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Biocontrol of phytopathogenic fungi relevant for cacao- and olive-derived economy

Lopes, Pedro Ferraz Ramos · 2020

Abstract

Plant diseases caused by fungal pathogens constitute an increasing threat to food production and security. This is the case of Witches’ Broom Disease (WBD) of cacao, caused by the basidiomycete Moniliophthora perniciosa, and of Olive Anthracnose (OA), caused by a consortium of Colletotrichum gloeosporioides and C. acutatum species complexes, which seriously impact the economy. Both share the lack of effective containment methods. The use of chemical fungicides is increasingly more precarious and restricted. More effective eco-friendly methods are required, such as the use of biocontrol agents. This work aimed to evaluate the ability of yeasts to antagonize the fungal causal agents of both diseases. A group of yeasts originating from Brazilian sugarcane-based fermentation industries were tested against M. perniciosa. Two isolates from cachaça production fermentation and one of the most used strains in bioethanol production, efficiently kill six strains of M. perniciosa in vitro. Antagonistic ability is maintained in non-optimal conditions. Microscopy analysis showed that fungal cells die upon contact with these yeasts, which physically attach and fuse to the mycelium and drain the cells, in what appears like a yeast predacious behaviour. Two further observations revealed the formation of connections between yeast and fungal cells, as well as fimbriae-like connections between yeast cells. These results agree in that, at a distance, only the proliferation of mycelia is diverted through the probable secretion of a non-volatile agar diffusible compound. Several proteins were identified specifically secreted by fungi and yeasts in single culture and co-culture. The methodology now developed was further used to study the antagonism of yeasts against the OA-causing Colletotrichum sp.. The same group of fermentative yeasts were tested along with others originating from the olive biome in Portuguese orchards. One of these last strains showed a promising antagonistic potential, by inhibiting the growth of the three fungal strains in all the conditions tested. Moreover, the possibility of using vinasse, a waste product from sugarcane bioethanol production process, to control the development of M. perniciosa was also evaluated. Immersing or spraying the mycelium with vinasse either kills the fungus or impedes its proliferation. This effect is not extensive the OA-causing fungi. The results support the exploration of microbial biodiversity of the infected plant-associated biome to generate greener and more sustainable alternatives to contain phytopathogens like those causing WBD and OA, contributing to alleviate the socio-economic impact of these diseases.

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Biocontrol of phytopathogenic fungi relevant for cacao- and olive-derived economy

Author: Lopes, Pedro Ferraz Ramos
Year: 2020
Source: https://repositorium.uminho.pt/bitstreams/b9f46313-f15f-481f-baf0-4e938bd455a1/download
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS
Es e é um abalho académico que pode se u ilizado po e cei os desde que espei adas as eg as e
boas p á icas in e nacionalmen e acei es, no que conce ne aos di ei os de au o e di ei os conexos.
Assim, o p esen e abalho pode se u ilizado nos e mos p e is os na licença abaixo indicada.
Caso o u ilizado necessi e de pe missão pa a pode aze um uso do abalho em condições não p e is as
no licenciamen o indicado, de e á con a a o au o , a a és do Reposi ó iUM da Uni e sidade do Minho.
A ibuição-NãoCome cial-SemDe i ações
CC BY-NC-ND
h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/
iii
ACKNOWLEDGEMENTS
Em p imei o luga , que o ag adece à P o esso a Cândida Lucas, po me e acolhido no seu g upo de
in es igação e po me e dado a conhece o ema que me apaixonou desde o p imei o minu o. Ag adeço
odo empo, paciência e conselhos aliosos, p incipalmen e nos momen os di íceis, que mui o me
ajuda am a consegui alcança es e ma co na minha ida pessoal e p o issional. À P o esso a Fe nanda
Cássio, po oda a ajuda, disponibilidade e pelas pala as de incen i o ao longo des es qua o anos.
A odos os colegas do Labo a ó io de Biodi e sidade e do LBM II po oda a ajuda e boa disposição, em
especial às minhas pa cei as de bancada. À Giulia e à Joana, po me e em ajudado desde o p imei o
minu o em udo e me e em in oduzido ao mundo da mic obiologia, po oda a aleg ia e amizade. À
Ma iana e à Co alie, as minhas pupilas, po oda a ajuda em an os momen os essenciais, p incipalmen e
nos mais complicados e po oda a boa disposição que o na am o ambien e no labo a ó io ão bom.
A odos os colegas do P og ama Dou o al, em especial à Guida, Má io, Rosana e Rui po e em pa ilhado
comigo o es a u o de cobaias e po odo o companhei ismo que semp e demons a am.
A odas as pessoas que pa ilha am comigo a ho a de almoço e as pausas pa a ca é nes es qua o anos,
em especial à Ca a ina, Cláudia, Dá io, Diana, Gab iel, Giulia, Joana Pe ei a, Joana Tulha, Ma ia, Má io
e Rosana, po odos os momen os bons e po se em amigos que ica ão pa a a ida.
A odas as pessoas do Depa amen o de Biologia e do IB-S que, de uma o ma ou de ou a, me ajuda am
quando necessá io. Um ag adecimen o especial à Lídia, Manuela e S . Luís po oda a disponibilidade.
Aos meus pais e i mãos, po e em o nado possí el es e meu sonho e po me e em semp e apoiado
incondicionalmen e ao longo da minha ida. Mui o ob igado po udo!
À Ca a ina, po odo o amo , ca inho e paciência, p incipalmen e po e a u ado es es úl imos empos
s essan es da esc i a da ese, eu sei que não o am áceis. Ob igado po es a es semp e lá pa a udo!
This wo k was suppo ed by he s a egic p og amme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) unded by
na ional unds h ough he FCT I.P. and by he ERDF h ough he COMPETE2020 - POCI, and he p ojec EcoAg iFood (NORTE-
01-0145-FEDER-000009), suppo ed by he NORTE 2020 unde he PORTUGAL 2020 Pa ne ship Ag eemen h ough he
Eu opean Regional De elopmen Fund (ERDF). PF is a s uden o he Doc o al P og amme in Applied and En i onmen al
Mic obiology (DP_AEM) and FCT g an ee PD/BD/113810/2015. We hank Ma iana Amo im-Rod igues (FCT g an
PD/BD/145354/2019) o he p ecious con ibu ions wi h he esea ch o he Chap e 4. We hank he B azilian companies,
Ce le , Lda., Ou o P e o, MG, and Fe men ec, Lda. Soluções Tecnológicas e Indus iais, Pi acicaba, SP, as well as P o esso
João Paulo Sampaio om he Po uguese Yeas Cul u e Collec ion/UCIBIO, NOVA, Po ugal o kindly supplying he yeas
s ains used in his wo k. We also hank P o esso Ped o Talhinhas, om LEAF - Linking Landscape, En i onmen , Ag icul u e
and Food, ISA, Uni e sidade de Lisboa, Po ugal, o kindly supplying wo o he ungal s ains used in his wo k.

i
STATEMENT OF INTEGRITY
I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used
plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o
i s elabo a ion.
I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o Minho.
ABSTRACT
Biocon ol o phy opa hogenic ungi ele an o cacao- and oli e-de i ed economy
Plan diseases caused by ungal pa hogens cons i u e an inc easing h ea o ood p oduc ion and
secu i y. This is he case o
Wi ches’ B oom Disease
(WBD) o cacao, caused by he basidiomyce e
Monilioph ho a pe niciosa
,
and o Oli e An h acnose (OA), caused by a
conso ium
o
Colle o ichum
gloeospo ioides
and
C. acu a um
species complexes, which se iously impac he economy. Bo h sha e
he lack o e ec i e con ainmen me hods. The use o chemical ungicides is inc easingly mo e p eca ious
and es ic ed. Mo e e ec i e eco- iendly me hods a e equi ed, such as he use o biocon ol agen s.
This wo k aimed o e alua e he abili y o yeas s o an agonize he ungal causal agen s o bo h diseases.
A g oup o yeas s o igina ing om B azilian suga cane-based e men a ion indus ies we e es ed agains
M. pe niciosa
. Two isola es om
cachaça
p oduc ion e men a ion and one o he mos used s ains in
bioe hanol p oduc ion, e icien ly kill six s ains o
M. pe niciosa
in i o
. An agonis ic abili y is main ained
in non-op imal condi ions. Mic oscopy analysis showed ha ungal cells die upon con ac wi h hese
yeas s, which physically a ach and use o he mycelium and d ain he cells, in wha appea s like a yeas
p edacious beha iou . Two u he obse a ions e ealed he o ma ion o connec ions be ween yeas and
ungal cells, as well as imb iae-like connec ions be ween yeas cells. These esul s ag ee in ha , a a
dis ance, only he p oli e a ion o mycelia is di e ed h ough he p obable sec e ion o a non- ola ile aga
di usible compound. Se e al p o eins we e iden i ied speci ically sec e ed by ungi and yeas s in single
cul u e and co-cul u e. The me hodology now de eloped was u he used o s udy he an agonism o
yeas s agains he OA-causing
Colle o ichum
sp.. The same g oup o e men a i e yeas s we e es ed
along wi h o he s o igina ing om he oli e biome in Po uguese o cha ds. One o hese las s ains
showed a p omising an agonis ic po en ial, by inhibi ing he g ow h o he h ee ungal s ains in all he
condi ions es ed. Mo eo e , he possibili y o using
inasse
, a was e p oduc om suga cane bioe hanol
p oduc ion p ocess, o con ol he de elopmen o
M. pe niciosa
was also e alua ed. Imme sing o
sp aying he mycelium wi h
inasse
ei he kills he ungus o impedes i s p oli e a ion. This e ec is no
ex ensi e he OA-causing ungi. The esul s suppo he explo a ion o mic obial biodi e si y o he in ec ed
plan -associa ed biome o gene a e g eene and mo e sus ainable al e na i es o con ain phy opa hogens
like hose causing WBD and OA, con ibu ing o alle ia e he socio-economic impac o hese diseases.
Keywo ds: An agonism; Biocon ol; Oli e An h acnose;
Wi ches’ B oom Disease
; Yeas s
i
RESUMO
Biocon olo de ungos i opa ogénicos impo an es pa a a economia do cacau e da oli ei a
Fi opa ologias causadas po ungos são uma ameaça c escen e à p odução e segu ança alimen a es. É
o caso da doença da Vassou a-de-b uxa (VB) do cacau, causada pelo ungo
Monilioph ho a pe niciosa
, e
da An acnose da oli ei a (AO), causada pelos complexos de espécies
Colle o ichum gloeospo ioides
e
C. acu a um
, que se o na am um isco pa a a economia, po não ha e mé odos e icazes de con enção.
O uso de ungicidas químicos é cada ez mais ine icaz e mais es i o, pelo que é necessá io encon a
al e na i as mais amigas do ambien e, como po exemplo a u ilização de biocidas. Es e abalho e e
como obje i o a alia a capacidade de le edu as pa a an agoniza os ungos causado es des as doenças.
Um g upo de le edu as p o enien e da e men ação de caldo-de-cana no B asil oi es ado con a
M.
pe niciosa.
Dois isolados do p ocesso de p odução de cachaça e uma das le edu as mais u ilizadas na
p odução indus ial de bioe anol ma am e icien emen e 6 es i pes de
M. pe niciosa
,
in i o
. O
an agonismo man ém-se em condições ad e sas. Obse ações de mic oscopia con i ma am que o ungo
mo e em con ac o com as le edu as que ade em e se undem com o micélio, es aziando-o, no que
pa ece se um compo amen o p eda ó io. Além disso, obse ou-se a exis ência de conexões en e
células de le edu a e do micélio, e en e células de le edu a en e si. Is o conco da com o ac o de, à
dis ância, ha e apenas edi eccionamen o do desen ol imen o do micélio, a a és da di usão de um
compos o não- olá il, endo sido e i icado que os ungos e as le edu as seg egam p o eínas di e en es
em cul u a simples ou em co-cul u a. O mé odo desen ol ido nes e abalho oi aplicado pa a e i ica o
an agonismo de le edu as con a os agen es causado es da AO. O mesmo conjun o de le edu as
e men a i as oi es ado, em con on o com es i pes p o enien es do bioma de oli ais po ugueses. Uma
des as úl imas le edu as mos ou g ande po encial an agonis a con a 3 es i pes de
Colle o ichum
sp.
em odas as condições es adas. Adicionalmen e, oi ensaiada com sucesso a u ilização da inhaça, o
esíduo denso e líquido que esul a da des ilação do e anol após a e men ação do caldo-de-cana, no
con olo do desen ol imen o do
M. pe niciosa
. A ime são ou a pul e ização do micélio com inhaça ma a
ou impede d as icamen e a p oli e ação do ungo. Es e e ei o não oi ex ensí el aos ungos causado es
da AO. Em suma, a explo ação da biodi e sidade mic obiana do bioma associado às plan as in e adas
pode pe mi i desen ol e me odologias al e na i as, mais amigas do ambien e e mais sus en á eis, pa a
a con enção da p esen e p og essão dos agen es i opa ogénicos, como os que p o ocam a VB e a AO,
con ibuindo pa a ali ia o impac o socioeconómico des as doenças.
Pala as-cha e: An agonismo; An acnose da Oli ei a; Biocon olo; Le edu as;
Vassou a-de-b uxa
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................................................ .iii
STATEMENT OF INTEGRITY ........................................................................................................................ i
ABSTRACT .............................................................................................................................................
RESUMO .............................................................................................................................................. i
LIST OF FIGURES .................................................................................................................................... xi
LIST OF TABLES .................................................................................................................................... xi
LIST OF COMMON ABBREVIATIONS ............................................................................................................ x i
LIST OF PUBLICATIONS .......................................................................................................................... iii
CHAPTER 1 - Gene al In oduc ion
INTRODUCTION ...................................................................................................................................... 2
WITCHES’ BROOM DISEASE OF CACAO ........................................................................................................ 3
OLIVE ANTHRACNOSE .............................................................................................................................. 7
FUNGAL DISEASES MANAGEMENT ............................................................................................................ 12
YEASTS AS BIOCONTROL AGENTS .............................................................................................................. 16
Kille oxins .................................................................................................................................. 17
Ly ic enzymes and mycopa asi ism ............................................................................................... 20
Vola ile compounds ...................................................................................................................... 20
Compe i ion o nu ien s and space ............................................................................................. 21
Quo um sensing ........................................................................................................................... 21
En i onmen al a iables ................................................................................................................ 22
ADVANTAGES AND CHALLENGES OF MICROBIAL WARFARE ............................................................................... 23
THESIS OUTLINE ................................................................................................................................... 26
REFERENCES ....................................................................................................................................... 29
xi
LIST OF TABLES
CHAPTER 1 - Gene al In oduc ion
Table 1. Success cases o using yeas s o an agonize he spoilage o ui s by ilamen ous ungi ...... 18
CHAPTER 2 -
Saccha omyces ce e isiae
and
Wicke hamomyces anomalus
a e able
o kill
Monilioph ho a pe niciosa
, he causal agen o cacao
Wi ches’ B oom Disease
Table 1. S ains o
Monilioph ho a pe niciosa
om CBS-KNAW (www.wi.knaw.nl), hei p imi i e o igin,
and he assigned le e code used in his wo k .................................................................................. 56
Table 2. Yeas s ains used in his wo k, hei p imi i e o igin, and hei assigned code used ............ 57
Table 3. Resul s o he an agonism assays be ween
M. pe niciosa
and yeas s in solid medium ........ 63
Table 4. An agonism assays in liquid medium using he combina ions be ween he e men a i e yeas
s ains and
M. pe niciosa
s ains F and G.......................................................................................... 67
Table 5. An agonism assays using selec ed combina ions o yeas and ungal s ains, challenged wi h
en i onmen al s esso s. ................................................................................................................... 72
CHAPTER 4 -
Wicke hamomyces anomalus
om oli e o cha ds mic obiome
e icien ly an agonizes he Oli e An h acnose’s causal agen s,
Colle o ichum
spp.
Table 1. Phy opa hogenic ilamen ous ungi and yeas s ains and hei o igin ................................ 107
Table 2. Resul s o an agonism assays in solid medium, using he yeas s ains om Table 1 agains
Colle o ichum gloeospo ioides
s.s.,
C. gode iae
and
C. nymphaeae
................................................ 109
Table 3. An agonism be ween he OA causa i e agen s
Colle o ichum gloeospo ioides
s.s.,
C. gode iae
and
C. nymphaeae
and he yeas s ains in Table 1: esul s a e pe cen age o inhibi ion in solid media ......... 110

x
Table 4. An agonism be ween he phy opa hogenic ungal s ains
Colle o ichum gloeospo ioides
s.s.,
C. gode iae
and
C. nymphaeae
and he yeas s ains in Table 1 in liquid media. .............................. 112
Table S1. G ow h a es o he phy opa hogenic ungal s ains
Colle o ichum gloeospo ioides
s.s.,
C.
gode iae
and
C. nymphaeae
a 25 and 30 °C, in wo cul u e media and a di e en pH alues. ....... 115
SUPPLEMENTARY MATERIAL
Table 1. P o eins om yeas s/ ungal s ains co-cul u es and con ol ungal single cul u e. SDS-PAGE
bands we e analyzed by PMF and agmen a ion. ............................................................................ 149
x i
LIST OF COMMON ABBREVIATIONS
µg G ow h Ra e (yeas )
ANOVA Analysis o Va iance
AOX Al e na i e Oxidase
ATP Adenosine T iphospha e
COD Chemical Oxygen Demand
cy c Cy och ome
c
DNA Deoxy ibonucleic Acid
dsDNA Double-s anded Deoxy ibonucleic Acid
Eno2 Enolase 2
ER Endoplasmic Re iculum
GAPDH Glyce aldehyde 3-phospha e dehyd ogenase
Gd G ow h diame e
G G ow h Ra e ( ungi)
GRAS Gene ally Recognized As Sa e
kDa kilodal on
K e1 Kille oxin Resis an 1
KT Kille Toxin
MB Me hylene Blue
MDA Malondialdehyde
ME Mal Ex ac
MEA Mal Ex ac -Aga
MOX Me hanol Oxidase
OA Oli e An h acnose
OD Op ical Densi y
PBS Phospha e Bu e ed Saline
PDA Po a o-Dex ose-Aga
PI P opidium Iodide
PMF Pep ide Mass Finge p in ing
QSI Quo um Sensing Inhibi o
QSM Quo um Sensing Molecule
x ii
RNA Ribonucleic Acid
ROS Reac i e Oxygen Species
pm Ro a ions pe minu e
RPS Ribosomal P o eins o he Small subuni
Scw4 Soluble Cell Wall 4
SDS Sodium Dodecyl Sulpha e
SDS-PAGE Sodium Dodecyl Sulpha e - Polyac ylamide Gel Elec opho esis
SEM Scanning Elec on Mic oscopy
SOD Supe oxide Dismu ase
TCA T ica boxylic Acid
Tdh3 T iose-phospha e Dehyd ogenase 3
TEM T ansmission Elec on Mic oscopy
/ olume pe olume
V-ATPase Vacuola - ype H+-ATPase
VOC Vola ile Compound
w/ weigh pe olume
WBD Wi ches’ B oom Disease
Ynk1 Yeas Nucleoside diphospha e Kinase 1
YPD Yeas Ex ac -Pep one-Dex ose
YPDA Yeas Ex ac -Pep one-Dex ose-Aga
x iii
LIST OF PUBLICATIONS
The wo k pe o med du ing his PhD esul ed in he ollowing pee - e iewed publica ions:
Fe az, P., Cássio, F. and Lucas, C. (2019) Po en ial o Yeas s as Biocon ol Agen s o he Phy opa hogen
Causing Cacao
Wi ches’ B oom Disease
: Is Mic obial Wa a e a Solu ion? F on ie s in Mic obiology 10:
1766
Fe az, P., Amo im-Rod igues, M., Cássio, F. and Lucas, C.
Saccha omyces ce e isiae
and
Wicke hamomyces anomalus
a e able o kill
Monilioph ho a pe niciosa
, he causal agen o cacao
Wi ches’ B oom Disease
(submi ed)
Fe az, P., Cássio, F. and Lucas, C. Mic oscopic assessmen o he an agonism e ec o yeas s agains
Monilioph ho a pe niciosa
, he ungal causal agen o
Wi ches’ B oom Disease
in cacao (manusc ip in
p epa a ion)
Amo im-Rod igues, M., Fe az, P., Cássio, F. and Lucas, C.
Wicke hamomyces anomalus
om oli e
o cha ds mic obiome e icien ly an agonizes he Oli e An h acnose’s causal agen s,
Colle o ichum
spp.
(manusc ip in p epa a ion)
Fe az, P., Amo im-Rod igues, M., Cássio, F. and Lucas, C.
Vinasse
was e om suga cane-based
bioe hanol p oduc ion plan s kills
Monilioph ho a pe niciosa,
he causa i e agen o cacao
Wi ches’ B oom
Disease
. (submi ed)
CHAPTER 1
Gene al In oduc ion
Pa o his chap e has been p e iously published in:
Fe az, P., Cássio, F. and Lucas, C. (2019) Po en ial o Yeas s as Biocon ol Agen s o he Phy opa hogen Causing
Cacao
Wi ches’ B oom Disease
: Is Mic obial Wa a e a Solu ion? F on ie s in Mic obiology 10: 1766

2
INTRODUCTION
A g ea numbe o plan diseases a e esponsible o majo c op losses wi h huge socio-economic impac ,
causing each yea a wo ldwide es ima ed losses o 40 billion dolla s (Syed Ab Rahman
e al.
, 2018).
Pa icula ly, he diseases caused by ungal pa hogens a e inc easingly ecognized as a global h ea o
ood p oduc ion and secu i y. In ac , since 2000 he numbe o new ungal plan pa hogen ale s has
inc eased by mo e han 7- old (Fishe
e al.
, 2012). P esen ly, ungal-gene a ed diseases cons i u e 64-
67% o he o al c op diseases epo ed globally (Fishe
e al.
, 2012; Fishe
e al.
, 2018), and accoun o
20% o he losses a he le el o p oduc ion and a u he 10% a pos ha es le el (Fishe
e al.
, 2018).
These au ho s es ima e ha ungal diseases a e sp eading no hbound a a a e o almos 8 km/yea .
This could de i e om inc easingly common ag icul u al p ac ices, such as he ex ensi e monocul u es
and he use o a es ic ed numbe o plan cul i a s, as well as he inc eased global in e na ional ade
p opo iona ing disease sp eading o e g ea dis ances (Fishe
e al.
, 2012). Clima e change adds a
bu den o ha equa ion, po en ia ing he de elopmen o mic obes and ec o s in unp eceden ed egions
(Robe
e al.
, 2015; Fishe
e al.
, 2018).
To p e en plan diseases and p o ec c ops om pes s and pa hogens, widely sp ead me hodologies
mainly co espond o applying chemical ungicides. The con inued use o chemical ungicides leads o
he de elopmen o ungicide esis ance in he ungal pa hogen (Syed Ab Rahman
e al.
, 2018) and, in
he absence o o he con ol measu es, o he e-eme gence o i ulence (Fishe
e al.
, 2018). The e o e,
in spi e ha he use o pes icides b ough clea imp o emen s in c op quali y and quan i y du ing mo e
han hal a cen u y, hei p og essi e ine icacy o ea some o he mos ha m ul plan diseases equi es
he u iliza ion o highe dosages each yea (Medei os
e al.
, 2010; Syed Ab Rahman
e al.
, 2018). The
use o ungicides hea ily impac s on he mic o lo a o ag a ian ecosys ems, des oying bene icial
mic obes, such as endophy ic bac e ia and ungi, as well as animals impo an o he quali y o he soils
(Syed Ab Rahman
e al.
, 2018). Ul ima ely, he sys emic use o hese d ugs leads o he pe sis ence o
chemical esidues in he en i onmen , p opo iona ing low dosage oxici y and con amina ing species
ac oss ophic le els (Ca alho, 2006; Duka e
e al.
, 2018).
Due o his scena io, new e icien and eco- iendly s a egies o con ol ungal diseases a e equi ed, such
as he use o biological con ol agen s, pa icula ly an agonis ic yeas s. This possible solu ion o e s some
ad an ages compa ing o he use o chemical ungicides, such as sa e me hods o applica ion and he
ac o being en i onmen ally iendly (Duka e
e al.
, 2018). The in oduc ion o he yeas -based biocon ol
p oduc s does no ha m he ecosys ems due o he absence o oxic esidues, and he low le els o oxici y
3
in combina ion wi h he high le el o biodeg adabili y make hem sui able sus ainable ag icul u e
p ocedu es and o human consump ion (Ocampo-Sua ez
e al.
, 2017). Besides he use in he ood and
ag icul u e, an agonis ic yeas can also ha e an impo an ole in medical applica ions, including i s use
as an imyco ics o he apeu ic ea men o human ungal in ec ions, e.g., in he comba o pa hogenic
Candida
in ec ions (Ha oum
e al.
, 2012)
WITCHES’ BROOM DISEASE OF CACAO
One o he ungal diseases wi h ecognized high nega i e socio-economic impac is he pa hology o cacao
plan and ui known as
Wi ches’ B oom Disease
(WBD). This is caused by he basidiomyce e ungus
Monilioph ho a pe niciosa
(Aime and Phillips-Mo a, 2005) ( o me ly designa ed
C inipellis pe niciosa
).
The se e i y and ex en o i s mani es a ion is endange ing he apidly expanding and e y quali y-
demanding chocola e ma ke . Acco ding o da a om he In e na ional Cacao O ganiza ion
(www.ICCO.o g), mo e han 4 million ons o cacao beans a e p oduced annually (Wick amasu iya and
Dunwell, 2018). Cacao beans a e he co e aw ma e ial o he chocola e indus y, al hough o he cacao-
de i ed p oduc s also ha e impo an wo ld ma ke s, such as cacao bu e o liquo (Pohlan and Pé ez,
2010; Wick amasu iya and Dunwell, 2018). The economic global ma ke o chocola e eached US$ 110
billion in 2015, and he wo ld demand is expec ed o g ow exponen ially in he nex decade due o he
globaliza ion o consump ion s yles in expanding economies such as China and India (Squiccia ini and
Swinnen, 2016). Cacao is p oduced in coun ies loca ed app oxima ely in he same la i ude in e al o
equa o ial clima e, o ming he so-called Cacao Bel (Pohlan and Pé ez, 2010). The bigges p oduce s
a e he e o e coun ies om Cen al and Sou h Ame ica and A ica. The cacao plan is a ec ed by se e al
diseases, he mo e h ea ening o which is WBD (Pu dy and Schmid , 1996; Pe ei a, 1999; G i i h
e al.
,
2003; Aime and Phillips-Mo a, 2005; Teixei a
e al.
, 2015) (Figu e 1). I has se e ely a ec ed Sou h and
Cen al Ame ica coun ies, whe e i has been esponsible o majo i e e sible c op losses. The highes
economic and social consequences o WBD a e desc ibed o ha e occu ed in B azil. In he en yea s
a e he onse o he disease in 1989, WBD educed he cacao p oduc ion in mo e han 70% (Pe ei a
e
al.
, 1989; San os Filho
e al.
, 1998; T e izan and Ma ques, 2002; Meinha d
e al.
, 2008; Pi es
e al.
,
2009; Teixei a
e al.
, 2015), causing B azil o shi om being he 2nd wo ld p oduce o becoming a ne
impo e o cacao beans (Bowe s
e al.
, 2001; Ma elli
e al.
, 2009; Teixei a
e al.
, 2015). Du ing ha
4
pe iod, he mos a ec ed egion o Bahia su e ed losses a ound 90%, con igu ing a se e e social c isis
om losing mo e han 200,000 a m jobs (T e izan and Ma ques, 2002; Teixei a
e al.
, 2015).
Figu e 1.
M. pe niciosa
basidioca ps and he eleased spo es on a d y b oom o a cacao plan (A), a heal hy cacao pod (B)
and he damage caused by he pa hogen in he in ec ed cacao pods (B). Adap ed om Meinha d
e al.
, 2008 (A) and Bowe s
e al.
, 2001 (C).
The se e i y o WBD de i es om se e al ac o s.
Monilioph ho a pe niciosa
does no o m specialized
in ec ion s uc u es such as
app esso ia
like o he ungal pa hogens. Since i is a hemibio ophic ungus,
he ull in ec ious cycle un olds h ough wo dis inc phases: (i) bio ophic and (ii) sap o ophic. (i) The
ini ial in ec ion occu s in young me is ema ic issues and suscep ible ac i ely g owing issues (e.g. buds,
young lea es, lowe cushions, young ui s). The ungus pene a es h ough he s oma al openings, he
bases o damaged ichomes and he husk o young ui s (Aime and Phillips-Mo a, 2005). A e he ini ial
in ec ion, he ungus induces hype ophy and hype plasia, causing he loss o apical dominance. This
co esponds o a diso ganized p oli e a ion o he in ec ed ege a i e me is ems o axilla y shoo s ha
esul s in he o ma ion o a b oom-like s uc u e o abno mal s ems called a
g een b oom
(Aime and
Phillips-Mo a, 2005; Meinha d
e al.
, 2008; Pi es
e al.
, 2009). Sho ly a e he ini ial in ec ion, he
ungus s a s g owing in e cellula ly, o ming a monoka yo ic and pa asi ic mycelium wi hou clamp
connec ions, es ablishing a bio ophic ela ionship wi h he hos ha co esponds o i s li e cycle bio ophic
phase. (ii) Usually 4 o 6 weeks a e he de elopmen o he
g een b ooms
, a conce ed se ies o in ec ed
plan cells dea h e en s occu s, and he in ec ed issues become nec o ic o ming a s uc u e called
d y
A
C
B
5
b oom
. Nec o ic o dead hos cells a e hen colonized by he ungus (E ans, 1980; Meinha d
e al.
,
2008), which a his poin , su e s majo mo phological changes en e ing i s sap o ophic phase
(Law ence
e al.
, 1991; Meinha d
e al.
, 2008). The hyphae become dika yo ic, clamp connec ions a e
o med, and he ungus begins o g ow in acellula ly, as well as be ween cells. The exac mechanisms
and signaling ac o s ha igge he swi ch om he bio ophic phase o a sap o ophic phase, con olling
he de elopmen al al e a ions, emain unknown (Meinha d
e al.
, 2008). A e he ungus p oli e a ion
and coloniza ion o he dead hos issues, pink-colo ed basidioca ps (small mush ooms) a e p oduced on
any in ec ed nec o ic issue. Upon al e na e we and d y pe iods, each basidioca p can p oduce 2 o 3.5
million spo es (basidiospo es), his way comple ing he ungus li e cycle (Rocha and Wheele , 1985;
Almeida
e al.
, 1997). The elease o he spo es occu s mainly a nigh and is ela ed o a high le el o
humidi y and a o able empe a u e (20-30 ºC). The spo es a e dissemina ed locally by wa e and o e
long dis ances by wind and can endu e la en in he soil o inside p uned b anches o he plan s o long
pe iods (Meinha d
e al.
, 2008; Pohlan and Pé ez, 2010). An o e iew o he disease p og ession in he
plan and he pa allel e en s o he ungus li e cycle desc ibed abo e a e summa ized in Figu e 2. The
abili y o
Monilioph ho a
pe niciosa
o in ec he plan in all s ages o i s li e-cycle and he ac ha i ually
all he plan issues can be in ec ed, unde lie his pes excep ional i ulence. This, allied o he ungus
high p e alence in he soil and plan dead ma e ial, explains why once a single plan de elops symp oms
he whole plan a ion can be comp omised.
The esilience o
Monilioph ho a pe niciosa
elies essen ially on i s capaci y o colonize bo h ali e and
dead plan issue, he bio ophic and nec o ophic li e cycle phases abo e men ioned. The shi be ween
he wo phases in ol es a d as ic mo phological and li es yle change. Al e na i e Oxidase (AOXp)-
espi a ion was associa ed wi h his ansi ion (Thomazella
e al.
, 2012). Possibly, his ype o espi a ion
allows he ungal cell o o e come he plan hos de enses gene a ed in he i s s ages o he WBD, as
obse ed wi h be e s udied model ungus
Us ilago maydis
(Cá denas-Mon oy
e al.
, 2017). The plan
de enses include he p oduc ion o high amoun s o NO, which a ec he ungal mi ochond ia, namely
inhibi ing espi a ion complex IV, his way inducing he p oduc ion o ROS (Thomazella
e al.
, 2012). AOXp
is an al e na i e mi ochond ial oxidase ha cons i u es alone a bypass o espi a o y chain complexes III
and IV, which unc ion p e en s collapse om d ugs ha a ge hese complexes like cyanide o An imycin-
A (Maxwell
e al.
, 1999; Ruy
e al.
, 2006; Vanle be ghe
e al.
, 2009). A he same ime, AOXp- espi a ion
con ibu es o cope wi h he elec on lux o e low wi hou phospho yla ion, and he e o e wi hou
p oducing ATP (Van Aken
e al.
, 2009) lowe ing he global ene gy yield o me abolism. AOX-encoding
gene sequences a e ound in many o ganisms (including yeas s and ungi) (El hon and McIn osh, 1987;
12
epidemic (Cacciola
e al.
, 2012). Mo eo e , he se e i y o he disease a ies acco dingly o
phy opa hogens ha a e in ec ing he oli e o cha ds (Talhinhas
e al.
, 2018).
C. acu a um
s.s. and
C.
nymphaeae
a e ound o be mo e i ulen , he e o e mo e equen ly ela ed wi h epidemic ou b eaks
(Schena
e al.
, 2014; Talhinhas
e al.
, 2015). F ui ipeness is also a c ucial ac o , since he ipening
a es a e highe and as e in ees wi h ewe ui s, consequen ly inc easing he hos suscep ibili y. Since
an epidemic ou b eak o OA dec eases he ui load in he subsequen yea , he p obabili y o ano he
epidemic e en inc eases, con ibu ing o he se e i y o his disease (Mo al and T ape o, 2012). The
nec o ophic s age o he pa hogens o en causes ui o , which in u n esul s in p ema u e ui
mummi ica ion. The majo i y o he mummi ied d upes all o he g ound and a e easily decomposed,
while he ew ha emain on he ee a e capable o eleasing iable conidia a a cons an a e du ing
se e al mon hs, p obably se ing as inoculum o sp ing in ec ions (Mo al and T ape o, 2012; Se gee a,
2014; Talhinhas
e al.
, 2018). These la en sp ing in ec ions can, in u n, se e as he main inoculum
sou ce o au umn in ec ions (Mo al
e al.
, 2009; Talhinhas
e al.
, 2011). I has also been epo ed ha
in con olled condi ions, one in ec ed ui pe ee can a ec up o 100% o he ui s in a suscep ible
cul i a i a o able wea he condi ions pe se e e du ing au umn (Mo al
e al.
, 2009). All hese ac o s
combined explain why once a single plan in an oli e o cha d s a s o de elop symp oms, he whole
plan a ion can be endange ed.
FUNGAL DISEASES MANAGEMENT
The chemical ungicides gene ally used a e ei he coppe -based compounds, such as cup ous oxide, o
azole-con aining molecules, pa icula ly ebuconazole (Oli ei a and Luz, 2005; Medei os
e al.
, 2010).
These a e usually used o con ol he sp ead o o he ungal plan diseases, such as g ape ine downy
mildew and oli e peacock spo , bu showed e y low e iciency agains
M. pe niciosa
(Medei os
e al.
,
2010) .as well as he abo e desc ibed
C. gloeospo ioides
and
C. acu a um
species complexes (Cacciola
e al.
, 2012). Coppe is
pe se
a non-speci ic an i-mic obial agen able o des oy na u ally occu ing
mic oo ganisms, including ungi, ha is o decades applied as olia sp ays (Yang
e al.
, 2011; Husak,
2015). The le hal ac ion o coppe -based ungicides de i es om hei abili y o ee coppe ions ha a e
massi ely in e nalized by he ungal cells. In acellula ly, hey bind a ious chemical g oups (imidazoles,
phospha es, sul hyd yls, hyd oxyls) namely in p o eins, causing hei dena u a ion and loss o unc ion
(Husak, 2015; Mi ko ić
e al.
, 2015). Ul ima ely, his leads o i e e sible cell damage and memb ane

13
leakage (Husak, 2015). Ye some ungi a e esis an o coppe ions. The mechanisms unde lying his
esis ance a e no well unde s ood, al hough se e al s udies ha e sugges ed ha hey migh exe a
combined ac ion: he ex acellula chela ion and cell wall seques e o coppe ions, and hei dec eased
in ake and in acellula complexing by me allo hioneins o o he p o eins (Ce an es and Gu ie ez-
Co ona, 1994). This las case includes he o e -exp ession o he supe oxide dismu ase (SOD) ha uses
coppe as ino ganic co- ac o (Naiki, 1980). SOD has been desc ibed o display he abili y o bu e coppe
excess independen ly o i s supe oxide sca enging unc ion (Culo a
e al.
, 1995). Addi ionally, he coppe -
induced accumula ion o glyce ol was also desc ibed o be in ol ed in i s ex usion (Gadd
e al.
, 1984).
Nei he o hese mechanisms we e e e desc ibed in associa ion wi h
Monilioph ho a pe niciosa
, al hough
inc eased le els o SOD would con ibu e he highe esis ance o he abo e-men ioned plan -gene a ed
ROS.
On he o he hand, ebuconazole, as o he azole- ungicides, ac s on he syn hesis o e gos e ol, al e ing
he s uc u e and unc ionali y o he ungal cell memb ane (P ice
e al.
, 2015) as well as acuola ion
homeos asis h ough -ATPase unc ion (Zhang
e al.
, 2010b). In consequence o e gos e ol syn hesis
dis up ion, mi ochond ial unc ion is also a ec ed in i s abili y o o m i on-sul u clus e s, which esul s
in he deposi o insoluble i on inside mi ochond ia and concomi an adical o ma ion and mi ochond ial
loss (Wa d
e al.
, 2018). Cy C ha bo s a
heme
g oup which a ailabili y o his enzyme p ope assembly
and unc ion would be a ec ed by i on homeos asis dis up ion, consequen ly a ec ing Complex IV
unc ion in espi a ion. This would jus i y why ungi ha can espi e h ough he AOXp could be esis an
o azoles. S ill, he e is no e e ence o his possibili y in he li e a u e. Ra he , in
Candida
species, he
esis ance o azole- ungicides implica es o he ypes o mechanisms (Whaley
e al.
, 2017). Ne e heless,
hese a e human commensals and pa hogens, ha ing he e o e speci ici ies ha a e no common wi h
o he yeas s o ungi.
Fungicides ail o con ol he sp ead o he WBD bu he mechanisms unde lying he esis ance o
Monilioph ho a pe niciosa
o hese d ugs a e no s udied. The use o ungicides is he e o e no a ou ine
p ac ice in mos cacao-p oducing coun ies also due o hei high cos , and he isks associa ed wi h
cacao chemical con amina ion which hinde s comme cializa ion. The easons unde lying his include he
inc easingly conside ed nega i e impac o ungicides on human heal h and he en i onmen . Public
conce ns ega ding he p e alence o ag onomic pes icide esidues in ood, and hei ela ion wi h he
inc easing ad en o pes icide esis an pa hogens, no only in plan s bu also in humans (D oby, 2006;
Pal and McSpadden Ga dene , 2006; Ma elli
e al.
, 2009; Ve weij
e al.
, 2009; Nunes, 2012) led o
es ic ions in Eu ope. The e o e, he mos commonly used me hods o con ol he WBD a e exclusi ely
14
ag onomic, h ough phy osani a y p uning, emo ing as much as possible he in ec ed ma e ial, which is
hough o en impossible, due o hidden ungal inoculum in he soil and cu b anches and lea es
2
.
The e o e, mo e e ec i e and eco- iendly me hods and s a egies a e needed o sa is y he consume
demands.
In he case o OA managemen , i is mos ly based on he use o chemical ungicides, which should be
applied in a p e en i e manne , since he disease is nea ly impossible o con ol once symp oms eme ge
(Se gee a, 2011; Talhinhas
e al.
, 2011; Cacciola
e al.
, 2012; Mo al
e al.
, 2012). Few ungicides ha e
shown some e icacy in con olling he disease, including coppe -based ungicides, di hioca bama e,
azoles (hexaconazole and ebuconazole) and s obilu ins (azoxys obin and i loxys obin) (Pennisi
e al.
,
1993; Se gee a, 2011; Mo al
e al.
, 2014). The e ec i eness o he ea men wi h hese d ugs depends
on se e al ac o s, such as he se e i y o he disease, he plan cul i a and en i onmen al condi ions,
being he e o e egionally a iable. Fu he mo e, he iming, equency and numbe o ungicide
applica ions, as well as he p e en i e o cu a i e na u e o he ea men , a e ac o s o ake in o accoun
o he success o a pa icula chemical-based s a egy (Cacciola
e al.
, 2012; Landum
e al.
, 2016).
Rega dless wi h he le el o e iciency o chemical ungicides, o he p oblems associa ed wi h hei use
also a ise. The wash-o o he ungicides om oli e c ops due o ains leads o esidue accumula ion in
soil and adjacen wa e a eas, which could ha e nega i e and ha m ul e ec s on he su ounding
ecosys em and e en in human heal h (Komá ek
e al.
, 2010; Lamichhane
e al.
, 2018). Addi ionally,
since he ac i e cons i uen s o ungicides comme cially exploi ed o disease managemen a e chemically
e y simila and gene ally belong o he same molecula amily, ungal phy opa hogens easily adap and
acqui e esis ance (Ma and Michailides, 2005). The e o e, he o e use o hese ypes o d ugs e en ually
leads o hei inc easing ine icacy ollowed by loss o e ec , lea ing c ops wi hou possible ea men
(Cacciola
e al.
, 2012). The gene al public awa eness o ood and en i onmen al con amina ion caused
by he misuse o chemical pes icides, as well as he isks associa ed o human heal h a e pushing
Eu opean policies o es ic he employmen o ungicides in he con ol o phy opa hogenic diseases.
Consequen ly, OA managemen is nowadays inc easingly based in ag icul u al s a egies, which include
he eplacemen o suscep ible cul i a s wi h esis an ones, ea ly ha es ing, s a egic p uning and e en
o cha d design (Talhinhas
e al.
, 2011; Cacciola
e al.
, 2012; Mo al
e al.
, 2012; Leoni
e al.
, 2018). The
e iciency o hese s a egies is hough limi ed. The use o esis an cul i a s should a e m be he bes
s a egy, al hough he comple e eplacemen o he o cha ds akes a signi ican lag- ime in p oduc ion
2
A ailable a : h ps://www.icco.o g/abou -cocoa/pes -a-diseases.h ml [Accessed Feb ua y 18, 2019]
15
and is al oge he e y expensi e (Landum
e al.
, 2016). Addi ionally, in cases whe e ideal condi ions o
ungal g ow h a e me , appa en ly he in ec ion occu s anyway (Cacciola
e al.
, 2012). The e o e, new
mo e e ec i e and sus ainable me hods o con ol OA a e essen ial.
A concep ha has gained conside able p ominence in he ag icul u e sec o in ecen yea s is he use o
nanoag opa icles, which a e nanopa icles designed o mi iga e ag icul u e- ela ed p oblems, including
plan pa hologies (Pa izi
e al.
, 2014; Pa isi
e al.
, 2015). These nanoag opa icles include sil e , coppe ,
sul u , zinc oxide and magnesium oxide nanopa icles (Bake
e al.
, 2017), and can ac e icien ly as
ungicides, pes icides, he bicides and also insec icides. They can easily en e in o he ungal cell wall.
Once inside he cell, hey ac h ough di e en modes, which include (i) causing he dis up ion o
me abolism, o o he cell memb ane, wi h consequen loss o cellula con en (Bake
e al.
, 2015; Bake
e al.
, 2017), (ii) p omo ing he elease o oxic ions (Cd2+, Zn2+ and Ag+) ha bind o sul u -con aining
p o eins, (iii) a ge ing he pa hogen DNA, his way inducing cell dea h, (i ) in e up ing elec on anspo ,
his way causing he collapse o memb ane po en ial, ( ) p omo ing he gene a ion o ROS, o ( i)
in e e ing wi h nu ien up ake (Alghu haymi
e al.
, 2015). Mo e han one o hese mechanisms can occu
simul aneously, con e ing he abili y o nanopa icles o be e ec i e agains di e en plan pa hogens
(Alghu haymi
e al.
, 2015). Recen ly, nanopa icles we e conjuga ed wi h some biomolecules (including
biocide/kille oxins), o ming bionano-hyb id ag opa icles (Bake
e al.
, 2017). The ee u iliza ion o
hese p omising phy opa hology managemen ools s ill equi es no only cy o oxicology s udies o
e alua e po en ial ha m o human and animal heal h, bu e en mo e impo an , ex ensi e eco oxicology
and biodeg adabili y s udies o e alua e hei p e alence in he en i onmen and ood chains and hei
e ec on he long un in he mic o lo a o plan , soil and wa e . P esen ly ew in o ma ion on his ega d
is a ailable (Alghu haymi
e al.
, 2015). None heless, he p ospec i e o being able o use such a nano ool
o e ec i ely deli e a oxin and kill a phy opa hogenic ungus is a ac i e, especially i ca ying a bio-
de i ed killing agen .
16
YEASTS AS BIOCONTROL AGENTS
One possible app oach o ungal diseases in plan s migh be he use o biocides o biological con ol
agen s. In phy opa hology, his e m designa es he use o in oduced o esiden li ing o ganisms o
con ain o supp ess popula ions o pa hogens (Pal and McSpadden Ga dene , 2006). The e a e a ew o
hese agen s in he ma ke , mos ly used in he con ol o pes s a small scale. They co espond o d y
biomass o bac e ial o ilamen ous ungal s ains isola ed om he endosphe e o he hizosphe e o
plan s (O’B ien, 2017) ha a e e-hyd a ed and used as ali e ep oduc i e mic oo ganisms. These include
a axonomically and biologically di e se g oup o endophy ic ungi ha a e cha ac e ized by colonizing
in e nally he plan hos issues wi hou causing any ex e nal disease symp oms (Wilson, 1995; Rubini
e
al.
, 2005). These endophy es can p e en pa hogen in ec ion and p opaga ion di ec ly by compe i ion,
mycopa asi ism o an ibiosis, o indi ec ly by inducing esis ance esponses in he plan (Bailey
e al.
,
2006). Despi e his, he biocon ol agen s ha could be applied in phy opa hology a e no es ic ed o
hese wo g oups o o ganisms.
Endophy ic mic oo ganisms also include Ascomyco a and Basidiomyco a yeas s, ound in many species
o ees om e y di e se clima es, bu also in ag icul u al species ( e iewed by Do y, 2013). Ascomyco a
yeas s ep oduce exclusi ely by budding, as he mos well-known yeas
Saccha omyces ce e isiae
.
Basidiomyco a g ow dimo phically, shi ing om a monoka yo ic yeas - o m o a dika yo ic ilamen ous
o m (Choudha y and Joh i, 2009). This is he case o
Rhodo o ula
and
C yp ococcus
sp. (Table 1).
Gene ally, endophy ic yeas s appa en ly h i e symbio ically o mu ualis ically, i ually colonizing di e se
plan issues ( e iewed by Do y, 2013), in which hey may cause s uc u al changes (Luna, 2017). They
consume suga s and assimila e amino acids gene a ed by he plan and con ibu e o he plan wellbeing
and s ess esponse in many di e en ways, including he p oduc ion o phy ophe omones, ca alase o
side opho es ( e iewed by Joube and Do y, 2018). Impo an ly, endophy ic like epiphy ic yeas s can
an agonize phy opa hogenic ilamen ous ungi, ei he by occupying hei niche o by an agonizing hem
in mo e complex ways.
The an agonis ic in e ac ion o yeas s wi h pa icula phy opa hogenic ungi has been desc ibed in he
li e a u e. Fo example, Suzzi
e al.
(1995) obse ed ha na u al wine yeas s ains o
Saccha omyces
and
Zygosaccha omyces
inhibi ed
in i o
he g ow h o 10 species o soil-bo ne ungal plan pa hogens,
namely
Cladospo ium a iabile
,
Rhizoc onia aga iae
,
Phomopsis longicolla
,
Colle o ichum acu a um
,
Aspe gillus nige
,
Scle o inia scle o io um
,
Penicillium digi a um
,
Mac ophomina phaseolina
,
T ichode ma i ide
and
Bo y is squamosa
. Also, Walke
e al.
(1995) epo ed ha s ains o
17
Saccha omyces ce e isiae
and
Pichia anomala
(know designa ed
Wicke hamomyces anomalus
) inhibi ed
in i o
he g ow h o se e al wood decay basidiomyce es including
Se pula lac ymans
,
Pos ia placen a
,
Len inus lepideus
and
Ophios oma ulmi
and phy opa hogenic ungi, such as
Rhizoc onia solani
,
Fusa ium
equise i
,
Bo y is abae
and
Phy oph ho a in es ans
. Impo an ly, Rosa-Mag i
e al.
(2011) desc ibed he
an agonism e ec o he yeas
To ulaspo a globosa
agains he phy opa hogenic mold
Colle o ichum
g aminicola
, he causal agen o an h acnose disease in maize. All o hese cases we e epo ed as
in
i o
s udies, none we e pe o med
in plan a
o
in ield
. O he wise, yeas s ha e o en been p oposed and
used o he con ol o mic obial con amina ions a he pos ha es phase (Table 1).
The possibili y o using yeas s as biocon ol agen s o ungal o bac e ial p oli e a ion associa ed wi h ood
spoilage has been ecognized since he ea ly 1960s, when i was ound ha Saccha omyces ce e isiae
s ains sec e ed oxins ha killed o he yeas s ains bu a e immune o hei own oxin (Be an and
Makowe , 1963). Kille oxins (KTs) can be encoded by cy oplasm-inhe i ed double-s anded RNA i uses
(Schmi and B einig, 2002) o linea dsDNA plasmids (Scha a h and Meinha d , 2005), bu hey can
also be ch omosomally encoded (Suzuki, 2005). The kille phenomenon is well cha ac e ized and s udied
in Saccha omyces ce e isiae. In his yeas species, KTs ha e been g ouped in o ou ypes, K1, K2, K28,
and Klus, based on hei killing p o iles and lack o c oss-immuni y (Schmi and B einig, 2006; Rod íguez-
Cousiño
e al.
, 2011). Each s ain p oducing one speci ic oxin kills s ains om he o he g oups bu has
sel -p o ec i e immuni y (Schmi and B einig, 2006).
Kille oxins
The modes o ac ion o he
Saccha omyces ce e isiae
KTs a e well known, wi h he excep ion o he
ecen ly ound Klus oxin (Schmi and B einig, 2002). K1 and K2 oxins kill sensi i e yeas cells in a
ecep o -media ed wo-s ep p ocess. The i s s ep in ol es a as , ene gy-independen binding o a
p ima y oxin ecep o (R1), consis ing o β-1,6-D-glucan (Lukša
e al.
, 2015). Though a second ene gy-
dependen s ep, he oxin is ansloca ed om he cell wall o he plasma memb ane, whe e i in e ac s
wi h a seconda y memb ane ecep o (R2), iden i ied in he case o K1 oxin as K e1p, an
O
-glycosyla ed
p o ein o he yeas cell su ace (B einig
e al.
, 2002; 2004). A e eaching he plasma memb ane, K1
and K2 oxins dis up i s unc ion by o ming ca ion-selec i e channels, and p omo ing he elease o ATP
and o he me aboli es, hus causing a le hal e ec on he a ge cell (Liu
e al.
, 2015).

18
Table 1. Success cases o using yeas s o an agonize he spoilage o ui s by ilamen ous ungi.
Yeas an agonis
Hos
Fungal phy opa hogen(s)
Re e ences
P eha es applica ion1
Candida (Pichia) guillie mondii
Che y oma o
F ui decay agen s
Zhao
e al.
, 2011
Candida sake
Apple
Penicillium expansum
Teixidó
e al.
, 1999
Pos ha es applica ion
Au eobasidium pullulans
Pea
Penicillium expansum
Robiglio
e al.
, 2011
Apple
Bo y is cine ea
,
Colle o ichum acu a um
and
Penicillium expansum
Ma i
e al.
, 2012
Candida (Pichia) guillie mondii
Chilli
Colle o ichum capsici
Chanchaichao i a
e al.
, 2007
Toma o
Rhizopus nig icans
Zhao
e al.
, 2008
Rhizopus s oloni e
Celis
e al.
, 2014
Kiwi ui
Bo y is cine ea
Sui and Liu, 2014
Papaya
Colle o ichum gloeospo ioides
Lima
e al.
, 2013
Candida oleophila
Banana
Colle o ichum musae
,
Fusa ium
monili o me
and
Cephalospo ium
sp.
Lassois
e al.
, 2008
Apple
Penicillium expansum
and
Bo y is cine ea
Liu
e al.
, 2012
Candida pelliculosa
Toma o
Bo y is cine ea
Dal Bello
e al.
, 2008
Candida sake
Apple
Penicillium expansum
Mo ales
e al.
, 2008
C yp ococcus in i mo-minia us
Swee che y
Monilinia uc icola
Spo s
e al.
, 2002
C yp ococcus lau en ii
S awbe y
Bo y is cine ea
Wei
e al.
, 2014
Swee che y
F ui decay agen s
Tian
e al.
, 2004
Deba yomyces hansenii
Peach
Rhizopus s oloni e
Mandal
e al.
, 2007
Manda in, o ange
Penicillium digi a um
Taqa o
e al.
, 2008
Me schnikowia uc icola
Apple
Penicillium expansum
Liu
e al.
, 2011
G ape ui
Penicillium digi a um
He shko i z
e al.
, 2013
Meye ozyma ca ibbica
Mango
Colle o ichum gloeospo ioides
Bau is a-Rosales
e al.
, 2013
Pichia memb ane aciens
Apple
Monilinia uc icola, Penicillium expansum
and
Rhizopus s oloni e
Chan and Tian, 2005
Rhodospo idium paludigenum
Che y oma o
Bo y is cine ea
Wang
e al.
, 2010
Rhodo o ula mucilaginosa
Pea
Penicillium expansum
Hu
e al.
, 2015
Rhodo o ula ub a
Toma o
Bo y is cine ea
Dal Bello
e al.
, 2008
Wicke hamomyces (Pichia) anomalus
Banana
Colle o ichum musae
,
Fusa ium
monili o me
and
Cephalospo ium
sp.
Lassois
e al.
, 2008
O ange
Penicillium digi a um
Aloui
e al.
2015;
Pla ania
e al.
, 2012
Papaya
Colle o ichum gloeospo ioides
Lima
e al.
, 2013
Comme cial yeas -biocon ol p oduc s2
Au eobasidium pullulans
Pome
Penicillium, Bo y is, Monilinia
Boni P o ec ®, Bio-Fe m, AT
Candida oleophila
Pome
Penicillium, Bo y is
Nexy®, Lesa e, BE
Me schnikowia uc icola
Pome, able g ape, s one ui s,
s awbe y, swee po a o
Penicillium, Bo y is, Rhizopus, Aspe gillus
Sheme ®, Baye /Koppe , NL
1 P eha es applica ion o p e en pos ha es spoilage.
2 Wisniewski
e al.
, 2016.
19
The K28 KT mode o ac ion is e y di e en , since i en e s a sensi i e a ge yeas cell by endocy osis, in
a cell wall ecep o -media ed manne (Schmi and B einig, 2006). The cell wall ecep o o K28 oxin
has been iden i ied as a mannop o ein wi h high molecula mass (Liu
e al.
, 2015). The K28 oxin is
in e nalized h ough he sec e o y pa hway ( ia Golgi and ER), and a e en e ing he cy osol he β-subuni
is ubiqui ina ed and deg aded in he p o easome. The subsequen ly ee small α-subuni has been
sugges ed o en e he nucleus wi hou he help o an ac i e nuclea impo machine y, he e o e by a so-
called passi e di usion (Schmi and B einig, 2006). Once inside he nucleus, he K28 oxin kills he hos
cell by i e e sibly blocking he DNA syn hesis. The a ge cells a es in ea ly S phase o he cell cycle,
o ming a medium-sized bud and a single, p e- eplica ed nucleus in he mo he cell, e en ually dying
(Schmi and B einig, 2006).
The kille phenomenon, despi e being bes cha ac e ized in
Saccha omyces ce e isiae
, is no con ined o
his yeas species, a he i is o en ound in o he yeas species and gene a (Magliani e al., 1997; Schmi
and B einig, 2002). Some o hese we e desc ibed o damage he plasma memb ane, e y simila ly o
he
Saccha omyces ce e isiae
K1 oxin. This is he case o he KTs p oduced by
Pichia kluy e i
(Ahmed
e al., 1999),
Pichia memb ani aciens
(San os and Ma quina, 2004; San os e al., 2009),
Pichia a inosa
(Suzuki e al., 2001) and
Zygosaccha omyces bailii
(Weile and Schmi , 2003). O he kille mechanisms
include he damage o he cell wall upon he inhibi ion o he syn hesis o β-glucans. Examples o his
mode o ac ion a e he oxins p oduced by
Hansenula m akii
(p e iously
Williopsis m akii
) (Ma quina e
al., 2002), Wicke hamomyces anomalus ( o me ly designa ed
Pichia anomala
o
Hansenula anomala
)
(Wang e al., 2007),
Williopsis sa u nus
(Guya d e al., 2002; Peng e al., 2010) and
Kluy e omyces
pha ii
(Comi ini e al., 2009). Ye o he yeas KTs ac by blocking he cell cycle, namely he one p oduced
by
Kluy e omyces
lac is
(Klassen e al., 2004), and by igge ing DNA damaging and he induc ion o
apop osis, which is he case o he oxins sec e ed by
Pichia acaciae
(Klassen and Meinha d , 2005) and
Wingea obe siae
(Klassen and Meinha d , 2002). The blocking o calcium up ake was also desc ibed as
kille mode o ac ion, namely in he case o
Us ilago maydis
(Gage e al., 2001). Al hough he e a e plen y
o epo s in he li e a u e ega ding he in e ac ion o non-
Saccha omyces
kille yeas s wi h a as a ie y
o sensi i e a ge s, he ac ual mechanisms in ol ed emain mos ly unknown o supe icially s udied a
he molecula le el.
20
Ly ic enzymes and mycopa asi ism
Se e al o he mechanisms o yeas an agonism ha e been p oposed ha do no in ol e he sec e ion o
a pep ide/p o ein ha may be classi ied as a KT. O he p o eins ha a e sec e ed by he yeas and
an agonize ilamen ous ungi a e ly ic enzymes ha des oy he ungal cell wall (Spada o and Gullino,
2004). This kind o an agonism is conside ed a o m o mycopa asi ism. An example is he manne in
which
Pichia guillie mondii
an agonizes
Bo y is cine ea
(Wisniewski
e al.
, 1991). The au ho s obse ed
ha he ungus cell wall glucans and a yeas -sec e ed β-(1–3) glucanase o m a lec in-like in e ac ion
esul ing in a s ong a achmen o he an agonis o he ungal pa hogen which culmina es wi h he lysis
o ungal cells. Besides he sec e ion o hese an i ungal compounds, o he modes o ac ion can be
in ol ed in he mycopa asi ism beha io . Yeas s can a ack he ungi pa hogens by di ec physical con ac ,
eaching he ungal cells and killing hem, al hough no necessa ily h ough an in asion o he a ge cell
(Mims
e al.
, 2007). Addi ionally, yeas s can ac as p eda o y mycopa asi es, physically pene a ing hei
p ey cell walls, h ough haus o ia o pene a ion pegs (Junke
e al.
, 2019).
Vola ile compounds
Yeas s ha an agonize o he yeas s can also p oduce se e al ola ile compounds agains ilamen ous
ungi ha inhibi he a ge g ow h (Ma i
e al.
, 2016). These compounds include alkenes, alcohols,
ke ones, benzenoids, py azines, sul ides and e penes (Schulz-Bohm
e al.
, 2017). These ha e in
common he ac o ha ing small molecula weigh and physicochemical p ope ies which acili a e
e apo a ion and di usion in soil and hizosphe e en i onmen s (Ma i
e al.
, 2016; Schulz-Bohm
e al.
,
2017). This is he case o he yeas
Au eobasidium pullulans
ha p oduces 2-me hyl-1-bu anol, 3-me hyl-
1-bu anol, 2-phene hyl alcohol and 2-me hyl-1-p opanol, wi h inhibi o y e ec agains
Bo y is cine ea
,
Colle o ichum acu a um
,
Penicillium expansum
,
Penicillium digi a um
and
Penicillium i alicum (Di
F ancesco e al., 2015)
. In e es ingly, i appea s ha an agonizing yeas s may ope a e in di e en ways
also because in some cases hey s ongly a ach o he ungus hyphae. This was desc ibed in de ail o
he case o he yeas s
Pichia memb ane aciens
and
C yp ococcus albidus
when challenged wi h h ee
phy opa hogenic ungi causing he pos ha es de e io a ion o nec a ines and apples
(
Monilinia uc icola,
Penicillium expansum
and
Rhizopus s oloni e
)
(Chan and Tian, 2005).
21
Compe i ion o nu ien s and space
Ano he e ec i e mechanism o an agonism and possibly he mos common is compe i ion. Mic obes
compe e o space, o oxygen and o cou se o nu ien s, such as ca bohyd a es, i amins, mine als and
amino acids (Spada o and D oby, 2016). Yeas s g ow much as e han ilamen ous ungi, being hus
able o quickly colonize he niches ha ungi can occupy, such as plan wounds o issue lesions, o ming
colonies o bio ilms (And ews
e al.
, 1994). Inc easingly bigge yeas popula ions educe he amoun o
nu ien s a ailable o ungi and make hem di icul o access (Zhang
e al.
, 2010a). In he case o
mic onu ien s, i on plays a c ucial ole in he g ow h, de elopmen and i ulence o he ungal pa hogens
(Sa a anakuma
e al.
, 2008). To compe e wi h he pa hogens o i on, yeas s sec e e side opho es ha
deple e i on om he g ow h medium such as pulche imin, p oduced by
Me schnikowia pulche ima
o
compe e wi h
Bo y is cine ea
,
Al e na ia al e na a
and
Penicillium expansum
(Sa a anakuma
e al.
,
2008).
Quo um sensing
The p oduc ion and sec e ion o quo um sensing molecules can be also in ol ed in yeas an agonism.
The concep o quo um sensing was in oduced in 1994 (Fuqua
e al.
), and consis s in he cell- o-cell
communica ion p ocess h ough which each indi idual cells can adjus i s pheno ype in esponse o he
p esence o ex acellula quo um sensing molecules (QSM) a a speci ic concen a ion (Wus e and Babu,
2007). When QSM each hose concen a ions, hey bind wi h a ecep o , link wi h p omo o sequences
and ac i a e he ansc ip ional egula o s o speci ic genes (Mehmood
e al.
, 2019). Examples o hese
kind o molecules desc ibed in yeas s a e 2-phenyle hanol, yp ophol and y osol (A belj
e al.
, 2016).
Fungi can also p oduce a pa icula ype o seconda y me aboli es which can ac as an i-mic obial agen s
known as quo um sensing inhibi o s (QSI). These mic oo ganisms colonize se e al habi a s in e ac wi h
o he o ganisms, such as o he mic obes, animals and plan s. Due o he need o compe e o nu ien s
and space, hey deal wi h compe i o o ganisms by p oducing seconda y me aboli es, enzymes and
chemicals (Padde
e al.
, 2018). These compounds can ac as QSI by deg ading QSM, delaying hei
p oduc ion o e en blocking he ecep o s ia homologs o QSM (Padde
e al.
, 2018; Mehmood
e al.
,
2019). An example o QSI is a nesol, a seconda y me aboli e sec e ed by many dimo phic yeas s
(Wongsuk
e al.
, 2016; Mehmood
e al.
, 2019).
28
Chap e 3 p esen s a mic oscopy analysis o he in e ac ions be ween he an agonis ic yeas s and he
phy opa hogenic ungi in he an agonism assays pe o med in Chap e 2. Scanning Elec on Mic oscopy
echnique was used. The yeas a achmen o he pa hogen hyphae, he occu ence o cons ic ion o he
ungal cells and an appa en d aining o he ungal cellula con en was obse ed. Mo eo e , yeas usion
wi h he hyphae and imb iae-like connec ions be ween he yeas and he ungal cells and also be ween
an agonis ic yeas s a e desc ibed. The possibili y o a p edacious-like beha io exe ed by he an agonis ic
yeas s and i s ole in he an agonism exe ed agains he WBD ungal pa hogens is discussed.
Chap e 4 comp ises he assessmen o he capabili y o he g oup o yeas s ains o igina ing om he
indus ial e men a ions o
cachaça
and om he indus ial p oduc ion o bioe hanol used on Chap e 2
o an agonize
Colle o ichum gloeospo ioides
and
C. acu a um
, he ungal causa i e agen s o Oli e
An h acnose. The abili y o a g oup o yeas s om he oli e biome in an agonizing he oli e an h acnose
ungal pa hogens is also explo ed. A
Wicke hamomyces anomalus
, isola ed om oli e o cha ds in
Po ugal, is he mos p omising s ain, being able o inhibi he g ow h o all
Colle o ichum
sp. es ed.
The alida ion o he applica ion o he p o ocol de eloped and op imized in Chap e 2 o he e alua ion
o yeas an agonism is also p esen ed.
Chap e 5 explo es he possibili y o using
inasse
, he main was e p oduc om suga cane bioe hanol
p oduc ion p ocess, o con ol he de elopmen o he
Wi ches’ B oom Disease
causal agen s. The abili y
o
inasse pe se
o inhibi he g ow h and de elopmen and e en kill
M. pe niciosa
is epo ed. The
po en ial o a solu ion based on he e i iga ion o cacao plan a ions wi h
inasse
, which could con ibu e
o con ain he p e alence and sp ead o he disease is p esen ed.
Chap e 6 is a conclusion chap e which summa izes all he esea ch pe o med in he scope o his
hesis. The esul s and indings o he di e en expe imen al s udies a e he e consolida ed. The u u e
pe spec i es in his a ea ela ed wi h he esea ch wo k pe o med is also p oposed and discussed.
Supplemen a y Ma e ial
An explo a o y assessmen and iden i ica ion o he p o ein con en o he yeas
s
ungus co-cul u e
supe na an s om he an agonism assays o Chap e 2 ha could be in ol ed in he an agonism e ec
agains
M. pe niciosa
is p o ided.

29
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CHAPTER 2
Saccha omyces ce e isiae
and
Wicke hamomyces anomalus
a e able o kill
Monilioph ho a pe niciosa
, he causal
agen o cacao
Wi ches’ B oom Disease
The wo k p esen ed in his chap e has been submi ed o publica ion:
Fe az, P., Amo im-Rod igues, M., Cássio, F. and Lucas, C.
Saccha omyces ce e isiae
and
Wicke hamomyces
anomalus
a e able o kill
Monilioph ho a pe niciosa
, he causal agen o cacao
Wi ches’ B oom Disease
52
ABSTRACT
BACKGROUND: Cacao
Wi ches’ B oom Disease
(WBD) is caused by
Monilioph ho a pe niciosa,
a ungus
ha in ec s all he plan issues, a any s age o i s li e cycle. Chemical ungicides a e ine ec i e agains
M. pe niciosa.
P esen ly, WBD is en a i ely con olled using a biocide,
T ichode ma s oma icum
,
expensi ely p oduced by demand, and he e o e economically unsus ainable.
RESULTS: A g oup o wild yeas s om se e al species, isola es om B azilian e men a ion indus ies,
we e es ed o hei abili y o an agonize
M. pe niciosa
s ains om Sou h and Cen al Ame ica in ec ed
o cha ds. Tes s we e done
in i o,
in solid and liquid media. Th ee yeas s we e ound ha e icien ly kill
6 s ains o
M. pe niciosa
. Dea h was con i med by me hylene blue and p opidium iodide s aining and
equi es he physical con ac be ween he yeas s and he mycelium. Two yeas s ains we e chosen,
Wicke hamomyces anomalus
#1105 and
Saccha omyces ce e isiae
#1112 om spon aneous
e men a ions o he p oduc ion o
cachaça,
he B azilian spi i , and he
S. ce e isiae
PE2 om bioe hanol
indus ial e men a ions in B azil. They a e e ec i e a h ee empe a u es, unde s a a ion, a di e en
cul u e s ages o g owing old.
CONCLUSIONS: These esul s a e he ini ial s ep owa ds he o mula ion o a new eco- iendly and
e ec i e al e na i e o con olling WBD, based on he applica ion o li e yeas wi hou he need o he
pu i ica ion o a speci ic an i ungal pep ide/compound. Mo eo e , esul s sugges ha spon aneous
e men a ions may p o ide mic obial biodi e si y o be locally applied in he con ol o he disease,
imp o ing he sus ainabili y o small cacao p oduce s in emo e a eas.
Keywo ds
Cacao;
Wi ches B oom Disease
;
Monilioph ho a pe niciosa
;
yeas ; an agonism
53
INTRODUCTION
The cacao plan (
Theob oma cacao
L.) is one o he mos aluable c ops wo ldwide (Pohlan and Pé ez,
2010; Teixei a
e al.
, 2015), esponsible o impo an ac ions o he economic e enue o he coun ies
om Cen al and Sou h Ame ica and A ica wi hin he Cacao Bel .
T. cacao
is a ec ed by se e al diseases,
he mos se e e being he
Wi ches’ B oom Disease
(WBD) caused by he basidiomyce e ungus
Monilioph ho a pe niciosa
( o me ly
C inipellis pe niciosa
) (Pu dy and Schmid , 1996; Aime and Phillips-
Mo a, 2005; Teixei a
e al.
, 2015). This disease is esponsible o majo c op losses wi h la ge social-
economic consequences, pa icula ly in B azil which cacao p oduc ion dec eased mo e han 70% in a
pe iod o 10 yea s a e he onse o he disease (T e izan and Ma ques, 2002; Meinha d
e al.
, 2008;
Teixei a
e al.
, 2015).
The se e i y o WBD is closely ela ed o he i ulence o
M. pe niciosa
, which comes om i s abili y o
i ually in ec all cacao plan issues a all s ages o he plan li e cycle (Meinha d
e al.
, 2008; Fe az
e
al.
, 2019). As a hemibio ophic ungus,
M. pe niciosa
has wo dis inc phases: a bio ophic and a
sap o ophic ( e iewed by Fe az
e al.
, 2019). A e he ini ial in ec ion, he pa hogen induces hype ophy
and hype plasia, causing a diso ganized p oli e a ion o he in ec ed ege a i e me is ems o axilla y
shoo s, which esul s in he o ma ion o g een b ooms, a s uc u e composed o abno mal s ems. Se e al
weeks a e he de elopmen o hese s uc u es, he in ec ed plan issues become nec o ic due o a
se ies o cell dea h e en s, o ming a s uc u e named d y b oom (Meinha d
e al.
, 2008).
M. pe niciosa
hen colonizes hose nec o ic plan cells and gene a es pink-colou ed basidioca ps p oducing 2 o 3.5
million spo es each (Almeida
e al.
, 1997). The spo es a e mainly eleased a nigh unde op imal
condi ions o empe a u e and humidi y, being dissemina ed by wa e and wind, and can endu e and
emain la en in he soil o inside p uned plan b anches o long pe iods o ime (Meinha d
e al.
, 2008;
Pohlan and Pé ez, 2010). All hese ac o s con ibu e o he excep ional i ulence o
M. pe niciosa
and
explain why a whole plan a ion is comp omised a e he ini ial in ec ion o one cacao plan .
The con en ional chemical ungicides used o con ol he sp ead o ungal plan diseases, such as coppe
o azole-based compounds, a e ine ec i e agains
M. pe niciosa
(Medei os
e al.
, 2010). In addi ion, he
use o chemical ungicides has been es ic ed in mos cacao p oducing coun ies due o hei high cos ,
and o he isks associa ed wi h chemical con amina ion o he cacao ui s and chocola e (Ma elli
e al.
,
2009; Ve weij
e al.
, 2009; Nunes, 2012). Cu en ly, WBD has only one managemen me hod
implemen ed in B azil, consis ing in sp aying he in ec ed plan s wi h T ico ab®, a li e suspension o
60
RESULTS AND DISCUSSION
Op imiza ion o
M. pe niciosa
cul i a ion condi ions
M. pe niciosa
s ains used in his wo k (Table 1) we e o iginally isola ed om cacao plan s and ui s
in ec ed wi h WBD in Sou h Ame ican coun ies. This species o ilamen ous ungus is e y agg essi e
and esilien bu g ows p e e ably wi hin a ela i ely na ow ange o empe a u es, be ween 20-30 ºC
(ICCO.o g; Pu dy and Schmid , 1996). Conside ing ha he majo i y o yeas s a e bes cul u ed a 30 ºC,
his empe a u e was chosen o cul i a e he ungal s ains. Op imal pH, on he o he hand, was
de e mined by quan i ying ungal g ow h a es in MEA, a well-known ungal g ow h media, adjus ed o pH
4.0, 4.5, 5.0, 5.5 o 6.0. Resul s (Figu e 1) showed ha
M. pe niciosa
s ains a y conside ably be ween
each o he . The as es g owing s ain was F om B azil wi h a speci ic g ow h a e o 2.7 mm.day-1 a pH
5, while he s ain B om Ecuado was he slowes , g owing a G =0.47 mm.day-1 a pH 4.5. No s a is ically
signi ican di e ences we e obse ed be ween he g ow h a pH 5 o 6. G ow h a es we e gene ally lowes
a pH 4.0, wi h a la ency phase las ing 1 day (s ains C, D, F and I) o mo e ( emaining s ains). Simila ly,
a la ency phase o 1 day was also obse ed o s ains C, E, H and I a pH 4.5.
Figu e 1
.
Va ia ion o he g ow h a e o
M. pe niciosa
s ains in Table 1 de e mined in MEA a 30°C a di e en pH.
Di e en le e s ep esen signi ican di e ences ac oss di e en pH alues. § ep esen s a la ency phase p io o he ini ial
mycelia g ow h (§ 1 day o la ency; §§ >1 day o la ency). Resul s a e da a om a leas 3 independen eplica es.

61
These assays we e epea ed in PDA and no s a is ically signi ican di e ences we e obse ed be ween he
g ow h a es in ei he media (no shown). Fungal s ains we e hus u he cul i a ed on MEA o PDA a
30°C and pH 5.5 o a oid la ency. F om he same assays, i was also es ablished ha 10 days is enough
o es ungal g ow h pheno ypes.
Op imiza ion o yeas s ungi an agonism assays
The yeas s chosen o his wo k (Table 2) can be g ouped in wo se s. The i s con ains s ains o
Wicke hamomyces anomalus
(p e iously
Pichia anomala
o
Hansenula anomala
) ob ained om a
c edi ed cul u e collec ion (PYCC - Po uguese Yeas Cul u e Collec ion), and he second con ains isola es
om e men a i e p ocesses in B azil. These yeas s we e chosen based on hei dominan na u e in
mic obial mix u es, pa icula ly he wild yeas s isola ed om he spon aneous e men a ions used o he
p oduc ion o
cachaça
, he B azilian spi i (da Conceição
e al.
, 2015), o used in a mo e con olled
indus ial p ocess o suga cane juice e men a ion o bioe hanol p oduc ion (Lopes
e al.
, 2016).
Mo eo e ,
W. anomalus
s ains we e chosen conside ing hei o igin om di e se na u al niches, possible
sou ces o s ong an agonism (Baysal and Silme, 2018), and he ac ha his is a kille species ac i e
agains a la ge a ie y o o he yeas s (Ab anches
e al.
, 1998; F edlund
e al.
, 2002). B oad kille s a e
known o be also e ec i e agains bac e ia and ilamen ous ungi, including phy opa hogens (Suzzi
e al.
,
1995; Walke
e al.
, 1995; Pla ania
e al.
, 2012; Lima
e al.
, 2013; Pa a a i
e al.
, 2015).
W. anomalus
s ains in Table 2 ha e o long been kep in a cul u e collec ion, hey do no o igina e
di ec ly om na u e. Bea ing in mind ha hey migh ha e los hei kille abili y, hey we e i s ly assayed
in ha ega d agains a b oad sensi i e s ain o
Meye ozyma guillie mondii
(p e iously
Pichia
guillie mondii
) (PYCC 2734) (Aguia and Lucas, 2000; da Sil a
e al.
, 2008)
.
W. anomalus
s ains we e
cul i a ed in he media and g ow h condi ions es ablished o
M. pe niciosa,
i.e.
on MEA wi h MB a pH
5.5 and 30 °C, in which condi ions hey displayed egula g ow h, and we e he e o e subsequen ly es ed
o hei kille pheno ype in hese same condi ions. All he s ains o
W. anomalus
caused a blue inhibi ion
halo on
M. guillie mondii
indica i e o cell dea h (no shown), so none was excluded om he assessmen
o inhibi o y e ec o e ungal g ow h. Fo his pu pose, he
M. pe niciosa
as es g owing s ains F and
G om B azil and A om Ecuado we e used. The de elopmen o mycelium was ollowed up o 10 days.
All he
W. anomalus
s ains a ec ed he ungal g ow h al hough o a di e en ex en . The mycelia
de eloped eely in he opposi e di ec ion o he yeas s ikeou , while he ex en o i s de elopmen a ied
62
in he space be ween he yeas and he ungal plug. Based on his a ia ion, h ee le els o esponse we e
iden i ied, which we e con e ed in o an empi ical scale o yeas / ungus in e ac ion (Figu e 2): le el 0,
co esponds o a high ungal esis ance, in which he mycelium o e g ows he yeas e en ually illing he
en i e pla e; le el 1, co esponds o a weak inhibi o y e ec , whe ein he mycelium ills he gap be ween
he yeas and he plug and s ops g owing upon con ac wi h he yeas cul u e; and le el 2 co esponds o
a clea inhibi o y e ec , wi h he o ma ion o a blue inhibi ion halo acing he yeas . The esul s o each
combina ion o yeas / ungal s ain a e p esen ed in Table 3 acco ding o his scale. As can be seen,
M.
pe niciosa
s ains om B azil we e mo e esis an han he s ain om Ecuado , which was inhibi ed by
mos o he
W. anomalus
s ains. These esul s we e ob ained in MEA supplemen ed wi h MB. As con ols,
iden ical assays we e made on PDA, wi h and wi hou MB, and MEA wi hou MB, pla ing all he
s ains o
W. anomalus
agains he mo e sensi i e s ain o
M. pe niciosa
, A om Ecuado . Resul s we e iden ical
o he ones in Table 3, showing ha nei he he medium no he p esence o MB a ec ed he yeas - ungus
an agonism (no shown). Assays we e subsequen ly ex ended o he indus ial yeas s in Table 2. Resul s
(Table 3) showed a mo e i egula esponse om he ungus in he p esence o hese s ains han wi h
W. anomalus
, which was weake han p edic ed acco ding o hei o igin (Ab anches
e al.
, 1998; Alonso-
del-Real
e al.
, 2019; Ha oum
e al.
, 2012; Mannazzu
e al.
, 2019). Mo eo e , he assays we e also done
using he ungal s ains B and C, absen om Table 3. The g ow h o hese ungi in he an agonism assays
was ex emely i egula gene a ing un eliable esul s, eason why hese ungi we e excluded om u he
assays. In o al, mos e men a ion yeas s s ains we e less able o an agonise
M. pe niciosa
han
W.
anomalus.
No iceably,
ungal
s ain F om B azil s ood ou o displaying he highes esis ance o all
yeas s es ed.
Figu e 2
.
Empi ical classi ica ion scale o an agonis ic esponse in solid media. 0 ep esen s he absence o inhibi ion o
any kind, he ungus e en ually g owing on op o he yeas cul u e; 1 ep esen s a weak inhibi o y esponse in which case
he ungus g ows up o he limi o he yeas cul u e wi hou o e g owing i , and 2 ep esen s a clea an agonis ic e ec .
63
Table 3. Resul s o he an agonism assays be ween
M. pe niciosa
and yeas s in solid medium. The as es -g owing ungal s ains, A om Ecuado and F and G
om B azil we e es ed agains all he yeas s in Table 2. Fu he assays we e done using all he emaining ungal s ains agains he yeas s o igina ing om
e men a ion p ocesses. Resul s using
M. pe niciosa
s ains D and E om Ecuado and H om Venezuela a e p esen ed. The inhibi o y e ec was a ed om 0 o
2, acco ding o he empi ical scale in Figu e 2. Resul s p esen ed we e iden ical in h ee independen eplica es. A able cell displaying 3 numbe s (e.g. 1/1/2)
ep esen s a si ua ion whe e each pla e had a di e en esul .
M. pe niciosa
s ains
Yeas s
Wicke hamomyces anomalus
#2495
#2505
#3294
#4121
#4380
#4554
#5008
A
2
2
2
2
1
2
1
F
1
1
1
2
1
1
1
G
1
1
1
1
2
1
0
Fe men a i e yeas s ains
#1
#2
#3
#4
#5
#6
#7
CAT1
PE2
A
1
2
2
1
1
0
1
2
2
F
1
0
1
0
0
0
0
0
0
G
0
1
1/1/2
1
0
0
1
2
1
D
1
2
1
1
1
0
1
2
1
E
1
1/1/2
1
2
0
1
2
2
1/1/2
H
1
1
1
1
0
0
1
1
1
64
Is
M. pe niciosa
g ow h inhibi ion caused by a ola ile compound?
Mycelia s ong g ow h inhibi ion occu ed a a ce ain dis ance be ween he yeas and he ungal cells
(
e.g.
Figu e 2, an agonism le el 2). This sugges s ha p obably he yeas s ains sec e ed some kind o
compound ha signals he ungal cells. Yeas s can sec e e a soluble molecule which di uses h ough he
aga , e en ually eaching he mycelium (Schmi and B einig, 2006; Lopes
e al.
, 2015; Liu
e al.
, 2018),
o a ola ile compound ha may a ec he ungus a some dis ance (Fialho
e al.
, 2016; Schulz-Bohm
e
al.
, 2017). To e alua e whe he he an agonis ic esponse obse ed agains
M. pe niciosa
s ains
co esponded o a ola ile compound sec e ed by he yeas , he abo e desc ibed an agonism assays we e
epea ed using sep a e Pe i dishes. These p e en he di usion o molecules h ough he aga bu allow
he o ganisms in he wo sides o he pla e o sha e he a mosphe e.
M. pe niciosa
s ain G was challenged
wi h he p esence o he yeas s ains #3 and CAT1 (Table 3). The yeas s ain #1 was used as nega i e
con ol. Unlike be o e, no inhibi o y esponse was obse ed o any o he combina ions used (
e.g.
Figu e
3), indica ing ha he ungal g ow h inhibi ion p obably in ol es he di usion o a non- ola ile compound
h ough he aga . Ne e heless, he compe i ion o nu ien s and space canno be disca ded a his poin .
Figu e 3. Example o an an agonism assay in di ided Pe i dishes (le pic u e), wi h he compa ison wi h he p e ious
es s in solid medium using he same yeas
s
ungus combina ion ( igh pic u e).
65
An agonism assays in liquid media
Since he ungi ha e a slowe g ow h a e in compa ison wi h yeas s, i is necessa y o incuba e he pla es
o 10 days o sco e he an agonism e ec . This is hough un a ou able o he main enance o a ully
iable yeas cul u e. Hence, blue halo o cell dea h a ound he yeas biomass can appea , o in some
cases he whole biomass can become blue. This could be a eason unde lying he di e se and i egula
esponse depic ed in Table 3, since each yeas s ain will s ay ali e and me abolically heal hy o di e en
pe iods o ime. Fo his eason, he possibili y o assaying an agonism mo e e icien ly in liquid medium,
which p o ides a di ec con ac be ween hyphae and yeas s, was conside ed. Mo eo e , he e men a ion
yeas s we e chosen o p oceed wi h he an agonism in liquid media, based on ha (i) he se also includes
one s ain o
W. anomalus
isola ed om spon aneous e men a ions o
cachaça
p oduc ion and his way
he species is ep esen ed, (ii) hese s ains should be mo e esilien bea ing in mind hei pu a i e u u e
u iliza ion in he ield, and (iii) hey o igina e om de same geog aphic egion o he WBD, which in case
o
in ield
applica ion, could a oid he po en ial imbalance o he cacao plan a ions ecosys em caused by
he in oduc ion o an alien mic obe. Impo an ly, since hese a e s ains used by he indus y, hei
legaliza ion, comme cializa ion and accep ance should be acili a ed.
To op imize he condi ions o he assays in liquid media, ungi and he indus ial yeas s we e i s ly
cul i a ed in ME o YPD, pH 5.5 and 30 °C.
M. pe niciosa
p oduced abundan mycelia in he o m one
o se e al co on ball-like la ge conglome a es g owing in size o e ime (Figu e 4A). Yeas s globally
pe o med sligh ly be e on YPD han on ME (Figu e 4A), which is consis en wi h hese yeas s ains
p e e ence o glucose. S ill, hei g ow h a es allowed o conside he use o ME.
P elimina y an agonism assays we e pe o med choosing in Table 3 he s onge an agonizing yeas s
agains he as e g owing ungal s ains F and G: s ains #1 and #3
s
F, using CAT1 as nega i e con ol,
and #3 and CAT1
s
G, using #1 as nega i e con ol. Sepa a e con ols o ungi and yeas s g owing alone
we e also pe o med. The ungi we e inocula ed h ough a mycelium aga plug placed in a glass ube
inocula ed wi h 108 cells/mL om an exponen ially g owing yeas cul u e. As expec ed, he yeas s g ew
as e han he ungi, illing he g ow h medium. A e 10 days o co-incuba ion he medium was decan ed
o check o he p esence o absence o mycelium. In e es ingly, unlike in solid medium, yeas s inhibi ed
ungal g ow h in all he combina ions es ed, including he nega i e con ols (no shown). Based on hese
p elimina y esul s, an agonisms assays in liquid medium we e expanded o he ull se o combina ions
be ween all he e men a ion yeas s ains and he ungal s ains F and G.

66
67
Figu e 4. (A) Fungal cul i a ion o
M. pe niciosa
s ain F in ME and YPD a 30 °C, wi h he de elopmen o
abundan mycelia in he o m one o se e al co on ball-like la ge conglome a es (le panel) and he g ow h cu es
o he indus ial yeas s ains in ME and YPD a 30 °C, de e mined by OD a 600 nm ( igh panel). (B) Empi ical
classi ica ion scale o an agonis ic esponse in liquid media. 0 ep esen s he absence o inhibi o y e ec , he
ungus g ows h ee-dimensionally p oducing a la ge conglome a e o hyphae; 1 ep esen s a limi ed inhibi o y
esponse in which case some small mycelium is s ill o med om he aga plug, and 2 ep esen s a s ong inhibi o y
e ec in which case no mycelium is o med. The cul u e media was emo ed and subs i u ed o esh ME medium
o allow a be e isualiza ion o he esul s, since he u bidi y esul ing om yeas g ow h made i di icul o
isualize he deg ee o inhibi ion a he end o he assays.
Resul s a ied, allowing o es ablish a second an agonism empi ical scale (Figu e 4B), in which le el 0
ep esen s high ungal esis ance, wi h he o ma ion o he co on ball-like conglome a es o mycelium
iden ical o he ungal-alone con ol; le el 1 co esponds o a weak inhibi o y esponse, wi h he
de elopmen o some mycelia a ound he ungal aga plug; and le el 2 ep esen s a s ong inhibi ion wi h
he o al absence o ungal de elopmen . Resul s a e sco ed in Table 4 acco ding o his scale. A la ge
inc ease in he inhibi o y esponse compa ed o ha ob ained in solid medium was ob ained. In ac , a
s ong inhibi o y esponse was obse ed in 9 ou o 18 combina ions (50%), in opposi ion o he 2 ou o
18 (11%) obse ed in solid media (Table 3). The yeas s ains #1, #5, #6 and PE-2 elici ed a s ong
inhibi o y esponse, while CAT1 inhibi o y e ec was s ong (le el 2) o weak (le el 1) in he case o s ains
G and F espec i ely.
Table 4. An agonism assays in liquid medium using he combina ions be ween he e men a i e yeas s ains and
M.
pe niciosa
s ains F and G. The p esence o he inhibi o y e ec was e alua ed on a scale om 0 o 2 acco ding o Figu e 2.
Resul s we e iden ical in h ee independen eplica es.
Yeas s
M. pe niciosa
s ains
#1
#2
#3
#4
#5
#6
#7
CAT1
PE2
F
2
0
1
0
2
2
0
1
2
G
2
0
1
0
2
2
0
2
2
68
The di e ence in esponses obse ed in solid and liquid media may de i e om se e al ac o s. Liquid
medium, in opposi ion o solid, p opi ia es he as g ow h o he yeas popula ion, as e han he ungal
mycelium. The inc easing yeas popula ion possibly compe e wi h he mycelium o nu ien s. This was
epo ed o occu be ween yeas s and phy opa hogenic ungi, as one o he main modes o an agonism
ac ion (And ews
e al.
, 1994; Sa a anakuma
e al.
, 2008; Zhang
e al.
, 2010; Spada o and D oby, 2016),
showing ha he inhibi ion o ungal g ow h does no always implica e dea h, being o en e e sible since
he ungal cells can e ain hei iabili y (Spada o and D oby, 2016). Addi ionally, liquid medium also
allows he homogenous sp ead o he yeas cells and hei con ac wi h he hyphae. Finally, yeas s emain
me abolically ac i e o a longe pe iod o ime han in solid medium, and hei pe manen con ac wi h
he mycelia may igge he p oduc ion and sec e ion o pep ides o o he compounds wi h an i ungal
p ope ies. This means ha an agonism may co espond o ungal dea h o jus an inhibi ion o i s
eplica ion.
E alua ion o ungal dea h by s aining wi h Me hylene Blue and P opidium
Iodide
To e i y i he an agonism obse ed in liquid medium co esponds o ac ual ungal dea h, wo well-known
ma ke s o cell dea h we e used, Me hylene Blue (MB) and P opidium Iodide (PI). MB is a ca ionic dye
which can pene a e bo h li e and dead cells, inside which i binds o nega i ely cha ged molecules such
as nucleic acids. Li ing cells a e able o educe he dye and consequen ly emain colou less. PI can only
en e he cells which memb ane is dis up ed; he e o e, only nec o ic cells a e s ained wi h ed
luo escence. Cells s ained wi h MB bu no wi h PI los iabili y bu p ese e he in eg i y o he plasma
memb ane, which means ha hey a e possibly dying o apop osis (Kwolek-Mi ek and Zad ag-Tecza,
2014). Resul s a e exempli ied in Figu e 5. All he yeas / ungal s ain combina ions e alua ed ha
demons a ed a s ong inhibi o y esponse (le el 2) p esen ed an almos ully s ained mycelium wi h bo h
MB and PI, while in all he combina ions wi h a weake inhibi o y esponse (le el 1) he mycelium was
only pa ially s ained. These esul s showed ha he e a e wo mechanisms unde lying
M. pe niciosa
an agonism by yeas s. Yeas s kill he ungi, bu in some cases, he ela i e amoun s o yeas
cells/mycelium do no appea su icien o induce he dea h o he whole ungal cul u e in he ime window
o 10 days in which he assays ake place. In his case, yeas s jus delay ungal g ow h. In e es ingly, i is
possible o obse e ha isola ed o clus e ed yeas cells appea o a ach o he killed ungal hyphae,
69
sugges ing ha con ac be ween he wo o ganisms may be necessa y o cause he e ec and may e en
be in ol ed in he mechanisms ha cause he ungal cells dea h.
Figu e 5. Example o he yeas / ungal s ains cul u es s aining wi h MB (uppe panel) and PI (middle and lowe panels,
co esponding o he obse ed b igh ield and luo escence o he same pic u e, espec i ely). Empi ical classi ica ion scale
based on he in ensi y o he s aining: 0 ep esen s an absence o s aining; 1 co esponds o a weak s aining; and 2
ep esen s a s ong s aining o he mycelia. Scale ba : 20 μm.
0
1
2
MB
PI
76
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CHAPTER 3
Mic oscopic assessmen o he an agonism e ec o yeas s
agains
Monilioph ho a pe niciosa,
he ungal causal
agen o
Wi ches’ B oom Disease
in cacao
The wo k p esen ed in his chap e is in p epa a ion o publica ion:
Fe az, P., Cássio, F. and Lucas, C. Mic oscopic assessmen o he an agonism e ec o yeas s agains
Monilioph ho a pe niciosa
, he ungal causal agen o
Wi ches’ B oom Disease
in cacao

82
ABSTRACT
Mycopa asi ism o pa hogenic ungi by yeas s could be associa ed wi h di ec physical con ac . P edacious
yeas s can pene a e he ungal cell walls killing he ungal pa hogen and/o eed on hem, aking up
nu ien s om he ungal cells. Con ac be ween yeas s and ungal phy opa hogens was obse ed in
an agonism yeas s ains agains
Monilioph ho a pe niciosa
, he ungal causal agen o he
Wi ches’
B oom Disease
o cacao (Fe az
e al
., submi ed 2). Cell analysis by Scanning Elec on Mic oscopy (SEM)
e ealed yeas binding and using wi h he hyphae o he phy opa hogen, causing a cons ic ion and
de o ma ion o i s cells, and e en a d aining o he ungal cellula con en . The o ma ion o eil-like
s uc u es possibly associa ed wi h in asion o hyphae by yeas s was also obse ed. Mo eo e , SEM
mic og aphs showed he occu ence o imb iae-like connec ions be ween an agonis ic yeas cells and o
physical ube-like connec ion be ween yeas and ungal cells, which can be ela ed wi h yeas cell-cell
communica ion and o yeas / ungus ecogni ion o e en yeas p edacious beha io , espec i ely. These
esul s cons i u e he i s desc ip ion o a possible p edacious-like beha io in non-
Saccha omycopsis
yeas s, which can be he basis o he an agonism exe ed by yeas s agains he ungal pa hogens ha
cause WBD o cacao.
Keywo ds
SEM, Cacao;
Wi ches B oom Disease
;
Monilioph ho a pe niciosa
;
yeas ; an agonism; p eda ion
83
INTRODUCTION
Yeas s can an agonize o he mic oo ganisms such as o he yeas s, molds, and bac e ia (Ha oum
e al.
,
2012). The an agonis ic e ec o yeas s is based on se e al mechanisms o ac ion, including he
compe i ion o nu ien s and space (Pa a a i
e al.
, 2015) and/o he p oduc ion and sec e ion o kille
oxins (Schmi and B einig, 2006) and o he an i ungal compounds such as hyd oly ic enzymes (Lopes
e al.
, 2015; Liu
e al.
, 2018), ola ile compounds (Schulz-Bohm
e al.
, 2017) and quo um sensing
molecules (A belj
e al.
, 2016). In all hese mechanisms, yeas s a e able o sec e e compounds and exe
hei an agonis ic e ec a a dis ance. Despi e his, some yeas s ha e he abili y o a ach o he hyphae
o ungal pa hogens and p oduce di e se ex acellula cell wall ly ic enzymes. This ype o in e ac ion is
known as mycopa asi ism (Duka e
e al.
, 2018). Some sequen ial e en s a e equi ed so ha a
mic oo ganism can pa asi ize a ungus. Fi s he e mus be close con ac be ween he an agonis and he
ungal pa hogen cells ollowed by mu ual ecogni ion. Subsequen ly he an agonizing mic oo ganism
sec e es ly ic enzymes and ul ima ely, he e is ac i e g ow h o he an agonis in o he ungal hos cells
h ough a kind o pene a ion peg (Spada o and Gullino, 2004). The ungal cell wall is o en a ge ed. The
ungal cell wall is composed o polysaccha ides, glucans and chi in, and glycop o eins, which oge he
p o ide mechanical s eng h and s uc u al in eg i y (Spada o and D oby, 2016). The an agonis sec e es
hyd oly ic enzymes, including chi inases, chi osanases, glucanases, cellulases o p o eases which may
ac independen ly o in combina ion wi h each o he o o he enzymes (Spada o and D oby, 2016).
Ul ima ely, he des uc ion o cell wall leads o se e e cy ological damage, in mos ci cums ances causing
he lysis o he cell and subsequen ly dea h (Di F ancesco
e al.
, 2015; Duka e
e al.
, 2018).
Some examples o mycopa asi ism and i s associa ion wi h he biocon ol o ungal phy opa hogens ha e
been desc ibed in he li e a u e, pa icula ly he mycopa asi ism exe ed by yeas s. This is he case o he
an agonism o
Bo y is cine ea
by he yeas
Meye ozyma guillie mondii
( o me ly known as
Pichia
guillie mondii
), in which he yeas cell a aches s ongly o he hos and sec e es a β-(1–3) glucanase,
which esul s in he lysis o he ungal cell (Wisniewski
e al.
, 1991). Is has also been epo ed ha , his
ype o ly ic enzymes also inhibi s he ge mina ion o he pa hogen spo es and he elonga ion o i s ge m-
ube (El-Ta abily and Si asi hampa am, 2006). Ano he example o mycopa asi ism is he biocon ol o
Penicillium expansum
by he yeas
Candida oleophila
in ha es ed apples, associa ed wi h he sec e ion
o an exo-β-1,3-glucanase, which esul s in he inhibi ion o conidial ge mina ion and mycelia g ow h
(Tamayo-U bina
e al.
, 2016).
84
The mycopa asi es can be classi ied in o di e en g oups, acco ding o hei modes o ac ion. The
nec o ophic mycopa asi es a e e y agg essi e o ganisms wi h a b oad ange o p eys, and can a ack
ungi a a dis ance by he sec e ion o oxins o ly ic enzymes in o he en i onmen (Junke
e al.
, 2019).
Con ac nec o ophs a ack he ungal cells by di ec physical con ac and can each and kill he ungal
pa hogen using hyphae, al hough hey do no necessa ily in ade he a ge cell (Mims
e al.
, 2007). On
he o he hand, in asi e nec o ophs, also known as p eda o y mycopa asi es, physically pene a e hei
p ey ungal cells h ough haus o ia o pene a ion pegs (Junke
e al.
, 2019). The p eda o y o ganism can
kill he ungal p ey, o simply ake up nu ien s om i s cells, o bo h (Je ies, 1995; Junke
e al.
, 2019).
The majo i y o he well-s udied mycopa asi es a e ilamen ous ungi, pa icula ly species om he
T ichode ma
genus (Schmoll
e al.
, 2016). Al hough many yeas species a e able o sec e e an agonis ic
pep ides such as kille oxins (Schmi and B einig, 2006), o ly ic enzymes (Spada o and Gullino, 2004),
he only yeas species desc ibed as a nec o ophic mycopa asi e belong o he
Saccha omycopsis
clade
(Lachance and Pang, 1997). These yeas s a e able o p eda e a wide ange o o he yeas s. All sha e a
me abolic peculia i y, he inabili y o use sul a e as sole sul u sou ce. Despi e no being well s udied,
hei mode o p eda o y ac ion in ol es he in asion o he p ey cells, media ed by small haus o ia-like
pene a ion pegs, leading he subsequen dea h o he p ey cells (Lachance
e al.
, 2012).
S. schoenii
is
conside ed he mos agg essi e p edacious yeas in his clade, and i was epo ed o a ack and kill
in
i o
se e al clinical isola es o pa hogenic
Candida
species, including he mul i-d ug esis an isola es o
C. au is
, hus ha ing a g ea po en ial in medical biocon ol applica ions (Junke
e al.
, 2018). Addi ionally,
some
Saccha omycopsis
species, ha e been desc ibed as success ul po en ial biocon ol agen s, namely
S. schoenii
, agains plan pa hogens de eloping in he su ace o o anges (Pimen a
e al.
, 2008), and
S.
ibulige a
agains oxic molds on po k speck (Iacumin
e al.
, 2017).
The mos a o able en i onmen al condi ions ha p omo es p eda ion a ies signi ican ly, depending on
he p edacious species. Some species a e s imula ed by he p esence o ich ni ogenous nu ien s and
high concen a ions o ammonium ni ogen, as well as by he p esence o o ganic sul u compounds,
while o he species may be inhibi ed unde he same condi ions (Lachance
e al.
, 2000). Mo eo e , he
p edacious yeas s a e able o pene a e he cells o o he desc ibed p edacious species, and in e es ingly
young cul u es can pene a e olde cul u es o he same p edacious s ain (Lachance
e al.
, 2000). This
is a e y poo ly s udied phenomenon which has no been desc ibed o o he yeas gene a and species,
and which p eda ion modes o ac ion ha e no been u he s udied.
85
In a ecen s udy he causal agen o cacao
Wi ches’ B oom Disease
(WBD), he ilamen ous ungus
Monilioph ho a pe niciosa
, was shown o be killed by yeas isola es om spon aneous e men a ions o
cachaça
p oduc ion and s ains used in bioe hanol indus ial p ocesses (Fe az
e al.
, submi ed 2). Dea h
o he ungal cell occu ed a e he co-incuba ion o bo h o ganisms and de ec ed by s aining wi h
me hylene blue and p opidium iodide. The wo k he eby p esen ed aimed a explo ing mic oscopically he
in e ac ion be ween he wo ypes o cells, un eiling ha he yeas s physically a ach o he hyphae,
appa en ly using hei walls su ace, he wo cells becoming as one, and ha he hyphae a e d ained.
Mo eo e , he s udy also showed ha yeas s communica e physically be ween each o he du ing he
p ocess. Resul s we e ob ained obse ing samples s ained wi h me hylene blue, as well as using Scanning
Elec on Mic oscopy (SEM). Fu he assessmen by s aining he cy oskele on wi h a speci ic luo escen
dye, and he use o T ansmission Elec on Mic oscopy (TEM), will allow in he u u e he elucida ion o
he cy ology o he ac ual me ge be ween he wo cells.
92
Figu e 5. SEM mic og aphs o he yeas / ungal s ains combina ions #1105
s
CBS 441.80 and PE2
s
CBS 441.80,
showing in close de ail di e en mo phological s uc u es o he in e ac ion: yeas pushing he hyphae and usion be ween
he cells o bo h o ganisms (whi e a ows); connec ions be ween he yeas and he ungal cells (blue a ows); and o ma ion
o a eil-like s uc u e ( ed a ows).

93
1
µm
0.5
µm
Figu e 6. SEM mic og aphs o he connec ion be ween yeas cells. Le panels - De ails o wo yeas cells ha appa en ly
we e connec ed and hei small imb iae-like s uc u es on he cell su ace. Righ panels - Fimb iae-like s uc u es in he
su ace o he yeas cells (blue a ows) and loca ions o p e ious connec ion s uc u es (yellow a ows).
94
Fimb iae and pili ha e abundan ly been desc ibed in bac e ia (Ca e
e al.
, 2016) as hai -like s uc u es
p o uding om bac e ia cell wall. They a e in ol ed in adhe ence o ine su aces o li ing issues, in
cell-cell communica ion, as well as in he conjuga ional ans e o DNA be ween wo cells ( e iewed by
Be ne e al., 2018). In he case o yeas s, connec ion ib ils be ween wo cells om he same species and
s ain ha e been epo ed in bio ilms (Mam u a
e al.
, 2017) o in s a ed cell (Va on and Chode , 2000).
To hese ib ils ha e been a ibu ed a ole in he colony o ma ion o some
Candida
species (Va gas
e
al.
, 2004; Fu lane o
e al.
, 2012). In o ma ion is hough e y sca ce, and he e a e no de ailed s udies
on which yeas s a e able o no o o m hem, on how o when hey a e o med, o on hei s uc u e and
composi ion.
95
CONCLUSIONS
The p esen s udy allowed he isualiza ion o (i) he an agonizing yeas cell binding and using o he
dead hyphae o he phy opa hogen, (ii) he cons ic ion and de o ma ion o he ungal cells caused by
yeas a achmen , (iii) he d aining o he ungal cellula con en in a eas adjacen o he yeas a achmen .
Mo eo e , he analysis by SEM e ealed (i) he o ma ion o a eil-like s uc u e ha could be ela ed wi h
a possible in asion o hyphae by yeas s, (ii) he occu ence o a physical ube-like connec ion be ween
yeas and ungal cells which can be ela ed o yeas / ungus ecogni ion o e en associa ed wi h yeas
p edacious beha io , and (iii) he exis ence o imb iae-like connec ions be ween an agonis ic yeas cells,
which can be associa ed wi h a mechanism o yeas cell-cell communica ion. Al hough nei he o he
an agonis ic yeas s es ed belong o he
Saccha omycopsis
clade, hese cha ac e is ics esemble hose
o he p edacious yeas s.
This wo k p esen s he i s desc ip ion o a possible p edacious-like beha io in non-
Saccha omycopsis
yeas s and migh p o ide a basis o unde s and why he indus ial yeas s a e so esilien o ha sh
en i onmen s and p e ail o e o he s ains in hei na u al/indus ial habi a . Ne e heless, a p edacious
beha io may no be independen o he sec e ion o some an i ungal compounds by hese yeas s, which
may ac syne gis ically, causing he an agonism e ec and leading o he pa hogen’s dea h. Fu u e
analysis by TEM and luo escence mic oscopy can imp o e he unde s anding o his an agonism and
cla i y hese yeas s mode o ac ion.
ACKNOWLEDGEMENTS
This wo k was suppo ed by he s a egic p og amme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569)
unded by na ional unds h ough he FCT I.P. and by he ERDF h ough he COMPETE2020 - P og ama Ope acional
Compe i i idade e In e nacionalizacão (POCI), and he p ojec EcoAg iFood (NORTE-01-0145-FEDER-000009) om
No e Po ugal Regional Ope a ional P og amme (NORTE 2020) unde he PORTUGAL 2020 Pa ne ship
Ag eemen h ough he Eu opean Regional De elopmen Fund (ERDF). PF is a PhD s uden o he Doc o al
P og amme in Applied and En i onmen al Mic obiology (DP-AEM), (FCT g an PD/BD/113810/2015). We hank
he B azilian companies, Ce le , Lda., Ou o P e o, MG, and Fe men ec, Lda. Soluções Tecnológicas e Indus iais,
Pi acicaba, SP o kindly supplying he yeas s ains used in his wo k.
96
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CHAPTER 4
Wicke hamomyces anomalus
om oli e o cha ds mic obiome
e icien ly an agonizes he Oli e An h acnose’s
causal agen s,
Colle o ichum spp.
The wo k p esen ed in his chap e is in p epa a ion o publica ion:
Amo im-Rod igues, M., Fe az, P., Cássio, F. and Lucas, C.
Wicke hamomyces anomalus
om oli e o cha ds
mic obiome e icien ly an agonizes he Oli e An h acnose’s causal agen s,
Colle o ichum
spp.
108
RESULTS AND DISCUSSION
Op imiza ion o ungal cul i a ion condi ions
Yeas s ains used in he p esen wo k (Table 1) can be di ided in wo g oups. The i s con ains s ains
which we e isola ed om he oli e biome o Po uguese o cha ds. The second g oup includes s ains
isola ed om suga cane-based spon aneous and indus ial e men a ion p ocesses. The ungal s ains
ha we e used ha e di e en o igins. The s ain o
C. gloeospo ioides
s.s. was o iginally isola ed om
in ec ed o cha ds in I aly. The s ains o
C. gode iae
and
C. nymphaeae
o igina e om in ec ed oli e ees
in Po ugal. P io o he an agonism assays, he
in i o
op imal cul u e condi ions o ungal and yeas s
s ains we e de e mined. Fi s ly, he ungal g ow h a es we e assessed in MEA and PDA, a di e en pH
(4.0, 4.5, 5.0, 5.5 and 6.0) and 30 °C. Only
C. gloeospo ioides
s.s. was able o g ow a his empe a u e.
The e o e, assays we e epea ed a 25°C. In ag eemen wi h he li e a u e (Wha on and Diéguez-
U ibeondo, 2004; Talhinhas
e al.
, 2005),
C.
gode iae
and
C. nymphaeae
( om he
C. acu a um
species
complex) g ew much slowe han
C. gloeospo ioides
s.s.
(Table S1). Al hough his las ac ually g ew be e
a 30 °C han a 25 °C, he di e ence was no signi ican (no shown). The selec ed empe a u e o
u he assaying all he ungi was he e o e de ined as 25 °C. Fu he mo e, di e ences in g ow h a es
be ween assays in MEA o PDA (no shown) and a di e en pH (Table S1) we e no signi ican . Based on
hese esul s, he op imal condi ions we e es ablished o all he ungal s ains as MEA o PDA, and pH
5.5 a 25 ºC. F om he same assays, i was also shown ha 4 o 8 days is enough o assess ungal
g ow h.
An agonism in solid media
An agonism was es ed using he yeas s and he phy opa hogenic ungal s ains in Table 1. Resul s we e
cap u ed a e 8 days o incuba ion (Table 2) and exp essed acco ding o he empi ical scale o le els 0,
1 and 2 desc ibed in Fe az
e al.
(submi ed). A mo e de ailed assessmen o an agonism ollowed,
de e mining he pe cen age o ungal g ow h inhibi ion (Table 3). As can be seen in hese Tables, all he
yeas s we e able o inhibi he g ow h o a leas one o he ungal s ains, in mos cases causing a low
inhibi o y esponse (le el 1). Acco dingly, some yeas s beha ed gene ally as weak an agonis s, like he
s ains #1 and PE2. In opposi ion, he yeas s ain causing he s onges inhibi o y esponse (le el 2) was
#18, which inhibi ed he g ow h o he h ee ungal s ains in mo e han 70 % (Table 3).

109
Table 2.
Resul s o an agonism assays in solid medium, using he yeas s ains om Table 1 agains
Colle o ichum
gloeospo ioides
s.s.
(C1)
, C. gode iae
(C2)
and
C. nymphaeae
(C3)
.
Assays we e pe o med in MEA a 25 °C. The numbe s
displayed ansla e esul s acco ding o he empi ical scale desc ibed in Fe az
e al.
(submi ed). Single numbe s ep esen
he inhibi o y esponse o 3 eplica es. A able cell displaying 3 numbe s (e.g. 1/0/0) ep esen s a si ua ion whe e each pla e
had a di e en esul .
Phy opa hogens
Yeas s
#1
#2
#3
#4
#5
#6
#7
CAT1
PE2
#11
#12
C1
0
1/0/0
1/0/0
0
2/1/1
1
0
1/1/0
0
0
1/1/0
C2
1
1/1/0
1/1/0
1/1/0
1
2/1/0
1
1
1/0/0
1
1
C3
1
1/0/0
1/0/0
1/1/0
1/1/0
1/1/0
1
1/0/0
0
1/1/1
1/1/0
#13
#14
#15
#16
#17
#18
#19
#20
#21
#22
C1
1/0/0
1/1/0
1/0/0
1/0/0
1/1/0
2/2/1
1/1/0
1
1/1/0
1
C2
1
2/1/0
1
1/1/0
1
2
1
1
1
1
C3
1
1/0/0
1/1/0
1/1/0
1
2
1
1/1/0
1
1/1/0
The inhibi ion o he ungal g ow h was obse ed o occu a a ce ain dis ance as exempli ied in Figu e
1A, sugges ing ha yeas s possibly sec e e a compound ha a ec s he ungal mul iplica ion. The e a e
wo ypes o possible compounds: (i) soluble, di using h ough he aga , such as kille oxins and ly ic
enzymes (Pa a a i
e al.
, 2015; O o
e al.
, 2018); and (ii) ola ile (To ano
e al.
, 2017). To assess which
migh be he case, he an agonism on solid media was epea ed in MEA using sep a e Pe i dishes,
equidis an ly inocula ed wi h he mycelial plug on one side o he sep um, and he yeas s eak on he
o he . This only allows he ungus and he yeas o sha e he a mosphe e inside he dish. No inhibi o y
esponse was obse ed up o 2 weeks o incuba ion, as exempli ied in Figu e 1B. Mo eo e , he ungal
s ains, excep
C. gode iae
, wen o e he
sep um
, e en ually g owing on op o he yeas and co e ing
he whole dish. This esul s ongly sugges ed ha he inhibi ion o ungal g ow h by his yeas equi es
he di usion o a molecule h ough he aga . Acco dingly, Lima and collabo a o s (2013) ound ha when
W. anomalus
and
M. guillie mondii
cul u es we e killed by au ocla ing and il e ed, hey los he abili y o
inhibi he spo e ge mina ion o
C. gloeospo ioides
s.s., which concu s wi h he need o yeas o be
me abolically ac i e abo emen ioned. A e 2 weeks o incuba ion, mos o he yeas popula ion is mos
p obably dying o dead, consis en ly wi h he MB colo a ion o he yeas s eak (no shown).
110
Table 3. An agonism be ween he OA causa i e agen s
Colle o ichum gloeospo ioides
s.s.
(C1)
, C. gode iae
(C2)
and
C. nymphaeae
(C3)
and he yeas s ains in Table 1. Resul s a e pe cen age
o inhibi ion in solid media, acco ding o Royse & Ries (1978). Values a e a e age ac oss eplica es N≥3 and s anda d de ia ion. Shaded cells show highes inhibi ion o all ungi.
Phy opa hogens
Yeas s
#1
#2
#3
#4
#5
#6
#7
CAT1
PE2
#11
#12
C1
3.2 ± 5.5
17.5 ± 7.3
19.1 ± 4.8
25.4 ± 15.3
70.4 ± 1.4
46.0 ± 22.5
23.8 ± 4.8
57.1 ± 24.7
19.1 ± 4.8
0.0 ± 0.0
28.6 ± 31.2
C2
40.9 ± 7.9
22.7 ± 25.3
33.3 ± 26.6
25.8 ± 9.5
28.8 ± 9.5
21.2 ± 11.4
59.1 ± 4.6
10.6 ± 14.6
9.1 ± 4.6
43.2 ± 18.4
46.1 ± 17.9
C3
25.9 ± 3.6
33.3 ± 24.7
47.2 ± 17.5
54.7 ± 2.1
62.9 ± 7.6
51.2 ± 22.8
25.3 ± 17.1
37.9 ± 20.3
14.6 ± 12.4
30.2 ± 2.8
55.7 ± 3.1
#13
#14
#15
#16
#17
#18
#19
#20
#21
#22
C1
22.2 ± 22.5
31.8 ± 28.7
11.1 ± 7.3
14.3 ± 12.6
33.3 ± 35.9
70.8 ± 7.2
22.2 ± 5.5
28.6 ± 0.0
25.4 ± 24.0
28.6 ± 4.8
C2
38.1 ± 5.7
32.1 ± 27.8
33.3 ± 11.6
48.2 ± 5.6
55.2 ± 18.9
70.5 ± 0.8
41.9 ± 7.3
44.1 ± 10.6
52.2 ± 10.6
55.9 ± 16.0
C3
56.3 ± 5.5
65.9 ± 3.0
54.0 ± 9.7
43.6 ± 19.4
52.8 ± 14.1
74.5 ± 4.3
49.1 ± 4.3
61.3 ± 5.2
62.2 ± 7.9
65.4 ± 3.8
111
Figu e 1. Yeas s ain #18 agains
Colle o ichum gloeospo ioides
s.s. s ain. Inhibi o y esponse a a dis ance (A) and
none in a sep a e pe i dish (B).
An agonism in liquid media
The an agonism assays we e subsequen ly epea ed in liquid medium. I a me aboli e o some kind is
p oduced and sec e ed by he yeas s ha inhibi s he ungal g ow h, i s di usion should be much mo e
e icien in liquid medium and he e o e migh cause s onge inhibi ion. Mo eo e , he p oduc ion o
some an i ungal me aboli es is g ea e a he exponen ial g ow h (Lopes
e al.
, 2015), which is only
possible in liquid cul u es. Mo eo e , hese also allow a g ea e nu ien a ailabili y p omo ed by he
cons an agi a ion, acco ding o which yeas s should s ay me abolically ac i e o longe pe iods o ime.
Finally, he e icien an agonism in some cases can ely on physical con ac be ween he inhibi o and he
inhibi ed s ains, as p e iously demons a ed wi h ano he phy opa hogen (Fe az
e al.
, submi ed).
no yeas
#18
no yeas
#18
A
B
112
The h ee OA ungal s ains we e challenged wi h a me abolically ac i e yeas inoculum and incuba ed o
4 days. Resul s o he an agonis ic esponse using empi ical scale wi h 0, 1, 2 le els o inhibi ion
p e iously desc ibed (Fe az
e al.
, submi ed) a e shown in Table 4, and ep esen he ypes o inhibi ion
exempli ied in Figu e 2.
Table 4. An agonism be ween he phy opa hogenic ungal s ains
Colle o ichum gloeospo ioides
s.s. (C1),
C. gode iae
(C2)
and
C. nymphaeae
(C3)
and he yeas s ains in Table 1 in liquid media. The numbe s displayed ansla e esul s acco ding
o a p e iously es ablished empi ical scale (Fe az
e al.
, submi ed; 0 = no inhibi ion; 1 = weak inhibi ion;2 = ull inhibi ion)
and ep esen a leas h ee independen eplica es wi h iden ical esul .
Phy opa hogens
Yeas s
#1
#2
#3
#4
#5
#6
#7
CAT1
PE2
#11
#12
C1
2
0
0
0
2
2
2
2
2
0
1
C2
2
1
0
0
2
2
2
2
2
0
1
C3
2
0
0
0
2
2
2
2
2
0
1
#13
#14
#15
#16
#17
#18
#19
#20
#21
#22
C1
2
1
2
0
1
2
2
2
2
2
C2
2
0
2
1
2
2
2
2
2
2
C3
2
1
2
0
1
2
2
2
2
2
In gene al, as expec ed, he inhibi o y esponse inc eased compa ing o ha ob ained in solid media. In
solid media, a s ong inhibi o y esponse (le el 2 in Table 2; >70% in Table 3) was only obse ed in 6 ou
o 63 combina ions, while in liquid medium, he ungi we e s ongly inhibi ed (le el 2) in 39 ou o 63
combina ions. Resul s in Table 4 also show ha 3 yeas s did no inhibi he g ow h o any o he ungal
s ains: #3, #4, and #11, and he #2 and #16 only weakly inhibi ed one o hem. Based on hese esul s,
hese yeas s we e no u he conside ed.
113
Figu e 2. Liquid media an agonism assay agains
Colle o ichum gloeospo ioides
s.s. s ain. Examples o he ype o inhibi o y
esponse.
Explo a o y assays o con i ma ion o he dea h o he ungal cells we e conduc ed. The ungal inoculum
plug, a e he co-incuba ion in liquid medium wi h he s onges an agonizing yeas s ains, was s ained
wi h Me hylene Blue (MB) and P opidium Iodide (PI) (no shown). The i s dye binds o nega i ely cha ged
molecules when i is no educed, hus s aining dead cells, and he second only en e s he cell when he
plasma memb ane is dis up ed (Kwolek-Mi ek and Zad ag-Tecza, 2014). In some combina ions bo h
mycelium and spo es we e well s ained, while o he combina ions showed no o weak s aining. These
p elimina y esul s sugges ha some yeas s ains could be killing he ungi, bu in he combina ions ha
we e no s ained, yeas s ains a e p obably jus inhibi ing ungal p oli e a ion, allowing hese o e ain
iabili y. This explo a o y mic oscopic inspec ion o all combina ions did no e eal yeas s a aching o he
hyphae as p e iously obse ed wi h ano he phy opa hogen (Fe az
e al.,
submi ed).
CONTROL
NO
inhibi ion
WEAK
inhibi ion
STRONG
inhibi ion
1
2
3

114
CONCLUSIONS
All aken, esul s sugges ed ha ungal g ow h inhibi ion migh occu h ough he sec e ion o a di usible
compound by an agonizing yeas s. Fo his o happen, yeas s ha e o be me abolically ac i e. Whe he
his compound causes he dea h o he ungi, o simply inhibi s hei eplica ion, emains o be con i med.
Mo eo e , esul s also showed ha yeas s ain #18, a
W. anomalus
isola ed om oli e o cha ds in
Po ugal, has a s ong abili y o an agonize he h ee
Colle o ichum
sp. s ains used in his wo k. These
esul s ag ee wi h o he s udies p oposing he po en ial o
W. anomalus
( o me ly known as
Pichia
anomala
o
Hansenula anomala
) as biocon ol agen (F iel
e al.
, 2007; O o
e al.
, 2014; Pa a a i
e al.
,
2015). This species possesses o he desi able cha ac e is ics, like he abili y o g ow unde high osmo ic
p essu e, a low pH, and a a b oad ange o empe a u es (F edlund
e al.
, 2002).
The ac ha he bes yeas candida e o igina es om he oli e ees biome sugges s ha , be e han
using well-known yeas s o long cap i e o labo a o y o indus ial p ocesses and acco dingly
domes ica ed, he exploi a ion o an app op ia e niche biodi e si y yields be e s ains, able o e icien ly
colonise
he oli e ee o cha ds. The niches whe e he na u al wild yeas and he phy opa hogens co-exis
a e mos p obably ideal. The esul s in his wo k sugges ed as much and can be conside ed a
s eppings one o he o mula ion o a new eco- iendly and e ec i e al e na i e o con olling OA. Fu he
s udies a e equi ed in o de o ully unde s and he mechanisms by which hese yeas s a e able o
an agonize he phy opa hogenic ungi.
ACKNOWLEDGEMENTS
This wo k was suppo ed by he s a egic p og amme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) unded by
na ional unds h ough he FCT I.P. and by he ERDF h ough he COMPETE2020 - P og ama Ope acional Compe i i idade e
In e nacionalização (POCI), and he p ojec EcoAg iFood (NORTE-01-0145-FEDER-000009), suppo ed by he No e Po ugal
Regional Ope a ional P og amme (NORTE 2020) unde he PORTUGAL 2020 Pa ne ship Ag eemen h ough he Eu opean
Regional De elopmen Fund (ERDF). PF and MA-R a e PhD s uden s o he Doc o al P og ammes in Applied and En i onmen al
Mic obiology (DP_AEM), (FCT g an PD/BD/113810/2015) and in Molecula and En i onmen al Biology (PDBMA), (FCT g an
PD/BD/145354/2019), espec i ely. We hank he B azilian companies, Ce le , Lda., Ou o P e o, MG, and Fe men ec, Lda.
Soluções Tecnológicas e Indus iais, Pi acicaba, SP, as well as P o esso João Paulo Sampaio om he Po uguese Yeas
Cul u e Collec ion/UCIBIO, NOVA, Po ugal o kindly supplying he yeas s ains used in his wo k. We also hank P o esso
Ped o Talhinhas, om LEAF - Linking Landscape, En i onmen , Ag icul u e and Food, ISA, Uni e sidade de Lisboa, Po ugal,
o kindly supplying wo o he ungal s ains used in his wo k.
115
SUPPLEMENTARY MATERIAL
Table S1. G ow h a es o he phy opa hogenic ungal s ains
Colle o ichum gloeospo ioides
s.s.
, C. gode iae
and
C.
nymphaeae
a 25 and 30 °C, in wo cul u e media and a di e en pH alues.
G ow h a e (mm.day -1)
pH 4.0
pH 4.5
pH 5.0
pH 5.5
pH 6.0
30 ºC
C. gloeospo ioides
PDA
3.90
4.43
5.31
5.13
5.86
MEA
4.10
5.89
6.14
5.76
6.14
25 ºC
C. gloeospo ioides
PDA
3.17
4.84
5.00
5.38
5.08
MEA
4.06
4.80
5.56
5.89
5.76
C. gode iae
PDA
1.88
2.77
1.77
2.68
2.56
MEA
1.77
2.21
2.76
2.25
2.46
C. nymphaeae
PDA
2.17
2.47
2.71
3.15
2.54
MEA
2.41
2.39
3.18
2.90
2.64
116
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a e able o kill
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124
B azil p oduces bioe hanol mos ly om yeas e men a ion o suga cane juice. The p ocess in ol es he
physical ex ac ion o he juice om he cane which is hen e men ed by yeas s. A e each e men a ion
cycle, he mus is cen i uged, and he yeas biomass sepa a ed and ecycled. The subsequen dis illa ion
p ocess yields a da k liquid was e called
inasse
(Ca ilho e al., 2016). B azilian indus y gene a es an
a e age 12 L o
inasse
pe li e o e hanol p oduced (Lopes
e al.,
2016), meaning ha each yea he
coun y gene a es a ound 300-400 billion li es o
inasse
(Ca ilho
e al.,
2016). This esidue has been
used o mo e han 50 yea s mos ly o e i iga ion ( e iliza ion + i iga ion) o he suga cane ields
(Ca ilho
e al.,
2016). This appea s o be a i uous cycle, since
inasse
is ich in po assium (Naspolini
e al.
, 2017), a oiding he need o chemical e iliza ion (P ado
e al.,
2016), which in he long un is
bene icial o soil quali y and suga cane p oduc ion yield (Naspolini
e al
., 2017). Bu in eali y, i
con igu es a se ious en i onmen al p oblem since he amoun s o
inasse
p oduced a e a bigge han
he ields can abso b, leading o hei sa u a ion. Addi ionally, he
inasse’s
low pH (3.5 – 5.0) and he
co osi e na u e (Fuess
e al.
, 2017) a e p omo ing undesi able changes o soil composi ion and
physicochemical p ope ies (Ch is o ole i
e al.,
2013), and se ious con amina ion o g ound wa e
(Bo elho
e al.,
2012). These p oblems a e exace ba ed by illici discha ges and inapp op ia e s o age.
The mos s udied possibili ies o
inasse
u iliza ion ela e wi h i s euse in he ene gy equa ion o he
e hanol plan (Naspolini
e al.
, 2017; Rod igues Reis and Hu, 2017). Ne e heless,
inasse
is ich in
mine als and o ganic ma e (Goldembe g
e al.,
2008; Ni aya a dhana
e al.,
2013; Ca ilho
e al.,
2016), a ound 100 – 130 g/L (COD) (Goldembe g
e al.,
2008), and includes suga s, o ganic acids,
e hanol and glyce ol (Ni aya a dhana
e al.,
2013; Ca ilho
e al.,
2016), which sugges
inasse
should
be i o mic obial g ow h. S udies p oposing his possibili y include he cul i a ion o economically
p omising ilamen ous ungi such as he edible
Rhizopus oligospo us
(Ni aya a dhana
e al.,
2013), o
he bio echnology ele an
Aspe gilus o yzae
(Money, 2016; Gmose
e al.,
2018) and
Neu ospo a
in e media
(Nai and Tahe zadeh
,
2016). In he p esen wo k, we explo ed he possibili y o using
inasse
o con olling he p oli e a ion o
M. pe niciosa
.
Th ee di e en
inasse
s we e es ed agains wo s ains o he ungus o igina ing om B azil
ia
a c edi ed
in e na ional cul u e collec ion. The ungi did no g ow on
inasse
. Ins ead, bo h died upon imme sing o
sp aying wi h wo o hem, al hough a a ying ime/dosage, and we e inhibi ed om p oli e a ing by he
hi d. One o he h ee es ed
inasses
inhibi ed he g ow h o he ungi. This killing e ec o
inasse
was
no sha ed by ano he gene ically dis an phy opa hogenic ungus sugges ing speci ici y. Resul s sugges
ha using
inasse
o e i iga ion o cacao plan a ions could, in ime, clean he esiden ungus inoculum.
I no e adica ing i , i could a leas educe he disease o manageable le els.

125
MATERIALS AND METHODS
Monilioph ho a pe niciosa
s ains CBS 441.80 and 442.80, and
Colle o ichum gloeospo ioides
CBS
100471 we e pu chased om CBS-KNAW Collec ions, The Ne he lands. They we e main ained a 4 °C
on MEA (20 g/L mal ex ac w/ 20 g/L aga ), and cul i a ed in he same medium a 30 °C.
Vinasse
was
ob ained om Fe men ec Lda (h ps://www. e men ec.com.b /), and o igina ed om h ee di e en
bioe hanol p oducing plan s in he S a e o São Paulo, B azil: Usina Al a Mogiana
(h p://www.al amogiana.com.b /), Usina Ba a ais (h p://www.usinaba a ais.com.b /) and Usina da
Ped a (h ps://www.ped aag oindus ial.com.b /).
Vinasse
was s o ed a 4 °C. P io o u iliza ion i was
cen i uged o 30 min a 13.000 x g and 4°C o elimina e pa icles in suspension, and au ocla ed (121
ºC, 1 a m, 20 min). G ow h assays we e pe o med inocula ing 20 mL o
inasse
in a glass ube (Ø 3 cm;
13 cm heigh ) wi h a one-week ME-g own ungus aga plug o app oxima ely 0.8x0.8 cm. Tubes we e
incuba ed a 30 ºC and 200 pm o bi al shaking. Cul u es iden ically inocula ed and incuba ed in liquid
ME medium we e used as con ol. G ow h was assessed a e 10 days, decan ing he cul u e supe na an
and checking o mycelium de elopmen . The pu a i e iabili y o any emaining ungal cells was assessed
incuba ing his aga plug in MEA a 30°C o 1 week. These assays we e epea ed in he same manne ,
using
inasse
supplemen ed wi h 2% o mal ex ac as a supplemen a y nu ien sou ce. Dea h along
incuba ion ime in
inasse
was assessed using a ully-g own ungal cul u e p e iously g own in liquid ME
medium in a glass ube (un il he o ma ion o a globula mycelia), which was hen ans e ed o a glass
ube con aining 20 mL o
inasse
, unde he same condi ions desc ibed abo e. Each sample/incuba ion
ime co esponding o an independen ube iden ically inocula ed om ully-g own ungal cul u es. The
incuba ion pe iod a ied be ween 1 and 10 days (T1 o T10), and a inc easing ime poin s, he mycelia
was aken om he
inasse
, gen ly washed wi h s e ile wa e and i s iabili y assayed in MEA a 30°C o
1 week. The dea h-inducing e ec wi hou imme sing he cells in
inasse
was pe o med by sp aying
inasse
on a MEA pla e con aining ully g own mycelia. The assay was pe o med du ing 10 days, applying
a single sp ay a T0, o epea ing i once each 24h. The pla es we e hen incuba ed a 30°C o 3 days.
All assays we e pe o med a leas in h ee independen eplica es (n ≥ 3).
126
RESULTS AND DISCUSSION
The
inasse
used in his wo k o igina ed om h ee bioe hanol plan s om he S a e o São Paulo in
B azil, all o which iden ically p oducing bioe hanol om a suga cane juice e men a ion p ocess.
Acco ding o he li e a u e, he composi ion and physical p ope ies o
inasses
do no di e signi ican ly
(e.g. Goldembe g
e al.,
2008; Ni aya a dhana
e al.,
2013). The h ee
inasses
used in his wo k ha e
±6 % solids in suspension (mos ly ashes), pH ≈ 4.5, 0.115 ± 0.060 % (w/ ) suga s and 0.9 % (w/ )
o ganic acids (in o ma ion kindly supplied by Fe men ec, Lda (h ps://www. e men ec.com.b /), alues
ha all wi hin published da a.
The pH o
inasse
is adequa e o suppo he g ow h o
M. pe niciosa
which op imal g ow h occu s a pH
be ween 4.5 and 5.5 (ou unpublished esul s), and he p esence o ca bon sou ce ( o alling ±0.5% (w/ ))
should allow some ungal mul iplica ion. The e o e,
inasse
was cen i uged, o emo e he solids in
suspension, and he emaining liquid ac ion was s e ilized and used o inocula e he wo
Monilioph ho a
pe niciosa
s ains. A e 10 days (Figu e 1A) he e was no isible g ow h o ei he ungus.
Figu e 1.
Two
M. pe niciosa
s ains o igina ing om con amina ed cacao ui s and ees in B azil we e g own in
unsupplemen ed
inasse
o igina ing om h ee B azilian bioe hanol plan s. (A) Resul s we e sco ed a e a 10 days incuba ion
a 30°C, be o e and a e decan ing he
inasse
showing he inoculum aga plug. Con ol g ow h in iden ical pe iod and
empe a u e in MEA showing he ungal biomass ball. (B) The plugs om (A) we e inocula ed in MEA solid medium. Only he
plugs o igina ing om he incuba ion in
inasse
om Ped a showed mycelia g ow h.
127
Mic oscopic inspec ion con i med he absence o hyphae associa ed wi h he aga plugs used as inocula.
To check whe he he e we e s ill esidual iable mycelia, he ungal plugs we e aken om he
inasse
(Figu e 1A), e-inocula ed in solid MEA medium and incuba ed o a u he 10 days a 30°C. As can be
seen in Figu e 1B, he plugs om he
inasses
o Al a Mogiana and Ba a ais did no de elop any g ow h,
sugges ing he ungus was killed. The ungal plug aken om he
inasse
om Ped a de eloped mycelia,
showing ha i did no exe a killing e ec , bu only a s ong inhibi ion o ungal g ow h.
Conside ing ha he ca bon sou ce p esen in
inasse
migh no be su icien o allow he ungal o de elop
gene ously, and/o ha he dea h o mycelia could occu a such di e en speed ha he 10 days con ac
wi h he
inasse
would no be enough o de ec dea h o he mycelia, wo di e en assays we e pe o med.
The i s consis ed in inc easing he amoun o ca bon sou ce o suppo su i al and g ow h in
inasse
by adding mal ex ac 2% (w/ ) and epea ing he incuba ion in he same condi ions used in he p e ious
assay. Resul s we e iden ical o he p e ious ones (no shown), indica ing ha he g ow h inhibi ion
obse ed when inocula ing he ungal plugs in
inasse
is no ela ed wi h nu ien dep i a ion. The second
app oach aimed o es ablish how as
M. pe niciosa
was dying on
inasse
. The assay consis ed in ha ing
ME-g own mycelia and eplacing ME wi h inasse (Figu e 2A). A inc easing ime poin s, he mycelia we e
aken om he
inasse
and hei iabili y was assayed in solid MEA medium as be o e. This was done
using he
inasse
o igina ing om Al a Mogiana plan , o ha ing p o ed able o kill he wo ungal s ains.
Resul s showed ha he comple e dea h o he mycelia om he wo ungal s ains indeed occu ed when
he ungi we e imme sed in
inasse
, bu a di e en imes. The s ain CBS 441.80 los ull iabili y
be ween day 6 and day 7 (Figu e 2B), while he s ain CBS 442.80 los ull iabili y as e , be ween day
2 and day 3 (Figu e 2B).
Vinasse
has hus a di e en ime/dosage e ec on he wo s ains o
M.
pe niciosa
, which mus be conside ed o he pu a i e applica ion in he ield.
In o de o check whe he he dea h-inducing e ec could be ob ained wi hou he need o imme se he
mycelium in
inasse
, his was used o sp ay MEA ully g own mycelia. The sp ay was applied once e e y
24h o 10 days and he e olu ion o he mycelium was pho og aphed. (Figu e 3). This ime he h ee
inasses
we e used. Mycelium p og essi ely sh unk, becoming da ke colou ed.
Vinasses
om he
di e en plan s had di e en ime/dosage e ec . The
inasse
om Ba a ais killed he wo s ains as e
han he one om Al a Mogiana, and Ped a’s p esen ed he weake inhibi o y e ec . Mo eo e , i was
also possible o e i y ha each
inasse
had a di e en ime-e ec on each o he
M. pe niciosa
s ains,
being CBS 442.80 mo e sensi i e since he mycelium disappea ed be ween days 5 and 10 (excep when
using he Ped a plan
inasse
).
128
Figu e 2.
(A) Scheme o he p ocedu e used o assay he e ec o
inasse
on he iabili y o he
M. pe niciosa
along ime. (B)
Rep esen a i e esul s o he u ning poin in he loss o iabili y which occu s a di e en ime poin s o each ungal s ain.
The con ols show he g ow h o he mycelium ball agmen ed, which occu s whene e he inoculum is la ge.
129
Figu e 3. Chosen examples o he e ec o sp aying
inasse
on he op o ully-g own mycelia on MEA once e e y 24h.
A ows indica e sh unk esidual mycelium a e 7 applica ions o
inasse
.
These esul s a e consis en wi h he ones ob ained by imme sing mycelium in
inasse
. Resul s u he
indica e ha he sp aying has o be epea ed o e ime in o de o obse e he desi ed e ec , co obo a ing
he hypo hesis ha he con inui y o he ea men could con ibu e o clean he ield o hidden mycelium
o e ime.
Finally, as an a emp o unde s and whe he he abili y o
inasse
o kill ilamen ous ungi was speci ic
o
M. pe niciosa
and/o he co esponden axa, he assays o ungal g ow h in
inasse
o 10 days we e
epea ed using ano he phy opa hogenic ungus,
Colle o ichum gloeospo ioides
(Wei e al., 2012). This
is one o he causa i e agen s o oli e an h acnose, a disease causing se ious economic losses in he
Medi e anean egion (Talhinhas e al., 2018) and i was chosen because i is a e y esilien ungal

130
species. Resul s (Figu e 4) showed ha
C. gloeospo ioides
was able o g ow wi hou he need o nu ien
supplemen a ion, ully occupying he olume o
inasse
wi h mycelium which hus became al oge he a
ha d-solid mass. These esul s indica e ha he abili y o
inasse
o kill
M. pe niciosa
does no co espond
o a gene alized an i ungal ac i i y, and he e o e depends on some speci ic ai o
M. pe niciosa
biology.
Vinasse
chemical composi ion is e y complex (Rod igues Reis and Hu, 2017). Fu u e app oaches migh
allow he iden i ica ion o he compounds o chemical g oups in
inasse
ha a e ac i e agains
M.
pe niciosa
, e en ually enabling he c ea ion o an enginee ed simple and mo e e icien solu ion which
conside s he en i onmen al e ec s in he long un. Mo eo e , he e alua ion o he po en ial spec um
o
inasse
applica ion as a speci ic ungicide will ha e o be add essed expe imen ally. In pa icula , he e
is one possibili y ha s ands ou , which is ha
inasse
migh a ec he p oli e a ion o
M. o e i
(Ba bosa
e al.,
2018)
,
ano he phy opa hogenic ilamen ous ungus gene ically e y close o
M. pe niciosa,
also
causing a se e e and economically h ea ening disease in cacao ui s known as
moniliasis
o os y pod
o (Bailey
e al.,
2018).
Figu e 4.
C. gloeospo ioides
g own in unsupplemen ed
inasse
o igina ing om h ee di e en bioe hanol plan s om
B azil. Inoculum was done using a ully-g own mycelia aga plug as used o
M. pe niciosa
. Resul s we e sco ed a e a 10
days incuba ion a 30°C (1), and a e decan ing he emaining
inasse
showing he ungal mycelium ha was o med (2).
Con ol g ow h was pe o med in MEA in iden ical pe iod and empe a u e.
C. gloeospo ioides
did no o m a mycelium
ball, ins ead i sp ead e en ually illing he ull liquid olume a ailable.
131
CONCLUSIONS
Vinasse
is he main was e p oduc om suga cane bioe hanol p oduc ion p ocess. I has a e y high
e iliza ion abili y, bu i applied in excess i becomes a c i ical pollu ion p oblem o soils and
g oundwa e . Many solu ions o
inasse
disposal ha e been sugges ed in li e a u e, mainly
bio echnological applica ions, including he g ow h o some economically in e es ing mic oo ganisms o
ae obic/anae obic diges ion. Addi ionally, many sugges ions in he li e a u e ega d solu ions ha
p omo e he educ ion o he amoun o
inasse
p oduced pe li e o e hanol, i s concen a ion, clea ing
o neu aliza ion. Fo now, he mos cos -e ec i e applica ion o
inasse
keeps being e i iga ion, in ime
imp o ing soil quali y and c op p oduc i i y, p o ided i is done ca e ully and esponsibly, gua an eeing
he con ol o e all he en i onmen al implica ions.
Resul s clea ly show he po en ial o
inasse
o con ol he ilamen ous ungus
M. pe niciosa
, cacao
Wi ches’ B oom Disease
causa i e agen . Conside ing he huge amoun s o
inasse
p oduced in B azil
e e y yea , and he ac ha his coun y was he mos a ec ed by he disease, he u iliza ion o
inasse
o i s con ainmen is an exp essi e possibili y. The e may be logis ic and cos p oblems hinde ing he
u iliza ion o
inasse
in he sho e m de i ing om he dis ances sepa a ing he bioe hanol plan s om
he cacao ields, associa ed wi h his was e physicochemical p ope ies, in pa icula i s pH and co osi e
na u e. Ne e heless, hese p oblems should in ime be echnically o e come in iew o he ad an age o
con ibu ing o sol e wo se ious and u gen p oblems wi h a single p ocedu e: in oducing a new
al e na i e o he disposal o high amoun s o
inasse
, educing he se ious dange o he en i onmen
and g oundwa e o unlaw ul discha ges and p eca ious s o age, and he con ainmen o cacao
Wi ches’
B oom Disease
. Toge he , he p ocedu e should impac posi i ely in he o e all economical equa ion and
con ibu e signi ican ly o he socio-economic eco e y o he egions o B azil ha mos su e ed wi h he
c isis caused by cacao p oduc ion all-ou in he las decades.
132
ACKNOWLEDGEMENTS
Funding: This wo k was suppo ed by he s a egic p og amme UID/BIA/04050/2013 (POCI-01-0145-FEDER-
007569) unded by na ional unds h ough he FCT I.P. and by he ERDF h ough he COMPETE2020 - P og ama
Ope acional Compe i i idade e In e nacionalização (POCI), and he p ojec EcoAg iFood (NORTE-01-0145-FEDER-
000009) om No e Po ugal Regional Ope a ional P og amme (NORTE 2020) unde he PORTUGAL 2020
Pa ne ship Ag eemen h ough he Eu opean Regional De elopmen Fund (ERDF). PF and MA-R a e PhD s uden s
o he Doc o al P og ammes in Applied and En i onmen al Mic obiology (DP_AEM), (FCT g an
PD/BD/113810/2015) and in Molecula and En i onmen al Biology (PDBMA), (FCT g an
PD/BD/145354/2019), espec i ely.
The images o
Wi ches’ B oom Disease
and cacao ee and ui used in he G aphical Abs ac we e ob ained a
h ps://www. o es yimages.o g/: c edi by Sco Baue , USDA Ag icul u al Resea ch Se ice, Bugwood.o g,
licensed unde a C ea i e Commons A ibu ion 3.0 License.
133
SUPPLEMENTARY MATERIAL
Figu e S1. G aphical abs ac .