identificationofsourcesofresistanceforpeanut aspergillus...

Research ArticleIdentification of Sources of Resistance for Peanut Aspergillusflavus Colonization and Aflatoxin Contamination

Richard Moise Alansou Dieme 1 Issa Faye2 Yedomon Ange Bovys Zoclanclounon3

Daniel Fonceka3 Ousmane Ndoye4 and Papa Madiallacke Diedhiou1

1Universite Gaston Berger BP 211 Saint-Louis Senegal2Centre National de Recherches Agronomiques de Bambey Institut Senegalais de Recherches Agricoles BP 211 Bambey Senegal3Centre drsquoEtudes Regional pour lrsquoAmelioration de lrsquoAdaptation a la Secheresse Route de Khombole BP 3320 +ies Senegal4Conseil Ouest et centre Africain pour la Recherche et le Developpement Agricoles Avenue Bourguiba BP 48 Dakar RP Senegal

Correspondence should be addressed to Richard Moise Alansou Dieme alansou7diemegmailcom

Received 25 May 2018 Revised 7 August 2018 Accepted 13 September 2018 Published 1 October 2018

Academic Editor Glaciela Kaschuk

Copyright copy 2018 Richard Moise Alansou Dieme et al is is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited

Peanut aflatoxin contamination caused by Aspergillus flavus is a serious constraint for food safety and human health in Senegale present study aimed to identify sources of resistance for A flavus colonization and aflatoxin contamination us seeds from67 peanut genotypes were tested under laboratory conditions Aqueous conidial suspension of an aflatoxinogenic strain of Aflavus was used for inoculation in Petri dishes containing ten seeds of each genotype and data on incidence and severity wererecorded Total aflatoxin concentration in seeds was determined on 15th day after inoculation using mReaderreg method Resultsshowed a significant (plt 0001) variation of aflatoxin incidence and severity among the tested peanut genotypes Incidenceranged from 0 to 70 with a mean of 2036plusmn 08 Out of the 67 genotypes eight showed incidence less than 10 Severity rangedfrom 0 to 44with a mean value of 882plusmn 045e genotype 12CS_104 showed aflatoxin concentration level in conformity withthe European standard (4 ppb) Out of three clusters revealed by hierarchical classification based on disease incidence and severitythe cluster 1 contained 33 genotypes characterised by low incidence and severity values ese genotypes can be tested under fieldconditions to confirm their resistance to A flavus

1 Introduction

Peanut (Arachis hypogaea L) is an important staple crop inSenegal e national peanut production was estimated at1050042 tons during the rainy season of 2016 [1] is cropis mainly produced in Fatick Kaolack Kaffrine Louga andies regions with more than 60 of the national peanutproduction [1] Peanut seeds are widely used for foodconsumption and play a significant economic role for small-scale farmers and food industries in Senegal [2] Howeverpre- and postharvest aflatoxin contamination in peanut isa serious threat for food safety and human health in Senegal[3] It is one of themajor constraints limiting sustainable andgood quality seed production in the world [4] Aflatoxincontamination is due to Aspergillus flavus (Link ex FriesTeleomorph Petromyces flavus) [5] Damages caused by this

facultative plant pathogen in maize peanut and sesamewere reported in Senegal [6] Considerable economic lossescaused by this bacterium are mainly due to crop qualityvalue and international trade restrictions on food stuffscharged in aflatoxin [7]

Aflatoxin is the name of a group of toxin known as G1G2 B1 B2 M1 and M2 that produced the plant pathogen[8] ese toxins occur naturally and have been found ina wide range of commodities including peanuts used foranimal and human consumption [9] Aflatoxins are toxicmutagenic and carcinogenic compounds [10] Dependingon their levels toxins can severely affect the liver and induceimmune-suppressing effects [9]

To handle this issue a wide range of preharvest aflatoxincontamination management methods were developedApplication of atoxinogenic isolates of A flavus [11] and

HindawiInternational Journal of AgronomyVolume 2018 Article ID 5468602 7 pageshttpsdoiorg10115520185468602

host genetic resistance were tested [12] In Senegal previousstudies reported that varieties 55-437 and 73-3 were resistantto A flavus [13] Identification of new sources of resistancemerits to be investigated for efficient peanut breedingprogram First step of host genetic resistance is the seedcolonization test erefore the present study was un-dertaken to identify promising peanut genotypes underlaboratory conditions

2 Materials and Methods

21 Plant Materials e plant material consisted of 67genotypes including 58 chromosomal substitutions lines[14] and nine national released varieties e chromosomalsubstitution lines belong to a cross between Fleur 11 anda synthetic amphidiploid parent (Table 1)

22 Isolation of Aspergillus flavus Sporangial SuspensionPreparation and Inoculation Aflatoxinogenic strain pro-vided from peanut seeds were purified by successive cultureson 52 agar medium e aflatoxin concentration level waschecked using the Revealreg Q+ Aflatoxin test kit (accessopeanut enterprise corporation USA) e spore suspensionofA flavuswas obtained by soaking colonized seeds in 50mlof sterile distilled water en one drop of Tween 20 wasadded to the solution and thoroughly mixed for 10 minutesInoculation was carried out by introducing 100 μl of thesupernatant of the spore suspension into each Petri dish

23 Seed Colonization Test e seed colonization test wasconducted following a modified Mehan and McDonaldprocedure For each genotype 50 seeds were sterilized andrinsed properly in sterile distilled wateren the seeds werehydrated to about 20 moisture content e 50 seeds ofeach genotype were placed in 5 Petri dishes containing 10seeds and each Petri dish was considered as a replicatione seeds were inoculated with a conidial suspension (60 microLcontaining approximately 1times 108mLminus1 conidia of the afla-toxigenic strain of A flavus) is preparation was kept atlaboratory conditions (25plusmn 012degC and 82plusmn 042 relativehumidity) for fifteen days

24 Data Collection e seedsrsquo colonization was observedduring two weeks and aflatoxin concentration was mea-sured using the Revealreg Q+ Aflatoxin test kit (accesso peanutenterprise corporation USA) e incidence was calculatedusing the following formula

incidence()

number of seeds showing pathogen colonizaton

total number of seeds

times 100

(1)

Table 1 Peanut material used in this study

No Genotypes Description Country of origin1 12CS_001 CSLlowast Senegal2 12CS_004 CSL Senegal3 12CS_006 CSL Senegal4 12CS_007 CSL Senegal5 12CS_008 CSL Senegal6 12CS_009 CSL Senegal7 12CS_010 CSL Senegal8 12CS_011 CSL Senegal9 12CS_012 CSL Senegal10 12CS_016 CSL Senegal11 12CS_018 CSL Senegal12 12CS_020 CSL Senegal13 12CS_021 CSL Senegal14 12CS_022 CSL Senegal15 12CS_023 CSL Senegal16 12CS_024 CSL Senegal17 12CS_027 CSL Senegal18 12CS_028 CSL Senegal19 12CS_031 CSL Senegal20 12CS_032 CSL Senegal21 12CS_033 CSL Senegal22 12CS_034 CSL Senegal23 12CS_036 CSL Senegal24 12CS_037 CSL Senegal25 12CS_039 CSL Senegal26 12CS_041 CSL Senegal27 12CS_042 CSL Senegal28 12CS_047 CSL Senegal29 12CS_048 CSL Senegal30 12CS_050 CSL Senegal31 12CS_051 CSL Senegal32 12CS_052 CSL Senegal33 12CS_053 CSL Senegal34 12CS_054 CSL Senegal35 12CS_055 CSL Senegal36 12CS_059 CSL Senegal37 12CS_060 CSL Senegal38 12CS_061 CSL Senegal39 12CS_062 CSL Senegal40 12CS_063 CSL Senegal41 12CS_066 CSL Senegal42 12CS_070 CSL Senegal43 12CS_072 CSL Senegal44 12CS_075 CSL Senegal45 12CS_076 CSL Senegal46 12CS_078 CSL Senegal47 12CS_079 CSL Senegal48 12CS_084 CSL Senegal49 12CS_085 CSL Senegal50 12CS_090 CSL Senegal51 12CS_091 CSL Senegal52 12CS_095 CSL Senegal54 12CS_096 CSL Senegal55 12CS_100 CSL Senegal56 12CS_111 CSL Senegal57 12CS_112 CSL Senegal58 12CS_118 CSL Senegal59 12CS_119 CSL Senegal60 55-33 Variety Senegal61 55-437 Resistant control Senegal

2 International Journal of Agronomy

e severity scale of aflatoxin on seeds was estimatedusing a modified Tonapi et al [15] scale It was defined asfollows 0 noninfected seeds 1 seeds whose surface coveredby the fungus is less than 20 2 20ndash40 seed surfacecovered by the fungus 3 40ndash60 seed surface covered bythe fungus 4 60ndash80 seed surface covered by the fungusand 5 80ndash100 seed surface covered by the fungus eseverity calculation based on Tonapi et al [15] formula wasas follows

severity() 1113936

ni1 Ni times i( 1113857

total of seeds times(nminus 1) (2)

where plt 0001 i is severity scale from 0 to 5 and Ni is thenumber of seed corresponding to scale i of severity

25 Data Analysis Data analysis was performed with theopen-source statistical software R version 345 [16] De-scriptive statistics of recorded data were generated withpastecs package [17] In order to find out variability ofincidence and severity according to tested genotypes datawere subjected to Poisson regression analysis using glm(generalized linear model) function of package statsimplemented in the R Spearmanrsquos rank correlation test wasperformed to highlight relationship between incidence se-verity and aflatoxin concentration levels using correlationtest function of package stats Identification of differentgroups of genotypes based on incidence and severity wasperformed based on a principal component analysis anda hierarchical clustering with the functions PCA and HCPCof package FactoMineR [18] respectively e Euclideandistance and Ward classification method were used toclassify tested genotypes e function fviz_pca_biplot [19]was used to plot the principal components analysis biplot indifferent clusters based on hierarchical classification

3 Results

31 Reaction of Peanut Genotypes to Aspergillus flavusAnalysis of variance revealed highly significant (plt 0001)variation of aflatoxin incidence and severity among thetested peanut genotypes (Table 2)

e severity ranged between 0 and 44 respectivelywith a mean of 882plusmn 045 e recorded incidence rangedfrom 0 to 70 with an average value of 2036plusmn 080(Table 3)

One genotype (12CS_104) showed aflatoxin concen-tration level less than 4 ppb A total of 34 genotypes

presented aflatoxin concentration level up to 2000 ppb(Figure 1)

Out of the 67 genotypes eight showed incidence lessthan 10 while 33 showed incidences between 10 and 20and 16 with incidences ranged from 20 to 30 (Figure 2)

32 Correlation between Incidence Severity and AflatoxinConcentration Level Spearmanrsquos rank correlation testrevealed a strong relationship (r 093 plt 0001) betweenincidence and severity of peanut genotypes Positive andsignificant correlations were detected between aflatoxinconcentration levels and disease incidence (r 028plt 001) and aflatoxin concentration levels and diseaseseverity (r 035 plt 005) (Table 4)

33 Classification of the Tested Genotypes according to Sen-sibility and Aflatoxin Concentration Level e factorial axes1 and 2 explained 605 and 395 of overall variabilityrespectively (Figure 3) Hierarchical classification performedon principal component analysis revealed three clusters ofgenotypes based on disease incidence and aflatoxin con-centration levels (Figure 3) e clusters 1 2 and 3 grouped33 20 and 14 genotypes respectively e incidence andaflatoxin concentration are significantly (plt 0001) associ-ated to cluster 1 (Table 4)

Mean values of these two variables in this cluster are lessthan the overall mean erefore cluster 1 is characterizedby desirable genotypes which combine low incidence valuesand aflatoxin concentration levels Cluster 2 is significantly(plt 0001) related to the aflatoxin concentration level(Table 5)

e mean value of aflatoxin concentration in cluster 2(40755 ppb) is 190 which is higher than the overall mean(21438 ppb) us this second cluster is characterized bygenotypes with high level of aflatoxin Incidence is linked tocluster 3 (Table 5) Mean value of this variable (35) incluster 3 is superior to overall mean (2035) us thecluster 3 encompasses the most susceptible genotypes to Aflavus

Based on the closest distance between each genotype andthe respective cluster centres 12CS_039 12CS_010 and12CS_050 were the first representative genotypes (paragon)of cluster 1 2 and 3 respectively (Table 4) Based on thefarthest distance from a genotype projected point in a clusterto the centres of the two others clustering revealed thatcluster 1 2 and 3 were characterised by the genotypes12CS_104 78-936 and 12CS_021 respectively (Figure 3Table 5) Based on results out of 67 genotypes 33 promisinggenotypes (cluster 1) were noted (Figure 3)

Table 2 Deviance values from the Poisson regression model onincidence and severity

Source of variation Degree of freedom Incidence SeverityReplication 4 339 (ns) 204 (ns)Genotype 66 72422lowastlowastlowast 19008lowastlowastlowastlowastlowastlowastSignificant chi-squared test at 0001 level of probability nsnotsignificant

Table 1 Continued

No Genotypes Description Country of origin62 73-30 CSL Senegal63 73-33 Resistant control Senegal64 78-936 Variety Senegal65 Fleur11 Susceptible control Senegal66 GC-8-35 Variety Senegal67 L27 Variety SenegallowastChromosomal substitution lines

International Journal of Agronomy 3

4 Discussion

In the present study a wide phenotypic variation was ob-served among the tested genotypes for incidence severityand aflatoxin concentrationsis variation can be explainedby the variability of seed coat structure of the tested ge-notypes In fact the seed coat can constitute a barrier to Aflavus seed invasion depending on its thickness andorpermeability [20] and Zhou and Liang [21] studiesshowed that genotypes seed coat with smaller hilum morecompact arrangement and thicker testa showed more re-sistance to A flavus In addition implication of wax andcutin layers of seed coat was demonstrated to be related togenotypes resistance [22] Another explanation of this widevariation in incidence severity and aflatoxin rate can bebiochemical compoundsrsquo differential variability in the testedseeds Lindsey and Turner [23] demonstrated that thepresence of polyphenol compounds specifically tannins inseed can have inhibitor effect against A flavus Amaya et al[24] and Liang et al [25] showed the difference among seedcoat biochemical compounds to determine sensibility to Aflavus Liang [22] demonstrated that the presence of trypsinin seeds can also be related to resistance to A flavus Turneret al [26] isolated and identified the 57-dimethoxyiso-flavone as an inhibitor for A flavus invasion in peanut seed

12CS_104 was the most resistant genotype to aflatoxincontamination with an aflatoxin level lower than the Eu-ropean Union standards (4 ppb) However except12CS_104 all the genotypes have their aflatoxin concen-tration level higher than the Chinese (20 ppb) standards

Table 3 Means of incidence and severity of the tested lines

LinesIncidence Severity

Mean Std deviation Mean Std deviation12CS_001 44 1342 102 04412CS_004 30 1225 074 02312CS_006 30 1581 046 02912CS_007 18 837 034 02112CS_008 12 1643 022 02712CS_009 24 894 058 03312CS_010 18 837 04 02712CS_011 18 1483 042 04812CS_012 32 1304 082 04312CS_016 34 894 082 05412CS_018 38 1304 066 03312CS_020 22 1095 05 03412CS_021 52 1095 164 04912CS_022 16 1517 036 04312CS_023 18 1304 028 02412CS_024 28 1095 05 02112CS_027 18 837 026 01912CS_028 18 837 03 02012CS_031 16 548 04 02812CS_032 14 1140 016 01112CS_033 10 1225 02 02312CS_034 14 1517 028 03412CS_036 14 548 026 01512CS_037 16 548 032 01912CS_039 14 894 022 02312CS_041 24 1517 064 03812CS_042 24 1817 042 04212CS_047 18 1304 04 02212CS_048 6 894 008 01312CS_050 34 1140 076 02912CS_051 34 2074 072 03612CS_052 30 1581 076 06712CS_053 36 1517 098 05912CS_054 26 1140 068 04412CS_055 18 837 048 05412CS_059 24 1673 048 04812CS_060 12 1304 04 03912CS_061 14 1140 024 01912CS_062 18 837 026 01512CS_063 18 1483 042 05312CS_066 32 2280 08 07612CS_070 22 1924 056 05312CS_072 14 1342 026 02812CS_075 28 1304 066 03612CS_076 12 447 018 00812CS_078 10 1225 016 01812CS_079 18 1643 034 03412CS_084 14 548 03 02112CS_085 22 837 05 03212CS_090 14 1342 034 05012CS_091 26 1140 064 04812CS_095 12 837 026 02712CS_096 16 894 048 05812CS_100 40 1225 096 03412CS_104 6 894 014 02612CS_111 6 894 01 01412CS_112 18 1643 026 03212CS_118 20 1000 03 02012CS_119 20 707 042 028

Table 3 Continued


Mean Std deviation Mean Std deviation55-33 4 548 004 00555-437 12 1095 018 01973-30 6 548 006 00573-33 16 1140 028 01978-936 12 447 024 031Fleur_11 28 1643 05 017GC-8-35 22 1095 06 024L27 10 000 016 013Mean 2036 882Standard error 080 045

Table 4 Spearmanrsquos rank matrix correlation performed on in-cidence severity and aflatoxin concentration levels data

Incidence Severity Aflatoxinconcentration levels

Incidence 100Severity 093lowastlowastlowast 100Aflatoxinconcentration levels 028lowast 035lowastlowast 100

lowastSignificant Spearmanrsquos rank correlation test at 005 level of probabilitylowastlowastSignificant Spearmanrsquos rank correlation test at 001 level of probabilitylowastlowastlowastSignificant Spearmanrsquos rank correlation test at 0001 level of probability


Indeed the highest aatoxin concentration level was ob-served with genotype 78-936 e contrasting genotypesobserved in this study can be used as positive and negativechecks respectively for accurate eld experiment Fur-thermore these contrasted genotypes can be used to developmapping population for genetic study such as inheritance ofaatoxin and identication of quantitative trait loci (QTL)e varieties 55-437 and 73-30 showed incidence less than15 as reported by the previous study realized 30 years agoby Zambettakis et al [13] but their aatoxin concentrationlevels were largely up to the European Union standards

e correlation test showed a positive relationship be-tween A avus colonization and aatoxin contamination

is conrmed that the presence of A avus induced af-latoxin production in seeds Hierarchical classicationhighlighted three clusters according to incidence severityand aatoxin concentration levels e relatively low valuesof incidence observed on the 33 genotypes belonged tocluster 1 should be conrmed under eld conditions esegenotypes can be evaluated in dierent locations on infestedelds

5 Conclusion

is study uncovered that the lines 12CS_104 exhibited lowvalues of incidence and severity Furthermore its aatoxin

0

5

10

15

20

25

4 ppble Afla 4 ppb lt Afla le20 ppb

20 ppb lt Afla le1000 ppb

1000 ppb ltAfla le 2000

ppb


ppb

Afla gt 4000 ppb

Num

ber o

f gen

otyp

es

Figure 1 Number of peanut genotypes according to aatoxin concentration level interval values

0

5

10

15

20

25

30

35

10 le Inc 10 lt Inc le20

20 lt Inc le30

30 lt Inc le40

40 lt Inc le50

Inc gt 50

Num

ber o

f gen

otyp

es

Figure 2 Number of peanut genotypes showing aatoxin presence according to incidence intervals


concentration level was smaller than standards is ge-notype represents a relevant tool for the breeding pro-gram for resistance to A avus as a potentially resistantgene donor

Data Availability

e data used to support the ndings of this study areavailable from the corresponding author upon request

73ndash3055ndash33

L27

78ndash936

55ndash437

12CS_031 12CS_02812CS_01112CS_076

12CS_02212CS_03412CS_010

12CS_031 12CS_02712CS_091

12CS_05212CS_05312CS_075

12CS_02412CS_085

12CS_04112CS_012

12CS_051

12CS_016

12CS_050

12CS_021

12CS_00112CS_100

12CS_018

12CS_006

12CS_004

12CS_05912CS_110

12CS_042

12CS_009

12CS_018

12CS_06312CS_055

12CS_070

12CS_06612CS_054

12CS_09612CS_07912CS_036

12CS_08412CS_072

12CS_07812CS_048

12CS_06012CS_095

12CS_00812CS_039 12CS_047

12CS_11212CS_007

12CS_03712CS_062

12CS_061

12CS_03212CS_104

12CS_11112CS_033

12CS_09073ndash33

GCndash8ndash3512CS_020

Fleur11

ndash2 0 2

Dim

2 (3

95

)

ndash3

ndash2

ndash1

0

1

2

Group 1

Group 1

Group 1

Dim1 (605)

Figure 3 Principal component analysis biplot showing peanut genotypes clustering based on their severity and incidence

Table 5 Description of each cluster based on incidence aatoxin concentration levels and paragon and singular genotype per cluster

Cluster 1 Cluster 2 Cluster 3

Mean Overallmean p valuedagger Mean Overall

mean p value Mean Overallmean p value

Description of clusters by variablesIncidence 1509 2035 lt10minus3 188 2035 gt005 3500 2035 lt10minus3Aatoxin 91565 214389 lt10minus3 407550 214389 lt10minus3 227957 214389 gt005Description of clusters by distancesTop 3 representativegenotypesdaggerdagger

12CS_039(011)

12CS_090(016)

73-33(029)

12CS_010(012)

12CS_079(022)

12CS_027(024)

12CS_050(015)

12CS_066(041)

12CS_016(042)

Top 3 characteristicgenotypesdaggerdaggerdagger

12CS_104(282)

55-33(275)

12CS_111(274)

78-936(368)

12CS_028(239)

12CS_031(228)

12CS_021(345)

12CS_001(300)

12CS_100(259)

daggerCorrelation signication of variables with a cluster daggerdagger Based on the closest distance between each genotype and the respective cluster centres daggerdaggerdagger Based on thefarthest distance from a genotype projected point in a cluster to the centres of the two others


Conflicts of Interest

e authors declare no conflicts of interest regarding thepublication of this paper

Acknowledgments

e authors sincerely thank the West Africa AgriculturalProductivity Program (WAAPP) for financial support andfacilities help

References

[1] ANSD Bulletin Mensuel des Statistiques Economiques ANSDDakar Senegal 2017

[2] D Clavel A Da Sylva O Ndoye andAMayeux ldquoAmeliorationde la qualite sanitaire de lrsquoarachide au Senegal un challengepour une operation de recherche developement participativerdquoCahiers Agricultures vol 22 no 13 pp 174ndash81 2013

[3] P M Diedhiou F Ba A Kane and N Mbaye ldquoEffect ofdifferent cooking methods on aflatoxin fate in peanutproductsrdquo African Journal of Food Science and Technologyvol 3 no 12 pp 53ndash58 2012

[4] W A Korani Y Chu C Holbrook J Clevenger and P OsiasAkins ldquoGenotypic regulation of aflatoxin accumulation but notAspergillus fungal growth upon post-harvest infection of peanut(Arachis hypogaea L) seedsrdquo Toxins vol 9 no 7 p E218 2017

[5] J C Fountain J Koh L Yang et al ldquoProteome analysis ofAspergillus flavus isoate-specific responses to oxidative stressin relationship to aflatoxin production capabilityrdquo ScienticReports vol 8 no 1 p 3430 2018

[6] P M Diedhiou R Bandyopadhyay J Atehnkeng andP S Ojiambo ldquoAspergillus colonization and aflatoxin con-tamination of maize and sesame kernels in two agro-ecoloicalzones in Senegalrdquo Journal of Phytopathology vol 159 no 4pp 268ndash275 2011

[7] X Q Liang M Luo and B Z Guo ldquoResistance mechanismsto Aspergillus flavus infection and aflatoxin contamination inpeanut (Arachis hypogea L)rdquo Plant Pathology Journal vol 5no 11 pp 115ndash124 2006

[8] S Amaike and N P Keller ldquoAspergillus flavusrdquo AnnualReview of Phytopathology vol 49 no 1 pp 107ndash133 2011

[9] J H Williams T D Phillips P Jolly J K Styles C M Jollyand D Aggarwal ldquoHuman aflatoxicosis in developingcountries a review of toxicology exposure potential healthconsequences and interventionsrdquo American Journal ofClinical Nutrition vol 80 no 5 pp 1106ndash1122 2004

[10] Y C Chen C D Liao H Y Lin L C Chiueh andD Y C Shih ldquoSurvey of aflatoxin contamination in peanutproucts in Taiwan from 1997 to 2011rdquo Journal of Food andDrug Analysis vol 21 no 3 pp 247ndash252 2013

[11] R Y KelleyW PWilliams J E Mylroie et al ldquoIdentificationof maize genes associated with host plant resistance or sus-ceptibility to Aspergillus flavus infection and aflatoxin accu-mulationrdquo PLoS ONE vol 7 no 5 Article ID e36892 2012

[12] H Q Xue T G Isleib G A Payne and G OBrian ldquoEval-uation of post-harvest aflatoxin production in peanutgermplasm with resistance to seed colonization and pre-harvest aflatoxin contaminationrdquo Peanut Science vol 31no 2 pp 124ndash134 2004

[13] C Zambettakis F Waliyar A Bockelee-Morvan andO de Pins ldquoResults of four years of research on resistance ofgroundnut varieties to Aspergillus flavusrdquo Oleagineux vol 36pp 377ndash385 1981

[14] D Fonceka H A Tossim R Rivallan et al ldquoConstruction ofchromosome segment substitution lines in peanut (Arachishypogea L) using a wild synthetic and QTL mapping for plantmorphologyrdquo Plos ONE vol 7 no 11 Article ID e48642 2012

[15] V K Mehan and D McDonald Screening for Resistance toAspergillus Invasion and Aflatoxin Production in GroundnutsICRISAT Groundnut Improvement Program OccasionalPaper2 Patancheru India 1980

[16] V A Tonapi R R Mundada S S Navi et al ldquoEffect oftemperature and humidity regimes on grain mold sporulationand seed quality in sorghum (Sorghum bicolor (L) Moench)rdquoArchives of Phytopathology and Plant Protection vol 40 no 2pp 113ndash127 2007

[17] R Core Team A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2018

[18] P Grosjean and F Ibanez PASTECS Package for Analysis ofSpace Time Ecological Series R package version 13-18 2014

[19] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25no 1 pp 1ndash18 2008

[20] A Kassambara and F Mundt factoextra Extract and visualizethe results of multivariate data analyses R package version104 2017

[21] R A Taber R E Pettit C R Benedict J W Dieckert andD L Kertrin ldquoComparison of Aspergillus flavus tolerant andsusceptible lines I Light microscope investigationrdquo in Pro-ceedings of the American Peanut Research Education Associ-ation vol 5 pp 206-207 Oklahoma USA 1973

[22] G Y Zhou and X Q Liang ldquoStudies on the ultramicroscopicstructure of seed coats between resistant and susceptible toAspergillus flavus invasion in peanutrdquo Chinese Journal of OilCrop Sciences vol 20 pp 32ndash35 1999

[23] X Q Liang G Y Zhou and R Z Pan ldquoStudy on the re-lationship of wax and cutin layers in peanut seeds and re-sistance to invasion and aflatoxin production by Aspergillusflavusrdquo Journal Of Tropical And Subtropical Botany vol 11pp 11ndash14 2003

[24] D L Lindsey and R B Turner ldquoInhibition of growth ofAspergillus flavus and Trichoderma viride by peanut em-bryosrdquo Mycopathologia vol 55 no 3 pp 149ndash152 1975

[25] F J Amaya C T Young A J Norden and A C MixonldquoChemical screening for Aspergillus flavus resistance inpeanutrdquo Oleagineux vol 35 no 5 pp 255ndash259 1980

[26] X Q Liang G Y Zhou and R Z Pan ldquoChanges of somebiochemical substances in peanut seeds under infection of As-pergillus flavus and their role in resistance to seed invasionrdquoChinese Journal of Oil Crop Sciences vol 23 no 2 pp 26ndash31 2001


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host genetic resistance were tested [12] In Senegal previousstudies reported that varieties 55-437 and 73-3 were resistantto A flavus [13] Identification of new sources of resistancemerits to be investigated for efficient peanut breedingprogram First step of host genetic resistance is the seedcolonization test erefore the present study was un-dertaken to identify promising peanut genotypes underlaboratory conditions

2 Materials and Methods

21 Plant Materials e plant material consisted of 67genotypes including 58 chromosomal substitutions lines[14] and nine national released varieties e chromosomalsubstitution lines belong to a cross between Fleur 11 anda synthetic amphidiploid parent (Table 1)

22 Isolation of Aspergillus flavus Sporangial SuspensionPreparation and Inoculation Aflatoxinogenic strain pro-vided from peanut seeds were purified by successive cultureson 52 agar medium e aflatoxin concentration level waschecked using the Revealreg Q+ Aflatoxin test kit (accessopeanut enterprise corporation USA) e spore suspensionofA flavuswas obtained by soaking colonized seeds in 50mlof sterile distilled water en one drop of Tween 20 wasadded to the solution and thoroughly mixed for 10 minutesInoculation was carried out by introducing 100 μl of thesupernatant of the spore suspension into each Petri dish

23 Seed Colonization Test e seed colonization test wasconducted following a modified Mehan and McDonaldprocedure For each genotype 50 seeds were sterilized andrinsed properly in sterile distilled wateren the seeds werehydrated to about 20 moisture content e 50 seeds ofeach genotype were placed in 5 Petri dishes containing 10seeds and each Petri dish was considered as a replicatione seeds were inoculated with a conidial suspension (60 microLcontaining approximately 1times 108mLminus1 conidia of the afla-toxigenic strain of A flavus) is preparation was kept atlaboratory conditions (25plusmn 012degC and 82plusmn 042 relativehumidity) for fifteen days

24 Data Collection e seedsrsquo colonization was observedduring two weeks and aflatoxin concentration was mea-sured using the Revealreg Q+ Aflatoxin test kit (accesso peanutenterprise corporation USA) e incidence was calculatedusing the following formula

incidence()

number of seeds showing pathogen colonizaton

total number of seeds

times 100

(1)

Table 1 Peanut material used in this study

No Genotypes Description Country of origin1 12CS_001 CSLlowast Senegal2 12CS_004 CSL Senegal3 12CS_006 CSL Senegal4 12CS_007 CSL Senegal5 12CS_008 CSL Senegal6 12CS_009 CSL Senegal7 12CS_010 CSL Senegal8 12CS_011 CSL Senegal9 12CS_012 CSL Senegal10 12CS_016 CSL Senegal11 12CS_018 CSL Senegal12 12CS_020 CSL Senegal13 12CS_021 CSL Senegal14 12CS_022 CSL Senegal15 12CS_023 CSL Senegal16 12CS_024 CSL Senegal17 12CS_027 CSL Senegal18 12CS_028 CSL Senegal19 12CS_031 CSL Senegal20 12CS_032 CSL Senegal21 12CS_033 CSL Senegal22 12CS_034 CSL Senegal23 12CS_036 CSL Senegal24 12CS_037 CSL Senegal25 12CS_039 CSL Senegal26 12CS_041 CSL Senegal27 12CS_042 CSL Senegal28 12CS_047 CSL Senegal29 12CS_048 CSL Senegal30 12CS_050 CSL Senegal31 12CS_051 CSL Senegal32 12CS_052 CSL Senegal33 12CS_053 CSL Senegal34 12CS_054 CSL Senegal35 12CS_055 CSL Senegal36 12CS_059 CSL Senegal37 12CS_060 CSL Senegal38 12CS_061 CSL Senegal39 12CS_062 CSL Senegal40 12CS_063 CSL Senegal41 12CS_066 CSL Senegal42 12CS_070 CSL Senegal43 12CS_072 CSL Senegal44 12CS_075 CSL Senegal45 12CS_076 CSL Senegal46 12CS_078 CSL Senegal47 12CS_079 CSL Senegal48 12CS_084 CSL Senegal49 12CS_085 CSL Senegal50 12CS_090 CSL Senegal51 12CS_091 CSL Senegal52 12CS_095 CSL Senegal54 12CS_096 CSL Senegal55 12CS_100 CSL Senegal56 12CS_111 CSL Senegal57 12CS_112 CSL Senegal58 12CS_118 CSL Senegal59 12CS_119 CSL Senegal60 55-33 Variety Senegal61 55-437 Resistant control Senegal



severity() 1113936





3 Results













Table 1 Continued



4 Discussion






Table 3 Continued











5 Conclusion


0

5

10

15

20

25




ppb


ppb

Afla gt 4000 ppb

Num

ber o

f gen

otyp

es


0

5

10

15

20

25

30

35


20 lt Inc le30

30 lt Inc le40

40 lt Inc le50

Inc gt 50

Num

ber o

f gen

otyp

es




Data Availability


73ndash3055ndash33

L27

78ndash936

55ndash437

12CS_031 12CS_02812CS_01112CS_076

12CS_02212CS_03412CS_010

12CS_031 12CS_02712CS_091

12CS_05212CS_05312CS_075

12CS_02412CS_085

12CS_04112CS_012

12CS_051

12CS_016

12CS_050

12CS_021

12CS_00112CS_100

12CS_018

12CS_006

12CS_004

12CS_05912CS_110

12CS_042

12CS_009

12CS_018

12CS_06312CS_055

12CS_070

12CS_06612CS_054

12CS_09612CS_07912CS_036

12CS_08412CS_072

12CS_07812CS_048

12CS_06012CS_095

12CS_00812CS_039 12CS_047

12CS_11212CS_007

12CS_03712CS_062

12CS_061

12CS_03212CS_104

12CS_11112CS_033

12CS_09073ndash33


Fleur11

ndash2 0 2

Dim

2 (3

95

)

ndash3

ndash2

ndash1

0

1

2

Group 1

Group 1

Group 1

Dim1 (605)







12CS_039(011)

12CS_090(016)

73-33(029)

12CS_010(012)

12CS_079(022)

12CS_027(024)

12CS_050(015)

12CS_066(041)

12CS_016(042)


12CS_104(282)

55-33(275)

12CS_111(274)

78-936(368)

12CS_028(239)

12CS_031(228)

12CS_021(345)

12CS_001(300)

12CS_100(259)





Acknowledgments


References





























Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy





severity() 1113936





3 Results













Table 1 Continued



4 Discussion






Table 3 Continued











5 Conclusion


0

5

10

15

20

25




ppb


ppb

Afla gt 4000 ppb

Num

ber o

f gen

otyp

es


0

5

10

15

20

25

30

35


20 lt Inc le30

30 lt Inc le40

40 lt Inc le50

Inc gt 50

Num

ber o

f gen

otyp

es




Data Availability


73ndash3055ndash33

L27

78ndash936

55ndash437

12CS_031 12CS_02812CS_01112CS_076

12CS_02212CS_03412CS_010

12CS_031 12CS_02712CS_091

12CS_05212CS_05312CS_075

12CS_02412CS_085

12CS_04112CS_012

12CS_051

12CS_016

12CS_050

12CS_021

12CS_00112CS_100

12CS_018

12CS_006

12CS_004

12CS_05912CS_110

12CS_042

12CS_009

12CS_018

12CS_06312CS_055

12CS_070

12CS_06612CS_054

12CS_09612CS_07912CS_036

12CS_08412CS_072

12CS_07812CS_048

12CS_06012CS_095

12CS_00812CS_039 12CS_047

12CS_11212CS_007

12CS_03712CS_062

12CS_061

12CS_03212CS_104

12CS_11112CS_033

12CS_09073ndash33


Fleur11

ndash2 0 2

Dim

2 (3

95

)

ndash3

ndash2

ndash1

0

1

2

Group 1

Group 1

Group 1

Dim1 (605)







12CS_039(011)

12CS_090(016)

73-33(029)

12CS_010(012)

12CS_079(022)

12CS_027(024)

12CS_050(015)

12CS_066(041)

12CS_016(042)


12CS_104(282)

55-33(275)

12CS_111(274)

78-936(368)

12CS_028(239)

12CS_031(228)

12CS_021(345)

12CS_001(300)

12CS_100(259)





Acknowledgments


References





























Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy




4 Discussion






Table 3 Continued











5 Conclusion


0

5

10

15

20

25




ppb


ppb

Afla gt 4000 ppb

Num

ber o

f gen

otyp

es


0

5

10

15

20

25

30

35


20 lt Inc le30

30 lt Inc le40

40 lt Inc le50

Inc gt 50

Num

ber o

f gen

otyp

es




Data Availability


73ndash3055ndash33

L27

78ndash936

55ndash437

12CS_031 12CS_02812CS_01112CS_076

12CS_02212CS_03412CS_010

12CS_031 12CS_02712CS_091

12CS_05212CS_05312CS_075

12CS_02412CS_085

12CS_04112CS_012

12CS_051

12CS_016

12CS_050

12CS_021

12CS_00112CS_100

12CS_018

12CS_006

12CS_004

12CS_05912CS_110

12CS_042

12CS_009

12CS_018

12CS_06312CS_055

12CS_070

12CS_06612CS_054

12CS_09612CS_07912CS_036

12CS_08412CS_072

12CS_07812CS_048

12CS_06012CS_095

12CS_00812CS_039 12CS_047

12CS_11212CS_007

12CS_03712CS_062

12CS_061

12CS_03212CS_104

12CS_11112CS_033

12CS_09073ndash33


Fleur11

ndash2 0 2

Dim

2 (3

95

)

ndash3

ndash2

ndash1

0

1

2

Group 1

Group 1

Group 1

Dim1 (605)







12CS_039(011)

12CS_090(016)

73-33(029)

12CS_010(012)

12CS_079(022)

12CS_027(024)

12CS_050(015)

12CS_066(041)

12CS_016(042)


12CS_104(282)

55-33(275)

12CS_111(274)

78-936(368)

12CS_028(239)

12CS_031(228)

12CS_021(345)

12CS_001(300)

12CS_100(259)





Acknowledgments


References





























Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy







5 Conclusion


0

5

10

15

20

25




ppb


ppb

Afla gt 4000 ppb

Num

ber o

f gen

otyp

es


0

5

10

15

20

25

30

35


20 lt Inc le30

30 lt Inc le40

40 lt Inc le50

Inc gt 50

Num

ber o

f gen

otyp

es




Data Availability


73ndash3055ndash33

L27

78ndash936

55ndash437

12CS_031 12CS_02812CS_01112CS_076

12CS_02212CS_03412CS_010

12CS_031 12CS_02712CS_091

12CS_05212CS_05312CS_075

12CS_02412CS_085

12CS_04112CS_012

12CS_051

12CS_016

12CS_050

12CS_021

12CS_00112CS_100

12CS_018

12CS_006

12CS_004

12CS_05912CS_110

12CS_042

12CS_009

12CS_018

12CS_06312CS_055

12CS_070

12CS_06612CS_054

12CS_09612CS_07912CS_036

12CS_08412CS_072

12CS_07812CS_048

12CS_06012CS_095

12CS_00812CS_039 12CS_047

12CS_11212CS_007

12CS_03712CS_062

12CS_061

12CS_03212CS_104

12CS_11112CS_033

12CS_09073ndash33


Fleur11

ndash2 0 2

Dim

2 (3

95

)

ndash3

ndash2

ndash1

0

1

2

Group 1

Group 1

Group 1

Dim1 (605)







12CS_039(011)

12CS_090(016)

73-33(029)

12CS_010(012)

12CS_079(022)

12CS_027(024)

12CS_050(015)

12CS_066(041)

12CS_016(042)


12CS_104(282)

55-33(275)

12CS_111(274)

78-936(368)

12CS_028(239)

12CS_031(228)

12CS_021(345)

12CS_001(300)

12CS_100(259)





Acknowledgments


References





























Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy





Data Availability


73ndash3055ndash33

L27

78ndash936

55ndash437

12CS_031 12CS_02812CS_01112CS_076

12CS_02212CS_03412CS_010

12CS_031 12CS_02712CS_091

12CS_05212CS_05312CS_075

12CS_02412CS_085

12CS_04112CS_012

12CS_051

12CS_016

12CS_050

12CS_021

12CS_00112CS_100

12CS_018

12CS_006

12CS_004

12CS_05912CS_110

12CS_042

12CS_009

12CS_018

12CS_06312CS_055

12CS_070

12CS_06612CS_054

12CS_09612CS_07912CS_036

12CS_08412CS_072

12CS_07812CS_048

12CS_06012CS_095

12CS_00812CS_039 12CS_047

12CS_11212CS_007

12CS_03712CS_062

12CS_061

12CS_03212CS_104

12CS_11112CS_033

12CS_09073ndash33


Fleur11

ndash2 0 2

Dim

2 (3

95

)

ndash3

ndash2

ndash1

0

1

2

Group 1

Group 1

Group 1

Dim1 (605)







12CS_039(011)

12CS_090(016)

73-33(029)

12CS_010(012)

12CS_079(022)

12CS_027(024)

12CS_050(015)

12CS_066(041)

12CS_016(042)


12CS_104(282)

55-33(275)

12CS_111(274)

78-936(368)

12CS_028(239)

12CS_031(228)

12CS_021(345)

12CS_001(300)

12CS_100(259)





Acknowledgments


References





























Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy






Acknowledgments


References





























Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy





Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy




identificationofsourcesofresistanceforpeanut aspergillus...

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