(cid:38)(cid:85)(cid:82)(cid:83) (cid:51)(cid:85)(cid:82)(cid:87)(cid:72)(cid:70)(cid:87)(cid:76)(cid:82)(cid:81) (cid:20)(cid:23)(cid:24) (cid:11)(cid:21)(cid:19)(cid:21)(cid:20)(cid:12) (cid:20)(cid:19)(cid:24)(cid:25)(cid:20)(cid:27)
`
`Contents!lists!available!at!ScienceDirect!
`
`Crop!Protection!
`
`journal homepage: www.elsevier.com/locate/cropro
`
`Multiple!resistance!to!DMI,!QoI!and!SDHI!fungicides!in!"eld!isolates!of!
`Phakopsora!pachyrhizi!
`
`Mˆonica!Anghinoni!M(cid:252)ller a,!Gerd!Stammler b,!Louise!Larissa!May!De!Mio a,*!
`
`a Universidade!Federal!do!Paran´a,!Rua!dos!Funcion´arios!1540,!80035-050,!Curitiba,!Brazil!
`b BASF!SE,!Speyerer!Straße!2,!67117,!Limburgerhof,!Germany!!!
`
`A R T I C L E !I N F O ! !
`
`A B S T R A C T ! !
`
`Keywords:!
`Asian!soybean!rust!
`Demethylation-inhibitors!
`Quinone-outside-inhibitors!
`Succinate-dehydrogenase!inhibitors!
`Fungicide!resistance!
`
`Asian!soybean!rust!(ASR),!caused!by!the!fungus!Phakopsora!pachyrhizi!Syd! &! P.!Syd,!is!the!main!disease!that!
`affects!soybean!crop!in!South!America.!Disease!control!is!based!on!the!use!of!site-speci"c!fungicides!(QoIs,!DMIs!
`and!SDHIs),!multisite!fungicides!and!host-free!period.!Continuous!and!intensive!use!of!fungicides!has!selected!
`isolates!of!P.!pachyrhizi,!with!reduced!sensitivity!to!DMIs,!QoIs!and!SDHIs.!This!work!includes!sensitivity!studies!
`for!prothioconazole!and!SDHIs!and!genetic!analysis!of!target!site!mutations!in!P.!pachyrhizi!related!to!sensitivity!
`reduction!to!DMIs,!QoIs!and!SDHIs!collected!from!different!regions!of!soybean!production!areas.!Different!CYP51!
`genotypes! with! distinct! point! mutations! known! to! in#uence! DMI! sensitivity! are! present! in! Brazilian! isolates.!
`Combined!mutation!F120L!+ Y131H!on!CYP51!gene!was!the!most!frequent!among!Brazilian!isolates.!Mutant!
`isolates!showed!higher!EC50!values!to!the!DMI!prothioconazole!compared!to!wild!type!isolates.!Regarding!QoI,!
`almost!all!Brazilian!isolates!carried!the!mutation!F129L!in!the!CYTB!gene.!Wild!type!of!the!SdhC!gene!was!the!
`most!frequent!genotype!to!SDHI!fungicides,!but!the!mutation!leading!to!the!I86F!amino!acid!exchange!has!also!
`been!detected.!Such!isolates!showed!higher!EC50!values!to!the!SDHIs!bixafen,!benzovindi#upyr!and!#uxapyroxad!
`compared!to!wild!type!isolates.!The!most!frequent!genotype!in!our!collection!presented!target!site!mutations!in!
`the!CYP51!and!CYTB!genes.!A!monouredinial!isolate!with!mutations!in!all!three!target!genes!was!detected!and!is!
`here!described!for!the!"rst!time.!Its!current!and!further!spread,!and!the!impact!on!"eld!performance!of!fungicides!
`with!these!modes!of!action!needs!further!evaluation.!!!
`
`1. Introduction!
`
`Soybean!is!one!of!the!most!produced!crop!in!the!world,!and!Brazil!is!
`responsible!for!a!third!of!the!global!production,!being!the!second!largest!
`producer!of!this!commodity!(CONAB,!2018;!USDA,!2018).!It!is!a!signif-
`icant!source!of!protein!for!livestock!feeding!and!is!also!used!for!indus-
`trial!purposes!(Hirakuri!and!Lazzaroto,!2014;!USB,!2017).!
`Asian! soybean! rust! (ASR)! is! the! main! soybean! disease! in! South!
`America!since!its!identi"cation!in!2001!(Yorinori!et!al.,!2005),!and!it!is!
`caused!by!the!fungus!Phakopsora!pachyrhizi!Syd!&!P.!Syd.!Its!infection!in!
`the!leaves!can!lead!to!complete!defoliation!and!yield!losses!up!to!80%!
`(Dalla!Lana!et!al.,!2015;!Hirano!et!al.,!2010;!Yang!et!al.,!1991).!
`Chemical!control!is!one!of!the!main!methods!to!manage!pathogens!
`and! it! is! performed! by! fungicide! application! that! aims! to! prevent!
`entrance!of!the!pathogen!and!the!secondary!cycle!of!diseases!(Mayton!
`et!al.,!2001;!Van!Der!Plank,!1963).!Another!very!important!method!is!
`the!establishment!of!the!host-free!period,!when!no!soybeans!are!grown!
`
`to!reduce!the!inoculum!potential!between!growing!seasons.!This!leads!to!
`a!delay!of!disease!onset!and!a!reduced!selection!pressure!by!the!various!
`modes!of!action!and!is!therefore!an!effective!and!important!tool!in!dis-
`ease!and!resistance!management!(Martin!Neto!and!Farias,!2014).!
`Site-speci"c! fungicides! belonging! to! the! demethylation! inhibitors!
`(DMIs)!and!quinone!outside!inhibitors!(QoIs)!effectively!controlled!the!
`disease!in!the!crop!for!years.!However,!ef"cacy!of!fungicides!with!these!
`modes!of!action!has!decreased!over!time!(Godoy!et!al.,!2014)!and!the!
`search!for!new!molecules!was!intensi"ed.!Succinate!dehydrogenase!in-
`hibitors! (SDHIs)! fungicides! were! incorporated! in! the! management! of!
`ASR,!with!the!release!and!registration!of!#uxapyroxad!in!2013!and!of!
`benzovindi#upyr!in!2014.!However,!a!reduction!of!SDHIs!ef"cacy!in!the!
`"eld!was!observed!and!reported!by!the!annual!monitoring!of!Embrapa!
`2–3!seasons!after!market!introduction!(Cl´audia!V.!Godoy!et!al.,!2017a).!
`Multisite!fungicides!were!also!incorporated!in!the!management!of!ASR,!
`in!order!to!control!the!disease!and!limit!the!increase!of!resistant!strains!
`within!populations!to!site-speci"c!fungicides!(FRAC,!2018;!Cl´audia!V.!
`
`* Corresponding!author.!
`E-mail!addresses:!maydemio@ufpr.br,!maydemio@gmail.com!(L.L.!May!De!Mio).!!
`
`https://doi.org/10.1016/j.cropro.2021.105618!
`Received!24!April!2019;!Received!in!revised!form!11!March!2021;!Accepted!13!March!2021!!!
`(cid:36)(cid:89)(cid:68)(cid:76)(cid:79)(cid:68)(cid:69)(cid:79)(cid:72) (cid:82)(cid:81)(cid:79)(cid:76)(cid:81)(cid:72) (cid:20)(cid:28) (cid:48)(cid:68)(cid:85)(cid:70)(cid:75) (cid:21)(cid:19)(cid:21)(cid:20)
`(cid:19)(cid:21)(cid:25)(cid:20)(cid:16)(cid:21)(cid:20)(cid:28)(cid:23)(cid:18)(cid:139) (cid:21)(cid:19)(cid:21)(cid:20) (cid:40)(cid:79)(cid:86)(cid:72)(cid:89)(cid:76)(cid:72)(cid:85) (cid:47)(cid:87)(cid:71)(cid:17) (cid:36)(cid:79)(cid:79) (cid:85)(cid:76)(cid:74)(cid:75)(cid:87)(cid:86) (cid:85)(cid:72)(cid:86)(cid:72)(cid:85)(cid:89)(cid:72)(cid:71)(cid:17)
`
`

`

`(cid:38)(cid:85)(cid:82)(cid:83) (cid:51)(cid:85)(cid:82)(cid:87)(cid:72)(cid:70)(cid:87)(cid:76)(cid:82)(cid:81) (cid:20)(cid:23)(cid:24) (cid:11)(cid:21)(cid:19)(cid:21)(cid:20)(cid:12) (cid:20)(cid:19)(cid:24)(cid:25)(cid:20)(cid:27)
`M.A.!M(cid:252)ller!et!al.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
`
`Godoy!et!al.,!2017b).!
`Decrease! of! fungicide! ef"cacy! in! the! "eld! can! be! conferred! by!
`different! mechanisms,! as! ef#ux,! overexpression,! detoxi"cation,! and!
`target! site! mutations! whereas! the! main! resistance! mechanisms! in!
`phytopathogenic! fungi! are! point! mutations! in! fungicide! target! genes!
`(Brent!and!Hollomon,!2007).!The!DMI!target!site!is!the!14-α demethy-
`lase! in! the! ergosterol! biosynthesis! pathway,! which! is! encoded! by! the!
`CYP51! gene! (Gisi! et! al.,! 2000).! The! target! protein! of! the! QoIs,! cyto-
`chrome! bc1-ubiquinol! oxidase,! is! encoded! by! the! mitochondrial! gene!
`CYTB!(Gisi!et!al.,!2002;!Grasso!et!al.,!2006).!The!SDHI!target!site!is!the!
`succinate! dehydrogenase! (SDH)! complex! in! the! respiratory! chain!
`(Yankovskaya! et! al.,! 2003).! This! enzyme! complex! is! codi"ed! by! Sdh!
`gene,!composed!by!subunits!A,!B,!C!and!D!(Gl¨attli!et!al.,!2009;!Stammler!
`et!al,!2008,!2011).!
`For!P.!pachyrhizi!there!are!reports!of!isolates!with!point!mutations!in!
`the!above!mentioned!genes!conferring!reduction!of!sensitivity!to!DMIs!
`(Schmitz! et!al.,! 2014),!QoIs!(Klosowski! et!al,!2016b,! 2018)! or! SDHIs!
`(Sim˜oes!et!al.,!2018)!independently.!Multiple!resistance!for!DMI!and!QoI!
`in! the! same! isolate! was! already! described! in! monouredinial! isolates,!
`collected!and!established!from!"elds!before!the!launch!of!SDHIs!(Klo-
`sowski!et!al.,!2018).!However,!there!are!no!reports!on!combined!SDHI,!
`DMI!and!QoI!resistance!in!individual!isolates!so!far.!In!Brazil,!with!the!
`high!selection!pressure!of!these!fungicides!in!soybean!production!areas,!
`isolates!may!present!multiple!resistance!to!the!three!main!groups!used!to!
`control!ASR.!
`In!this!work!it!was!evaluated!if!isolates!with!multiple!resistance!to!
`SDHIs,! DMIs! and! QoIs! can! already! be! detected! in! Brazil.! Therefore,!
`sensitivity!studies!were!performed,!and!genes!were!analysed!for!known!
`target!site!mutations!in!isolates!from!four!Brazilian!states.!
`
`2. Materials!and!methods!
`
`2.1. Phakopsora!pachyrhizi!isolates!
`
`Samples!of!P.!pachyrhizi!isolates!were!collected!from!soybean!"elds!
`from!different!locations!(Cascavel!and! Ponta!Grossa! municipalities!in!
`Paran´a!State,!Santo!Antˆonio!da!Posse!in!S˜ao!Paulo!State,!Diamantino!in!
`Mato!Grosso!State,!Vilhena!in!Rondonia!State)!and!seasons!(2013/14,!
`2014/15,! 2015/16! and! 2016/17)! (Fig.! 1).! Twenty-one! isolates! of!
`P.!pachyrhizi!collected!from!soybean!leaves!infected!by!ASR!were!used!in!
`
`N
`
`~~
`
`b p
`
`0
`
`\
`I
`
`en
`b
`b g
`
`67'30'0"W
`
`55°0'0"W
`
`42"30'0"W
`
`Legend
`Origin Of Isolates
`-~
`Cascavel
`£ Diamantino
`
`* PontaGrossa
`
`e SantoAntoniode rosse
`1:::> V~hen1;1
`Region s Of Brazil
`0
`Nml h RegionSta tes
`
`0Nor1 h EastR19ionStalH
`
`•
`
`O
`
`.
`
`Mid West Reg ion States
`
`southEost Rog ionSto tcs
`
`SoultlRegioflStetes
`
`Projection
`Universal Transverse Mercator
`Geog raphical Coord inate System
`Horizontal Datum SIRGAS 2000
`
`Source Data
`Politica l Division and Borders
`MMA , 2017; DIVA-GIS, 2017
`
`Organization: Felipe E. Jauch
`and Monica A. Muller (2018)
`
`the!experiments,!of!which!7!were!named!“population” and!14!“mono-
`uredinial!isolates”,!according!to!the!collection!methodology.!Population!
`were!composed!of!spores!collected!directly!from!"eld!soybean!leaves!
`infected!with!ASR!of!different!locations!with!the!aid!of!a!brush.!Mono-
`uredinial!isolates!were!composed!of!spores!collected!from!the!"eld!but!
`prepared!from!one!single!uredinia.!So,!from!this!single!uredinia!each!
`monouredinial! isolate! was! transferred! several! times! to! new! soybean!
`leaves!until!reaching!the!necessary!amount!to!perform!the!experiments.!
`In!order!to!obtain!an!uredinia!isolated,!spores!collected!from!the!"eld!
`were! inoculated! on! healthy! soybean! leaves! from! plants! grown! in! the!
`greenhouse.!After!15!days!spores!that!grew!in!one!uredinia!on!the!de-
`tached! leaf! were! collected! with! a! sterile! needle! and! passed! to! a! new!
`detached!leaf!for!the!establishment!of!the!monouredinial!isolate.!The!
`spores! produced! from! the! monouredinial! isolate! are! assumed! to! be!
`genetically!identical!since!most!probably!a!single!spore!gave!rise!to!the!
`collected!uredinia.!
`Monouredinial!isolates!and!populations!were!transferred!to!healthy!
`detached!leaves!every!two!weeks!so!the!isolates!were!maintained!and!
`multiplied!for!the!experiments!for!EC50!detection!and!for!genotyping!of!
`the!target!genes.!
`
`2.2. Sensitivity!test/EC50! assays/resistance!factor!
`
`Detached! leaves! assays! were! performed! to! calculate! the! effective!
`concentration!to!inhibit!50%!of!the!pathogen!activity!(EC50),!for!the!DMI!
`prothioconazole! and! the! SDHIs! bixafen,! benzovindi#upyr! and! #ux-
`apyroxad! according! to! the! leaf! methodology! proposed! by! FRAC!
`(Fungicide! Resistance! Action! Committee)! (Scherb! and! Mehl,! 2006).!
`Resistance!factors!(RFs)!were!calculated!by!dividing!the!EC50!value!for!
`each!isolate!with!around!50%!of!I86F!mutation!by!the!mean!EC50!value!
`of! all! the! sensitive! ones! (WT).! Commercially! available! formulated!
`products!were!used!for!the!DMI!prothioconazole!(Prolinefi! Bayer!AG,!
`Leverkusen,!Germany)!and!for!the!SDHIs!benzovindi#upyr!(Solatenolfi,!
`Syngenta! AG,! Basel,! Switzerland),! bixafen! (Thorefi! Bayer! AG,! Lev-
`erkusen,! Germany)! and! #uxapyroxad! (Xemiumfi,! BASF! SE,! Ludwig-
`shafen,!Germany).!
`Approximately!7!days!old!plants!of!the!soybean!cultivar!(cid:145)ES!Mentor’
`(Saatbau,! Linz,! Austria),! grown! in! the! greenhouse,! were! treated! with!
`different!fungicide!concentrations!(DMI:!0,!0.03,!0.1,!0.3,!1,!3,!10,!30!μg!
`mLࢤ1;!SDHIs:!0,!0.1,!0.3,!1,!3,!10,!30,!100!μg!mLࢤ1).!The!plants!were!
`treated!in!a!spray!chamber!and!the!leaves!were!evenly!covered!with!the!
`fungicide! solutions.! Afterwards! the! plants! were! placed! for! 24! h! in!
`growth!chambers!at!23!◦C!for!the!super"cial!drying!of!the!leaves.!Then,!
`each!unifoliolate!leaf!was!removed!and!placed!in!disposable!petri!dishes!
`containing!water!agar!medium!(1%)!with!streptomycin!(30!mg!Lࢤ1)!and!
`kinetin!(0.2!mg!Lࢤ1).!For!each!concentration!of!the!fungicide!there!were!
`three!Petri!dishes!containing!an!unifoliolate!leaf,!each!plate!represented!
`a!repetition.!
`A!spore!suspension!of!each!P.!pachyrhizi!isolate!was!inoculated!in!the!
`abaxial!side!of!the!unifoliolate!leaf.!The!spore!suspension!was!prepared!
`in! distilled! water! + Tween! 20! (0.01%)! with! 105! spores! mLࢤ1.! Petri!
`dishes!containing!treated!and!inoculated!leaves!were!incubated!for!12!h!
`in!the!dark!at!23!◦C!and!thereafter!for!15!days!at!the!same!temperature!
`with! 12! h! photoperiod.! Then! disease! severity! was! evaluated! by! dia-
`grammatic!scale!(Godoy!et!al.,!2006)!with!six!severity!levels,!varying!
`from!0.6!to!78.5%!of!the!leaf!area!with!lesions.!EC50!was!estimated!from!
`severity!data!at!different!fungicide!concentrations.!
`
`2.3. Point!mutation!analysis!
`
`Fig.!1. Brazilian!map!municipalities!(origin!of!the!isolates)!and!states!where!
`leaves! samples! infected! with! Asian-soybean! rust! were! collected.! Brazilian!
`States:! RO! – Rondonia,! MT! – Mato! Grosso,! SP! – Sao! Paulo! and! PR-! Parana.!
`Municipalities:! Cascavel.! Ponta! Grossa,! Santo! Antonio! da! Posse,! Diamantina!
`e!Vilhena.!
`
`2.3.1. DNA!extraction!
`P.!pachyrhizi!DNA!was!extracted!from!spores!using!the!NucleoSpin!
`DNA!Plant!II!extraction!Kit!(Macherey-Nagel!GmbH!&!Co.!KG,!D(cid:252)ren,!
`Germany)!according!to!the!manufacturer’s!instructions!for!CTAB-based!
`DNA!extraction!(Schmitz!et!al.,!2014).!
`
`(cid:21)
`
`

`

`(cid:38)(cid:85)(cid:82)(cid:83) (cid:51)(cid:85)(cid:82)(cid:87)(cid:72)(cid:70)(cid:87)(cid:76)(cid:82)(cid:81) (cid:20)(cid:23)(cid:24) (cid:11)(cid:21)(cid:19)(cid:21)(cid:20)(cid:12) (cid:20)(cid:19)(cid:24)(cid:25)(cid:20)(cid:27)
`M.A.!M(cid:252)ller!et!al.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
`
`2.3.2. Pyrosequencing!
`The!Pyrosequencing!technique!was!performed!using!PyroMark!Gold!
`96!Reagents!on!a!PSQ!96!MA!machine!(Qiagen,!Hilden,!Germany),!for!
`quantitative!detection!of!F129L!mutations!in!the!CYTB!gene!(Klosowski!
`et!al.,!2016b)!and!for!the!mutations!F120L,!Y131!H/F,!K142R,!I145F,!
`I475T! in! the! CYP51! gene! (Schmitz! et! al.,! 2014).! This! procedure! was!
`performed! in! the! Fungicide! Resistance! Laboratory! of! BASF! SE! (Lim-
`burgerhof,!Germany).!
`
`2.3.3. Real!time!qPCR!
`A!speci"c!quantitative!real-time!PCR!was!performed!for!P.!pachyrhizi!
`for!quantitative!detection!of!the!C–I86F!mutation!in!the!(cid:145)c’ subunit!of!the!
`Sdh!gene.!Takyon!qPCR!MasterMix!Plus!dTTP!No!Rox!reagents!(Euro-
`gentec,! K¨oln,! Germany)! were! used! for! the! ampli"cation,! and! the! re-
`actions! were! performed! on! a! Rotor-Gene! Q! cycler! (Qiagen,! Hilden,!
`Germany).!The!following!conditions!were!applied:!5!min!at!95!◦C!and!40!
`cycles!at!95!◦C!for!15!s!and!at!60!◦C!for!1!min!(Sim˜oes!et!al.,!2017).!The!
`relative! allele! frequencies! were! calculated! based! on! the! method!
`described!by!Germer!et!al.!(2000).!
`
`3. Results!
`
`3.1. Sensitivity!of!P.!pachyrhizi!to!DMI!fungicides!and!CYP51!gene!
`analysis!
`
`The! EC50! values! for! the! DMI! prothioconazole! ranged! from! 0.8! to!
`29.7!μg!mLࢤ1! for!the!21!P.!pachyrhizi!isolates!analysed!(Table!1).!The!
`monouredinial!isolates!of!the!Mid!West!region!of!Brazil!presented!higher!
`
`Table!1!
`Sensitivity!of!monouredinial!isolates!and!population!of!Phakopsora!pachyrhizi!to!
`prothioconazole!and!respective!mutations!in!CYP51!gene.!!
`
`Isolate/!
`Region!
`
`Season!
`
`EC50!(μg!mLࢤ1)!
`
`Mutations!in!CYP51!gene!(%)!
`
`Prothioconazole!
`
`F120L!
`
`Y131H!
`
`Y131F!
`
`I475T!
`
`values!of!EC50!than!isolates!from!the!other!regions.!Most!of!the!isolates!
`showed! mutations! in! the! CYP51! gene,! only! the! reference! population!
`GWH!and!149/17!were!the!only!samples!that!did!not!show!mutations!in!
`the!CYP51!gene.!
`Within! the! CYP51! gene,! genotypes! with! the! combined! mutation!
`(F120L! + Y131H)! were! most! frequent,! corresponding! to! 85%! of! the!
`isolates!analysed.!Monouredinial!isolates!with!this!combined!mutation!
`showed! frequencies! up! to! ~50%! of! the! mutation! F120L! and! Y131H.!
`Other!genotypes!were!also!found:!monouredinial!isolate!191!with!the!
`Y131F!and!I475T!mutations!and!the!population!named!193!with!F120L,!
`Y131H!and!I475T!mutations.!Isolates!with!mutations!in!the!CYP51!gene!
`had!higher!EC50!values!to!the!DMI!prothioconazole!(2.1–29.7!μg!mLࢤ1)!
`than!the!wild!type!genotypes!(0.8!and!1.5!μg!mLࢤ1).!
`
`3.2. CYTB!gene!analysis!
`
`Nearly! all! monouredinial! isolates! and! population! sampled! from!
`different! seasons! and! Brazilian! regions! showed! very! high! frequencies!
`(up!to!100%)!of!the!F129L!mutation!in!the!CYTB!target!gene!of!QoIs!
`(Table!2).!Only!the!monouredinal!isolates!109!and!191!from!the!south!
`and!southeast!were!wild!type!in!the!CYTB!gene!and!the!population!193!
`from!the!southeast!presented!a!lower!percentage!(58%)!of!the!F129L!
`mutation.!
`
`3.3. Sensitivity!of!P.!pachyrhizi!to!SDHI!fungicides!and!SDH-c!gene!
`analysis!
`
`The!EC50! values!of!P.!pachyrhizi!isolates!to!fungicides!of!the!SDHI!
`group!ranged!from!6.4!to!63.0!μg!mLࢤ1!for!bixafen;!0.2–71.4!μg!mLࢤ1!for!
`benzovindi#upyr;!and!1.6–20.6!for!#uxapyroxad!(Table!2).!The!isolates!
`sensitivity!to!fungicides!varied!without!any!speci"c!pattern!between!the!
`regions!and!seasons!sampled.!
`Among! the! samples! analysed,! two! of! them! (198! and! 203)! were!
`considered!completely!mutated!with!around!50%!I86F,!the!population!
`CV17!presented!11%!of!the!I86F!mutation!and!the!other!18!samples!did!
`not! present! the! mutation! I86F! on! the! SDH-C! gene.! Isolates! with!
`approximately!50%!of!the!mutation!C–I86F!(198,!203)!presented!higher!
`EC50!values!than!the!wild!type!isolates!and!the!population!sample!CV17!
`with!only!11%!I86F!(Fig.!2).!
`The!mean!EC50!for!WT!isolates!was!11.75!μg!mLࢤ1!for!bixafen;!2.31!
`μg!mLࢤ1!for!benzovindi#upyr!and!3.74!μg!mLࢤ1!for!#uxapyroxad,!while!
`for!isolates!with!~50%!I86F!mutation!(198,!203),!the!mean!EC50!values!
`were!47.05!μg!mLࢤ1! for!bixafen;!61.05!μg!mLࢤ1! for!benzovindi#upyr;!
`and!16.0!μg!mLࢤ1!for!#uxapyroxad.!Resistance!factors!for!the!population!
`198!were!5.36;!21.94!and!4.93!and!for!the!monouredinial!isolate!203!
`were!2.64;!30.90!and!5.50!for!bixafen,!benzovindi#upyr!and!#uxapyr-
`oxad,!respectively.!
`The!monouredinial!isolate!203!and!the!populations!198!and!CV17!
`that!presented!the!mutation!I86F!on!the!SdhC!gene!also!presented!mu-
`tations!on!the!genes!CYP51!and!CYTB!(Table!1;!Table!2).!
`
`4. Discussion!
`
`Multiple!selection!of!resistance!to!DMIs,!QoIs!and!SDHIs!fungicide!
`groups! was! detected! in! one! monouredinial! isolate! of! P.! pachyrhizi,!
`collected!in!the!2016/17!season,!in!the!north!region!of!Brazil.!Genotypes!
`with! mutations! in! the! target! genes! of! DMIs! and! QoIs! were! the! most!
`frequent!ones,!representing!76%!of!the!isolates.!Sensitivity!tests!showed!
`a! reduction! of! DMI! sensitivity! in! isolates! with! the! F120L! + Y131H!
`combined! mutation! in! the! CYP51! gene.! Isolates! with! the! SdhC! I86F!
`mutation!were!less!sensitive!to!SDHIs,!however!they!were!present!in!low!
`frequency! within! Brazilian! isolates! (14%)! in! our! study.! On! the! other!
`hand,! the! F129L! mutation,! which! causes! lower! QoI! sensitivity,! was!
`present!in!high!frequency!in!all!isolates!analysed.!
`Since! the! 2009/10! season,! F120L! + Y131H! mutant! isolates! were!
`detected! in! all! soybean! production! areas! in! Brazil! (Klosowski! et! al.,!
`
`7.3!
`14.7!
`11!!!!!
`
`5.9!
`3.9!
`11.9!
`7.2!!!!!
`
`North!(Vilhena!-RO)!
`3.2!
`202!
`16/17!
`11.0!
`203!
`16/17!
`7.1!!!!!
`Average!
`Mid!West!(Diamantino!-MT)!
`161!
`15/16!
`29.7!
`163!
`15/16!
`12.4!
`164!
`15/16!
`20.2!
`165!
`15/16!
`13.2!
`Average!
`18.9!!!!!
`South!(Cascavel!-PR)!
`174!
`16/17!
`CV!17a!
`16/17!
`Average!
`South!(Toledo!-PR)!
`205!
`16/17!
`TL!16a!
`15/16!
`TL!17a!
`16/17!
`Average!
`South!(Ponta!Grossa!– PR)!
`6.8!
`29!
`13/14!
`2.1!
`95!
`13/14!
`8.5!
`104!
`14/15!
`8.5!
`109!
`14/15!
`6.5!!!!!
`Average!
`South!East!(Santo!Antˆonio!de!Posse!– SP)!
`191!
`16/17!
`3.6!
`193a!
`16/17!
`5.6!
`Average!
`4.6!!!!!
`Reference!isolates!
`198a!!
`149/17a!!
`GWHa!!
`138!m3!!
`
`19.9!
`0.8!
`1.5!
`2.8!
`
`42!
`29!
`
`45!
`41!
`45!
`NA!b!
`
`45!
`35!
`
`43!
`45!
`28!
`
`30!
`49!
`25!
`22!
`
`0!
`25!
`
`35!
`0!
`0!
`49!
`
`40!
`28!
`
`47!
`41!
`43!
`NA!
`
`43!
`33!
`
`43!
`45!
`25!
`
`28!
`52!
`31!
`30!
`
`0!
`13!
`
`33!
`0!
`0!
`50!
`
`0!
`0!
`
`0!
`0!
`0!
`NA!
`
`0!
`0!
`
`0!
`0!
`0!
`
`0!
`0!
`0!
`0!
`
`47!
`0!
`
`0!
`0!
`0!
`0!
`
`0!
`0!
`
`0!
`0!
`0!
`NA!
`
`0!
`0!
`
`0!
`0!
`0!
`
`0!
`0!
`0!
`0!
`
`43!
`16!
`
`0!
`0!
`0!
`0!!
`
`a Population.!
`b Not!Analysed!(NA).!
`
`(cid:22)
`
`

`

`(cid:38)(cid:85)(cid:82)(cid:83) (cid:51)(cid:85)(cid:82)(cid:87)(cid:72)(cid:70)(cid:87)(cid:76)(cid:82)(cid:81) (cid:20)(cid:23)(cid:24) (cid:11)(cid:21)(cid:19)(cid:21)(cid:20)(cid:12) (cid:20)(cid:19)(cid:24)(cid:25)(cid:20)(cid:27)
`M.A.!M(cid:252)ller!et!al.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
`
`Table!2!
`Sensitivity! of! monouredinial! isolates! and! population! of! Phakopsora! pachyrhizi! to! fungicides!of! succinate! dehydrogenase!inhibitors! (SDHIs)! group! and! respective!
`mutation!in!two!different!genes.!!
`
`Isolate!
`
`Season!
`
`EC50!of!SDHI!fungicides!(μg!mLࢤ1)!
`
`Mutation!in!SdhC!gene!(%)!
`
`Mutation!in!CYTB!gene!(%)!
`
`Bixafen!
`
`Benzovindi#upyr!
`
`Fluxapyroxad!
`
`16/17!
`16/17!
`
`16/17!
`15/16!
`16/17!
`
`North!(Vilhena!-RO)!
`202!
`203!
`Average!
`Mid!West!(Diamantino!-MT)!!
`161!
`15/16!
`163!
`15/16!
`164!
`15/16!
`165!
`15/16!
`Average!
`South!(Cascavel!-PR)!!
`174!
`16/17!
`CV17a!
`16/17!
`Average!
`South!(Toledo!-PR)!
`205!
`TL!16a!
`TL!17a!
`Average!
`South!(Ponta!Grossa!– PR)!!
`11.2!
`29!
`13/14!
`9.8!
`95!
`13/14!
`11.2!
`104!
`14/15!
`17.6!
`109!
`14/15!
`12.4!
`Average!
`South!East!(Santo!Antˆonio!de!Posse!– SP)!!
`191!
`16/17!
`8.5!
`193a!
`16/17!
`8.8!
`Average!
`8.6!
`Reference!isolates!
`198a!!
`149/17a!!
`138!m3!!
`GWH1!!
`
`11.4!
`31.1!
`21.2!
`
`17.1!
`18.7!
`13.3!
`12.6!
`15.4!
`
`10.2!
`8.1!
`9.1!
`
`3.1!
`8.8!
`10.6!
`7.5!
`
`63.0!
`13.0!
`6.4!
`19.2!
`
`3.1!
`71.4!
`37.2!
`
`3.5!
`2.5!
`3.9!
`1.4!
`2.8!
`
`2.5!
`9.3!
`5.9!
`
`1.4!
`2.5!
`1.5!
`1.8!
`
`2.8!
`0.2!
`3.2!
`3.1!
`2.3!
`
`1.1!
`2.6!
`1.8!
`
`50.7!
`1.7!
`2.5!
`2.1!
`
`3.7!
`20.6!
`12.1!!!
`
`1.6!
`5.6!
`5.8!
`2.9!
`4.0!!!
`
`2.6!
`10.0!
`6.3!!!
`
`1.7!
`4.2!
`2.3!
`2.7!!!
`
`8.4!
`2.1!
`5.1!
`3.2!
`4.7!!!
`
`3.9!
`4.2!
`4.0!!!
`
`11.4!
`3.6!
`1.8!
`4.7!
`
`I86F!
`
`WT!b!
`50!
`
`WT!
`WT!
`WT!
`WT!
`
`WT!
`11!
`
`WT!
`WT!
`WT!
`
`WT!
`WT!
`WT!
`WT!
`
`WT!
`WT!
`
`47!
`WT!
`WT!
`WT!
`
`F129L!
`
`98!
`95!
`
`97!
`98!
`96!
`NA!
`
`94!
`94!
`
`93!
`100!
`94!
`
`96!
`96!
`96!
`WT!
`
`WT!
`58!
`
`52!
`93!
`91!
`99!!
`
`a Population.!
`b Wild!Type!(WT).!
`
`80
`
`•
`
`•
`8
`~
`
`0
`
`0
`
`•
`
`I
`
`WT Isolates I
`l86F isolates
`
`•
`
`•
`
`•
`• 0
`§
`
`,...__
`~
`oJl 40
`2,
`"'
`E c,; >
`u
`"'
`µ;:i
`
`60
`
`20
`
`0
`
`<1)
`
`0
`
`Bixafen
`
`Benzovinditlupyr
`
`Fluxapyroxad
`
`Fungicides
`
`Fig.!2. Sensitivity!of!isolates!of!Phakopsora!pachyrhizi!to!fungicides!of!succinate!
`dehydrogenase! inhibitors! (SDHIs)! group! by! effective! concentration! to! inhibit!
`50%! of! the! pathogen! (EC50)! value,! to! wild! type! (WT)! or!50%! mutant! (I86F)!
`isolates!(n!= 21!isolates!in!total)!in!the!SdhC!gene.!
`
`2018;!Schmitz!et!al.,!2014).!This!spread!of!mutated!isolates!is!the!result!
`of! the! long! time! and! continuous! use! of! different! DMI! fungicides! for!
`control!of!P.!pachyrhizi!since!season!2002/03!(Reis!et!al.,!2018).!With!
`such! a! high! selection! pressure! of! DMI! fungicides! over! the! years,! less!
`
`(cid:23)
`
`sensitive!individuals!emerged!and!remain!present!within!the!population.!
`Isolates!with!mutations!in!the!CYP51!gene!were!less!sensitive!to!the!DMI!
`used!in!our!study,!prothioconazole,!which!is!in!line!with!previous!re-
`ports!on!DMI!adaptation!(Klosowski!et!al.,!2016a;!Schmitz!et!al.,!2014).!
`Not!only!point!mutations!in!the!CYP51!gene,!but!also!overexpression!
`of!this!gene!was!described!for!Blumeriella!jaapii!(Ma!et!al.,!2006),!Mon-
`ilinia!fructicola! (Luo!and! Schnabel,!2008),! Puccinia! triticina!(Stammler!
`et!al.,!2009),!Venturia!inaequalis!(Schnabel!and!Jones,!2001)!and!also!the!
`up-regulation! of! ef#ux-transporter! in! Botrytis! cinerea! (Hayashi! et! al.,!
`2002),!Penicillium!digitatum!(Nakaune!et!al.,!1998),!Mycosphaerella!gra-
`minicola!(Zwiers!et!al.,!2002)!have!been!described!as!resistance!mech-
`anisms! which! reduce! DMI! sensitivity.! For! P.! pachyrhizi,! CYP51!
`overexpression! and! target! site! mutations! (F120L,! Y131H,! Y131F,!
`K142R,!I145F!and!I475T)!were!identi"ed!as!DMI!resistance!mechanisms!
`(Schmitz!et!al.,!2014),!while!no!reports!are!so!far!available!on!a!potential!
`in#uence!of!enhanced!ef#ux!activity.!
`In!previous!studies!all!isolates!of!P.!pachyrhizi!containing!the!F120L!
`+ Y131H! showed! overexpression! of! the! CYP51! gene,! and! those! con-
`taining! the! Y131F! + I145T! did! not! (Schmitz! et! al.,! 2014).! Over-
`expression!of!CYP51!can!also!compensate!for!reduced!enzyme!function!
`as!a!result!of!particular!target!site!alterations!(alone!or!in!combination).!
`This!suggests!that!in!the!present!work,!although!the!overexpression!has!
`not!been!evaluated,!it!may!be!present!in!those!isolates!with!F120L!+
`Y131H!and!may!be!responsible!for!the!stronger!DMI!adaptation!of!some!
`isolates,! such! as! 161! or! 164.! In! the! same! way,! the! isolate! 191,! with!
`Y131F! + I145T,! may! have! presented! higher! sensitivity! for! DMI! pro-
`thioconazole!due!to!the!lack!of!overexpression.!Because!of!the!interac-
`tion!of!target!gene!overexpression!on!DMI!sensitivity,!it!is!not!clear!so!far!
`
`

`

`(cid:38)(cid:85)(cid:82)(cid:83) (cid:51)(cid:85)(cid:82)(cid:87)(cid:72)(cid:70)(cid:87)(cid:76)(cid:82)(cid:81) (cid:20)(cid:23)(cid:24) (cid:11)(cid:21)(cid:19)(cid:21)(cid:20)(cid:12) (cid:20)(cid:19)(cid:24)(cid:25)(cid:20)(cid:27)
`M.A.!M(cid:252)ller!et!al.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
`
`if!the!F120L!+ Y131H!cause!stronger!DMI!adaptation!than!other!CYP51!
`genotypes!such!as!Y131F!+ I145T.!
`Interestingly,! the! CYP51! gene! of! the! isolates! from! 2015/16! and!
`2016/17!seasons!showed!higher!frequencies!of!the!mutation!F120L!+
`Y131H!than!isolates!from!previous!seasons.!The!CYP51!gene!has!several!
`copies!in!the!genome!and!the!number!of!copies!that!presented!the!mu-
`tation! can! in#uence! the! percentage! of! mutation! within! an! isolate!
`(Schmitz!et!al.,!2014).!Monouredinial!isolates!analysed!from!2009/2010!
`season!by!Schmitz!et!al.!(2014)!showed!a!mean!frequency!of!33%!for!
`both,! F120L! and! Y131H,! over! all! isolates! containing! these! mutations!
`included!in!their!study,!while!in!the!present!study!the!isolates!from!the!
`2015/2016!season!contained!around!50%!of!these!mutations.!However,!
`which!alleles!are!expressed!is!fundamental!and!Schmitz!et!al.!showed!
`that!the!mutated!ones!were!overexpressed!compared!to!the!wild!types,!
`which! consequently! led! to! higher! mutation! frequencies! in! the! cDNA!
`(Schmitz!et!al.,!2014).!That!might!explain!why!the!higher!percentage!of!
`the!F120L!+ Y131H!in!DNA!in!some!of!our!isolates!appeared!not!to!be!
`related!with!lower!DMI!sensitivity.!
`Point!mutations!located!on!subunits!B,!C!and!D!of!the!Sdh!gene!are!
`most!commonly!found!and!were!described!as!responsible!for!a!decrease!
`of!SDHI!ef"cacy!in!plant!pathogens!(Avenot!et!al.,!2012;!Rehfus!et!al.,!
`2018;!Sierotzki!and!Scalliet,!2013;!Veloukas!et!al.,!2011).!So!far,!the!
`mutation!I86F!located!in!the!subunit!C!of!the!Sdh!gene,!was!only!found!in!
`P.!pachyrhizi!in!season!2015/16!in!Rio!Grande!do!Sul,!the!southernmost!
`state! of! Brazil! (Sim˜oes! et! al.,! 2018).! In! this! work! one! monouredinial!
`isolate!and!a!population!with!the!I86F!mutation!were!found!in!season!
`2016/17!and!both!isolates!also!presented!mutations!in!the!target!genes!
`of! DMI! and! QoI! fungicides.! The! monouredinial! isolate! with! multiple!
`resistance!to!the!three!groups!of!fungicides!was!found!in!the!north!region!
`of!Brazil,!in!the!state!of!Rondˆonia,!that!borders!Bolivia.!Interestingly,!
`both!locations!allow!two!soybean!crop!cycles!in!one!season.!Selection!
`pressure!of!fungicides!in!the!case!of!successive!soybean!cultivation!is!
`high! and! can! be! the! reason! that! this! multiple! resistant! isolate! was!
`selected!for!the!three!modes!of!action.!Although!not!frequently!found!in!
`the!"eld,!these!mutant!isolates!can!be!present!in!other!regions,!since!they!
`were!found!in!states!that!are!geographically!distant.!
`Isolates!with!the!I86F!mutation!were!less!ef"ciently!controlled!in!our!
`assays! by! the! three! SDHIs! included! in! this! study! than! the! wild! type!
`isolates.!Calculation!of!resistance!factors!(RF)!was!done!by!division!of!
`mean!EC50!of!the!2!isolates!with!~50%!I86F!by!mean!EC50!values!of!all!
`wild!type!isolates.!These!were!4,!26!and!4!for!bixafen,!benzovindi#upyr!
`and! #uxapyroxad! (supplemental! table! 1).! Our! RF! values! for! benzo-
`vindi#upyr! and! #uxapyroxad! were! similar! to! those! of! Sim˜oes! et! al.!
`(2017),!but!lower!for!bixafen.!This!might!be!caused!by!the!mean!EC50!
`wild!type!value!to!bixafen,!which!is!lower!in!the!study!of!Sim˜oes!et!al.!
`(2017)!than!in!ours!(1.2!vs.!11.8).!Both!studies!show!that!all!three!SDHIs!
`were!affected,!but!a!dose!response!was!demonstrated!for!each!and!the!
`resistance! factors! are! rather! limited.! Compared! with! different! SDHI!
`resistance! cases! in! other! pathogens! e.g.! in! Botrytis! cinerea! caused! by!
`different!mutations!(Veloukas!et!al.,!2011),!the!resistance!factors!found!
`in!Phakopsora!pachyrhizi!caused!by!the!I86F!mutation!in!the!SDH-C!gene!
`are!lower.!
`The! detection! of! F129L! in! the! CYTB! gene! "rst! occurred! for!
`P.!pachyrhizi!isolates!in!samples!from!the!2013/14!season,!where!35%!of!
`the!Brazilian!isolates!presented!this!mutation!(Klosowski!et!al.,!2018).!In!
`the! present! study! almost! all! monouredinial! isolates! and! populations!
`presented!this!mutation!with!high!frequency.!The!F129L!single!mutation!
`is!so!far!the!only!mutation!in!the!CYTB!gene!described!in!P.!pachyrhizi!
`isolates!(Klosowski!et!al.,!2018).!In!other!plant!pathogenic!fungi,!the!
`G143A! and! rarely! the! G137R! mutation! in! the! CYTB! gene! have! been!
`detected!(FRAC,!2006).!The!G143A!is!described!as!the!most!common!
`mutation!and!the!mutation!which!causes!high!resistance!factors,!while!
`F129L! is! described! as! having! less! effect! on! QoI! sensitivity! reduction!
`(FRAC,!2006;!Kim!et!al.,!2003).!Because!of!the!gene!structure!(presence!
`of!intron!after!codon!143),!the!occurrence!of!the!G143A!mutation!seems!
`rather! unlikely! to! occur! in! rust! fungi,! including! P.! pachyrhizi! (Grasso!
`
`et!al.,!2006;!Schmitz!et!al.,!2014).!
`Competitiveness! and! "tness! of! isolates! might! be! reduced! by! the!
`presence! of! resistance! mechanisms.! The! emergence! and! spread! of!
`resistance!depend!on!whether!they!are!able!to!outcompete!pre-existing!
`sensitive! isolates! under! selection! pressure! and! under! non-selection!
`pressure.! Competitivity! assays! with! P.! pachyrhizi! resistant! isolates! to!
`DMIs!and!QoIs!and!wild!type!isolates!showed!that!the!"tness!might!be!
`reduced! by! DMI! resistance! mechanisms! (target! site! mutations! in! the!
`CYP51!and/or!CYP51!overexpression),!but!not!for!the!F129L!mutation!in!
`the!CYTB!gene!(Klosowski!et!al.,!2016a).!No!reports!on!competitiveness!
`and!"tness!are!so!far!available!for!SdhC!I86F!mutants!of!P.!pachyrhizi.!
`The!mutation!I86F!is!still!detected!at!low!levels!in!the!"eld!and!dy-
`namic!of!these!mutation!in!the!season!2017/18!showed!a!stabilization!in!
`the! frequency! in! Brazil! compared! to! previous! seasons! (FRAC,! 2019).!
`Therefore,!it!is!rather!likely!that!this!mutation!is!connected!to!"tness!
`penalties.! Studies! performed! with! other! pathogens! indicated! "tness!
`penalties! at! least! for! some! mutations.! SDHI-! resistant! Botrytis! cinerea!
`were!less!competitive!than!wild!type!isolates!and,!therefore,!the!alter-
`nation!of!groups!of!fungicides!application!should!reduce!the!frequency!
`of!SDHI!resistant!isolates!(Lal`eve!et!al.,!2014;!Veloukas!et!al.,!2014).!
`Studies!on!this!subject!with!P.!pachyrhizi!are!important!to!elucidate!the!
`dynamic! of! mutant! isolates! in! the! population! and! will! be! performed.!
`When!there!are!"tness!costs!connected!with!speci"c!resistance!mecha-
`nisms,!resistance!management!strategies!can!be!ef"cient.!
`
`5. Conclusion!
`
`Although! isolates! with! mutations! on! genes! that! confers! reduced!
`sensitivity!to!three