prevalenceandphylogeneticdiversityofpathogenic fusarium

Research ArticlePrevalence and Phylogenetic Diversity of Pathogenic FusariumSpecies in Genotypes of Wheat Seeds in Three Rift ValleyRegions Kenya

Otieno P Kheseli 1 Imbahale S Susan 1 Okoth Sheila 2 Miriam Otipa3

and Wekesa V Wafula 4

1Department of Applied and Technical Biology Technical University of Kenya PO Box 52428ndash00200 Nairobi Kenya2School of Biological Sciences University of Nairobi PO Box 30197ndash00100 Nairobi Kenya3Kenya Agricultural Research and Livestock Organization Kabete PO Box 14733-00800 Nairobi Kenya4Flamingo Horticulture (K) Limited (Dudutech) PO BOX 1927ndash20117 Naivasha Kenya

Correspondence should be addressed to Otieno P Kheseli phanicekheseliyahoocom

Received 9 July 2020 Revised 23 December 2020 Accepted 8 January 2021 Published 30 January 2021

Academic Editor Jiban Shrestha

Copyright copy 2021 Otieno P Kheseli et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Wheat is a source of nutrients for around 40 worldrsquos population and the second most important cereal crop in Kenya HoweverFusarium head blight (FHB) hinders sustainable sufficient production of the crop causing both economic and health losses Withthe emerging unfavorable climatic changes effective disease management strategies and adequate seed system are necessary tomeet the deficiency Current information on prevalence of the causative pathogens in varieties of wheat genotypes is a criticalprerequisite to such strategies is study aimed at determining the prevalence of pathogenic Fusarium species in seeds ofdeveloped varieties of wheat genotypes in three major wheat-producing regions in Kenya A total of 260 samples of 18 wheatgenotypes from 123 farms were collected Peptone pentachloronitrobenze agar was used for fungal isolation while identificationof Fusarium spp was based on the gene encoding translation elongation factor 1-α (tef1-alpha) sequence analysis Fusarium sppisolated include Fusarium poae F tricinctum F heterosporum F culmorum F equiseti Fusarium sp F verticillioides andF oxysporum ere was no significant difference in prevalence of Fusarium spp pathogens among the three regions studiedFusarium spp diversity index for Nakuru was 2008 Narok was 14603 and Uasin Gishu was 12337 Wheat produce from farm-saved seeds yielded 6625 of the isolates while the produce from certified commercial wheat seeds yielded 3375 of the isolatese significant finding of the study is that Fusarium spp associated with mycotoxins that contaminate the wheat food chain seemto be flourishing in all the sampled wheat seed genotypes from the regions studied Information on the prevalence and diversity ofthe pathogens on persistence of the disease in the crop is critical in advancing integrative FHB control measures

1 Introduction

Fusarium spp is one of the most significant filamentouspathogenic fungal genera that is ubiquitous around theglobe ey are field or soil fungi causing wilts seedlingblights rots and cankers in susceptible plants [1] Fusariumspp contamination is a major agricultural problem thatreduces the quality and yield of agricultural products whileproducing mycotoxins that have virulence factors respon-sible for many diseases in humans and farm animals [2]

Wheat (Triticum aestivum) is a staple source of nutrients forapproximately 40 of the worldrsquos population and is the mostwidely grown crop in the world cultivated on more than 218million ha In addition its world trade is greater than allother crops combined [3 4] However infestation of fungalpathogens in the crop is a serious worldwide problem due tovarious factors Unfavorable climatic conditions andagronomy-related issues have been reported to be critical inthis respect [5ndash8] Pathogenic Fusarium spp is the maincause of Fusarium head blight (FHB) that causes devastating

HindawiAdvances in AgricultureVolume 2021 Article ID 8839147 13 pageshttpsdoiorg10115520218839147

wheat grain destruction with resultant accumulation ofmycotoxins

Globally various control measures are under investi-gation by researchers with the aim of effectively protectingthe crop from FHB is has involved the use of differentpre-harvest and post-harvest methods starting from agro-technical methods that limit the source of primary infectionPost-harvest control measures include physical methodssuch as proper harvesting that does not damage the grainscrop rotation tillage fertilization use of appropriate qualityseedmaterial and the timely sowing period [9 10] educationand training of agricultural producers in application of goodagricultural practices Although fungicide efficacy againstFHB has been inconsistent due to the short time frameworkfor application as well as limited number of available fun-gicides [10] their role in control of FHB cannot be ignoredamong pre-harvest control measures Chemical control ofFHB in wheat influences the disease intensity in spikes anddeoxynivalenol contamination in grains [11] Timely use ofthe correct dosage of the effective type of fungicides [12ndash14]has been reported to be an applicable FHB control measureespecially during plant flowering Also included in thiscategory is the use of resistance inducers including bio-preparations based on antagonistic microbial agents en-dophytes and other biologically active ingredients andnonchemical fungicides and plowing [15ndash18] Selection anduse of cultivars with a high level of resistance to Fusariumspp infestation [10 17] and to lodging [19] cannot beoveremphasized Use of a combination of preharvest mea-sures when conditions are favorable for Fusarium spp in-festation is more effective [9 20] since they limit the survivalof the pathogens in debris reducing their presence in fieldand infestation severity However the ever-changing farmand climatic conditions require application of consistenteffective FHB control measures such as the use of wheatgenotypes that are highly resistant to Fusarium spp path-ogens e current study investigated the prevalence anddiversity of pathogenic Fusarium spp in varieties of wheatgenotypes from three major wheat-producing regions in theKenyan Rift Valley in line with factors determining selectionof the cultivated genotypes

11 Background of the Study Wheat is Kenyarsquos second mostimportant cereal crop after maize and an economicallyimportant crop among the large and small-scale farmers[21] It is grown in areas above 1500 metres above sea levelin southern and upper Rift Valley regions (Nakuru Narokand Uasin Gishu) and in the eastern part of Kenya (Nanyukiand Meru) Wheat production is carried out by smallmedium and large-scale farmers and the industry supportedby about 20 millers contributes 14 and 30 to overall andcereal GDP [22] respectively e high costs of farm inputsand land fragmentation among other factors have led to ashift in production from large-scale and medium-scalecommercial farms using capital-intensive technology toproduction mostly by small-scale farmers [23]

Wheat requires a fine planting bed for uniform germi-nation In this view farmers are encouraged to have the land

thoroughly ploughed and harrowed free from growingweeds and weed seeds at least 4 weeks before planting toensure that the land does not have fresh compost (plantmaterial that is not fully rotten) during planting Fertilizerapplication is recommended during planting depending onthe soil type as per the soil analysis report During plantingdiammonium phosphate (DAP) at 200ndash250 kg Haminus 1 isrecommended as determined by factors such as the stage ofthe crop since availability of nitrogen is important duringtilling stem extension and ear emergence

Diseases pre-harvest and post-harvest losses are themajor recurring challenges hindering optimal production ofwheat Occurrence of wheat devastating fungal diseases suchas wheat rust [24] and FHB [25ndash27] has been reported as themajor setback to wheat production in Kenya Generallyother than good farm management practices the mainwheat crop protection regime used by farmers in Kenyaagainst all fungal foliar wheat diseases such as wheat rust isthe use of fungicides Foliar diseases can increase the sus-ceptibility of wheat spikes to FHB Consequently fungicidesbecome crucial in reducing the effects of the diseases onwheat However the low efficacy and limited spectrum ofsuch fungicides create a gap in control of resistant fungaldiseases such as FHB Although disease resistance or tol-erance remains a driving goal of Kenya Agricultural andLivestock Organizationrsquos (KALROrsquo) wheat research pro-gram it remains a challenge for the recommended wheatvarieties [21] Hence there is consistent development ofwheat genotypes with high stable yields tolerance to bioticand abiotic stresses pre-harvest sprouting diseases andinsect pests so that farmers from different growing regionsin the country can select the most suited variety [21]erefore current information on prevalence and diversityof Fusarium spp in the existing and cultivated wheat ge-notypes is important prerequisite information in integratedmanagement of FHB and the improvement or developmentof other genotypes with better disease resistant qualities ecurrent study evaluated the occurrence and diversity ofpathogenic Fusarium spp in the developed wheat genotypesin three major wheat-producing regions within the KenyanRift Valley and the factors considered by farmers in selectingthe cultivated wheat varieties

2 Materials and Methods

21 Study Area e study was carried out in three regionssituated within the Kenyan Rift Valley the main commercialwheat-growing zone Figure 1 shows the location of each sitegenerated using the geographical positioning system (GPS)e average rainfall range was between 20mmndash40mm inNarok 60mmndash80mm in Uasin Gishu and 40mmndash60mmin Nakuru whereas the average temperature range in Narokwas 18degCndash22degC in Usain Gishu lt18degC and 18degCndash22degC inNakuru during the study period ese areas have annualrainfall varying between 800mm and 2000mm with theamounts occasionally rising up to 2500mm in higher al-titudes Research on smallholder wheat technologies isperformed by the National Plant Breeding Station based inNakuru e commonly cultivated crops on large-scale

2 Advances in Agriculture

Narok

Kajiado

Nakuru

Baringo

Laikipia

Nandi

Bomet

NyeriNyandarua

Kisii

Uasin Gishu

Kiambu

Kericho

Kisumu

Kakamega

Bungoma

Muranga

Samburu

Nyamira

Nairobi

Homa Bay

Vihiga

Isiolo

Trans Nzoia

Kiyo-Marakwete

Machakos

Migori

Turkana

Key

Uasin Gishu GPS coordinatesNakuru GPS coordinatesNarok GPS coordinates

NakuruUasin GishuNarokCounty boundary

0 25 50 75125Km

N

KENYA

Figure 1 e location of each sampling site generated using the geographical positioning system (GPS)

Advances in Agriculture 3

farms in the study regions are wheat barley and maizeOther subsistence crops such as legumes (beans) cabbageskales and Irish potatoes are also cultivated on small-scalebasis Farms barely stay fallow for 1-2 years beforecultivation

22 Field Sampling ofWheat Seeds e sampling took placebetween September 2016 and October 2017 as followsNarok July 28th to early September 2016 Uasin Gishuregion November 8 2016 to early December 2016 Nakururegion Nakuru-Naivasha area February 2017 and Nakuru-Njoro area October 2017 In Nakuru region most of thesampling was carried out in Njoro the main wheat-pro-ducing and research centre Cross-sectional purposefulsampling was followed whereby farms were randomlyselected depending on the day of harvesting Laboratoryand file samples were taken from bulks of freshly harvestedwheat grains using spear sampling technique [28 29] enumber of samples collected per farm varied as determinedby farm size and ranged from 3 to 21 samples per site Eachwheat variety was sampled in replicates of three persampling site and every sample weighed approximately500 gm Samples were packed in khaki papers and labelledand transported in cooler boxes to the laboratory wherethey were stored at 4degC for isolation of Fusarium speciese sampling process included gathering information fromwheat farmers on the genotypes of wheat seeds cultivatedand criteria used in selection of the wheat genotypes theuse of farm-saved and certified commercial wheat seedsand last common fungal diseases observed on the crop inthe fields

23 Preparation of Wheat Samples for Isolation and Char-acterization of Fusarium spp To obtain representativesamples from multiple samples procedures described by theauthors of [28 29] were used but with slight modificationsMultiple samples from specific farms and sites were asep-tically poured into a sterile container (polythene paper bag)and manually mixed to make one homogenous compositesample e composite sample was then manually reducedinto portions of 500 gm and labelled and randomly selectedfor use as test and file samples Samples that weighed 500 gmfrom collection sites were used directly as test samples aftermixing e methods developed by the authors of [30 31]were adopted for use in the process of isolation and mor-phological characterization of Fusarium species Approxi-mately one hundred and fifty seeds of each test sample wererandomly picked and soaked in 2 of NaOCl for 2 minutese seeds were then rinsed three times each time in freshsterile distilled water and dried on sterile muslin cloth in alaminar flow Ten sterile seeds from each of the three rep-licates of every sample were randomly picked and plated intriplicates on selective media peptone penta-chloronitrobenze agar and incubated at 25degC for 4ndash7 daysese constituted 90 plated seeds for each sample Germi-nating fungal colonies were subcultured on tap water agar at25degC for a period of 7ndash14 days for sporulation Spores of eachisolate were examined using a microscope to ascertain

distinctive characteristic features unique to Fusarium spe-cies To obtain pure colonies spores of each Fusarium sppisolate were subjected to 10minus 4 fold serial dilution and cul-tured on water agar at 25degC for 18 hours Hyphal tips of eachfungal colony formed were subcultured in triplicates onthree different media potato dextrose agar (PDA) syntheticagar (SNA) and carnation leaf agar (CLA) under two dif-ferent temperatures 25degC and 30degC with alternating 12hours of darkness and 12 hours of fluorescent light is wascarried out for further morphological characterization fol-lowing procedures by the authors of [32 33] Pure Fusariumspp isolates were then stored as spore suspensions in 15glycerol at minus 80degC in Kenya Agricultural and Livestock Re-search Organization (KALRO) biotechnology laboratory formolecular characterization

24 Molecular Characterization of Fusarium spp

241 DNA Extraction Spores of each isolate were culturedon PDA at 25degC and alternating 12 hours of fluorescent lightand darkness DNA of each isolate was extracted betweenday 7 and day 14 depending on the quantity of mycelia usinga Zymo Research FungalBacterial DNA Mini Prep Kit(Epigenetics Hatfield South Africa) and according to themanufacturerrsquos instructions For optimal performance beta-mercaptoethanol (user supplied) was added to the genomiclysis buffer to a final dilution of 05 (vv) ie 500 microl per100ml In the first step approximately 10mgndash20mg (wetweight) of fungal cells that had been resuspended in up to200 microl of isotonic buffer (eg PBS) were added to a ZRBashingBeadtrade lysis tube (01mm and 05mm) and 750 microlBashingBeadtrade buffer was added to the tube before cappingtightly e preparation was then secured in a bead beaterfitted with a 2ml tube holder assembly and processed at amaximum speed of ge5 minutes Processing time variedbased on sample input e ZR BashingBeadtrade lysis tube(01mm and 05mm) was then centrifuged in a micro-centrifuge at 10000 x g for 1 minute Up to 400 microl of thesupernatant was transferred to the Zymo-Spintrade III-F filterin a collection tube and centrifuged at 8000 x g for 1 minuteand the Zymo-Spintrade III-F filter was discarded Next 1200 microlof genomic lysis buffer was then added to the filtrate in thecollection tube and mixed well 800 microl of the mixture wasadded to a Zymo-Spintrade IC column 2 in a collection tube andcentrifuged at 10000 x g for 1 minute e flow through fromthe collection tube was discarded and the previous step (step6) was repeated 200 microl DNA pre-wash buffer was then addedto the Zymo-Spintrade IC column in a new collection tube andcentrifuged at 10000 x g for 1 minute 500 microlmiddotgminus 1 DNA WashBuffer was added to the Zymo-Spintrade IC column andcentrifuged at 10000 x g for 1 minute e Zymo-Spintrade ICcolumnwas then transferred to a clean 15mlmicrocentrifugetube and 20 microl (10 microl minimum) of DNA elution buffer wasadded directly to the column matrix and incubated for 1minute is was followed by centrifugation at 10000 x g for30 seconds to elute the DNA using a Zymo Research FungalBacterial DNA Mini Prep Kit (Epigenetics Hatfield SouthAfrica) and according to the manufacturerrsquos instructions


242 Polymerase Chain Reaction (PCR) Amplification ofFusarium spp DNA A set of primers targeting the geneencoding translation elongation factor 1-α (tef1-α)- Ef1(forward primer 5rsquo-ATGGGTAAGGA (AG) GACAA-GAC-3prime) and EF2 (reverse primer 5rsquo-GGA (GA) GTAC-CAGT (GC) ATCATGTT-3prime) was used to amplify 700 bpfragment [34] e PCR was performed in 25 μl reactionswhich included 1 μl of a 1 10 or 1 100 DNA dilution 1 URedTaq DNA polymerase (SigmandashAldrich Company MilanItaly) 2 μl RedTaq buffer supplemented with 17 μl of 22mMMgCl2 for a final concentration of 30mM 10 mM de-oxyribonucleotide) and 10 μM of each primer reverse andforward (ef1 and ef2) Reactions were run in a Mastercyclerep-gradient (BioRad California USA) with a thermal profileof 4min at 94degC followed by 35 cycles of 60 s at 94degC 60 s at57degC and 1min at 72degC followed by 72degC for 5min eamplified DNA was electrophoresed in 15 (wv) Tris-acetate ethylene diamine tetraacetic acid (EDTA) agarosegels Amplicons were visualized at 700 bpndash710 bp using a1 kb bp DNA ladder (Bioline) as a size standard A con-centration of 1 agarose gel was used To make the gel theagarose was boiled at 100degC for 5 minutes in a conical flaskand left to cool to 55degCen 03 μl of ethidium bromide wasadded while swirling the flask to enable the gel mix withethidium bromide e mixture was poured into a gel tankwith the combs on and left to solidify Molecular marker(2 μl) was added to one well and DNA (4 μl) plus the loadingdye was added to other wells and the arrangement was notedDistilled water was used as a control e gel was run for 45minutes at 80V and viewed under Geldoc (BioRad Mo-lecular Imager Gel Doc XR-CLASS Imaging System Cal-ifornia and USA) A qPCR to estimate the severity ofinfection for each given Fusarium spp per sample was notperformed since our interest for DNA analysis was to as-certain the species identity of the isolates

243 Nucleotide Sequencing and Analysis of tef1-α Nucle-otide Sequences Amplification products of TEF-1 gene werepurified using a QIAquick Gel Extraction Kit according tomanufacturerrsquos instructions (Qiagenmanual) Sequencing ofboth strands was performed in the ABI 3700 DNA Se-quencer in the Sequencing Service Unit of Macrogen(Netherlands) e ef1-α (tef1-α) raw nucleotide sequenceswere assembled contigs generated and consensus sequencesretrieved using Geneious version 1115 software BasicAlignment Search Tool for Nucleotide Sequences(BLASTN) httpsblastncbinlmnihgovBlastcgito wasused in retrieving sequences with high similarity index withthose from National Centre for Biotechnology Informatics(NCBI) database e retrieved sequences were alignedtogether with the consensus sequences of the isolates usingClustalW a matrix-based algorithm build in Geneiousversion 1115 software A phylogenetic tree was generated toinfer relatedness of the aligned 49 sequences Maximumlikelihood (ML) analysis was performed and a phylogenetictree (Figure 2) was built through the TamurandashNei modelOne thousand bootstrap replicates were performed for as-surance of stability and robustness of each of the branch

[35 36] e tree with the highest log likelihood (minus 3127016)was shown e percentage of trees in which the associatedtaxa clustered together was shown next to the branchesInitial trees for the heuristic search were obtained auto-matically by applying Neighbor-Join and BioNJ algorithmsto a matrix of pairwise distances estimated using themaximum composite likelihood (MCL) approach and thenselecting the topology with the superior log likelihood valueere were 4167 positions in the final dataset Evolutionaryanalysis was conducted in MEGA X [35]

25 Data Analysis on Prevalence and Diversity of Fusariumspp Data was analyzed in SPSS using one-way ANOVA tocompare prevalence of each of the Fusarium spp isolatede analysis was done with respect to overall occurrence andtypes of Fusarium spp in each of the sampled wheat ge-notypes among the three regions at 005 significance levelFusarium spp with significantly different means among theregions were subjected to further analysis using the LSD testfor multiple comparisons to identify any differences inoverall occurrences between any two regions To determinespecies richness and their relative abundance in each regiondiversity indices were computed using Simpsonrsquos andShannonrsquos diversity equations Evaluation of the prevalenceof Fusarium spp between wheat seeds arising from farm-saved seeds and certified commercial seeds was also per-formed using descriptive statistics

Simpsonrsquos diversity index (D) is given by

D 1 minus 1113944 pi( 11138572 (1)

While Shannonrsquos Weiner diversity index (H) is given by

H minus 1113944 pi lnpi (2)

where ldquoDrdquo stands for Simpsonrsquos diversity index ldquoHrdquo standsfor Shannonrsquos diversity index ldquo1113936rdquo is the sum of and ldquopirdquo isthe relative abundance of each species and ldquolnrdquo is the naturallogarithm

3 Results

31 Wheat Genotype Preference by Farmers in the Rift ValleyTwo hundred and sixty (260) samples of different genotypesof wheat seeds (Table 1) were collected from 123 wheatfarms Eighteen wheat seed genotypes were classified out ofthe 260 samples of wheat seeds collected Sixty-six percent(6625) was the produce of certified commercial wheatseeds and 3375was the produce of farm-saved wheat seeds(Table 2) Agro-economic factors that determined farmersrsquochoice of wheat seed genotype to cultivate varied frompotential weight of wheat yield resistance of wheat grains topests in storage affordability of wheat seed genotype re-sistance of wheat genotype to wheat rust availability of theseeds of genotypes released in the market and free wheatseed genotypes offered for research purposes or for pro-duction of certified wheat seeds Only 16 of the farmerswhose wheat seeds were sampled cited FHB as a seriousproblem that affects the crop However the disease effects


IV

III

II

I

Fusarium equiseti

Fusarium oxysporum

Outgroup to node IV

Fusarium equiseti

Fusarium verticillioides

Fusarium equiseti

Outgroup to nodes I and II

Fusarium poae


Fusarium heterosporum

Fusarium tricinctum

Fusarium culmorum

Fusarium sp (unitalize)

Fusarium equiseti

Fusarium equiseti

10098

98100100

100

100

8478

96

80

64

94

84

100 100

100

100

100

100

74

42

9850

28

9210

6

050

20

18

76

82

100

PT189 ef NarokPT89 ef Nakuru

PT25 ef Narok

PT74 ef Narok

PT42 ef Narok

PT71 ef Narok

PT15 ef Uasin Gishu

PT23 ef Uasin GishuPT61 ef Uasin Gishu

PT195 ef Uasin Gishu


PT53 ef Uasin Gishu

PT20 ef Uasin Gishu

PT70 ef Uasin Gishu



PT54 ef NarokPT36 ef Narok

PT46 ef Narok

PT12 ef Narok

PT10 ef Nakuru

PT34 ef Nakuru

PT193 ef Nakuru

PT51 ef NakuruPT21 ef Nakuru

PT47 ef Nakuru

KT224312

LS398491PT44 ef Nakuru

PT1 ef Nakuru

PT191 ef Nakuru

KR909339

MH464151

KU554687MH582332

MH936002

LT548347GQ505595

PT14 ef Nakuru

PT40 ef Nakuru

PT01 ef Nakuru

PT02 ef Nakuru

PT38 ef Nakuru

DQ465946

MH582324

MK168567

MK729605

KU671036

Figure 2 Phylogenetic tree of Fusarium spp isolated from freshly harvested genotypes of wheat seeds in three Rift Valley regions Kenyae analysis was based on tef1-α gene sequences and by the maximum likelihood method with 1000 bootstraps


were incidentally observable on some farms and some wheatgrains All (100) farmers whose wheat seeds were used inthis study cited stem rust as the major fungal disease af-fecting sustainable wheat production

32 Fusarium spp Complex in Genotypes of Wheat Seeds atHarvest Time Based on the identification key the authors of[33] 80 fungal isolates belonging to the genus Fusarium wereidentified Distinctive morphological features of myceliapigment formation and conidial cellsrsquo formation were used todetermine both similarities and differences among the isolatesof Fusarium spp Results following PCR amplification of DNAfor each positive isolate exhibited single clear amplified bandwith fragment total size of between 700 bp and 710 bp(Figure 3) Analysis and comparison of the resultant se-quences with sequences from National Centre for Biotech-nology Informatics (NCBI) database using Basic AlignmentSearch Tool for Nucleotide Sequences (BLASTN) httpsblastncbinlmnihgovBlastcgito established species identi-ties of the fungal isolates as Fusarium poae F tricinctumF heterosporum F culmorum F equiseti Fusarium spF verticillioides and F oxysporum Mean prevalence ofFusarium spp among the three counties was not significantly(all pgt 005) different (Figure 4) However exceptions inprevalence were observed at the species level with respect to Fverticillioides F oxysporum F tricinctum F culmorum andFusarium sp among the 3 regions after LSD pairwise com-parisonsese exceptions were observed in the occurrence ofthe following species F verticillioides between Nakuru andUasin Gishu (p 0591) F culmorum between Nakuru andUasin Gishu (p 100) F tricinctum between Nakuru andNarok (p 100) and Fusarium sp between Narok andUasin Gishu (p 0078) Diversity indices for Fusarium sppin the three regions were as follows Nakuru County 2008Narok 14603 and Uasin Gishu 12337 Based on descriptivestatistics wheat produce of certified seeds had a minimum of0 amounts of Fusarium spp while the maximumwas 16 witha mean of 342 Wheat produce of farm-saved seeds had aminimum of 0 amounts of Fusarium spp isolates while themaximum amount was 22 with a mean of 631 e overall

Table 2 Produce of certified and farm-saved genotypes of wheatseeds and overall occurrence of Fusarium spp

SN Wheat genotype

Occurrence of certifiedand farm-saved wheat seed

produce

Percentage()

occurrenceof Fusarium

sppCT FS CT FS CT FS CT FS

1 Njoro II + + + + + + 25 252 Unidentified minus + minus + minus + minus 6253 Eagle 10 + minus minus + + minus 375 754 Robin + minus minus + + minus 125 55 Mwamba minus minus + minus minus minus 25 minus

6 K Korongo + minus minus minus minus minus minus 6257 Ruiru minus minus minus + minus minus minus 58 Hawk minus minus minus + minus minus 125 3759 K Ibis minus + minus + minus minus minus 62510 Duma + minus minus minus minus minus 5 minus

11 Kwale + minus minus + minus minus 375 minus

12 Fahari minus minus + minus minus minus 25 2513 Kingbird + minus minus minus minus minus 125 minus

14 K Tai + minus minus minus minus minus 125 minus

15 Yombi + minus minus minus minus minus 25 minus

16 K Wren + minus minus minus minus minus 0 minus

17 Simba + minus minus minus + minus 25 minus

18 Sunbird + minus minus minus minus minus 125 minus

19 Ngamia + minus minus minus minus minus 25 minus

Overall occurrence of Fusarium spp 3375 6625K Kenya T total ldquo+rdquo produce of wheat genotype sampled ldquominus rdquo produce ofwheat genotype not sampled CT certified commercial wheat seeds FSfarm-saved wheat ldquoseedsrdquo

Table 1 Wheat genotypes sampled in three Rift Valley regions Kenya

Wheat genotypeRegion of sampling and no of samples

Total no of samplesNarok Uasin Gishu Nakuru

1 Robin 15 9 9 362 Eagle 10 9 3 6 183 Ibis 3 0 3 64 Njoro II 30 82 15 1275 Korongo 6 0 3 96 Farasi 3 0 0 37 Mwamba 6 0 3 98 Hawk 3 0 3 69 Ruiru 6 0 0 610 Simba 0 3 0 311 Ngamia 0 0 3 312 Duma 0 0 3 313 Kingbird 0 0 3 314 K Tai 0 0 3 315 Yombi 0 0 3 316 K Wren 0 0 3 317 Kwale 0 0 3 318 K Sunbird 0 0 3 319 Undisclosed 8 4 4 16Total 89 101 70 260


amounts of Fusarium spp per wheat variety in the category offarm-saved seeds were widely spread (SD= 6626) and henceimplicative of a higher prevalence of Fusarium spp comparedto those resulting from varieties of certified commercial wheatseeds (Table 2)

321 Phylogenetic Analysis of the Fusarium spp IsolatesA phylogenetic tree generated to infer relatedness of thealigned sequences was constructed based on bootstrap valuesof over 75 while the identity of the isolates was based onidentity matrix values lying between 95 and 100 insimilarity with reference sequences in the NCBI isanalysis further confirmed the identity of the species andphylogenetic relations revealing interspecies variation andintraspecies variation among members of F verticillioidesand F equiseti while some other fungal isolates emerged asoutgroups e phylogenetic tree divided the strains intofour major clusters namely I II III and IV (Figure 2)Fusarium strains identified under cluster I includedF verticillioides strain CNRG455 GeneBank accession (cc)no MH936002 Fusarium sp strain MCR2228 GeneBankaccession no MH582332 and F verticillioides strain F28

GeneBank accession no KU554687 Identity matrix valuesfor the strains ranged between 9785 and 100 and were allsupported by the bootstrap value of 100 Group II com-prised of five Fusarium spp (F verticillioides strain CM-CNRG455 GeneBank accession no MH936002F tricinctum strain PPRI20693 GeneBank accession noMH464151 F heterosporum GeneBank accession noDAOMC235644 F culmorum strain E24 GeneBank ac-cession No LT548347 Fusarium sp isolate NRRL 20722GeneBank accession no GQ505595 and F equiseti strainURM 6788 GeneBank accession no LS398491)e highestidentity matrix value in this cluster was 9985 supportedby bootstrap values of 100 for all the species Four (4)Fusarium spp isolates (PT20 PT21 PT53 and PT7) in thiscluster had low identity matrix and hence were treated asoutgroups to node I and node II Group III was supported bya bootstrap value of 100 and constituted of subclusters thatdivided the members into 3 Fusarium species F poae strainMontana II MK729605 F oxysporum KU671036 andF equiseti isolate LQ144 MK168567 with similarity matricesof 9767 8915 and 100 respectively In this clusterFusarium spp isolate PT42 and PT51 emerged as out groupsunder the identity of F equiseti isolate LQ144 MK168567

700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27

Figure 3 Agarose 1 gel electrophoresis of PCR-amplified elongation factor 1-alpha gene of Fusarium spp isolated from differentgenotypes of wheat seeds in three Rift Valley regions Kenya Amplification band of 700-710 bp shows positive results for identity ofFusarium spp


Group IV Fusarium spp strains had a bootstrap value of100 and comprised of one-subcluster and one outgroupPT61 whose reference accession identity was F equisetiisisolate was so prevalent in Uasin Gishu region e sub-cluster was further divided into two groups both of whichhad bootstrap values of 100 One of the groups comprisedof F equiseti isolate SAT73 with identity reference Gene-Bank accession no DQ465946 while the other groupconsisted of F verticillioides strain MRC929 GeneBankaccession no MH582324

322 Diversity of Pathogenic Fusarium spp in the WheatSeed Varieties e phylogenetic analysis shown in Figure 2shows that F verticillioides F equiseti Fusarium poaeF oxysporum F tricinctum Fusarium sp and F culmorumcomprised the pathogenic Fusarium spp complex diversityprevalent in the sampled genotypes of wheat seeds (Table 3)Fusarium verticillioides and F equiseti isolates were dis-tributed across more than one cluster in the phylogenetictree and were isolated from over 70 of all the wheat ge-notypes studied Percentage prevalence of F verticillioidesF equiseti and F poae in all the wheat seed genotypes was asfollows 94 77 and 50 respectively compared to theprevalence of the other Fusarium spp isolated All the eightFusarium spp were more prevalent in Njoro II and Eagle 10wheat seed genotypes while Kenya Wren is the only wheatgenotype that was not infected with Fusarium species (Ta-ble 3) Other than Robin wheat genotype both Njoro II andEagle 10 wheat genotypes were sampled from all the threeregions of study in the following proportions 44 15 and95 for Njoro II Eagle 10 and Robin respectively All ofthem were infected with over 50 of the isolated Fusariumspecies Fusarium graminearumwas not isolated from any ofthe wheat samples

4 Discussion

e findings of this study suggest that freshly harvestedwheat produce of both farm-saved and commercially pro-cessed wheat seeds was significantly infected with patho-genic Fusarium spp that may inherently affect plant growthfrom germination When such Fusarium spp infected grainsare used as seed for the next crop without decontaminationpoor stands poor vigor andor seedling blights can occursubstantially reducing optimal production of the cropFusarium spp infected grains may as well be contaminatedwith mycotoxins that affect both human beings and do-mesticated animals that consume wheat products and wheatremains in the form of fodder respectively Farm-savedwheat seeds had a higher prevalence of Fusarium sppcompared to certified commercial wheat seeds despite thefact that they formed only 227 of the wheat grainssampled Fusarium spp infected grains in wheat debris alsoform an initial point for FHB contamination on farms andmay contribute to propagation of FHB in the next cropespecially in case of poor preharvest and postharvest wheatcultivation practices erefore farmers should consistentlyuse good quality certified and decontaminated seeds toavoid initial fungal contamination by infected seeds [37 38]and the subsequent disease effects [39] Decontamination ofseeds has been reported to increase grain yield and reduceamount of deoxynivalenol (DON) in wheat [40]

Ninety-four percent (94) of the wheat seed genotypesstudied were infected with Fusarium spp the major caus-ative agents of FHB is high prevalence indicates thesusceptibility of some of the developed wheat genotypes tothe disease However the prevalence and distribution of thefungal pathogens within the eighteen wheat genotypessampled varied Njoro II Kenya Eagle 10 and Robin wheatgenotypes were dominant in all the three regions and they

75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

F verticillioides F equiseti F poae F oxysporum F heterosporum F culmorum F tricinctum Fusarium sp

0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)

Figure 4 Means on prevalence of pathogenic Fusarium spp in genotypes of wheat seeds at harvest time in three Rift Valley regions Kenya


were all infected with over 50 of the isolated pathogenicfungi Worth noting is the finding that all wheat genotypes(Kenya Tai Kenya Sunbird Kenya Wren and KenyaKingbird) developed for resistance against fungal diseasessuch as stem rust and yellow rust had less than 50 or hadzero overall occurrence of Fusarium spp However Eagle 10wheat genotype was an exception in this respect Njoro IIfarmersrsquo most preferred wheat genotype recorded thehighest (25) rate of Fusarium sp infestation in the categoryof farm-saved seeds compared to 375 that occurred incertified commercial genotypes (Eagle 10 and Kwale) ofwheat seeds e occurrence of F culmorum F poae Fequiseti F tricinctum Fusarium sp and F verticillioides inalmost all the wheat genotypes studied confirms the problemof pathogenic Fusarium spp in the wheat crop in the re-gions Fusarium oxysporum and F heterosporum are amongthe less frequently or sporadically isolated Fusarium spp incereals such as maize [41] and hence their occurrence in thestudied wheat genotypes should be a concern for furtherresearch Of significance was the absence of F graminearumthe main causative agent of FHB in the isolated populations ofFusarium spp isolates is finding contradicts with existingresearch findings on occurrence of Fusarium spp on wheatkernels in both Narok and Nakuru counties in whichF graminearum has been previously reported [26 27] eabsence of F graminearum might be due to the prolongeddrought that existed in years 2016 and 2017 [42] when thecrop was in season and did not favor the growth of the speciesIt may also be due to the decline in the population of thisstrain in the areas studied e high prevalence ofF verticillioides one of the causative agents of pink ear rot inall the three regions of the study is in agreement with theexisting reports on the wide distribution of the species [32 33]and the report that it is the most common species isolatedworldwide from diseased maize [43] Its high prevalence inwheat is alarming since it is a globally important pathogen ofagriculture and livestock [44] and harmful fumonisins eoccurrence of Fusarium spp in Uasin Gishu is significant dueto scanty or lack of similar published data in the regioncompared to Narok and Nakuru counties

e prevalence and diversity of the Fusarium spp in thethree regions was similar despite the fact that the regions arelocated in fairly different agro-ecological zones is could

be partly explained by the similarity in unfavorable changingclimatic conditions agronomy-related factors and farmpractices in wheat production e factors included cultivationof succeeding crops such as maize barley and wheat sharing ofwheat farm tillage and harvesting equipment without strictobservation of disease preventive measures poor managementof weeds less fertilization continuous land use unskilledmanpower and use of unprocessed low quality farmersrsquo savedwheat seeds Similar role of agro-economic factors in occur-rences and propagation of FHB pathogens in wheat has beenreported [45ndash48] Infected wheat barley and maize debrisusually act as the initial FHB inoculum [46 49] onwheat farmsConsequently consistent cultivation of these succeeding cropswithout proper farm management predisposes the wheat cropto the pathogens Nakuru region recorded all the isolatedFusarium spp which might be explained by two main reasonsFirst there are many potentially susceptible wheat varietiescultivated in the area for research purposes Second the rel-atively warm and wet humid climatic conditions that char-acterize the region are favorable to FHB infestation

Njoro II was the farmersrsquo most preferred wheat varietybecause of its potential to produce high yields and to resist wheatrust Only 163 of the farmers whose wheat seeds weresampled had knowledge about other fungal diseases such asFHB while the majority had no idea Over 90 of farmersrecognized all fungal wheat diseases as ldquoWheat Rustrdquo evenwheresymptoms were for a different fungal infection In Uasin Gishuthe farmersrsquo minimal adoption of new wheat seed varieties wasevident Only four wheat genotypes (Njoro II Robin Simbaand Eagle 10) were cultivated Existing reports suggest thatlimited knowledge on wheat seed management and adoption ofnew seed varieties [50] technical inefficiencies such as lack offarm equipment lack of capital for farm inputs and applicationof less than recommended levels of fertilizers and use of lowquality farm-saved seeds [22] hinder maximum potential ofwheat farms erefore awareness creation and education offarmers on the control of fungal wheat diseases and the relatedeffects of mycotoxins in wheat production remains critical

5 Conclusion and Recommendations

In conclusion Fusarium spp associated with mycotoxinsthat contaminate the wheat food chain seem to be rampant

Table 3 Pathogenic Fusarium spp diversity in genotypes of wheat seeds in three Rift Valley regions Kenya

Fusarium speciesWheat genotypes and the occurrence of Fusarium spp

A B C D E F G H I J K L M N O P Q R S T1 F verticillioides mdash 182 F equiseti mdash mdash mdash mdash 153 F poae mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash 94 F oxysporum mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash 85 F tricinctum mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash 76 F heterosporum mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash 47 Fusarium sp mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash 48 F culmorum mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash 3No of F species 8 8 4 4 4 4 5 4 3 2 4 3 2 4 2 3 2 1 0A Njoro II B Eagle 10 C unidentified wheat genotype D Robin E Kenya Ibis F Hawk G Duma H Ruiru I Kenya Korongo J Kwale K Kingbird LSimbaM Ngamia NMwamba O Fahari P Yombi Q Kenya Tai R Sunbird S KenyaWren T total number of affected wheat varieties ldquordquo isolated ldquomdashrdquonot isolated


in the genotypes of wheat studied with no significant dif-ference in both prevalence and diversity in the three regionsof study However the variation in percentages of Fusariumspp isolated from each wheat genotype provides evidencethat some of the developed wheat genotypes might be moresusceptible to pathogenic Fusarium species complex efindings from this study also emphasize the impact of un-processed and low quality farm-saved wheat grains inproduction of the wheat crop and its products Wheatfarmers are much more informed about wheat rust ascompared to FHBere is therefore a need for consolidativeeffort by stakeholders in wheat production to createawareness and educate farmers on control and managementof all fungal diseases for sustainable wheat crop productionere is a need for increased extension services to farmerswith the aim of inclusion in controlling FHB infestation inwheat crop and awareness on its effects from both economicand health perspective Since only 10 of wheat genotypesdeveloped for various purposes in Kenya is in use there is aneed for constant feedback from farmers on the criteria forselection of wheat genotypes is information is helpful towheat seed-producing companies in improving the resis-tance potential of existing varieties andor development ofnew wheat genotypes with stronger resistance

Data Availability

e data used to support the research findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that there are no conflicts of interest

Authorsrsquo Contributions

OPK OS andWVWconceptualized and designed the studyOPK carried out field data collection ISS and MO assistedOPK with sample collection and data analysis OPK and ISSdrafted the manuscript OS ISS and WVV criticallyreviewed the manuscript All authors read and approved themanuscript for publication

Acknowledgments

e authors appreciate Mr Wakoli Mrs Owiti Anne andKevin Ochwedo who assisted with technical work ey areindebted to Kenya Agricultural and Livestock ResearchOrganization Kabete for facilitating this work by providingworking space e research work was funded by the gov-ernment of Kenya National Commission for ScienceTechnology and Innovation (NACOSTI) under researchgrant number (NACOSTIRCDSTamp7TH CALLPHD215)

References

[1] A Logrieco A Moretti G Perrone and G Mule ldquoBiodi-versity of complexes of mycotoxigenic fungal species asso-ciated with Fusarium ear rot of maize and Aspergillus rot of

graperdquo International Journal of Food Microbiology vol 119no 1-2 pp 11ndash16 2007

[2] L Gong Y Jiang and F Chen ldquoMolecular strategies fordetection and quantification of mycotoxin-producing Fusa-rium species a reviewrdquo Journal of the Science of Food andAgriculture vol 95 no 9 pp 1767ndash1776 2015

[3] P Giraldo E Benavente F Manzano-Agugliaro andE Gimenez ldquoWorldwide research trends on wheat and barleya bibliometric comparative analysisrdquo Agronomy vol 9 no 7p 352 2019

[4] P R Shewry and S J Hey ldquoe contribution of wheat tohuman diet and healthrdquo Food and Energy Security vol 4no 3 pp 178ndash202 2015

[5] A Bernhoft M Torp P-E Clasen A-K Loslashes andA B Kristoffersen ldquoInfluence of agronomic and climaticfactors onFusariuminfestation and mycotoxin contaminationof cereals in Norwayrdquo Food Additives amp Contaminants PartA vol 29 no 7 pp 1129ndash1140 2012

[6] C C Dweba S Figlan H A Shimelis et al ldquoFusarium headblight of wheat pathogenesis and control strategiesrdquo CropProtection vol 91 pp 114ndash122 2017

[7] V Scala G Aureli G Cesarano et al ldquoClimate soil man-agement and cultivar affect Fusarium head blight incidenceand deoxynivalenol accumulation in durum wheat ofSouthern Italyrdquo Frontiers in Microbiology vol 7 no JUNpp 1ndash10 2016

[8] A Wenda-Piesik G Lemanczyk M Twaruzek A Błajet-Kosicka M Kazek and J Grajewski ldquoFusarium head blightincidence and detection of Fusarium toxins in wheat in re-lation to agronomic factorsrdquo European Journal of Plant Pa-thology vol 149 no 3 pp 515ndash531 2017

[9] M Mcmullen S Halley B Schatz S Meyer J Jordahl andJ Ransom ldquoIntegrated strategies for fusarium head blightmanagement in the United Statesrdquo Cereal Research Com-munications vol 36 no Supplement 6 pp 563ndash568 2008

[10] S N Wegulo P S Baenziger J Hernandez NopsaW W Bockus and H Hallen-Adams ldquoManagement ofFusarium head blight of wheat and barleyrdquo Crop Protectionvol 73 pp 100ndash107 2015

[11] E Bonfada D Honnef M T Friedrich W Boller andC C Deuner ldquoPerformance of fungicides on the control offusarium head blight (Triticum aestivum L) and deoxy-nivalenol contamination in wheat grainsrdquo Summa Phytopa-thologica vol 45 no 4 pp 374ndash380 2019

[12] D L D Angelo P Pathology and T Ohio ldquoDrsquoAngelo 2014FHB DON fungicide timing post-anthesis prosaro metco-nazolerdquo Journal of Plant Diseases vol 60 no 10 pp 1387ndash1397 2014

[13] M Haidukowski M Pascale G Perrone D PancaldiC Campagna and A Visconti ldquoEffect of fungicides on thedevelopment of Fusarium head blight yield and deoxy-nivalenol accumulation in wheat inoculated under fieldconditions with Fusarium graminearum and Fusarium cul-morumrdquo Journal of Plant Pathology vol 85 pp 191ndash198

[14] M Yoshida T Nakajima K Tomimura F Suzuki M Araiand AMiyasaka ldquoEffect of the timing of fungicide applicationon Fusarium head blight and mycotoxin contamination inwheatrdquo Plant Disease vol 96 no 6 pp 845ndash851 2012

[15] M Beyer M B Klix H Klink and J-A Verreet ldquoQuantifyingthe effects of previous crop tillage cultivar and triazolefungicides on the deoxynivalenol content of wheat grain - areviewrdquo Journal of Plant Diseases and Protection vol 113no 6 pp 241ndash246 2006


[16] M Blandino M Haidukowski M Pascale L PlizzariD Scudellari and A Reyneri ldquoIntegrated strategies for thecontrol of Fusarium head blight and deoxynivalenol con-tamination in winter wheatrdquo Field Crops Research vol 133pp 139ndash149 2012

[17] M Blandino V Scarpino M Sulyok R Krska andA Reyneri ldquoEffect of agronomic programmes with differentsusceptibility to deoxynivalenol risk on emerging contami-nation in winter wheatrdquo European Journal of Agronomyvol 85 pp 12ndash24 2017

[18] L Shah A Ali M Yahya et al ldquoIntegrated control offusarium head blight and deoxynivalenol mycotoxin inwheatrdquo Plant Pathology vol 67 no 3 pp 532ndash548 2018

[19] T Nakajima M Yoshida and K Tomimura ldquoEffect oflodging on the level of mycotoxins in wheat barley and riceinfected with the Fusarium graminearum species complexrdquoJournal of General Plant Pathology vol 74 no 4 pp 289ndash2952008

[20] E M Janssen M C M Mourits H J van der Fels-Klerx andA G J M O Lansink ldquoPre-harvest measures againstFusarium spp infection and related mycotoxins implementedby Dutch wheat farmersrdquo Crop Protection vol 122 pp 9ndash182019

[21] J Kamwaga G Macharia L Boyd et al ldquoKenya wheatproduction handbook 2016rdquo Experimental Agriculturevol 37 no 1 2016

[22] S Mburu C Ackello-Ogutu and R Mulwa ldquoAnalysis ofeconomic efficiency and farm size a case study of wheatfarmers in Nakuru district Kenyardquo Economics Research In-ternational vol 2014 Article ID 802706 10 pages 2014

[23] S Alessandro J Caballero J Lichte and S Simpkin ldquoKenyanagricultural sector risk assessmentrdquo p 126 2015 AgriculturalGlobal Practice Technical Assistance Paper World BankGroup Washington DC USA 96289

[24] N T Beatrice P O Pascal O Daniel and O MauriceldquoWheat stem rust disease incidence and severity associatedwith farming practices in the Central Rift Valley of KenyardquoAfrican Journal of Agricultural Research vol 11 no 29pp 2640ndash2649 2016

[25] J W Muthomi S L Musyimi J M Wagacha andR D Narla ldquoOccurrence of Fusarium species and associatedT2-toxin in Kenyan wheatrdquo Journal of Agricultural Sciencesvol 3 no 1 pp 24ndash34 2012

[26] J W Muthomi J K Ndungrsquou J K Gathumbi E W Mutituand J M Wagacha ldquoe occurrence of Fusarium species andmycotoxins in Kenyan wheatrdquo Crop Protection vol 27 no 8pp 1215ndash1219 2008

[27] J M Wagacha N K Njeru O O Okumu J W Muthomiand C K Mutegi ldquoOccurrence of Fusarium head blight ofwheat and associated mycotoxins in Narok and NakuruCounties Kenyardquo World Journal of Agricultural Researchvol 4 no 4 pp 119ndash127 2016

[28] H El-Shall and B M Moudgil ldquoDesign of optimum samplingplans for dry powders and slurriesrdquo KONA Powder andParticle Journal vol 31 no 1 pp 82ndash91 2014

[29] L Freese J-W Chen and R D Shillito ldquoSampling of grainand seed to estimate the adventitious presence of biotech-nology-derived seeds in a lotrdquo Cereal Foods World vol 60no 1 pp 9ndash15 2015

[30] P E Nelson A E Desjardins and R D Plattner ldquoFumo-nisins mycotoxins produced by fusarium species biologychemistry and significancerdquo Annual Review of Phytopa-thology vol 31 no 1 p 233 1993

[31] H I Nirenberg ldquoA simplified method for identifying Fusa-rium spp occurring on wheatrdquo Canadian Journal of Botanyvol 59 no 9 pp 1599ndash1609 1981

[32] L W Burgess Laboratory Manual for fusarium ResearchFusarium Research Laboratory Dept of Crop SciencesUniversity of Sydney Royal Botanic Gardens SydneyAustralia 3rd edition 1994

[33] J F Leslie and B A Summerrell Fe fusarium LaboratoryManual Blackwell Pub Hoboken NJ USA 1st ed edition2006

[34] D M Geiser M del Mar Jimenez-Gasco S Kang et alldquoFUSARIUM-ID v 10 a DNA sequence database for iden-tifying Fusariumrdquo European Journal of Plant Pathologyvol 110 no 56 pp 473ndash479 2004

[35] S Kumar G Stecher M Li C Knyaz and K Tamura ldquoMegaX molecular evolutionary genetics analysis across computingplatformsrdquo Molecular Biology and Evolution vol 35 no 6pp 1547ndash1549 2018

[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evolutionary genetics analysis version60rdquo Molecular Biology and Evolution vol 30 no 12pp 2725ndash2729 2013

[37] S A Inch and J Gilbert ldquoSurvival of Gibberella zeae inFusarium-damaged wheat kernelsrdquo Plant Disease vol 87no 3 pp 282ndash287 2003

[38] W E May M R Fernandez and G P Lafond ldquoEffect offungicidal seed treatments on the emergence developmentand grain yield of Fusarium graminearum-infected wheat andbarley seed under field conditionsrdquo Canadian Journal of PlantScience vol 90 no 6 pp 893ndash904 2010

[39] W E Nganje S Kaitibie W W Wilson L F Leistritz andD A Bangsund ldquoEconomic impacts of fusarium head blightin wheat and barley 1993-2001rdquo Agribusiness amp AppliedEconomics no 538 pp 1ndash62 2004

[40] A G Xue W Guo Y Chen et al ldquoEffect of seed treatmentwith novel strains of Trichoderma spp on establishment andyield of spring wheatrdquo Crop Protection vol 96 pp 97ndash1022017

[41] A Bottalico ldquoFusarium diseases of cereals species complexand related mycotoxin profilesrdquo Journal of Plant Pathologyvol 80 no 2 pp 85ndash103 1998

[42] W Schmidt A Peter Uhe J Kimutai F Otto and H Cullene drought in Kenya 2016-2017 Climate and developmentknowledge Network and world weather attribution initiativepaper April 1ndash8 2017

[43] G P Munkvold and A E Desjardins ldquoFumonisins in maizecan we reduce their occurrencerdquo Plant Disease vol 81 no 6pp 556ndash565 1997

[44] D Nagaraj and M Sreenivas ldquoFusarium verticillioides aglobally important pathogen of agriculture and livestock areviewrdquo Journal of Veterinary Medicine and Research vol 4no 4 p 1084 2017

[45] X Guo D Fernando P Bullock and H SapirsteinldquoQuantifying cropping practices in relation to inoculum levelsof fusarium graminearum on crop stubblerdquo Plant Pathologyvol 59 no 6 pp 1107ndash1113 2010

[46] M D Keller A B Baudoin W E omason andB A Vinatzer Fe contribution of within-field inoculumsources of gibberella zeae to fusarium head blight in winterwheat and barley PhD esis Virginia Polytechnic InstituteBlacksburg VA USA 2011

[47] N Njeru M Wagacha J W Muthomi and C Mutegi ldquoRoleof soil and crop residues as sources of inoculum for Fusariumhead blight of wheatrdquo in Proceedings of the Fourth RUFORUM


Biennial Regional Conference Maputo Mozambiquepp 213ndash217

[48] N K Njeru J WMuthomi C K Mutegi and J MWagachaldquoEffect of cropping systems on accumulation of fusarium headblight of wheat inocula in crop residues and soilsrdquo Journal ofPlant Sciences vol 11 no 1 pp 12ndash21 2016

[49] J M Wagacha U Steiner H-W Dehne et al ldquoDiversity inmycotoxins and fungal species infecting wheat in Nakurudistrict Kenyardquo Journal of Phytopathology vol 158 no 7-8pp 527ndash535 2010

[50] P Gamba and C Ngugi ldquoWheat farmersrsquo seed managementand varietal adoption in Kenyardquo Journal of Plant Breedingpp 53ndash62 2003


wheat grain destruction with resultant accumulation ofmycotoxins

Globally various control measures are under investi-gation by researchers with the aim of effectively protectingthe crop from FHB is has involved the use of differentpre-harvest and post-harvest methods starting from agro-technical methods that limit the source of primary infectionPost-harvest control measures include physical methodssuch as proper harvesting that does not damage the grainscrop rotation tillage fertilization use of appropriate qualityseedmaterial and the timely sowing period [9 10] educationand training of agricultural producers in application of goodagricultural practices Although fungicide efficacy againstFHB has been inconsistent due to the short time frameworkfor application as well as limited number of available fun-gicides [10] their role in control of FHB cannot be ignoredamong pre-harvest control measures Chemical control ofFHB in wheat influences the disease intensity in spikes anddeoxynivalenol contamination in grains [11] Timely use ofthe correct dosage of the effective type of fungicides [12ndash14]has been reported to be an applicable FHB control measureespecially during plant flowering Also included in thiscategory is the use of resistance inducers including bio-preparations based on antagonistic microbial agents en-dophytes and other biologically active ingredients andnonchemical fungicides and plowing [15ndash18] Selection anduse of cultivars with a high level of resistance to Fusariumspp infestation [10 17] and to lodging [19] cannot beoveremphasized Use of a combination of preharvest mea-sures when conditions are favorable for Fusarium spp in-festation is more effective [9 20] since they limit the survivalof the pathogens in debris reducing their presence in fieldand infestation severity However the ever-changing farmand climatic conditions require application of consistenteffective FHB control measures such as the use of wheatgenotypes that are highly resistant to Fusarium spp path-ogens e current study investigated the prevalence anddiversity of pathogenic Fusarium spp in varieties of wheatgenotypes from three major wheat-producing regions in theKenyan Rift Valley in line with factors determining selectionof the cultivated genotypes

11 Background of the Study Wheat is Kenyarsquos second mostimportant cereal crop after maize and an economicallyimportant crop among the large and small-scale farmers[21] It is grown in areas above 1500 metres above sea levelin southern and upper Rift Valley regions (Nakuru Narokand Uasin Gishu) and in the eastern part of Kenya (Nanyukiand Meru) Wheat production is carried out by smallmedium and large-scale farmers and the industry supportedby about 20 millers contributes 14 and 30 to overall andcereal GDP [22] respectively e high costs of farm inputsand land fragmentation among other factors have led to ashift in production from large-scale and medium-scalecommercial farms using capital-intensive technology toproduction mostly by small-scale farmers [23]

Wheat requires a fine planting bed for uniform germi-nation In this view farmers are encouraged to have the land

thoroughly ploughed and harrowed free from growingweeds and weed seeds at least 4 weeks before planting toensure that the land does not have fresh compost (plantmaterial that is not fully rotten) during planting Fertilizerapplication is recommended during planting depending onthe soil type as per the soil analysis report During plantingdiammonium phosphate (DAP) at 200ndash250 kg Haminus 1 isrecommended as determined by factors such as the stage ofthe crop since availability of nitrogen is important duringtilling stem extension and ear emergence

Diseases pre-harvest and post-harvest losses are themajor recurring challenges hindering optimal production ofwheat Occurrence of wheat devastating fungal diseases suchas wheat rust [24] and FHB [25ndash27] has been reported as themajor setback to wheat production in Kenya Generallyother than good farm management practices the mainwheat crop protection regime used by farmers in Kenyaagainst all fungal foliar wheat diseases such as wheat rust isthe use of fungicides Foliar diseases can increase the sus-ceptibility of wheat spikes to FHB Consequently fungicidesbecome crucial in reducing the effects of the diseases onwheat However the low efficacy and limited spectrum ofsuch fungicides create a gap in control of resistant fungaldiseases such as FHB Although disease resistance or tol-erance remains a driving goal of Kenya Agricultural andLivestock Organizationrsquos (KALROrsquo) wheat research pro-gram it remains a challenge for the recommended wheatvarieties [21] Hence there is consistent development ofwheat genotypes with high stable yields tolerance to bioticand abiotic stresses pre-harvest sprouting diseases andinsect pests so that farmers from different growing regionsin the country can select the most suited variety [21]erefore current information on prevalence and diversityof Fusarium spp in the existing and cultivated wheat ge-notypes is important prerequisite information in integratedmanagement of FHB and the improvement or developmentof other genotypes with better disease resistant qualities ecurrent study evaluated the occurrence and diversity ofpathogenic Fusarium spp in the developed wheat genotypesin three major wheat-producing regions within the KenyanRift Valley and the factors considered by farmers in selectingthe cultivated wheat varieties

2 Materials and Methods

21 Study Area e study was carried out in three regionssituated within the Kenyan Rift Valley the main commercialwheat-growing zone Figure 1 shows the location of each sitegenerated using the geographical positioning system (GPS)e average rainfall range was between 20mmndash40mm inNarok 60mmndash80mm in Uasin Gishu and 40mmndash60mmin Nakuru whereas the average temperature range in Narokwas 18degCndash22degC in Usain Gishu lt18degC and 18degCndash22degC inNakuru during the study period ese areas have annualrainfall varying between 800mm and 2000mm with theamounts occasionally rising up to 2500mm in higher al-titudes Research on smallholder wheat technologies isperformed by the National Plant Breeding Station based inNakuru e commonly cultivated crops on large-scale


Narok

Kajiado

Nakuru

Baringo

Laikipia

Nandi

Bomet

NyeriNyandarua

Kisii

Uasin Gishu

Kiambu

Kericho

Kisumu

Kakamega

Bungoma

Muranga

Samburu

Nyamira

Nairobi

Homa Bay

Vihiga

Isiolo

Trans Nzoia

Kiyo-Marakwete

Machakos

Migori

Turkana

Key



0 25 50 75125Km

N

KENYA















D 1 minus 1113944 pi( 11138572 (1)




3 Results



IV

III

II

I

Fusarium equiseti

Fusarium oxysporum

Outgroup to node IV

Fusarium equiseti


Fusarium equiseti


Fusarium poae



Fusarium tricinctum

Fusarium culmorum


Fusarium equiseti

Fusarium equiseti

10098

98100100

100

100

8478

96

80

64

94

84

100 100

100

100

100

100

74

42

9850

28

9210

6

050

20

18

76

82

100


PT25 ef Narok

PT74 ef Narok

PT42 ef Narok

PT71 ef Narok

PT15 ef Uasin Gishu




PT53 ef Uasin Gishu

PT20 ef Uasin Gishu

PT70 ef Uasin Gishu




PT46 ef Narok

PT12 ef Narok

PT10 ef Nakuru

PT34 ef Nakuru

PT193 ef Nakuru


PT47 ef Nakuru

KT224312


PT1 ef Nakuru

PT191 ef Nakuru

KR909339

MH464151

KU554687MH582332

MH936002

LT548347GQ505595

PT14 ef Nakuru

PT40 ef Nakuru

PT01 ef Nakuru

PT02 ef Nakuru

PT38 ef Nakuru

DQ465946

MH582324

MK168567

MK729605

KU671036






SN Wheat genotype


produce

Percentage()






















700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27





4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References
























































Narok

Kajiado

Nakuru

Baringo

Laikipia

Nandi

Bomet

NyeriNyandarua

Kisii

Uasin Gishu

Kiambu

Kericho

Kisumu

Kakamega

Bungoma

Muranga

Samburu

Nyamira

Nairobi

Homa Bay

Vihiga

Isiolo

Trans Nzoia

Kiyo-Marakwete

Machakos

Migori

Turkana

Key



0 25 50 75125Km

N

KENYA















D 1 minus 1113944 pi( 11138572 (1)




3 Results



IV

III

II

I

Fusarium equiseti

Fusarium oxysporum

Outgroup to node IV

Fusarium equiseti


Fusarium equiseti


Fusarium poae



Fusarium tricinctum

Fusarium culmorum


Fusarium equiseti

Fusarium equiseti

10098

98100100

100

100

8478

96

80

64

94

84

100 100

100

100

100

100

74

42

9850

28

9210

6

050

20

18

76

82

100


PT25 ef Narok

PT74 ef Narok

PT42 ef Narok

PT71 ef Narok

PT15 ef Uasin Gishu




PT53 ef Uasin Gishu

PT20 ef Uasin Gishu

PT70 ef Uasin Gishu




PT46 ef Narok

PT12 ef Narok

PT10 ef Nakuru

PT34 ef Nakuru

PT193 ef Nakuru


PT47 ef Nakuru

KT224312


PT1 ef Nakuru

PT191 ef Nakuru

KR909339

MH464151

KU554687MH582332

MH936002

LT548347GQ505595

PT14 ef Nakuru

PT40 ef Nakuru

PT01 ef Nakuru

PT02 ef Nakuru

PT38 ef Nakuru

DQ465946

MH582324

MK168567

MK729605

KU671036






SN Wheat genotype


produce

Percentage()






















700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27





4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References




































































D 1 minus 1113944 pi( 11138572 (1)




3 Results



IV

III

II

I

Fusarium equiseti

Fusarium oxysporum

Outgroup to node IV

Fusarium equiseti


Fusarium equiseti


Fusarium poae



Fusarium tricinctum

Fusarium culmorum


Fusarium equiseti

Fusarium equiseti

10098

98100100

100

100

8478

96

80

64

94

84

100 100

100

100

100

100

74

42

9850

28

9210

6

050

20

18

76

82

100


PT25 ef Narok

PT74 ef Narok

PT42 ef Narok

PT71 ef Narok

PT15 ef Uasin Gishu




PT53 ef Uasin Gishu

PT20 ef Uasin Gishu

PT70 ef Uasin Gishu




PT46 ef Narok

PT12 ef Narok

PT10 ef Nakuru

PT34 ef Nakuru

PT193 ef Nakuru


PT47 ef Nakuru

KT224312


PT1 ef Nakuru

PT191 ef Nakuru

KR909339

MH464151

KU554687MH582332

MH936002

LT548347GQ505595

PT14 ef Nakuru

PT40 ef Nakuru

PT01 ef Nakuru

PT02 ef Nakuru

PT38 ef Nakuru

DQ465946

MH582324

MK168567

MK729605

KU671036






SN Wheat genotype


produce

Percentage()






















700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27





4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References
























































IV

III

II

I

Fusarium equiseti

Fusarium oxysporum

Outgroup to node IV

Fusarium equiseti


Fusarium equiseti


Fusarium poae



Fusarium tricinctum

Fusarium culmorum


Fusarium equiseti

Fusarium equiseti

10098

98100100

100

100

8478

96

80

64

94

84

100 100

100

100

100

100

74

42

9850

28

9210

6

050

20

18

76

82

100


PT25 ef Narok

PT74 ef Narok

PT42 ef Narok

PT71 ef Narok

PT15 ef Uasin Gishu




PT53 ef Uasin Gishu

PT20 ef Uasin Gishu

PT70 ef Uasin Gishu




PT46 ef Narok

PT12 ef Narok

PT10 ef Nakuru

PT34 ef Nakuru

PT193 ef Nakuru


PT47 ef Nakuru

KT224312


PT1 ef Nakuru

PT191 ef Nakuru

KR909339

MH464151

KU554687MH582332

MH936002

LT548347GQ505595

PT14 ef Nakuru

PT40 ef Nakuru

PT01 ef Nakuru

PT02 ef Nakuru

PT38 ef Nakuru

DQ465946

MH582324

MK168567

MK729605

KU671036






SN Wheat genotype


produce

Percentage()






















700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27





4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References



























































SN Wheat genotype


produce

Percentage()






















700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27





4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References



























































700bp

700bp

700bp

L

700500

20075

L

L

L

700500

200

200

1000

1kb ladder

75

O 1 10 12 14 15 16 19A 19B 20A 20B

28 34 36 37 38 40 42 44 46 47A 47B

L 44 47a 47b 53 54 61 71 71b 89 94 150 152 153

21 23 25 27





4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References


























































4 Discussion



75

1625

625 75 65

1125

375

75

375125

75

25 375

0 125375

0 025 375

0

375125 0

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u

Naku

ru

Naro

k

Uasin

Gish

u


0

2

4

6

8

10

12

14

16

18

Mea

n fre

quen

cy (

)














Data Availability






Acknowledgments


References



































































Data Availability






Acknowledgments


References
























































prevalenceandphylogeneticdiversityofpathogenic fusarium

Documents