Efficacy of entomopathogenic nematodes and fungi as biological control agents of woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) under South African conditions

Thesis (PhD)--Stellenbosch University, 2016.

ENGLISH ABSTRACT: The woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) is an important pest of apples, (Malus domestica Borkh.). Severe infestations by WAA can lead to colonies near spurs that can deposit honeydew on fruit, which serve as a substrate for sooty mould. If not controlled, it can lead to destruction of developing buds in the leaf axils and a reduction in tree vigour or formation of galls. Increasing interest in environmentally sustainable farming has increased the demand for environmentally friendly pest control methods. Therefore the use of naturally occurring biocontrol agents and more environmental friendly methods are needed. The study aims to determine the potential of entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPF) to control WAA. EPNs and EPF are naturally occurring and environmentally friendly microbials that have the potential to be developed into bio-pesticides. Research on the use of EPNs and EPF as biocontrol agents against E. lanigerum showed that they have potential to be used to control WAA. This is the first report on the use of EPNs and EPF to control WAA in South Africa. These findings are significant, especially to those interested in integrated pest management (IPM). The first objective of this study was to evaluate the virulence of endemic EPN species against WAA by conducting laboratory bioassays and to determine the effect of imidacloprid on the infectivity of EPNs. A total of seven entomopathogenic nematode isolates were evaluated for their potential as biological control agents for WAA in the laboratory, using a 24-well bioassay protocol screening method. From these, the two most virulent nematode isolates were selected. In all cases studied the degree of infection associated with mortality was less than 50% and the other tested nematode isolates were less successful. Although penetration, recovery of the non-feeding Infective Juveniles (IJ) to the feeding stage and development into the adult stage was observed in some cases, the nematodes failed to complete their life cycle inside the WAA. When this failure to develop and reproduce was investigated using direct screening with the associated symbiotic bacteria and insect extract, it became evident that the WAA haemolymph contains an inhibitory factor that prevented the symbiotic EPN bacteria from growing. WAA size was also shown to have an effect on insect mortality, with higher mortality observed in adults and low mortality for intermediates, while no mortality was observed for the crawlers. The two most virulent nematode isolates, Nguyen, Tesfamariam, Gozel, Gaugler & Adams, 2004 and Heterorhabditis zealandica Poinar, 1990 were not affected by the addition of imidacloprid, leading to the conclusion that EPNs and imidacloprid could potentially be applied as tank mix. The second objective of the study was to evaluate the use of two EPF, Beauveria bassiana and Metarhizium anisopliae, for the control of WAA in the laboratory. The five fungal isolates tested were pathogenic to WAA and they caused significant WAA mortality compared with the control. The commercial isolates Beauveria bassiana (Eco- Bb strain R444) and Metarhizium anisopliae (ICIPE 69) caused the highest rate of WAA mortality and were selected for further evaluation. When the effect of EPF concentration and exposure time of these two most virulent isolates were evaluated, it became evident that increased EPF concentration (1 × 107 to 1 × 1010 conidia ml-1) and exposure time increased WAA mortality. This lead to the conclusion that a relatively long incubation period and high EPF concentration are needed to achieve full efficacy of the these fungal isolates for controlling WAA. The third objective of the study was to evaluate the interaction between EPNs and EPF for the control of WAA and the effect of mulching on EPN efficacy in pot trials. Joint use of EPNs and EPF increased insect mortality when compared to treatments with only EPNs or only EPF, indicating an antagonistic effect. Combining S. yirgalamense with B. bassiana (Eco- Bb® strain R444) and M. anisopliae (ICIPE 69) provided no advantage for improved efficacy against WAA, which lead to the conclusion that M. anisopliae (ICIPE 69) or B. bassiana (Eco- Bb® strain R444) could be used for suppression of WAA. When the environment was manipulated by adding mulches, WAA mortality was slightly higher compared to the unmulched soil. However, these differences were not statistically significant. The fourth objective of the study was to evaluate the efficacy of S. yirgalamense, B. bassiana and M. anisopliae against WAA in the field. A single application of S. yirgalamense (80 IJs/cm2) B. bassiana (Eco- Bb® strain R444) (1g/L) and M. anisopliae (ICIPE 69) (200 ml/ha) effectively controlled WAA under South African field conditions. The EPF performed better than EPNs. Based on these results the success in controlling the root colonies resulted in greatly reduced infestations of the aerial parts of the apple trees in the trial. These studies attempted to determine if local EPNs and EPF are pathogenic to WAA and if they could be used to control it in the field. It was found that all the isolates tested were pathogenic to WAA in the laboratory and that S. yirgalamense, B. bassiana and M. anisopliae were the most effective isolates. They were able to control WAA under field conditions, although the degree of control was low. Therefore future studies should be designed to investigate the application and post-application conditions required for EPF to be effective with respect to WAA control and to ensure that the application of the fungi is economically viable. The role of the wetting agent in improving soil saturation, penetration and uptake of EPNs and EPF also requires to be investigated.

AFRIKAANSE OPSOMMING: Appelbloedluis, Eriosoma lanigerum, is ʼn belangrike pes van appels (Malus domestica Borkh.). Swaar besmettings van appelbloedluis lei tot die vorming van kolonies op lootaansluitings. Dit veroorsaak dat heuningdou die vrugte besmet wat bydra tot die groei van poeieragtige meeldou. Indien appelbloedluis nie beheer word nie, kan infestasies lei tot die vernietiging van oksellêre knoppe en ʼn afname in boomgroeikrag deur die vorming van voeding galle op die wortels. ʼn Toename in belangstelling in volhoubare omgewingsvriendelike boerderypraktyke het gelei tot ʼn toename in die behoefte vir volhoubare omgewingsvriendelike beheer metodes. Gevolglik word meer sisteme wat natuurlik voorkom en omgewingsvriendelik is, benodig. Die doel van hierdie studie was om die potensiële bydrae van entomopatogeniese nematodes (EPNs) en entomopatogeniese fungi (EPF) tot die beheer van appelbloedluis te bepaal. EPNs en EPF kom natuurlik in grond voor en is omgewingsvriendelike mikrobes wat oor die potensiaal beskik om as biologiese insekbeheermiddels ontwikkel te word. Navorsing op die gebruik van EPNs en EPP as biologiese beheeragente het bewys dat hulle potensiaal toon vir die beheer van appelbloedluis. Hierdie is die eerste verslag rakende die gebruik van EPNs en EPF vir die beheer van appelbloedluis in Suid-Afrika. Die bevindinge van die studie is van belang, veral vir diegene wat in geïntegreerde plaagbeheer (IPB) belang stel. Die eerste doelwit van die studie was om die virulensie van endemiese EPN spesies teen appelbloedluis deur middel van laboratoriumtoetse te evalueer en om die uitwerking van imidaclopried op die infestasievermoë van die nematodes te bepaal. ʼn Totaal van sewe EPN isolate is geëvalueer deur middel van ʼn laboratorium siftings metode wat hulle potensiaal as biologiese beheeragente teen appelbloedluis toets. Hieruit is die twee mees virulente isolate geselekteer. Die graad van infestasie gekoppel aan mortaliteit, was in alle gevalle minder as 50%. Alhoewel dit waargeneem is dat die nematodes, nadat hulle die appelbloedluis penetreer het, wel ontwikkeling het tot volwasse stadia, maar daarna almal dood gegaan het. Verdere ondersoek gedoen deur middel van direkte-toetsing met bakterieë en insekekstrak, het getoon dat ʼn onderdrukkende faktor in appelbloedluis haemolymph die EPF bakterieë verhoed het om te groei. Daar is ook bewys dat die grootte en die ontwikkeling stadium van die appelbloedluis ʼn effek op insekmortaliteit het. Hoër mortaliteit is in die volwasse stadium, laer mortaliteit in die intermediêre stadiums en geen mortaliteit het onder die kruipers voorgekom nie. Die twee mees virulente nematode isolate, Steinernema yirgalamense en Heterorhabditis zealandica was nie geaffekteer deur die byvoeging van imidaclopried nie, wat beteken dat EPN en imidakloried potensieel as tenkmengsels toegedien kan word. Die tweede doelwit van hierdie studie was om die gebruik van twee EPF, Beauveria bassiana en Metarhizium anisopliae, te evalueer vir die beheer van appelbloedluis in die laboratorium. Die vyf getoetsde fungi isolate was patogenies teenoor appelbloedluis en het betekenisvolle mortaliteit veroorsaak. Die kommersiële isolate B. bassiana (Eco-Bb isolaat R444) en M. anisopliae (ICIPE 69), het die hoogste appelbloedluis mortaliteit veroorsaak en is geselekteer vir verdere evaluering. Laboratoriumtoetse het bewys dat ʼn verhoging in EPF konsentrasie, sowel as die periode van blootstelling, ʼn verhoging in appelbloedluis mortaliteit teweeg gebring het. ʼn Relatiewe lang inkubasieperiode van, en hoë EPF konsentrasie is dus nodig om doeltreffende beheer van appelbloedluis te verkry. Die derde doelwit van die studie was om die interaksie tussen EPNs en EPF vir die beheer van appelbloedluis, sowel as die effek van ʼn deklaag op EPN effektiwiteit in potproewe te bepaal. Gesamentlike toediening van S. yirgalamense met B. bassiana (Eco-Bb® isolaat R444) en M. anisopliae (ICIPE 69) het nie effektiwiteit teenoor appelbloedluis verhoog nie, en die gevolgtrekking is gemaak dat slegs M. anisopliae (ICIPE 69) of B. bassiana (Eco-Bb® isolaat R444) aangewend kan word vir die onderdrukking van appelbloedluis. Manipulasie van die omgewing, deur die gebruik van deklae, het die mortaliteit van appelbloedluis effens verhoog in vergelyking met grond sonder deklae, maar die verskille waargeneem was nie statisties betekenisvol nie. Die vierde doelwit van die studie was om die effektiwiteit van S. yirgalamense, B. bassiana en M. anisopliae teenoor appelbloedluis in veldproewe te evalueer. ʼn Enkel toediening van S. yirgalamense, B. bassiana (Eco-Bb® isolaat R444) en M. anisopliae (ICIPE 69) het appelbloedluis effektief onder Suid-Afrikaanse toestande beheer. Die EPF isolate het beter gevaar as die EPNs. Hierdie studie het gewys dat suksesvolle beheer van appelbloedluis se wortelkolonies grootliks kan bygedra het tot ʼn verlaging in die voorkoms van bogrondse kolonies op die appelbome. Bogenoemde studies het gepoog om te bepaal of plaaslike EPNs en EPF patogenies is teenoor appelbloedluis en of dit gebruik kan word vir die beheer van appelbloedluis in die veld. Dit is bevind dat getoetse isolate wel patogenies was in die laboratorium teenoor appelbloedluis met S. yirgalamense, B. bassiana en M. anisopliae as die mees effektiewe isolate. Alhoewel beheer van appelbloedluis onder veldtoestande waargeneem is, was die graad van beheer relatief laag. Metodes van toediening en toedieningstoestande mag egter die resultaat beïnvloed het. Verdere studies is nodig om die toediening en na-toedieningstoestande benodig deur EFF met betrekking tot appelbloedluis beheer, te ondersoek en om te verseker dat die toediening van die fungi ekonomiese volhoubaar is. Die rol van ʼn benattingsagent ter bevordering van grondversadiging en grond deurdringbaarheid, verdien verdere ondersoek. Van kern belang is egter die verdere soektog na meer virulente isolate van beide EPN en EPF as potensiële toekomstige kandidate vir die gebruik as biologiese beheer agente.

Doctoral