Plant-pathogen interaction can be diverse depending on the nature of the pathogens. Briefly, biotrophic plant pathogens stay inside living host tissues in a long-term feeding relationship. In contrast, necrotrophic pathogens are parasitic organisms that kill the living cells of their host and then feed on the dead matter. Hemibiotrophic pathogens have a biotrophic phase as the initial stage of infection and switch to the necrotrophic in the last steps of the disease or pathogen life cycle (Göhre & Robatzek 2008; Davidsson et al. 2013; Fatima & Senthil-Kumar 2015; Potrykus et al. 2018; Huang et al. 2020).
Soft-rot disease (SRD) caused by a necrotrophic pathogen is an obstacle to
Most
Constitutive features influence the plant response to biotic and abiotic stresses and modify plants’ defense mechanisms. The defense mechanisms are of a physical and biochemical nature (Mithöfer & Maffei 2017). They act in a constitutive or induced manner. Most of the physical defenses such as cell walls, waxy epidermal cuticles, and bark protect the plant as barriers that keep out infection and give strength and rigidity. The biochemical defenses are commonly induced after the plant detects the pathogen infections and response (Freeman & Beattie 2009). However, there are also some induced physical defenses such as callose deposition and lignification (Bhuiyan et al. 2009; Luna et al. 2011; Wang et al. 2021) and constitutive biochemical defenses known as phytoanticipins (various antimicrobial and repellents) (Pedras & Yaya 2015).
Induced defense is activated when the host plant recognizes the presence of the pathogen, which leads to the molecular and biochemical cascades as a response. ROS, as the earliest response (including hydrogen peroxide, H2O2) after the perception of pathogen-associated molecular patterns (PAMPs), has been reported in many plants (Bolwell & Wojtaszek 1997; Bindschedler et al. 2006; Gill & Tuteja 2010; Wang et al. 2020).
At a low level, ROS is essential in cellular proliferation and differentiation (Mittler 2017). Under biotic or abiotic stress, plants generate many ROS involved in the regulation of pathogen defense, programmed cell death (PCD), and stomatal behavior (Gill & Tuteja 2010; Schippers et al. 2016). High ROS accumulation is often referred to as oxidative bursts (Singh et al. 2021) that are toxic to both of host and the pathogen. To deal with the high ROS level, the host plant produces enzymatic and non-enzymatic antioxidants (Hasanuzzaman et al. 2020) as the ROS scavenger that works synergistically and interactively to neutralize free radicals (Huang et al. 2019). Enzymatic antioxidants mainly include superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), while the non-enzymes antioxidant includes glutathione, ascorbic acid (AsA), and flavonoids (Gechev et al. 2006; Huang et al. 2019; Kapoor et al. 2019).
POD is one of the ROS scavengers that convert toxic hydrogen peroxide to water and oxygen (Giorgio et al. 2007). POD activities indicate signal transduction to the remote sites, resulting in necrosis and cell death (Simons & Ross 1971). PAL is an essential enzyme in the phenylpropanoid biosynthesis pathway, producing non-enzymatic antioxidants like flavonoids and salicylic acid (SA) (Zhang & Liu 2015; Lefevere et al. 2020). SA, jasmonic acid (JA), and ethylene (ET) are well-known biochemical compounds that act as signal transducers in the plant to induce defense response genes (Andersen et al. 2018). Both POD and PAL are involved in early resistance response in plants. High POD and PAL activities were found in infected resistant barley seedling, cotton, and
Analysis of biochemical components as POD and PAL in
Four
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); 1 (0.1–1 mm); 2 (1.1–2 mm); 3 (2.1–3 mm); 4 (3.1–4 mm); 5 (4.1–5 mm); 6 (5.1–6 mm); 7 (6.1–7 mm); 8 (7.1–8 mm); 9 (8.1–9 mm); 10 (> 9 mm).
Symptom scoring was used to calculate the disease severity (DS) following the formula by Sanjaya et al. (2020):
ni – 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; vi – disease score in the score of I; Z – maximum score value; N – number of samples observed.
The plant resistance level to DS of 0–20% – resistant (R); DS of 21–40% – moderate resistant (MR); DS of 41–60% – moderate susceptible (MS); DS of 61–80% – susceptible (S); DS of > 80% – very susceptible (VS).
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:
The area under the disease progress curve (AUDPC) was calculated by using the formula below:
To evaluate the relationship between POD and PAL activities with resistance ability, we performed quick screening for these four species (
To confirm the defense mechanism, we choose two species with a distinct resistance to
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).
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.
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:
Lignin contents in infected leaves were evaluated in three samples from three biological repeats of a – dry sample weight; d – dry sample post refluxed and washed weight; e – final ash 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
The reaction of four
Species | 6 HPI | 12 HPI | 18 HPI | ||||||
---|---|---|---|---|---|---|---|---|---|
SrSD (mm) | DS (%) | RC | SrSD (mm) | DS (%) | RC | SrSD (mm) | DS (%) | RC | |
0.5 ± 0.1d | 7 ± 0.0 | R | 0.6 ± 0.5d | 7 ± 0.1 | R | 1.3 ± 0.8c | 20 ± 0.1 | R | |
2.7 ± 1.3c | 30 ± 0.1 | MR | 4.4 ± 3.2c | 50 ± 0.4 | MS | 7.9 ± 7.1b | 70 ± 0.5 | S | |
4.4 ± 0.6b | 50 ± 0.0 | MS | 10.1 ± 2.5b | 93 ± 0.2 | VS | 15.4 ± 4.7a | 100 ± 0.2 | VS | |
7.9 ± 0.4a | 83 ± 0.1 | VS | 14.3 ± 1.4a | 100 ± 0.0 | VS | 20.0 ± 1.5a | 100 ± 0.0 | VS |
The leaf cuts were inoculated with
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
The development of soft-rot disease symptoms among four species: (A) SrSD on infected leaves at 0–18 hours post-inoculation (HPI),
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
Interestingly, POD and PAL activities were the highest in the very susceptible species (
As shown in Table 2, POD and PAL activities in
POD, PAL, and SA content in
very susceptible | 15.30 ± 6.93a | 12.20 ± 1.60a | 5.26 ± 0.22a | |
14.96 ± 11.78a | 10.78 ± 7.59a | 5.32 ± 0.46a | ||
resistant | 2.35 ± 0.22b | 1.54 ± 0.16b | 12.56 ± 2.83b | |
1.64 ± 0.83b | 1.26 ± 0.44b | 12.88 ± 2.14b |
Percentage of lignin content in dry weight of
Soft-rot disease (SRD) is a very devastating disease in the production of essential crops such as potato (Łojkowska & Kelman 1994; Pérombelon 2002; Motyka et al. 2017; Rossmann et al. 2018),
SRD is caused by
Necrotrophic pathogens manipulate the ROS-mediated defense response of plants to facilitate cell death. ROS accumulation is also a virulence factor for disease development in necrotrophs such as
ROS accumulation could be predicted by the activities of antioxidant enzymes, POD and PAL. However, in the present results, there is no significant difference in POD and PAL activities between the non-inoculated and inoculated leaves in each species, suggesting that pathogen inoculation did not induce POD and PAL activities at 12 HPI. This result is different from the quick screening, where POD and PAL increased in
Both POD and PAL are inducible enzymes that react under stress (Fu et al. 2012; Prasannath 2017; Tsers et al. 2020). Besides the scavenging role, both POD and PAL are also involved in lignin biosynthesis (Xu et al. 2011; Liu et al. 2018). Lignin facilitates the host cell wall’s physical barrier and prevents the pathogen infection by spatial restriction, which is disturbed by defects in lignin deposition, trapping pathogens and thereby terminating their growth (Lee et al. 2019). However, there was no difference in lignin contents between the two species with distinctly differing resistance levels to
The resistance mechanism in
PAL is the first enzyme reported in SA biosynthesis (Huang et al. 2010), but lower PAL and higher SA were found in resistant