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Int.J.Curr.Microbiol.App.Sci (2020) 9(5): 1871-1878 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 5 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.905.212 Unclogging Seed Borne Pathogens to Prevent Diseases in Capsicum Suryapal Singh1* and Harshita Singh2 1 Department of Seed Science and Technology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (HP) 173 230 2 Department of Vegetable Science, CCSHAU, Hisar, India *Corresponding author ABSTRACT Keywords Hot water seed treatment, Bell pepper, Seed microflora, Disease incidence and Capsicum annuum Article Info Accepted: 15 April 2020 Available Online: 10 May 2020 The present investigations was carried out to study the effect of hot water seed treatment comprised of different ranges of temperature (47-49, 50-52 and 53-55 °C) and discrete duration of time (30, 45 and 60 min.). The seeds of bell pepper and their most important seed-borne pathogens (Alternaria spp., Curvuleria spp., Penicillium spp., Fusarium spp. Colletotrichum spp.) have been investigated in laboratory. Thee numbers of infected seeds were observed and recorded daily and per cent incidence was evaluated. Furthermore, per cent ungerminated seeds and incidence of various diseases like, damping-off, anthracnose, wilt, cercospora leaf spot and virus attack was recorded in nursery as well as in field under protected cultivation. Under in vitro conditions, the hot water treated seed with temperature 50-52°C for 30 min. showed significantly lower seed microflora (%) as compared to untreated seed (control). In nursery condition, the hot water treated seed, same temperature 50-52°C for 30 minutes showed lower damping-off and virus incidence as compared to control with 22.75 per cent post emergence damping-off and 5.56 per cent respectively. Though, the incidence of damping off and virus attack is minimum in case of the hot water treated seed same temperature 53-55°C for 60 min. i.e. 5.47 per cent and 1.11 per cent respectively but this high temperature for such long time have strongly affected germination. That’s why ungerminated seed percentage in case of is hot water treated with 53-55°C for 60 min. is 41.11 which is significantly very high compared to the hot water treated with 50-52°C for 30 minutes i.e. 12.22 per cent. Under protected condition the hot water treated with 50-52°C for 30 min. showed lower incidence of diseases like anthracnose, cercospora leaf spot, wilt and virus as compared to control. It may be concluded that hot water seed treatment at 50-52°C for 30 min. proved effective in reducing the incidence of diseases like damping–off, anthracnose, cercospora leaf spot and viruses in bell pepper cultivar Solan Bharpur without any ill effect on the germination of seeds. Introduction Bell pepper (Capsicum annuum L.), also known as sweet pepper, capsicum or Shimla mirch, belongs to family solanaceae. It is a high value vegetable and an important cash crop grown throughout the world. It has attained a status of high value crop in recent 1871 Int.J.Curr.Microbiol.App.Sci (2020) 9(5): 1871-1878 years because of its delicacy and pleasant flavour coupled with rich content of ascorbic acid and other vitamins and minerals (Agarwal et al., 2007). Bell pepper is mainly cultivated as summer and rainy season crop. Because of high humidity and soil moisture coupled with moderate temperature, the incidence of various diseases is high. Some diseases are seed borne in nature viz., anthracnose (Colletotrichum capsici), Cercospora leaf spot (C. capsici), bacterial spot (Xanthomonas campestris pv. vesicatoria), Bacterial wilt (Pseudomonas solanacearum), Bacterial canker (Clavibacter michiganensis) and viruses like Tomato spotted wilt virus (TSWV). To avoid the occurrence of such diseases, seed treatment with various chemicals has been recommended from time to time (Gupta and Thind, 2006). But in present day agriculture, use of chemicals for crop production is discouraged. Seed borne diseases of bell pepper are considered as alarming problem in organic farming systems because of the nonecofriendly chemical control methods. Hence, other alternative treatments for disease control have been developed and hot water treatment is one of them. Hot water soaking is a very old practice but has revived in this era of organic farming to control many seed-borne diseases by using temperatures hot enough to kill the organism but not quite hot enough to kill the seed and it is still being used as a very effective alternative (Floyd, 2005; Muniz, 2001). Hot water seed treatment is thermo physical method of plant protection. At the end of the 19th century, for control of control loose smut (Ustilago nuda) the hot water seed treatment was applied to in cereals (Jensen, 1888). During storage of oak seed, hot water seed treatment has been recommended against the fungus Ciboria batschiana (Natzke, 1997). Further examples for application of the method are shown by Baker (1962), Gabrielson (1983), Hoffmann et al., (1994) and Jahn et al., (2000). Hot water treatment is of more importance for organic farming (Trueman and Wick, 1996). It could also become an alternative method for conventional farming especially in case of failure of chemicals permitted for seed treatment. The present investigation was designed to study the effect of different temperature and time combinations of hot water seed treatment on incidence of diseases and seed microflora in bell pepper. Materials and Methods The present experiment was carried out at Experimental Farm and Laboratory of Department of Seed Science and Technology, Dr YS Parmar University of Horticulture and Forestry, Solan. The experimental farm of Department of Seed Science and Technology is located at an altitude of 1183 meters above mean sea level with latitude of 30.51ºN and longitude of 77.09ºE the mid- hill zone of Himachal Pradesh, India observed with GARMIN’S GPS 12 Personal Navigator. The soil texture of polyhouse was loam to clay loam having pH ranging from 6.85-7.05. The healthy, disease free, bold and uniform seeds of bell pepper cv. Solan Bharpur, were obtained from Department of Seed Science and Technology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.). The obtained seeds were treated with hot water in automatically controlled hot water bath tub at different temperature range for discrete time period (Table 1). The in vitro experiment was laid in Completely Randomized Block Design with four replications taking 50 seeds per replication. The experiments in nursery condition and protected condition were both laid in Randomized Block Design with ten treatments replicated three times taking 30 seedlings and 6 plants per replication respectively. 1872 Int.J.Curr.Microbiol.App.Sci (2020) 9(5): 1871-1878 Hot water bath works automatically controlling temperature with time. Firstly seeds were soaked in normal water for 15 min. wrapped in muslin cloth. Then, poured about 3 l. water in the device and it was connected with electricity. With the help of heater coil, the device was heated, with the time and thermostat bulb the device was regulated to the desired temperatures such as (47- 49), (50-52) and (53-55) °C was maintained. With the thermometer the desired temperature was denoted. Fixing temperature and time Thermostat bulb was regulated to fix the desired temperature. At the end of the treatment, seeds were taken out of the hot water bath and spread on blotter paper. After that the blotter paper with seeds was placed in shade for drying of seeds. Then, the seeds were used for test. Seed microflora (%) was observed by following standard Petri plate method as per the ISTA. The seeds of bell pepper were kept in Petri plates (50 seeds per replication). These plates with seeds incubated at 25°C temperatures for 14 days. Numbers of infected seed were observed and per cent incidence was observed by following the method:Seed microflora % = (No. of infected seed / Total no. of seed) x 100 Ungerminated seed (%) was also recorded in nursery by following formula: Ungerminated seed (%) = (Ungerminated seeds / Total number of seeds planted) x 100 Incidence of damping-off, virus attack and disease incidence was recorded in nursery and field under protected condition by using the following formula: Disease incidence (%) = (Number of diseased seedlings per plot / Total number of seedlings per plot) x 100 The statistical analysis of the data generated was done as per design of the experiment as suggested by Gomez and Gomez (1984). Results and Discussion Seed contamination with microflora (Alterneria spp., Curvuleria spp., Penicilium spp., Fusarium spp., Colletotrichum spp.) and total microflora was predominant in control group (10.00, 3.00, 12.00, 11.00, 7.00 and 43.00 %, respectively), whereas the least predominant microflora per cent (1.00, 1.00, 3.00, 2.00, 0.00 and 7.00, respectively) were recorded in seeds soaked at 53-55 °C for 60 min (T3t3). Seeds soaked at 50-52°C (T2t1/ T2t2/ T2t2) showed intermediate values between the two extremes. The temperatures and durations of hot water treatment resulted in significant reduction in the mean incidence of various fungi as the fungi did not tolerate the higher temperature range. Reported 50-52°C temperature for 1530 min. most suitable against important seed microflora in solanaceous crops like brinjal. Similarly Nega et al., (2003) reported that heating carrot seed to 54oC in water for 20 minutes completely eradicated A. dauci found that hot water treatment at 53oC for 10 to 30 min of carrot, cabbage, celery, parsley, and lamb’s lettuce seed controlled Alterneria dauci, A. radicina, A. alternata, and A. brassicicola. Hermansen et al., (1999) reported that heating carrot seed to 54oC in water for 20 min completely eradicated A. dauci. Temple et al., (2013) found treatment at 50oC for 20 significantly reduce incidence (Cladosporium spp., Fusarium Alternaria spp.) (Table 2 and 3). 1873 hot water min. can of fungus spp., and Int.J.Curr.Microbiol.App.Sci (2020) 9(5): 1871-1878 Treatment T1t1 T1t2 T1t3 T2t1 T2t2 T2t3 T3t1 T2t2 T3t3 T0 (control) Table.1 Treatment details Temperature (ºC) Time (min) 47-49 30 47-49 45 47-49 60 50-52 30 50-52 45 50-52 60 53-55 30 53-55 45 53-55 60 Untreated seeds Table.2 Effect of hot water seed treatment on seed microflora in bell pepper cv. Solan Bharpur Treatments Seed microflora (%) Alterneria Curvuleri Peniciliu Fusarium Colletotrichum Total Seed spp. a spp. m spp. spp. spp. microflora 7.00 2.00 9.00 8.00 4.00 30.00 T1t1 7.00 2.00 7.00 6.00 3.00 25.00 T1t2 6.00 2.00 5.00 5.00 3.00 21.00 T1t3 6.00 2.00 4.00 3.00 1.00 16.00 T2t1 4.00 1.00 4.00 4.00 1.00 14.00 T2t2 4.00 1.00 4.00 3.00 1.00 13.00 T2t3 4.00 1.00 4.00 3.00 0.00 12.00 T3t1 2.00 1.00 4.00 2.00 0.00 9.00 T3t2 1.00 1.00 3.00 2.00 0.00 7.00 T3t3 10.00 3.00 12.00 11.00 7.00 43.00 T0 1.07 NS 0.59 0.94 0.65 3.74 CD at 5% Table.3 Effect of hot water seed treatment on disease incidence in bell pepper cv. Solan Bharpur under nursery conditions Treatments Characters Ungerminated seeds (%) Damping–off (post emergence) (%) Virus (%) 31.11 54.84 15.55 T1t1 25.56 35.77 13.33 T1t2 17.78 25.53 10.00 T1t3 12.22 22.75 5.56 T2t1 23.33 23.16 4.44 T2t2 27.78 15.34 3.33 T2t3 26.67 18.07 3.33 T3t1 35.55 7.034 2.22 T3t2 41.11 5.47 1.11 T3t3 30.00 63.59 23.33 T0 3.65 7.00 0.86 CD at 5% 1874 Int.J.Curr.Microbiol.App.Sci (2020) 9(5): 1871-1878 Table.4 Effect of hot water seed treatment on disease incidence in bell pepper cv. Solan Bharpur under protected conditions Treatments T1D1 T1D2 T1D3 T2D1 T2D2 T2D3 T3D1 T3D2 T3D3 T0 CD at 5% Disease incidence (%) Anthracnose Cercospora Wilt leaf spot 23.18 38.89 0.00 19.79 27.78 0.00 11.27 11.11 0.00 9.59 16.67 0.00 10.63 11.11 0.00 7.59 5.56 0.00 7.62 5.56 0.00 4.97 0.00 0.00 3.16 0.00 0.00 24.23 44.44 5.56 0.30 19.55 NS Disease incidence and ungerminated seed (%) under nursery conditions Significantly higher ungerminated seeds (41.11%) were recorded, when seeds were soaked at 53-55 °C for 60 min (T3t3) and lowest proportion of ungerminated seeds (12.22%), when soaked at 50-52°C for 30 min (T2t1). Seedlings kept as control had intermediate proportion of ungerminated seeds (30.00%). In contrast, the per cent damping off (post-emergence) was highest in control groups (63.59 %) and lowest (5.47%) in seeds were soaked at 53-55°C for 60 min (T3t3). Seeds soaked at 50-52°C (T2t1/ T2t2/ T2t2) showed intermediate values between the two extremes. Similarly, the presence of virus (%) was shown to be highest in control (23.33 %) and lowest (1.11 %) in seeds soaked at 5355°C for 60 min (T3t3). In case of T2D1 (seeds soaked at 50-52 °C for 30 min) incidence recorded due to post emergence damping-off and virus (%) was 22.75 per cent and 5.56 per cent respectively which was significantly less as compared to control. Hot water seed treatment has been used to control many seed borne diseases by using Virus 27.78 22.22 16.67 11.11 11.11 5.56 5.56 5.56 5.56 44.44 20.46 temperature hot enough to kill organisms but not quite hot to kill the seed (Miller,2005). It is believed that hot water treatment may activate pathogenesis-related (PR) proteins. PR proteins coded by host plant genes are induced by pathogen infection or related situations, and are thought to play a major role in plant defence responses against a wide variety of pathogens Van Loon and Van Strien, 1999). Among the PR proteins, the most characterized enzymes are those of group 2 that have β-1, 3-glucanase activity (Kauffmann et al., 1987) and group 3 that have chitinase activity and both hydrolyze polymers of fungal cell walls and are, therefore, thought to be involved in the plant defence mechanism against fungal infection (Schlumbaum et al., 1986, Collinge et al., 1993). These enzymes were capable of inducing plant resistance against pathogen infection in transgenic plants which over express chitinase and β-1, 3-glucanase genes (Zhu et al., 1994; Jach et al., 1995). Hot water temperature at 50-53 °C has found as the optimum ranged for reducing various seed borne diseases including fungi, bacteria and viruses of vegetable crops and other crops by 1875 Int.J.Curr.Microbiol.App.Sci (2020) 9(5): 1871-1878 earlier workers (Nega et al., 2003; Nandini and Kulkarni, 2015). The use of hot water treatments to control seed-borne diseases is regarded as very efficient in destroying pathogens borne both, outside the testa, such as TSWV (Edmund and Pottorff, 2009) and inside the seed testa (Miller and Ivey, 2004;). Zitter et al., (1989) have reported that hot water seed treatment at 50-55 °C for 25-30 min. controlled viruses affecting tomato, such as the Tomato mosaic virus (TMV), Pepino mosaic virus (PMV), Tobacco mosaic virus (YMV) and Tomato spotted wilt virus (TSWV). Winter et al., (1997) compared hot water treatments (52 °C for 10 min.) of cereal seeds were with seed fungicides and found that it was equally effective in controlling damping-off disease. The mode of action of hot water seed treatment could be direct killing of seed borne inoculum in and on the seed. Disease incidence conditions under protected Hot water treatment of seeds has significant effect on disease incidence under protected condition (Table 4). Incidence (%) of antracnose, cercospora leaf spot, wilt and presence of virus was highest in control (24.23, 44.44, 5.56, and 44.44, respectively) and lowest in T3t3 (4.97, 0.00, 0.00 and 5.56, respectively). However, in the remaining treatment-time combinations, the values remained intermediate between two extremes. The probable reasons behind the reduced incidence of various diseases after hot water seed treatment were found to be the killing of seed borne inoculum of these pathogens due to increased temperature. Although, elimination of seed microflora was effective, seed quality i.e. percent ungerminated seeds deteriorated with higher temperature-time combinations. Muniz (2001) reported Hot water treatment as potent practice to neutralise the seed borne organisms but not quite enough to kill the seed. Similarly, Miller and Ivey (2004) and Miller and Ivey (2005) reported reduction in the occurrence of anthracnose, bacterial canker, bacterial spot, bacterial wilt and bacterial speck after treatment of seeds of tomato and bell pepper with hot water at 50 to 55°C for 25 to 30 minutes. Treatments of bell pepper seed with hot water at 45oC for 15 min or 53oC for 4 min prior to storage at 8oC reduced the incidence of fungal infections (Aguilar et al., 1998). This method is more eco-friendly and effective compared to chemical treatment, however, they can cause the loss of seed viability (Meah, 2004). Seed of okra (Abelmoschus esculentus) when treated with hot water at 52oC for 30 min. resulted in the improvement of crop, both in greenhouse and field conditions (Begum and Lokesh, 2012). Enotomo et al., (2002) described hot-water treatment as an alternative method to hypochlorite treatments for disinfecting pathogenic bacteria in seeds for alfalfa. 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Int.J.Curr.Microbiol.App.Sci. 9(05): 1871-1878. doi: https://doi.org/10.20546/ijcmas.2020.905.212 1878