1 CHAPTER-1 INTRODUCTION Capsicum (Capsicum annuum L. var. grossum Sendt), an important vegetable crop belongs to family Solanaceae. It is also known by common names such as bell pepper, sweet pepper, shimla mirch, and green pepper. It is considered as one of the important vegetable crop in the world. Broadly, on their level of pungency domesticated peppers could be classified into sweet and hot types. Sweet peppers differ from common hot peppers in size and shape of the fruits, capsaicin content and usage. They are usually called colored capsicum in India. Bell pepper is one of the cash crop vegetable cultivated in most parts of the world including European countries, Central and South America, tropical and subtropical regions of Asia i.e. India and China (Sreedhara et al., 2013). According to one believe, capsicum was originated in new world tropics and subtropics (Greenleaf, 1986), and in 19 th century, it was introduced by Britishers in Himachal Pradesh (Shimla) and Tamilnadu (Nilgiri Hills) (Greenleaf, 1986). However, capsicum is extensively produced in various states of India viz. Himachal Pradesh, Andhra Pradesh, Uttrakhand, Darjeeling district of West Bengal during summer months and in Karnataka, Maharashtra, Tamil Nadu and Uttar Pradesh as autumn crop (Singh et al., 1993; Sreedhara et al., 2013). China is the major producer of capsicum and contributes 36 per cent of the worlds cultivated area with a production of 12.53 million tonnes. In world ranking, India stands 4 th in capsicum production and has attained a status of high value crop in India in recent years. This includes annual production of 0.9 million tons from an area of 0.885 million hectare with a 1266 kg per hectare productivity (Anonymous, 2007). In Himachal Pradesh, it is cultivated both in open fields and protected environment in various agro climatic zones (I, II and III) and covers an area of 2,503 hectares with a production of 33,923 metric tonnes including hot pepper (Anonymous, 2009). Bell pepper contains high content of antioxidants such as ascorbic acid, beta carotene and flavonoids (quercetin and luteolin) having anti-cancerous properties (Villalon, 2002). Capsicum has been found to be rich in vitamin A (beta carotene) and C (ascorbic acid) as compare to tomatoes. One medium green bell pepper contains 177 percent of the Recommended Daily Allowance (RDA) for vitamin C, as they mature vitamin C content almost doubles while vitamin A content rises by a factor of nine. It has been observed from every 100 gm of edible capsicum 24 k cal energy, 1.3 gram of protein, 4.3 gram of carbohydrate and 0.3 gram of fat is provided (Rubio et al., 2010). Bell pepper is also loaded with various minerals like iron, magnesium, potassium, calcium, phosphorus,
2 sodium and selenium (Agarwal et al., 2007). The mature fruits of sweet pepper are produced in different colours (green, red and yellow). There are several uses of bell pepper viz., used as raw, cooked as vegetable, pickled or processed and used in bakings, stuffings, burger and pizza preparations. Due to heavy demand in urban areas, its consumption in India is increasing these days. Capsicum has great potential for export also, which require good quality fruit with various characteristics like medium size, longer shelf life, mild pungency and good taste (Brandt and Mabgaard, 2001). In India, there is high demand for this crop but there is less production due to various biotic (pest and diseases), abiotic (rainfall, temperature, relative humidity and light intensity) and crop factors (flower and fruit drop). Due to unreliable behavior of weather, the crops grown in open field are often exposed to fluctuating levels of temperature, humidity, wind flow etc. which ultimately affect the crop productivity adversely (Ochigbo and Harris, 1989). Globally, pests and diseases are main constraints for crop production. These can be categorized as viral, fungal, bacterial and nematodal. Plant viruses differ greatly from all other plant pathogens not only in size and shape, but also in simplicity of their chemical constitution and physical structure, methods of infection, multiplication, translocation within the host, dissemination, and the symptoms they produce on their host plants. Bell pepper is vulnerable to variety of diseases associated with viruses, fungi, bacteria, and phytomplasma (Singh, 2005). Among these, viral disease has become serious threat to capsicum as it was highly susceptible to viral diseases at all stages of development. These viruses transmit among host plants with the help of insect vectors. These viral diseases are difficult to diagnose by symptoms as there is much overlap in symptomatology. These symptoms difference can be attributed to many factors like age of the plant, environmental conditions (temperature, relative humidity), and host plant nutrition and strain differences in viral pathogens. Various viruses affecting capsicum includes aphid transmitted, thrips transmitted, nematodal transmitted and whitefly transmitted viruses. Thrips transmitted tospoviruses on capsicum are Tomato spotted wilt virus (TSWV), Groundnut bud necrosis virus (GBNV) and Capsicum chlorosis virus (CaCV) from family Bunyaviridae. The family Bunyaviridae includes animal-infecting viruses of the genera Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and the Tospovirus genus which is the only plant-infecting virus of the family (Whitfield et al., 2005; Pappu, 2008). Tomato spotted wilt virus (TSWV) was reported from almost all continents and is the type species of the plant viruses belonging to the genus Tospovirus within the arthropod-borne Bunyaviridae family (Van Regenmortel et al., 2000). It seems that disease caused by the TSWV was first observed in 1906 (Sakimura, 1962). The first characterization of
3 this virus as the causal agent of the disease was reported by Samuel et al. (1930), who gave it its current name Tomato spotted wilt virus. Since then it was reported in several tropical and temperate regions and it is considered worldwide in distribution. TSWV ranks among the top 10 of the most detrimental plant viruses worldwide and recently it was ranked second in the list (Prins and Goldbach, 1998; Scholthof et al., 2011). For a long time, the genus Tospovirus was thought to be consisted of only TSWV. However, Impatiens necrotic spot virus (INSV) discovered to be second virus of genus Tospovirus (Law and Moyer, 1990). It was followed by increasing number of different tospoviruses in different parts of the world. Till now, 23 distinct species have been discovered from different parts of the world, eight of them are considered as confirmed species by International Committee on Taxonomy of Viruses (ICTV) while rest 15 are considered as tentative (Fauquet et al., 2005; Plyusnin et al., 2011; King et al., 2012). But according to a recent report, list of tospoviruses have been increased to twenty eight (Charoenvilaisri et al., 2014). Out of 28, only five have been reported from India (Mandal et al., 2012). These include Groundnut bud necrosis virus (GBNV), Peanut yellow spot virus (PYSV), Iris yellow spot virus (IYSV), Watermelon bud necrosis virus (WBNV) and Capsicum chlorosis virus (CaCV). TSWV is one of the most economically important members of the genus Tospovirus (Peters and Goldbach, 1995; Moyer, 2000). Groundnut bud necrosis virus (GBNV) also known as Peanut bud necrosis virus (PBNV) as its disease was first described in peanut in 1968 (Reddy et al., 1968, 1992). It is the most ubiquitous virus among the five tospoviruses reported from the Indian subcontinent (Mandal et al., 2012). Capsicum chlorosis virus (CaCV) of Bunyaviridae family was first reported from Australia from Capsicum spp. (McMichael et al., 2002). It was a recently reported virus and very less information is available about the host range of this virus. Tospoviruses are enveloped viruses with 80-120 nm diameters. They are RNA viruses having tripartite genome consisting of three molecules of negative and ambisense RNA which are named as Large (L), medium (M) and small (S). RNA genome segment L encodes the RNA-dependent RNA polymerase (RdRP), M segment codes for two glycoproteins (Gn and Gc) and a non structural protein (NSm) and S segment codes for N protein and another non structural protein (NSs). Members of the Tospovirus genus are naturally transmitted by multiple species of thrips (Thysanoptera) in a circulative and propagative manner (Mound, 1996; Ullman et al., 1997; Jones 2005; Whitfield et al., 2005). Globally, tospoviruses cause significant losses in quality and yield from horticultural, agricultural crops and ornamental plants (Mumford et al., 1996; Pappu et al., 2009; Pearce, 2005; Persley et al., 2006). They can cause infection both in early and later stages
4 of crop growth leading to loss in yield and quality produce (Culbreath et al., 2003). Symptoms of tospovirus infection vary according to the developmental stage of the plant at the time of inoculation, the virus strain, plant age and environmental (growth) factors. Most plants respond to tospovirus infections with systemic symptoms. Symptoms vary with the host plant, time of year and environmental conditions and include severe stunting (groundnut), wilting, leaf distortion and top necrosis (tomato), chlorotic/necrotic ring/line pattern on leaves and plant death (lettuce), and tuber necrosis (potato). The symptoms on leaves and stems of infected crop plants include mosaic, mottle, ring spots and line patterns as well as wilting of leaves, leaf deformation, and stem and top necrosis (German et al., 1992; Mumford et al., 1996). The most striking symptoms of tospoviruses are found on fruits, e.g. tomatoes, which can be the only parts of the plant to show symptoms, especially when virus infections are introduced late in the crop cycle. Chlorotic and necrotic rings, blotches, fruit discoloration and deformation caused by various tospoviruses render affected fruits of tomato and pepper unmarketable. It has been observed that during the last few years due to direct and indirect human activities plant viruses are spreading worldwide like an epidemic causing a huge economic loss. Type member Tomato spotted wilt virus (TSWV) was responsible to cause one billion dollar loss annually. Economic and environmental damage can be prevented by proper diagnosis of tospoviruses and most important step in this regard is correct identification of these viruses in their host plants. This can be achieved by either visually or by using other tools (serological, molecular and arrays) available each with their particular advantages and disadvantages. For routine plant virus detection, serology based tests were preferred. The challenge with all types of tools is to develop a technique which is rapid, less time consuming and cheaper also. Recently, biosensor technology has taken a step forward in this direction. A novel technique Quartz Crystal Microbalance (Immunosensor) was used for the detection of plant viruses (Eun et al., 2002). It was first used for the detection of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). This technology was claimed to be rapid and economical as compare to ELISA. Immunosensor was used for the serological detection of proteins and antibodies in complex samples, e.g. infected plant samples, serum or blood. They are affinity biosensor based on the specific interaction between antigen and antibodies, this interaction could be monitored by several techniques including electrochemistry. Amperometric detection was one of the maximally used techniques in immunosensor research and development. This technique offer several advantages as compare to other methods, it is very simple to use, portable, fast analysis time and low cost of production (Holford et al., 2012). Huang et al. (2010) have developed disposable
5 electrochemical immunosensor for the detection of carcinoembryonic antigens. Several Immunosensors have also reported for Brucella (Wu et al., 2013). Biosensors based on antigenantibody reactions have an important application in agriculture sector (Vashpanov et al., 2008). Recently, there was a report showing use of cell biosensor for detection of plant viruses (Perdikanis et al., 2011). Immunosensor have also been used for the detection of Plum pox virus (PPV) and Prunus necrotic ringspot virus (PNRSV) affecting stone fruits and causing significant economic losses (Radecka et al., 2013). Amperometric biosensor has still not been used for detection of tospoviruses. Keeping the significance of tospovirus in world vegetable production and availability of tospovirus like symptoms on capsicum grown in various districts of Himachal Pradesh in mind present investigation was planned to identify the tospovirus causing disease in capsicum. The study was further taken to develop easy and more sensitive method for the detection of tospovirus infecting capsicum. Following objectives have been framed for this. 1.1 OBJECTIVES: 1. Survey of bell pepper grown in different districts of Himachal Pradesh for tospovirus infection. 2. Serological identification of tospoviruses in infected bell pepper plants. 3. Molecular identification and characterization of tospoviruses in infected bell pepper plants. 4. Development of immunosensor based efficient and sensitive method for tospovirus detection.