yeast diversity in crop-growing environments in cameroon
TRANSCRIPT
International Journal of Food Microbiology 127 (2008) 184–189
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International Journal of Food Microbiology
j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro
Short communication
Yeast diversity in crop-growing environments in Cameroon
Marzia Stringini, Francesca Comitini, Manuela Taccari, Maurizio Ciani ⁎Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
⁎ Corresponding author. Tel.: +39 071 2204150; fax: +E-mail address: [email protected] (M. Ciani).
0168-1605/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2008.07.017
a b s t r a c t
a r t i c l e i n f oArticle history:
In the present study, we h Received 27 May 2008Received in revised form 15 July 2008Accepted 16 July 2008Keywords:YeastBiodiversityEcologyEnrichment culturesPCR-DGGEMolecular methods
ave investigated the occurrence of yeast flora on several agricultural productscoming from crop-growing environments in Cameroon, to provide better knowledge of the biodiversity ofyeast flora, and to thus define the impact of this biodiversity on food products. The yeast biodiversity wasinvestigated using traditional culture-dependent methods, along with culture-independent methods. Theculture-dependent approach was carried out using both direct and enrichment procedures, to detect thebroadest possible presence of yeast species.A total of 151 strains belonging to 26 different yeast species were isolated and identified using restrictionpattern analysis of the internal transcribed spacer region 5.8S-ITS and sequence analysis of D1/D2 domain of26S rRNA gene. The enrichment isolation procedures carried out in high-sugar media allowed the recognitionof fermentative species such as Saccharomyces cerevisiae and Torulaspora delbrueckii, which have previouslynot been detected using direct isolation methodology.The results of culture-independent method using DGGE patterns and sequencing of the DNA bands revealeda lower number of yeast species when compared with the culture-dependent methodology even if theidentification of several yeast species not detected by traditional microbiological procedures such as Candidatropicalis and Hanseniaspora uvarum is allowed. Thus, these multiphasic approaches to study yeastbiodiversity (culture-dependent and -independent methods) have allowed us to get a more complete pictureof the microbial diversity in these natural environments.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Fruits, vegetables, drinks and other agricultural products are veryimportant microhabitats for a multiplicity of yeast species in nature. Asuccession of yeast populations is involved in a variety of biochemicaland ecological processes due to the ability of yeast to quickly use thesimple sugars present in these agricultural products (Kurtzman andFell, 1998). Recently, several investigations have been carried out indifferent natural and crop-growing environments so as to obtainbetter knowledge of yeast biodiversity and to define the impact thishas on food products. These studies were carried out in tropicalenvironments, including a Brazilian rain forest (soil, water, insect andplant materials) (Buzzini and Martini, 2002), and on tropical fruits,flowers and leaves (Santos et al., 1996; Trindade et al., 2002; Camotti-Sartori et al., 2005; da Silva et al., 2005), fresh orange juice fromFlorida (Arias et al., 2002), Nigerian sugar-cane peel (Olasupo et al.,2003), palm wine from Ghana (Amoa-Awua et al., 2006) and cocoabeans from Indonesia (Ardhana and Fleet, 2003). In these studies, theoccurrence of yeasts was investigated using traditional microbiologi-cal methods.
Over recent years, culture-independent methods based on mole-cular biology techniques have been developed to study microbial
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population dynamics, including fluorescence in-situ hybridization(FISH), real-time PCR, temperature gradient gel electrophoresis(TGGE), and denaturing gradient gel electrophoresis (DGGE). Thislast method has been used to investigate yeast population dynamics inmilk and fermented products, including wine, fermented cassava(manioc), maize dough and coffee beans (Cocolin et al., 2000, 2002;Masoud et al., 2004; Prakitchaiwattana et al., 2004). Other investiga-tions on the occurrence of yeasts in agricultural products have beencarried out to compare traditional microbiological methods andculture-independent methodologies (Masoud et al., 2004; Nielsenet al., 2005, Nielsen et al., 2007).
Today, culture-independent methods are particularly attractive asthey offer a good and rapid strategy for yeast detection and represent avalid alternative to classical microbiological analyses. In addition,culture-independent analysis offers the possibility of detectingspecies that may be present in the habitat in viable, but non-culturable, states (Head et al., 1997; Rappe et al., 2003; Ercolini, 2004).Indeed, the classical microbiological methods based on plate counts,and isolation and biochemical identification have been criticized sinceonly easily culturable microorganisms can be detected and membersof microbial community that need elective enrichments are notidentified. On the other hand, it has been demonstrated that themicrobiological patterns obtained after DGGE analysis are strictlyconnected to the numerically dominant species (Ampe et al., 1999;Prakitchaiwattana et al., 2004) and the detection limit of this
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technique is highly matrix dependent. Cocolin et al. (2000) demon-strated that during wine fermentation, PCR-DGGE did not detect yeastspecies at b103 cells ml−1.
In the present study, we describe the application of culture-dependent (traditional methods after direct and enrichment isolationprocedures) and culture-independent (DGGE analysis) methods tostudy the yeast ecology of several crop-growing environments inCameroon. Furthermore, over the last few decades, Cameroon hasbeen characterized by major economic stability, which has allowedthe development of agriculture and consequently of its products. Sinceyeast can have an impact on these typical crops, further knowledge ofthe yeast biodiversity is needed to obtain a more complete and correctpicture of their occurrence.
2. Materials and methods
2.1. Samples
Samples coming from two different geographic areas of Cameroon(Odza and Obala, in the central region) were collected during thesummer period (December–January) of 2005–2006. Two samplesrepresenting each collected product, were placed aseptically in sterileplastic bags and transferred in ice boxes (4 °C) to the laboratory at theDipartimento di Scienze degli Alimenti, Università Politecnica delleMarche (Italy), for the analysis. The yeast occurrence was investigatedin the following samples: corrosol (soursop) pulp and peel (Guanàbanoannona muricata L.); papaya fruit and leaf (Carica papaya L.,); cocoabeans (Teobroma cacao L.); sugar-cane juice (Saccharum officinarum L.);palmwine from a variety of palm trees (Elaeis guineensis J); palm nuts(Cocos nucifera L.); bananas (Musa acuminate L.); manioc (Manihotutilissima L.); and samples of soil: red clay soil (Sa), and modified soilfrom composting of agricultural wastes (Sb).
Within 24–36 h of their collection and transfer to the laboratory,the samples were subjected to the following procedures: (i) liquidsamples (1 ml) were placed in tubes containing 9 ml sterile water, andthen the standard procedure for serial dilutions was carried out;(ii) solid samples (1 g) were collected in sterile plastic bags andhomogenised using a Stomaker 400 Circulator (Seward, UnitedKingdom) for 5 min at 260 rpm, before standard viable-plate countingwas carried out; and (iii) the procedure described by Comitini andCiani (2006) was followed to detect the yeast population on thesurface of the papaya leaf sample.
2.2. Media and direct isolation procedures for the yeast
Yeast isolation and enumeration were performed in four differentmedia: (i) YPD medium (10 g l−1 Bacto Yeast Extract, 10 g l−1 BactoPeptone and20 g l−1 glucose) (Oxoid, Basingstoke, UK); (ii)WLnutrientagar (Oxoid); (iii) WL nutrient agar with 0.02% biphenyl (Fluka, USA)added after sterilization to reduce the growth of moulds; (iv) Rose-Bengal chloramphenicol agar base (Oxoid) added with chlorampheni-col to avoid bacterial growth. The plates were incubated at 30 °C for 3–8 days. After this, any colonies were counted and selected according totheir macro- andmicro-morphological aspects, and theywere isolatedin proportion to their frequencies (Martini et al., 1996). Representativecolonieswere picked randomly from the plates and pure culturesweresubjected to the next identification procedures.
2.3. Enrichment procedures for yeast isolation
The enrichment procedures to detect fermenting yeast specieswere carried out by inoculating 1ml or 1 g, depending on sample type,into high-sugar medium (Trebbiano toscano grape must, pH 3.2, withsugar added to a final concentration of 27%, w/v). All of themicrofermentations were carried out at 25 °C in 100-ml Erlenmeyerflasks containing 70ml pasteurisedmust. During fermentation (after 7
and 15 days), yeast isolation was performed on WL nutrient agar(Oxoid) using the procedures previously described in direct isolationprocedures.
2.4. Yeast identification
In all of the yeast isolates, after an initial micromorphologicallycharacterization, their DNA was extracted. Pure yeast cultures werepre-grown on YPD agar at 25 °C for 3 days. The cell mass was thentransferred to a screw-cap tube with 300 μl reaction buffer, containing0.1 M Trizma, pH 8, 50 mM EDTA, 1% SDS and 0.3 g glass beads (Ø of0.45–0.50mm) (Sigma-Aldrich, USA). The tubeswere vortexed 3 timesfor 1 min at the top setting. The mixtures were then boiled for 10 minand transferred to ice for 3 min. For each sample, 20 μl 1 M Tris–HCl,pH 8, 15 μl 0.5 M EDTA, pH 8, 50 μl 10% SDS and 200 μl 5 M potassiumacetate were added. The samples were then incubated for 30 min inice and finally centrifuged at 18,000 ×g for 10 min. The supernatantwas transferred to a new screw-cap tube, with the addition of an equalvolume of ice-cold isopropanol. The precipitated nucleic acids werecollected after an incubation of 5 min, and the pellet was washed in500 μl ice-cold 70% ethanol and centrifuged at 18,000 ×g for 5min. TheDNA obtained was dried overnight, resuspended in 100 μl 0.1 M TEbuffer, pH 8, and the DNA template was incubated at 45 °C for 15 min.After this incubation, the tubes were stored at −20 °C and used within2 weeks.
The isolates were then identified using the internal transcribedspacer (ITS)-PCR procedure, using the primers ITS1: 5′-TCC GTA GGTGAA CCT GCG G-3′; and ITS4: 5′-TCC TCC GCT TAT TGA TAT GC-3′. ThePCR mixture and the thermocycling protocol conditions and restric-tion endonucleases analysis were applied as described by Esteve-Zarzoso et al. (1999). The restriction patterns were compared with theresults of previously published studies (Esteve-Zarzoso et al., 1999,Esteve-Zarzoso et al., 2001; Sabate et al., 2002; de Llanos Frutos et al.,2004; Arroyo-Lòpez et al., 2006).
When the 5.8S-ITS restriction profile did not match with anyestablished restriction patterns, the amplicons obtained were purifiedand sequenced. Sequence similarities were performed on-line usingthe basic local alignment search tool (WU-BLAST2) programme on theEuropean Bioinformatics Institute (EBI) web page (http://www.ebi.ac.uk/bast2/index.htlm).
2.5. Yeast population detection from a matrix by PCR-DGGE
PCR-DGGE analysis was performed using the methodologiesdescribed by Cocolin et al. (2000).
The PCR products obtained from the direct DNA extractions wereanalysed using a specific apparatus, the DGGE-1 ELETTROFOR,(Elettrofor, RO, Italy). The DGGE for sequence-specific identificationwas performed in 8% polyacrylamide (acrylamide/bis-acrylamide mix37.5:1 wt/vol) (Eurobio, France) gels containing a 20–50% urea–formamide gradient. The electrophoresis was performed in 1×TAEbuffer at a constant voltage of 150 V for 6 h with a constant tem-perature of 60 °C. After electrophoresis the gels were stained in1.25×TAE buffer containing ethidium bromide, and then they werephotographed under UV transillumination using a Canon Power ShotG5 digital camera (Canon INC, Tokyo, Japan). For the soil samples, theDNA extraction was carried out using the specific “FastDNA Spin forsoil kit” DNA kit (MP Biomedicals, LLC, Ohio).
After the DGGE analysis, selected bands were excised from the gelswith a sterile pipette tip and the DNAwas eluted in 50 μl TE buffer, pH8, overnight at 4 °C. The eluted DNA was re-amplified with primerswithout the GC clamp, using the same reaction mixture describedabove. The PCR products were sequenced and DNA sequencesimilarity was performed on-line using the basic local alignmentsearch tool (WU-BLAST2) programme on the EBI web page (http://www.ebi.ac.uk/bast2/index.htlm).
Table 1Quantitative evaluation of the yeast isolated in this study
Samples Log CFU g−1±SD
Corrosol peel (Cp) 6.70± 0.170Corrosol pulp (Co) 5.89±0.247Papaya fruit (Pf) 7.13±0.267Papaya leaf (Pl) 6.49±0.790Palm nut (Pn) 6.84±0.611Cocoa beans (Cb) 6.47±0.715Banana fruit (Bf) 4.42±0.749Soil a (Sa) Uncountablea
Soil b (Sb) 5.75±0.322Manioc (Ma) 4.73±0.456Sugar-cane juice (Sc) 5.73±0.283b
Palm wine (Pw) 6.06±0.293b
a Extensive presence of moulds on plates.b Values expressed as Log CFU ml−1.
Table 2Identification of the yeast species found in the study: sizes as bp of PCR product andrestriction fragments
Species APa
(bp)Restriction fragments
HhaI HaeIII HinfI
Candida beechii (1) 650 300+300 400+140+90 330+330Candida diversa (1) 450 160+100+
100+90410 220+200
Candida montana (4) 525 250+230 400+125 270+250Candida tropicalis (5) 550 280+250 450+90 270+270Candida fructus/musae (3) 400 220+90+90 280+150 210+130+80Cryptococcus humicola (1) 520 265+255 425+55+40 230+230+60Cryptococcus skinneri (2) 565 260+200 400+110 220+150+
120+60Cryptococcus spp. (5) 500 200+180+120 400+100 200+150+150Dekkera anomala (4) 800 340+340+120 800 360+190+
160+80Dekkera bruxellensis (1) 485 255+140+90 375+95 270+215Hanseniaspora uvarum (2) 750 320+310+105 750 350+200+180Lodderomyces elongisporus (1) 600 330+250 550 300+270Pichia anomala (12) 650 575 600+50 310+310Pichia fermentans (22) 450 170+100+
100+80340+80+30 250+200
Pichia guillermondii (6) 625 300+265+60 400+115+90 320+300Pichia jadinii (1) 575 280+230 390+145+40 300+275Pichia kluyveri (1) 450 175+115+80+80 370+80 250+200Pichia membranifaciens (1) 500 175+110+90+75 330+90+50 275+200Rhodotorula mucilaginosa (1) 640 320+240+80 425+215 340+225+75Saccharomyces cerevisiae (56) 880 385+365 320+230+
180+150365+155
Torulaspora delbrueckii (11) 800 330+220+150+100
800 410+380
Yarrowia lipolytica (2) 380 210+170 380 190+190Pichia amylophila (1)b 650 300+270+50 400+250 325+270+40Pichia veronae (1)b 600 290+270+50 600 300+300Issachenkia orientalis (3)b 490 400+90 230+150+130 220+150+130Trichosporon asahii (1)b 530 270+270 500 250+220+50Cryptococcus spp. (1)b 580 270+220+90 500+60 240+160+
100+50Unidentified (1)b 630 300+250 380+250 320+260
a AP Amplified product (ITS1–ITS4).b Identification obtained with the sequence information from the ITS amplified
product and sequence similarity were performed on-line using the basic localalignment search tool (WU-BLAST2).
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2.6. Statistical methods
The DGGE DNA profiles were subjected to principal componentanalysis (PCA) using the Unscrambler 7.5 softwere (CAMO ASA) toinvestigate the relationships among yeast species and samples.
3. Results and discussion
3.1. Yeast enumeration, isolation and identification
The results of the enumeration of the yeasts isolated from thesamples collected are shown in Table 1, and they varied from 3.0×104
to 1.3×107 cell g−1. As expected, all of the samples contained a high-sugar content, and it was possible to detect a wide yeast populationthat was comparable with those reported in other studies (Nielsenet al., 2005; Nyanga et al., 2007). However, the banana and maniocsamples showed scarce colonization by yeast flora. These limitedyeast populations were probably due to the particular composi-tion; the underground development of a tuber for manioc andthe wrapped-round peel that hermetically encloses the fruit of thebanana.
The Sa soil sample (red clay soil) showed an extensive and fastcolonization of moulds and bacteria in all of the media used, but thesedid not allow the growth and the consequent enumeration of theyeast, while the Sb soil sample (composted soil) showed yeastcolonization comparable to that seen in other investigations (Kentand Triplett, 2002; Gomes et al., 2003; Jumpponen, 2003; Andersonand Cairney, 2004).
Using two different culture-dependent approaches (direct andenrichment isolation methods), a total of 151 isolates that were
Fig. 1. ITS-PCR amplification and Hha I, Hae III, Hinf I restriction analysis of 5.8S rDNA of(O'RangeRule™ 100 bp DNA Ladder); lanes 2–5, yeast isolated from Sa soil; lanes 6–9, yeast is14–17, yeast isolated from Sa soil; lanes 18–21, yeast isolated from palm wine (Pw).
representative of all of the samples were collected. Fig. 1 showsrepresentative data from restriction analysis of some of the isolates.The sizes of the PCR products and the restriction fragments of all of thespecies identified in this study are given in Table 2. In a few isolates,when the 5 .8S-ITS restriction profile did not match with anypublished restriction patterns, the amplicons obtained were purified
yeast strains isolated from different samples. Mk — molecular weight marker 100 bpolated from cocoa beans (Cb1); lanes 10–13, yeast isolated from cocoa beans (Cb1); lanes
Table 3Culture-dependent method: yeast species found in the samples using direct andenrichment procedures
Species identified with culture-dependent method
Samples Direct isolation No. ofisolates
Isolation withenrichment
No. ofisolates
Sugar-cane juice(Sc)
Pichia fermentans 5 Torulasporadelbrueckii
5
Pichia anomala 5 Saccharomycescerevisiae
3
Candida tropicalis 4Candida montana 4Yarrowia lipolytica 1
Palm wine (Pw) Saccharomyces cerevisiae 11 Saccharomycescerevisiae
5
Corrosol fruit (Co) Saccharomyces cerevisiae 3 Saccharomycescerevisiae
8
Pichia guillermondii 2 Pichia guillermondii 3Pichia fermentans 2 Torulaspora
delbrueckii1
Pichia anomala 3Dekkera bruxellensis 1
Corrosol peel (Cp) Cryptococcus skinneri 2 Saccharomycescerevisiae
4
Dekkera anomala 1Pichia kluyveri 1
Papaya fruit (Pf) Pichia fermentans 2 Pichia fermentans 3Issatchenkia orientalis 2 Issatchenkia
orientalis1
Hanseniaspora uvarum 1 Hanseniasporauvarum
1
Banana fruit (Bf) Pichia anomala 4 Not isolatedPapaya leaf (Pl) Torulaspora delbrueckii 2 Torulaspora
delbrueckii2
Pichia fermentans 2 Pichia fermentans 4Candida beechii 1 Saccharomyces
cerevisiae6
Rhodotorula mucilaginosa 1Pichia membranaefaciens 1Yarrowia lipolytica 1
Palm nut (Pn) Not isolated Saccharomycescerevisiae
4
Torulasporadelbrueckii
1
Cocoa beans (Cb) Dekkera anomala 2 Dekkera anomala 1Candida fructus/Candidamusae
3 Saccharomycescerevisiae
3
Pichia amylophila 1 Pichia fermentans 3Pichia jadinii 1Unidentified 1
Manioc (Ma) Cryptococcus spp. 2 Cryptococcus spp. 3Pichia fermentans 1Saccharomycescerevisiae
2
Soil a (Sa) Not isolated Saccharomycescerevisiae
7
Soil b (Sb) Pichia guillermondii 1Lodderomyceselongisporus
1
Cryptococcus humicola 1Cryptococcus spp. 1Candida tropicalis 1Candida diversa 1Pichia veronae 1Trichosporon asahii 1Total yeasts isolated 80 Total yeasts isolated 71
Fig. 2. Yeast DGGE profiles. The lane designations (top) indicate the samples. The bandsindicated by the numbers were excised, re-amplified and subjected to sequencing.
Table 4Sequencing results of the bands excised from the yeast DGGE gels
Band(s)a Size(bp)
Samples Closest relative %Identity
Sourceb
1 207 Sc, Pl1-2, Co, Sb1-2 Candida tropicalis 97 EU6518542 199 Pf Issatchenkia orientalis 98 AB4364473 194 Pf, Pl1, Pl2, Co Pichia fermentans 99 EU0192224 206 Pl1, Pl2, Co, Sb1 Torulaspora delbrueckii 99 AB4364665 210 Sb1, Sb2 Pichia guillermondii 99 EU1822166 182 Sb2 Pythium aphanidermatum 94 AY598622
a Bands are numbered as indicated on the DGGE gels shown in Fig. 2.b Accession number of the sequence of the closest relative found by a BLAST search.
187M. Stringini et al. / International Journal of Food Microbiology 127 (2008) 184–189
and sequenced, with the DNA sequence similarities investigated on-line.
The yeasts collected from the 12 samples revealedwide biodiversity,showing 26 different species belonging to 12 genera. Saccharomycescerevisiae was the most abundant yeast species isolated. However, itneeds to be emphasized that this yeast species was mainly found afterenrichment and after “auto-enrichment” procedures, such as with thepalmwine sample. Only in the corrosol fruit was S. cerevisiae found after
the direct isolation. The species identified at relatively high frequencieswere: Pichia fermentans, Pichia anomala and Torulaspora delbrueckii;while Pichia guilliermondii, Cryptococcus spp., Candida tropicalis, Can-dida montana, Dekkera anomala, Issatchenkia orientalis, Candida fructus/musae, Cryptococcus skinneri, Yarrowia lipolytica and Hanseniasporauvarum were all isolated at lower incidence. Finally, Candida beechii,Candida diversa, Cryptococcus humicola, Dekkera bruxellensis, Loddero-myces elongisporus, Pichia amylophila, Pichia jadinii, Pichia membranifa-ciens, Pichia veronae, Pichia kluyveri, Trichosporon asahii and Rhodotorulamucilaginosawere the species collected only once (Table 2). One isolateremains unidentified, since its patterns did not match with any of thereference strains.
3.2. Comparison of direct isolation and enrichment procedures
To detect the possible low presence of fermenting yeasts in theseagricultural environments, high-sugar-medium enrichment proce-dures were used. For each sample, a comparison of the direct data andthe data after the enrichment isolation procedures is showed inTable 3. As expected, a large number of different yeast species wasidentified after direct isolation (26 species), while only 8 species werefound using the enrichment procedures.
Fig. 3. Principal component analyses (PCA) of the data carried out as a function of the DGGE bands. The acronyms correspond to: (Hu) Hanseniaspora uvarum; (Pa) Pichia anomala;(Pg) Pichia guilliermondii; (Ct) Candida tropicalis; (Td) Torulaspora delbrueckii; (Pf) Pichia fermentans; (Ch) Cryptococcus humicola; (Sc) Saccharomyces cerevisiae; (Pya) Pythiumaphanidermatum; (Io) Issatchenkia orientalis; (NI) unidentified.
188 M. Stringini et al. / International Journal of Food Microbiology 127 (2008) 184–189
The palm wine provided a particular environment (Amoa-Awuaet al., 2006). In this substrate, S. cerevisiae was the only species foundusing both, direct and enrichment approaches. This substrate could beconsidered an “auto-enrichment” medium similar to grape mustduring fermentation, since a rapid increase in selective compounds,such as ethanol, was produced (Martini et al., 1996).
Notwithstanding the detection of a low yeast biodiversity, inseveral case enrichment procedures allowed the isolation species thatwere not found using direct isolation procedures. This was the case forS. cerevisiae, as with the enrichment procedures, it was found in all ofthe samples, with the exception of papaya fruit, banana fruit and inone soil sample (Sa). In addition to papaya leaf, enrichment methodsdetected T. delbrueckii in the pulp of corrosol and the juice of sugar-cane.
In the banana and manioc samples, according to the results of theviable cell counts (Table 1), a reduced yeast species diversity wasdetected. However, the enrichment procedures allowed the finding inthe manioc sample of yeasts besides Cryptococcus spp., the yeastspecies S. cerevisiae and P. fermentans, while in papaya fruit, there wasa complete overlap of the results between the two methods.
3.3. PCR-DGGE method
A range of samples were analysed in the DGGE investigations,including their DGGE profiles of the 26S rRNA gene fragments, ascompared with some selected species of reference (Fig. 2): sugar-canejuice (Sc), palm wine (Pw), papaya fruit and leaf (Pf, Pl1, Pl2), corrosol(Co) and the soil samples (Sb1, Sb2, Sa). For the remaining samples, itwas not possible to extract the DNA directly from the matrix, probablydue to their tough consistencies, and/or the scarcity of the yeast DNA.As shown in Fig. 2, some of the gel bands were easily attributed tospecific yeast species, such as H. uvarum and S. cerevisiae. However,when the DGGE bands did not match with any of the yeast ampliconsof reference and thus confirm their identity, the bands were excidedfrom the gel and sequenced (Table 4). Unfortunately, purification andreamplification of band 7, which was detected strongly in papaya leafand in corrosol samples, did not prove successful.
Band 1 in Fig. 2 corresponds to an isolate belonging to the speciesC. tropicalis, which was detected in all of the samples collected, with
the exception of palmwine and papaya fruit. Band 2 was present onlyin the papaya fruit sample and was identified as I. orientalis, whileband 3 was detected in papaya fruit, papaya leaf and corrosol fruit andwas identified as P. fermentans. Bands 4 (T. delbrueckii) and 7(unidentified) were detected in all of the papaya leaf and corrosolsamples. Band 5 corresponded to P. guillermondii, which includesseveral phenotypically indistinguishable taxa, including Candidafermentati and Pichia caribbica (Vaughan-Martini et al., 2005); thiswas detected in the soil samples, confirming its wide distribution innature as it has been routinely isolated from soil (Capriotti and Ranieri,1964). The DNA sequencing for band 6 showed a closely relativelyspecies with 94% of identity to Pythium aphanidermatum, whichshowed only aweak signal, and only appeared in one of soil b samples.
The data was further elaborated by PCA, which was carried outusing the matrix of all of the DGGE gel bands, and which allowed thedetermination of the relationships among the substrates in terms ofthe yeast species occurrence (Fig. 3). On the bases of these yeastspecies profiles, three sample groups could be distinguished: (i) palmwine (Pw), papaya fruit (Pf) and the juice of sugar-cane (Sc);(ii) corrosol (Co) and papaya leaf (Pl); and (iii) the Sb soil sample. Anexception to this grouping was seen for the Sa soil sample, which wascharacterized by the presence of an S. cerevisiaeDNA band that definesits collocationwithin the first group, while being differently related tothe other soil samples (Sb1 and Sb2).
Repeated DGGE analyses of different samples of papaya leaf (Pl1and Pl2) and the Sb soil sample (Sb1 and Sb2) confirmed thehomogeneous yeast species profile within the environment.
4. Conclusions
The yeast identification based on culture-dependent methodscarried out in this study allowed us to describe a wide biodiversity. Inparticular, the enrichment procedures allowed the detection offermenting yeasts, such as S. cerevisiae and T. delbrueckii, which areonly found with difficulty using direct isolation and the DGGEmethods. On the other hand, the DGGE methods confirmed thedominant culturable yeast population, and allowed detection of thepresence of yeasts species that are non-culturable because of thecultivation conditions used and/or the physiological state of the cells.
189M. Stringini et al. / International Journal of Food Microbiology 127 (2008) 184–189
It is interesting to note that the H. uvarum seen in palm wine andsugar-cane juice was revealed using the DGGE method, while it wasnot detected with the culture-dependent approach. This culture-independent approach allowed the detection of H. uvarum in four ofthe eight samples analyzed, while the use of classical methodologiesallowed the isolation of this species in only one sample (papaya fruit).
The most diffused species in the samples examined wasC. tropicalis. The use of DGGE analysis allowed the detection ofC. tropicalis in six samples, while in the culture-dependent methodsthis species was only seen in three samples of the eight examined. Thisyeast species was undetectable using both approaches in palm wineand papaya fruit, highlighting the particularity of these substrates.
Therefore, the combined use of culture-dependent and -indepen-dent approaches is indeed useful for a description of the biodiversityin these crop-growing environments. Indeed, the analysis of the D1/D2 domain (universally accepted in yeast taxonomy) that is associatedwith the DGGE technique and the available database has allowed us toidentify and differentiate yeast species without their isolation.
We thus conclude that PCR-DGGE can provide valid support for thestandard plating methods in studies of yeast biodiversity. For thesereasons, the investigations on yeast biodiversity using the combina-tion of culture-dependent microbiological methods and culture-independent methods can be profitably used to provide a morecomplete picture of the ecological distribution of yeasts.
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