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African Journal of Biomedical Research, Vol. 7, No. 3, Sept, 2004, pp. 139-143 Full Length Research Article BACTERIA ASSOCIATED WITH CULTURES OF PSATHYRELLA ATROUMBONATA (Pegler) AND SCHIZOPHYLLUM COMMUNE (Fr. Ex. Fr.) GBOLAGADE, JONATHAN SEGUN[*] Department of Botany and MicrobiologyUniversity of Ibadan. Ibadan. Nigeria. Received: September, 2003 Code Number: md04030 Studies were carried out on bacteria, which usually infect spawns and culture plates of Psathyrella atroumbonata (Pegler) and Schizophyllum commune (Fr. Ex. Fr.), two Nigerian edible mushrooms. During the vegetative propagation of these higher fungi, six different bacteria species were isolated and characterized from 14 day old spawns and mycelial ramified PDA culture plates. These bacteria include Bacillus licheniformis, Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus. The average bacteria count was 1.0 x 106 cfu/ml and these bacteria grew within pH range of 5.0 and 9.0. the optimum temperature range of growth lied between 30°C and 37°C. The significance of these findings to the cultivation of P. atroumbonata and S. commune in Nigeria were discussed. Key words: Physiological studies, bacteria, cultures, mushrooms, spawn INTRODUCTION In many countries of the world including Nigeria, edible mushrooms have been priced as delicacies for several years. Apart from their medicinal values, they constitute an important food source in a world that is threatened by energy food crisis. Mushrooms have been reported to be rich in protein, glycogen, vitamins, crude fibres and essential mineral compounds (Ogundana and Fagade, 1982; Fasidi, 1996; Jonathan, 2002; Stamets, 1993). Infact, Bano (1976) reported that nutrient contents of mushrooms lied between meat and vegetables. Mushrooms such as Flammulina velutipes, Lentinus edodes, Agaricus bisporus, Pleurotus oestratus, Volvariella volvacea and Agaricus campestris among others, have been cultivated for food in several countries of the world especially in America, Europe and few Asian countries. Artificial cultivation of mushrooms is new to many developing countries and there is a death of information on fundamental processes governing their growth. Because of this reason, people in Nigeria depends mainly mushrooms that are obtained from the wild. Spawns and composts used for mushroom growing are nutritionally and biologically active to support the growth of mushrooms and other unwanted microorganisms. Therefore, preparing mushroom composts, spawns and plates require skills and experience. This is because mushroom growing processes are always been intruded and spoilt by bacteria, fungi and nematodes leading to low yield and thereby causing economic loss for mushroom growers. Presently, the identity of bacteria contaminants of mushroom spawns and culture plates in Nigeria has not been well documented. Therefore, the objective of this study was to isolate and characterize such bacteria and make suggestions on how they could be controlled. MATERIAL AND METHODS Sample Preparation Rice straw spawns were prepared in 350ml jam bottles using the method of Fasidi and Ekuere (1993). The bottles after being filled with substrates were covered with aluminum foil and autoclaved at 1.02/kgcm-2pressure for 20 minutes. After cooling, the bottles were inoculated with 5 day old (actively growing) mycelial discs of P. atroumbonata and S. commune and incubated at 30 + 2°C for 21 days. The bacteria infected spawns were assessed visually for colony formation and selected for microbial analyses. For culture plates, healthy fruit bodies of P. atroumbonata and S. commune were tissue cultured to obtain mycelia using the method described by Jonathan and Fasidi (2001). The pure mycelial cultures of these fungi were maintained on plates of PDA supplemented with 0.5% yeast extract. The plates were assessed visually for colony formation after 4 days to verify contamination of mushroom spawn from unwanted bacteria. Isolation of Bacteria Ten grammes (10.0g) each of infected spawns of P. atroumbonata and S. commune were separately soaked in 90.0cm3 of sterile de-ionised water in 250cm3 conical flasks. The mixture was shaken properly and 1.0cm3 of it was transferred into a sterile McCartney bottle containing 9.0cm3 of sterile de-ionised water. This process was repeated for other sterile McCartney bottles so that at the end, dilution of 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 folds were obtained. Each dilution was then plated out by spread plate method on plate count agar (PCA) (Oxoid)and nutrient agar (NA) (Oxoid) (Okafor, 1977). A standard plate count was carried out on colonies obtained on solid PCA using electronic Gallenkamp model colony counter and the unit expressed in cfu/ml. All the bacterial isolates were streaked separately onto nutrient agar and, Eosine methylene blue agar (EMB) plates to obtain pure cultures and determine their colonial morphology respectively. For agar plates that contained mycelia of P. atroumbonata and S. commune, the bacteria infected plates were also streaked onto a new petri dishes containing sterile NA and EMB. They were incubated at 35°C for 48 hours. The bacteria isolated from spawns were subjected to Grams reaction, motility, catalase, oxidase, indole and other standard biochemical tests shown on Table 3 (Prescott et al, 1993. Bacterial identification and characterization were carried out by comparing the results obtained with the characterization definitions of Skinner and Lovelock (1979) and that of Bergey'sManual of systematic determinative bacteriology (2001). Effect of Temperature on Growth of Bacteria IsolatesFive milliliters of nutrient broth (NB0 (Oxoid) were dispensed into each of the 27 screw capped test tubes. They were autoclaved at 1.02kgcm-2 pressure for 15 minutes. After cooling, the test tubes were inoculated with 0.1ml of different bacterial isolates and incubated at 10, 20, 25, 30, 35, 37, 40 and 45°C respectively. Each treatment was replicated thrice. The control tubes were left without any inoculum. Growth was measured after 48 hours at 540nm using a Pye unicam spectrophotometer. Effect of pH on Growth of Bacteria IsolatesCitrate-Phosphate buffer solutions of pH 5.0, 6.0, 7.0, 8.0 and 9.0 were prepared (Ekunsanmi and Arojojoye 1997). Double strength nutrient broth (20.0cm3) was added to equal quantity of each buffer solution in separate 150.0cm3 conical flask to give 40.0cm3 nutrient broth of normal strength. Three replicates of these were prepared for each pH and the bottles were autoclaved at 1.02kgcm-2 pressure for 15 minutes. After cooling, the flasks were singly inoculated with the bacterial isolates and incubated at 35°C for 24 hours. Growth was assessed by turbidity after 48 hours using a Pye unicam Spectrophotometer at 540nm wavelength. A graph of growth against time was then plotted for the different pH levels. RESULTS AND DISCUSSION Six different bacteria species were isolated from mushroom spawns and culture plates used in these studies. The isolates consisted of one Gram-negative bacteria (Pseudomonas aeruginosa BI3) and five Gram positive bacteria (Leuconostoc mesenteroides BI1; Bacillus subtilis, BI2; Staphylococcus aureus, BI4; Bacillus cereus, BI5; and Bacillus licheniformis, BI6). They were identified by a series of morphological, biochemical as well as physiological characteristics using the standard characterization procedure of Skinner and Lovelock (1979) and that of Bergeys Manual of systematic bacteriology (2001). The frequency of occurrence of each bacteria isolate is represented on Table 1. Bacillus subtilis BI2 was isolated in all the five samples. Staphylococcus aureus BI4 was recorded four times while B.licheniforms BI6 was isolated three times (Table 1). The frequency of occurrence of B.subtilis is not a surprise because Corlett and Brown (1980) implicated this organism as one of the major bacterial contaminants of laboratory cultures. This organism has also been linked with the spoilage of various foods such as bread, fish, milk, soymilk and mayonnaise (Akinrele, 1984). Table 1: Percentage of Occurrence of Microbial Isolates From Five Isolation
Legend: MU = Medium used, NR = Number of times an organism is recorded. % 0cc. = Percentage of occurrence PCA = Plate Count Agar. Isolate BI2 was characterised as B.subtilis because it was Gram positive bacillus with central spores. The organism reduced nitrate, hydrolysed starch, gelatin and casein (Table 3). Morphologically, it formed creamy, circular and translucent colonies with 1.2mm diameter and its consistency was friable (Table 1). This result agrees with the standard descriptions of Prescott et al (1993). A simplified classification based on morphology, endospore formation, Voges proskuer test, gelating hydrolysis, indole production and sugar fermentation tests were used to classify mesophillic spore formers as B.cereus (Isolate BI5) and B.lichinifomis (BI6) respectively (Table 3). These properties agree with the standard characterization procedure of Bergeys Manual of determinative bacteriology (2001). Bacilli genera were also linked with the spoilage of a Nigerian local snack (dodo ikire) by Jonathan and Olowolafe (2001), and fermentation of oil been seeds by Kolawola and Okonkwo (1985). Isolate BI2 was characterized as L.mesenteroides (Table 2 and 3) because of its tiny colonies, dull dried surfaces, non-motility, hydrolysis of casein and its reaction on catalase and oxidase was negative. This organisms has been implicated in the spoilage of castol oil and Pineaple (Barber et al 1987). Bacterial isolate BI4 was identified as S. aureus. The cells of this organism were Gram-positive cocci in clusters. It fermented mannitol strongly with gas production and the coagulase test was positive. This bacteria could have been introduced into the mushroom spawns after sterilization during inoculum. The inability of BI3 to ferment most sugars, ability to hydrolyse starch and its motility polar flagellation were among the characteristics used to classify it as P. aeruginosa. Similar observation was made by Jonathan and Olowolafe (2001) who reported that P.aeruginosa can oxidize most sugars but cannot ferment them. The complete absence of coliforms from the mushroom spawns and culture plates indicate the absence of feacal contamination (Brock et al, 1986). Table 2: Colonial morphology of the isolates
Table 3: Microscopic and Biochemical Characteristics of Isolated Bacteria
Legend: -Ve = Negative reaction, +Ve = positive reaction, AG= Acid with gas production, A= Acid production only, AW = Weak acid production. Table 4: Effect of Temperature on Growth of Bacteria Isolates
Temperature had significant effect on growth of bacterial isolates (Table 4). Leuconostoc mesenteroides, B. subtillis and B. licheniformis had their optimum temperature of growth at 35°C.This result agrees with the suggestion of Brock et al (1986) that these microorganisms are mesophiles. Pseudomonas aeruginosa, S.aureus and B.cereus grew best at 37°C (Table 4). The ability of these bacteria, to grow optimally at 37°C probably enables them to survive well inside human body. The effect of pH on the growth of bacteria isolates was shown on figure 1. It was observed that all the bacterial isolates grew over a range of pH (5.0 9.0). This was in accordance with suggestion of Corlett and Brown (1980) that microorganisms could grow over a wide range of pH. The pH that supported the optimum growth of isolates B. licheniformis (BI6) and S.aureus (BI4) was 8.0 while L.mesenteroides (BI1), P.aeruginosa (BI3), and B.cereus (BI5) grew best at 7.0. These results agree with the report of Griffin (1994) that pH and temperature are the two most important environmental factors that control the growth of microorganisms. In Nigeria, the major obstacle of artificial mushroom production is contamination from unwanted microorganisms. This normally leads to discouragement and economic loss to mushroom growers. A mushroom farmer must be able to identify and eradicate these microbial contaminants which could affect mushroom yield. This could be achieved by proper sterilization and incorporation of appropriate antibacterial agents into the medium used for mycelial propagation of these mushrooms. An ideal sterility should also sbe maintained during the spawning processes. REFERENCES
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