Browsing by Author "Ferreira, Tiarin"

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    Characterisation of nematode symbiotic bacteria and the in vitro liquid culture of Heterorhabditis zealandica and Steinernema yirgalemense
    (Stellenbosch : Stellenbosch University, 2013-03) Ferreira, Tiarin; Malan, Antoinette P.; Addison, Pia; Addison, M. F.; Stellenbosch University. Faculty of AgriSciences. Dept. of Conservation Ecology and Entomology.
    ENGLISH ABSTRACT: Entomopathogenic nematodes have the potential to be outstanding biocontrol agents against agricultural pest insects. Combined with their bacterial symbionts, these biocontrol agents have proven to be very effective against numerous pests. The nematodes belong to the families Steinernematidae and Heterorhabditidae, and are ideal to be used in, and integrated with, pest management systems. There is a dire need for new and innovative methods to control agricultural pests, as numerous pest insects have developed resistance against broad-spectrum insecticides. Together with the environmental impact of these insecticides and the safety aspect regarding humans and animals, the need to develop new technologies, including entomopathogenic nematodes for pest management, is high. In this study, the associated symbiotic bacteria of three entomopathogenic nematodes species were isolated, and the potential of two nematode species to be successfully mass cultured in liquid medium was evaluated. Regarding the symbiotic bacteria, results from the study showed that bacteria species from all three nematode species, Heterorhabditis noenieputensis, Steinernema khoisanae and Heterorhabditis zealandica, were novel. Heterorhabditis noenieputensis was isolated in the Mpumalanga province during a previous survey conducted in citrus orchards. The bacterium isolated from this nematode belongs to the genus Photorhabdus, and bear closest similarity (98.6%) to the type strain of P. luminescens subsp laumondii (TT01T). Photorhabdus luminescens subsp. noenieputensis subsp. nov., derives its name from the area where the nematode was sourced, namely the farm Springbokvlei, near the settlement Noenieput close to the Namibian border. Thus far, 85 Steinernema spp. have been described worldwide, including S. khoisanae which was isolated in the Western Cape province of South Africa. Four S. khoisanae strains, namely SF87, SF80, SF362 and 106-C, were used for characterisating the new bacteria from different localities in South Africa. Using the neighbor-joining method, all the strains were aligned with 97% homology to the 16S rRNA sequences of several Xenorhabdus- type strains, indicating that they belonged to the same genus. The multigene approach was used to distinguish between the Xenorhabdus spp. and partial recA, dnaN, gltX, gyrB and infB gene sequences of the various strains were analysed. The bacterium species was named Xenorhabdus khoisanae sp. nov. after the nematode from which it was isolated. The results showed that the third bacterium species, which was isolated from H. zealandica, was new. The sequence of the bacteria strain clustered with the type strains of P. temperata and P. asymbiotica, indicate that it belonged to the genus Photorhabdus. This is the first study to show that H. zealandica associates with a luminescent Photorhabdus species, rather than with the known non-luminescent P. temperata. The potential of H. zealandica and Steinernema yirgalemense mass culture in liquid was investigated. Results illustrated that H. zealandica and its P. luminescens symbiont can be successfully cultured in liquid. However, two generations occurred during the process time, instead of the desirable one-generation. The growth curve of the symbiotic bacteria during the process time was measured, in order to determine when the stationary phase was reached, with the results showing this to occur after 36 h. Therefore, the optimum amount of time required for inoculating the IJs and for aiding in maximum infective juvenile (IJ) recovery is 36 h for adding the nematodes post pre-culturing of the bacteria. Future research goals should be to increase the percentage recovery in liquid culture, which would increase the number of nematodes produced per ml, which would, therefore, reduce the processing time significantly. The results from mass culturing the second nematode species, S. yirgalemense, indicated an asynchronous nematode development in the first generation. Growth curves were performed with the symbiotic bacteria that showed the exponential phase of Xenorhabdus started after 15 h, and that, after 42 h, the stationary phase was reached, with an average of 51 × 107 cfu·ml-1. Bioassays were performed to compare the virulence between in vitro- and in vivo-produced nematodes, with the results showing that the in vitro-produced nematodes were significantly less virulent than were the nematodes produced in vivo. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimising and upscaling of the commercial production of nematodes in industrial fermenters. The last aim of the current study was to determine when Xenorhabdus reached the stationary phase, when it is grown in a 20-L fermenter, as this would be the optimum time at which to add the IJs of S. yirgalemense. Such characteristics as the effect of stationary phase conditions on the bacterial cell density and on the DO2 rate in the fermenter were investigated. The results showed that the stationary phase of Xenorhabdus was reached after 36 h at 30˚C, which took 6 h less than did the same procedures followed with the Xenorhabdus sp. cultured in Erlenmeyer flasks on orbital shakers. This is the first step toward the liquid mass culturing of S. yirgalemense in industrial-size fermenters. Data from this study indicated the optimum amount of time that is required for adding nematodes to the bacterial culture in the fermenter, and for ensuring the optimum recovery of IJs, as well as a subsequent high yield of nematodes within a minimum processing time. This is the first report of its kind to investigate comprehensively the successful liquid culture of two South African entomopathogenic nematode species for the sole purpose of evaluating potential commercialisation. Results emanating from this study could be used as groundwork in future, in combination with similar research such as culturing nematodes intensively in large fermenters.
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    Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae
    (Society for General Microbiology, 2013-09) Ferreira, Tiarin; Van Reenen, Carol A.; Endo, Akihito; Sproer, Cathrin; Malan, Antoinette P.; Dicks, Leon Milner Theodore, 1961-
    Bacterial strain SF87T, and additional strains SF80, SF362 and 106-C, isolated from the nematode Steinernema khoisanae, are non-bioluminescent Gram-reaction-negative bacteria that share many of the carbohydrate fermentation reactions recorded for the type strains of recognized Xenorhabdus species. Based on 16S rRNA gene sequence data, strain SF87T is shown to be closely related (98 % similarity) to Xenorhabdus hominickii DSM 17903T. Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96–97 % similarity with Xenorhabdus miraniensis DSM 17902T. However, strain SF87 shares only 52.7 % DNA–DNA relatedness with the type strain of X. miraniensis, confirming that it belongs to a different species. Strains SF87T, SF80, SF362 and 106-C are phenotypically similar to X. miraniensis and X. beddingii, except that they do not produce acid from aesculin. These strains are thus considered to represent a novel species of the genus Xenorhabdus, for which the name Xenorhabdus khoisanae sp. nov. is proposed. The type strain is SF87T ( = DSM 25463T = ATCC BAA-2406T).
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    Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica
    (Society for General Microbiology, 2013-05) Ferreira, Tiarin; Van Reenen, Carol; Pages, Sylvie; Tailliez, Patrick; Malan, Antoinette P.; Dicks, Leon Milner Theodore, 1961-
    The bacterial symbiont AM7T, isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7T within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7T revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29T, P. luminescens subsp. akhurstii FRG04T and P. luminescens subsp. hainanensis C8404T, respectively.
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    Rearing of the banded fruit weevil, Phlyctinus callosus (Schonherr) (Coleoptera: Curculionidae) and control with entomopathogenic nematodes
    (Stellenbosch : University of Stellenbosch, 2010-03) Ferreira, Tiarin; Malan, Antoinette P.; Addison, Pia; University of Stellenbosch. Faculty of Agrisciences. Dept. of Conservation Ecology and Entomology.
    ENGLISH ABSTRACT: The banded fruit weevil, Phlyctinus callosus (Schönherr), is a key pest of apples, nectarines and grapevines in the southern areas of the Western Cape. The control of P. callosus is not satisfactory and the insecticides used to control this insect have not proved to be effective since the development of tolerance to pyrethroids and acephate. A control method that can be used, despite it being very labour-intensive, is that of tree trunk barriers. The use of such a method will prevent the weevils from reaching the fruit, as they are unable to fly. Alternative control options, such as the use of entomopathogenic nematodes, are urgently needed for the control of P. callosus. Entomopathogenic nematodes belonging to the Steinernematidae and Heterorhabditidae are ideal biocontrol agents for incorporation into an integrated pest management programme. In order to develop control strategies for P. callosus, large numbers and a predictable quantity of different weevil stages are needed. Especially large numbers of larvae are needed, as this is the stage that will be targeted with nematodes. One of the aims of the current study was to assess various artificial diets for rearing larvae of P. callosus. Though adult weevils were easily collected from orchards, it was very difficult to obtain large numbers of larvae. Modified versions of an agar diet, as well as different carrot based diets, were tested at 21°C. The highest percentage survival obtained for the agar diet was 50% and 60% for one type of carrot diet. A better rearing method proved to be that of planting full-grown carrots in pots, kept at 25°C, resulting in the attainment of the highest percentage survival rate of 90%. A study was undertaken to assess how long, and at what temperature, P. callosus eggs could be stored. A mean percentage hatch of 45.7% was obtained when eggs were stored at 4°C for 70 days. Eggs started hatching after 47 days and 10 days, when stored at temperatures of 11°C and 14°C, respectively. If the aim of the employment of such a method is only to delay egg hatching, the two temperatures (11°C and 14°C) will be suitable. For the following part of the study, several entomopathogenic nematode isolates were evaluated for their potential use as biological control agents against P. callosus. The susceptibility of P. callosus larvae and adults to nematode infection was assessed in the laboratory by screening for their mortality, using different nematode isolates. Larvae were found to be more susceptible to nematode infection than adults. Heterorhabditis isolates were found to cause higher levels of mortality than the Steinernema isolates during screening, when a concentration of 400 infective juveniles (IJ) per insect V was used. Biological characteristics, such as the effect of different temperatures on nematode activity and the minimum concentration of nematodes needed to obtain acceptable levels of control for P. callosus, were also investigated. The percentage mortality ranged from no infection to 75% after four days for the larvae, and the SF41 isolate of Heterohabditis zealandica was selected as the most promising isolate for further laboratory experiments. The vertical movement of nematodes in sand, compared with such movement in sandy loam soil, and the biology of H. zealandica in P. callosus larvae was also investigated in laboratory bioassays. After four days, the LD50 and LD90 values were 96 IJ/50 μl and 278 IJ/50 μl, respectively. Nematodes were found to be inactive at 11°C, with the highest mortality rate of P. callosus resulting from nematode infection being recorded at 25°C. A higher percentage mortality rate was obtained with the sandy loam soil (95.2%) than with the sand (77.5%). Heterorhabditis zealandica could successfully complete its life cycle in 6th instar P. callosus larvae. The study showed that P. callosus larvae are suitable hosts for H. zealandica, and that the control of P. callosus in the field by the selected isolate holds promise. The persistence of the SF41 isolate of H. zealandica at different concentrations was investigated in the last part of the study. The experiment took place in a blueberry orchard, subject to a high rate of infestation by P. callosus. Concentrations of 0, 20, 30 and 45 IJ/cm2 were topically applied, with persistence being evaluated for days 1, 35 and 84. Percentage persistence for 30 IJ/cm2 was calculated as 87.5% for days 35 and 84. The persistence of soil samples taken on day one, and kept in plastic containers at room temperature, was again evaluated on day 128, with the finding that both 30 IJ/cm2 and 45 IJ/cm2 caused 100% mortality of Tenebrio molitor (L.). Results indicated good persistence of H. zealandica after 84 days in field conditions, with a high maintenance of P. callosus populations. The study indicated the potential use of H. zealandica for the control of P. callosus, with the possibility of persistence for at least three months. Future research into the control of P. callosus with nematodes should aim to investigate the technical aspects of field application. The current study shows that entomopathogenic nematodes have potential for controlling the soil stages of P. callosus. The capacity to rear large numbers of P. callosus larvae in the laboratory, for later use in laboratory and field trials, is of key importance.