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An evaluation on the impact of fungi on the post-harvested stored wheat grains.

Introduction

Among the various seeds of cereals, the wheat grains provide more food to over one thousand million human beings of this earth than any other plant or animal products. According to an estimate (U.S. Year Book of Agriculture, 1961), about one fourth supply of human energy comes from the wheat grains in the United States; in Europe about one half and in far-east about three fourth. It is therefore, most appropriate that these wheat grain must be protected at all stages of handling, from the time of harvest, through storage, transportation and processing, up to the time they are ready to be consumed. Grain production in any country varies from year to year and hence the grains should be stored strategically from years of overproduction for the use in year of under production. Grain quality after harvest is influenced by a wide variety of biotic and abiotic factors and has been studied as a stored grain ecosystem. The post harvest losses at the farm level have been estimated to be 3.28 kg/q in wheat. The post harvest loss of wheat grain has been found to be highest during storage (Magan et al., 2003). Stored grains can have losses in both quantity and quality. Losses occur when the grain is attacked by microorganisms and other organisms including insects, mites, rodents and birds (Neetirajan et al., 2007).

The wheat grains come in association with the fungi from the time of grain maturity and also at the time of storage. Some of these fungi are in intimate association and are present as dormant mycelium under the pericarp or dormant spores on the surface of the kernel. However, there are a number of fungi which are only superficially associated with stored grains. The association of fungi with cereal grains starts from the field itself. Shortly after the grain reaches to maximum size, the lemma and palea protecting it are pushed apart exposing the grain to infection by fungi (Machacek and Greaney, 1938) and their extensive studies has been carried out in the laboratory on these aspects (Sankaran et al., 1975; Sankaran, 1976; Sankaran et al., 1976). An extensive microflora has been found to be associated with stored wheat grains (Duggeli, 1904; Kent- Jones and Amos, 1930; James et al., 1946; Christensen, 1956; Poisson and Guilbot, 1956; Inagaski and Ikeda, 1959; Field and King, 1962; Brook and White, 1966; Graves et al., 1967; Pelhate, 1968; Hesseltine, 1968; Wallace, 1973). Earlier in the laboratory a number of cereals have been screened with respect to microflora associated in storage grains (Basu, 1974; Mehrothra, 1974; Palni, 1975; Jayas, 1995). Fungal activity can cause undesirable effects in grains including discolouration, contribute to heating and losses in nutritional value, produce off-odours, losses in germinability, deterioration in baking and milling quality, and can result in contamination by mycotoxins (Hocking, 2003; Magan et al., 2003).

Materials and Methods

The investigation was done at Allahabad Agricultural Institute-Demmed University, Allahabad in India. The work was an attempt to correlate the fungal infestation and quality of wheat grain under storage. The wheat samples were collected separately in 3 replicates for each of the wheat (Triticium aestivum L.) varieties viz., U.P. 262 and H.D. 1982 from F.C.I. godown, Naini, Allahabad district, whole sale dealers from Naini and Muttiganj markets of Allahabad and from the local farmers. The samples were brought to the laboratory under aseptic condition where they were screened (before washing and after washing with water) for their associated fungal flora. Out of these, some samples were heavily infested with fungi, some were slightly infested and some were not at all infested. Toxins produced by the micromycetes are widespread in nature and when occurring in grains they often reduce both yield and the quality of the stored wheat grains. Micromycetes are the most important spoilage organism in wheat grains. The determination of fungi was done by Dilution plate technique and Colony forming unit. Fungi from each of the grain samples were determined both qualitatively and quantitatively by dilution plate technique (James et al., 1946) as described below:

(i) Ten grams of each grain samples were aseptically weighed and transferred separately to 250 ml Erlenmeyer flasks containing 100 ml of sterilized water and 10 g of sterilized sand (a dispersing agent).

(ii) The flasks were shaken for thirty minutes on a rotary shaker.

(iii) After the foam developed had subsided, serial dilutions were done for the original solution.

(iv) Triplicate plates of each dilution were made by pipetting out 1 ml solution to sterilized petridishes containing 20 ml Czapek's solution agar medium.

(v) The dishes were gently swirled to disperse the spores uniformly and after the medium had solidified it was incubated at 25 [+ or -] 1 [degrees]C for 7 days.

Each set of experiment had a control to differentiate the laboratory contaminants from the microflora actually associated with the sample. The fungal colonies were isolated and identified with the help of authentic literature. The frequency of occurrence of different fungi isolated from wheat samples was calculated by the following formula;

Percentage frequency = Total number of plates in which a particular fungus appeared/Total number of plates examined X 100

The various media used for the identification of fungi were Czepek's solution agar {NaN[O.sub.3] (3.0 g); [K.sub.2]P[O.sub.4] (1.0 g); KCL (0.5 g); MgS[O.sub.4] .7[H.sub.2]O (0.5 g); FeS[O.sub.4] .7[H.sub.2]O (0.01 g); Sucrose (30.0 g); Agar (20.0 g); Distilled water (1000.0 ml); [sub.P]H 6.5)}, Czepek's yeast agar {[K.sub.2]HP[O.sub.4] (1.0 gm); Czepek's concentrate (10.0 gm); Yeast extract (5.0 gm); Sucrose (30.0 gm); Agar (20.0 gm); Distilled water (1000.0 ml); [sub.P]H 6.5}, malt extract agar {Malt--Extract (20.0 g); Peptone (1.0 g); Dextrose (20.0 g); Agar (20.0 g); Distilled water (1000.0 ml) [sub.P]H 6.5}, Oat meal agar {Oat meal (20.0 g); Yeast extract (0.5 g); Agar (20.0 g); Distilled water (1000.0 ml); [sub.P]H 6.5}, Potatodextrose agar {Potato Dextrose (20.0 g); Agar (20.0 g); Distilled water (1000.0 ml); [sub.P]H 6.5}, Peptone agar {Peptone (10.0 g); Dextrose (20.0 g); Agar (20.0 g); Distilled water (1000.0 ml); [sub.P]H 6.5} and Synthetic mucor agar {Dextrose (20.0 g); Asparagine (2.0 g); K[H.sub.2]P[O.sub.4] (0.5 g); MgS[O.sub.4] x 7[H.sub.2]O (0.25 g); Thiamine chloride (0.5 g); Distilled water (1000.0 ml); [sub.P]H 6.5}. These media were sterilized at 121 [degrees]C and 15 lbs p.s.i. for 20 minutes.

Results and Discussion

A substantial number of stored fungi were associated with the wheat grains. i.e., a total number of 10 fungal organisms were isolated from the samples of stored wheat grains using dilution technique and colony forming unit as shown in Figure 1.1. The frequency of occurrence of fungi superficially associated with stored wheat grains is shown in Figure 1.2. As the temperature of incubation was 25 [+ or -] 2[degrees]C, only mesophilic fungi were obtained in both the wheat varieties. The count of mesophilic fungi in the wheat grain samples ranged from 350 to 700 (Table 1.1). The mycoflora of stored wheat grains predominantly consisted of ubiquitous mould genera Aspergillus and Penicillium, possibly because of their omnipresence, capacity to grow on all possible substrates and growth on a wide range of temperature and humidity (Figure 4.10).

[FIGURE 1.1 OMITTED]

[FIGURE 1.2 OMITTED]

The ten isolated fungal species belongs to seven different genera viz., Aspergillus, Alternaria, Cladosporium, Fusarium and Penicillium of the Phylum Ascomycota; Mucor and Rhizopus of the Phylum Zygomycota. The fungal colonies of various species isolated from the surface of stored wheat grains were grown in petriplates and its microscopic view are identified by authentic literature as shown in Figure 1.3. The frequency of Aspergillus. niger, A. fumigatus and Alternaria alternate were higher (33.76 to 40.34%) than the other fungal species identified. The frequency of A. niger was highest which is quite alarming because this strain can produce aflatoxins. Out of the Penicillium species isolated, the commonly found species was P. expansum, and P. citrinum with comparatively low percentage.

[FIGURE 1.3 OMITTED]

The safety and quality of the food grain supply are of paramount importance and are among the drivers of safe grain storage. The fungal contamination of stored grains may be a significant consideration for users because grains will not undergo further processing that includes a microbiocidal step. But problems caused by moulds and mycotoxins are more usually associated with grain storage. With an increased knowledge of the origin of mycotoxins, it is now understood that at least some mycotoxins in some commodities are formed either before harvest, or immediately after harvest (Neetirajan et al., 2007).

The presence of mycotoxins in grains is traditionally regarded as an indicator of poor storage conditions. Mycotoxins may already be present in grains coming into storage or may be produced as a result of poor storage only if there is sufficient moisture. Conversely, not all moulds that grow in stored commodities produce mycotoxins. The metabolic activity of these pioneer species raises the moisture content of the grain, which may allow growth of mycotoxigenic species and ultimately, the formation of mycotoxins. Alternaria, Aspergillus and Penicillium can act as pre or postharvest pathogens of grain and may form mycotoxins and however, the most frequent fungal species isolated in the investigation was Aspergillus viz., A. niger, A. fumigatus and A. citrinum. Among these, the frequency of A. niger was highest which is a matter of great concern because it produces aflatoxin which greatly contaminates the stored wheat grain quality. Reports from various countries show that this species is found to be the most common post harvest fungi. Saponaro and Madaluni (1960) reported the presence of Aspergillus in stored wheat grains in Italy, Wallace and Sinha (1962) in Canada, Kurata et al. (1968), Tsunado (1970) and Tsuruta (1970) in Japan. However, James and Smith, 1948 from Canada reported A. niger stood second with respect to frequency of occurrence of fungi in stored wheat grains. Conversely Aspergillus and Penicillium are more often considered as 'storage fungi'. They are known to form mycotoxins in stored grains and are usually not regarded as fungi that can produce mycotoxins before harvest (Frisvad, 1995; Wicklow, 1995; Hockings, 2003)

In the study it was found that members of mucorales are less frequent as compared to Aspergillus and Penicillium. The lesser number and infrequent occurrence may be because of the high humidity requirement of these fungi. Out of all species of mucorales, Rhizopus arrhizus and Mucor fragilis were more frequent. The present findings agree with that of Inagaki (1960). The average frequency of the fungi also varied with the nature of the substrate.

In the investigation, some species of Cladosporium was identified and that corroborate the observations of other workers from different countries. Tuite and Christensen (1957) have even noted that inoculation of ripe plants with spores of storage fungi did not increase the percentage of seed infected and they also found that storage fungi associated with 1 out of 300 samples of wheat grain collected. Flannigan (1974) also found Cladosporium species in 100% and Alternaria alternata in 78.5% of the wheat grains and also noted that two species of Cladosporium were the earliest established fungi and Alternaria alternata was among the ones which appeared later.

Acknowledgements

I gratefully acknowledge the cooperation, uninterrupted guidance, impeccable and valuable suggestions rendered to me by Prof. (Dr.) George Thomas during my research work.

References

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Shiju Mathew (1), George Thomas (2) and Tufail Ahmad (3)

(1) Assistant Professor, Department of Molecular Biology and Genetic Engineering Allahabad Agricultural Institute--Deemed University, Allahabad-211007, U.P. (India).

Correspondence Author E-mail: shijumathew_biotech@yahoo.com

(2) Professor, Department of Molecular Biology & Genetic Engineering, Allahabad Agricultural Institute--Deemed University, Allahabad 211007 (India)

(3) Professor and Head, Department of Agriculture Process and Food Engineering, Allahabad Agricultural Institute--Deemed University, Allahabad 211007 (India)
Table 1.1: Total number of fungal colonies isolated at 25 [+ or -]
2 [degrees]C from stored wheat grains.

S. No. Samples Total fungal colonies at 25 [+ or -] 2
 [degrees]C

1 Healthy 350
2 Slightly infested 450
3 Heavily infested 700
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Author:Mathew, Shiju; Thomas, George; Ahmad, Tufail
Publication:International Journal of Biotechnology & Biochemistry
Date:Dec 1, 2010
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