Investigating the potential of indigenous nematode isolates to control invasive molluscs in canola

Thesis (MScConsEcol)--Stellenbosch University, 2016.

ENGLISH ABSTRACT: Terrestrial molluscs (Mollusca: Gastropoda) are important economic pests worldwide, causing extensive damage to a variety of crop types, and posing a health risk to both humans and wildlife. In South Africa, the climate is favourable for invasive European molluscs, especially in the Western Cape province, where there are mild, damp winters. One crop that is particularly targeted by the pests concerned is canola (Brassica napus), which is a winter arable crop that is commercially produced for its use in cooking, food processing, fertilisers, fuels, pet food, plastics, and animal feed. Molluscs on canola in the Western Cape province are currently controlled using chemical molluscicide pellets. These chemicals have the potential to adversely affect the environment and non-target organisms. The use of mollusc-parasitic nematodes is a possible environmentally-friendly alternative. Current knowledge indicates that there are eight nematode families that associate with molluscs, including Agfidae, Alaninematidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogastridae, Mermithidae, and Rhabditidae. To date, Phasmarhabditis hermaphrodita is the only nematode that has been developed as a biological molluscicide. The nematode, which was commercially released in 1994 by MicroBio Ltd, Littlehampton, UK (formally Becker Underwood, now BASF) under the trade name Nemaslug®, is now sold in fifteen different European countries. Due to current legislation, Nemaslug® cannot be sold or used in South Africa. A survey was therefore conducted in the Western Cape province of South Africa to locate a local nematode isolate capable of causing mortality in invasive mollusc pests. A total of 1944 slugs were collected from 12 different study sites. On the identification of slugs, they were dissected alive, and examined for internal nematodes. Nematodes were identified using morphological and molecular techniques (18S rRNA). Seven of the 12 sites had nematodes present, with 8% of the slugs being found to be infected with nematodes. Six nematode species were identified, including Angiostoma margaretae, Angiostoma sp., Caenorhabditis elegans, a mermitid sp., and Phasmarhabditis spp. (SA3 and SA4). Of the six species mentioned, four were previously undescribed. The isolation of new Phasmarhabditis spp. indicates the importance of conducting further surveys of mollusc-parasitic nematodes in South Africa. Nematodes isolated in the survey were tested for their ability to reproduce on decaying organic matter (consisting of dead frozen slugs), with results demonstrating that one of the nematodes, Phasmarhabditis sp. SA4, could complete its life cycle under such conditions. In addition, pathogenicity tests illustrated that Phasmarhabditis sp. SA4 caused significant mortality of the slug D. panormitanum. Phasmarhabditis sp. SA4 was then fully described and characterised by the shape and length of the female tail, and by the presence of males. Phylogenetic analysis demonstrated that Phasmarhabditis sp. SA4 was placed in a monophyletic clade along with Phasmarhabditis sp. SA2, Phasmarhabditis papillosa, and the mollusc-parasitic nematode, Angiostoma dentiferum. The new species brings the total complement of the genus to seven species. Phasmarhabditis sp. SA4 was then established in monoxenic cultures. Five bacterial isolates were isolated from the intestine of slug hosts, identified using 16S rRNA gene sequences, and their pathogenicity tested by means of injecting directly into the haemocoel of D. reticulatum, and monitoring the mortality over time. Kluyvera sp., which was found to cause the highest mortality rate among the slugs concerned, was chosen for monoxenic culturing. Cultures containing Phasmarhabditis sp. SA4 and Kluyvera sp. were optimised using temperatures ranging from 15°C to 25°C, with results showing that 15°C was the optimum growth temperature.

AFRIKAANS OPSOMMING: Landlewende weekdiere (Mollusca: Gastropoda) is wêreldwyd belangrike ekonomiese plae wat aansienlike skade aan ‘n verskeidenheid landbou gewasse veroorsaak en kan ‘n nadelige effek op die gesondheid van mense en dier hê. Die Wes-Kaap provinsie van Suid-Afrika met sy matige, klam winters is veral ‘n gunstige omgewing vir indringer Europese slakke en naakslakke. Een gewas wat veral benadeel word deur die aktiwiteit van hierdie spesies is canola (Brassica napus). Canola word kommersieel produseer vir gebruik in die voorbereiding van voedsel, kunsmis, brandstowwe, voer vir troeteldiere, plastiek en veevoer. Slakke word tans beheer in canola in die Wes-Kaap, deur die gebruik van slakpille. Die chemikalieë in hierdie slakpille het die potensiaal om ‘n negatiewe effek op die omgewing en nie-teiken organismes te hê. Nematodes wat dien as natuurlike parasiete van slakke is ‘n moontlike omgewingsvriendelike biologiese beheer alternatief. Volgens kennis is daar tans agt nematode families wat assosieer met slakke, naamlik Agfidae, Alaninematidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogastridae, Mermithidae, en Rhabditidae. Tot op datum is Phasmarhabditis hermaphrodita die enigste nematode wat al ontwikkel is in ‘n biologiese beheermiddel vir slakke. Die nematode is in 1994 kommersieel vrygestel deur MicroBio Ltd, Littlehampton, UK (voorheen Becker Underwood, nou BASF) onder die handelsnaam Nemaslug® en word tans verkoop in vyftien verskillende Europese lande. Huidige wetgewing verbied die verkoop of gebruik van die produk in Suid-Afrika. ‘n Opname was daarom gedoen van die nematodes geassosieer met slakke in die Wes-Kaap van Suid-Afrika, om ‘n plaaslike nematode te vind met dieselfde biologiese beheer potensiaal. ‘n Totaal van 1944 naakslakke was versamel van 12 verskillende studie areas. Nadat hul geidentifiseer was, was hul lewend dissekteer en ondersoek vir interne nematodes. Nematodes was geidentifiseer deur gerbuik te maak van morfologiese en molekulêre tegnieke (18S rRNA). Nematodes was teenwoordig by sewe van die twaalf studie areas en 8% van naakslakke was geïnfekteer deur nematodes. Ses nematode spesies was geïdentifiseer, naamlik Angiostoma margaretae, Angiostoma sp., Caenorhabditis elegans, ‘n mermitid sp., en Phasmarhabditis spp. (SA3 and SA4). Van die ses spesies wat gevind is, was vier nog nie voorheen beskryf nie. Die ontdekking van die nuwe Phasmarhabditis spesies is ‘n aanduiding van die belangrikheid van verdere opnames vir die voorkoms van nematodes parasiete geassosieer met slakke in Suid-Afrika. Nematodes wat gevind was in die opname se vermoë om voort te plant op organiese materiaal (bestaande uit gevriesde, dooie naakslakke) was getoets. Phasmarhabditis sp. SA4 kon suksesvol sy lewenssiklus voltooi in laboratorium toestande en verdure patogenisiteit toetse het bewys dat die nuwe nematode ‘n merkwaardige effek gehad het op sterftes van die indringer naakslak, D. panormitanum. Phasmarhabditis sp. SA4 was toe volledig beskryf en word gekarakteriseer deur die vorm en lengte van die vroulike nematode se stert asook die teenwoordigheid van manlike nematodes. Filogenetiese analise het getoon dat Phasmarhabditis sp. SA4 geplaas is in ‘n monofiletiese klade saam met Phasmarhabditis sp. SA2, Phasmarhabditis papillosa, en die slak-parasitiese nematode, Angiostoma dentiferum. Die nuwe spesie bring die totale hoeveelheid spesies in die genus na sewe. Om die patogenisiteit van die nematode te verhoog, kan die nematode gegroei word op ‘n bakterieë wat dood veroorsaak van slakke. Vyf bakterieë spesies was geïsoleer vanaf die ingewandes van slak gashere en geïdentifiseer deur die 16S rRNA gene. Die patogenisiteit van die bakterieë was getoets deur dit direk in D. reticulatum in te spuit en sterftes te monitor. Kluyvera sp. het die meeste sterftes veroorsaak in slakke en was gekies vir verdere formulering met Phasmarhabditis sp. SA4. Kulture bestaande uit slegs Kluyvera sp. en Phasmarhabditis sp. SA4 was getoets by 15°C, 20°C en 25°C. Die optimale groeitemperatuur vir Phasmarhabditis sp. SA4 was 15°C.

Masters