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Horticultural Research Volume 8, Article Number: 13 (2021) Cite this article

Soft rot caused by Pectobacterium spp. It is the cause of serious agricultural losses of potatoes, vegetables and ornamental plants. The Zantedeschia genus includes two groups of ornamental tubers that are highly sensitive to this disease. Previous studies have shown that Z. aethiopica of the genus Zantedeschia has significantly higher resistance to Pectobacterium spp. Than a member of the same genus belonging to the Aestivae section. During the early infection period, we found different bacterial colonization patterns on the host leaves belonging to different parts. A similar pattern of bacterial colonization was observed on artificial inert replicas of polydimethylsiloxane (PDMS) on the leaf surface. In addition to biochemical plant-bacteria interactions, these replicas also confirmed the physical effects of leaf texture. Compared with species in the Zantedeschia group that are adapted to warm and marshy environments, the difference pattern may be related to the greater roughness of the back leaf surface of the Aestivae group, which has evolved to adapt to the mountain environment. Lateral leaf slices also showed compact aerated tissue and reduced the total volume of air space in the leaf tissue of Aestivae members. Finally, the analysis of defense marker genes revealed differential expression patterns in response to infection. Significantly higher levels of lipoxygenase 2 (lox2) and phenylalanine ammonia lyase were observed in the more resistant Z. aethiopica (pal), indicating a higher activation degree of induced systemic resistance (ISR) mechanism. The use of calla lily as a model plant reveals how natural ecological adaptability forms resistance to bacterial soft rot in cultivated agricultural environments.

Calla lily, commonly known as calla lily, is one of the most iconic ornamental plants in the world. It is endemic to Southern Africa, but it has been introduced into the world as a cut flower, potted plant, garden or landscape plant. The flower has developed a variety of colors, mainly in New Zealand, the Netherlands and the United States1. Calla lily is a genus of the Araceae family, with eight species divided into two parts: the calla part, which has two white species (Z. aethiopica Spreng. and Z. odorata Perry.), which bloom in late winter/spring, preferential Warm swamp habitat, and part of Aestivae with six main colored, summer-blooming calla lilies. They inhabit cool mountain environments and exhibit complete winter senescence (Z. albomaculata Baill., Z. elliottiana Engl. , Z. jucunda Letty., Z. pentlandii Wittm., Z. rehmannii Engl., and Z. valida Singh)2,3,4. Calla lily is easy to reproduce through vegetative reproduction, but due to its high sensitivity to bacterial soft rot and viral infections, further horticultural development is restricted.

Soft rot in Zantedeschia lily is mainly caused by P. aroidearum and Dickeya dadantii (former members of the Erwinia group 6,7). P. brasiliense (Pb) is an emerging pathogen in Pectobacteriaceae and has been reported in a variety of hosts, including ornamental plants and vegetables, such as Solanum tuberosum (potato), S. lycopersicum (tomato) and Cucumis sativus (cucumber) 8 The tolerance of lead to a wide temperature range has facilitated its spread to different climatic regions, from tropical regions such as Brazil to temperate regions such as Europe and North America9. Pectinella spp. are Gram-negative bacteria. They use the simultaneous production of plant cell wall degrading enzymes (PCWDE) as their main virulence properties8,10,11, and enter the host through stomata openings and wounds to colonize xylem blood vessels and thin walls. Tissues and raw xylem cells 12, 13, 14. Once the temperature, humidity, and pH are appropriate, the bacteria will proliferate to a critical number, and begin to produce and secrete large amounts of PCWDE, and finally impregnate plant tissues15. They attacked above-ground plants or underground storage institutions in fields and warehouses, causing serious losses16.

Studies have shown that there are differences in the susceptibility of the two parts of the calla lily to P. carotovorum infection2,3,4. No one proposed a mechanism as a basis for the observed variability. Since there is no treatment for the disease, including disease-resistant varieties into breeding programs may become a viable tool to overcome the disease. However, although the evergreen Z. aethiopica is more resistant to polybacterial infection than the winter dormant variety, the two groups cannot cross17,18. Interspecies hybrids are affected by plastid-genome incompatibility and produce degenerated, chlorophyll-deficient abnormal embryos4,16,18.

Here, we have characterized the morphological and biochemical differences, which can explain the higher resistance of Zantedeschia lilies to bacterial infections: anatomical differences and physical barriers restrict bacterial colonization, and the activation of immune response and defense-related genes to fight bacteria19. These characteristics are believed to be caused by different biotic and abiotic ecological pressures in the natural habitat of each Zantedeschia group. Ultimately, solving the protective function of resistant calla lily should open up new ways to improve tolerance and even resist the reproduction of soft rot.

The leaf disc assay was used to study the susceptibility of calla lily to soft rot infection, and to measure the development of necrotic areas after infection14,16. The results showed that white calla lily (ZA), white calla lily (ZA) showing less necrotic areas, and commercial colored hybrids (Aestivae) "Florex Gold" (FG, yellow), "Captain Romance" (CR, pink) And "Thermal Shot" (HS, orange) (Figure 1A, B). CR is a colored representative hybrid used for infection detection. It uses a GFP-labeled Pseudomonas brasiliensis strain (Pb) that allows the monitoring of bacterial colonization in tissues 24 hours after inoculation. Compared with ZA, confocal microscopy images showed denser and stronger colonization around the CR inoculation site. In addition, using ultraviolet excitation, a clear fluorescent ring around the bacterial penetration site was observed in ZA, but not in CR (Figure 1C). This fluorescence is a typical feature of the accumulation of phenolic compounds in the process of plant defense reactions, and is usually accompanied by the up-regulation of oxidases, mainly peroxidase (POD) and polyphenol oxidase (PPO). Compared with ZA, the POD and PPO analysis of the two Zantedeschia groups showed that the basal level of POD activity in CR was higher (Supplementary Figure S1); however, after Pb infection, POD activity was down-regulated in CR, while in ZA, Similar activity levels were observed in the treated samples and the control samples. After Pb inoculation, PPO activity in ZA increased significantly (Supplementary Figure S1), but not in CR.

A symptom was determined as the percentage of the immersed tissue in the total leaf disc area, 24 hours after inoculation with 10 µl bacterial suspension (108 CFU/ml, OD600 = 0.1), and incubation at 28 °C. The data represents the mean ± SE of three independent experiments, where four plates are taken from different plants, and each variety contains ten replicates. There are significant differences in the treatment of different markers (P <0.05). B A representative picture of a plate of each tested variety. C Confocal image of ZA and CR inoculated with GFP-expressing Pb or distilled water (control) as described above. The inoculation point is observed under a fluorescence microscope with GFP filter (left) and UV filter (right), 24 hours after inoculation

The transverse leaf sections of CR and ZA representing the two Zantedeschia groups were stained with Safranin and Fast Green and observed under an optical microscope. Lower tissue compactness and higher air space content were observed in the mesophyll tissue of ZA (Figure 2A). A similar pattern was observed in transverse petiole sections stained with toluidine blue (Figure 2B). The quantitative measurement of the cumulative air space per cross-sectional area showed that the air space volume in the leaves and petioles of ZA was much higher than that of CR (Figure 2C).

The transverse leaf sections of ZA and hybrid CR observed under an optical microscope were stained with Safranin-Fast Green. Resolution ×10, black bars 100 μm. B Transverse petiole sections of ZA and hybrid CR stained with toluidine blue and observed under an optical microscope. Resolution × 5, white bars 500 μm. C The air space ratio is calculated using ImageJ software as a percentage of the cross-sectional area. The data represents the mean ± SD of the six fully opened second leaves. The treatments marked with * were significantly different (P <0.01). Scanning electron microscope (SEM) images of the front (top) and back (bottom) leaf surfaces of D ZA and CR. Resolution×400, white bars 50 μm

Use a scanning electron microscope (SEM) to observe the paraxial (upper) and back (lower) leaf surfaces. The frontal leaf surface is covered with a cuticle that is indistinguishable between ZA and CR. However, the undersides of the leaves of the two groups are significantly different, with ZA showing a smooth surface and CR showing a ridge-like rough pattern (Figure 2D).

The bacterial colonization between the two calla lily varieties is very different, which is consistent with the difference in leaf surface patterns. Dense colonization of Pectobacterium cells was observed on the back leaf surface of CR using SEM, and scattered cells were only observed on ZA (Figure 3). The formation of biofilms and extracellular polymers observed in high-resolution scans follow the same trend. Sub-micron appendages were observed on the leaf surface of the infected CR but not on the ZA. The typical ridges and grooves on the surface of colored hybrids support the establishment of bacteria, and the orange hybrid HS is the densest colonization.

Z. aethiopica, ZA; "Captain Romance", CR; "Florex Gold", FG; "Hot Shots", HS. These photos were taken 3 hours after applying a 10 µl Pb bacillus (Pb) suspension (108 CFU/ml, OD600 = 0.1) to the leaf surface. The leaves were dried in a laminar flow hood at room temperature for 3 hours, then fixed in 70% ethanol overnight, and then dehydrated with 90, 95 and 100% ethanol. The scale of each image is in µm, and the resolution is specified at the top

In order to further test whether the differences in leaf surface patterns may be the basis of Pb population behavior and colonization, artificial leaf surfaces were produced using PDMS, a silicon-based polymer based on ZA and CR. Artificial surfaces with the same chemistry but different structures show unique colonization patterns that strictly depend on the structure of the leaf surface. Three hours after the lead was applied to the surface, the smooth pattern of ZA was covered by bacterial cells, which were apparently washed into the topographic grooves on the lower surface. Bacterial cells are scattered individually on the leaf plane (Figure 4A-D); at the same time, the artificial surface of CR encourages bacterial cells to attach and colonize on the rigid, notched leaf planes, which have already produced aggregation to the established leaf planes. Clusters of small cells in the biofilm (Figure 4E-G), as observed by SEM at higher resolution (Figure 4H).

A total of 10 µl bacterial suspension (108 CFU/ml, OD600 = 0.1) was applied to the surface of the replica. A copy of PDMS on the reverse leaf surface of ZA. BD ZA artificial surface after applying lead at resolutions of ×400, ×2000, and ×7000. A copy of E CR's PDMS, as above. FH higher resolution image of bacteria application after CR

The changes in the expression of representative plant defense genes were studied to further clarify the differences in the response of ZA and CR to Pb inoculation (Figure 5). Cloned lipoxygenase 2 (lox2), phenylalanine ammonia lyase (pal), aspartate aminotransferase (ast) and pathogenesis-related proteins (pr1), and designed primers for the qRT-PCR protocol (Table 1) for expression analysis. Under control conditions, the basal levels of lox2, pal, and ast, which represent the ISR signaling pathway, were lower in CR than ZA. After Pb inoculation, the expression of lox2 and pal increased significantly in ZA, but only pal increased in CR. These results indicate that the ISR signaling pathway is induced in both breeds, and the response of ZA is stronger. After Pb inoculation, the representatives of SAR signaling pathway pr1 in both plants were down-regulated.

Use 100 µl of bacterial suspension (106 CFU/ml, OD600 = 0.001) or distilled water (control, C) for leaf inoculation. Determine the relative transcription levels of lox2, ast, pal, and pr1 and normalize to tubulin. The bars represent the average relative expression ± SD of two independent experiments with three replicates. Bars not marked with the same mark indicate significant difference (P <0.05)

The Zantedeschia genus is divided into two plant parts: the Zantedeschia part and the Zantedeschia (white) are relatively resistant to bacterial soft rot, while the Aestivae part (colored hybrid) is highly sensitive2,17. The soft rot of the Zantedeschia genus is mainly caused by the fruit bacterium. Through all stages of on-site production and storage 20. We hypothesized that the difference in the response of the two Zantedeschia groups to Dopaella infection may indicate characteristics related to higher resistance to bacteria. Previous studies have characterized the Zantedeschia germplasm with relatively high resistance to P. carotovorum16,17,21, indicating the genetic background of resistance to soft rot based on genetic models. It is still unclear to emphasize the characteristics of these patterns2. Using P. brasiliense (Pb), a soft rot that occurs in warmer climates of potatoes and ornamental plants22, our results confirm that early research on P. carotovorum (formerly Erwinia carotovora subsp. carotovora) showed color Aestivae hybrids (FG, CR, and HS) are more susceptible to Pb infection than ZA because they all develop typical soft rot symptoms with large areas of necrosis2,4,17.

Plant canopy is the main factor for environmental survival, establishment and spread of bacterial diseases. Therefore, we focus on the leaf structure, which is the first layer of bacterial attachment, colonization and penetration. The cross-section of leaves and petioles allows characterization of the plant's internal environment. Surprisingly, the main difference in leaf surface texture between the two Zantedeschia groups was observed. The smoother and simpler structure of the back of the ZA is significantly different from the back of the CR (and other colored hybrids). On the other hand, compared with the denser CR tissue, the cross section shows a larger air space, occupying more parenchyma space in ZA. What is more obvious is the difference in air space in the petiole, which is almost twice that of CR in ZA. We speculate that these differences may be caused by the niche adaptation of the two plant parts. Although both originated in Southern Africa (Cape Province, Lesotho, Natal, Swaziland), ZA is almost entirely confined to the swampy wetlands of the southeast coastal zone up to 1,000 meters above sea level; the colored calla lily is limited to 1200 to 1200 meters above sea level. Mountain area of ​​2000 m1. ZA23 inhabits low-altitude wetlands and swamp valleys. The leaf surface is smoother, the stomata are larger, and the air space in the leaf and petiole is larger, while the Aestivae species prefers well-drained soil, tighter organization and compact life form, typical High altitude. These characteristics may also affect the availability of oxygen and may affect the early development of anaerobic conditions during bacterial attack. On the one hand, anaerobic organisms may affect oxygen-dependent host defenses, cell wall lignification and corkization, and on the other hand may affect bacterial virulence and the production of pectinase24,25. Therefore, a larger air space may determine the compatibility of bacteria and help improve the resistance of ZA. SEM micrographs taken immediately after the bacteria were applied to the leaf surface for 3 hours showed a large number of colonizations on the colored hybrids (CR, FG, and HS) with the behavioral structure of the typical bacterial population. In contrast, lower cell density and unusual colonies were observed on ZA. Since the spread of soft rot in agricultural systems is usually associated with irrigation water or aerosols26, it is clear that the leaf surface facing the ground has a significant impact on bacterial adhesion. After successful attachment, Pioneer bacteria penetrate the leaves through stoma openings, water sacs or wounds, colonize the intercellular spaces, and move to other tissues through the vascular system3,13,27. In order to independently study the effect of leaf structure on bacterial attachment and colonization, artificial polymer replicas of the leaf surface were constructed through PDMS. The colonization pattern was recorded 4 hours after the bacteria were applied to the artificial surface. The results revealed patterns comparable to those observed on the real leaf surface. The effect of surface structure on biofilm formation has been reported previously, using artificial nanostructure features. When using mesoscale patterned polymers, the surface morphology will also affect the development of biofilms, affecting the colonization, growth and persistence of species when exposed to antibiotics29. Here, the leaf structure is at least partly responsible for providing better conditions for the establishment of fruit flies on the colored calla lily, just as the formation of similar sub-micron appendages observed in E. coli during cell adhesion and biofilm formation As observed. Other factors that affect the susceptibility/tolerance of leaves to soft rot have previously been shown to involve antibacterial compounds, as well as innate or induced defense mechanisms14,31,32. Defense against polybacteria. Involving SAR and ISR signal pathways19. Here, the limited fluorescence of GFP-labeled Pb cells was observed on ZA after infection, accompanied by the accumulation of phenolic compounds around the infiltration site (observed under UV filters). The accumulation of phenolic compounds around the inoculation site may be part of the plant's defense response. The antibacterial properties of phenolic compounds34,35 may cause the growth restriction of ZA. The accumulation of phenols in ZA co-occurs with the higher activity of PPO, which is an enzyme that oxidizes phenols to quinones, which is believed to be involved in the defense of plants against microbial pathogens. The expression of defense-related genes lipoxygenase 2 (lox2), aspartate aminotransferase (ast), pathogenesis-related protein (pr1) and phenylalanine ammonia lyase (pal) were studied. lox2 encodes a key enzyme for the biosynthesis of the defensive signal hormone jasmonic acid, and is a representative of the ISR pathway in plants. The greatest increase in its expression was observed in ZA after Pb inoculation, which supports the induction of the ISR signaling pathway in this section. ast is a regulator of carbon and nitrogen metabolism and amino acid synthesis in the process of plant defense response to dead organisms. The higher expression of ast in Arabidopsis is associated with increased sensitivity to Botrytis cinerea. Here, ast expression was up-regulated in the CR response to Pb infection, while in ZA, its expression did not change after bacterial infection. Similarly, pr1 is the most recognized marker of SAR40, and it was down-regulated by Pb inoculation in both Zantedeschia species, supporting previous reports showing that SAR is less effective in resistance to Polybacteria spp14 and 33. Finally, pal expression is considered to be the first critical step in the phenylpropane pathway, leading to the synthesis of phenolic compounds. The expression level of this gene increased in both parts of Zantedeschia lily, and had a stronger response in ZA, confirming that it is involved in resistance to Pectobacterium spp36. This observation is consistent with the accumulation of phenolic compounds around the ZA infection site.

In conclusion, the less exploratory involvement of host-plant and soft-rot fungus Pb interaction is demonstrated. The emphasis on plant morphological characteristics and environmental adaptability reveals the higher resistance of Zantedeschia lilies to polybacteria. Infect. This resistance mechanism is a multifaceted phenomenon, involving multiple factors. In addition to different induced defense responses after pathogen infection, surface area structure and tissue compactness also play a role.

Commercially grown calla plants: white calla lily (Zantedeschia aetheopica) ZA and colored calla lily hybrids ("Captain Romance", CR; "Florex Gold", FG; "Hot Shot", HS) grown in a greenhouse (25/ 10 °C max/min, natural light). The youngest fully expanded leaf was cut at the base of the petiole and used fresh in all analyses. Lead isolated from potatoes and the same strain that carries green fluorescent protein-GFP (Pb) are used for research. The strain was cultured in Luria-Bertani (LB) medium (Difco Laboratories, MI, USA) at 28°C, and Pb was supplemented with 100 µg/ml ampicillin under continuous shaking at 200 rpm. P. brasiliense has a wide temperature range-20–39 °C, with an optimal temperature of 31–32 °C9. In short, 28 °C is the suboptimal temperature of Pseudomonas brasiliensis, and 28 °C was chosen to avoid drastic temperature changes in plants growing at 25 °C and to slow down the rapid response of highly sensitive colored hybrids.

Prepare leaf discs cut from different Zantedeschia species as described above, keep them on half-strength Murashige and Skoog (MS) basic medium, and challenge with 10 µl bacterial suspension (108 CFU/ml, OD600 = 0.1) or Distilled water was used as a control. The inoculated plant material was incubated at 28°C. Disease progression was assessed 24 hours after inoculation as a percentage of tissue decay relative to the total leaf disc area. Use the Threshold_Colour plug-in of imageJ software (NIH, MD, USA) to measure the attenuation area. Three independent experiments were conducted, each using 4 leaves, 10 replicates, and 40 leaf discs per cultivar (Pb per cultivar/control). Observe the inoculated leaf discs under a fluorescence microscope as described above.

The transverse hand slices cut from the petiole were placed in distilled water and stained with toluidine blue as described by Villodron 41. In addition, leaf slices 42 were prepared as explained by Ruzin et al. In short, a small leaf was fixed with FAA (formalin: glacial acetic acid: ethanol: 5: 5: 90). The fixation is followed by an ethanol dilution series, followed by a gradual exchange of ethanol with "Histoclear" (Xylem substitute, National Diagnostics GA, USA). The samples were then embedded in paraffin and cut into 20-micron-thick sections with an RM2245 microtome (Leica Biosystems, Germany). In addition, the sections were stained with Safranin and Fast Green and examined with a Leica DMLB microscope (Leica Microsystems, Germany) to observe the tissue morphology. The microscope is equipped with a DS-Fi1 camera (Nikon Instruments Inc., Japan).

Three hours after applying 10 µl of bacterial suspension (108 CFU/ml, OD600 = 0.1) to the leaf surface, the SEM observation of the leaf surface and the adhesion of bacteria to the back leaf surface of different cultivars were studied. The leaves were dried for 3 hours, then the samples were fixed in 70% ethanol overnight, and then dehydrated with 90%, 95%, and 100% ethanol for 1 hour. Finally, in accordance with the manufacturer's instructions (Quorum Technology Ltd., UK), the sample was dried at the critical point of K850 and coated with a gold-palladium alloy on a small sputter coater. The samples were observed under SEM, Jeol JSM 5410 (JEOL Inc, MA, USA). The leaves of SEM are taken from three different plants of each cultivar, with ten replicates for each plant. The image represents the bacterial colonization pattern of each calla lily cultivar/hybrid.

The polydimethylsiloxane (PDMS) prepolymer and curing agent in the Sylgard™ 184 silicone elastomer kit (The Dow Chemical Company, Michigan, USA) are mixed at a ratio of 10:1 w/w, fully stirred, and Degas under vacuum. Glue the calla lily leaf back to the petri dish. Pour the PDMS on the leaves, apply a vacuum for 2 hours, and then cure on a workbench overnight at room temperature. The next day, the PDMS layer (called a negative replica) was gently peeled off the leaves. The negative replica was activated by exposure to a plasma torch BD-20ACV, a high-frequency generator (Electro-Technic Products, IL, USA) for 30 seconds. The negative was then placed in a desiccator containing 100 µl of trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane (Sigma-Aldrich, MO, USA) and kept in vacuum for 3 hours. After activation, pour a mixture of PDMS and curing agent (10:1) on the negative. The negative film and liquid polymer were placed under vacuum for 2 hours and then cured overnight at room temperature. The newly formed polymer layer, called the positive replica, is gently separated from the negative replica and later used for bacterial colonization analysis and visualization. Three PDMS positive copies of ZA and CR leaves were used for SEM studies, 4 hours after inoculation with 10 µl bacterial suspension (108 CFU/ml, OD600 = 0.1). The experiment was repeated twice.

In order to observe the in situ reaction 24 hours after inoculation with Pb (Pb labeled with GFP), the leaf samples were examined under a Leica MZFLIII stereo microscope (Leica Microsystems, Germany) equipped with DS-Fi1 camera (Nikon, NY, USA) and NIS -Elements (Nikon, New York, USA) software (version 3.06). GFP is excited by the 440-520-nm light, and the emission is detected by the 520-600-nm GFP2 filter. To observe the autofluorescence of phenolic compounds, a 320-400 nm laser was used for excitation, and the emission was evaluated at 420 nm through an ultraviolet filter.

Using RNA buffer (10 mM Tris-HCl, pH 8.0; 1​​ mM LiCl, 0.2 mM EDTA, and 1% LiDS) and hot phenol, total RNA was isolated from the leaf segments of control and infected ZA or CR leaves. For each cultivar, 3 biological replicates were performed for each treatment, with 3 replicates for each. The leaves were infiltrated with 100 µl bacterial suspension (106 CFU/ml, OD600 = 0.001) and applied to four spots on each leaf. After 24 hours of inoculation with Pb, the leaf tissue, 200 mg fragments, was ground into a fine powder with a mixed grinding tissue lysator (Retch, Germany) under liquid nitrogen. Extract RNA and prepare cDNA as previously reported.

There is no full genome sequence of the calla lily; therefore, the cDNA sequences of the monocot species corn, wheat, rice, and the dicot model plant Arabidopsis were compared to identify conserved sites in all species. Based on these conserved sites, primers were designed and PCR was performed on the cDNA of the calla lily plant tissue (Table 1). The PCR products were then sequenced in the 3730 DNA Analyzer (Applied Biosystems, CA, USA) to obtain the cDNA sequence of specific genes in the Zantedeschia (Zantedeschia). These were further used to design qRT-PCR primers (Table 1) to quantify the expression of defense-related genes in this study. Use the pGEM®-T Easy Vector System kit (Promega, WI, USA) to clone the PCR product of the latter primer into the pGEM-T vector. In short, mix 2 µl of PCR product with 50 µl of competent E. coli (TOP10) DH5α on ice, then apply a heat shock protocol at 42°C for 45 seconds, and then add ice. The cells were incubated on a rotary shaker at 37°C for 1 hour, and then inoculated with 100 µg ml-1 ampicillin and another 100 µl isopropyl β-D-1-thiogalactopyranoside (IPTG) 0.1 M and 20 microliters of 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-GAL) 0.5 g ml-1 for blue and white screens. The transformed colonies were grown overnight in LB, supplemented with 100 µg ml-1 ampicillin, and plasmid DNA was extracted using PureYield™ Plasmid Miniprep System (Promega, Madison, Wisconsin, USA) and T7 primer 5' -TAATACGACTCACTATAGGG-3' is sequenced for verification 44.

SYBR® Green (Applied Biosystems, USA) qRT-PCR assay was used to determine the expression of defense-related genes in ZA and CR after Pb challenge infection. As previously reported, real-time PCR amplification was performed in the Step One Plus real-time PCR system (Applied Biosystems, CA, USA) using gene-specific primers (Table 1). The data was analyzed by the comparative CT (ΔΔCT) method, and the expression was normalized to the expression of the reference gene actin.

Use JMP software (SAS, Cary, NC, USA) to analyze the significance of the data by Student t test or ANOVA with Tukey-Kramer HSD. Unless otherwise stated, P <0.05 is considered statistically significant.

The gene fragment data set sequenced in this study is available on figshare.com (https://doi.org/10.6084/m9.figshare.12326495).

Funnell, KA'Calla Lily'. In the physiology of flower bulbs (eds. De Hertogh, A., & Le Nard, M.) 683–704. Elsevier, 1993.

Snijder, RC, Cho, H.-R., Hendriks, MMWB, Lindhout, P. & van Tuyl, JM Genetic variation of the calla lily. (Araceae) used to fight soft rot caused by Erwinia. Scavengers. Euphytica 135, 119–128 (2004).

Singh, US, Singh, RP & Kohmoto, K. Pathogenesis and host specificity of plant diseases: histopathology, biochemical, genetic and molecular basis. Pergamon, 1995.

Wei, Z. et al. Assess the genetic diversity and population differentiation of colored calla lilies (Zantedeschia hybrids) to develop effective breeding programs. Gene 8, 168 (2017).

Hu, W.-C., Huang, C.-H., Lee, S.-C., Wu, C.-I. & Chang, Y.-C. Using nad5 mRNA as an internal control, through multiple RT- PCR detects four calla viruses. EUR. J. Plant pathogens. 126, 43 (2009).

Ma, B. etc. The host range and molecular phylogeny of Pectobacterium and Dickeya in Enterobacter soft rot. Phytopathology™ 97, 1150–1163 (2007).

Yishay, M. etc. Differences in pathogenicity and genetic diversity between Pectobacterium carotovorum ssp. Carotovorum isolates from monocot and dicot hosts support early gene componentization within this taxa. environment. microorganism. 10, 2746–2759 (2008).

Charkowski, AO Facial change from bacterial soft rot. Anu. Pastor of plant pathogens. 56, 269–288 (2018).

du Raan, S., Coutinho, TA & van der Waals, JE Cardinal temperature difference, measured in vitro, determined between closely related species and subspecies of pectin-decomposing bacteria, causing black leg and soft rot on potatoes . EUR. J. Plant pathogens. 144, 361–369 (2016).

Toth, IK, Bell, KS, Holeva, MC & Birch, PRJ Soft rot rot erwiniae: From gene to genome. Mole. Plant pathology. 4, 17-30 (2003).

Charkowski, AO Soft Rot Ovenia. In Plant-Associated Bacteria (ed. Gnanamanickam, SS) 423–505 (Springer Netherlands, 2006).

Czajkowski, R., Pérombelon, MCM, Veen, J. Avan & Wolf, J. Mvander. Prevention and treatment of potato black leg disease and tuber soft rot caused by Fructobacillus and Bifidobacterium: a review. Plant pathology. 60, 999–1013 (2011).

Kubheka, GC, Coutinho, TA, Moleleki, N. & Moleleki, LN Brazilian strains in potato plants. Phytopathology 103, 1268–1279 (2013).

Luzzatto, T. etc. The plant activator methyl jasmonate provides effective and long-lasting resistance to the soft-rot fungus Pectobacterium carotovorum in the calla lily. Plant pathology. 56, 692–701 (2007).

De Boer, SH characterizes Erwinia pectinosa as a highly complex plant pathogen. EUR. J. Plant pathogens. 109, 893–899 (2003).

Snijder, RC, Brown, FS & van Tuyl, JM The role of plastid-genomic incompatibility and parental plastid inheritance in interspecific hybridization of the genus Calla (Araceae). Flower ornamental biotechnology. 8, 150–157 (2007).

Snijder, RC & van Tuyl, JM Test evaluation to determine the resistance of calla lily. (Araceae) Soft rot caused by Erwinia. Scavengers. EUR. J. Plant pathogens. 108, 565–571 (2002).

Yao, J.-L., Cohen, D. and Rowland, RE. Inheritance of plastid DNA and plastid-genomic incompatibility in interspecific hybrids of Calla lily (Araceae). theory. Application genes. 88, 255–260 (1994).

Davidsson, PR, Kariola, T., Niemi, O. & Palva, ET Pathogenicity and plant immunity of soft rot fungi. front. Plant science. 4, 191 (2013).

Wright, PJ, Burge, GK & Triggs, CM Effect of stopping irrigation and raising tuber time on bacterial soft rot of Zantedeschia (Zantedeschia) tubers. NZJ Crop Horticulture Science. 30, 265–272 (2002).

Cho, HR etc. The virulence variation of Erwinia carotovora subsp. 20 isolates. carotovora is on the Korean calla lily variety. Journal of Horticulture. 653-659. https://doi.org/10.17660/ActaHortic.2005.673.90 (2005).

Wolf, J. Mvander, etc. The virulence of Pectobacterium carotovorum subsp. In the prevailing climatic conditions in the Netherlands, the brasiliense on potatoes is compared with other Pectobacterium and Dickeya species. Plant pathology. 66, 571–583 (2017).

Cutler, DF, Botha, T. & Stevenson, D., Wm. Adaptive function. In Plant Anatomy: Applied Methods (Blackwell Publishing Co., Ltd., 2008).

Maher, E. & Kelman, A. The oxygen status of potato tuber tissue is related to the impregnation of Erwinia pectinase. Plant Pathology 73, 536–539 (1983).

Babujee, L. et al. The evolution of metabolic and regulatory networks related to the availability of oxygen in the two plant pathogenic Enterobacter bacteria. BMC Genomics 13, 110 (2012).

Perombelon, MCM & Kelman, A. The ecology of soft rot in Ovenia. Anu. Pastor of plant pathogens. 18, 361–387 (1980).

Pérombelon, MCM Potato disease caused by soft rot: an overview of pathogenesis. Plant pathology. 51, 1-12 (2002).

Hochbaum, AI and Aizenberg, J. Spontaneously form bacterial patterns on periodic nanostructure arrays. Nanolet. 10, 3717–3721 (2010).

Bhattacharjee, A., Khan, M., Kleiman, M. & Hochbaum, AI The effect of growth surface morphology on bacterial signal transduction in co-cultured biofilms. ACS application alma mater. Interface 9, 18531–18539 (2017).

El Abed, S., Ibnsouda, SK, Latrache, H. & Hamadi, F. Scanning electron microscope (SEM) and environmental SEM: suitable tools for studying adhesion stages and biofilm formation. In Scanning Electron Microscope (Kazmiruk, V. Ed.) IntechOpen, 2012.

Luzzatto-Knaan, T., Kerem, Z., Lipsky, A. & Yedidia, I. The protein expression pattern and signal molecule accumulation in Calla lily proves the systemic response of native plants. Plant Physiology. Biochemical. 71, 218–225 (2013).

Wegener, CB & Jansen, G. Resistance of colored potato varieties (Solanum tuberosum L.) to soft rot: the role of anthocyanins. Potato Resources 50, 31–44 (2007).

Luzzatto, T. etc. In the ornamental monocot plant Zantedeschia lily, it induces antibacterial phenolics during the process of inducing the resistance reaction against Fructus bacillus. J. Agriculture. Food Chemistry 55, 10315–10322 (2007).

Joshi, JR, Burdman, S., Lipsky, A. & Yedidia, I. The effect of plant antibacterial phenolic compounds on the virulence of Polybacteria. Reservoir microorganisms. 166, 535–545 (2015).

Joshi, JR, Burdman, S., Lipsky, A., Yariv, S. & Yedidia, I. Plant phenolic acids affect the virulence of Pectobacterium aroidearum and P. carotovorum ssp. Brasiliense is regulated by quorum sensing. Mole. Plant pathology. 17, 487–500 (2016).

Ngadze, E., Icishahayo, D., Coutinho, TA & van der Waals, JE Polyphenol oxidase, peroxidase, phenylalanine ammonia lyase, chlorogenic acid and total soluble phenols in potato resistance to soft rot In the role. Plant distribution 96, 186–192 (2012).

Yan, L. et al. The role of tomato lipoxygenase D in wound-induced jasmonic acid biosynthesis and plant immunity to insect herbivores. PLoS gene. 9. e1003964 (2013).

Zhou, Y. et al. Overexpression of the aspartate aminotransferase gene in rice leads to changes in nitrogen metabolism and an increase in amino acid content in seeds. theory. Application genes. 118, 1381–1390 (2009).

Brauc, S., De Vooght, E., Claeys, M., Höfte, M. & Angenon, G. Overexpression of cytosolic aspartate aminotransferase on the amino acid metabolism and defense response of Arabidopsis gray mold infection Influence. J. Plant Physiology. 168, 1813–1819 (2011).

Kiefer, IW & Slusarenko, AJ The relationship between the pattern of systemic acquired resistance induction and the pattern of translocation in the Arabidopsis rosette. Plant Physiology. 132, 840–847 (2003).

Willowden, AQ, etc. Characterization of Adventitious Root Development of Sweet Potato. Horticultural Science 44, 651–655 (2009).

Ruzin, SE Plant Microscopy and Microscopy, Vol. 198 (Oxford University Press, New York, 1999).

Khadka, N. etc. Host specificity and differential pathogenicity of Fructus strains from dicot and monocot hosts. Microorganism 8, 1479 (2020).

Mead, DA, Szczesna-Skorupa, E. & Kemper, B. Single-stranded DNA "blue" T7 promoter plasmid: a multifunctional tandem promoter system for cloning and protein engineering. Protein engineering. Germany. Sel. 1, 67–74 (1986).

The research was funded by the Chief Scientist of the Israeli Ministry of Agriculture (approval number: 20-01-0193).

The contributions of these authors are the same: Yelena Guttman, Janak Raj Joshi

Robert H. Smith School of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel

Yelena Guttman, Janak Raj Joshi, Nofar Chriker, Nirmal Khadka and Zohar Kerem

Institute of Plant Science, Agricultural Research Organization, Volcano Center, Rishon Lezion, Israel

Yelena Guttman, Nofar Chriker, Nirmal Khadka, Maya Kleiman, Noam Reznik, Zunzheng Wei and Iris Yedidia

Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, Colorado, USA

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YG, JRJ, NK and NR conducted experiments, analyzed data, and prepared papers. NT and ZW conducted experiments and analyzed micro data. MK, ZK, and IY coordinated experiments, data analysis and wrote papers.

The author states that the research was conducted without any commercial or financial relationships that could be interpreted as potential conflicts of interest.

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Guttman, Y., Joshi, JR, Chriker, N. etc. Ecological adaptation affects the sensitivity of Zantedeschia plants to the soft rot disease Pectobacterium spp. Hortic Res 8, 13 (2021). https://doi.org/10.1038/s41438-020-00446-2

DOI: https://doi.org/10.1038/s41438-020-00446-2

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