Heterorhabditis pakistanense n. sp. (Nematoda: Heterorhabditidae) a new entomopathogenic nematode from Pakistan.

A new entomopathogenic nematode species of Heterorhabditis, described as H. pakistanense n. sp., was isolated from soil samples around the roots of grass at Malir, Karachi, Sindh, Pakistan. The new species is characterized morphologically by features of males: body size 819 µm (720-1013 µm), D% ((distance from anterior end to excretory pore divided by pharynx length) × 100) 119 (110-126), SW% ((spicule length divided by anal body diameter) × 100) 156 (144-191), GS% ((gubernaculum length divided by spicule length) × 100) 58 (48-65) and variations in the number of bursal papillae of the terminal group: 8th and 9th papillae sometimes absent on both sides, sometimes eight papillae present on the right side whereas six papillae present on the left side. On the right side the arrangement of papillae is 1 + 2 + 3 + 2 whereas on the left side it is 1 + 2 + 3. The hermaphrodite has a prominent post-anal swelling and a conoid tail 82 µm (64-95 µm) long with a pointed terminus. Hermaphrodites of H. pakistanense n. sp. can be distinguished from all species of Heterorhabditis except H. downesi by having a mucronate tail. Infective juveniles have a medium-sized body (581 µm (558-624 µm)), long pharynx (117 µm (113-125 µm)), ensheathed tail (99 µm (95-110 µm)) and E% ((distance from anterior end to excretory pore divided by tail length) × 100) 100 (95-107). The new species can be distinguished from all species of Heterorhabditis by the absence of the 7th, 8th and 9th bursal papillae. Heterorhabditis pakistanense is further characterized by the internal transcribed spacer (ITS) and the D2D3 region of the 28S rDNA gene. The closest species H. indica, H. gerrardi, H. amazonensis and H. noenieputensis being separated by 9, 7, 66 and 15 bp, respectively, in the ITS region. Molecular phylogenetic trees based on sequences of ITS rDNA, D2D3 regions and the mitochondrial 12S rRNA gene support the description of H. pakistanense as a new species.

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).

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.

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.