David Walker (USDA-ARS & Univ. of Illinois, USA)

Transcription

1 Técnicas de Identificación y uso de Resistencia a la Roya en Variedades de Soja Identification techniques and use of rust resistance in soybean varieties David Walker (USDA-ARS & Univ. of Illinois, USA)

2 Phakopsora pachyrhizi in the United States First observed in the continental USA in November of 2004 near Baton Rouge, Louisiana (Ray Schneider et al.) Initially assumed that urediniospores from South or Central America were transported into North America by Hurricane Ivan in September 2004 Pathotype diversity already existed among the founding populations of P. pachyrhizi in 2004

3 Soybean rust confirmations by late October 2013

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5 Why hasn t SBR been a more serious problem in the United States? Virtually all of the soybean producing regions get freezing temperatures in the winter (November to March) => Winter survival bottleneck Major production regions are distant from areas where P. pachyrhizi can survive the winters Kudzu (Pueraria lobata) is not an ideal host for prolific reproduction of the fungus Popularity of Early Soybean Production System in the Midsouth production region

6 Nevertheless, SBR remains a concern in the southern USA: Disease-related costs in Georgia in 2012 & 2013 Disease 2012 % Reduction in value Damage ($ Million) Control ($ Million) Total ($ Million) Soybean rust Root-knot nematodes Total Soybean rust Root-knot nematodes Total 9.25

7 Concerns about complete reliance on fungicides to manage yield losses caused by SBR Producers risk becoming complacent about the threat of SBR in the southern USA Overuse of fungicides to reduce foliar disease caused by several fungal pathogens drives selection for pathogen tolerance to fungicide chemistries Fungicide application at optimal time for effective control might be hindered by rain Resistant cultivars would therefore be a valuable component of a more effective and durable disease management program

8 Steps in development of rust-resistant cultivars 1. Identify germplasm that are resistant to the P. pachyrhizi pathotypes in the production area 2. Cross resistant lines with agronomically elite cultivars and breeding lines adapted to target region 3. Inbreed or backcross lines to introgress resistance and to develop genetic mapping populations 4. Select rust-resistant plants and lines 5. Select against lodging, shattering & susceptibility to other diseases 6. Select best-yielding lines in replicated tests

9 Traditional classification of soybean reactions to rust infection (infection types) TAN lesions Susceptible response Abundant urediniospores Color is often that of the urediniospores TAN RB lesions Reddish-brown to chocolate brown or purple in color Incomplete resistance Generally little or no sporulation RB Immune response No visible lesions Disease severity

10 Challenges to identifying exotic sources of soybean rust resistance in the field Influence of environmental conditions on disease development Effect of plant maturity on disease development Temporal and geographic variation in pathogenicity of P. pachyrhizi within and among populations Lesion type (RB or tan) is not always obvious on field plants Other foliar diseases (especially bacterial pustule, Xanthomonas axonopodis) Bossier City, Louisiana, September 2007 MG VII uniform test MG VI uniform test Photo: J. Blair Buckley, Louisiana State Univ. AgCenter

11 USDA germplasm SBR resistance evaluations Bossier City Blackville Alexandria Attapulgus Baton Rouge Fairhope Quincy

12 Soybean rust resistance screening nursery Quincy, Florida, early November 2009

13 Reaction ratings of plant introductions (PIs) in the field Disease severity rated using a 1 to 5 scale (1 = no SBR lesions; 5 = >35% of leaf surface covered with SBR lesions) Sporulation rated using a 1 to 5 scale (1 = no sporulation lesions; 5 = profuse sporulation from >90% of lesions) Ratings made on 10 leaflets per plot, generally with two replications per nursery location Used when susceptible checks have clear and abundant soybean rust symptoms Approximately 580 PIs screened, with resistant PIs retested each year

14 Rust Index scores used to assess reactions Rust index (RI) = square root of (severity rating x sporulation rating) Range: 1.0 to 5.0 Walker et al Crop Sci. 54:1673 RI ~ 4.5 (out of 5.0)

15 Florida RI ratings 2009 to 2015 Gene Line MG S Check Williams 82 III Rpp1 Williams 82 + Rpp1 gene III 1.3 [2.3] Rpp1 PI VII Rpp2 PI VII Rpp3 PI VIII Rpp4 PI B VIII S Check NC-Raleigh VII S Check Kuell VIII LSD (0.05) Rpp-mediated resistance showed temporal variation - Moderate-low resistance from Rpp2 and Rpp4 remained steady until increase in Brackets [-] indicated means that were calculated from incomplete data sets

16 Florida RI ratings 2009 to 2015 Gene Line MG Rpp5 PI VIII Rpp5 PI VIII Rpp5 PI IX Rpp3 + Rpp5 Hyuuga VII Rpp6 PI B VIII [Rpp6] PI B VIII [1.6] S Check Kuell VIII LSD (0.05) PI and PI are more resistant in the U.S. than PI Hyuuga was less resistant in 2011 and 2012, but rebounded in Resistance of historically near-immune accessions PI B and PI B was also less effective in 2012, but recovered in 2013

17 RI ratings of some resistant accessions with unidentified Rpp genes Line MG DT2000 VI 1.6 [4.1] PI VIII PI B IX 4.2 [3.0] PI A or B IX PI A VIII PI C IX Kuell VIII LSD (0.05) Brackets [-] indicate means that were calculated from incomplete data sets.

18 Observations and conclusions Truly immune lines are extremely rare in the field Mixtures of RB and TAN lesions probably indicate that >1 pathotype was present in the field Selecting the best sources of resistance for breeding crosses requires data from several locations and/or years Only about 20% of the PIs that Miles et al. (2006) reported to be resistant to foreign isolates proved to be resistant to North American isolates

19 Restricted geographical origins of soybean PIs resistant to SBR in the U.S.

20 Approximate locations of known and mapped Rpp resistance gene loci Resistance gene Chromosome (linkage group) Approximate map position from end References Rpp1 18 (LG G) cm Hyten et al. 2007; Chakraborty et al. 2009; Kim et al Rpp4 18 (LG G) cm Silva et al Rpp6 18 (LG G) cm Li et al Rpp2 16 (LG J) cm Silva et al Rpp3 6 (LG C2) cm Hyten et al Rpp5 3 (LG N) cm Garcia et al. 2008; Pierozzi et al Rpp7 19 (LG L) cm Childs et al. 2018

21 Discovery of the Rpp7 locus in PI (Childs et al. 2018) PI has had moderate resistance to U.S. P. pachyrhizi isolates and field populations PI is a MG IX land race from Ha Giang, in northern Vietnam Most common reaction in the southern USA is development of RB lesions with limited sporulation

22 Discovery of the Rpp7 locus in PI (Childs et al. 2018) a: Williams 82 (susceptible parent) b: 5601T (susceptible parent) c and d: PI Most common reaction to U.S. isolates is RB lesions with low to moderate sporulation

23 Mapping of Rpp7 Two segregating populations used: Williams 82 x PI F 2 population (87 lines) 5601T x PI F 4:5 recombinant inbred lines (84 RILs) Mapping approach: Use bulked segregant analysis (BSA) on the F2 population, followed by Site-targeted linkage mapping on chromosome 19 using all of the lines from both populations

24 Principles of bulked segregant analysis (BSA) Susceptible Resistant Resistant progeny all inherited the same segment of DNA from their resistant parent

25 Fine mapping of the Rpp7 locus on Chromosome 19 a = Integrated genetic map generated with data from both populations b = Physical map of the region around the Rpp7 locus

26 Two Rpp genes were mapped in DT 2000 DT 2000 (PI ) is a soybean cultivar from Vietnam that has shown resistance to P. pachyrhizi populations in the U.S. as well as in Vietnam Originated as a selection from a breeding line developed in Taiwan at the Asian Vegetable Research and Development Center (AVRDC) Resistance in the southern U.S. has been moderately high most years

27 DT 2000 mapping population 250 F 6:7 RILs were developed from the cross of Williams 82 x DT 2000 RILs were evaluated for resistance in Ha Noi, Vietnam (to obtain AUDPC data) and Quincy, Florida, USA (to obtain disease severity and sporulation ratings) RILs were genotyped using the Universal Soy Linkage Panel 1.0 (queries 1,536 SNP marker loci) BSA results using Vietnam disease data implicated two genomic regions associated with rust resistance

28 Significant LOD (Likelihood of the Odds) peaks on Chromosomes 6 (A) and 18 (B) Location on Chromosome 6 corresponds to Rpp3 locus. Detected using VN and FL data. Location on Chromosome 18 corresponds to Rpp4 locus. Affected resistance only in VN.

29 Refined locations of genes on Chromosomes 6 and 18 based on composite interval mapping Location on Chromosome 6 corresponds to Rpp3 locus. Detected using VN and FL data. Location on Chromosome 18 corresponds to Rpp4 locus. Affected resistance only in VN.

30 A plausible explanation for why the gene at the Rpp4 locus was only detected using the disease data from Vietnam: The Rpp4 gene of PI B has not provided much resistance to U.S. populations of P. pachyrhizi, so if the Rpp4 gene in DT 2000 were similar, its segregation would not have been detected among the resistance of lines in Quincy, Florida Therefore, DT 2000 would have appeared to have only one resistance gene, located at the Rpp3 locus

31 Advantages and disadvantages of field and greenhouse assays for rust resistance Field assays Advantages Adult plant resistance evaluated Conditions closer to a production field Disadvantages Disease development is highly dependent on weather conditions Presence of other foliar diseases Lesions are likely to vary in age Mixed reactions may occur Greenhouse assays Advantages Much easier to distinguish infection types (RB, TAN, IM) Much more rapid than field assays Environmental conditions can be optimized for pathogen Disadvantages Seedling reaction after 2 weeks may not accurately indicate reaction of older plants

32 Evaluation of 45 PIs for resistance to six P. pachyrhizi isolates in greenhouse assays Isolate Year collected State Location AL08-Fairhope 2008 Alabama (AL) Fairhope (southern AL) AL09-Fairhope 2009 Alabama (AL) Fairhope (southern AL) LA09-Bossier City 2009 Louisiana (LA) Bossier City (NW Louisiana) FL09-Quincy 2009 Florida (FL) Quincy (north-central FL) FL11-Quincy 2011 Florida (FL) Quincy (north-central FL) FL12-Quincy 2012 Florida (FL) Quincy (north-central FL)

33 Mean disease severity (1-5 scale) on trifolialate leaflets of soybean genotypes after inoculation with six Phakopsora pachyrhizi isolates PI B Rpp4 A 4.9 Williams 82 Susceptible check A 4.8 PI Allele at Rpp5 locus A B 4.7 PI Rpp1b B C 4.4 PI Hyuuga C D 4.2 PI rpp5 allele D 4.1 PI Rpp2 D E 3.8 PI Rpp3 E F 3.7 PI Allele at Rpp5 locus E F G 3.6 PI F G H 3.4 DT2000 Rpp6 & Rpp4 alleles G H I 3.3 PI G H I 3.3 PI A - G H I 3.3 PI B Allele at Rpp3 locus G H I 3.3 PI A Allele at Rpp3 locus G H I 3.3 PI A - G H I J 3.2 PI Allele at Rpp3 locus H I J K 3.1 PI A Allele at Rpp3 locus I J K L 2.9 PI Allele at Rpp6 locus J K L M 2.9 PI A Allele at Rpp3 locus K L M N 2.8 PI B Allele at Rpp6 locus L M N 2.7 PI B Rpp6 M N 2.6 PI Rpp1 N 2.4

34 Mean number of uredinia per cm 2 on trifolialate leaflets of soybean genotypes inoculated with six Phakopsora pachyrhizi isolates Accessions Rpp genes Uredinia per sq cm Williams 82 Susceptible check A 59.4 LD P Susceptible check B 48.4 PI Allele of Rpp5 B C 42.9 PI rpp5 C D 37.8 PI Rpp1b D 35.1 PI B Rpp4 E 28.2 PI Rpp2 F 7.9 PI Rpp3 G 4.1 PI Rpp1 H 2.5 PI B Rpp6 H 2.5 PI A Allele at Rpp3 H I 2.2 PI H I 2.2 PI Allele at Rpp3 H I 2.1 PI H I J 2.0 PI B Rpp6 I J 1.9 PI Allele at Rpp3 I J K 1.8 PI I J K 1.8 PI Allele at Rpp6 I J K 1.8 PI Allele at Rpp3 I J K 1.8 PI Allele at Rpp5 J K L 1.6 DT2000 Rpp3 + Rpp4 alleles J K L 1.6 PI A Allele at Rpp3 J K L M 1.5 PI Allele at Rpp3 K L M 1.3 PI L M N 1.3 PI B Allele at Rpp3 L M N 1.2 PI A - L M N 1.2 PI A - M N 1.1 PI Rpp3 + Rpp5 allele N O 0.9 PI A - O P 0.6 PI B Allele at Rpp3 O P 0.6 PI A - P Q 0.5 PI A Allele at Rpp3 Q 0.2

35 Observations and conclusions There is no universally perfect resistance rating method Field and greenhouse assays each have their merits and limitations Beware of declarations of immunity based on greenhouse assays rated 2 weeks after inoculation (a longer latent period is possible) It is not uncommon to see lesions on field leaves that are not clearly RB or TAN

36 Reactions of soybean PIs with a soybean rust resistance allele at the Rpp3 locus to a pathogenically diverse set of Phakopsora pachyrhizi isolates from the southern United States Chandra Paul, Donna K. Harris, Zenglu Li, and David R. Walker Background information from Harris et al Identification of unique genetic sources of soybean rust resistance from the USDA Soybean Germplasm Collection. Crop Sci. 55: : 52 out of 75 SBR-resistant PIs (69%) were found to have a resistance gene at the Rpp3 locus on chromosome 6, originally identified in PI of the 52 PIs challenged with a panel of eight U.S. and international P. pachyrhizi isolates had the same reaction profiles as either PI or Hyuuga The degree of genetic similarity among these alleles was not known, though 37 PIs had a local DNA marker haplotype identical to that of PI

37 U.S. isolates used to inoculation soybean PIs with a gene at the Rpp3 locus State City County/ parish Year Source Code name Florida Quincy Gadsden 2007 Soybean FL07-1 Florida Quincy Gadsden 2009 Soybean FL09-2 Florida Quincy Gadsden 2011 Soybean FL11-Q Florida Quincy Gadsden 2012 Soybean FL12-Q Florida Unknown Jefferson 2013 Soybean FL13-Jeff Florida Unknown Leon 2015 Soybean FL15-Leon Florida Quincy Gadsden 2015 Soybean FL15-Q Texas Dayton, Hwy 90 Liberty 2013 Kudzu TX13-Hwy90 Louisiana Ville Platte Washington 2014 Kudzu LA14-Hwy67 Louisiana Bell City Calcasieu 2014 Kudzu LA14-Bell City Louisiana New Iberia Iberia 2014 Kudzu LA14-Iberia Louisiana Houma Terrebonne 2015 Kudzu LA15-Houma Alabama Elmore Co. Elmore 2014 Kudzu AL14-Elmore

38 Reactions of PIs with alleles at Rpp3 locus to eight U.S. P. pachyrhizi isolates Plant Introduction AL14- Elmore LA14- Hwy167 LA14-New Iberia LA15- Hwy90 LA15- Houma FL15-Quincy FL15-Leon PI (Rpp3) RB RB RB RB RB RB RB RB PI (Hyuuga) RB RB RB RB RB RB RB RB PI RB RB RB RB RB RB RB RB PI B RB RB RB IM RB RB RB RB PI A IM RB RB IM RB IM RB RB PI RB RB RB RB RB IM IM RB PI D RB RB RB RB IM RB RB IM PI C IM RB RB RB RB IM RB IM PI RB RB IM RB IM IM RB RB PI A RB IM IM IM RB RB RB RB PI IM RB RB RB RB IM IM IM PI B IM IM TAN IM RB IM RB RB PI B IM IM RB IM IM RB IM IM PI A IM IM IM RB IM IM RB RB FL13- Jefferson PI TAN RB TAN RB RB RB RB TAN PI A RB RB RB RB RB TAN TAN RB

39 Reactions of PIs with alleles at Rpp3 locus to 6 Florida and 2 foreign P. pachyrhizi isolates FL09- FL11- FL12- FL15- FL13- FL15- Accession Quincy Quincy Quincy Quincy Jefferson Leon ZM01-1 CO04-2 PI (Rpp3) RB2 TAN TAN RB1 RB1 RB1 TAN TAN PI (Hyuuga) RB1 Intermed Intermed RB1 RB1 RB1 RB RB PI RB1 RB2 TAN RB1 RB1 RB1 RB RB PI RB1 RB2 TAN RB1 RB1 RB1 TAN TAN PI RB1 RB2 TAN RB1 RB1 RB1 TAN TAN PI A RB2 RB1 TAN RB1 RB1 RB2 TAN TAN PI RB1 RB2 TAN RB1 RB1 RB1 TAN TAN PI RB1 TAN RB2 RB1 RB1 RB1 RB RB PI RB TAN RB RB RB RB TAN TAN PI A RB1 TAN RB2 RB1 IM IM - - PI B RB1 TAN RB2 RB1 IM IM Mixed Mixed PI RB1 RB1 RB2 RB1 TAN TAN RB RB PI A RB1 RB2 RB2 TAN RB1 TAN TAN RB PI TAN RB2 RB2 TAN TAN TAN RB RB Williams 82 (S) TAN TAN TAN TAN TAN TAN TAN TAN RB1 = reddish-brown lesion with little or no sporulation RB2 = RB lesion with moderate sporulation

40 Reactions of PIs with alleles at Rpp3 locus to 6 Midsouth and 2 foreign P. pachyrhizi isolates AL14- Elmore LA14- Bell City LA14- New Iberia LA14- Hwy 167 LA15- Hwy 90 LA15- Houma ZM01-1 CO04-2 Accession PI (Rpp3) RB2 RB2 RB2 RB2 RB2 RB1 TAN TAN PI (Hyuuga) RB1 RB1 RB2 RB1 RB2 RB1 RB RB PI RB1 RB1 RB1 RB1 IM RB1 RB RB PI RB2 RB2 RB1 RB1 RB1 RB1 TAN TAN PI RB1 RB2 RB1 RB1 RB1 RB1 TAN TAN PI A RB2 RB1 RB1 RB1 RB1 RB1 TAN TAN PI RB1 RB2 RB1 RB1 RB1 RB1 TAN TAN PI RB1 RB2 RB1 RB1 RB1 RB1 RB RB PI RB RB RB RB RB IM TAN TAN PI A IM IM IM RB1 IM IM - - PI B IM RB2 IM RB1 IM IM Mixed Mixed PI RB1 RB2 RB1 RB1 RB1 RB1 RB RB PI A RB1 RB2 RB1 RB2 RB1 RB2 TAN RB PI TAN TAN RB2 TAN RB2 RB1 RB RB Williams 82 (suscept.) TAN TAN TAN TAN TAN TAN TAN TAN RB1 = reddish-brown lesion with little or no sporulation RB2 = RB lesion with moderate sporulation

41 Infection type principal component analysis (PCA) of 55 PIs with an allele at the Rpp3 locus

42 Cluster analysis of 52 Rpp3 allele PIs for mean number of uredinia per cm 2 and uredinia per lesion to 13 U.S. P. pachyrhizi isolates Hyuuga & Rpp3 Analysis by Chandra Paul

43 Public sector soybean rust breeding and gene-mapping programs in the U.S. USDA-ARS, Urbana, IL - Walker, Hartman and Singh; Nelson (now retired) USDA-ARS, Stoneville, MS Smith, Ray and Li University of Georgia, GA - Li and Buck; Boerma and Phillips (now retired) Clemson University, SC - Fallen University of Illinois, IL - Diers USDA-ARS, Stoneville, MS -Smith, Ray, Li, & Gillen University of Missouri, MO - Vuong and Nguyen

44 SBR-Resistant lines yield-tested in Athens, GA in 2012 (MG VI-VII Test) Pedigree Line Bu/acre Days to mat (starting 9/1) LG (III) x PI B (IX) 34-A C B Dillon (VI) x PI A (IV) 121-B A Dillon Cv check AGS 787 RR Cv check AGS 758 RR CV check G RR Cv check LSD (0.05) 9.8

45 SBR-Resistant lines yield-tested in Athens, GA in 2012 (MG VIII Test) Pedigree Line Bu/acre Days to mat Dillon (VI) x PI A (IV) 64-b Dillon (VI) x PI A (VIII) 122-c a c c LG (III) x PI B (IX) 16-c Dillon Cv check AGS 787 RR Cv check AGS 758 RR CV check G RR Cv check LSD (0.05) 7.9

46 SBR-resistant parents of USDA breeding lines PI MG MG II-IV MG V-IX PI VIII x PI VIII x PI A VIII x PI VIII x PI VII x x PI VIII x x PI VII x PI B IX x PI MG MG II-IV MG V-IX PI A IV x x PI VI x PI V x PI IX x PI B V x PI A V x PI A IV x x PI VII x

47 Rust-resistant germplasm releases from U.S. breeding programs Developers Lines Genes Mat Groups Boerma et al. (2011; Univ. of Georgia) Diers et al. (2013; Univ. of Illinois) King et al. (2016; Univ. of Georgia) G01-PR16 Four lines with single Rpp genes in genetic background of LD (MG II); and four lines with genes in background of LD (MG IV) Four lines with single Rpp genes in the genetic background of line G Rpp3 (aka Rpp?(Hyuuga], Rpp1, Rpp1-b, Rpp3 (aka Rpp?(Hyuuga], and the Rpp5 allele from PI Rpp1, Rpp2, Rpp3 and Rpp4 VI II and IV VII

48 Acknowledgments USDA-ARS Esp. Randy Nelson United Soybean Board Kelly Whiting & Rich Joost North Florida Research and Education Center Esp. David Wright, Jim Marois, Kelly O Brien, Debbie Greene & Maynard Douglas University of Georgia Roger Boerma, Dan Phillips, Donna Harris, Billy Mills, Zach King, Zenglu Li, James Buck and Dale Wood Auburn University David Weaver, Ed Sikora and Malcomb Pegues Louisiana State University Blair Buckley & Ray Schneider