Prospects for using Entomopathogenic Nematodes as a biocontrol agent against Western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae)

Thesis (MSc)--Stellenbosch University, 2018.ENGLISH ABSTRACT: The western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thripidae: Thysanoptera), is one of the most economically important pests in greenhouses, with preference being exhibited towards feeding on flowers. WFT is a serious pest of greenhouse cultivation, because it damages plants directly by means of feeding and oviposition on foliage and flowers, and indirectly, by means of vectoring tospoviruses, such as impatiens necrotic spot virus and tomato spotted wilt virus. Approximately 7500 species of thrips have been identified to date, with 14 species being recognised as virus vectors, of which F. occidentalis is responsible for transmitting five species of tospoviruses. Chemical control has been the most frequently used method for the control of WFT in greenhouses. The high frequency of insecticide applications for WFT control, coupled with the short generation time of F. occidentalis, has led to an increasing incidence of insecticide resistance in WFT in recent years. An integrated pest management (IPM) programme offers a sustainable alternative control for WFT in undercover production. Biological control, especially the use of entomopathogenic nematodes (EPNs), has been identified as an environmentally friendly option. The use of other parasites and predators for biological control has shown only limited ability to reduce WFT populations, apparently because their movement is restricted when entering tight flower buds, meristem tissues, or narrow flower structures favoured by WFT, due to their large body size. This study investigated the potential use of indigenous EPNs for the control of WFT under laboratory and greenhouse conditions. To achieve the above, the development and survival rate of F. occidentalis on two host plants, as well as its biology, were studied under laboratory conditions to identify life stages targetable with EPNs. The efficacy of the local strains of EPNs to control the different life stages of WFT, and the optimum nematode concentrations required for the suppression of WFT under laboratory conditions, were investigated. Lastly, the potential of foliar and soil applications of different concentrations of locally isolated S. yirgalemense for controlling F. occidentalis in a commercial blueberry greenhouse was investigated. Laboratory studies were conducted to determine the life-history and host preference of adult WFT on chrysanthemum (Dendranthema grandiflora) leaflets and green bean pods (Phaseolus vulgaris). The identification of Frankliniella occidentalis was verified, using both morphological and molecular methods. Main morphological features included six to nine antennal segments, major setae on the head and pronotum dark, interocellar and postocular setae approximately the same length, the first vein of the anterior wing with a complete row of regularly spaced setae, and posteromarginal comb on tergite VIII of the female well-developed and complete. Molecular identification was based on amplification of the mtCOI gene sequences for the identification of four thrips species (F. occidentalis, Thysanoptera sp., Gynaikothrips ficorum and Pseudophilothrips ichini) collected from the study area. The F. occidentalis morphologically identified showed 100 % identity with sequences in the database from GenBank. One of the Thrips sp. could not be identified neither morphologically nor molecularly and could possibly be an unidentified species. Results from the life-history study showed that more first instar larva hatched on chrysanthemums, faster larval developmental rate and a higher survival rate on chrysanthemums indicating that chrysanthemum is a more attractive and more suitable host than green bean. Among the 12 EPN species tested against F. occidentalis in laboratory bioassays, virulence ranged from 11 % to 67 %. Generally, Heterorhabditis spp. were more virulent than the Steinernema spp. Heterorhabditis baujardi was found to be the most potent species, with a mortality of 67 %, although it was not significantly different from Steinernema yirgalemense (66 %). The study showed that the commercial nematode Steinernema feltiae did not perform better than the local EPN species. Bioassays to determine infectivity were performed using different life stages (larva, pupa and adult) of F. occidentalis exposed to infective juveniles (IJs) of S. yirgalemense, H. baujardi and Steinernema jeffreyense. The pupae of WFT were found to be more sensitive to nematode infection than either the larvae or the adults. The highest WFT mortality was recorded for the pupae (72 %) when applying 100 IJs/insect of H. baujardi, with the lowest being recorded when treated with S. jeffreyense (17 %). Steinernema yirgalemense and H. baujardi were tested at concentrations of 0, 10, 20, 40, 80, and 160 IJs/larva. Increasing EPN concentrations gave increased thrips mortality, with a probit analysis indicating S. yirgalemense to be 5.49 more potent than H. baujardi. Results from the temporal development study showed that both S. yirgalemense and H. baujardi were able to complete their life cycles in the host within 5 days, and were able to produce a new cohort of IJs. Relatively few IJs were found to penetrate the insect, due to the small size of the insect and the IJs recovered from the host were relative in number to the IJs penetrated. The field trial was initiated to determine the efficiency of different concentrations of S. yirgalemense in controlling F. occidentalis in a commercial blueberry greenhouse. A combination of foliar and soil applications of S. yirgalemense in two greenhouse trials, one at lower concentrations of 4.3, 8.6, and 17.2 IJs/cm2, and the other at higher concentrations of 25, 50, and 100 IJs/cm2 were applied. The results in both trials indicated thrips mortality < 50 % at the highest concentration of 100 IJs/cm2, at mean substrate temperatures < 15 °C, which was below optimum for S. yirgalemense infection. Increase in nematode concentration resulted in a decline in the number of thrips captured. The experiment with higher concentrations showed increased thrips mortality (53 %) in relation to the experiment with lower concentration (< 40 %). Steinernema yirgalemense was persistent for 4 weeks, with low mortalities when mealworms were used to monitor infectivity. The correct identification of thrips is important for further studies investigating biological control thereof. Research into the use of EPNs for the biological control of insects should not be restricted to laboratory conditions, as these conditions do not truly represent field performance. Steinernema yirgalemense showed potential for use as a biocontrol option for WFT, giving low to moderate results in the field trial, under suboptimal temperatures, at a concentration of 100 IJs/cm2. The application of S. yirgalemense to control WFT requires further investigation under relatively warmer substrate temperatures in the Haygrove tunnels under blueberry production. Application of nematodes should target WFT populations on new growth after post-harvest pruning, when WFT causes significant economic damage. Weekly follow-up applications should be investigated as a future alternative. The feasibility of applying S. yirgalemense in conjunction with other biological agents and insecticide–pathogen synergistic interactions in IPM systems should also be investigated.AFRIKAANSE OPSOMMING: Die westelike blomblaaspootjie (WBB), Frankliniella occidentalis (Pergande) (Thripidae: Thysanoptera), is een van die belangerikste ekonomiese peste in kweekhuise en toon ‘n voorliefde daarvoor om op blomme te voed. Die WBB is ‘n ernstige pes van plante in kweekhuise omdat dit plante direk beskadig deur voeding en die lê van eiers op blare en blomme, asook indirek deur die dra van tospovirusse soos “impatiens necrotic spot virus” en “tomato spotted wilt virus”. Ten minste 7500 spesies blaaspootjies is bekend, waarvan 14 geken is as draers. Frankliniella occidentalis is verantwoordelik vir die verspreiding van omtrent vyf tospovirus spesies. Chemiese beheer is die mees algemene metode wat toegepas word teen WBB in kweekhuise. Die hoë frekwensie waarteen insekdoders aangewend word vir die beheer van WBB, tesame met die kort generasietyd van die spesie, het gelei tot toenemende weerstand teen insekdoders in WBB in die afgelope paar jaar. ʼn Geïntegreerde pes beheer (GPB) program, bied ʼn volhoubare alternatief vir die beheer van WBB in onderdakproduksie. Biologiese beheer, veral die gebruik van entomopatogeniese nematodes (EPNs), is geïdentifiseer as ʼn omgewingsvriendelike beheer metode. Die gebruik van ander parasiete en predatore vir biologiese beheer het sover slegs beperkte sukses getoon, blykbaar omdat hul beweging beperk is wanneer hul stywe blomknoppe, meristeem weefsel of nou blomstrukture binnedring, as gevolg van hul liggaamsgrootte. Hierdie studie het die potensiaal ondersoek van die gebruik van inheemse EPNs vir die beheer van WBB in laboratorium en kweekhuis omstandighede. Om dit te bereik, was die ontwikkeling en oorlewingskoers van F. occidentalis op twee gasheer plante, sowel as sy biologie, bestudeer in laboratorium toestande, sodat lewensstadia wat vatbaar vir EPN infeksie is identifiseer kon word. Die vermoë van die plaaslike EPN spesies om die verskillende lewensfases van WBB te beheer, asook die optimale nematode konsentrasies vir die onderdrukking van WBB in laboratorium toestande, was ondersoek. In die laaste deel van die studie was die potensiaal van EPNs om F. occidentalis in ʼn kommersiële bloubessie kweekhuis te beheer, ondersoek deur plaaslike S. yirgalemense by verskillende konsentrasies aan te wend. Laboratorium studies om die lewensgeskiedenis en gasheer voorkeur van volwasse WBB te bepaal, was uitgevoer op krisant (Dendranthema grandiflora) blare en groenboontjies (Phaseolus vulgaris). Frankliniella occidentalis was geïdentifiseer en sy identiteit bevestig deur gebruik te maak van morfologiese en molekulêre metodes. Die hoof morfologiese kenmerke van F. occidentalis is ses tot nege antenna segmente, groot seta op die kop, donker pronotum, “interocellar” en post-okulêre seta omtrent dieselfde lengte, die eerste aar van die voorste vlerk met eweredig gespasieërde en volledige seta, asook ʼn goed ontwikkelde en volledige “posteromarginal comb” op die “tergite VIII” van die wyfie. Resultate van die lewensgeskiedenis eksperiment het vinniger ontwikkeling, meer eiers en larwes, asook ʼn hoër oorlewingskoers getoon op krisante, wat beteken dat dit ʼn meer gepaste gasheer is as die groenboontjie. Molekulêre identifikasie was gebaseer op die mtCOI geen vir die identifikasie van vier spesies (F. occidentalis, Thysanoptera sp., Gynaikothrips ficorum en Pseudophilothrips ichini) wat versamel was in die studie area. Die F. occidentalis was morfologies geïdentifiseer was, het 100% identiteit getoon met die inligting in die databasis van GenBank. Een van die blaaspootjie spesies kon nie morfologies of molekulêr identifiseer word nie en kan moontlik ʼn onbeskryfde spesie wees. Die virulensie van die 12 EPN spesies wat getoets was teen F. occidentalis in laboratorium biotoetse het gewissel van 11 % tot 67 %. Oor die algemeen het die Heterorhabditis spesies hoër virulensie getoon as die Steinernema spesies. Heterorhabditis baujardi was die dodelikste spesies, met ʼn mortaliteit van 67 %, alhoewel dit nie ʼn beduidende verskil getoon het teenoor die dodelikheid Steinernema yirgalemense (66 %) nie. Die studie het getoon dat die kommersiële nematode Steinernema feltiae nie beter gevaar het as die plaaslike EPN spesies nie. Biotoetse om infeksie te bepaal was uitgevoer op verskillende lewensstadia (larwes, papies en volwassenes) van F. occidentalis met die EPNs S. yirgalemense, H. baujardi en Steinernema jeffreyense. Die papies van WBB was meer vatbaar vir nematode infeksie as die larwes of die volwassenes. Die hoogste WBB mortaliteit was aangeteken met die aanwending van 100 Ils/insek van H. baujardi op WBB papies (72 %). Die laagste mortaliteit was aangeteken toe papies behandel was met S. jeffreyense (17 %). Steinernema yirgalemense en H. baujardi was getoets by konsentrasies van 0, 10, 20, 40, 80, en 160 ILs/larwe. ʼn Toename in EPN konsentrasies het gelei tot ʼn toename in die mortaliteit van blaaspootjies, met ʼn pro-bit analise wat getoon het dat S. yirgalemense 5.49 keer meer dodelik is as H. baujardi. Resultate van die temporale ontwikkeling studie het getoon dat beide S. yirgalemense en H. baujardi in staat was om hul lewenssiklusse te voltooi in die gasheer binne 5 dae en ook ʼn nuwe groep ILs kon produseer. Relatief min ILs het die insek gepenetreer, as gevolg van die klein liggaamsgrootte van die insek en die ILs wat gevind was in die gasheer was relatief tot die aantal ILs wat die insek gepenetreer het. Die doel van die veldproef was om die effektiwiteit van verskillende konsentrasies van S. yirgalemense te toets vir die beheer van F. occidentalis in ʼn kommersiële bloubessie kweekhuis. ʼn Kombinasie van blaar- en grondaanwending van S. yirgalemense was toegepas in twee kweekhuis proewe, een by laer konsentrasies van 4.3, 8.6, en 17.2 ILs/cm2, en ʼn ander by hoër konsentrasies van 25, 50, en 100 ILs/cm2. Albei proewe het mortaliteit van 40%). Steinernema yirgalemense het aangehou vir 4 weke, met lae mortaliteit toe meelwurms gebruik was om infektiwiteit te monitor. Die identifikasie van blaaspootjies is belangerik vir verdere navorsing oor hul biologiese beheer. Navorsing oor die gebruik van EPSs vir die biologiese beheer van insekte moet nie beperk word tot laboratoriumtoestande nie, omdat hierdie toestande nie werklik die prestasie van EPNs in die veld verteenwoordig nie. Steinernema yirgalemense het potensiaal getoon as ʼn biologiese beheer opsie vir WBB, met lae tot matige resultate in die veldproef, in suboptimale temperature, by ʼn konsentrasie van 100 ILs/cm2. Die aanwending van S. yirgalemense vir die beheer van WBB benodig verdere ondersoek, met relatief warmer substraat temperature in die Haygrove tonnels onder bloubessie produksie. Die aanwending van nematodes moet die piek in WBB populasies teiken gedurende die tydperk van nuwe groei, wanneer WBB aansienlike ekonomiese skade veroorsaak. Weeklikse opvolg aanwendings moet ondersoek word as ʼn toekomstige alternatief. Die moontlikheid van die aanwending van S. yirgalemense in samewerking met ander biologiese beheermiddels, asook insekdoder-patogeen sinergistiese interaksies in IPM sisteme, moet ondersoek word