Rinus Knoetze

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.

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.

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

During a survey for entomopathogenic nematodes in citrus orchards throughout South Africa, a new species of Steinernema was isolated from a citrus orchard on Rietkloof farm, near the town of Piketberg in the Western Cape Province, South Africa. The nematode was isolated from soil using the Galleria -baiting technique. Steinernema citrae n. sp. is characterised by the following morphological characters: third-stage infective juvenile with a body length of 754 (623-849) μ m, distance from head to excretory pore of 56 (49-64) μ m, tail length of 71 (63-81) μ m, and ratio E value of 110 (85-132). The lateral pattern for the new species is 2, 7, 8, 6, 4, 2 and is not typical for the genus. Steinernema citrae n. sp. is closely related the feltiae -group. The body length of the IJ is close to that of S. texanum and S. weiseri , though it differs in body diam., the length of the pharynx and E%. The male of S. citrae n. sp. differs from S. feltiae in the length and shape of the spicule and body diam. Steinernema citrae n. sp. differs from all species in the feltiae -group in the morphology of the vulva, as it has a single flapped, low, epiptygma. It also differs from the most closely related species, S. feltiae , as there is no interbreeding between the two species. In addition, the new nematode differs from other species of the feltiae -group by characteristics of the ITS and D2D3 regions of its rDNA.

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

A new species of entomopathogenic nematode, Steinernema sacchari n. sp., was isolated by trapping with the sugar cane borer, Eldana saccharina, from soil of a sugar cane field in the KwaZulu-Natal province of South Africa. The new species is morphologically characterised by the length of the infective juvenile (IJ) of 680 (630-722) μm, tail length of 64 (51-74) μm, ratio a = 19 (14-23), H% = 49 (43-57) and E% = 82 (70-109). The pattern of the lateral field of the IJ of the new species is 2, 5, 2 ridges (3, 6, 3 lines or incisures). The male of the first generation can be recognised by the long spicule of 83 (73-89) μm, gubernaculum of 61 (50-68) μm, D% = 67 (54-88) and GS% = 73 (66-81). The first generation male lacks a mucron, while the second generation male always has one. The first generation female can be recognised by the vulval lips not being raised, the possession of long double-flapped epiptygmata and the lack of a postanal swelling. Analysis of the ITS and D2D3 regions showed S. sacchari n. sp. to differ from all other Steinernema species and to belong to a new monophyletic group, the ‘Cameroonian’ clade, consisting of S. cameroonense, S. nyetense and S. sacchari n. sp. This group is closely related to the feltiae-kraussei-oregonense Clade III.

Morphological and molecular studies on Aphelenchoides arachidis Bos, 1977 (Tylenchina: Aphelenchoididae) from groundnuts in South Africa

Aphelenchoides arachidis is reported for the first time from South Africa and for the fourth time outside Nigeria. The A. arachidis-infested pods from South Africa showed the following symptoms: small seeds with the testa wrinkled and darker in colour than that of non-infested seeds; the pods showed dark lesions and some seeds within the pods showed early germination. Differences between the two South African and the Nigerian populations of A. arachidis include more lateral lines in some specimens (2-4 vs 2) and, on average, longer post-uterine sac length (extending for 74 (41-96) and 62 (33-82) vs about 50% of vulva to anus distance). Scanning electron micrographs of this species are presented for the first time. The ITS regions of ribosomal DNA were amplified, sequenced, aligned and compared with other sequences of Aphelenchoides species. Two pathogenic fungi, Thielaviopsis basicola and Neocosmospora vasinfecta, were also isolated from this material.

Techniques and Procedures

A wide range of generic, specialised and innovative techniques, and procedures used by local nematologists for their research on nematodes (plant-parasitic and non-parasitic), are presented. Practical approaches to identify damage symptoms caused by plant-parasitic nematodes on crop plants as well as sampling and storing of nematode samples are described. Various methods used to extract nematodes from soil and plant tissues for counting and identification purposes, as well as the principles upon which they are based are detailed. Information on the preparation of nematode specimens for morphological and molecular identification is furthermore presented. The preparation of infected plant tissue and nematodes for electronmicroscopy is also described. In addition, methods to culture plant-parasitic and entomopathogenic nematodes, both in vitro and in vivo, are presented. Most of the techniques described in this chapter are used routinely in nematology. An inventory of the basic materials and infrastructure needed to set up and run a small nematology laboratory is included. A list of the main activities when dealing with nematode samples, from the labelling and preparation of incoming samples to the compilation of a concluding report, is also given.

A survey of the Cape Floristic Region of South Africa for the presence of cyst nematodes (Nematoda: Heteroderidae).

A survey was performed to detect the presence of cyst nematodes in the Cape Floristic Region of South Africa. Soil was collected in the rhizosphere of the dominant plant species within blocks of indigenous vegetation and cysts were extracted from them. A total of 81 blocks of indigenous vegetation were sampled as described. Cysts were detected in 7 of these samples, representing 6 different vegetation types. One set of primers was used to amplify the ITS regions from these cysts, including the 5.8S ribosomal gene, as well as short parts of the 18S and 28S ribosomal genes. ITS-rDNA sequences from the indigenous isolates were aligned with selected sequences of other species from the Heteroderidae. Phylogenetic analyses to resolve the relationships between indigenous isolates and selected representatives of the Heteroderidae were conducted using the Maximum Parsimony method. The consensus tree resulting from alignment of the circumfenestrate cysts revealed that isolates SK18, WK1 and WK26 are included in a clade of Globodera species that parasitise non-solanaceous plants, forming a monophyletic group with G. millefolii, G. artemisiae, and an unidentified Globodera sp. from Portugal. In a tree resulting from the alignment of the Heterodera spp., isolates OK14 and WK2 are included in the Afenestrata group, forming a monophyletic group with H. orientalis.This survey unearthed at least four potentially new species of cyst nematodes, which may prove invaluable for the study of the evolution and biogeography of the group.