VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
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Research Article, Vegetos Vol: 30 Issue: 2

Evaluation of Biochemical Changes in Leaves of Tomato Infected with Alternaria Alternata and its Metabolites

Meena M1*, Prasad V2 and Upadhyay RS1
1Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi, India
2Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, India
Corresponding author : Mukesh Meena
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi-221005, India
Mob: 08726699266br
E-mail:
[email protected]
Received: February 06, 2017 Accepted: February 22, 2017 Published: February 24, 2017
Citation: Meena M, Prasad V, Upadhyay RS (2017) Evaluation of Biochemical Changes in Leaves of Tomato Infected with Alternaria Alternata and its Metabolites. Vegetos 30:2. doi:10.4172/2229-4473.2017.00020.9

 

Abstract

The present study was carried out to evaluate the biochemical changes occurring in tomato leaves following treatment with leaf spot disease causing pathogen Alternaria alternata and two of its phytotoxic metabolites. Results revealed that the pathogen treatment caused severe foliar necrosis and by 10th day of treatment almost 3/4th of the leaf area was necrotized. A significant decline in the chlorophyll content was observed in the leaves treated with the pathogen and its metabolites. Reducing sugars showed a visible decline in the treated leaves in comparison to the control leaves. When compared with the control an increase in total soluble protein was observed in leaves treated with the pathogen and its metabolites. In case of phenol, a significant enhancement in phenol content of the pathogen and metabolite treated leaves was observed than the amount detected in control leaves. The results indicate that infection of tomato by the leaf spot pathogen Alternaria alternata and its metabolites causes severe alterations in the amount of various biochemical components.

Keywords: Chlorophyll; Metabolites; Pathogen; Phenol; Reducing sugar; Total soluble protein

Keywords

Chlorophyll; Metabolites; Pathogen; Phenol; Reducing sugar; Total soluble protein

Introduction

Plant pathogenic fungi Alternaria alternata, generally known as a cosmopolitan saprophyte, produces a range of secondary metabolites belonging to a number of classes of phytotoxic chemicals, including either host-specific (host selective) or nonspecific toxins from different strains [1-3]. Alternaria is easily documented by the shape of their conidia, which are large, ovoid to obclavate, dark-colored (melanized), multicellular with longitudinal and transverse septations (phaeodictyospores). Conidia are produced in single or branched chains on petite conidiophores [4]. Pathogenic Alternaria has broad range (>380) of hosts including tomato, citrus, tobacco, potato, tangerine, apple, pear, pistachio, bean, garden plants, field crops and ornamental plants [5].
Members of the genus Alternaria are known to produce a wide range of phytotoxic metabolites which influence many plants on which the fungi are found [6]. These phytotoxins possessing a broad range of biological and metabolic effects include tenuazonic acid (TA), alternariol, alternariol monomethyl ether (AME), alternaric acid, altenuic acid, altenuene, tentoxin, AK-toxin and AAL-toxin [7]. It is reported that Alternaric acid (AA) plays a significant role in influencing host specificity and causative to disease development caused by Alternaria solani [8,9].
Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops grown worldwide [10]. It is cultivated essentially in all countries either in fields or in protected cultures. Leaf spot of tomato caused by Alternaria spp. is one of the common factors that reduce tomato production [11]. This pathogen infects the plant leaves at all stages of plant growth, and causes destructive necrotic symptoms, that lead to yield losses. The common biochemical constituents like chlorophyll, sugars and phenols are important in imparting resistance to the crop plants. Sometimes, host plant is induced to synthesize these compounds upon infection. The biochemical components like, phenols, sugars, chlorophyll and others are known to play an imperative role in susceptibility and or resistance against biotrophic, hemi-biotrophic and necrotic plant pathogens [12-14]. Such biochemical changes have been reported by many workers in different plants by different plant pathogens [13-19]. Here, the present investigation was undertaken with objectives to study the different biochemical changes in tomato, infected with Alternaria alternata and its metabolites.

Materials and Methods

Fungal culture, extraction of metabolite(s) from culture filtrate
The stock culture of the fungal pathogen Alternaria alternata previously isolated and preserved in the laboratory was used in the present work. The metabolites extraction from this isolate was carried out by the method of Janardhanan and Hussain [20]. Briefly, the fungal isolate was grown in potato dextrose broth medium for 20 days and was then processed. The fungal culture filtrate was collected by passing the broth culture through 4 layers of cheese cloth followed by filtration through filter paper (Whatman number 4) by using Buchner funnel. An equal volume of methanol was added to the fungal filtrate to precipitate proteins and kept in a refrigerator for 24 hours. The precipitate was filtered off and methanol recovered at 40-50°C in vacuum evaporator. The filtrate was then extracted with equal volume of ethyl acetate 3 times and the extract concentrated at 40-50°C in vacuum evaporator. An orange red viscous substance obtained was dissolved in methanol, absorbed on activated charcoal and then eluted with the same solvent. The methanol elute was concentrated in vacuum evaporator and was subjected to TLC fractionation. The TLC analysis done was similar as described by Meena et al. [21]. After TLC analysis two independent spots were visible on silica gel plates, which were further eluted in methanol. These two different metabolites eluted out were used in the present experimental work.
Plant growth and treatment
Seeds of tomato cv. Co-3 were obtained from the Indian Institute of Vegetable Research (Varanasi) and were grown in a greenhouse under a 14 h light and 10 h dark cycle at 27 ± 0.5°C. Autoclaved soil was used and the plants were allowed to attain a height of 15-20 cm before testing for sensitivity to the pathogens and the culture filtrate or for other experiments. Tomato plants were treated with the pathogen and its metabolites following the method described by Prasad and Upadhyay [22]. Briefly, the leaves of tomato plants were gently punctured with sterile sand paper followed by treatment with spore suspension of Alternaria alternata containing 2 × 106 spores/ ml by spraying until runoff. For metabolites treatment, each of the metabolite (2.0 ml) were applied to the leaves by infiltration of the leaf tissue with the help of a 1 ml syringe fitted with 25 guage needle in the midrib just above the petiole on the underside of the leaf. Control leaves were infiltrated with sterile distilled water. Leaves of the control and treated plants were sampled after 96 hours of treatment for various biochemical analyses. All the experimental treatments were carried out in triplicate.
Estimation of chlorophyll
Chlorophyll content of tomato plants treated with pathogen and their metabolites were extracted by using the method of Arnon [23]. The leaves were chopped into small pieces and extracted with 80% acetone. The absorbance was measured at 645 nm and 663 nm for chlorophyll a, b and total chlorophyll by using spectrophotometer.
Then chlorophyll a, b and total chlorophyll were calculated according to the Lichtenthaler and Wellburn [24] formula.
Chl a (mg g-1 leaf fresh weight) = [12.7 (OD663) - 2.69 (OD645)] × V/1000 × W
Chl b (mg g-1 leaf fresh weight) = [22.9 (OD645) - 4.68 (OD663)] × V/1000 × W
Total Chl (mg g-1 leaf fresh weight) = [20.2 (OD645) - 8.02 (OD663)] × V/1000 × W
(Where, OD = Optical Density, V = Volume of sample, W = Weight of sample)
Estimation of reducing sugar
Sugar was extracted from treated and control leaves by homogenising and boiling 200 mg of leaf tissue with 10 ml of 80% ethanol followed by centrifugation. The reducing sugars were estimated in the supernatant according to the Nelson-Somogyi method [25,26]. Briefly, 0.2 ml of sample was taken and made up to 1.0 ml with sterile distilled water. To this 1.0 ml of Somogyi’s copper reagent was added. The mixture was then kept in boiling water bath for 20 min. This was followed by cooling after which 1.0 ml of Nelson’s arsenomolybdate reagent was added with proper mixing. The intensity of the colour developed was measured at 540 nm. D-Glucose was used as standard.
Estimation of total soluble proteins
For estimation of total soluble protein, leaf and root tissues were extracted by the modified acetone-trichloroacetic acid (TCA) precipitation method of Damerval et al. [27]. Leaf samples were homogenized separately with cold 50 mM sodium phosphate buffer (pH 7.8) using pre-chilled mortar and pestle. The resulting homogenates were centrifuged at 4°C at 14,000 rpm for 15 min (Remi Instruments, India). The supernatants were mixed thoroughly with equal volume of 10% TCA, kept overnight at 4°C to facilitate complete precipitation of soluble proteins. The precipitates were then centrifuged at 12,000 rpm for 10 min and the pellets were washed with 100% acetone in order to remove the pigments. The pigmentfree pellets were washed successively with 80% ethanol, 3:1 (v:v) ethanol:chloroform, 3:1 (v:v) ethanol:diethyl ether and finally with diethyl ether to remove phenolic compounds. The washed pellets were air dried and solubilized by resuspending in a known volume of 0.1 N NaOH for 16 h at 37°C. The samples were centrifuged and supernatants were collected for protein estimation. Quantitative estimation of protein was done according to the method of Lowry et al. [28] using bovine serum albumin (BSA, Sigma) as standard.
Estimation of phenol
Leaf sample (1.0 g) were homogenized in 10 ml of 80% methanol and agitated for 15 min at 70°C according to method of Zieslin and Ben-Zaken [29]. The homogenate was centrifuged at 16,000 rpm at 4°C for 15 min. The reaction mixture contained 1.0 ml of the methanolic extract, 5 ml of distilled water and 250 μl of Folin-Ciocalteau phenol reagent (1N) and the solution was kept at 25°C. The absorbance of the developed blue colour was measured using a spectrophotometer (Systronics 2202, India) at 725 nm. Catechol was used as the standard. The amount of phenolics was expressed as μg catechol mg−1 protein
Statistical Analysis
Statistical analysis was performed by using IBM SPSS Statistics ver. 20 software via analysis of variance (one-way ANOVA) followed by Duncan’s multiple range test at the p ≤ 0.05 significance level. Data were expressed as mean ± standard deviation (SD) of at least three replicates of each metabolite.

Results

Disease severity of the pathogen
Upon artificial infection by the isolate the prominent visible symptoms on tomato leaves started appearing from 2nd day of infection and with the gradual increase of the treatment period increase in necrotic area were observed in comparison to the control leaves (Figure 1). By the 10th day of treatment almost 80% leaf area was necrotized in the treated leaves while only a slight degradation in the leaf area of control leaves was observed.
Figure 1: Infection of tomato plants by Alternaria alternata showing the area of necrotic lesions. The vertical bars indicate the ± SD of the mean.
Chlorophyll content
The chlorophyll content declined considerably after 96 hours following treatment with the pathogen and its two different metabolites in comparison to the control (Figure 2). A decline in the amount of both chlorophylls a and b was observed in all the three treatments. The pathogen treatment proved to be more destructive for decreasing the chlorophyll content and decreased it by 54% followed by the metabolite T2 and then T1 which produced a decline by 34% and 12% respectively (Figure 2). Total chlorophyll content was observed to be 23.28 mg g-1 FW in control samples while, in pathogen and metabolite T2 and T1 treated samples it was 10.79 mg, 15.50 mg and 20.58 mg g-1 FW respectively, after 96 hours of treatment.
Figure 2: Effect of treatment of Alternaria alternata and its metabolites on chlorophyll content in tomato. The results are expressed as the mean of three replicates and the vertical bars indicate the ± SD of the mean. Different letters indicate that the values are significantly different from each other (p ≤ 0.05).
Reducing sugar
The pathogen and its metabolites produced a significant decline in the amount of reducing sugar being synthesized. The results revealed that the total reducing sugar content was highest in control leaves in comparison to the treated ones. In the treatments the pathogen treatment produced a severe decline by almost 88% in the amount of reducing sugar followed by the metabolites T2 and T1 which were 47% and 30% respectively after 96 hours of treatment (Figure 3). The reducing sugar content was observed to be 0.62 mg g-1 FW in control samples while, in pathogen and metabolite T2 and T1 treated samples it was 0.08 mg, 0.33 mg and 0.43 mg g-1 FW respectively, after 96 hours of treatment.
Figure 3: Effect of treatment of Alternaria alternata and its metabolites on reducing sugar content in tomato. The results are expressed as the mean of three replicates and the vertical bars indicate the ± SD of the mean. Different letters indicate that the values are significantly different from each other (p ≤ 0.05).
Total soluble proteins
The results showed an increment in the amount of total soluble protein content in the leaves of tomato following the different treatments. A significantly increased amount of total soluble protein was observed in the pathogen treated leaves and it was by about 41% of control (Figure 4). Similar enhanced protein content was seen in the metabolites T2 (36% of control) and T1 (13% of control) treated samples also while the control leaves showed the lowest amount of total soluble protein present (Figure 4). The total soluble protein content was observed to be 899.64 μg g-1 FW in control samples while, in pathogen and metabolite T2 and T1 treated samples it was 1274.49 μg, 1224.51 μg and 1024.59 μg g-1 FW respectively, after 96 hours of treatment.
Figure 4: Effect of treatment of Alternaria alternata and its metabolites on total soluble protein content in tomato. The results are expressed as the mean of three replicates and the vertical bars indicate the ± SD of the mean. Different letters indicate that the values are significantly different from each other (p ≤ 0.05).
Phenol content
In the present investigation the phenol content was evaluated following the various treatments. The results revealed an increased phenol content following different treatments with the pathogen (925% of control) and the two metabolites T2 (844% of control) and T1 (812% of control). Higher phenol content was observed in the pathogen treated leaves followed by the metabolite T2 and T1 treated leaves, while the lowest content of phenol was observed in the control leaves (Figure 5). The phenol content was observed to be 0.014 mg equivalents of catechol mg-1 protein in control samples while, in pathogen and metabolite T2 and T1 treated samples it was 0.145 mg, 0.132 mg and 0.128 mg equivalents of catechol mg-1 protein respectively, after 96 hours of treatment.
Figure 5: Effect of treatment of Alternaria alternata and its metabolites on phenol content in tomato. The results are expressed as the mean of three replicates and the vertical bars indicate the ± SD of the mean. Different letters indicate that the values are significantly different from each other (p ≤ 0.05).

Discussion

This study is a report on physiological and biochemical changes occurring in tomato following treatment with leaf spot disease causing fungus Alternaria alternata and its metabolites. A. alternata f. sp. lycopersici produces in still culture a toxin that causes symptoms on tomato leaves which are indistinguishable from those that develop on naturally infected plants [30,31]. The first reported example of an Alternaria-produced phytotoxin exhibiting the same host specificity as the pathogen was from the black spot disease of Japanese pear caused by A. kikuchiana [32]. A. mali, the causal organism of apple blotch, is also reported to produce a host-specific toxin [33]. In the present work both the pathogen as well as it two metabolites were observed to produce severe necrosis in treated leaves.
mThere was a substantial difference in the chlorophyll levels of control and treated leaves in the present study. Various plant pathogens are known to produce toxic metabolites, which may destroy the chloroplast resulting into decrease of chlorophyll pigments. The reduction in chlorophyll may be associated to the toxic metabolites produced by pathogen which may destroy the chloroplast [21,34] or to the inhibition chlorophyll synthesis rather than the degradation of pre-existing pigments [35,36].
Results obtained here showed that following treatment, sugar level decreased in all the treatments. Reducing sugars were significantly higher in control than in treated samples. Post-treatment decrease in sugar levels may be caused by rapid hydrolysis of sugars during pathogenesis through enzymes secreted by the pathogen [37]. The invading pathogens may utilize the sugar leading to decrease in its content. The co-existence of free-sugars and phenols results in glycosylation of phenols by sugars, forming phenolic glycosides, which are more soluble in cell sap, thus provide more efficiency in the resistance expression [38].
The concentration of total soluble protein increased in leaves treated with the pathogen or its metabolites. During host-pathogen interaction, amino acids act as a substrate for the pathogen [39] or they may have a fungistatic effect through their involvement in metabolic reactions associated with disease resistance [40]. Changes in protein occur when the pathogen penetrates the host cells resulting in disturbances in protein and related metabolisms. Proliferation of microorganism synthesize several enzymes, proteins and sometimes cause rearrangement of nutritional composition of substrate due to formation of several degradation products thereby increasing its protein content [41].
Plants have developed an arsenal of defence mechanisms to protect themselves against pathogen attacks. Infection by A. alternata and its metabolites was associated with a marked increase in the phenolic contents of treated tissues. A gradual increase in total phenolic content was noticed with the progress of lesion development. The total phenols were higher in infected leaves than healthy. The post inflectional increase in phenolic contents could be due to their release from glycosidic esters by the enzymatic activity of host or pathogen, or due to migration of phenols from non-infected tissues.

Conclusion

Based on the present findings, it may be concluded that high level of total phenols, and high protein content of tomato appeared to be the important biochemical constituents, which may impart resistance against infection caused by A. alternata and its phytotoxic metabolites. Such studies may provide information regarding hostpathogen interaction which can be utilized for resistance breeding for the development of desirable trait by incorporating resistance in promising crop genotypes.

Acknowledgment

The author Mukesh Meena is thankful to Indian institute of vegetable research (IIVR), Varanasi, for providing Co-3 variety of tomato seeds. The author is also thankful to RGNF-(UGC) for providing financial assistance in form of JRF and SRF during the course of this study. The author is also thankful to the Head Department of Botany and Programme Coordinator, Centre of Advanced Study in Botany, Banaras Hindu University for providing necessary research facilities for the experiments.

References

 

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