The Mycoflora Of Two Ghanaian Maize (Zea Mays L) Varieties (Abeleehi And Obaatanpa)

The Mycoflora Of Two Ghanaian Maize (Zea Mays L) Varieties (Abeleehi And Obaatanpa)

Andrew Amegbedzi Minamor

Science Laboratory Technology Department, Accra Polytechnic, P. O. Box GP 561, Accra.

The mycoflora of two recently-developed maize (Zea mays L) varieties Abeleechi and Obaatanpa have been studied under varying ambient equilibrium relative humidities ERHs (55, 60, 65, 70, 75, 80, 85, 90, and 95%) representative of the Ghanaian ambient condition. About thirty (30) and twenty-eight (28) species of fungi belonging to the genera Aspergillus, , Clasdosporium, Penicillium, Curvularia, Chaetomium, Emericella, Eurotium, Fusarium, Paecilomyces, Mucor, Neurospora and Rhizopus were isolated from Abeleehi and Obaatanpa varieties respectively at ERHs 55 95%. Aspergillus species (Aspergillus candidus, A. effusus,

A. fumigatus, A. giganteus, A. niger, A. ochraceus, (= A. alutaceus), A. sulphureus, A . tamari,

A. ustus, A. versicolor, A. wentii, and Aspergillus species) predominated over the others followed by Penicillium (Penicillium brevi-compactum, P. critinum, P. verrucosum, P. glabrum and P. nigricans). Fungi belonging to the other genera encountered were Curvularia, Paecilomyces, Chaetomium, Cladosporium, Emericella, Eurotium. Fusarium, Mucor. The species diversity was influenced by grain variety and the ERH at which the grains were stored. Aspergillus flavus was ubiquitous and was encountered in all grains stored at 55 95% ERH. Fusarium moniliforme was isolated from some grains incubated at 65 95% ERH. Xerophillic or xerotolerant fungal species like Aspergillus fumigatus, A. alutaceus (= A. ochraceus), A. giganteus, Paecilomyces carneus, P. puntoni, and P. varioti, were isolated at 55 65% ERH in both grain varieties.

Maize (Zea mays L) is an important cereal grains in Africa ranking as high as rice as a staple food. In Ghana, maize is an important crop cultivated throughout the country with varying degrees of success depending on edaphic and climatic factors.

Being a seasonal crop, especially in West Africa, maize is stored as dry grains and forms an enormous reserve of food.

The microflora of cereal grains is varied and includes moulds, yeasts and bacteria. Generally, bacteria are not significantly involved in the spoilage of dry grain and become a spoilage factor only after extensive deterioration of the grain has occurred and high moisture content exist. (Bullerman, L.B. and Bianchini, A. 2009).

There are more than 150 species of filamentous fungi and yeasts on cereal grains. But the most important of these are the filamentous fungi or moulds. The filamentous fungi that occur on cereal grains are divided into two groups, depending on when they predominate in grain in relation to available moisture in the grain. These groups have been referred to as field fungi and storage fungi. Field fungi invade grain in the field when the grain is high in moisture (i.e. 18 to 30%) and at high relative humidities (90 to 100%). Field fungi include species of Alternaria, Alternaria, Clasdosporium, Fusarium and Helminthosporium. Storage fungi on the other hand invade grain in storage at lower moisture content (14 to 16%), and lower relative humidities (65 to 90%).These main storage fungi are species of Eurotium, Aspergillus and Penicillium.

The major effects of fungal deterioration of grains include decreased germination, discolouration, development of visible mould growth, musty or sour odour, dry matter loss and nutritional heating, caking and the potential for production of mycotoxins in the grain. (Bullerman, L.B. and Bianchini A. 2009).

To survive in seed/grain, most fungi must be able to survive dehydration, yet there are two ecologically distinct groups of fungi forming a contrast regarding survival and longevity in seed; hydrophilic fungi those unable to produce resting spores; oospores or those doing so sparsely thus being dependent on a constant humid (85% ERH) and xerophillic fungi those fungi which are characteristically capable of producing xerotolerant propagules, often abundant such as chlamydospores, including dormant mycelium, sclerotia and microsclerotia (Neergaard, 1983). Most xerotolerant conidia become quiescent if the Environmental Relative Humidity remains too low for germination usually 80% ERH.

In an attempt by man, to cultivate new species and varieties of crops suited to his needs, the Crops Research Institute of the Council for Scientific and Industrial Research (CSIR) of Ghana has through the Grains and Legumes Improvement Programme, developed high Lysine content maize grains including Abeleehi and Obaatanpa, Okomasa, Dobidi, to name but a few which are being sold to the local farmers. However, there is hardly any information on the mycoflora associated with these grains which have to be stored for prolonged periods as seed grains for the

next planting season. This paper is an attempt to document the mycoflora associated with Abeleehi and Obaatanpa and show their pathological effect on the crop under greenhouse and field conditions.

The maize varieties used Abeleehi and Obaatanpa were purchased from Aglow Seed Company, Accra ,Ghana.

Maize samples of Abeleehi and Obaatanpa were kept at 55, 60, 65, 70, 75, 80, 85, and 95% Equilibrium Relative Humidity (ERH) provided by glycerol; water mixtures and at temperature of 28 – 31°C for 36 days.

The maize grains were surface-sterilized by washing in Miltons reagent (1% sodium hypochlorite + 16.5% sodium chloride) for 5min and then rinsed with three changes of sterile water.Sodium hypochlorite treatment was used with the aim of reducing or removing completely external saprophytes which compete with pathogens. Ten surface-sterilized grains were placed on wither Sabourand Dextrose Agar (Oxoid CM 41) Dichloran Glycerol Agar DG 18 (Oxoid CM 727) in Petri plates without further treatment. Plates were incubated until fungi grew.

There were 25 replicates for each variety.

A 10g sample of the grains was weighed and transferred aseptically into 100ml 0.1% Peptone in 250ml Erlenmeyer flasks and then shaken in Gallenkamp Model Orbital shaker at 140 rev/min for 30 mins. From this stock suspension serial dilution method was employed up to 1:10v/v.

Spores were raised in Sabourands Agar(Oxoid CM 41) or Oxytetracycline Glucose Yeast Extract Agar(Oxoid CM 545). The objective of using two media was to recover a wider range of fungal species from the incubated grainS

The plates were incubated at 28 – 31°C until fungi grew (7-14 days)

Fungi encountered in these investigations were identified by their colour, culture and morphological characteristics using the conventional identification manuals of fungi by;

Barnett and Hunter (1972), Ellis and Ellis (1988). Funder (1953), Gilman (1957), Samson and Reenen Hoeskstra (1988) and Smith (1960).

Where necessary, the identification of the fungi were confirmed by G.T Odamtten professor of mycology, Botany Department, University of Ghana, Legon.

The list of seed-borne fungi isolated from the two maize varieties is presented in Table 2 below:

List of eed-borne Fungi isolated from maize varieties Abeleehi and Obaatanpa incubated at 55 95% Equilibrium Relative Humidity for 30 days at 28 – 31°C.

2 Obaatanpa Variety

Seed-borne fungi isolated from Abeleehi and Obatanpa are being recorded for the first time in these varieties in Ghana. About thirty different seed-borne fungal species were isolated from Abeleehi and twenty-eight from Obaatanpa varieties at the varying equilibrium relative humidities (55, 60, 65, 70, 75, 80, 85, 90 and 95%) at which the grains were stored.

Aspergillus species (A. candidus, A. effuses, A. fumigatus, A. niger, A. alutaceus (= A. ochraceus)

A. sulphureus, A. tamari, A. ustus, A. versicolor, A. wentii, and Aspergillus sp. 1) predominated over other species encountered followed by Penicillium (P. brevi-compactum, P. citrinum, P. glabrum, P. nigricans and Penicillium sp.) Fungi of other genera (Curvularia, Paecilomyces, Chaetomium, Cladosporium, Emericella, Eurotium, Fusarium, Mucor, Neurospora and Rhizopus were also isolated.

The species diversity was influenced by grain variety and the ERH at which they were incubated.

A. flavus was ubiquitous and was isolated from Abeleehi and Obaatanpa stored at ERH 55 95%, Fusarium moniliforme was encountered at ERH 65 95%. Xerophilic species like A. fumigatus, A. giganteus, A. alutaceus, (=A. ochraceus), Paecilomyces carneus, P. puntoni and P. varioti were isolated at ERHs 55 65% in both grain varieties.

Paecilomyces varioti produces patulin (Frisvad, 1988) but the nature of the mycotoxins from P. carneus, P. puntoni, have not clearly elucidated. Fusarium moniliforme is one of the most prevalent fungi associated with maize, a basic human and livestock dietary staple. (Marasas, et al 1984) Experimental studies in South Africa (Marasas, et al, 1984) and in China (Li and Cheng, 1984. Lin and Tang, 1980, Yang, 1980) have shown that cultures of F. moniliforme on maize and maize products can cause cancer in rats; hepatocaricinogens produce by one strain can survive drying of the maize at 45 – 50°C for 24 hours. (Marasas et al, 1984). At least two classes of mutagens are formed by F. moniliforme namely Fusarin C (Wiebe and Bjeldanes, 1981; Gelderblom et al 1982; Scott et al, 1986). Fusaric acid is a phytoxic compound (Marasas et al, 1984 a,b). Fusariocins A and C (Arai and Ito, 1970) have also been isolated from the metabolites of F. moniliforme. Thus, the danger to human and animal of F. moniliforme-infected maize is self evident. Furthermore, it is well established that F. moniliforme can be internally seed-borne in symptomless apparently healthy maize kernel, (Foley, 1962; Marasas et al 1979; Thomas and

Buddenhagen, 1980). Aspergillus ochraceus ( = A. alutaceus) forms ochratoxin A in maize (Frisvad, 1988) as well as other mycotoxins such as emodin, kojic acid, neospergillic acids, penicillic acid, secalonic acid A. Viomellenin and Xanthomegnin (Frisvad, 1988). Many other fungal flora encountered in Abeleehi and Obaatanpa in this investigation produce mycotoxins in foods and may have serious public health implication if ingested by the consumers. It is therefore, imperative that future investigations are carried out to quantify production of potent mycotoxins such as aflatoxins (A. flavus) cyclopiazonic acid (A. tamari) ochratoxin and Penicillic acid (A. alutaceus = A. ochraceus), patulin (Paecilomyces species) to mention but a few in maize stored for prolonged period for human and animal consumption.

Aria, T and Ito, I (1970) Cytotoxicity and antitumour activity of fusariocins, mycotoxin from Fusarium moniliforme. In: H. Umezawa (Ed), Progress in antimicrobial and anti cancer chemotherapy. Vol 1, University of Tokyo Press, Tokyo pp. 87 92

Barnettt H.L and Hunter B B (1972) Illustrated Genera of Imperfect fungi (Third edition) Burgess Publishing Company.

Bullerman L.B and Branchini, A. (2009) In: Microbiologically Safe Foods. Edited by Norman Hereclia, Irene, Wesley and Santos Garcia. Publishers John Wiley and Sons.

Cole, R.J., Kirksey, J.W Cutter, H.G Doupnik, B.L and Peckham J.C (1973)

ECDXII Maize Stackburn Project Report No1. 1994 No 2 1995 EC, Brussels, Belgium

Ellis M.B and Ellis J.P (1998) Micro fungi on miscellaneous substrates. An Identification Handbook Timber Press Portland Oregon.

Foley, D.C (1962) Systematic infection of corn by Fusarium Moniliforme. Phytopathology. 52: 870 872

Frisvad, J.C., (1988) Fungal species and their specific production of mycotoxin. In: Samson,

R.A. and Reenen-Hoekstra E.S. chapter 4. Introduction to food-borne fungi. CBS. Institute of the Royal Netherlands Academy of Arts and Sciences Pp 239 249

Funder, S. (1953) Practical Mycology. Manual for Identification of fungi Broggers Boktr Forlag Oslo-Norway.

Gelderblom, W.C., Thiel, P.G., Van der Merwe K.J., Marasas, W.F.O and Spies, H.S.C. (1983). A mutagen produced by Fusarium moniliforme. Toxicon, 21; 467 473.

Li, M. and Cheng, S.J. (1984). Etiology of carcinoma of the esophagus. In: G.J Huang and Y. K; Wu (editors), Carcinoma of the esophagus and gastric cardia Springer-Verlag. New York pp. 26

51.

Li, P and Tang, W., (1980) Ziir Epidomiologie and Antiologie des Oesophagus carcinomas in China J. Cancer Res. Clin Oncol., 96:121 130.

Marasas, W.F.O., Kriek, N.P.J., Wiggens, V.M., Stey P.S., Towers D.K and Hastie T.J., (1979). Incidence, geographic distribution and toxigenicity of Fusarium species in South African corn Phytopathology, 69: 1181 1185

Marasas, W.F.O., Kriek, N.P.J., Fincham, J.E and Van Rensburg. S. J. (1984a). Primary Liver cancer and oesophageal basal cell hyperplasia in rats caused by Fusarium moniliforme Int. J. Cancer, 34, 383 387

Marasas, W.F.O., Kriek, N.P.J., Fincham, J. E and Van Rensburg. S. J. (1984b) Toxigenic Fusarium species: Identify and Mycotoxicology. The Pennysylvania State Univ. Press. Univ. Park, 328 pp.

Neergaard, P. (1983) Seed Pathology Vol 1. The MacMillian Press Ltd. London Basingstoke Companies 283 297

Rabie, C. J., Marasas W. F. O., Thiel, P. G., Lubben, A and Vleggaar, R. (1982) Moniliformin production and toxicity of different Fusarium species from Southern Africa. ppl. Environ. Microbial, 43: 517, 521

Samson, R. A. and Van Reenen Hoekstra E. (1988). Introduction to food-borne fungi 3rd

Edition Centraalbureau Voor Schimmel Baar. Institute of the Royal Netherlands Academy of Arts and Sciences.

Scott, P.M., Lawrence, G. A. and Matilda, T. I (1986). Analysis of toxins of Fusarium moniliforme. In: Mycotoxins and Phycotoxins Eds Steyn, P. S. and Vleggaar, R. Volume (1): 305 316.

Smith, G. (1960) An Introduction to Industrial Mycology. Edward Arnold (Publishers) Ltd. Thomas, M.D. and Buddenhageb, I.W (1980) Incidence and persistence of Fusarium moniliforme in symptomless maize kernels and seedlings in Nigeria. Mycologia, 75: 882 887 Wiebe, L.A. and Bjeldanes, L.F., (1981). Fusarin C, a mutagen from Fusarium moniliforme grown on corn. J. Food Sci., 46: 1424-1426.

Yang, C. S., (1980) Research on oesophageal Cancer in China: a review. Cancer Res., 40: 2633 2644