13 J. Hortl. Sci. Vol. 14(1) : 13-19, 2019 Original Research Paper Evaluation of Solanum species and eggplant cultivated varieties for bacterial wilt resistance T.H. Singh1*, D.C. Lakshmana Reddy2, C. Anand Reddy1, A.T. Sadashiva1 P. Pandyaraj1 and Y.B. Manoj1 Division of Vegetable crops, Indian Institute of Horticultural Research (IIHR), Hessaraghatta Lake Post, Bengaluru - 560 089, India 1Division of Vegetable crops, Indian Institute of Horticultural Research (ICAR-IIHR), Bengaluru, India 2Division of Biotechnology, Indian Institute of Horticultural Research (ICAR-IIHR), Bengaluru, India *Email : Singh.HT@icar.gov.in ABSTRACT Bacterial wilt caused by Ralstonia solanacearum is one of the major diseases in Solanum species including cultivated Eggplant (Solanum melongena L.). Bacterial wilt (BW) disease management in eggplant is difficult due to high survival rate of pathogen in soil and chemical application is not eco-friendly. The best way to avoid bacterial wilt in eggplant is using disease-resistant varieties. However, only a limited number of bacterial wilt resistant varieties are available and, there is a necessity to identify and/or develop new resistant varieties. In the current study, wild Solanum species, and eggplant cultivated varieties were evaluated against Ralstonia solanacearum, and disease incidence was recorded. The cultivated varieties IIHR-108, Pusa Purple Long and Rampur Local were identified as susceptible, whereas, IIHR-7 and CARI-1 were identified as resistant to bacterial wilt. These resistant wild and cultivated varieties can be used as a root-stock in bacterial wilt disease resistant breeding programmes. Keywords: Wild Solanum species, Eggplant, Ralstonia solanacearum, Disease scoring, Grafting INTRODUCTION Eggplant (Solanum melongena L.) (2n=24) is one of the most popular Solanaceous vegetable crop cultivated globally. Asia is the major producer (93%) of eggplant (FAO, 2016). It is originated from India and South China (Da unay and Ha zra , 2012). Eggplant is generally cultivated in open fields with hot and humid conditions. Eggplant is susceptible to numerous diseases viz., bacterial wilt; fusarium wilt; verticillium wilt, early blight, leaf spot, potato virus-Y (PVY), tobacco ring spot virus, tomato spotted wilt virus (TSWV), phytoplasma, and root-knot nematode. Duo to these diseases, quality and quantity of eggplant production is adversely affected. Eggplant bacterial wilt (Ralstonia solanacearum) is a major concern in India. The pathogen is race specific and has five different races. Ralstonia solanacearum strains are divided into five different biovars based on the biochemical analysis. In India, eggplant bacterial wilt is mainly caused by Race 1 and Biovar 3 (Gopalakrishnan et al., 2005). The pathogen (Ralstonia solanacearum) can survive in soil upto ten years without any host plant and have the ability to colonize in non-host plants including a vast range of symptomless weeds (Gopalakrishnan et.al., 2014). The pathogen enters into the plants through wounds or secondary root initiation points leading to pathogen colonization in the vascular parenchyma and cell wall breakage (Ramesh, 2008). The initial wilt symptoms are leaf drooping, followed by full-plant wilting and vascular discoloration. When cut ends of wilted plant placed in water, milky white ooze out can be observed. There are different methods of artificial inoculation/ screening of bacterial wilt like root cut inoculation, leaf or stem pricking and natural field infestation (Ramesh, 2008). These pathogenic spores remain viable and active in the soil for several years making the disease control almost impossible through any means of chemical treatments especially in the regions 14 Singh et al J. Hortl. Sci. Vol. 14(1) : 13-19, 2019 where repeated eggplant cultivation is taken up. Many of the commer cial va rieties/hybrids ar e highly susceptible to bacterial wilt. Chemical management often leads to the presence of chemical residues in the fruits, thus, raises the concern of food safety. The most economical way of bacterial wilt control is to develop bacterial wilt resistant varieties/hybrids. Recently, grafting strategy using resistant root-stock has been proposed for soil-borne pathogens like bacterial wilt, and it will help in bacterial wilt resistant varieties/hybrids development with the different genetic backgrounds. Hence, identification of a bacterial wilt root-stock will be of higher use and any preferable variety/hybrid can be grafted on it. Here, we evaluated wild solanum species and cultivated varieties for bacterial wilt resistance through artificial inoculation, and these can be used as root-stock in grafting. MATERIALS AND METHODS Plant Material and Phenotypic Characters In this study, five eggplant cultivated varieties viz., IIHR-7 (IC395334), IIHR-662 (CARI-1, IC0585684), IIHR-108 (Arka Kusumakar), IIHR-663 (Rampur Local), and seven wild species viz., Solanum gilo (RS-3), Solanum indicum (RS-4), Solanum viarum (RS-6), Solanum aethiopicum (RS-7), Solanum mammosum (RS-8), and Solanum torvum (RS-9 and RS-9a) were used for bacterial wilt screening (Fig.1). These germplasm accessions were maintained at Indian Institute of Horticultural Research, Bengaluru. Fig. 1. Eggplant cultivated varieties [IIHR-7 (IC395334), IIHR-662 (CARI-1, IC0585684), IIHR-108 (Arka Kusumakar), IIHR- 663 (Rampur Local)] and seven wild species [Solanum gilo, S. indicum, S. viarum, S. aethiopicum, S. mammosum, and S. torvum used for bacterial wilt screening. 15 Evaluation of eggplant for bacterial wilt resistance T ab le 1 . P he no ty pi c tr ai ts o f el it e ge rm pl as m l in e S; . N o. 1 2 3 4 5 6 7 8 9 10 11 N am e of th e lin e IIH R -1 08 (A rk a K us um ak ar ) IIH R - 7 (IC 39 53 34 ) IIH R -6 62 (C A R I-1 , I C 05 85 68 4) II H R - 6 63 (R am pu r L oc al ) Pu sa P ur pl e L on g So la nu m g ilo S ol an um i nd ic um So la nu m vi ar um So la nu m ae th io pi cu m So la nu m m am m os um So la nu m t or vu m Ph en ot yp ic C ha ra ct er s A cc es si on II H R -1 08 , i s a pu re li ne s el ec tio n fr om II H R -1 93 (A lo ca l c ol le ct io n fr om K ar na ta ka ). Ph en ot yp ic c ha ra ct er s vi z. , Pl an t h ei gh t: m ed iu m /ta ll; g ro w th h ab it: s pr ea di ng ; s te m a nd fo lia ge c ol or : g re en ; f lo w er c ol or : w hi te ; f ru it: s m al l ( m ed iu m - lo ng ) gr ee n; c oo ki ng q ua lit ie s: g oo d; Y ie ld : 40 t/h a; c ro p du ra tio n: 1 40 -1 50 d ay s an d ba ct er ia l w ilt i nc id en ce : hi gh (s us ce pt ib le ).Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 85 -1 00 c m ); fr ui t l en gt h (1 3- 14 c m ); fr ui t d ia m et er (3 .0 -3 .5 c m ); av er ag e fr ui t w ei gh t ( 30 -4 0 g) . It is a n ad va nc ed b re ed in g lin e de ri ve d fr om c ro ss b et w ee n A rk a K es ha v, a nd II H R -3 22 . P he no ty pi c ch ar ac te rs v iz ., pl an t he ig ht : t al l; gr ow th h ab it: s pr ea di ng ; s te m a nd fo lia ge c ol or : g re en ; f lo w er c ol or : p ur pl e; fr ui t: lo ng -g re en ; c oo ki ng q ua lit ie s: go od a nd b ac te ri al w ilt in ci de nc e: lo w (h ig hl y re si st an t) .Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 10 0- 12 0 cm ); fr ui t l en gt h (1 8- 20 cm ); fr ui t d ia m et er (3 .2 -3 .5 c m ); av er ag e fr ui t w ei gh t ( 40 -4 5 g) . T hi s ge rm pl as m li ne w as c ol le ct ed fr om A nd am an & N ic ob ar (P or t B la ir ) i sl an ds . P he no ty pi c ch ar ac te rs v iz ., Pl an t h ei gh t: m ed iu m /ta ll; g ro w th h ab it: s pr ea di ng ; f ru it: r ou nd -g re en ; b ac te ri al w ilt in ci de nc e: lo w (h ig hl y re si st an t). Q ua nt ita tiv e tr ai ts : pl an t h ei gh t ( 11 5- 13 0 cm ); fr ui t l en gt h (1 0- 12 c m ); fr ui t d ia m et er (1 4- 16 c m ); a ve ra ge fr ui t w ei gh t ( 30 0- 35 0 g) . T hi s ge rm pl as m li ne w as c ol le ct ed fr om R am pu r vi lla ge o f U P, In di a. P he no ty pi c ch ar ac te rs v iz ., Pl an t h ei gh t: ta ll; g ro w th ha bi t: sp re ad in g; fr ui t: lo ng -v io le t p ur pl e; fr ui t b ea ri ng : m ix ed (f ru its a re b or ne in c lu st er s an d so lit ar y) ; c oo ki ng q ua lit ie s: go od a nd b ac te ri al w ilt in ci de nc e: h ig h (h ig hl y su sc ep tib le ).Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 11 5- 13 0 cm ); fr ui t l en gt h (2 0- 24 c m ); fr ui t d ia m et er (4 .0 -4 .5 c m ); a ve ra ge fr ui t w ei gh t ( 70 -7 5 g) . T hi s va ri et y w as d ev el op ed t hr ou gh p ur e- lo ne s el ec tio n by I A R I, N ew D el hi . P he no ty pi c ch ar ac te rs v iz ., Pl an t he ig ht : M ed iu m ; g ro w th h ab it: s pr ea di ng ; f ru it: lo ng -p ur pl e (g lo ss y) ; c oo ki ng q ua lit ie s: g oo d an d ba ct er ia l w ilt in ci de nc e: h ig h (h ig hl y su sc ep tib le ). Fr ui ts a re b or n in c lu st er s. Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 30 -4 0 cm ); fr ui t l en gt h (1 5- 18 c m ); y ie ld ab ou t 4 0- 45 to nn es /h a. Pl an t h ei gh t: ta ll; g ro w th h ab it: e re ct ; f ru it: s m al l- r ou nd (l oo k lik e to m at o an d ha ve b ot h ri dg e/ ri dg e- le ss tr ai ts ) co ok in g qu al iti es : b ad a nd b ac te ri al w ilt in ci de nc e: lo w (h ig hl y re si st an t) . Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 10 0- 11 5c m ); fr ui t l en gt h (3 .5 -4 .0 c m ); fr ui t d ia m et er (3 .0 -3 .5 c m ); a ve ra ge fr ui t w ei gh t ( 25 -3 0 g) . Pl an t h ei gh t: m ed iu m /ta ll, g ro w th h ab it: s pr ea di ng (t ho rn s on s te m s an d m id r ib s) ; f ru it: s m al l a nd p ur pl e (p ea s iz e, a nd r ed to o ra ng e w he n ri pe ) an d ba ct er ia l w ilt in ci de nc e: lo w ( hi gh ly r es is ta nt ).Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 80 -9 0 cm ); fr ui t le ng th (1 .0 -1 .5 c m ); fr ui t d ia m et er (0 .9 -1 .0 c m ); a ve ra ge fr ui t w ei gh t ( 2- 3 g) . Pl an t h ei gh t: ta ll, g ro w th h ab it: s pr ea di ng (d en se th or ns a re p re se nt o n th e st em m id r ib s) ; f ru it: s m al l- ro un d (w hi te s tr ip es du ri ng im m at ur e st ag e an d tu rn y el lo w a fte r ri pe ni ng ) a nd b ac te ri al w ilt in ci de nc e: lo w (h ig hl y re si st an t). Q ua nt ita tiv e tr ai ts : pl an t h ei gh t ( 10 0- 12 0 cm ); fr ui t l en gt h (2 .5 -3 .0 c m ); fr ui t d ia m et er (2 .0 -3 .0 c m ); a ve ra ge fr ui t w ei gh t ( 5- 8 g) . Pl an t h ei gh t: m ed iu m /ta ll, g ro w th h ab it: s pr ea di ng (h av e pu rp le ti ng e) ; f ru it: s m al l- re d, o bl at e ro un d (l oo k lik e to m at o) a nd ba ct er ia l w ilt in ci de nc e: lo w (h ig hl y re si st an t) .Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 85 -9 5 cm ); fr ui t l en gt h (5 .5 -6 .5 c m ); fr ui t di am et er (4 .5 -5 .0 c m ); a ve ra ge fr ui t w ei gh t ( 40 -5 0 g) . Pl an t h ei gh t: ta ll, g ro w th h ab it: s pr ea di ng ( de ns e th or ns a re p re se nt o n th e st em m id r ib s) ; f ru it: c ow ’s u dd er s ha pe (t ur n ye llo w o n ri pe ni ng ) an d ba ct er ia l w ilt in ci de nc e: lo w (h ig hl y re si st an t) .Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 12 5- 13 5 cm ); fr ui t le ng th (5 .5 -6 .0 c m ); fr ui t d ia m et er (3 .5 -4 .0 c m ); av er ag e fr ui t w ei gh t ( 15 -2 0 g) . Pl an t h ei gh t: ta ll, g ro w th h ab it: s pr ea di ng (d ar k gr ee n fo lia ge ); fl ow er c ol or : w hi te ; f ru it: d ar k gr ee n at im m at ur e st ag e an d tu rn y el lo w o n ri pe ni ng ) a nd b ac te ri al w ilt in ci de nc e: lo w (h ig hl y re si st an t) .Q ua nt ita tiv e tr ai ts : p la nt h ei gh t ( 13 0- 15 0 cm ); fr ui t l en gt h (1 .5 -2 .5 c m ); fr ui t d ia m et er (1 .0 -1 .5 c m ); a ve ra ge fr ui t w ei gh t ( 10 -1 5 g) . J. Hortl. Sci. Vol. 14(1) : 13-19, 2019 16 Basic information of Solanum species and eggplant varieties were described in Table 1. Artificial Screening of Bacterial Wilt R. solanacearum inoculum was prepared according to Urquhart et al. (1998) from bacterial wilted eggplants. The bacterium colonies were collected from wilted plant using ooze out test, and streaked on Triphenyl Tetrozolium Chloride (TZC or TTC) plate (Fig. 2). The plates were incubated at 28oC - 30oC for 48 hours, and the TZC plates were checked for development of avirulent and virulent colonies. The sepa r ated vir ulent colonies wer e selected a nd suspended in sterile distilled water. The concentration of inoculum was recorded using a spectrophotometer and stored at 4oC for further use. Soil drenching method was used for inoculating the 25 days seedlings with R. solanacearum suspension, with concentration of 1.0 x108 CFU / ml (OD 600 nm = 0.3) (Rashmi et al., 2012). A) Resistant B) Susceptible Observation and Bacterial Wilt Disease Screening According to Hussain et al. (2005), bacterial wilt symptoms and total number of wilted plants per germplasm/variety were recorded on a 0-5 scale, with minor modifications. Based on the percentage of wilted plants, accessions were categorized as highly resistant to highly susceptible. Disease scoring scale S.No. Percentage of disease Scale (0-5) Incidence (%) 1 No wilt symptom (0%) Highly Resistant (HR) (0) 2 1 - 10% wilted plants Resistant (R) (1) 3 11 -20% wilted plants Moderately Resistant (MR) (2) 4 21-30% wilted plant Moderately Susceptible (MS) (3) 5 31- 40% wilted plants Susceptible (S) (4) 6 > 40% wilted plants Highly Susceptible (HS) (5) RESULTS AND DISCUSSION Ralstonia solanacearum race 1 Biovar 3 is more devastating and causes a severe problem in eggplant cultivation mainly in hot & humid areas like India (Markose, 1996). Controlling the bacterial wilt disease is practically not possible with the help of chemicals, and hence growing the resistant variety/hybrid is the best a ppr oa ch. In the br eeding or gr a fting, identification of best-durable resistant root-stock is the first step. In various studies, high level of Ralstonia solanacearum resistance is reported in IIHR-7, CARI-1 and wild Solanum species Solanum torvum, LS 174, Solanum sisymbriifolium, Solanum viarum, Solanum mammosum, Solanum nigrum, Solanum maroniense and Solanum stramonifolium (Mochizuki and Yamakawa, 1979, Gousset et al., 2005; Reddy et al., 2015; Bainsla et al., 2016). Eggplant cultivated varieties and wild Solanum species were artificially inocula ted with R. solanacearum inoculum (Concentration ~1.0 x 108 cfu/ml). After 25-35 days post-inoculation period, wilt incidence was observed on IIHR-108, Rampur Local and Pusa Purple Long. Up to 7-8 weeks, no wilting symptoms were observed on IIHR-7, CARI-1 and all wild Solanum species. In the present study, susceptible check variety IIHR- 108 has shown ~100% bacterial wilt incidence on artificial inoculation. In the test accessions, bacterial wilt incidence ranged from 0 to 99.33 %. In general, the resistance level of eggplant cultivated varieties to bacterial wilt in is limited (Bhavana and Singh, 2016). Resistant source from cultivated eggplant accessions will be highly useful in breeding due to their advantage in crossing, and no negative effects of fruits will hinder the br eeding. Among the cultiva ble eggpla nt a ccessions, IIHR-7 (IC-395334) a nd IHR-662 Figure 2. Ooze out test and R. Solanacearum virulence identification in TZC plate. J. Hortl. Sci. Vol. 14(1) : 13-19, 2019 Singh et al 17 T ab le 2 . M ea n pe rf or m an ce o f ge no ty pe s fo r ba ct er ia l w ilt i nc id en ce a nd y ie ld a tt ri bu ti ng t ra it s Sl . N o. o f Pl an t Fr ui t Fr ui t N o. o f A ve ra ge Fr ui t % N am e of th e ge no ty pe pr im ar y he ig ht le ng th di am et er fr ui ts / fr ui t yi el d B ac te ri al w ilt N o. br an ch es (c m ) (c m ) pl an t w t. (g ) (t /h a) in ci de nc es 1 IIH R -1 08 6. 63 94 .6 7 14 .3 2 3. 09 37 .3 3 42 .6 7 6. 22 99 .3 3 (8 5. 70 *) 2 IIH R -7 5. 74 10 6. 00 19 .6 7 3. 50 39 .6 7 42 .6 7 37 .4 7 0. 00 (0 .0 0* ) 3 II H R -6 62 (C A R I- 1) 6. 64 95 .1 7 13 .8 4 10 .4 8 18 .0 0 29 1. 67 29 .1 5 0. 00 (0 .0 0* ) 4 IIH R -6 63 ( 6. 37 91 .1 6 17 .2 7 3. 61 31 .2 2 36 .7 2 27 .5 6 21 .5 4 (2 7. 64 *) 5 Pu sa P ur pl e L on g 4. 03 94 .7 8 19 .4 9 2. 94 6. 79 32 .8 4 4. 52 92 .3 2 (7 5. 46 *) 6 So la nu m g ilo 3. 57 93 .4 5 5. 13 3. 86 27 .6 7 25 .6 7 4. 56 26 .4 5 (3 0. 93 *) 7 So la nu m i nd ic um 3. 71 84 .8 1 1. 61 0. 38 17 4. 33 0. 29 0. 79 0. 00 (0 .0 0* ) 8 So la nu m v ia ru m 7. 13 12 3. 33 3. 42 2. 46 17 6. 00 19 .8 4 1. 80 0. 00 (0 .0 0* ) 9 So la nu m a et hi op ic um 3. 20 76 .0 0 5. 57 3. 31 26 .0 0 33 .1 7 8. 22 4. 38 (1 2. 03 *) 10 So la nu m m am m os um 5. 32 11 1. 99 5. 65 4. 05 35 .0 0 30 .8 3 4. 39 4. 50 (1 2. 19 *) 11 So la nu m to rv um 3. 70 14 3. 33 2. 12 1. 52 22 3. 33 0. 19 0. 82 0. 00 (0 .0 0* ) G. M EA N 5. 10 10 1. 34 9. 83 3. 56 72 .3 0 50 .6 0 11 .4 1 6 6. 53 SE M 0. 35 3. 79 0. 67 0. 27 4. 93 2. 69 0. 80 1 .2 4 SD 0. 49 5. 37 0. 94 0. 38 6. 97 3. 80 1. 14 1. 76 CD 1. 49 16 .2 7 2. 85 1. 16 21 .1 5 11 .5 4 3. 45 5. 18 C V (% ) 9. 64 5. 29 9. 57 10 .7 2 9. 64 7. 52 9. 97 2. 65 *V al ue s i n pa re nt he si s a re a ng ul ar tr an sf or m ed v al ue s J. Hortl. Sci. Vol. 14(1) : 13-19, 2019 Evaluation of eggplant for bacterial wilt resistance 18 (CARI-1) were found to have high resistance with no incidence of bacterial wilt. Among the wild species, bacterial wilt incidence ranged from 0 (Solanum torvum, Solanum viarum, and Solanum indicum) to 26.45 % (Solanum gilo). Wild species are hardy in nature, and will have the special advantage of strong root system for combating other abiotic stresses. These wild species cannot be used in breeding pr ogr amme ea sily because of negative fruit traits. However, these can be used as a root-stock for eggplant and tomato cultivation in bacterial wilt prone areas. These wild species can be effectively used for grafting the commercial varieties and hybrids of eggplant and tomato. Gopalakrishnan et al. (2005) identified high phenolic content, and some special root cortical cells in bacterial wilt resistant cultivar root system, and are thought to be responsible for controlling of R. solanacearum spreading and multiplication. Already, the success of using Solanum torvum in grafting eggplant for bacterial wilt and yield advantage has been proved. The same can be used for tomato and other Solanum cultivation. Solanum torvum have graft advantages and compatibilities with cultivated eggplant, hence, it can be effectively used in grafting programs of bacterial wilt resistant eggplant varieties/hybrids (Ashok et al., 2017). Ashok et al. (2017) reported Solanum torvum resistance with 5.7% susceptibility, whereas, in our study it was found highly resistant with no incidence. This variation in level of resistance may be due to the variability in the Solanum torvum accessions or difference in Ralstonia solanacearum races. ACKNOWLEDGEMENT Authors are thankful to the Director, Indian Institute of Horticultural Research (ICAR-IIHR), Bengaluru, India for providing research facilities. J. Hortl. Sci. Vol. 14(1) : 13-19, 2019 Singh et al REFERENCES Ashok, K.B., Raja, P., Pandey, A.K. and Ranindro, P. 2017. Evaluation of Wilt Resistance of Wild Solanum Species through Grafting in Brinjal. Int. J. Curr. Microbiol. App. Sci., 6(9): 3464-3469. Bainsla, N.K., Singh, S., Singh, P.K., Kumar, K., Singh, A.K. and Gautam, R.K. 2016. Genetic Behaviour of Bacterial Wilt Resistance in Brinjal (Solanum melongena L.) in Tropics of Andaman and Nicobar Islands of India. American Journal of Plant Sciences, 7: 333- 338. 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