i Investigating the above-ground application of EPNs for the control of the vine mealybug Planococcus ficus by Thomas Platt Thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Stellenbosch University Department of Conservation Ecology and Entomology, Faculty of AgriSciences Supervisor: Prof. Antoinette P Malan Co-supervisor: Dr. Nomakholwa F Stokwe December 2017 Stellenbosch University https://scholar.sun.ac.za ii DECLARATION By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third-party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: December 2017 Copyright  2017 Stellenbosch University All rights reserved Stellenbosch University https://scholar.sun.ac.za iii ACKNOWLEDGEMENTS I would like to thank the following people and institutions: My supervisors Prof. Antoinette Malan and Dr. Nomakholwa Stokwe for their expertise, kindness, attention, and patience; Prof Daan Nel, for his invaluable statistical expertise; The Department of Conservation Ecology and Entomology at Stellenbosch University, as well as my fellow Nematologists, for their help and support; In particular, Nicholas Kagimu, for providing me with nematodes; The project’s sponsors, SATI and Winetech, for enabling this project and allowing me this opportunity; All the staff at the Agricultural Research Council (ARC)-Infruitec-Nietvoorbij, Stellenbosch, for their provision of mealybugs and the use of their premises to conduct research; My family, with whose support I was able to pursue this degree; And Damian Hacking, who makes every day better and without whom I would have gone hopelessly, irreversibly mad. Stellenbosch University https://scholar.sun.ac.za iv ABSTRACT The table and wine grape industries in South Africa are of major economic importance, particularly within the Western Cape Province, making the pest control of grapevines a priority. The vine mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) is a key pest of South African grapevines, damaging vines by phloem feeding, by disfiguring grapes with waxy residues, by encouraging the growth of sooty moulds, and by serving as a vector for viruses. Chemical insecticides like chlorpyrifos have traditionally been used in their control, though the cryptic habitats on the vine chosen by the most economically significant mealybug life stage complicates pesticide application. Additionally, mealybugs excrete a waxy coating that repels liquids, and their short generation time allows the rapid development of resistance to chemical pesticides. Consequently, alternatives are sought for the control of mealybugs on grapevines. One candidate for their control is entomopathogenic nematodes (EPNs), which are nematode parasites of soil-based insect life stages. Of major interest in this respect are the EPNs of the families Steinernematidae and Heterorhabditidae, the infective juveniles (IJs) of which have been successfully applied to control soil-based insect pests. However, the maladaptation of IJs to non-soil environments (such as foliage) has limited their use as biocontrol agents above ground, due to their susceptibility to extremes of temperature and to prolonged exposure to ultraviolet light (UV), as well as their generally low tolerance for desiccation. The aim of this study was to investigate EPN candidates for the control of P. ficus, and to develop methods for overcoming the weaknesses of EPNs in foliar application. As new species of EPNs are constantly being described, laboratory-based bioassays were performed, screening three newly described EPN species (Steinernema jeffreyense, Heterorhabditis noenieputensis, and Steinernema spp. WS9), as well as Steinernema yirgalemense, for their control of P. ficus. Heterorhabditis noenieputensis was the most effective, causing 90%  3% mortality, followed by S. yirgalemense (63%  7%), with both mortalities being significantly greater than was Stellenbosch University https://scholar.sun.ac.za v that of the control. The presence of the nematodes within the body cavities of P. ficus cadavers was confirmed. Steinernema yirgalemense was selected as the EPN candidate of choice for experiments going forward, due to the difficulty in mass-producing H. noenieputensis. However, developments in the formulation methods of the Heterorhabditid species will warrant the re-examination of H. noenieputensis in future. On performing a laboratory bioassay to determine the minimum amount of time required for the optimal infectivity of P. ficus by S. yirgalemense, the mortality of P. ficus was found not to improve significantly for individuals exposed to S. yirgalemense for longer than 3h. Subsequently, the effects of varying temperature and relative humidity (%RH) on the ability of S. yirgalemense to cause mortality in P. ficus were tested. The mortality of P. ficus was greatest at 25°C (72% ± 3%), and at 100% RH, during the humidity trial. Each result established targets for the optimal application of S. yirgalemense. The ability of two adjuvants, Zeba® and Nu-Film-P®, to improve the efficacy of S. yirgalemense applications was tested under semi-controlled conditions. The combination of Zeba® and Nu-Film- P® in suspension with S. yirgalemense was shown to deposit significantly more EPNs (30.8 ± 4 IJs / 4 cm2) onto grapevine leaves in the laboratory than did formulations with EPNs and water alone, or with EPNs and Nu-Film-P®, though not significantly more than with EPNs and Zeba® alone. A growth chamber bioassay was conducted to assess the effect of the addition of the adjuvants to S. yirgalemense suspensions on P. ficus mortality. The addition of Zeba® and Nu-Film-P® to S. yirgalemense caused significantly higher mortality (84% ± 5%) in P. ficus in the growth chamber than did any other treatment, including EPNs + Zeba® (47% ± 3%), after 48h. A bioassay carried out in the greenhouse showed similar results, with the S. yirgalemense treatment containing Zeba® and Nu-Film-P® causing 88% ± 3% mortality after 48h, which was significantly higher than was that which was attained with any other EPN treatment. The treatments were then assessed under semi-field conditions that would be capable of inflicting the harshest environmental stress. Application of S. yirgalemense (at a concentration of Stellenbosch University https://scholar.sun.ac.za vi 4000 IJs/ml) + Zeba® + Nu-Film-P® to P. ficus individuals on grapevine leaf discs hung on grapevines resulted in 66% ± 4% P. ficus mortality after 48h, which was significantly higher (p < 0.01) than was achieved using either S. yirgalemense + Zeba® alone, or EPNs + water alone, though overall less than the control obtained in the glasshouse. A bioassay to assess the impact of reducing EPN concentration was performed, resulting in predictable reductions in P. ficus mortality when progressively lower concentrations of S. yirgalemense (3000, 2000 and 1000 IJs/ml) were applied with Zeba® and Nu- Film-P® to P. ficus on grapevine leaf discs. The control obtained by the formulation containing 3000 IJs/ml was significantly greater than was that which was achieved with each other treatment after 48h (44% ± 4%), though the control overall was lower than was attained with the 4000 IJs/ml concentration used in the previous bioassay. This demonstrates that the EPN concentration remains important to the efficacy of EPN applications. So as to assess the effects of climatic conditions on EPN longevity, a time-of-day application bioassay was performed. Steinernema yirgalemense was formulated with Zeba® and Nu-Film-P® and applied directly to grapevines, the leaves of which were removed and rinsed at timed intervals, whereupon the live nematodes present on them were counted. The experiment was carried out at 8:00 (with conditions being 14.6C and 93.2% RH at application), and repeated at 14:00 (with conditions being 31.0C and 39.9% RH at application). Higher numbers of living nematodes were recorded on the grapevine leaves at all of the time intervals concerned during the 8:00 trial when compared with the same intervals during the 14:00 trial, indicating that the higher percentage RH had a greater effect on IJ survival than did the more optimal temperature (but lower % RH) during the afternoon trial. This study represents an additional step towards the successful utilization of EPNs (in this case, S. yirgalemense) as biocontrol agents of P. ficus on grapevines in South Africa. Steinernema yirgalemense can achieve > 66 % mortality of P. ficus under semi-field conditions, when the humidity (which is the critical factor for IJ survival on foliage) is effectively managed. Future work should examine S. yirgalemense in full-field application, as well as available methods (such as the use of Stellenbosch University https://scholar.sun.ac.za vii irrigation, or shade netting) for maximizing the relative humidity immediately following IJ application. Stellenbosch University https://scholar.sun.ac.za viii OPSOMMING Die tafel- en wyndruif industrie is van groot ekonomiese belang in Suid-Afrika, veral in die Wes- Kaap provinsie. Die beheer van wingerd peste is daarom uiters belangerik. Die wingerd witluis, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), is een van die belangrikste peste in Suid- Afrikaanse wingerde en veroorsaak skade deur te voed op die floëem van die plant, deur die druiwetrosse te besmet met wasagtige afskeidings, deur swart swamgroei aan te moedig en dien ook as ‘n draer van virusse. Chemiese insekdoders soos chlorpirifos word tradisioneel gebruik vir die beheer van die wingerd witluis. Die aanwending van sulke insekdoders word egter bemoeilik deur benutting van kriptiese lewenswyse op die wingerd van die mees skadelike witluis lewensfase. Boonop skei wingerd witluise ‘n waslagie af wat vloeistowwe afweer en hul kort generasie tyd stel hul in staat om weerstand te ontwikkel tot chemiese plaagdoders. Daarom word daar alternatiewe metodes vir die beheer van wingerd witluise ondersoek. Entomopatogeniese nematodes (EPNs) is parasiete van grondlewende lewensfases van insekte en een van die kandidate vir die beheer van wingerd witluis. Van groot belang in hierdie nematodes is die EPNs van die families Steinernematidae en Heterorhabditidae, waarvan die infektiewe larwes (IJs) al suksesvol aangewend is om grondlewende insek peste te beheer. IJs is egter nie aangepas om bo grondvlak te oorleef nie, aangesien hul sensitief is vir uiterste temperature en langdurige blootstelling van UV strale, asook ʼn lae toleransie het vir uitdroging. Dit beperk die gebruik van IJs as biologiese beheermiddels in omgewings bo grondvlak, soos op die blare van die wingerd. Die doel van hierdie studie was om EPN kandidate te identifiseer vir die beheer van P. ficus, en metodes te ontwikkel om die probleme van EPNs in die aanwending op blare te oorkom. Omdat nuwe spesies van EPNs voortdurend beskryf word, was drie nuut beskryfde species is gebruik vir biotoetse in die laboratorium (Steinernema jeffreyense, Heterorhabditis noenieputensis, Steinernema spp. WS9), asook Steinernema yirgalemense. Hul vermoë om P. ficus te beheer was ondersoek. Resultate toon dat Heterorhabditis noenieputensis die mees effektief was met 90%  3% Stellenbosch University https://scholar.sun.ac.za ix mortaliteit, gevolg deur S. yirgalemense (63%  7%), albei se mortaliteit was beduidend groter as die van die kontrole. Die aanwesigheid van nematodes in die liggaamsholtes van P. ficus kadawers was bevestig. Steinernema yirgalemense was gekies as die EPN kandidaat vir toekomstige eksperimente, en mootlike probleme met die massaproduksie van H. noenieputensis. Alhoewel, toekomstige ontwikkeling in die massatelings metodes van Heterorhabditid spesies sal beteken dat H. noenieputensis heroorweeg sal kan word as ʼn belowende biobeheer agent. Met biotoetse in die laboratorium om te bepaal wat is die minimum tydperk vir S. yirgalemense om P. ficus optimaal te infekteer, was daar gevind dat die mortaliteit nie beduidend verbeter het na 3 h van blootstelling aan S. yirgalemense nie. Gevolglik was die effek van verskillende temperature en relatiewe humiditeit (%RH) op die vermoë van S. yirgalemense om mortaliteit in P. ficus te veroorsaak, getoets. Die mortaliteit van P. ficus was die hoogste by 25°C (72% ± 3%), en by 100% RH, gedurende die humiditeit toets. Elke resultaat het mikpunte gelewer vir die optimale aanwending van S. yirgalemense. Die vermoë van twee byvoegingsmiddels, Zeba® en Nu-Film-P®, om die doeltreffendheid van S. yirgalemense aanwendings te verhoog, was getoets onder semi-beheerde toestande. Die kombinasie van Zeba® en Nu-Film-P® in suspensie met S. yirgalemense het beduidend meer EPNs (30.8 ± 4 IJs / 4 cm2) op die wingerdblare in die laboratorium tot gevolg gehad as die suspensies met slegs EPNs, slegs water of met EPNs en Nu-Film-P®, alhoewel nie beduidend meer as die suspensies met slegs EPNs en Zeba® nie. ʼn Groeikamer biotoets was uitgevoer om die effek van die byvoeging van byvoegingsmiddels tot die S. yirgalemense suspensies op P. ficus mortaliteit te bepaal. Die byvoeging van Zeba® en Nu-Film-P® tot S. yirgalemense het beduidend hoër mortaliteit (84% ± 5%) in P. ficus in die groeikamer veroorsaak as enige ander behandeling, insluitend EPNs + Zeba® (47% ± 3%), na 48 h. ‘n Biotoets wat uitgevoer was in die glashuis het soortgelyke resultate gelewer, met die behandeling wat Zeba® en Nu-Film-P® bevat, wat 88% ± 3% mortaliteit veroorsaak het na 48 h. Dit was beduidend hoër as met enige ander EPN behandeling. Stellenbosch University https://scholar.sun.ac.za x Die toediening van S. yirgalemense was toe getoets onder semi-veld toestande, wat in staat sou wees om die ongunstige omgewingstoestande te veroorsaak. Aanwending van S. yirgalemense (teen ʼn konsentrasie van 4000 IJs/ml) + Zeba® + Nu-Film-P® tot P. ficus individuele op wingerdblaar skyfies wat gehang is op wingerde, het gelei tot 66% ± 4% insek mortaliteit na 48 h, wat beduidend hoër was as die resultate van die aanwending van slegs S. yirgalemense + Zeba® of slegs EPNs en water, alhoewel minder as vir die kontrole in die glashuis. ʼn Biotoets was ook uitgevoer om die impak van ʼn laer EPN konsentrasie te bepaal. Soos verwag, was P. ficus mortaliteit verlaag met verminderde konsentrasies van S. yirgalemense (3000, 2000 en 1000 IJs/ml) aangewend is met Zeba® en Nu-Film- P® op P. ficus op wingerdblaar skyfies. Die mortaliteit in die kontrole van die suspensies van 3000 IJs/ml was beduidend meer as die van enige ander behandeling na 48 h (44% ± 4%), alhoewel die kontrole laer was as die mortaliteit wat bereik was met ʼn 4000 IJs/ml konsentrasie wat gebruik was in die vorige biotoets. Die resultate toon dat die konsentrasie van EPNs belangerik bly in die doeltreffendheid van EPN aanwendings. Om die effek van klimaatstoestande op EPN oorlewing is getoets gebaseer was op die tyd gedurende dit dag wanneer die biotoets uitgevoer is. Steinernema yirgalemense was geformuleer met Zeba® en Nu-Film-P® en direk aangewend op wingerdblare. Die blare was dan verwyder en afgespoel op sekere intervalle en die nematodes aanwesig op die blare getel. Die eksperiment was uitgevoer om 8:00 (met toestande van 14.6C en 93.2% RH by aanwending), en herhaal om 14:00 (met toestande van 31.0C en 39.9% RH by aanwending). Hoër getalle lewende nematodes was waargeneem op die wingerdblare by alle intervalle van die 8:00 proewe in vergelyking met dieselfde intervalle by die 14:00 proewe, wat aandui dat die hoër persentasie RH ʼn groter effek gehad het op die oorlewing van die nematodes as die meer optimale temperatuur (maar laer % RH) van die middag proef. Die studie bied In addisionele stap nader aan die suksesvolle gebruik van EPNs (in hierdie geval, S. yirgalemense) as biologiese beheermiddel van P. ficus op wingerde in Suid-Afrika. Steinernema yirgalemense kan > 66 % mortaliteit van P. ficus tot gevolg hê onder semi-veld