IN VITRO SELECTION FOR FUSARIUM WILT TOLERANCE IN POMEGRANATE BY SCREENING AGAINST FUSARIC ACID

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1 Journal of Cell and Tissue Research Vol. 16(2) (2016) (Available online at www. Tcrjournals.com) ISSN: ; E-ISSN: Original Article IN VITRO SELECTION FOR FUSARIUM WILT TOLERANCE IN POMEGRANATE BY SCREENING AGAINST FUSARIC ACID GHOGHARE, D. S., CHIMOTE, V. P.,? PAWAR, B. D., KALE, A. A., RAGHUWANSHI, K. S. 1 AND JADHAV, A. S. State Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri (Maharashtra); 1 Department of Plant Pathology, Post Graduate Institute, Mahatma Phule Krishi Vidyapeeth, Rahuri (Maharashtra). E. mail: vivekchimote@rediffmail.com; Received: April 8, 2016; Accepted: May, 20, 2016 Abstract: Fusarium wilt of pomegranate causes severe losses in established orchards with disease going undetected at early stages. The objective of this study is to standardize in vitro screening protocol for Fusarium wilt tolerance in pomegranate using seedlings and detached leaves for identification of Fusarium wilt tolerant pomegranate genotypes. In the current investigation four different levels of fusaric acid were attempted in pomegranate to identify critical levels for toxin bioassay. In 10 ppm treatment genotype specific responses were observed in seedlings of 16 genotypes assayed. In vitro assayed seedlings produced symptoms similar to Fusarium wilt infection. Kabul and Code-429 exhibited resistance on in vitro seedling assay, while nine varieties exhibited complete susceptibility. In detached leaf assay of 58 genotypes, highest tolerance was recorded in Kabul Yellow, Shirin Anar and Nana genotypes, followed by Daru, Kabul and S-1 (Soft). On 24 th day of assay complete wilting was observed in forty two genotypes. No pomegranate genotype was immune to Fusarium wilt disease. In vitro screening will enable in identification of wilt resistant pomegranate genotypes. Key words: Pomegranate, Fusarium wilt, Fusaric acid. INTRODUCTION Pomegranate is an important fruit in tropical and subtropical countries. It was originated in Central Asian region of Iran, Afghanistan and Baluchistan [1] and is extensively cultivated in Mediterranean and Central Asian countries, India, China, Japan, Russia and U.S.A. India accounts for over half of the world s total pomegranate production. Pomegranate is commercially cultivated mostly in Maharashtra and Karnataka and to a smaller extent in Gujarat, Andhra Pradesh, Tamil Nadu and Rajasthan. -Pomegranate is commercially grown for its acid sweet fruits, mainly, used for dessert purpose. The edible part of the pomegranate fruit contains sugars, pectin, organic acids, and bioactive compounds such as phenolics and anthocyanins [2]. The fresh fruit is of exquisite quality, while its processed products are highly appreciated. Wild pomegranate grains are dried and commercially marketed as Anardana. The sweet type are said to be mainly laxative, while the less sweet types are believed to be good in inflammation of stomach and in heart pains. In addition, pomegranate has shown great importance for human due to its nutritional and medicinal properties for preventing the cancer and cardiovascular disease [3]. Pomegranate is a versatile crop adaptable to a wide range of agro-climatic conditions. In recent years severe incidences of Fusarium wilt and bacterial 5621

2 J. Cell Tissue Research wilt disease on pomegranate have started emerging as serious threats to its cultivation. Fusarium wilt is a major pomegranate disease with its infection going undetected ultimately leading to complete collapse of well-established orchards. Development of varieties resistant to Fusarium wilt is likely to play a significant role in tackling the wilt problem. Pomegranate being perennial fruit crop it is difficult to identify source for breeding wilt resistance. Limited information is available on wilt tolerance in pomegranate. Fusaric acid is known to be produced by various Fusarium species and is one of the toxins responsible for wilt symptoms [4]. In vitro selection using fusaric acid was found to be a very useful method for obtaining Fusarium wilts tolerance, although the tolerance mechanism of the selected plants may be different from that of existing tolerant cultivars [5]. Keeping this view in mind, the present investigation was undertaken with the objectives to standardize in vitro screening protocol and to identify pomegranates genotypes tolerant to Fusarium wilt. MATERIALS AND METHODS Plant materials: Fruits of 22 pomegranate genotypes (Table 1) and leaves of 58 pomegranate genotypes (Table 2) were collected from orchard of All India Coordinated Research Project on Arid Zone Fruits, Mahatma Phule Krishi Vidyapeeth, Rahuri. Assays for toxin activity were carried out on young (two month old) seedlings and detached leaves. Preparation of fusaric acid solution for in vitro screening: In order to evaluate the Fusarium wilt tolerance in various pomegranate genotypes fusaric acid (99% pure) procured from M/s Across Organics Ltd, USA was used. Fusaric acid solutions of concentration of 10, 20, 30, 40 ppm were prepared, filter sterilized and stored at 4 C till further usage. In vitro seedling assay: Seeds were extracted from fresh fully ripened fruits by squeezing out of pulp, followed by washing and drying. These seeds were sown in pot containing soil: cow dung manure (90:10). Seed germination started approximately one month after sowing. Forty five old seedlings were transferred to sterile autoclaved soilrite medium for 15 to minimize carryover contamination in subsequent screening assay. Out of twenty two genotype used for seedling germination, three genotypes (S1,Jyoti GKVK and Gul-E-Shah Red) failed to germinate, whereas three genotypes (Damani, Yercaud local and Kandhari) showed very low seed germination and therefore were excluded in subsequent analysis. Only sixteen genotypes yielded seedlings in number enough for subsequent screening assays. These seedlings were washed in sterile distilled water and transferred to plastic trays containing quarter strength Murashige and Skoog (MS) [6] liquid basal media supplemented with different concentration (0 ppm, 10 ppm, 20 ppm, 40 ppm) of fusaric acid. The symptoms like leaf curl, tip curl and wilting of the plantlets were recorded daily. Detached leaf assay: For detached leaf assay terminal new season shoots along with leaves were collected in black coloured bags to prevent and minimize light induced phenolic compound accumulation. The leaves along with their petiole were surface sterilized in 0.1% HgCl 2 (w/v). They were further rinsed five times with sterile distilled water to remove the traces of HgCl 2 and then transferred in test tube containing ¼ MS liquid medium supplemented with 10 ppm toxin as standardized during in vitro seedling assay and incubated at 27 C and 16 hr photoperiod in culture room. The susceptibility of detached leaves to toxin was recorded up to 24 (4, 8, 12 and 24 after assay). Detached leaves dipped in ¼ MS liquid medium were considered as controls. RESULTS AND DISCUSSION In vitro seedling assay: In the present study, all seedlings were found healthy in control treatment till the last day of observation (Fig. 1a). However, in 40 ppm treatment, effect of toxin started appearing on the 2 nd day and all plants irrespective of their genotype wilted by 5 th day (Fig. 1d). Similar symptoms were observed in 20 ppm treatment, but in four genotypes (Ganesh, Kabul, Code-429 and Code-303) few seedlings showed delayed wilting response (Fig. 1c). Most of the in vitro screenings assayed reported earlier are based on inducing genetic variation either mutagenesis induced during in vitro culture [7-9] or somaclonal variation during callus/ protoplast culture [10-14]. However, seedlings can also prove to be good source of variation for in vitro 5622

3 Ghoghare et al. Fig. 1a Fig. 1b Control 10 PPM Fig. 1c Fig. 1d 20 PPM 40 PPM Fig. 2a Fig. 2b Fig. 2c Fig. 2d Fig. 1: In vitro seedling assay on 5th day of treatment at different concentrations of fusaric acid. 1a. Control trial; 1b. 10 ppm treatment; 1c. 20 ppm treatment; 1d. 40 ppm treatment. Fig. 2: In vitro detached leaf assay on 12th day of treatment at 10 ppm fusaric acid concentration. 2a. Control, 2b. Moderately susceptible G-137genotype; 2c. Highly resistant Kabul Yellow genotype; 2d. Highly susceptible Dholka genotype 5623

4 J. Cell Tissue Research Table 1: In vitro seedlings assay of different pomegranate genotypes against 10 ppm fusaric acid (% incidence) Genotype 1 day 2 3 Healthy Mild leaf curl Moderate Leaf curl Tip Curl Bhagwa Acidic 1/ Acidic 1/ Acidic 2/ G Jalore seedless Amlidana Ganesh Kabul Code Dholka Code S Bedana Thin Skin KRS Mridula screening as reported previously [15-16]. On the first day of 10 ppm fusaric acid treatment none of the seedling of any genotype had any visible symptoms; however genotype specific responses were observed from the second day onwards (Table 1; Fig. 1b). In seven pomegranate genotypes (Bhagwa, S2, Mridula, Ganesh, Kabul, Code-429 and Code-303) all seedlings remained healthy, while in rest of genotypes intermediate response was observed. Quickest response to toxicity was observed in acidic 2/2324, Jalore seedless and Dholka with mild to moderate incidences of leaf curl symptom being recorded in all seedlings. On the third day after toxin treatment it was recorded that all the seedlings of the genotypes; S-2, Kabul, Code-429 and Code-303 were completely healthy. In the remaining genotypes, varying levels of tip and leaf curl infection symptoms were recorded. All the seedlings of genotype Kabul and Code-429 remain healthy even on 4 th and 5 th day of in vitro screening. Pomegranate being often cross pollinated with certain heterozygosity, seedlings of a same variety showed mixed response most likely due to segregation. It was observed that a 10 ppm level of fusaric acid was critical concentration for screening bioassays with different genotypes exhibiting differential response. The results from current investigation suggest that there is scope to utilise the resistance exhibited by seedlings of Kabul and Code-429. Further highly sensitive (to fusaric acid treatment) cultivars were identified i.e., acidic 2/2324, Jalore seedless and Dholka with symptom appearing from the second day of infection. Use of such varieties should be avoided for development of varieties for area having high incidence of wilt infection. Detached leaf assay: Earlier there were few reports involving use of detached leaf assay for screening Fusarium tolerant plant types. In tomato, the differences in sensitivity to fusaric acid of cultivated cells corresponded to the differences in plant susceptibility to Fusarium oxysporum f. sp. lycopersici [17]. In pineapple resistant response was induced both by Fusarium subglutinans (filtrate) and fusaric acid when infected on leaf segments and wounded tissue culture plantlets [18]. Susceptible cultivars were sensitive to culture filtrate whereas resistant ones showed tolerance, thereby using filtrate allowed them to select resistant plant to fungus itself. Seedlings as well as detached leaves of date palm were screened for bayoud disease resistance against Fusarium oxysporum f. sp. 5624

5 Ghoghare et al. Table 2: In vitro detached leaf assay of different pomegranate genotypes against 10 ppm fusaric acid (% incidence-rounded off) Genotype Healthy Petiole end wilting Mid vein wilting Chlorosis Complete wilted Ganesh Kabul Yercud Local Dholka A.K.Anar Speen Danedar Co-White G Bhoshka Linski Kandhari KRS Mridula Agah Bedana Sedana Damani Phule Arkta Kabul IIHR Kabul Cannor Jalore Seedless Bhagwa Jyoti (GKVK) Bedana Thin Skin Yercud Local HRS Muskat Tabasto Bassein Seedless Speen Sakarin P / Surk Anar Patna P Alah Dorsata Malas Bedana Suri Malta P Shirin Anar Kabul Yellow P Jodhapuri Red Continued on next page 5625

6 J. Cell Tissue Research Code Gul-E-Shah (Pink) Muskat Daru Turkey Nana American Type S-1 (Soft) Gul-E-Shah Red Amlidana Acidic 2/ Acidic 1/ Alandi S-3 (Soft) S-2 (Soft) Code Acidic 1/ albedinis [15]. They remarked that sensibility of date palm seedlings was correlated with that of detached leaf assay and therefore suggested use of detached leaf assay for rapid in vitro selection. Elsinoe ampelina (filtrate) and Fusarium oxysporum (filtrate) inhibited selected lines of grapes [19]. They also verified pathogen filtrate induced systemic resistance in greenhouse and detached leaf assay. In the present study, detached leaf assays were conducted on fifty eight genotypes at 10 ppm of fusaric acid in ¼ MS liquid medium. Readings were taken on 4 th, 8 th, 12 th and 24 th after treatment. No symptoms were observed in case of control treatment (without fusaric acid toxin) up to 24 (Fig. 2a). The result of detached leaf assay with 10 ppm toxin treatment are shown in Fig. 2b/2c/2d and Table 2. On the 4 th day of detached leaf assay, no incidence of infection was observed in thirty genotypes. None of the genotype studied had all its leaves wilted completely. Quickest infection symptoms were observed in Phule arakta. On the 8 th day of detached leaf assay all the detached leaves were completely healthy in eleven genotypes [Kabul, Jyoti (GKVK), Muskat, Tabasto, Speen Sakarin, Surk Anar, Shirin Anar, Kabul Yellow, Code-61, Acidic 2/2324 and Nana]. In 11 genotypes most of the leaves were having mild infection; while in 14 genotypes they showed moderate infection. Twenty two genotypes showed susceptibility with highest leaf wilting symptoms being observed in Dholka and Alandi (followed by Phule Arakta, Gul- E-Shah, Bhagwa, Bedana Suri, Kandhari, Kabul IIHR, KRS and S2). On the 12 th day of detached leaf assay only five genotypes had all detached leaves were green and healthy {Kabul Yellow, Kabul, Jyoti (GKVK), Shirin Anar, and Code-61}. Nine other genotypes showed high tolerance to fusaric acid were Speen Sakarin, Acidic 1/1920, Tabasto, Surk Anar, S-1 (Soft), Daru, Nana, Co-white and Jodhpuri Red in decreasing order of toxin resistance. On the other hand, moderate susceptibility to wilt was recorded in thirteen genotypes and twenty seven genotypes showed high susceptibility; with partial to complete wilting in all of their leaves. In detached leaf assay after 24 of treatment none of the variety had all their leaves green and healthy. Kabul Yellow variety exhibited highest resistance (24 after treatment) followed by Shirin Anar, Nana, Daru, Kabul and S-1 (Soft). There is a need to further extend the seedling assay both at in vitro as well as field level. Highest susceptibility was 5626

7 Ghoghare et al. observed in popular cultivars Dholka and Bhagwa. Moderate susceptibility was also exhibited by Alandi, Phule Arakta, Gul-E-Shah, Bedana Sedana, Patna-5, Alah, American Type, Code-61, Acidic 1/2324, P-16, Muskat-2 and Turkey. There is need to undertake extensive breeding programme to introduce disease resistance into these susceptible varieties to take advantage of their other desirable characteristics. Further it was observed that there was no clear correlation as such between varieties showing resistance in leaf assay and fruit acidity. Resistant genotypes, Shirin Anar, Kabul Yellow, Daru and Nana varied in their acidity with Kabul Yellow reportedly having low acidity ( %), while Shirin Anar had high acidity (3.5-4%) [20]. Similarly, susceptible pomegranate genotypes include those of both high acid types (Bedana Sedana, Gul E Shah) and low acid types (Bhagwa, Dholka, Alandi, Kandhari, Phule Arakta, P13, Kabul IIHR). From the results coming out from present study it is possible to carry out standardized assay against fusaric acid and select pomegranate genotypes exhibiting Fusarium wilt resistance. Use of detached leaves for phytotoxin assay allows simple and time saving method for screening tolerant types. The results of the present study will be very useful for pomegranate breeders for selecting wilt resistant types. [8] Mangal, M. and Sharma, D.R.: J. Horticul. Sci. Biotechnol., 77: (2002). [9] Chen, W.Q. and Swart W.: Euphytica, 127(1): (2002). [10] Chawla, H.S. and Wenzel, G.: Plant Breed., 99: (1987). [11] Bulk, R.W.: Euphytica, 56(3): (1991). [12] Evenor, D., Pressman, E., Ben-Yephet, Y. and Rappaport, L.: Plant Cell Tissue Organ Cult., 39(3): (1994). [13] Heath-Pagliuso, S., Pullman, J. and Rappaport, L.: Theoret. Appl. Genetics, 75: (1988). [14] Toyodo, H., Horikoshi, K., Yamano, Y. and Oochi, S.: Plant Cell Reports, 10: (1991). [15] Sedra, M.H., Lazrek, H.B., Lotfi, F. and Rochat, H.: Bayoud toxin isolation and use for screening of date palm plants for disease resistance. First International Conference on Date Palms, Al-Ain, UAE, pp (1998). [16] Huang, Y. H. and Hartman, G.L.: Plant Disease, 82: (1998). [17] Gapillout, I., Milat, M.L. and Blein, J.P.: European J. Plant Pathol., 102(2) : (1996). [18] Borras, O., Santos, R., Matos, A.P., Cabral, R.S. and Arzola, M.: Plant Breeding, 120: (2001). [19] Jayasankar, S., Li, Z.J. and Gray, D.J.: Planta, 211(2): (2000). [20] Ghoghare, D.S.: In vitro screening for Fusarium wilt resistance in pomegranate (Punica granatum L.). M. Sc. Thesis, Mahatma Phule Krishi Vidyapeeth, Rahuri. (2008). REFERENCES [1] De Candolle, A.: Origin of Cultivated Plants. Hafner Publishing Co. New York and London, 441: (1967). [2] Viuda-Martos, M., Fernandez-Lopez, J. and Perez- Alvarez, J.A.: Comprehen. Rev. Food Sci. Food Safety, 9(6): (2010). [3] Shishodia, S., Adams, L., Bhatt, I.D. and Aggarwal, B.B.: In: Pomegranates. Ancient Roots to Modern Medicine, 1st edition, Taylor and Francis Group, Boca Raton, FL, USA. pp (2006). [4] Davis, D.: Phytopathology, 59: (1969). [5] Matsmoto, K., Barbosa, M.L., Souza, L.A.C. and Teixeira, J.B.: Euphytica, 84: 67-71(1995). [6] Murashige, T. and Skoog, F.: Physiologia Plantarum, 15: (1962). [7] Pathania, N.S. and Misra, R.L.: In vitro mutagenesis studies in gladiolus for induction of resistance to Fusarium oxysporum F. sp. gladioli. ISHS Acta Hort: XXVI International Horticultural Congress: Elegant Science in Floriculture (Bloom T J and Criely, R. eds.), pp 624 (2003). 5627

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