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Effect of Subculturing and Culture Age on Sporulation and Morphological Characteristics of Bipolaris Sorokiniana

Zilkad Nasrin
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Zannat Ara Ferdousy
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Bidhan Chandro Sarker
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Sabiha Sultana
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Sanjoy Kumar Adhikary
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Chhoa Mondal*
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Abstract/ Executive Summary:

Two in vitro experiments were conducted to evaluate the effect of successive sub-culturing on sporulation, morphological characteristics and culture age for the production of conidia of three isolates of Bipolaris sorokiniana of which two were from wheat (BS-1 and BS-2) and one from barley (BS-3). The experiments were conducted following standard procedure. After every successive subculturing, number of spores gradually declined in all three isolates. In first two isolates spores were produced up to fourth subculture, but no spore was observed at fifth subculture. In isolate BS-3 the spores were produced up to second subculture and no spore was found at third subculture. There was significant variation among these three isolates for the production of spores in each subculture. Variation was found in margin, shape, color, texture, zonation and arial mycelium in top view and reverse view after 12 days of inoculation. Variation was also observed in conidia and conidiophore of the three isolates at 12 days age culture. In isolates BS-1, BS-2 and BS-3 the highest conidia production were obtained in 14 days (10620 x103 conidia/ml), 18 days (578 x103 conidia/ml) and 10 days (7.8 x103 conidia/ml) old culture, respectively. In the isolates BS-1 and BS-2, number of conidia was increased up to 14 days and 18 days, respectively and then decreased. In isolate BS-3 the highest number of conidia was found at 10 days old culture but decreased gradually with the age.

Keywords: Subculture, Culture age, sporulation, Bipolaris sorokiniana

Location: Agrotechnology Discipline, Khulna University, Khulna

Start Date:

End Date:

Program Area: Pest Management

Commodity: Fungal Disease

Objectives:

In practice, the changes that occur in the isolates come from the constant manipulation demanded due to the method of conservation which demands successive subculturing (Aparecido et al., 2007). Therefore, the necessity of maintenance and manipulation of the isolates demand studies that characterize the effect of the preservation in vitro of the pathogenic and reproductive characteristics of the isolate of B. sorokiniana. Considering the above facts, this study was undertaken to evaluate the effect of successive subculturing over sporulation and morphological characteristics of isolates of B. sorokiniana and to determine the effect of culture age on production of conidia.

Methodology:

Bipolaris sorokiniana was isolated following seed or tissue planting method (Dhingra and Sinclair 1985; Mian 1995). The pathogen was identified by placing the conidia adhered to a sterilized needle on a drop of Potato Dextrose Agar (PDA) (2%) kept in a sterilized petridish. After four days the slide was examined for germination of conidia. After that the fungal growth was observed and then identified. Purification of all isolates was made using the single spore method as stated by Ruppel (1972) with some modifications (Adhikary et al., 2004). Pathogenicity of the isolates was tested on excised leaves as in Dhingra and Sinclair (1985) and multiplication was done by inoculating the plates of three days old PDA culture of B. sorokiniana (5 mm discs) and keeping it in the growth chamber at a temperature of 26 _-+2⁰C for few days. Test tube slants with five to seven days old profuse mycelium and conidia of B. sorokiniana were stored in refrigerator at 4⁰C.

Two experiments were conducted to achieve the objective of the present study. Experiment-I continued of five treatments with five replications. Each petridish was considered as a replication. The treatments were T1 –first subculturing obtained from single spore isolate, T2 – sporulation obtained from the first subculturing of the original culture; T3 – sporulation obtained from the second subculturing; T4 – sporulation obtained from the third subculturing; T5 – sporulation obtained from the fourth subculturing. From first subculturing (T1) to second subculturing (T2) was done after 12 days of interval. Successively each subculture was done at 12 days interval.

For measuring sporulation ability, twelve days old culture grown on PDA was flooded with 100 ml of sterilized distilled water. Conidia along with mycelial mass were separated from the substratum by scrapping with a narrow edged sterilized glass slide. The suspension was sieved through double layer cheese cloth to discard mycelial mass. The volume was adjusted up to 100 ml using distilled water. It was then diluted 10 times by adding distilled water. The number of spore/ml was counted standard haemocytometer with Neubauer rulings and seen at 40x (4x objective). Counting was done five times for each replicated plate; morphological characteristics were recorded based on visual observation. Data were recorded on number of spores at 12 days intervals for each subculturing and morphological characteristics of mycelium and conidia of Bipolaris sorokiniana after 12 days of inoculation.

The experiment-II was laid out with six treatments and five replications using three single spore isolates of B. sorokiniana. The treatments were: D10 – colonies with 10 days of storage; D14 – colonies with 14 days of storage; D18 – colonies with 18 days of storage; D22 – colonies with 22 days of storage; D26 – colonies with 26 days of storage; and D30 – colonies with 30 days of storage. Number of spores was counted following the procedure of Experiment I and recorded at four days interval starting from 10 days up to 30 days. The both experiments were laid out under completely randomized design (CRD). Recorded data were transformed by using Square Root Method (Zaman, 1982) for exactly analyzing the data avoiding statistical complication. The data were analyzed statistically using MSTAT-C computer program and means were compared following Duncan’s Multiple Range Test (DMRT) and Least Significant Difference test (LSD).

Source of Information: Khulna University Studies Volume 14 (1 & 2) : 59-70 : January-December 2017

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The best sporulation was found in T1 stage in all isolates and no conidia were found in T5 stage for BS-1 and BS-2 and in T3 stage for BS-3 after successive subculturing. Remarkable variability was found in morphological characteristics of three isolates of Bipolaris sorokiniana after 12 days of inoculation. The highest conidia production was obtained in 14 days, 18 days and 10 days old culture in isolates of BS-1, BS-2 and BS-3, respectively after inoculation compared to other culture ages.

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