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Mycelial Growth and Biological Control Measures of Botrytis Cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh

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Mycelial Growth and Biological Control Measures of Botrytis Cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh Universal Journal of Microbiology Research 8(2): 13-18, 2020 http://www.hrpub.org DOI: 10.13189/ujmr.2020.080201 Mycelial Growth and Biological Control Measures of Botrytis cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh Sanjida Sultana, Md. Maniruzzaman Sikder, Md. Sabbir Ahmmed, Aireen Sultana, Nuhu Alam* Department of Botany, Faculty of Biological Sciences, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh Received May 22, 2020; Revised June 23, 2020; Accepted July 29, 2020 Cite This Paper in the following Citation Styles (a): [1] Sanjida Sultana, Md. Maniruzzaman Sikder, Md. Sabbir Ahmmed, Aireen Sultana, Nuhu Alam , "Mycelial Growth and Biological Control Measures of Botrytis cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh," Universal Journal of Microbiology Research, Vol. 8, No. 2, pp. 13 - 18, 2020. DOI: 10.13189/ujmr.2020.080201. (b): Sanjida Sultana, Md. Maniruzzaman Sikder, Md. Sabbir Ahmmed, Aireen Sultana, Nuhu Alam (2020). Mycelial Growth and Biological Control Measures of Botrytis cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh. Universal Journal of Microbiology Research, 8(2), 13 - 18. DOI: 10.13189/ujmr.2020.080201. Copyright©2020 by authors, all rights reserved. Authors agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abstract Fruit rot disease of strawberry caused by biological control. fungus is most economically important disease in Bangladesh, which reduces the production and quality of Keywords Botrytis cinerea, Fungal Biology, strawberry at both pre and post-harvest period. Present Molecular Identification, Strawberry study was conducted to identify the causal organism of the fruit rot disease of strawberry through classical and molecular techniques and its eco-friendly control measures. A fungal pathogen causing fruit rot was isolated and 1. Introduction identified as Botrytis cinerea through classical fungal taxonomy and molecular characterization based on their Strawberry is a stoloniferous perennial herb its internal transcribed spacer (ITS) region of ribosomal DNA cultivation and environmental conditions are very suitable (rDNA). Sequence analysis showed that 5.8S of rDNA in Bangladesh. Presently strawberry fruit is very popular in sequences were identical. The reciprocal homologies of our country due to its nutritional values, test and attractive the ITS region sequences ranged from 98 to 100%. colour. Flowering in strawberry plant is greatly influenced Experimental results suggested that studied fungus by day length and temperature. For better strawberry (MH371474.1) was genetically similar with MF521935.1 production photoperiod 10-20h days temperature 12-30°C Botrytis cinerea. Isolated fungus was evaluated on six and number of short days 12-24 are essential [1]. different culture media and at five different temperature Bangladesh day temperature is 15-25°C, photoperiod conditions. The optimum mycelial growth of B. cinerea 12-16h and short days about 30-50 days [2]. Bangladesh was found on Richard agar medium at 25°C temperature at Agricultural Research Institute (BARI) and Bangladesh 7days post inoculation (dpi), while mycelial growth was Agricultural Development Corporation (BADC) has drastically reduced the temperature 30°C and above. Three released strawberry variety namely, BARI strawberry-1 Trichoderma species viz., T. ressei, T. harzianum and T. and BADC strawberry. Therefore, Bangladeshi strawberry asperellum were used to assess the antagonistic effect on varieties are grown in different regions of the country isolated pathogenic fungus in which the mycelial growth of during winter season [3]. it was inhibited mostly by T. ressei. Experimental results Fungal disease of strawberry mainly attacks on leaves, revealed that Trichoderma ressei was the effective fruits, crown and roots. Colletotrichum gloeosporioides antagonistic fungus against Botrytis cinerea for the causing anthracnose leaf spot [4], Phomposis obscurans 14 Mycelial Growth and Biological Control Measures of Botrytis cinerea Isolated from Strawberry Fruit Rot Disease in Bangladesh causing phomopsis leaf blight [5]. A large number of obtain a pure culture. The pure culture of the isolated strawberries have been destroyed every year due to fungus was identified microscopically using standard strawberry pre and post-harvest diseases. Anthracnose methods based on colony morphology, mycelium and disease of strawberry caused by Colletotrichum conidia [11]. gloesporioides has been described in Bangladesh [6]. Fruit diseases are caused by Botrytis cinerea causing 2.3. Molecular Characterization fruit rot [7], Colletotrichum gloeosporioides causing For molecular characterization, the fungal DNA was anthracnose fruit rot [4]. Leaf blight and fruit rot disease of isolated by using Promega Wizard DNA Extraction Kit strawberry caused by Curvularia spp. had been discovered (USA). The ITS region of the rDNA of the fungus was in Kurdistan province, Iran, in 2013. Symptoms have been amplified by PCR using universal primers ITS4 found as small, brown to black, sunken lesions on fruits (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 and brown to black spots with irregular borders on the (5′-GGAAGTAAAAGTCGTAACAAGG-3′) according to leaves. Forty isolates have been recovered from infected White et al. [12]. GoTaq G2 Hot Start Green Master Mix plants, among them thirty one isolates have been identified which contains GoTaq G2 Hot Start Polymerase, dNTPs, as belonging to the genus Curvularia [8]. MgCl2 and reaction buffers; was used for PCR reaction. Molecular identification of fungi through DNA The PCR reaction was performed with 20ng of genomic barcoding has become important part of mycological DNA as the template in a 25µl reaction mixture by using research. It has recently been accepted as the official GoTaq G2 Hot Start Green Master Mix as primary barcoding marker for fungi. Primers like ITS1-F, follows-activation of Taq polymerase at 94°C for 5minute, ITS and ITS5 is biased towards the amplification of 35cycles of 94°C for 30Sec, 57°C for 30Sec, and 72°C for basidiomycetes and ITS2, ITS4, ITS5 for the ascomycetes 5minutes each were performed, finishing with a 10-minute [9]. Recent molecular phylogenetic studies have step at 72°C. The Maxwell® 16 DNA Purification Kits demonstrated that the ITS region of genomic DNA is very were used to purify the amplification products (Promega, useful for assessing phylogenetic relationships at lower USA). The purified PCR products of 541bp were taxonomic levels [10]. Considering the above facts, the sequenced by using 2primers in First BASE Laboratories present research has been under taken to isolate and SdnBhd (Malaysia). Sequencing data were assembled and identify of strawberry pathogen using morphological and compared with similar DNA sequences obtained from molecular techniques, to investigate the fungal biology NCBI GeneBank data base. The phylogenetic analysis was and to evaluate the antagonistic fungal effect against conducted using the multiple sequence alignment tools Botrytis cinerea. using MEGA6 software. Maximum likely hood and Neighbor-joining tree were generated using MEGA6 software. 2. Materials and Methods 2.4. Effect of Culture Media and Temperature 2.1. Sample Collection Six different culture media viz., potato dextrose agar Diseased strawberry fruits with characteristic symptoms (PDA), carrot agar (CA), potato sucrose agar (PSA), were collected in to labeled zip lock bag from the field of Richard agar (RA), Honey peptone agar (HPA), Honey Botanical Garden at Jahangirnagar University. agar (HA) were used to assay the mycelia growth of the Experiments were conducted in the Laboratory of fungus. Different temperatures (15, 20, 25, 30 and 35°C) Mycology and Plant Pathology, Department of Botany, were maintained for the mycelia growth of the fungus on Jahangirnagar University, Savar, Dhaka, Bangladesh. PDA in an incubator. The mycelia growth was recorded at 7 days post inoculation (dpi). The effect of culture media and temperature on the mycelia growth measurements was 2.2. Fungus Isolation and Morphological described by Sanjuti et al. [13]. Characterization Fungus was isolated through tissue planting method. 2.5. In vitro Mycelial Growth Inhibition of the Pathogen Infected parts of strawberry fruits were cut in to small Mycelial growth inhibition of the fungus was pieces about 0.5cm in length in such a manner so as to determined using dual culture technique in which include both fungal infected and non-infected tissues in biological control agents namely- Trichoderma asperellum piece. Then sterilization was done using NaOCl (5%) voucher JUF0025 (MH368120.1), T. reesei voucher solution for 5 minutes and rinsed with distilled water JUF0026 (MH368149.1) and T. harzianum voucher several times. Four pieces of samples were placed into JUF0028 (MH368147.1) were used. An agar disc (6-mm) potato dextrose agar (PDA) medium and were incubated of the antagonist, Trichoderma spp., was placed 2cm away under 12/12 hours dark and light condition at 25±2°C for from the periphery of the Petri dish, and a same sized agar 10days. Mycelial growth of growing fungus colony was disc of the test fungus, Botrytis cinerea was similarly transferred to fresh PDA plates as well as PDA slants to placed 2cm away from the edge of the Petri plate but on the Universal Journal of Microbiology Research 8(2): 13-18, 2020 15 end opposite of Trichoderma sample. All pairings were The ITS region
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