Diversity and detection of Kenyan and Nigerian populations of Fusarium oxysporum f. sp. strigae

Thesis (MSc)--Stellenbosch University, 2017.

ENGLISH ABSTRACT: The Genus Striga contains some of the most destructive plant-parasitic weed species in the world. These include S. hermonthica and S. asiatica, which attack important staple food crops in sub-Saharan Africa such as maize, sorghum and millet. Of the two, S. hermonthica is more devastating and can cause yield losses on average of 50% and, ultimately, land abandonment. The parasite produces up to 200 000 seeds per plant, which causes a rapid build-up of a seed bank in the soil. Germination of seeds happens after a ripening period and when strigolactones, exuded by the roots of susceptible hosts, are detected. After a haustorium connects to the host, it parasitizes the host for water and nutrients, and in effect stunts the development of the host plant. Striga infestations are difficult to manage due to its prolific nature and late emergence. The weed affects subsistence farmers most severely, as these growers have little access to, or the financial resources available, to control the pest with chemicals. Biological control, therefore, was proposed as a means of control, as this method can be integrated with existing farming practises, as its use would be safe to both the farmer and the environment. Fusarium oxysporum f. sp. strigae (Fos), a soil-borne fungal pathogen affecting Striga plants, was identified as a possible Biological Control Agent (BCA), as Fos was host specific and did not produce harmful secondary metabolites. Its population structure in Africa, and means to survive and disseminate in farmer fields, however, was unknown. The aims of this study, therefore, were to characterize Fos populations in two African countries, Kenya and Nigeria, and to develop molecular markers to rapidly and accurately identify the fungus. The diversity of Fos in Kenya and Nigeria was investigated by means of vegetative compatibility group analysis (VCGs) and phylogenetic analysis. VCG analysis showed that the Kenyan isolates consisted of a single VCG, and that the Nigerian isolates were divided into seven VCGs and eight SMV’s. A combined maximum likelihood tree of the translocation elongation factor (TEF) 1α and mitochondrial small sub-unit (MtSSU) gene areas revealed that Fos isolates from the two countries separated into two different clades. This suggested that there was two separate events of evolution, and that the Nigerian isolate group is older than the Kenyan group due to the greater number of VCGs present in Nigeria. Mating type analysis confirmed clonality within the Kenyan group, where all isolates in VCG 04708 contained only the MAT1-1 gene. However, a larger diversity was found in the Nigerian group, where both mating type idiomorphs were present in the different VCGs. Molecular markers that distinguish between the two Fos populations in Africa have been developed in this study. Primer-set Foxy2-F/R1 was developed form a single nucleotide polymorphism (SNP) in the TEF gene area, and primer set FK3-F/R from SNPs in the secreted-in-xylem 14 gene area. Both primer sets were tested against a collection of isolates that includes non-pathogenic F. oxysporum isolated from Striga, other Fusarium species associated with Striga, and other formae speciales of F. oxysporum. Sensitivity assays revealed that the Foxy2 primer only detected target Fos DNA at a concentration of 10 ng/μl in the presence of S. asiatica DNA. Primer-set FK3 on the other hand, could detect target Fos DNA at the low concentration of 0.1 ng/μl in the presence of S. asiatica DNA. The findings in this study suggest that pathogenicity evolved at two separate events in Fos. The molecular markers designed could compliment the primer-set designed by Zimmerman et al. (2015) to aid in diagnostics and monitoring of Fos after application as a BCA.

AFRIKAANSE OPSOMMING: Die Genus Striga word gesien as die mees verwoestende plant-parasietiese onkruid spesies in die wêreld. Daaronder val S. hermonthica en S. asiatica, wat belangrike stapel voedsel gewasse soos mielies, sorghum en millet aanval in sub-Sahara in Afrika. Striga hermonthica is die meer verwoestende plant parasiet uit die twee, en kan oeste verminder met ‘n gemiddeled van 50%, wat uiteindelik lei tot die opgee van die landbou grond. Hierdie plant-parasiet produseer tot en met 200 000 saad per plant, wat lei daartoe dat ‘n saad-bank baie vining op bou in die grond. Die saad ontkiem na ‘n ryp-wordings proses en wanneer strigolaktone, uit geskei deur die wortels, waar geneem word. ‘n Haustorium ontwikkel en verbind aan die gasheer plant, waar dit dan die gasheer plant parasiteer vir water en voedsel, wat lei tot vertraagde ontwikkeling van die gasheer. Striga besmettings is moeilik om te bestuur as gevolg van die onkruid se produktiewe aard en laat opkoms. Die onkruid beïnvloed bestaansboere die ergste, aangesien hierdie boere minder toegang het tot, of oor die beskikbare finansiële hulpbronne beskik, om die parasiet chemies te beheer. Biologiese beheer is dus voorgestel as 'n manier van beheer, aangesien hierdie metode met bestaande boerderypraktyke geïntegreer kan word,en die gebruik daarvan vir beide die boer en die omgewing veilig sal wees. Fusarium oxysporum f. sp. strigae (Fos), ‘n grond gedraagde patogeniese fungus wat Striga plante aanval, was geidentifiseer as ‘n moontlike Biologiese Beheer Agent (BBA), aangesien dit bewys was dat Fos gasheer spesifiek is en nie enige skadlike sekondêre metaboliete produseer nie. Die bevolkings struktuur van hierdie fungus in Afrika, asook die oorlewing en verspreiding in die bewerkbare lande, is egter onbekend. Die doelstellings van hierdie studie was dus om die Fos-bevolkings in twee Afrika-lande, Kenia en Nigerië te karakteriseer en molekulêre merkers te ontwikkel om die swam vinnig en akkuraat te identifiseer. Die diversiteit van Fos in Kenia en Nigerië was ondersoek deur om vegetatiewe verenigbaarheidsgroep (VVG) en filogenetiese analises te voltooi. Die VVG-analise het getoon dat die Keniaanse isolate bestaan uit 'n enkele VVG, en dat die Nigeriese isolate verdeel is in sewe VVG's en agt enkellid VVG's (EVVG’s). Die gekombineerde maksimum-waarskynlikheids filogenetiese analise van die translokasie-verlengings faktor (TEF) 1a en mitochondriale klein subeenheid (MtSSU) geengebiede het aan gedui dat die Fos populasie geskei kan word in twee verskillende afstammeling groepe. Hieruit kan afgelei word dat daar twee afsondelike evolusinêre ontstaans-gebeurtenisse was, en dat die Nigeriese isolaat-groep ouer is as die Keniaanse groepis, a.g.v. die groter aantal VVG's wat in Nigerië teenwoordig is. Die parings tipe analise bevestig klonaliteit in die Keniaanse groep, waar alle isolate in VVG 04708 slegs die MAT1-1 geen bevat. Daar is egter 'n groter diversiteit in die Nigeriese groep gevind, waar albei parings-tipe idiomorfe in die verskillende VVG's teenwoordig was. Molekulêre merkers is in hierdie studie ontwikkel wat tussen die twee Fos-populasies in Afrika kan onderskei. Inleier stel, Foxy2-F / R1 is ontwikkel in die TEF geen-area van 'n enkel nukleotied polimorfisme (ENP), en die inleier stel FK3-F / R van ENP's in die afgeskei-in-xileem 14 geen-area. Albei inleier stelle is getoets teen 'n versameling isolate wat nie-patogene van F. oxysporum bevat, geïsoleer vanaf Striga, ander Fusarium spesies wat met Striga geassosieer word, en ander formae spesiales van F. oxysporum. Sensitiwiteits studies het aan gedui dat die Foxy2-inleier slegs Fos DNA op 'n konsentrasie van 10 ng/μl in die teenwoordigheid van S. asiatica DNA kon amplifiseer. Die FK3 inleier, kon egter Fos DNS op die lae konsentrasie van 0,1 ng/μl in die teenwoordigheid van S. asiatica DNS amplifiseer. Hierdie studie dui daarop dat patogeniteit ontwikkel het tydens twee afsonderlike gebeurtenisse in Fos. Die molekulêre merkers wat ontwerp is in hierdie studie, kan die merker stel wat deur Zimmerman et al (2015) ontwerp is, komplimenteer, om te help met die diagnose en monitering van Fos in landbou grond na toediening.