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Mycological survey of ripped service tree fruits (Sorbus domestica L.) with an emphasis on toxinogenic fungi
International Journal of Food Microbiology 99 (2005) 215 – 223 www.elsevier.com/locate/ijfoodmicro
Mycological survey of ripped service tree fruits (Sorbus domestica L.) with an emphasis on toxinogenic fungi Roman Labudaa,*, Ladislav Kriva´nek Lb, Dana Tancˇinova´a, Silvia Ma´te´ova´a, Sonˇa Hrubcova´a a
Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic b Department of Animal Nutrition, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic Received 22 December 2003; received in revised form 9 July 2004; accepted 17 September 2004
Abstract To investigate a possible incidence of microscopic fungi in ripped service tree (Sorbus domestica L.) fruits, a mycological survey was carried out during October–November 2003 in Slovakia. This rare kind of fruit is renowned for its significant curative actions in various human and animal diseases. The study revealed that all 24 surface sterilized fruits analysed were contaminated with fungi. The most dominant endogenous contaminant encountered was Cladosporium cladosporioides (Fres.) de Vries followed by Alternaria alternata (Fr.) Keissler and Penicillium expansum Link with 88%, 63% and 54% frequency, respectively. Furthermore, 24 other fungal species were associated with the fruits as well. P. expansum (40), P. carneum (Frisvad) Frisvad/Penicillium paneum Frisvad (35) and P. griseofulvum Dierckx (6) isolates recovered from the fruits were screened by an agar plug method for production of mycotoxin patulin, all with positive results. In addition, citrinin, griseofulvin and zearalenon production by appropriate species isolates were detected. Despite of a limited number of samples of the service tree fruits examined during this study, it was concluded that the ripening of service tree fruits is accompanied with the presence of typical rotting-fungi as well as of some others, which have not previously been reported in connection with fruit. Furthermore, it was suggested that the ripped service tree fruits should be considered as a potential source of significant fungal secondary metabolites including mycotoxins. Secondary metabolite profiles of the species identified during the study were included here. D 2004 Elsevier B.V. All rights reserved. Keywords: Endogenous contaminants; Fusarium spp.; Mycotoxins; Patulin; Penicillium spp.
* Corresponding author. E-mail address: [email protected] (R. Labuda). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.09.002
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1. Introduction Small-sized apple- to pear-like fruits producing service tree (Sorbus domestica L., Rosaceae) represents a rare kind of fruit with only a limited state of domestication and distribution (Pagan and Paganova´, 2000). Aside from its wood of high quality, the service tree is also well known for the production of fruits with significant curative actions used in folk-medicine on various intestinal diseases, anaemia and emaciation. An extract of the fruits is used on haemorrhage cessation and also as diuretic agent at lithonephritis (Velgosˇova´ and Velgosˇ, 1988; Pagan and Paganova´, 2000). Interestingly, suitability of the fruits for direct consumption is reached when the peel becomes brown and the flesh gets a yellowish to brownish tint, so that the fruits are completely over-ripped or rotted. There is a similarity in the appearance of these ripped service tree fruits with other kind of pomaceous and/or stone fruits following fungal contamination resulting in a rot such as that of Penicillium expansum (Samson et al., 2002a, b; Filtenborg et al., 2002). Because of this, a question of possible connection among ripening of the fruits, presence of rotting-fungi and their metabolites had been raised. Therefore, the main goal of this pilot study was to explore service tree fruits from the mycological point of view with a special emphasise on characterisation of fungal species encountered as well as their mycotoxin-production potential.
2. Material and methods 2.1. Samples About 600 pieces (each about 4–5 g) of ripped service tree (S. domestica L.) fruits were collected from two locations Pozba and Jablonˇovce in Slovak Republic in October 2003. Twenty-four of them were used for endogenous fungi identification and the rest for identification of fungi in damaged fruits. Freshly collected fruits were immediately used for the analysis. 2.2. Isolation of endogenous fungi from undamaged fruits Each undamaged fruit representing 1 sample (in total 24 samples) was surface-disinfected with
sodium hypochlorite solution (1%) for 1 min and consequently rinsed in sterile water for three times. One half of the fruit was cut by means of a sterile scalpel and four small pieces of the fruit were transferred onto Dichloran Rose Bengal Chloramphenicol agar (DRBC) and Potato Dextrose agar (PDA) with 0.1% Chloramphenicol (Samson et al., 2002a, b). The expression a 100% contamination was used for those samples (fruits) when at least one of the fruit piece per plate showed fungal overgrowth. At the same time, the second half of the fruit was blended in 45 ml of saline (0.87% aqueous solution of NaCl) with horizontal shaker for 30 min. One milliliter of the diluents was then inoculated on both DRBC and PDA media in triplicate. Plates were then incubated at 25 8C for 5 days in the dark. Resulting pure colonies were transferred to appropriate media for species identification. 2.3. Isolation of fungi from damaged fruits A total of 8 samples, i.e. 4+4 samples representing each locality, were investigated. Each sample of 4 fruits (20 g) was blended in 180 ml of saline (0.87% aqueous solution of NaCl) with horizontal shaker for 30 min. The same diluents were used for subsequent serial dilution (up to 10 6). Spread plating of 1.0 ml of the appropriate dilution was carried out in triplicate on the same media as above. Agar with malt extract (MA, Bombay, India) was used here as well. Plates were then incubated at 25 8C for 5 days in the dark (Samson et al., 2002a, b). Resulting pure colonies were transferred to appropriate media for species identification. 2.4. Identification of Penicillium species Identification of Penicillium isolates from Aspergilloides, Furcatum and Biverticillium subgenera to the species level was performed following particular taxonomic schemes given by Pitt (1979). Conidial suspensions were inoculated at three equidistant points both on Czapek-yeast extract agar (CYA) and malt extract agar (MEA), and incubated in the dark at 25 8C. Sub-cultivation on CYA at 37 8C was used as well. In addition, Creatine Sucrose agar (CREA) was used for isolate belonging to P.
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glabrum and Penicillium raistrickii. Penicillium isolates from subgenus Penicillium were inoculated on CYA, MEA, CREA and Yeast Sucrose agar (YES), and incubated at 25 8C in the dark. The final identification was done after 7 days according to Pitt (1979, 2000), Pitt and Hocking (1997) and Samson et al. (2002a,b). 2.5. Identification of Fusarium species Potato Dextrose agar (PDA) was used for observation of colony characteristics and microconidia production only. bSynthetischer n7hrstoffarmer agarQ (SNA) was used for observation of micromorphological features including macroconidia, mono- and polyphialides and chlamydospores. Cultures were incubated at 25 8C in the dark (PDA and SNA) and UV-light 365 nm (SNA). The final identification was done after 7 days of incubation according to Nelson et al. (1983), Nirenberg (1981), Pitt and Hocking (1997) and Samson et al. (2002a,b). 2.6. Identification of Alternaria species Potato Carrot agar (PCA) was used for morphological examination (sporulation pattern and conidia morphology) and incubated under alternating light/ dark cycle consisting of 8 h of diffuse daylight at room temperature (22–25 8C) followed by 16 h darkness at 25 8C. Dichloran Rose Bengal Yeast extract agar (DRYES) was used for observation of growth rate and colour of colonies. The DRYES cultures were incubated at 20 8C for 7 days. Identification of Alternaria species was done according to Andersen et al. (2001), Simmons (1995, 1999) and Simmons and Roberts (1993). 2.7. Identification of other fungi CYA and MEA were used for species identification of all other fungi. As additional media, PDA was used for Epicocum nigrum identification. Cultures were incubated at 25 8C for 7 days. The MEA cultures of Geosmithia putterillii were incubated at 20 8C to initiate characteristic 5–6-mm-long mycelium ropes/funiculi. Identification was done according to Domsch et al. (1980), Klich (2002),
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Pitt and Hocking (1997) and Samson et al. (2002a,b). 2.8. Mycotoxin-screening in vitro An agar plug method according to Frisvad and Thrane (2002) was used to screen appropriate Penicillium species isolates for patulin, citrinin and griseofulvin, Aspergillus niger for ochratoxin A and Fusarium species for T-2 toxin, deoxynivalenol, 3acetyl deoxynivalenol, 15-acetyl deoxynivalenol, fusarenon X, nivalenol and zearalenon production. After 7 and 14 day cultivation on YES (for Penicillium, Aspergillus and Fusarium) and Potato Sucrose agar (PSA, for Fusarium species only), an agar plug was cut from the centre of a colony by means of sterile steel tube (i.d. 4 mm). The plug was then transferred on the TLC plate (Silicagel 60, Merck, Germany) with agar side (Penicillium and Aspergillus mycotoxins) down towards the plate 2.5 cm from the bottom edge and gently pressed. For Fusarium mycotoxins, the mycelium side was used and one or two drops of a chloroform–methanol mixture (2:1) were added to the mycelium prior to application onto TLC plate. After 5 to 10 s, the agar plug was removed and superimposed by another one in the same manner. A volume of 10 Al of patulin, citrinin, griseofulvin, ochratoxin A (Sigma, Germany), T-2 toxin, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, fusarenon X, nivalenol and zearalenon (Biopure, Austria) standards were applied onto the plate. Afterwards, the plates were dried for at least 30 min and consequently allowed to eluate by a chromatographic solvent consisting of toluene/ ethyl acetate/formic acid (5:4:1). After the solvent front reached 1.5 cm from the upper edge, the plate was removed and allowed to dry in the air in a fume hood for 60 min. Subsequently, the plate was placed under UV-light (366 nm) and citrinin was detectable by vivid yellow-green tailed spot and griseofulvin as blue spot. To visualise patulin, the plates were sprayed with 0.5% methylbenzothiazolone hydrochloride (MBTH, Merck, Germany) in water and heated at 130 8C for 8 min. Patulin was detectable as yellow-orange spot. Zearalenon was detectable under UV-light (265 nm) as blue spot. The other Fusarium mycotoxins were sprayed with
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20% AlCl3 in 60% ethanol and heated at 130 8C for 8 min and consequently seen under daylight as blue spots of different retention times.
both (Andersen et al., 2004; Frisvad, personal communication).
2.9. Detection of indole metabolites in P. expansum isolates
3. Results 3.1. Fungal contamination
Intracellular indole metabolites were detected by a filter paper method according to Lund (1995). To carry out the test, an agar plug was cut from the colony centre of 1-week-old cultures of P. expansum growing on CYA and then placed, mycelium side up, on a Petri dish lid. A strip (126 mm) of Whatman No. 1 filter paper was dipped in Ehrlich reagent (4-dimetylaminobenzaldehyde 2 g, 96% ethanol 85 ml, 10 N HCl 15 ml) and placed across the agar plug, with an agar plug in the centre of the strip. The arrangement was then placed in an air stream in a fume hood and allowed to dry. A violet ring in the strip within 10 min indicated a positive response for chaetoglobosins or communesins or
All 24 surface sterilised ripped service tree fruits showed fungal contamination, i.e. 100% contamination. In total, 22 endogenous fungal species were isolated and identified. The most dominant endogenous fungal species was Cladosporium cladosporioides (Fres.) de Vries followed by Alternaria alternata (Fr.) Keissler and P. expansum Link found in 21 (88%), 15 (63%) and 13 (54%) samples of the fruits, respectively. The amounts and frequency of the other fungal endogenous contaminants encountered are shown in Table 1. The most prevalent fungal population of the damaged fruits investigated was that of Penicillium
Table 1 The total frequency and amounts of the endogenous contaminants encountered in surface sterilised service tree fruit samples from Pozba and Jablonˇovce locations Species
Locality
Total Jablonˇovce
Pozba
Alternaria alternata Aspergillus niger Aureobasidium pullulans Cladosporium cladosporioides Cl. herbarum Cl. macrocarpum Clonostachys rosea Epicoccum nigrum Fusarium equiseti F. solani F. reticulatum Geosmithia putterillii Myrothecium roridum M. verrucaria Penicillium brevicompactum P. canescens P. carneum/P. paneum P. expansum P. glabrum P. implicatum P. variabile Phoma glomerata
Frequency
Number of isolates
Frequency
Number of isolates
Frequency
Number of isolates
66 0 17 100 42 17 8 25 33 17 42 8 8 8 25 0 17 83 8 8 8 25
57 0 2 256 120 4 5 6 12 4 20 61 1 1 7 0 19 196 1 1 6 5
58 8 0 75 42 0 0 8 0 0 25 0 0 0 8 8 8 25 0 0 0 8
19 1 1 80 378 0 0 2 0 0 4 0 0 0 2 1 1 14 0 0 0 1
63 8 8 88 42 17 8 17 33 17 33 8 8 8 17 8 13 54 8 8 8 17
76 1 13 336 498 4 5 8 12 4 24 61 1 1 9 1 20 210 1 1 6 6
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carneum/Penicillium paneum recovered from all samples of Pozba location in a huge number of more than 500106 CFU/g of sample followed by P. expansum and P. crustosum but in substantially lesser amounts of approximately 150–180 and 10– 25106 CFU/g of sample, respectively. In Jablonˇovce location, the most dominant population was represented by C. cladosporioides and Cladosporium herbarum of about 500 and 200106 CFU/g of sample, respectively. The amounts and frequency of the fungi recovered from damaged fruits are shown in Table 2.
isolates (n=6) also exhibited patulin production. In the P. griseofulvum isolates, griseofulvin production was detected as well. Two isolates of P. raistrickii and a single one of two P. canescens isolates produced griseofulvin. Five isolates of A. niger were screened for ochratoxin A-production but with negative results. Of the Fusarium toxins, only production of zearalenon was detected in four of the five Fusarium equiseti isolates. The other Fusarium species isolates tested did not produce any of the screened mycotoxins.
3.2. Mycotoxin potential of the Penicillium, Aspergillus and Fusarium species
4. Discussion 4.1. Mycobiota
All P. expansum isolates (n=40) produced patulin and citrinin. In addition, a positive reaction with Ehrlich reagent indicating a presence of the indole metabolites was observed in all P. expansum isolates tested. All P. paneum (n=35) and P. griseofulvum
Table 2 The total frequency and amounts of the isolated fungi encountered in damaged service tree fruits samples after 5 days of incubation and dilution 10 6 from Pozba and Jablonˇovce localities Species
Locality Jablonˇovce
Pozba
Frequency CFU/g of Frequency CFU/g of sample sample Alternaria alternata Aspergillus niger Cladosporium cladosporioides Cl. herbarum Epicoccum nigrum Fusarium sporotrichioides F. solani Geotrichum candidum Penicillium brevicompactum P. carneum/ P. paneum P. crustosum P. expansum P. griseofulvum P. raistrickii
25 50 25
5–20 1–4 300–350
100 nd 100
10–25 nd 450–500
nd nd 50
nd nd 5–15
50 25 nd
150–200 3–4 nd
50 25
5–8 1–3
25 nd
8–10 nd
75
3–8
25
2–5
100
500–550
25
1–3
75 75 nd 50
10–25 150–180 nd 3–7
50 50 25 nd
4–8 80–120 10–12 nd
nd—cfu not detected in all dilutions used.
C. cladosporioides, A. alternata and P. expansum were the most frequently isolated fungal species encountered in surface sterilised service tree fruits from both locations investigated. C. herbarum belonged also to rather frequently occurred species with the similar 42% frequency in the both locality and at the same time represented the most numerous fungal population found in the fruits from Jablonˇovce location. Less frequently isolated species encountered simultaneously in the samples from both localities were Aureobasidium pullulans, E. nigrum, Fusarium reticulatum, Penicillium brevicompactum, P. carneum/P. paneum and Phoma glomerata. Some species were isolated exceptionally only from one sample or only from some of the localities investigated. For instance, G. putterillii isolates were encountered in a single sample from Pozba locality, but in comparatively high amounts of 61 isolates. In agreement with Domsch et al. (1980), Pitt and Hocking (1997) and Samson et al. (2002a,b), a majority of fungal species isolated during the study have been previously reported from diverse plants including fruits or vegetables, either as primary or secondary invaders. Within the genus Penicillium, P. expansum, P. crustosum, P. solitum and P. brevicompactum are known as typical representatives of apple brown rot causing fungi (Pitt and Hocking, 1997). Two of them P. expansum and P. brevicompactum were encountered here as endogenous contaminants. Recently, Labuda et al. (2004) isolated a P. implicatum population causing a destruc-
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tive rot of pomegranate (Punica granata) fruits, and which had even been able to evoke a brown rot in some apple varieties after artificial infection of the fruits. However, only a single isolate of this species
was recovered here. The findings of P. carneum (or related P. paneum) in the service tree fruits is a newly revealed source of the mentioned species because rye bread and/or silage are the only known
Table 3 Fungi associated with fruits of service tree (Sorbus domestica L.) and their secondary metabolite profile (Frisvad and Filtenborg, 1989; Frisvad and Thrane, 2002; Pitt, 2000; Pitt and Hocking, 1997; Samson et al., 2002a,b) Species
Isolated from the fruits Undamaged
Damaged
Alternaria alternata (Fr.) Keissler
+
+
Aspergillus niger Van Tieghem Aureobasidium pullulans (De Bary) Arnaud Cladosporium cladosporioides (Fres.) de Vries Cl. herbarum (Pers.) Link Cl. macrocarpum Clonostachys rosea (Link: Fr.) Schroers et al. Epicoccum nigrum Link Fusarium equiseti (Corda) Sacc.
+ +
+
+
+
+ + +
+
+ +
+ +
F. sporotrichioides Sherb.
F. solani (Mart.) Appel et Wollenw. F. reticulatum Mont. Geosmithia putterillii (Thom) Pitt Geotrichum candidum Link Myrothecium roridum Tode ex Fries M. verrucaria (Albertini et Schweinitz: Fr.) Ditmar Penicillium brevicompactum Dierckx
+
+ + +
+
+ + + +
+
P. canescens Biourge P. carneum (Frisvad) Frisvad/P. paneum Frisvad P. crustosum Thom
+ +
+
P. expansum Link
+
P. glabrum (Wehmer) Westling P. griseofulvum Dierckx P. implicatum Biourge P. raistrickii G. Sm P. variabile Sopp Phoma glomerata (Corda) Wollenweber et Hochapfel
+
+ +
+ + + + +
Potential secondary metabolite production altenuen, alternariol, alternariol monomethyl eter, altertoxina naphto-4-pyrones, malformins, ochratoxin Ab nr N-methylcarbamatec, cladosporin and isocladosporind, calphostin Ce herbarin A and B, herbaric acid, citreoviridin Ab,f nr roselipinsg, glisopreninsh, glioroseini,j epicorazines A and Bk,l, rhodoxanthinm culmorin, enniatin, equisetin, chlamydosporol, chrysogine, fusarochromanoneb, trichothecenes Ab (diacetoxyscipenol, T-2, HT-2-toxin, and neosolaniol) and Bb (deoxynivalenol, 15-acetyl deoxynivalenol, fusarenone X and nivalenol), zearalenonb butenolide, fusarin C, trichothecenes Ab (HT-2 toxin, neosolaniol and diacetoxyscirpenol) and Bb ( deoxynivalenol, 15-deoxynivalenol, fusarenone X and nivalenol), zearalenoneb fusaric acid, naphthoquinone pigments nr nr nr hydroxymytoxin B and hydroxyroridin Eb,n, 12,13-deoxyroridin Eb,o trichoverroidsb,p, roridins and verrucarinq brevianamid A and B, botryodiploidinb, mycophenolic acid, Raistrick phenols griseofulvinb, penitrem Ab roquefortine Cb, patulinb, botryodiploidinb, penitrem Ab, isofumigaclavicine A and Bb, penicillic acidb roquefortine Cb, penitrem Ab, terrestric acid, cyclopenin, cyclopenol roquefortine Cb, patulinb, citrininb, chaetoglobosin A, B and Cb,r, communesins citromycetin roquefortin C,b cyclopiazonic acidb, patulinb, griseofulvinb nr griseofulvinb rugulosinb nr
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habitats from which these species have been reported (Samson et al., 2002a, b). Likewise, little is known about distribution and occurrence of F. reticulatum, although Nelson et al. (1983) regarded this species as cosmopolitan. Under the name F. reticulatum var. reticulatum, it has been reported from a bark lesion of Sophora japonica and from a root lesion of Paspalum dilatatum, under F. reticulatum form 1 from Beta vulgaris and timber pulp and under F. reticulatum var. negundinis as the causal agent of red stain of Acer negundo (Gerlach and Nirenberg, 1982). In the mycobiota composition recovered from the damaged fruit samples, P. carneum/P. paneum was dominating population in Pozba location with a huge number of isolates exceeding 50010 6 CFU/sample, whereas in the samples from Jablonˇovce C. cladosporioides was found to be the dominant one with the similar number of isolates. Of the species found here, Fusarium sporotrichioides, Geotrichum candidum, Penicillium crustosum, P. griseofulvum and P. raistrickii represented the fungal population isolated exclusively from the damaged fruit samples. Whereas, a more numerous group of species consisting of A. pullulans, Cladosporium macrocarpum, Clonostachys rosea, F. reticulatum, G. putterillii, Myrrothecium roridum, M. verrucaria, Penicillium canescens, P. glabrum, P. implicatum, P. variabile and P. glomerata was recovered only from surface sterilised fruits.
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With the exception of P. raistrickii, all species isolated and identified during this study fit with their descriptions given in appropriate identification keys. Population of this species was relatively different from its description by Pitt (1979, 2000) or Pitt and Hocking (1997). Isolates of P. raistrickii exhibited strong grey-green sporulation on both CYA and MEA without sclerotia formation. Nevertheless, based on its micromorphological (penicillus and conidia morphology) and biochemical (griseofulvin production and strong acid production on CREA) features it represents non-sclerotinogenic, well sporulating variant of the species. As to isolates placed to P. paneum or related P. carneum, their separation from the very close species P. roqueforti Thom was done primarily on colouration of colonies growing on CYA and YES and by the ability to produce patulin. The discrimination between P. carneum and P. paneum was not done in this study, because based on the traditional morphological and cultural characteristics they are very difficult or even impossible to separate (Boysen et al., 1996; Pitt, 2000; Samson et al., 2000). Placing of Alternaria isolates to A. alternata was done primarily on account of their sporulating pattern matching well with group 4 (Simmons and Roberts, 1993). Neither constriction nor dark septa in conidia were observed. Furthermore, a size of conidia beak did not exceed one third of conidial length of the Alternaria isolates treated here.
Notes to Table 3: +: present; : absent; nr: no report available. a Andersen et al. (2001). b Mycotoxins in the true sense; bold: mycotoxins detected during the study. c Suzuki and Takeda (1976). d Jacyno et al. (1993). e Kobayashi et al. (1989). f Jadulco et al. (2002). g Tabata et al. (1999). h Sterner et al. (1998). i Steward and Packter (1965). j Packter and Steward (1967). k Baute et al. (1978). l Deffieux et al. (1978). m Foppen (1969). n Alvi et al. (2002). o Namikoshi et al. (2001). p Amagata et al. (2003). q Murakami et al. (2001). r Andersen et al. (2004).
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4.2. Mycotoxin and other secondary metaboliteproduction potential As indicated in Table 3, it is obvious that the potential of the detected fungi to produce various kind of secondary metabolites including mycotoxins even into the fruits might be expected. This is supported by the ability of appropriate species isolates to make detectable amounts of patulin, citrinin, the indole metabolites griseofulvin and zearalenon under laboratory conditions. For example, patulin and citrinin co-occurrence in the pomaceous and stone fruits have been frequently observed (Filtenborg et al., 2002). In fact, P. expansum is considered as the most important patulin producer in apples (Frisvad and Thrane, 2002). Co-incidence patulin producers, namely P. expansum, P. carneum ( P. paneum) and P. griseofulvum, as it is in this case, may lead to high contamination of the fruits with the toxic metabolites. Amongst the other toxicologically significant mycotoxin producers undoubtedly belong P. crustosum and P. canescens, as potential producers of powerful neurotoxin penitrem A (Frisvad and Filtenborg, 1989; Pitt, 2000; Pitt and Hocking, 1997). In addition, F. equiseti isolated as endogenous contaminant should be take into consideration as a significant finding because of its potency to produce A and B trichothecens and zearalenon. However, only zearalenon production by the F. equiseti isolates was detected here. Apart from the possible mycotoxin production ability of the fungal species encountered in the fruits, pharmacologically significant secondary metabolites should be mentioned such as antibiotic epicorazins (Deffieux et al., 1978) or antitumoral roridins and verrucarins (Murakami et al., 2001) production by E. nigrum and Myrothecium verrucaria, respectively. Furthermore, numerous mycotoxins have shown their antibiotic properties against diverse bacteria as well as fungi (Betina, 1989). In conclusion, service tree fruits could be a source of significant fungal secondary metabolites including mycotoxins as it is indicated by the presence of specific fungal species. Nevertheless, more studies should be done to evaluate this fruit from the mycotoxicological point of view for further exploitation in human diet.
Acknowledgement The authors wish to express their gratitude to Dr. A. Kuba´tova´ for help in Myrothecium spp. identification, and Dr. M. Lemmens for help in Fusarium reticulatum and F. equiseti identification and for presubmission review of the manuscript. We are also grateful to the company BIOPURE, Referenzsubstanzen (Austria) for providing of Fusarium-mycotoxin standards as well as to Department of Plant Genetics and Plant Breeding, Slovak University of Agriculture for providing the fruit samples. This study was supported by Grant Committee of Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Slovak Republic. References Alvi, K.A., Rabenstein, J., Woodard, J., Baker, D.D., Bergthold, J.D., Lynch, J., Lieu, K.L., Braude, I.A., 2002. 14V-Hydroxymytoxin B and 16-hydroxyroridin E, two new cytotoxic trichothecenes from Myrothecium roridum. Journal of Natural Products 65, 742 – 744. Amagata, T., Rath, C., Rigot, J.F., Tarlov, N., Tenney, K., Valeriote, F.A., Crews, P., 2003. Structures and cytotoxic properties of trichoverroids and their macrolide analogues produced by saltwater culture of Myrothecium verrucaria. Journal of Medical Chemistry 46, 4342 – 4350. Andersen, B., Kroger, E., Roberts, R.G., 2001. Chemical and morphological segregation of Alternaria alternata, A. gaisen and A. longipes. Mycological Research 105, 291 – 299. Andersen, B., Smedsgaard, J., Frisvad, J.C., 2004. Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products. Journal of Agricultural and Food Chemistry 52, 2421 – 2428. Baute, M.A., Deffieux, G., Baute, R., Neveu, A., 1978. New antibiotics from the fungus Epicoccum nigrum: I. Fermentation, isolation and antibacterial properties. Journal of Antibiotics 31, 1099 – 1101. Betina, V., 1989. Mycotoxins: Chemical, Biological, and Environmental Aspects. Alfa, Bratislava. (in Slovak). Boysen, M., Skouboe, P., Frisvad, J.C., Rossen, L., 1996. Reclassification of the Penicillium roqueforti into three species on the basis of molecular genetic and biochemical profiles. Microbiology 142, 541 – 549. Deffieux, G., Filleau, M.J., Baute, R., 1978. New antibiotics from the fungus Epicoccum nigrum: III. Epicorazine B: structure elucidation and absolute configuration. Journal of Antibiotics 31, 1106 – 1109. Domsch, K.H., Gams, W., Anderson, T.H., 1980. Compendium of Soil Fungi. Academic Press, London.
R. Labuda et al. / International Journal of Food Microbiology 99 (2005) 215–223 Filtenborg, O., Frisvad, J.C., Samson, R.A., 2002. Specific association of fungi to foods and influence of physical environmental factors. In: Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O. (Eds.), Introduction to Food- and Airborne Fungi. Centraalbureau voor Schimmecultures, Utrecht, pp. 306 – 320. Foppen, F.H., 1969. On some pigments of Epicoccum nigrum Link: their isolation and structure elucidation and a biosynthetic pathway of rhodoxanthin. Annali dell’Istituto superiore di sanita 5, 439 – 513. Frisvad, J.I., Filtenborg, O., 1989. Terverticillate penicillia: chemotaxonomy and mycotoxin production. Mycologia 81, 837 – 861. Frisvad, J.C., Thrane, U., 2002. Mycotoxin production by common filamentous fungi. In: Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O. (Eds.), Introduction to Food- and Airborne Fungi. Centraalbureau voor Schimmecultures, Utrecht, pp. 321 – 330. Gerlach, W., Nirenberg, H., 1982. The Genus Fusarium, A Pictorial Atlas. Mitteilungen aus der Biologischen Bundesanstalt fuer Land- und Forstwissenschaftliche, Berlin-Dahlem, 206 – 208. Jacyno, J.M., Harwood, J.S., Cutler, H.G., Lee, M.K., 1993. Isocladosporin, a biologically active isomer of cladosporin from Cladosporium cladosporioides. Journal of Natural Products 56, 1397 – 1401. Jadulco, R., Brauers, G., Edrada, R.A., Ebel, R., Wray, V., Sudarsono, S., Proksch, P., 2002. New metabolites from sponge-derived fungi Curvularia lunata and Cladosporium herbarum. Journal of Natural Products 65, 730 – 733. Klich, M.A., 2002. Identification of Common Aspergillus Species. Centraalbureau voor Schimmelcultures, Utrecht. Kobayashi, E., Nakano, H., Morimoto, M., Tamaoki, T., 1989. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications 159, 548 – 553. Labuda, R., Hudec, K., Pieckova´, E., Mezey, J., Bohovic, R., Ma´te´ova´, S., Luka´cˇ, N., 2004. Penicillium implicatum causes a destructive rot in pomegranate fruits. Mycopathologia 157, 217 – 223. Lund, F., 1995. Differentiating penicillium species by detection of indole metabolites using a filter paper method. Letters in Applied Microbiology 20, 228 – 231. Murakami, Y., Okuda, T., Shindo, K., Roridin, L.M., verrucarin, M., 2001. New macrocyclic trichothecene group antitumor antibiotics, from Myrothecium verrucaria. Journal of Antibiotics 54, 980 – 983. Namikoshi, M., Akano, K., Meguro, S., Kasuga, I., Mine, Y., Takahashi, T., Kobayashi, H., 2001. A new macrocyclic trichothecene, 12,13-deoxyroridin E, produced by the marinederived fungus Myrothecium roridum collected in Palau. Journal of Natural Products 64, 396 – 398.
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Nelson, P.E., Tousson, T.A., Marasas, W.F.O., 1983. An Illustrated Manual for Identification of Fusarium Species. Pennsylvania State University Press. Nirenberg, H.I., 1981. A simplified method for identifying Fusarium spp. occurring in wheat. Canadian Journal of Botany 59, 1599 – 1609. Packter, N.M., Steward, M.W., 1967. Studies on the biosynthesis of phenols in fungi. Biosynthesis of 3,4-dimethoxy-6-methyltoluquinol and gliorosein in Gliocladium roseum I.M.I. 93 065. Biochemical Journal 102, 122 – 132. Pagan, J., Paganova´, V., 2000. Variability of service tree (Sorbus domestica L.) in Slovakia. Acta Facultatis Forestalis 42, 51 – 57. (Article in Slovak). Pitt, J.I., 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. Academic Press, London. Pitt, J.I., 2000. A Laboratory Guide to Common Penicillium Species, 3rd edn. Food Science Australia, North Ryde, N.S.W. Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage, 2nd edn. Blacklie Academic & Professional, London. Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O. (Eds.), 2002a. Introduction to Food- and Airborne Fungi. Centraalbureau voor Schimmecultures, Utrecht. Samson, R.A., Hoekstra, E.S., Lund, F., Filtenborg, O., Frisvad, J.C., 2002b. Method for the detection, isolation and characterisation of food-borne fungi. In: Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O. (Eds.), Introduction to Food- and Airborne Fungi. Centraalbureau voor Schimmecultures, Utrecht, pp. 283 – 297. Simmons, E.G., 1995. Alternaria themes and variations (112–144). Mycotaxon 55, 55 – 163. Simmons, E.G., 1999. Alternaria themes and variations (236–243). Mycotaxon 70, 325 – 369. Simmons, E.G., Roberts, R.G., 1993. Alternaria themes and variations (73). Mycotaxon 48, 109 – 140. Sterner, O., Thines, E., Eilbert, F., Anke, H., 1998. Glisoprenins C, D and E, new inhibitors of appressorium formation in Magnaporthe grisea, from cultures of Gliocladium roseum: 2. Structure determination. Journal of Antibiotics 51, 228 – 231. Steward, M.W., Packter, N.M., 1965. Biosynthesis of gliorosein in Gliocladium roseum I.M.I. 93065. Biochemical Journal 95, 26 – 28. Suzuki, T., Takeda, M., 1976. Microbial metabolism of Nmethylcarbamate insecticide: IV. 1 New metabolites of o-secbutylphenyl N-methylcarbamate by Cladosporium cladosporioides. Chemical and Pharmaceutical Bulletin 24, 1988 – 1991. Tabata, N., Ohyama, Y., Tomoda, H., Abe, T., Namikoshi, M., Omura, S., 1999. Structure elucidation of roselipins, inhibitors of diacylglycerol acyltransferase produced by Gliocladium roseum KF-1040. Journal of Antibiotics 52, 815 – 826. Velgosˇova´, M., Velgosˇ, S., 1988. Our Medicinal Plants (In Slovak). Slovakian Pedagogical Publishing-House, Bratislava.
Mycological survey of ripped service tree fruits (Sorbus domestica L.) with an emphasis on toxinogenic fungi Roman Labudaa,*, Ladislav Kriva´nek Lb, Dana Tancˇinova´a, Silvia Ma´te´ova´a, Sonˇa Hrubcova´a a
Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic b Department of Animal Nutrition, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic Received 22 December 2003; received in revised form 9 July 2004; accepted 17 September 2004
Abstract To investigate a possible incidence of microscopic fungi in ripped service tree (Sorbus domestica L.) fruits, a mycological survey was carried out during October–November 2003 in Slovakia. This rare kind of fruit is renowned for its significant curative actions in various human and animal diseases. The study revealed that all 24 surface sterilized fruits analysed were contaminated with fungi. The most dominant endogenous contaminant encountered was Cladosporium cladosporioides (Fres.) de Vries followed by Alternaria alternata (Fr.) Keissler and Penicillium expansum Link with 88%, 63% and 54% frequency, respectively. Furthermore, 24 other fungal species were associated with the fruits as well. P. expansum (40), P. carneum (Frisvad) Frisvad/Penicillium paneum Frisvad (35) and P. griseofulvum Dierckx (6) isolates recovered from the fruits were screened by an agar plug method for production of mycotoxin patulin, all with positive results. In addition, citrinin, griseofulvin and zearalenon production by appropriate species isolates were detected. Despite of a limited number of samples of the service tree fruits examined during this study, it was concluded that the ripening of service tree fruits is accompanied with the presence of typical rotting-fungi as well as of some others, which have not previously been reported in connection with fruit. Furthermore, it was suggested that the ripped service tree fruits should be considered as a potential source of significant fungal secondary metabolites including mycotoxins. Secondary metabolite profiles of the species identified during the study were included here. D 2004 Elsevier B.V. All rights reserved. Keywords: Endogenous contaminants; Fusarium spp.; Mycotoxins; Patulin; Penicillium spp.
* Corresponding author. E-mail address: [email protected] (R. Labuda). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.09.002
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1. Introduction Small-sized apple- to pear-like fruits producing service tree (Sorbus domestica L., Rosaceae) represents a rare kind of fruit with only a limited state of domestication and distribution (Pagan and Paganova´, 2000). Aside from its wood of high quality, the service tree is also well known for the production of fruits with significant curative actions used in folk-medicine on various intestinal diseases, anaemia and emaciation. An extract of the fruits is used on haemorrhage cessation and also as diuretic agent at lithonephritis (Velgosˇova´ and Velgosˇ, 1988; Pagan and Paganova´, 2000). Interestingly, suitability of the fruits for direct consumption is reached when the peel becomes brown and the flesh gets a yellowish to brownish tint, so that the fruits are completely over-ripped or rotted. There is a similarity in the appearance of these ripped service tree fruits with other kind of pomaceous and/or stone fruits following fungal contamination resulting in a rot such as that of Penicillium expansum (Samson et al., 2002a, b; Filtenborg et al., 2002). Because of this, a question of possible connection among ripening of the fruits, presence of rotting-fungi and their metabolites had been raised. Therefore, the main goal of this pilot study was to explore service tree fruits from the mycological point of view with a special emphasise on characterisation of fungal species encountered as well as their mycotoxin-production potential.
2. Material and methods 2.1. Samples About 600 pieces (each about 4–5 g) of ripped service tree (S. domestica L.) fruits were collected from two locations Pozba and Jablonˇovce in Slovak Republic in October 2003. Twenty-four of them were used for endogenous fungi identification and the rest for identification of fungi in damaged fruits. Freshly collected fruits were immediately used for the analysis. 2.2. Isolation of endogenous fungi from undamaged fruits Each undamaged fruit representing 1 sample (in total 24 samples) was surface-disinfected with
sodium hypochlorite solution (1%) for 1 min and consequently rinsed in sterile water for three times. One half of the fruit was cut by means of a sterile scalpel and four small pieces of the fruit were transferred onto Dichloran Rose Bengal Chloramphenicol agar (DRBC) and Potato Dextrose agar (PDA) with 0.1% Chloramphenicol (Samson et al., 2002a, b). The expression a 100% contamination was used for those samples (fruits) when at least one of the fruit piece per plate showed fungal overgrowth. At the same time, the second half of the fruit was blended in 45 ml of saline (0.87% aqueous solution of NaCl) with horizontal shaker for 30 min. One milliliter of the diluents was then inoculated on both DRBC and PDA media in triplicate. Plates were then incubated at 25 8C for 5 days in the dark. Resulting pure colonies were transferred to appropriate media for species identification. 2.3. Isolation of fungi from damaged fruits A total of 8 samples, i.e. 4+4 samples representing each locality, were investigated. Each sample of 4 fruits (20 g) was blended in 180 ml of saline (0.87% aqueous solution of NaCl) with horizontal shaker for 30 min. The same diluents were used for subsequent serial dilution (up to 10 6). Spread plating of 1.0 ml of the appropriate dilution was carried out in triplicate on the same media as above. Agar with malt extract (MA, Bombay, India) was used here as well. Plates were then incubated at 25 8C for 5 days in the dark (Samson et al., 2002a, b). Resulting pure colonies were transferred to appropriate media for species identification. 2.4. Identification of Penicillium species Identification of Penicillium isolates from Aspergilloides, Furcatum and Biverticillium subgenera to the species level was performed following particular taxonomic schemes given by Pitt (1979). Conidial suspensions were inoculated at three equidistant points both on Czapek-yeast extract agar (CYA) and malt extract agar (MEA), and incubated in the dark at 25 8C. Sub-cultivation on CYA at 37 8C was used as well. In addition, Creatine Sucrose agar (CREA) was used for isolate belonging to P.
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glabrum and Penicillium raistrickii. Penicillium isolates from subgenus Penicillium were inoculated on CYA, MEA, CREA and Yeast Sucrose agar (YES), and incubated at 25 8C in the dark. The final identification was done after 7 days according to Pitt (1979, 2000), Pitt and Hocking (1997) and Samson et al. (2002a,b). 2.5. Identification of Fusarium species Potato Dextrose agar (PDA) was used for observation of colony characteristics and microconidia production only. bSynthetischer n7hrstoffarmer agarQ (SNA) was used for observation of micromorphological features including macroconidia, mono- and polyphialides and chlamydospores. Cultures were incubated at 25 8C in the dark (PDA and SNA) and UV-light 365 nm (SNA). The final identification was done after 7 days of incubation according to Nelson et al. (1983), Nirenberg (1981), Pitt and Hocking (1997) and Samson et al. (2002a,b). 2.6. Identification of Alternaria species Potato Carrot agar (PCA) was used for morphological examination (sporulation pattern and conidia morphology) and incubated under alternating light/ dark cycle consisting of 8 h of diffuse daylight at room temperature (22–25 8C) followed by 16 h darkness at 25 8C. Dichloran Rose Bengal Yeast extract agar (DRYES) was used for observation of growth rate and colour of colonies. The DRYES cultures were incubated at 20 8C for 7 days. Identification of Alternaria species was done according to Andersen et al. (2001), Simmons (1995, 1999) and Simmons and Roberts (1993). 2.7. Identification of other fungi CYA and MEA were used for species identification of all other fungi. As additional media, PDA was used for Epicocum nigrum identification. Cultures were incubated at 25 8C for 7 days. The MEA cultures of Geosmithia putterillii were incubated at 20 8C to initiate characteristic 5–6-mm-long mycelium ropes/funiculi. Identification was done according to Domsch et al. (1980), Klich (2002),
217
Pitt and Hocking (1997) and Samson et al. (2002a,b). 2.8. Mycotoxin-screening in vitro An agar plug method according to Frisvad and Thrane (2002) was used to screen appropriate Penicillium species isolates for patulin, citrinin and griseofulvin, Aspergillus niger for ochratoxin A and Fusarium species for T-2 toxin, deoxynivalenol, 3acetyl deoxynivalenol, 15-acetyl deoxynivalenol, fusarenon X, nivalenol and zearalenon production. After 7 and 14 day cultivation on YES (for Penicillium, Aspergillus and Fusarium) and Potato Sucrose agar (PSA, for Fusarium species only), an agar plug was cut from the centre of a colony by means of sterile steel tube (i.d. 4 mm). The plug was then transferred on the TLC plate (Silicagel 60, Merck, Germany) with agar side (Penicillium and Aspergillus mycotoxins) down towards the plate 2.5 cm from the bottom edge and gently pressed. For Fusarium mycotoxins, the mycelium side was used and one or two drops of a chloroform–methanol mixture (2:1) were added to the mycelium prior to application onto TLC plate. After 5 to 10 s, the agar plug was removed and superimposed by another one in the same manner. A volume of 10 Al of patulin, citrinin, griseofulvin, ochratoxin A (Sigma, Germany), T-2 toxin, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, fusarenon X, nivalenol and zearalenon (Biopure, Austria) standards were applied onto the plate. Afterwards, the plates were dried for at least 30 min and consequently allowed to eluate by a chromatographic solvent consisting of toluene/ ethyl acetate/formic acid (5:4:1). After the solvent front reached 1.5 cm from the upper edge, the plate was removed and allowed to dry in the air in a fume hood for 60 min. Subsequently, the plate was placed under UV-light (366 nm) and citrinin was detectable by vivid yellow-green tailed spot and griseofulvin as blue spot. To visualise patulin, the plates were sprayed with 0.5% methylbenzothiazolone hydrochloride (MBTH, Merck, Germany) in water and heated at 130 8C for 8 min. Patulin was detectable as yellow-orange spot. Zearalenon was detectable under UV-light (265 nm) as blue spot. The other Fusarium mycotoxins were sprayed with
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20% AlCl3 in 60% ethanol and heated at 130 8C for 8 min and consequently seen under daylight as blue spots of different retention times.
both (Andersen et al., 2004; Frisvad, personal communication).
2.9. Detection of indole metabolites in P. expansum isolates
3. Results 3.1. Fungal contamination
Intracellular indole metabolites were detected by a filter paper method according to Lund (1995). To carry out the test, an agar plug was cut from the colony centre of 1-week-old cultures of P. expansum growing on CYA and then placed, mycelium side up, on a Petri dish lid. A strip (126 mm) of Whatman No. 1 filter paper was dipped in Ehrlich reagent (4-dimetylaminobenzaldehyde 2 g, 96% ethanol 85 ml, 10 N HCl 15 ml) and placed across the agar plug, with an agar plug in the centre of the strip. The arrangement was then placed in an air stream in a fume hood and allowed to dry. A violet ring in the strip within 10 min indicated a positive response for chaetoglobosins or communesins or
All 24 surface sterilised ripped service tree fruits showed fungal contamination, i.e. 100% contamination. In total, 22 endogenous fungal species were isolated and identified. The most dominant endogenous fungal species was Cladosporium cladosporioides (Fres.) de Vries followed by Alternaria alternata (Fr.) Keissler and P. expansum Link found in 21 (88%), 15 (63%) and 13 (54%) samples of the fruits, respectively. The amounts and frequency of the other fungal endogenous contaminants encountered are shown in Table 1. The most prevalent fungal population of the damaged fruits investigated was that of Penicillium
Table 1 The total frequency and amounts of the endogenous contaminants encountered in surface sterilised service tree fruit samples from Pozba and Jablonˇovce locations Species
Locality
Total Jablonˇovce
Pozba
Alternaria alternata Aspergillus niger Aureobasidium pullulans Cladosporium cladosporioides Cl. herbarum Cl. macrocarpum Clonostachys rosea Epicoccum nigrum Fusarium equiseti F. solani F. reticulatum Geosmithia putterillii Myrothecium roridum M. verrucaria Penicillium brevicompactum P. canescens P. carneum/P. paneum P. expansum P. glabrum P. implicatum P. variabile Phoma glomerata
Frequency
Number of isolates
Frequency
Number of isolates
Frequency
Number of isolates
66 0 17 100 42 17 8 25 33 17 42 8 8 8 25 0 17 83 8 8 8 25
57 0 2 256 120 4 5 6 12 4 20 61 1 1 7 0 19 196 1 1 6 5
58 8 0 75 42 0 0 8 0 0 25 0 0 0 8 8 8 25 0 0 0 8
19 1 1 80 378 0 0 2 0 0 4 0 0 0 2 1 1 14 0 0 0 1
63 8 8 88 42 17 8 17 33 17 33 8 8 8 17 8 13 54 8 8 8 17
76 1 13 336 498 4 5 8 12 4 24 61 1 1 9 1 20 210 1 1 6 6
R. Labuda et al. / International Journal of Food Microbiology 99 (2005) 215–223
219
carneum/Penicillium paneum recovered from all samples of Pozba location in a huge number of more than 500106 CFU/g of sample followed by P. expansum and P. crustosum but in substantially lesser amounts of approximately 150–180 and 10– 25106 CFU/g of sample, respectively. In Jablonˇovce location, the most dominant population was represented by C. cladosporioides and Cladosporium herbarum of about 500 and 200106 CFU/g of sample, respectively. The amounts and frequency of the fungi recovered from damaged fruits are shown in Table 2.
isolates (n=6) also exhibited patulin production. In the P. griseofulvum isolates, griseofulvin production was detected as well. Two isolates of P. raistrickii and a single one of two P. canescens isolates produced griseofulvin. Five isolates of A. niger were screened for ochratoxin A-production but with negative results. Of the Fusarium toxins, only production of zearalenon was detected in four of the five Fusarium equiseti isolates. The other Fusarium species isolates tested did not produce any of the screened mycotoxins.
3.2. Mycotoxin potential of the Penicillium, Aspergillus and Fusarium species
4. Discussion 4.1. Mycobiota
All P. expansum isolates (n=40) produced patulin and citrinin. In addition, a positive reaction with Ehrlich reagent indicating a presence of the indole metabolites was observed in all P. expansum isolates tested. All P. paneum (n=35) and P. griseofulvum
Table 2 The total frequency and amounts of the isolated fungi encountered in damaged service tree fruits samples after 5 days of incubation and dilution 10 6 from Pozba and Jablonˇovce localities Species
Locality Jablonˇovce
Pozba
Frequency CFU/g of Frequency CFU/g of sample sample Alternaria alternata Aspergillus niger Cladosporium cladosporioides Cl. herbarum Epicoccum nigrum Fusarium sporotrichioides F. solani Geotrichum candidum Penicillium brevicompactum P. carneum/ P. paneum P. crustosum P. expansum P. griseofulvum P. raistrickii
25 50 25
5–20 1–4 300–350
100 nd 100
10–25 nd 450–500
nd nd 50
nd nd 5–15
50 25 nd
150–200 3–4 nd
50 25
5–8 1–3
25 nd
8–10 nd
75
3–8
25
2–5
100
500–550
25
1–3
75 75 nd 50
10–25 150–180 nd 3–7
50 50 25 nd
4–8 80–120 10–12 nd
nd—cfu not detected in all dilutions used.
C. cladosporioides, A. alternata and P. expansum were the most frequently isolated fungal species encountered in surface sterilised service tree fruits from both locations investigated. C. herbarum belonged also to rather frequently occurred species with the similar 42% frequency in the both locality and at the same time represented the most numerous fungal population found in the fruits from Jablonˇovce location. Less frequently isolated species encountered simultaneously in the samples from both localities were Aureobasidium pullulans, E. nigrum, Fusarium reticulatum, Penicillium brevicompactum, P. carneum/P. paneum and Phoma glomerata. Some species were isolated exceptionally only from one sample or only from some of the localities investigated. For instance, G. putterillii isolates were encountered in a single sample from Pozba locality, but in comparatively high amounts of 61 isolates. In agreement with Domsch et al. (1980), Pitt and Hocking (1997) and Samson et al. (2002a,b), a majority of fungal species isolated during the study have been previously reported from diverse plants including fruits or vegetables, either as primary or secondary invaders. Within the genus Penicillium, P. expansum, P. crustosum, P. solitum and P. brevicompactum are known as typical representatives of apple brown rot causing fungi (Pitt and Hocking, 1997). Two of them P. expansum and P. brevicompactum were encountered here as endogenous contaminants. Recently, Labuda et al. (2004) isolated a P. implicatum population causing a destruc-
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tive rot of pomegranate (Punica granata) fruits, and which had even been able to evoke a brown rot in some apple varieties after artificial infection of the fruits. However, only a single isolate of this species
was recovered here. The findings of P. carneum (or related P. paneum) in the service tree fruits is a newly revealed source of the mentioned species because rye bread and/or silage are the only known
Table 3 Fungi associated with fruits of service tree (Sorbus domestica L.) and their secondary metabolite profile (Frisvad and Filtenborg, 1989; Frisvad and Thrane, 2002; Pitt, 2000; Pitt and Hocking, 1997; Samson et al., 2002a,b) Species
Isolated from the fruits Undamaged
Damaged
Alternaria alternata (Fr.) Keissler
+
+
Aspergillus niger Van Tieghem Aureobasidium pullulans (De Bary) Arnaud Cladosporium cladosporioides (Fres.) de Vries Cl. herbarum (Pers.) Link Cl. macrocarpum Clonostachys rosea (Link: Fr.) Schroers et al. Epicoccum nigrum Link Fusarium equiseti (Corda) Sacc.
+ +
+
+
+
+ + +
+
+ +
+ +
F. sporotrichioides Sherb.
F. solani (Mart.) Appel et Wollenw. F. reticulatum Mont. Geosmithia putterillii (Thom) Pitt Geotrichum candidum Link Myrothecium roridum Tode ex Fries M. verrucaria (Albertini et Schweinitz: Fr.) Ditmar Penicillium brevicompactum Dierckx
+
+ + +
+
+ + + +
+
P. canescens Biourge P. carneum (Frisvad) Frisvad/P. paneum Frisvad P. crustosum Thom
+ +
+
P. expansum Link
+
P. glabrum (Wehmer) Westling P. griseofulvum Dierckx P. implicatum Biourge P. raistrickii G. Sm P. variabile Sopp Phoma glomerata (Corda) Wollenweber et Hochapfel
+
+ +
+ + + + +
Potential secondary metabolite production altenuen, alternariol, alternariol monomethyl eter, altertoxina naphto-4-pyrones, malformins, ochratoxin Ab nr N-methylcarbamatec, cladosporin and isocladosporind, calphostin Ce herbarin A and B, herbaric acid, citreoviridin Ab,f nr roselipinsg, glisopreninsh, glioroseini,j epicorazines A and Bk,l, rhodoxanthinm culmorin, enniatin, equisetin, chlamydosporol, chrysogine, fusarochromanoneb, trichothecenes Ab (diacetoxyscipenol, T-2, HT-2-toxin, and neosolaniol) and Bb (deoxynivalenol, 15-acetyl deoxynivalenol, fusarenone X and nivalenol), zearalenonb butenolide, fusarin C, trichothecenes Ab (HT-2 toxin, neosolaniol and diacetoxyscirpenol) and Bb ( deoxynivalenol, 15-deoxynivalenol, fusarenone X and nivalenol), zearalenoneb fusaric acid, naphthoquinone pigments nr nr nr hydroxymytoxin B and hydroxyroridin Eb,n, 12,13-deoxyroridin Eb,o trichoverroidsb,p, roridins and verrucarinq brevianamid A and B, botryodiploidinb, mycophenolic acid, Raistrick phenols griseofulvinb, penitrem Ab roquefortine Cb, patulinb, botryodiploidinb, penitrem Ab, isofumigaclavicine A and Bb, penicillic acidb roquefortine Cb, penitrem Ab, terrestric acid, cyclopenin, cyclopenol roquefortine Cb, patulinb, citrininb, chaetoglobosin A, B and Cb,r, communesins citromycetin roquefortin C,b cyclopiazonic acidb, patulinb, griseofulvinb nr griseofulvinb rugulosinb nr
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habitats from which these species have been reported (Samson et al., 2002a, b). Likewise, little is known about distribution and occurrence of F. reticulatum, although Nelson et al. (1983) regarded this species as cosmopolitan. Under the name F. reticulatum var. reticulatum, it has been reported from a bark lesion of Sophora japonica and from a root lesion of Paspalum dilatatum, under F. reticulatum form 1 from Beta vulgaris and timber pulp and under F. reticulatum var. negundinis as the causal agent of red stain of Acer negundo (Gerlach and Nirenberg, 1982). In the mycobiota composition recovered from the damaged fruit samples, P. carneum/P. paneum was dominating population in Pozba location with a huge number of isolates exceeding 50010 6 CFU/sample, whereas in the samples from Jablonˇovce C. cladosporioides was found to be the dominant one with the similar number of isolates. Of the species found here, Fusarium sporotrichioides, Geotrichum candidum, Penicillium crustosum, P. griseofulvum and P. raistrickii represented the fungal population isolated exclusively from the damaged fruit samples. Whereas, a more numerous group of species consisting of A. pullulans, Cladosporium macrocarpum, Clonostachys rosea, F. reticulatum, G. putterillii, Myrrothecium roridum, M. verrucaria, Penicillium canescens, P. glabrum, P. implicatum, P. variabile and P. glomerata was recovered only from surface sterilised fruits.
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With the exception of P. raistrickii, all species isolated and identified during this study fit with their descriptions given in appropriate identification keys. Population of this species was relatively different from its description by Pitt (1979, 2000) or Pitt and Hocking (1997). Isolates of P. raistrickii exhibited strong grey-green sporulation on both CYA and MEA without sclerotia formation. Nevertheless, based on its micromorphological (penicillus and conidia morphology) and biochemical (griseofulvin production and strong acid production on CREA) features it represents non-sclerotinogenic, well sporulating variant of the species. As to isolates placed to P. paneum or related P. carneum, their separation from the very close species P. roqueforti Thom was done primarily on colouration of colonies growing on CYA and YES and by the ability to produce patulin. The discrimination between P. carneum and P. paneum was not done in this study, because based on the traditional morphological and cultural characteristics they are very difficult or even impossible to separate (Boysen et al., 1996; Pitt, 2000; Samson et al., 2000). Placing of Alternaria isolates to A. alternata was done primarily on account of their sporulating pattern matching well with group 4 (Simmons and Roberts, 1993). Neither constriction nor dark septa in conidia were observed. Furthermore, a size of conidia beak did not exceed one third of conidial length of the Alternaria isolates treated here.
Notes to Table 3: +: present; : absent; nr: no report available. a Andersen et al. (2001). b Mycotoxins in the true sense; bold: mycotoxins detected during the study. c Suzuki and Takeda (1976). d Jacyno et al. (1993). e Kobayashi et al. (1989). f Jadulco et al. (2002). g Tabata et al. (1999). h Sterner et al. (1998). i Steward and Packter (1965). j Packter and Steward (1967). k Baute et al. (1978). l Deffieux et al. (1978). m Foppen (1969). n Alvi et al. (2002). o Namikoshi et al. (2001). p Amagata et al. (2003). q Murakami et al. (2001). r Andersen et al. (2004).
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4.2. Mycotoxin and other secondary metaboliteproduction potential As indicated in Table 3, it is obvious that the potential of the detected fungi to produce various kind of secondary metabolites including mycotoxins even into the fruits might be expected. This is supported by the ability of appropriate species isolates to make detectable amounts of patulin, citrinin, the indole metabolites griseofulvin and zearalenon under laboratory conditions. For example, patulin and citrinin co-occurrence in the pomaceous and stone fruits have been frequently observed (Filtenborg et al., 2002). In fact, P. expansum is considered as the most important patulin producer in apples (Frisvad and Thrane, 2002). Co-incidence patulin producers, namely P. expansum, P. carneum ( P. paneum) and P. griseofulvum, as it is in this case, may lead to high contamination of the fruits with the toxic metabolites. Amongst the other toxicologically significant mycotoxin producers undoubtedly belong P. crustosum and P. canescens, as potential producers of powerful neurotoxin penitrem A (Frisvad and Filtenborg, 1989; Pitt, 2000; Pitt and Hocking, 1997). In addition, F. equiseti isolated as endogenous contaminant should be take into consideration as a significant finding because of its potency to produce A and B trichothecens and zearalenon. However, only zearalenon production by the F. equiseti isolates was detected here. Apart from the possible mycotoxin production ability of the fungal species encountered in the fruits, pharmacologically significant secondary metabolites should be mentioned such as antibiotic epicorazins (Deffieux et al., 1978) or antitumoral roridins and verrucarins (Murakami et al., 2001) production by E. nigrum and Myrothecium verrucaria, respectively. Furthermore, numerous mycotoxins have shown their antibiotic properties against diverse bacteria as well as fungi (Betina, 1989). In conclusion, service tree fruits could be a source of significant fungal secondary metabolites including mycotoxins as it is indicated by the presence of specific fungal species. Nevertheless, more studies should be done to evaluate this fruit from the mycotoxicological point of view for further exploitation in human diet.
Acknowledgement The authors wish to express their gratitude to Dr. A. Kuba´tova´ for help in Myrothecium spp. identification, and Dr. M. Lemmens for help in Fusarium reticulatum and F. equiseti identification and for presubmission review of the manuscript. We are also grateful to the company BIOPURE, Referenzsubstanzen (Austria) for providing of Fusarium-mycotoxin standards as well as to Department of Plant Genetics and Plant Breeding, Slovak University of Agriculture for providing the fruit samples. This study was supported by Grant Committee of Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Slovak Republic. References Alvi, K.A., Rabenstein, J., Woodard, J., Baker, D.D., Bergthold, J.D., Lynch, J., Lieu, K.L., Braude, I.A., 2002. 14V-Hydroxymytoxin B and 16-hydroxyroridin E, two new cytotoxic trichothecenes from Myrothecium roridum. Journal of Natural Products 65, 742 – 744. Amagata, T., Rath, C., Rigot, J.F., Tarlov, N., Tenney, K., Valeriote, F.A., Crews, P., 2003. Structures and cytotoxic properties of trichoverroids and their macrolide analogues produced by saltwater culture of Myrothecium verrucaria. Journal of Medical Chemistry 46, 4342 – 4350. Andersen, B., Kroger, E., Roberts, R.G., 2001. Chemical and morphological segregation of Alternaria alternata, A. gaisen and A. longipes. Mycological Research 105, 291 – 299. Andersen, B., Smedsgaard, J., Frisvad, J.C., 2004. Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products. Journal of Agricultural and Food Chemistry 52, 2421 – 2428. Baute, M.A., Deffieux, G., Baute, R., Neveu, A., 1978. New antibiotics from the fungus Epicoccum nigrum: I. Fermentation, isolation and antibacterial properties. Journal of Antibiotics 31, 1099 – 1101. Betina, V., 1989. Mycotoxins: Chemical, Biological, and Environmental Aspects. Alfa, Bratislava. (in Slovak). Boysen, M., Skouboe, P., Frisvad, J.C., Rossen, L., 1996. Reclassification of the Penicillium roqueforti into three species on the basis of molecular genetic and biochemical profiles. Microbiology 142, 541 – 549. Deffieux, G., Filleau, M.J., Baute, R., 1978. New antibiotics from the fungus Epicoccum nigrum: III. Epicorazine B: structure elucidation and absolute configuration. Journal of Antibiotics 31, 1106 – 1109. Domsch, K.H., Gams, W., Anderson, T.H., 1980. Compendium of Soil Fungi. Academic Press, London.
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