Entomopathogenic nematodes associated with organic honeybush (Cyclopia spp.) cultivation in South Africa.

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have demonstrated great potential as useful bio-control agents in the management of certain important soil-inhabiting insect pests of agricultural crops. In a survey of nematodes associated with organic honeybush cultivation, soil samples were obtained from nine organic honeybush plots, which are located in the Bredasdorp area of the Western Cape province of South Africa. The EPNs were isolated from soil by baiting with larvae of Galleria mellonella (greater wax moth) and identified by the amplification of the internal transcribed spacer region using the primer set TW81 and AB28. EPNs were abundant in the honeybush orchards, accounting for about 50% of the sampled fields and five EPN species were identified including Heterorhabditis bacteriophora, Heterorhabditis safricana, Steinernema khoisanae,. Steinernema nguyeni, Oscheius sp. and an unknown EPN, therefore suggesting a substantial diversity of EPNs in the sampled fields. A 100% mortality of infected G. mellonella larvae was recorded within 48 h of exposure to the nematodes. However, although these EPNs have been previously reported in South Africa, it is the first time they are found in such diversity on a conservative tillage management system in organic honeybush cultivation.

Colour of heterorhabditis zealandica-infected-Galleria mellonella dependent on the Photorhabdus symbiont, with two new nematode-symbiotic associations reported.

Bacterial symbionts associated with entomopathogenic nematodes (EPNs) play an important role in terms of the insecticidal properties of nematodes in pest control. Galleria mellonella larvae, shortly after being infected with three different strains of Heterorhabditis zealandica, which were isolated from South African soil, changed from pale white to steel grey-blue (blue), bright red, and yellow with a green tint (green), respectively. The genetic relatedness of the bacterial symbionts that were isolated from the three strains of H. zealandica was determined by means of comparing the 16S rRNA, recA, gyrB, dnaN, gltX and infB gene sequences. Subsequently, comparing the concatenated sequences revealed the presence of three distinct Photorhabdus species. The H. zealandica strain SF41, associated with Photorhabdus heterorhabditis, produced 'blue' G. mellonella larvae. The H. zealandica strain MJ2C, associated with Photorhabdus thracensis, yielded 'green' G. mellonella larvae, while the H. zealandica strain LLM associated with Photorhabdus laumondii subsp. laumondii yielded red larvae. The colour changes in G. mellonella larvae were found to have been instigated by a particular Photorhabdus species associated with H. zealandica. The red and 'green' phenotypes of G. mellonella larvae were found to represent new combinations of Heterorhabditis and Photorhabdus. In future studies, the colour of infected G. mellonella larvae needs to be reported as a phenotypic character, as it indicates the different bacterial species associated with the same nematode host, as shown in the case of H. zealandica.

Diversity and population distribution of nematodes associated with honeybush (Cyclopia spp.) and rooibos (Aspalathus linearis) in the Western Cape province of South Africa.

Nematodes are important soil organisms that constitute a key component of the soil ecosystem. A plant-parasitic survey was conducted to identify the diversity of nematodes associated with two endemic tea plants, honeybush (Cyclopia spp.) and rooibos (Aspalathus linearis) in the Western Cape province of South Africa. A total of 20 farmlands were surveyed and soil samples were collected from the rhizosphere of plants, for nematode isolation and identification based on morphological characters. Confirmation of the species of plant-parasitic nematodes was done using molecular-based tools. Nematodes were classified into various feeding groups based on their colonizer-persister (c-p) values. Plant-feeding nematodes identified from the honeybush tea plants include; Criconema mutabile, Meloidogyne hapla, M. javanica, and Xiphinema oxycaudatum, while Hoplolaimus sp., Neodolichorhynchus estherae and Pratylechus bolivianus were pathogenic on the rooibos monocultures. Bacterial and fungal feeders (Cephalobidae and Rhabditidae) were also abundant and frequently encountered in all samples. The study provides information on the diversity of nematodes associated with the indigenous herbal tea plants of South Africa.

Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape Province, South Africa.

Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be used as biological control agents against pest insects. EPF are capable of causing lethal epizootics in pest insect populations in agroecosystems. During a survey of the orchard soil at an organic farm, different EPF species were collected and identified to species level, using both morphological and molecular techniques. The EPF were trapped from soil samples taken from an apricot orchard. The traps, which were baited in the laboratory, used susceptible host insects, including the last-instar larvae of Galleria mellonella (wax moth larvae) and Tenebrio molitor (mealworm larvae). The potential pathogenicity of the local Metarhizium majus isolate was tested and verified using susceptible laboratory-reared last-instar T. molitor larvae. The identification of the M. majus isolated from South African soil was verified using both morphological and molecular techniques. The occurrence of M. majus in the South African soil environment had not previously been reported.

Molecular characterization of Helicotylenchus multicinctus and H. dihystera (Tylenchida: Hoplolaimidae) from Theobroma cacao in Nigeria.

The genus Helicotylenchus contains cosmopolitan, ubiquitous plant-parasitic nematodes with some species capable of causing significant economic damage to agricultural crops. Accurate species identification in this genus is essential in recognizing the damaging species and establishing effective management options. In a study on cocoa plantations in Nigeria, two species of spiral nematodes were found in high numbers from soil samples obtained from a six-decade old cocoa plantation at the Cocoa Research Institute of Nigeria. An integrated approach involving a combination of morphology, morphometrics, and molecular tools was used to identify the nematode species. Morphological data indicate the presence of both H. multicinctus and H. dihystera. There is congruence in the morphological and molecular data obtained for H. multicinctus. However, phylogenetic analysis of the 28S rDNA expansion segment revealed a high variability in the sequences of the Nigerian population of H. dihystera, suggesting the need for a careful appraisal and more comparative studies.

First report and molecular characterization of the dagger nematode, Xiphinema oxycaudatum (Nematoda, Dorylaimidae) from South Africa.

Plant-parasitic nematodes of the genus Xiphinema Cobb, 1913 comprise a complex group of nematode species, some of which are important vectors of plant viruses. During a field survey to determine the soil health of an abandoned honeybush (Cyclopia genistoides) monoculture, a high density of the dagger nematode, Xiphinema oxycaudatum Lamberti & Bleve-Zacheo, 1979 (Nematoda, Dorylaimidae), was observed in soil around the roots of honeybush plants in an abandoned farmland at Bereaville, an old mission station in the Western Cape province of South Africa. Soil samples were taken from the rhizosphere of plants and nematodes were extracted from the soil using a modified extraction tray method. Specimen of the dagger nematodes were processed for scanning electron microscopy, morphological and molecular analysis. Molecular profiling of the nematode species was done in order to give an accurate diagnosis and to effectively discriminate the nematode from other species within the Xiphinema americanum group. Phylogenetic analysis based on the D2D3 expansion segment of the 28S gene supported a close relationship of species within the americanum group, however, the protein-coding cytochrome oxidase (coxI) of the mitochondrial gene provided a useful tool for distinguishing the nematode from other species within the group. This study represents the first report of X. oxycaudatum from South Africa.

First Screening of Entomopathogenic Nematodes and Fungus as Biocontrol Agents against an Emerging Pest of Sugarcane, Cacosceles newmannii (Coleoptera: Cerambycidae).

Cacosceles newmannii (Coleoptera: Cerambycidae) is an emerging pest of sugarcane in South Africa. The larvae of this cerambycid beetle live within the sugarcane stalk and drill galleries that considerably reduce sugar production. To provide an alternative to chemical control, entomopathogenic nematodes and fungus were investigated as potential biological control agents to be used in an integrated pest management system. The nematodes Steinernema yirgalemense, S. jeffreyense, Heterorhabditis indica, and different concentrations of the fungus Metarhizium pinghaense were screened for efficacy (i.e., mortality rate) against larvae of C. newmannii. The different biocontrol agents used, revealed a low level of pathogenicity to C. newmannii larvae, when compared to control treatments.

Microbial control of phytophagous invertebrate pests in South Africa: Current status and future prospects.

Invertebrate pests pose a significant threat to food security on the African continent. In response, South Africa has become one of the largest importers of chemical pesticides in sub-Saharan Africa, with several hundred active ingredients registered. To address the over-reliance on such chemicals, the South African Department of Agriculture, Forestry and Fisheries (DAFF) has eliminated or restricted several pesticides since the late 1970s. The recent launch of the South African National Bio-Economy Strategy and establishment of the South African Bioproducts Organisation (SABO), together with new guidelines for registration of biopesticides in 2015, also support this endeavour. Concurrently, entomopathogen-related research and bioproduct development has increased over the past decade. Currently, 31 products (seven manufactured locally) are registered under the Fertilizers, Farm Feeds, Agricultural Remedies and Stock Remedies Act 36 of 1947. Commercially important microbes include Beauveria bassiana (Cordycipitaceae), Metarhizium anisopliae (Clavicipitaceae), Cydia pomonella granulovirus, Cryptophlebia leucotreta granulovirus, Helicoverpa armigera nucleopolyhedrovirus (Baculoviridae) and Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai (Bacillaceae). Both parasitic and entomopathogenic nematodes (EPNs) show potential for development as bioinsecticides with one commercial EPN product, based on Heterorhabditis bacteriophora (Heterorhabditidae), registered under the Act. Rapid scientific progression, supported by a favourable legislative environment, should facilitate further advances in microbial control of phytophagous invertebrate pests in South Africa.

Phasmarhabditis safricana n. sp. (Nematoda: Rhabditidae), a parasite of the slug Deroceras reticulatum from South Africa.

During a survey for mollusc-associated nematodes in South Africa, a new Phasmarhabditis species was isolated from the invasive slug, Deroceras reticulatum, collected from a nursery near George in the Western Cape province. The nematode was identified using a combination of morphological, morphometric, molecular, and phylogenetic techniques. The new species, P. safricana n. sp., is characterised by the cupola-shaped tail of the female with a spike, small, non-protruding phasmids, a fingerprint-like pattern of the cuticle covering the female tail, toothlike cephalic structures of the infective juveniles, and the distinct molecular characteristics of the species. The molecular phylogeny of the new species, as inferred from its SSU and LSU rRNA gene, places P. safricana n. sp. in close proximity to P. papillosa. Virulence tests were conducted, which demonstrated that P. safricana n. sp. caused significant mortality to the European invasive slug, D. reticulatum. The new species brings the total complement of the genus to eleven species.

Bacteria of the Genus Xenorhabdus, a Novel Source of Bioactive Compounds.

The genus Xenorhabdus of the family Enterobacteriaceae, are mutualistically associated with entomopathogenic nematodes of the genus Steinernema. Although most of the associations are species-specific, a specific Xenorhabdus sp. may infect more than one Steinernema sp. During the Xenorhabdus-Steinernema life cycle, insect larvae are infected and killed, while both mutualists produce bioactive compounds. These compounds act synergistically to ensure reproduction and proliferation of the nematodes and bacteria. A single strain of Xenorhabdus may produce a variety of antibacterial and antifungal compounds, some of which are also active against insects, nematodes, protozoa, and cancer cells. Antimicrobial compounds produced by Xenorhabdus spp. have not been researched to the same extent as other soil bacteria and they may hold the answer to novel antibacterial and antifungal compounds. This review summarizes the bioactive secondary metabolites produced by Xenorhabdus spp. and their application in disease control. Gene regulation and increasing the production of a few of these antimicrobial compounds are discussed. Aspects limiting future development of these novel bioactive compounds are also pointed out.

First report of a symbiotic relationship between Xenorhabdus griffiniae and an unknown Steinernema from South Africa.

Strain WS9, a mutualistic-associated bacterium, was isolated from an unknown entomopathogenic Steinernema nematode, collected from a litchi orchard in Friedenheim, Mpumalanga, South Africa. Based on phenotypic and phylogenetic data of the 16S rRNA, gltX, recA, dnaN, gyrB and infB gene sequences, strain WS9 is identified as X. griffiniae. Strain WS9 has antibacterial activity against Gram-positive and Gram-negative bacteria. This is the first report of an association between X. griffiniae and an unknown Steinernema species from South Africa.

Three Novel Xenorhabdus-Steinernema Associations and Evidence of Strains of X. khoisanae Switching Between Different Clades.

Xenorhabdus species are normally closely associated with entomopathogenic nematodes of the family Steinernematidae. Strain F2, isolated from Steinernema nguyeni, was identified as Xenorhabdus bovienii and strains J194 and SB10, isolated from Steinernema jeffreyense and Steinernema sacchari as Xenorhabdus khoisanae, based on phenotypic characteristics and sequencing of 16S rRNA and housekeeping genes dnaN, gltX, gyrB, infB and recA. All three strains produced antimicrobial compounds that inhibited the growth of Gram-positive and Gram-negative bacteria. This is the first report of associations between strains of the symbiotic bacteria X. bovienii with S. nguyeni, and X. khoisanae with S. jeffreyense and S. sacchari. This provides evidence that strains of Xenorhabdus spp. may switch between nematode species within the same clade and between different clades.

Divergent thermal specialisation of two South African entomopathogenic nematodes.

Thermal physiology of entomopathogenic nematodes (EPN) is a critical aspect of field performance and fitness. Thermal limits for survival and activity, and the ability of these limits to adjust (i.e., show phenotypic flexibility) depending on recent thermal history, are generally poorly established, especially for non-model nematode species. Here we report the acute thermal limits for survival, and the thermal acclimation-related plasticity thereof for two key endemic South African EPN species, Steinernema yirgalemense and Heterorhabditis zealandica. Results including LT50 indicate S. yirgalemense (LT50 = 40.8 ± 0.3 °C) has greater high temperature tolerance than H. zealandica (LT50 = 36.7 ± 0.2 °C), but S. yirgalemense (LT50 = -2.4 ± 0 °C) has poorer low temperature tolerance in comparison to H. zealandica (LT50 = -9.7 ± 0.3 °C), suggesting these two EPN species occupy divergent thermal niches to one another. Acclimation had both negative and positive effects on temperature stress survival of both species, although the overall variation meant that many of these effects were non-significant. There was no indication of a consistent loss of plasticity with improved basal thermal tolerance for either species at upper lethal temperatures. At lower temperatures measured for H. zealandica, the 5 °C acclimation lowered survival until below -12.5 °C, where after it increased survival. Such results indicate that the thermal niche breadth of EPN species can differ significantly depending on recent thermal conditions, and should be characterized across a broad range of species to understand the evolution of thermal limits to performance and survival in this group.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).

Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae.

Bacterial strain SF87(T), 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 SF87(T) is shown to be closely related (98% similarity) to Xenorhabdus hominickii DSM 17903(T). Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96-97% similarity with Xenorhabdus miraniensis DSM 17902(T). 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 SF87(T), 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 SF87(T) ( =DSM 25463(T) =ATCC BAA-2406(T)).

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica.

The bacterial symbiont AM7(T), 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 AM7(T) 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 AM7(T) revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97 % proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) ( = ATCC BAA-2407(T)  = DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov.

Potential of South African entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) for control of the citrus mealybug, Planococcus citri (Pseudococcidae).

Planococcus citri, the citrus mealybug, is the most important species of mealybug known to infest citrus in South Africa. Various laboratory bioassays were conducted to determine the potential of entomopathogenic nematodes to control P. citri. Adult female P. citri were screened for susceptibility to six indigenous nematode species. P. citri was found to be most susceptible to Steinernema yirgalemense and Heterorhabditis zealandica, causing 97% and 91% mortality, respectively. The development of nematodes after infecting adult female P. citri showed both H. zealandica and S. yirgalemense were able to complete their life cycles inside the host. Further bioassays illustrated a linear relationship between mealybug mortality and the concentration of nematodes applied, with the highest level of control using a concentration of 80 infective juveniles (IJs)/insect. As nematodes would be used as an above-ground application to control P. citri in citrus orchards, available water is a major limiting factor. Insecticidal activity proved to be dependent on the available surface moisture after nematode application. The water activity (a(w)) bioassay indicated that S. yirgalemense to be two times more tolerant to lower levels of free water, with a(w50)=0.96 and a(w90)=0.99, compared to H. zealandica with a(w50)=0.98 and a(w)90=1.0. After application, nematodes have a limited time frame in which to locate and infect hosts, as the level of available free water gradually decreases, as trees dry out. S. yirgalemense proved able to locate and infect P. citri quicker than H. zealandica. Nematode activity was not significantly affected when exposed to 15°C, 20°C and 25°C. IJs were able to infect P. citri at an exposure time as short as half an hour. Results also showed that the first 2-4h post application is the most decisive time for establishing successful infection of mealybugs. This is the first report on the potential use of nematodes for the control of P. citri.

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth.

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50IJs/FCM larva caused 100% mortality and 74% at a concentration of 200IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20IJs/cm(2). This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.

Susceptibility of the Mediterranean fruit fly (Ceratitis capitata) and the Natal fruit fly (Ceratitis rosa) to entomopathogenic nematodes.

The potential of entomopathogenic nematodes, Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema khoisanae, to infect pupariating larvae, pupae and adults of Ceratitis capitata and Ceratitis rosa was investigated in laboratory bioassays. Pupariating larvae and adult flies were susceptible to nematode infection, with no infection recorded for the pupae. Pupariating larvae of C. capitata were generally more susceptible to infection than those of C. rosa. Significantly more larvae of C. capitata were infected by H. bacteriophora. For C. rosa, highest infectivity of larvae was obtained with H. zealandica. In contrast, adults of both species were highly infected by S. khoisanae.