Investigating maize inbred line responses following infection by the Mycotoxigenic fungus Fusarium verticilliodes

Rose, Lindy Joy (2016-03)

Thesis (PhDAgric)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Fusarium verticillioides is an important fungal pathogen of maize and is associated with the crop wherever it is produced. It is the most common fungal contaminant of South African maize grain and causes Fusarium ear rot (FER). The presence of F. verticillioides can reduce grain yield and quality by the visible moulding and/or discolouration of the kernels which reduces its grading at silos. The fungus may also contaminate grain without visible symptoms and produce harmful secondary metabolites known as fumonisins. Fumonisins have been associated with a number of noxious effects on humans and animals. Their widespread occurrence in maize and maize-based products has led to numerous countries imposing maximum allowable limits of fumonisins in food and feed. Cultural practises, mainly focussed on optimal plant production as a disease management strategy, have been shown to reduce fumonisin contamination. However, host-plant resistance is considered the most feasible, economical and environmentally sound approach to manage F. verticillioides and its fumonisins. In this study the response of elite breeding lines to infection and fumonisin deposition by F. verticillioides was evaluated in a multi-environment trial over 2 years. These lines were evaluated with inbred lines previously characterised for their response to FER and fumonisin accumulation. The stability of the inbred line response across locations was also determined in order to identify lines with broad or specific adaptability for disease resistance. Inbred lines CML 390, RO 424W, US 2540W and VO 617y-2 consistently exhibited low FER severity (≤5%), fungal target DNA (≤0.1 ng μL-1) and fumonisin levels (≤5 mg kg-1) at most test locations. Line RO 424W was the most stable in its response to disease and fumonisin accumulation across environments. The elite inbred lines were highly susceptible to F. verticillioides and fumonisin accumulation with only CB-222 and CB-248 having intermediate resistance. These inbred lines may serve as sources of resistance in breeding programmes but can also be used in genomic and proteomic studies to better understand the genetic basis of resistance to F. verticillioides and its fumonisins. The response of Kenyan inbred lines, previously characterised for resistance to Aspergillus flavus and aflatoxins, to F. verticillioides and fumonisin contamination was evaluated in South Africa and Kenya. Common resistance mechanisms to these pathogens have been reported. The AER/aflatoxin-resistant lines were compared to South African lines previously characterised as resistant to F. verticillioides and fumonisin accumulation. Kenyan inbred lines CML 495, CML 264 and CKL05015 were most resistant to FER, F. verticillioides colonisation and fumonisin accumulation across localities. The inbred line CML 495 was also the most stable in its resistance response to F. verticillioides infection and fumonisin deposition, making it an attractive source of resistance for inclusion into a South African breeding programme. This study, therefore, provided further evidence that AER/aflatoxin-resistant lines appear to be a significant source of resistance to F. verticillioides and fumonisins. Genetic variability for resistance to F. verticillioides and fumonisin accumulation was generated by gamma irradiation of seven elite maize lines. Following mass irradiation, the lines were field planted and self-pollinated for four consecutive seasons (M0-M4 generations). The response of the M1 to M4 populations to F. verticillioides was evaluated visually for FER severity annually, and ears expressing less than 10% FER were advanced to the next breeding cycle. The M4 selections were also evaluated for F. verticillioides colonisation and fumonisin content. A number of selections derived from each inbred line were more resistant to FER, F. verticillioides colonisation and fumonisin accumulation when compared to their non-irradiated controls. However, no improved selections were obtained from inbred I-35. The transcriptional changes in maize induced upon infection by F. verticillioides were evaluated by next-generation RNA sequencing and monitored up to 7 days after inoculation (dai). Although an initial induction of defence-related transcripts associated with pathogen recognition, signalling molecules, pathogenesis-related genes, cell wall restructuring and secondary hormone-based signalling genes was observed 24 hours post inoculation (hpi), these were down-regulated 48 hpi. Plant responses did not prevent F. verticillioides from colonising maize kernels, as the target DNA of the pathogen continued to increase. At 72 hpi, genes involved in pathogenesis, G-coupled receptor signalling and response to oxidative stress were induced and may have resulted in the reduction of fungal contamination 7 dai. The transcriptional changes in maize suggest a delayed plant response to F. verticillioides infection and imply a pathogen-associated molecular pattern response characterised by a basal immunity. Several genes including PRRs, signalling molecules (protein kinases, calcium-dependant molecules, GTP-signalling and redox-associated molecules), PR protein-coding genes and those involved in secondary hormone signalling (auxins) that influence maize response to F. verticillioides warrant further investigation by genomic and proteomic approaches.

AFRIKAANSE OPSOMMING: Fusarium verticillioides is 'n belangrike patogeniese swam van mielies en word ge-assosïeer met die gewas in alle produksie areas wêreldwyd. Dit is die mees algemene swamsiekte van Suid-Afrikaanse mielie graan en die swam kan Fusarium kop vrot (FKV) veroorsaak. Die teenwoordigheid van F. verticillioides kan graanopbrengs en gehalte verlaag deur die sigbare besmetting en/of verkleuring van die pitte wat die gradering by silo's verlaag. Die swam kan ook graan besmet in die afwesigheid van sigbare simptome. Verder, kan dit skadelike sekondêre metaboliete, bekend as fumonisiens, produseer. Fumonisiens word ge-assosïeer met verskeie gesondheidsrisikos van mense en diere. Die wydverspreide voorkoms van fumonisiens in mielies en mielie produkte het gelei tot die instelling van maksimum toelaatbare vlakke van fumonisiens in voedsel en voer in talle lande. Bewerkings praktyke, veral gefokus op optimale plantproduksie as 'n siektebestuur strategie, het getoon dat dit fumonisiens besoedeling kan verminder. Dit is egter gasheerplant weerstand wat beskou word as die mees haalbare, ekonomiese en omgewingsvriendelike benadering tot F. verticillioides en sy fumonisiens. In hierdie studie is die reaksie van mielie teellyne teenoor infeksie en fumonisien neerlaag deur F. verticillioides in 'n multi-omgewing proef oor 2 jaar geëvalueer. Hierdie mielie lyne was vergelyk met lyne wat voorheen gekaraktiseerd is vir hul reaksie op FKV en fumonisiens. Die reaksie stabiliteit van die teellyn oor die verskillende lokaliteite is ook bepaal om lyne met 'n breë of spesifieke aanpasbaarheid vir siekte weerstand te identifiseer. Die lyne CML 390, RO 424W, VSA 2540W en VO 617y-2, voorheen bepaal as weerstandbiedend, het konstant lae FKV (≤5%), swam teiken DNA (≤0.1 ng μL-1) en fumonisien vlakke (≤5 dpm) by die meeste omgewings getoon. Lyn RO 424W was die mees stabiel in sy reaksie op die siekte en fumonisiens opeenhoping oor alle omgewings. Die ongekaraktiseerde teellyne was hoogs vatbaar vir F. verticillioides en fumonisiens opeenhoping met slegs CB-222 en CB-248 wat intermediêre weerstand getoon het. Die weerstandbiedende lyne wat in hierdie studie ge-identifiseer was kan dien as bronne van weerstand in teelprogramme, maar kan ook gebruik word in genomiese en proteomiese studies om die genetiese basis van weerstand teenoor F. verticillioides en sy fumonisins beter te verstaan. Die reaksie van Keniaanse mielie teellyne, voorheen gekenmerk vir weerstand teen Aspergillus flavus (wat Aspergillus kop vrot [AKV] veroorsaak) en aflatoksiene, om F. verticillioides en fumonisiens besmetting te weerstaan is in Suid-Afrika en Kenia geëvalueer. Algemene weerstand meganismes tot hierdie patogene is voorheen aangemeld. Hierdie AKV/aflatoksien-weerstandbiedende lyne was met die FKV/fumonisien- weerstandbiedende Suid-Afrikaanse lyne vergelyk. Keniaanse teellyne CML 495, CML 264 en CKL05015 was die mees weerstandbiedend teen FKV, F. verticillioides kolonisasie en fumonisiens opeenhoping oor lokaliteite. Die lyn CML 495 was ook die mees stabiel in sy weerstandreaksie op F. verticillioides infeksie en fumonisiens en maak dit 'n aantreklike bron van weerstand vir insluiting in 'n Suid-Afrikaanse teelprogram. Hierdie studie lewer dus verdere bewyse dat AKV/aflatoksien-weerstandige lyne 'n belangrike bron van weerstand teen F. verticillioides en fumonisins kan wees. Genetiese variasie van sewe mielie teellyne, met die oog om weerstand teen F. verticillioides en fumonisins opeenhoping te genereer, is aan gamma bestraling blootgestel. Die massa bestraalde lyne was in die veld geplant en self-bestuif vir vier agtereenvolgende seisoene (M0-M4 geslagte). Die reaksie van die M1 tot M4 generasies op F. verticillioides infeksie is visueel beoordeel vir FKV simptome en mielie koppe met minder as 10% FKV is tot die volgende teelsiklus oorgedra. Geselekteerde M4 koppe is ook vir kolonisasie en fumonisins inhoud geëvalueer. 'n Aantal geselekteerde M4 mieliekoppe, afgelei van elke teellyn, was meer bestand teen FKV, F. verticillioides kolonisasie en fumonisiens opeenhoping in vergelyking met hul onbestraalde oorspronklike ouer teellyn. Die bestraalde nageslag van lyn I-35 was nie meer siektebestand nie. Die transkripsionele veranderinge in mielies, as gevolg van infeksie deur F. verticillioides, is geëvalueer deur die volgende generasie RNA basispaarvolgorde en gemonitor vanaf nul tot 7 dae na inokulasie (dni). Aanvanklik is verdedigings gene wat verband hou met patogeen erkenning, sein molekules, patogenese verwante gene, herstrukturering van die selwand en sekondêre hormoon gebaseer sein gene 24 uur na inokulasie (uni), geïnduseer. Hierdie gene is egter 48 hni af-gereguleer. Hierdie plant reaksies het nie verhoed dat F. verticillioides mieliepitte koloniseer nie, soos aangedui deur die verhoging van die patogeen teiken DNA. Op 72 hni, was gene betrokke in patogenese, G-gekoppelde reseptorsein en reaksie op oksidatiewe stres geïnduseer en mag dit gelei het tot die vermindering van die swam besmetting op 7 dni. Die transkripsionele veranderinge in mieliepitte dui op 'n vertraagde reaksie van die plant tot infeksie deur F. verticillioides en impliseer 'n patogeen geassosieerde molekulêre patroon (PAMP) reaksie gekenmerk deur 'n basale immuniteit. Verskeie gene, insluitend herkennings molekules, sein molekules (proteïenkinases, kalsium-afhanklike molekules, GTP-sein en redoks ge-assosïeerde molekules), PR-proteïen koderende gene en die gene wat betrokke is in sekondêre hormoon sein (ouksiene) wat mielies se reaksie op F. verticillioides beinvloed, benodig verder ondersoek deur genomiese en proteomiese benaderings.

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