Cons i u i e and inducible oma o de enses con ibu e o Bacillus
hu ingiensis le hali y agains Spodop e a exigua
Ada F a ini
a
, Rosa M. Gonz´
alez-Ma ínez
a
, Juan M. Ga cía
b
, Zhi ko Minche
b
,
Ma ía J. Pozo
b
, Víc o Flo s
c
, C is ina M. C a a
a
, Sal ado He e o
a,*
a
Depa men o Gene ics and Ins i u o Uni e si a io de Bio ecnologia i Biomedicina (BIOTECMED), Uni e si a de Val`
encia, Bu jasso , Valencia, Spain
b
Depa men o Soil and Plan Mic obiology, Es aci´
on Expe imen al del Zaidín –Consejo Supe io de In es igaciones Cien í icas (CSIC), G anada, Spain
c
Depa men o Biology, Biochemis y and Na u al Sciences, Uni e si a Jaume I, Cas ell´
on, Spain
HIGHLIGHTS GRAPHICAL ABSTRACT
•Lea -based a i icial die s allow o s udy
he e ec o chemical de enses on
insec s.
•Toma o de enses enhanced he le hali y
o a bac e ial en omopa hogen.
•O e -accumula ed plan me aboli es
we e iden i ied a e elici a ion o
de enses.
•Plan de ense elici o s con ibu e o he
ac ion o Bacillus hu ingiensis.
ARTICLE INFO
Keywo ds:
Lepidop e a
Bac e ial en omopa hogen
Toma o sc eening
Plan de enses
T i ophic in e ac ions
ABSTRACT
In na u e, insec he bi o y exe s con inuous selec i e p essu e on plan s ha , in u n, ha e de eloped a wide
a ay o cons i u i e and inducible de enses o igh agains a acke s. Since plan de enses may a ec highe
ophic le els, including en omopa hogens, u he esea ch is equi ed o unde s and how plan compounds
in luence insec -pa hogens in e ac ions and hei implica ions o in eg a ed pes managemen p og ams. He e,
we e alua ed he impac o oma o de enses on he le hali y p oduced by he bac e ial en omopa hogen Bacillus
hu ingiensis (B ) agains second ins a la ae o Spodop e a exigua. We i s examined he e ec o cons i u i e
de enses om i een wild oma o species and o y cul i a ed a ie ies (Solanum lycope sicum) on S. exigua la al
g ow h and suscep ibili y o B . The esul s showed la ae ed on wild oma o species had a educed la al
g ow h compa ed o la ae ed on cul i a ed a ie ies, whe eas suscep ibili y o B was simila be ween bo h
g oups. We hen selec ed six cul i a ed a ie ies, including hose wi h high and low impac s on la al g ow h and
B -induced mo ali y, o u he explo e he e ec o inducible de enses. Elici a ion o de enses by me hyl
jasmona e (MeJA) educed la al g ow h and inc eased basal mo ali y. Addi ionally, when la ae we e in ec ed
wi h B , MeJA ea men u he inc eased hei suscep ibili y o he en omopa hogen. Me abolomic analysis
con i med a ep og amming o oma o lea me abolism ollowing MeJA elici a ion, wi h an induced accumu-
la ion o bioac i e compounds, such as saponins o la onoids, known o hei an i-he bi o y p ope ies in mos
* Co esponding au ho a : Uni e si a de Val`
encia. Depa men o Gene ics, D Moline 50, 46100 Bu jasso , Spain.
E-mail add ess: [email p o ec ed] (S. He e o).
Con en s lis s a ailable a ScienceDi ec
Biological Con ol
jou nal homepage: www.else ie .com/loca e/ybcon
h ps://doi.o g/10.1016/j.biocon ol.2024.105624
Recei ed 1 May 2024; Recei ed in e ised o m 19 Sep embe 2024; Accep ed 24 Sep embe 2024
Biological Con ol 198 (2024) 105624
A ailable online 26 Sep embe 2024
1049-9644/© 2024 The Au ho (s). Published by Else ie Inc. This is an open access a icle unde he CC BY-NC-ND license ( h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/ ).
oma o a ie ies. O e all, hese da a e eal ha bo h cons i u i e and inducible oma o de enses no only p o ec
he plan by di ec ly a ec ing he insec pes bu also enhance he e icacy o bac e ial en omopa hogens.
1. In oduc ion
The in e ac ion be ween plan s and he bi o ous insec s s a ed
a ound 400 million yea s ago, gi ing ise o a coe olu iona y p ocess
whe ein plan s ha e been compelled o acqui e di e en de ensi e
s a egies (Labandei a, 2013). These s a egies encompass he p oduc-
ion o a wide a ay o di ec de enses, such as physical ba ie s (i.e.
ichomes, cu icle, e c…) o oxic and an inu i ional compounds (i.e.
seconda y me aboli es, p o einase inhibi o s, e c…) (Wa e al., 2018;
Fe n´
andez de Bobadilla e al., 2022). Plan de enses may be cons i u-
i ely p oduced, o induced ollowing he bi o y damage o de ec ion
(Hilke and Meine s, 2006; Fel on e al., 2014; Wa s e al., 2023). In
esponse, insec s ha e de eloped mechanisms o coun e ac plan de-
enses, including seques a ion, de oxi ying o a oiding beha iou s
(Heidel-Fische and Vogel, 2015; Dussou d, 2017). Fu he mo e, plan s
encompass addi ional laye s o de ense, such as indi ec de enses ha
in ol e he emission o ola iles ac ing as p ey cues o a ac na u al
enemies o he he bi o es, including a h opod p eda o s o pa asi oids
(Aljbo y and Chen, 2018). Plan inducible de enses agains he bi o es
a e quickly igge ed upon he bi o y and inely egula ed by signaling
pa hways coo dina ed by jasmonic acid and ela ed compounds,
collec i ely known as jasmona es (JAs) (Tu ne e al., 2002; Howe and
Jande , 2008). Indeed, exogenous ea men wi h JAs induced p o ein-
ase inhibi o s, alkaloids and o he bioac i e seconda y me aboli es
(Thale e al., 1996; Wa e al., 2015; Flo es-Sanchez e al., 2016). Fo
example, exogenous applica ion o me hyljasmona e (MeJA) is a com-
mon s a egy used o igge inducible plan esponses agains insec
he bi o es, such as chewing he bi o es (Rod iguez-Saona e al., 2001;
Tian e al., 2014; Sen hil-Na han, 2019).
In na u e, insec he bi o es a e exposed o a wide ange o mic obial
en omopa hogens. This e m includes all he mic oo ganisms ha ha e
he abili y o in ec and kill di e en species o a h opods (Ruiu, 2018).
Due o hei hos speci ici y and lack o oxici y o non- a ge o ganisms
o he en i onmen , mic obial en omopa hogens a e widely used as
con ol agen s in in eg a ed pes managemen p og ams (Lacey e al.,
2015; I sad e al., 2023). Among en omopa hogens, bac e ia, baculo i-
uses, ungi, and nema odes a e he mos applied as biopes icides.
Cu en ly, hei comme cial use accoun s o abou 5 % o he en i e c op
p o ec ion ma ke wi h expec a ions ha his pe cen age will ise om
10 % o 20 % in he coming yea s (Sabbahi e al., 2022). The bac e ium
Bacillus hu ingiensis (B ) is he mos commonly used en omopa hogen in
pes con ol, wi h o mula ions based on i ep esen ing a ound 75–95 %
o he mic obial biopes icide ma ke (Ju a -Fuen es e al., 2021). B is a
g am-posi i e bac e ium which is p esen in di e en ecological niches
including soil, wa e , plan s and dead insec s, and i is e ec i e agains
se e al insec o de s such as Lepidop e a, Coleop e a and Dip e a
(Palma e al., 2014). The in ec ion p ocess begins wi h he o al inges ion
o he bac e ial spo ula ed cells, and/o hei o mula ed spo es and he
associa ed insec icidal c ys als, which a e p esen in he soil o on he
phylloplane. Once inside he insec ’s gu , he c ys als a e solubilized,
leading o he ac i a ion o he insec icidal p o eins ha o m he c ys al
(C y oxin). These p o eins bind o ecep o s in he midgu cell mem-
b ane, inducing po e o ma ion and ul ima ely causing he dea h o he
insec (B a o e al., 2011).
En omopa hogens may be a ge ed by he plan de ensi e s a egies,
which may p omo e he ac ion o hese he bi o es’enemies o hei
own bene i (Rasmann e al., 2005; Mohan e al., 2008a; Lin e al., 2016;
Hay e al., 2020). Fo example, maize de ensi e esponses a ge he
ca e pilla s’p o ec i e gu ba ie . This accele a es he in asion o gu
bac e ia in o he la al body ca i y whe e hey exace ba e he nega i e
impac o plan de enses on he insec (Mason e al., 2019).
I is known ha he e ec i eness o B o mula ions agains insec
pes s can be in luenced by a ious plan ai s. Fo ins ance, nu i ion
quali y o plan s can impac B e icacy (Shikano and Co y, 2014), as
well as plan -p oduced compounds modula e he insec in e ac ion wi h
he bac e ium (Macin osh e al., 1990; Mohan e al., 2008b; Gasmi e al.,
2019). Addi ionally, ce ain plan de enses a e known o in e e e wi h
he diges i e p ocess o he insec (Chen e al., 2005; Bhonwong e al.,
2009), impac on i s immune sys em (Shi e al., 2020; Chen e al., 2022)
o dis u b he in eg i y o he pe i ophic memb ane (Pechan e al.,
2002; Ka iya e al., 2017). These al e a ions can inc ease he suscep i-
bili y o he a ge insec o B. hu ingiensis, being he basis o he
con ibu ion o he hos plan geno ype in shaping insec -pa hogen
in e ac ion (Janmaa and Mye s, 2005; Ca i`
e e e al., 2005; Bi d and
Akhu s , 2007). In his con ex , Shikano e al. (2018) ound ha oma o
induced de enses enhanced he e icacy o B agains he polyphagous
ca e pilla Helico e pa zea. They sugges ed ha o hoquinones, esul ing
om he oxida ion o chlo ogenic acid by plan PPO enzymes, we e
esponsible o alkyla ing B p o o oxins, which inc eased hei solubi-
liza ion and/o p o eolysis, he eby enhancing he e ec i eness o he
oxins (Ludlum e al., 1991).
Gi en his, we hypo hesized ha plan de enses could modula e
insec -B in e ac ions by inc easing he suscep ibili y o he insec pes o
he bac e ial en omopa hogen. In he p esen wo k, we in es iga ed he
in e ac ion be ween cons i u i e and inducible olia de enses in oma o
(Solanum lycope sicum) and B in ec ion in he polyphagous ca e pilla
Spodop e a exigua. To explo e he di e si y o cons i u i e chemical de-
enses, we compa ed 55 oma o geno ypes, including i een wild oma o
species and o y cul i a ed oma o a ie ies. We es ablished a bioassay
sys em using lea -based a i icial die s o assess he impac o cons i u-
i e chemical de enses om oma o on S. exigua pe o mance, in he
p esence o absence o B . We hen selec ed six a ie ies showing con-
as ing le els o esis ance o S. exigua he bi o y and pe o med u he
expe imen s o assess he combined e ec s o cons i u i e and inducible
de enses on B -in ec ed la ae. To induce de enses, plan s we e ea ed
wi h MeJA o ac i a e JA-dependen esponses, and he e ec s on la al
g ow h and suscep ibili y o B we e eco ded. Finally, we cha ac e ized
he chemical changes in elici ed lea es ha may ha e con ibu ed o he
obse ed e ec s. Fo ha , we pe o med an un a ge ed me abolomic
analysis o iden i y me aboli es ha accumula ed a e elici a ion o
plan de enses and ha may play a ole in de ense agains S. exigua and
inc ease suscep ibili y o B . O e all, ou indings show ha bo h
cons i u i e and he bi o e-inducible oma o chemical de enses inc ease
he ac ion o B agains S. exigua, and we iden i ied key me aboli es ha
may egula e his in e ac ion.
2. Ma e ials and me hods
2.1. Insec s
The Spodop e a exigua ca e pilla s used in his s udy we e sou ced
om a labo a o y colony es ablished o e 10 yea s ago om eggs sup-
plied by Ande ma Biocon ol AG (G ossdie wil, Swi ze land). The
colony was main ained in a g ow h chambe unde con olled condi-
ions, wi h a empe a u e o 25 ±3◦C, ela i e humidi y o 70 ±5 % and
a pho ope iod o 16 h ligh : 8 h da k. La ae we e ea ed on a i icial
die (El i a e al., 2010), while adul s we e p o ided ad libi um access o
a 10 % suga solu ion.
2.2. Toma o a ie ies and lea -based die p epa a ion
The wild oma o species and cul i a ed a ie ies we e p o ided by
A. F a ini e al. Biological Con ol 198 (2024) 105624
2
he Ins i u e o he Conse a ion and Imp o emen o Valencian
Ag odi e si y (COMAV) o he Uni e si a Poli `
ecnica de Val`
encia
(Spain). The selec ion o he a ie ies o his s udy (Table 1,Supple-
men a y) was based on hei di e si y in ui me abolic composi ion
(An onio G anell, Pe sonal communica ion; Pons e al., 2022; 2023).
Toma o seeds we e sown in ji ys (0.4 cm), composed by p essed coconu
ibe , inside seedbeds (6.5 x 6.5 cm). They we e g own in a con olled
g eenhouse wi h a empe a u e anging 20–26 ◦C du ing he day and
19–22 ◦C a nigh , and wi h a diu nal pho ope iod o 16 h ligh and 8 h
da k (na u al ligh supplemen ed wi h luo escen ube ligh ing a dawn
and dusk). Plan g ow h was moni o ed, no phy osani a y ea men s
we e applied in he g eenhouse, and no auxilia y insec s we e eleased
o a oid un a ge ed e ec s on plan de enses. Toma o plan s wi h 6–8
expanded ue lea es we e ha es ed a e ou weeks o g ow h. Only
plan s wi h lack o disease symp oms we e employed. A minimum o 3
plan s pe a ie y we e p ocessed oge he , wi h an a e age o 8 plan s
pe sample. Lea es we e immedia ely ozen in liquid ni ogen and
s o ed a −80 ◦C. Lea samples we e g ounded in a mo a wi h liquid
ni ogen o ob ain a ine lea powde , which was hen lyophilized in 10
mL ubes using an Alpha1-2 lyophilize (Ch is , Ge many) o 48 h
(−52 ◦C, acuum p essu e 0.2 mm Hg), and s o ed a oom empe a u e
in he da k un il hei use o plan -based la al die p epa a ion.
Plan -based die s we e o mula ed by mixing 0.23 g o lyophilized
ma e ial wi h 4 mL o aga solu ion supplemen ed wi h 5 % o he
compounds used o he s anda d a i icial die (wi hou an ibio ics) o
ea ing S. exigua la ae (F a ini e al., 2022). To p e en hea inac i-
a ion o enzymes and me aboli es, he lea powde was mixed wi h he
aga once i had cooled o app oxima ely 35–40 ◦C.
2.3. S. exigua la al pe o mance
La al pe o mance was assessed by measu ing he ela i e g ow h o
newly mol ed second ins a S. exigua la ae (He e o e al., 2002). The
bioassay spanned 144 h, du ing which la ae we e ed o he i s 48 h
on he plan -based die , ollowed by a swi ch o a i icial die o he
emainde o he expe imen . Since he S. exigua la ae used in hese
bioassays we e sou ced om a labo a o y colony ea ed exclusi ely on
a i icial die , he eeding pe iod on plan die s was es ic ed o 48 h o
a oid la al mo ali y due o a lack o die a y adap a ion and o mini-
mize he in e e ence om o he ac o s, such as he nu i ional p op-
e ies o he di e en plan s. A he s a o he expe imen , la ae we e
weighed, indi idually placed in sepa a e wells o a bioassay ay
(p oduc no. 9074; F on ie Ag icul u al Sciences), which was sealed
wi h mic ope o a ed adhesi e ape (p oduc no. 9074-L; F on ie
Ag icul u al Sciences). The ays we e main ained in he g ow h
chambe unde he same condi ions o he labo a o y colony men ioned
abo e. A e 144 h, la ae we e weigh ed again, and he ela i e g ow h
was calcula ed as millig ams o biomass acqui ed pe millig am o ini ial
body weigh . La al weigh was de e mined using a p ecision balance
(Sa o ius MC-1 Analy ic AC 120S; G¨
o ingen, Ge many) wi h an accu-
acy o 0.1 mg. A o al o 16 la ae we e used pe oma o species/ a-
ie y, and wo biological eplica es o he expe imen we e pe o med a
di e en pe iods.
2.4. B. hu ingiensis suscep ibili y bioassays
B suscep ibili y assays we e conduc ed as desc ibed by F a ini e al.
(2022). Newly mol ed second ins a S. exigua la ae we e in ec ed wi h
he bac e ial en omopa hogen using he d ople eeding me hod. Fou
μ
L
d ople s con aining 10 % suc ose, phospha e-bu e ed saline (PBS; pH
7.4), 10 % ( / ) acking dye phenol ed, and a suble hal concen a ion
o B. hu ingiensis subsp. aizawai (XenTa i®GD, 15 million U.I./g;
Kenoga d S.A, Ba celona, Spain) we e p epa ed. The suble hal concen-
a ion o 0.6 mg/mL was de e mined on he basis o p e ious bioassays
conduc ed unde he same expe imen al condi ions (second ins a
S. exigua la ae, eeding on plan die o 48 h). Con ol la ae ecei ed a
mock ea men wi h he same p ocedu e bu wi hou B . La ae we e
allowed o eed on he d ople s o 15 min, and a e isually con i ming
inges ion, hey we e immedia ely placed indi idually in bioassay ays
and p o ided wi h plan -based die s. The ays we e kep o 48 h in a
g ow h chambe unde con olled condi ions. A e his pe iod, he
plan -based die was eplaced wi h a i icial die . Mo ali y was egis-
e ed e e y 24 h o a pe iod o 6 days. La al ela i e g ow h o la ae a
144 h was calcula ed and analyzed as desc ibed abo e. A o al o 16
la ae we e used pe species/ a ie y and ea men , and wo biological
eplica es we e pe o med a di e en ime pe iods. Fo s a is ical
analysis, di e ences in la al ela i e g ow h be ween wild oma o
species and cul i a ed a ie ies we e calcula ed using S uden ’s - es .
Mo ali y in con ol and B -in ec ed la ae we e compa ed using one-
way ANOVA ollowed by Tukey’s mul iple compa isons es (G aph-
Pad So wa e Inc., San Diego, CA). The Spea man and Shapi o-Wilk es s
we e applied o de e mine homoscedas ici y and no mali y o da a,
espec i ely. Addi ionally, he Pea son co ela ion coe icien was used
o measu e he linea co ela ion o g ow h and basal mo ali y alues o
non-in ec ed la ae wi h he mo ali y o B -in ec ed la ae.
2.5. Elici a ion o MeJA-inducible plan de enses and e alua ion o hei
e ec s on S. exigua
The esul s o he g ow h inhibi ion and B suscep ibili y bioassays
wi h plan -based die de i ed om he i een wild oma o species and
o y cul i a ed oma o a ie ies, allowed us o selec ed six cul i a ed
a ie ies wi h con as ing le els o cons i u i e esis ance (i.e., leading
o low o high ela i e la al g ow h and B -associa ed mo ali y in
plan -based die s, Suppl. Table 1). These we e chosen o u he in es-
iga e he e ec o JA-inducible de enses on la al g ow h and suscep-
ibili y o B (Table 1). To his pu pose, oma o seeds we e su ace-
s e ilized by imme sion in 4 % NaHClO (10 min), hen ho oughly
insed wi h s e ile dis illed wa e , and ge mina ed in s e ile e miculi e
a 25 ◦C in a phy o on o 10 days. Seedlings a wo-co yledon s age
we e hen ansplan ed o 250 mL po s con aining a s e ile sand:
e miculi e (1:1) mix u e (Ri e o e al., 2021). Plan s we e andomly
dis ibu ed on g eenhouse benches and g own in a g eenhouse a 24 ◦C/
16 ◦C wi h a 16 h/8h diu nal pho ope iod and 70 % humidi y. A e 6
weeks o g ow h, hal o he plan s we e ea ed by sp aying plan shoo s
wi h a solu ion o 100
μ
M MeJA (Sigma-Ald ich) in wa e , which was
p epa ed om a 100 mM MeJA s ock solu ion in 96 % ( / ) e hanol. To
ensu e uni o m applica ion, Silwe 77 (0.02 % / ) was added as a
su ac an , and lea es we e sp ayed un il un o using an ae og aph.
Con ol plan s we e simila ly sp ayed wi h a mock solu ion con aining
only he sol en and su ac an . This exogenous applica ion o 100
μ
M
MeJA has been shown o e icien ly elici plan de enses in a ious
species, including oma o (Ma ínez-Medina e al., 2017). Fi e plan s
we e ea ed pe a ie y. Lea es we e ha es ed 72 h a e MeJA o mock
Table 1
E ec o he selec ed cul i a ed a ie ies on S. exigua g ow h and suscep ibili y
o B .
Cons i u i e
esis ance
1
In e nal
code
Selec ed
a ie y
Rela i e g ow h ±
SEM
B mo ali y±
SEM
(%)
High 20 T 1 27.9 ±3.2 46.9 ±15.6
High 35 T 2 13.1 ±3.9 62.5 ±6.3
High 39 T 3 28.0 ±13.7 60.7 ±1.9
Low 3 T 4 55.1 ±15.5 12.1 ±0.4
Low 25 T 5 66.7 ±28.6 32.1 ±10.7
Low 16 T 6 109.9 ±35.0 6.3 ±0.0
1
High g oup co espond o cul i a ed oma o a ie ies (1, 2, 3) wi h a high
cons i u i e esis ance agains S. exigua (low la al g ow h and high mo ali y
wi h B ). Low g oup co espond o cul i a ed oma o a ie ies (4, 5, 6) wi h a
low cons i u i e esis ance agains S. exigua (high la al g ow h and low mo -
ali y wi h B ).
A. F a ini e al. Biological Con ol 198 (2024) 105624
3
applica ion o ensu e he accumula ion o he JA-inducible compounds.
The ha es ed lea es we e immedia ely ozen wi h liquid ni ogen and
s o ed a −80 ◦C.
Fo he S. exigua bioassays, ozen lea es we e pooled by ea men
and a ie y (5 plan s), g ounded, and lyophilized as desc ibed p e i-
ously o p epa e he plan -based die s. Bioassays o e alua e la al
g ow h and suscep ibili y o B we e conduc ed as desc ibed abo e, wi h
he only modi ica ion being he use o a suble hal concen a ion o
XenTa i®a 1 mg/mL. Th ee independen biological eplica es we e
pe o med, wi h 16 la ae used pe ea men , a ie y and eplica e.
Rela i e g ow h was calcula ed a e 48 h o eeding on plan -based die
and mo ali y was eco ded e e y 24 h o a pe iod o 7 days. S a is ical
di e ences in ela i e g ow h and mo ali y a e B in ec ion be ween
ea men s we e analyzed using wo-way ANOVA, wi h MeJA applica-
ion and cons i u i e esis ance le el as ac o s (G aphPad So wa e Inc.,
San Diego, CA). The Spea man and Shapi o-Wilk es s we e applied o
de e mine homoscedas ici y and no mali y o da a, espec i ely. The
Pea son co ela ion coe icien was used o measu e he linea co ela-
ion o g ow h and basal mo ali y alues o non-in ec ed la ae wi h
mo ali y alues o B -in ec ed la ae.
2.6. Un a ge ed me abolomic analysis
A compa a i e me abolomic analysis was conduc ed o iden i y
me aboli es ha we e o e -accumula ed in he selec ed a ie ies a e
MeJA ea men . Fo his pu pose, he apical lea le o he ou h ue
lea (young, ully expanded lea ) om each plan sp ayed wi h ei he
MeJA o he sol en was ha es ed and kep a −80 ◦C o molecula
analysis. In o al, he e we e i e biological eplica es pe ea men
(con ol o MeJA applica ion) o each o he six selec ed a ie ies,
esul ing in 60 samples in o al. Each lea le was indi idually g ound
wi h liquid ni ogen, lyophilized, and 10 mg o powde we e esus-
pended in 1 ml o he ex ac ion bu e (MeOH:H
2
O 30:70 con aining
0.01 % o HCOOH). A e incuba ing on ice o 40 min, he homogena e
was cen i uged a 15,000 ×g o 15 min a 4 ◦C. The supe na an was
il e ed wi h 0.2
μ
m cellulose il e s (Regene a ed Cellulose Fil e , 0.20
μ
m, 13mmØ. Pk/100; Teknok oma, S Cuga , Spain). The il e ed su-
pe na an s (50
μ
L) we e dilu ed 1:3 using he ex ac ion bu e , and an
aliquo o 20
μ
L o each sample was esol ed by an Acqui y UPLC sys em
(Wa e s, Mild o d, MA, USA) in posi i e (ESI+) and nega i e (ESI-) ion
modes o elec osp ay ioniza ion, coupled o a hyb id quad upole ime-
o - ligh equipmen (QTOF MS P emie , Wa e s, Mild o d, MA, USA) o
de ec ion o me aboli es. Iden i ica ion o he signals was pe o med by
in oducing a second agmen a ion unc ion in o he TOF analyze in a
-wa e anging om 5 o 45 eV.
Raw da a we e ob ained om Masslynx .4.2 so wa e (Wa e s, USA)
and ans o med in o.cd iles using he Da aB idge ool. Ch oma o-
g aphic signals om posi i e and nega i e ESI we e p ocessed sepa a ely
wi h R so wa e .4.3.2 (h ps://c an. -p ojec .o g), and he XCMS al-
go i hm was used o il e ing and peak iden i ica ion, g ouping, and
signal co ec ions. Me aboli e amoun s we e quan i ied by no malizing
peak a ea uni s o he d y weigh o each sample. Ma Vis Sui e 2.0
so wa e allowed signal compa a ions be ween ea men s using
K uskal-Wallis es (P<0.05) ollowed by adduc and iso ope co ec-
ion, and clus e ing. Only peaks wi h signal- o-noise a io >10 we e
included in he analysis. Compounds whose signal in ensi y was signi -
ican ly inc eased a minimum o 2- old a e MeJA ea men and we e
sha ed by a leas ou oma o a ie ies we e selec ed. Iden i ica ion o
me aboli es was based on hei exac mass, e en ion ime, and spec um
agmen a ion (Gami e al., 2014; Schymanski e al., 2014). Signals
we e iden i ied wi h he use o di e en online da abases such as
Massbank, MassBank o No h Ame ica, PubChem and Human Me ab-
olome da abases (www.massbank.jp;h ps://mona. iehnlab.ucda is.
edu;h ps://pubchem.ncbi.nlm.nih.go ;h ps://hmdb.ca).
In addi ion, da a om posi i e and nega i e ESI we e combined and
no malized by median, ans o med by cube oo and scaled by Pa e o
me hod using Me aboAnalys 5.0 so wa e (h ps://www.
me aboanalys .ca) o ob ain spa se pa ial leas squa es disc iminan
analysis (sPLSDA) plo s and hea maps o each a ie y. Hea maps we e
gene a ed wi h hose me aboli es which di e ed signi ican ly (P<0.05,
K uskal-Wallis es ) in signal in ensi y a e MeJA induc ion compa ed
o he con ol.
3. Resul s
3.1. Sc eening o cons i u i e de enses o oma o geno ypes on S. exigua
pe o mance and suscep ibili y o B. hu ingiensis
To explo e he e ec s o na u al di e si y in cons i u i e chemical
de enses in oma o on S. exigua pe o mance and mo ali y by B , we
pe o med a plan -based sc eening. Speci ically, we assessed bo h di ec
e ec s by measu ing he S. exigua ela i e g ow h and mo ali y, and
indi ec e ec s by measu ing he suscep ibili y o S. exigua la ae o B
when eeding on di e en oma o geno ypes, including wild species and
cul i a ed a ie ies.
We measu ed he ela i e g ow h o S. exigua la ae a e 48 h o
con inuous eeding on plan -based die , ollowed by eeding on a i icial
die un il 144 h. When la ae we e ed exclusi ely on he a i icial die ,
hei ela i e g ow h a 144 h a e aged 130. In con as , eeding on
plan -based die s esul ed in no iceably educed la al ela i e g ow h,
anging om 10 o 110 wi h a median o 32.1 (Suppl. Table 1). A sig-
ni ican in luence o plan geno ype on S. exigua g ow h was de ec ed
(ANOVA, F
54,55
=1.7, P=0.026). The mean ela i e g ow h eco ded
om la ae ed on wild species was lowe compa ed o ha o la ae ed
on cul i a ed a ie ies (S uden ’s - es , P=0.0383) (Suppl. Table 1,
Fig. 1A).
We nex e alua ed he e ec o plan geno ypes on B pe o mance.
Feeding mock-in ec ed la ae on he di e en plan die s esul ed in
a ound 20 % mo ali y. This inc eased up o 40 % a e B in ec ion in
bo h wild (Tukey HSD es , P =0.0003) and cul i a ed (Tukey HSD es ,
P<0.0001) a ie ies (Suppl. Table 1,Fig. 1B), con i ming he e icacy o
B . Howe e , no signi ican di e ences we e obse ed be ween wild
species and cul i a ed a ie ies in e ms o la al mo ali y, ei he wi h
(Tukey HSD es , P =0.7282) o wi hou B (Tukey HSD es , P =
0.9982) (Suppl. Table 1,Fig. 1B).
In addi ion, we ound a clea inc ease in he mo ali y o la ae ed
on plan -based a i icial die s compa ed wi h hose exclusi ely ea ed on
a i icial die , ei he wi h o wi hou B in ec ion (mock-in ec ed: 0–5 %,
B -in ec ed: 5–10 %). The la e mo ali y alues we e ob ained om
p e ious assays conduc ed mul iple imes, being hese da a consis en
among he bioassays.
Analyzing he combined da a, we ound ha bo h la al ela i e
g ow h and basal mo ali y igge ed by each geno ype co ela ed wi h
suscep ibili y o B (Fig. 2). Speci ically, ela i e g ow h and suscep i-
bili y o B showed an in e se co ela ion ( = − 0.6137, P<0.0001,
Fig. 2A), whe eas mo ali y induced by he plan die alone and mo -
ali y igge ed by B exhibi ed a posi i e co ela ion ( =0.5207, P<
0.0001, Fig. 2B). These esul s sugges ha cons i u i e chemical de-
enses in oma o, which nega i ely a ec la al de elopmen and su -
i al, also con ibu e o exace ba e he e ec o B .
3.2. E ec o inducible de enses on la al pe o mance and suscep ibili y
o B. hu ingiensis
We u he in es iga ed how he inducible de enses in six di e en
cul i a ed oma o a ie ies a ec ed la al g ow h and in luenced sus-
cep ibili y o B . The six oma o a ie ies selec ed o his se o expe -
imen s we e g ouped based on hei cons i u i e esis ance le els
obse ed in he p e ious sc eening esul s. Va ie ies exhibi ing low
la al g ow h and high mo ali y in esponse o B we e classi ied as
ha ing high cons i u i e esis ance ( a ie ies 1, 2, 3), while hose
showing high la al g ow h and low mo ali y o B we e classi ied as
A. F a ini e al. Biological Con ol 198 (2024) 105624
4
ha ing low cons i u i e esis ance ( a ie ies 4, 5, 6) (Table 1).
Inducible de enses we e igge ed by he applica ion o MeJA, he
ac i e o m o JA, which is well known o inducing plan de enses
(Tu ne e al., 2002). As expec ed, MeJA applica ion signi ican ly
educed la al ela i e g ow h in he absence o B (Fig. 3A), wi h no
obse ed di e ences be ween he high and low cons i u i e esis ance
g oups (MeJA: F
1,24
=17.64, P=0.0003; esis ance le el: F
5,24
=1.663,
P=0.1819; in e ac ion: F
5,24
=0.4834, P=0.7851; Fig. 3A). Rega ding
he mo ali y o B -in ec ed la ae, we obse ed ha he MeJA ea men
led o an o e all inc ease o app oxima ely 15 % compa ed o la ae
aised on non-induced plan -based die s (Fig. 3B), e ealing he con i-
bu ion o induced de enses o highe suscep ibili y o B . The combined
e ec o he plan cons i u i e esis ance g oup (high o low) and MeJA
applica ion on B suscep ibili y was no signi ican (MeJA: F
1,24
=12.13,
P=0.0019; esis ance le el: F
5,24
=4.193, P=0.007; in e ac ion: F
5,24
=0.1260, P=0.9851; Fig. 3B), indica ing ha inducible de enses had an
e ec ega dless he cons i u i e de enses. Hence, combining bo h ac-
o s (cons i u i e esis ance and induced de ense) had an addi i e, bu
no syne gis ic e ec on la al suscep ibili y o B . Once again, we ound
ha lowe ela i e g ow h and highe basal mo ali y, igge ed by he
e ec s o MeJA-elici ed chemical de enses, co ela ed wi h highe sus-
cep ibili y o la ae o B ( = − 0.8808, P=0.0002, Fig. 3C; and =
0.5876, P=0.045, Fig. 3D), highligh ing ha inducible oma o de enses
ha di ec ly impai la al g ow h and su i al also inc ease B oxici y
agains S. exigua.
3.3. Impac o MeJA elici a ion on olia me abolic p o ile
As ou da a show a clea nega i e e ec o JA-inducible de enses on
la al pe o mance, we aimed o explo e he changes in he lea chem-
ical composi ion igge ed by he MeJA ea men po en ially espon-
sible o he obse ed pheno ypes. Fo ha , we analyzed he
me abolomic p o iles by LC-MS/MS. MeJA applica ion led o a ea -
angemen o he lea me abolome in all he 6 a ie ies, as indica ed by
supe ised PCA analysis (Fig. 4A). Hea map plo s we e gene a ed o
isualize di e en ially abundan signals be ween he con ol and MeJA
ea men g oups. Depending on he a ie y, he numbe o signals
signi ican ly di e en ially accumula ed anged om 100 o 300, and he
clus e ing con i med he impac o MeJA ea men on he oma o
me abolic p o ile (Fig. 4B).
A mo e de ailed analysis o he me abolomic p o iles led o he
iden i ica ion o six compounds which we e signi ican ly o e -
accumula ed (o e 2- old) a e MeJA ea men in a leas ou ou o
he six selec ed oma o a ie ies (Fig. 5 and Table 2). Among hem, ou
compounds we e iden i ied in he posi i e ioniza ion mode: 5-hyd ox-
yindole-3-ace ic acid, (+)-ca echin, an uniden i ied compound (m/z
826.10) and soyasapogenol B base +O-HexA, Hex, dHex, 1malonyl
(SB). In he nega i e ioniza ion mode, he me aboli es sy ingaldehyde,
and phospha idylglyce ol 42:10 we e ound. Rema kably, SB was he
only one signi ican ly o e -accumula ed in he six cul i a ed a ie ies.
Fig. 1. E ec o cons i u i e chemical de enses om wild species and oma o cul i a ed a ie ies on S. exigua g ow h and mo ali y. (A) Rela i e g ow h o
la ae ed on plan -based die s o 48 h and eplaced by a i icial die un il 144 h. Mean alues we e analyzed wi h S uden s - es . Whiske s plo ep esen he
a e age and s anda d e o o he mean (SEM). (B) Mo ali y o la ae ea ed on plan -based die s and in ec ed o no wi h Bacillus hu ingiensis (144 h pos in ec ion).
One-way ANOVA ollowed by Tukey’s mul iple compa isons es was used o analyze di e ences. Whiske s plo ep esen he a e age and SEM. As e isks indica e
signi ican di e ences be ween g oups (*P<0.05; ***P<0.001; ****P<0.0001). Colo coding and abb e ia ions: W, wild species, a e depic ed in o ange; C,
cul i a ed a ie ies a e depic ed in aquama ine.
Fig. 2. Co ela ion be ween impac o plan compounds on S. exigua and i s suscep ibili y o B . Sca e ed plo and Pea son co ela ion analysis be ween
ela i e g ow h o non-in ec ed la ae (A) o basal mo ali y (B) and mo ali y alues o B -in ec ed la ae. Highligh ed poin s co espond o alues co esponding o
he selec ed cul i a ed a ie ies ( ed: high cons i u i e esis ance, a ie ies 1–3; g een: low cons i u i e esis ance, a ie ies 4–6).
A. F a ini e al. Biological Con ol 198 (2024) 105624
5

4. Discussion
Plan s ely on cons i u i e and inducible de enses o p o ec hem-
sel es om he bi o ous insec s, such as ca e pilla s. These de enses
se e a ious unc ions, including de e ence, an i eedan p ope ies, o
oxici y agains he he bi o e i sel , as well as a ac ion o na u al en-
emies, like p eda o s and pa asi oids (Mi h¨
o e and Boland 2012).
Howe e , hei in e ac ion wi h mic obial en omopa hogens, ano he
majo g oup o insec na u al enemies, has been poo ly add essed. In his
s udy, we demons a e ha bo h cons i u i e and inducible de enses in
oma o inc ease he le hali y o he bac e ial en omopa hogen Bacillus
hu ingiensis agains he ca e pilla Spodop e a exigua.
Unlike p e ious s udies ha ocused solely on he e ec s o indi-
idual plan de ensi e compounds on B ac i i y (Fel on and Dahlman
1984; K ischik e al., 1988; Ludlum e al., 1991; Si amani e al., 1992;
Appel and Schul z 1994), ou app oach in ol es he use o chemical
elici a ion o plan de enses and lyophilized lea ma e ial o assess he
in luence o o e all chemical de enses, including he con ibu ion o
bo h cons i u i e and inducible componen s. Physical de enses such as
lea hickness, shape, o ha dness we e excluded, as he lea ma e ial
was g ounded in o powde . Mo eo e , by compa ing di e en plan
geno ypes, we demons a e ha he e ec on la al g ow h is geno ype-
dependen . Ou esul s e eal he posi i e con ibu ion o oma o de-
enses o he e ec i eness o he en omopa hogen B. hu ingiensis agains
a gene alis ca e pilla .
Toma o plan s syn hesize a ple ho a o chemical compounds o
de end agains insec he bi o es, such as alkaloids, phenolic com-
pounds, o i e penoids, which possess an i eedan p ope ies and
di ec oxici y agains he bi o es (Howe and Jande 2008; Wa e al.,
2012; Paudel e al., 2019). Howe e , many o hese compounds a e no
p oduced cons i u i ely due o hei high cos o he plan (Ka ban and
Baldwin 1997). Ins ead, hei accumula ion is induced upon de ec ion o
he bi o y. The balance be ween cons i u i e and induced de enses
wi hin a plan is o en in luenced by he domes ica ion p ocess, which
can ei he enhance o educe he a es o inducibili y in esponse o
he bi o e a ack (Whi ehead e al., 2017; Fe e o e al., 2020; Szyma´
nski
e al., 2020). Fo ins ance, many o namen al plan species exhibi lowe
cons i u i e and highe induced esis ance in domes ica ed species
(Kempel e al., 2011). The indings o ou s udy, which assessed he
e ec o cons i u i e chemical de enses on he ela i e g ow h o
S. exigua using plan -based die bioassays, co obo a ed his end.
Indeed, non-induced cul i a ed oma o a ie ies allowed o a g ea e
ela i e g ow h o S. exigua compa ed o wild species. This may be a
consequence o selec i e b eeding o e he yea s selec ing o yield and
ui cha ac e is ics, bu no o esis ance (Be gougnoux 2014). In
con as , he mo ali y o B -challenged la ae ed on di e en plan
die s was simila , ega dless o whe he he plan s we e wild o cul i-
a ed. This sugges s ha while domes ica ion al e ed he le els o
cons i u i e chemical de enses agains he ca e pilla s, i did no a ec
hose de enses ha could inc ease he e icacy o B .
The clea co ela ion be ween he mo ali y p oduced by B and he
de imen al e ec s on la al pe o mance (g ow h dec ease and mo -
ali y) om non-induced plan -based die s e eals ha cons i u i e o-
ma o de enses enhance la al suscep ibili y o B . These inding was
u he co obo a ed by obse a ions om MeJA-elici ed plan s, which
exhibi ed an analogous pa e n. A simila end was obse ed in ano he
Fig. 3. E ec o inducible chemical de enses om he selec ed oma o a ie ies on S. exigua g ow h and suscep ibili y o B . (A) Rela i e g ow h o la ae ed
on plan -based die s o 48 h. (B) Pe cen age o mo ali y o la ae ea ed on plan -based die s and in ec ed wi h Bacillus hu ingiensis (168 h pos in ec ion). In bo h
g ow h and mo ali y plo s, mean alues we e analyzed wi h wo-way ANOVA using MeJA and cons i u i e esis ance as ac o s. E o ba s depic s anda d e o o
he mean (SEM). Sca e ed plo and Pea son co ela ion analysis be ween ela i e g ow h (C) and basal mo ali y (D) o non-in ec ed la ae and pe cen age o
mo ali y o B -in ec ed la ae. As e isks show signi ican di e ences (**P<0.01; ***P<0.001).
A. F a ini e al. Biological Con ol 198 (2024) 105624
6
pa hosys em: a oma o-based die exace ba ed he e ec o he oppo -
unis ic pa hogen Se a ia ma cescens in Helico e pa zea, ano he gene -
alis lepidop e an om he Noc uidae amily (Mason e al., 2023). Based
on hese da a, we hypo hesized ha he enhanced ac ion o B p o ides a
p o ec i e ad an age o he plan , allowing o mo e e ec i e con ol o
insec he bi o es.
Plan s may use di e en s a egies o weaken insec s and enhance B
ac i i y. B. hu ingiensis kills i s hos s h ough he p oduc ion o po e-
o ming oxins, namely Vip and C y p o eins (B a o e al., 2011).
These oxins bind o he gu epi helium o suscep ible insec s, whe e
hey inse in o he b ush bo de memb ane o columna cells, c ea ing
an osmo ic imbalance ha leads o gu des uc ion. The dea h o he
insec may be u he acili a ed by he ac ion o oppo unis ic gu
bac e ia, which bene i om he gu des uc ion p oduced by B o
in ade he hos body and cause sep icaemia (Mason e al., 2011; Caccia
e al., 2016). We hypo hesize ha plan de enses may con ibu e o B
ac ion in se e al ways. Fi s , se e al plan phy ochemicals di ec ly
a ge he diges ion p ocess, jeopa dizing he insec ’s abili y o ob ain
nu i ional esou ces. Fo example, phenolics a e oxidized by poly-
phenol oxidases (PPOs) esul ing in he o ma ion o o hoquinones ha
alkyla e aminoacids and p o eins (Fel on e al., 1992; Cons abel and
Ba behenn 2008). Lec ins bind o glycosyl g oups and damage he
luminal epi helial memb ane (Vandenbo e e al., 2011), and p o einase
inhibi o s obs uc he ac i i y o diges i e enzymes (Zhu-Salzman and
Zeng 2015). The in e ac ion be ween hese molecules, which limi he
bioa ailabili y o nu ien s, and he des uc ion o he gu epi helial cells
p omp ed by B , may u he diminish he insec ’s abili y o ob ain
nu i ional esou ces, hus accele a ing hei dea h. In line wi h his, a
p e ious obse a ion ela ed he B -mo ali y o H. zea in JA-induced
oma o wi h he plan PPO ac i i y (Shikano e al., 2018b). Secondly,
o he plan compounds like i e penoids, alkaloids, la onoids o lec ins
may di ec ly weaken he immune sys em o he insec , in e e ing wi h
i s abili y o emo e he pa hogen and e en ually enhancing he la al
suscep ibili y o B due o hei inhe en oxici y (Wa e al., 2012). In
his ega d, we p e iously showed ha S. exigua aised on plan -based
die om plan s al eady exposed o he bi o y impai ed ca e pilla
g ow h, inc eased suscep ibili y o B , and dec eased la al PPO ac i i y
in he hemolymph, a key ma ke o he insec immune s a us (F a ini
e al., 2022). Thi dly, some phy ochemicals and de ensi e p o eins like
annins, chi inases o p o eases dis up he pe i ophic memb ane
(Mason e al., 2019), a physical ba ie agains pa hogens and me-
chanical damage ha co e s he en i e la al gu (Ba behenn and Pe e
Cons abel 2011; Konno and Mi suhashi 2019), whose absence inc eases
he suscep ibili y o ca e pilla s o B (Guo e al., 2019; Güney e al.,
2024).
Al hough we obse ed an inc ease in he suscep ibili y o S. exigua
Fig. 4. Me abolomic p o ile o MeJA-elici ed oma o plan s om he selec ed a ie ies. A non- a ge ed me abolic analysis o he lea es was pe o med o assess
me abolic changes a e ea men o plan shoo s wi h MeJA. Signals om posi i e and nega i e elec osp ay ioniza ion we e combined. A) Spa se pa ial leas
squa es disc iminan analysis (sPLSDA) ep esen a ion o he majo sou ces o a iabili y om he ob ained signals. B) Hea map ep esen ing me aboli es ha
signi ican ly di e ed in signal in ensi y a e MeJA induc ion compa ed o he con ol (P<0.05, Ku skal-Wallis es ). Yellow: con ol plan s; blue: MeJA-elici ed
plan s. Va ie ies 1–3 (high cons i u i e esis ance), a ie ies 4–6 (low cons i u i e esis ance).
A. F a ini e al. Biological Con ol 198 (2024) 105624
7
la ae o B a e eeding on he oma o plan s, o he ac o s, such as
la al ins a , body size, o iming o exposu e o B (be o e o a e plan
die eeding), should be conside ed o a mo e comple e unde s anding
o he e ec s o plan de ense compounds on he ac i i y o B . P e ious
s udies ha e shown ha smalle la al body size o slowe la al g ow h
is associa ed o a highe suscep ibili y o pa hogens (Vogelwei h e al.,
2013; Shikano e al., 2018a), likely due o a lowe alloca ion o esou ces
o comba in ec ion and/o a p olonged pe iod o ulne abili y o he
na u al enemies. Based on his, i is plausible ha in ec ing younge
S. exigua la ae (neona es o i s ins a s) wi h B would esul in a
highe e icacy o he bac e ial en omopa hogen agains he ca e pilla .
Timing o pa hogen challenge is a c i ical ac o when e alua ing he
ole o plan s in he in e ac ion be ween insec s and pa hogens (Co y and
Hoo e 2006; Shikano 2017). Depending on he iming o pa hogen
inges ion and plan ma e ial inges ion, we may encoun e di e en
scena ios; (1) I plan ma e ial is inges ed well in ad ance o pa hogen-
challenge, his may esul in plan -media ed a ia ion in hos physi-
ology, o example he weakening o pe i ophic memb ane may acili-
a e he u he ac ion o he en omopa hogen; (2) I plan ma e ial and
he pa hogen a e co-inges ed, di ec in e ac ions be ween plan chem-
icals and he pa hogen may occu in he insec ’s gu , o example in he
case o he po en ial alkyla ion o B p o oxins igge ed by quinones
p oduced by oma o de ensi e enzyme PPO; and (3) I plan ma e ial is
inges ed a e pa hogen-challenge, he plan quali y may a ec he in-
sec ’s immune unc ion. In ou s udy, we in ec ed la ae wi h B p io o
eeding on plan die s, wi h a 15-minu e ime window be ween he
ea men s. A e 48 h o eeding on he plan die s, la ae we e shi ed
o an a i icial die . We hypo hesize ha he enhanced B mo ali y
Fig. 5. Rela i e abundance o oma o me aboli es elici ed ac oss selec ed oma o cul i a ed a ie ies. Rela i e abundances a e shown as no malized signal
in ensi ies o MeJA-elici ed me aboli e compa ed o he con ol wi hin each a ie y. Fou compounds we e iden i ied in posi i e mode ioniza ion (le ), while wo
o he s we e iden i ied in nega i e mode ioniza ion ( igh ). E o ba s depic s anda d e o o he mean (SEM). The colo code indica es con ol plan s in yellow and
MeJA-elici ed plan s in blue. As e isks indica e signi ican di e ences be ween MeJA-elici ed and he espec i e con ol plan s (K uskal-Wallis es , P<0.05, FC ≥2,
n=5).
Table 2
Me aboli es wi h signi ican induc ion ollowing MeJA applica ion ac oss he six selec ed oma o a ie ies.
Ion mode R (s)
1
Exac mass Pa en ion Majo agmen s Compound iden i y Va ie ies
2
Induc ion le el
3
Posi i e 4.81 1029.14 1030.02 528.95 >145.07 Soyasapogenol B base +O-HexA, Hex, dHex, 1malonyl 1–6 3.5–14.9
Posi i e 2.66 191.03 192.10 147.04 >117.03 5-Hyd oxyindole-3-ace ic acid 3–6 2.2–9.5
Posi i e 3.73 290.09 291.08 207.08 >123.09 (+)–Ca echin 1, 3, 5, 6 2.2–7.8
Posi i e 4.81 826.10 827.35 Unknown 1, 2, 4, 5 2.2–4.1
Nega i e 2.64 182.05 181.04 123.03 >151.01 Sy ingaldehyde 1, 3–6 2.8–23.5
Nega i e 2.41 843.31 842.24 153.01 >327.01 Phospha idylglyce ol 42:10 1, 3–6 3–8.6
1
R : e en ion ime.
2
S. lycope sicum cul i a ed a ie ies g ouped as high (1–3) o low (4–6) cons i u i e esis ance based on hei impac on S. exigua pe o mance and mo ali y.
3
Rela i e abundances o MeJA-elici ed me aboli es we e compa ed wi h hose om con ol ea men o ob ain he induc ion le el alues (K uskal-Wallis es , P<
0.05).
A. F a ini e al. Biological Con ol 198 (2024) 105624
8
ela ed o he inges ion o plan ma e ial, such as ha obse ed in MeJA-
induced plan , is likely ela ed o changes in he hos physiology. Fo
example, i could be associa ed wi h he opening o po es in he insec
gu by B po e- o ming oxins, which may allow plan chemicals o en e
he hemocoel and ha e g ea e oxic e ec . Howe e , u u e esea ch
should assess he e ec o lea compounds on B le hali y when plan
die s a e inges ed simul aneously wi h, o p io o, pa hogen challenge.
To da e, only ew indi idual de ensi e plan compounds ha e
demons a ed o enhance B ac ion (Fel on and Dahlman 1984; K ischik
e al., 1988; Si amani e al., 1992; Appel and Schul z 1994). To iden i y
bioac i e molecules po en ially esponsible o bo h de imen al e ec s
on la al g ow h and mo ali y and B suscep ibili y, we compa ed he
p o iles o he six MeJA-elici ed oma o a ie ies. Ou esul s e ealed a
subs an ial ea angemen o he lea me abolic p o ile in all a ie ies
a e MeJA ea men , ega dless o hei cons i u i e esis ance classi-
ica ion. This me abolic shi in lea es a e exogenous applica ion o
MeJA is a well-documen ed phenomenon in a ious plan s (Flo es-
Sanchez e al., 2016; Papazian e al., 2019; Ramabulana e al., 2020),
a ibu ed o JA-media ed induc ion o seconda y me aboli e biosyn-
hesis (Was e nack and Hause 2013; Was e nack 2014; Yu e al., 2019).
We looked o me aboli es consis en ly o e -accumula ed in elici ed
plan s ac oss he es ed a ie ies. Among hem, we iden i ied six me-
aboli es o e -accumula ed ac oss a ie ies, i e o hem wi h signi ican
inc eases in abundance in a leas ou ou o he six es ed oma o a-
ie ies: 5-hyd oxyindole-3-ace ic acid, (+)-ca echin, sy ingaldehyde,
phospha idylglyce ol 42:10 and an uniden i ied compound wi h a m/zo
826.10. Among hese, he la onoid ca echin showed an acu e oxici y
agains Spodop e a li u a la ae (Ru anaphan e al., 2023), and educed
he la al g ow h o he lepidop e an pes Ec opis g isescens (Li e al.,
2022) when supplemen ed in a i icial die . In e es ingly, he compound
la one, belonging o he la onoid amily, has been epo ed o in e ac
in a syne gis ic way wi h he B oxin C y1Ac when inducing mo ali y
agains Helico e pa a mige a (Wang e al., 2021). Simila ly, he poly-
phenol sy ingaldehyde nega i ely a ec ed he mobili y o he bean
wee il Acan hoscelides ob ec us and inc eased i s mo ali y (Regnaul -
Roge e al., 2004).
A single compound was signi ican ly o e -accumula ed in all six
oma o a ie ies: soyasapogenol B base +O-HexA, Hex, dHex, 1
malonyl. This compound co esponds o a i e penoid saponin, a sec-
onda y me aboli e widely dis ibu ed in plan s and whose p oduc ion is
in luenced by bio ic en i onmen al ac o s (Szakiel e al., 2011; de Cos a
e al., 2013). Saponins play a ole in plan de enses agains he bi o es,
pa icula ly due o hei amphipa hic p ope ies, which lead o he o -
ma ion o s ong complexes wi h choles e ol, igge ing cellula oxici y
and al e ing insec mol ing (Weng e al., 2011). Mo eo e , saponins
dis u b insec diges ion by inac i a ing diges i e enzymes and al e ing
insec gu mic obio a (Singh and Kau 2018). P e ious s udies whe e
saponins we e added o he a i icial die ha e shown a nega i e e ec
on g ow h and/o mo ali y o o he lepidop e an insec s such as Spo-
dop e a li o alis (De Gey e e al., 2007), Os inia nubilalis (Nozzolillo
e al., 1997) and Helico e pa zea (Dowd e al., 2011). Mo eo e , a syn-
e gis ic in e ac ion be ween a ea saponin, a myco oxin om Me a-
hizium anisopliae and B a . ku s aki was de ec ed agains S. exigua in
e ms o mo ali y (Rizwan-Ul-Haq e al., 2009). The epo ed an i-
he bi o y e ec s o hese compounds suppo hei ole in he g ow h
educ ion obse ed in la ae ed on MeJA-induced plan s and may be
esponsible o he inc eased mo ali y upon B in ec ion.
In conclusion, ou s udy demons a ed ha bo h cons i u i e and
inducible oma o de enses inc ease he suscep ibili y o he gene alis
ca e pilla S. exigua o B. hu ingiensis. We iden i ied some compounds
which may con ibu e o hese oxic e ec s. These esul s s ongly sup-
po he no ion ha oma o de enses, which di ec ly a ec he insec
he bi o e, may also indi ec ly enhance he ac ion o a hi d ophic
le el, he en omopa hogen g oup. This inding has posi i e implica ions
o sus ainable c op managemen p og ams, which may combine he
selec ion o plan de ense ai s wi h he use o en omopa hogens.
CRediT au ho ship con ibu ion s a emen
Ada F a ini: W i ing –o iginal d a , So wa e, Resou ces, Me h-
odology, In es iga ion, Fo mal analysis, Da a cu a ion. Rosa M.
Gonz´
alez-Ma ínez: Resou ces, Me hodology. Juan M. Ga cía: Me h-
odology, Resou ces. Zhi ko Minche : W i ing – e iew &edi ing,
So wa e, Fo mal analysis. Ma ía J. Pozo: W i ing – e iew &edi ing,
Valida ion, Supe ision, P ojec adminis a ion, Me hodology, Funding
acquisi ion, Fo mal analysis, Concep ualiza ion. Víc o Flo s: W i ing –
e iew &edi ing, Valida ion, Supe ision, So wa e, Resou ces, P ojec
adminis a ion, Me hodology, Funding acquisi ion, Fo mal analysis,
Concep ualiza ion. C is ina M. C a a: W i ing – e iew &edi ing,
Valida ion, Supe ision, So wa e, P ojec adminis a ion, Me hodology,
Funding acquisi ion, Fo mal analysis, Concep ualiza ion. Sal ado
He e o: W i ing – e iew &edi ing, Valida ion, Supe ision, P ojec
adminis a ion, Me hodology, Funding acquisi ion, Fo mal analysis,
Concep ualiza ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
We hank Cla a Pons and An onio G anell om he Ins i u e o Plan
Molecula and Cellula Biology (IBMCP) o he ini ial in o ma ion
abou he me abolic di e si y o he oma o a ie ies used in he
sc eening. This wo k was suppo ed by g an s PID2021-124813OB-C33,
-C31, -C32 unded by MCIN/AEI/10.13039/501100011033 and by
‘ERDF A way o making Eu ope’, by he Eu opean Union and by g an
om he Gene ali a Valenciana (g an numbe CIPROM/2023/56) and
by g an PID2020-118787RA-I00 unded by MICIU/AEI/10.13039/
50110001103. AF was ecipien o a PhD g an om he Spanish Min-
is y o Educa ion (No. FPU16/02363). CMC was suppo ed by a Ram´
on
y Cajal g an (RYC2021-033098-I) unded by MICIU/AEI/10.13039/
501100011033 and Nex Gene a ionEU/PRTR.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.biocon ol.2024.105624.
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