Joumal of Biological Sciences 4 (1): 271-298, 2004 ISSN 1727-3048 © 2004 Asian Network for Seientifie Information Mycobiota Associated with Rice Grains Marketed in Uganda HK. Taligoola, °M.A. Ismail and *S.K. Chebon Department of Botany, Faculty of Science, Makerere University, P.O, Box 7062, Uganda ‘Department of Botany, Faculty of Science, Assiut University, P.O, Box 71516, Assiut, Egypt "Moi High School-Kabarak, P.O. Box, 7049, Nakuru, Kenya Abstract: A comparative investigation was made for the natural fungal contamination of imported and locally grown milled rice grains. Twenty four samples of each type of rice wete obtained from various market centers ‘within and around Kampala capital city, Contaminating fixigi were enumerated by direct plating method on two isolation media including dichloran rose-bengal chloramphenicol agar (DRBC) and dichloran 18% glycerol agar (DL) media, The locally grown rice recorded a total of 62 species belonging to 34 genera while the imported rice hal 61 species belonging to 31. genera, on both isolation media, The broaclest species spectrum on both types of rice grains were from the genera Aspergillus, Penicilium, Eurotium followed by Fusarium, Cladosporium and Cochliobolus. Both types of rice grains were predominantly contaminated by Aspergillus candidus, 4. flavus, A. niger, Eurotium amstelodami, R. rubrum, Penicillium citrinn, P.axalicum and Talaromyces spp. on the two isolation media wsed. The incidence levels of xerophilic fingi especially A.carelidus and Eurotiwn spp. were hovvever, comparatively higher on the imported riee than the local rie. Field fungi including Acremonium strictum, Fusarium moniliforme, F. solani, Scytalidivas lignicola and Trichorderma harzianum ocourted only on the local rice while xerophilic fungi including -spergillus penicillioides, A. wenti, Eurotium repens and Paecilomyces lilacinus oocuered only on the imported rice, on both isolation media, The imported rice grain samples recorded comparatively higher moisture contents thant the local siee whereby 15.50% was the highest level forthe import while for the local it was 14.60%. A half of the imported rice samples had moisture content above the recommended level for safe storage of milled rice while for the loca rice only 12.5% of the samples moisture content were above this level Key words: Local rice, imported rice, millod rice grains, storage fungi, xerophilic fungi, Uganda INTRODUCTION Consumption of rice (Oryza sativa L.) has been inoreasing in Uganda for the last 1S years. The domestic output is however, inadequate forthe countries demand, ‘Thos, importation has been a supplementary measure. Its high mutitional value tozether wit its ability to mix well with most local staple dicts including, matooke(banana) account for this dit shift", However, the gains nutritive value and its high hygroscopicity subjects it to natural contamination by fungi, particularly churing such postharvest operations as. drying, transportation. and storage. Fungal ‘may also occur before harvest®. These fungi have their spores suspended in the air, thus prevention of grain contamination cannot be totally eliminated”. However, by dying the grains to moisture content of 14.0%, the recommended level for storage of milled rie, the time taken for storage fungi 10 develop on the grains will delay"! Fungi cause organoleptic changes that results in the mins discoloration, mould odowr, mustness and reduced germinbilily by desuoying the embryo, Similarly, respiration ofthe stored nutrients by the fungal enzymes reduces not only the grains weight but also its nutritional and market value, Further, it will be unsafe for human consumption owing tots ability o cause food poisoning or mycotoxicoses!=", In the developing world sehere food availabilty still remains a serious problem, fungal contamination is of greater significance since it not only reduces the food availability, food quality is ako reduced"! The formation of some toxic fungal aetabolites (mycotoxins) isthe most deleterious effect of furgal invasion of cereals by fungi, The low moisture conten of the died grain is ideal for mycotoxin developmen by storage fungi including species of Aspergillus wn Eurotiwm axl rately, Penicillium arxl Emericella, all of which ate significant storage fungi in the tropics, Indeed, these fungi including Corresponding Author: Email: madyismail yahoo.com am MA. Ismail, Deparment of Botany, Faculty of Science, Assut University, Assit, EgyptJ. Biok Sei, 4 Aspergillus candids, A.restrictus, A, wentii and Eurotium spp. ae xerophiles capable of initiating spoilage of otherwise sufficiently dried grain! Studies on fangal and mycotoxin contamination of foods in Uganda have not been documented in the past 10 years despite prevalence of environmental conditions optimum for fungal and aflatoxin development characteristic of the tropics. Similarly, comparative sudies on the incidence of various fungiion loeal and imported foods including rice is yet undocumented. However, Sebunya and Yourtes™ established Ugandan staple foods (peanuts and com) and poultry feed to have contamination of Aspergillus flavus’ parasiticus and aflatoxin B, level of 20 ppb. Similarly, studies in Uganda and Kenya established a link between the highest incidence of liver cancer in the world in tis sub-Saharan region to the presence of high levels aflatoxins in foods and beverages", This implies that the conditions associated with the possibility of consuming myectoxin contaminated food exist inthis region, This include lack of food variety and food supply, agricultural practices ard environmental conditions which favour fimgal development in crops and foods and absence of reliable mycological and mycotoxin quality programmes!" In the present study, locally grovm milled rie grain and rice imported into Uganda from Pakistan were analyzed for fungal contamination with the aim of establishing those Which are potential producers of myeotaxins MATERIALS AND METHODS Sampling of rice grains: A total of 48 milled nce grein samples cach weighing 2k were priically bought from different retailers in shops and open-air markets within and around Kampala, Mukono and Mpigi districts of Uganda between Nov. 1988 and Aug. 1999. Random method of sampling™ was used for the collection of the rice samples such that an equal number of samples, & each, ofa locally grown rice variety called super and an imported rice grain from Pakistan were collected fea each of the three districts. Thus, 24 samples each of the two lypes of rice were involved in the study. These samples \Were analysed for fungal contamination and their moisture content. Isolation of fungi: The seed-plate (direct plating) method vas used to determine the seed-borne fungi on the rice grains. Prior to plating, a 500 g subsample was first surface sterilized using 70% ethanol pre-rinse prior to a (0.8% chlorine treatment for 2 mis, Excess disinfectant was drained off from the grains after which sterilized listlled water was used to rinse the grains three times, a 22 271-278, 2004 Excess water on the grains was mopped using sterile filter paper. The grains were then plated on a suitable isolation media ata plating rate of 10 rice grains per plate. A general purpose enumeration agar medium, dichloran rose-benaal chloramphenicol agar: DRBC™!, modified by Pitt and Hocking!” ancl a selective isolation medium, dichloran 18% glycerol agar: DGIS! were used to detect and isolate fungi, in general and xerophilic fungi, respectively. Fifty ‘grains per sub-sample were plated for DRBC, while 100 grains were plated on G18. All plates were incubated at under natural conditions of light and darkness for 7-8 days, but plates containing DGI8 were incubated for 14-20 days, Identification of isolated fungi: Fungi were identified on the basis of their macroscopic and microscopic features using the keys of Raper and Fennell; Booth", Elis"; Pit", Pit and Hocking! Five identification media were used including Czapek yeast extract agar supplemented with 20% sucrose: CY20S™, for the identification of Eurotium spp., Ceapek yeast extract agar: CYA, Malt extract agar: MEAP and 25% glycerol nitrate agar: G25N for identification of Penicillium spp, and Potato sucrose agar; PSA™' for identification of Fusarium spp. Determination of moisture content: The moisture content of each rice grain sample was determined by finding the Joss in weight ofthe rice grains upon heating for a 24 h period in an oven at 10°C and expressing it as a percentage of the fresh weight. Triplicate sub-samples of $0. per each rice sample were used. The average of the triplicates became the moisture content of a rice sample, Statistical analyses: Data wore subjected to analysis of variance (ANOVA), test and F-test, Statements of significance are based on P 0.05%, Correlation was used to determine the relationship between the various variables, RESULTS AND DISCUSSION Relationship between moisture content and incidence of fungi: The locally grown super tice had the lowest moisture content of 1] 69% while 14.60% was its highest level. Only 3 out of 24 (12.5%) ofits samples had moisture contents above 14.0%, the recommended level for safe storage of milled rice grains", In contrast, the imported Pakistan rice had the lowest moisture content of 11.60%, while 15.50% was its highest, in which ease, 12 out of 24 (5086) of its samples had moisture contents above the recommended level for safe storage of milled rice grains (Table 1). This difference in moisture contents of the twoJ. Biol. Sei, 4 (3): 271-278, 2004 Table: Moise content DC) and percentage incidence (616) offing fv 24 spl each of local lle super nice and ported riled Pakisor rice gains, en dchloran rovers {hlceampherice px (DREC) and ictloran 18% yee aa Doro\neda ‘Mile Paki ee 1.600036 1600.1 11932006 1238en06 12360 68 1250.06 1assenas 12100 12702010 intencs e000 Taesens 1256005 Iassn1s 1320 Tssoas 120.06 Tsaenes ragte1t0 Tus. 1209 17 igo. 10 ® o 56 4 2 2 % » 100 9s o 16 0 Las7s006 1497025 13208020, Isa7s006 0 types of rice is consistent with the difference in the incidence of fungi on the two rice, on both DREC and GIB agar media, The local rice had 13 out of 24 (54.20%) of its samples with incidence level of below 20%, with only 3 out of 24 samples (12.50%) having had incidence level of above 50%, on DRBC agar (Table 1), In contrast, the imported rice had 9 out of 24 (37.5026) of its samples with incidence level of below 20%, with 6 out of 24 samples (254%) having had ineidence level of above 50%, con DRBC agar. Similarly, the local riee had 11 out of 24 (45.80%) ofits samples with incidence level of below 20%6, with only 4 out of 24 samples (16.70%) having had incidence level of above 50%, on DG18 agar. In contrast, the imported rice had 6 out of 24 (25%) of its samples with incidence level of below 2006, with 11 out of 24 samples (45.80%) having had incidence level of above 50%, om DREC agar, Thus, the local rice had majority of its samples recording incidence levels of less than 20% on. both isolation media, while the percentage of samples which bad incidence levels of more than 50% were relatively higher on the imported rice than the local rice, ‘on both isolation media, Incidence of fungi on dichloran rase-bengal chloramphenicol agar (DRBC): Locally grown rice grain marketed in Uganda were found contaminated by 45 m3 species of fig that represented 29 genera while rice ‘imported from Pakistan had 51 species belonging to 26 genera, as determined on DREC agar medium, The broadest species spectrum on both types of rice grain was from the gems Aspergillus that had 7 species on the local rice and 9 species in the imported soe. Zurovium, Cladosporium, Fusarium and Penicillium genera ere ‘each represented by 3 species on the local rice, while on the imported rice, Eurotim, Cachliobolus and Fusarium each had 4 species. Cackliobolus and Rhizopus each had 2 species on the local rice while Cladasporiian and Peniciliton each had 3 species on the imported rice. The remaining genera were cach represented by only one species (Table 2), ‘The two types of rice grains were predominantly contaminated by Aspergillus candidus, Eurotium chevalier, B. rubrum, Mucor circinelloides and yeasts, ‘which oocusred on 11, 27, 1.4, 03 and 4.2%, respectively ‘onthe leal rice, while on the imported rie they occurred 053,68, 27,2 Sand 43% ofthe rice grains, respectively (Table 2). These fungi except. M. circinlloides were similarly the most frequently” recorded contaminants, oeusring on 208, 25,208, 4.2 and 54.1%, respectively of the local rice samples while on the imported rice they ocurred en 292, 1.7, 333, 3 and 58.3% ofthe samples, respectively (Table 2; Fig, 1). Thus, the imported rice had comparatively higher species diversity and percentage frequency of the predominant fungi, most af which were xsrophiles. Eurotium amstelodami, also a xerophile, cecurred more predominantly and frequently on the Imported rice whereby, it was occurred on 3.7% of the grains and 37.5% of the samples while onthe local rice st cceurred only on 0.2% of the grains and 8.3% of the samples. Together with these xerophilic fungi, other storage fing inchiding 2. repens, Penicillium citrinum and Talaromyees spp. occurred more frequently on the ‘mported ice than on the loal sce. In conta, field fangi including Acremonium strictum, Aspergillus niger, Aureobasidiwn pullulans, Chaetomium spp, Fusarium ‘moniliforme, F. solani, Geotriokn candid, Seytalidim —Hignicola. and Cladosporium sphaerospermum, a common field fungus, all occurred at a relatively higher frequency on the local rice than on the imported rice (Table 2). Chaetomiun spp. and Geotrichuom candidu, both of which oowred more frequently on the local rice require @ moisture content of 20-25% for their grovith?™, The local rice, having been fieshly harvested were thus ideal substrates far their growth, ‘The prevalence of -spergilius on both types of tice ‘rains, whereby it ranked the first genus with the widest species spectrum is consistent with other findings, inJ. Biol. Sei, 43): ‘uble2: Percrase incidene 610) Sequency (Fo soation (en 2b sarple) nlpecenage repay CoP) of varias of ng fn ea mle ser ces tmpoted milled ean nee Sone marketed in Usinda on dchcom Torben ‘Stpar ee Tak ee deremonio sircnom os 33s ‘cdremucor elegans os 1 a2 Semaiachamdspora 000 Aspergilis cans i$ 208 ens ooo O0 4 flows os 3 as ager os 4 exh O43 enaae oo A posi ot penile oo {Sant ano Atanas oot Sana 123 “wns ooo Auvectesidie puhdons 022 ‘yosoeamys ave reed Chaviontan giotoum = 000 Chaetontan os 5) ans Cupsospornfniniola OL 43 Clabsportonclabiporiiles 33128 Chern a1 sphaerospermun O88 as Cochliobe olor 00 9 a0 Chee 143 © mipaoom 2 C palescens © 00 please nium 1 42 ‘Bonion cmtodand 2 Hechevalon 6 280 Breen © 00 Emm S208 Fess fies 1 “42 Fuucruondbcemeslie = 0.000 Eimer oo 9 a0 E graniecrm ol 2 8s E mouliome os 3 Bs F owsponim oo 9 ‘00 sot’ os 2 88 Geomicumcantim = O44 163 Gitomer of or 1 42 Walrond Momgrephelanivais” 0328S ‘cor crimes, os 1 AB ‘cor op o2 1 42 ‘Neosarersa chert os 142 Pevclompecsimins = Pencillumcirsegenm = O21 A Peon oF 1 43 ikon oo 9 00 P exam os 3 1s Ppinphin or 1 az P ppg oo 0 0 Penilion p o7 3 135 Peseteropssguepini = LA Pramas or 1 43 ‘zope nam o2 1 42 Scapilanopisconta = 0090 ‘Syalhon lgntata os 3 88 Talwonyees pp. o4 3 ns Tiehedmakeianm = «9000 Tnehediecumvosim = «30M Ponta. oo 9 oo rae se OL 1 az Rcetorla meilegiosa 09-208 ma 271-278, 2004 which, stored cereal grains have been established to have frequent contamination by Aspergillus. Cereal grain in storage among them corn from Spain; barley. wheat, ‘maize and sorghum from Eaypé™ and stored maize in sub- Saharan Africa" were found to show predominant contamination by Aspergillus. ‘The comparatively higher incidence levels of storage fungi and species diversity on the imported rice than on the local rice may be attributed to the repeated handling of the imported grain from its harvesting in Pakistan until its arrival in Ugandan markets, Fungal population and diversity have been found to reflect the kind and efficiency of post harvest handling, comsitioning ard the storage environment and period that the arain is subjected to. The difference between the locally grown rice and the imported rice is that the imported rice has been subjected to prolonged transport over the seas during, which time the grains took up moistare from the atmosphere and from metabolic water of the respiring again!" Subsequently, the grains moisture content may have increased to levels suitable for growth of xerophilic fungi. Moisture contents ranging fom 12.5 to 16.0% have been found to allow occurence of an ecological suceession of fungi on stored cereal grains from extreme xerophiles to xerotolerants"* ‘The difference in moisture contents between the {imported andthe loeal rice (Table 1) i consistent with the higher incidence levels and frequencies of the predominant fungi, together with the broader species diversity on the imported sice as compared to the locally grown super rice (Table 2}, The comparatively low incidence levels of storage fungi on the loeal rice but prevalence of Cladosporium sphaerospermuon, a field angus, is similar to findings from Burundi whereby less than 5% of rice grains sampled from various markets were found contaminated with field fungi including Cladosporium spp. Aliernaria spp. and Pyricularia oryzae. Rice grains from Turkey and Iran were also foun to be comparatively free of fungal contamination 2s compared to other cereals including barley, wheat and com Incidence of fungi on dichloran 18% glycerol agar (DEAS): Locally grown rice marketed in Uganda was found contaminated by 11 species belonging to 6 genera of xerophilie fungi among other 52 species of xerotolerant fungi as determined on DG8 agar medium. The imported rice had a comparatively narrower species spectrum of 9 species belonging to 3 genera of xerophilic fungi among other 40 species of xerotolerant fungi. Among the xerophilie fungi, Aspergillus candids, E. chevalieri and E, rubrum were the most frequently isolated species,Teble3: Percentage incidence C4), eqvency (Ff iseation om 2 samples) and percentage regency CF) of xeric fg 30 sled supe snd mld Patan Sc pts ual en asa stakets on dafloan 18% hve aga naan DCI) serie akin | Toa sag Fs Saga Xerephiic ing Apes canes A pentileies 4 went Chopper rico (Chrssaporuon 90. ivothan amteleant chevalier repens Enum Pobpaveion olin Wallendasebt Serotlerant fa: Acimmcr ees ser clean i ‘fons 4 4 fonts 6 4 ccerene 0 1 tan oa om 0 A tere hoe om 2 8S A a oo os 1 A. vernal 030 oo tad ‘eaten pullans 010 oo aa Cheetomtan siobenon 00 oo 2 a3 hactonan Sp ato om Ooo Cladsportn ladoporioies 0.30 tat ie sao Cplecrspermn a7 tet 1 ase © Kenan alo om 2 4 Cachet mivatears O00 oo 2 83 (Celescene 50 bm 2 8a Swat perpendiytt 10 am 9 ao merce tans foo uo 143 ‘picoce nignm 00 ao 1 43 Flaca calmer Soe 2m 0 00 F monljorm O30 0 9 oo Fol oat om 9 ao Ecco oo oo 9 oo Fiscruen p ao 200 0 oo Goch ecaten ty 030 4 167 Muska ora oo ost aa Iesiedpotariectromae 00 os 1 a3 Memerphellznals” 0.00 oo 1 4a Macorcicinlas 030 S202 aa Mravemons foo am Tt 42 Pasclemyes aciner 00 aot 1 43 Ponellle crscgenon 0302 om 3 as P dbelonat fa i am 9 ao 2 iledtoun aot 1 oo 2 83 P mick alo 1 20 0 Oo 5 nesta a3 2 900 0 oo pain am 1 (42 ow 0 6 P purpurea Qo 6 00 ao aa Pana ao 8 oo aoe tad Peni 2% to a7 1208 ay Sepulanopnscamds = 02)! 42 Oo) DOD Strip am 9 oo 020 2&3 ‘Syed lola ao 2 83 ato i 42 Tatromycesinermedis = «10142 800d Tatromgces pp. o> 1 43 om 2 RS Prmoucwarntas 90 0 00 Oo 1 42 Diechodmakersonm = «O01 42 Ook Aa Reciaondamwlgioa 07 «$28 O80 $ MOB E niin loides Yeasts / Fig. 1: Percentage frequeney of the predominant fingi on Ical super and imported milled Pakistan rice grains marketed in Uganda, on dichloran sose-bengal chloramphenicol agar medium (DRBC) cceurring on S42, 50 and 45.8% of the local rice samples and 58,3, $33 and 66.7% of the imported rice grain samples, respectively. Aspergillus wentit and E, amstelodami were also frequently isolated on both types of rice occurring on 29:2 and 25% of the local rice samples, while on the imported riee they occurred on 25 and 58 3% of the samples, respectively (Table 3, Fig. 2). These xerophiles except d. wenti, were similarly the ‘most predominant species on oth types of rice, whereby A.candidus, Aswents, E. amstelodami, E, chevalieri and E, rubrum occured on 2.5, 08, 08, 2.7 and 3.6% of the local rice grains, respectively while on the impented rice they occurred on 3.9, 06, 74, 15.Oand 5.5% ofthe grains, respectively (Table 3), The remaining species, except E. repens which ocoured only on the imported rice, contaminating 1.5% of its grains and 25% of its samples, occurred only sparsely. However, other species of xxerophilicfiangi which were recorded on both types of rice include A. restrictus, E. oristatum and Wallemia sebi (Table 3). ‘The predominant xerophilic fungi occurring on both the local milled super and imported Pakistan rice wereJ. Biol. Sei, 43): Pereemtage equency of fang Fig. 2: Percentage frequency of the predominantxerophilie fungi on local super and imported milled Pakistan tice grains marketed in Uganda, on dichloran 18% glycerol agar medium (DG18) chevaltert I thus: Ewrotium spp. and Aspergillus spp. Among the Eurotium spp. Eurotiwn amstelodami, E. chevalieri and E. rubrum, followed by E. repens were predominant, while among the Aspergillus spp, A. candidus was the most predominant species followed by A. wentif and A. restricius (Table 3, Fig, 2). These observations are consistent with other findings whereby Zurortum chevalieri has been reported as the commonest spoilage fungi in stored cereal grains while E. amstelodami and E. rubrum have also been reported to aceus frequently, bat the latter is more common in warmer regions!” Foodstuffs in retail outlets and markets have been reported to be actually under storage and depending on the conditions and time that the commodities are stored awaiting customers, fimgal contamination may accu", As a biological produet, aging of the rice grain with subsequent increase in its hygroscopicity is highly significant in the storabilty of the rice, Absorption of atmospheric moisture by the rice grains increases its 76 271-278, 2004 moisture content. Subsequently, this enhances fungal growth on the rice# ‘The comparatively higher moisture contents and prevalence of xerophiles on the imported rice grain than the local tice grain (Tables 1 and 3; Fig. 2) may be attributed to the prolonged period of time involved in transporting the imported rice grain from Pakistan to Uganda. Further, more time is involved in distributing it within the various loeal wholesale and retail markets, Similarly malpractice among traders of withholding the rice aa they speculate for increased demand and higher prices may also provide more time for the grains! moisture content to increase resulting in further fungal contamination ofthe imported Pakistan rice In contrast, the locally grown super rice does not undergo prolonged transportation and thas comparatively litle time is available for the grains’ moisture content to ‘The current study revealed that both the locally grown and imported rice grains were predominantly contaminated by xerophilie fimgi including Euroriem amstelodami, E, chevalieri, E. rubrum and Aspergillus ‘candids, The presenee of these toxigenie furgi on the grain suggest contamination by mycotexins including sterigmatocystin by E.amstelodami and citrinin by A.candidus. The incidence of these xerophilic fungi was however, higher on the imported rice, Further, the imported rice had a relatively wider species spectrum of xerophilic fungi inckiding A. penicilloides, A. went E, repens and Pavcilomyces lilacinus, all of which were rnot encountered on the Ioeal rice, Instead, field fungi including Aeremonium strictum, Fusarium moniliforme, F. Seytalidium Tignicola and Trichoderma hharsiamun occurred only on the local rice, The presence of these fing including F. moniliforme anki F. solani on the rice grains also suggest presence of mycotoxins including fumonisins, which have been associated with esophageal cancex"?, Similarly, the imported rice, recorded a comparatively higher moisture contest, whereby, 15.5% was its highest level while for the local rice 14.6% was the highest level. Further, a half of the ‘imported rice samples had 1 the ideal level for storage of milled rice, while the local rice had only 12.5% of its samples with moisture above 14 The comparatively higher moisture contents of the imported rice comespond to its higher contamination levels by fungi, particularly storage fungi, mest of which are associated with the production of mycotoxins, Thus, there is a high possiblity that the imported rice could have more mycotaxin contamination than the local rice. the common solani oisture content above 14.0%,J. Biol. Sei, 4 (3): 271-278, 2004 ACKNOWLEDGMENTS ‘The authors are deeply indebted to Prof. Bukerya- Zisaba, The Head of Botany Department, Makerere University, Kampala for the facilites he provided during. this research. 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