Ci a ion: Fe ei a, I.; Dias, T.; C uz, C.
The Po en ial o Ec omyco hizal
Fungi o Modula e below and
Abo eg ound Communi ies May Be
Media ed by 1-Oc en-3-ol. J. Fungi
2023,9, 180. h ps://doi.o g/
10.3390/jo 9020180
Academic Edi o : Ka ina
Ma ia Ramonell
Recei ed: 22 Decembe 2022
Re ised: 13 Janua y 2023
Accep ed: 25 Janua y 2023
Published: 29 Janua y 2023
Copy igh : © 2023 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/).
Fungi
Jou nal o
A icle
The Po en ial o Ec omyco hizal Fungi o Modula e below and
Abo eg ound Communi ies May Be Media ed by 1-Oc en-3-ol
Inês Fe ei a , Te esa Dias * and C is ina C uz
cE3c—Cen e o Ecology, E olu ion and En i onmen al Changes & CHANGE—Global Change and
Sus ainabili y Ins i u e, Faculdade de Ciências, Uni e sidade de Lisboa, Campo G ande, Bloco C2,
1749-016 Lisboa, Po ugal
*Co espondence: [email p o ec ed]
Abs ac :
I is known ha ec omyco hizal (ECM) ungi can modula e below and abo eg ound
communi ies. They a e a key pa o belowg ound communica ion as hey p oduce a as a ay o
me aboli es, including ola ile o ganic compounds (VOCs) such as 1-oc en-3-ol. He e, we es ed i
he VOC 1-oc en-3-ol may be in ol ed in he ECM ungal mechanisms ha modula e below and
abo eg ound communi ies. Fo ha , we conduc ed h ee
in i o
assays wi h ECM ungi and he
1-oc en-3-ol ola ile o (i) explo e he e ec s o mycelium g ow h o h ee ECM species, (ii) in es iga e
he impac on he ge mina ion o six hos Cis aceae species, and (iii) s udy he impac on hos plan
ai s. The e ec s o 1-oc en-3-ol on mycelium g ow h o he h ee ECM species depended on he
dose and species: Bole us e icula us was he mos sensi i e species o he low (VOC) dose, while T.
lep ode ma was he mos ole an . In gene al, he p esence o he ECM ungi esul ed in highe seed
ge mina ion, while 1-oc en-3-ol esul ed in lowe seed ge mina ion. The combined applica ion o he
ECM ungus and he ola ile u he inhibi ed seed ge mina ion, possibly due o he accumula ion o
1-oc en-3-ol abo e he plan species’ h eshold. Seed ge mina ion and plan de elopmen o Cis aceae
species we e in luenced by ECM ungal ola iles, sugges ing ha 1-oc en-3-ol may media e changes
in below and abo eg ound communi ies.
Keywo ds: 1-oc en-3-ol; C-8 ola iles; ungal ola iles; ec omyco hizal ungi; Te ezia; Cis aceae
1. In oduc ion
Al hough i emains unclea when and how ec omyco hizal (ECM) ungi media e
he di ec ion and s eng h o eedback in plan communi ies, i is consensual ha hey can
modula e below and abo eg ound communi ies [
1
]. Se e al ac o s can con ibu e o he
explana ion o he e ec s o ECM ungi on communi ies, namely he media ion o plan –soil
eedbacks [
2
] and o plan de enses [
3
,
4
], plan acili a ion [
5
] and communica ion wi h
o he soil mic oo ganisms [6,7].
Vola ile o ganic compounds (VOCs) a e a i al communica ion s a egy among in-
di iduals o a species and o di e en kingdoms [
8
]. Among he mos common ungal
VOCs, he e a e he eigh ca bon compounds (C-8), which esul om he oxida ion o
linoleic acid [
9
]. These compounds a e also known as oxylipins, and play key oles in
egula ing ungal mo phogenesis, plan in e ac ions and biocon ol [
10
]. Among hese,
1-oc en-3-ol, also known as he “mush oom alcohol” due o i s cha ac e is ic mush oom
la o [
9
,
11
], is one o he mos abundan VOCs p oduced by ungi [
3
] and is conside ed a
signaling molecule, p oduced in di e en ungal s uc u es, conidial masses, spo oca ps
and hyphae [12,13].
The C-8 ola ile oxylipin 1-oc en-3-ol has an impo an ole in ungal in e ac ions,
p esen ing a egula o y e ec on he physiology and de elopmen o se e al ungi om
di e en gene a [12]. This compound can inhibi ungal g ow h and spo e ge mina ion o
Bo y is cine ea [
3
], Penicillium paneum [
13
], Penicillium ch ysogenum,Monilinia uc icola [
14
],
J. Fungi 2023,9, 180. h ps://doi.o g/10.3390/jo 9020180 h ps://www.mdpi.com/jou nal/jo
J. Fungi 2023,9, 180 2 o 14
Fusa ium icinc um and Fusa ium oxyspo um [
11
]. Fu he mo e, 1-oc en-3-ol also inhibi s he
g ow h o se e al bac e ial species (e.g., S aphylococcus au eus,Bacillus sub ilis,S aphylococcus
epide midis,Esche ichia coli,Pseudomonas ae uginosa) [
11
], while, in sap ophy ic species
such as Aga icus bispo us, his ola ile is in ol ed in bac e ial s imuli o p imo dium
o ma ion [15].
Besides being in ol ed in ungi– ungi and ungi–bac e ia in e ac ions, 1-oc en-3-
ol is also in ol ed in ungi–plan in e ac ions. Fo example, 1-oc en-3-ol inhibi s he
ungal pa hogen Bo y is cine ea and con ibu es o enhancing plan esis ance by he
induc ion o de ense signaling cascades [
9
,
10
]. Fo ins ance, ea ing Zea mays seeds wi h
100
µ
L o 1-oc en-3-ol in he o m o he pa en ed p oduc (Omega
™
) esul ed in season-
long imp o emen s in shoo and oo g ow h, simila o hose ob ained when applying
T ichode ma spp. [
16
]. Low concen a ion (0.05
µ
L/L) o his VOC also showed plan g ow h-
p omo ing ac i i y in oma o plan s [
17
]. The e o e, 1-oc en-3-ol can s imula e o inhibi he
g ow h o neighbo ing o ganisms (o he ungi, bac e ia and plan s) in a dose-dependen
manne [18].
1-oc en-3-ol is conside ed a bioma ke cha ac e is ic o symbio ic ungi, being emi ed
in high doses by hese ungi, while a low emission o his compound can be obse ed in
non-symbio ic ungi [
19
]. In ag eemen , and al hough 1-oc en-3-ol was i s iden i ied in
ec omyco hizal (ECM) ungi in hei ui bodies [
20
], mycelium [
21
] and du ing ec omyc-
o hiza syn hesis [
22
], i s e ec s on spo e ge mina ion, mycelium g ow h and p imo dia
ini ia ion o hese ungi ha e no ye been documen ed. Fu he mo e, he s udy o in e -
ac ions be ween ECM ungal VOCs, such as 1-oc en-3-ol, and hei hos plan s ha e been
insu icien ly s udied.
ECM ungi play an impo an ole in he ecology o Medi e anean sh ublands by
helping plan s o su i e in such nu ien -poo soils ha a e common in hese ecosys-
ems [
23
]. In his biome, ECM ungi ha e been ound o be associa ed wi h many plan
species such as Cis us,Que cus and Pinus [
24
–
26
]. Cis us sp. a e equen in Medi e anean
Basin plan communi ies and a e well adap ed o he ha sh condi ions o Medi e anean
sh ublands [
27
]. In addi ion, Cis us sp. ha e been ound o o m myco hizal ela ionships
wi h a ious ECM ungal species, including hose in he genus Te ezia,Lac a ius and Bole-
us [
28
–
32
]. The e o e, he p esen s udy aims o e alua e he e ec s o he ungal ola ile
1-oc en-3-ol in ECM ungi and hei hos plan s in di e en s ages o hei in e ac ions
owa ds symbiosis es ablishmen . We hypo hesized ha 1-oc en-3-ol has he po en ial o
modula e below and abo eg ound communi ies due o di e en h esholds o ECM ungal
and plan species. Fo ha , we pe o med h ee
in i o
assays o (i) explo e he e ec s
o 1-oc en-3-ol on mycelium g ow h o h ee ECM species: Te ezia lep ode ma,Lac a ius
deliciosus and Bole us e icula us; (ii) in es iga e he impac o 1-oc en-3-ol and he wo ECM
species on he ge mina ion o six hos Cis aceae species: Cis us albidus L., C. ladani e L., C.
sal ii olius L., C. psilosepalus Swee , Halimium halimi olium (L.) Willk. and Tube a ia gu a a
(L.) Fou .); and (iii) s udy he impac o 1-oc en-3-ol o ECM ungus (Te ezia a ena ia) on
hos plan Cis us sal ii olius ai s o nine mon hs.
2. Ma e ials and Me hods
2.1. Fungal Ma e ial
Ma u e Te ezia a ena ia,T. lep ode ma,Lac a ius deliciosus and Bole us e icula us spo o-
ca ps we e ha es ed om di e en loca ions in Po ugal (Alen ejo and Lei ia egions)
du ing au umn and sp ing seasons o 2019. Specimens we e eed om subs a e deb is a
he si e and u he cleaned in he labo a o y. The spo oca ps we e iden i ied by hei mo -
phological mac o and mic oscopic cha ac e is ics and by ollowing se e al au ho s [
33
,
34
]
and online keys (h p://www.mycokey.com/ accessed on 13 Ma ch 2019) [
35
]. In he case
o he Te ezia species, due o he simila i y be ween species, he samples we e u he
iden i ied by molecula analysis.
The mycelia o h ee ec omyco hizal species (T. lep ode ma,L. deliciosus and B. e icula-
us) we e isola ed om spo oca ps on po a o dex ose aga (PDA) (VWR), pH 5.5. Cul u es
J. Fungi 2023,9, 180 3 o 14
we e incuba ed a 25
◦
C, ans e ed on o esh medium e e y 3 mon hs and main ained a
4
◦
C. The ea e , he spo oca p samples we e d ied a 35
◦
C in a o ced en ila ion o en (Lab
Companion, Model OF-11E) un il cons an mass. D ied ungal ma e ials we e powde ed in
a po celain mo a and kep in b and-new sealed polye hylene bags unde d y condi ions.
Fo spo al inoculum p epa a ion, p e iously powde ed, 2.4 g o d ied T. a ena ia ascoca ps
was added o 400 mL o s e ile dis illed wa e and le o shake o e nigh a 23
◦
C
±
1
◦
C in
he da k be o e use. A 100-
µ
L aliquo was aken o coun he spo e’s concen a ion unde
he mic oscope, which was ound o be 1 ×108o spo es mL−1.
2.2. Plan Ma e ial
Seeds om Cis us sal ii olius L., C. psilosepalus Swee and Tube a ia gu a a (L.) Fou .
plan s g owing in he Lei ia egion (Po ugal) we e collec ed in Sep embe 2018 in a
Pinus pinas e o es a ea wi h a na u al sh ub unde s o y. The seeds we e d ied a oom
empe a u e and kep in he da k un il use. Seeds o C. albidus L., C. ladani e L. and
Halimium halimi olium (L.) Willk. we e pu chased om Semen es de Po ugal and kep in
he da k un il use.
To b eak seed do mancy, seed sca i ica ion was pe o med by ubbing he seeds o e a
ough su ace (i.e., sandpape ) and hen hea ing hem a 105
◦
C in an o en (Lab Companion,
Model OF-11E) o 10 min and lea ing hem o cool down un il oom empe a u e. The
seeds we e hyd a ed o 10 min in s e ile dis illed wa e , su ace s e ilized by imme sion
in 30 % H
2
O
2
o 30 min and washed h ee imes wi h s e ile dis illed wa e ollowed
by ano he imme sion in a 20% bleach solu ion wi h 3 d ops o Tween o 5–10 min.
A e wa ds, he seeds we e washed h ee imes in s e ilized wa e . Seeds we e hen used in
he ge mina ion es s.
2.3. VOC Assays
To unde s and he po en ial o 1-oc en-3-ol o modula e below and abo eg ound
communi ies, we conduc ed h ee
in i o
assays o es he e ec s o 1-oc en-3-ol on he
de elopmen o ECM ungal species and hos plan s, namely on ECM mycelium g ow h,
Cis aceae sp. seeds and in C. sal ii olius ai s.
In he ollowing assays, h ee doses o 1-oc en-3-ol we e es ed: 0
µ
g (designa ed as
CT); 0.17
µ
g (designa ed as VOC_low); and 1280
µ
g (designa ed as VOC_high). The doses
we e selec ed om he ange epo ed in se e al s udies using 1-oc en-3-ol [
12
,
36
–
39
]. The
low dose we used ook in o conside a ion he posi i e esul s ob ained in p e ious wo ks,
while he high dose was highe han all he doses epo ed.
2.3.1. Mycelium De elopmen
To e alua e he e ec o he 1-oc en-3-ol dose on he de elopmen o he mycelium
o he h ee ECM ungi (Te ezia lep ode ma,Lac a ius deliciosus and Bole us e icula us), we
es ed h ee doses pe pla e: 0
µ
g (CT); 0.17
µ
g (VOC_low); and 1280
µ
g (VOC_high). Fo
ha , we used PDA (VWR) wi h pH 5.5 as he cul u e medium (wi hou an ibio ics addi ion).
On he bo de o he Pe i dishes (9 cm diame e ) wi h he PDA medium, we added 10
µ
L
o 1-oc en-3-ol (VOC) in di e en doses.
The solu ion o 1-oc en-3-ol a 1
µ
M was p epa ed using 1-oc en-3-ol 98% (Al a Aesa
CAS 3391-86-4) dilu ed in Chlo o o m 99% (Me ck CAS 67-66-3) and s e ilized by il a ion
(0.2
µ
m, 47 mm memb ane il e , Minisa
®
NML, Sa o ius, Go ingen, Ge many). The
mycelial plugs wi h 0.25 cm
2
o T. lep ode ma,L. deliciosus and B. e icula us we e ans e ed
om 1-mon h pu e cul u es (in PDA) and placed in he cen e o he Pe i dishes o each
cul u e medium. Each ea men was eplica ed 10 imes (N = 10 Pe i dishes).
The ECM ungal cul u es we e placed in a g ow h chambe a 23
◦
C
±
1
◦
C in he da k.
Colonies’ g ow hs we e measu ed e e y 7 days o 63 days by measu ing mycelia adial
g ow h a he bo om o he Pe i dish.
J. Fungi 2023,9, 180 4 o 14
2.3.2. E ec o VOC and ECM Fungi on Cis aceae Species Ge mina ion
To e alua e he e ec o 1-oc en-3-ol (VOC) on he ge mina ion o plan hos s o he
ECM ungi, we used six Cis aceae species (Cis us albidus L., C. ladani e L., C. sal ii olius L.,
C. psilosepalus Swee , Halimium halimi olium (L.) Willk. and Tube a ia gu a a (L.) Fou .) and
wo ECM ungal species (Te ezia lep ode ma and Lac a ius deliciosus) whose g ow h was no
a ec ed by he low VOC dose. Gi en ha high concen a ions o 1-oc en-3-ol can damage
plan s [
19
], we used he lowe doses (i.e., 0
µ
g; 0.17
µ
g pe pla e). Fou ea men s we e
es ed o each Cis aceae sp., wi h i e eplica es (i.e., 9 cm Ø Pe i dishes) o each species
and ea men (Figu e 1).
J. Fungi 2023, 9, x FOR PEER REVIEW 4 o 15
The ECM ungal cul u es we e placed in a g ow h chambe a 23 °C ± 1 °C in he da k.
Colonies’ g ow hs we e measu ed e e y 7 days o 63 days by measu ing mycelia adial
g ow h a he bo om o he Pe i dish.
2.3.2. E ec o VOC and ECM Fungi on Cis aceae Species Ge mina ion
To e alua e he e ec o 1-oc en-3-ol (VOC) on he ge mina ion o plan hos s o he
ECM ungi, we used six Cis aceae species (Cis us albidus L., C. ladani e L., C. sal ii olius L.,
C. psilosepalus Swee , Halimium halimi olium (L.) Willk. and Tube a ia gu a a (L.) Fou .)
and wo ECM ungal species (Te ezia lep ode ma and Lac a ius deliciosus) whose g ow h
was no a ec ed by he low VOC dose. Gi en ha high concen a ions o 1-oc en-3-ol can
damage plan s [19], we used he lowe doses (i.e., 0 µg; 0.17 µg pe pla e). Fou ea men s
we e es ed o each Cis aceae sp., wi h i e eplica es (i.e., 9 cm Ø Pe i dishes) o each
species and ea men (Figu e 1).
PDA pH 5.5 cul u e medium (wi hou an ibio ics addi ion) was used o he ge mi-
na ion o Cis aceae seeds wi h ECM mycelium o he VOC. Fo all ea men s, in each Pe i
dish, we placed 4 seeds p e iously s e ilized, sca i ied and hea ed o b eak do mancy (as
p e iously explained in Sec ion 2.2). Fo he VOC ea men , on he cen e o he Pe i
dish, we added 10 µL o 1 µM 1-oc en-3-ol solu ion (0.17-µg dose pe pla e). In he CT, we
added only 10 µL o chlo o o m. Fo he ECM ungi ea men s, mycelium plugs (0.25
cm2) o T. lep ode ma (Tlep) o L. deliciosus (Ldel) we e placed in he cen e o he Pe i
dishes. In he con ol (CT) Pe i dishes, only he seeds we e used (i.e., ECM ungi and VOC
we e absen ). Fi e eplica es o each ea men we e p epa ed. The Pe i dishes we e
placed in a g ow h chambe a 23 °C ± 1 °C in he da k wi h 60 ± 5% ela i e humidi y,
and ge mina ion was e alua ed a e 35 days.
Figu e 1. Expe imen al design o e alua e he e ec o 1-oc en-3-ol and ECM mycelium on Cis aceae
species ge mina ion (N = 5).
2.3.3. E ec o VOC and ECM on Cis us sal ii olius Ge mina ion and De elopmen
Al hough we used T. lep ode ma in he p e ious assays, we did no ha e a iable spo e
inoculum o ca y ou his assay. Since bo h T. a ena ia and T. lep ode ma a e cha ac e is ic
o acid soils and a e able o o m in i o myco hizal associa ions wi h Cis us sal ii olius
[28,40], we used T. a ena ia in his hi d assay; based on he ole ance o T. lep ode ma and
o C. sal ii olius o he low VOC dose, we used Cis us sal ii olius as he plan hos and T.
a ena ia as he ECM ungus o e alua e he e ec o 1-oc en-3-ol (VOC) du ing symbiosis
es ablishmen on hos plan ai s. The ollowing ou ea men s we e implemen ed: CT—
con ol ea men only wi h Cis us sal ii olius seeds; VOC— ea men wi h 10 µL o 1-oc-
en-3-ol low dose (0.17 µg pe pla e); Ta— ea men wi h 10 mL o T. a ena ia spo al inoc-
ulum (108 mL−1); and TaVOC— ea men wi h 10 µL o 1 µM 1-oc en-3-ol solu ion (0.17
µg dose pe pla e) and 10 mL o T. a ena ia spo al inoculum (108 mL−1) (Figu e 2).
Figu e 1.
Expe imen al design o e alua e he e ec o 1-oc en-3-ol and ECM mycelium on Cis aceae
species ge mina ion (N = 5).
PDA pH 5.5 cul u e medium (wi hou an ibio ics addi ion) was used o he ge mina-
ion o Cis aceae seeds wi h ECM mycelium o he VOC. Fo all ea men s, in each Pe i
dish, we placed 4 seeds p e iously s e ilized, sca i ied and hea ed o b eak do mancy (as
p e iously explained in Sec ion 2.2). Fo he VOC ea men , on he cen e o he Pe i dish,
we added 10
µ
L o 1
µ
M 1-oc en-3-ol solu ion (0.17-
µ
g dose pe pla e). In he CT, we added
only 10
µ
L o chlo o o m. Fo he ECM ungi ea men s, mycelium plugs (0.25 cm
2
) o T.
lep ode ma (Tlep) o L. deliciosus (Ldel) we e placed in he cen e o he Pe i dishes. In he
con ol (CT) Pe i dishes, only he seeds we e used (i.e., ECM ungi and VOC we e absen ).
Fi e eplica es o each ea men we e p epa ed. The Pe i dishes we e placed in a g ow h
chambe a 23
◦
C
±
1
◦
C in he da k wi h 60
±
5% ela i e humidi y, and ge mina ion was
e alua ed a e 35 days.
2.3.3. E ec o VOC and ECM on Cis us sal ii olius Ge mina ion and De elopmen
Al hough we used T. lep ode ma in he p e ious assays, we did no ha e a iable spo e
inoculum o ca y ou his assay. Since bo h T. a ena ia and T. lep ode ma a e cha ac e is ic o
acid soils and a e able o o m
in i o
myco hizal associa ions wi h Cis us sal ii olius [
28
,
40
],
we used T. a ena ia in his hi d assay; based on he ole ance o T. lep ode ma and o
C. sal ii olius o he low VOC dose, we used Cis us sal ii olius as he plan hos and T.
a ena ia as he ECM ungus o e alua e he e ec o 1-oc en-3-ol (VOC) du ing symbiosis
es ablishmen on hos plan ai s. The ollowing ou ea men s we e implemen ed:
CT—con ol ea men only wi h Cis us sal ii olius seeds; VOC— ea men wi h 10
µ
L o
1-oc en-3-ol low dose (0.17
µ
g pe pla e); Ta— ea men wi h 10 mL o T. a ena ia spo al
inoculum (10
8
mL
−1
); and TaVOC— ea men wi h 10
µ
L o 1
µ
M 1-oc en-3-ol solu ion
(0.17 µg dose pe pla e) and 10 mL o T. a ena ia spo al inoculum (108mL−1) (Figu e 2).
J. Fungi 2023,9, 180 5 o 14
J. Fungi 2023, 9, x FOR PEER REVIEW 5 o 15
Ge mina ion was pe o med in polyp opylene anspa en mic oboxes (90 mm Ø
and 120 mm in heigh ) wi h a co e wi hou il e s. Each box con ained 100 mL o s e i-
lized subs a e (pea :pe li e mix u e, 3:1 / ) and 50 mL o dis illed wa e was au ocla ed
a 121 °C o 60 min. In a low chambe , en p e iously disin ec ed seeds (as desc ibed in
Sec ion 2.2) we e placed in each con aine .
Fo he ea men s wi h he ECM ungus (Ta and TaVOC), a e he en seeds we e
dis ibu ed in he mic oboxes, 10 mL o Te ezia spo al inoculum was dis ibu ed in each
mic obox o he wo ea men s wi h Te ezia. In he ea men s wi h he VOC (VOC and
TaVOC), 10 µL o 1-oc en-3-ol 1 µM was pul e ized inside each mic obox. As con ol, we
used mic oboxes only wi h Cis us sal ii olius seeds (CT), and we added only 10 µL o chlo-
o o m.
The mic oboxes (N = 18 pe ea men ) we e kep in a g ow h chambe wi hou di ec
ligh , a 16 h da k/8 h ligh pho ope iod and 25 °C/20 °C (±2 °C) day/nigh empe a u e.
The bo om pa o he mic oboxes, con aining he subs a e wi h he seeds and he spo es,
was co e ed wi h aluminum oil o dec ease ligh incidence in his a ea. The numbe o
ge mina ed plan s was coun ed e e y mon h o h ee mon hs. In he hi d mon h, oo
samples om six mic oboxes pe ea men we e collec ed o con i m myco hiza ion by
mic oscopic cha ac e iza ion. A six and nine mon hs, six mic oboxes pe ea men we e
selec ed andomly, and he ollowing plan ai s we e measu ed: shoo leng h, esh shoo
weigh , oo leng h, esh oo weigh , numbe o b anches pe shoo and numbe o lea es
pe shoo . The oo and shoo esh weigh we e measu ed on an analy ical scale (Radwag)
wi h 0.0001 g esolu ion. The shoo and oo leng h we e measu ed wi h a ule , and he
numbe o numbe o b anches and numbe o lea es we e coun ed pe shoo .
2.4. S a is ical Analysis
S a is ical analysis was conduc ed using Mic oso Excel 2019/XLSTAT-P emium
(Ve sion 2021.4.1, Addinso , Inc., B ooklyn, NY, USA). Since ou nume ical a iables did
no ollow a no mal dis ibu ion, he K uskal–Wallis es was selec ed. Di e ences be-
ween he ea men s we e compa ed using he K uskal–Wallis one-way analysis o a i-
ance. Mul iple pai wise compa isons we e pe o med using he Dunn’s es (p < 0.05).
Figu e 2. Expe imen al design o e alua e he e ec o 1-oc en-3-ol and Te ezia spo es on Cis us
sal ii olius ge mina ion and plan ai s. (a) Con ol ea men only wi h Cis us sal ii olius seeds; (b)
ea men wi h 10 µL o 1-oc en-3-ol low dose; (c) ea men wi h 10 mL o Te ezia spo al inoculum
(108 mL−1); (d) ea men wi h 10 µL o 1-oc en-3-ol low dose and 10 mL o Te ezia spo al inoculum
(108 mL−1). Ten seeds p e iously s e ilized we e dis ibu ed in each con aine (N=18 pe ea men ).
Figu e 2.
Expe imen al design o e alua e he e ec o 1-oc en-3-ol and Te ezia spo es on Cis us
sal ii olius ge mina ion and plan ai s. (
a
) Con ol ea men only wi h Cis us sal ii olius seeds;
(
b
) ea men wi h 10
µ
L o 1-oc en-3-ol low dose; (
c
) ea men wi h 10 mL o Te ezia spo al inoculum
(10
8
mL
−1
); (
d
) ea men wi h 10
µ
L o 1-oc en-3-ol low dose and 10 mL o Te ezia spo al inoculum
(10
8
mL
−1
). Ten seeds p e iously s e ilized we e dis ibu ed in each con aine (N=18 pe ea men ).
Ge mina ion was pe o med in polyp opylene anspa en mic oboxes (90 mm Ø and
120 mm in heigh ) wi h a co e wi hou il e s. Each box con ained 100 mL o s e ilized
subs a e (pea :pe li e mix u e, 3:1 / ) and 50 mL o dis illed wa e was au ocla ed a
121
◦
C o 60 min. In a low chambe , en p e iously disin ec ed seeds (as desc ibed in
Sec ion 2.2) we e placed in each con aine .
Fo he ea men s wi h he ECM ungus (Ta and TaVOC), a e he en seeds we e
dis ibu ed in he mic oboxes, 10 mL o Te ezia spo al inoculum was dis ibu ed in each
mic obox o he wo ea men s wi h Te ezia. In he ea men s wi h he VOC (VOC and
TaVOC), 10
µ
L o 1-oc en-3-ol 1
µ
M was pul e ized inside each mic obox. As con ol,
we used mic oboxes only wi h Cis us sal ii olius seeds (CT), and we added only 10
µ
L o
chlo o o m.
The mic oboxes (N = 18 pe ea men ) we e kep in a g ow h chambe wi hou di ec
ligh , a 16 h da k/8 h ligh pho ope iod and 25
◦
C/20
◦
C (
±
2
◦
C) day/nigh empe a u e.
The bo om pa o he mic oboxes, con aining he subs a e wi h he seeds and he spo es,
was co e ed wi h aluminum oil o dec ease ligh incidence in his a ea. The numbe o
ge mina ed plan s was coun ed e e y mon h o h ee mon hs. In he hi d mon h, oo
samples om six mic oboxes pe ea men we e collec ed o con i m myco hiza ion by
mic oscopic cha ac e iza ion. A six and nine mon hs, six mic oboxes pe ea men we e
selec ed andomly, and he ollowing plan ai s we e measu ed: shoo leng h, esh shoo
weigh , oo leng h, esh oo weigh , numbe o b anches pe shoo and numbe o lea es
pe shoo . The oo and shoo esh weigh we e measu ed on an analy ical scale (Radwag)
wi h 0.0001 g esolu ion. The shoo and oo leng h we e measu ed wi h a ule , and he
numbe o numbe o b anches and numbe o lea es we e coun ed pe shoo .
2.4. S a is ical Analysis
S a is ical analysis was conduc ed using Mic oso Excel 2019/XLSTAT-P emium
(Ve sion 2021.4.1, Addinso , Inc., B ooklyn, NY, USA). Since ou nume ical a iables did
no ollow a no mal dis ibu ion, he K uskal–Wallis es was selec ed. Di e ences be ween
he ea men s we e compa ed using he K uskal–Wallis one-way analysis o a iance.
Mul iple pai wise compa isons we e pe o med using he Dunn’s es (p< 0.05).

J. Fungi 2023,9, 180 6 o 14
3. Resul s
3.1. Mycelium G ow h
Al hough no an ibio ics we e added o he PDA medium, con amina ions we e no ob-
se ed on he pu e cul u es o he ECM ungi. While he high 1-oc en-3-ol dose (VOC_high)
ully inhibi ed he mycelium g ow h o all h ee ECM species, he low 1-oc en-3-ol dose
(VOC_low) esul ed in di e en esponses in he ECM species (Figu e 3). When compa ed
o he con ol, he mycelium g ow h o B. e icula us was inhibi ed (Figu e 3a), ha o
L. deliciosus showed a non-signi ican endency o inhibi ion (Figu e 3b) and ha o T.
lep ode ma was no a ec ed (Figu e 3c). The e o e, he mycelium g ow h o hese h ee ECM
species showed a sensi i i y g adien o he 1-oc en-3-ol low dose (VOC_low), anging
om no e ec o g ow h inhibi ion. F om he es ed ECM species, Bole us e icula us was
shown o be he mos sensi i e species o he low VOC dose, while T. lep ode ma was he
mos ole an .
J. Fungi 2023, 9, x FOR PEER REVIEW 7 o 15
Figu e 3. E ec s o he 1-oc en-3-ol doses on mycelium g ow h o Bole us e icula us, Lac a ius deli-
ciosus and Te ezia lep ode ma (N = 10). (a) Mycelium g ow h o B. e icula us; (b) mycelium g ow h
o L. deliciosus; (c) mycelium g ow h o T. lep ode ma, measu ed o e 63 days. T ea men s: CT—black
line; VOC_high— ed line; VOC_low—blue line. Da a we e compa ed using a K uskall–Wallis es .
Pos hoc compa isons we e made using a Dunn’s es . Da a a e mean ± s anda d e o . Di e en
le e s show signi ican di e ences (p < 0.05) be ween ea men s o each ECM ungus.
Figu e 3.
E ec s o he 1-oc en-3-ol doses on mycelium g ow h o Bole us e icula us,Lac a ius deliciosus
and Te ezia lep ode ma (N = 10). (
a
) Mycelium g ow h o B. e icula us; (
b
) mycelium g ow h o L.
deliciosus; (
c
) mycelium g ow h o T. lep ode ma, measu ed o e 63 days. T ea men s: CT—black line;
VOC_high— ed line; VOC_low—blue line. Da a we e compa ed using a K uskall–Wallis es . Pos
hoc compa isons we e made using a Dunn’s es . Da a a e mean
±
s anda d e o . Di e en le e s
show signi ican di e ences (p< 0.05) be ween ea men s o each ECM ungus.
3.2. Cis aceae Species Ge mina ion
The ge mina ion o he six Cis aceae species (Cis us albidus,C. ladani e ,C. psilosepalus,
C. sal ii olius,Halimilium halimi olium and Tube a ia gu a a) a e 35 days in co-cul u e wi h
Te ezia lep ode ma, Lac a ius deliciosus o VOC (lowe 1-oc en-3-ol dose) showed dis inc
esponses o he ola ile and ECM mycelium (Figu e 4, Table S1). We obse ed seed
J. Fungi 2023,9, 180 7 o 14
ge mina ion o all Cis aceae sp. in he ou ea men s. Ge mina ion wi hou he ola ile
and ECM mycelium (i.e., CT) did no esul in highe ge mina ion a es o any o he
Cis aceae sp., and, in T. gu a a, i esul ed in he lowes ge mina ion a e (40%).
J. Fungi 2023, 9, x FOR PEER REVIEW 8 o 15
Figu e 4. E ec s o 1-oc en-3-ol 1 µM (VOC) and ECM mycelium (T. lep ode ma and L. deliciosus) on
Cis aceae ge mina ion a es (%, N = 5). Da a we e compa ed using a K uskall–Wallis es . Pos hoc
compa isons we e made using a Dunn’s es . Da a a e means. Di e en le e s show signi ican di -
e ences (p < 0.05) be ween ea men s o each Cis aceae species.
3.3. Cis us sal ii olius T ai s
Al hough he e we e no di e ences in C. sal ii olius ge mina ion be ween he low 1-
oc en-3-ol dose (VOC) and he con ol (CT) a e h ee mon hs, he combined addi ion o
he ola ile wi h he ECM ungus (TaVOC) inhibi ed he ge mina ion by ~60% (in ela ion
o he CT). By con as , adding he ECM ungus alone (Ta) s imula ed C. sal ii olius ge -
mina ion by ~35% (in ela ion o he CT) (Figu e 5).
F om he analysis o he oo samples in he hi d mon h, o he ea men s wi h T.
a ena ia inoculum, myco hizal s uc u es we e de ec ed in he ini ial s age . F om he six
C. sal ii olius ai s e alua ed six and nine mon hs a e he beginning o he assay, only
he numbe o la e al shoo s and he numbe o lea es showed di e ences be ween he
ea men s (Table 1). A mon h six, he combined addi ion o he ola ile wi h he ECM
ungus (TaVOC) s imula ed he numbe o la e al shoo s (p < 0.05), and no la e al shoo s
could be obse ed in he CT plan s. S ill a mon h six, he addi ion o he ola ile (VOC)
o he ECM ungus (Ta) esul ed in lowe numbe s o la e al shoo s, simila o he CT.
Nine mon hs a e he beginning o he assay, TaVOC s ill esul ed in he highes
numbe o la e al shoo s, and so did Ta, and bo h we e highe han he CT (p < 0.05). The
numbe o lea es, which was simila o all ea men s a mon h six, showed di e ences
a mon h nine: Ta and TaVOC plan s had mo e lea es ha he CT plan s (p < 0.05). In
addi ion, o he shoo and oo esh weigh , he e was a endency o he TaVOC plan s
o show highe alues.
Figu e 4.
E ec s o 1-oc en-3-ol 1
µ
M (VOC) and ECM mycelium (T. lep ode ma and L. deliciosus)
on Cis aceae ge mina ion a es (%, N = 5). Da a we e compa ed using a K uskall–Wallis es . Pos
hoc compa isons we e made using a Dunn’s es . Da a a e means. Di e en le e s show signi ican
di e ences (p< 0.05) be ween ea men s o each Cis aceae species.
The low VOC dose had con as ing e ec s on he Cis aceae sp. ge mina ion. Fo hal
o he Cis aceae sp. es ed (C. albidus,C. psilosepalus and H. halimi olium), he p esence
o he low VOC dose esul ed in he lowes ge mina ion a es, anging om 20% in C.
albidus and C. psilosepalus o 30% in H. halimi olium. In C. ladani e and T. gu a a, he e was
a non-signi ican endency o s imula e ge mina ion, esul ing in high ge mina ion a es
o hese species (60% and 85%, espec i ely). In C. sal ii olius, ge mina ion was simila in
all ea men s, including he low VOC dose. Al oge he , he es ed Cis aceae sp. showed
di e en sensi i i ies o he low 1-oc en-3-ol dose.
Finally, he p esence o he ECM ungi ne e had a nega i e e ec on he Cis aceae sp.
ge mina ion; i had no e ec (e.g., C. ladani e and C. sal ii olius) o i s imula ed ge mina ion
(e.g., C. albidus,C. psilosepalus and T. gu a a wi h Ldel and H. halimi olium wi h Tlep).
3.3. Cis us sal ii olius T ai s
Al hough he e we e no di e ences in C. sal ii olius ge mina ion be ween he low 1-
oc en-3-ol dose (VOC) and he con ol (CT) a e h ee mon hs, he combined addi ion o he
ola ile wi h he ECM ungus (TaVOC) inhibi ed he ge mina ion by ~60% (in ela ion o he
CT). By con as , adding he ECM ungus alone (Ta) s imula ed C. sal ii olius ge mina ion
by ~35% (in ela ion o he CT) (Figu e 5).
J. Fungi 2023,9, 180 8 o 14
J. Fungi 2023, 9, x FOR PEER REVIEW 10 o 15
Figu e 5. Cis us sal ii olius seeds ge mina ion o e h ee mon hs, showing he e ec o Te ezia a e-
na ia inoculum and 1-oc en-3-ol on Cis us sal ii olius (N = 18 pe ea men ). T ea men s: CT—con-
ol; VOC—1-oc en-3-ol 1 µM; Ta—Te ezia a ena ia inoculum; TaVOC—T. a ena ia inoculum and 1-
oc en-3-ol 1 µM. Da a we e compa ed using a K uskall–Wallis es . Pos hoc compa isons we e
made using a Dunn’s es . Da a a e mean ± s anda d e o . Di e en le e s show signi ican di e -
ences (p < 0.05) be ween ea men s.
4. Discussion
The lowe dose o 1-oc en-3-ol inhibi ed he mycelium g ow h o B. e icula us bu no
o L. delicious and T. lep ode ma. This may e lec dis inc esponses o ECM species o 1-
oc en-3-ol, whe e ECM species may di e in hei h esholds o he ola ile. The e o e, he
1-oc en-3-ol p oduced by soil mic oo ganisms, including ECM ungi, by in e e ing in
ungal g ow h may modula e he s uc u e and composi ion o ECM ungal communi ies
belowg ound. ECM ungi p oduce 1-oc en-3-ol mainly on hei ui bodies, an a ea wi h
a high numbe o spo es, simila ly o wha occu s in he conidial masses o mic o ungi
[12]. I is known ha 1-oc en-3-ol can inhibi ungal g ow h and spo e ge mina ion o se -
e al mic o ungi [3,11,12,14]. Fo example, in Penicillium paneum, 1-oc en-3-ol is a sel -in-
hibi o ola ile ha blocks he ge mina ion p ocess, and, in Monilinia uc icola, 1-oc en-3-
ol des oys he hyphae mo phology and cell s uc u e [14]. I is sugges ed by Chi a a and
colleagues [12] ha his C-8 ola ile has a common unc ion as an inhibi o o p ema u e
spo e ge mina ion. In mic o ungi, spo es and conidial masses a e di ec ly exposed o he
ai , so a ola ile p oduced in he ui body o by conidia could be a mo e e icien sel -
inhibi o compound o he ge mina ion un il mo e app op ia e en i onmen al condi ions
p e ail [12]. Mo eo e , in Aga icus bispo us, i is an e ec i e au o egula o o ui ing body
de elopmen [41].
Ou expe imen al design did no allow us o dis inguish be ween a sel -inhibi o y
beha io o a lowe h eshold, bu clea ly showed B. e icula us is mo e sensi i e o 1-
oc en-3-ol han L. deliciosus and T. lep ode ma. The e ec s o 1-oc en-3-ol on myco hizal
spo oca ps ha e been s udied o a lesse ex en compa ed o o he ungal gilds. Howe e ,
a ecen s udy showed ha VOCs can ac as an a ac an o ce ain insec s, which is
Figu e 5.
Cis us sal ii olius seeds ge mina ion o e h ee mon hs, showing he e ec o Te ezia a ena ia
inoculum and 1-oc en-3-ol on Cis us sal ii olius (N = 18 pe ea men ). T ea men s: CT—con ol;
VOC—1-oc en-3-ol 1
µ
M; Ta—Te ezia a ena ia inoculum; TaVOC—T. a ena ia inoculum and 1-oc en-3-
ol 1
µ
M. Da a we e compa ed using a K uskall–Wallis es . Pos hoc compa isons we e made using a
Dunn’s es . Da a a e mean
±
s anda d e o . Di e en le e s show signi ican di e ences (p< 0.05)
be ween ea men s.
F om he analysis o he oo samples in he hi d mon h, o he ea men s wi h T.
a ena ia inoculum, myco hizal s uc u es we e de ec ed in he ini ial s age. F om he six
C. sal ii olius ai s e alua ed six and nine mon hs a e he beginning o he assay, only
he numbe o la e al shoo s and he numbe o lea es showed di e ences be ween he
ea men s (Table 1). A mon h six, he combined addi ion o he ola ile wi h he ECM
ungus (TaVOC) s imula ed he numbe o la e al shoo s (p< 0.05), and no la e al shoo s
could be obse ed in he CT plan s. S ill a mon h six, he addi ion o he ola ile (VOC) o
he ECM ungus (Ta) esul ed in lowe numbe s o la e al shoo s, simila o he CT.
Nine mon hs a e he beginning o he assay, TaVOC s ill esul ed in he highes
numbe o la e al shoo s, and so did Ta, and bo h we e highe han he CT (p< 0.05). The
numbe o lea es, which was simila o all ea men s a mon h six, showed di e ences
a mon h nine: Ta and TaVOC plan s had mo e lea es ha he CT plan s (p< 0.05). In
addi ion, o he shoo and oo esh weigh , he e was a endency o he TaVOC plan s o
show highe alues.
J. Fungi 2023,9, 180 9 o 14
Table 1.
E ec s o Te ezia a ena ia inoculum and 1-oc en-3-ol on Cis us sal ii olius plan ai s: shoo
leng h (SL), shoo weigh (SW), oo leng h (RL), oo weigh (RW), numbe o la e al shoo s and
numbe o lea es obse ed six and nine mon hs a e he inocula ion. (N = 6 pe ea men ). T ea -
men s: CT—con ol; VOC—1-oc en-3-ol 1
µ
M; Ta—Te ezia a ena ia inoculum; TaVOC—T. a ena ia
inoculum and 1-oc en-3-ol 1 µM.
6 Mon hs 9 Mon hs
T ai T ea men Mean SD Mean SD
Shoo leng h
(cm)
CT 4.34 ±3.14 8.91 ±4.09
Ta 5.63 ±3.26 10.38 ±4.33
TaVOC 4.83 ±3.04 11.40 ±4.69
VOC 4.35 ±2.61 7.54 ±4.29
Roo leng h
(cm)
CT 6.11 ±3.31 9.28 ±4.13
Ta 4.83 ±2.25 7.23 ±2.88
TaVOC 3.81 ±1.74 6.94 ±2.47
VOC 4.75 ±3.22 5.94 ±2.20
Shoo esh weigh
(mg)
CT 52.11 ±46.14 99.75 ±74.21
Ta 86.35 ±84.16 146.93 ±144.51
TaVOC 149.33 ±129.22 309.02 ±245.66
VOC 53.80 ±45.01 89.45 ±82.70
Roo esh weigh
(mg)
CT 16.84 ±14.76 42.35 ±40.05
Ta 21.65 ±20.46 46.16 ±43.21
TaVOC 32.35 ±31.13 58.74 ±34.40
VOC 12.02 ±13.01 44.85 ±40.45
B anches (No/shoo )
CT 0.00 ±0.00 b 0.81 ±0.61 b
Ta 0.32 ±0.07 b 4.05 ±3.52 a
TaVOC 1.57 ±0.61 a 5.00 ±4.90 a
VOC 0.38 ±0.09 b 1.05 ±0.74 a,b
Lea es
(No/shoo )
CT 10.90 ±5.58 a 11.39 ±5.96 b
Ta 13.71 ±5.05 a 28.10 ±19.35 a
TaVOC 13.55 ±5.97 a 49.43 ±39.12 a
VOC 12.24 ±5.64 a 13.24 ±4.88 a,b
Da a we e compa ed using a K uskall–Wallis es . Pos hoc compa isons we e made using a Dunn’s es . Da a a e
mean ±s anda d de ia ion (SD). Di e en le e s show signi ican di e ences (p< 0.05) be ween ea men s.
4. Discussion
The lowe dose o 1-oc en-3-ol inhibi ed he mycelium g ow h o B. e icula us bu
no o L. delicious and T. lep ode ma. This may e lec dis inc esponses o ECM species o
1-oc en-3-ol, whe e ECM species may di e in hei h esholds o he ola ile. The e o e,
he 1-oc en-3-ol p oduced by soil mic oo ganisms, including ECM ungi, by in e e ing in
ungal g ow h may modula e he s uc u e and composi ion o ECM ungal communi ies
belowg ound. ECM ungi p oduce 1-oc en-3-ol mainly on hei ui bodies, an a ea wi h a
high numbe o spo es, simila ly o wha occu s in he conidial masses o mic o ungi [
12
].
I is known ha 1-oc en-3-ol can inhibi ungal g ow h and spo e ge mina ion o se e al
mic o ungi [3,11,12,14]
. Fo example, in Penicillium paneum, 1-oc en-3-ol is a sel -inhibi o
ola ile ha blocks he ge mina ion p ocess, and, in Monilinia uc icola, 1-oc en-3-ol de-
s oys he hyphae mo phology and cell s uc u e [
14
]. I is sugges ed by Chi a a and
colleagues [
12
] ha his C-8 ola ile has a common unc ion as an inhibi o o p ema u e
spo e ge mina ion. In mic o ungi, spo es and conidial masses a e di ec ly exposed o he
ai , so a ola ile p oduced in he ui body o by conidia could be a mo e e icien sel -
inhibi o compound o he ge mina ion un il mo e app op ia e en i onmen al condi ions
p e ail [
12
]. Mo eo e , in Aga icus bispo us, i is an e ec i e au o egula o o ui ing body
de elopmen [41].
Ou expe imen al design did no allow us o dis inguish be ween a sel -inhibi o y
beha io o a lowe h eshold, bu clea ly showed B. e icula us is mo e sensi i e o 1-oc en-