Interactive effect of busseola fusca and fusarium verticillioides on ear rot and fumonisin production in maize

Thesis (PhD)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Maize is a crop of great economic importance in southern Africa, and is widely consumed as a staple food and animal feed. Production of maize, however, is hampered by pathogens and pests such as Fusarium verticillioides and the African stem borer Busseola fusca, respectively. Fusarium verticillioides infection results in Fusarium ear rot (FER) and contamination of maize kernels with fumonisin mycotoxins, while B. fusca, causes significant damage to maize tissues during larval feeding. Despite attempts to control F. verticillioides, fungal infection and fumonisin production remains a threat to maize production due to a lack of resistant maize cultivars and the inability to target the pathogen with fungicides and biocontrol products. Planting Bt maize hybrids have become an important mechanism for the management of stem borers of maize. However, the recent discovery of B. fusca resistance to Bt maize with a single crystal protein MON810 gene, indicates that care should be taken not to solely rely on this technology for the management of B. fusca. The interactive effect of B. fusca and F. verticillioides on FER and fumonisin production in maize was investigated in this study. Maize ears were inoculated with F. verticillioides alone, with both F. verticillioides and B. fusca, and with B. fusca alone. Fusarium verticillioides isolate MRC826 was inoculated by injecting a spore suspension of the fungus into the silk channel of each primary ear at the blister stage. For B. fusca infestation, aliquots of 10-15 neonate larvae were deposited into the whorl of each plant at the 12-13th leaf stage before tasselling using a mechanical applicator. Maize ears were also mechanically wounded at the blister stage with a cork borer (different sizes and number of wounds) to mimic hail damage, and half of the wounds infected with F. verticillioides. Results from this study indicated that the impact of B. fusca infestation on FER varied seasonally, possibly due to its sporadic damage to maize ears. Busseola fusca, however, did not result in a significant increase in fumonisin production. The severity of wounding of maize ears was an important contributor to FER development and fumonisin production. The effect of host plant genetic modification and pesticide application on FER and fumonisin production in maize was investigated by studying the response of a Bt hybrid and its non-Bt isohybrid to F. verticillioides infection and B. fusca infestation; and by treating plants with Beta-cyfluthrin (non-systemic) and Benfuracarb (systemic) insecticides. The field trials were conducted over three seasons using a randomised complete block design with six replicates per treatment. Uninoculated, uninfested and undamaged control treatments were included. All ears were harvested at physiological maturity and FER, total fumonisin concentration, stem borer cumulative tunnel length (B. fusca damage) and target DNA of fumonisin-producing Fusarium spp. were quantified. Busseola fusca infestation had no effect on fungal colonisation and fumonisin production in maize. Bt and non-Bt kernels were equally contaminated with fungal DNA, but FER and fumonisin production were reduced in the Bt hybrid under natural farming conditions. Despite the evidence found in this study and others that Bt maize indirectly reduces FER and fumonisin production, this was also inconsistent over seasons. Benfuracarb controlled stem borers, and therewith indirectly reduced FER and fumonisin production. FER development and fumonisin production by F. verticillioides varied over seasons, indicating the importance of environmental conditions on FER and fumonisin production. A survey was also conducted at two sites in the North West province and one site in the Free State province of South Africa to analyse mycoflora in B. fusca frass. The exposure of B. fusca larvae to F. verticillioides in stem borer frass was also evaluated in both greenhouse and field trials. Maize whorls were inoculated with a spore suspension of F. verticillioides MRC826 4 weeks after plant emergence and infested with aliquots of 5-10 neonate B. fusca larvae 2 days later. The control treatment consisted of B. fusca infestation only. Several fungal species were associated with stem borer frass, including Acremonium zeae, Aspergillus flavus, A. niger, F. chlamydosporum, F. incarnatum-equiseti species complex, F. oxysporum, F. subglutinans, F. verticillioides, Mucor circinelloides, Rhizopus oryzae and Talaromyces flavus. The occurrence of A. niger in the frass suggests that further studies need to be conducted to determine the effect of A. niger infection on fumonisin production in maize in South Africa. DNA quantity of fumonisin-producing Fusarium spp. was significantly more in frass collected from greenhouse plants inoculated with F. verticillioides and infested with B. fusca larvae than in frass collected from the uninoculated and infested control, whilst the field trial showed no significant differences in quantity of target DNA in frass from inoculated and uninoculated plants infested with B. fusca larvae. This indicates that plants in the field were naturally infected with F. verticillioides. This study showed that Bt maize had no effect on infection of maize ears by fumonisinproducing Fusarium spp. and the subsequent production of fumonisin in F. verticillioidesinoculated maize ears, indicating that the effect of Bt maize on fumonisin production in maize ears is indirectly associated with its control of severe stem borer damage. Busseola fusca frass was a reservoir of different fungal species; some pathogenic to maize, and others antagonistic to maize pathogens. Moreover, B. fusca infestation of maize stems was associated with higher levels of fumonisin-producing Fusarium spp. in larval frass when F. verticillioides was present on the plant. Multiple large wounds created by cork borers resulted in significantly more FER symptoms and fumonisin production, irrespective of artificial F. verticillioides inoculation of maize ears whereas B. fusca infestation resulted in a significant increase in FER in only one of the three seasons, moreover, it had no effect on fumonisin production in all three seasons. This indicates that severe wounds that opens up husk coverage and exposes maize kernels; caused by factors such as insects, hail and bird damage, and damage by implements; are important entry points for F. verticillioides that may lead to the transition from symptomless infection to necrotrophic pathogenicity resulting in FER and concomitant fumonisin production in maize kernels. However, climatic conditions are also important in FER and fumonisin production in maize. Moreover, Acremonium zeae endophytes occurring in frass can be used for the biological control of F. verticillioides resulting in the management of FER and subsequent fumonisin production.AFRIKAANSE OPSOMMING: Mielies is ‘n gewas van groot ekonomiese belang in suidelike Afrika wat wyd as stapel voedsel en veevoer dien. Mielieproduksie word egter belemmer deur onderskeidelik patogene en peste soos Fusarium verticillioides en die Afrika stamboorder, Busseola fusca. Fusarium verticillioides infeksie lei tot Fusarium kopvrot (FKV) en die produksie van fumonisien mikotoksiene, terwyl die Afrika stamboorder, Busseola fusca, beduidende skade verrig aan mielieweefsel tydens larf voeding. Ondanks pogings om F. verticillioides te beheer, bly infeksie en fumonisienproduksie ‘n bedreiging vir mielieproduksie weens die tekort aan weerstandbiedende mieliekultivars en die onvermoë om die patogeen te teiken met swamdoders en biologiese beheerprodukte. Die aanplanting van Bt mieliebasters is ’n belangrike meganisme vir die bestuur van mieliestamboorders. Die onlangse ontdekking van B. fusca weerstand teen Bt mielies met ‘n enkele kristal proteïen geen, dui egter daarop dat daar nie slegs op hierdie tegnologie gesteun moet word vir die bestuur van B. fusca nie. Die interaktiewe effek van B. fusca en F. verticillioides op FKV en fumonisienproduksie in mielies is tydens hierdie studie ondersoek. Mieliekoppe is slegs F. verticillioides geïnokuleer, met beide F. verticillioides en B. fusca, en met slegs B. fusca. Fusarium verticillioides isolaat MRC826 is geïnokuleer deur ‘n spoorsuspensie van die fungus in te spuit in die sy kanaal van elke primêre kop tydens die blaas fase. Vir B. fusca infestasie, is afmetings van 10-15 neonaat larwe gedeponeer in die krans van elke plant by die 12-13de blaarfase voor pluimvorming, deur gebruik te maak van ‘n meganiese toediener. Mieliekoppe is ook meganies gewond tydens die blaasfase met ‘n kurkboorder (verskillende groottes en hoeveelhede wonde), om sodoende haelskade na te boots, en die helfde van die wonde is geïnfekteer met F. verticillioides. Resultate van hierdie studie het aangedui dat die impak van B. fusca infestasie op FKV seisoenaal variasie getoon het, moontlik weens die sporadiese skade aan mieliekoppe. Busseola fusca infestasie het egter nie gelei tot ‘n beduidende toename in fumonisienproduksie nie. Die graad van verwonding van die mieliekoppe was ‘n belangrike bydraer tot FKV ontwikkeling en fumonisienproduksie. Die effek van gasheer plant genetiese verandering en plaagdoder toediening op FKV en fumonisienproduksie in mielies is ondersoek deur die reaksie te bestudeer van ‘n Bt kruising en sy nie-Bt iso-kruising tot F. verticillioides infeksie en B. fusca infestasie; en deur die behandeling van plante met Beta-cyfluthrin (nie-sistemies) en Benfuracarb (sistemies) insekdoders. Die veldproewe is oor ‘n tydperk van drie seisoene uitgevoer met die gebruik van ‘n ewekansige volledige blokontwerp met ses herhalings per behandeling. Ongeïnokuleerde, nie-geïnfesteerde en onbeskadigde kontrole behandelings is ingesluit. Alle koppe is geoes by fisiologiese volwassenheid en FKV, totale fumonisien konsentrasie, stamboorder kumulative tonnel lengte (B. fusca skade) en die hoeveelheid teiken DNA van fumonisien-produserende Fusarium spp. is gekwantifiseer. Busseola fusca infestasie het geen effek gehad op swam kolonisasaie en fumonisienproduksie in mielies nie. Bt en nie-Bt pitte is eweveel gekontamineer met swam DNA, maar FKV en fumonisienproduksie het afgeneem in die Bt kruising. Ten spyte van die bewyse gevind in hierdie en ander studies dat Bt mielies indirek FKV en fumonisienproduksie verminder, was dit ook strydig oor seisoene. Benfurakarb het stamboorders beheer, en saam met dit ook indirek FKV en fumonisienproduksie verminder. FKV ontwikkeling en fumonisienproduksie deur F. verticillioides het oor seisoene gevarieer, wat dui op die belangrikheid van omgewingstoestande vir FKV en fumonisienproduksie. ‘n Opname is ook by twee areas in die Noordwes Provinsie en een area in die Vrystaat Provinsie van Suid-Afrika uitgevoer, om mikoflora in B. fusca wurmboorsel te analiseer. Blootstelling van B. fusca larwe teenoor F. verticillioides in staboorder wurmboorsel is ook geëvalueer in beide kweekhuis- en veldproewe. Mieliekranse is met ‘n spoorsuspensie van F. verticillioides MRC 826 geïnokuleer 4 weke na opkoms en 2 dae later geïnfesteer met afmetings van 5-10 neonaat B. fusca larwe. Die kontrole behandeling het bestaan uit slegs B. fusca infestasie. Verskeie swamspesies is geassosieer met stamboorder wurmboorsel, insluitend Acremonium zeae, Aspergillus flavus, A. niger, F. chlamydosporum, F. incarnatumequiseti spesiekompleks, F. oxysporum, F. subglutinans, F. verticillioides, Mucor circinelloides, Rhizopus oryzae en Talaromyces flavus. Die voorkoms van A. niger in die wurmboorsel dui daarop dat verdere studies uitgevoer moet word om die effek van A. niger infeksie op fumonisienproduksie in mielies in Suid-Afrika te bepaal. DNA hoeveelheid van fumonisien-produserende Fusarium spp. was betekenisvol hoër in wurmboorsel wat versamel is van kweekhuisplante geïnokuleer met F. verticillioides en geïnfesteer met B. fusca larwe as in wurmboorsel versamel vanaf die ongeïnokuleerde kontrole terwyl die veldproef geen betekenisvolle verskille in kwantiteit teiken DNA in wurmboorsel vanaf geïnokuleerde en nie-geïnokuleerde plante geïnfesteer met B. fusca larwe getoon het nie. Dit dui daarop dat plante in die veld natuurlik deur F. verticillioides geïnfekteer is. Die studie het aangedui dat Bt mielies geen effek op infeksie van mieliekoppe het by fumonisien produserende Fusarium spp. en vervolgens fumonisienproduksie in geïnokuleerde F. verticillioides mieliekoppe nie. Dit toon dat die effek van Bt mielies op fumonisienproduksie in mieliekoppe indirek met die beheer van stamboorder skade geassosieer is. Hierdie studie het getoon dat B. fusca wurmboorsel as reservoir gedien het vir verskeie swamspesies; sommiges patogenies vir mielies, en ander antagonisties tot mieliepatogene. Met infestasie van B. fusca op mielie stamme word ondervind dat hoër vlakke van fumonisien produserende Fusarium spp. in larwe wurmboorsel geassosieer word met teenwooidigheid van F. Die studie het aangedui dat Bt mielies geen effek op infeksie van mieliekoppe het by fumonisien produserende Fusarium spp. en vervolgens fumonisienproduksie in geïnokuleerde F. verticillioides mieliekoppe nie. Dit toon dat die effek van Bt mielies op fumonisienproduksie in mieliekoppe indirek met die beheer van stamboorder skade geassosieer is. Hierdie studie het getoon dat B. fusca wurmboorsel as reservoir gedien het vir verskeie swamspesies; sommiges patogenies vir mielies, en ander antagonisties tot mieliepatogene. Met infestasie van B. fusca op mielie stamme word ondervind dat hoër vlakke van fumonisien produserende Fusarium spp. in larwe wurmboorsel geassosieer word met teenwooidigheid van F. verticillioides op plante. Verskeie groot wonde veroorsaak deur kurkboorders het gelei tot beduidend meer FKV simptome en fumonisienproduksie, ongeag kunsmatige F. verticillioides inokulasie van mieliekoppe, terwyl B. fusca infestasie gelei het tot ‘n beduidende toename in FKV in slegs een van die drie seisoene. Verder het dit geen effek gehad op fumonisienproduksie in enige van die drie seisoene nie. Dit dui daarop dat wonde wat dopbedekking oopmaak en pitte blootstel; veroorsaak deur faktore soos insekte, haelskade, voëlskade, en implimentskade; belangrike toegangspunte is vir F. verticillioides wat mag lei tot die oorgang van simptoomlose endofitisme na nekrotrofiese patogenisiteit wat lei tot FKV en gepaardgaande fumonisienproduksie in mieliepitte. Klimaatstoestande is egter ook belangrik in FKV en fumonisienproduksie in mielies. Verder, kan Acremonium zeae endofiete wat in wurmboorsel voorkom vir die biologiese beheer van F. verticillioides gebruik word wat lei tot die bestuur van FKV en gevolglike fumonisienproduksie.National Research Foundation (NRF