Biological control of the banded fruit weevil, Phlyctinus callosus (Schönherr), using entomopathogenic nematodes and fungi

Thesis (PhDConsEcol)--Stellenbosch University, 2018.

ENGLISH ABSTRACT: The overall aim of this study was to establish guidelines, from research undertaken mainly in the laboratory, and from field application, for the use of entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPF) to control the banded fruit weevil (BFW), Phlyctinus callosus (Schönherr), in vineyards and apple orchards in the Western Cape province. Discovering new species of entomopathogens is important, as more virulent, locally adapted species can be used to increase the potential of microbial control. The first objective of this study was to survey for EPNs and EPF strains/species in orchards and vineyards of the Western Cape, and to investigate their potential to control the BFW. The most virulent EPN species for the biological control of the BFW were selected in laboratory bioassays, and a field trial using Steinernema yirgalemense at different concentrations was conducted. In a further study, the best EPF strains for the biological control of the BFW were selected in laboratory bioassays. The most virulent EPN and EPF isolates were applied, in combination, to determine their interaction-effect on the mortality of BFW in the laboratory, as the final objective. In total, 70 soil samples were collected from deciduous fruit orchards and vineyards in the Western Cape. The soil samples were baited with mealworms, Tenebrio molitor (Coleoptera: Tenebrionidae) to trap EPNs and EPF, which were characterised by using morphological and molecular techniques, and evaluated for their potential to control the BFW adults. EPNs were isolated from 17 % (12) of the samples, with Heterorhabditis bacteriophora Poinar and Heterorhabditis safricana as the only two EPN species isolated. Heterorhabditis bacteriophora (53 % mortality) resulted in significantly higher (p < 0.05) mortality of adult BFW, compared to H. safricana (37 % mortality), in laboratory trials. EPF were trapped from 37 % (26) of samples, consisting of a total of 14 Beauveria bassiana isolates and 12 isolates from the Metarhizium anisopliae complex. A Metarhizium isolate (79 % mortality) resulted in significantly higher (p < 0.05) mortality of BFW adults, compared to B. bassiana (63 % mortality) in laboratory trials. Results obtained from the study indicate that the soil samples from the deciduous fruit orchards and vineyards from the Western Cape contained both EPNs and EPF that can be used to control BFW. Different EPN species were evaluated by laboratory screening for virulence against the different life stages of the BFW. A field trial to determine the performance of Steinernema yirgalemense, applied at different concentrations, followed. Results from a probit analysis showed S. yirgalemense to be six times more potent than Heterorhabditis noenieputensis, giving 95 % mortality of BFW larvae at a concentration of 400 infective juveniles (IJ) per insect, which was significantly higher (p < 0.05) compared to other concentrations. At a concentration of 100 IJs/insect, Steinernema yirgalemense, H. noenieputensis, and Steinernema feltiae resulted in significantly higher (p < 0.05) mortality of BFW larvae compared to the other EPNs, with no significant difference between each other. Heterorhabditis indica (70 % mortality) and H. baujardi (67 % mortality) resulted in significantly higher (p < 0.05) mortality of the BFW pupae, compared to H. noenieputensis (55 % mortality). In the case of adult BFW, Heterorhabditis indica (95 % mortality) and S. yirgalemense (94 % mortality) gave significantly (p < 0.05) higher mortality, compared to three other EPN species evaluated. In the field trials, S. yirgalemense, at 20 and 40 IJs/cm2, gave 69 % and 78 % mortality of BFW larvae, respectively. The results showed that all EPNs screened controlled the juvenile and adults stages of BFW. Steinernema yirgalemense was also shown to be capable of controlling BFW under field conditions, even at low concentrations. Different Different EPF isolates were tested at 1 × 106 conidia ml-1 for their virulence against the different life stages of the BFW. Results showed that Broadband® (Beauveria bassiana strain PPRI5339) (97 %) and Meta 69 (Metarhizium anisopliae) (93 %) gave significantly higher (p < 0.05) mortality of BFW larvae compared to Eco-Bb® (B. bassiana) (58 %), with no significant difference in mortality between Broadband® and Meta 69. In the case of pupae, Broadband® (92 %) gave significantly higher (p < 0.05) mortality, compared to Eco-Bb® (67.5 % mortality) and Meta 69 (65.8 % mortality). Broadband® (90 % mortality) gave significantly higher (p < 0.05) mortality of adult BFW compared to Eco-Bb® (69.2 % mortality) and Meta 69 (65 % mortality), of which neither differed from each other. When compared to a local EPF (M. anisopliae EA2), Broadband® (91.7 % mortality) gave significantly (p < 0.05) higher mortality of BFW adults, compared to EcoBb® (65 % mortality), Meta 69 (59.5 % mortality) and M. anisopliae isolate EA2 (64.2 % mortality), which did not differ significantly from one another. In a sand bioassay, Broadband® (85 % mortality) resulted in significantly (p < 0.05) higher mortality of BFW larvae compared to Eco-Bb® (55 % mortality) and Meta 69 (70 % mortality). The results indicated that all the EPF isolates tested were effective against juvenile and adults stages of the BFW, with Broadband® showing the most promise in controlling the BFW under laboratory conditions. The last objective of the study was to evaluate the combined use of the EPF; Eco-Bb®, Broadband®, Meta 69 and a local isolate M. anisopliae EA2, with the EPN, S. yirgalemense against BFW larvae and adults. The EPF were either applied alone, or at the same time as S. yirgalemense, or S. yirgalemense was introduced 1 and 2 weeks after fungal application; the EPNs were also applied alone. Results showed that 100% larval and adult mortality was obtained when S. yirgalemense was applied 1 or 2 weeks after Eco-Bb® and BroadBand® application. Synergistic interactions were noted when Eco-Bb® and S. yirgalemense, BroadBand® and S. yirgalemense, Meta 69 and S. yirgalemense, and M. anisopliae isolate EA2 and S. yirgalemense when applied 1 or 2 weeks after application of the EPF. Additive interactions were observed when the EPF and S. yirgalemense were applied simultaneously. Future research into the biological control of the BFW, using EPNs and EPF, should be focused on conducting large-scale field trials to demonstrate their potential use as biocontrol agents, within an integrated pest management programme. Both entomopathogens have shown outstanding potential to control the BFW when used alone, and in combination, could provide an economically viable control strategy against the BFW.

AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om riglyne vas te stel, vanuit die resultate van navorsing in die laboratorium, asook veldproewe, vir die gebruik van entomopatogeniese nematodes (EPNs) en entomopatogeniese swamme (EPS) vir die beheer van die gebande vrugtekalander (GVK) in wingerde en appelboorde in die Wes-Kaap provinsie. Die ontdekking van nuwe spesies van entomopatogene is belangrik, omdat spesies wat plaaslik aangepas en effektief is vir gasheer spesies, gebruik kan word om die potensiaal van mikrobiese beheer te verhoog. Die eerste doel met hierdie studie was dus om ʼn opname te doen van EPN en EPS in boorde en wingerde van die Wes-Kaap en om dan hul potensiaal om die GVK te beheer, te ondersoek. Biotoetse in die laboratorium het getoon dat Steinernema yirgalemense die effektiefste EPN spesie is vir die biologiese beheer van die GVK en is daarna gebruik vir veldproewe by verskillende konsentrasies. In ʼn verdere studie is die beste EPS spesies vir die biologiese beheer van die GVK deur middel van biotoetse in die laboratorium geselekteer. As die laaste doel van die studie, is die effektiefste EPN en EPS isolate in kombinasie aangewend om die effek van hul interaksie te bepaal op die mortaliteit van die GVK in die laboratorium. ʼn Totaal van sewentig grondmonsters is versamel vanuit sagtevrugte boorde en wingerde in die Wes-Kaap. Meelwurms, Tenebrio molitor (Coleoptera: Tenebrionidae), is gebruik as lok gashere en geplaas op die grondmonsters om EPN en EPS te isoleer. Hierdie spesies was dan gekarakteriseer deur gebruik te maak van morfologiese en molekulêre tegnieke en hul potensiaal vir die beheer van die volwassene GVK, geëvalueer. EPNs is geïsoleer vanuit 17 % (12) van die grondmonsters, met Heterorhabditis bacteriophora Poinar en Heterorhabditis safricana Malan, Nguyen, De Waal & Tiedt as die enigste twee EPN spesies wat gevind is. Gedurende proewe in die laboratorium, het H. bacteriophora (53 %) het aansienlik beter resultate gelewer (p < 0.05) in die mortaliteit van volwasse GVKs, in vergelyking met H. safricana (37 %). EPS is geïsoleer vanuit 37 % (26) van die grondmonsters en was geïdentifiseer as 14 Beauveria bassiana isolate en 12 isolate van die Metarhizium anisopliae kompleks. ʼn Metarhizium isolaat (79 %) het aansienlik beter resultate getoon (p < 0.05) in mortaliteit van volwasse GVKs, in vergelyking met B. bassiana (63 %) in laboratorium proewe. Resultate van die studie toon dat die grondmonsters van die sagtevrugte boorde en wingerde in die Wes-Kaap beide EPNs en EPS bevat het wat gebruik kan word vir die beheer van grondlewende, asook nie-grondlewende fases van GVKs. Die effektiwiteit van verskillende EPN spesies is vergelyk deur hul vermoë om die verskillende lewensfases van die GVK te beheer, in die laboratorium te toets. Daarna is ʼn veldproef uitgevoer om die sukses van Steinernema yirgalemense by verskillende konsentrasies te ondersoek. Resultate van ʼn probitanalise het getoon dat S. yirgalemense ses keer meer effektief is as Heterorhabditis noenieputensis. Steinernema yirgalemense het 95% mortaliteit van GVK larwes getoon teen ʼn konsentrasie van 400 infektiewe larwes (ILs)/insek, wat aansienlik hoër was (p < 0.05) as die vlak van beheer by ander konsentrasies. Steinernema yirgalemense, H. noenieputensis, en Steinernema feltiae het aansienlik beter gevaar (p < 0.05) in die mortaliteit van GVK larwes teen ʼn konsentrasie van 100 ILs/insek wanneer hul vergelyk was met ander EPNs, met geen beduidende verskil tussen mekaar nie. Heterorhabditis indica (70 %) en Heterorhabditis baujardi (67 %) het aansienlik beter gevaar (p < 0.05) in die mortaliteit van GVK papies, in vergelyking met H. noenieputensis (55 %). Heterorhabditis indica (95 %) en S. yirgalemense (94 %) het aansienlik beter resultate getoon (p < 0.05) in die mortaliteit van volwassene GVKs, in vergelyking met die ander drie EPN spesies wat getoets was. In die veldproewe het S. yirgalemense 69% mortaliteit veroorsaak in GVK larwes teen ʼn konsentrasie van 20 ILs/cm2 en 78% mortaliteit teen ‘n konsentrasie van 40 ILs/cm2. Die resultate toon dat al die EPNs wat getoets was, die vermoë het om verskillende lewensfases van GVK te beheer. Steinernema yirgalemense het ook die vermoë getoon om die GVK te beheer in die veld, selfs teen lae konsentrasies. Die vermoë van verskillende EPS isolate om die verskillende lewensfases van GVK te dood, was getoets teen ʼn konsentrasie van 1 × 106 spore ml-1. Resultate het getoon dat daar geen beduidende verskil was tussen die vermoë van Broadband® (Beauveria bassiana isolaat PPRI5339) (97 %) en Meta 69 (Metarhizium anisopliae) (93 %) om larwes van GVK te dood nie, maar dat die twee produkte wel aansienlik beter gevaar het (p < 0.05) as Eco-Bb® (B. bassiana) (58 %). Broadband® (92 %) het ook aansienlik beter gevaar (p < 0.05) as Eco-Bb® (67.5 %) en Meta 69 (65.8 %) in die mortaliteit van GVK papies. Daar was ʼn beduidende verskil (p < 0.05) tussen Broadband® (90 %) se vermoë om volwasse GVKs te dood in vergelyking met Eco-Bb® (69.2 %) en Meta 69 (65 %), wat nie beduidend verskil het van mekaar nie. Toe die vermoë van die EPS om volwasse GVKs te dood vergelyk was met In plaaslike isolaat, was daar weereens ʼn beduidende verskil (p < 0.05) tussen die resultate van Broadband® (91.7 %) in vergelyking met Eco-Bb® (65 %), Meta 69 (59.5 %) en M. anisopliae isolaat EA2 (64.2 %), wat nie beduidende verskille getoon het onder mekaar nie. In ʼn sandbiotoets, was daar ʼn beduidende verskil (p < 0.05) in die vermoë van Broadband® (85 %) om GVK larwes te dood, in vergelyking met die vermoëns van Eco-Bb® (55 %) en Meta 69 (70 %). Die resultate het getoon dat al die EPS isolate wat getoets was, effektief was teen die verskillende lewensfases van GVK, met Broadband® wat die meeste potensiaal getoon het vir mortaliteit van die GVK in die laboratorium. Die laaste doel van die studie was om die gebruik van die Eco-Bb®, Broadband®, Meta 69 en M. anisopliae EA2, ‘n plaaslike isolaat, gekombineer met S. yirgalemense, te toets teen die larwes en volwassenes van die GVK. Die EPS was of alleen, of saam met S. yirgalemense óf 1 en 2 weke na behandeling met swamme, aangewend. EPNs was ook op hul eie aangewend. Resultate het 100% mortaliteit van GVK larwes en volwassenes getoon in die behandelinge waar S. yirgalemense 1 of 2 weke na Eco-Bb® en BroadBand® aangewend is. Sinergistiese interaksies was opgemerk by behandelinge waar Eco-Bb® en EPN, BroadBand® en S. yirgalemense, Meta 69 en EPN, en M. anisopliae isolaat EA2 en S. yirgalemense aangewend is 1 of 2 weke na die aanwending van die S. yirgalemense. Bykomende interaksies is opgemerk toe die EPS en S. yirgalemense gesamentlik aangewend is. Toekomstige navorsing op die biologiese beheer van die GVK deur die gebruik van EPNs en EPS, moet fokus op die uitvoer van grootskaalse veldproewe om hul potensiaal as biologiese beheermiddels te demonstreer, tesame met ʼn geïntegreerde pes beheer program. Beide entomopatogene het uitmuntende potensiaal getoon om die GVK op hul eie te beheer en kan in kombinasie ‘n ekonomies lewensvatbare beheerstrategie bied teen die GVK.

Doctoral