Investigating biological control agents for the management of Ceratitis capitata (Wiedemann)

Thesis (MScConsEcol)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Persistent fruit damage and loss caused by fruit flies (Diptera: Tephritidae) has occasioned the reliance on chemical control methods for their management in the fruit industry. However, social, environmental and economic consequences associated with such control methods have necessitated the need for the exploration of alternative, more sustainable and eco-friendly options. This study investigates the use of entomopathogenic nematodes (EPNs), entomopathogenic fungi (EPF) and parasitoid wasps, as biological control agents against one of the most widespread and dominant fruit flies in South Africa, the Mediterranean fruit fly or Medfly (Ceratitis capitata). Different methods were used in order to (i) isolate and identify local EPNs and EPF from fruit orchard soils; (ii) evaluate the pathogenicity of local EPN and EPF isolates against the third larval instar stage of Medfly under controlled laboratory conditions, and selected species of each in a more natural (sand) environment; (iii) estimate the lethal concentration/dose needed to result in 50% C. capitata mortality (LD50) using selected EPN isolates; and (iv) survey for and identify fruit fly parasitoid species occurring in the Western Cape, Mpumalanga and Limpopo Provinces. Soil sampling yielded a number of local entomopathogenic isolates, including EPNs with new bacterial associations. Similarly, an EPF, Metarhizium robertsii (MJ06), was also trapped using Medfly larvae as bait. Initial EPN screenings (100 IJs / 50 μl) showed all tested EPNs to be highly pathogenic against third instar Medfly larvae, while at the lower concentration (50 IJs / 50 μl), Heterorhabditis noenieputensis, was the most virulent EPN species. This species, as well as Steinernema yirgalemense, which is currently in the process of being mass cultured and formulated for commercial use, was further, tested in sand bioassays. H. noenieputensis caused significantly higher mortality (94-100%) as most Medfly infected as larvae pupated, but died within the puparium. S. yirgalemense also offered good control, with 58-79% of exposed larvae dying as adults. The LD50 of H. noenieputensis was 37 IJs / insect, which was 14 times more effective than that of S. yirgalemense. Local EPF isolates and commercial products tested against third instar larvae, using the dipping method, were pathogenic and caused visible fungal infection (mycosis) of 57-74%. Reduction of humidity also reduced overall mycosis, with the highest mycosis of 55% due to the local isolate, MJ06. Third instar Medfly larvae added to sand and sprayed with the soil-collected EPF M. robertsii (MJ06) and Beauveria bassiana (6756), died and mycosed as adults (62-86%). Parasitoid wasps were obtained during fruit sampling, but difficulties with low DNA extraction, amplification and limited available barcodes of local fruit fly parasitoids, restricted their species identification. The use of sentinel traps - setting out apples infested with Medfly eggs, larvae and exposed pupae - did not trap any wasps during this study, but provides a simple and inexpensive method to be used in future studies. This study documents an EPN (H. noenieputensis SF669) and EPF (M. robertsii MJ06), virulent against the soil-life stages of Medfly, which could be the focus of future studies as potential biocontrol agents. Moreover, this study provides novel data on additional biological control agents that could be incorporated into an overall integrated pest management system (IPM) system, to sustainably and effectively manage the Mediterranean fruit fly.AFRIKAANSE OPSOMMING: Die voortdurende skade en verliese veroorsaak deur vrugtevlieë (Diptera: Tephritidae), het die vrugtebedryf forseer om afhanklik te wees van chemiese beheermetodes. Negatiewe sosiale, omgewings en ekonomiese gevolge wat met hierdie metodes gekoppel is, het die soektog na alternatiewe, vir meer volhoubare en omgewingsvriendelike, beheermiddels genoodsaak. Hierdie studie ondersoek die gebruik van entomopatogeniese nematodes (EPNs), entomopatogeniese swamme (EPF) en parasitiese wespe, as biologiese beheeragente vir die wydverspreide vrugtevlieg in Suid-Afrika, die Mediterreense vrugtevlieg of Medvlieg (Ceratitis capitata). Verskeie metodes was gebruik om (i) plaaslike EPNs en EPF van vrugteboord grond te isoleer en identifiseer; (ii) die patogenisiteit van inheemse EPN en EPF isolate teen die derde larwe stadium van Medvlieg te evalueer onder optimum laboratorium toestande en van die effektiefste isolate te selekteer en te toets in ʼn meer natuurlike (sand) omgewing; (iii) te bepaal watter konsentrasie benodig word om 50% mortaliteit (LD50) te veroorsaak in die C. capitata populasie deur die gebruik van geselekteerde isolate; en (iv) ʼn opname van wespe, wat parasiete is van vrugtevlieë, in die Wes-Kaap, Mpumalanga en Limpopo provinsies te identifiseer. Die versameling van grondmonsters het ʼn groot opbrengs van inheemse entomopatogeniese isolate voortgebring, insluitende EPNs met nuwe bakteriële assosiasies. Die EPF, Metarhizium robertsii (MJ06), was ook geïsoleer direk vanuit ʼn Medvlieg larwe. Tydens die bepaling van die graad van vatbaarheid van die derde larwe stadium van die Medvlieg vir EPN infeksie (100 IJs / 50 μl), is daar bevind dat al die EPNs hoogs effektief teen die vlieg-pes is, maar met laer konsentrasie (50 IJs / 50 μl), was Heterorhabditis noenieputensis die effektiefste EPN. Laasgenoemde spesie, sowel as Steinernema yirgalemense, wat tans in die proses is om geformuleer te word vir kommersiële gebruik, het verdere toetse ondergaan deur die gebruik van ʼn sand bioassessering-sisteem. Heterorhabditis noenieputensis het ʼn wesenlike hoër mortaliteit (94-100%) getoon, en alhoewel die meeste Medvlieg larwes papies geword het, was die meerderheid geïnfekteer met nematodes. Steinernema yirgalemense het effektiewe beheer getoon met 58-79% van die larwes wat as volwassenes doodgegaan het. Die LD50 van H. noenieputensis was 37 IJs / insek, wat 14 keer meer effektief was as S. yirgalemense. Plaaslike EPF isolate en kommersiële produkte is teen die derde instar larwes getoets deur gebruik te maak van die dip metode. Al die EPF isolate was patogenies en het sigbare infeksie (mikose) van 57-74% veroorsaak. Verlaagde humiditeit het mikose laat daal, en veroorsaak dat die inheemse isolaat, MJ06, die hoogste mikose van 55% getoon het. Derde instar larwes is by sand gevoeg en behandel met die EPF, M. robertsii (MJ06) en Beauveria bassiana (6756) (wat gedurende die plaaslike grond opname geïsoleer was), het as volwassenes doodgegaan en mikose ondergaan (62-86%). Parasitiese wespe was deur vrugte versameling gevind, maar weens uitdagings van lae DNA konsentrasies, versterkings en beperkte beskikbaarheid van die strepie-kodes van Suid-Afrikaanse wespe wat parasities is tot vrugtevlieë, het spesies identifisering ingeperk. Die gebruik van sentinel lokvalle – wat die uiteensetting van appels wat infesteer is met Medvlieg eiers, larwes en papies is – het geen wespe gelok gedurende die studie nie, maar voorsien ʼn eenvoudige en goedkoop metode om van gebruik te maak in toekomstige studies. Die studie dokumenteer potensiële EPN (H. noenieputensis SF669) en ʼn EPF (M. robertsii MJ06) kandidate, wat effektiewelik beheer toon van die grond stadia van Medvlieg, en kan in toekomstige studies ʼn fokuspunt as biologiese beheermiddels gebruik word. Die meesterstudie voorsien nuwe opwindende navorsing op potensiële biologiese beheermaatreëls wat gebruik kan word in ʼn geïntegreerde plaagbeheerprogram, om die Mediterreense vrugtevlieg op ʼn volhoubare en doeltreffende manier te bestuur