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RESEARCH ARTICLE
Identification of Fungus Resistant WildAccessions and Interspecific Hybrids of theGenus ArachisMarcos Doniseti Michelotto1, Waldomiro Barioni, Jr.2, Marcos Deon Vilela de Resende3,Ignácio José de Godoy4, Eduardo Leonardecz5¤, Alessandra Pereira Fávero2*
1 Pólo Centro Norte, Agência Paulista de Tecnologia dos Agronegócios, Pindorama, São Paulo, Brazil,2 Embrapa Southeast Livestock, São Carlos, São Paulo, Brazil, 3 Embrapa Forestry,Colombo, Paraná,Brazil, 4 Agronomic Institute of Campinas, Campinas, São Paulo, Brazil, 5 Laboratory of ScientificComputing, Campus Planaltina, University of Brasília, Planaltina, Federal District, Brasília
¤ Current address: Laboratory of Computational Biology, Department of Genetics and Evolution, FederalUniversity of São Carlos,–São Carlos, São Paulo, Brazil* [email protected]
AbstractPeanut, Arachis hypogaea L., is a protein-rich species consumed worldwide. A key improve-
ment to peanut culture involves the development of cultivars that resist fungal diseases
such as rust, leaf spot and scab. Over three years, we evaluated fungal resistance under
field conditions of 43 wild accessions and three interspecific hybrids of the genus Arachis,as well as six A. hypogaea genotypes. In the first year, we evaluated resistance to early and
late leaf spot, rust and scab. In the second and third years, we evaluated the 18 wild species
with the best resistance scores and control cultivar IAC Caiapó for resistance to leaf spot
and rust. All wild accessions displayed greater resistance than A. hypogaea but differed in
their degree of resistance, even within the same species. We found accessions with as
good as or better resistance than A. cardenasii, including: A. stenosperma (V15076 and Sv
3712), A. kuhlmannii (V 6413), A. kempff-mercadoi (V 13250), A. hoehnei (KG 30006), and
A. helodes (V 6325). Amphidiploids and hybrids of A. hypogaea behaved similarly to wild
species. An additional four accessions deserve further evaluation: A.magna (V 13751 and
KG 30097) and A. gregoryi (V 14767 and V 14957). Although they did not display as strong
resistance as the accessions cited above, they belong to the B genome type that is crucial
to resistance gene introgression and pyramidization in A. hypogaea.
IntroductionThe oil and protein reach peanut (Arachis hypogaea L.) is consumed both in natura and pro-cessed as oil, constituting the fifth largest oleaginous crop worldwide [1]. his plant, which is na-tive from South America, belongs to a genus with 81 described species distributed in ninetaxonomic sections [2,3]. The Arachis section includes 31 species including the commercialpeanut.
PLOSONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 1 / 17
OPEN ACCESS
Citation: Michelotto MD, Barioni W, Jr., de ResendeMDV, de Godoy IJ, Leonardecz E, Fávero AP (2015)Identification of Fungus Resistant Wild Accessionsand Interspecific Hybrids of the Genus Arachis. PLoSONE 10(6): e0128811. doi:10.1371/journal.pone.0128811
Academic Editor: Randall P. Niedz, United StatesDepartment of Agriculture, UNITED STATES
Received: January 5, 2015
Accepted: April 30, 2015
Published: June 19, 2015
Copyright: © 2015 Michelotto et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: APF received funds of Embrapa for thiswork, and IJG received funds of CNPq for this work.The funders provided support in the form of salariesfor authors [APF, WBJ], but did not have anyadditional role in the study design, data collection andanalysis, decision to publish, or preparation of themanuscript. The specific roles of these authors arearticulated in the ´author contributions´ section.
The development of fungus resistance represents one of the main challenges for the im-provement of cultivated peanuts. Some of the most severe fungal foliar diseases include leafspot (Cercosporidium personatum Berk & Curtis Deighton and Cercospora arachidicolaHorii),rust (Puccinia arachidis Speg.), web blotch (Phoma arachidicolaMarasas, Pauer & Boerema),and scab (Sphaceloma arachidis Bit & Jenk). The genus Arachis has long been studied with re-gards to the introgression potential of resistance genes in peanut cultivars [4–9]. Extensivestudies have shown that the A. cardenasii accession GKP 10017 is resistant to diseases [10,8].However, these studies were conducted in greenhouses or laboratories, with detached leaves.Obstacles associated with field research such as the low availability of seeds of wild species, ana-lytical difficulties, and inoculum natural pressure are common in wild Arachis bioassays. Fieldstudies of ancient and recently-collected accessions are necessary, especially in areas close toproduction centers.
The state of São Paulo accounts for 80% of peanut production in Brazil. Leading phytosani-tary threats in the state include late leaf spot (Cercosporidium personatum), early leaf spot(Cercospora arachidicola), rust (Puccinia arachidis), and scab (Sphaceloma arachidis). Further-more, inoculum pressure in São Paulo is consistently high [9], making this a good site for theassessment of genotype resistance to prevailing pathogens.
We evaluated 43 accessions and three interspecific Arachis hybrids with regards to resis-tance to foliar diseases under field conditions in the state of São Paulo. Accessions might belater crossed generating amphidiploids (artificially doubled interspecific hybrids with distinctgenomic backgrounds that might be AABB or might have other genomic combinations) to befurther crossed with cultivars or elite lines of A. hypogaea, generating segregated populationsthat can be selected and backcrossed in a breeding program.
Materials and Methods
Plant cultureBioassays were conducted at the Pólo Apta Centro Norte experimental area in Pindorama, SãoPaulo, Brazil. Seeds were originally provided by the Arachis Germplasm Bank, Embrapa Genet-ic Resources and Biotechnology. Seeds of different genotypes (Table 1) were treated with thefungicide Plantacol
1
(10g/100kg of seeds) and germinated in paper towels in a room with ade-quate temperature, air humidity and light. Seedlings were transplanted to 200-ml plastic cupsfilled with soil and sand (3:1) and placed in a greenhouse. When plants reached a height of 10to 15 cm they were transplanted to the field in soil previously prepared with 250 kg/ha of 8-28-16 NPK.
During the first year, we evaluated 43 accessions belonging to 10 wild species, six A. hypo-gaea genotypes and three interspecific hybrids, including amphidiploids and segregating popu-lations (Table 1). Twenty-five F2 individuals of the progenie by the cross between IAC Caiapóand the amphidiploid An 2 were evaluated. The average of the experimental unit were used foranalyses of variance. In the second and third years, we selected the 18 most resistant accessionsand the IAC Caiapó cultivar as control.
The experiment design was random uncompleted delineated block with four replications.Each block was initially composed of four meters with five plants spaced one meter apart andwith a separation of 1.5 meters between lines. This spacing was needed because of the amplegrowth of these plant species. Just three plants in the middle of the experimental unit were eval-uated. Every block was sprayed twice-monthly with insecticides to avoid infestation. Weedcontrol was performed with the pre-transplantation application of commercially available Tri-fluralin (2.5 l/ha). During plant growth, weed control was performed manually. The
Fungi Resistance of Wild Arachis Species and Hybrids
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Competing Interests: The authors have declaredthat no competing interests exist.
Table 1. Arachis spp. accessions included in the present study.
Accessions Code Species BrazilianAccessionsCode
CollectionsitesCity
State in Brazilor Country
Lat(W)
Long(S)
Alt(m)
Genome
K 9484 A. batizocoi Krapov. & W. C. Gregory 013315 Parapeti BOL 20°05’
63°14’
700 K
KG 35005 A. benensis Krapov. & W.C. Gregory 037206 Trinidad BOL F
GKP 10017 A. cardenasii Krapov. & W. C.Gregory
013404 Roboré BOL 18°20’
59°46’
200 A
K 7988 A. duranensis Krapov. & W. C.Gregory
013307 Campo Duran ARG 22°19’
63°13’
500 A
VSGr 6389 A. gregoryi C. E. Simpson, Krapov.&Valls
012696 Vila Bela da Ssa.Trindade
MT 15°19’
60°06’
210 B
VOfSv 14760 A. gregoryi C. E. Simpson, Krapov.&Valls
038792 Vila Bela da Ssa.Trindade
MT 16°08’
59°47’
B
VOfSv 14767 A. gregoryi C. E. Simpson, Krapov.&Valls
038814 Vila Bela da Ssa.Trindade
MT 16°05’
59°58’
290 B
VS 14957 A. gregoryi C. E. Simpson, Krapov.&Valls
040002 Vila Bela da Ssa.Trindade
MT 15°22’
60°14’
B
CoSzSv 6862 A. helodes Martius ex Krapov &Rigoni
018619 MT 15°22’
56°13’
175 A
VSGr 6325 A. helodes Martius ex Krapov &Rigoni
012505 S. Antonio doLeverger
MT 15°52’
56°04’
150 A
KG 30006 A. hoehnei Krapov. & W. C. Gregory 036226 Corumbá MS 18°15’
57°28’
A
VRcMmSv 14546 A. hoehnei Krapov. & W. C. Gregory 022641 Corumbá MS 19 °15’
57 °22’
100 A
cv. BR1 A. hypogaea subsp. fastigiata var.fastigiata
033383 AB
cv. IAC Caiapó A. hypogaea 037371 AB
2562 A. hypogaea 037354 AB
IAC Runner 886 A hypogaea subsp. hypogaea var.hypogaea
037389 AB
cv. IAC Tatu-ST A. hypogaea subsp. fastigiata var.fastigiata
011606 Campinas SP AB
V 12549 A. hypogaea subsp. hypopaea var.hypogaea
030716 AB
KGPScS 30076 A. ipaënsis Krapov. & W. C. Gregory 036234 Ipa BOL 21°00’
63°25’
650 B
V 13250 A. kempff-mercadoi Krapov., W. C.Gregory & C. E. Simpson
030643 Sta. Cruz de laSierra
BOL 17°45’
63°10’
280 A
VKSSv 8979 A. kuhlmannii Krapov. & W. C.Gregory
020354 Cáceres MT 15°35’
57°13’
210 A
VPoBi 9243 A. kuhlmannii Krapov. & W. C.Gregory
022560 Corumbá MS 18°52’
56°16’
100 A
VPoJSv 10506 A. kuhlmannii Krapov. & W. C.Gregory
024953 N. Sra. doLivramento
MT 15°48’
56°21’
A
VRGeSv 7639 A. kuhlmannii Krapov. & W. C.Gregory
017515 Miranda MS 20°15’
56°23’
125 A
VSGr 6351 A. kuhlmannii Krapov. & W. C.Gregory
012602 Cáceres MT 15°56’
57°48’
130 A
VSGr 6413 A. kuhlmannii Krapov. & W. C.Gregory
012688 Cáceres MT 15°47’
57°25’
200 A
VSW 9912 A. kuhlmannii Krapov. & W. C.Gregory
022900 Aquidauana MS 20°26’
55°54’
210 A
(Continued)
Fungi Resistance of Wild Arachis Species and Hybrids
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Table 1. (Continued)
Accessions Code Species BrazilianAccessionsCode
CollectionsitesCity
State in Brazilor Country
Lat(W)
Long(S)
Alt(m)
Genome
KGSSc 30097 A. magna Krapov., W. C. Gregory &C. E. Simpson
036871 San Ignacio deVelasco
BOL 16°22’
60°58’
370 B
VPzSgRcSv 13761 A. magna Krapov., W. C. Gregory &C. E. Simpson
036218 Vila Bela da Ssa.Trindade
MT 15°21’
60°04’
380 B
VSPmSv 13751 A. magna Krapov., W. C. Gregory &C. E. Simpson
033812 Vila Bela da SsaTrindade
MT 16°16’
59°27’
530 B
VOa 14165 A. monticola Krapov. & Rigoni 036188 Yala, Jujuy ARG 24°07’
65°23’
AB
VSPmSv 13710 A. simpsonii Krapov. & W. C. Gregory 033685 Porto Esperidião MT 15°58’
58°31’
270 A
HLK 408 A. stenosperma Krapov. & W. C.Gregory
013366 Antonina PR 25°24’
48°44’
3 A
Lm 5 A. stenosperma Krapov. & W. C.Gregory
036013 Antonina PR A
SvW 3712 A. stenosperma Krapov. & W. C.Gregory
035254 Cocalinho MT 14°22’
51°00’
220 A
VSStGdW7805-AR
A. stenosperma Krapov. & W. C.Gregory
032476 São Felix doAraguaia
MT 11°38’
50°48’
240 A
VKSSv 9010 A. stenosperma Krapov. & W. C.Gregory
020176 Santo Antonio doLeverger
MT 15°52’
56°04’
150 A
VMiSv 10229 A. stenosperma Krapov. & W. C.Gregory
023001 Cananéia SP 25°01’
47°55’
10 A
VS 13670 A. stenosperma Krapov. & W. C.Gregory
018104 Araguaiana MT 15°33’
52°12’
350 A
VSMGeSv 7379 A. stenosperma Krapov. & W. C.Gregory
016063 Antonina PR 25°26’
48°42’
3 A
VSPmSv 13832 A. stenosperma Krapov. & W. C.Gregory
033961 S. M. doAraguaia/LuizAlves
MT 13°13’
50°34’
280 A
VSPmW 13824 A. stenosperma Krapov. & W. C.Gregory
033936 S. M. doAraguaia/LuizAlves
MT 13°13’
50°34’
280 A
VSSv 13258 A. stenosperma Krapov. & W. C.Gregory
016128 São Sebastião SP 23°45’
45°24’
5 A
VSv 10309 A. stenosperma Krapov. & W. C.Gregory
024830 Rondonópolis MT 16°28’
54°39’
215 A
VArLf 15076 A. stenosperma Krapov. & W. C.Gregory
040266 Matinhos PR A
WPz 421 A. stenosperma Krapov. & W. C.Gregory
033511 Alvorada TO 12°36’
49°20’
310 A
WiDc 1118 A. williamsii Krapov. & W. C. Gregory 036897 Trinidad BOL B
An 2 = (V 6389 x V9401)4x
(A. gregoryi x A. linearifolia)4x AB
An 4 = (KG 30076x V 14167)4x
(A.ipaënsis x A. duranensis)4x AB
IAC Caiapó x An2 A. hypogaea x (A. gregoryi x A.linearifolia)4x
AB
* Collectors: = Ar = A.R. Custodio, Bi = L. B. Bianchetti, Co = L. Coradin, Dc = D. Claure, G = W. C. Gregory, Gd = I. J. Godoy, Ge = M. A. N. Gerin,
Gr = A. Gripp, H = R. Hammons, J = L. Jank, K = A. Krapovickas, L = W.R. Langford, Lf = L. G. Faria,Lm = L. Monçato, M = J. P. Moss, Mi = S.T.S.Miotto,
Mm = M. Moraes, Oa = O.Ahumada, Of = F. O. Freitas, P = J. R. Pietralli, Pm = R. N. Pittmann, Po = A. Pott, Pz = E. Pizarro, R = V. R. Rao, Rc = R.C.
Oliveira, S = C. E. Simpson, Sc = A. Schinini, Sg = A. K. Singh, St = H. T. Stalker, Sv = G. P. Silva, Sz = R. Schultze-Kraft, V = J. F. M. Valls, W = W. L.
Werneck, Wi = D. E. Williams.
doi:10.1371/journal.pone.0128811.t001
Fungi Resistance of Wild Arachis Species and Hybrids
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experimental design was the same for all three years of evaluations apart from the number ofaccessions analyzed.
Resistance testingIn the first year, fungal diseases evaluated included early leaf spot (Cercospora arachidicola),late leaf spot (Cercosporidium personatum), rust (Puccinia arachidis.) and scab (Sphacelomaarachidis). All diseases except for scab were also evaluated in the second and third years to con-firm resistance of wild species accessions (18 genotypes and IAC Caiapó control). Scab was notevaluated in the second and third years due to its low incidence. A 1–9 visual grade scale fordamage caused at the end of the plant cycle was used in all evaluations.
Resistance data for early leaf spot, late leaf spot and rust were analyzed following the SASGLM procedure [11] taking into account the model cultivar effect (1 to 19) and time (years 1, 2and 3). Data from early and late leaf spot were transformed 1/x and log10(x), respectively, assuggested for the normalization of residues and cultivar variance homogeneity. In the compari-son of averages from cultivars, we adopted Duncan’s test at a significance of 5%. Software Sele-gen-Reml/Blup [12] were used for Restricted Maximum Likelihood/ Best, Linear, UnbiasedPrediction (REML/BLUP) analysis (Model 20 for first year data and Model 29 for three-yeardata).
Data were also subjected to grouping analysis (GA) complemented with principal compo-nent analysis (PCA) to group genotypes according to the variables: late leaf spot, early leafspot, scab, and rust. Genotype GA was performed according to Ward’s method [13], and Eu-clidian distance was considered a measure of dissimilarity. Dendrogram and connection graphswere used to interpret GA results. In PCA the two first principal components (PC1 and PC2)were considered the most important in their respective contributions to total variability. PC1and PC2 allowed for simultaneous visualization of variable and genotype projections as well asdeduction of the linear correlation among the variables: late leaf spot, early leaf spot, scab andrust. The software used for PCA and GA was STATISTICA [14], other analyses were con-ducted with SAS [11] and MS Office Excel.
Analysis of Variance conducted on data for the three different years showed significant dif-ferences between years for all three diseases and the interaction accessions x years for disease(late leaf spot and rust). Therefore, average measurements were used for GA.
Results and DiscussionIn the first year of study, we evaluated resistance to late leaf spot, early leaf spot, rust and scab.Within 50 accessions evaluated at the first year (Table 2) there was a large difference in resis-tance to late leaf spot, with averages ranging from 1.75 to 9. On the other side, for early leafspot, scab and rust, the variation among wild accessions was less significant. It is possible toverify either the difficulty to select accessions based on ANOVA and Duncan test. These resultsjustify the utilization of PCA and grouping analysis.
REML/BLUP analysis were shown in Table 3 for 50 genotypes in first year field assay. Theselection accuracy of genotypes had a high value, as well as PEV value was low for all variables.All CVgi% were higher than CVe% values except for scab variable indicating that the environ-ment had a important effect in the phenotypic pattern of this disease.
Resistance rank of each accession was obtained in individual BLUP analysis, as well as ageneral rank was observed by the sum of all ranks of the three diseases. The highest values arethose with best resistance to the three diseases. Ranks of genotypes in BLUP analysis were verysimilar to Duncan test results (Table 2).
Fungi Resistance of Wild Arachis Species and Hybrids
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Table 2. Duncan test results for Arachis spp. accessions for resistance to late leaf spot (LLS), early leaf spot (ELS), scab (S) and rust (R) in fieldassay (first year).
Accessions Code Species LLS ELS S R
2562 A. hypogaea 9.00 a1 - - 5.50 b
IAC Tatu-ST A. hypogaea 8.75 a 2.00 efg 3.00 a 5.00 bc
IAC Runner 886 A hypogaea 8.69 a 1.67 efgh 2.33 abc 8.00 a
BR1 A. hypogaea 8.00 ab 5.00 a 2.50 ab 4.67 cd
IAC Caiapó A. hypogaea 7.50 bc 5.19 a 2.44 ab 4.17 d
K 35005 A. benensis 6.75 c 1.00 h 1.00 e 1.00 f
K 9484 A. batizocoi 5.33 d 1.67 efgh 1.67 bcde 1.00 f
K 7988 A. duranensis 5.33 d 3.67 b 1.00 e 1.00 f
V 14165 A. monticola 5.33 d 1.33 fgh 2.33 abc 2.00 e
V 12549 A. hypogaea 5.00 de 3.25 bc 2.50 ab 5.00 bc
V 13761 A. magna 5.00 de 1.00 h 1.00 e 1.33 f
K 30097 A. magna 4.67 def 1.00 h 1.33 de 1.33 f
V 7805-AR A. stenosperma 4.67 def 1.33 fgh 1.00 e 1.00 f
An 4 (A.ipaënsis x A. duranensis)4x 4.50 defg 1.75 efgh 1.50 cde 1.00 f
V 10506 A. kuhlmannii 4.00 efgh 2.25 def 1.00 e 1.33 f
V 8979 A. kuhlmannii 4.00 efgh 3.00 bcd 1.00 e 1.50 ef
V 14767 A. gregoryi 4.00 efgh 1.50 fgh 1.25 de 1.00 f
V 7639 A. kuhlmannii 4.00 efgh 1.33 fgh 1.00 e 1.33 f
IAC Caiapó x An2 A. hypogaea x (A. gregoryi x A. linearifolia)4x 3.78 efghi 1.63 efgh 1.48 de 1.55 ef
K 30076 A. ipaënsis 3.75 efghi 1.25 gh 2.50 ab 2.00 e
V 9243 A. kuhlmannii 3.75 efghi 1.25 gh 1.00 e 1.00 f
V 6351 A. kuhlmannii 3.75 efghi 2.25 def 1.25 de 1.00 f
An 2 (A. gregoryi x A. linearifolia)4x 3.75 efghi 1.00 h 2.00 bcd 1.00 f
W 421 A. stenosperma 3.50 fghij 2.00 efg 1.00 e 1.00 f
Wi 1118 A. williamsii 3.50 fghij 1.50 fgh 1.00 e 2.00 e
V 14546 A. hoehnei 3.50 fghij 2.50 cde 1.25 ed 1.00 f
V 13832 A. stenosperma 3.33 ghijk 1.33 fgh 1.33 de 1.00 f
Co 6862 A. helodes 3.25 ghijk 1.75 efgh 1.00 e 1.00 f
V 9912 A. kuhlmannii 3.25 ghijk 1.00 h 1.00 e 1.00 f
V 13751 A. magna 3.25 ghijk 1.25 gh 1.00 e 1.00 f
V 14957 A. gregoryi 3.00 hijkl 1.00 h 1.25 de 1.00 f
V 6389 A. gregoryi 3.00 hijkl 1.00 h 1.75 bcde 1.00 f
H 408 A. stenosperma 3.00 hijkl 1.67 efgh 1.33 de 1.00 f
V 13824 A. stenosperma 3.00 hijkl 1.67 efgh 1.33 de 1.00 f
V 10309 A. stenosperma 3.00 hijkl 1.67 efgh 1.67 bcde 1.50 ef
V 14760 A. gregoryi 2.75 hijkl 1.00 h 1.50 cde 1.00 f
G 10017 A. cardenasii 2.67 hijkl 1.67 efgh 1.00 e 1.33 f
V 13710 A. simpsonii 2.67 hijkl 2.00 efg 1.00 e 1.00 f
V 7379 A. stenosperma 2.67 hijkl 1.67 efgh 1.67 bcde 1.33 f
V 13670 A. stenosperma 2.50 ijkl 2.00 efg 1.50 cde 1.00 f
V 15076 A. stenosperma 2.33 jkl 1.67 efgh 1.00 e 1.00 f
V 6325 A. helodes 2.33 ijkl 1.33 fgh 1.00 e 1.33 f
Lm 5 A. stenosperma 2.33 jkl 1.67 efgh 1.33 de 1.00 f
V 9010 A. stenosperma 2.33 jkl 1.67 efgh 1.33 de 1.00 f
V 10229 A. stenosperma 2.25 jkl 1.75 efgh 1.50 cde 1.00 f
V 13258 A. stenosperma 2.25 jkl 1.50 fgh 1.50 cde 1.00 f
(Continued)
Fungi Resistance of Wild Arachis Species and Hybrids
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Genetic correlation between the variables LLS, ELS, S and R for the first year assay and forthree years data, based on the REML/BLUP analysis, are shown in Table 4. In the first year,LLS and R was genetically correlated. In the analysis of three years for the 18 wild accessions se-lected as resistant and the control, all variables were correlated.
A first GA was conducted with the 50 genotypes (Fig 1). At cut-off point 10 of this dendro-gram, genotypes are divided into two groups: Group 1 encompasses all six accessions of A.hypogaea, A.monticola (V 14165) and A. ipaënsis (KG 30076). Interestingly, A.monticola is atetraploid species closely related to, and most likely a direct ancestor of, A. hypogaea [15]. Evi-dence also suggests that A. ipaënsis was the B genome species that originated A. hypogaea [16–18]. Group 2 encompasses all other wild genotypes included in the study, indicating that themajority of wild species are very distinct from cultivated peanut with regards to resistance toevaluated fungal diseases. This finding also suggests that many unexplored genes may be pres-ent in these pools that could be introduced into the genome of A. hypogaea. Another importantGA outcome is the grouping of three hybrids (two amphidiploids—An 2 and An 4—and the F2progeny individuals of Caiapó x An 4) in Group 2, as they all kept resistance patterns similar tothose of wild species. This finding shows that resistance is maintained after interspecificcrossings.
If the more susceptible accessions are removed from the analysis, a more detailed picture ofwild accession differentiation emerges (Fig 2): a cut-off point of 11 separated accessions ac-cording to their resistance to scab, whereas a cut-off point of 7 then discriminated between fivegenotype groups. The genotypes with least resistance to scab were subdivided as to their resis-tance to rust. The ones more resistant to scab were further subdivided as to their resistance toearly leaf spot. Among the early leaf spot resistant genotypes, another division was possiblewith regard to resistance to late leaf spot. Therefore, the joint evaluation of four diseases indi-cates that Group 2 of Fig 2 provides the most resistant accessions and might be the best one formultiple selections. The seven accessions that comprise this group are V 15076 (A. stenos-perma), V 6413 (A. kuhlmannii), V 13250 (A. kempff-mercadoi), Sv 3712 (A. stenosperma), KG30006 (A. hoehnei), V 6325 (A. helodes), and GKP 10017 (A. cardenasii). All of these accessionsare of A genome type, and apparently A genome species are more resistant to fungal diseasesthan species with other genomes in the Arachis section. Validating this observation is difficultdue to the smaller number of B genome sensu lato accessions evaluated in this report, whichmakes it difficult to investigate true variability when compared to the number of A genomeaccessions.
Another important aspect of resistance is the variability observed among accessions of a sin-gle species. Pande and Rao [8] have previously emphasized the importance of evaluating reac-tions at the individual level. We show that A. stenosperma accessions are present in everygroup, whereas A. kuhlmannii are present in three, A. hoehnei in two, and A. gregoryi in twogroups. A wider distribution of A. stenospermamay be a result from a larger number of
Table 2. (Continued)
Accessions Code Species LLS ELS S R
V 13250 A. kempff-mercadoi 2.00 kl 1.50 fgh 1.00 e 1.00 f
V 6413 A. kuhlmannii 2.00 kl 1.67 efgh 1.00 e 1.00 f
Sv 3712 A. stenosperma 2.00 kl 1.00 h 1.00 e 1.00 f
K 30006 A. hoehnei 1.75 l 1.25 gh 1.25 ed 1.00 f
1.Distinct letters indicate significant differences among accessions according to Duncan’s test (p<0,05)
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Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 7 / 17
Tab
le3.
Estim
atives
ofc
omponen
tsofv
ariance
(Individual
REML)a
ndtheco
mponen
tsofa
verage(Individual
BLUP)fortheva
riab
lesresistan
ceto
late
leaf
spot(LLS),ea
rlyleaf
spot(ELS),sc
ab(S)a
ndrust
(R)a
nd50
gen
otypes
,infirsty
earoffield
assa
y.
Componen
tsofav
erag
e(Individual
BLUP)
Gen
otype
LLS
ELS
SR
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
GR
2562
14.89
168.80
544.89
168.80
54-
--
--
--
--
-2
3.76
215.41
594.97
416.62
793
IAC
Tatu-ST
24.65
128.56
54.77
148.68
526
1.00
182.78
281.72
153.50
241
1.03
192.47
081.03
192.47
084
3.14
404.79
794.09
675.75
0513
IAC
Run
ner86
63
4.59
168.50
544.71
158.62
535
1.00
182.78
281.86
543.64
647
0.64
52.08
390.78
812.22
71
6.18
617.84
006.18
617.84
0016
BR
14
3.86
187.77
564.49
918.41
292
2.54
424.32
522.75
514.53
605
0.72
472.16
360.83
612.27
498
0.31
371.96
762.81
044.46
4319
IAC
Caiap
00F3
53.44
907.36
284.28
908.20
291
2.96
594.74
692.96
594.74
694
0.77
582.21
470.86
392.30
286
2.47
494.12
873.63
815.29
2016
V12
549
101.04
464.95
852.91
796.83
184
1.27
883.05
982.08
133.86
232
0.82
392.26
280.92
792.36
683
3.29
454.94
844.41
426.06
8119
K94
847
1.32
065.23
443.64
197.55
5719
-0.150
21.63
080.65
752.43
8410
0.20
261.64
150.63
972.07
8620
-0.643
81.01
010.96
962.62
3456
V14
165
91.32
065.23
443.12
617.03
9935
-0.432
71.34
830.24
282.02
386
0.69
162.13
050.81
202.25
097
0.34
081.99
473.16
714.82
0957
V89
7918
-0.038
23.87
561.79
455.70
837
0.87
472.65
571.60
053.38
1531
-0.242
81.19
610.18
091.61
9811
-0.193
01.46
082.04
033.69
4167
IAC
Caiap
óxAn2
19-0.132
53.78
131.69
35.60
6818
-0.134
91.64
610.70
242.48
3318
0.02
881.46
770.39
391.83
2810
-0.144
11.50
982.26
363.91
7565
K30
076
20-0.157
53.75
631.60
055.51
4338
-0.462
21.31
870.18
801.96
893
0.82
392.26
280.89
332.33
215
2.96
724.62
103.87
085.52
4666
K79
888
1.32
065.23
443.35
187.26
563
1.53
633.31
722.34
884.12
9734
-0.288
31.15
060.13
951.57
8422
-0.643
81.01
010.82
292.47
6867
V10
309
35-0.976
2.93
780.66
284.57
6617
-0.121
81.65
920.75
162.53
2612
0.17
761.61
640.56
472.00
3612
-0.193
01,46
081,85
423,50
876
An4
140.56
384.47
762.29
446.20
8215
-0.026
91.75
400.86
172.64
2715
0.04
751.48
630.46
451.90
3334
-0.643
81.01
010.30
521.95
9178
V14
546
24-0.398
03.51
581.29
755.21
138
0.62
602.40
691.47
873.25
9627
-0.146
71.29
220.23
661.67
5526
-0.643
81.01
010.59
732.25
1185
V10
506
160.08
293.99
672.01
795.93
189
0.40
832.18
931.35
983.14
0747
-0.340
81.09
810.01
451.45
3416
-0.316
41.33
751.31
152.96
5488
Wi1
118
26-0.507
13.40
671.16
295.07
6731
-0.327
31.45
360.33
002.11
0930
-0.242
81.19
610.19
511.63
399
0.29
671.95
062.53
114.18
596
V73
7939
-1.218
22.69
560.47
244.38
6227
-0.150
21.63
080.41
822.19
9111
0.20
261.64
150.59
992.03
8819
-0.316
41,33
751,05
452,70
8396
V76
3917
0.05
123.96
501.90
235.81
6132
-0.432
71.34
830.30
622.08
7133
-0.288
31.15
060.15
251.59
1414
-0.316
41.33
751.54
413.19
7996
V63
5122
-0.157
53.75
631.44
075.35
4510
0.40
832.18
931.26
463.04
5629
-0.146
71.29
220.21
021.64
9036
-0.643
81.01
010.25
251.90
6497
K30
097
130.68
714.60
092.42
756.34
1348
-0.713
1.06
790.01
491.79
5820
-0.043
81.39
510.35
021.78
918
-0.316
41.33
751.13
062.78
4599
V14
767
150.08
293.99
672.14
696.06
0828
-0.244
61.53
640.39
452.17
5528
-0.146
71.29
220.22
291.66
1829
-0.643
81.01
010.46
892.12
2710
0
GKP10
017
37-1.218
22.69
560.56
384.47
7620
-0.150
21.63
080.61
712.39
8032
-0.288
31.15
060.16
631.60
5213
-0.316
41,33
751,68
723,34
1110
2
V78
05-AR
120.68
714.60
092.57
256.48
6333
-0.432
71.34
830.28
382.06
4735
-0.288
31.15
060.12
731.56
6227
-0.643
81.01
010.55
132.20
5110
7
V13
832
27-0.584
73.32
911.09
815.01
1936
-0.432
71.34
830.22
412.00
5024
-0.043
81.39
510.28
451.72
3448
-0.643
81.01
010.02
851.68
2313
5
K35
005
62.72
776.64
154.02
887.94
2641
-0.679
81.10
110.13
511.91
6042
-0.340
81.09
810.05
681.49
5721
-0.643
81.01
010.89
272.54
6611
0
Co68
6228
-0.638
43.27
541.03
614.94
9914
-0.026
91.75
400.92
522.70
6243
-0.340
81.09
810.04
761.48
6428
-0.643
81,01
010,50
862,16
2511
3
V13
761
111.00
394.91
772.74
396.65
7747
-0.713
1.06
790.03
031.81
1339
-0.288
31.15
060.08
471.52
3617
-0.316
41.33
751.21
582.86
9611
4
V13
670
40-1.445
2.46
890.42
454.33
8313
0.07
341.85
430.99
852.77
9413
0.09
511.53
400.52
861.96
7549
-0.682
80,97
100,01
391,66
7811
5
V63
8932
-0.878
93.03
50.81
184.72
5644
-0.679
81.10
110.07
951.86
059
0.24
161.68
050.68
822.12
7130
-0.643
81,01
010,43
182,08
5611
5
V14
760
36-1.119
32.79
450.61
334.52
7143
-0.679
81.10
110.09
721.87
8114
0.04
751.48
630.49
421.93
3124
-0.643
81,01
010,70
072,35
4511
7
HLK
408
33-0.901
43.01
240.75
994.67
3724
-0.150
21.63
080.48
922.27
0221
-0.043
81.39
510.33
141.77
0341
-0.643
81,01
010,14
321,79
7111
9
V10
229
45-1.600
22.31
360.20
514.11
8916
-0.026
91.75
40.80
622.58
7116
0.04
751.48
630.43
841.87
7342
-0.643
81,01
010,12
451,77
8311
9
V14
957
31-0.878
93.03
50.86
634.78
0142
-0.679
81.10
110.11
571.89
6625
-0.146
71.29
220.26
731.70
6123
-0.643
81,01
010,75
912,41
3012
1
Lm5
43-1.537
32.37
650.28
764.20
1423
-0.150
21.63
080.51
702.29
8019
-0.043
81.39
510.37
091.80
9839
-0.643
81,01
010,18
361,83
7412
4
W42
125
-0.398
03.51
581.22
975.14
3511
0.13
021.91
111.16
152.94
2449
-0.340
81.09
810.00
001.43
8940
-0.643
81.01
010.16
291.81
6812
5
V63
2541
-1.537
32.37
650.37
664.29
0434
-0.432
71.34
830.26
272.04
3636
-0.288
31.15
060.11
581.55
4715
-0.316
41,33
751,42
003,07
3912
6
An2
23-0.157
53.75
631.37
125.28
5046
-0.679
81.10
110.04
651.82
748
0.43
571.87
460.74
412.18
3050
-0.682
80.97
100.00
001.65
3912
7
V13
824
34-0.901
43.01
240.71
14.62
4826
-0.150
21.63
080.44
002.22
1023
-0.043
81.39
510.29
881.73
7746
-0.643
81,01
010,05
771,71
1512
9
V90
1044
-1.537
32.37
650.24
614.15
9925
-0.150
21.63
080.46
372.24
4622
-0.043
81.39
510.31
431.75
3243
-0.643
81,01
010,10
661,76
0513
4
V13
710
38-1.218
22.69
560.51
694.43
0712
0.13
021.91
111.07
562.85
6540
-0.288
31.15
060.07
541.51
4244
-0.643
81,01
010,08
961,74
3413
4
V92
4321
-0.157
53.75
631.51
685.43
0639
-0.462
21.31
870.17
131.95
2245
-0.340
81.09
810.03
031.46
9232
-0.643
81.01
010.36
462.01
8413
7
V13
258
46-1.600
22.31
360.16
594.07
9730
-0.244
61.53
640.35
192.13
2817
0.04
751.48
630.41
541.85
4345
-0.643
81,01
010,07
331,72
7113
8
K30
006
50-2.081
1.83
280.00
003.91
3837
-0.462
21.31
870.20
551.98
6526
-0.146
71.29
220.25
131.69
0225
-0.643
81,01
010,64
692,30
0813
8
V15
076
42-1.537
32.37
650.33
114.24
4922
-0.150
21.63
080.54
742.32
8338
-0.288
31.15
060.09
451.53
3438
-0.643
81,01
010,20
541,85
9214
0
V64
1348
-1.854
2.05
980.08
23.99
5821
-0.150
21.63
080.58
062.36
1537
-0.288
31.15
060.10
481.54
3735
-0.643
81,01
010,27
811,93
2014
1
V99
1229
-0.638
43.27
540.97
844.89
2245
-0.679
81.10
110.06
261.84
3646
-0.340
81.09
810.02
221.46
1133
-0.643
81,01
010,33
401,98
7915
3
V13
751
30-0.638
43.27
540.92
454.83
8340
-0.462
21.31
870.15
551.93
6448
-0.340
81.09
810.00
711.44
637
-0.643
81,01
010,22
831,88
2215
5
V13
250
47-1.840
62.07
320.12
324.03
729
-0.244
61.53
640.37
252.15
3444
-0.340
81.09
810.03
871.47
7631
-0.643
81,01
010,39
712,05
0915
1
(Con
tinue
d)
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 8 / 17
Tab
le3.
(Con
tinue
d)
Componen
tsofav
erag
e(Individual
BLUP)
Gen
otype
LLS
ELS
SR
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
GR
Sv37
1249
-1.854
2.05
980.04
253.95
6349
-0.713
01.06
790.00
001.78
0941
-0.288
31.15
060.06
651.50
5447
-0.643
81,01
010,04
281,69
6618
6
Com
pone
ntsof
varia
nce
(Ind
ividua
lREML)
Vg=3.26
25Vg=0.71
96Vg=0.19
00Vg=2.12
06
Ve=0.64
88Ve=0.53
51Ve=0.27
35Ve=0.13
28
Vf=
3.91
13Vf=
1.25
47Vf=
0.46
35Vf=
2.25
35
h2g=0.83
41+-
0.17
34h2
g=0.57
35+-
0.14
64h2
g=0.41
00+-
0.12
38h2
g=0.94
11+-
0.20
06
h2mc=0.96
18h2
mc=0.87
05h2
mc=0.77
65h2
mc=0.98
46
Acclon=0.98
07Acclon=0.93
30Acclon=0.88
12Acclon=0.99
23
CVgi%
=46
.150
7CVgi%
=47
.630
6CVgi%
=30
.293
9CVgi%
=88
.051
1
CVe%
=20
.579
8CVe%
=41
.074
9CVe%
=36
.343
5CVe%
=22
.037
4
CVr=2.24
25CVr=1.15
96CVr=0.83
355
CVr=3.99
55
PEV=0.12
48PEV=0.09
317
PEV=0.04
247
PEV=0.03
27
SEP=0.35
33SEP=0.30
52SEP=0.20
608
SEP=0.18
08
GA=3.91
38GA=1.78
09GA=1.43
89GA=1.65
39
*g:
geno
typicefec
t,u+g:
geno
typicav
erag
e,GG:g
eneticga
in,N
a=ne
wav
erag
e,GR:g
eneral
rank
,Vg:
geno
typicva
rianc
e,Ve:
residu
alva
rianc
e,Vf:ph
enotyp
icva
rianc
e,h2
g=plot
heritab
ility
inbroa
dse
nse,
h2mc=av
erag
ege
notype
heritab
ility,A
cclon=se
llectionge
notype
accu
racy,C
Vgi%
=individu
alco
efficien
tofa
dditive
varia
nce,
CVe%
=co
efficien
tof
expe
rimen
talvariatio
n,CVr=co
efficien
tofrelativeva
riatio
n,PEV=pred
ictio
nerrorva
rianc
eof
geno
typicva
lues
,SEP=stan
dard
deviationof
geno
typicva
lue,
GA=ge
nerala
verage
doi:10.1371/journal.pone.0128811.t003
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 9 / 17
accessions in this species. If there were more accessions in the other species, we might have ob-served a similarly ample distribution. As it was observed the wide variability at the specieslevel, research efforts are necessary in the identification of resistances in accessions, notin species.
The two amphidiploids were grouped in Group 5. Amphidiploid An 2 remained very closeto one of its progenitors, V 6389 (A. gregoryi). The other progenitor, V 9401 (A. linearifolia),was not included in the study due to an insufficient number of seeds. Amphidiploid An 4 re-sulted from a cross between A. ipaënsis x A. duranensis V 14167 followed by artificial polyploi-dization. The female progenitor fell into Group 1, along with accessions of A. hypogaea. The A.duranensis accession was not included in the study for lack of seeds. Interestingly, some ramifi-cations within Group 5 included amphidiploids and other B and K genome species, but no Agenome accessions. In another subdivision of Group 5, only A genome accessions were segre-gated. Non-A genomes were also concentrated in Groups 3 and 4. The F2 progeny of IACCaiapó x An 4, such as A. stenosperma V 10309, were situated in Group 4, exhibiting partial re-sistance when compared to wild genotypes.
Four accessions that showed special potential for future studies are the A.magna accessionsV 13751 and KG 30097 and the A. gregoryi accessions V 14767 and V 14957. While they werenot the best in terms of resistance, they belong to the B genome type that is crucial for resis-tance-gene introgression and pyramidization in A. hypogaea.
Table 4. Genetic correlation between variables (resistances to late and early leaf spots–LLS, ELS–scab–S, and rust—R) in first year for 50 accessions and in three years field assay for 18 selected ac-cessions and one control.
Genetic correlation
1st year 3 years
Variable LLS ELS S LLS ELS
LLS
ELS 0.3986 0.9519
S 0.2779 0.5447
R 0.7058 0.3095 0.4062 0.9615 0.9801
doi:10.1371/journal.pone.0128811.t004
Fig 1. Distribution of wild Arachis genotypes and A. hypogaea controls with respect to resistance toearly leaf spot, late leaf spot, rust, and scab in the first year of study.Cut-off point = 10 (arrow) indicatesgenotype segregation into two groups.
doi:10.1371/journal.pone.0128811.g001
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 10 / 17
Similarly to the GA with a cut-off point of 10, a two-group division was observed throughPCA (Fig 3), where the two first components explained 81.41% of variation. Again, Group 1(red circle) was formed by the same eight genotypes as in Fig 1, whereas the other wild acces-sions were tightly connected in Group 2 (green circle). Arrows point towards accessions, in-cluding those of Group 1, which were more susceptible to the diseases evaluated. Some Group1 genotypes such as IAC-Caiapó and BR-1 were more susceptible to early leaf spot, whereascultivars IAC-Tatu-ST and IAC-Runner 886 were more strongly associated with late leaf spot.V12549 was more susceptible to scab. Finally, accessions of A. hypogaea 2562, A.monticolaV14165 and A. ipaënsis 30076 were more strongly correlated with rust. Accession V14165 wasalmost equidistant from Group 1 and 2 accessions.
Group 2 encompassed accessions and hybrids that were opposite to the arrows, indicating atrend to multiple resistances of wild genotypes. Again, the two amphidiploids (An 2 and An 4)and the F2 progeny individuals of Caiapó x An 4 grouped with wild species. Because Group 2genotypes were very closely associated, a more refined analysis to define which one would bepreferred for genetic improvement required re-running PCA without Group 1 accessions.
Fig 4 shows the PCA re-run without Group 1 accessions. The two main components explain57.17% of variation. When the accessions in Fig 4 were divided into the five GA groups ob-tained from Fig 2, these groups tended to disperse, with few intersections. Group 1 genotypes,in green, showed less resistance to early leaf spot; and Group 4, in yellow, was the least resistantto rust. Group 3, in black, was more closely associated to late leaf spot and rust; and Group 5,in blue, was in the same direction as the scab arrow, but showing a fair amount of internal vari-ation. For example, Group 5 amphidiploid An 2 had lower resistance to scab whereas Lm 5 wason the border with Group 2, distant from each one of the arrows. In fact, amphidiploids An 2and An 4 had lower resistance to scab but were more resistant to early leaf spot, rust and lateleaf spot. On the other hand, the F2 of Caiapó x An 2 showed reduced resistance to rust, lateleaf spot and scab, but greater resistance to early leaf spot. An important obstacle to the selec-tion of progenies from interspecific crossings targeting disease resistance is the risk of losingimportant alleles as a result of backcrossing.
Accessions in Group 2 of Fig 2, in red, were distant from all arrows, and therefore are morelikely to have multiple resistances. Overall, PCA validated the GA results.
Fig 2. Distribution of wild Arachis genotypes according to resistance to late leaf spot (LLS), early leafspot (ELS), rust and scab, in the first year of study, excluding susceptible groups (accessions of A.hypogaea and two closely related wild species).Cut-off point = 7 (arrow) indicates genotype segregationinto five groups.
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PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 11 / 17
Table 5 shows average values from the studies conducted in three consecutive years evaluat-ing resistance to fungal diseases in 18 wild accessions and in the cultivar IAC Caiapó. Therewas a difficulty of selection of the best accessions for the three diseases, justifying again thePCA and grouping analysis utilization. It was also observed that there were differences amongyears. ANOVA results showed the interaction between accessions x years.
Of the seven accessions identified as the most resistant (Group 2 of Fig 2), six are shown inbold in Table 5; only accession KG 30006 was not included, because at the time it was not be-lieved to be an A genome species [2]. Additionally, previous tests had failed in crossing andgenerating fertile amphidiploids from this species. Currently, it is known that this species hasthe A genome [19], and further work is needed to validate its potential as a male progenitor ininterspecific crossings and generation of new amphidiploids.
During GA with all 18 genotypes and the control, only two groups were obtained, becauseIAC Caiapó was considered susceptible when compared to the wild accessions. Therefore, weremoved the control from the analysis to evaluate isolated behavior among the accessions.
Fig 5 shows GA where a cut-off point of 0.7 forms three groups. All species has A genome,except A. gregoryi accession V 14767. Rust resistance was not relevant to discriminate between
Fig 3. Distribution of wild Arachis accessions and A. hypogaea controls according to their resistanceto late leaf spot, early leaf spot, rust and scab in the first year of study. PCA with the two firstcomponents explaining 81.41% of variation. Group 1 (red circle) and Group 2 (green circle) includesusceptible and resistant accessions respectively.
doi:10.1371/journal.pone.0128811.g003
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 12 / 17
accessions, as they all had low grades, i.e., low infection rates. Comparing data from Table 5with Fig 5, we may conclude that Group 1 accessions had lower resistance to late leaf spot,whereas Groups 2 and 3 showed greater resistance to this disease. The distinguishing featurebetween Groups 2 and 3 was that the former included accessions with lower resistance to earlyleaf spot, compared to the latter.
Therefore, our data suggest that Group 2 accessions (Fig 5) have the greatest potential foruse in genetic improvement programs. However, given that late leaf spot is the most importantdisease in the field, and that the difference in resistance to early leaf spot was small betweenGroups 2 and 3, both of these groups should be considered in improvement programs. Again,only A genome species were selected, except for one B genome accession, A. gregoryi V 14767,which segregated to Group 1. V14767 may not be considered the best resistance genotype, butmight prove to be an excellent allele donor for gene pyramiding. We must again point out thatdifferences observed among data from Table 5, Figs 2 and 5 result from the fact that, in the firstyear, we evaluated scab resistance whereas in later years the disease occurred at a very low rateand could not be quantified. Overall, the data show that the best accessions regarding multipleresistance to diseases in this study conditions are V 15076 (A. stenosperma), V 6413 (A. kuhl-mannii), V 13250 (A. kempff-mercadoi), Sv 3712 (A. stenosperma), V 6325 (A. helodes), GKP10017 (A. cardenasii) (Table 5 - bold).
The individual REML analysis (Table 6) of 19 genotypes used in three years assays detectedthat the environmental variance value was low, allowing the discrimination of genotypes.Based on individual BLUP (Table 6), resistance ranking of each accession was obtained, as wellas a general ranking was observed by the sum of all ranks of the three diseases. The highest val-ues are those with the best resistance to the three diseases. Accessions in bold in Table 5 hadGR values higher than 32 in Table 6, corroborating the results of Duncan Test, PCA and GA.
Variance analysis showed that A. kuhlmannii (V 6413) had the lowest average degrees of ob-servation of late leaf spot, whereas A. stenosperma (Sv 3712) and A. kuhlmannii (V 9912) hadthe lowest incidence (lowest grade) of early leaf spot. All wild genotypes showed resistance torust at the natural inoculum pressure used (Table 5).
Fávero et al. [9] utilized detached leaves to show that Arachis hypogaea and Arachis monti-cola were susceptible to late leaf spot, early leaf spot and rust, as we reproduced here in the
Fig 4. Distribution of wild Arachis accessions according to their resistance to late leaf spot, early leafspot, rust and scab in the first year of study. PCA with the two first components explaining 57.17% ofvariation. Green, red, black, yellow and blue groups means groups 1, 2, 3, 4 and 5 of Fig 2 respectively.
doi:10.1371/journal.pone.0128811.g004
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 13 / 17
Table 5. Average grades of resistance to late leaf spot, early leaf spot and rust of genotypes evaluatedduring three consecutive years and differences among years averages.
Species/Accessions Late Leaf Spot Early Leaf Spot Rust Fig 2 Group Fig 5 Group
A. simpsonii V 13710 2.22 bcdefg1 1.83 b 1.00 b 1 2
A. helodes Co 6862 2.17 defgh 1.53 bcd 1.03 b 1 2
A. kuhlmannii V 6413 1.70 h 1.64 bcd 1.00 b 2 2
A. stenosperma V 15076 1.91 gh 1.46 bcd 1.12 b 2 2
A. kempff-mercadoi V 13250 1.97 fgh 1.44 bcd 1.00 b 2 2
A. cardenasii GKP 10017 2.27 bcdefg 1.27 cd 1.09 b 2 3
A. helodes V 6325 2.18 bcdefg 1.36 cd 1.09 b 2 3
A. stenosperma Sv 3712 2.00 efgh 1.27 d 1.27 b 2 3
A. gregoryi V 14767 2.83 b 1.58 bcd 1.03 b 3 1
A. kuhlmannii V 9912 2.57 bcd 1.31 d 1.00 b 3 1
A. stenosperma V 13832 2.65 bc 1.51 bcd 1.00 b 3 1
A. stenosperma V 10309 2.17 cdefg 1.79 bc 1.10 b 4 2
A. stenosperma V 7379 2.00 fgh 1.64 bcd 1.09 b 4 2
A. stenosperma V 13670 2.57 bcde 1.43 cd 1.00 b 5 1
A. stenosperma HLK 408 2.27 bcdefg 1.54 bcd 1.03 b 5 2
A. helodes Lm5 2.29 bcdefg 1.39 cd 1.24 b 5 3
A. stenosperma V 9010 2.33 bcdef 1.27 cd 1.00 b 5 3
A. stenosperma V 13258 2.31 bcdefg 1.42 bcd 1.00 b 5 3
A. hypogaea IAC Caiapó 6.82 a 5.63 a 5.92 a * *
Year 1 3.00 a 1.79 b 1.25 b
Year 2 2.31 c 1.32 c 1.33 b
Year 3 2.97 b 2.62 a 2.01 a
Accessions displaying multiple resistance in bold.1.Distinct letters indicate significant differences among accessions according to Duncan’s test (p<0,05)
* Not included in GA
doi:10.1371/journal.pone.0128811.t005
Fig 5. Wild Arachis genotypes segregated according to their resistance to late leaf spot (LLS), earlyleaf spot (ELS), and rust after three years of study, excluding IAC Caiapó control.Cut-off point = 0.7(arrow) indicates genotype distribution into three groups.
doi:10.1371/journal.pone.0128811.g005
Fungi Resistance of Wild Arachis Species and Hybrids
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Tab
le6.
Estim
atives
ofc
omponen
tsofv
ariance
(Individual
REML)a
ndtheco
mponen
tsofa
verage(Individual
BLUP)fortheva
riab
lesresistan
ceto
late
leaf
spot(LLS),
earlyleaf
spot(ELS)a
ndrust
(R)a
nd19
gen
otypes
,duringthreeco
nse
cutive
years.
Componen
tsofav
erag
e(Individual
BLUP)
Gen
otype
MP
MC
F
Ran
kg*
u+g
GG
Na
Ran
kg
u+g
GG
Na
Ran
kg
u+g
GG
Na
GR
IAC
Caiap
ó1
4.37
416.88
284.37
416.88
281
3.59
135.28
93.59
135.28
901
4.30
855.62
534.30
855.62
533
V14
767
20.31
232.82
102.34
324.85
196
-0.106
21.59
160.58
802.28
5710
-0.269
01.04
780.26
571.58
2518
V10
309
13-0.321
82.18
690.24
122.74
983
0.07
871.77
641.25
072.94
845
-0.204
71.11
210.76
502.08
1921
Lm5
8-0.202
12.30
660.55
863.06
7214
-0.284
61.41
310.13
081.82
863
-0.063
31.25
351.40
362.72
0525
V13
832
30.16
742.67
611.61
84.12
667
-0.129
21.56
860.48
552.18
3318
-0.298
11.01
880.01
671.33
3628
V73
7916
-0.479
22.02
940.11
562.62
434
-0.059
01.63
870.92
322.62
108
-0.210
11.10
670.39
931.71
6228
HLK
408
10-0.216
92.29
180.40
362.91
228
-0.143
51.55
420.40
692.10
4611
-0.269
01.04
780.21
701.53
3929
V13
710
11-0.275
32.23
340.34
192.85
052
0.08
211.77
981.83
673.53
4416
-0.298
11.01
880.05
611.37
2929
Co68
6214
-0.329
02.17
970.20
052.70
919
-0.158
41.53
940.34
412.04
189
-0.269
01.04
780.32
511.64
1932
V15
076
18-0.564
61.94
400.04
272.55
1410
-0.228
1.46
980.28
691.98
464
-0.181
11.13
571.00
742.32
4332
V99
124
0.11
192.62
061.24
153.75
0116
-0.327
51.37
030.07
451.77
2313
-0.298
11.01
880.13
781.45
4633
GKP10
017
9-0.216
02.29
260.47
252.98
1219
-0.397
81.29
990.00
01.69
786
-0.210
11.10
670.60
251.91
9334
V63
2512
-0.303
22.20
550.28
812.79
6815
-0.312
51.38
530.10
131.79
917
-0.210
11.10
670.48
641.80
3234
Sv37
1215
-0.477
52.03
120.15
532.66
3918
-0.397
1.30
080.02
211.71
992
-0.034
31.28
262.13
713.45
3935
V13
670
50.06
012.56
881.00
523.51
3812
-0.252
31.44
540.19
831.89
6019
-0.301
11.01
570.00
001.31
6836
V13
258
7-0.195
92.31
270.66
723.17
5913
-0.262
81.43
50.16
281.86
0617
-0.298
11.01
880.03
521.35
2137
V90
106
-0.159
42.34
930.81
113.31
9717
-0.397
01.30
080.04
681.74
4515
-0.298
11.01
880.07
971.39
6538
V64
1319
-0.769
41.73
920.00
002.50
875
-0.059
01.63
870.72
682.42
4614
-0.298
11.01
880.10
671.42
3538
V13
250
17-0.515
81.99
290.07
852.58
7111
-0.237
11.46
070.23
921.93
7012
-0.298
11.01
880.17
411.49
140
Com
pone
ntsOfV
arianc
e(Ind
ividua
lREML)
Vg=1.24
44Vg=0.83
51Vg=1.13
22
Vpe
rm=0.06
27Vperm
=0.07
51Vperm
=0.04
45
Ve=0.52
81Ve=0.62
29Ve=0.37
77
Vf=1.83
52Vf=1.53
31Vf=1.55
45
h2g=0.67
81+-
0.15
96h2
g=0.54
47+-0.14
27h2
g=0.72
84+-0.16
78
r=0.71
22+-
0.16
36r=0.59
37+-
0.14
90r=0.75
70+-
0.17
10
c2perm
=0.03
414
c2perm
=0.04
897
c2perm
=0.02
86
h2mg=0.86
47h2
mg=
0.78
35h2
mg=
0.89
07
GA=2.50
87GA=1.69
78GA=1.31
68
*g:
geno
typicefec
t,u+g:
geno
typicav
erag
e,GG:g
eneticga
in,N
a=ne
wav
erag
e,GR:g
eneral
rank
,Vg:g
enotyp
icva
rianc
e,Vperm:v
arianc
eof
thepe
rman
ente
nviro
men
tal
effects,
Ve:res
idua
lvarianc
e,Vf:ph
enotyp
icva
rianc
e,h2
g=plot
heritab
ility
inbroa
dse
nse,
r:plot
repe
atab
ility,c
2perm
=en
viromen
tdeterminationco
efficien
t,h2
mg:g
enotyp
e
averag
ehe
ritab
ility,G
A-Gen
eral
Ave
rage
doi:10.1371/journal.pone.0128811.t006
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 15 / 17
field. Similarly, our results agree with those of Fávero et al. [9] with regards to V9243 suscepti-bility to late leaf spot, and Wi 1118 and V13824 susceptibility to rust. However, in contrast tothat previous work, we show that in three years of field evaluation the Sv 3712 accession was re-sistant to rust. Yet another distinct new finding of our study is the susceptibility of A. batizocoito scab; Fávero et al. [9] found this accession to be highly resistant for late and early leaf spotsbut did not test it for scab.
Pande and Rao [8] also identified late leaf spot resistance in an A. hoehnei accession collect-ed at a site near the collection site of the species used in our study, and they reported the sameresult for their KG 30006 accession from the same region. In both studies, A.monticola acces-sions were susceptible to late leaf spot and rust.
ConclusionsWe have found accessions with greater resistance to disease than A. cardenasii. The mostpromising accessions with multiple resistance to late leaf spot, early leaf spot, rust and scab inour study conditions were V 15076 (A. stenosperma), V 6413 (A. kuhlmannii), V 13250 (A.kempff-mercadoi), Sv 3712 (A. stenosperma), KG 30006 (A. hoehnei), V 6325 (A. helodes) andGKP 10017 (A. cardenasii). Amphidiploids and A. hypogaea x amphidiploid hybrids behavedsimilarly to wild species. Four accessions that should be further evaluated are the A.magna ac-cessions V 13751 and KG 30097 and the A. gregoryi accessions V 14767 and V 14957. Althoughthey did not show specifically high resistance, they belong to the B genome type that is crucialto resistance gene introgression and pyramiding in A. hypogaea.
Supporting InformationS1 Table. Raw data of 50 Arachis genotypes evaluated for resistance to late leaf spot, earlyleaf spot, rust and scab in field assays.(DOC)
Author ContributionsConceived and designed the experiments: MDM IJG APF. Performed the experiments: MDMIJG APF. Analyzed the data: WBJ EL MDVR. Contributed reagents/materials/analysis tools:IJG APF. Wrote the paper: APF MDMWBJ.
References1. United State Department of Agriculture (USDA). 2015. Available: http://usda.mannlib.cornell.edu/
MannUsda/viewDocumentInfo.do?documentID=1290.Table46.xls.
2. Krapovickas A, Gregory WC Taxonomía del género Arachis (Leguminosae). Bonplandia 1994; 8(1–4):1–186. PMID: 8091443
3. Valls JFM, Simpson CE New species of Arachis L. (Leguminosae) from Brazil, Paraguay and Bolivia.Bonplandia 2005; 14(1–4): 35–63.
4. Foster DJ, Stalker HT, Wynne JC, Beute MK Resistance of Arachis hypogaea L. and wild relatives toCercospora arachidicolaHori. Oléagineux 1981; 36(3): 139–143.
5. Subrahmanyam P, Smith DH, Simpson CE Resistance to Didymella arachidicola in wild Arachis spe-cies. Oléagineux 1985; 40(11): 553–556.
6. Stalker HT, Moss JP Speciation, citogenetics and utilization of Arachis species. Adv agron 1987; 41:1–40.
7. Varman PV, Ganesan KN, Mothilal A Wild germplasm: potential source for resistance breeding ingroundnut. Journal of Ecobiology 2000; 12(3): 223–228. PMID: 10963041
8. Pande S, Rao R Resistance of wild Arachis species to late leaf spot and rust in greenhouse trials. PlantDis 2001; 85(8): 851–855.
Fungi Resistance of Wild Arachis Species and Hybrids
PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 16 / 17
9. Fávero AP, Simpson CE, Valls JFM, Vello NA Characterization of rust, early and late leaf spot resis-tance in wild and cultivated peanut germplasm. Sci Agric 2009; 66: 110–117.
10. Stalker HT Utilizing Arachis cardenasii as a source of Cercospora leafspot resistance for peanut im-provement. Euphytica 1984; 33(2): 529–538.
11. SAS Institute Inc., Cary, NC, USA, Software 9.3, 2002–2010.
12. Resende MDV Selegen-Reml/Blup, 2002.
13. Ward JH Jr Hierarchical Grouping to Optimize an Objective Function. J Am Stat Assoc 1963; 58: 236–244.
14. StatSoft, Inc. STATISTICA (data analysis software system), version 7. www.statsoft.com, 2004.
15. Seijo G, Lavia GI, Fernández A, Krapovickas A, Ducasse DA, Bertioli DJ, et al. Genomic relationshipsbetween the cultivated peanut (Arachis hypogaea, Leguminosae) and its close relatives revealed bydouble GISH. Am J Bot 2007; 94(12): 1963–1971. doi: 10.3732/ajb.94.12.1963 PMID: 21636391
16. Kockert G, Stalker HT, Gimenes M, Galgaro L, Lopes CR, Moore K RFLP and cytogenetic evidence onthe origin and evolution of allotetraploid domesticated peanut, Arachis hypogaea (Legumiinosae). Am JBot 1996; 83: 1282–1291.
17. Seijo G, Lavia GI, Fernándes A, Krapovickas A, Ducasse DA, Moscone EA Physical mapping of the 5Sand 18S-25S rRNA genes by fish as evidence that A. duranensis and A. ipaënsis are the wild diploideprogenitors of A. hypogaea (Leguminosae). Am J Bot 2004; 91: 1294–1303. doi: 10.3732/ajb.91.9.1294 PMID: 21652361
18. Fávero AP, Simpson CE, Valls JFM, Vello NA Study of the evolution of cultivated peanut through cross-ability studies among Arachis ipaënsis, A. duranensis and A. hypogaea. Crop Sci 2006; 46: 1546–1552.
19. Robledo G, Seijo G Species relationships among the wild B genome of Arachis species (section Ara-chis) based on FISHmapping of rDNA loci and heterochromatin detection: new proposal for genome ar-rangement. TAG 2010; 121: 1033–1046. doi: 10.1007/s00122-010-1369-7 PMID: 20552326
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