Advances in understanding the epidemiology, molecular biology and control of net blotch and the net blotch barley interaction

Full text

Achieving durable
disease resistance in
cereals
Edited by Professor Richard Oliver, formerly Curtin University,
Australia
BURLEIGH DODDS SERIES IN AGRICULTURAL SCIENCE
E-CHAPTER FROM THIS BOOK

http://dx.doi.org/10.19103/AS.2021.0092.31
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Advances in understanding the
epidemiology, molecular biology and
control of net blotch and the net blotch
barley interaction
AnkeMartin1, BarshaPoudel and Buddhika AmarasingheDahanayaka, Centre for Crop
Health, University of Southern Queensland, Australia; Mark S.McLean, Agriculture Victoria,
Victorian Department of Economic Development, Jobs, Tourism and Resources, Australia;
LisleSnyman, Queensland Department of Agriculture and Fisheries, Australia; and Francisco
J.Lopez-Ruiz, Centre for Crop and Disease Management, Curtin University, Australia
1 Introduction
2 Hybrids
3 Molecular markers to accurately diagnose P. teres isolates
4 Genetic variation and population genetics of P. teres
5 Pathogenic variation and changes in virulence
6 Differential sets
7 The P. teres genome
 8 Identicationofgenesassociatedwithvirulence/avirulencebyQTLand
association mapping
9 Managing the net blotches
10 Conclusion and future trends
11 Where to look for further information
12 Acknowledgements
13 References
1 Introduction
The net blotches are stubble-borne diseases in which primary infection is
derived from barley crop stubble or residue. Infection can be derived from
eitherair-borneconidiawhichareproduced on conidiophores that growon
1 All authors contributed equally.
Epidemiology, molecular biology and control of net blotch
Chapter taken from: Oliver, R. (ed.), Achieving durable disease resistance in cereals,
BurleighDoddsSciencePublishing,Cambridge,UK,2022,(ISBN:9781786766014;www.bdspublishing.com)

Epidemiology, molecular biology and control of net blotch2
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Epidemiology, molecular biology and control of net blotch
the stubble surface or from ascospores derived from pseudothecia. The asexual
morphconsistsofconidiaofvariablelengths,25–300µmlong,7–11µmthick,
straight,cylindricalinshape,smoothwithroundedends,inconspicuoushilum,
and have 1–14 (usually 4–6) pseudosepta (Smedegård-Petersen, 1971). Conidia
are dispersed over short distances with the majority wind dispersed within
100cmandapproximately50%dispersedwithin25cm(DeadmanandCooke,
1989).The sexual morph is produced in pseudothecia which are 1–2 mm in
diameter,globosetoelongatedandcoveredwithdarkseptatesetaeatmaturity
(Steffenson,1997). Pseudothecia contain asci with 3–8 ascospores which are
10–13 µm long and 20–23 µm thick (Steffenson,1997; Van den Berg, 1988).
Ascospores are air-borne and are able to travel greater distances to infect
neighbouringbarleyelds(Steffenson,1997).Netformnetblotchcanalsobe
seed-borne.Laboratoryandglasshousetestshaveshownthatmyceliuminthe
caryopsis grows to infect the developing coleoptile that then penetrate the
leaves.Spot form net blotch has not been shown to beseed-borne(Jordan,
1981).
Disease cycles of spot form and net form of net blotch of barley are illustrated
inFigs1and2.Primaryinfectionoccurswhenconidiaorascosporeslandonthe
surfaceoftheleaf,sheathesororalbractsandcoolwetweatherconditionsare
present for a prolonged period. Infection takes place at temperatures ranging
Figure 1Diseasecycleofspotformnetblotchofbarley.

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Epidemiology, molecular biology and control of net blotch 3
from5°C to25°Cwith optimumtemperaturesof15°C–22°C(Hargreavesand
Keon,1983).Theinfectionprocessisslowerduringcoolertemperatures,taking
6hat10°Candisfasterinwarmertemperatures,takingaslittleas1hat25°C
(Hargreaves and Keon, 1983). The infection process consists of ascospores or
conidia germinating and producing appressoria and penetration pegs that
penetrate the epidermal cell wall, forming an intracellular infection vesicle
(Hargreaves and Keon, 1983). After initial penetration, the fungus grows
throughout the epidermal cell layer, colonising the apoplast of the mesophyll
tissue(HargreavesandKeon,1983).Symptomsstarttoappearwithin24–48h
as dark pin-point spots.These develop into lesions with varying amounts of
necrosis and chlorosis depending on the climatic conditions and resistance/
susceptibility of the host. On susceptible hosts, spot form net blotch develops
asdarkbrownnecroticspotsthatincreaseinsizetoformellipticalorfusiform
lesionsmeasuringfrom3mmto6mm.Thesearesurroundedbyachloroticzone
ofvaryingwidth(Smedegård-Petersen,1971).Symptomsofnetformnetblotch
are initially very similar to those of spot form net blotch, but on susceptible
varietiesdevelopasnedarkbrownlinesextendingacrosstheleafsurfaceto
producea network patternthat maylatercoalesceand formirregularstripes
(EllisandWaller,1973),whichcanextendand coalesce to destroytheentire
leaf. Symptoms of spot form net blotch and net form net blotch on resistant
varietiesaresmallpin-point,darkbrownnecroticlesionsthatdonotincreasein
sizebutmaydevelopasmallchlorotichalodependingonthetypeofresistance
Figure 2Diseasecycleofnetformnetblotchofbarley.

Epidemiology, molecular biology and control of net blotch4
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present.Thedifferencesobservedbetween netandspotform symptomsare
duetodifferencesinfungalgrowth.Spotformofnetblotchinitiallygrowsasa
biotrophformingintracellularvesicleswithinepidermalcellsbeforeswitching
tonecrotrophic(intercellular)growthinthemesophyll.Netformofnetblotch
does not have the biotrophic stage and penetrates the mesophyll more quickly
(LightfootandAble,2010).
Secondary conidia development occurs from 14 days to 22 days after initial
infection.Conidiadevelopreadilyfromlargelesionswithseverenecrosisand
chlorosis and less frequently from small lesions, especially on resistant varieties.
Conidia development occurs during the night and conidia are typically released
followingstrongwinds(Jordan,1981).Secondaryinfectionoccursasoftenas
cool, wet conditions are repeatedthroughout the growing season. Infection
can occur in the head and grain of plants if conditions are favourable late in
the season.
Pyrenophora teres survives from one season to the next on crop stubble
orresidue.Oncethecrophassenesced,mycelialgrowthcolonisesthesurface
duringwetconditions.Myceliumandpseudotheciaareviableaslongasbarley
stubbleispresentonthesoilsurface.Therateatwhichpseudotheciadevelop
depends on the environment. They have been found to develop rapidly in the
rstorsecondseason,withdevelopmentreducinginthethirdseason(Duczek
et al., 1999).
Pyrenophora teres can also infect and survive on other cereals such as
barley grass (H. leporinum),wheat (Triticum aestivum) and oat (Avena sativa)
(Van den Berg, 1988; Uranga et al., 2020) and can infect a wide host range
of other cereal grasses such as Agropyron, Bromus, Elymus, Hordelymus, Stipa
and other Hordeum spp. (Sampson and Watson, 1985; Brown et al., 1993).
However,theseareminorhostsandgeneratelittleinfection.
2 Hybrids
In vitro sexual recombination has been observed within and between the
two forms of P. teres. McDonald (1963) was able to induce ascospores in
culture when two compatible single-spore P. teres isolates were crossed.
He indicated that P. teres are heterothallic in nature. In heterothallic species,
sexualreproductionoccursbetweenindividualsoftwodifferentmatingtypes.
Pyrenophora tereshasa single matingtypelocus (MAT)withtwoidiomorphs
designated as MAT1-1 and MAT1-2.
Smedegård-Petersen (1971) produced viable hybrid progeny from crosses
betweenP. teres f. teres and P. teres f. maculata. Hybrids inoculated on barley
leaves produced symptoms similar to those of net or spot form net blotch or
intermediate to those of the parents (Smedegård-Petersen, 1971; Smedegård-
Petersen, 1976; Crous et al., 1995). They are genetically stable and can retain

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Epidemiology, molecular biology and control of net blotch 5
their virulence and fertility over generations (Campbell and Crous, 2003).
Hybridsproducedinalaboratoryhadreducedsensitivitytotriazolefungicides
as compared to the parents (Campbell et al., 1999). Different virulence patterns
were observed when comparing the progeny with either of the parental
isolates,withsomehybridsbeingvirulentonbarleylinesonwhichbothparents
wereavirulent(Jalli,2011).
Pyrenophora teres hybrids are relatively rarely found in the eld and
appear to be reproductively isolated (Rau et al., 2003; Serenius et al., 2007;
Ellwoodetal.,2012;Poudelet al., 2017,2019a).The reproductive isolation
couldariseduetopre-or post-mating barriers and has been reviewedina
paper by Poudel et al. (2019a). The two most likely reasons suggested for
the reproductive isolation include sexual preferences for the same form or
reducedtness compared totheirparents.Thending of P. teres hybrids in
nature,however,suggests thatreproductivebarriers undersomeconditions
can be permissive, thereby allowing hybrids to be formed more readily.
Seven hybrids have been identied under eld conditions to date, one in
SouthAfrica(Campbelletal.,2002),twointheCzechRepublic(Leisovaetal.,
2005b),oneinHungary(Dahanayakaetal.,2020),oneinJapan(Dahanayaka
et al., 2020) and two in Australia(McLean et al., 2014; Turo et al., 2021).A
hybrid collected from elds in Western Australia was highly resistant to
some Group 3 (azole or demethylase inhibitor) fungicides and was found
to rapidly propagate by asexual reproduction (Turo et al., 2021). Although
rare, P. teres hybrids are of concern as they can rapidly evolve acquiring
virulence and fungicide resistance from both net blotch forms due to sexual
recombination, thus producing more resistant strains. There is therefore a
need to constantly monitor P. terespopulations in elds forthepresenceof
hybrids.RandomampliedpolymorphicDNA(RAPD)andampliedfragment
length polymorphism (AFLP) markers (Campbell et al., 1999, 2002) have
been used to identify hybrids, but these techniques are demanding and time-
consuming. Recently, more efcient and accurate PCR-based markers have
been developed to identify hybrids using six unique regions from each P. teres
form (Poudel et al., 2017).
3 Molecular markers to accurately diagnose P. teres isolates
It is important to accurately characterise pathogens for disease management,
plant breeding and epidemiological studies. As the spore morphology of the
twoforms ofP. teres is very similar, symptom expression is the usual means of
identication.Itcan,however,bedifculttodistinguishbetweenthetwoformsof
P. teres, especially at early stages of symptom development. Spot form net blotch
symptomsarealsodifculttodistinguishfromspot blotch symptomscaused
by Bipolaris sorokiniana Shoemaker 1959 (Williams et al., 2001; Lehmensiek

Epidemiology, molecular biology and control of net blotch6
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et al., 2010). The most reliable characterisation of the blotches is thus by using
molecular markers. A number of molecular assays have been developed, and
therstofthesewasdevelopedin2001byWilliamsetal.(2001).
Williamsetal.(2001)clonedarandomampliedpolymorphicDNA(RAPD)
markerthatwaspresentinP. teres f. maculata isolates and absent from P. teres
f. teres isolates. The cloned segment was sequenced and primers designed
whichampliedthesamesizefragmentinbothforms.Theampliconsfromboth
formswerethussequencedandprimersdesignedspecictoeachformofnet
blotch (Table 1). Testing of the developed primer sets across 27 P. teres isolates
collectedinAustralia,Canada,theUSAandGermanyconrmedthattheywere
specictothetwoforms.ThemarkersdidnotamplifyonDNAofB. sorokiniana
but did amplify DNA from P. graminea. In European countries it is important to
beabletodifferentiatebetweenP. teres and P. graminea as the seed infection
threshold for P. gramineaislowerthanthatfor P. teres and is applied for both
speciesifthespecieshasnotbeendiagnosed(Justesenetal.,2008).
In2005,Leisovaetal.(2005a)developedform-specicmarkersthatcould
distinguish the net blotches from P. graminea as well as distinguish between
thetwoforms.Theyusedsequencedampliedfragmentlengthpolymorphism
(AFLP)markerstoidentifyform-specicmarkersthatwerealsodistinctamongst
different species, including P. graminea, P. tritici-repentis and Helminthosporium
sativum. The form-specic markers were cloned and sequenced and primer
sets were designed. Primers were validated across 66 P. teres isolates, ve P.
graminea, three P. tritici-repentis, one P. avispora and four B. sorokiniana isolates.
Twoprimersetsforeachformwerebothspeciesandformspecic(Table1).
AfurthersetofdiagnosticmarkersweredevelopedbyKeiperetal.(2008).
Thesenine microsatelliteorsimplesequence repeat(SSR)markers amplied
loci in one form only,amplied loci with different allele size ranges in each
form,oramplieddifferentnumbersoflociineachform.Oftheninemarkers,
threewereform specic,onetoP. teres f. maculataandtwotoP. teres f. teres
(Table1).TheprimerswereonlytestedacrosssixP. teres isolates of each form
andspeciesspecicitywasnotdetermined.
In2010,mating type locus-specicmarkersweredeveloped by Luetal.
(2010)whichcouldbeusedtodifferentiatebetweenthetwoformsofP. teres.
The mating type loci were cloned and sequenced and form- and species-
specic primers were designed across single nucleotide polymorphism
differences.Twosetsofprimersforeachmatingtypeweredeveloped(Table1).
Specicityofeachprimersetwasvalidatedacross54P. teres isolates and DNA
from P. graminea, P. tritici-repentis and other ascomycetes. Unfortunately, the
specicityofthemarkerswithB. sorokiniana,whichproducesdiseasesymptoms
very similar to those of P. teres f. maculata,wasnot tested.The advantage of
using the mating type markers is that the pathogenicity and mating type can be
simultaneouslycharacterisedwhilstconrmingthespeciesandP. teres form.

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Epidemiology, molecular biology and control of net blotch 7
Sequence-specic PCR markers were produced by Poudel et al. (2017)
toidentify thetwo formsofP. teres and progeny derived from recombination
betweenthetwoforms(hybrids).Hybridsnormallyhavesimilarlesiontypesto
those of their parents and can therefore only be diagnosed using molecular
markers.Previouslydevelopeddiagnosticmarkerswerenotidealforidentifying
hybrids and could therefore not be used to monitor the occurrence of hybrids
in natural environments. Whole-genome assemblies and RNA-seq derived
assembled and aligned transcripts were utilised to identify unique P. teres f.
teres and P. teres f. maculataregions.Twelveform-specicexpressed regions
wereidentiedandsixsetsofprimersdesignedforeachP. teres form (Table 1).
Thespecicityoftheprimerssets was validated across 78Australianand46
South African P. teres isolates, seven P. teres isolates obtained from barley
grass (Hordeum leporinum), six B. sorokiniana isolates and one isolate each
of Exserohilum rostratum and P. tritici-repentis.The primer sets were specic
toeach formwithallsixmarkerspresentandnoamplication onDNAofthe
otherspeciestested.Hybridshadbetweentwoandelevenmarkersbutalways
at least one marker specic to each form. Interestingly the P. teres isolates
collectedfrombarleygrassonlyampliedfourofthesixP. teres f. teres-specic
markers and none of the P. teres f. maculata-specicmarkers.AFLPanalysisof
both P. teres isolates collected from barley and barley grass revealed different
ngerprints for the two groups of isolates (Poudel et al., 2017). Only 2–4
markersoutofthe12needtobeampliedtoconrmthespeciesandformof
P. teres.However,todetecthybridsall12markersneedtobeamplied.Asthe
fragmentsproducedbythemarkersarerelativelysmall,theycanbeamplied
anddetectedusingaquantitativeassaywithoutneedingtorunanagarosegel
(Dahanayaka et al., 2021).
In conclusion, a number of molecular markers have been developed to
correctly diagnose P. teres isolates. Some markers have an added advantage
overotherssuchastheLuetal.(2010)matingtypemarkerswhichsimultaneously
characterisethematingtypeoftheisolateandthePoudeletal.(2017)markers
whichcansimultaneouslyidentifyhybrids.Thechoiceofmarkersdependson
the aim of the diagnosis.
4 Genetic variation and population genetics of P. teres
Geneticvariationreferstothenaturallyoccurringgeneticdifferencesbetween
individualsof the same species that dene population structures (Milgroom,
1996).Thepopulationstructureofapathogencanbeusedtopredicthowrapidly
a pathogen can evolve and overcome host or fungicide resistance. Molecular
markerssuchasRAPD,AFLP,SSRandDiversityArraysTechnology(DArT)have
been applied effectively to understand genetic diversity in P. teres populations
worldwide,includingCanada,Germany,USA(PeeverandMilgroom,1994;Liu

Epidemiology, molecular biology and control of net blotch8
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Table 1ListofprimersdevelopedbydifferentgroupstodistinguishthetwoformsofP. teres and identify hybrids
Species
Primername(F=forward;
R = reverse) Sequence 5’-3’
Amplicon
size(bp) Reference
P. teres f. teres PTT-F
PTT-R
CTCTGGCGAACCGTTC ATG ATGG AAAA GTAA T TTGTA 378 (Williams et al.,
2001)
P. teres f.
maculata
PTM-F
PTM-R
TGCTGAAGCGTAAGTTTC ATG ATGG AAAA GTAA T TTGTG 411 (Williams et al.,
2001)
P. teres f. teres DTT471h-F
DTT471h-R
CCT GAGT AACT TGCCC CACC
GAA AAGA GATG ATGC G GACAC
91 (Leisovaetal.,
2005a)
P. teres f. teres DTT339i-F
DTT339i-R
TGA TGCG CTGG AGTG A GACAC TGT ACAT ACGC CGCAT CACG 81 (Leisovaetal.,
2005a)
P. teres f.
maculata
DTM494d-F
DTM494d-R
TAT TCTG CTAA GAGC TAGC ATCCTA ACT GCGT ACCA ATTC TCTA
CAACTA
161 (Leisovaetal.,
2005a)
P. teres f.
maculata
DTM348j-F
DTM348j-R
CTT GATG CGCT GGAGT GAGA
TGC ATTT CCAC CTAC TGGT ATGTA C
66 (Leisovaetal.,
2005a)
P. teres f. teres hSPT2_4agac-F
hSPT2_4agac-R
CCT TGGT GGTT TCTG TGG TTTCT AGA GAGA GAGA GAGA CA CACAC 128–132 (Keiper et al.,
2008)
P. teres f. teres hSPT2_24tcac-F
hSPT2_24tcac-R
ACT TCGC TGAG TGTT AGTT GCATC
TCT CTCT CTCT CTCA CA CACAC
90–106 (Keiper et al.,
2008)
P. teres f.
maculata
hSPT2_24agac-F
hSPT2_24agac-R
ATA CTTG TGGT AGCC TACT TTGCA
AGA GAGA GAGA GAGA CA CACAC
126–168 (Keiper et al.,
2008)
P. teres f. teres Ptt-MAT1-F
Ptt-MAT1-R
Ptt-MAT2-F
Ptt-MAT2-R
ATG AGAC GCTA GTTC AG AGTCT GATGCCCAGCCAAGGACAA
TAC GTTG ATGC AGCT TTC TCAAT AACACCGTCCAAAGCACCT
1143
1421
(Luetal.,2010)
P. teres f.
maculata
Ptm-MAT1-F
Ptm-MAT1-R
Ptm-MAT2-F
Ptm-MAT2-R
TGT TAGA GACC CCAC C AGCGT
CAG CTTT CTTG GCCT T CTGAA ACG CAAG GTAC TCTG T ACGCA GAC
GTCG AGGG AGTCC ATTT
149
939
(Luetal.,2010)

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 9
P. teres f. teres PttQ1-F
PttQ1-R
GGA TGAT GACC TCGCC AGAT
GCG ATGG TATG TTCTG CGAA
70 (Poudel et al.,
2017)
P. teres f. teres PttQ2-F
PttQ2-R
AAC ACTC TGAA CGTGG TTGC
TTC AGTT GTAA GCTGC GTGG
110 (Poudel et al.,
2017)
P. teres f. teres PttQ3-F
PttQ3-R
CCT CGTC CTAA GTTG A CTCGA
TTA CACG GGTT CCCTC CATC
130 (Poudel et al.,
2017)
P. teres f. teres PttQ4-F
PttQ4-R
CGT CCCG CCGA AATTT TGTA
CAA GGAC TTAC GCGCT CAAA
150 (Poudel et al.,
2017)
P. teres f. teres PttQ5-F
PttQ5-R
GCA TTGT CTAG CACTC GTCG
CGC GGAC TCAG AAGAC ATTG
173 (Poudel et al.,
2017)
P. teres f. teres PttQ6-F
PttQ6-R
TCA GAAT ACTC CGCGG ACTC
GTC CGCA TTGT CTAGC ACTC
188 (Poudel et al.,
2017)
P. teres f.
maculata
PtmQ7-F
PtmQ7-R
GTA GAGG CTGT AGGA GATG TGATT
CAT GGCA AATT GTTC GTAA TCCTG
140 (Poudel et al.,
2017)
P. teres f.
maculata
PtmQ8-F
PtmQ8-R
ACG CTAA GACC ACCTC GTTT
TCG ACCA GAGA GAGCA CAAA
161 (Poudel et al.,
2017)
P. teres f.
maculata
PtmQ9-F
PtmQ9-R
AAT GCTC AATT CTGGT GGCG
TGT TCGA GTGC AAACT TGGG
201 (Poudel et al.,
2017)
P. teres f.
maculata
PtmQ10-F
PtmQ10-R
TGC TGTG GACT TAGAC GAGG
TGG GGAT CCTT GACCA ACTC
220 (Poudel et al.,
2017)
P. teres f.
maculata
PtmQ11-F
PtmQ11-R
GAT TAGA CCAT TACC ACAC TAGCG
ACC ACCA CATC TTTC CTAC TAACT
260 (Poudel et al.,
2017)
P. teres f.
maculata
PtmQ12-F
PtmQ12-R
CTA ACCA AAGA ACTT CACA GACGA
CCT TATT AGCC AATT CCAT GTCGA
279 (Poudel et al.,
2017)

Epidemiology, molecular biology and control of net blotch10
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
et al., 2012; Akhavan et al., 2015), Finland (Peltonen et al., 1996; Serenius et al.,
2005,2007),Sweden(Jonssonetal., 2000),Italy (Rauetal.,2003),the Czech
andSlovakRepublic (Leisovaetal.,2005b;Leisovaet al.,2014), SouthAfrica
(Campbell et al., 2002; Lehmensiek et al., 2010), Iceland (Stefansson et al.,
2012) and Australia (Serenius et al.,2007; Bogacki et al., 2010; Lehmensiek
etal.,2010;McLeanetal.,2010b,2014;Poudeletal.,2019b).Asthegenetic
markers used in these studies were different, the outcomes are not directly
comparable.Nevertheless,highpopulationgeneticdiversity hasalwaysbeen
reported for P. terespopulations.
Inoneoftheearliestsuchstudies,usingRAPDmarkers,veP. teres f. teres
populationsoriginatingfrom Canada,Germany and the USAwereexamined
(Peever and Milgroom, 1994). These populations shared a high number of
alleles implying that P. tereswas derived fromacommon source population;
however, a signicantly high degree of variability was detected within four
of the populations. This implied that sexual reproduction occurred regularly
in eld populations and was consistent with most of the other population
studies from a wide range of geographical locations, in which the genetic
structure of P. teres f. teres and P. teres f. maculatawereinvestigated(Akhavan
etal.,2015; Campbelletal.,2002; Rau etal.,2003; Liu et al.,2012;Bogacki
etal.,2010;McLeanetal.,2010b;Jonssonet al.,2000; Sereniusetal.,2005;
Leisovaetal.,2005b,2014;Peltonenetal.,1996).Thegeneticvariationwithin
apopulationcanoccurwithinaeldoraplant(Peltonenetal.,1996;Jonsson
et al., 2000; Liu et al.,2012; Bogacki et al., 2010; Poudel et al., 2019b) and
thepopulationstructure can change substantially eachyear(Liuetal., 2012;
Poudel et al., 2019b). Diversity in P. teres populations is strongly associated
withgeographicaldistances. Distinct geneticlineagesweredetected among
populationsseparatedby wide geographical areas (such as states,countries
and continents) due to limited gene ow (Serenius et al.,2007; Lehmensiek
etal.,2010).Withinaeld oratclosely locatedelds, highgeneticvariability
was observed due to sexual reproduction and/or gene exchange through
migration(Serenius etal.,2005; Bogacki etal.,2010; Liuetal.,2012; Poudel
etal.,2019b).In contrast,some studieshaveindicatedlowgenetic variability
andoccurrenceofclonalpopulationswithinaeldindicatingthatreproduction
withinP. terespopulationsoccursasexually(Jonssonetal.,2000; Lehmensiek
et al., 2010; Rau et al., 2003; Serenius et al., 2007). The prevalence of mode
of reproduction and genetic similarity between geographical regions can
be highly variable depending on environmental conditions, specic barley
varietiesgrownintheregions,agriculturalpracticessuchasstubbleretention,
close barley rotation and seed exchange (Rau et al., 2003; Serenius et al., 2007;
Liuetal.,2012;Fowleretal.,2017).
Collectively,frequentsexualrecombination withineachform,generation
of conidia by asexual reproduction and gene ow via air-borne spores and/

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 11
or infected seeds are important in epidemic development of the disease. The
rapid change in P. teres populations due to sexual reproduction can lead to
therapiddevelopmentofnewvirulences,whichhasthepotentialtoovercome
majorhostresistancegenesandfungicides.
5 Pathogenic variation and changes in virulence
The net blotch pathogens pose considerable threats to the barley industry as
theyareabletofrequentlyovercometheresistance inbarleyvarieties(Linde
and Smith, 2019). These virulence changes are particularly devastating if several
barleyvarietieswith common resistancesaregrown (Martinetal., 2020).Itis
widely accepted that widespread intensive cultivation and the development
of genetically homogeneous crops lead to selective pressure on fungal
pathogenpopulations(Ellwoodetal.,2012).Manycountrieshavereportedan
increasedincidenceofnetform,probablycausedbythe practiceofgrowing
continuous barley crops in the same paddock (Arabi et al., 2003) and no-till
farmingpractices(McLeanetal.,2009).Douiyssietal.(1998)indicatedisolates
collectedfromregionswherebarleyiswidelygrowntobemorevirulentthan
isolatesoriginatingfromregionswithlimitedbarleyproduction.
Changes in resistance can be attributed to the appearance of new
physiologic forms or races (Christensen and Graham, 1934). Fungi that
reproduce both asexually and sexually are considered to pose a higher
evolutionaryriskand are predictedtoovercomenewly deployed resistances
more rapidly than those reproducing only asexually or sexually (McDonald and
Linde,2002).Duringsexualreproduction,virulenceallelesfromtwoindividuals
combineintothesamegeneticbackgroundandthesenewgenecombinations
arerapidlypropagatedthroughasexualreproduction(LindeandSmith,2019).
Pathogens that exhibit mixed reproduction exhibit higher genotype diversity as
a result of recombination and have greater potential for local adaptation to a
changingenvironment(McDonaldandLinde,2002).
Most studies on host resistance have focussed on resistance identied
at the seedling stage leading many to conclude that seedling tests can be
condently used to identify resistance and offering many advantages over
eldscreening(BuchannonandMcDonald,1965).A numerical scale (Fig.3)
developed to classify reactions of barley to P. teres at the seedling stage is
widely used (Tekauz, 1985). Resistance expressed at the seedling stage was
reported to also express in adult plants (Arabi et al., 1990). It was however
noted that several barley lines screened for resistance to P. teres f. teresshowed
nocorrelationbetweenseedlingtestsintheglasshouse(onetotwoleafstage),
disease reactions on the fourth and ag leaves of plants grown in growth
chambersanddiseaselevelsobservedundereldconditions,whileforothers,
thecorrelationwassignicant(Jonssonetal.,1998).

Epidemiology, molecular biology and control of net blotch12
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Theoccurrenceofthetwoformsofnetblotchvariesbetweengeographical
regionswithoneformusuallydominatinginanarea(McLeanetal.,2009).The
netform was identied to be dominant in Canada(Tekauz,1990), California
(SteffensonandWebster,1992a),Sweden(Jonssonetal.,1997)andMorocco
(JebboujandElYous,2010),whereasthespotformismoreprevalentinFrance
(Arabietal.,1992).Fewerand lessin-depthstudiesonvirulenceprolesof P.
teres f. maculatahavebeenpublished(Liuetal.,2011).
PhysiologicspecializationinP. teres f. tereswasrstreportedin1949with
the identication of different races and differencesin pathogenicity towards
varieties (Pon, 1949). Plant pathologists and breeders can utilise knowledge
about the variation in virulence of pathogen populations in the deployment
of resistant sources that are likely to be effective against the spectrum of
pathotypes in the area (Steffenson and Webster, 1992a).
In California, no distinct geographical differences in the distribution of
pathotypes were identied (Steffenson andWebster, 1992a).Despite clusters
of pathotypes being identied in some areas, others contained mixtures of
pathotypes, identied even within a single crop. Sixteen pathotypes were
identied from the 91 Californian isolates. A small number of isolates from
Minnesota,MexicoandEngland includedinthis studyweredistinctlydifferent
from each other with only the Mexican pathotypes being similar to those
from California in virulence to specic differentialgenotypes (Steffenson and
Webster, 1992a). A study using 48 Algerian isolates screened across a set of 22
differentials indicated high levels of variation in the virulence of the pathogen
and12pathotypeswereidentied.SimilartotheCalifornianisolates,themost
common isolate did not display a high level of virulence (Boungab et al., 2012).
The P. teres f. teres population in Algeria was highly variable from region to
region,possiblyinresponsetolocallygownvarieties(Boungabetal.,2012).
Asetof18differentialgenotypes wereused topathotype25Swedishand
twoCanadianisolatesinto14pathotypes.Hostselectionon thepathogenwas
evidentwiththesamepathotypecollectedfromthesamevariety(Jonssonetal.,
1997);isolatescollectedfromsusceptiblehostswerereportedtobemorevirulent
than those collected from less susceptible hosts (Robinson and Jalli,1996). It
wasalsoconcludedthatP. teres f. teresdominatesinSwedenasonlyoneofthe
isolatescollectedwereidentiedtobeP. teres f. maculata(Jonssonetal.,1997).
Twohundredandnineteennetformisolates,collectedinwesternCanada
were characterised on a set of 12 differential barley genotypes,resulting in
82%identiedtobeP. teres f. teresand18%P. teres f. maculata. For P. teres f.
teres,45pathotypeswereidentiedand20forP. teres f. maculata. Net form net
blotch was generally distributed throughout the collection area; however, P.
teres f. maculatawasmostprevalentinSaskatchewan(Tekauz,1990).
Twenty-ve barley genotypes were usedtopathotype23P. teres f. teres
and eight P. teres f. maculataisolatescollectedfrom12differentbarley-growing

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 13
regions of the world. Fifteen P. teres f. teres pathotypes and four P. teres f.
maculatapathotypeswereidentied,withabroaderspectrumandhigherlevel
of virulence observed in the P. teres f. teres compared to the P. teres f. maculata
isolates (Wu et al., 2003).
Figure 3The infection response eight days after inoculation, scores based onTekauz
(1985) scoring scale. (a) 10-point scale for net form net blotch and (b) 9-point scale for
spot form net blotch.

Epidemiology, molecular biology and control of net blotch14
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Highlevelsofpathogenic variability wereidentiedin 15 P. teres f. teres
isolates collected in Morocco and assessed at seedling and adult plant stage
on a set of 38 differential genotypes, with none of the 15 isolates being
identical (Douiyssi et al., 1998). A collection of 61 P. teres f. teres and 21 P.
teres f. maculataisolatescollected inthesemi-arid regionsofMorocco,were
pathotyped on differential sets of 22 P. teres f. teres and 20 P. teres f. maculata
genotypes, identifying ten pathotypes of P. teres f. teres and nine of P. teres f.
maculata(JebboujandElYous,2010).
Twenty pathotypes were identied on 14 differential genotypes in a
collection of 104 P. teres f. teresisolatescollectedinTunisiaandSyria(Bouajila
etal.,2011).Asuccessivestudy,usingthesamedifferentiallines,identied23
pathotypes in 85 P. teres f. teresisolatescollectedacrossTunisia,withvariability
within groups higher than between groups observed (Bouajila et al., 2012).
Signicantvariationinpathogenicitywas alsodemonstratedinP. teres f. teres
isolatescollectedinFranceandSyriawithveclustersidentiedin23isolates
screened on a set of 11 genotypes (Arabi et al., 2003).
Astudy in New Zealandidentied11 pathotypes in29isolatesusing 31
differentialgenotypes.LowlevelsofvariationintheP. teres f. teres population
was identied, with low levels of virulence observed for the most common
collected pathotype, despite it being recorded throughout the surveyed area
(Cromey and Parkes, 2003).
Pathotype variations of P. teres f. teres in Western Australia (WA) have been
extensivelystudied(KhanandBoyd,1969a;Khan,1982;GuptaandLoughman,
2001;Fowleretal.,2017).TherstevidenceofphysiologicspecializationinP.
teres f. teresisolatesinAustraliawerereportedin1969(KhanandBoyd,1969a)
with the identication of three physiologic races in WA.A major shift in the
pathogenpopulation was reportedinWAin1982,with adeclineinthe area
growntothesusceptiblevarietyBeecher.Dampier,alsosusceptible,remaineda
popularvarietyuntil1976,whereastheresistantvarietyClipperwasintroduced
intheearly1970sbecomingthemajorvariety.Isolatescollectedpost1976in
WAidentiedanewgroupofisolates,avirulentonthevarietyBeecher,whereas
Dampierwassusceptibleto all 52 isolates.Itwasconcludedthattheshiftin
thepathogenpopulationwasdueto changes in commercially grownbarley
varieties(Khan,1982).Intheperiod1995–1996,netformnetblotchwasfound
tobewidelydistributedacrossbarleygrowingareasofWA,withP. teres f. teres
frequentlyobservedinsurveyedcrops(GuptaandLoughman,2001).Seventy-
nine P. teres f. teresisolatescollectedduringthisperiodwereclassiedintotwo
distinctgroups,basedonvirulencetothevarietyBeecher,withthemajorityof
isolates avirulent on Beecher. They concluded that virulence to P. teres f. teres
in WA remained stable over a period of at least 19 years.
The pathogenic variation of P. teres f. maculata in Western Australia
between2001and2002wasexploredbyscreeningacollectionof99isolates

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 15
across 26 differential barley lines (Gupta et al., 2012). Seven isolate groups
wereidentiedandthe26differentiallineswereclassiedintofourlinegroups
according to their differential responses to the spot form net blotch isolates.
The P. teres f. teres populations in South Australia have evolved over time in
responsetocultivationofbarleyvarieties(Wallworketal.,2016).Priorto1993,
the main barley varieties cultivated, Clipper and Schooner had good levels of
adultplantresistance.Afteraperiodoflowdetection,P. teres f. teres re-emerged
in South Australia in 1994 after the release of the susceptible variety Franklin.
Increasedlevelsofnetformnetblotch incropsresultedinthebreakdownof
seedlingresistanceinthevarietySkiff,followedbyvirulenceinKeelin2007and
Maritimein2009(Wallworketal.,2016).VirulenceonFleetwasalsodetected
in2009withvirulenceonthevarietyOxforddetectedin2012.Itisunknownif
thevirulencesevolvedlocally or were the resultofpathogenmigrationfrom
other Australian barley growing regions (Linde and Smith, 2019). Cluster
analysis suggests that the virulence on varieties Maritime and Keel evolved in
WAandwasintroducedtoSA,possiblymediatedbyhumans(LindeandSmith,
2019).
Amorerecentcomprehensive study by Fowler et al.(2017) identied P.
teres f. teres isolates to be different between easternand Western Australia,
suggesting regional evolution of pathotypes, dependent on varieties prevalent
in each region. This study reported virulence of 123 P. teres f. teres isolates
collected across Australia over a period of 27 years. Phenotype cluster
analysisidentiedsevenlinegroups,whichclusteredintofourdistinctgroups,
indicated by differential virulence to four key barley genotypes: Maritime, Prior,
SkiffandTallon.Distinctdifferenceswereobservedbetweenisolategroupsin
easternAustraliaandWA,whereasallisolategroupsweredetectedinsouthern
Australia(Fowleret al.,2017).Results indicatedP. teres f. teres populations of
each state to be quite unique and reect the cultivation of locally adapted
varieties.Virulencetosupersededvarietieswasdetectedinallstates,indicating
that virulences remain in the pathogen population, despite the varieties not
beingpresent(Fowleretal.,2017).
6 Differential sets
Virulences in P. teres f. teres populations have been studied extensively in
many regions using sets of differential lines (Arabi et al., 2003; Cromey and
Parkes,2003;Douiyssietal.,1998;Fowleretal.,2017;GuptaandLoughman,
2001;Guptaetal.,2003;Khan,1982;KhanandBoyd,1969a;Platzetal.,2000;
Steffenson and Webster, 1992a; Tuohy et al., 2006; Wallwork et al., 2016).
However,duetothelackofauniversalsetofgenotypes,pathogenpopulations
cannot be compared globally and this hampers monitoring of pathogen
populations across regions and over time.

Epidemiology, molecular biology and control of net blotch16
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Afanasenkoetal.(1995)identiedasetof12differentialsforcharacterising
P. teres f. teres populations that could be used to compare results of population
studiesbetweencountriesandregionsandextendknowledgeofevolutionin
pathogenpopulations.Ongoingattemptstodenethesetforinternationaluse
byscreening1000isolatesfromRussia,Europe,AustraliaandCanadasawthe
proposed standard, international differential set reduced to nine genotypes
(Afanasenko et al., 2009).
For a differential set to have local benet, the inclusion of regional
differential cultivars is required, enabling breeders and pathologists to detect
localvirulencesnotidentiedbythestandardset(CromeyandParkes,2003).
Duetotheever-changingvirulenceproleofP. teres f. teres populations, there
willbeanongoingneedtoincludebarleygenotypesrepresentingnewsources
of resistance (Afanasenko et al., 2009).
Thirty-onebarleygenotypeswereusedbyFowleretal.(2017)todetermine
the diversity of 123 Australian P. teres f. teres pathotypes, consisting of a
combination of genotypes used in international diversity studies, Australian
diversitystudiesandsixAustralianvarieties.Twentyofthedifferentialsusedby
Fowleretal.(2017)wereusedtophenotype188Australianisolatesforgenome-
wideassociationmappingtoidentifyregionsassociatedwithvirulenceinthenet
formnetblotchpathogen(Martinetal.,2020).These20genotypes,togetherwith
ten current commercial varieties (Bass, Compass, Explorer, Flinders, Navigator,
Oxford, RGT Planet, Rosalind, Shepherd and Urambie) constitutes the current
differential set used for pathotyping Australian P. teres f. teres isolates.
TodeterminethepathogenicvariationintheworldwideP. teres f. maculata
populationsaninitialdifferentialsetconsistingof19barleylineswasproposed
byMcLeanet al. (2012).This set was later validatedforreactionresponseto
60 P. teres f. maculata isolates collected throughout the Australian barley-
growingregion(McLeanetal.,2014).Theneedforfurtherstudiestodevelopa
consolidatedspotformnetblotchdifferentialsetwasexpressed.
Resistanceexpression,suchaslesionsizeandthepresenceofnecrosisand/
orchlorosis,canbeinuenced byfactorsotherthan pathogenvirulenceand
hostresistance,includinggrowthstage,temperatureandlight(Douiyssietal.,
1998;KhanandBoyd,1969a;Liuetal.,2011),complicatingthedevelopment
of international differential sets.
7 The P. teres genome
The rst genome sequencing and assembly of the P. teres f. teres isolate
0-1 provided a signicant resource for understanding the necrotrophic
lifestyle and pathogenicity of P. teres (Ellwood et al.,2010).Thisgenome was
sequenced using short-read paired-end Illumina sequencing resulting in an
assemblywith6412contigsandatotal size of 41.95 Mb (Table2). Recently,

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 17
using Pacic Biosciences (PacBio) single-molecule real-time sequencing, the
genome sequence of isolate 0-1 was improved to generate a high-quality
reference genome assembly and annotation (Wyatt et al., 2018). The updated
genomeof0-1has86contigswithatotalgenomesizeof46.5Mb(Table2).The
qualityofthe genomeassemblywasimprovedduetothe useoflongreads,
whichcanassemble low complexity,repeat dense regions.Geneannotation
was performed with an evidence-based approach that included assembled
RNA-seq transcripts sequenced using an Illumina platform, multiple ab initio
gene predictions,protein evidence from the closely related species P. tritici-
repentis(Manningetal.,2013)andanavailableP. teres f. teres0-1annotation
(Ellwoodetal.,2010).Secretedproteinsandpotentialcandidateeffectorswere
predictedwhicharelikelytobeinvolvedinthebarley–P. teresinteraction.
A large-scale genomic comparison between P. teres f. teres and P. teres
f. maculata was conducted to explore structural variations, co-linearity and
orthology (Syme et al., 2018). Five P. teres f. teres (W1-1, Stir9-2, NB29, NB73
and NB85) and four P. teres f. maculata (SG1, Cad6-4, M2 and FGOB10Ptm-1)
isolateswereincludedinthestudy.Toderiveacomprehensivelandscapeofthe
P. teres genome structure, a combination of long and short DNA reads along
with RNA reads and optical genetic mapping (isolateW1-1) were employed.
SinglemoleculePacBiolongreadsandIlluminashortreadswerecombinedto
assemble the reference genomes of the P. teres f. teres (W1-1) and P. teres f.
maculata (SG1) isolates. Both P. teresformscomprisedof12chromosomeswith
nochromosomalfusionorrearrangementsbetweenthem.ThegenomesizeofP.
teres f. teresisolatesrangedfrom46.31Mbpto51.76MbpandP. teres f. maculata
isolatesrangedfrom39.27Mbpto41.28Mbp(Table2).Thechromosomal-scale
differencesbetweenP. teresformswereentirelyduetoexpansionorinsertion
of repetitive repertoires, more particularly transposable elements (TEs) with
Line-likeTad1andDNAtransposonTc1/MarinerTEsmainlyassociatedwiththe
differentiation.Similarnumber of geneswaspredictedbetweenP. teres forms
andthemajorityof thegenes withinGC-rich syntenicregionswerestableand
conserved. Genic differences found between the forms were mainly located
in gene-sparse regions near or within TE-rich regions and often harboured
fungal effectors. Gene interruptions by TE insertions resulting in pseudogenes
were also observed in P. teres f. teres, which is an effective mechanism for
removingavirulencegenesandproducingneweffector-likegenes.Secondary
metabolites, which are associated with adaptation to a particular niche and
virulence,werecomparedbetweenP. teresforms (Moolhuijzenet al.,2020).In
all P. teres isolates compared, non-ribosomal peptides synthases (NRPS), NRPS-
like, type I polyketide synthase (T1PKS), type III PKS (T3PKS), hybrid T1PKS-
NRPS and terpene secondary metabolites were detected. Pyrenophora teres
f. teres had almost twice the number of NRPS and polyketide synthase (PK)
clusters compared to P. teres f. maculata (Table 2). The NRPS expanded regions

Epidemiology, molecular biology and control of net blotch18
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
werelocatedin repeat regionsof chromosomes1,2 and 3 ofP. teres f. teres
and on chromosome 3 for P. teres f. maculata. These differences highlight the
evolutionary divergence, host-pathogen interactions and adaption of P. teres.
ThediversityingenomicarchitectureandgenecontentwithinP. teres f.
teresisolateshasbeencomparedtoidentifycoreandisolate-speciceffectors
using a pan-genome approach (Wyatt et al., 2020). Using this pan-genome
approach, the full gene repertoires including genes common to all individuals
(core) and those unique to individuals (accessory) could be dened. When
comparedbetweenP. teres f . teresisolates,thegenomicsyntenywas mainly
conservedexceptfor oneinstancewherefusionbetweenchromosome1and
2wasdiscovered.Theauthorsconsideredthisfusiontobearecenteventand
Table 2GenomesequencingandassemblystatisticsofcurrentlysequencedP. teres f. teres and
P. teres f. maculata isolates
P. teres f. teres P. teres f. teres
Isolate name 0-1 W1-1 NB29 NB73 NB85 15A 6A FGOH04Ptt-21
Collection site Ontario, Canada Western Australia Western Australia Queensland,
Australia
Queensland,
Australia
California, USA California, USA North Dakota, USA
Collection date Before 1998 2009 1985 1994 1995 1984–1986 Unknown Before 2017
Sequencing information
Sequencing platform Solexa;
PacBio
PacBio;
Illumina; HiSeq;
Optical map
PacBio PacBio PacBio PacBio PacBio PacBio
RNA-Seq Illumina
Nextseq
Illumina;
Hiseq
NA NA NA Illumina;
Nextseq
Illumina;
Nextseq
Illumina;
Nextseq
Genetic maps 0-1x15A ;
FGOH04 × BB25
NA NB29 × NB85 NA NB29 × NB85 15Ax6A 15Ax6A FGOH04 × BB25
Genome assembly statistics
Genomesize(Mb) 46.50 51.76 50.12 48.03 49.03 45.3 48.60 49.7
Total contigs 86 74 55 43 47 163 52 42
Gene number 11541 11245 11214 11199 11193 12183 11551 11557
GC%content 46.68 45.21 45.57 45.94 45.71 NA NA NA
Repeats(%) 31.90 35.80 NA NA NA 27.50 34.30 31.80
Secondary
Metabolites
72 82 38 39 59 77 79 76
Predicted effectors 282 NA NA NA NA 205 201 207
Genome data source
NCBIBioProject PRJNA50389;PRJNA392275 PRJEB18107 PRJNA577527 PRJNA577527 PRJNA577527 PRJNA434142 PRJNA434143 PRJNA434144
NCBI GenBank
accession
GCA_000166005.1;
GCA_006112615.1
GCA_900232045.2 GCA_009728665.1 GCA_009728675.1 GCA_009728655.1 GCA_008086755.1 GCA_008086725.1 GCA_008086845.1
References Ellwoodetal.,2010.
Wyatt et al., 2018, 2020;
Moolhuijzenetal.,2020;
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Wyatt et al., 2020;
Moolhuijzenetal.,
2020
Wyatt et al., 2020;
Moolhuijzenetal.,
2020
Wyatt et al., 2020;
Moolhuijzenetal.,
2020

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 19
nottheresultof ancestral inheritance or an interspecies hybridization event.
Orthologousgeneanalysisidentiedmultiple copies of orthologous groups
representingparalogsofgene family expansions within ve P. teres isolates,
and several of these orthologous groups contained genes responsible for the
productionofsecondarymetabolites.Comparativeanalysisofeffectorproteins
between ve P. teres genomes identied isolate-specic effectors that were
found to be expressed in planta and possibly responsible for isolate’s differential
response on different barley genotypes. In P. teres genomes, accessory regions
were identied on the ends of chromosomes within sub-telomeric regions
wherebreaksinsyntenyandsyntenybetweennon-homologouschromosomes
occurred. These regions clustered nearest to TEs and contained higher number
Table 2
(Continued)
Table 2GenomesequencingandassemblystatisticsofcurrentlysequencedP. teres f. teres and
P. teres f. maculata isolates
P. teres f. teres P. teres f. teres
Isolate name 0-1 W1-1 NB29 NB73 NB85 15A 6A FGOH04Ptt-21
Collection site Ontario, Canada Western Australia Western Australia Queensland,
Australia
Queensland,
Australia
California, USA California, USA North Dakota, USA
Collection date Before 1998 2009 1985 1994 1995 1984–1986 Unknown Before 2017
Sequencing information
Sequencing platform Solexa;
PacBio
PacBio;
Illumina; HiSeq;
Optical map
PacBio PacBio PacBio PacBio PacBio PacBio
RNA-Seq Illumina
Nextseq
Illumina;
Hiseq
NA NA NA Illumina;
Nextseq
Illumina;
Nextseq
Illumina;
Nextseq
Genetic maps 0-1x15A ;
FGOH04 × BB25
NA NB29 × NB85 NA NB29 × NB85 15Ax6A 15Ax6A FGOH04 × BB25
Genome assembly statistics
Genomesize(Mb) 46.50 51.76 50.12 48.03 49.03 45.3 48.60 49.7
Total contigs 86 74 55 43 47 163 52 42
Gene number 11541 11245 11214 11199 11193 12183 11551 11557
GC%content 46.68 45.21 45.57 45.94 45.71 NA NA NA
Repeats(%) 31.90 35.80 NA NA NA 27.50 34.30 31.80
Secondary
Metabolites
72 82 38 39 59 77 79 76
Predicted effectors 282 NA NA NA NA 205 201 207
Genome data source
NCBIBioProject PRJNA50389;PRJNA392275 PRJEB18107 PRJNA577527 PRJNA577527 PRJNA577527 PRJNA434142 PRJNA434143 PRJNA434144
NCBI GenBank
accession
GCA_000166005.1;
GCA_006112615.1
GCA_900232045.2 GCA_009728665.1 GCA_009728675.1 GCA_009728655.1 GCA_008086755.1 GCA_008086725.1 GCA_008086845.1
References Ellwoodetal.,2010.
Wyatt et al., 2018, 2020;
Moolhuijzenetal.,2020;
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Wyatt et al., 2020;
Moolhuijzenetal.,
2020
Wyatt et al., 2020;
Moolhuijzenetal.,
2020
Wyatt et al., 2020;
Moolhuijzenetal.,
2020

Epidemiology, molecular biology and control of net blotch20
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
of accessory genes. Five P. teres f. teresisolatesincludedinthisstudywereused
in at least one bi-parental mapping study (0-1 × 15A, 15A × 6A, FGOH04P. teres
f. teres-21 × BB25) that reported a total of 15 virulence/avirulence quantitative
traitloci(QTL)(Weilandetal.,1999;Laietal.,2007;Shjerveetal.,2014;Koladia
etal.,2017).Outof15uniqueQTLidentied,14spannedaccessorygenomic
regionsand10werelocalisedinsub-telomericregions.Thissuggeststhatthe
sub-telomericaccessorygenomicregionsharbourmostoftheknownvirulence
loci and can undergo rapid evolution.
8 Identication of genes associated with virulence/
avirulence by QTL and association mapping
Bi-parental and genome-wide association mapping studies have been
conducted to identify QTL/genes associated with virulence/avirulence in
P. teres f. teres P. teres f. maculata
BB25 HRS09122 HRS09139 SG1 FG0B10Ptm-1
Denmark NewSouthWales,
Australia
South Australia Western Australia North Dakota,
USA
Before 2017 2009 2009 1996 2010
PacBio PacBio PacBio PacBio; Illumina;
HiSeq
PacBio
Illumina ;
Nextseq
NA NA Illumina;
Hiseq
NA
FGOH04 × BB25 NA FGOB10 × SG1 FGOB10 × SG1
50.60 47.98 50.89 41.28 39.27
115 44 91 47 46
11986 10555 10579 11165 11080
NA NA NA 46.86 47.37
35.60 NA NA 21.00 NA
75 61 36 47 45
201 NA NA NA NA
PRJNA434145 PRJNA577527 PRJNA577527 PRJEB18107 PRJNA417860
GCA_008086785.1 GCA_009728645.1 GCA_009728635.1 GCA_900231935.2 NA
Wyatt et al., 2020;
Moolhuijzenetal.,
2020
Moolhuijzenetal.,
2020;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzenetal.,
2020
Syme et al., 2018;
Moolhuijzen
et al., 2020
Table 2
(Continued)

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 21
P. teres(Beattieet al.,2007;Carlsenetal., 2017; Kinzer,2015; Koladia etal.,
2017;Laiet al.,2007;Martin etal.,2020; Shjerve etal.,2014; Weilandet al.,
1999). Comparisons between QTLregions identied in the different studies
has been difcult as different markersystems were used and chromosomes
werenotconsistentlynumbered,withsomestudiesusingtheP. tritici-repentis
genome sequence as a reference to allocate numbers to P. teres chromosomes.
Reference genomes for both P. teres f. teres and P. teres f. maculata have
recentlybecome available and enable us nowtolocatepreviouslyidentied
QTLregionsontheP. teres genomes (Syme et al., 2018; Wyatt et al., 2018). We
havesummarisedthe QTL regionsidentiedin previous studies andlocated
them on the reference genomes for convenient comparison in future studies.
The chromosomes of P. teres f. teres and P. teres f. maculata referred to in this
sectionareclassiedaccordingtothereferencegenomesW1-1(Table2)and
SG1 (Table 2), respectively.
Allpublished QTLregionshavebeensummarised inTables 3and4 and
Figs 4 and 5.The rst mapping study in P. teres was conducted by Weiland
et al. (1999) who used 82 progeny froma cross between the two P. teres f.
teres isolates 0-1 from Ontario, Canada and 15A from California, USA. The locus
AvrHarwasidentiedin15A,conferringlowvirulence/avirulenceonthebarley
lineHarbin(Table3,Fig.4)andwascloselylinkedtothreeco-localisedRAPD
markers on chromosome 5. As the barley line Harbin possesses a dominant
resistance gene against net form net blotch (Mode and Schaller, 1958), it
was suggested that AvrHar could be the respective dominant avirulence
geneaccordingto the gene-for-gene model(Laietal.,2007).Anotherstudy
implementingAFLPmarkerson78isolatesfromthesameP. teres f. teres 0-1/15A
crossandphenotypingtheseonPrato,Tifang,CanadianLakeShore(CLS)and
Ming reported a locus (AvrPra2)avirulentonCLSandTifang.AccordingtoLai
etal.(2007)theidentiedlocus AvrPra2co-segregatedwith theAvrHar locus
onchromosome5describedbyWeilandetal.(1999).However,theavirulence
of AvrPra2 and AvrHarwereconferredbyparent0-1and15A,respectively.Lai
etal.(2007)suggestedthattheco-segregationmightbeduetodifferentalleles
of the same gene or different genes located close together. According to the
QTL locations on the W1-1 reference genome (Table 3, Fig. 4), AvrHar and
AvrPra2aresituatedapproximately1.2Mbapart,conrmingthattheseQTLare
represented by different genes.
A unique avirulence gene, AvrHeartland, was reported for the barley line
HeartlandusingacrossbetweentwoCanadianisolatesWRS1906andWRS1607
with 67 progeny (Beattie et al.,2007). Unfortunately this gene could not be
locatedonthereferencegenome.AnotherQTLmappingstudyreportedfour
virulent loci VK1, VK2, VR1 and VR2,withVK1 and VK2 virulent on barley Kombar
and VR1 and VR2virulentonRika(Shjerveetal.,2014).Amappingpopulation
of118progenyderivedfromtwoUSAP. teres f. teresisolates,15Aand6Awas

Epidemiology, molecular biology and control of net blotch22
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Table 3SummaryofQTL/genesreportedforP. teres
Locus
Marker
type
No.
proaCross
Vir/
AvibChroc
Positiond
Marker at
peakofQTL Genotypee
LOD
scorefR2 g
Parent
contributing
theQTL ReferenceStarting Ending
QTLidentiedin P. teres f. teres
AvrHar RAPD 82 0-1/15A Avi 5 4193688 - - Harbin 36 72 15A Weiland
etal.(1999)
AvrPra2 AFLP 78 0-1/15A Avi 5 3008702 - M11E13190-
M12E11250
Tifang,
CanadianLake
Shore
5.3 - 0-1 Laietal.
(2007)
AvrPra1 AFLP 78 0-1/15A Avi 9 - 1256349 M15E20400-
M12E11250
Prato 7.2 - 0-1 Laietal.
(2007)
AvrHeartland AFLP 67 WRS1607/
WRS1906
Avi 1 - - GTTA285-
CGAA1600
Heartland - - WRS1906 Beattieetal.
(2007)
VR1 SNP, SSR,
AFLP
118 6A/15A Vir 2 2066532 3939100 07628_18 Rika 5–10 35 6A Shjerve
etal.(2014)
VR2 SNP, SSR,
AFLP
118 6A/15A Vir 10 1516021 2300448 10177_27 Rika 10–15 20 6A Shjerveetal.
(2014)
VK1 SNP, SSR,
AFLP
118 6A/15A Vir 3 1041300 1650040 18850_67 Kombar 15–20 26 15A Shjerve
etal.(2014)
VK2 SNP, SSR,
AFLP
118 6A/15A Vir 2 442489 507296 03948_8 Kombar 10–15 19 15A Shjerve
etal.(2014)
PttTif1* SNP 109 BB25/
FGOH04Ptt-21
Vir 1 1519813 2279473 1579_4251 CI4822,Tifang,
Manchurian
11,30,35 45,67,74 FGOH04Ptt-21 Koladia
etal.(2017)
PttTif2* SNP 109 BB25/
FGOH04Ptt-21
Vir 8 - 593132 547_32651 Tifang 4.4 3 FGOH04Ptt-21 Koladia
etal.(2017)
PttBee1* SNP 109 BB25/
FGOH04Ptt-21
Vir 1 - 2776486 1588_12100 Beecher 24.0 56 FGOH04Ptt-21 Koladia
etal.(2017)

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 23
PttBee2* SNP 109 BB25/
FGOH04Ptt-21
Vir 5 316575 525281 752_3220 Beecher 7.0 17 FGOH04Ptt-21 Koladia
etal.(2017)
PttPin1* SNP 109 BB25/
FGOH04Ptt-21
Vir 3 5804230 - 1667_1175 Pinnacle 14.0 49 BB25 Koladia
etal.(2017)
PttPin2* SNP 109 BB25/
FGOH04Ptt-21
Vir 12 1044631 1438885 2428_2378 Pinnacle 3.1 11 FGOH04Ptt-21 Koladia
etal.(2017)
PttCel1* SNP 109 BB25/
FGOH04Ptt-21
Vir 8 2843621 3029139 1454_3802 Tifang,
Celebration
3.2,7.0 7,17 FGOH04Ptt-21 Koladia
etal.(2017)
PttCel2* SNP 109 BB25/
FGOH04Ptt-21
Vir 9 2562601 2806018 994_25330 Celebration 5.1 17 FGOH04Ptt-21 Koladia
etal.(2017)
PttHec1* SNP 109 BB25/
FGOH04Ptt-21
Vir 8 2652633 - 252_25719 Hector, Stellar 3.1,6.5 11,18 FGOH04Ptt-21 Koladia
etal.(2017)
PttSki_3 DArTseq 78 NB29/
HRS09122
Vir 3 114611 - 28946459 Skiff 6.6 24 HRS09122 Martinetal.
(2020)
PttBee_5 DArTseq 78 NB29/
HRS09122
Vir 5 5183980 5208563 28948016 Beecher 4.0 15 NB29 Martinetal.
(2020)
PttSki_5 DArTseq 78 NB29/
HRS09122
Vir 5 3980200 4457075 28948170 Skiff 4.8 19 HRS09122 Martinetal.
(2020)
PttBee_9 DArTseq 78 NB29/
HRS09122
Vir 9 1073073 1122817 28945299 Beecher 3.0 11 NB29 Martinetal.
(2020)
PttBee_3 DArTseq 72 NB29/NB85 Vir 3 796216 970812 28945535 Beecher 12.0 36 NB29 Martinetal.
(2020)
PttBee_7 DArTseq 72 NB29/NB85 Vir 7 2166986 2928737 28946946 Beecher 3.9 11 NB85 Martinetal.
(2020)
PttPri_7 DArTseq 72 NB29/NB85 Vir 7 2166986 2928737 28949493 Prior 3.6 18 NB85 Martinetal.
(2020)
PttBee_8 DArTseq 72 NB29/NB85 Vir 8 1883966 1972858 28949931 Beecher 3.0 7 NB29 Martinetal.
(2020)
(Continued)

Epidemiology, molecular biology and control of net blotch24
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
QTLidentiedin P. teres f. maculata
VQTL1A
VQTL1B
VQTL1C
SNP 105 SG1/
FGOB10Ptm-1
Vir 1 117776
170676
316665
117958
170836
316851
SNP_11381_87
SNP_2207_88
SNP_16439_27
TR326, Skiff
81-82/033
PI 392501
8.4,5.8
5.5
9.4
21,23
21
34
FGOB10Ptm-1 Carlsen
etal.(2017)
VQTL2 SNP 105 SG1/
FGOB10Ptm-1
Vir 3 3154965 3155149 SNP_12879_149 Skiff 5.5 22 FGOB10Ptm-1 Carlsen
etal.(2017)
VQTL3 SNP 105 SG1/
FGOB10Ptm-1
Vir 5 2709841 2710025 SNP_41831_15 Skiff 5.3 20 FGOB10Ptm-1 Carlsen
etal.(2017)
VQTL4 SNP 105 SG1/
FGOB10Ptm-1
Vir 2 1108007 1108161 SNP_21264_143 81-82/033
PI 392501
8.0,11.0 30,37 FGOB10Ptm-1 Carlsen
etal.(2017)
VQTL5 SNP 105 SG1/
FGOB10Ptm-1
Vir 3 2419962 2420099 SNP_2673_169 81-82/033,
TR326,
PI 392501
6.0,6.6,
9.3
33,26,
34
FGOB10Ptm-1 Carlsen
etal.(2017)
VQTL6 SNP 105 SG1/
FGOB10Ptm-1
Vir 4 1618736 1618905 SNP_26064_6 PI 392501 5.0 20 FGOB10Ptm-1 Carlsen
etal.(2017)
a Number of progeny, bvirulence nature of theallele(Vir= virulent; Avi=avirulent), c,d chromosome location according to W1-1 reference genome, ename of the
genotype that the allele is virulent/avirulent on, flogarithm of odds, gpercentageofphenotypicvariationexplainedbytheQTL.
Locus
Marker
type
No.
proaCross
Vir/
AvibChroc
Positiond
Marker at
peakofQTL Genotypee
LOD
scorefR2 g
Parent
contributing
theQTL ReferenceStarting Ending
Table 3(Continued)

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 25
usedwiththegeneticmapconsistingofAFLP,SSRandsinglenucleotiderepeat
(SNP)markers.Virulence loci VK1 and VK2 werelocated on chromosomes3
and2with 26% and 19% of the phenotypicvarianceexplained,respectively,
whileVR1 and VR2werepositionedonchromosomes2and10with35%and
20%ofthephenotypicvarianceexplained,respectively.
AQTLmapping study conductedbyKoladiaet al. (2017) reported nine
QTLassociatedwitheightbarleygenotypesusingaP. teres f. teres population
consistingof 109 progenydevelopedbycrossingDanish isolate, BB25,with
the USA isolate FGOH04Ptt-21.Three QTL were reported to be major QTL
accountingfor morethan45% of thephenotypicvariation.Two oftheseQTL
werelocatedonchromosome1andoneonchromosome3.OneoftheQTLon
chromosome 1 conferred virulence on the commonly used differential cultivars
Manchurian,Tifang and CI4922, while the other QTL on chromosome 1 was
virulentonBeecher.Thechromosome3QTLwasassociatedwithvirulenceon
Pinnacle.
A genome-wide association mapping study of 188 Australian P. teres f.
teresisolatescollectedfromvestatesinAustraliaandassessedon20barley
linesidentied thepresenceof14 differentgenomic regionsassociatedwith
virulence,mainlyonchromosome3and5withoneeachonchromosomes1,6
and9(Martinetal.,2020)(Table4,Fig.4).Inordertoconrmtheseidentied
genomicregions,QTLanalysisoftwobi-parentalmappingpopulations,NB029/
HRS09122 (78 progeny) and NB029/NB085 (72 progeny) was undertaken.
ProgenywerephenotypedonBeecher,SkiffandPrior.Fourregionsidentied
byGWASwereconrmedby bi-parentalQTLmapping.The DArTseqmarker
system was implemented for both GWAS and QTL mapping. Martin et al.
(2020)reportedthatQTLVK1onchromosome3whichconferredvirulenceon
Kombar(Shjerveetal.,2014),wasinasimilarlocationtoPttBee_3andQTL3,
witha distanceof58kb betweentheirankingmarkers.Thisissimilar tothe
70 kb distance observed on the W1-1 reference genome map (Fig. 4). The
SkiffvirulentQTL,PttSki_5onchromosome5identiedbyMartinetal.(2020)
in the bi-parental mapping population NB29/HRS09122 seems to be in the
samelocationastheQTLAvrHar,which Weilandetal.(1999)identied tobe
avirulenton Harbin.Co-localising of theseQTLsuggeststhatthisregionmay
containmultiplecloselylinked genes associated withvirulence/avirulenceor
thatdifferentalleles of the samegeneconfervirulence/avirulence.Both QTL
associatedwiththe Beecher virulence contributed by a USA isolate (Koladia
etal.,2017)areinadifferentlocationtotheBeechervirulentQTLidentiedby
Martinetal.(2020)inisolatesfromAustralia.ThissuggeststhatQTLassociated
with virulence on the same cultivar may not be conserved between isolates
from different geographical regions.
Only one QTL mapping study in P. teres f. maculata has been reported
(Carlsen et al., 2017). The P. teres f. maculata parent FGOB10Ptm-1 from the

Epidemiology, molecular biology and control of net blotch26
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Table 4SummaryofQTLreportedforP. teres f. teresbygenome-wideassociationmapping(Martinetal.,2020)
QTLIDaGenotypebMarker Chroc
Positiond
LODeR2 f
Starting Ending
QTL1 Beecher 28947393 1 679123 6.07 14.58
QTL2 Fleet, Skiff Herta 41806492|F|0-7:A>G-7:A>G 3 102867 114611 5.02–6.7 18.93–16.33
QTL3 Beecher, Kombar,
Maritime, Beecher
28946135 3 788584 796281 5.26–9.77 12.07–23.58
QTL4 Beecher, Maritime 36352701 3 1028769 - 5.34–7.46 12.71–17.48
QTL5 CIho 11458 100133310 3 1480380 - 5.41 12.46
QTL6 Herta, Skiff 28945761 3 1905727 - 6.71–6.95 16.12–16.93
QTL7 Beecher 28949829|F|0-28:C>T-28:C>T 3 3985143 - 5.15 11.03
QTL8 Commander 28945998 3 5556417 - 5.05 12.69
QTL9 Fleet,Yerong 28948035 5 4110850 - 7.59–8.14 18.72–21.47
QTL10 Fleet,Yerong 36347132 5 4456299 - 7.27–8.89 17.55–22.98
QTL11 Harbin,Prior 28950148|F|0-5:A>C-5:A>C 5 5195933 5209123 5.11–8.55 13.5–21.66
QTL12 Corvette, Harbin,
Orior
36352140|F|0-22:T>C-22:T>C 5 5243799 - 4.79 –8.34 14.19–21.61
QTL13 Tallon 100139818 6 410017 - 5.04 10.9
QTL14 Beecher 28949038 9 839959 - 4.82 10.19
aQTLidentication,bmarkeratQTL,c,d chromosome location on W1-1 reference genome, e logarithm of odds, f percentage of phenotypic variation explained.

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 27
USAwascrossedwithSG1fromWesternAustraliaand105progenyproduced.
SixQTLwere detected and explained 20–37% of the phenotypic variance in
barley lines Skiff, 81-82/033, TR326, and PI 392501 (Table 3, Fig. 5).
MultipleQTLwithbothmajorandminoreffecthavebeenidentiedwithin
the P. teres genomes. Studies conducted using bi-parental mapping populations
and GWAS suggest that P. teres f. tereschromosomes3and5harbourQTL/gene-
richregions(Fig.4)associatedwith virulence/avirulence.Withtheexceptionof
chromosomes 4 and 11, all other chromosomes of the P. teres f. teres genome
hadatleast onevirulence/avirulenceQTL demonstratingthecomplex genetic
basis of P. teres f. teresvirulence.Virulencesondifferentbarleygenotypesare
governed by different genes and genes that confer virulence on one barley
genotypemaybeavirulentonanother.Geneswhichareresponsibleforvirulence
on one barley genotype (e.g. Beecher) in one geographical area (e.g. USA) may
notbeassociatedwiththesamevirulencegenesforthesamebarleygenotype
from another geographical area (e.g. Australia) (Koladia et al., 2017; Martin et al.,
2020). Thus, in order to better understand this complex pathosystem, more
studiesarewarranted as improvingourunderstandingofthegeneticbasis of
the P. teres-barley pathosystem is crucial for developing cultivars.
9 Managing the net blotches
Netblotchesareeconomically important diseasesandareregarded amajor
constrainttobarleyproductioninmostbarley-growingregionsworldwide(Liu
etal.,2011). Netformnetblotchofbarleyis common throughout the major
barley-growingregionsoftheworld,includingMorocco(Amezrouetal.,2018;
Douiyssi et al., 1998), the USA (Buchannon and McDonald, 1965; Steffenson
andWebster,1992a),Australia(Cakiretal.,2003;Ellwoodetal.,2019;Fowler
etal.,2017;Khan,1973;Lehmensieketal.,2007;Shipton,1966;Wallworketal.,
2016), South Africa (Campbell et al., 2002), Canada (Tekauz, 1990; Xi et al.,
1999),Sweden(Jonssonetal.,1997),Finland(RobinsonandJalli,1996),Syria
andTunisia(Bouajilaetal.,2011),RussiaandBelarus(Novakazi etal.,2019a),
Sardinia(island west of Italy) (Rau etal.,2003), Germany(Vatteret al.,2017),
Norway(Wonnebergeretal.,2017),NewZealand(CromeyandParkes,2003),
andFrance(Youcef‐Benkada et al., 1994). Spot form net blotch is a prevalent
disease of barley in many countries (McLean et al., 2009),including Canada
(Tekauz,1990),Denmark(Smedegård-Petersen,1971),Finland(Mäkelä,1975),
France(Arabi et al., 1992),SouthAfrica(Louw,1996; Campbell et al.,1999),
Turkey(OğuzandKarakaya,2017)andtheUSA(LiuandFriesen,2010;Lartey
etal.,2013).ItwasrstreportedinWesternAustraliain1977(KhanandTekauz,
1982), however, was not detected in eastern Australia until 1990 (Wallwork
et al., 1992; Wallwork, 1995) and has since spread through southern and
easternAustralia(Wallwork,1995;McLeanetal.,2009).

Epidemiology, molecular biology and control of net blotch28
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Yieldlosses associatedwithnet formnetblotchtypically rangebetween
10%and 40% (SteffensonandWebster,1992a).Under Australian conditions
yieldlossesabove20%arecommon(Shipton,1966;Khan,1987)withlossesin
excessof60%reportedinQueensland(Qld)incropsofGilbert(Poulsenetal.,
1999)andupto70%onthesusceptiblevarietyMaritimeinSouthAustralia(SA)
under epidemic conditions (Wallwork,2011). Yield loss reductions areoften
associated with signicant reductions in seed weight and grain size (Khan,
1987; Shipton, 1966; Sutton and Steele, 1983; Poulsen et al., 1999; Rees et al.,
1999). Yield loss response curve trials conducted over a three-year period
(2014–2016) in Qld, Australia indicated increased yield loss with increased
susceptibility to net form net blotch. Yield loss ranged between 9.5% and
Figure 4 Map of the Pyrenophora teres f. teres reference genome W1-1 indicating
genomicregionsassociatedwithvirulence/avirulenceidentiedthroughQTLmapping
and GWAS (green). Map distance in bps is indicated on the left of the bar. Created using
MapChart2.32v(Voorrips,2002).

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 29
18.6%inthevarietyCompass(moderatelyresistanttomoderatelysusceptible),
whereas yield loss in the very susceptible variety Charger werein excess of
40%.Disease levels did not haveabigimpacton grain quality,with biggest
differencesobserved inseedretentionlevels(%ofseed >2.5mm) (Snyman
et al., 2017).Yield loss due to spot form net blotch is not well documented
withmostinformationreportedforAustralia(McLeanetal.,2009).InWestern
Australia,yieldlosses upto 44%as aresultofspotformnetblotchinfections
Figure 5 Map of the P. teres f. maculata SG1 reference genome indicating genomic
regions associated with virulence/avirulence identied through QTL mapping. Map
distance in bps is indicated on the left of the bar. Created using MapChart 2.32v
(Voorrips,2002).

Epidemiology, molecular biology and control of net blotch30
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
wererecorded, dependant on season, sowing dateandvariety(Khan,1989;
Jayasenaetal.,2007).
Several methods are applied to manage P. teres, including fungicides,
culturalpracticesand host resistance (Liu et al.,2011).Rotation to reduceor
eliminate primary inoculum is an important factor in cultural management, as
is chemical control using either seed dressing to reduce primary inoculum or
foliar application to lower disease levels (Liu et al., 2011).As the net blotch
pathogens persist on plant residue, the adoption of reduced- or zero-till
practices has signicantly increased the incidence in recent years (McLean
et al., 2009). Crop rotation forms an integral part of the successful cultivation of
barleyandgrowersareadvisednottoplantbarleyonbarley(Reesetal.,1999).
Planting successive barley crops in the same paddock increases the incidence
of the net blotch diseases and cultivation of the same variety will lead to an
increase in the presence of pathotypes virulent on that particular variety and
put increased pressure on effective resistance genes. Best practice includes
crop rotation with non-host crops. During prolonged periods of drought,
stubblebreakdownoccursoveralongerperiodoftimeandstubblefromcrops
grownafewyearsearliercouldserveasasourceofinoculum,emphasisingthe
need for crop rotation.
In Australia, all current barley varieties and varieties considered for release
are rated for resistance to a suite of diseases and pathogens through the
National VarietyTrial disease screening process (https://www.nvtonline.com
.au). They are categorised in nine resistance categories rating from resistant (R)
toverysusceptible(VS).Thesegenotypesarescreenedannuallyinnationwide
diseasenurseries,withdiseaseratingsassignedandreviewedonayearlybasis.
Growingahigh-yieldingwell-adapted resistantvariety providesthe most
economic and environmentally friendly means of disease control. A study
conductedinQldbetween2014and2017onyieldlossassociatedwith net
form net blotch indicated that under disease pressure representative of that
expected in commercial paddocks, the level of net form net blotch disease in
varietieswithsomelevelofresistancedidnotreachthelevelsanticipatedwhen
screeningunder high diseasepressures.Thisindicates thatgrowingvarieties
withatleastsomelevelofresistancetonetformnetblotchcanaidinlimiting
yield and quality loss (Forknall et al., 2019).
The spread of pathotypes depends on the importance of long distance
dispersalofseed-borneinoculumorwind-borneascospores.Therelativetness
ofpathotypeswilldependonbarleyvarietiesgrown,cultivationpracticesand
chemicalcontrolmeasures(Jonssonetal.,1999).Anincreaseinthecultivation
of varieties susceptible to the net blotch diseases would see an increase in
incidenceandseverity(McLeanetal.,2010a;McLean,2016).
Pyrenophora teres f. teresisaseed-bornepathogenthatcanspreadwith
infected seed and could aid the spread of isolates resistant to fungicides. It

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 31
is important that growers use an appropriateseed treatment, registered for
net blotch control at the recommended rate and to ensure that seed treatment
is effectively applied, reducing the effect of seed-borne inoculum (Rees et al.,
1999). Foliar fungicides are applied routinely in most barley crops and should
beaimedatprotectingthetoptwoleaflayers.Toensurethatfungicidesremain
effective,it is important to limit fungicide application by spraying only when
necessary,rotatefungicideswithdifferentmodesofactionandusefungicidesat
recommended rates. Fungicide applications are more effective if applied before
the disease becomes established in the crop. This requires regular monitoring
toensurecropscanbesprayedattherstsignofdisease.Whenconditionsare
favourablefordiseasedevelopment,morefrequentcropinspections willbe
needed and repeat fungicide applications may be necessary.
9.1 Fungicide resistance
Along with cultural practices, the application of fungicides and the use of
resistant varieties are the main control strategies available for the management
of fungaldiseases.However,duetothe lack of adequate genetic resistance,
fungicides are frequently solely relied upon for the control of many fungal
diseases. Despite a considerable body of research that has been developed
overthelast50years,fungicideresistancecontinuestobeamajorconcernto
agricultureworldwidemirroringasituationthatclinics andanimal production
facilities are facing as a result of overuse of particular fungicide modes of action
(MOA)(Leadbeater,2014;Talbotetal.,2018;Builetal.,2019).Whenafungal
disease develops resistance to one fungicide, often many other fungicides
that share the same MOA are also at risk due to a phenomenon called cross-
resistance(Brent,1995). This frequentlyleadsto scenarios where pathogens
survivetheapplicationofmultiplefungicideswithdifferentMOA(Ruppetal.,
2017).Unfortunately,the rateat which new MOA arereleasedtothemarket
cannot match that of practical fungicide resistance development, resulting in a
progressive reduction in the number of available effective fungicides. This arms
racehasexacerbatedinrecentyearswiththeintroductionofbansonspecic
agricultural fungicides (e.g. chlorothalonil) due to health risk concerns (Arena
et al., 2018).
Fungicides used against the net blotch diseases are predominantly of the
azoleor demethylaseinhibitor(DMI, group3) class,althoughthereareother
groupslikethequinoneoutsideinhibitor(QoI,group11)and,morerecently,the
succinate dehydrogenase inhibitor (SDHI, group 7), that also play an important
roleinthecontrolofthesetwodiseases(Sierotzkietal.,2007;Mairetal.,2016).
Despite their relatively widespread use since their introductionin 1996,
QoI fungicides have traditionally provided good levels of control over the
net blotch diseases and no occurrences of crop protection failure have been

Epidemiology, molecular biology and control of net blotch32
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
reportedtodate.ThemutationG143AattheCYTbtargetsitehasbeenfoundin
allpathogensisolatedfromcropswhereeldresistancetoQoIswasreported.
This mutation is associated with cross-resistance to all QoI compounds and
controlfailureistobeexpectedincropswhereisolatescarryingthismutation
dominatethepopulation (FRAC,2020b).However,the presence of anintron
that interrupts the codon responsible for this mutation in the Cytb gene of net
blotch pathogens makes the evolution of this mutation quite unlikely, as codon
143ispartofthesplicesite(Sierotzkietal.,2007;OliverandHewitt,2014;Grasso
et al., 2006). This phenomenon, that seems to provide an apparent advantage
of QoIs overotherMOAs,needs to be considered carefullywhendesigning
adequate disease management strategies given that other lesser mutations
couldstilldevelop.Since2003,reducedsensitivitytoQoIshasbeendetected
inseveral Europeanbarley-growing regionsand, inallcases,mutationF129L
wasfoundtobewidespread,butnotassociatedwithsignicantreductionsin
thecontrolofnetblotches(Table5)(Semaretal.,2007;Sierotzkietal.,2007;
Rehfus, 2018).
Reduced sensitivity to SDHI fungicides in net blotch diseases was rst
reported in Europe in 2012 (Stammler et al., 2014). At that time, analysed
isolateswerefoundtocarrymutationSdhB-H277Y.Sincethen,aplethoraof
mutations affecting subunits C (N75S, G79R, H134R, S135R) and D (D124N,
D124E, H134R, D145G, E178K) in the Sdh complex have been described and
cross resistance between the different SDHI members established (Rehfus
etal.,2016;FRAC,2020c).Mutationsassociatedwithvaryinglevelsoflower
sensitivitytothemajorityofSDHIsarenowwidespreadinregionsofGermany,
France and Australia (Rehfus et al., 2016). Prior to 2019, crop protection failure
had not been observed and in vivoglasshouseexperimentshadshownthat
control of the mutant genotypes was always achieved when foliar SDHIs
wereapplied at fullrates(Stammler et al.,2014;Rehfus et al.,2016;FRAC,
2020c).However,awidespreadoutbreakofresistancetoauxapyroxadseed
dressingformulationinabarley-intensivegrowingregionofSouthAustralia
in 2019 seems to challenge these results. The high frequency at which
mutations SdhC-H134R and SdhD-D145G were found in South Australia,
togetherwiththe SDHI treatmentused,being aseeddressingformulation,
might have contributed to the lack of control seen in several farms across this
region.
For many years, DMI fungicides have been a key component in many
spray programs aimed at controlling the net blotch diseases. Introduced in
the70s,thisgroupoffungicideshasbeenusedagainstnetblotcheswithonly
onesignicant fungicideresistanceoutbreakbeingreported inthetwentieth
century. In 1983, seed dressing application with DMI triadimenol failed to
controlnetblotchinregionsoftheNorthIslandinNewZealand(Sheridanetal.,
1985). Interestingly, isolates collected from barley elds in 1984 seemed to

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 33
have similar levels of resistance to triadimenol and nuarimol to that of isolates
originating from UK and Denmark. These ndings suggest that net blotch
DMIresistantgenotypeswerealreadypresentinEuropepriorto thendings
reportedinNewZealand.
In2013,reducedsensitivitylevelstoseveralDMIswerefoundduringthein
vitro analysis of P. teres f. teres isolates collected across the central region of the
WesternAustralianwheatbelt(Mairetal.,2016).Themolecularcharacterisation
of these isolates revealed an overexpressed copy of the Cyp51A gene carrying
mutationF489Lwasthemechanismresponsiblefortherecordedreductionin
sensitivity(Table 5).Crop protectionfailureto differentDMIs was reportedin
2016inbarleycropsaffectedwithspotformnetblotchinthesouthernregion
of the Western Australian wheatbelt (Mair et al., 2020). The in vitro analysis
showed very high resistance levels to different DMIs in P. teres f. maculata
isolatescollectedfromtheseelds.Althoughtheresistantisolatescarriedthe
samemutationdiscoveredafewyearsearlierinP. teres f. teres, the high levels
ofresistancefound, seemedtobedue to the combinationofF489Land the
presenceofa134bpinsertioninthepromoteroftheCyp51A gene (Table 5).
Isolatescarryingonlythepromoterinsertionwerealsofound,butinthiscase,
thelevelofresistancewassignicantlylowerandcouldnotbecorrelatedwith
crop protection failure (Mair et al., 2020).
P. teresspp.areclassiedas‘medium-risk’pathogensintermsoffungicide
resistance development (FRAC, 2020a). However, the surprising capacity of
thesetwopathogenstoevolveresistancetoseveralSDHIandDMIfungicides
throughdifferentmechanismsisareectionofhowtheirhighlyplasticgenomes
can respond to adapt to the selection pressure applied by the use of fungicides.
Thisprobablychallengesthe validityofthecurrentclassicationand callsfor
arevisionoftheirplacementwithintheexistingfungicideresistanceriskscale.
Selection pressure is the driving force responsible for the evolution of
pathogen populations able to overcome management strategies. Disease
management requires a balanced combination of multiple effective control
methodologies in order to be sustainable and successful in the long run, and
in the case of net blotch diseases this includes the strategic use of adequate
geneticresistanceandfungicides(Waltersetal.,2012).Thelackofasufciently
diversepoolofstrategieswillinevitablyresultinsomestrategiesbeingunder
higher selection pressure than others. The time required to breed a new
resistant cultivar is often comparable to that of developing a new fungicide
(MortonandStaub,2008). Thisindicateshowimportantthemanagementof
timeis inthis armsracescenarioandhowdamagingit canbe toloosethese
resources too quickly due to poor disease management practices.
Despitetheavailabilityof somebarley varietieswithresistancetothenet
blotches, their lack of suitability for all barley growing regions and market
demand for susceptible varieties has led to growers developing a chronic

Epidemiology, molecular biology and control of net blotch34
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
over-reliance on chemical control. This has resulted in increased selection
pressureand fungicideresistancedevelopment (Sierotzkietal.,2007;Tucker
etal.,2015;Mairetal.,2016;Rehfusetal.,2016).Withthethreemajorfungicide
MOAcompromised in net blotch diseases, it is paramountthatgrowersand
advisers have up-to-date fungicide resistance information that can guide their
variety choices to produce optimal combinations of these management tools
suitable for particular crop environments. The deployment of monitoring
programmes capable of providing information on the resistance status of
regional and local net blotch pathogen populations, to determine best
fungicide treatments according to frequency of resistant mutations, seem to be
the most logical approach. To some extent, such programmes already exist in
cereal-growingregionsofEurope.AnexampleistheEuroWheatproject,which
has been collecting information on fungicide-resistant mutations affecting
DMI and SDHI fungicides in European populations of the wheat pathogen
Zymoseptoria tritici over a number of years (EuroWheat, 2020). Although less
current, similar information has been made available for the spread of mutations
affecting SDHI performance in European populations of net blotches (Rehfus
et al., 2016). Despite the enormous advantage of having these resources, no
monitoring strategy has yet addressed the need to provide within-season
fungicideresistanceinformationtogrowersandadvisers.Ultimately,theuseof
themosteffectivefungicidescancontributetoslowingdowntherateatwhich
netblotchdiseasesovercome genetic resistance,whichin turn will lead to a
reduction in fungicide use and a delay of fungicide resistance development.
9.2 Breeding strategies
Breeding for resistance to multiple diseases has been an objective of many
breeding programs and should remain a priority (Platz, 2005). High-risk
pathogens, such as P. teres, require great effort to achieve durable resistance
as virulence mutations can be recombined into many genetic backgrounds
and can be dispersed over long distances. Breeding efforts should focus
on quantitative resistance in an effort to achieve durability. If quantitative
resistancecannotbeachieved,majorgeneswillprovideprotection,butshould
be managed aggressively (McDonald and Linde, 2002). Pyramiding major
resistance genes will prolong the durability of each gene; however, higher
levels of resistance could be attained by the accumulation of resistance genes
withminoreffects(Jonssonetal.,1999;Qamaretal.,2008;Grewaletal.,2008).
The P. teres pathogens are very adaptive, highlighting the need for barley
breeders to use multiple sources of resistance to safeguard against mutations
(Poudel et al., 2019b). Ideally, resistance should be effective to all pathotypes
of P. terespresentinallareaswhereawidelyadaptedbarleyvariety isgrown
(BuchannonandMcDonald,1965).Resistance thatiseffective againstawide

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 35
Table 5SummaryoffungicideresistancemechanismsdescribedinP. teres f. teres and P. teres
f. maculata(TablemodiedfromIRELAND,2021)
Mechanism
Phenotypes by pathogen
and affected MOAs
Examples of resistant
mutations
Target-site mutation Reduced
sensitivity
P. teres f. teres –
DMI, Group 3
Cyp51A-F489L
P. teres f. teres –
SDHI, Group 7
SdhD-D145G
P. teres f. maculata
– Group 3
Cyp51A-F489L
P. teresa–QoI,
Group 11
Cytb-F129L
P. teresa – Group 7 SdhC-N75S
Resistant P. teres f. teres –
Group 7
SdhC-H134R
Multiple target-site
mutations
Reduced
sensitivity
P. teres f. teres –
Group 7
SdhD-D145G for Group 7
Resistant P. teres f. teres –
Group 7
SdhC-H134R for Group 7
Target-site
over-expression
Reduced
sensitivity
P. teres f. maculata
– Group 3
134-bp insertion at different
positions in the Cyp51A
promoter
Target-site mutation +
gene over-expression
Resistant P. teres f. maculata
– Group 3
134-bp insertion in
the Cyp51 promoter +
Cyp51A-F489L
aNodistinctionwasmadebetweenP. teres f. teres and P. teres f. maculata.

Epidemiology, molecular biology and control of net blotch36
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
spectrum of pathotypes may last longer than resistance exhibiting strong
differential reactions to different genotypes of the pathogen (Steffenson and
Webster, 1992b).
The P. teres populations are diverse and ever changing and will be
inuenced by current varieties, environmental conditions and cultivation
practicesfavouringinfection(Wallworketal.,2016).Thereportedevolutionof
these pathogens emphasise the importance of screening breeding material
with multiple isolates from different growingregions to account for varying
virulences (McLean et al., 2014). Considerable variation observed between
seedlingglasshouseandeldresponseshighlighttheriskassociatedwiththe
useofsingleisolatesforglasshousescreeningwhichmayresultinsusceptibility
inthe eld(Douiyssietal.,1998).Thisis alsosupportedby thedevelopment
of adult plant virulent pathotypes, emphasising the need for multi-pathotype
screeningatadultplantstageunder eldconditions(Platzetal.,2009).Lines
withresistancetoawiderangeofAustralianisolatesatbothseedlingandadult
plantstagehavebeenidentied(Guptaetal.,2003;Wallworketal.,2016)and
could be used as diverse resistance sources.
Population genetic analysis to date suggests that sexual reproduction
occurs within the worldwide P. teres population, but the contribution of
sexual and asexual reproduction varies between regions, possibly based on
environmentaldifferences(Liuetal.,2011).Sexualrecombination/hybridization
betweenandwithinformscanleadtotheformationofnovelpathotypes.Thisin
turnwouldincreasegeneticdiversityinthepopulationandincreasechallenges
in the host plant to prevent disease (Syme et al., 2018).
Wild relatives and landraces represent valuable reservoirs of traits left
behind as a consequence of domestication and may prove useful for crop
improvement. Varying levels of resistance to various pathogens, including
powderymildew(Blumeria graminis), scald (Rhynchosporium secalis), leaf rust
(Puccinia hordei) and net form net blotch (P. teres f. teres)havebeenidentied
in the Spanish barley core collection (Silvar et al., 2010) and resistance to P.
teres f. teres and spot blotch (Bipolaris sorokiniana) in the Vavilovcollection
(Novakazi et al., 2019a,b). Non-host resistance in barley grass should be
exploredto combat thediseasein cultivated barley(LindeandSmith, 2019).
Breedingstrategies,however,willneedtoberenewedregularlytostayahead
ofeverchangingpathogenpopulations(McDonaldandLinde,2002).
10 Conclusion and future trends
It is clear that there is a high variability in P. teres with respecttopopulation
genetics,phenotypic interactionswithdifferentbarleylinesandthe genomic
location and number of virulence/avirulence genes. The complex nature of the
P. teres-barley pathosystem indicates that a number of different factors need

© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
Epidemiology, molecular biology and control of net blotch 37
to be taken into account when breeding for durable resistance against this
pathogen.PhenotypingandgeneticdiversityandQTLmappingstudiesfurther
conrmthat therearecleardifferencesbetweenP. teres f. teres and P. teres f.
maculata and that these pathogens need to be studied as separate entities. To
date more studies have involved the form teres than the form maculata.
AlthoughsexualreproductionbetweenthetwoformsofP. teres have been
reportedtoberare,therecentidenticationofafungicide-resistanthybridin
Western Australia that has spread very quickly via asexual conidia production
andisvirulentonthewidelygrownbarleycultivarOxfordillustratesthatsexual
reproductionbetween the forms should not be ruled out and that thereisa
need to monitor hybrid occurrences.
Anumberofmarkersassociatedwithvirulence/avirulenceinP. teres have
beenidentiedandthereferencegenomesforboth formsarenowavailable.
Thiswill aidinthe characterizationoftheunderlyingmechanismsinvolvedin
thepathogen/hostinteractionsandwillbethenextsteptowardsbreedingfor
resistance against this pathogen.
Aswithotherpathogens,themonitoringofP. teres is a continual process as
these pathogens are constantly evolving due to frequent sexual recombination
withineachformandpossiblybetweenforms.Thesechangescanleadtothe
rapid development of new virulences with the potential to overcome major
host resistance genes and fungicides.
11 Where to look for further information
11.1 Further reading
AthoroughreviewofbothformsofP. tereshasbeenpublishedbyLiuetal.(2011).
A good review specically on spot form of net blotch is McLean et al.
(2009b).Resistance/susceptibilitylociassociatedwiththehostandeffectorloci
identiedinP. tereshaverecentlybeenreviewedbyClareetal.(2020).
11.2 Key conferences
TheInternationalWorkshoponBarleyLeafDiseasesisattendedbybarleyfoliar
disease researchers from many different countries.
TheInternationalBarleyGeneticsSymposiumiswellattendedbymembers
of the barley community.
12 Acknowledgements
TheauthorswouldliketothankDrNathanWyatt,FriesenLab,USDA-ARSCereal
CropResearchUnit,whohelpedtondQTLlocationson theW1-1 reference
genome.

Epidemiology, molecular biology and control of net blotch38
© Burleigh Dodds Science Publishing Limited, 2022. All rights reserved.
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