Prospects for using entomopathogenic nematodes as a biocontrol agent against fungus gnats, bradysia spp. (Diptera: Sciaridae) in nursery and glass house crops

Katumanyane, Agil (2017-12)

Thesis (MScConsEcol)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Fungus gnats of the genus Bradysia, have become important pests to undercover, cultivated crops. Commonly referred to as mushroom fly, nuisance flies, or dark-winged flies, they have a global distribution. Their larval stage, which is the main feeding stage of the insect, feeds on the radical system of the plant, as well as tunnels into the stems, resulting in direct physical damage to the plant. Coupled herewith is the indirect damage that is caused through the creation of entry points for soil-borne pathogens. In South Africa, Bradysia impatiens, which is one of the most important species of fungus gnats that specialises in feeding on living plants, has recently been identified in association with major tree nursery beds. In the course of this study, the same species, B. impatiens was found to be living in association with greenhouse-grown cucumbers, mushrooms, blueberries, tomatoes, herbs, and chrysanthemums. Bradysia impatiens was concluded as being a well-established pest species in South African undercover farming systems. Lycoriella sativae was reported for the first time, as a pest on mushroom, for South Africa, and the Afro tropical region. The use of entomopathogenic nematodes (EPNs) for the control of Bradysia spp. has proved to be relatively advantageous, since sciarids are mainly pests of protected crops that are either too close to harvest, or for which, otherwise, the application of pesticides is unsuitable. The control of Bradysia spp. using EPNs has been dominated by the use of Steinernema feltiae, which, despite having a global distribution, has, to date, not yet been isolated either from South Africa, or from the rest of the African continent, except for Algeria. Steinernema feltiae has also been cited as being biologically constrained at relatively high temperatures, corresponding to most greenhouse temperatures in South Africa. The objective of this study was to identify the Bradysia spp. causing problems in greenhouses in the Western Cape province, as well as to test locally isolated EPN species for their potential to control Bradysia spp. Using molecular techniques and morphological observations, the species involved were identified as Bradysia impatiens Johannsen and Lycoriella sativae Johannsen. A laboratory culture for B. impatiens was established using a 3:1:1 mixture of pine sawdust, cornmeal and soy meal. The number of eggs laid per single adult female was between 100 and 250. A new phenomenon of egg laying, in the form of chains, was observed for the first time, for B. impatiens. In length, the first instar larvae measured between 1.2 and 2.5 mm, the second instars between 2.6 and 4.5 mm, and the third instars between 4.6 and 6.5 mm, while the fourth instar larvae measured about 6.5 to 7.2 mm. The life cycle of B. impatiens lasted for approximately three weeks in a growth chamber, at 25°C in the dark. Eight South African local EPN species, and a foreign S. feltiae, were tested for their ability to kill fourth instar larvae of B. impatiens. Bioassay protocols were performed to determine the insecticidal activity, the lethal dose, and the efficacy of the EPNs at different temperatures. The results showed four local EPN species, Steinernema yirgalemense, Heterorhabditis noenieputensis, Heterorhabditis indica, and Heterorhabditis zealandica, achieving higher than 80% mortality, at 25°C and 30°C, from the different bioassays performed. In contrast, S. feltiae achieved higher percentage mortality at 13°C, (82%), at which temperature, none of the local isolates was able to infect fourth instar larvae of B. impatiens. Steinernema feltiae maintained a high percentage mortality at 25°C, (72%) but registered a much lower mortality for B. impatiens at 30°C, (47%). Heterorhabditis zealandica had the lowest LD50 of 2.6 infective juveniles (IJs) per larva, and an LD90 of 18.68 IJs/larva, while S. yirgalemense and H. indica had LD50 and LD90 of 9; 64, and 9; 64, respectively. Steinernema yirgalemense, H. indica and H. zealandica were all able to reproduce inside the fourth instar larvae of B. impatiens, and to produce a new cohort of IJs. The relatively bigger nematodes, S. jeffreyense, S. khoisanae, and Steinernema sp. (WS9), were unable to infect the fourth instar larvae of B. impatiens, which showed that the size of the EPNs affected their ability to infect fungus gnat larvae. The locally isolated EPNs were concluded as having high potential for use against fungus gnats in South Africa. The effect of different concentrations of a local entomopathogenic nematode, S. yirgalemense, for its potential to control a natural infestation of B. impatiens in a commercial cucumber greenhouse was tested. Additionally, the effect of potting media on nematode movement to control an artificial population of fungus gnats was tested. The concentrations used were based on the recommended concentration of S. feltiae (a commercial product) for the control of sciarids (5 × 105 IJs per m2). Doubling the number of nematodes in the recommended dosage reduced the number of fungus gnats by 77% after 14 days, and by 76% after 21 days. However, the fungus gnat populations were observed to restore quickly to their original levels, after a period of approximately three weeks, emphasising the short life cycle of the fly, as well as the importance of the pre-treatment, and of consecutive applications, of EPNs throughout the whole production process. The three different types of potting media, consisting of pine sawdust, coco coir, and a mixture of both bale coir and vermiculite, all had a positive effect on the infection of S. yirgalemense. Mortality of >75 % was achieved for the fungus gnat larvae that were added to the media, as indicated by the number of adult fungus gnats caught on the yellow sticky cards used. The results of the current study show that, under optimum conditions, the locally isolated EPNs, namely H. zealandica, S. yirgalemense, and H. indica, have great potential for use as biocontrol agents for B. impatiens. However, additional research should be undertaken into the effect of timing, concentrations, and nematode application techniques, among other factors, so as to obtain further critical information regarding the field applications of the nematodes. Further studies should look at different factors that might affect the optimum field performance of S. yirgalemense. The factors involved could include the pre-treatment of the substrates, and the use of higher concentrations of EPNs, and follow-up applications, the timely applications of EPNs, and other factors, such as pre-treatment with cadaver application, versus the aqueous drench application of the EPN species. However, the application of nematodes should not be seen as a stand-alone, but it should, rather, be implemented as part of an integrated pest management system, with sanitation being the most important consideration.

AFRIKAANSE OPSOMMING: Muggies van die genus Bradysia, het ’n ernstige plaag geword wat bedekte, verboude gewasse betref. Hulle staan ook as donkervlerkswammuggies bekend en kom wêreldwyd voor. Gedurende die larvale fase, wat die insek se hoofvoedingsfase is, voed dit op die plant se wortelstelsel en boor dit in die stamme in, wat tot fisiese skade aan die plant lei. Gepaardgaande indirekte skade word deur die skepping van toegangspunte vir grondgedraagde patogene veroorsaak. In Suid-Afrika is Bradysia impatiens, een van die belangrikste muggiespesies wat daarin spesialiseer om lewende plante te voed en is onlangs in assosiasie in boomkwekerye geïdentifiseer. In die verloop van hierdie ondersoek is daar bevind dat B. impatiens in assosiasie met kweekhuiskomkommers, sampioene, bloubessies, tamaties, kruie en krisante voorkom. Lycoriella sativae is vir die eerste keer aangemeld as ʼn plaag van sampioene vir Suid-Afrika en die Afro tropiese streek. Daar is reeds getoon dat die gebruik van entomopatogenies nematodes (EPNs) betreklik voordelig is in die bestryding van Bradysia, aangesien die Sciaridae meestal ʼn plaag by beskermde gewasverbouing is, wat óf byna geoes word óf waarvoor die gebruik van plaagdoders andersins nie moontlik is. Die beheer van Bradysia met behulp van EPNs word deur die gebruik van Steinernema feltiae oorheers, wat, ten spyte van wêreldwye verspreiding, tot op datum nog nie in Suid-Afrika of in die res van die vasteland geïsoleer is nie, behalwe in Algerië. Daar word genoem dat S. feltiae biologies minder effektief is met betreklik hoë temperature, wat ooreenstem met die meeste kweekhuise temperature in Suid-Afrika. Die doel van hierdie studie was om die Bradysia spesies te identifiseer wat probleme in kweekhuise in die Wes-Kaap skep en om plaaslik geïsoleerde EPN-spesies te toets vir hul potensiaal om Bradysia te bestry. Deur molekulêre tegnieke en morfologiese waarnemings te gebruik, is die spesie as B. impatiens geïdentifiseer. ʼn Laboratoriumteling is met behulp van ʼn 3:1:1-mengsel bestaande uit dennehoutsaagsels, mieliemeel en sojameel gevestig. Die aantal eiers per wyfie was tussen 100 en 250. ʼn Nuwe verskynsel is vir die eerste keer vir B. impatiens waargeneem: die lê van eiers in ʼn ketting. Die lengte van die eerste larvale stadium is tussen 1.2 en 2.5 mm, die van die tweede tussen 2.6 en 4.5 mm, die derde tussen 4.6 en 6.5 mm en die vierde tussen 6.5 en 7.2 mm. Bradysia impatiens se lewensiklus duur by 25°C ongeveer drie weke in ʼn groeikamer. Daar is vasgestel dat B. impatiens as plaagspesie reeds goed gevestig is in Suid-Afrikaanse bedekteboerderystelsels. Agt plaaslike Suid-Afrikaanse EPN-spesies en ʼn uitheemse S. feltiae is getoets vir hul vermoë om die vierde larvale stadium van B. impatiens te beheer. Laboratorium toetse is uitgevoer om insekdodende aktiwiteit, dodelike dosis (LD) en die doeltreffendheid van die EPN by teen verskillende temperature vas te stel. Die resultate toon dat, van die verskillende laboratorium toetse wat uitgevoer is, vier plaaslike EPN-spesies nl. Steinernema yirgalemense, Heterorhabditis noenieputensis, Heterorhabditis indica, en Heterorhabditis zealandica, mortaliteit van meer as 80% behaal het teen 25°C en 30°C. Heterorhabditis zealandica het die laagste LD50 van 2.6 nematodes per insek larwe en ʼn LD90 van 18.68 nematode per larwe gehad, terwyl S. yirgalemense en H. indica LD50 en LD90 van onderskeidelik 9; 64 en 9; 64 gehad het. Steinernema yirgalemense, H. indica en H. zealandica kon almal in B. impatiens se vierde larvale staduim reproduseer en ʼn nuwe kohort IJ’s vorm. Nematodes wat betreklik groter is, soos in die geval van S. jeffreyense, S. khoisanae en Steinernema sp. (WS9), was nie daartoe in staat om B. impatiens se vierde larvale stadium te infekteer nie, wat aantoon dat die grootte van die EPNs hul vermoë om swammuggielarwes te infekteer, beïnvloed. Die effek van verskillende konsentrasies van ʼn plaaslike, S. yirgalemense, is getoets om vas te stel wat die potensiaal vir die bestryding van ʼn natuurlike besmetting van B. impatiens in ʼn kommersiële komkommerkweekhuis is. Daarbenewens is die effek van potmedia op nematodebeweging getoets om ʼn kunsmatige populasie muggies te bestry. Die konsentrasies is gebaseer op die aanbevole konsentrasie S. feltiae (ʼn kommersiële produk) vir die bestryding van Sciaridae (5 ×105 IJ’s per m2). Die verdubbeling van die aantal nematodes in die aanbevole dosis het die aantal B. impatiens met 77% na 14 dae verminder en na 21 dae met 76%. Daar is egter waargeneem dat die muggiepopulasies baie vinnig tot hul oorspronklike vlakke herstel, wat die kort lewensiklus van die vlieg en die belangrikheid van voorafbehandeling en die herhaalde toediening van EPNs beklemtoon. Die drie verskillende tipes potmedia, bestaande uit dennehoutsaagsels, kokosklapperhaar en ʼn mengsel van baalklapperhaar en vermikuliet, het almal ʼn positiewe effek op die S. yirgalemense-infeksie gehad. Vir die muggielarwes wat by die media gevoeg is, is mortaliteit van > 75% behaal, soos aangetoon deur die aantal volwasse muggies wat op geel plakkaarte gevang is. Die resultate van die huidige studie toon aan dat die plaaslik geïsoleerde EPN, d.w.s. H. zealandica, S. yirgalemense en H. indica, onder optimale toestande belowende potensiaal toon as biobestrydingsagense vir B. impatiens in te hou. Bykomende navorsing moet onderneem word na die impak van, onder andere, tydsberekening, konsentrasie en nematodetoedieningstegnieke, om verdere belangrike inligting in te win oor die veldtoepassings van nematodes. Verdere ondersoeke moet ook vasstel watter verskillende faktore die optimale veldprestasie van S. yirgalemense kan beïnvloed. Die betrokke faktore sou, onder andere, die voorafbehandeling van substrate, die gebruik van hoër konsentrasies EPN, opvolgtoedienings, die tydsberekening van EPN-toedienings, of voorafbehandeling met kadawertoediening teenoor die watersuspensiestoediening van die EPN kon insluit. Die toediening van nematodes moet egter nie as ʼn alleenstaande behandeling gesien word nie, maar eerder as deel van ʼn geïntegreerde bestuurstelsel vir plaagbestryding beskou word waar sanitasie as die belangrikste oorweging geag word.

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