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Screening Cucumber Plant Introduction Accession Lines for Resistance against Cucumber Strain of Papaya ringspot virus (PRSV)

Cucumber is a popular fruit vegetable widely cultivated in Odukpani and Akamkpa local government areas of Cross River State, Nigeria, supplying the consumption needs of the nearby metropolitan Calabar City dwellers. However, its cultivation is threatened by infection by Papaya ringspot virus watermelon strain (PRSV-W). This study provides the result of screen house evaluation of USDA Plant Introduction (PI) accessions lines of cucumber for resistance to a Nigerian cucumber strain of PRSV-W. The response of the lines showed a spectrum of reactions to the virus strain from the extremely susceptible ones, that could not withstand the virus infection to the immune that were uninfected by the virus. Out of the 187 accession lines screened, 22 immune lines (PI 391570, PI 419010, PI 419017, PI 432873, PI 432877, PI 432878, PI 432895, PI 489752, PI 137845, PI 211979, PI 211984, PI 605924, PI 606010, PI 227207, PI 390253, PI 223437, PI 118279, PI 422200, PI 525152, PI 406473, PI 390952 and PI 357867) were identified. Besides, 17 others (PI 118807, PI 163217, PI 165509, PI 175121, PI 177364, PI 179263, PI 212985, PI 215589, PI 217644, PI 220791, PI 234517, PI 249562, PI 267746, PI 271326, PI 344384, PI 358813 and PI 358814) showed high tolerant and eight others were moderately tolerant to the virus. The remaining 140 PI lines were very susceptible to the virus. The identification of completely immune and highly tolerant PI accession cucumber lines in this study points the availability at the USDA germ plasm collections of potential materials that could be included in breeding programs to develop high resistant varieties of cucumber to PRSV-W.


INTRODUCTION
Cucumis sativus L. (Cucurbitaceae), commonly known as cucumber, is believed to have originated in India (Renner et al., 2007), where a great many varieties have been observed and has now been introduced to other parts of the world. It is cultivated for its fruits which are eaten fresh or used in the preparation of sandwiches and salads. It is also reported to be a rich source of phytonutrients such as vitamins B1, C, K and essential minerals (Abiodun and Adeleke, 2010). Cucumber varieties have also been reported to provide valuable antioxidant, anti-inflammatory and anti-cancer substances in the diet (Lee et al., 2010;Kumar et al., 2010;Nema et al., 2011).
Papaya ringspot virus (PRSV), previously described as Watermelon mosaic virus-1 (WMV-1), (Purcifull et al., 1984) is a potyvirus transmitted by a number of aphid species in a style-borne manner (Gonsalves and Ishii, 1980) and considered a major production constraint to papaya and cucurbit production worldwide (Gonsalves et al., 2010;Mohammed et al., 2012). Two biotypes are recognized, the papaya infecting biotype (PRSV-P) which infects papaya and cucurbits and the PRSV-W that infects only cucurbits (Tripathi et al., 2008). PRSV-W which has been reported to as a major limiting factor for watermelon production worldwide Strange et al., 2002), has been found to be transmitted in a stylet-borne manner by 24 aphid species in 15 genera with Myzus persicae, Aulacorthum solani, Aphis craccivora and Macrosiphum euphorbiae as natural vectors (Purcifull et al., 1984). Reports of the occurrence of PRSV in cucurbits from Africa abound. The PRSV-P has been reported on Cucurbita pepo in Egypt (Omar et al., 2011), on C. maxima in Sudan (Mohammed et al., 2012), in Carica papaya in Nigeria (Taylor, 2001) and Cote d'Ivoire Diallo et al. (2007) while the PRSV-W biotype has been reported in cucumber in Nigeria (Owolabi et al., 2008).
The cultivation of C. sativus has become a thriving business in Odukpani and Akamkpa local government areas of Cross River State, Nigeria, providing jobs for the local folks and meeting the consumption needs of the nearby metropolitan Calabar city dwellers. However, all the local varieties available for cultivation are susceptible to a PRSV-W strain isolated from cucumber in which it elicited mosaic, leaf malformation, rugosity, conspicuous green vein-banding and stunting (Owolabi et al., 2008).
Although, some of these approaches may make valuable contributions to virus disease management in specific favourable situations, they sometimes are of little practical benefits. Planting resistant varieties, in many cases, appears to offer a more practical option in controlling plant virus diseases. The aim of the present study was to evaluate 187 accession lines of C. sativus with a view to identifying resistant lines that could provide genetic material for breeding resistance against PRSV-W in Nigeria.  Int. J. Virol., 11 (2): 66-76, 2015 Maryland (USA). The accessions lines had their origin from Africa, Asia, Europe and the Americas (33 countries in all). Countries with most accessions included India with 40, China 25, Turkey 17, Japan 16 and Iran 13. Other had less than 10 or fewer.

PRSV virus isolate:
The PRSV-W strain used in the study was that described by Owolabi et al. (2008) and maintained on Cucumeropsis manni (Naudin) in the screen house.
Planting and inoculation procedure: Five seeds of each of the accession lines were sown in 20×16 cm polyethylene bags containing heat-sterilized garden soil. The cotyledonary leaves (8-day old seedlings), pre-dusted with 600-mesh carborundum were mechanically inoculated with the PRSV strain in 0.03 mM Na 2 HSO 4 , pH 8.00 inoculation buffer. Leaf to buffer ratio was 1:5 (1 g infected leaf to 5 mL of buffer). Three buffer-inoculated seedlings of each accession line served as controls.
The inoculated seedlings were promptly rinsed with water, kept in an insect-proof screen house at 27±°C in the University's Botanical Garden and left for symptom development for over a period of 21 days. Plants that did not show symptoms of infection were back-indexed on C. manni that had been reported to be readily susceptible to the virus (Owolabi et al., 2008) to check for latent infection.
Disease rating/determination of degree of susceptibility to PRSV: The germ plasm accession lines were evaluated against their response to the virus on a scale of 0-4 according to Bashir et al. (2002) with some modifications on the basis of viral symptoms where, 0 = immune (inoculated plants did not show symptoms and no virus recovered after back-indexing, 1 = highly tolerant (inoculated plant showed mild mottle and no apparent reduction in plant growth), 2 = moderately tolerant (inoculated plant showed mosaic, green vein-banding, leaves not malformed and no apparent reduction in leaf size), 3 = susceptible (stunting of inoculated plants, leaf malformed and accompanied by reduction in leaf size) and 4 = highly susceptible (apical necrosis leading growth cessation).

Reaction of accession lines to PRSV:
The reaction of the cucumber accession lines to PRSV inoculation showed a spectrum of responses from complete lack of symptom induction to severe disease reactions ( Fig. 1a-i and Table 1) and in some cases, growth cessation occasioned by shoot tip necrosis and eventual death of inoculated seedlings. Some of the symptoms observed included severe leaf malformation and reduction in leaf size ( Fig. 1a-e), rugosity (blistering), (Fig. 1f) mosaic and green-vein banding and (Fig. 1h) no symptom at all (Fig. 1i).

DISCUSSION
Cucumis sativus is an important fruit vegetative in Cross River, Nigeria. All available varieties under cultivation are readily susceptible to the PRSV-W strain, with concomitant poor growth. In this study, 187 USAD-ARS Plant Introductions of C. sativus var. sativus were screened against PRSV-W isolate from Nigeria for possible discovery of varieties that could provide materials for resistance breeding programme.
The results of this investigation showed a varying degree of reactions to the cucumber isolate of PRSV-W among Cucumis sativus var. sativus germ plasm collection from the USDA-ARS. A preponderance (about 67.02%) of the accession lines was susceptible to the virus. Similar observations have been made with respect to some cucurbits screened against viruses of economic importance. Strange et al. (2002) found that a greater majority of 1248 accession lines of watermelon were susceptible to PRSV-W. The report of Ling and Levi (2007)  and Yugoslavia (Slovania) (PI 357867). Completely immune and moderately resistance PI accession lines have been detected among some cucurbit germ plasm sourced from USDA. Strange et al. (2002) reported the existence of PRSV-W resistance in eight PI accessions: three accessions from South Africa (PI 244017, PI 244018 and PI 244019), in three accessions from Zimbabwe (PI 482342, PI 482318 and PI 482379), one accession from Botswana (PI 485583) and one accession from Nigeria (PI 595203) in watermelon germ plasm collection from the USDA. Ling and Levi (2007) also found that of the 190 Lagenaria siceraria PIs screened, 30 were completely immune to the Florida strain of ZYMV while Kousik et al. (2009) reported the existence of moderate resistance in two C. colocynthis (PI 386015 and PI 386024), a Praecitrullus fistulosus (PI 381749) and two C. lanatus var. lanatus PIs (PI 482266 and PI 392291 to Squash vein yellowing virus (SqVYV). On the other hand, Habib et al. (2007) reported that none of 254 lines of mungbean was found to be resistant to MYMV while 247 lines were highly susceptible to the virus.
The USDA watermelon and other cucurbit germ plasm collections have been extensively screened for resistance to PRSV-W and ZYMV. This is the first report of screening cucumber accessions line for resistance against PRSV-W.

CONCLUSION
The result of the present study demonstrates that there is significant genetic resistance to PRSV-W among USDA C. sativus var. sativus germ plasm collections. The twenty two accessions so identified are potential sources of genetic materials for cucumber breeding against the virus.