View of Pectinases Synthesis by Ralstonia solancearum is influenced by various nutritional and environmental factors

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Pectinases Synthesis by Ralstonia solancearum is influenced by various nutritional and environmental factors

Asrar Siraj, B. Y. Gottigalla, M. M. V. Baig

Department of Botany and Department of Biotechnology, Yeshwant Mahavidyalaya, Nanded - 431 602, India.

*corresponding author: [email protected]

Abstract:

Ralstonia solancearum was isolated from four host plants viz. Turmeric, Banana, Tomato and Brinjal. From these samples total twenty strains were isolated and screened for further study. Out of twenty strains, one strain is recorded as more effective for the pectinases (PG, PmG, PGL, PnL) production. This strain was further used for the study of various factors like time of incubation, temperature, pH, sources of carbon and nitrogen affecting pectinases production. The optimum time, temperature and pH for pectinases production was 48hrs, 30 °C and 7 respectively. In the experiment of effect of various sources of carbon (pectin, sucrose, glucose, maltose, starch) and sources of nitrogen (yeast extract, ammonium nitrate, ammonium sulphate, potassium nitrate, peptone) it was found that pectin was most suitable carbon source and yeast extract was most suitable sources of nitrogen for optimum production of pectinases.

Introduction:

Pectin is complex hetero polysaccharide and present in the primary cell wall of plant as a major component (William et al., 2000). In the cell walls, dicotyledonous plants have about 35 percent pectin, 30 percent cellulose, 30 percent hemicellulose, and 5 percent protein, whereas grasses have 2–10 percent pectin. (Voragen et al., 2009). Porosity, surface charge, pH, ion balance, and ion transport are all controlled by pectin in the cell wall. (McNeil et al., 1984). The pectinases are classified into PG, PMG, PgL, PnL depending upon their mode of action. PG degrades pectate. It hydrolyses o-glycosyl linkage and form a- 1,4-polygalacturonic residues. PMG hydrolyse highly esterified pectin without affecting the ester content of remaining polymers (Itziar et al.,1998). PnL cleaves esterified pectin and form oligo or monomethyl galacturonate without disturbing ester group (Sangeeta et al.,2009). PgL cleaves the de-esterified pectin molecule (Sharma et al.,2011).

Materials and methods:

i) Isolation of Ralstonia solancearum:

Twenty strains of Ralstonia solancearum isolated from Turmeric, Banana, Tomato and Brinjal wilted plant samples. The infected leaves with typical wilting symptoms were collected during mid rainy season were used in this study. These strains were isolated and purified by following standard method and maintained on KZC media (Peptone 10gm;

Hydrolyzed Casein - 1gm; Glucose - 0.5gm (Filter sterilized); Agar - 17gm; Distilled water- 1000ml; 3ml of TZC) (Hi-media chemicals, Mumbai India) slants.

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ii) Morphological Characterization:

Morphological characteristics like colony characters, Gram staining, cell morphology, cell motility etc. were recorded for all these twenty strains (Bradbury et al.,1984).

iii) Biochemical test:

These twenty strains were further characterized by using specific biochemical test described by Goszczynska et al., (2000) such as fermentation, Catalase production, Oxidase production, Starch hydrolysis, additionally the tests such as Levan production, Arginine utilization, Urease productions, Tyrosine utilization Tween 80 hydrolysis, Gelatin liquification, Aesculin hydrolysis

.

iv) Production media for pectinases

The media used for production of pectinases contained, Pectin-5gm / 200 ml, KH2PO4- 1.6gm, Na2HPO4- 1.6gm, MgSO4.7H2O-0.2gm, CaCl2.2H2O- 0.1 gm, Yeast extract- 5gm, Total volume - 800 ml, pH- 7.

v) Optimization of cultural parameters for pectinases production:

i) Effect of incubation time on pectinases production:

Twenty ml of medium was taken in each 50 ml Erlenmeyer flasks and all flasks were autoclaved at 15 lbs for 20 min. These flasks were kept for different incubation time period varied from 1-6 days at 30°C after inoculation with 1x10⁷cells/ml.

ii) Effect of temperature on pectinases production:

For the determination of optimum temperature for pectinases production, flasks (50ml) containing 20 ml of production medium were autoclaved at 15 lbs for 20 min. All flasks were then inoculated with the 1x10⁷cells / ml and incubated at different temperature ranges from 25 °C to 45 °C by keeping all process parameters constant.

iii) Effect of pH on pectinases production:

To determine the optimum pH for pectinases production, different pH media 6.5, 7, 7.5, 8 and 8.5 were adjusted by using 1N HCL or 1N NaOH. All flasks were autoclaved at 15 lbs for 20 min. and then inoculated with 1x10⁷ cells /ml. All flasks were incubated at 30

°C for 48hrs by keeping all other process parameters constant.

iv) Effect of different carbon sources on pectinases production:

By keeping all process parameters constant, the flasks containing production medium with different carbon-sources as glucose, sucrose, starch, maltose and pectin were used at a concentration of 0.5% w/v. These flasks were autoclaved at 15 lbs for 20 min. and further inoculated with 1x10⁷ cell/ml culture of Ralstonia solancearum.

v) Effect of different nitrogen sources on pectinases production:

Effect of nitrogen sources on pectinases production was studied by keeping all other process parameters constant. Different nitrogen sources such as yeast extract, ammonium sulphate, ammonium nitrate, potassium nitrate and peptone were used in the production media at a concentration of 0.5 % w/v. All these flasks were autoclaved at 15 lbs for 20

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min. Then same culture of Ralstonia solancearum was inoculated in each flask as 1x 10⁷ cells/ml and all flasks were incubated at 30 °C for 48 hrs.

vi) Extraction of pectinases:

After the experiment of optimization of parameters for pectinases production, all flasks were taken for filtration, the culture filtrate was collected and further centrifuged at 6000 rpm for 15 min. After centrifugation supernatant was collected and cell pellets were discarded. The supernatant was filtered through Whatman no. 1 filter paper and then precipitated by adding cold acetone. (Weber and Parola, 1984).

vi) Assays for pectinases:

To determine activity of polygalacturonases (PG), the increase in absorbances at 240 nm was measured. For this activity 2 ml of 1% polygalacturonic acid in 1% pectin in 0.1M acetate buffer of pH 6.5 as a substrate was hydrolyzed by 1 ml of enzyme solution (Gewali et al., 2007). Poly methyl Galacturonase (PMG) activity was determined by measuring the increase in absorbance at 240 nm of substrate concentration of 2 ml of 1%

pectin in 0.1M acetate buffer of pH 6.5 hydrolyzed by 1 ml of enzyme solution (Mahadevan and Sridhar, 1986). Activity of PnL was measured at 240 nm by recording the increase in absorbance of substrate concentration as 2 ml of 1% pectin in 0.1 M citrate phosphate buffer of pH 4.5 is hydrolysed by 1 ml of enzyme solution (Ramanujam and Palani, 2008). For the study of activity of pectate lyases (PgL), the reaction mixture was prepared containing 2 ml of 1% polygalacturonic acid in 0.1M citrate phosphate buffer of pH 4.5 which is hydrolysed by 1% enzyme solution, this reaction was measured by recording increase in absorbance at 240 nm (Viviani et al., 2010; Visser and Voragen, 1996). One unit of enzyme activity was defined as the amount of enzyme which released 1umol of reducing sugar per minute.

Result:

The various strains of Ralstonia solancearum were isolated from infected leaves of Tomato, Brinjal, Turmeric and Banana collected from different regions for the production of pectinases. Pectinases production is affected by various biochemical parameters like time, temperature, pH, and other medium components like carbon-sources (glucose, sucrose, starch and maltose) and nitrogen-sources (yeast extract, ammonium sulphate, ammonium nitrate, potassium nitrate and peptone).

Optimization of cultural parameters for pectinases production:

i) Optimum time for pectinases production:

The incubated flasks were collected at different time intervals as 24hrs, 48hrs, 72hrs, 96hrs, 120hrs and 144 hrs. The production of pectinases was found to be increase from 24hrs to 48 hrs and after 48 hrs to 6o hrs there was slight decrease in production till 120 hrs. Thus the maximum production of pectinases as PG (0.039 U/ml), PMG (0.045 U/ml), PgL (0.049 U/ml) and PnL(0.064 U/ml) on 48hrs of incubation. The results are given in Fig.1

Table 1: Effect of incubation time on pectinases production

Sr. No. Incubation Time PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

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1 12 hrs 0.029 0.035 0.041 0.054

2 24 hrs 0.032 0.037 0.044 0.057

3 36 hrs 0.034 0.041 0.047 0.061

4 48 hrs 0.036 0.045 0.049 0.064

5 60 hrs 0.039 0.042 0.049 0.059

6 72 hrs 0.032 0.038 0.043 0.055

7 82 hrs 0.032 0.032 0.040 0.049

8 96 hrs 0.029 0.029 0.036 0.046

9 108 hrs 0.027 0.026 0.032 0.044

10 120hrs 0.025 0.024 0.030 0.043

Figure 1: Effect of incubation time on pectinases production

ii) Optimum temperature for pectinases production:

The effect of temperature on production of pectinases was studied by using temperature ranges from 25 °C to 45 °C. It was found that as temperature increased from 25 °C to 35

°C the production of pectinases also increased but further increase in temperature there was decrease in pectinases production. Hence, it was concluded that the optimum temperature was 30°C and the maximum production at 30 °C was PG (0.036 U/ml), PMG (0.037 U/ml), PgL (0.059 U/ml) and PnL (0.065 U/ml) as shown in Fig. 2.

Table 2: Effect of temperature on pectinases production

Sr. No. Temperature PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

1 25 °C 0.025 0.030 0.048 0.059

2 30 °C 0.035 0.034 0.054 0.065

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

12 hrs 24 hrs 36 hrs 48 hrs 60 hrs 72 hrs 82 hrs 96 hrs 108 hrs 120hrs

Enzyme production (U/ml)

Incubation Time

PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

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3 35 °C 0.036 0.037 0.059 0.064

4 40 °C 0.028 0.026 0.043 0.051

5 45 °C 0.020 0.022 0.037 0.047

Table 2: Effect of temperature on pectinases production

Figure 2: Effect of temperature on pectinases production

iii) Optimum pH for pectinases production:

The effect of pH on pectinases production was studied by conducting an experiment by using different pH ranges from 6.5 to 8.5 with all other process parameters constant.

Initially as pH was increased from 6.5 to 7, the production of pectinases was also increased but further increase in pH from 7 to 7.5, 8, 8.5 there was decrease in pectinases production. Thus maximum production of pectinases as PG(0.049 U/ml), PMG(0.037 U/ml), PgL(0.054 U/ml) and PnL(0.060U/ml) as shown in Fig. 3 was found in the medium having pH 7. Hence optimum pH value was 7.

Table3: Effect of pH on pectinases production

Sr. No. pH PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

1 pH 6.5 0.042 0.033 0.044 0.058

2 pH 7.0 0.049 0.037 0.054 0.060

3 pH 7.5 0.045 0.035 0.052 0.056

4 pH 8.0 0.036 0.025 0.037 0.044

5 pH 8.5 0.03 0.022 0.027 0.036

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

25 °C 30 °C 35 °C 40 °C 45 °C

Temperature

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Figure 3: Effect of pH on pectinases production

iv) Effect of carbon sources on pectinases production:

The production of pectinases was also studied with various carbon sources as glucose, sucrose, starch, maltose and pectin by keeping all other process parameters constant. The maximum production of pectinases as PG (0.054 U/ml), PMG(0.043 U/ml), PgL(0.064 U/ml) and PnL(0.072 U/ml) was observed in media containing pectin as a carbon-source (Fig. 4)

Table4: Effect of carbon sources on pectinases production

Sr. No. Carbon sources PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

1 glucose 0.042 0.028 0.052 0.068

2 sucrose 0.049 0.041 0.056 0.061

3 starch 0.041 0.035 0.052 0.057

4 maltose 0.046 0.04 0.046 0.054

5 pectin 0.054 0.043 0.064 0.072

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

pH 6.5 pH 7.0 pH 7.5 pH 8.0 pH 8.5

pH

PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

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Figure 4: Effect of carbon sources on pectinases production

v) Effect of nitrogen sources on pectinases production:

The production of pectinases, extractedby culture of Ralstonia solancearum was influenced by different nitrogen sources like yeast extract, ammonium sulphate, ammonium nitrate, potassium nitrate and peptone when other process parametersconstant. By this study it was cleared that the maximum production of pectinases like PG (0.051 U/ml), PMG (0.037 U/ml), PgL(0.054 U/ml) and PnL(0.066 U/ml) was observed in media containing yeast extract as a nitrogen-source. Thus yeast extract is suitable nitrogen source for maximum production of pectinases. The results are given in Fig.5.

Table 5: Effect of nitrogen sources on pectinases production

Sr. No. Nitrogen sources PG (U/ml) PMG (U/ml) PgL (U/ml) PnL (U/ml)

1 Yeast extract 0.051 0.037 0.054 0.066

2 Amm. Sulphate 0.042 0.033 0.047 0.049

3 Amm. Nitrate 0.035 0.035 0.042 0.045

4 Pott. Nitrate 0.036 0.029 0.035 0.045

5 Peptone 0.045 0.034 0.051 0.057

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

glucose sucrose starch maltose pectin

carbon source

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Figure 5: Effect of nitrogen sources on pectinases production

Discussion

The characterisation of R. solancearum pectinases is crucial since these enzymes have a role in plant disease. Plant pathogenicity, plant-microbe interaction, and the breakdown of dead plant material are all influenced by microbial pectinases(Lang and Domenburg, 2000).. Saprophytic fungi, plant pathogenic fungi, and a number of bacteria all generate pectinases in large amounts (Gummadi and Panda, 2003; Hasunuma et al., 2003).

The mode of action and biochemical properties of these microbial pectolytic enzymes differ(Favela-Torres et al., 2005; Gummadi and Panda, 2003).. Alkaline pectinases are produced by bacteria, while acid pectinases are produced by fungus (Hoondal et al., 2002; Jayani et al., 2005; Kashyap et al., 2001).

R. Solanacearum synthesizes one pectin methyl esterase (PME) and one multimedia (PGs). PME eliminates methyl groups from pectin, allowing PGs to break down this key cell wall component, degrading the pectin polymers in the process(Schell et al., 1988;

Allen et al., 1991),. There are two forms of PG in R. solanacearum: an endo-PG called PglA or PehA that cleaves the pectin polymer at random, releasing large fragments, and two exo-PG called exo-poly-α-D-galacturonosidase PehB (Allen et al., 1991) and exopolygalacturonase PehC that release galacturonic acid dimers and monomers, respectively(Gonzalez & Allen, 2003).

These enzymes make only a minor contribution to R. solanacearum virulence. PME inactivation on several host plants does not affect virulence (Tans-Kersten et al., 1998). A triple mutant strain that is completely non-pectinolytic and slightly reduced virulence in tomatoes following soil inoculation assays, defective for pehA, PehB, and PehC was recently generated (Gonzalez & Allen, 2003). Comparatively, exuT lies immediately downstream from the Galacturonate transporter gene. A mutant, exuT, which still manufactures all three PG isozyms, but can not use PG degradation products, was

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

Yeast extract Amm. Sulphate Amm. Nitrate Pott. Nitrate Peptone

Nitrogen source

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developed for evaluation of the nutritional role of planta of the PGs (Gonzalez & Allen, 2003).

This exuT mutant had wild-type tomato virulence, demonstrating that galacturonic acid metabolism does not significantly contribute to bacterial multiplication within the plant (Gonzalez & Allen, 2003). These findings support the theory that one of the primary virulence functions of PGs is the maceration of plant pectic substances during the early stages of interaction, allowing R. solanacearum invasion and spread.

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