0051-01-0021-0004-2.pdf511.5KB

Introduction

 Calla lily (Zantedeschia spp., Araceae), is a very attractive plant that consists of 8 species in 2 sections originated from South Africa (Dole and Wilkins 1999; Letty 1973; Singh et al. 1996). Section Zantedeschia consists of Z. aethiopica, further called Aethiopica-group. Most of Zantedeschia hybrids with colored flowers have developed after crossing within section Aestivae (Singh et al. 1996). Calla lilies are indeed a commercial high value crop with great economic potential but this crop is facing increased risk of disease during the calla lily production cycle (Yao et al. 1995). Calla lily commercial cultivation is facing detrimental effects of an anaerobic pathogen E. carotovora (currently known as Pectobacterium carotovorum) that causes bacterial soft rot disease. Involvement of Paenibacillus polymyxa in pathogencity of calla lily was also reported by Mikicinski et al. (2010). E. carotovora causes rotting and maceration of parenchymatous leaf tissues and tubers that result in complete mortality of calla plants (Snijder et al. 2004). During storage of calla lily tubers, 15-20% loss can occur due to this disease, whereas, in field conditions this pathogen enters the plant through wounds or exposed area and multiplies rapidly within plant body. This result in total decay of calla plant and death losses can approach 100% in severe infestation (Clemens and Welsh 1993; Corr et al. 1993; Kuehny 2000). Different approaches like, cultivation environment, biological control and by using disinfectants and antibiotics have been attempted to control this pathogen. Cultural practices can partially reduce the extent of this disease (Wright et al. 2002), but any chemicals or hot water treatment were not found affected against this pathogen (Snijder et al. 2004; Tzeng et al. 1999). Calcium and silicate application on pathogenicity of E. carotovora proved to be effective (Cho et al. 2013) but this not the absolute control of this pathogen; therefore, selection of resistant cultivars against this disease is only the possible option. But, lack of efficient screening methods for resistant varieties impedes the breeding for soft rot resistance in lily. Different experiments were conducted to determine resistance level between Zantedeschia genotypes (Corr and Widmer 1990; Darling et al. 2000; Funnell and Mackay 1999) however, they were not able to estimate the resistance level because there was no identified screening method for this pathogen in calla lily (Snijder et al. 2004). Identification of the most effective screening method for a specific pathogen is very important in resistance breeding (Amodu and Akpa 2012). Until now, a few screening methods, which have clear results, have been developed in different plants like, in beans and calla lily, detached leaf test method was developed for rust resistance evaluation (Herath et al. 2001; Joung et al. 2013).

 The status of calla industry is growing all over the world and farmers are showing eagerness in its cultivation and trade. It is therefore need of the time to develop the subsequent soft rot disease management innovations to enable this industry to establish commercial reputation with better stability. Advancements in disease management caused by E. carotovora during cultivation and storage of tuber would possibly leads to minimize the farmers’ losses and help to establish calla cut flower industry in the country. To get information on the level of E. carotovora resistance of calla lily genotypes and on the virulence variation between isolates of E. carotovora is a prerequisite for E. carotovora resistance breeding in Zantedeschia. The objective of present study was to compare effectiveness of four different screening methods for detecting resistance against E. carotovora.

Materials and Methods

Plant materials

 In present study, all cultivars from section Zantedeschia and from section Aestivae were obtained from commercial sources (Table 1). To conduct tuber test, tubers werecollected from 1 year old plants that were produced in vitro (T₁-tubers). For tuber slice test, tubers were collected from T₂-plants (two teams from in vitro propagated plants). All collected tubers were stored at 10℃ with 70% relative humidity. All tubers were treated with 100 mg · L¹ gibberellic acid for 10 min (GA₃, Berelex, Bayer) before sowing to enhance the germination (Funnell and Mackay 1993). T₁-tubers were planted in a greenhouse by maintaining 20-30℃ day temperature and 15℃ night temperature.

Table 1. Zantedeschia genotypes within section Zantedeschia and Aestivae that were evaluated for their resistance to soft rot caused by Erwinia carotovora.

Bacterial isolates

 All isolates of E. carotovora were stored at -80℃ using solid medium (0.8% Lab-Lemco Broth, 86 mM NaCl₂, 1.5% bacteriological agar) adding Glycerol 20% on single colony isolation. The bacterial concentration was measured by using a haemocytometer. 0.01 mm depth, 0.0025 mm² for 4 boxes, 0.04 mm² for 5 boxes of a haemocytometer standard was used for the E. carotovora counting method. Before counting, the haemocytometer and the cover glass were dipped in 70% ethyl alcohol and cleaned with lens paper. 10 μL of E. carotovora suspension was dropped in the space between the haemocytometer and the cover glass. Under the microscope, concentration of E. carotovora isolate was counted, calculated and fixed to the 1 × 10⁵, 10⁷, 10⁹ bacteria/mL density by experiment condition.

 Four methods i.e. leaf disk, petiole test, tuber test, and tuber slice test were used to determine the resistance level in calla lily. Conditions in all four methods were arranged as done by Snijder and Tuyl (2002).

Fig. 1. Leaf disk test was used for determining resistance levels of Zantedeschia plants. A, The leaf punching position of the Zantedeschia leaf of leaf disk test B, Each disk was submerged in the 5 mL bacterial inoculum and incubated in a moist chamber controlled with 100% relative humidity, 20℃, constantly.

Fig. 2. Petiole test, Petioles were placed into 5 mL inoculum (1 × 10⁵ cfu/ mL) in plastic test tubes (=2.0 cm) and incubated in an environmental chamber at 100% RH for five days.

Fig. 3. Photograph of Erwinia tuber slice test. A, Tuber slice test, 1 × 10⁵ E. carotovora subsp. carotovora inoculum soaked disk of lab paper ϕ 5 mm was inoculated on the surface of tuber slice, B, tuber slices were placed on the layer of sterile water soaked lab paper spread out in a sawing box.

Data collection

 Observations were recorded after 3 to 6 days after incubation. The percentage of the macerated surface area was estimated on a light-box visually. Each replication was comprised of 2 leaves with 12 disks from each leaf.

Statistical analysis

 Iterative reweighted residual maximum likelihood algorithm (IRREML, assuming a binomial distribution using a logit link) was used for leaf disk and petiole test. Estimation of differences between healthy tissue (LH) and percentage of age of macerated disk area (P), respectively were done by using IRREML. In this method data can be set to a Generalized Linear Mixed Model (GLMM) which enables to arrange unbalanced data sets with random components of variance that are not distributed normally (Keen and Engel 1998). Whereas, in tuber and tuber slice test, ANOVA and Duncan’s multiple range (DMR) test was used for estimation of differences between cultivars by using SAS (Statistical Analysis System, V. 9.1, Cary, NC, USA) software (Haynes et al. 1997).

Results and Discussion

 In present study, E. carotovora isolates Ecc NHRI-3 and PD 1784 which were identified as most aggressive isolates were used for screening variation in resistance. Eleven genotypes from section Zantedeschia were used for variation studies against E. carotovora by using the leaf disk test in two trials (Table 2). It was observed that in both trials all genotypes showed variation in resistance level and could be categorized in 3 groups on the basis of their resistance level. Four of the genotypes were resistant against this pathogen, whereas, 6 genotypes were moderately resistant and only one genotype was susceptible. Z. aethiopica wild species exhibited strong resistance, but other wild species showed moderately resistance. Variation in resistance levels within section Aestivae was also investigated and 21 cultivars were tested to identify their resistance level to E. carotovora (Table 3). It was observed that in both trials the resistance levels among cultivars from different sources are similar. ‘Galaxy’, ‘Florex Gold’, ‘Mango’ and ‘Treasure’ cultivars were very susceptible whereas, ‘Coral Sunset’, ‘Hazel Marie’ and ‘Neroli’ were categorized in susceptible cultivar group. It is clear from present findings that most of the cultivars that belong to section Aestivae were susceptible to bacterial soft rot and ‘Florex Gold’ was identified as susceptible control cultivar. Wild germplasm of Zantedeschia spp. were also tested for E. carotovora resistance. It was found that almost all wild genotypes were susceptible to E. carotovora except Z. rehmannii and Z. albomaculata which were moderately resistant (Table 4). Twenty five genotypes of Zantedeschia spp. were also tested with inoculation of Ecc NHRI-3 (Table 5). It was recorded that among 25 genotypes, 3 were resistant and 3 were moderately resistant, whereas, 13 and 6 genotypes were categorized as susceptible and very susceptible respectively.

Table 2. Maceration percentage of leaf disk tissue by Erwinia carotovora subsp. carotovora PD1784 among twelve genotypes of section Zantedeschia.

Table 3. Maceration percentage of leaf disk tissue in Zantedeschia spp. of section Aestivae cultivars after inoculation by Erwinia carotovora subsp. carotovora PD 1784.

Table 4. Maceration percentage of leaf disk tissue in Zantedeschia spp. of section Aestivae wild genotypes after inoculation by Erwinia carotovora subsp. carotovora PD 1784.

Table 5. Maceration percentage of leaf disk tissue by inoculation of Erwinia carotovora subsp. carotovora ‘Ecc NHRI-3’ among 25 genotypes of Zantedeschia 4 days after inoculation.

 To determine the efficacy of four methods for screening resistance level against E. carotovora, 8 genotypes were used. Among these genotypes ‘Crowborough’ was already categorized as resistant cultivar, whereas, ‘Pink Persuasion’, ‘Best Gold’, ‘Black Magic’, ‘Sensation’ and ‘Treasure’ as susceptible cultivars. Cultivars ‘Galaxy’ and ‘Forex Gold’ were categorized as very susceptible. It was observed that all the four methods used in this study resulted in almost similar groups for resistance level (data not shown). ‘Crowborough’ proved to be the least susceptible cultivar, whereas, ‘Treasure’ and ‘Florex Gold’ were identified as highly susceptible cultivars in four screening tests. ‘Pink Persuasion’ and ‘Black Magic’ exhibited lower susceptibility against E. carotovora. It was also revealed that the cultivars ‘Best Gold’, ‘Sensation’, and ‘Galaxy’ showed a non-consistent behavior in all methods applied. The cultivar ‘Sensation’ exhibited as a relatively susceptible genotype in three screening methods, but in leaf disk method it seemed to be relatively resistant cultivar. ‘Galaxy’ was classified as highly susceptible cultivar in leaf disk method but it behaved less susceptible in petiole and tuber slice methods. In leaf disk test the cultivar ‘Best Gold’ recorded as relatively resistant but this cultivar was designated moderately susceptible according to tuber and petiole screening methods. This showed that the both tuber tests were very reproducible and a non-significant correlation was observed between leaf disk and other methods. Leaf disk test was more appropriate to conduct and usually had lesser coefficients of variation when compared with other methods but it cannot be correlated with field results (Paul et al. 1995). Whereas, Jan and Honma (1976) tested many methods and reported that petiole inoculation was more reliable that give more differentiation of resistant and susceptible plants than other methods.

 Difference in resistance level against E. carotovora pathogenicity was observed within sections Zantedeschia and Aestivae. It is important to note that characterization of plant resistance against pathogens can be established after observation of cultivated species and their wild relatives (Simms 1996). Some genotypes show resistance against some specific pathogens because they can identify and exclude it through the hypersensitive response, whereas some are susceptible because they cannot recognize pathogen until after the disease is well-spread (Staskawicz et al. 1995). Inoculation of pathogen isolates on test cultivars will establish a resistance prole for each genotype. Different methods showed variation in resistance level and this variation can be control by inoculating plants with large equal doses of the pathogen’s isolates (Morrison 1996). Although these findings are more accurate for identification of resistance qualitatively, whereas, quantitative reactions to natural infestation are mostly uncorrelated with those perceived after pathogen inoculation (de Nooij et al. 1995). Before establishing a solid conclusion about resistance level in different genetic makeup it is important to survey intensity of the infection on same host genotypes in different locations and time of the year (Jason et al. 2004). It is recommended that for selection of resistant genotypes against E. carotovora in any breeding program to perform pre-screening of seedlings by using leaf disk method. Whereas, tuber slice test should be used for screening of subsequent genotypes at a later growth stage in any breeding program. This pre-screening evaluation would helpful to categorize resistant and susceptible cultivars. Tuber slice test can be used to find out resistance in selected clones efficiently. Leaf disk test can be used for the determination of resistance level as it is non-destructive test. Leaf disk test had more advantages compared with other screening methods such as multi-race or multi-pathogen testing without facing difficulties due to systemic acquired resistance, the capability to assess and keep susceptible genotypes (which is essential in genetic testing) and large number of replication when testing single plants of rare genotypes.