Prunus Necrotic Ringspot Virus in Peach

International Journal of Chemical Studies 2018; 6(3): 486-494

P-ISSN: 2349–8528 E-ISSN: 2321–4902 IJCS 2018; 6(3): 486-494 Prunus necrotic ringspot virus in peach- A bird’s © 2018 IJCS Received: 02-03-2018 eye view on detection and production of virus free Accepted: 04-04-2018 plants Shelly Kapoor Plant Virology Laboratory, Dr. Y S Parmar University of Shelly Kapoor, Anil Handa and Abhilasha Sharma Horticulture and Forestry, Nauni, Solan, (HP), India Abstract

Anil Handa Peaches are widely cultivated as fruit and ornamental crops all over the world. Virus diseases in peach Plant Virology Laboratory, however causes significant economic losses not only in terms of yield but also results in the production Dr. Y S Parmar University of of poor quality virus infected plants as the majority of the viruses infecting peach are graft transmissible Horticulture and Forestry, and are passed on to the budwood from infected mother trees. Members of the genus Ilarvirus Nauni, Solan, (HP), India infect peach trees and are distributed worldwide. Among these, prunus necrotic ringspot virus (PNRSV) is the most economically important and prevalent viruses causing significant losses in cultivated peach Abhilasha Sharma crop as the virus has a wide host range affecting all Prunus species hop and rose. Early information on Plant Virology Laboratory, sanitary status of peach tree health and detection of PNRSV can facilitate the control of prunus ringspot Dr. Y S Parmar University of disease through proper management strategies such as the use of virus free planting material produced by Horticulture and Forestry, regular serological and molecular indexing of mother trees by ELISA and RT-PCR. Additionally, use of Nauni, Solan, (HP), India biotechnological approaches such as in vitro micropropagation using axillary and terminal buds as explants. A proper well defined system devised for the purpose of raising PNRSV free peach trees using

advanced detection and in vitro techniques can lead to the development of a foolproof certification

programme. The present review recognizes the need for developing a rapid, cost-effective, and reliable health monitoring system that would facilitate advancements in peach industry. It describes the currently used serological, molecular and in vitro techniques that can be used for for this purpose. These technologies include the review compares the benefits and limitations of these potential methods for producing PNRSV free peach plants.

Keywords: Peach, PNRSV, ELISA, RT-PCR, in vitro techniques, virus free plants

1. Introduction

Graft transmissible pathogens (GTPs) including viruses, viroids and phytoplasma of peach trees incite over 20 different diseases, including some that result from mixed infections of different viruses. Agents associated with these GTPs most likely are also involved in other diseases of unknown etiology. Some of the diseases caused by these GTPs are economically important, particularly when they affect fruit quality or induce severe tree decline. On the other

hand, some viruses and viroids that produce conspicuous symptoms on specific peach cultivars or other Prunus species do not cause phenotypic alterations on other peach cultivars or hosts. Most peach viruses do not spread naturally, although some viruses may be spread by aphid and nematode vectors or through pollen. The propagation of infected sources of plant material and their exchange over long distances is the main cause of the distribution of viruses in the peach

industry worldwide. The use of symptomless infected trees for grafting purposes considerably contributes to the spread of viruses and related pathogens. Reliable techniques based on serological and molecular methods are available today for the detection of most peach viruses and related pathogens. These are gradually and successfully replacing the conventional biological indexing methods and are contributing directly to the control of the peach diseases

based on the exclusive use of virus free propagation material. Peach diseases, particularly virus and virus like diseases have a major impact on peach production worldwide and often become dominant yield limiting factor in orchard Correspondence management. Viral diseases are of vital importance and a number of viruses have been Shelly Kapoor reported by different workers from different peach growing areas of the world. As the Plant Virology Laboratory, Dr. Y S Parmar University of designation "latent" implies, these viruses are symptomless in most commercial cultivars, but Horticulture and Forestry, may cause symptoms in certain cultivars. A critical screening of the available literature on the Nauni, Solan, (HP), India occurrence of various plant viruses infecting peach under natural conditions in different parts ~ 486 ~ International Journal of Chemical Studies

of the world revealed that this crop is infected by about 12 the inoculum for infection. This peculiarity, together with the viruses and their strains. Majority of these viruses are apple characteristic lability of the virus particles in tissue extracts chlorotic leaf spot, cherry mottle leaf, peach rosette mosaic, delayed the development of knowledge on the molecular plum pox, plum american line pattern, prune dwarf, prunus biology of ilarviruses in comparison with other less necrotic ringspot, strawberry latent, and tobacco ringspot and economically relevant viruses. In the recent past, a large tomato ringspot representing 6 genera viz., Begomovirus, number of ilarviruses have been studied and critically Closterovirus, Ilarvirus, Nepovirus, Potyvirus, Trichovirus reviewed with particular reference to temperate fruits (Kapoor from bromoviridae, betaflexiviridae, closteroviridae, et al., 2018) [11]. Molecular characterization of ilarviruses comoviridae, geminiviridae and potyviridae families (Brunt et allowed initial approaches to understand several steps in the al., 1996) [1]. In addition to these viruses, peach has also been viral life cycle of this group of viruses. However, in most reported to be infected by some graft transmissible pathogens cases, these achievements were in the shadow of the research like hop stunt viroid from dapple fruit disease of plum and completed using ApMV. The enormous progress made with peach in Japan (Teruo et al., 1989) [2] and peach latent mosaic the ApMV model meant that some properties shared with viroid (Hassan et al., 2006) [3]. As pathogenic agents, viroids ilarviruses, such as genome expression, viral replication, and are limited in range of crop species they affect compared to genome activation, were addressed bearing in mind the need viruses (Mathews, 1992) [4]. PLMVd directly affects the peach for confirmatory research. fruit and leads to the development of symptoms like Out of the different ilarviruses that have been reported on retardation in the opening of buds from 4-5 days with peach plants from different parts of the world, some are well consequent delay in foliation, flowering and ripening and characterized (Pallas et al., 2012) [12]. ELISA is being colourless fruit with cracked structures which affects the routinely used in the indexing, certification and quarantine marketability of the produce. Phytoplasma are another class programme of different temperate fruit growing schemes of the major pathogens which are graft transmissible and there (NRSP-5(IR-2)-USA, EPPO, NAPPO, SEAPPO etc.) in are several reports of phytoplasma infecting stone fruits and developed countries for many years (Nemeth, 1986) [13]. With Candidatus Phytolasma prunorum is among the most the advent of nucleic acid based molecular detection common one in Europe associated with European stone fruit techniques there has been a shift in the use of these techniques yellows (ESFY) diseases (Anfoka and Fattash, 2004) [5]. for the characterization and detection of viruses and other Thakur et al., (1998) [6] reported peach yellow leaf roll disease pathogens. Print and spot capture PCR, semi-nested PCR and from Northwestern Himalayas of India whereas Blomquist PCR-ELISA are recently developed techniques used for the and Kirkpatrick (2002) [7] reported the same disease from detection and characterization of the ilarviruses (Cambra et USA. Among all ilarviruses associated with stone fruits, al., 1998; Candresse et al., 1998) [14, 15]. In case of mixed PNRSV is the most widely spread ilarvirus infecting all infections, multiplex PCR and non-isotopic molecular Prunus species around the globe. Since stone fruits stands hybridization has also been used (Saade et al., 2000; Menzel next to apples only in Himachal Pradesh in terms of economic et al., 2002) [16, 17]. According to EPPO certification schemes, returns, the role of PNRSV in commercial stone fruit PNRSV is of quarantine importance in most of the developed production becomes more relevant. The literature pertaining countries like Australia and New Zealand. The virus naturally to PNRSV in respect of geographic distribution, economic infects and cause disease not only in peach but also in all importance, transmission, host range, particle morphology, pome and stone fruits. Relevant literature pertaining to this serological and molecular detection and in vitro production of virus encompassing the information in relation to the present virus free plants is presented in this chapter. level of research with a special focus on molecular and biotechnological aspects has been reviewed and presented in Bromoviridae this chapter. Bromoviridae is known as one of the most important families of plant RNA viruses whose members are distributed Prunus Necrotic Ringspot Virus (PNRSV) throughout the world. It has a wide host range (more than Taxonomic position of Prunus necrotic ringspot virus 10000 species) and is the major cause of agronomically Family : Bromoviridae important diseases. The genome of the family Bromoviridae Genus : Illarvirus is composed of three segments of positive-sense, single- Species : Prunus necrotic ringspot virus stranded RNA of approximately 8kb. RNAs1 and 2 are Acronym : PNRSV monocistronic and encode the protein involved in virus Synonyms : European plum line pattern virus, replication, whereas RNA3 encodes the movement (MP) and hop B virus, red currant coat (CP) proteins, the latter of which was translated from the xnecrotic ringspot virus, rose vein bending virus, rose yellow subgenomic RNA4 (Ji and Ding, 2001; Shi et al., 2002) [8, 9]. vein mosaic virus and Some members of the family, mainly cucumoviruses and sour cherry necrotic ringspot virus some ilarviruses encode a fifth protein (P2b) that is located in RNA2 and is part of the c-terminal region of the P2 protein PNRSV was first reported in peach (Prunus persica L.) from this peptide is involved in silencing (Froncisco et al., 2008) USA by Cochran and Hutchins (1941) [18] and named by Allen [10]. (1941) [19]. It is a member of the subgroup 3 of the genus Ilarvirus in the family Bromoviridae (Bujarski et al., 2012) Ilarviruses [20]. The virus is also known by various names like European Ilarviruses are distributed worldwide and affect a large plum line pattern virus, hop B virus, red currant necrotic number of agronomically relevant crop species including fruit ringspot virus, rose vein bending virus, rose yellow vein trees, vegetables, and ornamentals. In the 1970s, it was mosaic virus and sour cherry necrotic ringspot virus (Fulton, recognized that ilarviruses, together with the closely related 1985) [21]. It has also been reported from many countries viz., ApMV, were unique among plant viruses due to their Jordan (Salem et al., 2003) [22], India (Kapoor and Handa requirement of the presence of a few molecules of the CP in 2017a; Kulshrestha et al., 2005) [23, 24], Egypt (Salam et al.,

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2007) [25], Croatia and Czech Republic (Sucha and some are very well characterized. However, in Indian Svobodova, 2010) [26], Poland (Cichal et al., 2011) [27], scenario, not much systematic work has been done on this Albania, Bosnia, Herzegovina, Montenegro, Sebia, Turkey, virus. Chandel et al., (2013) [39] observed that shoot growth, Ukraine (EPPO PQR, 2012) [28] and Brazil (Fajardo et al., fruit set, fruit weight, yield/tree and fruit ascorbic acid content 2015) [29]. In India, prunus necrotic ringspot virus has been of PNRSV infected peach trees were reduced in comparison reported in Begonia (Verma et al., 2002) [30] and Pelargonium to virus free plants. Kapoor and Handa (2017b) [40] found (Kulshrestha et al., 2005) [24]. The characteristic symptoms PNRSV to be transmissible through grafting on the basis of evoked by the prunus necrotic ringspot virus include brown direct DAS-ELISA conducted for the detection of PNRSV in lines, rings and leaf curling (Fulton 1970; Brunt et al., 1996; peach cv. July Elberta. Hammond, 2011) [31, 1, 32]. Almaraz et al., (2014) [33] made observations in commercial peach orchards in a four year Symptomatology period between 2008-2012 in Estado de Mexico and reported Regardless of the host, infection by PNRSV remain leaf damage in the form of yellow mottle, chlorotic rings, symptomless as it is a latent virus. The virus is however linear pattern and mosaic. The virus is transmitted mostly by characterized by a wide variety of symptoms that have been mechanical inoculation and grafting (Brunt et al., 1996; observed by a number of workers from different parts of the Fulton, 1970; Hammond 2011) [1, 31, 32] via seeds and pollen in world (Almaraz et al., 2014; Kapoor and Handa 2017 a and b; several natural hosts, including Prunus spp, hops and roses Fulton 1970; Brunt et al., 1996; Hammond 2011) [33, 23, 40, 31, 1, and in some experimental hosts like Cucurbita maxima (Card 32]. These include leaf damage in the form of yellow mottle, et al., 2007; Hammond, 2011) [34, 32]. Natural host range of the chlorotic rings, linear pattern and mosaic. chlorotic bright virus includes Prunus cerasus, Prunus persica, Rosa, Prunus yellow discolourations of the leaves in the form of blotching, domestica (plum), Prunus dulcis (almond), Humulus (hops) mottling, vein banding or yellowing, ringspots, shot holes, and Cucumis sativus (Brunt et al., 1996) [1]. Instance of line and oak leaf patterns seldom accompanied by evident natural infection of Rubus ellipticus (Yellow Himalyan deformation of the leaf blades. Raspberry) by PNRSV has been reported from India (Sharma et al., 1998) [35] and the experimental host range of PNRSV was found to be comprising of plants of more than 21 dicotyledonous families (Fulton, 1970[31]; Kapoor and Handa 2017a) [23].

Geographical distribution and economic importance A comprehensive description of PNRSV was made by Fulton (1970) [31] and a review of the virus was prepared by Hammond (2011) [32]. Turkey is one of the most important stone fruit suppliers as it produces around 1.3 million tonnes

[36] of stone fruits annually (Gumus et al., 2007) reported the Fig 1: Discolouration of petals in PNRSV infected peach tree occurrence and distribution of all the stone fruit viruses and viroids in commercial plantings of Prunus species in Western Anatolia province of Turkey and the studies concluded that PNRSV along with other viruses and viroids like PDV, PPV, ApMV, ACLSV and PLMVd were causing major losses to the growers of Prunus species. A total of 1732 specimens of stone fruits were tested and the overall infection level with these graft transmissible agents was found to be 30 percent with the PDV as the predominant one followed by PPV and PNRSV. PNRSV was the most common virus detected in peach and apricot trees grown throughout Algeria (Aouane, 2003) [37]. It is a common pathogen in commercial cultivars of peach and is economically important because considerable losses have been reported in peach due to its infection. Scott [38] Fig 2: Shot holes in leaves of PNRSV infected peach (2014) studied the effect of prunus necrotic ringspot virus on the growth, yield and quality of peach and found a In sweet cherry trees, PNRSV results in the development of reduction in tree growth between 12 to 70 percent and yield chlorotic ringspots that turn dark brown necrotic areas in both loss of 5 to 70 percent with fruits having lower soluble sugar secondary veins and interveinal regions of the leaf (Sanchez content. PNRSV has also been reported to cause significant et al., 2004) [41]. Smith et al., (1988) [42] also observed crop losses depending on the host with 15 percent yield loss chlorotic and necrotic spots on the leaves of sweet cherry in sweet cherry and upto 100 percent in peach. PNRSV can trees infected by PNRSV but the centers of these necrotic reduce bud-take in nurseries, decrease growth of fruit from 10 spots often disappeared leading to shot hole-effect. The to 30 percent and fruit yield reduction from 20 to 60 percent [38] [12] presence of PNRSV was also reported by (Scott, 2014) with delayed fruit maturity (Pallas et al., 2012) . who found the infection initially causing shock and later developing chronic symptoms in most woody hosts. The Status of PNRSV in India symptoms of PNRSV on apricot trees were in the form of In India, some of the virus and related pathogens of peach mosaic spots on leaves and distribution of symptoms was have been detected on the basis of symptomatology, observed to be very erratic on the tree, often showing on a transmission, serology and molecular studies. Presently, few branches only. Considerable stunting and decline were PNRSV has been reported from several parts of the world and also observed, as well the necrosis of many buds causing ~ 488 ~ International Journal of Chemical Studies

stripping and scarce renewal of branches (Aouane, 2003) [37]. fruits, PNRSV bearing pollen must be deposited onto plant In the same studies, symptoms on peach included a scattered surfaces on which thrips were also occurring. The studies mosaic pattern with yellowing of leaf blade and large yellow concluded that this could probably be a means of PNRSV and creamy spots were also observed on a large proportions of transmission to new trees and cause infection. Prunus necrotic the tree canopy. ringspot virus transmission takes place in nature during flowering and pollination and is thought to be transmitted through pollens and seeds although the exact mechanism is unknown (Amari et al., 2004) [46]. The vertical transmission of PNRSV by gametes in apricot to check the intensity of virus on this crop was also studied by using dot blot hybridization technique to confirm the virus and the presence of PNRSV outside as well as inside the apricot pollen grains.

Partical Morphology and Host Range of PNRSV Virions are isometric, non-enveloped, 23-27 nm in diameter, Fig 3: Creamy yellow spots and shot holes in plum rounded in profile and without a conspicuous capsomere arrangement (Brunt et al., 1996) [1]. RNAs are encapsidated in icosahedral particles of 22-30 nm in diameter (Fulton 1970, Bujarski et al., 2012) [31, 20]. PNRSV isolated from rugose mosaic showing trees could be separated into three serotypes (Mink et al., 1987) [47]. Analysis of PNRSV isolates from rose indicated that the most frequent serotype in rose was different from that in Prunus suggesting that there may exist some

level of host adaptation or barrier to interspecific transmission Fig 4: Irregular necrotic spots and shot holes on cherry leaves (Moury et al., 2001) [48].

Transmission and host range Diagnosis and detection The virus is not transmissible through insect vector but is Serological detection (protein based detection) transmitted by pollen, vegetative propagation from infected Although many variants of agar gel immunodiffusion tests trees or by mechanical sap inoculation to herbaceous hosts [1, 31, 32] were commonly used for detection of serological relationships (Brunt et al., 1996; Fulton, 1970; Hammond 2011) , among ilarviruses (Casper 1973; Mink et al., 1987; Crosslin through seeds and pollen in several natural hosts, including and Mink 1992; Kapoor and Handa 2017 a and b) [49, 47, 50, 23, Prunus spp, hops and roses and in some experimental hosts 40] sometimes they failed to detect these viruses in infected like Cucurbita maxima (Card et al., 2007; Hammond, 2011) [33, 32] plants due to very low concentrations in infected woody . PNRSV has a rather limited natural host range plants or the presence of inhibitors (Thomas, 1980) [51]. comprising of a number of Prunus species Including all the Despite all these problems, serological reactions among cultivated species, hops (Humulus species) and rose (Fulton, [31, 32] ilarviruses could be easily confirmed by DAS-ELISA. A 1970; Hammond 2011) . In addition, an instance of preliminary survey was conducted in Tunisia to identify stone natural infection of Rubus ellipticus (Yellow Himalayan fruit virus diseases occurring in orchards and mother block raspberry) by PNRSV has been reported in India (Sharma et [35] stands. Two ilarviruses namely prunus necrotic ringspot virus al., 1998) . The experimental host range of PNRSV (PNRSV) and prune dwarf virus (PDV) were detected by includes plants of more than twenty one dicotyledonous [52] [31] [43] DAS-ELISA (Boulila and Marrakchi, 2001) . families (Fulton, 1970) . Yuan et al., (1990) studied the transmission of PNRSV via Criconemella xenoplax handpicked from the root zone of infected peach trees. The work concluded that Criconemella xenoplax failed to transmit the virus to cucumber or peach seedlings as rootstocks remained symptomless and ELISA also showed negative results for these. Greber et al., (1991) [44] tested the transmission of PNRSV to cucumber and peach seedlings using thrips as vector. Iinfected plum pollens were dusted onto cucumber and peach seedlings and the plants were caged with 8-10 thrips on each of them for 24 hours. Fifty six percent of transmission rate on to both seedlings with thrips tabaci and 66 percent with a

mixture of five thrip species were recorded when pollens were taken from highly infected flower buds whereas only 7 Fig 5: DAS- ELISA detection of PNRSV in peach flowers percent transmission rate was reported when pollen was taken from flowers with less infectivity. Hence, high rate of To evaluate the effect of PNRSV and PDV on peach, Scott infection in pollen was observed to be very important for the and Bachman (2001) [53] conducted field trials in different transmission of virus to the plants. parts of the world and named the disease caused by combined Transmission of PNRSV through thrips and virus-bearing effect of PNRSV and PDV as peach stunt disease. The pollen has been reported by Milne and Walter (2003) [45] when integrity of the viral treatments was assessed using ELISA. thrips and pollens were placed together onto the tested plants. Salem et al., (2003) [22] inspected the level of PNRSV It was also observed that for transmission to occur on stone infection in almond, peach and plum cultivars over the course

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of entire year by testing different plant parts of naturally higher than the longer (785 base pair) product and it was infected trees using the double antibody sandwich-enzyme concluded that PNRSV was detected better in plant tissues linked immunosorbent assay (DAS-ELISA). The studies with low virus concentrations in the dormant trees using RT- concluded that spring was the best time of year for PNRSV PCR. detection in flowers, active growing buds and young leaves. Real-time fluorescent reverse-transcriptase polymerase chain Similar results were reported by Kapoor and Handa (2017a) reaction (RT-PCR) assay using a short fluorogenic 3’ minor [23] on peach cv. July Elberta. groove binder DNA hydrolysis probe for the detection of Serological reactions among 19 prunus necrotic ringspot virus PNRSV in stone fruit trees was developed by Morbot et al., (PNRSV) isolates originating from different plants was (2003) [61]. Real-time PCR assay results were correlated with studied by Szyndel et al., (2006) [54]. The symptoms on conventional RT-PCR results by using bark tissues of PNRSV infected plants ranged from none or very mild dormant cherry and plum trees. The experiment reveled the (mosaic, ringspots and line patterns) to severe (necrosis and method to be the best as it eliminates risk of contamination by rugosity) including a reduction in fruit yield and quality. performing the whole test in a single closed tube and this DAS-ELISA was found capable of detecting PNRSV in plants system is also capable of replacing the common diagnostic with these type of symptoms thereby proving it to be the best techniques like woody indicators and immunological tests to detection method among all methods because concentration of detect PNRSV in test samples. Torre-Almaraz et al., (2008) virus does not affect its sensitivity as plant viruses with very [55] surveyed three locations of commercial peach orchards in mild symptoms were also easily detected. Almaraz et al., Mexico on the basis of symptoms after performing DAS- (2008) [55] reported PNRSV for the first time in peach located ELISA. The virus was further inoculated onto Chenopodum in the temperate regions of Mexico during surveys conducted quinoa, C.amaranticolor, Nicotiana tabacum, N. glutinosa in the commercial peach orchards during 2006 and observed and Datura stramonium and RT-PCR was performed using folliar symptoms on the infected trees. Samples (flowers, these indicator plants. An expected size amplicon of young shoot tips and leaves) were collected from 120 such approximate 450bp was generated from all these test plants. symptomatic trees and the virus was detected successfully in In a detailed study conducted by Yu et al., (2013) [62] in all plant parts using DAS-ELISA. China, a total of 505 peach leaf samples were brought to the Presence of plum pox virus, prunus necrotic ringspot virus laboratory and tested using RT-PCR. The presence of nine and prune dwarf virus particles from the infected plum major viruses infecting peach was revealed in RT-PCR. The (Prunus domestica L.) shoots and leaves using DAS-ELISA results varied for each virus in context of concentration of the were detected by Jakab-Iiyefalvi et al., (2011) [56]. DAS- infection they were causing in leaves with infection rates of ELISA was found to be a reliable and quick method for the different viruses as follows: ACLSV (71.8 percent), PNRSV detection of viruses in plum. Kilic et al., (2016) [57] carried out (20.2 percent), cherry green ring mottle virus (8.1 percent) serological detection of Prunus necrotic ringspot virus on rose and apricot pseudo-chlorotic leaf spot virus (1.6) percent. The in Turkey using DAS-ELISA and concluded that DAS-ELISA overall average of virus infection was found to be 24.6 is one of the most reliable methods for the detection of percent though apple mosaic virus, plum pox virus, prune PNRSV. dwarf virus, cherry virus A and cherry leaf roll virus were not Chandel et al., (2013) [39] characterized PNRSV infecting present in the peach trees. Chandel et al., (2013) [39] studied stone and pome fruits in India at serological and molecular the diversity of PNRSV on the basis of coat protein gene level. Different stone fruits such as almond, apple, cherry, using bioinformatics tools such as ClastalW, DNA Data Bank nectarine, peach, plum and wild cherry were tested and the of Japan, MultiAlin and Recombination Detection results concluded that DAS-ELISA is the best detection programme. PNRSV was detected in plum, peach, cherry, method as PNRSV was found in all of these stone fruits. almond, nectarine, wild cherry and apple. It was also found Sanchez-Perez et al., (2017) [58] investigated the status of sour that the isolates showed identity levels from 82-100 percent and duke cherry genetic resources in the Iberian Peninsula for with previously reported isolates from other countries. the presence of PNRSV using DAS-ELISA as the detection method and reported a high infection rate of 46 percent in the Micropropagation of peach leaf samples. Similar type of serological surveys conducted Tissue culture techniques have long been used as alternative by Kapoor and Handa (2017a) [23] in peach, almond, cherry, propagation methods for raising disease free plants (Martinez- plum, nectarine and apricot resulted the presence of PNRSV Gomez et al., 2005) [63]. In vitro protocols have a history of in all the hosts tested except for apricot and plum. over half a century of their use for this purpose and dates back to as early as 1960. Micropropagation is a technique which Molecular Characterization (Nucleotide based detection) allows the production of large number of plants from small The relationship of PNRSV in peach with other ilarviruses explants in relatively short period of time and limited space. was studied by Guo et al., (1995) [59]. The RNA3 of PNRSV With the realization of its advantages and unprecedented was cloned and its entire sequence was determined. The applications in producing virus free plants, this technique has studies concluded that RNA3 consisted of 1943 nucleotides received great attention all over the world. Micropropagation and possesses two large ORF’s. PNRSV RNA3 showed a of peach is well documented and a number of tissue culture high degree of similarity with those of tobacco streak virus protocols have been developed for many peach cultivars (TSV), prune dwarf virus, apple mosaic virus and additionally (Hammerschlag et al., 1985; Ahmad et al., 2003; Alsalihy et some motifs showed similarity with alfalfa mosaic virus as al., 2004; Couto et al., 2004; Sotiropoulos and Fotopoulos, well. 2005; Alanagh et al., 2010) [64-69]. Beside, cell culture Reverse transcriptase polymerase chain reaction (RT-PCR) technique has also been developed to regenerate peach plants. for the detection of PNRSV in dormant peach and almond The technique has however failed to get positive response in trees by the application of two different pairs of primers many cultivars and rootstocks of peach particularly during yielded a short and a long product (Rosner et al., 1997) [60]. multiplication and in vitro rooting (Hammerschlag et al., The relative amount of the short (200 base pair) product was 1985) [64].

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Acclimatization High mortality is often considered to be a major limitation in large scale application of micropropagation technology faced by in vitro raised plants in laboratory and subsequently transferred to field (Poole and Conover, 1983) [83]. Tissue cultured plants are susceptible to transplantation shocks leading to high mortality in the final stage of micropropagation (Dhawan and Bhojwani, 1986) [84]. Transferring micropropagated plants to the field conditions often expose the plants to altered temperature, light intensity and water stress conditions and these factors play a crucial role in initial stages and need acclimatization for successful establishment and survival (Pospisilova et al., 1999; Chandra et al., 2010) [85, 86]. Quality of the in vitro raised plants depend largely on the successful acclimatization in commercial micropropagation units and determines the economic viability of these production houses (Conner and Thomas, 1982) [87].

Control

Fig 7: in vitro culture of peach The use of certified virus-tested planting material is the preferred strategy for protection from viruses in plants. Explant Meristem culture and heat therapy can be used to eliminate A wide variety of explants like embryo, cotyledon, shoot tips the viruses from propagation material of rose infected with and axillary buds have been used for in vitro propagation of PNRSV for the production of virus free plantlets. Heat peach. Success of micropropagation largely depends upon the treatment of potted plants of rose infected with PNRSV at 0 type of explant, its size, source and physiological age and 38 C for three weeks resulted in complete virus elimination as [88] these factors have great influence on morphological activity indicated by ELISA assays (Chahardehi et al., 2016) . 0 and degree of differentiation (Ozaslan and Aytekin, 2005; Complete virus inhibition was also recorded after a 22 C hot Isikalan et al., 201) [70, 71]. Nodal stem explants have been water treatment of infected budwood for 30 minutes. [89] found to be an excellent explant source for inducing direct Cieslinska (2007) used thermotherapy for producing plum 0 organogenesis Ozaslan et al., (2005) [70]. rootstock myrobalan by keeping the plants for a week at 28 C 0 temperature and further keeping at 36̊ C for four weeks. For Axillary bud initiation and multiplication chemotherapy, antiviral compound used was virazole at 10, -1 The most common practice used to produce virus free elite 25, 50 and 100 mgl applied for four weeks after initiation of [90] plants in temperate fruits is the use of meristem culture or shoot tips. Kudelkova et al., (2017) used ribavirin and axillary buds as it ensures genetic stability of clones. Apical acyclovir as antiviral for producing virus free peach plants 25 -1 and axillary shoots are known to bear active meristems. Such and 50 mgl concentration were used in MS medium. Shoots observations were made up by many workers (Gomez and were tested after two months using ELISA and PCR and the Segura 1995; Rahman and Bhadra 2011) [72, 73] who have products were separated electrophoretically on 1.5 percent found good response in micropropagation of peach by using agarose gel. The use of acyclovir was found to be more nodal stem explant. effective than ribavirin. Shoot initiation is a pre-requisite for micropropagation of plants and cytokinins are important for shoot initiation and References multiplication. The success of initiation and multiplication of 1. Brunt AA, Crabtree K, Dallwitz MJ, Gibbs AJ, Watson explants largely depend upon types of cytokinins and their L, Zurcher EJ. Apple mosaic Ilarvirus. Plant Viruses concentrations used for different varieties and even tissues Online: Descriptions and Lists from the VIDE Database, and organs of plants. There are suitable culture media and 1996. concentrations of plant growth regulators for peach explants 2. Teruo S, Hataya T, Terai Y, Shikata E. Hop stunt viroid (Yeh-jin et al., 2007; Casanova et al., 2008; Kakani et al., strains from Dapple fruit disease of plum and peach in 2009) [74-76]. Cytokinins, particularly BAP, are known to Japan. Journal of General Virology. 1989; 70:1311-1319. overcome apical dominance and help in proliferation of lateral 3. Hassan M, Rysanek P, Malfitano M, Alioto D. First buds to promote shoot formation (George, 1993) [77]. Couto et report of peach latent mosaic viroid infecting peach in al., (2004) [67] obtained maximum number of shoots per Egypt. Plant Disease in press, 2007. explant by using vegetative buds from nodal explants in case 4. Mathews REF. Fundamentals of Plant Virology. San of in vitro propagation of peach. Diego: Academic Press, Inc, 1992. 5. Anfoka GH, Fattash I. Detection and identification of Root induction aster yellows (16Sr1) phytoplasma in peach trees in Prunus species Have been reported by number of workers to Jordan by RFLP analysis of PCR-amplified products have a significantly positive correlation with Indole-3-butyric (16srDNAs). Journal of Phytopathology. 2004; 152:210- acid (IBA) at concentration of 1–5 mg/l for stimulating 214. rooting percentage, root length and root number per shoot 6. Thakur PD, Handa A, Chawfla SC, Krczal G. Outbreak (Ahmad et al., 2003; Alsalihy et al., 2004; Alanagh et al., of a phytoplasma disease in peach in the Northwestern 2010; Espinosa et al., 2006; Mansseri-Lamrioui et al., 2011; Himalayas of India. International Society for Deepa et al., 2011, Naghmouchi et al., 2008; Ndoye et al., Horticultural Science. 1998; 17:472-498. 2003) [65, 66, 69, 78-82]. ~ 491 ~ International Journal of Chemical Studies

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