Effects of 3-methylthiopropionic acid (MTPA) phytotoxin produced by Rhizoctonia solani on reactive oxygen species (ROS) metabolism of potato plants

Volume07-2019
Advances in Agricultural Science 07 (2019), 01: 11-23

Effects of 3-methylthiopropionic acid (MTPA) phytotoxin produced by Rhizoctonia solani on reactive oxygen species (ROS) metabolism of potato plants

Frederick Kankam 1, 2*, Pu Lumei 3, Qiu Huizhen 2

College of Resources and Environmental Sciences, Gansu Agricultural University/Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, China.
Department of Agronomy, University for Development Studies, P.O. Box 1882, Tamale, Ghana.
College of Science, Institute of Agricultural Resources Chemistry and Application, Gansu Agricultural University, Lanzhou 730070, China.

ABSTRACT

Studies were conducted to investigate the effects of 3-methylthiopropionic acid (MTPA) produced by Rhizoctonia solani on antioxidant enzyme activities of potato in causing stem canker. The experiments were laid out in a completely randomized design with six treatments replicated five times. The treatments were 1, 2, 4, and 8 mM concentrations of MTPA while sterilized soil only (CK1) and sterilized soil with R. solani inoculum (CK2) served as control treatments. Results showed that compared with control plants (CK1), the activities of enzyme antioxidants such as superoxide dismutase (SOD), peroxidases (POD), and catalase (CAT) in potato stems treated with 8 mM MTPA increase by 86.95, 203.64, and 123.28%, respectively. Similarly, the activity of chitinase in the roots, stems and that of leaves increases by 120.22, 145.45, and 143.91%, respectively compared to CK1. However, the levels of malondiadehyde (MDA) in the roots, stems and leaves of 8 mM MTPA-treated plants decreased by 167.80, 156.52, 158.30%, respectively, compared to CK1. The study revealed that MTPA increased the activities of the anti-disease effective antioxidant enzymes of the potato plant due to the higher levels of ROS.

KeywordsPotatoPhytotoxinRhizoctonia solani3-Methylthiopropionic acidStem cankerAntioxidase activity


How to Cite: Kankam, F., Lumei, P., & Huizhen, Q. (2018). Effects of 3-methylthiopropionic acid (MTPA) phytotoxin produced by Rhizoctonia solani on reactive oxygen species (ROS) metabolism of potato plants. Advances in Agricultural Science7(1), 11-23.   

Introduction

The relationships between plants and pathogenic fungi are very intricate. Plant pathogenic fungi have developed wide defensive mechanisms to defend themselves when under stress and competition. This enables them to ameliorate the effects of toxic compounds or to overcome other microorganisms in terms of scavenging nutrients and altering the environment (Duffy et al., 2003). Rhizoctonia solani is most studied species of the complex genus of Rhizoctonia. R. solani produces phytotoxins and releases them in the plant tissue to expedite penetration and colonization of the plant. These phytotoxic metabolites can selectively damage host-plants. In response to anticipated infection, plants activate complex defense mechanisms that enable them to resist the pathogen attack. There are numerous reports of plant growth-regulating activity and phytotoxicity of 3-methylthioproprionic acid (MTPA) on various species of plants. Carboxylic acid, including MTPA and 3-methylthioacrylic acid, isolated from culture filtrates of the stem canker pathogen, R. solani, have been reported to possess phytotoxic activity against potato seedlings in the form of necrosis or canker (Kankam et al., 2016a).

R. solani can defend itself against oxidative stress caused during confrontation with microorganisms through antioxidant production and degradation of xenobiotics and environmental alteration (Gkarmiri et al., 2015). It has been demonstrated that potato plants respond to infection by virulent R. solani strains by systemic activation of an array of defense genes such as of chitin-hydrolyzing enzymes and 1,3,-β-glucanase, which are involved in hydrolyzing fungal cell walls, and that this decreases the likelihood of infection of subsequent shoot tips (Lehtonen et al., 2008b). Kim et al. (2007) observed that when plants are attacked by pathogens, their defense enzymes [peroxidase (POD), polyphenol oxidase (PPO) and chitinase] are activated. These enzymes participate in various physiological metabolic processes, so that plant disease resistance reactions are stimulated for preventing the invasion of pathogens (Kim et al., 2007). Malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are closely related with plant disease resistance. Dou et al. (2010) stated that SOD is associated with lignin synthesis thereby enhancing plant resistance to pathogens. POD can also maintain the balance of active oxygen metabolism and protects the membrane structure in plants (Joseph et al., 1998).

Knowledge on how phytotoxins interfere with the plant’s defense mechanisms should bring clarity of their role in the penetration and colonization of plants by pathogenic fungi particularly R. solani. An increased understanding of the disease causing activity of the phytotoxin produced by the fungus will help in designing appropriate and effective management practices to reduce crop damage in the future.

Though there is considerable information about the bioactive role played by several fungal toxins, little information is available concerning the impact of R. solani phytotoxins on the functioning of potato stems. Hence, this study investigates the involvement of antioxidant enzymes in inducing stem canker of potato.

 

Materials and Methods

Soil sterilization, potato cultivars, fungal isolates and MTPA

The experiment was conducted in a greenhouse of the Gansu Provincial Key Lab of Aridland Crop Science of the Gansu Agricultural University, which is located in Lanzhou of China. The sandy loam soil was collected from the surface of cultivation plots from Tiaoshan farm of Jingtai County, Gansu Province, China. The soil was autoclaved at a temperature of 121 oC and a pressure of 1.02 kg/cm3 for 30 min. Diseased-free seeds of potato cv. Leshu and 3-methylthiopropionic acid (MTPA) were obtained from the Gansu Provincial Key Lab of Aridland Crop Science, Gansu Agricultural University, China.

R. solani AG-3 was isolated and identified from infested potato tuber in our laboratory. It was incubated in potato dextrose agar (PDA) at 25 ˚C for 7 days to obtain spore suspension. Isolates was maintained on sterilized barley seeds stored at 4 ˚C.

Pathogen inoculum was produced on media, wheat bran+V8 juice (1:1 w/v) which was mixed in 250 ml conical flasks and then autoclaved. Flasks were inoculated with the respective R. solani isolates from PDA cultures and placed in an incubator for 13 days at 25 ˚C.

 

Experimental design and treatment

Six treatments were applied in the greenhouse experiment and each treatment was replicated five times. Each pot (20 cm diameter, 25 cm height) was filled with 5 kg of autoclaving soil with 1, 2, 4 or 8 mM of MTPA phytotoxin with the exceptions of CK1 (sterilized soil only) and CK2 (Sterilized soil with R. solani inoculum) and planted with seven minitubers per pot. The treatments were applied to the base of the potato stems using an aseptic 1 cc syringe and 26-gauge needle. The experiment was laid out in a randomized block design. The plants were grown in a greenhouse at a maximum of 32 ˚C (day) and minimum of 18 ˚C (night) at a relative humidity of 60-80% under daylight conditions in a greenhouse of Gansu Agricultural University, China. The experiment was conducted twice. The detailed treatment descriptions were as follows: Sterilized soil only without R. solani inoculum (CK1); Sterilized soil + R. solani inoculum (CK2); Sterilized soil + 1.0 mM of MTPA phytotoxin (1.0 mM MTPA); 2.0 mM of MTPA phytotoxin (2.0 mM MTPA); 4.0 mM of MTPA phytotoxin (4.0 mM MTPA); and 8.0 mM of MTPA phytotoxin (8.0 mM MTPA). The choice of the range of concentrations of MTPA used in the experiments was based on the protocol of Bartz et al. (2012) and Kankam et al. (2016b).

 

Samples

At 90 days after planting, the plants were uprooted carefully by loosening the soil around them with a cutlass before pulling them out gently with the hand making sure to collect the few tubers that got detached from the roots in the process. The roots were then severed from the plants, washed carefully on a 2 mm sieve under a jet of tap water to remove any adhering soil and organic debris after which the tubers were detached from the roots and counted. Data were collected on disease severity assessment, MDA), SOD, POD, CAT, and chitinase levels.

 

Disease severity assessment

The bioassay of stem canker incidence was performed at 90 days after the experiment was carried out in a greenhouse. Plant roots were washed free of growing medium and for each plant; the stem canker (SC) was assessed and compared to the non-inoculated control.

The stem canker incidence was assessed on a 0-5 visual disease rating scale as described by Tsror and Peretz-Alon (2005) using the following formula:

SCI (%) = [Number of stems in each rating × rating] / [Total number of stems] X100

Where: 0 = no lesion;  1 = a single lesion of less than 25 mm; 2 = a single lesion of 25-50 mm (or a composite of small lesions totaling less than 50 mm); 3 = a single lesion > 50 mm (or a composite of small lesions totaling > 50 mm but not girdling the stem; 4 = lesion(s) less than 25 mm that are girdling the stem; 5 = lesion(s) more than 25 mm that are girdling the stem.

 

Sample preparation for assays

Sample (400 mg) of roots, stems and leaves were separately crushed and ground to a fine powder in a mortar under liquid nitrogen. One portion of powder was suspended in 50 mM-1 potassium phosphate buffer (PBS), pH 7.5, in a final volume of 5 ml. The suspension was homogenized and centrifuged. The supernatant was diluted twice and divided into aliquots, frozen in liquid nitrogen and stored at -70 ˚C for analysis to be done. The afore-mentioned operations were carried out at 0-4 ˚C. The levels of the various antioxidant enzymes activities and MDA content in the plant extracts were determined spectrophotometrically. It was repeated twice.

 

Lipid peroxidation (LPO) activity

The level of LPO products in the roots, stems and leaf extract were measured, according to the method described by Hartman et al. (2004) with minor modifications, as 2-thiobarbituric acid (TBA) reactive substances, which were mainly MDA, by testing the increase in A532 owing to formation of the red TBA-MDA complex. Absorbance values of the supernatant at A532, A600 and A450, respectively, were measured. The results were calculated from the formula: MDA = [6.45(A532 – A600) – 0.56 A450]/plant fresh weight.

 

SOD activity

The activity of superoxide dismutase (SOD) was measured using a method described by Manoranjan and Dinabandhu (1976) and Garcia-Limones et al. (2002). The reaction mixtures were started by adding riboflavin under 4000 lux of fluorescent light, and A560 was measured after 20-min incubation at room temperature under continuous light. One SOD unit was defined as the amount of enzyme that inhibits the rate of nitro blue tetrazolium (NTB) reduction by 50% under the above assay conditions.

 

POD activity

The activity of POD using guaiacol as substrate (in the presence of H2O2) was measured using a method described by Manoranjan and Dinabandhu (1976) and Garcia-Limones et al. (2002). The oxidation of guaiacol was determined based on the increase of absorbance in A470. The increase produced by H2O2 breakdown was recorded, with 1 U of POD activity being defined as an increase of 0.01 in reading per minute under these assay conditions. The amount of H2O2 formed was determined from the standard curve made earlier with known concentrations of H2O2 and expressed as µmol g-1 FW.

 

CAT activity

CAT activity was measured using a method described by Manoranjan and Dinabandhu (1976) and Garcia-Limones et al. (2002). The reaction medium consist of 50 mM-1 potassium phosphate buffer, pH 7.5, 15 mM H2O2 and 20 µl of enzyme extract. The reaction was initiated by adding H2O2. The decrease of the readings in A240 produced by H2O2 breakdown was recorded.

 

Chitinase assays

One gram of chitin was added to 4 ml of acetone before it was grounded. 40 ml HCl was gradually added during the process. With the aid of glasswool, filtration of colloid chitin was done. The filtrate was added slowly into strongly agitated fivefold greater volumes of 50% (v/v) alcohol to precipitate the sample afore-mentioned. The topmost level of the liquid was discarded, and the colloid precipitation was collected, washed several times with distilled water.

The chitinase activity was measured using the method described by Fink et al. (1988) and Schraudner et al. (1992). A standard curve of N-acetyl-amino-glucosamine absorbance (A) was prepared. The difference between the A420 of the standard control and the A420 of the samples was represented as N-acetyl-amino-glucosamine enzyme lysed from chitin by chitinase. One unit chitinase was defined as the amount of enzyme that produced 1 µg N-acetyl-amino-glucosamine per hour from chitin under the described assay conditions.

 

Statistical analysis

All statistical analyses were performed with IBM SPSS 22.0 software for Windows (Chicago, IL) using two-way ANOVA. The values of stem canker, black scurf indices and antioxidant enzymes were represented as the means of five replicates (mean ± SD) from five pots. Duncan’s multiple range tests was used to separate the means (P ≤ 0.01). The relationship between the stem canker and the antioxidant enzyme activities were analyzed using Bivariate (two-tailed) Pearson-ranked correlation coefficient.

 

Figure 1. Effects of MTPA phytotoxin on stem canker

Figure 1. Effects of MTPA phytotoxin on stem canker

 

 

Figure 2. Effects of MTPA phytotoxin on malondialdehyde (MDA) activity of potato. Error bars represent the standard deviation of the mean. Bars with the same letter(s) are not significantly different (P < 0.01), according to Duncan’s LSD test

Figure 2. Effects of MTPA phytotoxin on malondialdehyde (MDA) activity of potato. Error bars represent the standard deviation of the mean. Bars with the same letter(s) are not significantly different (P < 0.01), according to Duncan’s LSD test

 

Results

Effect of MTPA phytotoxin on stem canker

Generally, plants treated with MTPA phytotoxin recorded significantly higher (P < 0.05) incidence and severity of stem canker. Stem canker development in the CK1 and CK2 treatments were lower than in the other treatments, with the incidence rate in the treatments increasing with increasing amounts of the phytotoxins. Stem canker incidence observed on plants exposed to the highest phytotoxin tested (8.0 mM MTPA) was increased by 450% and 100% compared with CK1 and CK2, respectively (Fig. 1).

Figure 3. Effects of MTPA phytotoxin on superoxide dismutase (SOD) activity of potato. Error bars represent the standard deviation of the mean. Bars with the same letter are not significantly different (P < 0.01), according to Duncan’s LSD test

Figure 3. Effects of MTPA phytotoxin on superoxide dismutase (SOD) activity of potato. Error bars represent the standard deviation of the mean. Bars with the same letter are not significantly different (P < 0.01), according to Duncan’s LSD test

 

Figure 4. Effects of MTPA phytotoxin on guaiacol peroxidase (POD) activity of potato. Bars with the same letters are not significantly different at P < 0.01 according to post hoc tests (Duncan’s LSD test. Error bars indicate the standard deviation of the means

Figure 4. Effects of MTPA phytotoxin on guaiacol peroxidase (POD) activity of potato. Bars with the same letters are not significantly different at P < 0.01 according to post hoc tests (Duncan’s LSD test. Error bars indicate the standard deviation of the means

 

Effect of MTPA phytotoxin on MDA content

The content of MDA in the leaves, roots and stems of control plants (CK1 and CK2) were highest in the stems, followed by the roots, and the lowest in the leaves. However, MDA content was lowest in the roots, and followed by in the leaves and the highest in the stems in 8.0 mM MTPA treatments. When corresponding tissues were compared, MDA content was higher in the CK1 plants than in the plants subjected to treatments. The MDA content in tissues of potato plants decreased gradually in the order CK1 > CK2 > 1.0 mM MTPA > 2.0 mM MTPA > 4.0 mM MTPA > 8.0 mM MTPA (F5,24 = 62.98, P < 0.01 for root; F5,24 = 93.34, P < 0.01 for stem; F5,24 = 51.09, P < 0.01 for leaf). The lowest content of MDA was found in the roots, stems and leaves of plants subjected to the 8.0 mM MTPA treatment. MDA content in the roots, stems and leaves of 8.0 mM MTPA treatment was decreased by 167.80, 156.52 and 158.30%, respectively, compared with CK1 (Fig. 2).

 

Effect of MTPA phytotoxin on SOD activity

SOD activity was highest in stems and lowest in roots of potato plant. The SOD activities in corresponding tissues of all treatments were significantly higher than CK1 (F5,24 = 248.09, P < 0.01 for root; F5,24 = 174.40, P < 0.01 for stem; F5,24 = 181.17, P < 0.01 for leaf). The highest activities were found in treatment of 8.0 mM MTPA, being increased by 86.95%, 112.50%, and 219.42% in stems, leaves and roots as much as those in CK1 (Fig. 3).

 

Effect of MTPA phytotoxin on POD activity

The guaiacol–POD activity was diversely distributed in roots, stems and leaves of potato plant, and there was a great difference between different treatments. For plants treated with MTPA treatments, guaiacol–POD activity gradually increased in the order roots, stems and leaves. The activities of POD in stems and roots for all treatments were much lower than those in the CK1. The guaiacol–POD activities were the greatest in the 8.0 mM MTPA treatment, with the roots being increased by 204% and the stems showing a 203.64% increase comparable to the control treatment. However, the leaves showed an increase of 214.95 relative to the control tissues (Fig. 4).

Effect of MTPA phytotoxin on CAT activity

Catalase activity was lower in roots, greater in leaves and greatest in stems. The CAT activities in the leaves of CK2, 1.0 mM MTPA, 2.0 mM MTPA, 4.0 mM MTPA and 8.0 mM MTPA treatments were higher than in the leaves of CK1. Catalase activity was significantly higher in the stems than that in the roots (F5,24 = 43.43, P < 0.01 for root; F5,24 = 53.43, P < 0.01 for stem; F5,24 = 89.65, P < 0.01 for leaf). The highest CAT activity was recorded in plants treated with 8.0 mM MTPA; these plants showed an increase of 114.20%, 123.28% and 166.85%, respectively, in CAT activity in the leaves, stems and roots, compared with those in CK1 (Fig. 5).

 

Effect of MTPA phytotoxin on chitinase activity

Chitinase activity was low in the roots, high in the stems and highest in the leaves. It was significantly higher in the tissues of all treated potato plants than in the corresponding tissues of the CK1 plants and increased with increasing application rates of MTPA phytotoxin (F5,24 = 13.39, P < 0.01 for root; F5,24 = 68.74, P < 0.01 for stem; F5,24 = 116.84, P < 0.01 for leaf). The highest activities were found in plants treated with 8.0 mM MTPA. Chitinase activities in the stems, leaves and roots of 8.0 mM MTPA plants were 145.45, 143.91 and 120.22% higher than those of the CK1 plants (Fig. 6).

 

Relationship between the antioxidant enzyme activities and stem canker of potato

There was significant difference between stem canker and MDA activities in the roots, stems and the leaves. Stem canker was negatively correlated with MDA in the roots, stems and the leaves (r = -0.954, -0.970, -0.925, P < 0.01), respectively (Fig. 7a).

 

Figure 5. Influence of MTPA phytotoxin on catalase (CAT) activity of potato. Bars with the same letter are not significantly different (P < 0.01), according to Duncan’s LSD test

Figure 5. Influence of MTPA phytotoxin on catalase (CAT) activity of potato. Bars with the same letter are not significantly different (P < 0.01), according to Duncan’s LSD test

 

 

Figure 6. Effects of MTPA phytotoxin on chitinase activity of potato plants. Bars with the same letter are not significantly different at P < 0.01 according to post hoc tests (Duncan’s LSD test). Error bars represent the standard deviation of the mean

Figure 6. Effects of MTPA phytotoxin on chitinase activity of potato plants. Bars with the same letter are not significantly different at P < 0.01 according to post hoc tests (Duncan’s LSD test). Error bars represent the standard deviation of the mean

 

As a consequence of the significant effect of SOD content and stem canker, when data of both treatments were combined a strong positive relationship was found between SOD and stem canker 90 days after planting (Fig. 7b). The relationship between SOD and stem canker in the roots, stems and leaves were highly significant (r = 0.968, 0.983, 0.977, P < 0.01), respectively (Fig. 7b).

Figure 7. Relationship between antioxidant enzyme activities of potato and stem canker; (a) MDA content and stem canker (b) SOD activity and stem canker. *, ** indicate relationships that differ significantly from the two interactions at P < 0.05 and P < 0.01, respectively

Figure 7. Relationship between antioxidant enzyme activities of potato and stem canker; (a) MDA content and stem canker (b) SOD activity and stem canker. *, ** indicate relationships that differ significantly from the two interactions at P < 0.05 and P < 0.01, respectively

 

Discussion

3-Methylthiopropionic acid (MTPA) is a compound involved in R. solani disease development (Kankam et al., 2016b). Potato plants treated with MTPA phytotoxin resulted in Rhizoctonia disease at all growing stages which agree with Ahvenniemi et al. (2007) that Rhizoctonia disease developed in four phases. The first and second phases result in infection of stolon tips prior to and after seeding. Contrary, the 3rd and 4th stages lead to scabby and black scurf development on progenies respectively (Ahvenniemi et al., 2007; Woodhall et al., 2008). There is also one published report that diseased stolons may cause severe damaged to the potato stems and tubers and also hinders the development of aerial tubers (Atkinson et al., 2010). Generally, plants treated with the MTPA phytotoxin recorded significantly higher (P < 0.05) stem canker incidence. Treatment with 8.0 mM MTPA was the most affected. This showed that development of lesions increased with an increase in concentration of the phytotoxins and the duration of dip treatment. The 8.0 mM MTPA treatment which was the highest concentration may have released more chemicals into the cells and tissues causing damage to the stem of the plant as reported by Gommers et al. (1982).

MDA is a marker of the production of lipid peroxide inside of cell membranes (Qiu et al., 2013). Its content could reflect the degrees of the lipid peroxide of pathogen under attack and was enhanced under plants treated with without MTPA phytotoxin (CK1). From the results obtained, MDA content in tissues and organs of plants treated with MTPA phytotoxin was much lower than that in corresponding tissues of the CK1 and CK2 plants. It also decreased with increasing application rates of MTPA, with the lowest MDA content found in the 8.0 mM MTPA treatment. In the CK1 and CK2 treatments, MDA levels were highest in the stem, followed by the root and then by the leaf in that order. In contrast with the CK1, the 8.0 mM MTPA treatment reduced MDA content in the root, stem and leaf by 167.80, 156.52 and 158.30%, respectively (Fig. 2). This finding disagrees with the work of Mao et al. (2012) who observed that the MDA content in transgenic rice increase gradually upon R. solani invasion, suggesting that lipid peroxidation might have been triggered due to the generation of OH generated in response to pathogen infection. Plants have developed an intricate and delicate antioxidants defense system to efficiently counteract the toxic effects of reactive oxygen species (ROS), by reducing ROS-induced injuries, aiding in resistance towards stresses. This change in MDA content demonstrates the phytotoxin used triggered the potato plant by decreasing its MDA content in order to resist the effects of the MTPA phytotoxin.

The SOD activity is one of the important indexes of plant stress resistance (Oliveira et al., 2014) which plays an important role in protecting the cell against the oxygen damage. Following the extension of stress time, the SOD activity of potato tissues continuously increased under plants with MTPA (Fig. 3). Besides, the SOD activity of 8.0 mM MTPA in the roots, stems and the leaves increased sharply by 219.42%, 86.95% and 112.50%, respectively compared with the CK1 after 90 days after planting. It was obvious that the increase extent of the SOD activity in 8.0 mM MTPA was more remarkable than that of the CK1. Therefore, plants treated with MTPA phytotoxin could adapt to stress caused by the phytotoxin by keeping a higher level of the SOD activity to alleviate lesion degrees of cell membrane. The findings agree with Azevedo et al. (2008) who reported a corresponding increase in SOD when rice was exposed to R. solani and maritime pine suspension cells exposed to Bacillus cinerea spore elicitation. Therefore, these results imply that SOD activity in roots, stems and the leaves can be associated with resistance to R. solani phytotoxin in potato.

The POD is also one of important protective enzymes, which could protect cell membranes against lipid peroxidation (Arici and Sanli, 2014). Plants contain pathogenesis-related protein chitinase, which can degrade the polymers known to be in cell walls of fungi (Nakazaki et al., 2006). The results from our experiments demonstrate that MTPA significantly affected the activities of POD, CAT and chitinase of potato plants. Compared to CK1 potato plants, the tissues of plants treated with 8.0 mM MTPA showed the following changes: POD activities increased by 204% in the root, 203.64% in the stem and 214.95% in the leaf; CAT activity increased by 166.85% in the root, 123.28% in the stem and 114.20% in the leaf; chitinase activity increased by 120.22% in the root, 145.45% in the stem and 143.91% in the leaf. All of these antioxidases and pathogenesis-related proteins were found to be differentially present in the roots, stems and leaves.

Generally, lower activities of CAT were found in the roots and leaves and higher activities in stems. On the contrary, higher activities of chitinase were located in the leaves whereas lower activities in the roots and stems. In terms of Rhizoctonia disease of potato, although the pathogen invades from root, the main damaged parts are the stems due to lack of water and their sensitivity to stress. It could be presumed that the activities of antioxidases in leaves are important than those in roots and stems because the former are directly related to the resistance of the plant to biotic stress. All of these enzymes prompt the resistance and defense reaction of a plant, whether local or systemic, to the invasion of a pathogen (Kavroulakisa et al., 2005). Wu et al. (2009) reported that the activity of antioxidative enzymes (SOD, CAT, POD) and chitinase in both the root and leaf increased in watermelon plants subjected to Fusarium wilt. The CAT provides a cellular defense mechanism by scavenging O2-, which constitutes one of the major defense mechanisms of cells against oxidative stress (Chen et al., 2015).

In the present study, the activities of SOD, POD, CAT and chitinase in all treatments were much higher than that in CK1, especially the 8.0 mM MTPA treatment. This revealed that MTPA increased the activities of the anti-disease enzymes of the potato plant, resulting in an increased induction of systemic acquired resistance of the plant to the phytotoxin and thus leading to the reduction of the stem canker/black scurf. This increased induction of the pathogenesis- related proteins and antioxidase activities caused by MTPA improved the resistance of the plant to pathogen. When the plants invaded by pathogens, the resistant-related enzymes (SOD, POD and CAT) are activated and participate in many physiological processes, such as oxidation, lignification, response to pathogenic toxin, to prevent the invasion and reproduction of the pathogens. Therefore, SOD, POD and CAT are closely related to the plant disease resistance.

The present study showed that MDA is negatively correlated with stem canker (Fig. 7a). On the other hand, due to the significant increases observed in antioxidant enzyme activities and stem canker as a result of MTPA phytotoxins, we found strong positive correlations between SOD and stem canker (Fig. 7b). These results confirm that antioxidant enzyme activity is one of the factors determining potato disease, as previously reported (Lehtonen et al., 2008a; Gkarmiri et al., 2015).

 

Conclusions

The study demonstrated that the activities of SOD, POD, CAT and chitinase in all treatments were much higher than that in CK1, especially at the 8.0 mM MTPA treatment. The study also revealed that increased levels of AOS, built up by either enhanced production and decreased scavenging potential, may contribute to the resistance reaction in potato to stem canker.

 

Acknowledgments

This work was funded by a grant from the Natural Science Foundation of China (Project Number 31360500). We would like to thank Dr. Fu Guo Rui of the Department of Chemistry, Gansu Agriculture University, for his technical assistance in obtaining 3-methylthioproprionic acid. The authors have no conflict of interest to declare.

 

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