**Abstract**

Rice (*Oryza sativa*) is cultivated in swampy geographical locations of tropical Nigeria, West Africa. Here it is infected by a host of fungal pathogens on the field or contaminated at postharvest. This has led to its loss and reduction in its production in both the national and global market. *Lasiodiplodia theobromae* and *Rhizoctonia solani* have recently been identified as the major fungal phytopathogens causing the deterioration of this grain on the field and at postharvest and affecting its production in Nigeria leading to gross capital loss. Hence the need to determine physiological control measures for the eradication of both phytopathogens on the field and at postharvest. In this study, tropical strains of *Lasiodiplodia theobromae* and *Rhizoctonia solani* obtained from deteriorated rice (*Oryza sativa*) were grown in a growth nutrient medium composed of MgSo4.7H20, K2HPO4, FeSO4.7H20, potassium nitrate and pectin at 30°C. Endo-Polygalacturonase activities were produced by the fungal isolates in the growth medium within ten days. The endo-polygalacturonases from both fungi were purified by a combination of ammonium sulphate precipitation, dialysis, gel filtration (on Sephadex G-100 column) and ion-exchange chromatography (on CM-Sephadex C-50 and CM-Sephadex C-25 columns). The molecular weight of endo-polygalacturonase from the *Lasiodiplodia theobromae* using Sephadex G-100 was estimated as 124,000 Daltons while that of the *Rhizoctonia solani* was estimated as 92,000 Daltons. The purified endo-polygalcuronase from the *Lasiodiplodia theobromae* exhibited optimum activity at 30°C and at pH 4.5 while that from the *Rhizoctonia solani* exhibited optimum activity at 32°C and at pH 5.0. The purified endo-polygalacturonases from both fungi exhibited optimum activities at 0.2% pectin concentration. They were stimulated by Ca2+ but 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 endo-polygalacturonase 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* while 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*. In conclusion, the conditions inhibiting endo-polygalacturonases from *Lasiodiplodia theobromae* and *Rhizoctonia solani* capable of degrading the pectin portion of rice (*Oryza sativa*) 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 while temperature and pH extreme from 32°C and pH 5.0 will inhibit growth of *Rhizoctonia solani* on the grain. These physiological conditions will preserve pectin in rice (*Oryza sativa*) from degradation by these two fungal phytopathogens.

**Keywords:** rice (*Oryza sativa*), phytopathogen, fungi, microbial enzymes, polygalacturonase, endo-polygalacturonase, purification, characterisation

#### **1. Introduction**

*Oryza sativa* (Asian rice) and *Oryza glaberrima* (African rice) are species of rice grown all over the world [1]. Rice is the species of the seed of grass. It is a cereal grain consumed mostly in Asia and Africa [2]. It is an agricultural grain which has the third-highest worldwide production only after sugarcane and maize [3, 4] and the world's most consumed staple food [4]. It is rich in starch, protein, minerals and vitamins but low in calories and fats [5]. Rice is grown in all the geographical zones of Nigeria, West Africa depending on the variety [6]. The area of land used for rice cultivation in Nigeria, West Africa is about 2 million hectares. Nigeria however has the potentials of cultivating about 5 million hectares [7, 8].

Rice is affected by a host of fungal pathogens which include: *Magnaporthe grisea* which causes Rice blast; *Rhizoctonia solani* which causes Rice sheath blight; *Cochliobolus miyabeanus* (an Ascomycete) which causes brown spot disease in rice [1]. In Nigeria, the major fungal pathogens of rice include: *Fusarium moniliforme* Sheldon which causes Bakanae foot rot disease; *Cercospora oryzae* Miyake which causes Narrow brown leaf spot; *Rhynchosporium oryzae* Hashioka and *Rhynchosporium oryzae* Yokogi which cause Leaf scald; *Rhizoctonia solani* Kühn which causes Basal sheath rot; *Pyricularia oryzae* Cav. which causes Rice blast; *Cochliobolus miyabeanus* Ito Dreschler ex Dastur and *Cochliobolus miyabeanus* Kuribayashi Dreschler ex Dastur which cause Rice brown spot; *Lasiodiplodia theobromae* which causes Root rot disease complex in rice [9, 10].

Endo-Polygalacturonase (EC: 3.2.1.15) also known as Pectin depolymerase, PG, Pectolase, Pectin hydrolase, and Poly-alpha-1,4-galacturonide glycanohydrolase, is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It degrades pectin by hydrolyzing the O-glycosyl bonds yielding alpha-1,4-polygalacturonic residues [11–14]. This enzyme has multiple parallel beta sheets which form a helical shape that is called a beta helix. This highly stable structure has numerous hydrogen bonds and disulfide bonds between strands common to all pectin degrading enzymes. The interior of the beta helix is hydrophobic [14, 15]. Exo-Polygalacturonases and Endo-Polygalacturonases have differing hydrolytic modes of action. Endo-Polygalacturonases hydrolyze pectin in a random fashion along the polygalacturonan chain resulting in oligogalacturonides. Exo-Polygalacturonases hydrolyze pectin at the non-reducing end of the polymer resulting in monosaccharide galacturonic acid [14]. Fungal Polygalacturonases

**163**

K+

at pH 4.0.

but inhibited by Ag<sup>+</sup>

**2. Materials and methods**

**2.1 Source and identification of isolates**

**2.2 Inocula and their culture conditions**

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

polygalacturonase from *Aspergillus flavus* isolated from orange peel with maximum activity in the presence of polygalacturonic acid at 35°C and pH 4.5. Carrasco *et al*. [19] reported from their studies the expression of a polygalaturonase in *Pichia pastoris* with an optimum activity 15°C higher than its mesophilic counterpart. According to Thakur [20], *Mucor circinelloides* was able to produce an extracellular polygalacturonase in a growth medium with pectin methyl ester (1% w/v) as carbon source and a combination of casein hydrolysate (0.1% w/v) and yeast extract (0.1% w/v) as nitrogen source. Optimum polygalcturonase activity was obtained

The aim of this study was to determine the physiological conditions that will inhibit the growth of major and specific fungal phytopathogens of rice (*Oryza sativa*) in Nigeria, West Africa. The present study will establish the contributions of endo-polygalacyuronases produced by *Lasiodiplodia theobromae* and *Rhizoctonia solani* to the deterioration of rice (*Oryza sativa*) cultivated in Nigeria. Control measures which include conditions inhibiting endo-polygalacturonases from these specific fungal phytopathogens can be adapted in the cultivation (pre-harvest) and

The tropical fungal strains of *Lasiodiplodia theobromae* and *Rhizoctonia solani* for this investigation were isolated from deteriorated rice (*Oryza sativa*) obtained from Cocoa Research Institute, Ibadan, Nigeria. The isolates were identified at the International Institute of Tropical Agriculture, Ibadan, Nigeria. The isolates were

*Lasiodiplodia theobromae* and *Rhizoctonia solani* isolated from deteriorated rice (*Oryza sativa*) were grown in a fungal growth medium with defined specific nitrogen and carbon sources for growth. The isolates were cultured on plates and slants containing potato dextrose agar at 30°C. Ninety six-hour-old cultures of isolates were used in the investigation [21]. Isolates were cultured in a growth medium composed of MgSO4.7H20 (0.1 g), K2HPO4 (2 g), KH2PO4 (0.5 g), FeSO4.7H2O (1 mg), KNO3 (9.9 g) and pectin (10 g) source (Sigma-Aldrich, USA) per 1 litre of distilled water (The pH of the medium was adjusted to pH 5.0 using 0.1 N HCl and 0.1 N NaOH). Conical flasks (250 ml) containing 100 ml growth medium were inoculated

spores

with 1 ml of an aqueous spore suspension containing approximately 6 x 105

storage (post-harvest) of rice (*Oryza sativa*) globally.

cultured on potato dextrose agar on plates and slants.

, Hg2+ and Ca2+. Doughari and Onyebarachi [18] produced

are affected by a variety of factors which include: pH, substrate concentration, substrate specificity, and temperature [13]. Phytopathogenic fungi expose plant cell walls to Cell Wall Degrading Enzymes (CWDEs) such as Polygalacturonases [14]. Siddiqui [16] purified a monomeric polygalacturase with molecular weight of 32 kDa and optimum activity at 55°C and at pH 5.0 using Sephadex G-200 and Sephacryl S-100 from thermophilic *Rhizomucor pusillus* isolated from decomposting orange peels. Anand *et al*. [17] purified an endo-polygalacturonase using acetone precipitation and gel filtration from a strain of *Aspergillus* fumigatus. They determined the molecular weight to be 43.0 kDa, stable at a pH range of 7–10 and with optimum activity at 30°C. The polygalacturonase was stimulated by Cu2+ and

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

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

are affected by a variety of factors which include: pH, substrate concentration, substrate specificity, and temperature [13]. Phytopathogenic fungi expose plant cell walls to Cell Wall Degrading Enzymes (CWDEs) such as Polygalacturonases [14]. Siddiqui [16] purified a monomeric polygalacturase with molecular weight of 32 kDa and optimum activity at 55°C and at pH 5.0 using Sephadex G-200 and Sephacryl S-100 from thermophilic *Rhizomucor pusillus* isolated from decomposting orange peels. Anand *et al*. [17] purified an endo-polygalacturonase using acetone precipitation and gel filtration from a strain of *Aspergillus* fumigatus. They determined the molecular weight to be 43.0 kDa, stable at a pH range of 7–10 and with optimum activity at 30°C. The polygalacturonase was stimulated by Cu2+ and K+ but inhibited by Ag<sup>+</sup> , Hg2+ and Ca2+. Doughari and Onyebarachi [18] produced polygalacturonase from *Aspergillus flavus* isolated from orange peel with maximum activity in the presence of polygalacturonic acid at 35°C and pH 4.5. Carrasco *et al*. [19] reported from their studies the expression of a polygalaturonase in *Pichia pastoris* with an optimum activity 15°C higher than its mesophilic counterpart. According to Thakur [20], *Mucor circinelloides* was able to produce an extracellular polygalacturonase in a growth medium with pectin methyl ester (1% w/v) as carbon source and a combination of casein hydrolysate (0.1% w/v) and yeast extract (0.1% w/v) as nitrogen source. Optimum polygalcturonase activity was obtained at pH 4.0.

The aim of this study was to determine the physiological conditions that will inhibit the growth of major and specific fungal phytopathogens of rice (*Oryza sativa*) in Nigeria, West Africa. The present study will establish the contributions of endo-polygalacyuronases produced by *Lasiodiplodia theobromae* and *Rhizoctonia solani* to the deterioration of rice (*Oryza sativa*) cultivated in Nigeria. Control measures which include conditions inhibiting endo-polygalacturonases from these specific fungal phytopathogens can be adapted in the cultivation (pre-harvest) and storage (post-harvest) of rice (*Oryza sativa*) globally.

#### **2. Materials and methods**

*Grain and Seed Proteins Functionality*

fungal phytopathogens.

**1. Introduction**

hectares [7, 8].

pectin as carbon source supported highest activity of endo-polygalacturonase by the *Rhizoctonia solani*. In conclusion, the conditions inhibiting endo-polygalacturonases from *Lasiodiplodia theobromae* and *Rhizoctonia solani* capable of degrading the pectin portion of rice (*Oryza sativa*) 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 while temperature and pH extreme from 32°C and pH 5.0 will inhibit growth of *Rhizoctonia solani* on the grain. These physiological conditions will preserve pectin in rice (*Oryza sativa*) from degradation by these two

**Keywords:** rice (*Oryza sativa*), phytopathogen, fungi, microbial enzymes, polygalacturonase, endo-polygalacturonase, purification, characterisation

Nigeria however has the potentials of cultivating about 5 million

*theobromae* which causes Root rot disease complex in rice [9, 10].

*Oryza sativa* (Asian rice) and *Oryza glaberrima* (African rice) are species of rice grown all over the world [1]. Rice is the species of the seed of grass. It is a cereal grain consumed mostly in Asia and Africa [2]. It is an agricultural grain which has the third-highest worldwide production only after sugarcane and maize [3, 4] and the world's most consumed staple food [4]. It is rich in starch, protein, minerals and vitamins but low in calories and fats [5]. Rice is grown in all the geographical zones of Nigeria, West Africa depending on the variety [6]. The area of land used for rice cultivation in Nigeria, West Africa is about 2 million hectares.

Rice is affected by a host of fungal pathogens which include: *Magnaporthe grisea* which causes Rice blast; *Rhizoctonia solani* which causes Rice sheath blight; *Cochliobolus miyabeanus* (an Ascomycete) which causes brown spot disease in rice [1]. In Nigeria, the major fungal pathogens of rice include: *Fusarium moniliforme* Sheldon which causes Bakanae foot rot disease; *Cercospora oryzae* Miyake which causes Narrow brown leaf spot; *Rhynchosporium oryzae* Hashioka and *Rhynchosporium oryzae* Yokogi which cause Leaf scald; *Rhizoctonia solani* Kühn which causes Basal sheath rot; *Pyricularia oryzae* Cav. which causes Rice blast; *Cochliobolus miyabeanus* Ito Dreschler ex Dastur and *Cochliobolus miyabeanus* Kuribayashi Dreschler ex Dastur which cause Rice brown spot; *Lasiodiplodia* 

Endo-Polygalacturonase (EC: 3.2.1.15) also known as Pectin depolymerase, PG, Pectolase, Pectin hydrolase, and Poly-alpha-1,4-galacturonide glycanohydrolase, is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It degrades pectin by hydrolyzing the O-glycosyl bonds yielding alpha-1,4-polygalacturonic residues [11–14]. This enzyme has multiple parallel beta sheets which form a helical shape that is called a beta helix. This highly stable structure has numerous hydrogen bonds and disulfide bonds between strands common to all pectin degrading enzymes. The interior of the beta helix is hydrophobic [14, 15]. Exo-Polygalacturonases and Endo-Polygalacturonases have differing hydrolytic modes of action. Endo-Polygalacturonases hydrolyze pectin in a random fashion along the polygalacturonan chain resulting in oligogalacturonides. Exo-Polygalacturonases hydrolyze pectin at the non-reducing end of the polymer resulting in monosaccharide galacturonic acid [14]. Fungal Polygalacturonases

**162**

#### **2.1 Source and identification of isolates**

The tropical fungal strains of *Lasiodiplodia theobromae* and *Rhizoctonia solani* for this investigation were isolated from deteriorated rice (*Oryza sativa*) obtained from Cocoa Research Institute, Ibadan, Nigeria. The isolates were identified at the International Institute of Tropical Agriculture, Ibadan, Nigeria. The isolates were cultured on potato dextrose agar on plates and slants.

#### **2.2 Inocula and their culture conditions**

*Lasiodiplodia theobromae* and *Rhizoctonia solani* isolated from deteriorated rice (*Oryza sativa*) were grown in a fungal growth medium with defined specific nitrogen and carbon sources for growth. The isolates were cultured on plates and slants containing potato dextrose agar at 30°C. Ninety six-hour-old cultures of isolates were used in the investigation [21]. Isolates were cultured in a growth medium composed of MgSO4.7H20 (0.1 g), K2HPO4 (2 g), KH2PO4 (0.5 g), FeSO4.7H2O (1 mg), KNO3 (9.9 g) and pectin (10 g) source (Sigma-Aldrich, USA) per 1 litre of distilled water (The pH of the medium was adjusted to pH 5.0 using 0.1 N HCl and 0.1 N NaOH). Conical flasks (250 ml) containing 100 ml growth medium were inoculated with 1 ml of an aqueous spore suspension containing approximately 6 x 105 spores

per ml of each isolate. These were the experimental flasks. Control flasks contained un-inoculated medium. Experimental and control flasks were incubated without shaking at 30°C. Protein content was determined [22].

#### **2.3 Enzyme extraction**

Contents of flasks were carefully filtered through glass fibre filter paper (Whatman GF/A) on the tenth day of inoculation of growth medium. Protein content of the filtrates was determined [22]. The filtrates were also assayed for polygalacturonase activity [23, 24].
