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Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 2632-2638 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 05 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.705.305 Survival of Didymella bryoniae Incitant of Ridge Gourd Blight Under Temperate Conditions Z.A. Bhat*, M.A. Bhat, M.A. Ahanger, Z.A. Badri, G.H. Mir and F.A. Mohi-u-Din Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir Shalimar Campus-190025, India *Corresponding author ABSTRACT Keywords Didymella bryoniae, Perpetuation, Plant debris, Ridge gourd Article Info Accepted: 20 April 2018 Available Online: 10 May 2018 Studies on role of infected seed and crop debris in the survival of Didymella bryoniae incitant of blight in ridge gourd were conducted under temperate conditions of Kashmir valley. The studies revealed that the pathogen perpetuated in the form of spores on seeds and plant debris stored indoors under ambient laboratory conditions as these produced viable spores throughout the observation period of twelve months. The pathogen was also observed to survive in the form of spores on plant debris stored indoors under ambient laboratory conditions, however, perpetuated as a dormant mycelium on plant debris left on the soil surface in open and was unable to survive on plant debris buried 7.5 cm deep in soil. Introduction Didymella bight caused by Didymella bryoniae (Aures.) Rehm. (anamorph: Phoma cucurbitacearum (Foutry) Sacardo) is an important disease of all the cucurbitaceous crops and reduces their yield as a result of defoliation, fruit infection and subsequent decay (Schenck, 1968). The disease is widely distributed in tropical countries and also reported frequently in areas having temperate climate (Sitterly, 1969). The disease was first reported from France in 1891 on cucumber (Roumeguere, 1891) and since then it has been reported from many countries namely, USA, Britain, Germany, Japan, New Zealand, Brunei, Mexico, Netherlands, Republic of Ireland, Salvadar (Puninthalingam and Holliday, 1972). In India, the disease was first reported from Mysore on leaves of Scheum edule (Sohi and Prakash, 1972) and then subsequently on Cucumis sativus (Kumar and khan, 1984), Mamordicha charanta (Kulwant and Shetty, 1996), Benincasa hispida (Pandey and Pandey, 2003) and Cucumis melo (Sudisha et al., 2004). Yield losses due to this disease on watermelon have been estimated up 100 per cent in Trindad (Bala and Hosein, 1986), 43 per cent in USA (Kienath and Duthie, 1998), 30 per cent on muskmelon in Australia (Mc Grath et al., 1993) and 35 per cent on cucumber in Polland (Leski, 1984). 2632 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 2632-2638 The disease has emerged in the recent past as one of the major constraints in the successful cultivation of the crop in Kashmir valley and has been responsible for causing a marked reduction in both quality and quantity of the fruit yield. For an effective management of the disease mode of perpetuation of the pathogen during the crop-less off-season is imperative. The present study was therefore, conducted under temperate conditions of Kashmir valley to find out the role of infected crop debris and seeds in perpetuation of the pathogen. Materials and Methods Perpetuation in/on seed The seeds collected at harvest from severely infected ridge gourd plants were stored in cotton cloth bags in laboratory and assessed for presence of the pathogen through blotter and agar plate methods at monthly intervals. Blotter method Random sample of sixty seeds were taken at monthly intervals from harvest and surface sterilized by immersing in 0.1% mercuric chloride for one minute followed by three subsequent rinses with sterilized distilled water. The seeds were placed in sterilized Petri dishes on three folds of sterilized blotter papers moistened with sterilized distilled water. Twenty seeds were placed aseptically in each Petri dish maintaining three replications for each plate. The plates were incubated at 24±1oC. The blotter paper in Petri dishes was kept moist by carefully pouring a few drops of sterilized distilled water in the plates as and when required. The plates were examined from 4th up to 15th day after incubation for the appearance of fungal colonies. Agar plate method Sixty seeds were taken from the seed lot and surface sterilized. Twenty surface sterilized seeds were aseptically placed on 2 per cent water agar in Petri dishes and incubated at 24±1oC. The plates were examined from 4th to 10th day after incubation for recording the percentage of seeds showing D. bryoniae growth. The number of seeds showing D. bryoniae growth in blotter and agar plate method were recorded and per cent infected seeds estimated by using the formula: Number of seed infected Per cent infected x seeds = Total number of 100 seeds examined Spore viability Twenty ridge gourd seeds were taken randomly at monthly intervals from harvest and 1 cm2 hilum portion from each seed carved out and crushed in a pestle and mortar in 40 ml of distilled water. The crushed material was strained through a double layer of cheese cloth. Twenty milliliter of the filtrate was centrifuged at 6000 rpm for 15 minutes. After centrifugation, supernatant was discarded and pellet was made 5 milliliter by adding sterilized water (Fi1ajdic and Sutton, 1995). Number of pycnidiospores/ ascospores was counted with the help of haemocytometer and the average spore load of three replications was estimated. To estimate the pycnidiospore/ ascospore germination one drop of 50 µl from processed sample was placed on a glass slide and incubated in a moist chamber at 24±1oC. After 24 hour incubation, the slide was viewed under binocular microscope to record spore germination index of spore viability. The spore viability was recorded using the formula: 2633 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 2632-2638 Spore viability = Number of spores germinated Total number of spores viewed cent viability of pycnidiospores/ascospores the method as described above for spore viability test of infected seeds was adopted. x 100 Results and Discussion Perpetuation through plant debris The infected plant debris including fruit husk and leaves were collected at harvest from the diseased crop and kept separately in nylon mesh bags. The bags were divided into three sets of 12 bags each. One set, each of leaves and fruit husk, was placed on the soil surface in vacated ridge gourd fields. The other set was buried 7.5 cm deep in such soil and third set was stored under ambient laboratory conditions. Each set was replicated three times. One month after placement of the bags at appropriate positions, twenty bits of 1 cm2 area were randomly taken from one randomly selected bag of each leaf and fruit husk and examined for the presence of pycnidia/pseudothecia. These leaf and fruit discs were then crushed separately in 40 ml of sterilized distilled water and for spore suspension preparation and estimation of per Survival on seed The seeds were found infected with D. bryoniae and the infection was found to persist from harvest up to 12 months of storage under ambient room conditions by using blotter and agar plate methods (Table 1). Similarly, the seeds were found to harbour viable spores of D. bryoniae throughout the observation period of twelve months starting from one month after harvest (in November during both the crop seasons of 2004 and 2005). The extent of viability constantly decreased with the advancement of storage period such that after 12 months of storage seeds harboured 18.6 and 20.3 per cent viable conidia in 2004 and 2005, respectively (Table 1). Table.1 Survival of Didymella bryoniae in/on ridge gourd seeds observed at monthly intervals after harvest in 2004 and 2005 Month Spore viability (%) Seeds showing D. bryoniae growth (%) Blotter method Agar plate method 2004 2005 2004 2005 2004 2005 November 2004 93.9 (76.0) 93.7 (75.9) 45.0 (42.1) 48.3 (44.0) 41.6 (40.1) 45.0 (42.1) December 95.4 (77.8) 92.9 (74.7) 41.6 (40.1) 45.0 (42.1) 38.3 (38.2) 43.3 (41.1) January 2005 81.2 (64.3) 86.9 (68.9) 38.3 (38.2) 41.6 (40.1) 36.6 (37.2) 38.3 (38.2) February 72.0 (58.0) 80.8 (64.0) 33.3 (35.2) 38.3 (38.2) 33.3 (35.1) 35.0 (36.2) March 61.3 (51.5) 78.0 (62.0) 31.6 (34.2) 35.0 (36.2) 31.6 (34.2) 28.3 (32.1) April 55.9 (48.4) 73.2 (58.8) 30.0 (33.1) 31.6 (34.2) 28.3 (32.0) 25.0 (29.9) May 32.5 (34.7) 60.8 (51.2) 28.3 (32.1) 30.0 (33.1) 25.0 (29.9) 23.3 (28.8) June 29.8 (33.0) 46.6 (43.0) 26.6 (30.9) 26.6 (30.9) 21.6 (27.6) 21.6 (27.5) July 28.0 (31.9) 45.9 (42.6) 23.3 (28.8) 25.0 (29.9) 20.0 (26.4) 21.6 (27.5) August 28.35 (32.0) 32.6 (34.8) 20.0 (26.4) 21.6 (27.5) 16.6 (24.0) 20.0 (26.5) September 23.3 (28.8) 24.1 (29.4) 20.0 (26.5) 20.0 (26.4) 11.6 (19.8) 15.0 (22.5) October 18.6 (25.5) 20.3 (26.7) 16.6 (24.0) 18.3 (25.2) 8.3 (16.5) 11.6 (19.8) CD (P=0.05) 4.69 3.88 4.32 4.43 4.92 4.58 Figures in parentheses are arc sine transformed values 2634 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 2632-2638 Table.2 Fructification and pycnidiospore/ ascospore production and viability on infected ridge gourd leaves kept under different conditions after harvest in 2004 and 2005 November 2004 *No. of pycnidia/ pseudothecia cm-2 fruit husk area Ambient Leaf burial at soil depth (cm) conditions 0 7.5 2004 2005 2004 2005 2004 2005 47.6 49.8 45.6 49.8 17.2 23.6 *No. of pycnidiospores/ ascospores cm-2 fruit husk area Ambient Leaf burial at soil depth (cm) conditions 0 7.5 2004 2005 2004 2005 2004 2005 5166 4643 4100 3956 0.0 0.0 2004 90.4 (72.1) 2005 87.6(69.3) 0 2004 92.2 2005 90.1 7.5 2004 NA December 40.1 46.9 34.2 39.5 0.0 0.0 3646 3916 3453 3126 0.0 0.0 76.5 (61.0) 73.2(58.8) 81.2 86.9 NA January 2005 38.0 44.9 21.2 18.0 0.0 0.0 3166 2926 0 0 0.0 0.0 64.0 (53.1) 58.6(49.9) NA NA NA February 31.2 39.9 13.2 10.2 0.0 0.0 2643 2336 0 0 0.0 0.0 48.4(44.1) 49.0(44.4) NA NA NA March 23.5 33.0 19.4 20.7 - - 1833 2173 1846 2100 - - 30.3 (33.5) 38.4(38.3) 85.2 82.6 NA April 20.6 29.0 29.6 20.0 - - 1413 1856 2533 2836 - - 23.3 (28.8) 35.0(36.2) 94.4 80.2 - May 18.5 21.3 24.5 17.4 - - 1046 1643 2013 2126 - - 20.3 (26.7) 32.7(34.9) 84.8 80.0 - June 12.1 16.4 19.5 11.6 - - 1026 1393 1226 1640 - - 20.0 (26.5) 28.9(32.5) 78.6 73.4 - July 10.9 15.1 - - - - 956 876 - - - - 16.4 (23.9) 24.2(29.4) - - - August 12.5 12.6 - - - - 646 630 - - - - 10.4 (18.8) 19.0(25.9) - - - September 8.5 9.5 - - - - 576 646 - - - - 9.6 (18.0) 13.7(21.7) - - - October 6.5 8.3 - - - - 416 343 - - - - 8.8(17.2) 7.2(15.6) - - - 2.90 3.07 3.28 2.54 2.95 3.23 2.59 3.18 2.60 3.29 Month CD(P=0.05) Figures in parentheses are arc sine transformed values - Material perished; NA Spores not available 2635 *Pycnidiospore/ ascospore viability (%) Ambient conditions Leaf burial at soil depth Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 2632-2638 Table.3 Fructification and pycnidiospore/ ascospore production and viability on infected ridge gourd fruit husk kept under different conditions after harvest in 2004 and 2005 November 2004 *No. of pycnidia/ pseudothecia cm-2 fruit husk area Ambient Leaf burial at soil depth (cm) conditions 0 7.5 2004 2005 2004 2005 2004 2005 78.1 71.8 76.6 71.8 13.4 24.6 *No. of pycnidiospores/ ascospores cm-2 fruit husk area Ambient Leaf burial at soil depth (cm) conditions 0 7.5 2004 2005 2004 2005 2004 2005 6926 6576 6126 4936 0.0 0.0 2004 94.2 (66.5) 2005 93.2 (74.9) 0 2004 92.05 2005 91.1 7.5 2004 NA 2005 NA December 70.8 69.2 63.2 53.6 0.00 0.0 6200 5963 4533 3863 0.0 0.0 84.6 (59.7) 89.4 (71.0) 87.33 88.7 NA NA January 2005 62.4 65.7 36.7 30.6 0.00 0.0 5036 5376 0 0 0.0 0.0 72.5 (52.2) 77.1 (61.4) NA NA NA NA February 54.4 57.8 22.2 10.5 0.00 0.0 4520 4656 0 0 0.0 0.0 51.2 (45.7) 53.2 (46.8) NA NA NA NA March 43.2 52.1 24.2 19.4 0.00 - 3865 4226 2946 1453 - - 43.2 (41.1) 45.2 (42.2 89.55 90.6 - - April 37.1 47.1 41.0 37.2 - - 3353 3623 3756 2100 - - 41.5 (40.1) ) 33.2 (35.2) 88.05 82.4 May 30.2 37.2 25.1 18.2 - - 2850 3150 2253 1353 - - 38.4 (38.2) 29.8 (33.0) 85.16 80.2 - - June 24.1 31.4 18.4 12.5 - - 2630 2413 1676 1000 - - 32.0 (34.4) 23.4 (28.9) 80.38 75.1 - - July 20.2 21.5 13.2 9.2 - - 1746 1853 1240 813 - - 28.4 (32.2) 24.2 (29.4) 78.25 - - - August 14.1 19.6 - - - - 1573 1356 - - - - 22.5 (28.3) 21.0 (27.3) - - - - September 10.1 13.5 - - - - 1256 923 - - - - 18.5 (25.5) 16.2 (23.7) - - - - October 8.2 10.5 - - - 983 780 - - - 13.2 (21.3) 11.8 (20.1) - - - - 2.27 2.83 3.25 3.20 3.29 3.64 0.91 1.57 Month CD(P=0.05) 3.10 Figures in parentheses are arc sine transformed values - Material perished; NA Spores not available 2636 2.41 *Pycnidiospore/ ascospore viability (%) Ambient conditions Leaf burial at soil depth (cm) - Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 2632-2638 These findings provide sufficient grounds to infer that the pathogen D. bryoniae perpetuates in/on the seeds as spores and the pathogen inoculum reaches the cropping fields along with seeds. Similar observations have also been made by several workers. Rankin (1954) found the invasion of D. bryoniae in the epidermis and sclerenchyma layers and isolated the fungus from cotyledons and embryo. Chen and Bao (1990) noted the survival of the fungus both in and on the seeds of infected fruits up to 21 months of storage at room temperature. Sudisha et al., (2006) also reported the seed borne nature of D. bryoniae and noted mean incidence of 31 per cent in seed coat, 11 per cent in cotyledons and 4 per cent in embryo while evaluating the different components of infected seeds. Kienath (2002) could not recover D. bryoniae beyond 6 to 7 months of storage in buried infected watermelon debris which he attributed to the antagonistic activities of saprophytic soil microorganisms that seemed to have eliminated the pathogen resident in the watermelon debris. Van Steekelenburg (1983) observed old pseudothecia with ascospores and pycnidia with some pycnidiospores, even after storage of diseased plant debris for 18 months, whereas on debris kept on soil surface in open only empty fruiting bodies were observed during winter months when average monthly temperature was below 5oC. Similarly, Chiu and Walker (1949) found empty pycnidia and pseudothecia in the overwintered crop debris but could isolate the fungus readily and suggested the overwintering of fungus in winter as dormant mycelium. Survival on plant debris References The periodic examination of the infected debris placed indoors, on soil surface and buried under soil indicated the presence of fruiting bodies as well as viable spores throughout the observation period of twelve months when placed indoors under roof protection and only up to four to five months when left on soil surface as debris perished beyond this period (Table 2 and 3). 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Int.J.Curr.Microbiol.App.Sci. 7(05): 2632-2638. doi: https://doi.org/10.20546/ijcmas.2018.705.305 2638