Relationship of Resistance-Related Enzyme Activity and Salicylic Acid Content in Phalaenopsis Species with Different Levels of Resistance to Dickeya dadantii

Four Phalaenopsis species, namely P. amboinensis, P. pantherina, P. schilleriana, and P. amabilis, were collected from nurseries in West Java. The plants of each species were adapted to experimental conditions in a plastic, shading net house under 50–80% relative humidity, at 27–30 °C, with 80% shading. Fertilizers and vitamin B1 were applied twice a week, while fungicides, acaricides, and pesticides twice a month for three months.

D. dadantii isolate was obtained from Plant Molecular Biology Laboratory (PMB Lab), Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University. The isolate was previously confirmed as pathogenic to Phalaenopsis (Lubis et al. 2020; Sanjaya et al. 2020; Putri et al. 2021). Isolate preparation was made, according to Sanjaya et al. (2020). Briefly, a single colony of D. dadantii was cultured in 15 mL of lactose broth medium and shaken (100 rpm) overnight at room temperature. The culture was then centrifuged at 8,000 rpm for 6 minutes. The pellet was diluted with sterilized water to the concentration OD₆₀₀ = 0.2. The inoculation was performed using detached leaves following Putri et al. (2021) protocol. The leaf of each plant was cut into 2 cm long parts. The abaxial sites were gently pricked using a sterilized needle. The wound was dropped with 10 µL of D. dadantii culture (inoculum concentration 6 × 10⁶ CFU). The leaf cuts were then incubated in covered plastic boxes lined with wet tissue paper at the humidity of 75%.

Diameters of rot lesions (SrSD) on inoculated leaves were measured every six hours until 18 hours post-inoculation (HPI) using an automatic caliper. The results were scored on the scale from 0 to 10:

0 (no symptoms);

Symptom scoring was used to calculate the disease severity (DS) following the formula by Sanjaya et al. (2020): (1) DS (%)=Σ (ni ×vi)Z×N×100% {\rm{DS}}\,(\%) = {{\Sigma \,({\rm{ni}}\, \times {\rm{vi}})} \over {{\rm{Z}} \times {\rm{N}}}} \times 100\%

ni – 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

The plant resistance level to D. dadantii was classified based on DS value:

DS of 0–20% – resistant (R);

Colony-forming units (CFU) from infected leaves were observed at 18 hours post-inoculation. The 50 mg leaf samples were taken from the area around the soft-rot symptom. The samples from three infected leaves from each species were mixed and ground together in 1 mL of sterile distilled water. The suspensions were diluted into seven serial dilutions. Each dilution (3 µL) was dropped into NA medium, incubated at room temperature overnight, and the growing colonies were counted and normalized as CFU per mg following the formula: (2) infected leaf sample (CFU⋅mg−1)=number of colonies3 μl×1000 μl50 mg×dilution factor \matrix{{{\rm{infected}}\,{\rm{leaf}}\,{\rm{sample}}\,({\rm{CFU}} \cdot {\rm{m}}{{\rm{g}}^{- 1}}) =}\cr{{{{\rm{number}}\,{\rm{of}}\,{\rm{colonies}}} \over {3\,\mu {\rm{l}}}} \times {{1000\,\mu {\rm{l}}} \over {50\,{\rm{mg}}}} \times {\rm{dilution}}\,{\rm{factor}}}\cr}

The area under the disease progress curve (AUDPC) was calculated by using the formula below: (3) AUDPC=∑sympton diameter difference2×observation time interval \matrix{{{\rm{AUDPC}} =}\cr{\sum {{{{\rm{sympton}}\,{\rm{diameter}}\,{\rm{difference}}} \over 2} \times {\rm{observation}}\,{\rm{time}}\,{\rm{interval}}}}\cr}

To evaluate the relationship between POD and PAL activities with resistance ability, we performed quick screening for these four species (P. amboinensis, P. pantherina, P. schilleriana, and P. amabilis). Three leaf samples from three biological repeats of each species were inoculated with D. dadantii (inoculum concentration of 3 × 10⁶ CFU) and taken for protein extraction at 12 and 16 HPI. Three leaves of each species were mixed into one sample for protein extraction and then used to quickly screen PAL and POD activities.

To confirm the defense mechanism, we choose two species with a distinct resistance to D. dadantii to perform POD and PAL activities and SA contents. This step used four biological repeats for each species inoculated by D. dadantii with detached leaf inoculation methods (inoculum concentration of 6 × 106 CFU) and compared them to non-inoculated leaves (mock inoculation) using sterile distilled water. Leaf samples from inoculated and non-inoculated plants were taken at 12 HPI and ground for protein extraction.

Proteins were extracted following the protocol by Sukma et al. (2012). The 0.5 g of leaf tissue was ground in cold phosphate buffer (50 mM, pH 7) at a 1 : 4 (w : v) ratio and centrifuged at 5,000 rpm and 4 °C for 10 minutes. The supernatant was then taken, and total dissolved protein was determined using the protocol outlined in Lowry et al. (1951). Buffer A (0.9 mL, 2% Na₂CO₃ in 100 mL of 1 N NaOH) was added to 1 mL of protein and homogenized by vortex, and then incubated at 50 °C for 10 minutes. The 0.1 mL of buffer B (0.5% CuSO₄·5H₂O in 100 mL of 1% Na₂Tartrate·2H₂O) was added to the solution, followed by homogenization, and incubated at room temperature for 10 minutes. Then 1 mL of buffer C (50 mL of buffer A + 1 mL of buffer B) was added, followed by homogenization and incubation at room temperature for 10 minutes. As much as 1 mL of buffer D (50% Folin–Ciocalteu dissolved in H₂O) was added, homogenized, and incubated at room temperature for 10 minutes. The absorbance of the solution was measured on a spectrophotometer at a wavelength of 500 nm. The total dissolved protein was determined by using a standard curve from bovine serum albumin (BSA). Tissue protein content was determined by dividing the total dissolved protein value by the weight of the sample used, while the percentage was determined by calculating the total weight of protein (mg) per 100 mg of leaf sample.

POD activity was measured based on the Kar and Mishra (1976) protocol. A total of 100 μl of protein extract was mixed with 2.5 mL of pyrogallol 0.2 M and 250 mg of 1% H₂O₂. The absorbance values after the reaction were measured using a spectrophotometer at a wavelength of 420 nm every 30 seconds for 150 seconds. The blank used was a mixture of the same solution without protein extract, replaced with phosphate buffer. POD activity was calculated as an increase in absorbance value per unit time per protein weight (∆_A420·min⁻¹·mg⁻¹ protein). (4) POD activity=difference in absorbance of blank and samplesquantity of protein (mg) \matrix{{{\rm{POD}}\,{\rm{activity}} =}\cr{{{{\rm{difference}}\,{\rm{in}}\,{\rm{absorbance}}\,{\rm{of}}\,{\rm{blank}}\,{\rm{and}}\,{\rm{samples}}} \over {{\rm{quantity}}\,{\rm{of}}\,{\rm{protein}}\,({\rm{mg}})}}}\cr}

PAL activity was measured based on Dangcham et al. (2008). Briefly, 0.1 mL protein extract was combined with 2.4 mL of L-Phe buffer (0.5 M Tris-HCL containing 6 μM L-phenylalanine) pH 8.5 and incubated at 37 °C for 1 hour. The incubation results were then combined with 0.5 mL of 5 M HCl. The absorbance value of the solution was measured at a wavelength of 290 nm. The blank used was 2.5 mL of L-Phe solution with 0.5 mL of 5 M HCl. (5) PAL activity=difference in absorbance of blank and samplesquantity of protein (mg) \matrix{{{\rm{PAL}}\,{\rm{activity}} =}\cr{{{{\rm{difference}}\,{\rm{in}}\,{\rm{absorbance}}\,{\rm{of}}\,{\rm{blank}}\,{\rm{and}}\,{\rm{samples}}} \over {{\rm{quantity}}\,{\rm{of}}\,{\rm{protein}}\,({\rm{mg}})}}}\cr}

SA contents were measured using the modified Tenhaken and Rübel protocol (1997). A total of 1 g leaf sample was ground using a pestle and mortar while adding 10 mL methanol and acetone (1 : 1, v : v) buffer. The suspension was then transferred to an Eppendorf tube and centrifuged at 5,000 rpm for 10 minutes. The supernatant was separated from the pellet. The pellet was resuspended in 3 mL methanol buffer and acetone (1 : 1, v : v) and centrifuged at 5,000 rpm for 10 minutes. The supernatant from this step was mixed with the previous supernatant and vacuum-dried in a freeze dryer. Methanol (5 mL of 30%) was added to the dry residue. The suspension was then centrifuged at 5,000 rpm for 10 minutes. The pellets were discarded, and the resulting supernatant was used for further analysis. The SA contents in sample plants were analyzed qualitatively and quantitatively using HPLC. The mobile phase used was methanol-sodium acetate buffer 50 mM (30 : 70) pH 4.5 (methanol and sodium acetate buffer as much as 500 mL prepared by homogenizing for 10 minutes on a magnetic stirrer) with a flow rate of 0.6 mL per minute. First, the sample and the solution for the mobile phase were filtered off using a 0.45 µm RC cellulose acetate filter membrane. The Chromatopac used was Shimadzu C-R7A plus. The wavelength used in this analysis was 280 nm, while the column used was a VP-DS Ultrasphere (a UV detector at a wavelength of A 280). The SA retention time is 2.549. The formula for calculating the SA contents: (6) SA contents=sample areastandard area×standard concentration×dilution factorsample weight \matrix{{{\rm{SA}}\,{\rm{contents}} =}\cr{{{{\rm{sample}}\,{\rm{area}}} \over {{\rm{standard}}\,{\rm{area}}}} \times {\rm{standard}}\,{\rm{concentration}} \times {{{\rm{dilution}}\,{\rm{factor}}} \over {{\rm{sample}}\,{\rm{weight}}}}}\cr}

Lignin contents in infected leaves were evaluated in three samples from three biological repeats of P. amabilis and P. amboinensis at 12 HPI. It was measured following Chesson protocol (Datta 1981). Briefly, 150 ml H₂O was added to 1 g of dry sample (a) and then refluxed at 100 °C in a water bath for 1 hour. The resulting mixture was filtered, and the residue was washed with 300 mL hot water and then dried in an oven until constant weight (b). As much as 150 mL of 1 N H₂SO₄ was added to the residue, then refluxed with a water bath for 1 hour at 100 °C. The mixture was filtered and washed to neutral pH with 300 mL of hot water and dried (c). Ten milliliters of 72% H₂SO₄ were added to the dry residue, soaked at room temperature for 4 hours. As much as 150 mL of 1 N H₂SO₄ was added and refluxed in a water bath for 1 hour at 100 °C. The solid was washed with 400 mL H₂O, strained, and heated in an oven at a temperature of 105 °C, and the result was weighed (d). Finally, the residue was heated into ash at 600 °C and weighed (e). The formula for calculating the lignin contents: (7) Lignin (%)=(d−e)a×100% {\rm{Lignin}}\,(\%) = {{({\rm{d}} - {\rm{e}})} \over {\rm{a}}} \times 100\%

a – dry sample weight;

The experiments were performed three times. The data obtained were analyzed by analysis of variance (ANOVA) using the Statistical Tool for Agricultural Research (STAR) application. If the treatment was found to have a significant effect on the parameter, the data were further tested with the Tukey test at p = 0.05.

The results of the disease assessment of four Phalaenopsis species are presented in Table 1. SRD symptoms first appeared clearly on 6 HPI. The diameters of soft-rot lesions extended with time in all species and were significantly different between the species on 6 and 12 HPI. However, on 18 HPI, there was no significant difference in soft-rot lesion diameter between P. schilleriana and P. amabilis. The most prominent lesions were found in P. amabilis, followed by P. schilleriana, P. pantherina, and then P. amboinensis, respectively. P. amabilis had the fastest soft-rot symptom development, from 7.9 ± 0.4 mm on 6 HPI to 20.0 ± 1.5 on 18 HPI. Conversely, lesions on P. amboinensis developed more slowly from 0.5 ± 0.1 on 6 HPI to 1.3 ± 0.8 on 18 HPI. From Table 1, we can see that the highest (80%) disease severity (1) was on P. amabilis on 6 HPI. In contrast, P. amboinensis showed the lowest disease severity (7%) on 6 HPI. On 12 HPI, P. amabilis reached 100% disease severity, while P. amboinensis, P. pantherina, and P. schilleriana showed 7%, 50%, and 93% disease severity, respectively. The resistance category of each species was determined based on the disease severity as described in the methods section. On 6 HPI, P. amabilis was classified as very susceptible, while P. schilleriana was moderately susceptible, and P. pantherina was moderately resistant. On 12 HPI, P. schilleriana was in the very susceptible category, while P. pantherina was moderately susceptible; on 18 HPI, they became susceptible and very susceptible, respectively. In contrast, P. amboinensis was in the resistant category from the beginning until the end of the observation. These results showed that P. amabilis is the most susceptible and P. amboinensis is the most resistant species among the four evaluated species.

The soft-rot disease symptoms of the four species are shown in Figure 1A. The symptoms are characterized by necrosis, brownishness, rottenness, and wet areas around the infection site. Figure 1A also provides an average colony-forming unit (CFU) value from each inoculated leaf of the species on 18 HPI. The highest number of CFU (2 × 10⁶) were found in P. amabilis, followed by P. schilleriana (1 × 10⁶), P. pantherina (3 × 10⁵), and P. amboinensis (5 × 10⁴), respectively. This data revealed the trend of CFU among the species. The species with the heaviest soft-rot symptoms (P. amabilis) had a more significant number of CFU than the species with the slightest soft-rot symptoms (P. amboinensis), which means a higher number of bacterial cells in the infected leaves of P. amabilis than P. amboinensis. A higher CFU value seemed to correlate with broader SRD symptoms in susceptible species (Table 1), showing that the pathogen successfully broke the host cell wall as the physical barrier. Besides the CFU value, Figure 1B also shows the area under the disease progress curve (AUPDC). The highest position of AUPDC is from P. amabilis, and the lowest position is from P. amboinensis. The rapid increase of AUPDC happened on 6 to 12 HPI, as shown by P. amabilis, P. pantherina, and P. schilleriana. In contrast, there was no significant increase in AUPDC on P. amboinensis. Soft-rot disease symptoms, disease severity, CFU, and AUPDC values suggest different levels of resistance in the four species and P. amboinensis and P. amabilis as being very distinct in their response to D. dadantii infection.

Figure 1

We performed quick screening for POD and PAL activities of these four species by using mixed samples of three biological repeats of infected leaves from each species on 12 and 16 HPI. The results showed that POD activities of P. amboinensis, P. pantherina, P. schilleriana were 2.0 × 10⁻⁴, 0.77 × 10⁻⁴, 7.25 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein on 12 HPI, respectively, while those of P. amabilis was 8.73 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein. On 16 HPI, POD activities of P. amboinensis, P. pantherina, and P. schilleriana were 1.14 × 10⁻⁴, 0.57 × 10⁻⁴, and 4.74 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein. In contrast, P. amabilis POD activities increased sharply to 72.49 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein. PAL activities in P. amboinensis, P. pantherina, and P. schilleriana on 12 HPI were 1.20 × 10⁻⁴, 0.95 × 10⁻⁴, and 13.75 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein, while in P. amabilis, they were 13.41 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein. On 16 HPI, PAL activities in P. amboinensis, P. pantherina, and P. schilleriana were 1.82 × 10⁻⁴, 1.11 × 10⁻⁴, and 14.73 × 10⁻⁴ u·min⁻¹·mg⁻¹ protein; conversely, in P. amabilis, they increased to 123.51 u·min⁻¹·mg⁻¹ protein.

Interestingly, POD and PAL activities were the highest in the very susceptible species (P. amabilis), increasing around nine-fold from 12 to 16 HPI. In contrast, the resistant species, P. amboinensis, showed much lower POD and PAL activities. The higher increase in POD and PAL activities in P. amabilis at 16 HPI suggested it is induced by D. dadantii infection. For more detailed observation of POD and PAL activities, we selected the two extreme species for their resistance to D. dadantii (P. amabilis vs. P. amboinensis) and observed the POD and PAL activities on 12 HPI and compared to the non-inoculated and inoculated leaves. We chose P. amabilis, not P. schilleriana because, from the quick screening, P. amabilis had extreme POD and PAL, even though both are included in the very susceptible class to D. dadantii.

As shown in Table 2, POD and PAL activities in P. amabilis and P. amboinensis had a pattern similar to the previous results from the screening of four species. POD and PAL activities in P. amabilis (very susceptible) were significantly higher than those in P. amboinensis (resistant). SA contents from P. amabilis and P. amboinensis are also presented in Table 2. The data shows that P. amabilis, the very susceptible species, had twice lower SA content than P. amboinensis, the resistant species. However, there is no significant difference in the SA contents between inoculated and uninoculated leaves in each species. Similar SA content level indicated that SA did not increase from 0 to 12 HPI. Lignin contents measured in the infected leaves of P. amboinensis and P. amabilis at 12 HPI showed no significant difference between the two species (Figure 2).

Figure 2