Academic Edi o : Da id S. Pe lin
Recei ed: 24 No embe 2024
Re ised: 19 Decembe 2024
Accep ed: 30 Decembe 2024
Published: 1 Janua y 2025
Ci a ion: Ma ínez-Ríos, M.;
Ma ín-To ijos, L.; Diéguez-
U ibeondo, J. T achemys sc ip a Eggs as
Pa o a Po en ial In Vi o Model o
S udying Sea Tu le Egg Fusa iosis. J.
Fungi 2025,11, 23. h ps://doi.o g/
10.3390/jo 11010023
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
T achemys sc ip a Eggs as Pa o a Po en ial In Vi o Model o
S udying Sea Tu le Egg Fusa iosis
Ma ía Ma ínez-Ríos , Lau a Ma ín-To ijos and Ja ie Diéguez-U ibeondo *
Depa men o Mycology, Real Ja dín Bo ánico CSIC, Plaza Mu illo 2, 28014 Mad id, Spain;
[email p o ec ed] (M.M.-R.); [email p o ec ed] (L.M.-T.)
*Co espondence: [email p o ec ed]
Abs ac : The ungal pa hogens Fusa ium ke a oplas icum and Fusa ium alci o me a e e-
sponsible o he eme ging in ec ious disease named sea u le egg usa iosis (STEF). This
disease a ec s all sea u le species h oughou he wo ld, causing low ha ching success
and mass mo ali ies. In his s udy, we in es iga ed he po en ial use o widely a ailable
and easy- o-handle eggs o he in asi e alien ed-ea ed slide u le, T achemys sc ip a, as
pa o an
in i o
hos model o imp o e ou knowledge o he biological p ope ies o
he pa hogens esponsible o he STEF. Speci ically, we pe o med
in i o
expe imen s,
in which T. sc ip a eggs we e challenged wi h conidia o F. ke a oplas icum isola ed om
diseased sea u le eggs. We ound ha he pa hogen could colonize and de elop simila
signs o hose obse ed in na u e and ul ill Koch’s pos ula es. The pa hogen showed
high i ulence p ope ies (e.g., high disease incidence, se e i y, and low ha ching success)
and i s abili y o modi y he pH in bo h he egg su ace and cul u e media, con i ming
p e iously desc ibed ungal pa hogen models. These esul s suppo he use o T. sc ip a
as an expe imen al
in i o
hos model o s udying he biological cha ac e is ics o STEF,
hus p o iding aluable insigh s in o he mechanisms unde lying he eme gence o his
ungal disease.
Keywo ds: pa hogen; eme ging ungal diseases; ascomyce es; pH; u le; Fusa ium
ke a oplas icum; mul ilocus sequence ypes (MLSTs)
1. In oduc ion
O e he pas ew decades, he incidence and i ulence o ungal pa hogens ha e
inc eased along wi h an expansion in hei geog aphic dis ibu ion and hos ange [
1
–
3
].
Di e se ungal axa a e esponsible o eme ging ungal diseases (EFDs) and o some o he
wo s declines in wildli e species, especially endange ed animal species [
1
,
2
,
4
–
6
]. Some o
he common key biological p ope ies o eme ging ungal pa hogens allow hem o mani es
as EFDs [
1
,
7
]. Fo example, hey (i) can show high i ulence, i.e., he ela i e capaci y o
he pa hogen o cause damage o he hos [
8
]. This capaci y is associa ed wi h apid in a-
hos g ow h a es [
9
,
10
] and/o modula ed pH changes in he hos ha allow o ungal
coloniza ion [
11
,
12
]. P e ious s udies ha e shown ha ce ain ascomyce e ungi, such as
Colle o h ichum acu a um [
13
], Fusa ium oxyspo um [
14
], and Penicillium expansum [
15
], can
locally modi y he hos pH in esponse o ca bon a ailabili y, he eby modula ing he ac i-
a ion o genes in ol ed in pa hogenici y [
11
]. (ii) They possess long-li ed en i onmen al
s ages and he abili y o su i e as sap obes ou side hei hos , acili a ing hei su i al in
di e se en i onmen s wo ldwide as cosmopoli e o ganisms [
1
]. (iii) They a e oppo unis ic
( hey depend on en i onmen al o hos condi ions o in ec ions) and gene alis pa hogens
J. Fungi 2025,11, 23 h ps://doi.o g/10.3390/jo 11010023
J. Fungi 2025,11, 23 2 o 12
( hey can in ec a wide ange o hos s), which allows he pa hogen o easily dispe se, in ec ,
and gene a e high loads o inoculum. When pa hogen h esholds o in ec ion a e eached
and en i onmen al condi ions o immune sys em ailu e leads o he de elopmen o he
disease, he pa hogen mani es s as an EFD [16].
The sea u le egg usa iosis (STEF) is a ecen ly desc ibed EFD [
17
] ha a ec s
sea u le eggs wo ldwide. The disease is caused by he ascomyce ous ungi Fusa ium
ke a oplas icum and Fusa ium alci o me [
17
,
18
], which belong o he Fusa ium solani species
complex (FSSC). These species ha e been desc ibed ecen ly [
19
,
20
], and hei iden i ica ion
equi es a mul ilocus sequence ype (MLST) app oach based on he combined analysis o
se e al loci [
20
]. These pa hogens a e globally dis ibu ed [
21
] and a e associa ed wi h bo h
low ha ching success [
18
,
22
–
24
] and se e e mass emb yonic mo ali ies in he wild [
18
,
22
].
The cha ac e is ic signs o STEF in a ec ed eggs include he p esence o ungal mycelium
g ow h on he egg su ace and/o abno mal discolo a ions (e.g., yellowish, bluish, g ayish,
and eddish) on he eggshell, as desc ibed in Sa mien o-Ramí ez e al. [
18
,
22
]. These
pa hogens appea o g ow slowly on he egg su ace and apidly colonize in e nally when
condi ions a e a o ed [
18
]. Howe e , much emains unknown abou hei biological
p ope ies, in ec ion p ocesses, o i ulence. Resea ch on hese aspec s is challenging
o conduc in si u due o ad e se en i onmen al condi ions in opical egions and he
logis ical di icul ies o ob aining esea ch pe mi s o s udies on h ea ened species [
25
].
Thus, iden i ying sui able model o ganisms is a signi ican ad an age o his ype o
esea ch. Fo example, he zeb a ish has been a model o s udy he bac e ial pa hogenesis
o S ep ococcus spp. [
26
], Paci as acus leniusculus o Aphanomyces as aci pa hogenici y and
in e eb a e immuni y [
27
], o Galle ia mellonella o he ish pa hogenici y o Sap olegnia
pa asi ica [28].
In asi e alien species a e ideal o ganisms o his pu pose since hey mee mos o he
equi emen s equi ed o model o ganisms; i.e., hey a e widely a ailable, easy o main ain
and inexpensi e, and assays can be easily eplica ed. Fo example, he ed-ea ed slide
u le T achemys sc ip a is a semiaqua ic u le lis ed among he Wo ld’s 100 Wo s In asi e
Alien Species [
29
] and as an in asi e alien species o Eu opean Union conce n [
30
]. Du ing
he las ew decades, he e a e p og ams used o he con ol and e adica ion o his ep ile
in some Eu opean s a es [
31
–
33
], which make eggs o his in asi e species easily a ailable
o expe imen al s udies.
In his s udy, we assessed he u ili y o he eggs o he in asi e species ed-ea ed slide
u le, T. sc ip a, as a hos model o
in i o
expe imen s on he biological p ope ies associ-
a ed wi h he coloniza ion o STEF-causing pa hogens. Fo his pu pose, we speci ically
(i) conduc ed an inocula ion expe imen in T. sc ip a eggs wi h F. ke a oplas icum o in es-
iga e hei pa hogenic p ope ies on he hos model o ganism; (ii) calcula ed he disease
incidence, disease se e i y, and ha ching a e o e alua e he use ulness o he po en ial
model o ganisms; (iii) checked he compliance o Koch’s pos ula es; and (i ) cha ac e ized
he pH changes du ing ungal g ow h as one o he main biological p ope ies o i ulence.
2. Ma e ials and Me hods
2.1. Sample Collec ion
Du ing he mon hs o May o July (laying season o he species [
34
]) o 2019, a o al
o 564 T. sc ip a eggs om 94 nes s we e collec ed om El Ma jal de Almena a we land
(Figu e 1) in he p o ince o Cas ellón (Spain) by he Depa men o In as uc u e, Te i o y
and En i onmen o he Gene ali a de Valencia as desc ibed in Ma ínez-Ríos e al. [
21
]
and, subsequen ly, anspo ed and main ained in an a i icial incuba o a 29.5
±
0.5
◦
C
o one day be o e s a ing he challenging expe imen s.
J. Fungi 2025,11, 23 3 o 12
J. Fungi 2025, 11, x FOR PEER REVIEW 3 o 13
[21] and, subsequen ly, anspo ed and main ained in an a i icial incuba o a 29.5 ± 0.5
°C o one day be o e s a ing he challenging expe imen s.
Figu e 1. Sampling a ea o T achemys sc ip a nes s, El Ma jal de Almena a we land, Cas ellón (Spain).
2.2. Inocula ion o T. sc ip a Eggs wi h F. ke oplas icum
In o de o eplica e he STEF disease signs, we inocula ed T. sc ip a eggs wi h a F.
ke a oplas icum isola e (RJB-FCR34A) ob ained om a lea he back u le (De mochelys co i-
acea) a Pacua e Na u e Rese e, Cos a Rica, in 2018. We selec ed a o al o 45 eggs om
30 nes s wi hou symp oms o STEF as desc ibed in Sa mien o-Rami ez e al. [18,22]. The
eggs we e andomly dis ibu ed in h ee plas ic boxes (i.e., h ee expe imen eplica es)
wi h s e ile we e miculi e as an incuba ing subs a e. In hese boxes, he eggs we e
placed in h ee ows wi h i e eggs each (i.e., 15 eggs o each expe imen al eplica e)
(Figu e 2). The i s and second ows we e used as con ols. Eggs in he i s ow (con ol
1 he ea e ) we e no inocula ed wi h he pa hogen no we e hey washed wi h dis illed
wa e be o e he expe imen . Eggs in he second ow (con ol 2 he ea e ) we e no inocu-
la ed wi h he pa hogen, bu hey we e washed wi h dis illed wa e (pH = 7) o emo e
aces o clay om he su ace be o e he s a o he incuba ion pe iod conside ing he
ac ha he spo es o he ungus can pe sis in he soil o se e al yea s [35]. The hi d
ow (inocula ed eggs he ea e ) con ained eggs ha we e i s washed and hen inocula ed
wi h a 6 mm diame e pep one glucose aga (PGA) disc wi h an ac i ely g owing ungal
inoculum o F. ke a oplas icum. The plas ic boxes we e incuba ed in wo a i icial incuba-
o s a 29.5 ± 0.5 °C, which co espond o he pi o al empe a u e o T. sc ip a eggs [36].
The eggs we e checked daily du ing wo mon hs o de ec ungal g ow h, and we sp ayed
s e ile wa e (pH = 7) o keep he e miculi e mois u e. Du ing he en i e incuba ion pe-
iod, we mac oscopically looked o he p esence o common signs o STEF disease on
eggs, i.e., he p esence o ungal mycelium g ow h on he ou side o he eggs and/o a yp-
ically colo ed a eas (e.g., yellowish, bluish, g ayish, eddish) on he eggshell as desc ibed
in Sa mien o-Ramí ez e al. [18,22].
Figu e 1. Sampling a ea o T achemys sc ip a nes s, El Ma jal de Almena a we land, Cas ellón (Spain).
2.2. Inocula ion o T. sc ip a Eggs wi h F. ke oplas icum
In o de o eplica e he STEF disease signs, we inocula ed T. sc ip a eggs wi h a
F. ke a oplas icum isola e (RJB-FCR34A) ob ained om a lea he back u le (De mochelys
co iacea) a Pacua e Na u e Rese e, Cos a Rica, in 2018. We selec ed a o al o 45 eggs om
30 nes s wi hou symp oms o STEF as desc ibed in Sa mien o-Rami ez e al. [
18
,
22
]. The
eggs we e andomly dis ibu ed in h ee plas ic boxes (i.e., h ee expe imen eplica es)
wi h s e ile we e miculi e as an incuba ing subs a e. In hese boxes, he eggs we e
placed in h ee ows wi h i e eggs each (i.e., 15 eggs o each expe imen al eplica e)
(Figu e 2). The i s and second ows we e used as con ols. Eggs in he i s ow (con ol 1
he ea e ) we e no inocula ed wi h he pa hogen no we e hey washed wi h dis illed wa e
be o e he expe imen . Eggs in he second ow (con ol 2 he ea e ) we e no inocula ed
wi h he pa hogen, bu hey we e washed wi h dis illed wa e (pH = 7) o emo e aces
o clay om he su ace be o e he s a o he incuba ion pe iod conside ing he ac
ha he spo es o he ungus can pe sis in he soil o se e al yea s [
35
]. The hi d ow
(inocula ed eggs he ea e ) con ained eggs ha we e i s washed and hen inocula ed
wi h a 6 mm diame e pep one glucose aga (PGA) disc wi h an ac i ely g owing ungal
inoculum o
F. ke a oplas icum
. The plas ic boxes we e incuba ed in wo a i icial incuba o s
a
29.5 ±0.5 ◦C
, which co espond o he pi o al empe a u e o T. sc ip a eggs [
36
]. The
eggs we e checked daily du ing wo mon hs o de ec ungal g ow h, and we sp ayed
s e ile wa e (pH = 7) o keep he e miculi e mois u e. Du ing he en i e incuba ion pe iod,
we mac oscopically looked o he p esence o common signs o STEF disease on eggs,
i.e., he p esence o ungal mycelium g ow h on he ou side o he eggs and/o a ypically
colo ed a eas (
e.g., yellowish
, bluish, g ayish, eddish) on he eggshell as desc ibed in
Sa mien o-Ramí ez e al. [18,22].
2.3. Disease Incidence, Disease Se e i y, and Ha ching Ra e
Du ing he wo-mon h incuba ion pe iod, we examined he p esence o common signs
o ungal in ec ion on eggs. A he end o he incuba ion pe iod, we assessed (i) he disease
incidence, i.e., he numbe o in ec ed eggs ou o o al o e he incuba ion pe iod [
37
];
and (ii) he disease se e i y, i.e., he le el o he in ec ion caused in he eggs, using a scale
om 0 o 5 based on he pe cen age o egg shell su ace a ec ed, i.e., he in ec ion co e age:
0 = no signs,
1 =
≤
20%, 2 = 21 o 40%, 3 = 41 o 60%, 4 = 61 o 80% and 5 = >80% o he egg
shell su ace was diseased [
38
]; and (iii) he ha ching a e as he numbe o eggs ha ched
ou o he o al numbe o eggs.
J. Fungi 2025,11, 23 4 o 12
J. Fungi 2025, 11, x FOR PEER REVIEW 4 o 13
Figu e 2. Design o he expe imen al in i o hos model using eggs o T achemys sc ip a. Real (le )
and schema ic ( igh ) image o an expe imen ca ied ou in plas ic boxes wi h s e ile we e micu-
li e as an incuba ing subs a e. In each expe imen , 15 eggs a anged in h ee columns we e used
( i e eggs/ ow). Each ow ep esen s one ype o ea men (le o igh ): (i) con ol 1, which con ains
eggs as hey came om he sample collec ion; (ii) con ol 2, which con ains washed eggs wi h dis-
illed wa e o emo e aces o clay om he su ace; and (iii) washed and inocula ed eggs wi h
g owing ungal inoculum o Fusa ium ke a oplas icum.
2.3. Disease Incidence, Disease Se e i y, and Ha ching Ra e
Du ing he wo-mon h incuba ion pe iod, we examined he p esence o common
signs o ungal in ec ion on eggs. A he end o he incuba ion pe iod, we assessed (i) he
disease incidence, i.e., he numbe o in ec ed eggs ou o o al o e he incuba ion pe iod
[37]; and (ii) he disease se e i y, i.e., he le el o he in ec ion caused in he eggs, using a
scale om 0 o 5 based on he pe cen age o egg shell su ace affec ed, i.e., he in ec ion
co e age: 0 = no signs, 1 = ≤20%, 2 = 21 o 40%, 3 = 41 o 60%, 4 = 61 o 80% and 5 = >80%
o he egg shell su ace was diseased [38]; and (iii) he ha ching a e as he numbe o eggs
ha ched ou o he o al numbe o eggs.
2.4. Reisola ion o F. ke a oplas icum om Diseased T. sc ip a Eggshell
In o de o de e mine whe he he in ec ion signs we e caused by he F. ke a oplas i-
cum isola e (RJB-FCR34A) used in he inocula ion expe imen , we e i ied ha Koch’s pos-
ula es we e ul illed. Koch’s pos ula es a e a se o c i e ia o iden i y whe he a pa icula
o ganism is he causa i e agen o a pa icula disease, based on he isola ion o he pa h-
ogen om a diseased issue, in ec ion o a heal hy hos wi h he isola ed pa hogen, and
he eisola ion o he pa hogen o p o e causa ion o his in ec ion p ocess [39]. Thus, wo
mon hs a e he inocula ion and coinciding wi h he pe iod needed o he emb yonic
de elopmen o T. sc ip a [40], we selec ed om each inocula ed egg a shell piece showing
ypical signs o STEF. In addi ion, we also ook shell pieces om some o he con ol eggs
o check he na u al p esence o F. alci o me and F. ke a oplas icum in he eggs. F om he
selec ed a eas o he eggs, we cu shell pieces (0.5 × 0.5 cm) and placed hem on o PGA
Pe i dishes supplemen ed wi h ampicillin (100 mg/L) as desc ibed by Ma ínez-Ríos e al.
[21,41]. The samples we e incuba ed a 25 °C o 2–5 days un il a mycelium was o med.
Figu e 2. Design o he expe imen al
in i o
hos model using eggs o T achemys sc ip a. Real (le )
and schema ic ( igh ) image o an expe imen ca ied ou in plas ic boxes wi h s e ile we e miculi e
as an incuba ing subs a e. In each expe imen , 15 eggs a anged in h ee columns we e used ( i e
eggs/ ow). Each ow ep esen s one ype o ea men (le o igh ): (i) con ol 1, which con ains eggs
as hey came om he sample collec ion; (ii) con ol 2, which con ains washed eggs wi h dis illed
wa e o emo e aces o clay om he su ace; and (iii) washed and inocula ed eggs wi h g owing
ungal inoculum o Fusa ium ke a oplas icum.
2.4. Reisola ion o F. ke a oplas icum om Diseased T. sc ip a Eggshell
In o de o de e mine whe he he in ec ion signs we e caused by he F. ke a oplas icum
isola e (RJB-FCR34A) used in he inocula ion expe imen , we e i ied ha Koch’s pos ula es
we e ul illed. Koch’s pos ula es a e a se o c i e ia o iden i y whe he a pa icula
o ganism is he causa i e agen o a pa icula disease, based on he isola ion o he
pa hogen om a diseased issue, in ec ion o a heal hy hos wi h he isola ed pa hogen,
and he eisola ion o he pa hogen o p o e causa ion o his in ec ion p ocess [
39
]. Thus,
wo mon hs a e he inocula ion and coinciding wi h he pe iod needed o he emb yonic
de elopmen o T. sc ip a [
40
], we selec ed om each inocula ed egg a shell piece showing
ypical signs o STEF. In addi ion, we also ook shell pieces om some o he con ol eggs o
check he na u al p esence o F. alci o me and F. ke a oplas icum in he eggs. F om he selec ed
a eas o he eggs, we cu shell pieces (0.5
×
0.5 cm) and placed hem on o PGA Pe i dishes
supplemen ed wi h ampicillin (100 mg/L) as desc ibed by
Ma ínez-Ríos e al. [21,41]
. The
samples we e incuba ed a 25 ◦C o 2–5 days un il a mycelium was o med.
2.5. Molecula Iden i ica ion o F. ke a oplas icum Isola es om Expe imen ally-In ec ed T. sc ip a
Egg Shell
Molecula cha ac e iza ion o he cul u ed ungal eisola es was pe o med by am-
pli ying he nuclea DNA loci ITS n DNA, LSU n DNA, RPB2 nDNA, and TEF nDNA
o each isola e, as used in he MLSTs app oach [
20
] o iden i ying FSSC axa, ollowing
he p o ocol desc ibed in Ma ínez-Ríos e al. [
21
]. A single ound o PCR was pe o med
acco ding o Sa mien o-Ramí ez e al. [
18
]. As a posi i e con ol, we used DNA ex ac ed
om an axenic cul u e o RJB-021FUS F. ke a oplas icum om he Real Ja dín Bo ánico-
CSIC cul u e collec ion. Dis illed MiliQ wa e was used as a nega i e con ol. Ampli ied
p oduc s we e isualized by elec opho esis in a 1% aga ose gel s ained wi h SYBR
®
Sa e
J. Fungi 2025,11, 23 5 o 12
DNA (In i ogen
®
, Mad id, Spain). Posi i e ampli ied p oduc s we e sequenced using
an au oma ed sequence (MACROGEN, Inc., Mad id, Spain). The esul ed sequences
we e isualized and edi ed wi h Geneious .10.2.3 [
42
]. Finally, we conduc ed a BLAST
sea ch o each o he ob ained sequences om each isola e o e i y hei iden i ies in he
GenBank da abase.
2.6. Physiological Cha ac e iza ion: pH Change
In o de o check whe he his ungal pa hogen esponsible o STEF p oduces pH
changes acco ding o he a ailabili y o ca bon based on he ungal pa hogen model
desc ibed by [
11
], we measu ed he pH in a pep one glucose liquid media (PGl), which
has high ca bon concen a ions, and in he inocula ed egg shell su ace, which has low
ee-ca bon concen a ions. Fo his pu pose, we selec ed h ee isola es o F. ke a oplas icum
(RJB-FUS34A, RJB-FUS39A, and RJB-FUS37C) ob ained om a lea he back u le om
Pacua e Na u e Rese e. Fo measu ing he pH in PGl, we ans e ed a 6 mm diame e
aga disc wi h ac i e ungal g ow h in o 15 mL ubes con aining 10 mL o PGl media o
each isola e. We measu ed he pH o he medium o he ollowing 7 days using a pH me e
(mic opH 2001, C ison
®,
Fishe Manu ac u e , Mad id, Spain). The pH alues measu ed o
each ungal pa hogen species we e plo ed g aphically, including he mean alue o h ee
eplica es pe ungal isola e, using he “ggplo 2” package [
43
] in he so wa e R .3.4.1 [
44
].
2.7. S a is ical Analysis
The alues o he disease incidence, disease se e i y, and ha ching a e om he
selec ed ea men s (con ol 1, con ol 2, and inocula ed eggs) we e s a is ically analyzed
by a one-way ANOVA and subsequen ly compa ed wi h a pos hoc Tukey es p o ided
in he so wa e R .3.4.1 [
44
]. Fu he mo e, we compa ed whe he he e we e signi ican
di e ences be ween he ea men s, i.e., inocula ed eggs and con ols using S uden ’s - es
in he so wa e R .3.4.1 [44].
The di e ences in pH alues o each species we e analyzed applying S uden ’s - es
in R .3.4.1 [
44
]. Fo measu ing pH alues in egg shells, we used pH indica o s ips (Me k
KGaA, Da ms ad , Ge many) on en egg shells showing STEF signs and ano he en egg
shells ee o STEF.
3. Resul s
3.1. Inocula ion Expe imen Using Eggs o T. sc ip a: Disease De elopmen
A he ime o collec ion, none o he eggs showed signs o STEF in ec ion caused
by F. alci o me and F. ke a oplas icum, namely he p esence o ungal mycelium and/o
a ypical colo ed spo s on he egg shell as desc ibed below. A e he i s week ollowing
he inocula ion, FSSC in ec ion de eloped and he i s signs o STEF disease appea ed.
3.2. Disease Incidence, Disease Se e i y, and Ha ching Ra e in T. sc ip a Eggs
Di e ences in disease incidence be ween he expe imen ea men s (i.e., inocula ed
eggs, con ol 1, and con ol 2) we e s a is ically signi ican (p- alue < 0.001). A o al o
14 eggs ou o 15 inocula ed wi h F. ke a oplas icum showed de elopmen o Fusa ium
mycelium on he egg shell and/o ypical colo ed spo s o he STEF de elopmen (Figu e 3).
In con ol 1 (unwashed eggs), 5 o he 15 eggs showed signs o he in ec ion, and in
con ol 2 (washed eggs), 8 o he 15 eggs show signs o in ec ion. Howe e , he e we e no
s a is ical di e ences be ween he eplica es (p- alue = 0.075) no be ween bo h con ols
(p- alue = 0.23
). The e o e, esul s indica ed ha disease incidence in inocula ed eggs was
s a is ically highe han in con ol eggs (Table 1).
J. Fungi 2025,11, 23 6 o 12
J. Fungi 2025, 11, x FOR PEER REVIEW 6 o 13
3.2. Disease Incidence, Disease Se e i y, and Ha ching Ra e in T. sc ip a Eggs
Diffe ences in disease incidence be ween he expe imen ea men s (i.e., inocula ed
eggs, con ol 1, and con ol 2) we e s a is ically signi ican (p- alue < 0.001). A o al o 14
eggs ou o 15 inocula ed wi h F. ke a oplas icum showed de elopmen o Fusa ium myce-
lium on he egg shell and/o ypical colo ed spo s o he STEF de elopmen (Figu e 3). In
con ol 1 (unwashed eggs), 5 o he 15 eggs showed signs o he in ec ion, and in con ol 2
(washed eggs), 8 o he 15 eggs show signs o in ec ion. Howe e , he e we e no s a is ical
diffe ences be ween he eplica es (p- alue = 0.075) no be ween bo h con ols (p- alue =
0.23). The e o e, esul s indica ed ha disease incidence in inocula ed eggs was s a is i-
cally highe han in con ol eggs (Table 1).
Figu e 3. Signs o sea u le egg usa iosis on eggs o T achemys sc ip a. (A–C) In ec ed eggs showing
whi e ungal mycelium g ow h a ound he egg shell (a ow) (scale ba = 1 cm). (B,D–F) In ec ed eggs
showing he g ay spo s (a ow) ypically associa ed wi h his disease (scale ba = 1 cm).
J. Fungi 2025,11, 23 7 o 12
Table 1. Disease incidence, disease se e i y, and ha ching success o he
in i o
expe imen o
T achemys sc ip a eggs wi h Fusa ium ke a oplas icum (Fk).
T ea men Disease Incidence (%) aDisease Se e i y bHa ching Success c
Con ol 1 (non- ea ed eggs) 33.3 0.87 ±1.3 2/15
Con ol 2 (washed eggs) 53.3 1.6 ±1.55 4/15
Inocula ed eggs wi h Fk 93.3 * 3.87 ±1.73 * 3/15
a
Disease incidence is exp essed as he pe cen age o he numbe o in ec ed eggs ou o he o al eggs.
b
Disease
se e i y is ep esen ed as a scale om 0 o 5 based on he pe cen age o egg shell su ace a ec ed by he in ec ion
wi h 0= no signs and 1 =
≤
20, 2 = 21 o 40, 3 = 41 o 60, 4 = 61 o 80 and 5 = >80%.
c
Ha ching success is ep esen ed
as he numbe o ha ched eggs ou o he o al eggs. * An as e isk deno es signi ican di e ences om o he
ea men s a p- alue > 0.05.
The esul s ega ding disease se e i y we e also signi ican ly di e en (p- alue < 0.001)
be ween he expe imen ea men s (i.e., inocula ed eggs, con ol 1, and con ol 2). The inocula ed
eggs showed di e ences in in ec ion co e ages: eigh eggs had a 5- alue in he scale (100% o
he egg shell su ace a ec ed), ou eggs had a 4- alue (60–80% o he egg shell su ace a ec ed),
and wo eggs had a 1- alue (less han 20% o he su ace a ec ed). The con ol eggs showed also
di e en in ec ion co e age. Thus, in con ol 1, nine eggs had a 0- alue (no signs o in ec ion);
one egg had a 1- alue (less han 20% o he egg shell su ace a ec ed); wo eggs had a 2- alue
(20–40% a ec ed), one egg had a 3- alue (40–60% a ec ed), and one egg had a 4- alue (60–80%
a ec ed). In con ol 2, eigh eggs had a 0- alue (no signs o in ec ion); one egg had a 1- alue
(less han 20% o he egg shell su ace a ec ed); wo eggs had a 2- alue (20–40% a ec ed),
h ee eggs had a 3- alue (40–60% a ec ed), and wo eggs had a 4- alue (60–80% a ec ed).
Howe e , no con ols we e ound wi h a 100% egg shell su ace co e age by he pa hogen.
The e we e no signi ican di e ences be ween con ols 1 and 2 (p- alue = 0.75) no be ween he
eplica es (p- alue = 0.59). The e o e, esul s indica ed ha disease se e i y in inocula ed eggs
was s a is ically highe han in con ol eggs (Table 1).
The e we e no signi ican di e ences (p- alue = 0.68) in ha ching success be ween he
expe imen ea men s (i.e., inocula ed eggs, con ol 1, and con ol 2). O he 45 eggs used
in he expe imen , only nine ha ched (Figu e 4): h ee eggs in inocula ed eggs, wo eggs in
con ol 1, and ou eggs in con ol 2 (Table 1). Se en o he nine ha ching eggs had no signs
o in ec ion (p- alue < 0.01), while he o he wo had less o 20% o hei egg shell su ace
a ec ed by in ec ion.
J. Fungi 2025, 11, x FOR PEER REVIEW 8 o 13
Figu e 4. Non-in ec ed eggs o T achemys sc ip a. (A) An egg ha shows no signs o ungal in ec ion
(scale ba = 1 cm). (B) Non-in ec ed unha ched (le ) and ha ched ( igh ) eggs (scale ba = 1 cm).
3.3. Koch’s Pos ula es: Fungal Isola ion and Molecula Cha ac e iza ion
We eisola ed Fusa ium colonies om all inocula ed egg shells wi h signs o STEF
in ec ion and om wo egg shells om con ol 1 and wo om con ol 2, showing simila
signs. BLAST sea ch esul s o he ou sequenced loci (i.e., ITS n DNA, LSU n DNA,
RPB2 nDNA, and TEF nDNA) o he inocula ed eggs showed ha he isola es had a 100%
BLAST iden i y o he F. ke a oplas icum isola e used in he expe imen (RJB-FCR34A)
(GenBank accession numbe OL348272, OL348368, OL416122 and OL416123, espec-
i ely), excep o some o hem which showed a 100% iden i y o a F. alci o me sequence.
In he con ols, all he ob ained sequences om he isola es had a 100% iden i y o F. alci-
o me sequences.
3.4. Physiological Cha ac e iza ion o F. ke a oplas icum: Induced Changes in he pH
Colonies o F. ke a oplas icum g own on PG1 (pH = 7) acidi ied he medium o a mean
pH alue 4.35 (sd ± 0.05) (Figu e S1) a e 7 days o incuba ion. When moni o ing pH al-
ues in egg shells, we obse ed ha he pH o all in ec ed egg shell su aces anged om
8.0 o 9.0, while he pH o he non-in ec ed egg shells anged om 7.0 o 8.0.
4. Discussion
In his s udy, we demons a ed ha eggs o in asi e alien u le species T. sc ip a can
be used as an in i o expe imen al hos model o in es iga e key biological p ope ies o
he ungal pa hogens esponsible o STEF. We showed ha STEF signs could be ep o-
duced using his hos model and con i med he ul illmen o Koch’s pos ula es in he
inocula ion expe imen s. The pa hogen exhibi ed high i ulence p ope ies wi h a high
disease incidence, se e i y, low ha ching success, and he abili y o modi y he pH o bo h
he egg su ace and cul u e media [1,11,12,45]. The e o e, hese esul s con i med ha
STEF pa hogen F. ke a oplas icum possesses key biological ai s, such as high i ulence,
ha enable i o mani es as an EFD [21].
Howe e , we ound ha eggs used o he expe imen we e na u ally exposed o F.
alci o me ei he as a ca ie o due o con ac wi h spo es in he soil. This explains why
some con ol eggs showed signs o STEF, likely as a esul o na u al in ec ions by F. alci-
o me in T. sc ip a eggs, simila o wha occu ed in he inocula ed eggs. These ungal spe-
cies a e ound wo ldwide, p ima ily as soil-associa ed species [18], and unde sui able
Figu e 4. Non-in ec ed eggs o T achemys sc ip a. (A) An egg ha shows no signs o ungal in ec ion
(scale ba = 1 cm). (B) Non-in ec ed unha ched (le ) and ha ched ( igh ) eggs (scale ba = 1 cm).
J. Fungi 2025,11, 23 8 o 12
3.3. Koch’s Pos ula es: Fungal Isola ion and Molecula Cha ac e iza ion
We eisola ed Fusa ium colonies om all inocula ed egg shells wi h signs o STEF
in ec ion and om wo egg shells om con ol 1 and wo om con ol 2, showing simila
signs. BLAST sea ch esul s o he ou sequenced loci (i.e., ITS n DNA, LSU n DNA, RPB2
nDNA, and TEF nDNA) o he inocula ed eggs showed ha he isola es had a 100% BLAST
iden i y o he F. ke a oplas icum isola e used in he expe imen (RJB-FCR34A) (GenBank
accession numbe OL348272, OL348368, OL416122 and OL416123, espec i ely), excep o
some o hem which showed a 100% iden i y o a F. alci o me sequence. In he con ols, all
he ob ained sequences om he isola es had a 100% iden i y o F. alci o me sequences.
3.4. Physiological Cha ac e iza ion o F. ke a oplas icum: Induced Changes in he pH
Colonies o F. ke a oplas icum g own on PG1 (pH = 7) acidi ied he medium o a mean
pH alue 4.35 (sd
±
0.05) (Figu e S1) a e 7 days o incuba ion. When moni o ing pH
alues in egg shells, we obse ed ha he pH o all in ec ed egg shell su aces anged om
8.0 o 9.0, while he pH o he non-in ec ed egg shells anged om 7.0 o 8.0.
4. Discussion
In his s udy, we demons a ed ha eggs o in asi e alien u le species T. sc ip a can
be used as an
in i o
expe imen al hos model o in es iga e key biological p ope ies o
he ungal pa hogens esponsible o STEF. We showed ha STEF signs could be ep o-
duced using his hos model and con i med he ul illmen o Koch’s pos ula es in he
inocula ion expe imen s. The pa hogen exhibi ed high i ulence p ope ies wi h a high
disease incidence, se e i y, low ha ching success, and he abili y o modi y he pH o bo h
he egg su ace and cul u e media [
1
,
11
,
12
,
45
]. The e o e, hese esul s con i med ha
STEF pa hogen F. ke a oplas icum possesses key biological ai s, such as high i ulence, ha
enable i o mani es as an EFD [21].
Howe e , we ound ha eggs used o he expe imen we e na u ally exposed o
F. alci o me ei he as a ca ie o due o con ac wi h spo es in he soil. This explains
why some con ol eggs showed signs o STEF, likely as a esul o na u al in ec ions by
F. alci o me
in T. sc ip a eggs, simila o wha occu ed in he inocula ed eggs. These ungal
species a e ound wo ldwide, p ima ily as soil-associa ed species [
18
], and unde sui able
en i onmen al condi ions (mois u e and empe a u e) [
22
], hey can colonize he hos [
16
].
In his s udy, he incuba ion condi ions seem o ha e p o ided an op imal en i onmen
o he de elopmen o his na u ally occu ing ungal pa hogen on he eggshells. As a
esul , he disease incidence and se e i y ob ained in hese in ec ed con ol eggs we e
signi ican ly lowe han hose o he inocula ed. Al hough some con ol eggs we e washed
and cleaned o p e en hese na u ally occu ing in ec ions, his ea men was insu icien
o ully p e en i . Fo u u e expe imen s using his model, we ecommend collec ing
eggs om he u les du ing he o iposi ion, p e en ing hem om ouching he soil as
desc ibed by Sa mien o-Rami ez e al. [18].
The physiological cha ac e iza ion o F. ke a oplas icum e ealed e idence o ad an a-
geous biological p ope ies ha acili a e hos coloniza ion and i ulence. We showed ha
he pa hogen can induce pH changes in he hos en i onmen , which is a cha ac e is ic o
se e al ungal pa hogens [
11
,
12
]. Speci ically, alkaliniza ion o he egg shell (a subs a e
wi h poo ca bon a ailabili y) was obse ed in in ec ed a eas, while acidi ica ion was
de ec ed in
in i o
expe imen s unde high ca bon supply. This physiological beha io
suppo s he pH modula ion hypo hesis p oposed by P usky e al. [
11
], which sugges s ha
ca bon a ailabili y in he en i onmen is a key ac o ha igge s he sec e ion o small
pH-modula ing molecules, i.e., alkali unde limi ed ca bon o acids unde excess ca bon.
These molecules ac i a e some ungal genes encoding pa hogenici y ac o s, depending
J. Fungi 2025,11, 23 9 o 12
on he en i onmen al pH, hus enabling he pa hogen o adap o he hos . En i onmen al
alkaliniza ion in ungi is commonly media ed by ammonia. This p ocess has been desc ibed
in he pa hogens Colle o h icum acu a um [
13
] and Colle o h icum gloeospo ioides [
46
], which
causes an h acnose ui o ; he ice blas ungus Magnapo he o yzae, which causes high
losses in ice c ops wo ldwide [47]; o C yp ococcus neo o mans, which in ec s humans and
a ious animals [
48
]. In con as , many o he ungi acidi y he en i onmen o acili a e
hos issue in asion. Fo example, Pennicilium sp. and Aspe gillus sp. sec e e gluconic and
ci ic acids [
15
,
49
], while Fusa ium oxyspo um acidi ies plan su aces by p oducing usa ic
acid [
14
]. Simila mechanisms may occu in F. ke a oplas icum and F. alci o me, as hese
beha io s ha e been documen ed in o he Fusa ium species [11].
Fu he s udies using T. sc ip a eggs as an expe imen al
in i o
hos -model will help
un a el he molecula mechanisms unde lying he in ec ion p ocess o hese wo pa hogens.
As a model o ganism, T. sc ip a o e s se e al ad an ages o e o he u le species: (i) i is
widely dis ibu ed, (ii) easily a ailable, and (iii) can be main ained unde labo a o y condi-
ions. In Eu ope, his in asi e species coexis s and compe es wi h he h ea ened na i e
eshwa e u le, such as he Eu opean pond u le (Emys o bicula is) and he Medi e -
anean pond u le (Mau emys lep osa) [
40
,
50
–
53
]. One o he main pu poses o conse a ion
p og ams o hese na i e species [
54
] is o con ol and e adica e he in asi e ed-ea ed
slide u le by emo ing g a id emales and hei eggs [
55
]. These conse a ion measu es
ep esen a sou ce o T. sc ip a eggs, enabling a as - eplica i e model sys em ha is widely
dis ibu ed, easily accessible and can be main ained unde con olled condi ions.
Finally, he use o his model sys em could enhance ou unde s anding o STEF
and acili a e he de elopmen o new ea men s, p e en i e me hods, and managemen
p o ocols o add ess his disease.
5. Conclusions
1.
T achemys sc ip a eggs can be used as an expe imen al
in i o
hos model o ganism o
s udying he biological p ope ies and he in ec ion p ocess o he ungal pa hogens
esponsible o STEF disease.
2.
Fusa ium ke a oplas icum is capable o modula ing he hos en i onmen pH o allow
egg coloniza ion.
3.
Using his model o ganism, we can now en ision u he s udies ocused on imp o -
ing ou unde s anding o ac o s in ol ed in he pa hogenici y o hese ungal species
and he en i onmen al condi ions conduci e o sea u le egg usa iosis de elopmen .
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps://www.
mdpi.com/a icle/10.3390/jo 11010023/s1, Figu e S1: Acidi ica ion induced by Fusa ium ke a oplas icum
isola es cul u ed on pep one glucose medium (ini ial pH = 7). The eg ession line was de i ed om
he mean alues o iplica e measu emen s aken daily. The shaded g ay egion ep esen s he
s anda d de ia ion (SD). No s a is ically signi ican di e ences in pH changes we e obse ed among
isola es (p> 0.05).
Au ho Con ibu ions: Concep ualiza ion: M.M.-R., L.M.-T. and J.D.-U.; Me hodology: M.M.-R. and
J.D.-U.; So wa e: M.M.-R.; Valida ion: J.D.-U.; Fo mal Analysis: M.M.-R.; In es iga ion: M.M.-R.;
Resou ces: J.D.-U.; Da a cu a ion: M.M.-R.; W i ing—O iginal D a P epa a ion: M.M.-R.; W i ing—
Re iew and Edi ing: L.M.-T. and J.D.-U.; Visualiza ion: M.M.-R., L.M.-T. and J.D.-U.; Supe ision:
L.M.-T. and J.D.-U.; P ojec Adminis a ion: J.D.-U.; Funding Acquisi ion: J.D.-U. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
