Description of Paramylonchulus iranicus sp. n. (Nematoda: Mononchida) from Iran.

During a survey of soil nematodes in Iran, a population of a species belonging to the order Mononchida was recovered. The new species, Paramylonchulus iranicus sp. n. is characterized by body length (1292-1535 µm in females and 1476-1670 µm in males), c (20.2-29.0 in females and 19.9-27.4 in males), buccal cavity length (23.0-26.0 µm), post vulval uterine sac length (135-162 µm), spicule length (46.0-50.0 µm), gubernaculum length (8.0-11.0 µm), and tail length (49.0-70.0 µm in females and 55.0-73.0 µm in males). Canonical discriminant analysis clearly separated P. iranicus sp. n. from the closely related species Paramylonchulus based on the important morphometric characters of females and males. A molecular study of the 18S rDNA region of P. iranicus sp. n. places this population in a well-supported clade with other species of the genus.

Bacterial communities associated with Zeldia punctata, a bacterivorous soil-borne nematode.

Soil inhabiting organisms are important determinants of agroecosystem productivity. Understanding the composition, the abundance, and the type of interactions established by soil microorganisms is therefore crucial to design strategies to improve agricultural practices and agroecosystem management. In this study, we collected Zeldia punctata nematodes in maize fields in South Africa and profiled their associated bacterial communities using next-generation sequencing. We observed that Z. punctata nematodes establish associations with ecologically diverse bacterial species. The most abundant species observed are Pseudomonas syringae, a phytopathogenic bacterial complex; Lactobacillus paraplantarum, a broadly distributed bacterial species that is present in soils, water bodies, and animal intestinal tracts and has certain probiotic and antimicrobial properties; and Melissococcus plutonius, a serious pathogenic bacterial species that causes brood disease in honeybees. Our study contributes to a better understanding of the soil bacterial communities associated with nematodes in maize agricultural soils in South Africa and unravels the presence of diverse detrimental and beneficial nematode-associated bacteria.

Steinernema Africanum n. Sp. (Rhabditida, Steinernematidae), a New Entomopathogenic Nematode Species Isolated in the Republic of Rwanda.

Alternatives to hazardous insecticides are urgently needed for an environmentally friendly and effective management of insect pests. One such option is the use of entomopathogenic nematodes (EPN). To increase the availability of EPN with potential for biocontrol, we surveyed agricultural soils in the Republic of Rwanda and collected two Steinernema isolates. Initial molecular characterization showed that they represent a new species, for which we propose the name S. africanum n. sp. To describe this new species, we reconstructed phylogenetic relationships, calculated sequence similarity scores, characterized the nematodes at the morphological level, conducted crossing experiments, and isolated and characterized their symbiotic bacteria. At the molecular level, S. africanum n. sp. is closely related to S. litorale and S. weiseri. At the morphological level, S. africanum n. sp. differs from closely related species by the position of the nerve ring and also because the stoma and pharynx region is longer. The first-generation males have ventrally curved spicules with lanceolate manubrium and fusiform gubernaculum and the second-generation males have rounded manubrium and anteriorly hook-like gubernaculum. Steinernema africanum n. sp. does not mate or produce fertile progeny with any of the closely related species.

First report of Mesocriconema sphaerocephalum (Taylor, 1936) Loof, 1989 associated with wild grass in Botswana.

During a survey on the biodiversity of plant-parasitic nematodes of natural areas in Botswana, Mesocriconema sphaerocephalum was discovered around the rhizosphere of the wild grass. The nematodes were extracted using the tray method and then fixed according to the available protocols. The morphological characters fit well with the M. sphaerocephalum. Besides, molecular aspects using 18S and 28S rDNA were studied. The phylogenetic analysis of 18S and 28S rDNA placed the examined population with other populations of M. sphaerocephalum in a group. According to the knowledge, this is the first report of M. spaherocephalum from Botswana.

Morphological and molecular characters of Scutellonema brachyurus () from South Africa.

During a survey on plant-parasitic nematodes from South Africa, Scutellonema brachyurus was recovered from soil samples collected around the rhizosphere of wild grass in the North West and Limpopo provinces. This species characterized by a hemispherical lip region with four to six annuli, basal lip's annuli with longitudinal incisures, body length 696-904 µm (a = 25.1-33.5; b = 5.0-7.2; c = 48.9-75.3; c' = 0.5-0.9; V = 55-60), stylet 21-27 µm length, tail rounded with 10-19 µm length and spermatheca nonfunctional and male absent. The nblast analysis based on the D2-D3 segment of 28 S rDNA placed South African populations of S. brachyurus with 98% similarity to Greece (KU059494) and 99% similarity to South African (JX472052) S. brachyurus. Besides, nblast of COI of mtDNA showed 98% similarity of the test species with South African populations of S. brachyurus (JX472096; JX472097). The phylogenetic analysis put the South African populations of S. brachyurus together with other S. brachyurus with a 100 posterior probability support. Besides, the measurements, line illustration, and scanning electron microscopy photographs are provided for S. brachyurus from South Africa.

First report of Bitylenchus ventrosignatus () associated with wild grass in Botswana.

During a survey on the biodiversity of plant-parasitic nematodes of natural areas in Botswana, Bitylenchus ventrosignatus was discovered around the rhizosphere of wild grass. The nematodes were extracted using the tray method and then fixed according to the available protocols. The morphological characters fit well with the description of B. ventrosignatus. In addition, molecular analysis using 18 S and 28 S rDNA indicated 98% (KJ461617) and 95% (KJ461567) similarity with the Spanish population of B. ventrosignatus. The phylogenetic analysis of 18 S and 28 S rDNA placed the examined population with other populations of B. ventrosignatus in a group with a posterior probability support value of 100. According to published literature, this is the first report of B. ventrosignatus from Botswana.

First report of Meloidogyne hapla on kiwifruit in South Africa.

Kiwi is becoming one of the most important fruit in subtropical regions of South Africa with altitudes that confer sufficient chilling requirements. During a survey on biodiversity of plant-parasitic nematodes of kiwi in Magoebaskloof in Limpopo Province, several plant-parasitic nematodes were discovered, with Meloidogyne species occurring at the highest frequency. Nematodes were sampled from roots and the rhizosphere of one stunted Kiwi tree, extracted using the tray method and then fixed. The morphological characters fit well with those of M. hapla. The molecular approach using ITS and 28S rDNA, along with the related phylogenetic analysis, placed the examined population in a group with other populations of M. hapla. Kiwi is being reported as a new host for M. hapla in South Africa.

New data on known species of Hirschmanniella and Pratylenchus (Rhabditida, Pratylenchidae) from Iran and South Africa.

Hirschmanniella anchoryzae from Iran and Pratylenchus hippeastri from South Africa were recovered during a survey of plant-parasitic nematodes belonging to the family Pratylenchidae. Both species were studied using morphological and molecular techniques. Hirschmanniella anchoryzae is identified based on the flattened head, short stylet (19-22 µm), excretory pore position (anterior to pharyngo-intestinal junction), spicule length (27-30 µm), and existence of an axial mucro at the tail end. Phylogenetic analysis using 28S rDNA showed monophyly of Hirschmanniella which Iranian H. anchoryzae placed close to H. halophila (EU620464; EU620465). This result was supported by the principal component analysis of Hirschmanniella species. SEM observation of the South African population of P. hippeastri showed the presence of two annuli in the lip region. Morphometric characters resembled those of specimens earlier reported from South Africa. Hierarchal cluster using morphometrical criteria showed that the Floridian (USA) and South African populations form a group. However, the principal component analysis showed variation within this species. The molecular study of P. hippeastri populations using 18S, ITS, 28S rDNA, and COI of mtDNA showed that all P. hippeastri cluster in one group and confirmed the identification of the species using both morphological and molecular techniques. In addition, the results indicated that South African populations group close to the USA populations. Illustrations of both species including light and scanning electron microscopy observations for P. hippeastri 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.

Bacterial communities associated with Acrobeles complexus nematodes recovered from tomato crops in South Africa.

The productivity of agricultural ecosystems is heavily influenced by soil-dwelling organisms. To optimize agricultural practices and management, it is critical to know the composition, abundance, and interactions of soil microorganisms. Our study focused on Acrobeles complexus nematodes collected from tomato fields in South Africa and analyzed their associated bacterial communities utilizing metabarcoding analysis. Our findings revealed that A. complexus forms associations with a wide range of bacterial species. Among the most abundant species identified, we found Dechloromonas sp., a bacterial species commonly found in aquatic sediments, Acidovorax temperans, a bacterial species commonly found in activated sludge, and Lactobacillus ruminis, a commensal motile lactic acid bacterium that inhabits the intestinal tracts of humans and animals. Through principal component analysis (PCA), we found that the abundance of A. complexus in the soil is negatively correlated with clay content (r = -0.990) and soil phosphate levels (r = -0.969) and positively correlated with soil sand content (r = 0.763). This study sheds light on the bacterial species associated to free-living nematodes in tomato crops in South Africa and highlights the occurrence of various potentially damaging and beneficial nematode-associated bacteria, which can in turn, impact soil health and tomato production.

The genera Nothacrobeles Allen & Noffsinger, 1971 and Zeldia Thorne, 1937 (Nematoda: Rhabditida: Cephalobidae) from southern Iran, with description of N. abolafiai sp. n.

A new species of the genus Nothacrobeles Allen & Noffsinger, 1971 is described from a natural area in Kerman Province, Iran. Nothacrobeles abolafiai sp. n. is characterized by its body length (518-655 µm in females), "single" cuticle, lateral field with three incisures, labial probolae 8.5-9.4 µm long, bifurcated and without tines, bearing a minor curvature at the tip, pharyngeal corpus 3.3-4.1 times isthmus length, R(ex) = 25-32, spermatheca 22-30 µm long or 0.8-1.2 times the corresponding body diameter, postuterine sac 15-18 µm long or 0.5-0.7 times the corresponding body diameter, female tail conical with rounded terminus (31-43 µm, c = 11.9-18.1, c' = 1.7-2.4), and phasmid at 38-43% of tail length. In addition, two species of the genus Zeldia Thorne, 1937: Z. punctata (Thorne, 1925) Thorne, 1937 and Z. spannata Waceke, Bumbarger, Mundo-Ocampo, Subbotin & Baldwin, 2005, were recovered. The latter is recorded for the first time from Iran. Description, measurements, illustrations and LM pictures are provided for these three species. Furthermore, comparative morphometrics for the species of Nothacrobeles are given. Molecular analysis based on 28S rDNA (D2-D3 expansion) places this new species of Nothacrobeles in a different clade to other Nothacrobeles species. The results suggest that the genus Nothacrobeles may be a paraphyletic taxon.

Study of mononchids from Iran, with description of Mylonchulus kermaniensis sp. n. (Nematoda: Mononchida).

During a survey of soil nematodes in Iran, three species of predatory nematodes, including a new species of the genus Mylonchulus Cobb, 1916 were recovered. Mylonchulus kermaniensis sp. n. is characterised by its body length (1.2-1.4 mm), six rows of rasp-like denticles, the sixth line consisting of four denticles, female tail slightly sigmoid, sharply bent ventrad with digitate posterior portion slightly but clearly bent dorsad, (37-49 µm long, c=27.9-38.9, c'=1.2-1.7) with a terminal opening of spinneret. Two advulval papillae present, one is pre-vulval and the other one is located posterior to vulva. Furthermore, two other mononchid species namely M. cf. hawaiiensis (Cassidy, 1931) Goodey, 1951 and Mononchus truncatus Bastian, 1865 were also recovered from soil in the province of Kerman, Iran, the former representing a new geographical record for Iran. Measurements and illustration are provided for these three species. Molecular study of 18S rDNA region of M. cf. hawaiiensis demonstrated that the Iranian population compared with the nearest populations identified as M. hawaiiensis from Japan, shows 5 to 8 nucleotide differences. In addition, phylogeny of Mylonchulus is discussed and a checklist of the species of Mononchida from Iran is provided.

Morphometric and total protein responses in Meloidogyne incognita second-stage juveniles to Nemafric-BL phytonematicide.

After hatch, second-stage juveniles (J2) of root-knot (Meloidogyne species) nematodes could spend at least 12 weeks in soil solutions searching for penetration sites of suitable host plants. The external covering of nematodes, the cuticle, consists of various layers that contain glycoproteins, lipids, soluble proteins (collagens) and insoluble proteins (cuticulins). Generally, cucurbitacins are lipophilic, but there is scant information on how cuticular proteins relate to these complex terpenoids. A study was conducted to investigate the nature and extent of damage post-exposure of J2 to a wide range of Nemafric-BL phytonematicide concentrations. Post-72 h exposure to Nemafric-BL phytonematicide, nematode morphometrics versus phytonematicides exhibited either negative quadratic, positive quadratic, or negative linear relations, with the models explained by significant (P < 0.05) associations (R-squared). Similarly, total proteins versus phytonematicide exhibited significant negative quadratic relations. The principal component analysis indicated that concentration level of 2-4% of Nemafric-BL phytonematicide have the highest impact on the morphometric changes of J2. In conclusion, the nature and extent of damage suggested that Nemafric-BL phytonematicide was highly nematicidal as opposed to being nematostatic, thereby explaining its potent suppressive effects on nematode population densities.

Molecular and Phenotypic Characterization of Heterorhabditis indica (Nematoda: Rhabditida) Nematodes Isolated During a Survey of Agricultural Soils in Western Uttar Pradesh, India.

INTRODUCTION: Entomopathogenic nematodes (EPNs) are important biocontrol agents of insect pests. To increase the availability of locally adapted entomopathogenic nematode isolates for biocontrol programs, a survey of several agricultural soils in Western Uttar Pradesh, India was conducted. MATERIALS AND METHODS: Eight hundred and sixty soil samples from the districts Meerut, Bulandshahr, Baghpat, and Bijnor were collected and examined for the presence of entomopathogenic nematodos using the "Galleria baiting method". Steinernema and Heterorhabditis nematodes were recovered. The isolated Heterorhabditis nematodes were molecularly, and morphologically characterized, and their biocontrol potential was evaluated against Spodoptera litura. Finally, the geographical distribution of entomopathogenic nematodes was studied based on the analysis of ITS GenBank records. RESULTS: A small proportion of the collected soil samples were positive for Heterorhabditis and Steinernema nematodes. Twelve soil samples were positive for the presence of Heterorhabditis nematodes, and 29 samples were positive for Steinernema. The Heterorhabditis nematodes were identified as Heterorhabditis indica based on morphological, morphometrical and molecular analyses. No other species of Heterorhabditis were isolated from the soil samples analyzed, suggesting that this species is dominant in the western part of Uttar Pradesh, India. The morphology of the nematode isolates was somewhat similar to the morphology of the H. indica isolate used for the original description of this species, with a notable exception mucrons were present in the hermaphrodite and female specimens we collected, but this structure was not observed in the specimens used for the original description of the species. Principal component analyses (PCA) show small inter- and intraspecific morphological variability between the nematodes species of the "Indica" clade. The insecticide properties of one isolate, CH7, were evaluated against Spodoptera litura, and the results show that this isolate effectively killed this pest under laboratory conditions, demonstrating its potential as a biocontrol agent. CONCLUSION: This study sets the basis for establishing new biocontrol agents to be used in future pest management programs in India.

Entomophilic nematodes, Diploscapter coronatus and Oscheius tipulae from Afghanistan.

Several soil samples from different habitats in Badakhshan province of Afghanistan were collected to isolate and characterize bacteria feeding nematodes. The Galleria mellonella-baiting method was used for the isolation of the Afghan insect-associated nematodes. The nematodes were studied using morphological and morphometric data. The Oscheius specimen was characterized by a longer body (630-820 µm) and shorter pharynx (125-145 µm), whereas other morphological characters were not unusual. The Diploscapter specimen had an annulated cuticle, with lip region width 1.5 times shorter than the stoma, and had separated pharyngeal corpus from the isthmus and vulva located in the middle of the body. The molecular data were derived using three loci; 18S, 28S (D2/D3 segment), and ITS rRNA region, which were utilized to measure the genetic distance. The phylogenetic analysis was conducted to reconstruct the relationship tree. Both morphological and molecular approaches confirmed the identity of nematode isolates as Oscheius tipulae and Diploscapter coronatus. This is the first report of insect-associated nematodes from the soil of Afghanistan. Both species were capable of infecting and killing G. mellonella larvae in less than 96 h.

Morphological adjustment in free-living Steinernema feltiae infective juveniles to increasing concentration of Nemafric-BL phytonematicide.

Third-stage larvae (L3) of Steinernema feltiae exist as free-living infective juveniles (IJ), with suspended development activities. In contrast, parasitic stages (L1, L2, L4, adult) have mutualistic relations with Xenorhabdus species bacteria, along with unique morphological changes and development inside the cadaver of host insects and/or plant-parasitic nematodes. Commercial IJ strains are tolerant to cucurbitacin-containing phytonematicides, but we have scant information on how morphological adjustments in IJ are achieved. In this study, we investigated the nature of morphological adjustments in commercial S. feltiae IJ strains to Nemafric-BL phytonematicide, which contains cucurbitacin B as active ingredient. Post-72 h exposure to phytonematicide concentration, IJ specimens were fixed on mounting slides. Length (body, excretory pore to anterior end, pharynx, rectum, stoma, tail), diameter (head width, neck base, mid-body, anal body), cuticle thickness and De Man ratios were measured with a computer software programme attached to Omax light microscope. Morphometric data against increasing phytonematicide concentration exhibited either density-dependent quadratic, linear or neutral relations. Increase in body length at low phytonematicide concentration was accompanied by decrease in tail length and pharynx length during muscle contraction when IJ were still alive. After death at high phytonematicide concentration, the opposite morphometric effects ensued due to muscle relaxation. The observed changes in morphometric structures were explained on the basis of morphological adjustments that modulated volumes of pseudocoelom cavity in IJ. The modulation is intended to maintain hydrostatic pressure within permissible upper limits in order to avoid structural damage to internal organs embedded in the pseudocoelom fluids.

Shelf-life in cucurbitacin-containing phytonematicides: Non-conformity to Arrhenius model.

Shelf-life information provides end-users with the assurance that the product is still in compliance with label claims. Behavioral reaction orders of the Arrhenius model had been consistently used under fixed conditions to provide shelf-life in food products. Due to non-conformity of the cucurbitacin-containing phytonematicides to the Arrhenius behavioral reaction orders, an alternative quadratic model consistent with the behavioral reaction orders of cucurbitacins was developed under chilled (5°C at 95-98% RH) and fixed tropical (38°C at 90% RH) conditions, while room temperature constituted unfixed conditions. Sampling for cucurbitacins was done at time-frames compliant with geometric series, with cucurbitacin analysis regularly performed using high-performance liquid chromatography techniques. Under chilled conditions, neither the Arrhenius nor the quadratic model could predict the shelf-life for Nemarioc-AL phytonematicide, whereas Nemafric-BL phytonematicide had shelf-life of 35 weeks. In contrast, under tropical conditions, the positive quadratic models showed that Nemarioc-AL and Nemafric-BL phytonematicides had shelf-life of 35 and 825 weeks, respectively. In conclusion, the two phytonematicides could be stored under fixed tropical conditions to enhance the shelf-life of their active ingredients.

Redescription of Basiria gracilis (Thorne, 1949) Siddiqi, 1963 (Rhabditida, Tylenchidae) from South Africa.

Basiria gracilis is redescribed from North West Province, South Africa, associated with willow (Salix sp.), which is a dominant ornamental tree species in the province. B. gracilis is characterised by its female body length (692-994 µm), lateral field with two longitudinal incisures, weakly cuticularised labial framework, high lip region lacking annuli, stylet 8-13 µm long, axial spermatheca filled with small rounded sperm, conical-elongated female tail with round to pointed terminus (103-146 µm, c=6.1-7.4, c'=8.8-12.1). Morphologically, B. gracilis is closely related to B. paragracilis but the taxa are differentiated on the basis of lip region and stylet morphology. In addition, B. gracilis is reported for the first time from South Africa. Line drawings, LM and SEM illustrations are given for the species.

Molecular and morphological characters of Helicotylenchus Steiner, 1945 species from Iran with a note on the identity of H. pseudorobustus (Steiner, 1914) Golden, 1956.

Helicotylenchus species were recovered from natural areas of three provinces (Mazandaran, Kerman and Guilan) in Iran, with morphological and molecular characteristics suggesting that they were Helicotylenchus minzi and Helicotylenchus pseudorobustus. A phylogenetic analysis of the two Helicotylenchus species using 28S rDNA placed H. pseudrobustus and H. minzi in a clade with 1.00 posterior probability. Results suggest that the Helicotylenchus population identified using molecular characteristics as H. pseudorobustus is a paraphyletic group, with hierarchical clustering analysis indicating a close relationship between the Iranian and the Chinese populations of H. pseudorobustus. In conclusion, further molecular studies within the genus Helicotylenchus are needed, especially for morphologically similar populations that have been recognized as separate species.

Morphological and molecular characterization of Heterorhabditis bacteriophora isolated from Indian soils and their biocontrol potential.

Agricultural soils and open fields from Western Uttar Pradesh (India) were surveyed to determine the presence of entomopathogenic nematodes. From the entomopathogenic nematodes isolated, Heterorhabditis isolates were selected and further characterized using morphological, morphometrical and molecular approaches. The results showed that three isolated nematodes were Heterorhabditis bacteriophora and were associated with Photorhabdus laumondii subsp. clarkei bacteria, while the rests were identified as Heterorhabditis indica. The biocontrol potential of H. bacteriophora against three agricultural pests was evaluated. Nematode infectivity experiments showed that the nematode isolates DH7 and DH8 were highly pathogenic against cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura), and less pathogenic against white grub (Holotrichia serrata) larvae. This study sets the basis for establishing new biocontrol agents to be used in pest management programs in India.