Female Nematodes Research Articles

Parasite load effects on sex ratio, size, survival and mating fitness of Heleidomermis magnapapula in Culicoides sonorensis.

Heleidomermis magnapapula parasitizes the blood-feeding midge Culicoides sonorensis. Most (84%) single mermithid infective second stage juveniles (J2) developed into adult females, while parasitism by multiple J2 yielded 97% male adults. Nematodes emerged from the midge larval host as adults and mated immediately; females were ovoviviparous. Host larvae were exposed to nematode J2 and examined intact microscopically to score initial parasite load. Midge hosts were reared individually. Premature midge death, nematode survival within the host, and emerging adult nematode sex ratio and size as a function of load and host size were all tracked. Higher nematode loads produced smaller adult nematode males. The higher loads also increased and accelerated premature host death. Emergence of > 7-9 adult nematode males was rare, but up to 19 tiny males emerged from a single host. Larger midges supported higher parasite loads and a larger total volume of emerged nematode biomass. Virgin adult nematode males then were paired with females of variable, known sizes (volume) and held to determine size effects on fertility (egg hatch), and male survival (longevity). Tested adult males ranged in size from 0.0025 - 0.0334 mm3 and females from 0.0121 - 0.1110 mm3. Logistic regression indicated female nematode fertility was positively influenced by male nematode size, while nematode load and female nematode size had no significant effect. While fertility was reduced statistically in smaller males, even some of the smallest male and female individuals could be fertile. Findings are related to field studies in this system.

First Report of Meloidogyne javanica Infecting Strawberry (Fragaria × ananassa) in the United States.

Strawberry (Fragaria × ananassa) is native to temperate regions. However, it has been produced in tropical areas, as a seasonal crop including in Florida, USA during the winter months. In March 2022, root galls resembling those induced by root-knot nematodes (Meloidogyne spp.) were observed in declining strawberry plants 'WinterstarTM FL 05-107' growing in an organic-certified research site in Hillsborough County, Florida, USA. To our knowledge, M. hapla is the only root-knot species reported to infect strawberry in Florida. Preliminary molecular analyses, including newly synthesized DNA sequences (TW81/AB28 = OQ469833 - OQ469836; D2A/D3B= OQ473043 - OQ473047) using extracted nematode females from the strawberry roots, initially identified the RKN as M. javanica. Nematode species confirmation was further performed using the morphology of the female perineal patterns and isozyme analysis, mainly esterase (EST) and malate dehydrogenase (MDH), DNA sequencing, (NAD5-F/NAD5-R) and the SCAR primer set (Fjav/Rjav), species-specific for M. javanica. Isozyme analyses, EST= J3, which is specific for M. javanica and MDH=N1, as well as the morphology of female perineal patterns, agreed with data previously reported for M. javanica. A pathogenicity test on strawberry 'WinterstarTM FL 05-107' transplants was performed using 10,000 eggs of the original M. javanica population, which induced galls on strawberry plants (Gall index, GI = 4.1) with egg masses clearly visible outside of the roots, producing an average of 1,344 eggs/gram of fresh root and 9,201 ± 4,206 eggs/root system. No galls or egg masses were observed on non-inoculated plants. Tomato 'HM 1823' was used as a control for the viability of the inoculum and showed numerous galls and egg masses (GI=5.0;). The newly obtained DNA sequences using NAD5-F/NAD5-R (OQ474970 - OQ474972) were compared with other sequences available in the GenBank and were shown to be 100% identical to five M. javanica populations from Polk County, Florida, USA (OM418745 - OM418749) and the complete mitochondrion genome of M. javanica (NC026556). To our knowledge, this is the first report of M. javanica infecting strawberry in the United States.

Six Genera of Plant-Parasitic Nematodes From Bangladesh

The presence of plant-parasitic nematodes causing crop loss every year had been overlooked in the existing fruits, vegetables and flowers cultivation system in Bangladesh. Therefore, the study was conducted to isolate and identify plantparasitic nematodes from soil and infected plant-parts of fruits (banana, orange), vegetables (pointed gourd, tomato and brinjal) and flower (gerbera) from the districts of Jashore, Jhenaidah, Gazipur and Sylhet during 2017-18 and 2018-19 cropping seasons. A total of 68 samples of which 20 fruit samples, 45 vegetables samples and 3 flower samples were collected and observed for nematode infestation. Baermann funnel method was used to extract the active nematodes from plant parts and Cobb’s method was used for the isolation of nematodes from soil samples. Different morphological structures of the nematodes viz. esophagus, median bulb, basal bulb, intestine, reproductive structures (vulba, bursa), etc. were used to identify the nematodes. To identify Meloidogyne spp. from root knot of tomato, brinjal and gerbera, juvenile larvae J2 and perineal pattern of female nematodes were used. A total of six genera of nematodes were identified and all of them were under the order Tylenchida. The nematode genera were Tylenchorhynchus sp., Tylenchus sp., Hoplolaimus sp., Helicotylenchus sp., Pratylenchus sp. and Meloidogyne spp. The root-knot nematode, Meloidogyne incognita was recorded for the first time in flower crop gerbera in Bangladesh.
 Bangladesh J. Agril. Res. 46(3): 275-284, September 2021

Identification of the root-knot nematode species associated with Carica papaya

Abstract. Plena SNG, Indarti S, Widiastuti A, Putra NS. 2022. Identification of the root-knot nematode species associated with Carica papaya.Biodiversitas 23: 6212-6217. Root-knot nematodes (Meloidogyne spp.) are important economic pests causing low production and yield losses in horticultural crops. Meloidogyne spp. is widespread and regarded as a major pest since they are found all over the world in a variety of geographic locations, most frequently found in tropical and subtropical climate zones. They are obligate parasites that feed on various plants, including papaya and other horticultural plants. They are root-galling in host plants. Many species of root-knot nematodes may be found in a suitable environment. Therefore, accurate identification of the root-knot nematode species in papaya plants is important for developing and applying control and management measures. Surveys and sampling of nematodes on papaya plants were performed at the Agrotechnology Innovation Center, Universitas Gadjah Mada, the Special Region of Yogyakarta, Indonesia, and its surroundings. Identification was performed both morphologically (based on the perineal pattern of female nematodes) and molecularly (using root-knot nematode species-specific primers). The results revealed that the species of root-knot nematode associated with papaya plants was Meloidogyne javanica based on the perineal pattern of female nematodes, specifically the lateral lines. This finding was also supported by the results of the DNA amplification at 670-bp product using specific primers for M. javanica (FJav/RJav). Consequently, plant protection efforts will need to focus on minimizing M. javanica damage in papaya cultivation.

Disinvasive activity of “Hemastal BIO” and “Staldren” against non-invasive eggs of Trichuris skrjabini nematodes parasitizing sheep

Geohelminths parasitizing sheep are widespread throughout the world and have a significant pathogenic effect on the animal body. Among them, one of the leading places in terms of distribution is occupied by helminths of the genus Trichuris. This is due to the high resistance of trichurises to the effects of chemicals and adverse environmental factors. The presence of viable eggs of trichurosis pathogens in the surface layer of the soil and a sufficiently long period of their storage in the environment represents a potential threat of parasitic invasion on the territory of sheep farms. The aim of the research was to determine the disinfestation activity of modern ecologically safe means for dry disinfection against eggs of non-invasive nematode eggs of Trichuris skrjabini parasitizing sheep. An experimental test of the preparations “Hemastal BIO” (AS – calcium hydroxide, calcium sulfate, calcium sulfite-semihydrate, calcium chloride) and “Staldren” (AS – calcium carbonate, pine oil, magnesium silicate, iron oxide) was carried out in laboratory conditions. In the experiment, test cultures of eggs isolated from the gonads of T. skrjabini nematode females were used. The results of the conducted research established a high level of disinfestation efficiency of “Hemastal BIO” and “Staldren” (DE – 100 %) relative to non-invasive eggs of T. skrjabini on the 54th day of cultivation – the period of formation of invasive eggs in the control culture. During the embryogenesis of trichurises, the effectiveness of dry disinfectants gradually increased with “Hemastal BIO” up to 88.84 % – on the 18th day and up to 98.36 % – on the 36th day, and “Staldren” up to 82.79 % – on the 18th day and up to 95.90 % – for 36th days. Their harmful effect was characterized by specific changes in the eggs of nematodes in the form of sticking of crystals of the agents around the causative agents, as a result of which there was a stop in growth and development and the gradual decay and resorption of the embryos. Such changes are also confirmed by the metric parameters of the width and length of T. skrjabini eggs. The obtained data allow to recommend dry disinfectants “Hemastal BIO” and “Staldren” for the purpose of effective maintenance of sanitary and epidemiological well-being in relation to trichurosis in sheep farms.

Coated Cu-doped ZnO and Cu nanoparticles as control agents against plant pathogenic fungi and nematodes

In the current study, coated copper nanoparticles with polyethylene glycol 8000 (Cu@PEG NPs) and copper-doped zinc oxide nanoparticles with diethylene glycol (Cu-doped ZnO@DEG NPs) have been synthesized via solvothermal and microwave-assisted process, physicochemical characterized, and studied as nano-fungicides and nano-nematicides. Spheroidal Cu-doped ZnO@DEG NPs and urchin-like Cu@PEG NPs have been isolated with average crystallite sizes of 12 and 21 nm, respectively. The Cu doping (11.3 wt%) in ZnO lattice (88.7 wt%) was investigated by Rietveld refinement analysis and confirmed by X-ray Diffraction (XRD) and X-ray Photoelectron Spectroscopy (XPS). The Cu-doped ZnO@DEG and Cu@PEG NPs revealed a growth inhibition of fungi Botrytis cinerea (B. cinerea) and Sclerotinia sclerotiorum (S. sclerotiorum) and nematode paralysis of Meloidogyne javanica in a dose-dependent manner. Cu-doped ZnO@DEG NPs were more effective against M. javanica (EC50 = 2.60 μg/mL) than the Cu@PEG NPs (EC50 = 25 μg/mL). In contrast, the antifungal activity was approximately similar for both NPs, with EC50 values at 310 and 327 μg/mL against B. cinerea, respectively, and 260 and 278 μg/mL against S. sclerotiorum, respectively. Lettuce (Lactuca sativa) plants were inoculated with S. sclerotiorum or M. javanica and sprayed with either Cu-doped ZnO@DEG NPs or Cu@PEG NPs. The antifungal effect was evaluated based on a disease index (DI), and nematicidal activity was assessed based on the total number of galls and nematode females per root gram. NPs successfully inhibited the growth of both pathogens without causing phytotoxicity on lettuce. The DI were significantly decreased as compared to the positive control (DI = 5.2), estimated equal to 1.7, 2.9 and 2.5 for Cu@PEG NPs, Cu-doped ZnO@DEG NPs and the chemical control (KOCIDE 2000), respectively. The reduction in galling and population of M. javanica ranged from 39.32% to 32.29%, statistically like chemical control. The treatment of lettuce plants with Cu-doped ZnO@DEG NPs increased the leaf net photosynthetic value at 4.60 and 6.66 μmol CO2−2 s−1 in plants inoculated with S. sclerotiorum and M. javanica, respectively, as compared to the control (3.00 μmol CO2−2 s−1). The antioxidant capacity of NPs treated lettuce plants was evaluated as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in leaf extracts. Plants inoculated with S. sclerotiorum and sprayed with Cu-doped ZnO@DEG and Cu@PEG NPs, exhibited a 34.22% and 32.70% increase in antioxidant capacity, respectively, higher than the control. Similarly, an increase in antioxidant capacity was measured (39.49 and 37.36%) in lettuce inoculated with M. javanica and treated with Cu-doped ZnO@DEG and Cu@PEG NPs, respectively. Moreover, an increase of phenolic compounds in lettuce leaf tissue treated with NPs was measured as compared to the control. Overall, foliar applied Cu and Cu-doped ZnO NPs could be a promising tool to control phytopathogenic fungi and nematodes contributing to sustainability of agri-food sector.

Modeling Root-Knot Nematode Regulation by the Biocontrol Fungus Pochonia chlamydosporia.

Two models of increasing complexity were constructed to simulate the interactions between the root-knot nematode (RKN) Meloidogyne incognita and the biocontrol fungus Pochonia chlamydosporia var. catenulata in a rhizosphere microcosm. The models described discrete population dynamics at hourly rates over a 6-month period and were validated using real parasitism and nematode or fungus data. A first, general Pochonia-nematode-root model (GPNR) used five functions and 16 biological constants. The variables and constants describing the RKN life cycle included the rates of egg production, hatching, juvenile (J2), and mature female development, including root or nematode self-density-dependent factors. Other constants accounted for egg parasitism, nematode-induced root losses, growth, and mortalities. The relationship between nematodes and fungal propagules showed density dependence and cyclic variations in time, including an attractor on the propagules and J2 phases space. The simulations confirmed a P. chlamydosporia optimal initial density of 5 · 103 propagules · cc soil-1, as usually applied in assays. The constants used in GPNR showed adherence to the nematode biology, with 103 eggs per egg mass, a 10-day average lifespan of J2, with 2 days required to enter roots, and adult lifespan lasting 24 days. The fungus propagule lifespan was 25 days, with an average feeder root lifespan lasting around 52 days. A second, more complex Pochonia-nematode-root detailed model (GPNRd) was then constructed using eight functions and 23 constants. It was built as GPNR did not allow the evaluation of host prevalence. GPNRd allowed simulations of all RKN life stages and included non-parasitic and parasitic fungus population fractions. Both GPNR and GPNRd matched real J2 and fungus density data observed in a RKN biocontrol assay. Depending on the starting conditions, simulations showed stability in time, interpreted as effective host regulation. GPNRd showed a fungus cyclic relationship with the J2 numbers, with prevalence data close to those observed (38.3 vs. 39.4%, respectively). This model also showed a further density-independent nematode regulation mechanism based on the P. chlamydosporia switch from a non-parasitic to a parasitic trophic behavior. This mechanism supported the biocontrol of M. incognita, also sustained by a concomitant increase of the root density.

First record and morphological contributions of Procyrnea leptoptera (Rudolphi, 1819) (Nematoda: Habronematidae) in the kestrel Falco tinnunculus Linnaeus (Falconiformes: Falconidae) from Egypt

BackgroundThe kestrel Falco tinnunculus, Linnaeus, 1758 is widely distributed in Europe, Asia, Africa, and East coast of North America. It is a predator species that belongs to family Falconidae. Nematode species of the genus Procyrnea infects the proventriculus and gizzard of the birds and may cause serious health hazards including inflammation, erosions, and ulcers for the infected gastric mucosa, general weakness and may lead to death. The present study aimed to improve the limited information regarding the endoparasites of Falco tinnunculus in Egypt.ResultsNine males and twenty-one female nematodes were identified as Procyrnea leptoptera, Rudolphi 1819, Chabaud 1958, isolated from the proventriculus of common kestrel; Falco tinnunculus gathered from El-Faiyum governorate, Cairo, Egypt, as a new geographical record. The isolated species has been studied morphologically by light and scanning electron microscopy. It was revealed that it is characterized by two lateral pseudolabia with dorsal and ventral labia. The pseudolabium is divided into 2 lobes at the internal side, a number of medium-size teeth (4–6) in the buccal cavity on the interior border of pseudolapia, in addition to the presence of transverse striations. Also, the histopathological effects of the isolated nematode on the liver of the infected host revealed severe damage in the bird's tissues and morphological alterations including infiltration of inflammatory cells, congestion, focal necrosis, and degeneration of hepatic cells.ConclusionsRaptors like Falco tinnunculus are infected with parasites via prey ingestion, so they are serving as intermediate hosts for many species of parasites. Due to the scarcity of studies for the helminth parasites of raptors in Egypt, further studies are needed to get additional precise data on the helminthic infection of raptors and their histopathological effects.

Strongyloidiasis (Strongyloides papillosus) in lambs in Uruguay.

Here, we describe a spontaneous outbreak of strongyloidiasis (Strongyloides papillosus) in sheep in Uruguay. In a flock of 150 lambs, 60 animals became ill and 12 died within 3weeks. Affected animals showed weakness, weight loss, enophthalmos and, in some cases, sudden death. Postmortem examination revealed muscle atrophy, dehydration, and small intestines with a foamy and liquid content. Histological examination of the duodenum and the jejunum showed a thick mucosa with severe villous atrophy, mononuclear mixed infiltrate in the lamina propria, and a large number of nematodes embedded in the mucosa, with numerous granulomas in the lamina propia. Small intestinal content analysis showed a total of 13,200 female nematodes in the duodenum and 2000 in the jejunum, identified as Strongyloides papillosus (Strongyloididae). This parasite has long been considered to behave as a commensal parasite or at least to cause significant disease only when present in large numbers. This report shows that Strongyloides papillosus can be highly pathogenic in sheep.

FACTORS INFLUENCING THE BIOLOGICAL AND MORPHOMETRIC PARAMETERS OF HETERAKIS GALLINARUM EGGS (GMELIN, 1790)

The article presents the results of the research on the peculiarities of embryo and morphometric development
 of the eggs isolated from the gonads of female nematodes Heterakis gallinarum that parasitize turkeys and hens at different
 intensities of invasion in case of mono-and mixed forms of helminth infections. It was established that the eggs of H. gallinarum became invasive at 30 °C for 7 days, their survival rate in the laboratory conditions was: 80,67±2,08 % (turkeys), 84±3 % (chickens). The morphometric parameters of eggs excreted by mature H. gallinarum females are objectively influenced by such factors as the stage of biological cycle, the intensity of invasion, the type of host.

Obtaining immunodiagnostic preparations from <i>Toxocara canis</i> antigens for serodiagnostics of toxocariasis in human

Toxocariasis is a human helminthic invasion that has a wide range of hosts with epizootic distribution. The populational seropositivity in countries with a temperate climate comprises about 37%, whereas in regions with tropical climate up to 92%. Almost all age groups of the population are at risk of invasion by toxocars. The immunological method currently retains diagnostic significance for toxocariasis, because the reaction of immune system against helminths is accompanied by developing sensitization, and techniques as well as methods of collecting clinical material to identify the migratory form of toxocarosis in humans are time-consuming and invasive. The purpose of this study is to develop an immunobiological preparation based on excretory-secretory antigens from Toxocara canis larvae for serodiagnostics of larva migrans syndrome in human by using enzyme-linked immunoassay (ELISA). Antigens were obtained from Toxocara canis larvae by culturing its eggs isolated from the uterus of female nematodes in the glutamine-supplemented medium. The preparation was purified from ballast substances by centrifugation at 8000 g for 20 minutes followed by filtration through microfiltration membrane with a pore diameter of 0,050,15 microns, type MFAS-P-1 (manufactured by CJSC STC Vladipor). Blood serum of student volunteers from countries with high incidence rate of toxocariasis were tested to detect T. canis-specific antibodies. ELISA with commercial antigens Toxocara-IgG-ELISA-BEST kit was used for selection of seropositive and seronegative sera also used as a control. Diagnostic antigenic preparations with different protein concentrations were prepared to examine seropositive sera for determining optimal dose of the antigenic drugs. Results. ELISA with standard test kit allowed to detect Toxocara-IgG antibodies in 20 sera (8%) from 250 samples, what indicates about antigenic stimulation of the immune system by helminths and suspected former disease. Analyzing experimental samples allowed to find the number of seropositive data correlated with antigenic protein concentration in preparations: at a concentration of 1,96 g/ml 5,2% of positive results were detected, 1,71 g/ml 3.2%, 0.33 g/ml 0.8% (Pearsons correlation coefficient 0,94). The number of positive sera detected with commercial antigen in ELISA kit was identical to that of positive sera detected by an experimental antigen sample with a protein concentration of 2,49 g/ml. Thus, the immunodiagnostic preparations obtained by the experimental method based on the excretory-secretory antigens from Toxocara canis are characterized by high sensitivity and can be used to detect Toxocara-IgG antibodies in ELISA. The antigen preparation protein concentration of at least 2.49 g/ml is optimal and correlates whit sensitivity of the commercial antigen of the standard ELISA.

First application of two distinguishment techniques: Using Linear Discriminate Function method and Artificial Neural Networks approach according to the ovary types for some plant parasitic nematodes

In this study mono and dual ovaries, which belonged to female individuals of different plant parasitic nematode species that were obtained from the quince (Cydonia oblonga Mill.) (Rosales: Rosaceae) cultivated areas in Sakarya Province (Turkey), were classified. The total number of 109 and 121 female nematodes, which were taken from the soil, were used in 2016, July and 2017, July, respectively. Overall body length (L), spear length (Stylet) and tail/distance from vulva to anus (T/VA) parameters belonged to these nematodes were measured and examined. The mono and dual ovary groups were distinguished by using the Linear Discriminate Function (LDF) method (Fisher’s method) and Artificial Neural Networks (ANNs) approach taking correlation between those parameters into consideration. The pair of parameters L and (T/VA) had higher accuracy percentage (as 97% for LDF method and 100% for ANNs approach) than the pair of parameters L and Stylet (as 91% for LDF method and 97% for ANNs approach) for the classification using 2017, July data set. The second approach was more successful than the first method. This research is the first study that was used these method and approach together at the nematology study area in Turkey and the World. The taxonomical studies may be improved using different statistical methods and artificial neural networks approaches together at the nematology.

First Report of the Parasitic Nematode Pseudoterranova spp. Found in Mediterranean Monk Seal (Monachus monachus) in Greece: Conservation Implications

The Mediterranean monk seal (Monachus monachus) is classified as an endangered species by the IUCN, with a global population that does not exceed 800 individuals. There is limited understanding around the biology and health status of the species, rendering possible parasitic infections grave for its conservation efforts. The aim of the current study was the molecular identification of a parasitic nematode found in the digestive system of a sub-adult Mediterranean monk seal individual, that was found stranded in the area of Pagasitikos Gulf, Greece in 2019. Analysis of the stomach contents revealed the presence of two intact female nematode individuals. Standard protocols were followed as DNA extraction of the parasites was conducted and PCR amplification of the cytochrome oxidase subunit I (COI) mitochondrial gene was implemented. Sequencing analysis of a 585 bp-amplified product displayed a 96% similarity of the screened nematodes to the Pseudoterranova bulbosa species. Bayesian inference was implemented for the subsequent tree reconstruction. The phylogenetic tree revealed a clear genetic similarity between our parasitic nematode individuals named as Pseudoterranova spp. and Pseudoterranova bulbosa (bootstrap value: 82%), which is indicated for the first and only time as such, to be found in the waters of the Mediterranean Sea and also in the stomach of a Mediterranean monk seal.

Bovine parafilariosis - New autochthonous cases from Germany and summary of recent reports from Europe.

Bovine parafilariosis is an emerging fly-borne disease in central Europe, characterized by seasonal occurrence of hemorrhagic exudations ('bleeding spots') from the end of winter to end of summer. In two cases from Germany reported here, one animal of a small herd in Bavaria and 20 animals on a farm in Baden-Württemberg presented bleeding spots from late March and late April 2020, respectively. Exudate samples from both cases were positive for larvated Parafilaria eggs. Examination of the skin and trimmed tissue after slaughter of the animal from Bavaria resulted in the collection of 11 nematodes (two males, eight females, one specimen in fragments). The animal's carcass presented typical yellow-greenish areas and bloody spots on the subcutaneous tissue of the flesh side of the skin. The nematodes were microscopically determined as Parafilaria bovicola. Basic morphometric measurements of two (one intact) male and six female nematodes are within the ranges of published data; length (male/female) 28.8/48.0-64.5mm; width, 397.6μm/430.7-527.6μm; distance of cervical papillae to anterior end, 177.6/248.9-337.4; left spiculum/right spiculum (male), 365.3-379.4/149.5-180.3μm; gubernaculum 45.0-48.1μm; distance of vulva to anterior end (female), 37.3-66.0mm. In order to gain information on P. bovicola in its vector, 91 cattle-visiting Musca autumnalis flies were collected from the affected animal in Bavaria (36 flies) and from co-pastured animals (55 flies) for PCR analysis and sequencing. A total of 14 flies were PCR-positive for filarial DNA, and sequencing of a fragment of the cox1 gene resulted in identification of P. bovicola (n=10) and Thelazia gulosa (n=5). This report presents further cases of bovine parafilariosis in Germany, provides morphometric data on male and female P. bovicola nematodes retrieved from cattle and identified DNA of P. bovicola and T.gulosa in M. autumnalis flies collected at a site of occurrence of bovine parafilariosis.

Study on burrowing nematode, Radopholus similis, pathogenicity test system in tobacco as host

Radopholus similis (Cobb 1893) Thorne (1949) is a destructive migratory endoparasitic plant nematode. In this study, the pathogenic process of R. similis infection in Nicotiana benthamiana (tobacco) was studied using quartz sand culture in laboratory. The results showed that R. similis mainly parasitised the root cortex, leading to cortical cell decomposition and tissue decay. We optimised the inoculation conditions to establish a method for determining the pathogenicity of R. similis as follows: (1) a glass culture tube was filled with quartz sand (about 1/3 of the height) and sterilised twice; (2) 20-day-old N. benthamiana seedlings were transplanted into test tubes and cultivated for 10 days at (25±1)°C; (3) R. similis female nematodes were inoculated in the root rhizosphere at a rate of 150 nematodes per plant; (4) the number of nematodes, disease severity, and growth of the plant at 30 days post-inoculation (dpi) were determined. The pathogenicity of eight R. similis populations from different hosts was determined, which proved the feasibility of this method.

The Predation Behaviour of Fictor composticola on Parasitic Nematodes of Button Mushroom, Agaricus bisporus

In this study, the predation behaviour of male and female predatory nematode, Fictor composticola, was studied on five prey nematode species, Aphelenchus avenae, Aphelenchoides swarupi, Ditylenchus myceliophagus, Bursilla sp. and Panagrolaimus sp., found in the white button mushroom compost. The period of the study is of six months. The data recorded on number of encounters, part of the body of prey attacked, stage of the prey attacked, duration of feeding etc. The strike rate and prey susceptibility were calculated. The average number of encounters on all the five preys done by female F. composticola was 3.0 and that of the male was 6.0. Male F. composticola had more number of encounters on the prey nematode species than the females. Both the sexes preferred juvenile stages over adults as prey. The most attacked part by both females and males predator, was the posterior part of the prey body. In 80% of cases, female predators fed on the first encountered prey while males attacked the first encountered prey in 30% of cases only. The strike rate of female F. composticola was more (78.6%) than the male (48.2%). Mycophagous nematodes were more susceptible to predator’s attack than the microbivorous nematodes. The strike rate of the predator on different prey nematode species was found more on mycophagous nematodes than on microbivorous nematodes and minimum on Panagrolaimus sp. The average feeding duration of female F. composticola was 8 min and 31 sec and in the case of males it was 4 min and 11 sec.

Exploring Citrus Nematodes and its Antagonists in Citrus Growing Areas of Punjab, Pakistan

Citrus nematode (Tylenchulus semipenetrans) is a major threat to the citrus industry in Pakistan. During 2019-20, a survey was carried out for citrus nematode and its antagonists in citrus orchards (Both healthy and declining trees were samples) of Punjab, Pakistan. Bhalwal, Shahpur, Sillanwali, Kotmomin, Sargodha, Quaidabad, Jhang, and Rahim Yar Khan were surveyed for disease incidence, prevalence, and association of T. semipenetrans. Maximum disease prevalence (100%) was recorded in Rahim Yar Khan while minimum (20%) in Bhalwal and Kotmomin. Maximum slow decline with 80% field incidence was observed in Rahim Yar Khan while a minimum of 20% in Quaidabad. Soil and root samples were used for nematode extraction using Baerman's funnel method and the Whitehead Hemming tray method. A maximum number of J2s/100ml of soil were observed in soil samples taken from Rahim Yar Khan (1674) followed by Shahpur (1534), Qaidabad (1432), Sargodha (1347), and Bhalwal (1172). The highest number of nematode females per gram of root were recorded in Rahim Yar Khan (652) followed by Quaidabad (611), Bhalwal (490), Kotmomin (421), Sillanwali (387). Isolation of different fungal and bacterial antagonist isolates was also done by soil dilution plate technique using nutrient agar media and potato dextrose agar media. Identification of fungal antagonists was made on colony growth, color, and spore structure. Bacterial identification was also done on morphological characters and gram tests. Trichoderma harzianum, Trichoderma viride, Trichoderma koningii Trichoderma atroviride, Pseudomonas fluorescens, Pseudomonas putida, Bacillus cereus and Bacillus subtilis were found associated.

Parasitylenchus myiophagus n. sp. (Nematoda: Parasitylenchidae), a tylenchid nematode parasite of long-legged flies (Diptera: Dolichopodidae).

A tylenchid nematode parasite of a male long-legged fly, Tachytrechus sanus Osten Sacken (Diptera: Dolichopodidae) from Montana, USA is described as Parasitylenchus myiophagus n. sp. (Nematoda: Parasitylenchidae). The new species is characterized by the presence of an extremely long first generation female and numerous short and wide second generation males and females produced in enclosed clusters in the hosts body cavity. Both generation female nematodes are ovoviviparous, with short stylets lacking knobs and simple tails lacking spikes, palps or mucrons. The second generation males have paired, separate spicules, short stylets, and a bursa but no visible gubernaculum. The fly host shows evidence of demasculinization, which is attributed to nematode parasitism. The gonads of the second generation adults are infected with a microsporidium (Microsporidia), which is a new host record for tylenchid nematodes. A tylenchid-infected Baltic amber dolichopodid shows that associations between these two organisms extend back at least to the Eocene.

First report of Meloidogyne arenaria on roots of Grona triflora in Guangdong Province, China.

Grona triflora (Desmodium triflorum), a perennial herbaceous legume, is widely distributed in southern China. G. triflora has antipyretic, antiseptic and expectorant properties and can therefore be used as a phytomedicine (Ghosal et al. 1973). In July 2020, roots of G. triflora were investigated for nodules and rhizobia collection at the Shibaluohan Mountain Forest Park of Guangzhou. Root galls induced by a root-knot nematode were observed on 90% of the G. triflora samples (in a 200 m2 plot) and the infested plants had yellow, small and withered leaves compared with the healthy ones. The galls number on a G. triflora root ranged from 43 to 92 and the population densities of second stage juveniles (J2s) ranged from 573 to 894 per 100 cm3 soil surrounding the plant. The female perineal patterns showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between the anus and tail terminus, which matched with the description of Meloidogyne arenaria (Hartman and Sasser 1985). The J2s had the following morphometric characters (n = 15): body length = 501.05 ± 23.71 µm; body width = 17.14 ± 1.23 µm; DGO = 3.13 ± 0.27 µm; stylet length = 12.97 ± 1.38 µm; tail length = 58.02 ± 4.77 µm; hyaline tail terminus = 10.08 ± 0.65 µm. DNA from four female nematodes was isolated for PCR-based diagnostic analyses. A fragment between the COII and LrRNA genes of the mitochondrial DNA was amplified with primers C2F3/1108 (Powers and Harris 1993). In addition, a 28S ribosomal DNA D2/D3 region was amplified with primers MF/MR (Hu et al. 2011). The amplicons were sequenced (GenBank No. MW315989 and MW307358). Nucleotide BLAST results indicated that both sequences show 100% identity with corresponding M. arenaria sequences of isolates from various countries such as Brazil, China, Myanmar and Vietnam (e.g., MK033428, JQ446377, KY293688 and MK026624). For further confirmation, sequence characterized amplified region (SCAR) PCR was employed using the M. arenaria specific primers Far/Rar (Zijlstra et al. 2000). The amplicon was also sequenced (GenBank No. MW315990). The Nucleotide BLAST results showed >99% identity with M. arenaria isolates from Indonesia and Argentina (KP234264, KP253748 and MK015624). Greenhouse tests were conducted to analyze the capacity of M. arenaria to induce galls on G. triflora roots. The G. triflora seeds were collected from the sampling plot and germinated on 0.8% (W/V) agar plates. Then the seedlings were planted in 14 cm deep and 15 cm diam pots filled with sterilized soil from sampling plot. Every seedling was inoculated with 2,000 J2s (n = 15) and plants without J2s were used as a control. Two months later, galls were observed for inoculated roots while no galls were formed on roots of control plants. An average of 13,300 J2s and eggs of M. arenaria (reproduction factor = 6.65) were recovered from the root. Stanton and Rizo (1988) found that G. triflora was susceptible to M. javanica in Australia, and Ogbuji (1978) reported that a population of M. incognita reproduced on roots of G. triflora in Nigeria after artificial inoculation. To our knowledge, this is the first report on G. triflora parasitized by M. arenaria in Guangdong province. M. arenaria has potential to infest local, economically important plants like citrus, pomelo, sugarcane, maize and peanut. As G. triflora is widely distributed in southern China, there is the risk of spreading M. arenaria into agricultural and horticultural systems, that will cause yield loss and economic impacts.

Human Ocular Thelaziasis: A Case Report

Thelaziasis is a zoonotic disease which affects the eye of domestic and wild carnivores caused by the nematode Thelazia. It is transmitted to humans by secretophagous arthropod-borne zoophilic nonbiting flies of the family Drosophilidae. Human thelaziasis is rare and occurs in poor socio-economic families of the rural locations where people live in close proximity with animals. A one and halfyear-old was presented to the outpatient ophthalmology clinic after her mother noticed a whitish, thread-like worm in her right eye. A total of four worms were mechanically removed from her right eye. All the collected worms were gravid female nematodes of Thelazia species. The present case of human ocular thelaziasis from Palpa, Nepal is presented for its rarity.