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First Report of Alternaria Brassicae Leaf Spot Disease of Aloe Vera and It's Disease Intensity in West Bengal

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First Report of Alternaria Brassicae Leaf Spot Disease of Aloe Vera and It's Disease Intensity in West Bengal European Journal of Biotechnology and Bioscience 2014; 2 (1): 37-43 Ramakrishna Mission Vivekananda entenary College, Rahara, Kolkata 700118, India. Biotechnology, SVPUA&T, Meerut, UP, India. ISSN: 2321-9122 www.biosciencejournals.com EJBB 2014; 2 (1): 37-43 First report of Alternaria brassicae leaf spot disease of Received: 30-6-2014 Accepted: 15-7-2014 Aloe vera and it’s disease intensity in West Bengal Swapan Kr Ghosh Molecular Mycopathology, PG Swapan Kr Ghosh, Subhankar Banerjee Department of Botany, Ramakrishna Mission Vivekananda Centenary Abstract College, Rahara, Kolkata 700118, Aloe vera (L.) Burm.f. is a medicinally and commercially important plant. A destructive leaf spot disease India. in this plant is recently observed in West Bengal, India. It’s causal organism was isolated and phenotypically identified as Alternaria and molecular identification (ITS 1- 5.8s- ITS2) shows as Subhankar Banerjee Alternaria brassicae (Berk.) Sacc. This is the first report of the leaf spot disease on Aloe vera in West Molecular Mycopathology, PG Bengal. Once in a week survey was conducted to note the occurrence of this disease throughout the year of Department of Botany, Ramakrishna 2013 in ten places of 24 Parganas District, West Bengal. In all ten places, this disease was present from Mission Vivekananda Centenary College, Rahara, Kolkata 700118, January – December, 2013. Intensive research was conducted in order to record the disease intensity by India monitoring Aloe cultivation at 4 selected areas of West Bengal throughout this year. The disease intensity was recorded maximum during July (95.71%) in State Pharmacopical Laboratory and Pharmacy for Medicine, Govt. of W.B, April in Nilgunj (95.45%), May at Rajarhat (87.77%), June at Basirhat(83.28%) and whereas the Disease Intensity was lowest in all four areas surveyed extensively during the month of December were recorded as 25.71%, 32.85%, 35.77% and 50.00%, respectively in Basirhat, State Pharmacopical Laboratory and Pharmacy for Medicine, Govt. of W.B., Nadia, Nilgunj(24-Parganas), Rajarhat. This study indicates that Aloe vera is the new host of A. brassicae and this disease is present in moderate to severe form throughout the year causing crop damage. Keywords: Aloe vera, Alternaria brassicae, Destructive disease. 1. Introduction Aloe vera (L.) Burm.f., ( Aloe barbadensis ) belongs to family Aloeaceae,is a perennial, succulent plant with a height of 60-100 cm. About 400 species of Aloe have been reported so far. But the most extensively cultivated as medicinal plant among them is A. vera. The margin of the leaves is serrated with soft and small teeth. The species is widely adapted all over the world, ranging from tropical to temperate regions. Aloe leaves are filled with gel which is the most important constituent of the plant and has great medicinal value. Aloe vera contains amino acids, anthraquinones, enzymes, lignin, minerals, mono and polysaccharides, salicylic acid, saponins, sterols and vitamins [1]. Many herbal drugs and drinks have been formulated from A. vera plants for the maintenance of good health. In cosmetic industries Aloe is used in the production of soap [2] for bathing, shampoo, hair wash, tooth paste and body creams . Beside that, A. vera gel has been reported to be very effective for the treatment of sore and wounds, skin cancer, skin disease, cold and cough, constipation, piles and fungal infection [2][3][4]. A. vera plants can also be used for treatment of asthma, ulcer and diabetes [5]. Aloe vera is found as wild herb in coastal areas of South India. It is now under cultivation in fairly large areas in many parts of India viz Chhattishgarh, Maharashtra, Madhya Pradesh, Gujarat etc. In West Bengal Aloe vera is grown for commercial purpose in different pharmacopical gardens and also grown as ornamental plants by many people. A destructive “leaf spot disease” of Aloe plant recently came into limelight in West Bengal. As the Aloe plants are generally used in medicine and cosmetics [4, 6], the contamination with fungal pathogen will have a huge impact on human health. Some fungal pathogens and non-pathogens produce mycotoxins [7] Correspondence: in their infected hosts and substrates on which they grow . Mycotoxins are hazardous to human Swapan Kr Ghosh and animal health [8]. The main objectives of this work is to report the leaf spot disease of A vera, Molecular Mycopathology, PG to identify the causal organism both phenotypically and at molecular level, to record the Department of Botany, occurrence and disease intensity of this disease in 24 – Parganas (N) district, West Bengal. 2. Materials and Methods A) Study of Symptoms ~ 37 ~ The infected leaves were carried to the laboratory in sterilized Genomic DNA from fungal isolate (FAq) was extracted and the biodegradable polythene bags and the symptoms studied under internal transcribed spacer (ITS1-5.8S-ITS2) regions were hand lens and simple microscope. amplified by PCR technology using with DNA amplification reagent kit manual (GeNei) along with fungus specific forward B) Isolation and purification of pathogen from diseased primer ITS-1 F (CTTGGTCATTTAGAGGAA-GTAA) [13] and parts the reverse primer ITS-4 (TCCTCCGCTTATTGATA-TGC) At first the diseased leaves were carried into the laboratory in [14]. PCR was carried out using the following protocol, modified air tight sterilized biodegradable polythene bags. The collected from [13]; initial denaturation at 95 0C for 85s, followed by 37 leaf samples were washed in sterile distilled water and soaked in cycles of denaturation at 95 0C for 35s, annealing at 55 0C for alcohol to remove the surface impurities. After that the leaf 55s, extension at 72 0C for 10 min. The reaction was held at 4 samples were cut into small pieces of 3-5 mm in size from the 0C. Amplicon was electrophoresed in a 1% Agarose gel and diseased portion. Then they are passed through 0.1% of HgCl2 visualized under UV. Concentration of the amplicon was solution for one minute for surface sterilization and washed checked in a Nanodrop ND 2000. The amplicon was purified three times in three changes of sterile distilled water. These leaf using GeneJET gel Extraction Kit (Thermo Scientific). cuttings were blotted between sterile filter papers and aseptically plated on Potato Dextrose Agar (PDA). In each plate C) Gene Sequencing a single piece was placed and incubated at BOD (28± 2 °C) for 7 Sequencing of PCR product was carried out [15] in Sci Genome days. After appearance of mycelial growth it was transferred on Labs Pvt Ltd, Kerala, India and the sequence obtained was to fresh PDA slant. For purification of isolated pathogen, single submitted to NCBI GeneBank and the accession number was hyphal tip method was taken. The purified isolate of the fungal obtained. The sequence analysis was carried out using pathogen was labelled as FAq. The entire procedure for bioinformatic tool BLAST of NCBI. isolation of the disease was done under laminar air flow. Based on maximum identity score first few sequences were selected and aligned using multiple sequence alignment C) Pathogenecity test of the pathogen software ClustalW2 and phylogram was constructed. Pathogenecity test was done following the Koch postulate. D) Study of the disease occurrence and intensity (PDI) D) Characterization and Identification of the pathogen We have selected two areas from each of the five subdivisions The identification of the pathogen was done phenotypically as of the district North 24 Parganas, West Bengal to record the well as at molecular level through gene sequencing. disease occurrence of leaf spot disease in Aloe vera. Fields were visited once in a week to record the disease occurrence from the E) Phenotypical Identification month of January, 2013 to December 2013. It was done by cultural and microscopical characteristics with For the study of Per cent of Disease index (PDI) we have the help of published fungal keys and books [9, 10, 11]. The carried out extensive survey in four selected fields namely identification was confirmed by IMTECH scientist (Chandigar, Rajarhat, Nilgunj (24-Parganas), State Pharmacopical India). Laboratory and Pharmacy for Medicine, Govt. of W.B., Nadia and Basirhat. For this purpose 3 plots from each of the four 2.1. Molecular Identification by PCR method areas were taken and per plot 50 plants were randomly selected. A) Genomic DNA extraction+ Fields were visited once in a week to record the PDI by using Genomic DNA of fungus was extracted by modified CTAB the following formula. method [12]. Mycelia was taken from liquid medium and washed with help of sterile water and transferred into 1.5 ml eppendorf tubes. To the mycelia sterile white quartz sand and 500 µl PDI = sterilized extraction buffer(100mM Tris HCL, 1.4 M NaCl, 20 mM EDTA, pH 8.0) were added and the sample was grounded A=Total Number of infected plant; B=Total Number of selected and incubated at 65 oC for 60 min. 500 µl of chloroform- plants. isoamyl alcohol (24:1) was added to each tube and mixed. The mixture was centrifuged at 13000 rpm for 30 min, and the 3. Results and Discussion aqueous extraction layer was transferred into a new eppendorf 3.1 Symptom of the disease tube. The addition of chloroform-isoamyl alcohol (24:1) and The symptoms appeared on the leaves in form of small dark centrifugation was repeated two times until no interface was brown necrotic spots on both sides which gradually enlarge to visible. 800 µl of cold absolute ethanol was added to each tube cover up an area of 2-8 cm in diameter. The infected area and left at -20 0C for 5-10 min, and centrifuged at 13000 rpm transforms from dark brown to black. Gradually the leaf surface for 15 min at 4 0C.
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