Introduction
Calla lily (Zantedeschia spp. Araceae), comprises eight species, all of them native to southern Africa (Singh et al. 1996). This group is judged by consumers to be a very attractive plant and having multiple uses: as bedding plants, as commercial cut flowers and, recently, as potted flowering plants (Kuehny 2000). This plant has been cultivated worldwide for many years as a cut-flower crop but its commercial production has decreased dramatically in recent years (MAFF 2008). The number of calla lily growers and the cultivation area per grower has decreased during the last decade due to severe attack by bacterial soft rot. This has resulted in substantial plant losses during field cultivation, due principally to infection of the flowers and loss of tubers during storage.
Bacterial soft rot is caused by the pathogen Pectobacterium carotovorum subsp. carotovorum (syn. Erwinia carotovora) and poses a major threat to commercial calla lily production worldwide (Ni et al. 2010). In the field, this pathogen enters the aboveground plant body or the belowground tuber through wounds or exposed areas and causes rotting of parenchyma tissues (P’erombelon and Kelman 1980). The plants first yellow, then release a foul smell as the tissues rapidly macerate and the plants die within few days (Wright and Burge 2000). A number of factors such as the unavailability of an effective bactericide (Blom and Brown 1999), the wide range of alternate hosts for the pathogen, a broad range of virulence and a wide genetic variability (Avrova et al. 2002; Gardan et al. 2003) makes this disease a major challenge for pathologists. This pathogen is endemic in the soil, making it extremely difficult to control even through re-planted with disease-free seed tubers (Funnell and MacKay 1999). Low soil aeration, high relative humidity, high temperature and wounding of tubers during storage all exacerbate the spread of this disease (Wright and Burge 2000; Wright and Triggs 2011). Preventive control measures for bacterial soft rot include appropriate watering, mulching and substrate ventilation (Funnell and MacKay 1999; Wright and Burge 2000). Application of calcium to the soil has been positively correlated with the control of soft rot to some extent (Funnell and MacKay 1999). Meanwhile, foliar application of methyl jasmonate (10 mM) has proved effective bestowing longlasting resistance against the disease (Luzzatto et al. 2007). Due to the necrotrophic nature of this pathogen (it feeds on dead or dying plant parts) and because it has salicylic acid dependent signaling pathways (Glazebrook 2005) it is difficult to control this disease completely and thus no efficient control method has yet been reported. However, a combination of different cultural practices combined with the growing of resistant cultivars, seems to be the most appropriate way to control this disease. However, most commercial cultivars of calla lily are highly susceptible to Pectobacterium and, till now, no work has been done to identify resistant cultivars within the Zantedeschia spp group. Some preliminary work have shown that Z. aethiopica is much less susceptible to this disease than genotypes from the section Aestivae (Yao and Cohen 2000), although this observation has not been further investigated. Therefore, the aim of this study was to explore genetic variation in resistance to bacterial soft rot in the cultivated varieties and in some crossable varieties and also to attempt to develop any Zantedeschia aethiopica cultivars showing high-level resistance against soft rot. The leaf-disk test (LDT) was used to quantify resistance against this disease.
Materials and Methods
Plant material
Plant materials of Zantedeschia were imported from Silver Hill Seeds Co., South Africa and a breeding company in Korea including Z. aethiopica ‘Childsiana’, ‘Crowborough’, ‘White Dream’, ‘Wedding March’, ‘Speckled’, ‘Green Godness’, ‘Pink Mist’ and Z. odorata. These genotypes were used to develop breeding lines and were evaluated for the selection of the parents for soft-rot resistance breeding. Despite its known susceptibility to soft rot, ‘Childsiana’ was the only cultivar selected for use as the maternal parent in crosses because of its inherited multi-flowering characteristics and popularity with consumers. ‘Pink Mist’ and Z. odorata were unable to produce viable pollen which prevented their use as paternal parents.
Crosses of Zantedeschia genotypes were made and six months after fertilisation the progenies obtained from each cross combination were propagated in 72-cell plug trays filled with an amended peat-base media. After leaf emergence, the seedlings were transplanted and grown on in plastic pots.
Bacterial strain
Pectobacterium carotovorum subsp. carotovorum (syn Erwinia carotovora) was isolated from a Zantedeschia accession (Ecc NHRI) that was imported from Plant Research International, Wageningen University, Netherlands. This bacterial strain was cultured on solid media (LB broth) and was sequentially diluted with Ecc NHRI 1 to 7 (1 × 101 to 1 × 107). The aggressiveness level of seven Pectobacterium isolates (Ecc NHRI-1 to -7) of were tested for resistance in Zantedeschia cultivars. Of the seven isolates, Ecc NHRI-3 showed most aggressive virulence to our selected Zantedeschia cultivars. Therefore, this isolate was used in this study to evaluate soft rot resistance in Zantedeschia.
Leaf disk test (LDT)
Two newly-developed leaves of each selected genotype were harvested. After folding the leaf blade, twelve disks per blade were cut using a 22 mm diam. cork borer. Discs were immediately placed, one per well, in a 12-well plate, each well containing 5 mL of inoculum (1 × 107 cfu mL−1). Incubation was carried out in a controlled environment chamber at 20℃ and 100% RH (Snijder and Van Tuyl 2002). Data were recorded after three, four and six days of incubation to quantify disease symptom levels. The fraction (%) of diseased surface area was estimated visually using a light box. The test was carried out with three leaf-disc replications per seedling genotype.
The resistance level of each genotype against Pectobacterium was measured and compared relative to the maceration percentage at the stage (3, 4, and 6 days) of 0 ~ 10% maceration range of the leaf-disk test against the resistant control cultivar ‘Wedding March’. One of four levels of resistance was assigned to each genotype, where R = highly resistant (1 ~ 10%), MR =moderately resistant (11 ~ 30%), S = susceptible (31 ~ 90%) and VS = very susceptible (91 ~ 100%). All genotypes rated MR and/or R by the LDT were retained and all others (i.e. those rated S and/or VS) were discarded.
All F1 hybrids were cultivated along with the control cultivar under the same conditions in a plastic house for three years (2004-2006). At the end they were again compared with the control cultivar for resistance level (LDT) and for plant survival ratio. During the three-year trial, any breeding lines found to be severely infested with Pectobacterium were discarded.
Statistical analysis
For the LDT, cultivar differences in healthy tissue (LH) and in the fraction (%) of macerated disk area (P), were assessed using an ‘iterative reweighted residual maximum likelihood’ algorithm (IRREML). This assumes a binomial distribution and employs a logit link. In this procedure, datasets can be arranged in a Generalised Linear Mixed Model (GLMM). This model can be used to fit unbalanced data that are not normally distributed, and have random components of variance (Keen and Engel 1998). Spearman’s rank correlation coefficients of the estimates of means of the cultivars were calculated. All analyses were done using the statistical analysis software package Genstat 6 (Genstat 6 Committee 2002).
Selected lines were evaluated according to the Manual for Agricultural Investigation (RDA 1995) and the guidelines for the conduct of tests for distinctness, homogeneity and stability (UPOV 1991). Preference ratings (excellent, good, satisfactory, poor, very poor) for the selected lines were then obtained from growers and from flower-market experts at NIHHS. These discontinuous, categorical ratings were analysed by assigning to each a numerical value (where excellent = 5; good = 4; satisfactory = 3; poor = 2; very poor = 1) which allowed them to be combined and averaged to create a semi-continuous rating.
Results
Selection of resistance line for crossing
From the evaluation of results, the three cultivars ‘Wedding March’, ‘White Dream’ and ‘Crowborough’ emerged as showing strong resistance to Pectobacterium, one cultivar, ‘Speckled’ was found to be moderately resistant and three cultivars, ‘Childsiana’, ‘Green Goddess’ (Greenish spathe) and ‘Pink Mist’, were found to be susceptible. Wild-type plants of the species, Z. odorata and Z. aethiopica from South Africa were also found to be susceptible to Pectobacterium (Table 1).
Hybrid selection by LDT
Table 2 shows segregation by resistance level of Zantedeschia plants obtained after crossing of various Zantedeschia genotypes. A total of 703 plants were evaluated in comparison with ‘Wedding March’ (the standard resistant cultivar for maceration percentage) using the LDT. Natural mortality was recorded in 39 of these plants. Of the remainder, 544 plants were rated as susceptible to Pectobacterium (31 ~ 90% macerated) and were discarded.
Field selection
Field selection was carried out to assess the survival over three years of 120 selected hybrid plants (out of the original 703 plants). After the first year's field observations and further LDT tests, only 54 plants remained. The number of plants remaining continued to fall each year leaving 32 plants (end year 2) and 15 plants (end year 3) (see Table 3).2
Most (12) of the lines remaining after field selection were observed and evaluated for a range of characteristics according to the Manual for Agricultural Investigation (RDA 1995). Fig. 1 shows that survival percentage of lines no. 50 and 86 were highest among those in the trial. These two lines were finally selected after evaluation of their resistance levels, flowering characteristics and following the guidelines for the conduct of tests for distinctness, homogeneity and stability of calla lily (Zantedeschia spp.) (UPOV 1991). A pedigree diagram of these newly-developed calla lily lines is presented in Fig. 4 where they are now named as ‘Silky White’ and Mont Blanc’. Morphological characteristics of ‘Silky White’, ‘Mont Blanc’ and ‘Childsiana’ were recorded and are presented in Table 4. Flower colour was similar in all three cultivars but the leaf colour of ‘Mont Blanc’ (Fig. 3) was brighter than of the other two cultivars. Spadix colour of ‘Silky White’ and ‘Mont Blanc’ was darker yellow than in ‘Childsiana’. Spathe size of ‘Silky White’ and ‘Mont Blanc’ was bigger than in ‘Childsiana’. Flowering and growth traits of the three cultivars are presented in (Table 5). ‘Silky White’ and ‘Mont Blanc’ flowered 6 days and 1 day earlier, respectively, than ‘Childsiana’. ‘Silky White’ plants were 33 cm taller, and ‘Mont Blanc’ were 16 cm taller than ‘Childsiana’. ‘Silky White’ average number of flowers per plant (7.3), exceeded ‘Childsiana’ (5.5) and ‘Mont Blanc’ (4.8). Larger flowers were observed in ‘Silky White’ and ‘Mont Blanc’ compared with ‘Childsiana’ (Table 5).
Preference evaluation of the newly-developed cultivars was also carried out. Growers, consumers, researchers and agricultural advisors NIHHS were selected randomly and their preferences are recorded (Table 6). Multiplication characteristics of all three cultivars were also carried out. It was found that cultivar ‘Silky White’ ranked excellent in the preference evaluation, followed by ‘Mont Blanc’ and ‘Childsiana’.
Discussion
To identify the most aggressive isolate for this study, seven Pectobacterium (E. carotovora) isolates were compared. Variation in aggressiveness in different isolates was also observed by other researchers (Darling et al. 2000; Ren et al. 2001; Smith and Bartz 1990). This emphasises the wide genetic variation for this pathogen (Avrova et al. 2002; Seo et al. 2002). In Zantedeschia, variation was observed in resistance level against bacterial rot in the different cultivars under study. A reason for this could be that, Z. aethiopica is a variable species that is widely distributed around the world in a range of agro-climatic zones (Letty 1973). Environmental variables are one of the possible causes for resistance variation against Pectobacterium. This contrasts with Z. odorata, which is found to be susceptible to this pathogen and also has a much less-varied distribution, being limited to South Africa (Perry 1989). It is also noted that each plant has its own defense system against pathogen attack (McDowell and Dangl 2000; Thomma et al. 2001). Other studies on bacterial soft rot resistance in different plant species also showed low reproducibility (Lojkowska and Kelman 1994; Schober and Vermeulen 1999) but the exact reasons for this are not clear. It may be related to the complex mechanisms involved in the activation of the pathogen, E. carotovora, as demonstrated by Mukherjee et al. (2000). However, this has not yet been examined in relation to infection of calla lily under field conditions. Conventional breeding requires estimation of genetic variation within the cultivated species as well as in its crossable cultivars. The potential for resistance breeding against soft rot has not previously been explored in the genus Zantedeschia. Moreover, selection and identification of resistant genotypes requires sensitive tests to confirm disease resistance. However, these have not yet been thoroughly investigated and no truly resistant cultivars are available. For breeding Zantedeschia spp. for resistance to soft rot, information based on findings from other crops could be useful, e.g. potato (Lojkowska and Kelman 1994; Tzeng et al. 1990), cabbage (Ren et al. 2001) and broccoli (Darling et al. 2000). During breeding of calla lily genotypes for resistance against Pectobacterium, it is suggested that plants be prescreened using LDT. This pre-screen test should allow discrimination between the most susceptible and resistant genotypes. Results of the present study are in agreement with previous work (Cho et al. 2008; Snijder et al. 2002, 2004). There is an urgent need to research the tolerance mechanisms involved in the resistance of Zantedeschia to Pectobacterium car otovorum. This information is essential if we are to make rapid advances in resistant breeding in this genus.
After confirmation of LDT, scientific analysis of growth and flowering characteristics and preference evaluation, application documents were submitted to the Korea Seed and Variety Service for registration of the newly developed varieties ‘Silky White’ and ‘Mont Blanc’. ‘Silky White’ and ‘Mont Blanc’ are the first resistant cultivars of a planned breeding programme which aims to produce a range of new hybrid Zantedeschia cultivars resistant to Pectobacterium. It is confidently expected that the commercial release of these two new, fairly-resistant cultivars will significantly reduce the impact of soft rot disease on the Zantedeschia industry and will encourage existing growers to remain in the calla lily industry and for new participants to join it.
