Activity of peroxidase and cucurbitacin D in melon genotypes following challenge by the fungal pathogen Fusarium oxysporum f. sp.

Transcription

1 International Research Journal of Applied and Basic Sciences. Vol., 3 (8), , 2012 Available online at www. irjabs.com ISSN X 2012 Activity of peroxidase and cucurbitacin D in melon genotypes following challenge by the fungal pathogen Fusarium oxysporum f. sp. melonis race 1 Esmaeil Madadkhah 1, Mostafa Nasertorabi 1, Amanollah Soleimani 2, Ecehagh moghbeli 3 1- Department of Horticulture, College of Aboureihan, University of Tehran, Tehran, Iran. 2- Department of Plant Science, Faculty of Agriculture, University of Jiroft, Jiroft, Iran. 3- Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. *Corresponding Author madadkhah@ut.ac.ir Abstract Melon (Cucumis melo) production is significantly and economically affected by Fusarium wilt worldwide. Cucurbitacins are bitter tetracyclic triterpenoids that are toxic to most organisms and occur widely in wild and cultivated Cucurbitaceae. In order to study the effect of cucurbitacin D content and activity of peroxidase (POX) on the resistance of melon genotypes to Fusarium oxysporum, five susceptible and five resistant genotypes were inoculated with the race 1 of the causal agent. Root samples were taken daily from the inoculated plants for 8 days and were used for quantification of cucurbitacin D by High performance liquid chromatography. Analysis of variance showed that there was a direct relation between cucurbitacin D with resistance to the disease. No cucurbitacin D was found in susceptible genotypes in day 0 (no infection), but in resistant genotypes in the same period was higher than other days. Analysis of variance demonstrated a direct relation between cucurbitacin D with resistance to the pathogen. Although the POX activitiy in both groups increased in response to infection by the pathogen, this significant increase was more prominent in resistant genotypes. The enzyme activity approached to pick at 4 days after inoculation. The results suggested that the increased levels of POX activity in melon genotypes play an important role in the induction of resistance to this disease. Keywords: Melon, Fusarium wilt, defense responses, triterpenoids. Abbreviations: POX, Peroxidase; F.o.m, Fusarium oxysporum f.sp. melonis; HPLC, High performance liquid chromatography Introduction Fusarium wilt, caused by Fusarium oxysporum f.sp. melonis, is one of the most devastating diseases in melon production worldwide. Epidemics of Fusarium wilt can result in approximately 100 percent yield losses. The fungal soil-borne pathogen remains viable on non-host crop residues and roots grown in rotation or as chlamydospores for decades. Thus, the control of this vascular wilt disease carries out its own difficulties (Gordon and Martyn, 1997). In 1976, Risser et al. designated physiological races of F. oxysporum f. sp. Melonis including 0, 1, 2, and race 1, 2 (1,2w and 1,2y). They were designated based on the host resistance genes overcome by the variants of the pathogen. In melons, two wilt resistance genes, including Fom-1 and Fom-2, have been identified till now. Development of an antioxidant defense system protects the plant by either partial suppression of the ROS production, or the scavenging of ROS which have already been produced (Torres et al., 2006). According to Avdiushko et al. (1993) POX catalyzes the formation of lignin and other oxidative phenols that contribute to the formation of defense barriers to reinforce the cell structure. The activity of polyphenol oxidase and peroxidase has increased in response to infection by pathogen. There was an increase in the activity of

2 peroxidase and polyphenol oxidase in rose (Rosa centifolia) plants reacting to infection by Alternaria tenuis, the causal agent of black spot (Khatun et al., 2009). Morkunas and Gemerek (2007) reported that POX participates in defense systems of yellow lupins in response to pathogen attack such as Fusarium oxysporum. Cucurbitacins are highly oxygenated tetracyclic triterpenes which are predominantly found in the family Cucurbitaceae and are also found in other plants. They are responsible for the characteristic bitter taste of most wild species of Cucurbitaceae (Chen et al. 2005). These chemicals have a broad range of potential biological activities such as cytotoxicity, antitumor properties, antimicrobial and anthelmintic activities which are also involved in plants resistance against insects (Aharoni et al. 2005). In 1981, Nes and Patterson reported that cucurbitacins have an inhibiting effect on the growth of Phytophthora cactorum, causal agent of root rot. It has been shown that cucurbitacin I has antifungal properties against Botrytis cinerea (Bar-Nun and Mayer, 1990). The objective of this study was to evaluate the effects of cucurbitacin D and POX activity on the resistance of melon to wilt causal agent, Fusarium oxysporum f.sp. melonis race1 (Fom 1). Material and Methods Plants and cultivation Five resistant genotypes (Ogen, Chorok-Zard, Khatooni, Mahalli, Zard-Shotori) and five susceptible genotypes (Shahabadi, Yellow Chanery, Spanish, Dastanbu 1, Dastanbu) to Fom 1 race M263 were used in our trials (Madadkhah et al., 2012). The seeds were kindly provided by the Iranian National Gene Bank Department, Seed and Plant Improvement Institute, Karaj, Iran. Before sawing, seeds were surfacedisinfected with sodium hypochlorite (1%) for 1 min and then rinsed in sterile distilled water. Seeds were sown in cell-type plastic growing trays; filled with a pasteurized potting mix of peat, perlite and sand, and were grown under greenhouse conditions (24 28 C, 16 h day/8 h night). Pathogen isolates Six isolates of Fom1 (dominant race in Iran), supplied from the collection of Plant Protection Department, Shiraz University, were used in this study. Single spore cultured on potato dextrose agar (PDA), were incubated with 22±1ºC and a photoperiod of 14h (L:D) for 7 days. After inoculating the susceptible variety of melon under greenhouse conditions, two isolates of Fom1, M263 and Msh were determined showing high degrees of aggressiveness and were used for experiments. Plant inoculation At first true leaf stage, seedlings were removed from soil and roots were washed under running tap water, pruned to approximately half length, and dipped in the conidial suspension to obtain a concentration of spores/ml, for 1-2 minutes and transplanted into new pots (Moretti et al. 2002). Extraction of POX and activity assay Enzymatic activities and total phenol content were investigated in resistant and susceptible genotypes at 0 (control), 2, 4, 6 and 8 days after inoculation. Root samples (1g) were ground with mortar and pestle in liquid nitrogen and homogenized in 1 ml sodium phosphate buffer (ph 7.0). The extracts were transferred into Eppendorf tubes 2 milliliter and centrifuged at 14,000 rpm for 20 min at 4 C in a refrigerated centrifuge. The supernatant were stored at 80 C for later analyzing the activity of POX enzyme and protein concentration determination. The bovine serum Albumin, guaiacol and prolin were obtained from Sigma-Aldrich Co. Protein concentration was determined according to Bradford (1976) with bovine serum Albumin as a standard. POX activity was measured by monitoring the change in absorbance of enzyme extracts (containing 40g of total protein) with 20 l guaiacol and 10 l H 2 O 2 (30) in 2ml citrate phosphate buffer (ph 5/4). Following the absorbance variation at 475 nm for 1 min at 25 ºC in a UV-VIS spectrophotometer (Perkin Elmer- lambda 25) (Janda et al., 2003). Extraction and assay of cucurbitacin D content In the last experiment, the content of cucurbitacin D present in the melon genotypes was determined by high performance liquid chromatography (HPLC) analysis. The target contents were measured in resistant and susceptible genotypes at 0 (control), 2, 4, 6 and 8 days post-inoculation. Since highest levels of cucurbitacin concentrations are normally found in roots, the root samples were used for the purpose. The extraction and purification were done according to Balkema-Boomestra et al. (2003). The quantitative 1591

3 analysis of cucurbitacin was determined by HPLC (Analytical, Knauer, Germany), with UV detector at 235 nm (after much trial and error) and column C18 (125nm 4nm, Nucleosil-100, 5m). Statistical analyses Analysis of variances and mean comparisons were performed using SAS software (version 9). A Duncan test with a probability of 0.05 was used to show significant differences between treatments. All data are presented as means. Results and Discussion Determination of POX activity Analysis of variance showed that peroxidase activity is significantly different between the susceptible and resistant genotypes. Comparison of means indicated that the structural POX activity has been higher in resistant genotypes than in susceptible ones. This difference became even sharper soon after inoculation, as the inoculation induced the enzyme activity. POX activity decreased sharply in resistant genotypes four to six days after inoculation, in such a way that in 6 th to 8 th day after inoculation its activity was even less than those of susceptible genotypes. In both resistant and susceptible genotypes, the highest POX activity was observed on the fourth day, while the lowest activity was detected in six days after inoculation (Fig. 1 a). Highest amount of POX activity was observed in Ogen 9, Mahali Mazandaran, and Zardshotori and lowest in Shahabadi and Dastanboo Aranbidgol (Table 1). Figure 1. POX activity (a) and cucurbitacin D content (b) at 0 (control), 2, 4, 6 and 8 days after inoculation with Fusarium oxysporum f.sp. melonis race 1 isolate M263 in resistant and susceptible melon genotypes. Data are average of three replicates. Error bars indicate ±SE. (P 0.01). Variations of cucurbitacin D content between resistant and susceptible genotypes Comparison of means indicated that cucurbitacin D content was higher in roots of resistant genotypes than susceptible ones. There was no detectable content of cucurbitacin D in susceptible genotypes Yellow Chanery, Dastanbu and Spanish (Table 1). The production of cucurbitacin D decreased in resistant genotypes until six post inoculation day and then demonstrated increase until day eight but in susceptible ones it was continuously increased during the same period (Fig. 1 b). The highest content of cucurbitacin D belonged to Chorok-Zard (0.54 ppm) and Zard-Shotori (0.5 ppm) (Table 1). 1592

4 Table 1. POX activity and cucurbitacin D content in the resistant and susceptible melon genotypes inoculated Cucurbitacin D POX mean (!OD Genotype content mean (ppm) 475/min/mg protein) Mahali Mazandaran (R) 0.31 B 0.27 C Khatoni (R) 0.2 E 0.26 C Zardshotori (R) 0.29 C 0.5 AB Ogen 9 (R) 0.46 A 0.48 B Chorok zard (R) 0.27 C 0.54 A Yellow Chanery (S) 0.24 D 0 F Shahabadi (S) 0.17 F 0.18 D Dastanbu Dezfool (S) 0.25 D 0 F Dastanboo Aranbidgol (S) 0.19 EF 0.13 E Spanish (S) 0.23 D 0 F Different letters indicate significant differences among means according to Duncan s multiple range test (*P 0.05). The main objects of the present study were to detect possible relations between resistance to the disease and plant biochemical responses. Two isolates, M263 and Msh, of Fom race 1 were used in the preliminary greenhouse assay. Our results revealed that resistance to Fom race 1 in melon is significantly correlated with induced host defense responses including increased peroxidase activity and cucurbitacin content. There were also significant differences of disease severity between resistant and susceptible genotypes as well as total phenol levels, and activity of the current enzymes. It has been suggested that resistance to several phytopathogens could be attributed to the presence of higher levels of phenol (Kushwaha and Narain, 2005). Peroxidase is involved in the production of reactive oxygen species, which are directly toxic to the pathogen. The current obtained results showed a significant correlation between the resistance and POX activity. Although the POX activity in resistant genotypes declined significantly at 6 th day after inoculation, the earlier induced activity might have a role on limiting the number of successful infections. However, this significantly different induction of peroxidase might be a reaction of host to pathogen attack in resistant and susceptible genotypes. But, it could be an effort to replace damaged tissues as the enzyme is responsible for lignification and suberization in plant tissues. Our results revealed that resistant genotypes of melon demonstrate increased cucurbitacin D content and accumulation of its in response to Fom 1 isolate. Cucurbitacins B and D have been identified as antagonists to insect steroid hormones acting at the ecdysteroid (regulation of the pupation) receptor in Drosophila melanogaster cells. Ecdysteroids are known to be involved in the molting process (Dinan et al., 1997). There was no cucurbitacin D in susceptible genotypes at day 0 (no infection) in comparison with resistant genotypes (Fig. 1 b). Interestingly, cucurbitacin D was not detected in Yellow Chanery, Dastanbu and Spanish roots which were susceptible genotypes (Table 1). Differences in cucurbitacin D content (after inoculation) occurred between resistant and susceptible groups implied that this responses is closely associated with defense responses in host, after penetration of the pathogen. The results of cucurbitacin D are strong evidence to suggest their participation in a direct defense mechanism of melon against Fom 1. The cucurbitacin A, B, C, D. and E strongly inhibited growth of Phytophthora cactorum. Growth inhibition of Phytoptora cactorum caused by Pentacyclic and various tetracyclic triterpenoids indicates that these compounds may be naturally occurring fungistatic agents (Nes and Patterson, 1981). Bar-Nun and Mayer (1990) suggested that the ability of Cucurbitacins I and D to inhibit induction of laccase formation by Botrytis cinerea is responsible for its protective effect. The biochemical reactions occurred during resistance and susceptibility interactions between genotypes and Fom1 is understood. In agreement with Shahbazi et al. (2010), this understanding will assist us to differentiate actual resistant germplasm from those probably containing late-maturity traits that have indirect effect on pathogen growth. Conclusion The results obtained during our study imply that the elements of the whole array of the enzymes supporting plant defense are certainly constituted by the involved enzymes. Although the levels of cucurbitacin D 1593

5 content increased in both susceptible and resistant melon plants after infection, this increase was more considerable in resistant genotypes. Importantly, it could be considered as a biochemical indicator for determining resistance in melon genotypes. The resistance to Fom1 observed in some melon genotypes can be attributed to antibiotic effects, the mechanism of which may be due to certain biochemical properties. References Aharoni A, Jongsma MA, Bouwmeester HJ, Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Science 10: Avdiushko SA, Ye XS, Kuc J, Detection of several enzymatic activities in leaf prints cucumber plants. Physiol Mol Plant Pathol 42: Balkema-boomstra AG, Zijlstra S, Verstappen FWA, Inggamer H, Mercke PE, Jongsma MA, Bouwmeester HJ, Role of cucurbitacin c in resistance to spider mite (Tetranychus urticae) in cucumber (Cucumis sativus L.). J. Chem. Ecol 29: Bar-Nun N, Mayer AM, Cucurbitacins protect cucumber tissue against infection by Botrytis cinerea. Phytochem 29: Bradford MM, A rapid and susceptible method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: Chen JC, Chiu MH, Nie RL, Cordell GA, Qiu SX, Cucurbitacins and cucurbitane glycosides: structures and biological activities. Natural Product Reports 22: Dinan L, Whiting P, Girault JP, Lafont R, Dhadialla TS, Cress DE, Mugat B, Antoniewski C, Lepesant JA, Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor. Biochem. J 327: Gordon TR, Martyn RD, The evolutionary biology of Fusarium oxysporum. Annu Rev Phytopathol 35: Janda T, Szalai G, Rios-Gonzales K, Veisa O, Paldi E, Comparative study of frost tolerance and antioxidant activity in cereals. Plant Sci 164: Khatun S, Bandyopadhyay PK, Chatterjee NC, Phenols with their oxidizing enzymes in defense against black spot of rose (Rosa centifolia). Asian J. Exp. Sci 23: Kushwaha KPS, Narain U, Biochemical changes to pigeon pea leaves infected with Alternaria tenuissinia. Ann. Pl. Protec. Sci 13: Madadkhah E, Lotfi M, Nabipour A, Rahmanpour S, Banihashemi Z, Shoorooei M, Enzymatic activities in roots of melon genotypes infected with Fusarium oxysporum f. sp. melonis race 1. Scientia Horticulturae 135: Moretti A, Belisario A, Tafuri A, Ritieni A, Corazza L, Logrieco A, Production of beauvericin by different races of Fusarium oxysporum f. sp. melonis, the Fusarium wilt agent of muskmelon. Eur. J. Plant Pathol 108: Morkunas I, Gemerek J, The possible involvement of peroxidase in defense of yellow lupin embryo axes against Fusarium oxysporum. Plant Physiol 164: Nes WD, Patterson GW, Effects of tetracyclic and pentacyclic triterpenoids on growth of Phytophthora cactorum. Journal of Natural Products 44: Risser G, Banihashemi Z, Davis DW, A proposed nomenclature of Fusarium oxysporum f. sp. melonis race and resistance genes in Cucumis melonis. Phytopathol 66: Shahbazi H, Aminian H, Sahebani N, Halterman DA, Biochemical evaluation of resistance responses of potato to different isolates of Alternaria solani. Phytopathol 100: Torres MA, Jonathan DG, Dangl JL, Reactive oxygen species signaling in response to pathogen. Plant physiol 141: