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.

Screening of Rhizosphere Bacteria and Nematode Populations Associated with Soybean Roots in the Mpumalanga Highveld of South Africa.

Soybean is among South Africa's top crops in terms of production figures. Over the past few years there has been increasingly more damage caused to local soybean by plant-parasitic nematode infections. The presence of Meloidogyne (root-knot nematodes) and Pratylenchus spp. (root lesion nematodes) in soybean fields can cripple the country's production, however, little is known about the soil microbial communities associated with soybean in relation to different levels of Meloidogyne and Pratylenchus infestations, as well as the interaction(s) between them. Therefore, this study aimed to identify the nematode population assemblages and endemic rhizosphere bacteria associated with soybean using Next Generation Sequencing (NGS). The abundance of bacterial genera that were then identified as being significant using linear discriminant analysis (LDA) Effect Size (LEfSe) was compared to the abundance of the most prevalent plant-parasitic nematode genera found across all sampled sites, viz. Meloidogyne and Pratylenchus. While several bacterial genera were identified as significant using LEfSe, only two with increased abundance were associated with decreased abundance of Meloidogyne and Pratylenchus. However, six bacterial genera were associated with decreased Pratylenchus abundance. It is therefore possible that endemic bacterial strains can serve as an alternative method for reducing densities of plant-parasitic nematode genera and in this way reduce the damages caused to this economically important crop.

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.

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 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.

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.

Nematodes and the effect of seasonality in grassland habitats of South Africa.

Nematodes in South Africa have mainly been studied for their diversity and agricultural importance. However, the ecological status of nematodes and the effect of seasonal variation in local grasslands remain unknown. For this reason, a nematode study was conducted in the Telperion Nature Reserve and represented the first ecological study in a natural grassland area in South Africa. In total, 104 soil samples were collected during four consecutive seasons from 2015 until 2016 in three habitats, viz. (i) open grassland, (ii) shrubland with rocky outcrops, and (iii) riparian zone. From these the nematode community structure and soil ecosystem status were studied. In total, 93 genera from 50 families were recorded with herbivores and bacterivores being the most abundant trophic groups in all three habitats. Linear mixed models revealed that season had an overwhelmingly dominant impact on the condition, food web status, and functioning of the soil ecosystems with pairwise comparisons indicating that significantly higher values were recorded during winter. Interestingly, this seasonal shift can largely be attributed to fluctuations in the populations of only a few nematode groups (namely Aporcelaimellus, Dorylaimidae, Iotonchus, and Mononchus) with high colonizer-persister values. Although the reason for the higher abundance of specific nematode groups recorded during the winter is not explicitly clear, it is possibly linked to reduced competition from other soil fauna. This study clearly shows that further investigations are required to better understand the dynamics of grassland ecosystems.

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.

Description of two new species, Aphanolaimus strilliae n. sp. (Nematoda:br />Aphanolaimidae) and Makatinus africanus n. sp. (Nematoda: Aporcelaimidae), and SEM observations on three known species from freshwater sources in the Telperion Nature Reserve (Mpumalanga, South Africa).

Aphanolaimus strilliae n. sp. and Makatinus africanus n. sp. are described from freshwater sources in the Telperion Nature Reserve, Mpumalanga, South Africa. Aphanolaimus strilliae n. sp. is characterised by a body length of 1240-1613 µm, more than 800 body annules, lateral field originating between the first and second lateral body pore at the 34th-46th annule, first lateral body pore located at the 25th-35th annule, vagina V-shaped and bent anteriorly, 142-195 µm long uterus and 165-207 µm long tail with spinneret offset. This species is ovoviviparous and no males were found. Makatinus africanus n. sp. is characterised by a large, thick body (3228-4128 µm long, a = 30-39 wide), slightly set off lip region with amalgamated lips; 31-34 µm long odontostyle; long tongue-shaped cardia, and stout, short tail (30-43 µm long) with a small peg / digitate extension and male absent. Populations of three known species, Chronogaster africana, Eutobrilus annetteae and Neotobrilus ampiei, from fresh water at the Telperion Nature Reserve are described and scanning electron microscope graphs of these species published for the first time.

Terrestrial Non-Parasitic Nematode Assemblages associated With Glyphosate-tolerant and Conventional Soybean-Based Cropping Systems.

Information about the effects of glyphosate on nematodes is limited and contradictory, while none existing for South African agricultural fields. The abundance and identity of non-parasitic nematodes in the rhizospheres of commercial glyphosate-tolerant and conventional (non-glyphosate-tolerant), soybean cultivars from cultivated fields, and adjacent natural vegetation (reference system) were obtained for two growing seasons. The impact of glyphosate was also investigated on non-parasitic nematodes in a 2-year soybean-maize cropping system. Thirty-two non-parasitic nematode genera were identified from soils of the three field ecosystems, with most of the genera occurring in natural vegetation (28), and less in conventional (23) and glyphosate-tolerant soybean (21). Bacterivores had the greatest diversity in soils of all three ecosystems during both seasons, while fungivores tended to be more abundant in glyphosate-tolerant soybean fields especially during the second season. Soils from the three ecosystems were disturbed and degraded with low abundance and diversity of omnivores and predators. Of the 14 genera identified from the soybean-maize cropping experiment, bacterivores dominated in terms of diversity in non-treated, and fungivores in glyphosate-treated plots. Soils from glyphosate-treated plots were degraded, less enriched and fungal-mediated, while those from non-treated plots were disturbed, enriched, and bacterial-mediated.

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.

Aphelenchoides spp. (Nematoda: Aphelenchida) and Panagrolaimus leperisini (Nematoda: Rhabditida) found associated with grass seeds in the Telperion Nature Reserve, South Africa.

As part of the South African Plant-Parasitic Nematode Survey (SAPPNS), an extensive nematode diversity survey was conducted in the Telperion Nature Reserve, Mpumalanga, South Africa. During this survey, samples of grass seeds were collected. Some of these seeds yielded specimens of the genera Aphelenchoides, Aphelenchus and Panagrolaimus. Identified species include Aphelenchoides africanus, A. besseyi, A. lichenicola, A. rutgersi, A. spicomucronatus and Panagrolaimus leperisini. Four of these species are new records for South Africa and the South African populations of these species are described in this paper.

Ditylenchus sarvarae sp. n. (Tylenchina: Anguinidae) from Iran.

During a survey of soil nematodes in Iran, a new species of Ditylenchus Filipjev, 1936 was discovered. Ditylenchus sarvarae sp. n. is characterised by its body length (1.0-1.4 mm), lateral field with seven incisures and without areolation, long postuterine sac (1.8-2.8 times the corresponding body diameter) and conical female tail with pointed tip (68-89 µm long, c = 13.7-18.2, c' = 4.2-5.1). Male specimens have conical tails (64-70 µm long, c = 14.5-16.7, c' = 4.0-4.5), spicules (22-26 µm long) and gubernaculum (8-10 µm long). Measurements and illustrations are provided for this new species. A molecular study of the 28S rDNA region of D. sarvarae sp. n. demonstrates that the Iranian species belongs to a separate group compared with the other molecularly characterized species of genus Ditylenchus.

Morphological and molecular characterization of three Helicotylenchus species from Iran.

Three Helicotylenchus species, namely Helicotylenchus digonicus, Helicotylenchus pseudorobustus and Helicotylenchus vulgaris were identified during a nematode survey conducted in Iran. The morphological and molecular characteristics of the species were defined. In addition, phylogenetic analysis of five Iranian Helicotylenchus populations based on 28S rDNA were done and showed the unresolved position of molecularly identified H. pseudorobustus and H. digonicus. However, populations of H. vulgaris and H. digonicus were placed close to each other. Phylogenetic analysis confirmed that molecularly characterized Helicotylenchus species formed a monophyletic clade. A detailed revision of the species is, however, needed. Illustrations and measurements for all species are provided.

Butlerius butleri Goodey, 1929 (Rhabditida) from Iran with the Phylogenetic Position of the Species.

A population of Butlerius butleri Goodey, 1929 was isolated from vermicompost in Kerman in the Kerman Province of Iran during a nematode survey that was conducted during 2014. This population of B. butleri is characterized by the presence of a dorsal thorn-like tooth (4 to 5 µm long), long spicules (44 to 47 µm long), gubernaculum (33 to 37 µm or more than half of the spicule length), three pairs of precloacal papillae, five pairs of postcloacal papillae (papillae V3 and V5 comprising three small papillae), and a long filiform tail (304 to 409 µm in females, 312 to 380 µm in males). Molecular and phylogenetic analysis of B. butleri individuals from this Iranian population based on 18S ribosomal deoxyribonucleic acid (rDNA) sequence placed this species close to Pseudodiplogasteroides compositus (AB597237) and an unidentified Pseudodiplogasteroides species (AB597238). Measurements, illustrations, and the phylogenetic tree, including the position of B. butleri are provided.

Relationships between initial population densities of Meloidogyne incognita race 2 and nematode population development in terms of variable soybean resistance.

The effect of increasing initial population density levels (Pi) of Meloidogyne incognita race 2 on nematode population development and yield of a susceptible (Prima2000) and resistant (LS5995) soybean cultivar was investigated. Two experiments, one in a hail net cage and one in microplots, were conducted one each during two consecutive growing seasons at Potchefstroom in the North West Province of South Africa. Nematode reproduction was assessed by determining the number of eggs and second-stage juveniles (J2) in the rhizosphere and roots, egg masses, egg-laying females (ELF) and reproduction factor (Rf) values per root system at harvesting 110 days after planting. Percentage yield reduction in the two cultivars was also calculated. Strong non-linear relationships existed between all nematode variables as well as between Pi and percentage yield loss in both cultivars for both experiments in this study. Significantly higher numbers of eggs and J2, egg masses and ELF were maintained in the roots of the nematode-susceptible Prima2000 than in the resistant LS5995 from Pi = 100 and higher in both experiments. Rf values were inversely related to Pi for both cultivars and were lowest on LS5995, with Prima2000 maintaining significantly higher Rf values in both experiments. Yield loss in LS5995 was at least six times higher than that of Prima2000. The difference in monetary terms is demonstrated, although it is suggested that host plant resistance to plant-parasitic nematodes may not be sufficient as the only management tool in highly infested soils or in rotation systems including nematode susceptible crops.