Abstract
Gummy stem blight is a major disease affecting cultivation of cucurbitaceous vegetable crops all over the world. In India, this disease has been reported in several cucurbits such as ash gourd, bitter gourd, chow-chow, cucumber, muskmelon and ridge gourd. Our report represents first time identification, isolation and molecular characterisation of gummy stem blight fungus in watermelon at Indian Institute of Horticultural Research, Bangalore, India. As it infects several curcurbits and is a seed borne pathogen, seed importation may have been the source of introduction to the region. To our knowledge, this is the first report of gummy stem blight affecting watermelon in India.
Gummy stem blight (GSB) is a major production constraint in growing cucurbitaceous vegetable crops worldwide. In India, this disease was first reported on chow-chow (Sohi and Prakash 1972) in Mysore and then subsequently on other cucurbit crops viz., cucumber (Kumar and Khan 1984), bitter gourd (Kulwant and Shetty 1996), ash gourd (Pandey and Pandey 2003), muskmelon (Sudisha et al. 2004), and ridge gourd (Bhat et al. 2018) which represent a major share of vegetable production in India. All these reports indicate that Didymella bryoniae was the causal organism of this disease. Aveskamp et al. (2010) described the new species Stagonosporopsis cucurbitacearum as a synonym of D. bryoniae infecting cucurbits. Though the reports on occurrence of gummy stem blight in watermelon in other countries are available, there has been no report of this disease in watermelon from India.
Watermelon (Citrullus lanatus) is an important commercial, cucurbitaceous vegetable cultivated throughout the year in Bengaluru, India. Recently, GSB has become a threat for the production of watermelon that has not been reported earlier in this region. Disease symptoms initially start with foliar marginal brown discolouration, spreading towards the inside leaf lamina. Later, the pathogen spreads to cover complete leaf, turning it dark brown and dry affecting photosynthesis and yield. In severe cases, the pathogen enters into the vascular bundles of plant and clogs the phloem tissue and later damaging the xylem. It causes complete wilting of plant with an estimated yield loss up to 100% (Bala and Hosein 1986). We attempted to isolate and characterise the gummy stem blight pathogen of watermelon at the Indian Institute of Horticultural Research, Bengaluru, India from diseased plants observed in our research farm. Samples were collected from the infected watermelon plant from the symptomatic region of stem. These bits of 5 mm length and 2 mm width were washed in normal water followed by surface sterilisation with 0.1% NaOCl for 2 min and rinsed twice with sterile distilled water to make it free from any surface contamination. Samples were kept on pre-sterilised blotting paper for 5 min for air-drying. The dried pieces of infected stems were placed on potato dextrose agar (PDA) and kept incubated at ambient conditions. After two days when growth was observed on plates, the growing tip of the fungal colony was transferred to another freshly prepared PDA plate for purification. A thick layer of mycelium appeared just seven days after transfer without any sporulation. Initially mycelium was bright white in colour later turning into light olive-green colour as reported by Keinath et al. (1995). The fungus has been characterised molecularly using ITS primers viz., ITS 1 and ITS 4 (White et al. 1990). Genomic DNA was extracted by CTAB method (Mishra et al. 2014) and PCR reactions were carried out in a total volume of 25 μL. Reaction components consisted of 2.5 μL of 10X PCR buffer with 25 mM MgCl2 (GeNei, India), 0.5 μL10 mM dNTPs (GeNei, India), 0.5 μL of10 μM forward and reverse primers, 0.3 μL 3 U/μL.
Taq (GeNei, India), and one μL DNA template. PCR conditions included an initial denaturation step at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s., annealing at 52 °C for 30 s., extension at 72 °C for 30 s., followed by a final extension at 72 °C for 5 min. PCR product of 5 μL volume was loaded on to 1% (w/v) agarose gel and electrophoresed at 75 V for 1.5 h. The ethidium bromide stained agarose gel was visualised under UV light (UVITEC FIREREADER V.10, Cambridge, U.K). Remaining PCR product was purified and sequencing was performed in ABI-3710 Prison automated DNA analyser (Eurofins, India). The sequence of the isolate was compared with the NCBI – GenBank database using the blast algorithm (www.ncbi.nlm.nih.gov). Results confirmed that the fungal isolate was sharing 99% homology with the S. cucurbitacearum isolate (NCBI accession no.KC460840.1) available in the database submitted by Liu et al. (2013). Isolated DNA was also used to amplify with SCAR primers (RG-I-F 5’-TGTCGTTGACATCATTCCAGC-3′ and RG-I-R 5’-ACCACTCTGCTTAGTATCTGC-3′) which are specific to S. cucurbitacearum (Babu et al. 2015). The amplicon obtained with this primer was of 735 bp size. The sequence of the amplified product was deposited at NCBI with accession No. MN396362. The live culture has been deposited at National Agriculturally Important Microbial Culture Collection (NAIMCC), Mau, Uttar Pradesh, India, which is one of the culture collections registered by the World Federation for Culture Collections (http://www.wfcc.info/collections/). The accession number of the culture is NAIMCC-TF 2438 (Figs. 1 and 2).
The same fungal isolate was used to re-inoculate watermelon seedlings to confirm pathogenicity. The mycelium mat of a seven-day-old culture was used for this purpose. Four mm round discs using a cork borer were placed on leaf lamina of 17-day-old seedlings at 2 true leaf stage. The inoculated seedlings were kept under constant moist condition (>80% relative humidity) to create conducive environment for disease development. Two days after inoculation, dark brown, water soaked and necrotic lesions spreading around the place of inoculation could be observed. No changes were detected in the control seedlings. The same fungus could be successfully re-isolated from the infected seedling, fulfilling Koch’s postulates.
Gummy stem blight has been reported in watermelon worldwide in several places viz., southern United States (Sherbakoff 1917; Schenck 1962; Power 1992), South Carolina (Keinath et al. 1995), Antalya and Turkey (Basım et al. 2016). S. cucurbitacearum has been reported in several cucurbitaceous vegetables viz., cucumber and watermelon in France (Chester 1891), Italy (Chiu and Walker 1949) and United States (Farr and Rossman 2010); melon in Brazil (Dalcin et al. 2017) and Cucurbita maxima in northeast China (Zhao et al. 2018). With the emergence of free trade order over the last two decades, there is an increased movement and exchange of seed material. This has probably led to the introduction of this disease in Bengaluru, India where the vegetable seed industry is active. Though occurrence of gummy stem blight by S. cucurbitacearum on other cucurbitaceous crops has already been reported, this is the first report of gummy stem blight by S. cucurbitacearum on watermelon in India.
References
Aveskamp MM, de Gruyter J, Woudenberg JH, Verkley GJ, Crous PW (2010) Highlights of the Didymellaceae: a polyphasic approach to characterise Phoma and related pleosporalean genera. Stud Mycol 65:1–60. https://doi.org/10.3114/sim.2010.65.01
Babu B, Kefialew YW, Li PF, Yang XP, George S, Newberry E, Paret ML (2015) Genetic characterization of Didymella bryoniae isolates infecting watermelon and other cucurbits in Florida and Georgia. Plant Dis 99:1488–1499
Bala G, Hosein F (1986) Studies on gummy stem blight disease of cucurbits in Trinidad. Trop Agric 63:195–197
Basım E, Basım H, Abdulai M, Baki D, Öztürk N (2016) Identification and characterization of Didymella bryoniae causing gummy stem blight disease of watermelon (Citrullus lanatus) in Turkey. Crop Prot 90:150–156
Bhat ZA, Bhat MA, Ahanger MA, Badri ZA, Mir GH, Mohi-u-Din FA (2018) Survival of Didymella bryoniae incitant of ridge gourd blight under temperate conditions. Int J Curr Microbiol App Sci 7:2632–2638. https://doi.org/10.20546/ijcmas.2018.705.305
Chester FD (1891) Notes on three new or noteworthy diseases of plants. Bulletin of the Torrey Botanical Club 18:371–374
Chiu WF, Walker JC (1949) Physiology and pathogenicity of cucurbit black-rot fungus. JAgr Res 78:589–615
Dalcin MS, Tschoeke PH, Aguiar RW, Fidelis RR, Didonet J, Santos GR (2017) Severity of gummy stem blight on melon in relation to cultivars, use of fungicides and growing season. Hortic Bras 35:483–489
Farr DF, Rossman AY (2010) Fungal databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. http://nt. ars-grin.gov/fungaldatabases/
Mishra IG, Tripathi N, Tiwari S (2014) A simple and rapid DNA extraction protocol for filamentous fungi efficient for molecular studies. Indian J Biotechnol 13:536–539
Keinath AP, Farnham MW, Zitter TA (1995) Morphological, pathological, and genetic differentiation of Didymella bryoniae and Phoma spp. isolated from cucurbits. Phytopathology 85:364–369
Kulwant S, Shetty KPV (1996) Didymella black fruit rot of bitter gourd. Indian Phytopathology 49:294–296
Kumar R, Khan MT (1984) Leaf spot disease of Cucumis sativus. Indian Phytopathology 37:374
Pandey KK, Pandey PK (2003) First report of some new fungal diseases on wax gourd (Benincasa hispida) in the world from India. Journal of Mycology and Plant Pathology 33:439–441
Power HJ (1992) South Carolina 1991 vegetable statistics. South Carolina Agriculture Experimental Station Bulletin 472
Schenck NC (1962) Mycosphaerella fruit rot of watermelon. Phytopathology 52:635–638
Sherbakoff CC (1917) Some important diseases of truck crops in Florida. Florida Agriculture Experimental Station Bulletin 139:269–273
Sohi HS, Prakash O (1972) New records of fungal diseases from India. Indian J Mycol Plant Pathol 2:139–142
Sudisha J, Kumar TV, Niranjana SR, Shetty HS (2004) First report of gummy stem blight caused by Didymella bryoniae on muskmelon (Cucumis melo) in India. Plant Pathol 53:533
White TJ, Bruns T, Lee SJWT, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. PCR protocols: a guide to methods and applications, pp.315–22
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mahapatra, S., Rao, E.S., Sandeepkumar, G.M. et al. Stagonosporopsis cucurbitacearum the causal agent of gummy stem blight of watermelon in India. Australasian Plant Dis. Notes 15, 7 (2020). https://doi.org/10.1007/s13314-020-0376-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13314-020-0376-z