It is possible to identify resistance genes of common bunt with a range of different virulence races of the pathoge

Borgen, Anders

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Purifying virulence races of common bunt (Tilletia caries) to identify resistance genes in wheatBy:Anders Borgen, Agrologica, Houvej 55, 9550 Mariager, Denmark E-mail: borgen@agrologica.dkIntroductionBreeding wheat with resistance to common bunt (Tilletia caries) is an important part of a strategy to control  the disease in organic farming. Many wheat varieties are described as resistant to the disease, and several major genes are described able to control the disease. However, there is a lack of consistency in field trials assessing wheat varieties for their susceptibility to the disease (Blažkova and Bartoš 1997).No doubt, genetic markers will be a powerful tool to identify resistance genes in breeding lines, also when it comes to resistance against bunt in wheat, but in order to develop markers, reliable phenotypic assessments are needed. To get there, we need tools to distinguish between different resistances in wheat lines.Most trials published on susceptibility of wheat varieties to bunt are done by adding spores to a seed sample before sowing, at assessing the frequency of of infected heads. This method is a more or less adapted method of trials testing for the effect of seed treatments (EPPO 1997). When testing for resistance, the virulence of the spores are of crucial importance, since a trial will not be able to identify resistance genes, if the spores used are virulent to the gene. Often, the origin and virulence of the spores in trials are more or less unknown, and most spore samples found in practical farming are genetic diverse. The may therefore be a mixture of virulent and avirulent spores against the  resistances in question. Using such spores in a trial may result in data difficult to interpret, as a low infection level can be a result of a low frequency of virulent spores in the spore sample. Next year, the composition of spores may have changed, which will lead to a different result in the same varieties.In this situation, I felt a need to work with less diverse spore samples in my bunt trials, and to achieve this, I developed a simple technique to develop more uniform and well described spores. Already Weston (1932) and Roemer og Bartholly (1933) showed that susceptibility of a variety increased when it was infected by spores from the same variety compared with spores from other varieties. Woolman (1930) showed that only up to 4 spores were able to infect the wheat meristem. Therefore, working with spores from a single plant will decrease diversity in a spore collection, and if this plant is resistant, it will after a few regenerations of such a variety be selective to a specific race, virulent to the resistance gene in question.Materials and methodsSpores were collected in the ORGSEED project in 2001-5 by Bent Nielsen. Spores were collected from a broad range of fields grown with different varieties in different regions of Denmark. It is likely that they pretty much represent the genetic diversity of the pathogen present in Danish agriculture by the time of collection. The collection was maintained on susceptible varieties until 2011 and hereafter on a composite cross population.From 2012 and onwards, resistant varieties were infected with spores collected on their own variety if infected spikes were found. In this way, a collection of 98 virulence races were build up specific to the 98 wheat varieties.In 2013-14 and in 2014-5, spores from selected varieties were used to infect other varieties. The varieties were sown in 0.5 rows without replication with 5g seed per row. Results and discussionSome of the varieties that had low infection in previous trials(Steffan 2014 , Steffan et al. 2014), and therefore were recordedas resistant, could indeed be infected if they were re-inoculatedwith the spores from the few infected plants of the variety. Forexample, varieties with the resistance gene Bt10 had no or lowinfection when infected with the diverse spore collection, and itwas concluded that Danish spores were avirulent to Bt10.However, when varieties with Bt10 were inoculated with sporesfrom their own variety, they turned out to be highly susceptible.The spore collection is therefore a mixture of virulent andavirulent spores against Bt10. The same is the case for Bt2, Bt7,Bt13 and BtZ.When varieties were inoculated with spores that had beenpurified on other varieties, they were normally either moreresistant or more susceptible compared with inoculation with thespores mixture. It is likely that varieties that react in a similar wayto different origins of spores may have the same resistance genes.However, this is not always the case. Some (or maybe most)varieties carry more than on gene affecting the susceptibility, andcertainly most virulence races of the pathogen are virulent tomore than one resistance gene. For example, spores virulent toBt10 are in this study also virulent to BtZ, and I have been unableto distinguish between these two genes.Some varieties have so far been resistant I all studies, and even ifa few plants were infected, spores from these plants have beenunable to create a high infection level. Some of these varietiesmay have a resistance gene to which no virulence have beenfound, and some varieties may have a combination of genes. Ihave been unable to develop virulence races specific to theresistance genes Bt3, Bt4, Bt5, Bt6, Bt8, Bt9, Bt11, Bt12.It is possible that the varieties NGB9014, NGB-9015, Tambor,Kuban, Begra, Maribos, Fold, Monopol, Tarso, Torrild, Cardos,Kranich, Türkis, Gluten, Folke have Bt7, since they react in asimilar way to the 7 different sources of spores used in 2014-5. It is possible that the varieties Format, Curier, Complet, Solstice,Bussard, Paroli, Dream, Butaro, Ochre, PG3540 and Herewardhave Bt2. The varieties Korrund, Aron, Karat, Tulsa, Xenos, Tataros,Erbachshofer Braun and Indigo have in some trials shownresistance, but in 2014-15, they have been susceptible to most orall virulence races.Spores harvested on the variety Tommi by Svalöf/SLU in 2014were able to give a high infection in the Tommi, Globus andSegor, and a medium infection was achieved with spores from BOKU, even though these varieties have so far been resistant or shown low susceptibility to spores from Denmark. The variety Quebon could be infected by spores purified from Czech Republic, but was resistant to all other spores tested on other varieties. This indicates that Tommi, Globus and Segor may carry the same resistance gene, which is different to the gene in Quebon, and different to the known Bt-genes.Pi554-121 (Bt3) 0,0 0,0 0,0 0,0 0,0 0,0 0,0Ridit  (Bt3) 11,1 0,0 5,0 3,4 0,0 0,0 0,0 0,0PI 554-116 (Bt3 33,3 0,0 0,0 11,1 7,1 0,0 0,0 0,0Nebred (Bt4) 16,7 0,0 0,0 14,3 0,0 3,7 0,0 2,3PI-554-115 (Bt4) 66,7 0,0 4,8 0,0 14,8 3,8 0,0 0,0Carlton 0,0 6,7 0,0 60,0 0,0 7,8 0,0PI-554-117 (Bt6) 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0Erythrosperum 5221 (Bt14) 0,0 2,6 0,0 3,0 9,8 0,0 0,0 4,8Promesse (Bt5) 0,0 0,0 0,0 2,7 0,0 0,0 0,0 0,0Hohenheimer  (Bt5)Ci-11458 50,0 27,6 16,7 0,0 0,0 6,9 0,0 12,5Lars 0,0 8,3 35,3 0,0 0,0 2,4 6,3Triple Dirk 0,0 3,2 13,0 0,0 0,0 4,8 0,0 0,0Hypnos 0,0 2,9 18,2 3,7 3,4 9,7 0,0Skagen 4,5 37,8 20,0 9,1 0,0 3,3 1,6 0,0Skotte 0,0 26,7 0,0 0,0 0,0 0,0 0,0 0,0PI-554-110 (Bt8) 0,0 0,0 20,0 33,3 40,0 0,0 0,0 0,0PI-554-111 (Bt8) 50,0 0,0 100,0 20,0 47,1 0,0 0,0Thule III (Bt13) 71,4 0,0 0,0 0,0 0,0 2,6 30,4 13,3Inna 22 0,0 84,0 0,0 0,0 0,0 4,2 0,0Nemchinovskaya 25 (BtZ) PI 0,0 3,4 55,6 3,8 7,1 0,0 0,0 2,9Pi173437 (Btp) 2,0 18,8 0,0 0,0 0,0 0,0 13,3Weston 33,3 0,0 52,6 0,0 0,0 0,0 0,0 13,6PI-554-118 (Bt10) 45,5 0,0 53,6 0,0 0,0 0,0 0,0 0,0PI-554-109 (Bt10) 33,3 0,0 50,0 10,5 7,1 15,8 0,0 0,0Intaler 47,4 15,7 9,1 13,8 2,4 10,5 1,8 2,0Lutescens 6028 (PI 591884) 0,0 12,5 25,0 30,0 10,0 17,1 4,0 4,8Penta 20,0 9,5 0,0 0,0 0,0 4,8 0,0 0,0PI-554-103 (Bt2) 25,0 8,8 0,0 45,0 0,0 0,0 0,0PI-554-097 (Bt2) 57,1 61,3 5,9 31,6 0,0 0,0 0,0 0,0Format 76,9 44,4 0,0 22,2 0,0 4,7 0,0 0,0Curier 54,5 41,9 11,8 0,0 0,0 2,4 0,0 0,0Complet 43,8 69,7 0,0 6,3 0,0 0,0 0,0 0,0Solstice 75,0 40,3 0,0 12,5 2,4 4,2 0,0 0,0Bussard 77,3 63,4 0,0 12,5 2,3 0,0 0,0 0,0Paroli 80,8 53,3 0,0 14,8 2,0 4,8 0,0 0,0Dream 61,5 28,1 0,0 16,7 0,0 2,3 0,0 0,0Butaro (154) 37,5 64,7 7,1 25,0 0,0 3,0 2,6 0,0Ochre 50,0 30,0 0,0 24,2 0,0 1,7 0,0 0,0PG3540 50,0 10,0 0,0 15,0 0,0 0,0 0,0 0,0Hereward 60,0 14,8 12,0 4,2 0,0 0,0 0,0 7,7PI-554-108 (Bt1) 0,0 0,0 0,0PI-554-101 (Bt1) 100 11,1 0,0 37,5 7,7 12,5 0,0 0,0Bill 16,7 0,0 3,8 0,0 2,0 25,0 0,0 2,8NGB9014 57,1 3,2 15,6 0,0 0,0 30,0 10,0 0,0NGB-9015 33,3 22,5 23,1 4,2 0,0 18,0 4,7 0,0Tambor 0,0 31,6 12,5 0,0 2,0 19,7 4,0 3,0Kuban 11,1 18,5 12,1 3,0 32,8 11,9 0,0 0,0Begra 0,0 27,8 44,4 4,5 17,6 27,5 5,4 7,3Maribos 12,1 21,4 3,2 37,5 8,6 2,3 10,0Fold 16,7 12,2 14,6 5,6 8,6 9,8 0,0 4,3Monopol 50,0 90,9 0,0 40,0 11,1 3,4 0,0 0,0Tarso 7,1 23,4 34,4 16,0 28,6 6,3 8,7 2,1Torrild 14,3 38,5 35,0 13,9 14,3 17,5 2,1 3,2Cardos 17,6 26,2 57,9 31,3 16,0 8,9 0,0Kranich 19,0 35,0 10,5 33,3 23,7 22,7 11,7 0,0Türkis 45,0 40,0 25,0 11,1 42,2 28,6 6,8 0,0Gluten 28,6 16,7 46,2 34,6 61,0 22,5 14,3 0,0Folke 37,0 32,6 25,0 33,3 70,6 24,3 5,6 0,0PI-554-114 (Bt7) 50,0 19,2 22,2 30,0 0,0 25,0 15,2 3,7PI-554-100 (Bt7) 33,3 28,6 45,7 4,5 0,0 35,5 10,9 9,1Korrund 20,0 25,0 55,6 0,0 4,2 17,0 14,8 35,7Aron 33,3 50,0 55,6 15,8 2,9Karat 84,6 55,6 12,5 0,0 20,0Tulsa 21,1 54,8 42,9 51,1 34,4 36,2 45,7Xenos 70,7 62,0 66,7 39,4 18,4 13,2 26,3Tataros 12,5 34,6 52,0 26,7 29,7 26,2 16,7 26,3Erbachshofer Braun 55,6 75,0 45,5 55,0 46,4 57,1 12,5 0,0Indigo 76,6 68,0 77,5 88,2 75,7 59,0 30,0 4,4Colour legends: 0,0% <10% 10-40 >40%Infecting 65 varieties of winter wheat with 7 sources of common bunt inoculum”Own spores” means spores harvested previous year on the tested varietylow plant numberAcknowledgementThe research was part of the COBRA project (2013-15)funded by Core Organic II and Fonden for ØkologiskLandbrug, and based on spores and germplasm selected inprevious work in the BIOBREED project (2010-13) fundedby Fødevareforskningsprogrammet and the project“Stinkbrandresistens i gamle hvedesorter” funded by Fondenfor Økologisk Landbrug. Thank you also to VeronikaDumalasova and Bent Nielsen for delivering spores for thestudy.ReferencesEPPO 1997: Guidelines for the efficacy evaluation of plantprotection products. Fungicides Bactericides. Europeanand Mediterranean Plant Protection Organization.Roemer, T. og R. Bartholly 1933: Die Aggressivitätverschiedener “Steinbrandherkünfte” [Tilletia tritici.(Bjerk.) Wint.] und ihre Veränderung durch dieWirtssorte. Phytopathologische Zeitscrift 6(5):469-504Steffan, P, 2014: Biotechnology Assisted Wheat Breeding forOrganic Agriculture. PhD  thesis, CopenhagenUniversity. 95 p.Steffan, P, G.Backes, S.Rasmussen and Borgen, A. 2014:Genome Wide Association Study for Common BuntResistance in Wheat and Creation of Common BuntResistant Composite Cross Populations. Abstract for theProceedings of the XVIII Biennial International Workshop on the Smuts and Bunts Tune/Copenhagen 3rd-5th February 2014.Weston, D. 1932: The relative resistance of some wheat varieties to Tilletia caries (DC.) TUL. (=T.tritici (Bjerk.)Wint.). The Annals of Applied Biology 19:35-54Woolman, H.M. 1930: Infection phenomena and host reaction caused by Tilletia tritici in susceptible and nonsusceptible varieties of wheat. Phytopathology 20:637-653.Quebon 24,0 0,0 6,7 0,0 39,3 6,3Tommi 26,7 17,1 14,3 10,3Globus 0,0 55,6 14,3 0,0Segor 43,8 0,0 13,3Huslop 0,0 0,0 0,0Winridge 0,0 0,0 0,0 0,0Wasatch 0,0 0,0 0,0 0,0Sprague 0,0 0,0 0,0PI-554-104 (Bt5) 0,0 0,0 0,0PI-554-112 (Bt9) 0,0 0,0 0,0Pi 554099 (Bt9) 0,0 0,0Pi 554099 (Bt9) 0,0Andrews (Bt-9?) 0,0 0,0 0,0BB118 (Philipps) 2,9 0,0 0,0BB152 (Philipps) 0,0 0,0 2,9 0,0PI-554-098 (Bt11) 0,0 0,0 0,0PI-554-119 (Bt11) 0,0 0,0 0,0PI-554-105 (Bt4)+5 0,0 0,0 0,0Hansel (Bt-8+9+10?) 2,4 0,0 0,0PI-119-333 (Bt12) 0,0PI-119-333 (Bt12) 7,4 0,0 0,0 0,0PI-554-106 (Bt12) 9,1 6,5 44,4Rio (Bt6) 0,0 0,0 8,3 0,0Sam 0,0 9,1 0,0 0,0Golda (Zarya-res.) 31,0 0,0 0,0Bill 10,4Marin (Ridit-res.) 43,8 27,0 0,0 4,0PI-554-120 (Bt8) 50,0 0,0Yayla 50,0 0,0 33,3Turkey (Pi11610) Bt4) 41,2 19,0 34,8Heines VII (no Bt genes) 33,3 50,0 30,0Rola (Trebelir-res.) 45,5 2,9 68,0 44,4PI 172201 (Doubbi resistens) 0,0PI 172201 (Doubbi resistens) 50,0Doubbi (Bt-14) 25,0 50,0Doubbi (Bt-14) 0,0Infecting 34 highly resistant varieties of winter wheat with 4 sources of common bunt inoculum”Own spores” means spores harvested previous year on the tested variety

Purifying virulence races of common bunt (Tilletia caries) to identify resistance genes in wheat

https://orgprints.org/34182/3/Virulence_Vingsted2015_34182.pdf

Purifying virulence races of common bunt (Tilletia caries) to identify resistance genes in wheat

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