Natural infection of white grubs (Coleoptera: Scarabaeidae) with entomopathogenic nematodes in the KwaZulu-Natal province of South Africa.

White grubs are root feeding larvae of beetles (Coleoptera: Scarabaeidae) that are sporadic pests in agriculture and can lead to economic damage. The grubs feed on the roots of plants, while the adult beetle can bore into underground stems, as well as cause defoliation of plants. Sporadic incidence of larvae with symptoms of nematode infections were detected in wattle and sugarcane plantations in the KwaZulu-Natal province of South Africa. The larvae with infection symptoms were isolated, washed, and put on water traps to collect infective juveniles of possible nematode infections. Three species of entomopathogenic nematodes (EPNs) were isolated from the white grub larvae. These included Steinernema bertusi isolated from Maladera sp. 4., Oscheius myriophila from Maladera sp. 4 and Schizonchya affinis, and Steinernema fabii isolated from Maladera sp. 4., Pegylis sommeri, and S. affinis. Of these S. fabii was the most common species in the sample (87%). This is the first report of such a high diversity of locally occurring EPNs found naturally associated with white grub species in this region of South Africa.

In vitro liquid culture of the mollusc-parasitic nematode Phasmarhabditis (Rhabditida: Rhabditidae).

The success of the mollusc-parasitic nematode, Phasmarhabditis hermaphrodita (Schneider) Andrássy (Rhabditida: Rhabditidae), as a biological control agent in Europe has led to worldwide interest in phasmarhabditids as biocontrol agents. In this study, the mass culture potential of three phasmarhabditids, namely Phasmarhabditis papillosa, Phasmarhabditis kenyaensis and Phasmarhabditis bohemica, was assessed. In addition, ten bacterial candidates, consisting of seven associated with slugs and three associated with entomopathogenic nematodes, were investigated. The bacteria were tested for their ability to cause mortality to Deroceras invadens, as well as to support nematode growth. Initial mortality studies demonstrated that Kluyvera, Aeromonas and Pseudomonas spp. (AP3) caused 100% mortality when they were injected into the haemocoel of D. invadens. However, in growth studies, Pseudomonas sp. (AP4) was found to be the most successful bacterium, leading to recovery and reproduction in almost all nematode species, except for P. kenyaensis. In flask studies, P. bohemica, which showed exceptional growth with Pseudomonas sp. (AP1), was chosen for further investigation. The effect of inoculating flasks with different concentrations of Pseudomonas sp. (AP1), as well as with different concentrations of P. bohemica, was evaluated by assessing the nematode populations for 14 days. The results indicated that the lowest, 1% (v/v), bacteria inoculation led to higher total nematode and to infective juvenile (IJ) yield, with flasks with the highest IJ inoculum (3000 IJs/ml) having a positive effect on the total number of nematodes and IJs in cultures of P. bohemica. This study presents improvements for the mass-culturing of nematodes associated with molluscs.

Ex vivo development of Phasmarhabditis spp. associated with terrestrial molluscs.

The success of Phasmarhabditis hermaphrodita (Schneider) Andrássy (Rhabditida: Rhabditidae) as a biological control agent of molluscs has led to a worldwide interest in phasmarhabditids. However, scant information is available on the lifecycle development of species within the genus. In the current study, the development of P. hermaphrodita, Phasmarhabditis papillosa, Phasmarhabditis bohemica and Phasmarhabditis kenyaensis were studied using ex vivo cultures, in order to improve our understanding of their biology. Infective juveniles (IJs) of each species were added to 1 g of defrosted homogenized slug cadavers of Deroceras invadens and the development monitored after inoculated IJ recovery, over a period of eight-ten days. The results demonstrated that P. bohemica had the shortest development cycle and that it was able to produce first-generation IJs after eight days, while P. hermaphrodita, P. papillosa and P. kenyaensis took ten days to form a new cohort of IJs. However, from the perspective of mass rearing, P. hermaphrodita has an advantage over the other species in that it is capable of forming self-fertilizing hermaphrodites, whereas both males and females are required for the reproduction of P. papillosa, P. bohemica and P. kenyaensis. The results of the study contribute to the knowledge of the biology of the genus and will help to establish the in vitro liquid cultures of different species of the genus.

Occurrence and Seasonal Changes in the Population of Root-knot Nematodes on Honeybush (Cyclopia Sp.).

Root-knot nematodes in the genus Meloidogyne are an important group of plant-parasitic nematodes causing severe damage on agricultural crops worldwide. A study was conducted to identify the species of root-knot nematodes causing damage on honeybush monocultures and to assess the seasonal variations in the nematode population. Soil samples were collected from six experimental sites in Genadendal, Western Cape province of South Africa from 2016 to 2017. DNA was extracted from single-second stage juveniles and species identifi cation was done using species-specifi c sequence-characterised amplifi ed regions (SCAR) primers. Meloidogyne hapla and M. javanica were identifi ed from the sites. Mean population density of the nematodes varied significantly (p < 0.05) in the six sites, with the peak population being recorded in summer of 2017. The study suggests that seasonal variation in temperature and moisture could contribute to changes in the population density of root-knot nematodes in the soil.

Nematodes that associate with terrestrial molluscs as definitive hosts, including Phasmarhabditis hermaphrodita (Rhabditida: Rhabditidae) and its development as a biological molluscicide.

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. Current knowledge indicates that there are eight nematode families that associate with molluscs as definitive hosts, including Agfidae, Alaninematidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogastridae, Mermithidae and Rhabditidae. To date, Phasmarhabditis hermaphrodita (Schneider, 1859) Andrássy, 1983 (Rhabditida: Rhabditidae) 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 tradename Nemaslug®, is now sold in 15 different European countries. This paper reviews nematodes isolated from molluscs, with specially detailed information on the life cycle, host range, commercialization, natural distribution, mass production and field application of P. hermaphrodita.

First Report of the Isolation of the Symbiotic Bacterium Photorhabdus luminescens subsp. laumondii Associated with Heterorhabditis safricana from South Africa.

Photorhabdus luminescens subsp. laumondii is closely associated with the entomopathogenic nematode Heterorhabditis bacteriophora and has, to date, not been isolated from other nematode species. This study is the first report of P. luminescens subsp. laumondii from two South African isolates of entomopathogenic nematodes, Heterorhabditis safricana SF281 and H. bacteriophora SF351. Both symbiotic bacterial strains are phenotypically closely related to P. luminescens subsp. laumondii previously isolated and described from H. bacteriophora. The genetic relatedness between P. luminescens subsp. laumondii strains SF281B and SF351B was confirmed by comparing 16S rDNA, recA, gyrB and gltX sequences with sequences of P. luminescens subsp. laumondii, including the type strain (TT01T) and strain E21.

Development and population dynamics of Steinernema yirgalemense (Rhabditida: Steinernematidae) and growth characteristics of its associated Xenorhabdus indica symbiont in liquid culture.

Entomopathogenic nematodes have become a valuable addition to the range of biological control agents available for insect control. An endemic nematode, Steinernema yirgalemense, has been found to be effective against a wide range of key insect pests. The next step would be the mass production this nematode for commercial application. This requires the establishment of monoxenic cultures of both the nematode and the symbiotic bacterium Xenorhabdus indica. First-stage juveniles of S. yirgalemense were obtained from eggs, while X. indica was isolated from nematode-infected wax moth larvae. The population density of the various life stages of S. yirgalemense during the developmental phase in liquid culture was determined. The recovery of infective juveniles (IJs) to the third-stage feeding juveniles, was 67 ± 10%, reaching a maximum population density of 75,000 IJs ml- 1 on day 13 after inoculation. Adult density increased after 8 days, with the maximum female density being 4600 ml- 1 on day 15, whereas the maximum male density was 4300 ml- 1 on day 12. Growth curves for X. indica showed that the exponential phase was reached 15 h after inoculation to the liquid medium. The stationary phase was reached after 42 h, with an average of 51 × 107 colony-forming units ml- 1. Virulence tests showed a significant difference in insect mortality between in vitro- and in vivo-produced nematodes. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimizing and upscaling of the commercial production of nematodes in fermenters.

Entomopathogenic nematodes for the control of the codling moth (Cydia pomonella L.) in field and laboratory trials.

Three commercially available entomopathogenic nematode (EPN) strains (Steinernema feltiae and Heterorhabditis bacteriophora Hb1 and Hb2) and two local species (S. jeffreyense and S. yirgalemense) were evaluated for the control of the codling moth (Cydia pomonella). In field spray trials, the use of S. jeffreyense resulted in the most effective control (67%), followed by H. bacteriophora (Hb1) (42%) and S. yirgalemense (41%). Laboratory bioassays using spray application in simulated field conditions indicate S. feltiae to be the most virulent (67%), followed by S. yirgalemense (58%). A laboratory comparison of the infection and penetration rate of the different strains showed that, at 14°C, all EPN strains resulted in slower codling moth mortality than they did at 25°C. After 48 h, 98% mortality was recorded for all species involved. However, the washed codling moth larvae, cool-treated (at 14°C) with S. feltiae or S. yirgalemense, resulted in 100% mortality 24 h later at room temperature, whereas codling moth larvae treated with the two H. bacteriophora strains resulted in 68% and 54% control, respectively. At 14°C, S. feltiae had the highest average penetration rate of 20 IJs/larva, followed by S. yirgalemense, with 14 IJs/larva. At 25°C, S. yirgalemense had the highest penetration rate, with 39 IJs/larva, followed by S. feltiae, with 9 IJs/larva. This study highlights the biocontrol potential of S. jeffreyense, as well as confirming that S. feltiae is a cold-active nematode, whereas the other three EPN isolates tested prefer warmer temperatures.

First report of the symbiotic bacterium Xenorhabdus indica associated with the entomopathogenic nematode Steinernema yirgalemense.

The entomopathogenic nematode Steinernema yirgalemense is considered a promising agent in the biocontrol of insects. However, little is known about the bacteria living in symbiosis with the nematode. In this study, we have identified the only available bacterial strain (157-C) isolated from S. yirgalemense, as a member of the species Xenorhabdus indica. Identification was based on 16S rDNA, recA, dnaN, gltX, gyrB and infB gene sequence analyses. The relatedness of strain 157-C to the type strain of X. indica (DSM 17 382) was confirmed with DNA-DNA hybridization. The phenotypic characteristics of strain 157-C are similar to those described for the type strain of X. indica. This is the first report associating X. indica with S. yirgalemense.

Steinernema jeffreyense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Africa.

During a non-targeted survey for entomopathogenic nematodes in South Africa, a new species of Steinernema was isolated from a soil sample collected from underneath a guava tree, close to the shore at Jeffrey's Bay. The nematode was isolated by means of the insect-baiting technique using last-instar larvae of Galleria mellonella. It is described herein as Steinernema jeffreyense n. sp. The nematode can be separated from other described, closely related species in terms of the morphological and morphometric characteristics of the different life stages, and in terms of the characterization and phylogeny of DNA sequences of the internal transcribed spacer (ITS) rDNA of the 18S gene, and of the D2D3 region of the 28S rDNA gene. The new species is placed molecularly in the arenarium-glaseri-karii-longicaudatum group characterized by the following morphological characters: infective third-stage juvenile with a body length of 926 (784-1043) µm, distance from head to excretory pore of 87 (78-107) µm, tail length of 81 (50-96) µm, with an E% of 109 (86-169), and eight evenly spaced ridges (i.e. nine lines) in the middle of the body. First-generation males have a spicule length of 88 (79-95) µm and gubernaculum length of 57 (51-61) µm. Male mucron is absent in both generations. First-generation females have an asymmetrical protuberance and a short, double-flapped epiptygmata, with both flaps directed to the front. The tail of the first-generation female is shorter than the anal body width, with a mucron on the dorsal tail tip, with D% = 78 (59-99). Cross-hybridization with S. khoisanae, S. tophus and S. innovationi showed the new species to isolate reproductively from the others. The analyses of ITS rDNA and D2D3 sequence of the 18S and 28S rDNA genes support the studied nematode isolate to be a valid new species belonging to the 'glaseri' group (Clade V).

Adjuvants to improve aerial control of the citrus mealybug Planococcus citri (Hemiptera: Pseudococcidae) using entomopathogenic nematodes.

The citrus mealybug, Planococcus citri, is a highly destructive pest of citrus, occurring only in the aerial parts of plants. Humidity will be one of the key factors to consider when using entomopathogenic nematodes (EPN) as biological control agents. Different adjuvants can be added to suspensions of EPNs, to improve control as a foliar application. An aqueous suspension containing Heterorhabditis zealandica and 0.3% Zeba® significantly increased P. citri mortality by 22% at 80% relative humidity (RH) with a temperature cycle starting at 22°C for 14 h and 11°C for 11 h. The same polymer formulation was tested for Steinernema yirgalemense and mortality of P. citri increased by 21% at 60% RH and by 27% at 80% RH. The addition of Nu-Film-P® and Zeba® to H. zealandica suspensions did not significantly retard application run-off on citrus leaves. The combination of Nu-Film-P® and Zeba®, however, was able to significantly retard sedimentation, increasing the average number of nematodes deposited on 2-cm2 leaf discs by 10 nematodes. In an aqueous suspension, nematodes settle rapidly to the bottom, resulting in an uneven distribution of nematodes. Xanthan gum, at a concentration of 0.2%, was highly effective at retarding sedimentation, with 72% of the initial nematode number still in suspension after 1 h. Zeba®, at a concentration of 0.3%, despite not being as effective as Xanthan gum, nevertheless still retarded sedimentation significantly. This is the first report of the potential of Nu-Film-P® and Zeba® to improve EPN performance against P. citri when used above ground in citrus orchards.

Heterorhabditis noenieputensis n. sp. (Rhabditida: Heterorhabditidae), a new entomopathogenic nematode from South Africa.

A new entomopathogenic nematode in the genus Heterorhabditis is described from South Africa, from two singular isolates found 1000 km from each other, from beneath a fig tree and in a citrus orchard, respectively. Morphological and molecular studies indicate both isolates to be the same and a new undescribed Heterorhabditis species. Comparison of sequences of the internal transcribed spacer (ITS) rDNA and the D2D3 region of the 28S rDNA gene with available sequences of other described species within the genus, indicate the two isolates as a new species. Phylogenetic analysis of the sequence data concerned placed the new species, H. noenieputensis n. sp., closest to H. indica and H. gerrardi in the indica-group. The new species, H. noenieputensis n. sp., is distinguished from other species in the genus by a combination of several morphological traits of the males and the infective juveniles (IJs). The new species differs from all other species previously described, as regards the body length of the IJs, except for H. indica and H. taysearae, in which the IJ is smaller. The IJ also differs from that of H. indica in the length of the oesophagus, the body diameter, the length of the tail and the E%. In addition, males of H. noenieputensis n. sp. differ from their closest relative, H. indica, in the position of the excretory pore, SW% and D%; and from H. gerrardi in the length of the oesophagus and SW%. The seventh pair of genital papillae of H. noenieputensis n. sp. are normally developed, while for H. indica they are often branched or swollen at the base, while 8 and 9 are usually absent in both species.

Potential of entomopathogenic nematodes for the control of the banded fruit weevil, Phlyctinus callosus (Schönherr) (Coleoptera: Curculionidae).

Entomopathogenic nematodes (EPN) were evaluated for their potential use as biological control agents against Phlyctinus callosus, the banded fruit weevil (BFW). The susceptibility of larvae and adults to EPN was evaluated using 400 infective juveniles (IJ) per insect after 4 days in 24-well bioassay trays. The nematode isolates used were all able to infect BFW, although the larvae were found to be more susceptible than were the adults. The percentage mortality for BFW larvae ranged from 41 to 73% and for BFW adults from 13 to 45%. The most effective isolate, SF41 of Heterorhabditis zealandica, was used to investigate the effect of vertical movement of nematodes in sand and sandy loam soil, at specified concentration and temperature. A higher (82.2 ± 0.084%) percentage mortality rate was obtained with the sandy loam soil, than with the use of sand (67.5 ± 0.12%). The LD50 and LD90 values after 4 days of incubation were 96 and 278 IJ/50 µl, respectively. Nematodes were inactive below 15 °C, with the highest mortality of 74 ± 0.081% for BFW larvae recorded at 25 °C. Heterorhabditis zealandica was able to complete its life cycle successfully in sixth-instar BFW larvae after a period of 22 days. The study showed BFW larvae not to be as susceptible to nematode infection as they need a high concentration (400 IJ/larva) and 4 days to give effective control.

ORGANIC VS CONVENTIONAL: SOIL NEMATODE COMMUNITY STRUCTURE AND FUNCTION.

Global increases in human population are creating an ever-greater need for food production. Poor soil management practices have degraded soil to such an extent that rapidly improved management practices is the only way to ensure future food demands. In South Africa, deciduous fruit producers are realising the need for soil health, and for an increased understanding of the benefits of soil ecology, to ensure sustainable fruit production. This depends heavily on improved orchard management. Conventional farming relies on the addition of artificial fertilizers, and the application of chemicals, to prevent or minimise, the effects of the soil stages of pest insects, and of plant-parasitic nematodes. Currently, there is resistance toward conventional farming practices, which, it is believed, diminishes biodiversity within the soil. The study aimed to establish the soil nematode community structure and function in organically, and conventionally, managed deciduous fruit orchards. This was done by determining the abundance, the diversity, and the functionality of the naturally occurring free-living, and plant-parasitic, nematodes in deciduous fruit orchards in the Western Cape province of South Africa. The objective of the study was to form the basis for the use of nematodes as future indicators of soil health in deciduous fruit orchards. Orchards from neighbouring organic, and conventional, apricot farms, and from an organic apple orchard, were studied. All the nematodes were quantified, and identified, to family level. The five nematode-classified trophic groups were found at each site, while 14 families were identified in each orchard, respectively. Herbivores were dominant in all the orchards surveyed. Organic apples had the fewest herbivores and fungivores, with the highest number of carnivores. When comparing organic with conventional apricot orchards, higher numbers of plant-parasitic nematodes were found in the organic apricot orchards. The Maturity Index (MI) indicated that all orchard soils had values below 1.5, indicating disturbed conditions. The conventionally managed apricot orchard had the highest MI value, of 1.48. The Plant Parasitic Index (PPI) value was highest in the organically managed apricot orchard. In order to determine the existing enrichment, structural, and basal conditions, the nematode faunal analysis was applied to each site. All the sites indicated enriched and structured conditions. Regarding the diversity, the richness, and the evenness of the distribution, soil from the conventional apricot orchard had the highest species richness, whereas the organic apple orchard soil had the most even distribution of families. Different management practices in fruit orchards did not show marked differences in terms of community composition and structure.

Diversity and distribution of nematodes associated with terrestrial slugs in the Western Cape Province of South Africa.

A survey of nematodes associated with native and introduced species of terrestrial slugs was conducted in the Western Cape Province of South Africa, in order to gather new data regarding diversity and distribution. A total of 521 terrestrial slugs were collected from 35 localities throughout the Western Cape. All slugs were dissected and examined for the presence of internal nematodes. Extracted nematodes were identified using a combination of molecular (18S rRNA gene sequencing) and morphological techniques. Nematodes were found parasitizing slugs at 14 of the 35 sites examined, amounting to 40% of sample sites. Of all slugs, 6% were infected with nematodes. A total of seven species of nematode were identified in the province, including Agfa flexilis, Angiostoma sp., Phasmarhabditis sp. SA1, Phasmarhabditis sp. SA2, Caenorhabditis elegans, Panagrolaimus sp. and Rhabditis sp. Of these species, four were thought to be parasitic to slugs (A. flexilis, Angiostoma sp., Phasmarhabditis sp. SA1 and Phasmarhabditis sp. SA2), as opposed to forming necromenic or phoretic associations. Three new species of slug-parasitic nematode were identified during this study (Angiostoma sp., Phasmarhabditis sp. SA1 and Phasmarhabditis sp. SA2).

Angiostoma margaretae n. sp (Nematoda: Angiostomatidae), a parasite of the milacid slug Milax gagates Draparnaud collected near Caledon, South Africa.

Angiostoma margaretae n. sp. (Angiostomatidae) is described from the oesophagus of the slug Milax gagates Draparnaud collected near Caledon in the Western Cape Province of South Africa. The new species closely resembles another parasite of a milacid slug, A. milacis Ivanova & Wilson, 2009, with a similar head, stoma and spicule shape, the presence of distally outstretched ovaries, coiled oviducts, the same number of caudal papillae and enlarged rectal glands. However, A. margaretae differs from the latter by having: a shorter, wider tail with a rounded vs pointed tip; the distal parts of both ovaries with a particular hook-like shape due to an expansion closely following the short initial zone; ovoviparous females; and a different arrangement of male papillae. A. margaretae is comparable with A. limacis Dujardin, 1845, A. asamati (Spiridonov, 1985), A. coloaense (Pham Van Luc, Spiridonov & Wilson, 2005) and A. stammeri (Mengert, 1953), which have a similar stoma shape and size, but can be readily differentiated by the presence of distally outstretched vs reflexed ovaries and the presence vs lack of enlarged rectal glands. The new species has a similar arrangement of the ovaries to A. kimmeriense Korol & Spiridonov, 1991 and A. zonitidis Ivanova & Wilson, 2009, but is clearly differentiated by the lack of an off-set lip region and presence of a large bowl-shaped vs tubular stoma and less numerous male caudal papillae (seven pairs vs nine in A. kimmeriense and 10 in A. zonitidis).

First Report of the Root-Knot Nematode Meloidogyne javanica on Buchu (Agathosma betulina) in South Africa.

Agathosma betulina, commonly known as buchu, has been used for centuries by the indigenous people of South Africa for medicinal purposes. Currently, the essential oils from buchu are used in medicine, food flavorings, and aromatic oils. Increased exploitation of natural growing buchu in the Fynbos biome and a worldwide shortage of buchu oil encouraged commercial cultivation in South Africa. The root-knot nematode (Meloidogyne spp.) is one of the most common plant-parasitic nematodes found on commercial crops grown in the Western Cape. It has also been isolated from the soil and roots of plants in the natural Fynbos vegetation (2). In June 2003, a nursery propagating buchu plants experienced problems with poor growth. Examination of the buchu roots under a stereo microscope showed extensive galling with large numbers of female root-knot nematodes with eggsacs. Nematode extractions of the soil were also done. Only second-stage juveniles of Meloidogyne spp. (311 per 250 ml of soil) were recovered. A polymerase chain reaction (PCR)-based diagnostic method (1) was used for the identification of the root-knot nematode species. Ten intact females were dissected from the roots and individually placed directly in 5 µl drops of 1× PCR reaction buffer (16 mM [NH4]2SO4, 67 mM tris-HCL, pH 8.8, 0.1% vol/vol Tween 20) ontaining 60 µg/ml of proteinase K. The tube was kept at -80°C for a minimum of 10 min. The tube was incubated at 60°C for 15 min and 5 min at 95°C. The PCR amplifications were then prepared directly in the same tube. Amplified DNA fragments were digested with HinfI and DraI. The digested DNA was loaded on a 2% agarose gel, separated by electrophoresis, and detected by ethidium bromide staining. The digested amplified DNA fragments correspond to those of Meloidogyne javanica. Morphological characteristics were used to verify the PCR-based identification of the nematode. To our knowledge, this is the first report of M. javanica causing extensive galling on the roots of Agathosma betulina. Visual damage to the roots indicates the root-knot nematode to be an important threat to the commercial cultivation of buchu. References: (1) R. Knoetze. Potential of the polymerase chain reaction for the identification of plant-parasitic nematodes. M.Sc. thesis. University of Stellenbosch, Stellenbosch, South Africa, 1999. (2) A. J. Meyer, S. Afr. J. Enol. Vitic., 20:75, 1999.