73
ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS
Volume LVIII 8 Numbe 4, 2010
THE DETERMINATION OF MICROSCOPIC
FUNGI FROM CHESTNUT (Cas anea sa i a Mill.)
FRUITS, LEAVES, CRUST AND POLLEN
M. Kačánio á, J. Sudzino á, M. Kádasi-Ho áko á, M. Valšíko á, S. K áčma
Recei ed: Feb ua 5, 2010
Abs ac
KAČÁNIOVÁ, M., SUDZINOVÁ, J., KADÁSI-HORÁKOVÁ, M., VALŠÍKOVÁ, M., KRÁČMAR, S.:
The de e mina ion o mic oscopic ungi om Ches nu (Cas anea sa i a Mill.) ui s, lea es, c us and pollen. Ac a
uni . ag ic. e sil ic. Mendel. B un., 2010, LVIII, No. 4, pp. 73–78
The plan -mic obial in e ac i e ela ions wi h espec o de e mina ion o he mycofl o a o he Cas-
anea sa i a Mill. nu s, c us , lea es and pollen and hei eff ec on he hos o ganism in ou Slo ak e-
gions we e s udied. In he expe imen s we e isola ed 7 gene a and 10 species o mic oscopic ungi
om he nu , c us and lea es. I was ound, ha isola es om he Cas anea sa i a Mill. pollen we e ep-
esen ed by 8 gene a and 11 species o mic oscopic ungi. Al e na ia, Cladospo ium, Muco and Rhizopus
appea ed o be he mos equen ly occu ing gene a o nu s, lea es and c us . Ac emonium, Al e na ia,
Cladospo ium, Fusa ium, Penicillium and T ichode ma a e he mos equen ly ungi o pollen. On he base
o u he axonomic de e mina ion om he gene a Aspe gillus we e isola ed and iden ifi ed ep e-
sen a i es o species A. fl a us, A. umiga us, A. och aceus, A. e eus and A. e sicolo . F om he gene a Fu-
sa ium was isola ed F. oxyspo um and om Penicillium gene a we e isola ed P. c us osum and P. glab um. I
is necessa y o unde line ha he isola ed gene a Aspe gillus, Fusa ium and Penicillium a e conside ed as
he mos impo an p oduce s o myco oxins.
Cas anea sa i a Mill., nu s, lea es, c us , pollen, mic oscopic ungi
The ches nu ui s p oduced in Slo akia a e ge-
ne al ly o small size and o lowe quali y because
o less a o able clima ic condi ions and absence o
b eeding and selec ion wo k.
The Eu opean ches nu ees sp ead all o e
he wo ld and p oduce wood and ches nu s ha
ha e conside able economical alue. Specifi c li e-
a u e desc ibes some u iliza ion o ches nu s in
Eu ope being emphasized he ma ons glaces p o-
duc ion; some e e ences also ela e hei use in bak-
ing, mainly unde economically diffi cul condi ion
(Demia e e al., 2001). Also i is gene ally known,
ha he main componen o ches nu s is s a ch,
he e a e a li le s udies ela ed o chemical compo-
si ion o he ches nu s. Many s udies a e o ien ed o
he ag i cul u al ea u es o he ees and especially
o he phy opa hology (Ru e e al., 1991).
Moulds a e dis ibu ed in na u e ubiqui ously and
hei spo es can be ound in he a mosphe e e en
a high al i udes. They a e d i e and dissemina e by
wind and ai cu en s, as well as can be sp ead by
insec s, oden s, and o he animals. The me abolic
ac i i ies accompanying he g ow h and mould de-
elopmen decomposes o ganic subs a e ensu -
ing he ecycling o elemen s ha comp ise o ganic
ma e s. Food p oduc s, being o ganic subs ances
and con aining essen ial nu ien s, a e e y sui able
subs a es o he mould g ow h (Leoni e al., 2001;
Sa lee mul lah e al., 2006; Ca mo e al., 2008). Be-
cause o hei hyd oly ic enzymes powe ul a senal,
moulds may cause a high deg ee o de e io a ion
when hey occu in/on oods and can be in cha ge o
conside able economic losses (O e y e al., 2003; Ka-
washima and Valen e Soa es, 2006). Besides he pos-
sible ood decaying caused by moulds and ul ima e
changes in i nu i ional and o ganolep ic cha ac-
e s, he moldiness in oods uff s is oxicologically
signifi can since he mould species g owing on such
p oduc s is known as po en ially myco oxicogenic
(Ba ilani e al., 2006; Ca mo e al., 2008). My co o-
74 M. Kačánio á, J. Sudzino á, M. Kádasi-Ho áko á, M. Valšíko á, S. K áčma
xins a e seconda ily oxic me aboli es p oduced by
fi lamen ous ungi ha ha e been de ec ed in se e al
ood commodi ies. Le els o myco oxins and myco-
oxicogenic moulds, which may cause isk o pop-
ula ion, a e e used by consume s and many coun-
ies ha e se egula ions in a ious ag icul u al
oods. The consump ion o mouldy p oduc s may
cause human o animal myco oxicoses, and some o
mo e impo an myco oxins a e s ong ca cinogens
(Rasooli and Abyaneh, 2004; Wangika e al., 2005;
Kawashima and Valen e Soa es, 2006; Ca mo e al.,
2008).
Fo he be e unde s anding o ecological and
p ac ical in e ac ions occu ing on he nu s, c us ,
lea es and pollen o Cas anea sa i a Mill., some ana-
ly ses including he s udy o he di e si y and exac
species iden ifi ca ion a e needs. Ou s udy was in-
ended on he su ey o ungal communi y compo-
si ion occu ing on ou diff e en pa s o C. sa i a
Mill. species om ou diff e en egions o Slo akia.
MATERIALS AND METHODS
The samples we e aken om ou diff e en pa s
o Cas anea sa i a Mill. in locali ies o K upina, Mly-
ňany, P íbelce and Radošiná. The samples as lea es,
c us and pollen we e collec ed in June and July o
yea 2007. The samples o nu we e collec ed in Sep-
embe and Oc obe o yea 2007.
1. Nu sample numbe : N1–N13.
2. Lea es sample numbe : L1–L13.
3. C us sample numbe : C1–C13.
4. Pollen sample numbe : P1–P43.
The samples o nu s, lea es and c us numbe ed
1–4 we e aken om locali y K upina, 5–9 we e
aken om Radošiná and 10–13 we e aken om lo-
cali y P íbelce. Sample o pollen numbe ed 1–14
we e aken om Radošiná, 15–22 we e aken om
K upina, 23–32 we e aken om P íbelce and 33-43
we e aken om Mlyňany.
All samples we e aken wi h scalpel p e iously
cleaned wi h 70% e hylalcohol, ans e ed o s e ile
es - ubes and di ec ly anspo ed o he labo a o y
o u he mic obiological p ocessing.
Isola ion and mo phological cha ac e iza ion
o ungi
One g am o samples was ans e ed o 10 ml
0.01% Tween 80 solu ion and shaken o 30 min.
Coa se-gained ma e ial was allowed o p ecipi a e
and 50 μl we e homogeneously dispe sed on o se-
lec i e aga media in Pe i dishes and he pla es we e
incuba ed a oom empe a u e. The isola es we e
pu ifi ed in wo o h ee s eps by ans e o esh me-
dium; pu i y was checked and main ained by e pe-
i i e ligh mic oscopic obse a ion. The ungi we e
g own in Pe i dishes and slide cul u es on 2% mal
ex ac aga (MEA) and Czapek – Dox aga (CDA)
(Imuna Ša išské Michaľany, Slo akia) o he mo -
phological s udies. A e 14–20 days o cul i a ion
in da kness a oom empe a u e, slide cul u es
and p epa a ions we e made in solu ion o lac ic
acid. The pu e cul u es we e iden ifi ed acco ding
o me hod o diagnosis o ungi (Hoog e al., 2000;
Klich, 2002; Samson e al., 2002).
RESULTS AND DISCUSSION
The s udy o mould spo es ha e majo impo -
ance because many ungi may cause conside able
economic losses wo ldwide ac ing as plan pa ho-
gens o igge ing espi a o y diseases and alle genic
p ocesses in humans (Rod iguez-Rajo e al., 2005).
The con amina ion o ood p oduc s wi h mic o-
myce es o he Penicillium, Aspe gillus, Fusa ium, Al e -
na ia, Paecilomyces, T icho ecium, gene a should ecei e
pa icula a en ion. Some o he s ains g owing on
a pa icula ype o ege able o ui could syn he-
size and exc e e diff e en oxic seconda ily me abo-
li es (Lugauskas e al., 2005; Piecko á and Jesenská,
1999).
The mos equen ly ungi ound in nu s o Cas-
anea sa i a Mill. we e Al e na ia al e na a, Cla do spo i-
dium cladospo oides, Fusa ium oxyspo ium, Muco mucedo
and Rhizopus s oloni e . The less abundance was de-
e mined by Aspe gillus umiga us, Aspe gillus e eus,
Muco acemosus, Penicillium c us osum and Penicil-
lium glab um (Tab. I). Cladospo ium cladospo oides was
he mos common spoile o nu samples con ami-
na ing 43.7%.
The na ionwide su ey was ca ied ou o assess
mould spoilage o Cas anea sa i a nu s sold in Ca-
nadian g oce y s o es. Th ee myco oxigenic ungi
we e domina ed (Penicillium c us osum, Penicillium gla-
b um/spinulosum and Penicillium discolo ) and isola ed
a equencies o 67.1%, 18.6% and 17.7%, espec-
i ely, om a o al sample size o 350 nu s. Aspe gillus
och aceus was also isola ed, bu a a much lowe e-
quency (O e y e al., 2003). Al e na ia al e na a aff ec
diff e en cul i a ed and wild plan s. Inhibi o om
buckwhea also supp essed spo e ge mina ion and
mycelium g ow h o phy opa hogenic ungi Al e -
na ia al e na a and Fusa ium oxyspo ium (Value a and
Mosolo , 2004).
The mos equen ly ungi ound in lea es o Cas-
anea sa i a Mill. we e Al e na ia al e na a, Cla do spo-
i dium cladospo oides, Muco mucedo, Muco acemosus
and Rhizopus s oloni e . The less abundance was de-
e mined by Aspe gillus umiga us, Aspe gillus e eus,
Fusa ium oxyspo um, Penicillium c us osum and Penicil-
lium glab um (Tab. I). Al e na ia al e na a was he mos
common spoile o lea e samples con amina ing
49.7%. The lea li e decomposi ion in s eams is
an impo an ecosys em le el p ocess, which de-
pends on he ac i i y o in e eb a es and mic o-
o ganisms. Bo h ungi and bac e ia con e lea
ca bon in o mic obial biomass, enhancing lea pa-
la a bi li y o sh edding in e eb a es (Pascoal and
Cássio, 2004).
Al e na ia al e na a (100%), Cladospo ium cla do spo-
ioi des (100%), Mycelia s e ilia (67%) and Penicilium spp.
(67%) we e he mos equen ly isola ed species o
ungi encoun e ed on he su ace o he So bus do-
The de e mina ion o mic oscopic ungi om Ches nu (Cas anea sa i a Mill.) ui s, lea es, c us and pollen 75
I: F equency o ungi species o Cas anea sa i a Mill.
Species o ungi Con amina ed samples
%
Numbe o isola es
( ange)
Nu s
Al e na ia al e na a (F .) Keissl. 36.4 0–45
Aspe gillus umiga us F esen. 16.5 0–30
Aspe gillus e eus Thom. 12.0 0–34
Cladospo ium cladospo ioides (F esen.) G.A. de V ies 43.7 0–65
Fusa ium oxyspo ium Schl dl. 23.8 0–34
Muco mucedo F esen. 21.4 0–27
Muco acemosus F esen. 8.6 0–10
Penicillium c us osum Thom. 14.0 0–27
Penicillium glab um (Wehme ) Wes ling 15.7 0–39
Rhizopus s oloni e (Eh enb.) Vuill. 22.2 0–40
Lea es
Al e na ia al e na a (F .) Keissl. 49.7 0–39
Aspe gillus umiga us F esen. 12.4 0–56
Aspe gillus e eus Thom. 8.4 0–50
Cladospo ium cladospo ioides (F esen.) G.A. de V ies 34.7 0–87
Fusa ium oxyspo ium Schl dl. 21.3 0–34
Muco mucedo F esen. 30.0 0–56
Muco acemosus F esen. 25.8 0–35
Penicillium c us osum Thom. 15.4 0–60
Penicillium glab um (Wehme ) 17.5 0–45
Rhizopus s oloni e (Eh enb.) Vuill. 30.5 0–25
C us
Al e na ia al e na a (F .) Keissl. 35.1 0–60
Aspe gillus umiga us F esen. 12.7 0–27
Aspe gillus e eus Thom. 12.7 0–56
Cladospo ium cladospo ioides (F esen.) G. A. de V ies 35.9 0–50
Fusa ium oxyspo ium Schl dl. 23.6 0–45
Muco mucedo F esen. 15.4 0–70
Muco acemosus F esen. 12.7 0–47
Penicillium c us osum Thom. 10.7 0–7
Penicillium glab um (Wehme ) 12.5 0–20
Rhizopus s oloni e (Eh enb.) Vuill. 25.9 0–30
Pollen
Ac emonium ch ysogenum (Thi um. & Sukap.) W. Gams 38.9 0–87
Al e na ia al e na a (F .) Keissl. 54.8 0–48
Aspe gillus fl a us Link 23.5 0–90
Aspe gillus umiga us F esen. 12.7 0–58
Aspe gillus och aceus G. Wilh. 17.9 0–67
Aspe gillus e sicolo (Vuill.) Ti ab. 12.6 0–37
Cladospo ium cladospo oides (F esen.) G.A. de V ies 50.9 0–57
Fusa ium oxyspo ium Schl dl. 23.7 0–35
Muco acemosus F esen. 18.5 0–7
Penicillium c us osum Thom. 28.9 0–10
T ichode ma ha zianum Ri ai. 21.9 0–20
76 M. Kačánio á, J. Sudzino á, M. Kádasi-Ho áko á, M. Valšíko á, S. K áčma
mes ica L. lea samples (Kačánio á and Fikselo á,
2007).
Al e na ia was consis en ly isola ed om disease
lea es and ui s. Spo s did no de elop on lea es
inocula ed wi h isola e om Cas anea sa i a Mill. o
nec osis sp ead e y slowly on lea blades ea ed
wi h isola e om Chamaecypa is lawsoniana (O -
likowskij and Oszako, 2005). In C oa ia esea ch was
ca ied ou on he p esence and a ia ion in occu -
ence o ungal gene a on plan samples collec ed
om h ee diff e en clima ic egions in ela ion o
empe a u e and ela i e humidi y. The spo es be-
longing o 15 ungal gene a and s e ile mycelium
we e iden ifi ed. Cladospo ium (65.3–74%), Penicillium
(4.43–13.9%), Al e na ia (2–4.7%), and s e ile myc-
elium (7.4–15%) we e he mos p e alen ungi in
he ai o all h ee clima ic egions, while Fusa ium
(20.3–36.2%) was he mos p e alen ungal gene a
on plan samples especially in he modes con inen-
al clima e (Pepeljnjak and Šeg ič, 2003).
The mos common ungi ound in c us s o Cas-
anea sa i a Mill. we e Al e na ia al e na a, Cla do spo-
i dium cladospo oides, Fusa ium oxyspo ium and Rhizo-
pus s oloni e . The less abundance was de e mined by
Aspe gillus umiga us, Aspe gillus e eus, Muco mucedo,
Muco acemosus, Penicillium c us osum and Penicillium
glab um (Tab. I). Cladospo idium cladospo oides was by
a he mos common spoile o c us samples con-
amina ing 35.9%. Al e na ia al e na a (89%), Cladospo-
ium cladospo ioides (89%), Mycelia s e ilia (89%), Penicil-
lium sp. (89%) and T ichode ma sp. (67%) we e he mos
equen ly isola ed ungal species encoun e ed on
he su ace o he So bus domes ica L. c us samples
(Kačánio á and Fikselo á, 2007).
Some scien is iden ifi ed in he ches nu s Pho-
mopsis cas anea, which occu ed endophy ically in
plan pa s o Eu opean ches nu including fl o we s,
lea es, shoo s and ui s. The appa en ly heal hy
ees o ou cul i a s we e sampled se en imes
h oughou 1 yea . This ungus is associa ed wi h
he impo an pos -ha es disease Phomopsis nu o
(Washing on e al., 1999). I was ound, ha he un-
gus Phomopsis cas anea could be associa e wi h bo h
o en and heal hy nu s in all he No h Island sam-
ples, al hough he e was a la ge a ia ion in inci-
dence be ween a ie ies and loca ions. O he ungi
isola ed om o en nu s we e Bo y is cine ea and
species belonging o Penicillium, Fusa ium and Pes alo-
ia (Wadia e al., 1999).
The mos common ungi ound in pollen o Cas-
anea sa i a Mill. we e Ac emonium ch ysogenum, Al-
e na ia al e na a, Aspe gillus fl a us, Cladospo idium
cladospo oides, Fusa ium oxyspo ium, Penicillium c us o-
sum and T ichode ma ha zianum. Less common we e
Aspe gillus umiga us, Aspe gillus och aceus, Aspe gillus
e sicolo and Muco acemosus (Tab. I). Cladospo idium
cladospo oides was by a he mos common spoile
o pollen samples con amina ing 35.9%. Six mic o-
scopic ungal species we e isola ed om So bus do-
mes ica L. pollen samples: Al e na ia al e na a, Aspe gil-
lus fl a us, Aspe gillus nige , Cladospo ium cladospo ioides,
Fusa ium sp. and Penicillium sp. The mos equen ly
species ound in he pollen samples we e Penicil-
lium sp. (100%), Aspe gillus nige (67%) and Cladospo-
ium cladosp ioides (67%) (Kačánio á and Fikselo á,
2007).
Fungi belong o he genus Aspe gillus we e de-
ec ed in 80% o pollen samples being clea ly p e-
dominan Aspe gillus sec ion Nig i. Species o his
sec ion we e ound in 66.6% o he pollen samples.
The g oup A. fl a us plus A. pa asi icus was ound
in 23.3% o he samples. O he species o Aspe gil-
lus we e ound bu only in 10% o he samples.
Among hese species i is ema kable he p esence
o A. och aceus, which was isola ed in 6% o he pol-
len samples. The genus Penicillium con amina ed
90 % o he samples, al hough P. e ucosum was iso-
la ed in only 6.6% o he samples. O he Penicillium
species we e ound con amina ing 83.3% o he sam-
ples. Species o he genus Fusa ium we e less com-
mon. They we e isola ed in 53.3% o he pollen sam-
ples. Al e na ia sp. was ound in 86.6% o he pollen
samples. O he ungi o low in e es ega ding my-
co oxin p oduc ion we e isola ed. Cladospo ium and
Rhizopus we e ound in 90% and 80% o he samples,
espec i ely. Muco was isola ed in 46.6% (González
e al., 2005).
SOUHRN
S ano ení mik oskopických hub z ořechů, lis í, ků y a pylu kaš ano níka jedlého
(Cas anea sa i a Mill.)
Ve č yřech slo enských egionech byly s udo ány z ahy medzi os linou a mik oo ganismy s ohle-
dem na s ano ení mykofl o y u Cas anea sa i a Mill. (ořechy, ků a, lis í a pyl) a jejich li na hos i el-
ský o ganismus. V expe imen ech bylo izolo áno 7 odů a 10 d uhů mik oskopických hub z oře-
chů, ků y a lis í. Bylo zjiš ěno, že izolá y z pylu Cas anea sa i a Mill. byly zas oupeny osmi ody a je-
denác i d uhy mik oskopických hub. U izolá ů ořechů se nejčas ěji ysky o aly d uhy Al e na ia al e -
na a (36,4%), Cladospo ium cladospo ioides (43,7%), Fusa ium oxyspo ium (23,8 %) a Rhizopus s oloni e (22,2%).
U izolá ů lis ů se nejčas ěji ysky o aly d uhy Al e na ia al e na a (49,7%) a Cladospo ium cladospo ioi-
des (34,7%) a ků y se nejčas ěji ysky o aly d uhy Al e na ia al e na a (35,1%) a Cladospo ium cladospo i-
oides (35,9%). D uhy Al e na ia al e na a (54,8%) a Cladospo ium cladospo ioides (50,9%) byly nejčas ěji y-
sky ující se mik oskopické houby pylu. Na základě dalších axonomických u čení z odů Aspe gillus
byly izolo ány a iden ifi ko ány zás upci d uhů A. fl a us, A. umiga us, A. och aceus, A. e eus a A. e si-
The de e mina ion o mic oscopic ungi om Ches nu (Cas anea sa i a Mill.) ui s, lea es, c us and pollen 77
colo . Z odu Fusa ium byl izolo án d uh F. oxyspo um a z odu Penicillium byly izolo ány d uhy P. c us o-
sum a P. glab um. Zdů azňujeme, že izolo ané ody Aspe gillus, Fusa ium a Penicillium, jsou po ažo ány
za nej ýznamnější p oducen y myko oxínů. Typič í p oducen i myko oxínů z odů Penicillium, Al-
e na ia, Fusa ium a Aspe gillus byli de eko áni na če s ých ořechách, lis ech, kůře a pylu při labo a-
o ní eplo ě. U šech es o aných čás í Cas anea sa i a Mill. bylo zaznamenáno ysoké p ocen o plís-
ňo é kon aminace. Rody Al e na ia, Cladospo ium, Penicillium a Fusa ium byly pře ládající e šech izo-
lá ech os liny. Něk e é z odů (např. Cladospo ium, Al e na ia a Penicillium) jsou po enciálními ale geny,
za předpokladu ysoké p odukce konidií. Pok očilé kažení ořechů by mohlo mí nepřízni ý účinek
na zd a í pe sonálu, k e ý nakládá s ěmi o komodi ami. Z láš ní opa ření by měla bý přija a během
sklizně, čiš ění, řídění, balení, přep a ě, sklado ání a u ádění zboží na h. Chlazení je důleži é p o
ud žení k ali y a p odloužení anli os i, i když něk e é d uhy mohou ůs při nízkých eplo ách.
Plody, k e é jsou ci li é na nízké eplo y, by měly bý u áděny na h ychle, aby se zab ánilo houbo é
kon aminaci a po enciální o bě myko oxinů a ím sou isejícímu zniku zd a o ního izika.
Cas anea sa i a Mill., ořechy, ků a, pyl, mik oskopické houby
The au ho s hank M. Tonko a o excellen labo a o y assis ance om Depa men o Mic obiology,
Slo ak Uni e si y o Ag icul u e in Ni a and Depa men o Plan Gene ics and Plan B eeding, Slo-
ak Uni e si y o Ag icul u e in Ni a o p o iding he samples.
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Add ess
doc. Ing. Mi osla a Kačánio á, Ph.D., Ing. Mi iam Kádasi-Ho áko á, PhD. Ka ed a mik obiologie, Fakul a
bio echnológie a po a iná s a, p o . Ing. Magdaléna Valšíko á, PhD., Ka ed a zeleniná s a, Fakul a záh-
adníc a a k ajinného inžinie s a, Slo enská poľnohospodá ska uni e zi a Ni e, T . A. Hlinku 2, 949 71
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um ýskumu ži očíšnej ý oby Ni a, Hloho ecká 2, 951 41 Lužianky, Slo enská epublika, p o . Ing. S a-
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