Virulence Variation in 20 Isolates of Erwinia carotovora subsp. carotovora on Zantedeschia Cultivars in Korea

Transcription

1 Virulence Variation in 20 Isolates of Erwinia carotovora subsp. carotovora on Zantedeschia Cultivars in Korea H.R. Cho, J.H. Lim, K.J. Yun, R.C. Snijder 1, D.H. Goo, H.K. Rhee, K.S. Kim 2, H.Y. Joung and Y.J. Kim National Horticultural 1 Plant Research International 2 Seoul National University Research Institute P.O. Box 16 Suwon RDA, Suwon, AA Wageningen Korea Korea The Netherlands Keywords: Zantedeschia, Erwinia carotovora, calla, breeding, resistance, isolate Abstract Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in Zantedeschia spp. Chemicals or modified cultivation methods only provide partial control of this disease. So highly resistant cultivars are needed to produce high quality calla flowers. Experiments were conducted to screen for Erwinia resistance in Zantedeschia spp. by a leaf disk test. To determine variation for aggressiveness, 20 Erwinia isolates collected from sites at Jeonju, Pyeongchang, and Suwon were inoculated onto five Zantedeschia cultivars: including Wedding March as a resistant genotype, Little Suzy as a moderately resistant genotype, and Chianti as a susceptible genotype. Ecc NHRI-3 and Ecc NHRI-6 were the most virulent isolates among the 20 isolates of Erwinia carotovora subsp. carotovora tested. Within 25 Zantedeschia genotypes, including Z. rehmannii and Z. albomaculata, variation of Erwinia resistance ranged from almost completely resistant to extremely susceptible. Wedding March was the most resistant, while Galaxy was the most susceptible. INTRODUCTION Calla is a very attractive plant native to Africa extending from Cape Province to Eastern Orange Free State, Natal, Lesotho, Swaziland, Transvaal, Rhodesia, Malawi, Zambia, Angola, and into northern Nigeria (Traub, 1948; Letty, 1973). The calla lily (Zantedeschia spp., Araceae), consists of eight species in two sections, which all originated from Southern Africa (Letty, 1973; Singh et al., 1996). Section Zantedeschia consists of Z. aethiopica (Singh et al., 1996), further called Aethiopica-group. Most of Zantedeschia hybrids with colored flowers are developed from crosses within section Aestivae (Singh et al., 1996). Calla may be grown as outdoor garden plants, commercial cut flowers and more recently as flowering potted plants (Kuehny, 2000). Zantedeschia aethiopica has been cultivated for 30 years in Korea, and Zantedeschia Aestivae group (colored calla lily) have been co-cultivated during the last five years. However, expansion of production of Aestivae group has resulted in severe outbreaks of soft rot during cultivation and storage of tuber. Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in this crop. This anaerobic pathogen causes maceration and rotting of parenchymatous tissue of the affected organ, resulting in loss of the whole plant (Pérombelon and Kelman, 1980). Under favorable conditions, 15-20% of stored calla tubers can be destroyed by an outbreak of the disease. In the field, the soft rot bacteria enter the plant or tuber through natural openings or wounds. Once in the plant tissue, the bacteria multiply rapidly especially under warm and moist conditions (temperatures around 25 C and free water). The final stage is total collapse of the plant and death; losses can approach 100% in a bad outbreak. There is no chemical treatment against the disease. Cultural practices can partly decrease the problem (Wright, 1996), but the development of resistant cultivars is the most desired approach in overcoming problems caused by soft rot (Hogenboom, 1993). Efforts are also underway to identify effective biological control techniques to control this disease Proc. IX th Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS

2 (Vanneste, 1997). To obtain information on the level of Erwinia resistance in Zantedeschia genotypes and the virulence variation between isolates of Erwinia, which is a prerequisite for calla breeding in Korea, several experiments were conducted to screen for Erwinia resistance in Zantedeschia spp. by a leaf disk test. MATERIALS AND METHODS Plant Material Calla plants were cultivated in a heated greenhouse and maintained at an average night and day temperature of 15±5 C and 25±5 C, respectively. Plants were planted in sandy soil amended with 5% manure. All plants were watered until five months after planting. Twenty-five genotypes, containing relatively resistant cultivar Zantedeschia aethiopica Wedding March, partially resistant cultivar Z. rehmannii, and relatively susceptible cultivar Florex Gold, were tested in this experiment. Erwinia Inoculum Concentration Initially beads of isolate s cells cultured from the diseased plants were placed at -80 C for cryoperservation using solid medium (0.8% Lab-Lemco Broth, 86 mm NaCl 2, 1.5% bacteriological agar, Oxoid B.V.) adding Glycerol (20%) to the medium (Fig. 1). To obtain inoculum for testing, each isolate s beads were removed from -80 C storage and were placed in suspension cultured at 20 C for 48 hr. Condensing bacteria masses was centrifuged at 3,500 rpm for 10 minutes. The supernatant was discarded, 20 ml sterile water added and thoroughly shaken to suspend the bacterial pellet (Fig. 1). Bacteria numbers were counted using a haemocytometer. A 0.01 mm depth, mm 2 in four boxes and 0.04 mm 2 for five boxes on the haemocytometer was used for the standard Erwinia count. Before counting, the haemocytometer and the cover glass were dipped in 70% ethyl alcohol and cleaned with lens paper. 10 µl Erwinia suspension was dropped in the space between the haemocytometer and the cover glass. Under the microscope, Erwinia concentration was calculated. A final Erwinia concentration to screen the section Aestivae is 1 x 10 7 bacteria/ml. However, 1 x 10 9 bacteria/ml inoculum concentration is needed for screening the more resistant section Zantedeschia. Virulence Test To determine variation for aggressiveness, 20 Erwinia isolates collected (Table 1) from three sites near Jeonju, Pyeongchang, and Suwon were inoculated to five Zantedeschia cultivars. The cultivars included Wedding March as a resistant genotype, Little Suzy as a moderately resistant genotype, and Chianti as a susceptible genotype. Disease Resistance Test A leaf disk test was used for determining resistance levels of Zantedeschia plants. Six disks of at least two young leaves were harvested just after unfolding and used for the test. Each disk was submerged in a 5 ml suspension of 1 x 10 7 bacteria/ml or 1 x 10 9 bacteria/ml (section Zantedeschia) in sterilized, demineralised water and incubated in a moist chamber (20 C, 100% relative humidity). During three to seven days after inoculation, the % of the leaf disk infected by Erwinia was observed. The percentage of infected surface area and water soaking were estimated per leaf disk using a view box. Resistance levels were calculated per leaf. All genotypes were classed by macerated leaf disk area relative to the very susceptible control Florex Gold. Ratings were: 5, highly resistance; 6-30, moderately resistance; 31-90, susceptible; and , very susceptible. 654

3 RESULTS AND DISCUSSION Variation in Aggressiveness of Erwinia Isolates To identify a representative isolate to screen resistance level of genotypes, 20 isolates of Erwinia carotovora subsp. carotovora were screened for their virulence. Ecc NHRI-3 and Ecc NHRI-6 were the most virulent among the 20 isolates tested (Table 2). All isolates could be used to discriminate the three representative genotypes tested. Z. aethiopica Wedding March was the most resistant genotype to all isolates and section Aestivae Chianti was most susceptible. Section Aestivae Little Suzy was the most resistant genotype to all isolates. The reaction of the three genotypes was similar for all isolates; there are no interactions between the isolates and the Zantedeschia genotypes (Snijder et al., 2002). Disease Measurement In this experiment, Erwinia resistance levels in 25 Zantedeschia genotypes including Z. rehmannii and Z. albomaculata were significantly different (Table 3). Section Zantedeschia Wedding March was the most resistant, while all other cultivars in Section Zantedeschia were resistant, except Pink Mist, which was moderately resistant. While, section Aestivae Galaxy was the most susceptible cultivar, Golden Affair, Pot of Gold, Mango, and Flame were very susceptible to Ecc NHRI-3 isolate. However, in section Aestivae, Z. rehmannii, Neroli and Hazel Marie were moderately resistant genotype. All the other cultivars in section Aestivae were susceptible. Within the 25 Zantedeschia genotypes, variation in Erwinia resistance ranged from almost completely resistant to extremely susceptible (Snijder et al., 2002, 2003). Literature Cited Hogenboom, N.G Economic importance of breeding for disease resistance. Durability of Disease Resistance 5-9. Kuehny, J.S Calla history and culture. HortTech. 10(2): Letty, C The genus Zantedeschia. Bothalia 11:5-26. Perombelon, M.C.M. and Kelmon, A Ecology of the soft rot Erwinias. Ann. Rev. Phytopathol. 18: Singh, Y., van Wyk, A.E. and Baijnath, H Floral biology of Z. aethiopica. J. S. African Bot. 62(3): Snijder, R.C. and van Tuyl, J.M Evaluation of tests to determine resistance in Zantedeschia spp. (Aracaea) to soft rot caused by Erwinia carotovora ssp. carotovora. Eur. J. Plant Pathol. 108: Snijder, R.C., Cho, H.-R., Margriet, M.W.B.H., Lindhout, P. and van Tuyl, J.M Genetic variation in Zantedeschia spp. (Araceae) for resistance to soft rot caused by Erwinia carotovora subsp. carotovora. Euphytica (in press). Traub, H.P The genus Zantedeschia. Plant Life 4:3-32. Vanneste, J Research to control calla soft rot. Crop & Food research: Wright, P Research to control calla soft rot. Crop & Food research: 655

4 Tables Table 1. Isolates of Erwinia carotovora subsp. carotovora that were evaluated for variation in aggressiveness. Isolate No. Year Area Source Ecc NHRI Suwon Pink Persuasion Ecc NHRI Suwon Black Magic Ecc NHRI Suwon Z. albomaculata Ecc NHRI Suwon Mango Ecc NHRI Suwon Treasure Ecc NHRI Suwon Childsiana Ecc NHRI Suwon Aneke Ecc NHRI Suwon Nelolri Ecc NHRI Suwon Black Magic Ecc NHRI Jeonju Orange Elite Ecc NHRI Jeonju Sun light Ecc NHRI Suwon Black Magic Ecc NHRI Suwon Z. aethiopica Ecc NHRI Suwon Golden Affair Ecc NHRI Suwon Pot of Gold Ecc NHRI Suwon Z. rehmannii Ecc NHRI Pyeongchang Black Magic Ecc NHRI Pyeongchang Mango Ecc NHRI Suwon Pacific Pink Ecc NHRI Suwon Z. aethiopica Table 2. Percentage of leaf disk area from three cultivars macerated by 20 Erwinia isolates five days after inoculation. Isolate No. Wedding March Little Suzy Chianti Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI Ecc NHRI

5 Table 3. Percentage of leaf disk area macerated by Erwinia isolate Ecc NHRI-3 following inoculation of 25 Zantedeschia genotypes. Genotype % macerated Class Z. aethiopica Wedding March 1 R Z. aethiopica Childsiana 4 R Z. aethiopica Green Goddess 5 R Z. aethiopica Pink Mist 15 MR Z. rehmannii 18 MR Neroli 25 MR Hazel Marie 22 MR Black Eyed Beauty 45 S Majestic Red 50 S Pink Persuasion 52 S Best Gold 65 S Schwarzwalder 77 S Black Magic 78 S Celeste 80 S Hot Shot 80 S Sensation 85 S Cameo 88 S Chianti 87 S Treasure 86 S Flame 92 VS Mango 95 VS Pot of Gold 92 VS Golden Affair 97 VS Florex Gold 100 VS Galaxy 100 VS * -5: highly resistance (R); 6-30: moderately resistance (MR); 31-90: Susceptible (S); : very susceptible (VS) 657

6 Figures Fig. 1. Long term storage at -80 C and growing bacteria by suspension culture. Fig. 2. Growth of isolates of Erwinia carotovora from infested tubers collected from three calla-cultivation sites in Korea. 658

7 % of macerated ar day 4day 5day 6day 7day % of macerated ar Anneke c1 BEB c1 BG c1 BM c1 Flam e c1 GA c1 MR c1 PoG c1 Schwarz c1 Z albo c1 WM c1 Chids c1 0 3day 4day 5day 6day 7day Fig. 3. Macerated leaf disk area of 12 Zantedeschia genotypes three to seven days after inoculation with Ecc NHRI-3 (upper) and Ecc NHRI-1 (lower). 659