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 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

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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 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. The upper layer was discarded and the DNA is covered with numerous such lesions which become rotten and pellet was dried overnight. The dried pellet was re-suspended in dried within 4-7days with a central cup shaped depression 30 µl of TE buffer (10mM tris-HCL, 1 mM EDTA, pH 8.0) having a depth of 5-8 mm. (Fig 1-3). These disease symptoms containing 20 µg/ml RNase and incubated at 37 0C for an hour. indicate that it is leaf spot. Along with such manual method fungal DNA also extracted with the help of HiPurATM Fungal DNA Purification Kit (HiMedia Laboratories Pvt. Ltd.). The concentration of DNA was checked by electrophoresis in 1% agarose gel stained with ethidium bromide under UV light.

B) PCR of ITS1-5.8S- ITS-2 of r DNA

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(1) (2)

(3)

Fig 1-3: Disease symptoms on Aloe vera.

The leaf spot disease on Aloe plant first came into limelight as 3.3 Phenotypical characterization and identification purple spot disease on Aloe arboescens Mill. caused by The pure cultures were analyzed macroscopically and Fusarium phyllophilum [16] and later Haematonectria microscopically. Growth of the fungus is very slow (around 2-4 haematococca (anamorph: Fusarium sp.) causing ring spot mm per day). The fungal colony shows puffy growth at first disease on Aloe barbadensis [17]. with white colouration, gradually becomes green with white From other states of India the disease with same type of margin and finally greyish black in appearance, covering full symptoms was reported by other workers. Leaf spot disease was plate in 8-10 days (Fig 4). With the growth of the fungal colony found (caused by Alternaria alternata) in Aloe barbadensis the reverse plate turns black. Slide was prepared from the Mill. in Southern India [18]. The disease was also reported from margin of the colony. Size, shape and feature of spore, hyphal Osmanabad district, Maharashtra [19]. From outside India this patterns were recorded by preparing numerous slides from disease of Aloe vera has been reported from Lousinia [20] and different stages of the colonial growth. Conidiophores pale from Pakistan [21]. Beside that a Fusarium rot disease of Aloe brown, cylindrical with monopodial branching, hyphae 80- vera was reported from Bali [22]. Some instances of anthracnose 100×4.75-6.25µm (Fig 5). Conidia solitary, straight or curved, disease of Aloe vera caused by Colletotrichum sp was also obclavate to ellipsoidal, tapering gradually into paler beak. Size reported from Lucknow [23]. of conidia ranges from 21×10.5-57.75×15.75µm, frequently with a beak attaining a size of 8.75×3.5-31.5×5.25µm (Fig 6). 3.2 Characterisation and Identification of the causal Comparing of all the cultural and microscopical characteristics organism of this fungal isolate with the published key [9, 10, 11], the fungus The identification of the pathogen was done phenotypically as was phenotypically identified as Alternaria sp and also well as through gene sequencing. confirmed by IMTECH scientist (Chandigar, India).

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Fig 5: Microscopic view of Alternaria brassicae showing Fig 4: Front view (A) and reverse view (B) of culture plate hyphal characters

--- (Bar = 5 µm) Fig 6: Conidia and hyphae of Alternaria brassicae (Bar = 5 µm)

Fig 7: Amplicon photograph of PCR product (FAq)

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3.4 Molecular identification by ITS 1-5.8S-ITS 2 of r DNA Genomic DNA from mycelium of isolated fungal pathogen Reverse primer 5´CAGACTT(G/A)TA(C/T)ATGGTCCAG 3´) (FAq) was extracted and the internal transcribed spacer (ITS1- in Sci Genome Labs Pvt Ltd, Kerala, India. 18s-ITS4) regions of r DNA were amplified by PCR and BLAST analysis of a 510-bp sequence resulted in 100% purified using Gene JET Gel Extraction Kit (Thermo Scientific) homology with Alternaria brassicae strain HYMS01 (GenBank , viewed in Gel electrophoresis (Fig7) and sequenced using Accession No. JX857165.1) from NCBI. Gene sequence has primers ITS1(Forward primer 5 been submitted in NCBI gene bank and obtained Gene bank ´CTTGGTCATTTAGAGGAAGTAA 3´ and Reverse primer 5´ accession No. is KJ022772 and it has been published in NCBI (t/a)TG GT(c/t) (a/g/t)(t/c)AGAGGAAGTAA 3´) & ITS4 gene bank on 23rd, March, 2014. (Forward primer 5´TCCTCC GCTTATTGATATGC 3´ and

3.5 Phylogram

3.6 Study of the disease occurrence and Intensity The data presented in the table -1 showed that this disease was Maharashtra during 2011. They reported three pathogens were found to present in all studied zones (Naihati, Halishahar, associated with the disease namely Alternaria alternata, A. Hasnabad, Hingalganj, Badu, Nilganj, Duttafulia, Gopalnagar, tenuissima and Fusarium sp. According to them the disease was Rajarhat and Mohishbathan ) and it was present throughout the found during rainy season and winter but absent in summer [19]. year (Jan.-Dec.2013). Similarly a survey conducted on fungal But in our survey we found the occurrence of disease all diseases of medicinal plants in Maharashtra state, scientists through the year. This may be due to differences in observed leaf spot disease of Aloe vera in Osmanabad district of geographical and climatic factors among survey areas.

Table 1: Month wise occurrence of disease in Aloe vera at our survey zones in North 24 Parganas

Occurrence of leaf spot disease Places Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec Naihati + + + + + + + + + + + +

Halishahar + + + + + + + + + + + +

Hasnabad + + + + + + + + + + + +

Hingalganj + + + + + + + + + + + +

Badu + + + + + + + + + + + +

Nilganj + + + + + + + + + + + +

Duttafulia + + + + + + + + + + + +

Gopalnagar + + + + + + + + + + + +

Rajarhat + + + + + + + + + + + +

Mohishbathan + + + + + + + + + + + +

*(+) indicates occurrence of disease.

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Table 2: Percentage of disease intensity (PDI) in Aloe vera at our selected fields.

PDI of leaf spot disease (*) State

Months Pharmacopical Nilgunj Rajarhat Basirhat Garden, Nadia 77.13 61.11 70 65.71 January (61.41) (51.41) (56.79) (54.15) 84.09 71.11 77.14 67.14 February (66.42) (57.48) (61.41) (55.00) 89.77 76.66 84.28 70.00 March (71.23) (61.07) (66.58) (56.79) 95.45 80.00 74.28 72.85 April (77.61) (63.44) (59.47) (58.56) 94.31 87.77 75.71 80.00 May (76.19) (69.47) (60.47) (63.44) 89.77 68.88 83.28 84.28 June (71.23) (56.04) (65.80) (66.58) 95.71 86.36 66.66 65.71 July (78.03) (68.28) (54.70) (54.15)

82.95 64.44 61.42 94.28 August (65.57) (53.37) (51.59) (76.06) 73.86 61.11 64.28 88.57 September (59.21) (51.41) (53.25) (70.18) 63.63 55.55 47.14 74.28 October (52.89) (48.16) (43.34) (59.47) 54.54 53.33 37.14 35.71 November (47.58) (46.89) (37.52) (36.69) 35.77 50.00 25.71 32.85 December (36.69) (45.00) (30.46) (34.94) CD(p=0.05) 19.07 9.80 18.00 22.18 SEM± 9.21 4.73 8.69 10.71

(*) In each field we have surveyed over 50 plant samples to record the disease intensity. The value within parentheses denotes the angular transformation value.

The data presented in Table 2 showed that the disease intensity and mustard crops have been reported from many countries, was recorded maximum during July (95.71%) in State viz., India [26], Italy [27], USA, UK and several other European Pharmacopical Laboratory and Pharmacy for Medicine, Govt. countries [28], Canada [29][30]), Iran [31] etc. In Australia fields of of W.B. April in Nilgunj (95.45%), May at Rajarhat (87.77%), Crambe abissycinia, the source of biodesel is highly invaded by June at Basirhat (83.28%) and whereas the Disease Intensity A. brassicae [32]. Leaf blight caused by Alternaria brassicae was lowest in all four areas surveyed extensively during the (Berk.) Sacc. is an important disease of mustard ( month of December. They were recorded as 25.71%, 32.85%, juncea (L.) and (B. campestris L. var toria) in the 35.77% and 50.00%, respectively for Basirhat, State Indian sub-continent and losses of crop ranges from 15 to 71% Pharmacopical Laboratory and Pharmacy for Medicine, Govt. [26][33][34]. In West Bengal state of India, leaf spot of Aloe vera of W.B., Nadia, Nilgunj (24-Parganas), Rajarhat. This study caused by Alternaria brassicae was not reported earlier. This indicated that maximum disease intensity was from April to study is first of its kind in this State. This investigation globally July 2013 in all four studied areas. It was probably due to high reveals Aloe vera as a new host of Alternaria brassicae. temperature and moisture. This pathogen is mostly active in wet seasons and in areas with relatively high rainfall [24]. Moreover 4. Conclusion minimum percentage of disease intensity (25.71-50.00%) was In conclusion, this work indicates that leaf spot of Aloe vera recorded in December, 2013 in all four studied areas. In occurs in 24 Parganas, West Bengal throughout the year December temperature and moisture are low and not so (January-December, 2013). It’s causal fungal pathogen is favorable to the fungal pathogen. Conidiophores of A. brassicae Alternaria brassicae. The disease intensity was at it’s peak from produce asexual spores (conidia), and conidia germinate over a April to July (83.28%-95.71%) in four places of our survey. wide range of temperature (8-31 °C) but this spore germination Whereas the minimum percentage of disease was recorded in can occur more quickly (within three hours) when the December (25.71%-50%) in all four study areas. This work not temperature is between 21-28 °C (when 98% spores are only globally establishes Aloe vera as a new host of Alternaria germinated). With the lowering of temperature the amount of brassicae but also this disease of Aloe vera is first reported from time it takes for 98% spore germination also increases [25]. This the district of West Bengal.It is expected that this work may is the primary work to report the occurrence and intensity of this encourage other workers to study this disease, its severity, crop disease in the district of 24- Parganas, West Bengal. loss and its proper control management to combat this disease. Alternaria brassicae (Berk.) Sacc. is a filamentous fungus and Infection of Aloe vera by Alternaria brassicae not only reduces basically a necrotrophic member of family under the crop loss and market value but it may reduce antioxidant the phylam Ascomycotina. Several hosts of this pathogen are so property and other medicinal efficacy of the herb [35]. far reported. Black spot of oil seed rape, , cauliflower ~ 42 ~

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