Pyrenophora Teres F. Teres Isolates and Its Implications for Resistance Breeding

The virulence of Finnish Pyrenophora teres f. teres isolates and its implications for resistance breeding

Doctoral Dissertation MTT is publishing its research findings in two series of publications: MTT Science and MTT Growth. Marja Jalli

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The virulence of Finnish Pyrenophora teres f. teres isolates and its implications for resistance breeding

Doctoral Dissertation

Marja Jalli

Academic Dissertation: To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Viikki, Auditorium C1 (Latokartanonkaari 5), on 23rd April 2010, at 12:00 o’clock. Supervisor: Professor Aarne Kurppa Plant Production Research MTT Agrifood Research Finland Jokioinen, Finland

Pre-reviewers: Associate Professor Lisa Munk Department of Plant Biology University of Copenhagen ISBN 978-952-487-273-7 (Print) Copenhagen, Denmark ISBN 978-952-487-274-4 (Electronic) ISSN 1798-1824 (Printed version) Associate Professor Erland Liljeroth ISSN 1798-1840 (Electronic version) Department of Plant Protection Biology Swedish University of Agricultural http://www.mtt.fi/mtttiede/pdf/mtttiede9.pdf Sciences Copyright MTT Agrifood Research Finland Alnarp, Sweden Marja Jalli Distribution and sale Opponent: MTT Agrifood Research Finland, Adjunct Professor Andrej Tekauz Media and Information services, Agriculture and Agri-Food Canada FI-31600 Jokioinen, phone +358 3 41881, Cereal Research Centre e-mail [email protected] Winnipeg, Canada Printing year 2010 Custos: Cover picture Marja Jalli: Helena and net blotch resistant barley landraces. Professor Minna Pirhonen Printing house Plant Pathology Laboratory Tampereen Department of Agricultural Sciences Yliopistopaino Juvenes Print Oy University of Helsinki, Finland

2 MTT SCIENCE 9 The virulence of Finnish Pyrenophora teres f. teres isolates and its implications for resistance breeding

Marja Jalli MTT, Plant Production Research, Jokioinen, FI-31600 Jokioinen [email protected]

Abstract

n Finland, barley, Hordeum vulgare L., barley seedlings under greenhouse condi- covers 50 % of the total acreage devot- tions is a feasible and cost efficient meth- ed to cereal cultivation. The most com- od to describe the virulence spectrum of Imon disease of barley in Finland is net the pathogen. However, the environmental blotch, a foliar disease caused by the asco- conditions of temperature, light and hu- mycete Pyrenophora teres Drechsler. Dis- midity, need to be stable to achieve reliable ease resistance based on plant genes is an and comparable results between different environmentally friendly and economical studies. Inoculum concentration and the way to manage plant diseases caused by bi- seedling leaf used to gauge virulence had otic stresses. Development of a disease re- significant effects. Barley grain size, mor- sistance breeding programme is dependent phological traits of P. teres isolates, spore on knowledge of the pathogen. In addi- production and growth rate on agar did tion to information on the epidemiology not affect the expression of virulence. A and virulence of a pathogen, knowledge on common barley differential set to charac- how the pathogen evolves and the nature terize the P. teres virulence was developed of the risks that might arise in the future and is recommended to be used globally: are essential issues that need to be taken c-8755, c-20019, CI 5791, CI 9825, Cana- into account to achieve the final breed- dian Lakeshore, Harbin, Prior, Skiff, and ing aims. Harrington.

The main objectives of this study were The virulence spectrum of FinnishP. teres to establish reliable and efficient testing f. teres isolates collected in 1994-2007 was methods for Pyrenophora teres f. teres viru- constant both within and between the lence screening, and to understand the role years. The results indicated differences in of virulence of Pyrenophora teres f. teres in the pathogen’s aggressiveness and in barley Finland from a disease resistance breed- genotypes resistance. However, differenc- ing point of view. The virulence of P. teres es in virulence were rarely significant. No was studied by testing 239 Finnish P. teres virulent reactions were recorded on barley f. teres isolates collected between 1994 – genotypes CI 5791 and CI 9819. Unlike 2007 originating from 19 locations, and in laboratory conditions, no indications of 200 P. teres progeny isolates originating changes in virulence caused by the sexual from artificially producedP. teres matings. reproduction have been observed in Finn- ish barley fields. According to the results of this study, screening for P. teres f. teres isolates on

MTT SCIENCE 9 3 In Finland, durable net blotch resistance to the P. teres f. teres isolates collected glo- has been achieved by introducing resist- bally and all of them showed excellent re- ance from other barley varieties using tra- sistance to Finnish P. teres f. teres isolates. ditional crossing methods, including wide crossing, and testing the breeding material at early generations at several sites under natural infection pressure. Novel resist- Keywords: ance is available, which is recommended Hordeum vulgare, barley, Drechslera to minimize the risk of selection of vir- teres, net blotch, net form of net blotch, ulent isolates and breakdown of current- virulence testing, differential set, sex- ly deployed resistance. Barley genotypes ual reproduction, evolution, tillage c-8755, CI 9825 and CI 5791 are poten- method, disease resistance tial resistance sources to be used in Finn- ish barley. They differed in their reaction

4 MTT SCIENCE 9 Suomalaisten Pyrenophora teres f. teres - isolaattien virulenssi ja sen huomioiminen taudinkestävyysjalostuksessa

Marja Jalli

MTT, Kasvintuotannon tutkimus, 31600 Jokioinen [email protected]

Tiivistelmä ilmankosteuden tulee olla testeissä va- kioidut, jotta eri testien tulokset ovat kes- uolella Suomen viljanviljelyalas- kenään vertailukelpoiset. Tartukkeen itiö- ta kasvaa ohraa. Yleisin ohran kas- pitoisuus sekä havainnoitavan ohralehden vitauti on lehtiä vioittava verkko- ikä vaikuttivat merkittävästi virulenssin Plaikku, jonka aiheuttaja on kotelosieniin ilmenemiseen. Sitä vastoin ohran sieme- kuuluva Pyrenophora teres Drechsler. Tau- nen koko, P. teres -isolaattien morfologi- dinkestävyys on ympäristöystävällinen ja set ominaisuudet, itiöntuottokyky tai kas- taloudellinen keino hallita pieneliöiden ai- vunopeus agar-maljalla eivät vaikuttaneet heuttamia kasvitauteja. Taudinaiheuttajan virulenssiin. Tutkimuksessa määritettiin tuntemus on taudinkestävyysjalostuksen ohragenotyypit, joiden reaktiot ilmentä- perusta. Taudinaiheuttajan yleisyyden li- vät P. teres -sienen virulenssia eri maissa säksi on huomioitava taudinaiheuttajan ja joita suositellaan käytettäväksi kansain- virulenssi (taudinaiheuttamiskyky) ja sen välisesti. Virulenssia tehokkaasti mittaa- muuntelun todennäköisyys. Taudinaiheut- viksi ohragenotyypeiksi osoittautuivat: tajan kyky muuntua tuo mukanaan riske- c-8755, c-20019, CI 5791, CI 9825, Ca- jä, joiden ennakoiminen on oleellinen osa nadian Lakeshore, Harbin, Prior, Skiff, ja onnistunutta taudinkestävyysjalostusta. Harrington.

Tutkimuksen tavoitteena oli kehittää Suomalaisten P. teres f. teres -isolaattien luotettavat ja tehokkaat menetelmät Py- virulenssi on pysynyt muuttumattoma- renophora teres f. teres – virulenssin testaa- na vuosien 1994 ja 2007 välillä. Tulokset miseksi ja selvittää, mikä on suomalaisten osoittivat eroja taudinaiheuttajan aggressii- P. teres f. teres –isolaattien virulenssi ja mi- visuudessa mutta erot isolaattien virulens- ten se huomioidaan taudinkestävyysjalos- sissa olivat harvoin merkittäviä. Yksikään tusohjelmassa. Aineisto koostui vuosina testatuista P. teres f. teres -isolaateista ei ol- 1994–2007 Suomesta 19 paikkakunnalta lut virulentti ohragenotyypeillä CI 5791 ja kerätyistä 239 P. teres f. teres –isolaatista CI 9819. Toisin kuin laboratoriotesteissä, sekä 200 laboratoriossa tuotetusta suvullis- Suomen ohrapelloilla ei havaittu suvulli- ten P. teres –risteytysten jälkeläisisolaatista. sesta lisääntymisestä johtuvaa virulenssin muuntelua. Tutkimus osoitti, että kasvihuoneessa to- teutettava virulenssitesti on luotettava ja Tutkimustulokset osoittivat, että suoma- taloudellinen menetelmä P. teres f. teres laisissa ohralajikkeissa esiintyy pitkäkes- -sienen virulenssin kuvaamiseksi. Ympä- toista verkkolaikunkestävyyttä. Tämä on ristöolosuhteiden, lämpötilan, valon ja saavutettu laajoilla perinteisiin menetel-

MTT SCIENCE 9 5 miin perustuvilla risteytyksillä, sekä tes- sa ohranjalostuksessa. Näillä on toisistaan taamalla jalostusaineistoa jo ensimmäis- poikkeava yhdysvaikutus eri P. teres f. te- ten sukupolvien aikana eri ympäristöissä res -isolaattien kanssa, ja kaikki genotyypit luonnon tartunnalle altistettuna. Tutki- ovat erityisen kestäviä suomalaisia P. teres muksessa valikoitui jalostustyössä aiem- f. teres -isolaatteja vastaan. min käyttämätöntä, hyvin verkkolaikun kestävää ohramateriaalia. Laaja-alaisuudel- taan ja tehokkuudeltaan poikkeavien tau- dinkestävyyslähteiden käyttö on suositelta- Asiasanat: vaa, jotta P. teres -isolaattien muuntumista Hordeum vulgare, ohra, Drechsle- sekä tästä aiheutuvaa taudinkestävyyden ra teres, verkkolaikku, verkkolaikun murtumisen riskiä voidaan hillitä. Ohra- verkkotyyppi, virulenssin testaus, dif- genotyypit c-8755, CI 9825 ja CI 5791 ferentiaalisetti, suvullinen lisääntymi- ovat suositeltavia uusia verkkolaikun kes- nen, evoluutio, muokkausmenetelmä, tävyyslähteitä käytettäväksi suomalaises- taudinkestävyys

6 MTT SCIENCE 9 List of original publications

This thesis is based on the following original articles, which are referred to in the text by their Roman numerals (I-IV).

I Robinson, J. and Jalli, M. 1996. Diversity among Finnish net blotch isolates and resistance in barley. Euphytica 92: 81-87.

II Jalli, M. and Robinson, J. 2000. Stable resistance in barley to Pyrenophora teres f. teres isolates from the Nordic-Baltic region after increase on standard host genotypes. Eu- phytica 113: 71-77.

III Afanasenko, O. S., Jalli, M., Pinnschmidt, H.O., Filatova, O and Platz, G.J. 2009. Devel- opment of an international standard set of barley differential genotypes for Pyrenophora teres f. teres. Plant Pathology 58: 665–676.

IV Jalli, M. Sexual reproduction and soil tillage effects on virulence of Pyrenophora teres in Finland. (Manuscript, submitted).

Reprints of the original articles I and II are published with the kind permission of Spring- er and article III with the kind permission of Wiley and Sons.

MTT SCIENCE 9 7 Contents

1 Introduction...... 9 1.1 Role of virulence in host-pathogen interactions...... 9 1.2 Plant disease resistance...... 12 1.3 Breeding for disease resistance...... 13 1.4 Pyrenophora teres as a pathogen...... 14 1.5 Net blotch resistance in Finnish barley...... 15 1.6 Objectives of the thesis...... 18

2 Materials and methods...... 19 2.1 Pyrenophora teres f. teres isolates (I-IV)...... 19 2.2 Mating studies with Pyrenophora teres isolates (IV)...... 19 2.3 Set of differential barley genotypes used in P. teres f. teres virulence studies (I-IV)...... 19 2.4 Inoculation and scoring (I-IV)...... 20 2.5 Data analysis (I-IV)...... 20

3 Results and discussion...... 22 3.1 Pyrenophora teres f. teres virulence testing methods...... 22 3.2 Diversity of Pyrenophora teres f. teres isolates in Finland...... 25 3.2.1 Diversity in virulence...... 25 3.2.2 Change in virulence over years...... 28 3.2.3 Effect of sexual reproduction on virulence...... 29 3.2.4 Effect of cultivation practices on virulence ...... 31 3.3 Implications of P. teres f. teres virulence for net blotch resistance breeding .. 32

4 Conclusions...... 36

5 Acknowledgements...... 38

6 References...... 40

8 MTT SCIENCE 9 1 Introduction

1.1 Role of virulence in n 2007, barley, Hordeum vulgare L., host-pathogen interactions was the fourth most important cereal crop both in terms of quantity pro- Infectious plant diseases result from an in- Iduced and global area of cultivation (FA- teraction between at least two organisms, OSTAT 2009). In Finland, barley covered a host plant and a pathogen. The outcome 50 % (601 000 ha) of the total acreage in each host-pathogen combination is de- used for cultivation of cereals in 2009 termined by the genetic constitution of the (Matilda 2009). Plant pathogens can cause host and the pathogen. Host plants vary in an average loss of 20 % of the average an- resistance and tolerance while pathogens nual value of the barley crop (Murray and are variable in their ability to infect a host Brennan 2010). (Barrett et al. 2009). Plant-pathogen interactions are due to continuous long-term The most common disease of barley in co-evolution of plants and the microbes. Finland is net blotch, a foliar disease Plants have evolved systems to recognize caused by the ascomycete Pyrenophora teres pathogens while pathogens have evolved Drechsler. The imperfect state of Pyreno- effectors to turn off plant defences (Stuke- phora teres is Drechslera teres (Sacc.) Shoe- nbrock and McDonald 2009). maker (syn. Helminthosporium teres Sacc.). Two forms of the pathogen exist: Pyren- According to Ebert and Hamilton (1996), ophora teres Drechs. f. teres Smedeg., the the impact that parasites have on the evo- causal agent of the net form of net blotch, lution and ecology of their hosts depends and Pyrenophora teres f. maculata Smedeg., on their virulence, which is a product of the causal agent of the spot form of net the host-parasite interaction. Pathogens blotch. These two forms are morpholog- produce compounds termed pathogen as- ically indistinguishable in culture media sociated molecular patterns (PAMPs) that but they produce different types of symp- are molecules unique to pathogens and toms on barley leaves (Smedegård-Petersen conserved across many pathogen species 1971). Rau et al. (2007) studied the mat- (Navarro et al. 2004). PAMPs have a key ing type genes in net and spot form isolates role in the basal defence system of the host. and found a long genetic isolation between Pathogenic species have developed special- the two forms. ized strategies to overcome the plant basal defence (Abramovitch and Martin 2004). In 1970 and 1971, net blotch was found Production of enzymes, toxins, and effec- in over 50 % (Mäkelä 1972) and in 1971- tor proteins can promote pathogen viru- 1973 nearly 60 % of the Finnish spring lence by interacting with the host. Effec- barley fields, and in nearly 90 % of the lo- tors do not usually have a function outside calities studied (Mäkelä 1975). In 1985, of the host. Pathogens may deliver various 84 % of the 234 inspected Finnish bar- effectors by different mechanisms (Bent ley fields had net blotch symptoms as ob- and Mackey 2007). Several of the fun- served on the two uppermost leaves of gal effectors are cloned and most of them plants (Avikainen and Isotupa 1986). The are small proteins of unknown function latest results from 2006-2007 showed that (Chisholm et al. 2006). However, with net blotch exists in 60 % of Finnish barley novel and sophisticated biotechnological fields (Manninen et al. 2009). tools the function of most fungal effec-

MTT SCIENCE 9 9 tors can be investigated experimentally and Contrary to non-specific resistance induced it is expected that their role in the preven- by PAMPs, the interaction between the tion of PAMPs triggered immunity is sig- products of R and Avr genes specify resist- nificant (De Wit et al. 2009). ance between a particular host and a pathogen (Stukenbrock and McDonald 2009). In a gene-for-gene model, the term viru- The gene-for-gene model is commonly con- lence is used for the pathogen genotype that nected to biotrophic fungal pathogens as a is able to overcome a resistance factor (Sac- result of long-term co-evolution (De Wit ristán and García-Arenal 2008). Gene-for- et al. 2009). However, several necrotroph- gene interaction was first described by Flor ic pathogens have been reported to pro- (1955) based on investigations on the host- duce proteins that may function as effec- parasite interaction between flax,Linum usi- tors in inducing necrosis in plants following tatissimum L., and rust, Melampsora lini var. a gene-for-gene model (Figure 1). Host spe- lini (Ehrenb.) Lév. Flor’s studies led to the cific toxins (HST) are defined as pathogen gene-for-gene hypothesis, which applies to effectors that are critical for the virulence cases in which resistance is controlled by in necrotrophic pathogens. They facilitate monogenic, dominant or semi-dominant the disease infection, for example, in spe- resistance genes. Resistance response re- cies of Alternaria, Cochliobolus and Pyreno- sults when plant resistance proteins (R pro- phora (Friesen et al. 2008). teins) recognize corresponding proteins of the pathogen, named avirulence (Avr) fac- Progress towards understanding the mo- tors. When a pathogen carrying an Avr gene lecular basis of the gene-for-gene interac- attacks the host carrying the correspond- tion has been made and numerous novel ing R gene, the Avr gene product can act Avr genes and R genes have been identi- as an elicitor, or it can direct the synthesis fied (Bent and MacKey 2007; De Wit et al. or modify a metabolite or protein that is a 2009). The role of avirulence determinants race-specific elicitor that can be recognized has been questioned in pathogen popula- by a receptor of a resistant plant (Figure 1). tions: why should a microbe keep a mol-

Avr gene-for-gene interaction HST gene-for-gene interaction

Host Host

R- rr S- ss

A- - + T- + - Pathogen Pathogen

aa + + tt - -

Lack of recognition = virulence Recognition = virulence A resistant plant (R) interacts with an Avr A toxin sensitive plant (S) interacts with the toxin factor (A) of a biotroph to induce a hypersensitive (T) of a necrotroph to induce a hypersensitive reaction and host cell death that prevents further reaction and host cell death that allows further pathogen propagation. pathogen propagation.

Figure 1. Genetic basis for gene-for-gene interactions based on avirulence proteins and host specific toxins (HST). Adapted from Stukenbrock and McDonald (2009).

10 MTT SCIENCE 9 ecule that allows it to be recognized? Avr Plant pathogens are difficult to control be- genes may perform some essential functions cause their populations are variable over for pathogen survival and must be main- time, space and genotype. Pathogen effec- tained (Skamnioti and Ridout 2005). In a tor genes, including Avr elicitors and host plant-pathogen system where evolution of a specific toxins, are diverse and local selection pathogen is directed towards avoiding rec- pressure plays an important role in their evo- ognition and induction of resistance, in- lution (Stukenbrock and McDonald 2009). creasing evidence implicates Avr genes in Barrett et al. (2009) summarized that envi- roles other than race-specificity (Leach and ronmental heterogeneity increases the po- White 1996). In some cases the products tential for variation in host-pathogen inter- of diverse Avr genes are not only for R-gene actions. They also stated that host range and recognition but have a functional role by traits associated with fitness vary, making encoding effectors that facilitate virulence broad ecological and evolutionary predic- (De Wit et al. 2009). In susceptible hosts tions difficult. Selection on the pathogen Avr genes may enhance the pathogen infec- population favours escape of host recognition (Stukenbrock and McDonald 2009). tion and the pathogen is pressured to keep updating its virulence factors (Does and Rep Different Avr genes do not share com- 2007). Thrall and Burdon (2003) investi- mon features (Sacristán and García-Arenal gated the Linum usitatissimum - Melamp- 2008). A number of avirulence genes have sora lini interaction and demonstrated that a measurable fitness function, and mutation virulent pathogens occurred more frequent- of these genes might impose a cost on vir- ly in highly resistant host populations and ulence. Huang et al. (2006) studied near- avirulent pathogens in susceptible popula- isogenic isolates of Leptosphaeria maculans tions. Gene loss and mutation in a patho- (Desm.) Ces. & De Not. (phoma stem can- gen population may be compensated for by ker) differing at the AvrLm4 avirulence lo- gene gain through horizontal gene trans- cus in oilseed rape, Brassica napus L., cul- fer (HGT) (Van der Does and Rep 2007). tivars that were lacking the resistance gene New combinations of Avr genes generated Rlm4, which corresponds to AvrLm4. They by HGT may evolve in unpredictable situa- determined that the isolates without the Avr tions (Skamnioti and Ridout 2005). Friesen gene incurred a fitness cost compared with et al. (2006) have provided strong evidence AvrLm4 isolates. In contrast, Schürch et al. on HGT, where a gene encoding a virulence (2004) showed how deletion of an aviru- factor (ToxA) was transferred from a wheat, lence gene may allow pathogens to escape Triticum aestivum L., pathogen, Stagonospo- recognition by the corresponding resist- ra nodorum (Berk.) E. Castell. & Germano, ance gene in the host. Virulence of a barley to another wheat pathogen, Pyrenophora trit- pathogen Rhynchosporium secalis (Oudem.) ici-repentis (Died.) Shoemaker. Davis isolate to a barley genotype carrying the Rs1 resistance gene was achieved The term virulence has a confused history through deletion and point mutations of and various usages for the term exist (Sac- the Avr gene NIP1 that produces a phy- ristán and García-Arenal 2008). In the cur- totoxic peptide which acts as an elicitor of rent study I use the term virulence as it is the defence response. Plant pathogens have defined by Sacristán and García-Arenal evolved Avr determinants in a complex en- (2008): virulence is the degree of damage vironment. Although Avr genes function as caused to a host by parasite infection. More effectors to promote virulence, not all have specifically, in gene-for-gene interactions vir- obvious function in pathogen-host interac- ulence is used for the pathogen genotype tion (Leach et al. 2001). that is able to overcome a resistance factor.

MTT SCIENCE 9 11 1.2 Plant disease resistance put of the plant innate immune system. In phase 1, PAMPs are recognized by pat- The majority of plants possess natural de- tern recognition receptors (PRRs) result- fence that enables them to avoid or resist ing in immunity that can halt further col- infection by the majority of plant patho- onization. In phase 2, successful pathogens gens present in their environment. Recog- deploy effectors that contribute to patho- nition of pathogen results in a massive re- gen virulence. Effectors can interfere with programming of the plant cell to activate PAMP-triggered immunity (PTI) which and deploy defence responses to interrupt results in effector-triggered susceptibility pathogen growth (Bolton 2009). Plants (ETS). In phase 3, a given effector is rec- have evolved two lines of defence. The first ognized by proteins resulting in effector- line is based on recognition of PAMPs, triggered immunity (ETI). ETI results in which provides basal defence against all disease resistance and usually, a hypersen- pathogens. The second line is based on sitive cell death response. In phase 4, nat- pathogens’ effector recognition by plant ural selection drives pathogens to avoid R (resistance) proteins. Jones and Dangl ETI. Natural selection results in new R (2006) introduced a four phased ‘zigzag’ specificities and the ETI can be triggered model to illustrate the quantitative out- again (Figure 2).

High

PTIETS ETIETS ETI

Threshold for HR e

Pathogen Pathogen

f defenc f effectors effectors e o

Amplitud Avr-R Avr-R

Threshold for effective resistance PAMPs

Low

Figure 2. A zigzag model that illustrates the quantitative output of the plant immune system. PAMP: pathogen-associated molecular pattern. PTI: PAMP-triggered immunity. ETS: effector-triggered susceptibility. ETI: effector-triggered immunity. Adapted from Jones and Dangl (2006).

12 MTT SCIENCE 9 Plants are resistant to certain pathogens tive disease resistance is still poorly under- either because they belong to taxonom- stood (Poland et al. 2008). ic groups that are outside the host range of these pathogens, or because they pos- 1.3 Breeding for disease sess genes for resistance directed against resistance the avirulence gene of the pathogen, or because they tolerate or escape infec- Breeding crops for disease and pest resist- tion by these pathogens. Van der Plank ance is one method used to protect them (1968) divided disease resistance into two from damage due to biotic factors. Inher- types: vertical (race-specific) and horizon- ited resistance is valuable because it is easy tal (non-specific). According to Stuthman for the grower and reduces the need for et al. (2007) Van der Plank’s terms were other methods of control. The breeder has more epidemiological than genetic. Verti- to choose weather to select for resistance, cal resistance reduces initial inoculum by or against high susceptibility. Surveys of screening out avirulent races but has no ef- disease incidence are useful in indicating fect on the rate of increase of the virulent the importance of diseases providing infor- races. Horizontal resistance is at least par- mation for breeders on priorities (Johnson tially effective against all races. and Jellis 1992). Stuthman et al. (2007) identified three aspects of disease resist- R-genes are important in many systems. ance relating to practical breeding strate- They typically provide high levels of resist- gies: inheritance, effectiveness and specif- ance and are easy to manipulate, but their icity of disease resistance. utility varies among pathosystems. Several R-genes have been isolated and their func- Special and increased interest in disease re- tions have been studied in detail. The lim- sistance breeding has emphasised durable itations of R-genes in crop protection are resistance that is cost effective, environ- a lack of durability in some systems and a mentally sound, and promotes the conser- lack of availability in others (Poland et al. vation of genetic resources. Durable resist- 2008). For highly specialized pathogens ance can be defined as the adequacy of the with narrow host ranges, monogenic race- resistance throughout the useful lifetime specific resistance has generally been un- expected from a variety. Keeping ahead reliable (Stuthman et al. 2007). of the changing pathogen populations is a continuous challenge for plant breed- Resistance breakdown is considered to be ers. The capacity to predict the durabili- less of a problem with multiple gene based ty of resistance genes would be desirable quantitative (horizontal) disease resistance, when making investments in plant breed- which leads to lower selection pressure on ing (Leach et al. 2001). the pathogen (Poland et al. 2008). Palloix et al. (2009) demonstrated that the fre- The durability of disease resistance is high- quency of breakdown of major gene rely influenced by the recognition method of sistance against Potato Virus Y was high R protein, by the capability of the path- when introgressed into a susceptible back- ogen to retain virulence after altering or ground and no breakdown occurred when eliminating the recognized effector, and by the same gene was introgressed into a par- the ability of a pathogen to evolve. Using R tially resistant genetic background. Even genes in rotation, with monitoring the cur- though quantitative disease resistance is rent races of pathogen, using R genes only generally considered being non-race-specif- when needed, removing them from use be- ic, race-specific quantitative disease resist- fore they become widely ineffective, keep- ances have also been identified. However, ing more than one effectiveR gene present the molecular mechanism of the quantita- in plant, combining use of fungicides and

MTT SCIENCE 9 13 R genes, and coordinating the system care- tion of P. teres f. teres occurs in dry soils at fully among pathologists, plant breeders, temperatures of 12 ºC (Youcef-Benkada et and growers, are examples of how to en- al. 1994). Infected volunteer crop species hance the durability of genetic resistance may also serve as sources of primary inocu- (Bent and Mackey 2007). The challenge lum. Brown et al. (1993) demonstrated the and main goal in plant breeding is to op- successful net form net blotch infection on timize the plant genotype by choosing the 65 gramineous species in California. One most promising resistance genes and gene of the volunteer crop species for net blotch combinations for durable disease resistance is couch grass, Elymus repens L. Gould, a (Palloix et al. 2009). common weed of cereal fields that exists in 66 % of the spring cereal fields in Fin- Although disease resistance is a valuable land (Salonen et al. 2001). Besides barley, tool to be used in plant breeding, the ac- Finnish P. teres f. teres isolates have shown tive defence processes in plants occur at pathogenicity on spring and winter wheat, the expense of the energy resources and winter rye and oats (Mäkelä 1972). may be a limiting factor in plant growth and yield. The use of energy for defence During the growing season, D. teres co- responses relies on several metabolic path- nidia produced on the surface of prima- ways (Bolton 2009). If resistance is costly ry lesions caused by ascospores or conidia and has a negative effect on yield, a breed- serve as secondary inoculum. Sporulation er’s most effective strategy may not be to occurs when relative humidity is near 100 select for excellent resistance but to select % for 10 – 30 hr and the temperature re- for moderate resistance and eliminate very mains between 15 and 25 ºC. Isolates of susceptible lines. The gene content, instead P. teres from areas where barley is grown of comprising single genes, determines a over mild winter periods have lower tem- cultivar’s value to end-users (Brown 2002). perature requirements for sporulation and infection (Shipton et al. 1973). In a suc- 1.4 Pyrenophora teres as a cessful penetration, a primary vesicle de- pathogen velops within the epidermal cell, followed by the formation of a secondary vesicle. The life cycle of P. teres f. teres involves Subsequently, infection hyphae grow from both an asexual and a sexual stage. The the secondary vesicle to penetrate the inner asexual lifecycle includes over-wintering cell wall of the epidermal cell and the apo- of the pathogen as mycelium in seed or plastic space of the mesophyll. No hyphae in crop debris. The sexual fruiting bod- penetrate the mesophyll cells during the ies, pseudothecia, are formed on barley early stages of infection. Cells within the straw after harvest in autumn. Asci and developing lesions exhibit various degrees ascospores are formed during the late au- of disruption (Keon and Hargreaves 1983). tumn and the following spring and sum- mer, depending on the climatic condi- In infected barley tissue, P. teres induces tions (Smedegård-Petersen 1972, Shipton long dark brown netted necrotic lesions et al. 1973). Infection of barley seedlings (the net form) or dark brown circular or is caused by D. teres conidia or P. teres elliptical spots (the spot form) surrounded ascospores, and is more efficient at cool by chlorosis (Smedegård-Petersen 1971). temperatures (10-15 ºC). The role of as- Smedegård-Petersen (1977) isolated two cospores in the life cycle of P. teres is con- toxins, toxin A and toxin B, from P. teres sidered more as a source of novel variation cultures that produced necrosis, chloro- than as an important source of primary in- sis and water-soaking on barley leaves. A oculum (Shipton et al. 1973). The most se- third aspergillomarasmine-derived toxin, vere damage caused by seed-borne infec- toxin C, was later identified by Bach et

14 MTT SCIENCE 9 al. (1979). Weiergang et al. (2002) found three major genes in P. teres f. teres in- a positive correlation between the reac- volved in avirulence/virulence of the fun- tions on detached barley leaves caused by gus: AvrHar conferring avirulence to Ti- the toxins A B, and C of both net and fang and Canadian Lake Shore, and two spot form P. teres strain cultures. Toxins A, epistatic genes AvrPra1 and AvrPra2 con- B and C induce water soaking, chlorosis, ferring avirulence to Prato. Beattie et al. and general necrosis but not well-defined (2007) identified six AFLP markers linked necrotic net or spot lesions. Sarpeleh et al. to a P. teres f. teres avirulence gene Avr- (2007) isolated phytotoxic low molecu- Heartland. Despite the identification of lar weight compounds and proteinaceous the P. teres f. teres avirulence genes their metabolites both from P. teres f. teres and function in the virulence of P. teres is still P. teres f. maculata which were responsible unclear. for symptoms on susceptible barley cultivar. Later Sarpeleh et al. (2008) found that 1.5 Net blotch resistance in the activity of toxins on barley is both tem- Finnish barley perature and light dependant. Toxins were only active in the presence of light and no Studies on barley net blotch resistance were necrotic lesions were induced when treated already conducted in 1948 in California plants were kept for 168 h at 4 ºC. when resistance of 4 526 barley varieties was tested in the field (Schaller and Wiebe The severity of net blotch is related to the 1952). Seventy-five of the tested varieties climatic conditions, to the cultivation were highly resistant to net blotch and most practice and to the susceptibility of culti- of the resistant varieties originated from vars used. The seed borneP. teres infection Manchuria. Later, Buchannon and Mc- reduces leaf area, plant height and total Donald (1965) studied the reaction of P. mass compared to healthy plants (Dead- teres infection on 6 174 barley genotypes in man and Cooke 1988). The net blotch in- Canada. Forty varieties, seventeen originat- fection of the uppermost three leaves of a ing from Ethiopia, showed good resistance barley plant has the main effect on yield, to a wide range of P. teres isolates. Already which may be reduced by 30 %. However, then it was concluded that P. teres isolates the effect of the disease on barley yield is from different regions, and even within the more than the pure effect of the destroyed same region, may differ in pathogenicity leaf area (Jebbouj and Yousfi 2009). Kernel and the results of the disease screening tests weight and size are the yield components are highly dependent on the methodology most affected by P. teres f. teres infection and isolates used. Later on, several exper- (Steffenson 1991). iments on screening for P. teres resistance have been conducted and a considerable The studies on avirulence genes in P. teres amount of valuable information is available f. teres provide information on the role of (e.g. Steffenson and Webster 1992b; Rob- virulence in the P. teres – barley interac- inson and Jalli 1996; Jørgensen et al. 2000; tion. Weiland et al. (1999) reported that a Gupta et al. 2003; Bonman et al. 2005). single major gene controls virulence in P. teres f. teres on ‘Harbin’ barley. They found Research on the net blotch resistance genes five random amplified polymorphic DNA indicates a complex host-pathogen inter- (RAPD) markers associated with the low action controlled by both quantitative and virulence on Harbin and designated the major resistance genes. The reports on the trait avrHar denoting avirulence to ‘Har- presence of qualitative net blotch resistance bin’. Lai et al. (2007) constructed an AFLP suggest the potential existence of a gene- (amplified fragment length polymorphism) for-gene interaction in the P. teres - barley based genetic linkage map and identified pathosystem (Afansenko et al. 2007). Stef-

MTT SCIENCE 9 15 fenson et al. (1996) identified three QTL The disease pressure by net blotch in Finn- (quantitative trait loci) for seedling resist- ish official trials is based on natural infec- ance and seven QTL for adult plant re- tion whose average level varied between sistance. Also several other studies on the 5 and 12 % over all tested cultivars and genetic background on the net blotch re- breeding lines in 2000-2008. The data sistance show that the resistance is control- were analyzed using linear mixed mod- led by a single or several genes depending els and the estimated means for the differ- on source of resistance, plant development ent varieties are comparable despite the dif- stage and P. teres isolate used (Manninen ferent trial period. The general level of the et al. 2000; Cakir et al. 2003; Manninen resistance against barley net blotch in the et al. 2006; Grewal et al. 2008). When fur- barley material tested in the Finnish official ther studying barley net blotch resistance, variety trials has improved. When com- molecular marker technology provides po- paring the barley genotypes to the stand- tential tools both for mapping disease re- ard cultivar Scarlett, a malting barley orig- sistance genes and for genotypic selection inating from Germany, 55 % of the tested and may promote considerable resource barley genotypes were more susceptible in savings (Grewal et al. 2008). 2002, and only 21 % in 2008. In neither of the years were any of the genotypes signifi- The Finnish barley breeding programme is cantly more resistant than Scarlett (Kangas working systematically on improving bar- et al. 2002, Kangas et al. 2008). In 2001- ley net blotch resistance. Special input has 2008, the average net blotch infection lev- been targeted to reduce the susceptibility el in Scarlett was 1.5 %. Thirteen of the 16 of six-rowed barley cultivars by selection Finnish barley cultivars that are on the of- of crossing parents that inherit high levels ficial variety list in Finland in 2009 express of quantitative resistance, which mainly resistance comparable to Scarlett. Nine of originates from two-rowed North Ameri- these cultivars are six-rowed barleys (Ta- can barley genotypes. The programme re- ble 1). None of the cultivars is known to lies on a multi-locational testing system carry any specific resistance genes against where the material is tested already in ear- net blotch. ly generations under a high disease pressure (Nissilä 2009, personal communication). Currently, plant breeding is business that has to respond rapidly to end-users’ needs. The level of net blotch infection in barley At the same time it represents a long term varieties has been monitored in the official project that has to look to the future. variety trials in Finland since 1989. The Building up a disease resistance breed- main purpose of the official variety trials ing programme relies on knowledge of the is to assess the value of new genotypes and pathogen. Besides information on the epi- make recommendations on yield and quali- demiology of the pathogen, knowledge on ty parameters. Plant disease resistance is an how the pathogen evolves and which risks increasingly important factor in the culti- might arise in the future are key points that vation value of cereal varieties (Kangas et need to be taken into account to achieve al. 2008). Robinson and Jalli (1997a) stud- the final breeding goals. ied the severity of net blotch in Finnish official barley variety trials over six years and MTT Agrifood Research Finland, in five sites. Net blotch occurred at all five close cooperation with the Finnish breed- sites. The disease severity differed between ing company Boreal Plant Breeding Ltd., years but was similar across sites. The data has concentrated on improving barley net indicated that differences in resistance to blotch resistance in several research projects net blotch existed among the 19 genotypes (Figure 3). The research has concentrat- studied. ed on understanding the function of the

16 MTT SCIENCE 9 Table 1. The net blotch infection % of the Finnish barley cultivars in the Finnish Official Vari- ety Trials in 2001-2008 compared to the standard cultivar Scarlett (Kangas et al. 2008).

Variety Head type Net blotch infection % in Breeder Year of Approval Finnish Official Variety on National List Trials 2001-2008 of Plant Varieties Harbinger two-rowed 0.6 Boreal Plant Breeding Ltd 2009 Elmeri six-rowed 0.7 Boreal Plant Breeding Ltd 2009 Edvin six-rowed 0.7 Boreal Plant Breeding Ltd 2008 Eerik six-rowed 0.8 Boreal Plant Breeding Ltd 2009 Minttu two-rowed 1.4 Boreal Plant Breeding Ltd 2005 Scarlett two-rowed 1.5 Saatzucht Josef Breun GmbH & Co 1998 Einar six-rowed 1.6 Boreal Plant Breeding Ltd 2008 Olavi six-rowed 2.2 Boreal Plant Breeding Ltd 2006 Saana two-rowed 2.4 Boreal Plant Breeding Ltd 1996 Jyvä six-rowed 2.4 Boreal Plant Breeding Ltd 2000 Kunnari six-rowed 2.5 Boreal Plant Breeding Ltd 2001 Erkki six-rowed 2.7 Boreal Plant Breeding Ltd 1998 Polartop six-rowed 3.0 Boreal Plant Breeding Ltd 2005 Pohto six-rowed 4.9 * 1) Hankkija Plant Breeding 1994 Rambler two-rowed 5.2 Boreal Plant Breeding Ltd 2009 Rolfi six-rowed 5.4 *** Boreal Plant Breeding Ltd 1997 Voitto six-rowed 25.4 *** Boreal Plant Breeding Ltd 2005 1) Significantly different from Scarlett: * at 5 % level, ** at 1 % level, *** at 0.1 % level

WORKING WITH NET BLOTCH IN FINLAND

Disease assessments in Finnish official variety trials since 1990

Developing methods Disease DNA-markers Durable disease Healthy good quality Genetic resources into for disease testing resistance breeding introduced to resistance barley: from genes use – a tool box on wheat and barley against barley net blotch introduced to practice for plant breeding leaf pathogens to barley and oat

Projects 1988-1993 1995-1997 1998-2000 2002-2005 2006-2008 2009- s •Basic tools to work •Knowledge on the •Mapping of net •Both mating types •Barley disease surveys •Cost of resistance with Pyrenophora teres net blotch resistance blotch resistance genes of net blotch exist in in Finland •Barley near-isogenic •Inoculation and of Finnish cultivars •Developing Finland •National & international lines with net blotch res. assessment methods • P. teres virulence markers for P. teres •Population diversity virulence studies •Economical &

Result for barley net blotch screening started resistance genes studies using AFLPs •Mapping of R genes environmental aspects of disease resistance

Barley disease resistance breeding programme in Boreal Ltd

Figure 3. The background of the barley net blotch research in Finland at MTT Agrifood Re- search Finland.

MTT SCIENCE 9 17 disease triangle that illustrates interac- discussed. In addition to the data already tions among the three components of net published in the individual papers I-IV, blotch: the pathogen, the environment and this work includes new results on com- the host. bined data (Finnish P. teres f. teres isolates in collection 1-4), which made it possible 1.6 Objectives of the thesis to study the virulence over a longer time period. Those results are published only in this work. The main objectives were to establish reliable and efficient testing methods forPyren - The specific aims were: ophora teres f. teres virulence screening, and to understand the role of virulence of the • To establish stable virulence testing Pyrenophora teres f. teres pathogen in Fin- methods under greenhouse conditions land from a disease resistance breeding (papers I, II, III, IV) point of view. The background material in this study includes four publications • To assess diversity of virulence among that are discussed together with related Finnish isolates of Pyrenophora teres f. scientific papers. The papers included in teres (papers I, II, III, IV) my thesis were chosen based on the results of P. teres virulence screening in Fin- • To identify the impact of P. teres f. land using greenhouse test methods. Only teres virulence on a net blotch resist- the parts of the articles that support the ance breeding programme (papers I, objectives of this study are presented and II, III, IV)

18 MTT SCIENCE 9 2 Materials and methods

2.1 Pyrenophora teres f. teres isolates (I-IV) long-term storage the isolates were deep- frozen as mycelium and conidia and stored Virulence of 239 Finnish P. teres f. teres in –80 ºC at MTT Agrifood Research isolates was tested in 1994 - 2008. The iso- Finland and Boreal Plant Breeding Ltd. lates that were collected in 1994, 1997, and 2000-2007 across 19 locations originat- 2.2 Mating studies with ed from four P. teres collections: 1) 20 P. Pyrenophora teres isolates teres f. teres isolates collected in 1994 from (IV) 11 sites in Finland originating from two barley cultivars, a net blotch susceptible Twenty-four P. teres isolates were used for cultivar Arve and a field resistant cultivar mating studies. One of the isolates rep- Pohto (Kangas et al. 2008). Three Swed- resented the spot form. Sterilized barley ish and one Canadian isolate were also in- straw was placed on Sachs’s agar (Sme- cluded in the studies. One of the Swedish degård-Petersen 1971) and inoculated at isolates represented P. teres f. maculata. 2) both ends with 100 µl of two P. teres sus- 120 Nordic-Baltic and Irish P. teres f. teres pensions. The plates were incubated in isolates collected in 1997. The collection darkness for six months at 15 ºC. The suc- consisted of 19 Estonian, 29 Finnish, 11 cess of crossings was studied under the Irish, 20 Latvian, 22 Norwegian, and 19 microscope by assessing the mature pseu- Swedish isolates. 3) 247 isolates originat- dothecia. The ascospores were released ing from 13 countries from Europe, North from the pseudothecia by breaking the asci America and Australia collected between with a thin needle. Single ascospores were 1984 and 2007. 153 of the isolates originat- placed on 2.3 % LB-agar. ed from Finland, collected in 2000-2007. 4) Six P. teres populations isolated from ex- Morphological character measurements perimental fields in Jokioinen Finland in were made on one successful crossing 2006-2007 (276 P. teres f. maculata and population between a net and a spot form 37 P. teres f. teres isolates). The fields had P. teres isolate. Colony growth, weight of a five to eight year history of barley mono- mycelium, conidia production, conidia culture. Three of the populations were col- length, width and amount of septa were lected from no-tillage and three from nor- measured on six subcultures from one mal tillage cultivation systems. hundred single ascosporic cultures.

Leaf samples with net blotch lesions were 2.3 Set of differential barley surface sterilized in 50 % ethanol for 15 genotypes used in P. teres f. s and in 2 % NaOCl for 30 s, rinsed in teres virulence studies (I-IV) distilled water and placed on 2.3 % lima- bean (LB) or 20 % V8-agar. Plates were The barley genotypes suggested by Stef- kept under near-UV light for 5-7 days. fenson and Webster (1992a) constituted Single spores were collected with a needle the net blotch differential set in the first and placed on LB- or V8-agar. For inocu- P. teres virulence study. In the later stud- lum production, single spore cultures were ies, that set formed the basis of barley gen- grown under near-UV-light in a 12-h pho- otypes used and was modified according toperiod at 16 ºC for 14 days. A spore sus- to the aim of the study. Barley genotype pension for inoculum was made by flood- CI 9819 was included in all test sets. Arve, ing Petri-dishes with sterile water. For Rolfi, or Pirkka was included in a test set

MTT SCIENCE 9 19 as a susceptible check (Table 2). In addi- with SAS PROC CORR (SAS Institute tion to the barley differential set, barley va- 2004). Hierarchical cluster analyses were rieties cultivated in Finland were included carried out to examine similarities and dis- in the virulence tests. The total amount of similarities in the reaction patterns of the barley genotypes in the ten virulence tests accessions with respect to the net-blotch was 46, including 16 varieties that have isolates. The clustering method of Ward been cultivated in Finland. (1963) and Simpson’s diversity index was computed to characterize pathotype diver- 2.4 Inoculation and scoring sity of the isolates. (I-IV)

The virulence studies were carried out in two greenhouses at MTT Agrifood Re- Table 2. The barley genotypes in barley search Finland and at Boreal Plant Breed- differential sets used for screening viru- ing Ltd. Barley seeds were sown in pots lence in P. teres f. teres. containing nutrient-supplemented peat Genotype CI-number Used in and placed in the greenhouse at 18-22 ºC studies with a 12-h photoperiod. The pots were Algerian CI 1179 I arranged in a split plot design; isolates as- Atlas CI 4118 I, II signed to main plots and barley genotypes Beecher CI 6566 I to sub plots with 2-4 replicates. At the two c-20019 c-20019 III to three leaf stage (14 days after the sow- c-8755 c-8755 III ing) relative humidity in the greenhouse Canadian Lake- CI 2750 I, II, III was raised to 100 % and plants were in- shore oculated with a conidial suspension at 0.1 Cape CI 1026 I, II – 0.2 ml / plant by using a pressurized CI 4922 CI 4922 I, II sprayer. Lights were switched off and high CI 5791 CI 5791 I, II, III, IV humidity was maintained for 24 h after in- CI 5822 CI 5822 I, IV oculation. The infection response was re- CI 7584 CI 7584 I, II corded 7 to 14 days after the inoculation CI 9214 CI 9214 III on the first, second or third seedling leaf CI 9819 CI 9819 I, II, III, IV using the 10-point scale of Tekauz (1985) CI 9825 CI 9825 III or the percentage of leaf area damaged. CI 11458 CI 11458 I, II The infection scores < 5 were classified as Coast CI 2235 I, II avirulent reactions and > 5 as virulent re- Corvette III actions (Figure 4). Harbin CI 4929 I, II, III Harrington III 2.5 Data analysis (I-IV) Haruna Nijo III Kombar CI 15694 I, II Manchuria CI 2330 I, II, IV The virulence data were analysed as split Manchurian CI 739 I, II, III plot designs by using SAS PROC ANO- Ming CI 4797 I VA, PROC GLM and PROC GLIMMIX. Pirkka III, IV Significantly different means were separat- Prato CI 15815 I ed with Tukey’s HSDs. SAS PROC FREQ Prior III was used to study the general association Rika CI 8069 I, II of virulence of the P. teres isolates and till- Rojo CI 5401 I, II age method. Kendall Correlation coeffi- cients between the morphological char- Skiff III acters and virulence data were analyzed Tifang CI 4407 I, II, III

20 MTT SCIENCE 9 55

Figure 4. The 10-point scale of Tekauz (1985) and the corresponding infection re- sponseFigure on 4. living The barley10-point genotypes scale of Tekauz 10 days (1985) after inoculationand the corresponding on the second infection seedling. response Photoon Marja living Jalli. barley genotypes 10 days after inoculation on the second seedling. Photo Marja Jalli.

MTT SCIENCE 9 21 3 Results and discussion

3.1 Pyrenophora teres f. teres virulence testing meth- Nijo, while the lowest frequencies of vir- ods ulent reactions was observed on CI 5791, CI 9819, CI 9214, Beecher, c- 8755 and CI In the first virulence study (paper I) it was 9825 (Table 3). Based on similarity anal- shown that the comparison and interpre- ysis, the genotypes CI 5791 and CI 9819 tation of results from the same laboratory were almost identical in the reaction to from different times, and between labora- the isolates. Also, Skiff and Corvette re- tories is made difficult by the variable -na sembled each other and were more sim- ture of the host-pathogen interaction. That ilar to susceptible cultivars Pirkka, Har- was also observed in the second study (pa- rington and Haruna Nijo than to other per II) where there were significant differ- cultivars. Excluding the cultivars Beecher, ences in the results between the replicates CI 5791, CI 9819 and Harrington from that were located in different greenhouse the test set had a minor effect on the rel- rooms. ative number of pathotypes and diversity

The environmental factors, temperature, light and humidity, were stable in all trials and their effect was not studied in this Table 3. Range and mean susceptible re- research. However, it has been shown that actions of P. teres f. teres isolates tested even the slight changes in environmen- on barley differential genotypes. tal conditions can significantly affect the expression of plant infection. Khan and Barley differential Range of Mean of Boyd (1969) indicated the importance of susceptible susceptible stable testing methods based on their re- reactions (%) reactions (%) search on P. teres resistance in Manchurian CI 5791 0-14 1.7 varieties, which was enhanced by providing either pre-inoculation high tempera- CI 9819 0-25 2.9 ture or high light intensity during the in- CI 9214 0-25 7.2 cubation period. Beecher 0-24 7.6 c-8755 0-25 9.7 The virulence of P. teres has been studied by using barley genotypes with specif- CI 9825 0-50 10.2 ic resistance as virulence markers (Tekauz c-20019 0-50 13.1 1990; Sato and Takeda 1993; Afanasenko Manchurian 0-35 17.9 et al. 1995). The lack of a universal barley differential set makes the comparison Tifang 0-75 22.4 of separate virulence studies difficult. The Prior 0-50 31.3 aim in paper III was to develop an inter- Harbin 0-64 41.1 national standard set of barley differential Canadian Lakeshore 0-75 41.5 genotypes to standardize the characteri- zation of P. teres f. teres virulence global- Skiff 0-97 63.6 ly. In the greenhouse tests, the virulence Corvette 13-86 71.4 of 174 P. teres f. teres isolates (part of col- Haruna Nijo 25-100 78.5 lection 3) was studied on 17 barley geno- Pirkka 50-94 80.9 types. Most of the P. teres isolates were virulent on Harrington, Pirkka and Haruna Harrington 50-100 89.4

22 MTT SCIENCE 9 index, whilst omission of cultivars Harbin in Arabidopsis. The possible inheritance of and Skiff markedly affected both the rela- a plant’s expression to stress from one gen- tive number of pathotypes and the diver- eration to another may cause unpredicta- sity index. The results of greenhouse tests ble interactions when measuring the plant- carried out in Finland were incorporat- pathogen interaction. Moreover, treating ed with the detached-leaf studies made by the seed with fungicides may affect infec- the All Russian Research Institute of Plant tion response at the seedling stage. There Protection (VIZR), Russia. Based on the is evidence that triadimenol is systemic and similarity analyses and the ability of in- could be found even in the third wheat dividual barley genotypes to discriminate leaves 21 days after sowing (Thielert et al. pathotypes and indicate pathotype diver- 2007). Triadimenol is an active ingredient sity, barley genotypes c-8755, c-20019, CI in fungicides Baytan I and Baytan Univer- 5791, CI 9825, Canadian Lakeshore, Har- sal (Finnish Food Safety Authority 2010), bin, Prior, Skiff and Harrington were iden- which are commonly used seed dressings tified to be the most appropriate to char- in Finland. Therefore, the seed used in acterize P. teres f. teres virulence globally. virulence studies should have been grown A further aim was to study the genetic in a completely stress-free environment to background of the resistance and to devel- avoid the effect of seed treatment products op near-isogenic lines that carry different and the possible inheritance of expression resistance genes in the same background, to stress factors. similarly as the Pallas-lines for powdery mildew (Kølster et al. 1986). If P. teres f. Passaging the P. teres isolates through bar- teres resistance that has not been identified ley might be necessary after several cycles earlier exists, it needs to be included in the of sub culturing on artificial culture me- test set. The composition of a barley dif- dia, the effect of which varies on different ferential set should be flexible when stud- isolates (McDonald 1967). A fall-off in co- ying a pathogen-host system that evolves nidia production capacity was also noticed and might change rapidly. in our studies, but was improved after passaging the isolate through a living barley The effect of barley seed size on the -in plant. However, we found no evidence that fection response via differences in growth passaging the P. teres f. teres isolates, which speed was recorded for biotrophic path- were used directly after the isolation from ogens (Jørgensen 1992). In our studies, field passaging once through barley cul- the barley seed size correlated positive- tivars differing in resistance, were affect- ly with plant growth. The seed size (sort- ed in terms of their virulence. As Lohan ing through sieves with mesh diameters and Cooke (1986) reported, when compar- 2.2 mm, 2.4 mm, 2.6 mm and 2.8 mm) ing different agar media for P. teres spore used in the virulence test affected the plant production, we found the spore produc- height at seedling stage. Larger seeds pro- tion to be most efficient in diurnal NUV duced larger plants for all tested geno- light on V8-juice agar. In our studies, in- types. Seed size, and its consequent ef- oculum concentration (1 250 ml-1, 2 500 fect on plant height, did not significantly ml-1, 104 ml-1 and 2 x 104 ml-1) had a sig- affect the infection response or percent- nificant effect on plant height, suppress- age leaf area damaged. Even though no ing it at higher concentrations. Infection correlation between the seed size and the response and percentage leaf area dam- P. teres infection response was found, the age were also influenced by inoculum con- seed may carry other elements that inter- centration. A higher concentration led to act with the disease infection. Molinier et higher infection response and proportion al. (2006) determined that stressed plants of leaf area damaged. The inoculum con- inherited the capacity for genomic change centration used in papers III and IV was

MTT SCIENCE 9 23 fixed at 4 x 104 ml-1 and the amount of in- lence or resistance that are not influenced oculum was adjusted to 0.2 ml/plant. This by other characters of P. teres isolates. quantity of spores per plant was sufficient to get an even infection and to differenti- The qualitative Tekauz scale provides a ate the infection response reactions of dif- common understanding on infection re- ferent barley genotypes. action types. In our studies the differential genotypes ranked similarly to the infec- Net blotch symptoms developed well in tion rate (Tekauz scale) and the percentage all our studies, lesions becoming appar- of leaf area damaged. However, the scale ent within two days post-inoculation. The based on the infection type is less sensitive differences in the degree of symptom ex- to the microclimatic conditions on a bar- pression on the inoculated first, second ley leaf at the time of inoculation and in- and third leaves were significant. The sec- cubation than the scale based on percent- ond seedling leaf was the most affected. age leaf area damaged. When measuring Regression lines for the means infection infection type, a few successful symptoms response indicated that barley germplasm on the second leaf are enough for prop- was more clearly differentiated into re- er investigations while for the scale based sistant and susceptible categories by scor- on percentage leaf area damaged it is as- ing the second seedling leaf rather than sumed that all spores have standard con- the first. The information on the seedling ditions for germination. Both scales are leaf from which the virulence scorings are based on visual observations, which might made is often missing in descriptions of cause some error between different studies. materials and methods. Even if the second The Tekauz scale has clear descriptions for seedling leaf seems to be the most com- all infection types and the risk for misscor- monly used for disease scoring in seedling ing is relatively low. tests (e.g. Gupta 2001; Wu et al. 2003, Beattie et al. 2007), it is an essential de- When comparing the studies made at tail to report when analysing and compar- the seedling stage in a greenhouse using ing P. teres virulence results. the Tekauz scale and the field adult plant test, Robinson and Jalli (1997b) found no The Tekauz 1-10 numerical scale for scor- correlation between the tests on six Nor- ing the infection response is based on dic barley varieties carrying no known P. symptom expression as it appears 7-9 days teres resistance genes. The contrast was ex- after inoculation (Tekauz 1985). Tekauz’s plained by the higher infection rate in the scale to classify barley reactions to Pyren- greenhouse than in the field. Therefore, for ophora teres is used worldwide both for quantitative disease resistance screening the P. teres virulence (e.g. Sato and Take- in the greenhouse it is necessary to opti- da1993; Jonsson et al. 1997; Gupta 2001; mize the test methods. In P. teres virulence Wu et al. 2003) and for resistance stud- screening studies, when a barley differen- ies (e.g. Sato and Takeda 1997; Cakir et tial set is used, a high positive correlation al. 2003; Ma et al. 2004; Manninen et between the reactions in greenhouse and al. 2006; Grewal et al. 2008). Our results in field is observed (Jalli 2009, unpub- showed that the infection response values lished data). based on Tekauz scale were not affected by any of the fungal isolates’ morphological Tekauz (1985) rated lesions below 5 as re- characters (growth rate on agar, spore pro- sistant reactions and greater than 5 as sus- duction ability, size of conidia or amount ceptible reactions. In different studies a of septa per conidium). That supports the score of 5 is considered either as a viru- uses of Tekauz scale for pathogen-host in- lent/susceptible reaction (e.g. Beattie et al. teraction studies either to measure viru- 2007) or avirulent/resistant reaction (e.g.

24 MTT SCIENCE 9 Jonsson et al. 1997; Wu et al. 2003). In our research that should be as carefully evalu- virulence studies, a value of 5 was consid- ated as other parts of the research and their ered to be a virulent reaction. However, value is often underestimated. the fixed limit between an avirulent and a virulent reaction over all tested barley The screening of P. teres virulence with genotypes might be misleading. For ex- molecular tools instead of resistant bar- ample, on barley genotypes CI 5791 and ley plants might be possible in the future. CI 9819, a score of five is clearly a sign of There already exist molecular methods to virulence, which may develop further in detect P. teres avirulence genes AvrHar, time. On genotypes Canadian Lakeshore AvrPra1, AvrPra2 (Lai et al. 2007) and and Manchuria it is more considered as an AvrHeartland (Beattie et al. 2007). How- avirulent, stable reaction. The expression of ever, the relatedness between avirulence defence differs slightly between different genes and virulence is variable between barley genotypes. On some genotypes, like different pathogens (Sacristán and García- CI 9819, no netting is observed and the Arenal 2008) and the role of the detect- symptoms are closer to symptoms caused ed P. teres avirulence genes in virulence by P. teres f. maculata. Therefore, when is still unclear (Beattie et al. 2007). Also, monitoring the P. teres f. teres virulence, the research on phytotoxic compounds iso- to follow the possible evolution in patho- lated from P. teres (Sarpeleh et al. 2007) gen population, defining the limit between and their role as pathogen effectors (Stuke- avirulent and virulent classes individually nbrock and McDonald 2009) may open for each barley genotypes instead of hav- novel possibilities to study virulence of ing a fixed limit over the entire barley dif- P. teres. However, for routine virulence ferential set may be a more sensitive tool. screening to monitor the changes of virulence in P. teres f. teres populations, green- An important part of the research is the house seedling tests still represent a valua- use of different statistical analyses and ble and efficient tool. their effect on the final conclusions. In our studies the statistical methods were 3.2 Diversity of Pyrenophora chosen based on the trial design, on the teres f. teres isolates in Fin- type of data (categorical or continuous land variable), on the distribution of data, and on the statistical methods available at the 3.2.1 Diversity in virulence time of analyses. Several different statistical methods are used when analysing the TheP. teres isolates used in this study were virulence or resistance data based on the originally collected for several different Tekauz scale: e.g. general linear models purposes. Some of the isolates were collect- (GLM) procedure, restricted maximum ed systematically from selected locations, likelihood (REML) procedure, best linear cultivars and fields with known cultiva- unbiased predictors (BLUPS), and protect- tion histories, while others were collected ed least significant difference test (PLSD) randomly from experimental or farmers’ (Grewal et al. 2008; Gupta et al. 2003; fields. The testing conditions in different Tuohy et al. 2006). Statistical methods studies were close to each other and all the evolve and give increased opportunities disease observations were made by the au- for better understanding the data. That de- thor, allowing the analyses and conclusions mands optimal trial design, close coopera- to be made over the separate trials. tion between researchers and statisticians, and careful comparison of studies with dif- In total, 239 Finnish P. teres f. teres iso- ferent statistical methods used. Definitive- lates were tested in 1994 – 2008. None ly, statistical methods are the part of the of the isolates was virulent on CI 5791.

MTT SCIENCE 9 25 On CI 9819, 1 % of the isolates scored 5, in Finland provided the basis for this re- the lowest value considered to be a viru- search (collection 1). The results were il- lent reaction. All other reactions on CI lustrated with regression plots and each 9819 were avirulent. The median of the isolate exhibited a unique pattern of re- infection score was above four (virulent sponse on the differential genotypes. The reaction) on 11 of 31 barley differential results indicated differences between the genotypes: Cape, Corvette, Harbin, Har- isolates and genotypes, but not always in rington, Haruna Nijo, Kombar, Manchu- slope of the regression line. All the isolates ria, Ming, Pirkka, Rika and Skiff (Figure were avirulent only on genotypes CI 5791 5). The average infection score of the tested and CI 9819 and virulent on the six Nor- isolates over the all tested barley genotypes dic barley genotypes that showed a range varied from 1.9 to 7.4 (Figure 6). of symptoms in the field.

The first virulence study on 20 P. teres f. We investigated if the host plant from teres isolates collected from 11 sites in 1994 which the isolate originated significant-

Figure 5. Box plot displaying the median, quartiles, and minimum and maximum observations for the symptom scores of the 239 Finnish Pyrenophora teres f. teres isolates on barley differential set genotypes. The medians are connected by a line.

26 MTT SCIENCE 9 score 10 9 8 7 6 5 4 3 2 1 0 isolate

Figure 6. The average and standard deviation for the symptom scores of the 239 Finn- ish Pyrenophora teres f. teres isolates over the tested barley differential genotypes.

ly affected the virulence of the isolate. to those of the isolates originating from The isolates from susceptible barley cul- Estonia, Latvia, Norway, Sweden and Ire- tivar Arve were more virulent than those land. The study identified large differenc- from less susceptible Pohto. That was con- es in response of barley genotypes to the firmed by Jalli & Robinson (1997) using P. teres f. teres isolates. Only little varia- 64 P. teres isolates originating from culti- tion was detected among the P. teres iso- vars Arve and Pohto. However, as Thrall lates from Nordic-Baltic countries on the and Burdon (2003) reported in their stud- tested genotypes Cape, CI 9819, Coast, ies on flax rust and flax, aggressiveness and Manchuria. Contrary to our stud- is favoured over virulence in susceptible ies, Tuohy et al. (2006) found significant host populations, whereas virulence is fa- variation in virulence among Northern voured in resistant populations, and it European P. teres f. teres isolates, includ- might be that instead of greater virulence ing isolates from Finland. However, the the issue in our studies was one of great- two studies included only one barley gen- er aggressiveness. The differences in defi- otype in common (Coast), which part- nitions between various terms are not al- ly explains the different conclusions from ways clear. Aggressiveness and virulence the two host-pathogen interaction studies. are even used as synonyms (Shaner et al. 1992). The same problem of nomenclature In paper III, the greenhouse test viru- exists when discussing virulence on the lence data on 174 P. teres f. teres isolates barley genotypes that are quantitatively (including 80 Finnish isolates) were an- resistant or susceptible to the P. teres. The alysed together with the virulence data proper term to use in such cases would be from detached leaf tests on 885 isolates aggressiveness (Pariaud et al. 2009). (including 150 Finnish isolates). Based on Afanasenko’s detached leaf studies on the In paper II, the virulences of 29 Finnish Finnish isolates (Afanasenko 2009, per- isolates collected in 1997 were compared sonal communication), the most resistant

MTT SCIENCE 9 27 barley genotypes identified were Tifang, four trials and nine of the barley genotypes CI 9819, Canadian Lakeshore, c-8755, in different trials were common: Beecher, and CI 5791. The results correlate well Canadian Lakeshore, CI 5791, CI 9819, with our greenhouse virulence data col- CI 11458, Harbin, Manchuria, Manchu- lected between 2000 and 2005. The high- rian, and Tifang. Based on the results of est frequency of avirulent reactions was GLIMMIX analyses made on the com- found on CI 9819 and CI 5791. 86 % bined data there were no significant av- of the isolates were avirulent on c-8755, erage differences in the virulence of the but only 47 % on Canadian Lakeshore. P. teres isolates among the years (P=0.10). In the seedling tests Canadian Lakeshore The pairwise analyses showed a slight dif- was mainly considered to be a medium ference in virulence of the studied P. teres resistant genotype and some isolates were isolates between 1994 and 2004 (P=0.04). highly virulent on it. The slight differenc- The estimated frequencies of virulent iso- es between Afanasenko’s and our stud- lates over the nine barley genotypes in dif- ies may have resulted from the origin of ferent years were 1994: 29 %; 2003: 40 %; the isolates and the testing methods. In 2004: 20 %; and 2005: 35 %. The results Afanasenko’s research the Finnish isolates of GLIMMIX analysis on 44 isolates col- originated from three fields while in our lected in 2000, 2003 and 2007 that were studies they were collected from 14 sites tested on five common barley genotypes and several cultivars. (Annabell, CI 9819, Kunnari, Manchu- ria, and Pirkka,) showed no significant in- In paper IV, the virulence of P. teres f. teres teraction between the collection year and isolates originating from a no-tillage and the virulence (P=0.52). normal-tillage cultivation system was studied. Surprisingly, only 37 of the 313 sin- The virulence studies on the combined da- gle spore isolates represented the net form taset provided novel possibilities for anal- of P. teres and the rest represented P. teres yses of the specific and overall virulence f. maculata. The virulence reactions of net among the isolates collected in 1994-2007. form isolates on barley differential gen- No clear virulence was found on CI 9819, otypes CI 9819 and Manchuria were al- which was the only barley genotype in- most identical and no influence of the till- cluded in all trials. The collecting year had age system was observed. All the isolates no significant effect on the infection re- were avirulent on CI 9819. Two of the 37 sponse when using the avirulent/virulent isolates were virulent on Manchuria, one classification. However, the expression of originating from a no-tillage and the oth- avirulence on CI 9819 varied while the er from a normal tillage plot. infection response ranged between scores from one to five. Some of the isolates were 3.2.2 Change in virulence over years highly aggressive, causing numerous leaf spots even when the spot size remained The combined dataset (Finnish isolates in small and no chlorosis was observed. The the collections 1–4) was used to study the same phenomenon was observed in field change in P. teres virulence over time. Chi- trials where CI 9819 seemed to be sensi- Square values that illustrated the associa- tive to physiological leaf spots. The physi- tion between the year of collection of the ological leaf spots appeared at later growth isolate and the virulence of all tested 239 stages and no physiological spots were ob- P. teres isolates on a susceptible barley gen- served in seedling tests. No virulence on otype and on CI 9819, indicated no signif- CI 9819 is recorded in P. teres virulence icant interaction (P= 0.97). The virulence studies made in other barley growing are- of 98 P. teres f. teres isolates collected in as (paper III Afanasenko’s studies; Gupta 1994, 2003, 2004 and 2005 was tested in et al. 2003; Tuohy et al. 2006).

28 MTT SCIENCE 9 Regarding the separate trials and the over- tion genetic studies of P. teres. Wu et al. all analyses, the virulence of Finnish P. (2003) combined the results of virulence teres f. teres isolates collected between 1994 and genetic variation studies based on re- and 2008 was fairly stable, both within striction fragment length polymorphism and among years. In Finland, the patho- (RFLP) analysis. The results indicated that gen has not been exposed to major resist- P. teres possesses a high degree of diversi- ance genes, and therefore it has not been ty at species and subspecies level (P. teres under a selective pressure, making coevo- f. teres and P. teres f. maculata). Serenius et lution of the pathogen-host interaction to al. (2005) studied the genetic variation of produce a more virulent pathogen unlike- two P. teres populations originating from ly. Similar results were reported by Gupta Finnish barley fields using AFLP analy- (2001). He concluded that the virulence sis. The mean genetic similarity among all among the isolates from Western Austral- the isolates was 93 % and most of the varia remained stable for 19 years due to the iation was observed within field popula- cultivars grown. More detailed knowledge tions. They concluded that FinnishP. teres on the evolution of Finnish P. teres f. teres isolates are genetically more variable than populations could be achieved by desig- expected based on virulence surveys in pa- nating pathotypes (Steffenson and Web- pers I and II. In later P. teres population ster 1992a) or isolate groups (Gupta 2001) studies Serenius et al. (2007) showed that that illustrate the similarities and differ- most of the total variation within Finnish ences between isolates in a very compre- P. teres f. teres isolates was within the col- hensive way. However, in our studies, the lection years rather among them and they rationale has been to serve the practical were genetically more different than those barley resistance breeding programme and collected from other Nordic areas. Vir- therefore emphasis has been put on follow- ulence is only one indicator used to de- ing the evolution of virulence on specific scribe the population structure. The com- barley genotypes instead of the general ev- bination of systematic population genetic olution of P. teres pathogen populations. and virulence studies would be an ideal To get a more comprehensive overview tool to study the factors affecting the P. of the changes in pathogen populations, teres – barley interaction and to predict larger numbers of isolates rather than sin- the risk for changes in the P. teres viru- gle isolates would have to be tested. Also lence spectrum. the number of barley genotypes included in virulence tests increases knowledge 3.2.3 Effect of sexual reproduction on on the population structure. The variabil- virulence ity of a population of P. teres depends on the number of barley cultivars examined Mating studies with pathogen isolates are a and the differences in their genetic back- valuable method for better understanding ground, the presence of both net and spot pathogen-host interactions. The effect of forms in studies, and the number of iso- sexual reproduction on virulence in P. teres lates examined (Tekauz 1990). was studied in three artificially produced populations (paper IV): in one crossing Not many comparable novel studies on P. between two net form isolates (isolates 14 teres virulence exist in the literature, which x 15) and in two crossings between a net together with the different methodologies form and a spot form isolate (isolates 14 x used weakens the comparison between the 24 and 16 x 24). The virulence of the P. P. teres virulence situation in Finland and teres f. teres parental isolates 14, 15 and 16 in other countries. Instead, the rapid de- were identical on five tested barley geno- velopment of molecular tools has made it types Arve, CI 5791, CI 5822, Manchu- possible to put more emphasis on popula- ria, and Pohto: avirulent on CI 5791 and

MTT SCIENCE 9 29 CI 5822 and virulent on Arve, Manchuria to the specific isolates and host genotypes, and Pohto. The virulence reaction of theP. and generalization of the results should be teres f. maculata parental isolate was oppo- done with caution. site to the net form isolates on CI 5791, CI 5822, and Manchuria. None of the cross- The sexual mating of heterothallic as- ing progenies was entirely identical to its comycetes is controlled by fast evolving parents in terms of virulence. The average mating type genes (MAT1 and MAT2) similarity of the progeny isolates to their (Turgeon 1998) that determine the sexu- parents was calculated by assessing the per- al compatibility between different haploid centage of similar reactions between the individuals. The heterothallism among progeny isolates and parental isolates over ascomycetes supports the importance of the five barley genotypes. The average sim- generating new genetic combinations. A ilarity of the net by net form mating prog- pathogen must choose between sexual and eny isolates to its parents was 92 %. The asexual development, and they use nutri- average similarity of the net by spot form tional, temperature and light clues to make mating progenies isolates was 58 % to the this decision (Nelson 1996). Serenius et al. net form parents and 73 % to the spot (2005) reported that both mating types form parent. (MAT1 and MAT2) exist in Finland at a ratio close to 1:1. Based on the AFLP stud- The P. teres crossing studies done in the ies of Finnish P. teres f. teres isolates Seren- laboratory showed the possible risk for ius (2006) concluded that sexual repro- changes in virulence on genotypes CI duction occurs in Finland. However, no 5791, CI 5822, and Manchuria if sexu- visual evidence of the naturally existing al mating exists in the field. In the cross- pseudothecia has been reported. In con- ing between the two net form isolates, the trast, pseudothecia were easily observed in changes in virulence were smaller than in Denmark and in southern Sweden (Sme- crossings between the net and spot form degård-Petersen 1972; Jonsson 2001). The isolates because of the different virulence artificially inoculated straw samples that profiles of the net and spot form parents. were stored at different soil depths over All three progenies included isolates that the winter 2006-2007 in Jokioinen, Fin- were either avirulent or virulent on each land had visible pseudothecia in spring barley genotype. Both net form parents but at the time of collection, just before were avirulent on CI 5791, but still 8 % of sowing time in early May, the ascospores their progeny isolates were virulent on it. were already ejected (Jalli 2007, unpub- Both the parents scored 2 while in proge- lished data). The equal amounts of mat- ny isolates the maximum score was 7. The ing types in Finnish populations (Seren- studies included two net and spot form ius et al. 2005), the conclusions reached matings, having the same spot form pa- on AFLP studies of Finnish isolates (Se- rental and two net form parental isolates renius 2006), and the success of artificial sharing the same virulence profile. How- mating in nature (Jalli 2007, unpublished ever, the inheritance of virulence was not data) indicate the high probability of the identical among the two net x spot form existence of sexual reproduction of P. teres matings. On CI 5822, 68 % of the 16x24 f. teres in Finland. However, the possible and 94 % of the 14x24 progeny isolates effects of sexual reproduction on the viru- were virulent. The unpredictable results lence of P. teres have not yet been noted in confirm that virulence of a pathogen is Finnish barley fields. result of several factors (Bent and Mack- ey 2007) and in the studied host-patho- Due to the different genetic structure (Rau gen interactions it seemed to be control- et al. 2007) and virulence profile of net led by several genes. Our study is related and spot forms of P. teres isolates (paper

30 MTT SCIENCE 9 IV) their recombination may lead to un- to the potential for enhanced productivity, predictable virulence. Our crossing stud- cost and environmental savings, most agri- ies in the laboratory were successful in pro- cultural institutions and governments are ducing large amounts of viable ascospores. promoting reduced tillage. However, in a There are several examples of the success minimum or no-tillage system the risk for in mating the two forms under labora- residue-borne plant diseases may increase tory conditions (e.g. Smedegård-Petersen (Joshi et al. 2007). There are a few studies 1971; Campbell et al. 1999). Campbell on the influence of no-tillage and reduced and Crous (2003) investigated the hybrid tillage on the incidence and severity of net progeny of a mating between net and spot blotch (e.g. Jordan and Allen 1984; Duc- form isolates, concluding that they re- zek et al. 1999). There is also evidence on tain their virulence and fertility over time the effect of both the no-tillage and crop and are genetically stable after two cycles rotation on pathogen population structure. of inoculation, mitosis and re-isolation. Almeida et al. (2008) showed that Macro- Evidence on the recombination between phomina phaseolina, an important disease net and spot form isolates in the field in of soybean, is a genetically variable spe- South Africa was reported (Campbell et cies and can be affected by crop rotation. al. 2002). In contrast, the results of Seren- Chulze et al. (2000) indicated that the ius et al. (2007) on the AFLP studies on Fusarium populations isolated from no-till P. teres populations, which also included maize are similar to those recovered from Finnish isolates, indicated that recombi- maize managed with conventional tillage. nation between the two forms hardly oc- However, no research has been done on curs in nature. Also the studies by Rau et the effect of cultivation system on P. teres al. (2007) confirm that the hybridization population structure and virulence. between the two forms is rare or absent under field conditions. In that regard our In this study the effect of no-tillage culti- results on the net and spot form crossings vation practices on the virulence of P. teres should be considered more as an example populations was studied among 313 iso- of possible changes in virulence when two lates that were obtained from three fields isolates of different virulence profile mate. that had five to eight year histories of barley monoculture either under no-tillage or 3.2.4 Effect of cultivation practices on in normal tillage cultivation (paper IV). virulence Of the isolates, 276 represented the spot form and only 37 the net form of P. teres. Reproduction and mating systems affect Based on Cochran-Mantel-Haenszel statis- how gene diversity is distributed with- tics, the tillage method (no-tillage or nor- in and among individuals in a pathogen mal tillage) had no effect either on the vir- population. Populations of sexual patho- ulence structure of the P. teres f. teres or P. gens usually exhibit a high degree of gen- teres f. maculata populations. otype diversity and represent greater risks to break down of resistance (McDonald Collections 1-3 in our studies represent and Linde 2002). One phenomenon that only the net form of P. teres and the low may affect the occurrence of sexual repro- frequency of net form isolates in collection duction is the cultivation system. Reduced 4 was not predicted. The fungicide treat- and no-tillage cultivation systems are be- ed seed and the net blotch infections ob- coming popular among farmers around served at the early tillering stage in no-till- the world. Reduced tillage improves a age blocks confirms that the infection was soil’s physical, hydro-physical and biolog- mainly straw-borne. Besides the possible ical properties, especially in a good crop effect of the microclimatic conditions in rotation system (Rusu et al. 2009). Due the fields, the high proportion of spot form

MTT SCIENCE 9 31 3.3 Implications of P. teres f. isolates may be explained by long mono- teres virulence for net blotch culture of barley cultivars Annabell and resistance breeding Saana in the fields from which the samples were isolated. Both cultivars are medium resistant against the net-form of the fungus Besides the differential sets that include (Kangas et al. 2008), which might have barley genotypes with different resistance caused selection towards the spot form. phenotypes, our virulence studies includ- Based on the virulence studies there were ed barley genotypes that are cultivated in no signs of increased variation among the the Nordic countries and whose resistance isolates originating from no-tillage com- background is unknown. The virulence pared to isolates originating from normal study results identified large differences in tillage plots. No recombinants between net response of barley genotypes to P. teres f. and spot form isolates were observed ei- teres. When infection scores from 1 to 4 ther; all the isolates produced clear net or were considered as resistant reactions, CI spot form symptoms. The AFLP analyses 5791 was the only genotype resistant to on some of the isolates support the results all tested isolates. Of the 46 barley geno- of virulence studies. No differences in the types, 23 were resistant to at least 50 % of genetic variation were found among the the isolates. Those genotypes included two populations originating from different cul- commonly grown malting barley cultivars tivation systems (Serenius 2008, unpub- in Finland: Scarlett (resistant to 80 % of lished data). Even though natural drift of the isolates) and Annabell (resistant to 72 isolates between blocks with different cul- % of the isolates), and a six-rowed culti- tivation systems is probable, there was no var Kunnari (resistant to 61 % of the iso- such variation in virulence either in the lates). The frequency of resistant reactions isolates originating from no-tillage or nor- was less than 10 % on cultivars Tiril, Rolfi, mal tillage field. Arve, WW 7977, Harrington and Agne- ta (Table 4). What was observed in the study on the effect of sexual mating on virulence, and Scarlett, Annabell and Kunnari have the sexual recombination of P. teres isolates shown good net blotch resistance also in could not have been proved based on the the Finnish official variety trials (Kangas virulence studies. The virulence of the net et al. 2008). Robinson and Jalli (1997b) form isolates originating from no-tillage found adequate levels of quantitative re- and normal tillage background was com- sistance in genotype H 6221 (‘Thule’) and parable to the results earlier presented in later (2001) in Bor 94007 (‘Jyvä’). These this work: all net form isolates were avir- genotypes are examples of widely adapted ulent on CI 9819 and virulent on Pirkka. genotypes that possess adequate levels of However, this research was a preliminary resistance to make them useful parents in study on the possible effects of cultivation crossing programmes. Testing the breed- methods on P. teres related to specific en- ing material at several sites over several sea- vironments, and to better understand the sons provides an opportunity to monitor long-term effect of no-tillage on sexual re- and develop adapted genotypes that exhib- combination and on the virulence of P. it stable resistance to P. teres. Based on the teres. The research continues, considering analysis made on Finnish official variety also the effects of monoculture and crop tests, Robinson and Jalli (2001) concluded rotation on the virulence and on popula- that there are no site-specific populations tion genetics. of net blotch that differ greatly in virulence

32 MTT SCIENCE 9 Table 4. Number of tested isolates, the minimum infection score, the maximum infection score, and the percentage of resistant reactions (score < 5) on barley genotypes tested with different Finnish P. teres f. teres isolates.

Genotype Number of tested Min infection Max infection % of resistant isolates score score reactions CI 5791 142 1 4 100 CI 9819 238 1 5 98 CI 7584 53 1 10 93 CI 5822 53 1 8 89 CI 9825 51 2 8 88 CI 9214 50 1 9 87 c-8755 51 1 6 86 Rojo 53 1 10 86 c-20019 51 1 8 83 Beecher 110 1 10 83 Scarlett 33 2 6 80 Manchurian 110 1 10 77 Coast 82 1 8 74 Annabell 22 1 10 72 Algerian 20 1 10 72 CI 11458 110 1 10 71 Tifang 110 1 10 63 Prato 20 1 10 63 Canadian Lakeshore 110 1 10 62 Kunnari 66 2 8 61 Prior 51 1 9 60 CI 4922 20 1 10 60 Atlas 20 1 10 51 Rika 20 1 10 48 Olavi 44 2 9 42 Skiff 51 1 9 37 Cape 49 1 10 36 Manchuria 160 1 10 33 Ming 20 1 10 30 Harbin 110 1 10 27 Kombar 20 1 10 24 Edel 43 3 9 18 Corvette 51 1 9 18 Pohto 49 3 9 17 Artturi 20 3 6 17 Pirkka 156 1 10 15 H 6221 20 1 10 14 Pilvi 44 2 10 13 Voitto 44 2 9 10 HarunaNijo 32 1 10 10 Tiril 44 2 9 9 Rolfi 33 1 10 9 Arve 93 2 10 5 WW 7977 20 1 10 4 Harrington 51 2 10 3 Agneta 20 4 10 1

MTT SCIENCE 9 33 in Finland. That was confirmed by the vir- net blotch (Arabi et al. 1992; Tuohy et al. ulence studies made during this research. 2006; Grewal et al. 2008). According to Cultivar Kunnari was released in 2000 the cluster analyses and the ability of indi- and Jyvä in 2001 (Table 1). Both cultivars vidual genotypes to discriminate P. teres f. are widely cultivated in Finland (Kunnari teres isolates, c-8755, CI 9825 and CI 5791 30 000 ha and Jyvä 36 000 ha in 2009) showed a different ability to illustrate vir- but no breakdown of the resistance has ulence in P. teres isolates. Therefore, it is been recorded (Kangas et al. 2008). The likely that these resistant genotypes carry durability of resistance may be explained at least partly different resistance genes. by the genetic background of the resistance The genetic studies made on the host- and the stability of Finnish P. teres f. teres pathogen interaction of these barley gen- populations. In the Danish studies on net otypes and P. teres f. teres isolates confirm blotch infection on barley during sever- that the resistance is mostly isolate-specific al years and at several locations, the total and controlled by one or two genes (Afa- variation in disease severity (61-81 %) was nasenko et al. 2007). explained by plant genotype and environment, and only a minor part of the total Diverse sources of resistance must be de- variation was assumed to be caused by vir- ployed to achieve long-lasting resistance. ulence characteristics of pathogen popula- Novel net blotch resistance sources are es- tions (Pinnschmidt and Hovmøller 2002). sential in environments that encourage the risks for changes in P. teres popula- The results of our experiments indicate tions. Even though this study demonstrat- that the barley genotypes used in the dif- ed the slow evolution of P. teres virulence ferential sets contain useful levels of resist- between 1994 and 2007, there are exam- ance to P. teres f. teres that could be used in ples on host-pathogen interactions where the Finnish barley disease resistance breed- the situation has changed rapidly resulting programme. Several genotypes were ing from recombination and the selection more resistant than most of the cultivat- imposed by resistance genes (McDonald ed barleys and could be used to strength- et al. 1989). en the level of resistance that exists among barleys as a result of the regular breeding Novel resistance sources and allelic vari- programme. ation could be found from landraces and wild genotypes originating from areas with Barley genotypes CI 5791 and CI 9819 high net blotch pressure. Robinson and were resistant to all Finnish P. teres f. teres Jalli (1996) showed that barley genotypes isolates. In paper III, where genotypes were originating from Latin America carry ef- tested against 1059 P. teres f. teres isolates ficient net blotch resistance against Finn- from 14 countries, the lowest mean fre- ish P. teres f. teres isolates. Bonman et al. quency of virulent isolates across popula- (2005) demonstrated that disease resist- tions and testing methods was observed ance varies depending on geographic ori- on CI 5791, CI 9819, c-8755 and CI 9825, gin. Adult plant net blotch resistance was which indicates a high level of resistance in most common in accessions from East- these cultivars. Genotypes c-8755 and CI ern Asia, China, South Korea and Japan. 9825 showed good resistance also against Accessions from Eastern Africa showed Finnish isolates. Despite the good poly- the highest levels of resistance at the seed- genic resistance of CI 5791 and CI 9819 ling stage. Wild barley, Hordeum vulgare against barley net blotch (Jonsson et al. spp. spontaneum, may represent a total- 1999; Manninen et al. 2006; Afanasenko ly novel input for improving barley net et al. 2007), it seems that they are ful- blotch resistance. However, the value of ly efficient only against the net form of genetic resources of wild barley will only

34 MTT SCIENCE 9 be realised when their resistance genes are ing for adult-plant resistance. Jonsson et transferred into cultivated barley. Sever- al. (1998) reported that net blotch resist- al H. vulgare spp. spontaneum and culti- ance in barley seedlings inoculated at the vated barley crosses have been developed one to two leaf stage correlated with net and resistance was successfully transferred blotch reactions on the fourth and flag into adapted barley germplasm (Steffen- leaves when barley was grown in growth son et al. 2007). Sato and Takeda (1997) chambers, as well as with disease levels re- found H. vulgare spp. spontaneum acces- corded on the three uppermost leaves in a sions from Afghanistan and Russia that field experiment. However, based on our showed high levels of net blotch resistance. virulence studies on the barley differen- Using wild barleys and landraces to in- tial set and the studies by Khan and Boyd crease biodiversity in barley breeding pro- (1969) on the sensitivity of resistance ex- grammes affords the development of plant pression to temperature and light intensity, varieties with novel genetic combinations resistance screening only under the green- that will be required to meet the challeng- house conditions can lead to false conclu- es arising from the changing environment, sions. The value of expression of resistance like drought and pests. in diverse climatic conditions is increasing due to climate warming. Climate change The net blotch disease infection level is can also affect the durability of resist- highly influenced by the environment ance. There is evidence that some forms and the reliability of the resistance screen- of disease resistance might be overcome ing results may decrease if they only rely more rapidly following changes in levels on non-inoculated survey data or on data of CO2, ozone and UV-B (Chakraborty from too few on non-representative envi- et al. 2000). The optimal way to get prof- ronments (Pinnschmidt and Hovmøller itable and long-lasting results in improv- 2002). The testing methods for screening ing net blotch resistance could be to find a virulence of P. teres f. teres on seedlings in balance between different breeding meth- the greenhouse with specific isolates seem ods most appropriate to the needs and re- to be efficient also in disease resistance sources: molecular tools to map and screen testing. Gupta et al. (2003) and Grewal specific resistance genes, greenhouse seed- et al. (2008) concluded there was strong ling tests to select against specific viru- agreement between seedling and adult- lence, and multi-environmental field tests plant reactions, which indicates that seed- to follow the resistance-environment inter- ling screening could be useful in select- actions and to secure durability.

MTT SCIENCE 9 35 4 Conclusions

creening for P. teres f. teres virulence The virulence spectrum of FinnishP. teres on barley seedlings under greenhouse f. teres isolates collected in 1994-2007 is conditions is a reliable and feasible stable both within and between years. The Smethod, especially in the absence of mod- results indicated differences between the ern molecular tools to monitor the com- isolates and genotypes but not always in plex P. teres f. teres – barley interaction. their interaction. There is no virulence to Because the method is based on visual barley genotypes CI 5791 and CI 9819. observations of disease symptoms on liv- The virulence of FinnishP. teres f. teres iso- ing plants it is also a comprehensive tool lates is highest on Cape, Corvette, Har- for studying the P. teres – barley patho- bin, Kombar, Manchuria, Ming, Rika, system. Besides the genetic backgrounds and Skiff of the barley differential geno- of the host and the plant, disease expres- type set. In Finland, there is no evidence of sion is related to the environmental con- any effect of sexual reproduction on viru- ditions, temperature, light and humidity, lence under natural conditions; not even in which need to be stable to achieve reliable barley monoculture no-tillage fields, which and comparable results among different should represent an optimal environment studies. Even slight changes in environ- for sexual recombination. However, based mental conditions may change the disease on artificial mating studies the virulence of expression of some barley genotypes. A a pathogen is a result of several factors that common barley differential set to charac- may be influenced significantly by sexual terize the P. teres f. teres virulence was de- recombination even when two isolates with veloped based on its ability to discriminate similar virulence profiles mate. The mat- pathotypes and indicate pathotype diver- ing and host-pathogen interactions studies sity, and is recommended for use globally: related to specific isolates and host geno- c-8755, c-20019, CI 5791, CI 9825, Cana- types, and generalization of the results, dian Lakeshore, Harbin, Prior, and Skiff. needs to be done with caution. Cultivar Harrington is recommended to be used as a susceptible check. These gen- Even though no changes in the Finnish otypes form the basis of the set that needs P. teres f. teres virulence spectrum were to be enlarged when novel resistance is in- observed, changes in climate and cultiva- troduced. The barley seed for virulence tion methods could change the situation tests should be produced in an environ- rapidly and breeders need to be prepared. ment that minimizes the risks for biot- Therefore, virulence surveys, concentrat- ic or abiotic stresses. Seed dressing treating on areas with high risk for evolu- ment is not recommended. Barley seed tion, should be an essential and contin- size, P. teres isolates morphological char- uous part of a disease resistance breeding acters, spore production or growth rate programme. Large differences in response on agar have no effect on the expression of barley genotypes to P. teres f. teres ex- of virulence. The Tekauz numerical scale ist. Finnish six-rowed cultivars Kunnari based on infection type is more appropri- and Jyvä are examples of durable resist- ate to describe the virulence of the isolate ance achieved through traditional breed- than the percentage of leaf area damaged. ing methods. Novel resistance that has not The second seedling leaf is best for differ- been introduced earlier into Finnish barley entiating the isolates. is also available. Barley genotypes c-8755,

36 MTT SCIENCE 9 CI 9825 and CI 5791 have excellent re- plant pathogens in barley production will sistance against Finnish P. teres f. teres iso- not decrease in the future. There is also ev- lates and it is assumed that their resist- idence that besides P. teres f. teres, the spot ance background is not entirely identical. form of the pathogen P. teres f. macula- The greenhouse virulence screening meth- ta will be an important pathogen of bar- ods are valuable for P. teres f. teres resist- ley in the future, which needs to be taken ance monitoring, especially when screen- into account in disease resistance breeding against specific virulence. However, ing programmes. The barley disease resist- when screening for quantitative and du- ance breeding represents a palette of sev- rable resistance, testing breeding material eral pathogens, resistance genes, positive at early generations in multi-environmen- and negative interactions, test methods, tal field tests is an efficient way to gauge and also compromises but has a common the effect of different environments andP. aim to develop a plant that is valuable for teres populations on the resistance expres- farmers, end-users, the environment, and sion. It is obvious that the importance of the breeder.

MTT SCIENCE 9 37 5 Acknowledgements

This study was carried out at MTT Agri- pio gave an impressive introduction to the food Research Finland and Boreal Plant world of plant pathogens together with Breeding Ltd in Jokioinen, Finland. The Prof. Risto Tahvonen and Timo Kauko- main part of the research was funded by ranta. My enthusiasm on plant pathogen- Ministry of Agriculture and Forestry. I ic fungi is due to their lessons. Besides, I truly thank them for all the resources and want to express my respect to Eeva Ta- support that made this long-term research pio for her positive encouragement on in- on barley net blotch possible. ternational cooperation and for pointing out that the success in research is great- I greatly appreciate my supervisor Prof. ly influenced by the relationships be- Aarne Kurppa and Prof. Minna Pirhonen tween researchers with different skills and who stayed as my tower of strength espe- backgrounds. cially during the final part of this work. Aatu’s continuous interest on the subject My very special thanks are due to the staff and his inspirational support on coopera- of MTT Plant Protection. I warmly thank tion between research and practical plant Dr. Jukka Salonen, the head of our re- breeding made possible to connect the sci- search team, for his invaluable and kind ence and the real life. Minna’s calm posi- pressure to finally concentrate on the the- tive attitude and skillful comments during sis and for the possibility to prioritize the the hectic moments together with her en- use of my time. I also want to thank Juk- couragement in keeping the timetable were ka for his supportive comments on the very remarkable. Prof. Jari Valkonen I thank for first drafts of this work. his valuable input. I greatly appreciate that he also led me to the most recent findings Researchers Asko Hannukkala, Kari Tii- in host-pathogen interactions. likkala, Peppi Laine, Päivi Parikka, Anne Lemmetty, Irmeli Markkula, Satu Lat- My sincere thanks are due to the pre-re- vala, Ulla Heinonen, Sanni Junnila, Isa viewers, Prof. Lisa Munk and Prof. Erland Lindqvist, and all the other scientists at Liljeroth, who made significant work in re- MTT Plant Protection I greatly appreciate viewing and giving the prompt and con- for their giving hands, thoughts and fruit- structive criticism on the thesis. ful discussions. Erja Huusela-Veistola (my personal trainer) and Heikki Jalli I deep- My supervisor at the beginning of this ly thank for teaching me that agriculture study, Dr Jonathan Robinson, is the god- carries many other important aspects than father of this work. His great skills on sci- only plant pathogens, and that also insects ence, and more specifically on plant dis- and weeds can be beautiful. ease resistance, have had enormous effect on disease resistance breeding work in Fin- The extremely skilful and well-orientat- land. Jonathan taught me the two most ed technical staff of our Plant Protec- important issues in research, the moral and tion team, Auli Kedonperä, Anne Muoti- the hunger to look for the truth. Thank la, Kirsi-Marja Palm, Hilkka Timonen, you Jonathan. Tuula Viljanen, Aila Siren, Marjaana Vir- tanen and Timo Jaska (and all you who The basic for this research was built in have been dealing with this work during 1980’s at the Department of Plant Pathol- the last almost twenty years not forget- ogy, University of Helsinki. Prof. Eeva Ta- ting the excellent students Elin Koho, Eli-

38 MTT SCIENCE 9 na Seppä and Aleksi Mäenpää) made this like plant pathogens; they do not recognize work possible. Thank you for your pa- frontiers which I am deeply grateful on. tience and flexibility to researcher’s some- times odd demands, and thank you for Luckily barley net blotch is only a small your kindness. The whole MTT Plant Pro- part of life. Without all the friends and tection team I want to thank for the fam- relatives who have shared the joys and sor- ily like, care taking atmosphere where joy rows I could not have manage throughout is present. these years. To our singing group Kööri avec I want to give a big hug in sharing It has been wonderful to learn that one the world of music and several other things person cannot be expert on all areas and during the last years. My piano students I how rewarding cooperation with people want to thank for giving childhood’s and with different skills can be. Dr. Outi Man- youth’s energy through their sincere and ninen, Dr. Marjo Serenius and Tarja Ho- heartened attitude. vivuori from MTT Genetics Research I thank for the good co-operation in the My dearest thanks are due to my mother barley disease resistance breeding work at Liisa and farther Esko. You have given me MTT. Lauri Jauhiainen and Timo Hurme the tools for the life. The respect on nature I thank for their kind and understanding and on Finnish agriculture, the impor- help in statistical analysis and Ritva Kala- tance and possibility to study, to improve koski and the whole MTT library team for and to think with opened eyes but the feet the excellent services. Hannu Känkänen I on the ground, I have inherited from you. warmly thank for sharing the world of sen- My sisters Ulla and Eeva, and Eeva’s fam- ior PhD students at MTT. ily Esa, Timo, Liisa and Hanna, I want to thank for staying beside. You have taken Boreal Plant Breeding Ltd., and especial- excellent care of your younger sister, keep- ly the people involved in the disease resist- ing my opera knowledge updated and re- ance breeding work during and after my minding through your own way of living stay at Boreal, have a significant effect on on the basic things important in life. The the contents of this work. I want to thank beauty and calm of the Rantamaa cottage Managing Director Markku Äijälä, Breed- where the most part of this work was writ- ing Director Eero Nissilä, barley breeder ten, I never forget. Reino Aikasalo, Mika Hyövelä, Dr. Nigel Kilby, Dr. Kirsi Peltoniemi, Tenho Lahti My heartfelt thanks are due to Heikki, and Aija Viljanen for their true interest on Osmo and Helena. Finally, we managed disease resistance breeding, for the good to finish the colourbook in which you gave lessons on practical plant breeding and for the colours. To be honest, the most impor- their special input in this work. tant things during this project I have learnt from you. Thank you for your love. Co-authors Dr. Jonathan Robinson, Prof. Olga Afanasenko, Dr. Hans Pinnschmidt, Kiitos & hali Greg Platz and Olga Filatova I warmly thank for the common understanding and Koski Tl 15.3.2010 open cooperation we have. Scientists are marja

MTT SCIENCE 9 39 6 References

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MTT SCIENCE 9 45 9

The virulence of Finnish Pyrenophora teres f. teres isolates and its implications for resistance breeding

Doctoral Dissertation MTT is publishing its research findings in two series of publications: MTT Science and MTT Growth. Marja Jalli

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