cabinet.

Changing the Media Composition for the Growth of Alternaria brassicicola
To prepare a modified PDA medium, different plant extracts were added to PDA @ given in the Table 1.
After cooking, the media were poured into conical flask and the mouth of the conical flask was closed with non-absorbent cotton plug and sterilized in an autoclave at 121 0 C under 15 PSI for 20 minutes.After autoclaving, 10-12 drops of lactic acid was added in the media for avoiding bacterial contamination and then all the media were transferred into petridishes and each of the petridishes contained 20 ml of media.

Changing the Media Composition for the Growth of Bipolaris sorokiniana
Media composition was changed by adding different types of host and non-host plant extracts with PDA medium.Potato wheat leaf agar medium was prepared by mixing 100ml of wheat leaf extract (1:5) with boiled water extract of 150ml sliced potato, 5g agar and 5g dextrose for 250ml of medium.On the other hand, Potato rice leaf agar medium was prepared by mixing 100ml of rice leaf extract (1:5) with boiled water extract of 150ml sliced potato, 5g agar and 5g dextrose for 250ml of medium.

Fungal Growth Measurement Technique
In case of all supplemented media, mycelial growth for both the fungi were determined directly by measuring the diameter of the colonies in the same axis.Mycelial growth of the colony was measured with the help of fine transparent plastic scale in cm and the sporulation for the both fungi were determined by spore counting using hemocytometer in spores/ml.

Measurement of Vegetative Growth Rate
After 10 days, mycelial growth of A. brassicicola and B. sorokiniana colony were recorded by measuring the average of two diameters at right angles to one another.The same procedures were repeated three times and mean number of mycelial growth was considered.

Measuring sporulation
The degree of sporulation for both fungi were determined by adding 5ml of double distilled water and 2.5 ml of 70% ethanol in each petridish containing 10 days old pure culture of those fungi.
Then the surface of the fungal growth on PDA was scraped gently with a camel hair brush to disperse the spores.After that 3 ml of the suspension and 22ml double distilled water were taken in a falcon tube and then shaken until it mixed well.From the final suspension 100 µl was placed in a hemocytometer and the number of conidia were counted under compound microscope and number of conidia/ml was estimated by using the key outlined by Tuite 28 .The same procedures were repeated three times for three replications and mean number of conidia/ ml was determined.

statistical analysis
The experiments were done under controlled laboratory condition, and the data were analyzed following completely randomized design (CRD) by using the SPSS statistical software.

Growth Characteristics of Alternaria brassicicola on pda Media supplemented with different plant Extracts
The growth characteristics like colour of substrate/ media, color of colony and margin of colony of Alternaria brassicicola on different culture media were observed in this study.The colony colour of A. brassicicola having dark brown centre and white margin in case of PDA media and PDA supplemented with mustard leaf extract and cabbage leaf extract.Whereas, PDA media supplemented with tomato fruit, bean fruit, carrot fruit, spinach leaf and sweetgourd fruit showed light brown, light brown centre and white margin, whitish, brown centre and white margin and brownish colony respectively (Table 2; Figure 2).This result indicating that PDA media supplemented with different plant extracts have an impact on the colony color of A. brassicicola.On the other hand, margin of the pure colony of A. brassicicola on PDA medium and PDA supplemented with different plant extracts showed mostly regular form.On sweet gourd and spinach leaf extract supplemented medium, the colony appeared irregular (Table 2; Figure 2).

Effect of Media Composition on the Vegetative Growth of Alternaria brassicicola
PDA media supplemented with different plant extracts showed better vegetative growth.PDA media supplemented with mustard leaf extract showed significantly highest vegetative growth (7.8 cm) of the fungus, followed by supplementation with tomato fruit (7.1 cm) and cabbage leaf (7.1 cm).PDA media with no supplementation i.e. control showed better vegetative growth of A. brassicicola (7.3 cm) compared to the PDA media supplemented with other plant extracts.The PDA media supplemented with carrot fruit, bean fruit and sweet gourd fruit also showed better vegetative growth of A. brassicicola of 7.0 cm, 6.8 cm and 6.4 cm respectively.On the other hand, PDA media supplemented with the combination (treatment No. 8, 9 & 10 in Figure 2) and spinach leaf extract showed poor vegetative growth compared to the other treatments (Figure 3).

influence on spore production of Alternaria brassicicola By The Media Composition
The data revealed that PDA media supplemented with combination of mustard leaf, tomato fruit, carrot fruit and cabbage leaf extract showed the highest sporulation (11×10 5 spores/ml) of A. brassicicola followed by PDA media supplemented with combination of mustard leaf and tomato extract (925×10 3 spores/ml) and combination of mustard leaf, tomato, carrot (875×10 3 spores/ml) (Table 3).This result indicating that PDA media supplemented with more than one plant extracts produced more spores compared to the PDA media supplemented with one plant extract.

Growth Characteristics of Bipolaris sorokiniana on pda Media supplemented with different plant Extracts
The colony color of B. sorokiniana having dark brown centre and white margin in case of PDA media and PDA media supplemented with carrot and tomato extract whereas, PDA media supplemented with mustard, bean, cabbage, spinach and sweet gourd showed light brown at center and white at margin, whitish colony with greenish center, whitish colony, cottony white and dark brown colony of B. sorokiniana respectively (Table 4; Figure 4).On the other hand, PDA media supplemented with wheat and rice leaf extract had brownish and dark brown center and greenish at margin of the colony of B. sorokiniana.
This result indicating that PDA media supplemented with different plant extracts have an impact on the colony colour of B. sorokiniana.On the other hand, margin of the pure colony of B. sorokiniana on PDA media and PDA supplemented with different plant extracts showed mostly irregular form (Table 4; Figure 4).However, colony on potato mustard leaf agar and potato bean agar was regular.

Effect of Media Composition on the Vegetative Growth of Bipolaris sorokiniana
The result explained that the PDA media supplemented with non-host extracts showed better vegetative growth compared to the host extracts and control.PDA media supplemented with mustard leaf extract showed significantly highest vegetative growth (7.4 cm) of the fungus, followed by PDA media supplemented with spinach leaf extract (7.3 cm) and bean (6.8 cm).The PDA media supplemented with host extracts such as wheat and rice showed less vegetative growth compared to the PDA media supplemented with non-host extracts.Non-host extract PDA media such as potato tomato agar medium, potato sweet gourd agar medium and potato carrot agar medium showed vegetative growth of 4.9 cm, 4.3 cm and 2.9 cm respectively.PDA media with no supplementation with host and non-host extract i.e. control showed poor vegetative growth compared to all other treatments (Fig. 5).

influence on spore production of Bipolaris sorokiniana by he Media Composition
To know the effect of media composition on the spore production of B. sorokiniana isolate, 10 different PDA media composition (PDA media supplemented with different host and non-host plant extracts) were used in this research experiment.The data revealed that PDA media supplemented with wheat leaf extract showed the highest sporulation (45×104 spores/ml) of B. sorokiniana followed by PDA media supplemented with rice leaf extract (30×10 5 spores/ ml) and PDA media supplemented with sweet gourd extract (1×10 5 ml spores/ml) (Table 5).This result indicated that PDA media supplemented with host extracts produced more spores compared to the PDA media supplemented with non-host extract.

discussion
The experiments were conducted for the evaluation of media composition for growth characteristics of

Conclusion
In the present investigation, maximum mycelial growth of both A. brassicocola and B. sorokiniana were obtained in PDA supplemented with mustard leaf while maximum sporulation of A. brassicocola and B. sorokiniana found in PDA supplemented with all plant extracts combination and PDA supplemented with wheat leaf respectively.Based on these findings it may be concluded that PDA supplementation with extract of single or combination of host plant extracts, the sporulation of the two fungi can be enhanced.

acknowledgement
The study was supported partly by the Bangladesh Agricultural University Research System (BAURES), Bangladesh Agricultural University, Mymensingh, Bangladesh.We also thank the Department of Plant Pathology and Seed Pathology Center, Bangladesh Agricultural University for providing research facilities.

Fig. 2 :
Fig. 2: Colour and appearance of pure colonies of A. brassicicola on pda media supplemented with different plant extracts.

Fig. 4 :
Fig. 4: Colony colour and appearance of pure colonies of Bipolaris sorokiniana on pda media supplemented with different plant extracts.

Table 1 : supplemented pda media with their composition and name
inoculation of the Culture Media plates With a. brassicicola and B. sorokiniana isolates PDA plates supplemented with different plant extracts were inoculated with pure culture of A. brassicicola and B. sorokiniana.In brief, mycelial blocks were cut out of 10 days old fungal colony near the margin by means of sterilized cork borer of 5 mm diameter.These blocks were transferred to the center of the petri plates by means of a sterilized inoculating needle.For each of the treatments three replications were maintained.All these were done under aseptic condition inside a laminar air flow

Table 5 : spore production of Bipolaris sorokiniana by the media composition
21apek's dox and oat meal broth media.In addition, Somappa et al.24reported that PDB is the best medium supporting good growth of A. solani infecting tomato.These results indicated that complex formulation of the media does not allow the fast mycelial growth of A. brassicicola.It might be due to PDA media with mustard leaf, tomato fruit, carrot fruit and cabbage leaf extracts contain all the major compounds for fungal sporulation i.e. carbon, nitrogen, phosphate, potassium, magnesium, sulphur elements including sugars.This finding also corroborates the findings of earlier researchers.Osman et al.18found Czapek's Dox Agar medium was best for the growth and sporulation of Alternaria alternata.Fancelli and Kimati 7 also observed that Czapek's Dox and host leaf extract medium yielded better sporulation of Alternaria dauci compared to other tested media.It might be due to PDA supplementation with plant extracts has the sufficient nutrient content, supporting the best mycelial growth of the fungus.On the other hand, PDA media supplemented with wheat leaf extract showed the highest sporulation followed by PDA media supplemented with rice leaf extract.These findings are indicating that Bipolaris fungus showed better sporulation on PDA media supplemented with graminaceous plant extracts compared to other plant extracts.These results corroborate the findings of former literatures such as Chattopadhyay and Dasgupta 4 found that production of conidia by B. oryzae was favored by media either rich in or made exclusively of plant parts.Coeman et al.5showed that methionine and ethylene both reduced mycelium growth of B. sorokiniana when grown on Czapek's medium or on media containing grass leaf infusion.Methionine and other amino acids reduced sporulation as did ethylene and precursors of ethylene, S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid.Cegielko 2 examined that faster growth of Drechslera avenae recorded in MPA (maltose peptone agar) medium and RA (rye agar) were the best media for the sporulation of D. avanae.Garraway and Evans 8 observed xylose (1.0-10.0g/)stimulates sporulation but not mycelial growth of B. maydis when added as a supplement to a basal agar medium containing glucose, sporulation on a xylose -supplemented medium was significantly higher than on a non-supplemented control medium.Sharma and Singh21found that rice husk agar and near ultraviolet radiation were required for production of conidia by Helminthosporium oryzae.
15ecorded maximum vegetative growth of A. brassicicola on host extract media.Mohapatra et al.15stated that maximum growth of Alternaria sesame (infecting sesame) on PDB compared to Richard's,