Download Reconstruction of the original mycoflora in pelleted feed by PCR

RESEARCH LETTER
Reconstruction of the original mycoflora in pelleted feed by
PCR-SSCP and qPCR
€ lzel1, Sebastian Wenz1, Isabella Hartwig1,
Samart Dorn-In1, Carmen Fahn2, Christina S. Ho
1,
1
Karin Schwaiger & Johann Bauer
1
Chair of Animal Hygiene, WZW, TUM, Freising, Germany; and 2Chair of Animal Nutrition, WZW, TUM, Freising, Germany
Correspondence: Samart Dorn-In, Chair of
Animal Hygiene, WZW, TUM,
Weihenstephaner Berg 3, 85354 Freising,
Germany.
Tel.: +49 8161 71 5503;
fax: +49 8161 71 4516;
e-mail: [email protected]
Present address: Karin Schwaiger, Chair of
Food Safety, Faculty of Veterinary Medicine,
€nleutnerstr. 8, 85764
LMU, Scho
Oberschleißheim, Germany
Received 12 May 2014; revised 30 July 2014;
accepted 30 July 2014. Final version
published online 21 August 2014.
MICROBIOLOGY LETTERS
DOI: 10.1111/1574-6968.12552
Editor: Michael Bidochka
Abstract
Ground feeds for pigs were investigated for fungal contamination before and
after pelleting (subsamples in total n = 24) by cultural and molecular biological
methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the
resulting acrylamide gel, more than 85% of DNA bands of ground feeds were
preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was
equivalent to 4.77–5.69 log10 CFU g1, calculated by qPCR using a standard
curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in
average 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10 CFU g1 culturable
fungi, while there was < 2.83 log10 CFU g1 detected in pelleted feeds. This
result shows that, while the process of pelleting reduced the amount of living
fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the
described methodology was able to reconstruct the fungal microbiota in feeds
and reflected a considerable fungal contamination of raw materials such as
grains.
Keywords
fungi; primer; DNA; PCR; SSCP; pelleted
feed.
Introduction
Fungi are often main problems for feed quality, as some
of them can produce mycotoxins, and in addition, their
growth activity may decrease the quality of grain and
reduce the content of nutrients by several methods like
respiratory heating (Maciorowski et al., 2007). Grains –
in which fungal growth can occur both before and after
harvesting – are a natural source for fungal contamination of feed. (Baliukoniene et al., 2005; Jouany, 2007;
Maciorowski et al., 2007). For the investigation of fungal
species and their level of contamination in feed or grains,
a culture-based method is normally used. However, the
application of this method for pelleted feed is limited, as
pelleting is an advanced processing, in which the living
fungi will be deactivated. Thus, the culture result of
pelleted feed does not reflect the original level of mycoª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
flora in raw materials but rather the recontamination
after pelleting or the residual fungi that survived the pelleting process (Chelkowski, 1991).
Although the deactivated fungi are not harmful for animals, their presence in high concentrations may indicate
hygienic problems of raw materials or inadequate storing
conditions as well as the risk of mycotoxin contamination. Additionally, the use of unhygienic or moldy grains
to produce feed is deceptive and should be prevented.
Therefore, to evaluate the fungal contamination in pelleted or heat-treated feed, a molecular biological based
method is necessary – such as qualitative or quantitative
polymerase chain reaction (PCR/qPCR). For this purpose,
a fungal-specific primer pair is required for the amplification of fungal DNA; afterward, the PCR products can be
used for further applications – such as single-strand
conformation polymorphism (SSCP) – and sequence
FEMS Microbiol Lett 359 (2014) 182–192
183
Reconstruction of the original mycoflora in pelleted feed
analysis. According to previous studies, many universal
fungal primer pairs also amplify plant DNA (White et al.,
1990; Zhou et al., 2000; Wu et al., 2002; Mitchell &
Zuccaro, 2006; Ihrmark et al., 2012; Dorn-In et al.,
2013). Those primer pairs cannot be applied for the
amplification and quantification of fungal DNA in feed,
as co-amplification with DNA from grains will occur. To
avoid this problem, a fungal cultivation from feed is often
applied as the first step before cultures are further processed for molecular biological tests (Zachova et al., 2003;
Degola et al., 2007; Yazeed et al., 2011). Otherwise,
instead of using universal primers, fungal species-specific
primers were developed, mostly targeting on mycotoxin
producing fungi, for example Fusarium spp. (Edwards
et al., 2001; Wilson et al., 2004; Nicolaisen et al., 2009;
Scauflaire et al., 2012), Alternaria spp. (Zur et al., 2002)
and Aspergillus spp. (Somashekar et al., 2004).
Nevertheless, according to Dorn-In et al. (2013), there
are some universal primer pairs such as ITS1F/ITS4
(Gardes & Bruns, 1993), U1/U2 (Sandhu et al., 1995) and
ITS1/ITS5.8R that amplify only DNA from fungi but not
from plants. The latter primer pair was used successfully
to reconstruct and to identify fungal flora in heatprocessed meat products, which were already spiced
(Dorn-In et al., 2013). However, the analysis of a pure
plant-based matrix poses greater difficulties than a meat
matrix in which plant material (spices) represents only a
small percentage. Therefore, this primer pair was further
validated in this study, which moreover aimed to apply
PCR-SSCP and qPCR for the detection, identification,
and quantification of the DNA of fungal flora in pelleted
feed, in which the application of the traditional culture
method is limited.
Materials and methods
Samples
Sixty kilogram of each four tested ground feed (feed A–D)
samples (containing barley, wheat bran, soybean meal,
corn, wheat, alfalfa meal, mineral supplement, animal feed
salt, and soybean oil) was collected from a pig farm; a subsample was pelleted on the day of sampling. Aliquots of
these eight samples (four ground and four pelleted feeds)
were processed directly on the day of preparation for fungal
cultivation and DNA extraction. Each of these eight samples was split into three subsamples for culturing, and each
of these subsamples was processed for two DNA extractions. Both DNA-extracted samples were pooled; thus,
there were in total 24 DNA-extracted feed samples prepared for PCR-SSCP analysis and qPCR. All reference fungal species shown in Fig. 3, including Aspergillus flavus for
qPCR were provided by the reference stock of the Chair of
FEMS Microbiol Lett 359 (2014) 182–192
Animal Hygiene, WZW, TUM, Germany. In addition, five
different pelleted feed types (field samples, feed E–I, containing barley, wheat, oat, corn, soybean, alfalfa meal, and
molasses) were also investigated for their fungal contamination by PCR-SSCP method.
Pelleting
The pelleting process was performed at the Chair of Animal Nutrition (WZW, TUM, Germany) using a customary horizontal ‘Flat Die Pelleting Presses, Amandus’
(Kahl, Germany). Ground feed was first mixed for 8 min.
During pelleting process, mixed ground feed was steamed
until it reached a temperature of 69 °C, then it was
pressed with a double-conical die. This process took
3–5 s. The size of pellets was 4 mm in diameter. After
pelleting, the pellets were left for 20 min at room
temperature for cooling and drying.
Culture method
Ground feed was processed directly for fungal cultivation,
while pelleted feed was first grinded with a mortar. Ten
gram feed in 90 mL peptone water in a stomacher bag
was shaken for 1 min in a stomacher. Then, 100 lL of
the 101–106 serial dilution was spread on two solid
media, which were selective for fungi: Sabouraud glucose
agar (SAB, 1 L contains antibiotic: 400 000 units penicillin G and 40 mg streptomycin; Sigma), and dichloran
glycerol agar (DG18, 1 L contains 20 mg chlortetracyclin
HCl; Sigma), and incubated at room temperature
(c. 25 °C). Fungal colonies were enumerated on the 3rd
and 7th day of incubation.
Ten morphologically different colonies from ground
feed A were further subcultured on SAB agar. After
5 days of incubation, the macroscopic and microscopic
morphology of fungal colonies and spores were investigated for primary species identification. Then, all 10 fungal cultures were harvested and processed for DNA
extraction, PCR-SSCP method, and sequencing analysis
for final species identification. For A. flavus, used as the
standard species for qPCR, spores were harvested at the
7th day after incubation using sterile distilled water and a
cotton net to separate hyphae from spores. The harvested
spores were serial diluted and enumerated by the spread
plate method as described above.
DNA extraction
The DNA extraction method followed the instructions of
the DNA extraction ‘PowerSoil Kit’ (MoBio, Germany).
Five hundred microlitre of feed suspension (containing
50 mg feed) from dilution 101 was processed for
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184
genomic DNA extraction. To prepare a standard for
qPCR, 10 mL of feed suspension in a tube (15 mL) was
irradiated with Gamma rays (450 kGy) to destroy the
existing DNA, thus representing only feed matrix. Then,
three aliquots with 450 lL irradiated feed suspension
were artificially contaminated with 50 lL solution containing A. flavus in a concentration of 5 9 106 CFU per
sample. After DNA extraction, these three aliquots were
pooled and then serially diluted, corresponding to 108–
102 CFU g1 feed. For cultured fungi, 50 lL spore solution was used for DNA extraction.
S. Dorn-In et al.
(a)
PCR-SSCP method and identification of fungal
species
A thermocycler instrument (Biometra, Goettingen, Germany) was used to amplify the target DNA. The forward
primer was ITS1 (TCC GTA GGT GAA CCT GCG G,
White et al., 1990) and ITS5.8R (GAG ATC CGT TGT
TGA AAG TT, Dorn-In et al., 2013) was the reverse primer, which was phosphorylated at the 50 end for PCR-SSCP
analysis. PCR-SSCP and DNA purification were carried out
as previously described (Dorn-In et al., 2013). The DNA
band profiles from all 24 subsamples of ground and pelleted feed (from feed A, B, C and D) were analyzed with
the GELCOMPAR II program (Applied Maths) to evaluate
their similarity. The selected DNA bands in acrylamide gel
(SSCP gel) were cut and purified by the crush-and-soak
method (Peters et al., 2000). The DNA was reamplified by
thermocycling. PCR products were purified and submitted
for sequencing (Sequiserve, Germany). The sequenced nucleotides were aligned with the BLAST program from NCBI
(National Center for Biotechnology Information, http://
blast.ncbi.nlm.nih.gov/Blast.cgi) for species identification.
Quantitative PCR (qPCR)
The qPCR assay was performed using a LightCycler 480
Instrument II (Roche). Each reaction contained a 20 lL mixture that included 6 lL H2O, 1 lL of each 20 lM primer,
10 lL of mastermix with SYBR Green I and 2 lL DNA template. The following thermocycling pattern was used: initial
denaturation at 95 °C for 10 min, followed by 40 cycles in a
series of denaturation at 95 °C for 10 s, annealing at 56 °C
for 10 s, and extension at 72 °C for 14 s.
Results
(b)
Fig. 1. (a) DNA band profiles in SSCP gel of ground (G) and pelleted
(P) feed from feed sample A, B, C, and D; (b) similarity of DNA band
profiles from ground and pelleted feed analyzed with program
GELCOMPAR II.
and three subsamples of pelleted (P) feed. In total, there
were > 16 DNA bands for each subsample. All intense
DNA bands from both feed types were at the same position. Figure 1b shows the similarity of DNA band profiles
of subsamples of ground and pelleted feed from each feed
sample and analyzed with the GELCOMPAR II program
including Pearson’s correlation analysis. The maximum
similarity of band profiles from both feed types (ground
vs. pellet) was 97.6%, 95.8%, and 95.5% and the minimum similarity was 93.1%, 92.0%, and 94.7% for feed A,
B and C, respectively. The minimum similarity of DNA
band profiles of feed D (ground vs. pellet) was 85.8%.
The minimum similarity within subsamples of one feed
type was 86.9% (ground feed from feed D).
PCR-SSCP
Figure 1a shows the DNA band profiles in the SSCP gel
resulting from four feed samples (A, B, C, and D). Each
feed sample consisted of three subsamples of ground (G)
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Sequencing result
Ten different fungal colonies from feed A were subcultured and processed for DNA extraction. The DNA of all
FEMS Microbiol Lett 359 (2014) 182–192
185
Reconstruction of the original mycoflora in pelleted feed
isolated colonies was submitted for sequencing. Figure 2
shows the DNA bands profile of pelleted feed A (lane 1)
compared with the mixture of DNA bands from 10 isolated fungal species (lane 2). Fourteen DNA bands of feed
A (lane 1) were also sequenced (Fig. 2). Eight of 10
(80%) of the DNA bands from isolated fungi (bands
15–23, lane 2) were also found in the DNA profile of feed
A (lane 1). DNA bands 1, 2, 3, and 10 from feed were at
the same position as bands 15, 16, 17, and 22 (from Colletotrichum acutatum, Penicillium brevicompactum, Penicillium freii, and Rhodosporidium babjevae, respectively)
from isolated colonies, but they were not sequenceable, as
the DNA bands were too faint. Three of four pairs of
sequenceable bands from feed and from isolated fungi
which were at the same position showed identical
sequencing result (band 4 vs. 18 = Aureobasidium pullulans, band 6 vs. 19 = Fusarium sp., and band 12 vs.
23 = Phoma sp.). The DNA sequence of the fourth band
from feed (lane 1) corresponded to Fusarium nivale, while
the DNA sequence from an isolated colony at the same
position in lane 2 corresponded to Cladosporium cladosporioides (band 20). DNA band 13 from feed (lane 1)
and DNA band 24 from an isolated colony (lane 2) were
not at the same position, but both of them were from a
yeast of the genus Cryptococcus. One band (No. 9, lane 1
vs. No. 21, lane 2) differed slightly in the position and
gave two different sequencing results: Udeniomyces pannonicus (band 9) and Eurotium spp. (band 21).
Figure 3 shows DNA band profiles of nine pelleted feed
samples compared to 12 fungal reference species in SSCP
gel. Eleven DNA bands (bands 15–25 from feed B–C)
were additionally purified and then submitted for
sequencing. All DNA bands turned out to be really from
fungal species and not from plants (Fig. 3). The four
most prevalent and most intense bands were on the
height of Fusarium sp., Alternaria sp., Eurotium spp., and
Epicoccum nigrum.
qPCR and cultivation results
All ground and pelleted subsamples from feed A, B, C,
and D were quantified by qPCR to determine the level of
fungal contamination. DNA extraction from irradiated
feed samples artificially contaminated with A. flavus in
concentrations of 108–102 CFU g1 feed was used as a
standard curve for the quantification of fungal DNA in
Fig. 2. DNA band profile of pelleted feed A (Lane 1: L1) compared to DNA bands from 10 isolated fungal colonies from ground feed A (Lane 2:
L2) and the sequencing result of DNA bands.
FEMS Microbiol Lett 359 (2014) 182–192
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Published by John Wiley & Sons Ltd. All rights reserved
186
S. Dorn-In et al.
Fig. 3. DNA band profiles from nine pelleted
feed samples (A–I) and further 11 DNA bands
(bands 16–25) which were submitted for
sequencing. The result of sequencing was 15:
Claviceps purpurea (99% identity to the
sequence in GenBank, NCBI); 16: Lewia
infectoria/Alternaria spp. (99%); 17:
Aspergillus penicillioides (99%); 18:
Pyrenophora tritici-repentis (100%); 19:
Ramularia collo-cygni (100%); 20:
Saccharomycopsis fibuligera/Pichia spp. (99%);
21: Pichia anomala (100%); 22: Issatchenkia
orientalis (99%); 23: Candida inconspicua/
Candida ernobii (98%); 24: Pichia burtonii
(100%); and 25: Candida rugosa (100%). For
sequencing results of DNA bands from feed A
(bands 1–14), see Fig. 2.
feed samples. The slope of the standard curve was 3.74,
with an error of 0.036 and PCR efficiency of 1.850. The
crossing points from the qPCR of feed samples were converted into log10 CFU g1 feed using a standard curve of
A. flavus. Thus, the level of fungal contamination in feed
A, B, C, and D is equivalent to 4.79 (standard deviation:
r = 0.13), 5.69 (r = 0.03), 5.48 (r = 0.14), and 4.77
(r = 0.05) log10 CFU g1 in ground feed and 4.75
(r = 0.02), 5.67 (r = 0.01), 5.58 (r = 0.02), and 4.66
(r = 0.05) log10 CFU g1 in pelleted feed, respectively.
Figure 4 shows the result of cultivation (log10 CFU g1
feed) compared with log10 CFU g1 equivalent as calculated from qPCR. The level of fungal contamination
determined by culture method in SAB, and DG18 agar
was in average 3.78 (r = 0.16), 4.00 (r = 0.18), and 4.51
(r = 0.03) log10 CFU g1 in ground feed A, B and C and
2.56 (r = 0.58) and 2.83 (r = 0.03) log10 CFU g1 in
pelleted feed B and C, respectively. In pelleted feed A as
well as in ground and pelleted feed D, there was no fungal growth (< 102 CFU g1 feed). The level of fungal
DNA from the same feed sample calculated by qPCR was
in average 0.07 (r = 0.04) log different between
ground and pelleted feed, while results from culturing
were in average 1.72 (r = 1.56) log different between
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Fig. 4. Log10 CFU g1 feed resulting from cultivation in SAB and
DG18 agar compared with log10 CFU g1 equivalent calculated from
qPCR by means of standard species Aspergillus flavus. G, ground
feed; P, pelleted feed.
both feed types. Comparing the results pairwise between
both methods, the level of fungal contamination
enumerated by plate counting in ground feed was
0.97–4.70 log10 lower than the level that was calculated
from qPCR; in pelleted feed, this difference was
FEMS Microbiol Lett 359 (2014) 182–192
Reconstruction of the original mycoflora in pelleted feed
2.78–4.75 log10. For pelleted feed samples E–H, the level
of fungal contamination calculated from qPCR was 5.2,
5.3, 5.2, 5.3, and 4.6 log10 CFU g1, respectively.
Discussion
Pelleted feed is widely used for animals, as it is convenient for handling, improves palatability and feed efficiency, and reduces the waste of feed as well as the risk of
microbial carry over from raw material (Winowiski, 1995;
Ziggers, 2012). However, in case that raw material (such
as grains) with a high level of fungal contamination was
used for the production of pelleted feed, it is rather
restricted using traditional culture methods to calculate
the status of the original fungal contamination from end
products. Therefore, to reconstruct, to identify, and to
quantify fungal DNA in these feed types, PCR-based
methods were applied.
The DNA bands in SSCP gel were analyzed with the
GELCOMPAR II program. The results show that the similarities of DNA band profiles from both feed types (ground
vs. pellet) were > 85% up to 97.6%. The within-group variability of ground feed was as high as the group-to-group
variability between ground feed and pelleted feed. All
intense DNA bands from both feed types were on the same
position. Some faint DNA bands were absent or present
either in ground or in pelleted feed. This might be the
result of an inhomogeneity of the samples. Additionally,
the DNA of highly concentrated fungi will have more
chance to be amplified by PCR, while the amplification of
fungi in low concentrations is low and fluctuating. Similar
results were found previously in heat-processed food
(Dorn-In et al., 2013). The results indicate that the dominant fungal DNA present in ground feed can be still
reconstructed after pelleting by the PCR-SSCP method.
Afterward, 25 DNA bands from feed samples A–D in
an acrylamide gel were sequenced for species/genera identification. As primer pair ITS1/ITS5.8R amplified a region
ITS-1 from an internal transcribed spacer (ITS) of ribosomal RNA, the identification of fungi in this study was
based on this sequence. The ITS region was reported as a
highly variable region and was often used to distinguish
taxonomic groups of fungi (Edwards et al., 2002; Suanthie et al., 2009). From 25 DNA bands, four were not sequenceable as they were too faint and relatively
heterogeneous. The other 21 bands represented fungal
DNA. Two identified fungi, A. pullulans and E. nigrum,
are known as ubiquitous (Bisht et al., 2000; Gaur et al.,
2010). The latter one is also known as saprophyte for
plants (Bisht et al., 2000; Favaro et al., 2011). Some
strains of Alternaria spp. and their teleomorph form such
as Lewia infectoria are also reported as plant pathogens
(Akamatsu et al., 1999; Zur et al., 2002; Perell
o &
FEMS Microbiol Lett 359 (2014) 182–192
187
Sisterna, 2008). Alternaria tenuissima is also able to
degrade and alter the nutrient profile of cereals (Fapohunda & Olajuyigbe, 2006). Fusarium nivale (syn. Microdochium nivale) is a cause of Fusarium seedling blight of
wheat and is responsible for seedling losses (Cockerell
et al., 2004; Glynn et al., 2006), while Fusarium avenaceum and Fusarium tricinctum are the main causes of
Fusarium head blight of cereal crops (Yli-Mattila, 2010).
Phoma medicaginis (syn. Mycosphaerella pinodes, Ascochyta
pinodes, and Phoma pinodella) is a pathogen of some
plants and causes black lesions of leaves and stems, resulting in losses in forage quality of feeds such as alfalfa, pea,
common bean, and lentil (Djebali, 2013). Udeniomyces
pannonicus is a ballistoconidium-forming yeast, which is
found in many plants (Niwata et al., 2002); unfortunately, there is only little further information about this
fungal species. Aspergillus penicillioides is an important
fungal species in storage grain with lower water activity
(Hocking, 2003). Claviceps purpurea, Pyrenophora triticirepentis, and Ramularia collo-cygni are important
pathogens of cereals such as wheat, barley, rye, and corn
(Lamari & Bernier, 1989; Tudzynski & Scheffer, 2004;
Walters et al., 2008). Pichia burtonii was also found in
storage grain while Pichia anomala was reported to be
used to prevent mold spoilage and to improve preservation of moist grain (Olstorpe et al., 2010b). Issatchenkia
orientalis (syn. Candida krusei, Pichia kudriavzevii) was
experimentally used as probiotic in feed to promote animal health (Lee et al., 2003; Koh & Suh, 2009) or as a
candidate for bioethanol production (Kitagawa et al.,
2010). However, the anamorph form of I. orientalis,
C. krusei, is reported as a notable pathogen especially for
immune-compromised patients (Samaranayake & Samaranayake, 1994) and as a causative agent of mycotic mastitis in cattle (Watts, 1988; Elad et al., 1995; Sheena &
Sigler, 1995). Dioszegia hungarica (syn. Cryptococcus hungaricus) was isolated from root plants and is found in
many habitats (Renker et al., 2004). Rhodosporidium babjevae and Rhodotorula glutinis are carotenoid producing
yeasts, which can be isolated from plants and natural
environments (Gadanho & Sampaio, 2002). Last, with
regard to the four identified yeasts, Saccharomycopsis fibuligera is a food-borne amylolytic yeast, which is able to
degrade starch and was also used as a starter for the fermentation process of starch products (Hostinova, 2002).
Candida rugosa was also found in cereal grain or wet
wheat distillers’ grain (Olstorpe et al., 2010a). Candida
inconspicua was reported as an important Candida species
which can be pathogenic in humans due to its poor susceptibility to fluconazole, an antifungal drug used in
human medicine (Guitard et al., 2013), while Candida
ernobii has an antifungal effect, for example against Penicillium expansum and Diplodia natalensis (Liu et al.,
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188
2014). Unfortunately, there is only little information published about the contamination of yeasts in feed or grains.
The result of sequence analysis corroborates the specificity
of the primer pair ITS1/ITS5.8R to amplify only fungal
DNA, but not DNA from plants such as grains (e.g. barley, wheat, corn, and soybean), which are the main components in animal feed.
After DNA was extracted from 10 isolated fungal species from feed A, eight of 10 (80%) DNA bands were
found also in the band profile of DNA directly extracted
from the same feed sample. The DNA band positions of
four isolated colonies (C. acutatum, P. brevicompactum,
P. freii, and R. babjevae) were probably also found in the
DNA profile of the feed sample, but they were not
sequenceable due to the heterogeneity of the bands.
Therefore, it could not be confirmed whether the
sequences were identical to those of the four isolated
species. Three of four pairs of sequenceable DNA bands
showed identical sequencing results. One isolated fungus,
C. cladosporioides, was not found in the band profile of
DNA directly extracted from the feed sample, as the DNA
band at the same position represented DNA from another
fungus, namely F. nivale. When a DNA band is heterogeneous, only the clearly dominant sequence will be edited
and sequenced, although the DNA of other fungi might
also be present in sample, but only at a low level. As
mentioned previously, DNA from certain fungi will have
less chance to be amplified if the fungi are present only
in a small amount in feed samples, compared with dominant fungi in the samples. This introduces a stochastic
error in the detection of rare fungi. However, this stochastic error applies also to the plate counting, and the
cultural method additionally introduces another sort of
bias as fungi which produce predominantly hyphae
instead of spores are limited to be detected by cultural
methods. This is also demonstrated by the fact that four
intense bands from the directly extracted DNA (feed sample) were not detectable by cultivation. The DNA
sequences of these fungi corresponded to L. infectoria,
Alternaria spp., F. nivale, and E. nigrum; all are known to
produce predominantly hyphae (Karakousis et al., 2006;
Taniwaki et al., 2006; Favaro et al., 2012). For the DNA
band of U. pannonicus, according to Niwata et al. (2002),
this yeast requires a special method for isolation and cultivation; therefore, it may be a reason that it was not
detectable in SAB and DG18 agar using in this study.
Fungi grown on crops have been traditionally divided
into field and storage fungi. However, some fungal species
such as A. flavus are equally found in both situations
(Pitt & Hocking, 1997). Field fungi are generally plant
pathogens, which invade the growing seeds before harvest.
They rarely play a significant role in further deterioration
of crop postharvest (Pitt & Hocking, 1997). Some genera
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S. Dorn-In et al.
found in feed samples in this study such as Alternaria
alterna, Cladosporium spp., E. nigrum, Penicillium spp.,
Aureobasidium pullurans, and Fusarium spp. were also
reported as the most frequently found field fungi in crops
worldwide (Pitt & Hocking, 1997), while other fungal
species such as Phoma spp., C. purpurea, P. tritici-repentis, R. collo-cygni, C. hungaricus, R. babjevae, and R. glutinis were also mentioned above as plant pathogens.
During storage, spoilage of crops usually results from
invasion by storage fungi, which are able to grow under
condition with a very low water activity (aw) such as
Eurotium spp., Aspergillus penicillioides, and some species
of Penicillium spp. such as P. brevicompactum (Pitt &
Hocking, 1997). These three fungal genera were also
found in this study.
In general, the reproduction in fungi occurs through
the production of spores. The distribution of fungal
spores can be airborne, moving by wind from field to
field, or may occur by use of contaminated trucks and
equipment. Insects and birds can serve as mechanical vectors, which then transport fungal spores from plant to
plant (USDA, 2006; Hawksworth & Wiltshire, 2011). The
concentration of fungal DNA in the air is normally low.
In most cases, fungal spores are rarely dispersed more
than 100–200 m horizontally from the sources (Hawksworth & Wiltshire, 2011). However, spores of certain species that occur abundantly in leaves or bark, for example
Alternaria spp. and Cladosporium spp., can be encountered in large numbers in air samples, particularly in late
summer or autumn (Hawksworth & Wiltshire, 2011).
Similar findings were also reported for Cladosporium spp.,
Alternaria spp., Penicillium spp., Aspergillus spp., Fusarium spp., and Aureobasidium spp. – fungal genera that are
often found in the air of poultry and dairy houses (Dalcero et al., 1998; Alvarado et al., 2009; Ajoudanifar et al.,
2011). In contrast, fungi with more restricted occurrences
rarely reach levels of even 1% of the total air spores
(Hawksworth & Wiltshire, 2011).
To control those field fungi, the refinement in agricultural practice is required; to prevent storage fungi, a storing condition with a very low aw is recommended (Pitt &
Hocking, 1997).
For the result of qPCR, the crossing points of both feed
types were converted into log10 CFU g1 feed by means
of a standard curve of A. flavus. The level of fungal DNA
in pelleted feed was slightly different from ground feed,
in average equivalent to 0.07 (r = 0.04) log level. That
means, the pelleting process did not affect the quality and
quantity of fungal DNA in feed.
In this study, one fungal species, A. flavus, was used as
the standard for the quantification of fungal contamination in feed samples by qPCR with SYBR Green I. The
level of detected fungal DNA in samples may alter, if
FEMS Microbiol Lett 359 (2014) 182–192
189
Reconstruction of the original mycoflora in pelleted feed
other fungal species were used as standards. Each fungus
or even cell type (spore/mycelial cell) has different characteristics, which may influence the efficiency of DNA
extraction methods and therefore the amount of extracted
DNA. For example, DNA from mycelia of Penicillium
spp. can be extracted easier than from spore (Le Drean
et al., 2010). Fungal spores of Alternaria spp. and Aspergillus spp., especially Aspergillus fumigatus, are resistant to
enzymatic lysis and can be better extracted by mechanical
disruption, while spores of Penicillium chrysogenum and
Candida albicans can be well lysed by both methods (Fredricks et al., 2005; Karakousis et al., 2006).
The level of fungal contamination in four ground feeds
as determined by qPCR was higher than the values calculated by the culture method, namely 0.97–4.70 log10 levels. This may be attributable to the growth characteristics
of fungi and the limitations of the culture method, as it
can detect only active living spores and maybe some parts
of hyphae. However, feeds or grains contain not only
active but also inactive or defective spores and hyphae,
which altogether form the total fungal biomass in the
sample. During fungal growth, some spores or parts of
hyphae are inactive or die, while the others can grow further. Therefore, the accumulation of inactive or dead
fungi is increasing, especially if the storing time of grains
is longer. In case that the stored grains are properly handled, fungal contaminants before and during harvesting
will die off, which results in a low level of fungal contamination calculated from the culture method (Scudamore
& Wilkin, 2000). However, repeated or continuous
growth of fungi during long-time-storage is undesirable,
particularly with regard to contamination with mycotoxins, irrespective of whether these fungi are still cultivable
at the moment of random sampling. Moreover, as mentioned above, some fungi found in this study such as
Alternaria spp., Fusarium spp., and Epicoccum spp. produce predominantly hyphae (Karakousis et al., 2006; Taniwaki et al., 2006; Favaro et al., 2012), which are limited
to be detected and enumerated by cultural methods.
Some raw materials or grains were treated by any process
such as dry heat to reduce the level of contaminating living fungi prior to be produced for animal feed such as
for feed D, in which no living fungi could be cultured
both from ground and pelleted feed. Thus, the use of
cultivation might lead to an underestimation of total
original fungal biomass in samples. On the contrary, a
PCR-based method can detect DNA from both living and
inactive fungal biomass. Hence, the level of fungal
contamination quantified by qPCR has a tendency to be
higher than the quantification results from culturing.
A high level of fungi in grains or feed may indicate the
risk of mycotoxin contamination produced from, for
example Fusarium spp., as these toxins are relatively
FEMS Microbiol Lett 359 (2014) 182–192
stable, even if the mycotoxin producing fungi died out
already and cannot be detected by cultural methods.
However, due to cost and precision of analysis, analytical
methods such as thin-layer chromatography (TLC), highperformance liquid chromatography (HPLC), gas chromatography, and enzyme-linked immunosorbent assay
(ELISA) would still be methods of choice for direct detection of mycotoxins in feed or grains (Zheng et al., 2006).
However, some fungal strains degrade the quality of feed
but do not produce mycotoxin. The application of qPCR
is suitable for monitoring the quality of feed or grains
during long-time storage as well as to assure the quality
of grains directly postharvest and of pelleted feed, for
which the application of cultural methods is limited.
Conclusion
The primer pair ITS1/ITS5.8R and the molecular biological methods PCR-SSCP and qPCR were successfully
applied to reconstruct, to identify and to quantify fungal
DNA in both ground and pelleted feed. All dominant
DNA bands in SSCP gel from both feed types are identical and represent only fungal DNA; plant DNA was not
amplified. The results of qPCR of pelleted feed reflect the
level of the original fungal microbiota in ground feed.
Therefore, this study provides the possibilities (1) to evaluate the diversity of the fungal flora in feed, (2) to monitor the quality of grains or feed, stored for a long period,
and (3) to control the end product such as pelleted feeds
without cultivation. This helps to establish monitoring
methods to ensure that pelleted feeds are produced from
qualified raw material in terms of fungal contamination.
Such feed monitoring might indeed seem advisable with
regard to the observed contamination level of feed samples with fungal DNA. Moreover, the methodology can
also be applied to human food which is produced under
similar (or less degrading) conditions, or for diagnostic of
plant diseases caused by fungi; moreover, it might even
be helpful in forensic mycology.
Acknowledgements
We would like to thank the technical assistants from the
Chair of Animal Hygiene and Chair of Animal Nutrition,
Center of Life and Food Sciences Weihenstephan
(WZW), Technical University Munich, for their helpfulness and hints on laboratory technique.
References
Ajoudanifar H, Hedayati MT, Mayahi S, Khosravi A &
Mousavi B (2011) Volumetric assessment of airborne indoor
and outdoor fungi at poultry and cattle houses in the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
190
Mazandaran province, Iran. Arh Hig Rada Toksikol 62: 243–
248.
Akamatsu H, Taga M, Kodama M, Johnson R, Otani H &
Kohmoto K (1999) Molecular karyotypes for Alternaria
plant pathogens known to produce host-specific toxins.
Curr Genet 35: 647–656.
Alvarado CS, Gandara A, Flores C, Perez HR, Green CF, Hurd
WW & Gibbs SG (2009) Seasonal changes in airborne fungi
and bacteria at a dairy cattle concentrated animal feeding
operation in the southwest United States. Environ Health 71:
40–44.
Baliukoniene V, Bakutis B & Gerulis G (2005) Feeding grains
contamination with fungi and mycotoxins after harvest.
Animals and Environment : Proceedings : XIIth International
Congress, ISAH, Vol. 2 (Krynski A & Wrzesien R, eds),
pp.376–379. Bel Studio, Warsaw.
Bisht V, Singh BP, Arora N, Sridhara S & Gaur SN (2000)
Allergens of Epicoccum nigrum grown in different media for
quality source material. Allergy 55: 274–280.
Chelkowski J (1991) Mycological quality of mixed feeds and
ingredients. Cereal Grain, Mycotoxins, Fungi and Quality in
Drying and Storage (Chelkowski J, ed.), pp. 217–227.
Elsevier, Amsterdam.
Cockerell V, Clark WS & Roberts AMI (2004) Cereal seed
health and seed treatment strategies: exploiting new seed
testing technology to optimise seed health decisions for
wheat, Technical paper No. 4: the effect of Microdochium
nivale, on the quality of winter wheat seed in the UK.
HGCA: 85–104.
Dalcero A, Magnoli C, Luna M, Ancasi G, Reynoso MM,
Chiacchiera S, Miazzo R & Palacio G (1998) Mycoflora and
naturally occurring mycotoxins in poultry feeds in
Argentina. Mycopathologia 141: 37–43.
Degola F, Berni E, Dall’Asta C, Spotti E, Marchelli R, Ferrero I
& Restivo FM (2007) A multiplex RT-PCR approach to
detect aflatoxigenic strains of Aspergillus flavus. J Appl
Microbiol 103: 409–417.
Djebali N (2013) Aggressiveness and host range of Phoma
medicaginis isolated from Medicago species growing in
Tunisia. Phytopathol Mediterr 52: 3–15.
Dorn-In S, H€
olzel CS, Janke T, Schwaiger K, Balsliemke J &
Bauer J (2013) PCR-SSCP-based reconstruction of the
original fungal flora of heat-processed meat products. Int J
Food Microbiol 162: 71–81.
Edwards SG, Pirgozliev SR, Hare MC & Jenkinson P (2001)
Quantification of trichothecene-producing Fusarium
species in harvested grain by competitive PCR to
determine efficacies of fungicides against Fusarium head
blight of winter wheat. Appl Environ Microbiol 67: 1575–
1580.
Edwards SG, O’Callaghan J & Dobson ADW (2002)
PCR-based detection and quantification of mycotoxigenic
fungi. Mycol Res 106: 1005–1025.
Elad D, Shpigel NY, Winkler M, Klinger I, Fuchs V, Saran A &
Faingold D (1995) Feed contamination with Candida krusei
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
S. Dorn-In et al.
as a probable source of mycotic mastitis in dairy cows. J Am
Vet Med Assoc 207: 620–622.
Fapohunda SO & Olajuyigbe OO (2006) Studies on stored
cereal degradation by Alternaria tenuissima. Acta Bot Mex
77: 31–40.
Favaro LCL, Melo FL, Aguilar-Vildoso CI & Ara
ujo WL (2011)
Polyphasic analysis of intraspecific diversity in Epicoccum
nigrum warrants reclassification into separate species. PLoS
ONE 6: e14828.
Favaro LCL, Sebastianes FLS & Ara
ujo WL (2012) Epicoccum
nigrum P16, a Sugarcane endophyte, produces antifungal
compounds and induces root growth. PLoS ONE 7: e36826.
Fredricks DN, Smith C & Meier A (2005) Comparison of six
DNA extraction methods for recovery of fungal DNA as
assessed by quantitative PCR. J Clin Microbiol 43: 5122–
5128.
Gadanho M & Sampaio JP (2002) Polyphasic taxonomy of the
basidiomycetous yeast genus Rhodotorula: Rh. glutinis sensu
stricto and Rh. dairenensis comb. nov. FEMS Yeast Res 2:
47–58.
Gardes M & Bruns TD (1993) ITS primers with enhanced
specificity for basidiomycetes – application to the
identification of mycorrhizae and rusts. Mol Ecol 2: 113–
118.
Gaur R, Singh R, Gupta M & Gaur MK (2010) Aureobasidium
pullulans, an economically important polymorphic yeast
with special reference to pullulan. Afr J Biotechnol 9: 7989–
7997.
Glynn NC, Ray R, Edwards SG, Hare MC, Parry DW, Barnett
CJ & Beck JJ (2006) Quantitative Fusarium spp. and
Microdochium spp. PCR assays to evaluate seed treatments
for the control of Fusarium seedling blight of wheat. J Appl
Microbiol 102: 1645–1653.
Guitard J, Angoulvant A, Letscher-Bru V et al. (2013) Invasive
infections due to Candida norvegensis and Candida
inconspicua: report of 12 cases and review of the literature.
Med Mycol 51: 795–799.
Hawksworth DL & Wiltshire PEJ (2011) Forensic mycology:
the use of fungi in criminal investigations. Forensic Sci Int
206: 1–11.
Hocking AD (2003) Microbiological facts and fictions in grain
storage. Proceedings of the Australian Postharvest Technical
Conference (Wright EJ, Webb MC & Highley E, eds), pp.
55–58. CSIRO, Canberra, ACT.
Hostinova E (2002) Amylolytic enzymes produced by the yeast
Saccharomycopsis buligera. Biologia 11: 247–251.
Ihrmark K, B€
odeker ITM, Cruz-Martinez K et al. (2012) New
primers to amplify the fungal ITS2 region – evaluation by
454-sequencing of artificial and natural communities. FEMS
Microbiol Ecol 82: 666–677.
Jouany JP (2007) Methods for preventing, decontaminating
and minimizing the toxicity of mycotoxins in feeds. Anim
Feed Sci Technol 137: 342–362.
Karakousis A, Tan L, Ellis D, Alexiou H & Wormald PJ (2006)
An assessment of the efficiency of fungal DNA extraction
FEMS Microbiol Lett 359 (2014) 182–192
Reconstruction of the original mycoflora in pelleted feed
methods for maximizing the detection of medically
important fungi using PCR. J Microbiol Methods 65: 38–48.
Kitagawa T, Tokuhiro K, Sugiyama H, Kohda K, Isono N,
Hisamatsu M, Takahashi H & Imaeda T (2010)
Construction of a b-glucosidase expression system using the
multistress-tolerant yeast Issatchenkia orientalis. Appl
Microbiol Biotechnol 87: 1841–1853.
Koh JH & Suh HJ (2009) Biological activities of
thermo-tolerant microbes from fermented rice bran as an
alternative microbial feed additive. Appl Biochem Biotechnol
157: 420–430.
Lamari L & Bernier CC (1989) Toxin of Pyrenophora
tritici-repentis: host-specificity, significance in disease, and
inheritance of host reaction. Phytopathology 79: 740–744.
Le Drean G, Mounier J, Vasseur V, Arzur D, Habrylo O &
Barbier G (2010) Quantification of Penicillium camemberti
and P. roqueforti mycelium by real-time PCR to assess their
growth dynamics during ripening cheese. Int J Food
Microbiol 138: 100–107.
Lee JH, Lim YB, Park KM, Lee SW, Baig SY & Shin HT
(2003) Factors affecting oxygen uptake by yeast Issatchenkia
orientalis as microbial feed additive for ruminants.
Asian-Australas J Anim Sci 16: 1011–1014.
Liu P, Shi Y-Y, Chen L & Long CA (2014) Farnesol produced
by the biocontrol agent Candida ernobii can be used in
controlling the postharvest pathogen Penicillium expansum.
Afr J Microbiol Res 8: 922–928.
Maciorowski KG, Herrera P, Jones FT, Pillai SD & Ricke SC
(2007) Effects on poultry and livestock of feed
contamination with bacteria and fungi. Anim Feed Sci
Technol 133: 109–136.
Mitchell JI & Zuccaro A (2006) Sequences, the environment
and fungi. Mycologist 20: 62–74.
Nicolaisen M, Supronien_e S, Nielsen LK, Lazzaro I, Spliid NH
& Justesen AF (2009) Real-time PCR for quantification of
eleven individual Fusarium species in cereals. J Microbiol
Methods 76: 234–240.
Niwata Y, Takashima M, Tornai-Lehoczki J, Deak T & Nakase
T (2002) Udeniomyces pannonicus sp. nov., a
ballistoconidium-forming yeast isolated from leaves of
plants in Hungary. Int J Syst Evol Microbiol 52: 1887–1892.
Olstorpe M, Axelsson L, Schn€
urer J & Passoth V (2010a)
Effect of starter culture inoculation on feed hygiene and
microbial population development in fermented pig feed
composed of a cereal grain mix with wet wheat distillers’
grain. J Appl Microbiol 108: 129–138.
Olstorpe M, Borling J, Schn€
urer J & Passoth V (2010b) Pichia
anomala yeast improves feed hygiene during storage of
moist crimped barley grain under Swedish farm conditions.
Anim Feed Sci Technol 156: 47–56.
Perell
o A & Sisterna M (2008) Formation of Lewia infectoria,
the teleomorph of Alternaria infectoria, on wheat in
Argentina. Australas Plant Pathol 37: 589–591.
Peters S, Koschinsky S, Schwieger F & Tebbe CC (2000)
Succession of microbial communities during hot
composting as detected by PCR-single strand conformation
FEMS Microbiol Lett 359 (2014) 182–192
191
polymorphism based genetic profiles of small-subunit rRNA
genes. Appl Environ Microbiol 66: 930–936.
Pitt JI & Hocking AD (1997) Fungi and Food Spoilage.
Academic & Professional, London.
Renker C, Blanke V, B€
orstler B, Heinrichs H & Buscot F
(2004) Diversity of Cryptococcus and Dioszegia yeasts
(Basidiomycota) inhabiting arbuscular mycorrhizal roots or
spores. FEMS Yeast Res 4: 597–603.
Samaranayake YH & Samaranayake LP (1994) Candida krusei:
biology, epidemiology, pathogenicity and clinical
manifestations of an emerging pathogen. J Med Microbiol
41: 295–310.
Sandhu GS, Kline BC, Stockman L & Roberts GD (1995)
Molecular probes for diagnosis of fungal infections. J Clin
Microbiol 33: 2913–2919.
Scauflaire J, Godet M, Gourgue M, Enard CL & Munaut F
(2012) A multiplex real-time PCR method using
hybridization probes for the detection and the
quantification of Fusarium proliferatum, F. subglutinans,
F. temperatum, and F. verticillioides. Fungal Biol 116: 1073–
1080.
Scudamore K & Wilkin R (2000) Mycotoxins in stored grain.
Home-Grown Cereals Authority (HGCA), Topic Sheet No.
34. Available at: http://adlib.everysite.co.uk/resources/000/
100/178/TS34.pdf (accessed on 9 April 2013).
Sheena A & Sigler L (1995) Candida krusei isolated from a
sporadic case of bovine mastitis. Can Vet J 36: 365.
Somashekar D, Rati ER, Anand S & Chandrashekar A (2004)
Isolation, enumeration and PCR characterization of
aflatoxigenic fungi from food and feed samples in India.
Food Microbiol 21: 809–813.
Suanthie Y, Cousin MA & Woloshuk CP (2009) Multiplex
real-time PCR for detection and quantification of
mycotoxigenic Aspergillus, Penicillium and Fusarium. J
Stored Prod Res 45: 139–145.
Taniwaki MH, Pitt JI, Hocking AD & Fleet GH (2006)
Comparison of hyphal length, ergosterol, mycelium dry
weight and colony diameter for quantifying growth of fungi
from foods. Adv Exp Med Biol 571: 49–67.
Tudzynski P & Scheffer J (2004) Claviceps purpurea: molecular
aspects of a unique pathogenic lifestyle. Mol Plant Pathol 5:
377–388.
United States Department of Agriculture (USDA), Grain
Inspection, Packer & Stockyards Administration (2006)
Grain fungal diseases & mycotoxin reference. Available at:
http://www.gipsa.usda.gov/publications/fgis/ref/mycobook.
pdf (accessed on 23 June 2014).
Walters DR, Havis ND & Oxley SJP (2008) Ramularia
collo-cygni: the biology of an emerging pathogen of barley.
FEMS Microbiol Lett 279: 1–7.
Watts JL (1988) Etiological agents of bovine mastitis. Vet
Microbiol 16: 41–66.
White TJ, Bruns T, Lee S & Taylor J (1990) Amplification
and direct sequencing of fungal ribosomal RNA genes for
phylogenetics. PCR Protocols: A Guide to Methods and
Applications (Innis MA, Gelfand DH, Sninsky JJ & White
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
192
TJ, eds), pp. 315–322. Academic Press, Inc., New York,
NY.
Wilson A, Simpson D, Chandler E, Jennings P & Nicholson P
(2004) Development of PCR assays for the detection and
differentiation of Fusarium sporotrichioides and Fusarium
langsethiae. FEMS Microbiol Lett 233: 69–76.
Winowiski TS (1995) Pellet quality in animal feeds. MITA (P)
NO. 195/11/95 (Vol. FT21-1995). Available at: http://www.
asasea.com.
Wu Z, Wang X-R & Blomquist G (2002) Evaluation of PCR
primers and PCR conditions for specific detection of
common airborne fungi. J Environ Monit 4:
377–382.
Yazeed HAE, Hassan A, Moghaieb REA, Hamed M & Refani
M (2011) Molecular detection of fumonisin-producing
Fusarium species in animal feeds using polymerase chain
reaction (PCR). J Appl Sci Res 7: 420–427.
Yli-Mattila T (2010) Ecology and evolution of toxigenic
Fusarium species in cereals in northern Europe and Asia. J
Plant Pathol 92: 7–18.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
S. Dorn-In et al.
Zachova I, Vytrasova J, Pejchalova M, Cervenkaa
L&
Tavcar-Kalcherb G (2003) Detection of aflatoxigenic fungi
in feeds using the PCR method. Folia Microbiol 48:
817–821.
Zheng MZ, Richard JL & Binder J (2006) A review of rapid
methods for the analysis of mycotoxins. Mycopathologia 161:
261–273.
Zhou G, Whong W-Z, Ong T & Chen B (2000) Development
of a fungus-specific PCR assay for detecting low-level fungi
in an indoor environment. Mol Cell Probes 14: 339–348.
Ziggers D (2012) The better the pellet, the better the
performance. AllAboutFeed. Available at: http://www.
allaboutfeed.net/Nutrition/Research/2012/2/Thebetter-the-pellet-the-better-the-performance-AAF012746W/
(accessed on 25 February 2013).
Zur G, Shimoni E, Hallerman E & Kashi Y (2002) Detection
of Alternaria fungal contamination in cereal grains by a
polymerase chain reaction-based assay. J Food Prot 65:
1433–1440.
FEMS Microbiol Lett 359 (2014) 182–192