SPH -JHS Coverpage December 2019 Number 2 130 J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 Original Research Paper Correlation of Leaf Parameters with Incidence of Papaya Ring Spot Virus in Cultivated Papaya and its Wild Relatives Linta Vincent1, Soorianathasundaram K.2 and Shivashankara K.S.1 1ICAR-Indian Institute of Horticultural Research, Bengaluru, India, 2Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, India, Corresponding Author: linta.vincent@icar.gov.in ABSRACT Papaya ring spot virus (PRSV) disease has been the major impediment in papaya cultivation. The disease is transmitted through three aphid vectors and field tolerance towards this disease varies among Carica papaya cultivars as well as within the Vasconcellea genus. Leaf morphological traits are known to have influence on the probing preferences of aphids. Hence, this study was conducted to know whether the leaf parameters could contribute to the incidence of PRSV possibly by influencing the probing or feeding behaviour of aphid vectors. Leaf parameters viz., leaf thickness, leaf epicuticular wax content, presence and type of trichomes, trichome density were correlated with disease incidence at field conditions. The result revealed that leaf thickness along with epicuticular wax content had significant negative correlation with disease incidence. Similarly, trichome density had negative impact on disease incidence at 99.92% significance level. High epicuticular wax content and high trichome density in V. cauliflora and V. cundinamarcensis were found to be negatively associated with low to very low infection indicating that these parameters may have limited the vector transmission significantly. Keywords: Epicuticular wax content, Papaya, Trichome density and Vasconcellea. INTRODUCTION Papaya is ranked as the third most traded tropical fruit (excluding bananas). The area and production of papaya is on the increase in recent years due to its wide ecological adaptability, easiness in cultivation, high palatability, early fruiting, year round bearing, higher productivity and economic returns. Papaya is a nutrition basket filled with vitamins (2020 IU of vitamin A, 40 mg of vitamin B1 and46 mg ofvitamin C per 100g of fruit), minerals, carbohydrates, proteins, iron, calcium and phosphorous (Dinesh, 2010). However, the production of papaya is hampered by a serious outbreak of viral disease caused by Papaya r ing spot vir us (PRSV-P). T his vir us a ffects pr oduction a nd pr oductivity by decr ea sing photosynthetic capacity of plant, and subsequently leading to stunted growth, deformed and inedible fruitsand early mortality. PRSV is transmitted by several species of aphids in a non-persistent manner. Gener a lly, aphids do not colonize pa pa ya and transmission of PRSV is through transient aphid vectors.Aphis gossypii is the predominant vector followed by A. craccivora and Myzus persicae. Recent study suggested that M. persicae is more efficient than the other two species with 52.5 % transmission after the first inoculation access period (IAP) (Kalleswaraswamy and Krishnakumar, 2008). Field tolerance towards PRSV varies between the Carica papaya varieties as well as the Vasconcellea species. Vasconcellea species viz. , V. cundinamarcensis,V. candicans, V. stipulata, V. cauliflora andV. quercifolia are repor ted to be resistant to PRSV. Even though the genetic variability within the Carica genus for PRSV resistance is very low, a few varieties have shown tolerance to PRSV. This might be due to the non-preference of aphids to these varieties or species in addition to the innate resistance mechanism in Vasconcellea genepool. Morphological traits such as higher density of simple and glandular trichomes, epicuticular waxes and leaf thickness are reported to hinder the aphid attack in plants (Bin, 1979; Guer rieri and Digilo, 2008; 131 Correlation of Leaf Parameters with Incidence of Papaya Ring Spot Virus Wojcicka, 2015). A preliminary study was carried out to explore the possibility of aphid tolerance in papaya varieties as well as Vasconcellea species. MATERIALS AND METHODS Plant material The experiment was carried outat ICAR- Indian Institute of Horticultural Research, Bengaluru during the period of June 2016 to August 2017 under open field conditions consecutively in two different locations during kharif and rabi seasons.The experiment plot surrounded by papaya crop with already established with PRSV incidence was selected. Six cultivars of Carica papaya namely Arka Surya, Arka Prabhath, Red Lady, Pusa Dwarf, Pusa Nanha and CO8, two intergeneric hybrids of Arka Surya and V. cauliflora (IGHI a nd IGHII) a nd four wild r ela tives V. cauliflora, V. goudotiana, V. cundinamarcensis and V. parviflora were used in this study. Twenty plants of each accession in three replicates were maintained as per the randomized block design. Observations Characters such as leaf thickness, leaf pubescence, epicuticular wax content and trichome density were recorded after three months of transplanting in the field, since symptoms a ppea r ed in susceptible genotypes after three months of transplanting. Leaf thickness was measured using Digital Vernier Caliper (Mitutoyo, Digimatic Caliper). Presence of leaf pubescence was visually recorded while the type of the pubescence wa s obser ved under ster eo- microscope (Leica M205A) and Scanning Electron Microscope (Hita chi, T M3030 plus, Ta bletop microscope). Epicuticular wax content was estimated as described by Ebercon et al. (1977). Trichome density was calculated as number of trichomes per centimetre area. These observations were correlated with per cent infection (PI) and per cent disease index (PDI). PDI was calculated after first symptom development in field condition at fortnightly intervals and calculated as per the formula. PDI = Where, n = individual ratings, N= total number of leaves/ plant; 5= maximum rating The individual ratings (n) were given using the scale adopted by Dhanam (2006) and ranged from 0 to 5 (0 = no disease symptoms; 1 = slight mosaic on leaves; 2 = mosaic patches and/or necrotic spots on leaves; 3 = leaves near apical meristem deformed slightly, yellow, and reduced in size; 4 = apical meristem with mosaic and deformation; 5 = extensive mosaic and serious deformation of leaves, or plant dead). RESULTS AND DISCUSSION Leaf thicknessat three months of transplanting ranged from 0.22 mm to 0.40 mm. The thickest leaf was observed in Pusa Nanha (0.40 mm) which was on par with V. cundinamarcensis (0. 38 mm) a nd V. cauliflora (0.37 mm), followed by V. parviflora (0.35 mm). Among the accessions, thinnest leaves were noticed in TNAU papaya CO8, Arka Parbhath and IGH2 (0.22 mm) which was on par with IGH1 and Arka Surya (0.23 mm). The accessions such as Arka Surya, Arka Prabhath, Red Lady, Pusa Dwarf, Pusa Nanha, TNAU Papaya CO8, IGH1, IGH2 and V. goudotiana lack leaf pubescence on both dorsal and ventral surfaces (Fig.1). However, the wild species viz., V. cauliflora, V. cundinamarcensis and V. goudotiana had leaf pubescence with higher trichome density on ventral surface than dorsal surface. Trichome density was highest in V. cundinamarcensis (192. 75/cm2) followed by V. cauliflora (25. 25/cm2) a nd V. goudotiana (14.88/cm2). The type of trichome on the leaves were also observed under sca nning electr on micr oscope. V. cundinamarcensis consisted of single celled non- glandular trichomes, whereas V. cauliflora and V. goudotiana comprised of multicellular glandular tr ichomes (Fig. 2). Tr ichomes wer e pr esent a s extension of veins in V. cauliflora and V. goudotiana, while these were distributed throughout the leaf surface in V. cundinamarcensis. Studies suggested that trichome density has more impact on entry of aphids rather than the type of trichomes, as higher trichome density blocked aphids (Musetti and Neal, 1997). It is the first feature affecting the selection behaviour of an aphid. Most of the r esista nt va r ieties or wild r ela tives ar e characterized by presence of trichomes (Bin, 1979). However, the gla ndula r tr ichomes might ha ve produced toxic exudates or acyl sugars that repel aphids (Goffreda et al., 1989). J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 132 Linta Vincent et al Table.1. Leaf thickness, leaf epicuticular wax content, trichome density, PRSV percentage infection, disease intensity score and PDI at field condition Accessions Leaf Leaf Trichome Per cent Disease Per cent thickness epicuticular density infection intensity Disease (mm) wax content (number/cm2) (%) score index (%) (g/cm2) Arka Surya 0.23de 95.00ef Nil 100.00a 4/5 65.71a (89.71) (54.16) Arka Prabhath 0.22e 109.38de Nil 100.00a 4/5 61.25b (89.71) (51.51) Red Lady 0.27cd 143.75c Nil 100.00a 4/5 51.04c (89.71) (45.60) Pusa Dwarf 0.29c 134.38c Nil 100.00a 4/5 62.50ab (89.71) (52.25) Pusa Nanha 0.40a 114.38d Nil 25.33e 1 1.56f (29.77) (9.34) TNAU Papaya CO 8 0.22e 96.25ef Nil 100.00a 3/4 35.64d (89.71) (36.65) IGH1 0.23de 94.38f Nil 81.33d 3/4 21.46e (64.66) (27.59) IGH2 0.22e 106.25def Nil 86.67c 3/4 25.24e (68.60) (28.24) V. goudotiana 0.26cde 114.38d Nil 87.66b 4 25.00e (70.35) (29.97) V. cauliflora 0.37ab 200.00a 25.25b 0.00f 0 0.00g (0.286) (0.286) V. cundinamarcensis 0.38ab 170.00b 192.75a 0.00f 0 0.00g (0.286) (0.286) V. parviflora 0.35b 105.00def 14.88b 23.62e 1 2.46f (29.32) (9.02) Mean 0.29 123.59 77.63 67.05 29.32 (59.32) (28.74) CV (%) 9.96 3.32 3.17 1.61 5.02 SE(d) 0.012 3.353 1.231 0.78 1.18 Tukey HSD at 1% 0.045 14.667 4.26 1.73 1.91 Values inparentheses are arc sine transformed values A correlation was drawn between the leaf parameters and disease scoring (Table.2). Epicuticular wax content in leaves was negatively and significantly correlated with percentage of infection. Epicuticular waxes are complex mixture of long chain aliphatic and cyclic components such as fatty acids, hydrocarbons, alcohols, aldehydes, ketones, esters, terpenoids, sterols, flavanoids and phenolic substances. Higher epicuticular wax content might be a reason for reduction in aphid landing or movement, which in turn could have contributed, to inhibition of the sap transmission of PRSV. Similar negative effects of leaf epicuticular waxes were reported on neonate larval movement of Spodoptera frugiperda on Zea mays (Ostrand et al., 2008), resistance in cabbage to aphids Bravicoryne brassicae, sor ghum to gr een bug (Schizaphis graminum) and winter wheat to English grain aphid (Sitobion avenae) (Shepherd et al., J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 133 1999). The presence of thick wax content might have affected the probing and feeding by aphids, thereby rejecting the particular variety or species. Leaf thickness was negatively correlated with PDI a nd infection per centa ge. Lea f thickness wa s positively contributed by the epicuticular wax content, which indirectly influences the disease tolerance or resistance. Thick cell wall has been attributed to resist the feeding activity of aphids (Guerrieri and Digilo, 2008). The association analysis reveals that there is a significant negative correlation between trichome density and infection percentage. Surface resistance is the first barrier against aphid attack (Wang et al., 2004). Either or both of epicuticular wax content or presence of trichomes is known to hinder the aphid movement and stylet insertion (Bin, 1979). Table.2. Correlation between disease incidence and leaf parameters Epicuticular Leaf Trichome Per centage Per centage Wax Thickness Density Disease Infection Content Index Epicuticular 1.00 0.62*0.031 0.76*0.004 -0.360.247 -0.61*0.033 Wax Content Leaf Thickness 1.00 0.390.201 -0.71*0.009 -0.90*<.0001 Trichome Density 1.00 -0.390.201 -0.53*0.076 Percentage 1.00 0.87*0.000 Disease Index Percentage 1.00 Infection First value represents ‘r’ value and second value represents p value. *<0.05 p value shows significant correlation. Higher field tolerance to PRSV was observed in ‘Pusa Nanha’, which had thicker leaves. The PRSV incidence was significantly low in both V. cauliflora and V. cundinamarcensis, which registered thicker leaves, higher epicuticular wax content and denser trichomes. The preliminary study broadly indicates that leaf thickness, presence of trichomes in higher density and epicuticular wax content in papaya is likely to play a definitive role towards reduction in PRSV incidence probably by restricting the aphid vector incidence. Higher leaf thickness, epicuticular wax content and trichome density in papaya is found to have negative impact on the incidence of Papaya ring spot virus in papaya and these factors may have a role to play in restricting the virus transmission by aphid vector. Futur e r esea r ch needs to be focussed on the biochemical constituents of glandular trichomes and its effect on aphids. ACKNOWLEDGEMENT Guida nce a nd technica l suppor t r ender ed by Dr. M. Krishna Reddy, Dr.C.Vasugi, Ms. Jayanthi Mala, and Dr. Kalaivannan, ICAR-IIHR are gratefully acknowledged. Correlation of Leaf Parameters with Incidence of Papaya Ring Spot Virus J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 134 Linta Vincent et al J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 Fig. 1: Stereo microscope view of leaf ventral surface pubescence 135 Correlation of Leaf Parameters with Incidence of Papaya Ring Spot Virus J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 Fig. 2: Scanning electron microscope view of leaf ventral surface pubescence at 500m a) V. cauliflora (b) V. cundinamarcensis (c) V. goudotiana 136 Linta Vincent et al J. Hortl. Sci. Vol. 14(2) : 130-136, 2019 Bin, F. 1979. Influenza dei peli glandolari sugli insetti in Lycopersicon spp. Frust Ent., 15:271-283. Kalleshwaraswamy, C.M and Krishnakumar, N.K. 2008. Transmission efficiency of Papaya ringspot virus by thr ee a phid species. Phytopathology, 98(5):541-546. Dinesh, M.R. 2010. Papaya breeding in India. Acta Hort., 851: 69-75 Ebercon, A., Blum, A and Jordan, W.R. 1977. A rapid colorimetric method for epicuticular wax content of sorghum leaves. Crop Sci., 17: 179- 180. Goffreda, J.C., Mutschler, M.A., Ave ́ D.A., Tingey, W.M and Steffens, J.C. 1989. 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