Morphological and molecular characterization of Butlerius butleri Goodey, 1929 (Nematoda: Diplogastridae) from South Africa: First report.

Two populations of a Butlerius species were recovered from compost in two gardens in Potchefstroom, North-West Province, South Africa. Although the genus has previously been reported from South Africa, no species of the genus has ever been identified in the country. Based on morphological, morphometric, and molecular studies, the specimens were identified as Butlerius butleri and are herein reported for the first time from South Africa. The South African specimens are 1,082 to 1,423 µm long, a = 40.8 to 47.6; b = 4.7 to 5.8; c = 4.0 to 6.0; c' = 117 to 16.3; V = 44 to 47%. Cuticle with evenly spaced punctations. Reproductive system didelphic, amphidelphic, both branches equal in length. Four large glands opening into proximal part of uterus. Males with prominent sphincter present in mid-region of vas deferens. Spicules 36 to 43 µm long, gubernaculum 23 to 31 µm long, nine pairs of genital papillae, three pre-cloacal and six post-cloacal, formula: v1, v2, v3d/v4, ad, ph, v5, 6, 7, pd. The v5, 6, 7 clusters greatly separated, left subventral group at level of phasmid, right subventral group at level of posterior dorsal papilla. Although there were some differences, the South African populations of the species compare well to all know descriptions of the species. Phylogenetic analysis based on partial small subunit (SSU) rDNA sequences showed that both South African populations of B. butleri are in a maximally supported sister relation with an Iranian population of this species. Based on large subunit (LSU) rDNA sequences, the two populations of B. butleri clustered together in a well-supported clade.

Description of Prionchulus jonkershoekensis n. sp. (Nematoda: Mononchida), a new predatory species from South Africa.

Prionchulus jonkershoekensis n. sp. is described from South Africa and illustrated using morphological, morphometric, and molecular techniques. This species is characterized by its body length (1.78-2.14 mm); the size of buccal cavity (38-44 × 24-31 µm), lower dorsal tooth position in relation to buccal cavity base, the position of amphidial aperture just above dorsal tooth apex, pars proximalis vaginae with almost straight walls, and tail 144-158 µm long with sickle shaped posterior third part. Phylogenetic analyses based on 18 S rDNA and 28 S rDNA of P. jonkershoekensis n. sp. revealed close relationships of the new species with Prionchulus punctatus and Prionchulus muscorum. This is also an additional geographical record for the genus from South Africa.

Laimaphelenchus africanus n. sp. (Tylenchomorpha: Aphelenchoididae) from South Africa, a morphological and molecular phylogenetic study, with an update to the diagnostics of the genus.

A newly recovered population of the genus Laimaphelenchus from a dead maritime pine wood sample in Potchefstroom, South Africa, representing a new species, named L. africanus n. sp., is herein described and illustrated based on morphological and molecular data. The new species is mainly characterized by the following: 750-987 µm long females; a cephalic region with no disc and six cephalic lobs not divided by ribs; a 10.0-12.5 µm long stylet; four incisures in the lateral field; secretory-excretory pore (SE-pore) at slightly posterior to the nerve ring; vulva with a well-developed anterior flap, vagina with two well-developed sclerotized pieces; post-vulval uterine sac (PUS) 63-125 µm long; tail conical, 30-44 µm long, ventrally curved with a subventral stalk in terminus, lacking tubercles, with six to nine small projections at the tip in scanning electron microscopy (SEM); and rare males with 17 µm long spicules. The new species was morphologically compared to those species of the genus with a stalk in tail terminus, lacking tubercles, a vulval flap and four incisures in the lateral field viz., L. liaoningensis, L. preissii, L. simlaensis, L. sinensis, L. spiflatus, and L. unituberculus. Phylogenetically, the new species was placed into the major Laimaphelenchus clade using partial large subunit ribosomal DNA (LSU rDNA D2-D3) sequences. An overall literature review corroborated the presence of the stalk (currently with two main groups) at the tail end is the main characteristic trait delimiting the genus. A compendium based on the characters of the stalk, presence/absence of a vulval flap in females and number of the lateral lines was also established.

Morphological and molecular characterization of Hoplolaimus pararobustus (Schuurmans Stekhoven and Teunissen, 1938) Sher 1963 with its first report on Zea mays roots in Namibia.

In the summer of 2018, specimens of a Hoplolaimus population were extracted from a maize root sample collected near Stampriet, Namibia. This population was identified as Hoplolaimus pararobustus and is described and illustrated based on its morphological, morphometric, and molecular characteristics. To our knowledge, this is the first report H. pararobustus from maize roots. Females of the population had a mean body and stylet length of 1,100 µm and 36 µm, respectively. Esophagus with three nuclei in three pharyngeal glands. Lateral field reduced, ranging from a very faint line to just breaks in striae. The males were shorter than the females with a mean body length of 925 µm and the stylet slightly shorter, with a mean length of 34 µm. Phylogenetic analyses using partial sequences of 18 S and the expansion fragment D2-D3 of 28 S rDNA genes showed the close relation of this species and H. columbus. This Namibian population of H. pararobustus is the first Hoplolaimus species from Africa to be molecularly characterized.

Cephalenchus driekieae n. sp. (Nematoda: Tylenchidae) from South Africa, a new member of the genus with a long pharyngeal overlap.

Cephalenchus driekieae n. sp. is described and illustrated based on its morphological, morphometric, and molecular characteristics. This new species is mainly characterized by its short stylet 11.5 to 13.0 µm, and 13.5 to 17.5 µm long pharyngeal overlap extending over the intestine. It could further be delimited by 451 to 526 µm long females with a prominently annulated cuticle, dorso-ventral amphidial openings as shown using scanning electron microscopy (SEM), four lines in the lateral field, anchor-shaped stylet knobs, empty spermatheca, elongate conoid tail with finely rounded tip and males absent. The shortest stylet and long pharyngeal overlap, distinguish this new species from previously described members and update the characteristics of the genus. With four lines in the lateral field, this new species was morphologically compared with four previously described species with this feature and another species with a short stylet. Molecular phylogenetic analyses using the partial small and large subunit ribosomal DNA gene (SSU and LSU rDNA D2-D3) sequences showed that it was clustered with other Cephalenchus spp. in both SSU and LSU trees, retaining the monophyly of the genus. This new species from South Africa updates the biogeography of the genus.Cephalenchus driekieae n. sp. is described and illustrated based on its morphological, morphometric, and molecular characteristics. This new species is mainly characterized by its short stylet 11.5 to 13.0 µm, and 13.5 to 17.5 µm long pharyngeal overlap extending over the intestine. It could further be delimited by 451 to 526 µm long females with a prominently annulated cuticle, dorso-ventral amphidial openings as shown using scanning electron microscopy (SEM), four lines in the lateral field, anchor-shaped stylet knobs, empty spermatheca, elongate conoid tail with finely rounded tip and males absent. The shortest stylet and long pharyngeal overlap, distinguish this new species from previously described members and update the characteristics of the genus. With four lines in the lateral field, this new species was morphologically compared with four previously described species with this feature and another species with a short stylet. Molecular phylogenetic analyses using the partial small and large subunit ribosomal DNA gene (SSU and LSU rDNA D2-D3) sequences showed that it was clustered with other Cephalenchus spp. in both SSU and LSU trees, retaining the monophyly of the genus. This new species from South Africa updates the biogeography of the genus.

Suppressive Effect of Soil Microbiomes Associated with Tropical Fruit Trees on Meloidogyne enterolobii.

Plant-parasitic nematodes are one of the main biotic factors limiting agricultural production worldwide, with root-knot nematodes (Meloidogyne spp.) being the most damaging group. This study was conducted to evaluate the efficacy of soil microbiomes, associated with various subtropical fruit trees, on the management of a Meloidogyne enterolobii population. Of 14 soil microbiomes tested for nematode suppression, 9 samples in the first experiment and 10 samples in the repeat experiment had significantly (p ≤ 0.05) lower numbers of eggs and J2 compared to the untreated control. The highest nematode suppression was recorded for SA12 extracted from a papaya orchard with a 38% reduction in the nematode population density. In addition, the presence of some bacteria (Bacillus aryabhattai, B. funiculus and B. simplex) and fungi (Metarhizium marquandii, Acremonium sp. and Mortierella sp.) was correlated to a higher suppression potential in some samples. Substantial variations were observed for the diversity of bacterial and fungal isolates among the samples collected from various crop hosts and regions. This suggests that the nematode suppression potential of different soil microbiomes highly depends on the abundance and diversity of fungal and bacterial strains present in the soil. The study confirmed that among all variables, soil dryness, pH, Fe, Zn, organic matter, altitude, and crop cultivar strongly influenced the soil microbial composition.

A Pilot Approach Investigating the Potential of Crop Rotation With Sainfoin to Reduce Meloidogyne enterolobii Infection of Maize Under Greenhouse Conditions.

Root-knot nematodes (RKNs) are one of the most important plant-parasitic nematodes of cereal crops in sub-Saharan Africa. This study was designed to evaluate the rotation effects of different cultivars of sainfoin (Esparsette, Perly, Taja and Visnovsky), soybean (DM-5953-RSF) and alfalfa (BAR 7) with maize (P-2432-R), on a Meloidogyne enterolobii population, compared to monoculture maize. The results showed that sainfoin (Perly and Esparsette) and alfalfa had significantly (P ≤ 0.05) lower numbers of M. enterolobii eggs and second stage juveniles (J2) compared to the monoculture maize in the first experiment. However, in the repeat experiment all treatments had significantly (P ≤ 0.05) lower numbers of eggs and J2 compared to monoculture maize. Rotation of sainfoin Esparsette/maize resulted in the lowest numbers of eggs and J2 (91 and 202, respectively) in the first and repeat experiments. Rotation of sainfoin Esparsette/maize reduced M. enterolobii population density by 81 and 60% in the first and repeat experiments, respectively, followed by alfalfa (54 and 43%, respectively). Ultimately, substantial variation was evident in terms of the efficacy of different sainfoin cultivars with regards to their effect on nematode reduction when used in rotation with maize.

Morphological and morphometrical identification of Meloidogyne populations from various crop production areas in South Africa with emphasis on M. enterolobii.

Accurate identification of Meloidogyne spp. is crucial and the first step to apply suitable management strategies to combat these nematode pests. Perineal-pattern morphology of female specimens is one of the most common characteristics used for identification. However, for some species various morphological characteristics are similar which makes it challenging to correctly identify species. In this study different morphological and morphometrical characteristics were used to identify 37 populations of Meloidogyne obtained during 2015 and 2016 from various crop production areas situated across different geographical regions in South Africa. A comprehensive study of females, males and second-stage juveniles (J2) of the 37 Meloidogyne populations isolated was conducted, revealing the presence of Meloidogyne enterolobii, M. hapla, M. incognita and M. javanica. Although three perineal-pattern characteristics proved to be useful in discriminating particularly between M. enterolobii and M. incognita females, most of the morphometric characters used to identify female, male and J2 individuals overlapped among the different species. Substantial intraspecies variation was also evident among different populations. The use of classical identification approaches alone could therefore not clearly distinguish among the 37 Meloidogyne populations studied. Therefore, the use of molecular techniques in combination with morphological and morphometrical analyses is suggested to be more accurate and reliable in discriminating between Meloidogyne spp.

Genetic diversity and phylogeny of South African Meloidogyne populations using genotyping by sequencing.

Meloidogyne species cause great crop losses worldwide. Although genetic host plant resistance is an effective control strategy to minimize damage caused by Meloidogyne, some resistant genes are ineffective against virulent species such as Meloidogyne enterolobii. Detailed knowledge about the genetic composition of Meloidogyne species is thus essential. This study focused on genotyping-by-sequencing (GBS) and Pool-Seq to elucidate the genetic relation between South African M. enterolobii, M. incognita and M. javanica populations. Hence, 653 common single nucleotide polymorphisms (SNPs) were identified and used to compare these species at genetic level. Allele frequencies of 34 SNPs consistently differed between the three Meloidogyne species studied. Principal component and phylogenetic analyses grouped the M. enterolobii populations in one clade, showing a distant relation to the M. javanica populations. These two species also shared genetic links with the M. incognita populations studied. GBS has been used successfully in this study to identify SNPs that discriminated among the three Meloidogyne species investigated. Alleles, only occurring in the genome of M. enterolobii and located in genes involved in virulence in other animal species (e.g. a serine/threonine phosphatase and zinc finger) have also been identified, accentuating the value of GBS in future studies of this nature.