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Isolation and physiological studies of fungus associated with rice grain (Oryza sativa) in Makurdi, Benue State, Nigeria.

Introduction

Rice (Oryza sativa) family poaceae, is probably originated in China but was taken to India very early, and now extensively grown throughout the warmer parts of Asia, and now in South America, Africa, USA [5]. However, there are speculations that rice might have originated from South Eastern Asia where wild species equally exists. The wild form of rice which is found in Africa includes: O. glaberrima and O. brevilligulata [2]. There are at least twenty different species of Oryza, but only O. satva (Asian rice) and O. glaberrima (African rice) are cultivated in many countries.

With the exception of maize (Zea mays), rice (Oryza sativa) is the second most important cereal crop in Nigeria in terms of area cultivated, output, consumption, nutritional value and foreign exchange earnings [11]. Rice is popular almost everywhere in Africa South of Sahara (FAO, 1998) and on the worldwide scale; half the world's arable land is under cereals of which one fifth produces rice [12].

It was reported that about 10 percent of the world acreage is 'hill' rice, grown dry like an ordinary cereal. 'Swamp' or 'pady' (paddy) rice is grown in shallow standing water, either impounded rain water or irrigation water, especially in most areas of northern Nigeria. In most countries the crop is largely for local consumption and grown on small holdings (Makurdi Local Government Area, Benue State Nigeria). But in some areas commercial production for export is encouraged by local growing conditions. The most important production area is the 'rice triangle' in Burma and Thailand, others include China and USA.

Nigeria has the highest acreage of rice in West Africa of which about 16 percent, or 105,000ha, is deep-water rice or floating rice. The small acreage of 16,000 ha of deep water rice in Niger represents 3-quarters of her total rice area. In both countries (Nigeria and Niger) deep water rice is grown by traditional method in the narrow, level grass covered flood plains along the Niger River and its tributaries and on the flood plains of island. In Nigeria the fadamas planted to deep water rice are situated along the Sokoto River near Birnin Kebbi and on the Hadeija River downstream of Kano.

Though measures have been put in place to halt importation and encourage local production, Nigeria still has not met the demand for rice through local production because of a number of factors such as climate, pest and disease attack. Among the diseases, those caused by fungi are most common and they infect rice crop from the field to storage; this reduces total output and availability of rice and grain quality for processing as well as storability for next planting season [6]. Field rice is contaminated with numerous fungi which include rice blast (Magnaporthe grisea), rice sheath blight (Rizoctonia solani), and brown spot (Cochliobolus miyabeanus) [8]. Among the storage fungi cited are Aspergillus flavus [10] and Penicillium sp [7,4]. The study is aimed at identifying the seed-borne fungal organism associated with local varieties of rice in Makurdi Local Government Area of Benue State, Nigeria.

Materials and methods

Collection of samples:

Infected seed samples of three (3) local varieties of rice grown in Benue State namely: "China", "Mars" and "Turn-2" were collected from six suppliers in markets namely: North Bank, Wurukum and Wadata markets.

Isolation of fungi:

Two methods of isolation were used (serial dilution and direct plating). Ten (10) grains of rice in test tube, 10ml of distilled water was added and shaken for 3 minutes to get a stock solution. 1ml of the stock was pipetted into 9ml of distilled water in a test tube to make a serial dilution of 10-1, 1ml of 10-1 serial dilution was pipetted into 9ml of distilled water in a test tube gave [10.sup.-2] serial dilution. Similar method was carried out to give final concentrations of [10.sup.-1], [10.sup.-2], [10.sup.-3], [10.sup.-4] and the stock (10[degrees]). While in the direct isolation, ten (10) grains (milled and unmilled each) were surface sterilized with 0.1% Hg[Cl.sub.2] for 30 seconds and rinsed in distilled water. After surface sterilization, the seeds were spaced out in the Petri dishes containing the medium (PDA). The plates were incubated at room temperature of 25[degrees]C[+ or -]1[degrees]C for five days.

Physiological studies (carbon and nitrogen supplemented):

This was determined by dry weight measurement. Mycelia dry weight determination was carried out on mycelia growing on different carbon sources following the method of Suleiman, [9]. The following carbon sources were incorporated into basal medium; glucose, starch and fructose. The basal medium consisted of: 1.0g, KCL; 0.5g, Mg S[O.sub.4] 7[H.sub.2]O; 3.0g, Ca [(N[O.sub.3]).sub.2]; 1.0g, [K.sub.2]HP[O.sub.4]; 0.01g, FeS[O.sub.4] 7[H.sub.2]O. The appropriate weight of each carbon source (10g dissolved into 100ml sterile distilled water) was dispensed into conical flasks. Ten (10ml) solutions of the different sugars (sterile) were aseptically added to the sterile basal medium in the flasks. Each conical flask contained 20ml sterile basal medium and 10ml sterile carbon source. The flasks were inoculated with 5mm diameter disc of test fungus of a seven day old culture growing on PDA such that the mycelia matt were uppermost and floated on the medium. Three replicates flasks were used for each carbon source. Similar procedure as above was carried out with liquid medium on nitrogen sources. The appropriate weight of each nitrogen source was dissolved separately before adding aseptically to the basal medium in the flasks. Twenty (20) g of glucose was the carbon source of the medium. The different nitrogen sources used were ammonium chloride (NH4Cl), sodium nitrate (NaNO3), urea [C [(N[H.sub.2]).sub.2] and calcium nitrate Ca [(N[O.sub.3]).sub.2]. The flasks were stopper with sterile non--absorbent cotton wool and incubated at room temperature for one week at 25 [+ or -] 2[degrees]C. Harvesting was carried out at five days interval.

The mycelia were filtered by suction through filter paper previously dried to a constant weight. Both filter paper and mycelia were then dried in an oven at 80[degrees]C to a constant weight on whatman's filter paper. The weight of the mycelia was determined by subtracting the initial weight of the filter paper from the weight of mycelia and filter paper.

Results and discussion

Makurdi, the administrative headquarters of Benue State lies approximately between latitude 7[degrees] [44.sup.1] N and longitude 8[degrees] [54.sup.1] E. The town is located along the coast of the River Benue. The climatic conditions influenced by two air masses; warm, moist South Westerly air mass. The South westerly air mass is a rain-bearing wind that brings about rainfall from the months of May to October. The dry North Westerly air mass blows over the region from November to April, thereby bringing about seasonal drought. The mean annual rainfall is about 1,290mm. The above weather conditions favour abundant rice production. Of course, the state is tagged "food basket of the nation". The nutritional content of rice is variable with respect to variety, milling and cooking methods as well as environmental conditions. About 85% of the kernel consists of carbohydrate, most of which is contained in the endosperm. Although rice protein ranks high in nutritional quality among cereals, protein content is modest. Unmilled (brown) rice average 9.5% protein content [8]. Awareness of the nutritional content of rice has raised its consumption in West Africa to 9.87 million tones between 2001 and 2005 and its consumption rate increased annually at 6.55%, well above production growth rate over the same period [12].

Suleiman and Taiga, [10] reported fungal pathogen as the major cause of reduction in the quality of stored rice when insects and rodents are controlled. Rice growing in the field is contaminated with numerous fungi which include rice blast caused by Magnaporthe grisea, rice sheath blight caused by Rhizoctonia solani, brown spot caused by Cochlioblus miyabeanus and false smut caused Ushlaginoidia virens [8]. Blight brown spots caused by Helminthosporium oryza was reported by Bhatti and Soomio, [3]. Stored rice grains are prone to fungal attack, especially at a moderate temperature and high humidity. Spoilage of stored rice is attributed to storage fungi which were introduced during the post harvest handling process [7]. Among the storage fungi cited are Aspergillus flavus [1] and Penicillium sp [7]; [4]. In the present study, Aspergillus niger was isolated from three samples collected from the three major markets of the study areas. This confirms that the fungus is widely associated with unmilled rice grain and in storage which may result in deterioration in form of discolouration and bad odours may occur with reduction in milling yield due to partly growth of mould and other micro organisms. The results, as shown in table 1, revealed that the isolated fungus was associated with stored rice with varying frequencies in the three markets sampled. The frequency of the fungal pathogen was generally high at Wadata market, followed by North Bank while the least was recorded at Wurukum market.

The results on physiological studies on the fungus obtained in the present study showed that growth was supported by all the carbon sources tested. Fructose supported the highest mycelia dry weight; while sporangia production was more in glucose. In the present study, glucose and starch were utilized with an average mycelia dry weight of 0.45g/30ml media and 0.68g/30ml media respectively at the end of the fifteen days of inoculation (Table 2). The highest mean dry weight of 0.96g recorded in Fructose showed that Aspergillus niger utilized fructose better than other carbon sources, ranked second was starch, and glucose had the least mean mycelia dry weight. With HSD of 4.04g, there was a significant difference between the control and the carbon sources. Sporangia production was more in glucose compared to starch and fructose.

On nitrogen utilization, it has been reported that the level as well as the nature of the nitrogen, an essential element used for both physiological as well as for structural purposes supplied are vital importance in determining fungal development. Likewise different fungi show differential ability in utilizing different nitrogen sources [9]. The results of the present study from four nitrogen sources namely, N[H.sub.4]Cl, urea, CaN[O.sub.3] and NaN[O.sub.3] showed that Aspergillus niger utilized urea better than other nitrogen sources. Calcium nitrate and sodium nitrate appeared to be poor nitrogen sources for the growth of the fungus. The best nitrogen source was urea with the highest mean mycelia dry weight of 0.67g, there was a significant difference (P 0.01<0.05) compared with control; this was closely followed by ammonium chloride with mean mycelia dry weight of 0.46g; while sodium nitrate and calcium nitrate had 0.39g and 0.36g respectively in that order, with no significant difference among the nitrogen sources supplemented at P [less than or equal to] 0.05%. Sporangial production also varies among the nitrogen sources. There was low production of sporangia in calcium nitrate and sodium nitrate (Table 3).

Conclusion:

This study provides information serving as a base line in establishing Aspergillus niger as the causal pathogen of biodeterioration of rice grain and very rampant in Makurdi Local Government Area, Benue state.

Acknowledgement

The contributions of members of the Department of Biological Sciences, Kogi State University, Anyigba, is highly appreciated. I am particularly grateful for the technical assistance of the technologists of the same Department.

References

[1.] Allexopoulos, C.J. and C.W. Mims, 1988. Introductory Mycology. Third Ed. Wiley Eastern Limited, New Delhi, pp: 632.

[2.] Amadioha, A.C., 2002. Fungicidal activity of leaf extracts of four plants against Pathogenic fungi of rice in Nigeria. Archives of Phytopathology and Plant Protection, 51: 50-61.

[3.] Bhatti, I.M. and A.M. Soomii, 1996. Micbiologie alimentair. Vol.1, Voisier, Paris, pp: 81-95.

[4.] Chiejina, N.V. and E.B. Ulobo, 2006. Fungi in soil cultivation with rice Oryza sativa to Rotation with tubers. Journ. of Bio Research, 4(2): 78-84.

[5.] Hill, D.S. and J.M. Waller, 1999. Pests and Diseases of Tropical Crops, 2: 275-300.

[6.] IRRI, 2002. International Rice Research Institute. Rice Almanac.

[7.] Khanzada, A.K. and S.A. Jamil Khan, 1987. Efficiency of some Detection Methods For seed Borne fungi in seed Health Testing Programme. Pakistan Journal of Agriculture, 8(2): 74-86.

[8.] Rola, A.C. and L.P. Prabhu, 1994. Pesticides rice productivity and farmers health International Rice Research Institute, Manila, Phillipines.

[9.] Suleiman, M.N., 2005. Growth and Nutritional Studies on Alternaria and Fusarium isolated from leaf spots of Codiaeum variegatum. Scientia Africana., 41(1&2): 121-130.

[10.] Suleiman, M.N. and A. Taiga, 2009. Efficacy of aqueous extracts of neem and Sharf from the control of fungi associated with milled and unmilled stored rice Grain. Proceedings of 5th AKURE--HUMBOLDT KELLOG/SAAT Annual Conference, 71-73.

[11.] WARDA. 2002. West African Rice Development Agency. Africa Rice Trends.

[12.] WARDA., 2007. West African Rice Development Agency. Africa Rice Trends.

Suleiman, M.N. and Akaajime, D

Department of Biological Sciences, Faculty of Natural Sciences Kogi State University, P.M.B. 1008, Anyigba, Nigeria.

Suleiman, M.N. and Akaajime, D: Isolation and Physiological Studies of Fungus Associated with Rice Grain (Oryza Sativa) in Makurdi, Benue State, Nigeria.: Adv. Environ. Biol., C(C): CC-CC, 2010

Suleiman, M.N., Department of Biological Sciences, Faculty of Natural Sciences Kogi State University, P.M.B. 1008, Anyigba, Nigeria. +2348050622702, E-mial: nasirums@yahoo.com
Table 1: The frequency of the fungal pathogen at three major markets

Markets       Frequency per 10

North Bank    08
Wurukum       06
Wadata        09

Table 2: Mean mycelia dry weight and sporangial production of
Aspergillus niger on different carbon sources

                                           Mean No. of sporangia
Carbon sources    Mean [+ or -] SE (g)     [+ or -] SE (x [10.sup.4])

Control (basal    0.32 [+ or -] 0.1 (a)    50 [+ or -] 1 (a)
  medium)
Glucose           0.45 [+ or -] 0.1 (b)    63 [+ or -] 0 (b)
Starch            0.68 [+ or -] 0.2 (c)    59 [+ or -] 2 (a)
Fructose          0.96 [+ or -] 0.4 (d)    57 [+ or -] 1 (a)

Means represented by the same letter are not significant different
(P < 0.05)

Table 3: Mean mycelial dry weight and sporangial production of
Aspergillus niger on different nitrogen sources

                                                 Mean No of sporangia
Nitrogen sources      Mean [+ or -] SE (g)       [+ or -] SE
                                                 (x [10.sup.4])

Control (basal        0.12 [+ or -] 0.0 (a)      56 [+ or -] 0 (a)
  medium)
Calcium nitrate       0.36 [+ or -] 0.0 (a b)    56 [+ or -] 1 (a)
Sodium nitrate        0.39 [+ or -] 0.1 (a b)    59 [+ or -] 1 (a)
Ammonium chloride     0.49 [+ or -] 0.1 (a b)    61 [+ or -] 0 (a)
Urea                  0.67 [+ or -] 0.1 (b)      64 [+ or -] 1 (b)

Means represented by the same letter are not significant different
(P < 0.05)
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Title Annotation:Original Article
Author:Suleiman, M.N.; Akaajime, D.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:6NIGR
Date:May 1, 2010
Words:2504
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