**3. Results**

When strains of *Lasiodiplodia theobromae* and *Rhizoctonia solani* isolated from deteriorated rice (*Oryza sativa*) were grown in a defined growth medium containing potassium nitrate as nitrogen source and pectin as carbon source, they expressed endo-polygalacturonase activities at 30°C within ten days. The enzymes were isolated and purified by ammonium sulphate precipitation, gel filtration and ionexchange chromatography. The purification steps are presented on **Tables 1** and **2**. Purification of the endo-polygalacturonase from the *Lasiodiplodia theobromae* by gel filtration using Sephadex G-100 gave four peaks of absorption designated A, B, C. Only the fractions with peak B expressed endo-polygalacturonase activity. The molecular weight estimate of endo-polyglacturonase produced by the *Lasiodiplodia theobromae* was 124,000 Daltons. Purification of the endo-polygalacturonase from *Rhizoctonia solani* by gel filtration (Sephadex G-100) gave two peaks of absorption designated D and E. Only the fractions of peak E expressed endo-polygalacturonase activity. The molecular weight estimate of endo-polygalacturonase from *Rhizoctonia solani* was 92,000 Daltons.


#### **Table 1.**

*Purification of endo-polygalacturonase from* Lasiodiplodia theobromae *isolated from deteriorated rice (*Oryza sativa*).*

**167**

*Characterisation of Endo-Polygalacturonases Activities of Rice (*Oryza sativa*) Fungal Pathogens…*

**Total Protein (mg)**

Crude extract 1615 32.2 50.1 100 1

Peak D 1121 3.1 361.6 69.4 7.2 Peak E 926 7.4 125.1 57.3 2.5

**Specific Activity (Units/mg protein)**

1328 22.1 60.1 82.2 1.2

**Yield (%)**

**Purification fold**

The purified endo-polygalcuronase from the *Lasiodiplodia theobromae* (ionexchange chromatographic fraction - CM-Sephadex C-50) exhibited optimum activity at 30°C and at pH 4.5 while the purified endo-polygalacturonase from the *Rhizoctonia solani* (ion-exchange chromatographic fractions - CM-Sephadex C-25)

Peak Eb 895 6.5 137.7 55.4 2.7

*Purification of endo-polygalacturonase from* Rhizoctonia solani *isolated from deteriorated rice (*Oryza sativa*).*

The purified endo-polygalacturonases from both the *Lasiodiplodia theobromae* and the *Rhizoctonia solani* exhibited optimum activities at 0.2% pectin

The purified endo-polygalacturonases from both fungi were stimulated by Ca2+ They were inhibited by ethlylenediamine tetracetic acid (EDTA) and

The purified endo-polygalacturonase from the *Lasiodiplodia theobromae* lost 80% of its activity within 20 minutes of heat at 80°C. While the purified endopolygalacturonase from the *Rhizoctonia solani* lost 82% of its activity within

Potassium nitrate as nitrogen source in the defined growth medium with pectin

Pectin is found in the tissues of rice (*Oryza sativa*) [28]. In this investigation, we observed that the fungal pathogens *Lasiodiplodia theobromae* and *Rhizoctonia solani* isolated from deteriorated rice (*Oryza sativa*) produced endo-polygalacturonases in a growth medium containing pectin as carbon source and potassium nitrate as nitrogen source of fungal growth at 30°C. In Nigeria, West Africa, *Lasiodiplodia theobromae* and *Rhizoctonia solani* are fungal pathogens of rice (*Oryza sativa*) in the field [9, 10, 29]. From the results of this study, we can therefore establish that *Lasiodiplodia theobromae* and *Rhizoctonia solani* are capable of causing the deterioration of rice (*Oryza sativa*) at 30°C by breaking down the grain's pectin portion.

as carbon source supported highest activity of endo-polygalacturonase by the *Lasiodiplodia theobromae* however, ammonium chloride as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of

*DOI: http://dx.doi.org/10.5772/intechopen.94763*

**Activity (Units)**

**Fraction Total** 

90% (NH4)2SO4 Precipitation

Sephadex G-100 Gel filtration Chromatography

CM-Sephadex C-25 Ion-Exchange Chromatography

exhibited optimum activity at 32°C and at pH 5.0.

endo-polygalacturonase by the *Rhizoctonia solani*.

concentration.

**Table 2.**

2,4-dinitrophenol.

**4. Discussion**

20 minutes of heat at 80°C.

**Fraction Total Activity (Units) Total Protein (mg) Specific Activity (Units/mg protein) Yield (%) Purification fold** Crude extract 1615 32.2 50.1 100 1 90% (NH4)2SO4 Precipitation 1328 22.1 60.1 82.2 1.2 Sephadex G-100 Gel filtration Chromatography Peak D 1121 3.1 361.6 69.4 7.2 Peak E 926 7.4 125.1 57.3 2.5 CM-Sephadex C-25 Ion-Exchange Chromatography Peak Eb 895 6.5 137.7 55.4 2.7

*Characterisation of Endo-Polygalacturonases Activities of Rice (*Oryza sativa*) Fungal Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.94763*

#### **Table 2.**

*Grain and Seed Proteins Functionality*

**2.16 Effects of specific nitrogenous compounds**

were the substrates.

recorded.

**3. Results**

*solani* was 92,000 Daltons.

90% (NH4)2SO4 Precipitation

Sephadex G-100 Gel filtration Chromatography

CM-Sephadex C-50 Ion-Exchange Chromatography

**Fraction Total** 

**Activity (Units)**

in 0.1% pectin (Sigma-Aldrich, USA) in citrate phosphate buffer at pH 5.0. These

The nitrogenous compounds used were potassium nitrate, ammonium sulphate and ammonium chloride. Pectin was the constant carbon source in the growth medium. Endo-polygalacturonase activity expressed by the fungal strains of *Lasiodiplodia theobromae* and *Rhizoctonia solani* were investigated. Endo-Polygalacturonase activity expressed on the tenth day of incubation at 30°C was

When strains of *Lasiodiplodia theobromae* and *Rhizoctonia solani* isolated from deteriorated rice (*Oryza sativa*) were grown in a defined growth medium containing potassium nitrate as nitrogen source and pectin as carbon source, they expressed endo-polygalacturonase activities at 30°C within ten days. The enzymes were isolated and purified by ammonium sulphate precipitation, gel filtration and ionexchange chromatography. The purification steps are presented on **Tables 1** and **2**. Purification of the endo-polygalacturonase from the *Lasiodiplodia theobromae* by gel filtration using Sephadex G-100 gave four peaks of absorption designated A, B, C. Only the fractions with peak B expressed endo-polygalacturonase activity. The molecular weight estimate of endo-polyglacturonase produced by the *Lasiodiplodia theobromae* was 124,000 Daltons. Purification of the endo-polygalacturonase from *Rhizoctonia solani* by gel filtration (Sephadex G-100) gave two peaks of absorption designated D and E. Only the fractions of peak E expressed endo-polygalacturonase activity. The molecular weight estimate of endo-polygalacturonase from *Rhizoctonia* 

> **Total Protein (mg)**

Crude extract 4120 60.2 68.4 100 1

Peak A 2139 10.6 201.79 51.9 2.95 Peak B 1023 8.3 123.25 24.8 1.80 Peak C 995 4.6 216.3 24.1 3.16

Peak Ba 966 2.8 345 23.4 5.04

*Purification of endo-polygalacturonase from* Lasiodiplodia theobromae *isolated from deteriorated rice* 

**Specific Activity (Units/mg protein)**

3228 40.3 80.0 78.3 1.16

**Yield (%)**

**Purification fold**

**166**

**Table 1.**

*(*Oryza sativa*).*

*Purification of endo-polygalacturonase from* Rhizoctonia solani *isolated from deteriorated rice (*Oryza sativa*).*

The purified endo-polygalcuronase from the *Lasiodiplodia theobromae* (ionexchange chromatographic fraction - CM-Sephadex C-50) exhibited optimum activity at 30°C and at pH 4.5 while the purified endo-polygalacturonase from the *Rhizoctonia solani* (ion-exchange chromatographic fractions - CM-Sephadex C-25) exhibited optimum activity at 32°C and at pH 5.0.

The purified endo-polygalacturonases from both the *Lasiodiplodia theobromae* and the *Rhizoctonia solani* exhibited optimum activities at 0.2% pectin concentration.

The purified endo-polygalacturonases from both fungi were stimulated by Ca2+ They were inhibited by ethlylenediamine tetracetic acid (EDTA) and 2,4-dinitrophenol.

The purified endo-polygalacturonase from the *Lasiodiplodia theobromae* lost 80% of its activity within 20 minutes of heat at 80°C. While the purified endopolygalacturonase from the *Rhizoctonia solani* lost 82% of its activity within 20 minutes of heat at 80°C.

Potassium nitrate as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of endo-polygalacturonase by the *Lasiodiplodia theobromae* however, ammonium chloride as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of endo-polygalacturonase by the *Rhizoctonia solani*.

#### **4. Discussion**

Pectin is found in the tissues of rice (*Oryza sativa*) [28]. In this investigation, we observed that the fungal pathogens *Lasiodiplodia theobromae* and *Rhizoctonia solani* isolated from deteriorated rice (*Oryza sativa*) produced endo-polygalacturonases in a growth medium containing pectin as carbon source and potassium nitrate as nitrogen source of fungal growth at 30°C. In Nigeria, West Africa, *Lasiodiplodia theobromae* and *Rhizoctonia solani* are fungal pathogens of rice (*Oryza sativa*) in the field [9, 10, 29]. From the results of this study, we can therefore establish that *Lasiodiplodia theobromae* and *Rhizoctonia solani* are capable of causing the deterioration of rice (*Oryza sativa*) at 30°C by breaking down the grain's pectin portion.

In this research, we observed that the molecular weight of endo-polygalacturonase from our strain of *Lasiodiplodia theobromae* using Sephadex G-100 has an estimate of 124,000 Daltons while that of our strain of *Rhizoctonia solani* has an estimate of 92,000 Daltons. Adejuwon *et al.* [11] reported from their studies a polygalacturonase from *Penicillium funiculosum* Thom. isolated from tomato fruits with molecular weight estimate of 89,100 Daltons. Ajayi *et al*. [12] in their own investigation purified polygalacturonase from *Rhizopus arrhizus* Fisher isolated from tomato fruits with molecular weight estimates of approximately 166,000 Daltons and 60,260 Daltons. Olutiola [13] reported from his investigation, an extracellular polyglacturonase complex from *Penicillium citrinum* associated with internal mouldiness of cocoa (*Theobroma cacao*) beans having molecular weights of 30,000 and 56,000.

The purified endo-polygalcuronase from our *Lasiodiplodia theobromae* exhibited optimum activity at 30°C and at pH 4.5 while that from our *Rhizoctonia solani* exhibited optimum activity at 32°C and at pH 5.0. Olutiola [30] reported that *Penicillium sclerotigenum* Yamamoto isolated from rotten yam tuber produced a polygalacturonase with optimum activity at pH 5.0.

The purified endo-polygalacturonases from both fungi exhibited optimum activities at 0.2% pectin concentration. The purified endo-polygalacturonases from both fungi were stimulated by Ca2+ but inhibited by ethlylenediamine tetracetic acid (EDTA) and 2,4-dinitrophenol. Ajayi *et al.* [31] purified polygalacturonase from *Rhizopus arrhizus* Fisher with optimum activity at pH 4.5, stimulated by Ca2+ but inhibited by ethlylenediamine tetracetic acid (EDTA) and 2,4-dinitrophenol. The purified endo-polygalacturonase from our *Lasiodiplodia theobromae* lost 80% of its activity within 20 minutes of heat at 80°C. While the purified endo-polygalacturonase from our *Rhizoctonia solani* lost 82% of its activity within 20 minutes of heat at 80°C. Potassium nitrate as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of endo-polygalacturonase by *Lasiodiplodia theobromae* while ammonium chloride as nitrogen source in the defined growth medium with pectin as carbon source supported highest activity of endo-polygalacturonase by *Rhizoctonia solani*. According to Olutiola [32], pectin as carbon source with L-asparagine as nitrogen source of a defined growth medium supported the growth and sporulation of *Fusarium oxysporum* Schlecht. In conclusion, the conditions inhibiting endo-polygalacturonases from *Lasiodiplodia theobromae* and *Rhizoctonia solani* capable of degrading the pectin portion of rice (*Oryza sativa*) observed in this study can be adapted as feasible control measures limiting the infection and contamination of rice (*Oryza sativa*) by these phytopathogens on the field and at postharvest. Temperature and pH extreme from 30°C and pH 4.5 will be feasible inhibitory control measures for the growth of *Lasiodiplodia theobromae* on rice (*Oryza sativa*) in Nigeria and particularly the preservation of pectin in rice (*Oryza sativa*) from degradation by this particular phytopathogen. Temperature and pH extreme from 32°C and pH 5.0 will inhibit growth of *Rhizoctonia solani* on rice (*Oryza sativa*) and preserve pectin in this grain from degradation by *Rhizoctonia solani*.

#### **Acknowledgements**

Authors are grateful to the British Mycological Society (BMS), Britain, United Kingdom; the National Academy of the Sciences (NAS) of Ukraine, Ukraine, East Europe; and the Institute of Bioorganic Chemistry and Petrochemistry (IBOCP), Kyiv, Ukraine, East Europe for research supports.

**169**

*Characterisation of Endo-Polygalacturonases Activities of Rice (*Oryza sativa*) Fungal Pathogens…*

**A.1 DNSA reagents for terminating endo-polygalacturonase activity**

*DOI: http://dx.doi.org/10.5772/intechopen.94763*

Authors declare no conflict of interest.

i.Sodium hydroxide – 10 g

ii.Potassium sodium tartrate – 200 g

iv. 3, 5-Dinitrosalicylic acid – 10 g

v. 5% Sodium sulphite – 10 ml

vi.0.03% Glucose – 10 ml

vii.Distilled water – 1Litre

**Conflict of interest**

**A. Appendix**

**Reagents [17]**

iii.Phenol – 2 g

*Characterisation of Endo-Polygalacturonases Activities of Rice (*Oryza sativa*) Fungal Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.94763*
