First Report of Collectotrichum Acutatum J.H. Simmonds Associated with Anthracnose Disease in Malayan Apple from India

Available online at www.ijpab.com Sonavane et al Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) ISSN: 2320 – 7051 DOI: http://dx.doi.org/10.18782/2320-7051.2712 ISSN: 2320 – 7051 Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) Research Article

First Report of Collectotrichum acutatum J.H. Simmonds Associated with Anthracnose Disease in Malayan Apple from India

Priti Sonavane, V. Venkataravanappa* and M. Krishna Reddy Division of Plant Pathology, Central Horticultural Experiment Station, Chettalli, Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore-560089, India *Corresponding Author E-mail: [email protected] Received: 28.03.2017 | Revised: 30.04.2017 | Accepted: 4.05.2017

ABSTRACT The genus Colletotrichum contains an extremely diverse number of fungi including both plant pathogens and saprophytes. Plant pathogenic species are important worldwide, causing diseases commonly known as anthracnose of vegetables, fruits, and perennial tree crops. Anthracnose diseases appear in developing fruit in the field (pre-harvest) and those damaging mature fruit during storage (post-harvest). The anthracnose disease infected samples of Malayan apple fruit and leaves were collected from Central Horticultural Experiment Station, Chettalli, Karnataka state of India. The fungus was isolated from infected leaves and fruits by standard fungal isolation method. The Koch’s postulates of the fungus were confirmed by re-inoculated on the fruit and leaves of the same host. Further the fungus was identified based on pathogenic, morphological and amplification of internal transcribed spacer (ITS) region by PCR using universal ITS specific primers. The amplified PCR product were cloned and sequenced. The sequence analysis showed that Malayan apple isolate was showed maximum nucleotide identity of 100% with Collectotrichum acutatum infecting Lupines albus flowering plant.

Key words: Fungi, PCR, Malayan apple, Species

INTRODUCTION presumably originated in Indo-Malaysia region Malayan apple (Syzygium malaccense Merr. & or Southeast Asia. The tree species is being Perry.) is a non-climacteric tropical fruit cultivated for long time in tropics especially in species. It is also known as red guava, Malay Indo-Malaysia, Southeast Asia, Melanesia, rose apple, jambu bol or ball guava. The fruits Polynesia and Macronesia26. There are three are pear-shaped usually pink, light-red, red, species of Syzygium, namely the Malay apple green, sometimes greenish white, or cream- (Syzygium malaccense), water apple (Syzygium colored and are generally crisp, with a subtly aquem) and wax apple (Syzygium sweet taste or aromatic flavor 15. The species samaragense) bear edible fruits.

Cite this article: Sonavane, P., Venkataravanappa, V. and Reddy, M.K., First report of Collectotrichum acutatum J.H. Simmonds associated with anthracnose disease in Malayan apple from India, Int. J. Pure App. Biosci. 5(5): 465-472 (2017). doi: http://dx.doi.org/10.18782/2320-7051.2712

Sonavane et al Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) ISSN: 2320 – 7051 The delicious fruit can be eaten directly and is Surface sterilized leaf spot pieces were then known to be beneficial for human health, due aseptically transferred into 9 cm Petri dishes to its significant levels of vitamins and containing potato dextrose agar (PDA) and minerals, organic compounds, flavonols, incubated at 25±2°C for seven days. phenolic compounds, and sequiterpenes, as Thereafter, the pure culture Collectotrichum well as limited amounts of carotenoids, all of acutatum was isolated by selecting growing which act as antioxidants11. The tree is also mycelia tip on PDA and aseptically transferred widely used in traditional medicines. into another Petri plate containing PDA Basically, it was grown as an ornamental tree medium, where it was grown for 15 days at in south east Asia, but due to increase in 23±2°C in the BOD incubator. On the basis of acceptance of exotic fruits in international fruit their conidiophore and conidial morphology as market the fruits crop has been grown in parts described by Simmons20 the pathogen was of West Bengal, Kerala, Karnataka and Tamil identified as Collectotrichum acutatum J.H. Nadu states of India. Simmonds, and purified by single spore A few selected superior germplasm of isolation method. Monoconidial culture fungus Malayan apple is maintained at experimental was maintained on potato dextrose agar (PDA) Plot in Central Horticultural Experiment slants at 4°C Station Chettalli, Karnataka, India are showing Fungal materials and microscopy: The symptoms of anthracnose on leaves, flowers anthracnose infected leaves and fruit samples and fruit. But, in the past two years, the of Malayan apple and purified pure culture of disease incidence was increasing sharply in fungus conidial morphology was examined severe farm on new flash of leaves, flowers under the light microscope (Nikon Eclipse and fruits. The level of incidence differed 50i). The 15 days old of culture of the fungus significantly about 50 to 60 % on different was grown PDA were scraped and placed on a accession of Malayan apple. Therefore the glass slide containing a drop of sterile water. present study aim was to identify the causal The specimen was observed at 400X agent of anthracnose observed on Malayan magnification under a microscope. apple in Karnataka. Pathogenicity test: Pathogenicity test of Collectotrichum MATERIAL AND METHODS acutatum was conducted on symptomless, Collection Isolation and maintenance of detached Malayan apple fruits and leaves. The Collectotrichum acutatum: The Malayan leaves and fruits were surface-sterilized with apple infected leaves, flowers and fruit ethanol (70%) and then washed three times samples with typical anthracnose symptoms with sterilized distilled water and dried with were collected from Malayan apple germplasm sterile tissue papers. The surface sterilized maintained at experimental Plot in Central fruits were inoculated with 15 µl of the Horticultural Experiment Station Chettalli, conidial suspension (106 conidia /ml, adjusted Karnataka, India (75.8°E longitude; 12.5°N with Haemocytometer) by creating the wound latitude with an annual rainfall of 1500 mm with sterile needle as described by Lin et al.14. which is spread over a period of more than 100 Control fruit and leaves were inoculated with days with peak period between July and 15µl of sterile distilled water. The fruits and September-2016). Pieces of the diseased leaves were then incubated in a plastic tissues were washed 3 to 4 times in sterilized container at 25°C and >95% relative humidity, distilled water and then surface sterilized by and examined for lesion development 5 days dipping in 4% sodium hypochlorite (NaOCl) after inoculation. After 7 days, anthracnose solution for 1 min, followed by several rinses symptoms development on fruit surfaces was with sterile distilled water for 4 to 5 times. re-isolated from the disease fruit and leaves Copyright © Sept.-Oct., 2017; IJPAB 466

Sonavane et al Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) ISSN: 2320 – 7051 and subcultured onto PDA plates, which were extraction kit (Qiagen) and sequencing was incubated at 25°C in darkness. The resultant done by Eurofins Genomics India Pvt. Ltd cultures were checked for colony and spore (Karnataka, India). morphology to confirm Koch’s postulates. The Sequence analysis test was repeated twice. Sequence similarity searches were performed Fungal genomic DNA purification: The pure by comparing sequence to sequences available culture of the fungus grown on PDB at 25 ± 2 in the database using BlastN1. The sequences ºC for 7 days. The fungal mycelium was showing highest scores with the present harvested by filtration through Whatman No.1 isolates were obtained from database and filter paper and washed with sterile distilled aligned using SEAVIEW program8 . The water and dried. Two grams of dried mycelium sequence identity matrixes for the big bud was used for total genomic DNA by following phytoplasmas were generated using Bioedit modified protocol of CTAB method3.The Sequence Alignment Editor (version 5.0.9)10 isolated DNA was dried under vacuum, and phylogenetic tree was generated by resuspended in 50 ml of TE buffer (10 mM MEGA 6.01 software24 using the neighbour Tris-HCl, pH 7.5; 1 mM EDTA, pH 8.0). The joining method with 1000 bootstrapped quality of the genomic DNA was checked on replications to estimate evolutionary distances 0.8% agarose gel and stored at -200C for between all pairs of sequences simultaneously. further use. PCR amplification RESULT AND DISCUSSION Furthermore to confirm the exact identity of Symptoms: The infected leaves showed a the pathogen, total genomic DNA was small black lesion that coalesces and gives amplified by PCR using universal primers scorched appearance to the canopy (Fig 1a). ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) On fruit sunken concentric rings, which are and ITS4 (5’-TCCTCCGCTTA pinkish in color around 5mm in diameter were TTGATATGC-3’)27 .The DNA amplification observed4 . These lesions may coalesce entire was performed with 35 cycles with cycling surface of the fruit, which leads to inside pulp conditions of denaturation for 1 min at 940C, become giving bad odour (Fig 1c). The area of primer annealing for 45 seconds at 550C, and lesions is firm at first, but eventually become primer extension for 90 seconds at 720C, with soft and putrid. The infection of anthracnose an initial denaturation at 940C for 3 min and a was also observed on emerging buds of final extension for 15 min at 720C. The PCR flowers, as small black lesions later coalesce to reactions were carried out in a Gene Amp PCR farm big lesion may leads to premature system 9700 (PE Applied Biosystems, Foster dropping flower at early stage, thereby City, CA) thermocycler. The final volume of reducing fruit set (Fig 1b). 25 μL PCR mix containing 2 μL DNA Morphological characterization template, 1.5 U Taq polymerase, 25 mM Colonies of Collectotrichum acutatum were

MgCl2, 2 mM dNTPs and 25 pmole of each dense aerial, initially white or cream white, primer were taken in a PCR tube. PCR becoming gray and then turning dark gray, as products were electrophoresed (1 h at 80 volts) the cultures aged on PDA (Fig 2 a). Colony in 0.8% agarose gel in Tris-borate-EDTA reverse was white to pinkish in colour. Bright buffer, pH 8.0. Gels were stained with orange spore masses were produced outward ethidium bromide (10 mg/mL) and were from the center of the colony. The cultures visualized and documented by Alpha developed black acervuli around the centre of digidoc1000 system (Alpha Innotech the colony. Fungal colonies on PDA medium Corporation, USA). The amplified PCR grew to 70-80 mm diameter in one week at products (550bp) were purified by gel room temperature (25 ± 2°C). The isolated Copyright © Sept.-Oct., 2017; IJPAB 467

Sonavane et al Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) ISSN: 2320 – 7051 pathogen produced orange spore mass on anthracnose and fruit fly under natural plates. Mycelial was branched, septate and condition. Therefore to effective management hyaline. Conidia were hyaline, one-celled, of the disease, one should know the straight, smooth, fusiform, (8-15 × 2.5-5 µm) morphogenic symptom of the disease on the with pointed ends (Fig 2 b). Conidiophores are plant as well as pathogen. In the present study branched at base, hyaline in early stage later Malayan apple showing the typical turning into brown colour. Acervuli are anthracnose symptoms was confirmed by superficial without setae. The fungus was infecting of Colletotrichum acutatum based on initially identified as Collectotrichum phenotypic traits (colony appearance, and acutatum J.H. Simmonds, based on characters character of vegetative and reproductive like culture morphology and conidium shape structures) and sequences of rDNA ITS region. and size21,22,6. The cultures did not form sectors after 10 days of incubation. The colour of cultures may vary considerably Molecular characterization within and between species of C. acutatum and The total genomic DNA of C. acutatum was C. gloeosporioides. Colonies of C. amplified by using universal intergenic gloeosporioides were usually gray in transcriber spacer region (ITS) specific appearance, while C. acutatum colonies had a primers. The expected PCR amplicon size of chromogenic (pink) or nonchromogenic (white 7,12,6,28 550bp was amplified (data not shown). The to gray) phenotype . Colletotrichum amplified PCR product was cloned and acutatum causes anthracnose diseases on 5 sequenced. The sequence is available in numerous host plants in worldwide . GenBank (NCBI) with the accession number, Originally the disease was described on KU933355. The ITS sequence of C. acutatum Papaya, Capsicum and Delphinium ajacis in 19 isolated from Malayan apple was compared Australia by Simmonds . Today C. with other Colletotrichum species available in acutatum species complex is known infect 9 16 13 the database2. The sequence analysis showed many fruits like strawberry , Citrus , Apple , 23 18 25 that the isolate showed 100% similarity with Olive , Cranberry and Blueberry . The Collectotrichum acutatum infecting Lupines pathogen also found to be infecting avocado, albus and Fragaria sp. The phylogenetic bread fruit, Carambola, citrus, fig, guava, 17 analysis was carried out by comparing the kiwifruit, lychee, mango and papaya . Internal transcribed spacer of C. acutatum with The information would be helpful for growers other corresponding region of different to manage the disease successfully in Malayan Colletotrichum species available in database2. apple. Anthracnose caused by C. acutatum is The result revealed that, the Malayan apple an emerging disease that may threaten the isolate formed closely cluster with C. profitability of Malayan apple as well other acutatum infecting Lupines albus and fruits crops considering its broad host range. Fragaria sp. (Fig 3). Therefore, the fungus More likely that the inoculum can disseminate that caused anthracnose on Malayan apple was from one crop to another and survive for long identified as Collectotrichum acutatum based duration in the under fields. Additional studies on morphological, pathogenicity test and on etiology and epidemiology of anthracnose amplification of ITS region. caused by C. acutatum are needed to define Malayan apple has become widely further the disease management strategies. To cultivated in recent years as a Indian our knowledge, this is the first report traditional herb in northeast and south India. of Collectotrichum acutatum J.H. Simmonds The main drawback of this production is infected Malayan apple in India. Copyright © Sept.-Oct., 2017; IJPAB 468

Sonavane et al Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) ISSN: 2320 – 7051

Fig. 1: Malayan apple showing the symptoms of anthracnose on (a) Leaf, (b) fruits, (c) flower under natural conditions

Fig. 2: (a)Pure culture of Colletotrichum acutatum on Potato dextrose agar, (b) Conidia having pointed ends of Colletotrichum acutatum

Sonavane et al Int. J. Pure App. Biosci. 5 (5): 465-472 (2017) ISSN: 2320 – 7051

Fig. 3: Phylogenetic trees based on sequences of ITS region of Colletotrichum acutatum with corresponding ITS regions of other Colletotrichum species using Neighbor-joining algorithm. Horizontal distances are proportional to sequence distances, vertical distances are arbitrary. The trees are unrooted. A bootstrap analysis with 1000 replicates was performed and the bootstrap percent values more than 50 are numbered along branches. The GenBank accessions of different Colletotrichum strains used for analyses in this study as given by Alvarez et al. (2014).

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