The restoration of biodiversity and functional tropical forests is critical to mitigating global biodiversity losses. Aboveground, increasing the connectivity of regenerating forest fragments facilitates the recolonization of tropical forest biodiversity. However, restoring functional ecosystems also requires the recovery of decomposition processes as these are essential in shaping aboveground biodiversity. Therefore, we investigate the role of forest connectivity in restoring the composition and functioning of fungal communities in the leaf litter layer during a chronosequence of forest restoration. In the Brazilian Atlantic Forest, we studied secondary forests regrown between 18 and 55 years after deforestation and different levels of forest connectivity and compared their litter to recently abandoned pastures and undisturbed primary forests. We quantified how forest age and connectivity between fragments influenced the litter fungi composition in relation to tree diversity, litter chemistry and litter isotopes. We show that fungal composition was highly heterogeneous in forest litter, whereas pasture litter exhibited a more homogeneous community. Moreover, forest connectivity had stronger effects on litter fungal composition compared to forest age. Connectivity promoted wood saprotrophs and endophytes, while suppressing soil saprotrophs, with its effects being more evident during later stages of restoration. Fungal guilds such as endophytes and saprophytes were primarily influenced by tree diversity and leaf litter chemistry. We conclude that forest connectivity promotes the re-establishment of saprophytic fungi capable of decomposing recalcitrant litter substrates, driven mainly by enhancing tree diversity and litter quality. Practical implications of increasing connectivity may relate to forest resilience in front of future climate change scenarios.
Amplicon sequencing-based community analyses offer an attractive alternative to microscopy-based nematode identification approaches where expert knowledge is required. However, the potential of analysing DESS-preserved samples in metabarcoding studies remained underexplored. Moreover, previous studies often omitted certain representative groups of nematodes. To fill this gap, a nematode metabarcoding experiment was conducted on two types of mock community sample sets: DESS pretreated and freshly acquired, both of them covering taxa of various feeding strategies. Additionally, the performance of two, 18S and 28S rDNA-based (the D2A/D2 primer pair for the first time in such studies), polymerase chain reaction primer combinations, and the efficiency of two DNA isolation methods were compared. The results of this study show that the DESS preservative can be successfully used in qualitative metabarcoding studies of soil nematode communities. All 24, initially morphologically identified, nematode taxa were recovered from the DESS-treated and fresh nematode samples, using both rDNA primer combinations and both DNA isolation methods tested. Each of the two isolation methods used had stronger effect on read recovery of slightly different taxa.
Deladenus longicaudatus n. sp. was recovered from decaying wood of a broad-leaf forest tree in the forests of Javaherdeh, Mazandaran province, northern Iran, and is herein described and illustrated based upon morphological, morphometric, and molecular characters. The new species is mainly characterized by its small to medium body size of free-living mycetophagous females (523-682 µm long) with an elongate conical tail (c = 9.3-11.0 and c' = 3.9-5.3), a relatively anterior vulval position (V = 84.5-89.0%), the pharyngo-intestinal junction at the level of the nerve ring, the secretory-excretory pore (S-E pore) posterior to the nerve ring, and at the same level as the hemizonid. It is further characterized by seven incisures in the lateral fields, a short stylet (5.0-6.5 µm), small lateral vulval flaps, and absence of a postvulval uterine sac (PUS). By having similarities in tail shape, c' ratio greater than 3, posterior position of the S-E pore and hemizonid to the nerve ring, and lack of PUS, the new species is comparable to four known species of the genus, namely, D. gilanica, D. hyrcanus, D. zyzyphus, and D. parvus. It is furthermore typologically comparable to mycetophagous phase of Hexatylus vigissi. In molecular phylogenetic analyses using partial small and large subunit (SSU and LSU D2-D3) rDNA sequences, the SSU sequences of the new species formed a moderately supported clade with sequences of Howardula spp. In LSU phylogeny, the newly generated sequences of the new species formed a clade with corresponding sequence of D. hyrcanus.
Pistachio is one of the most economically important horticultural crops worldwide, and its yield is annually reduced due to damages caused by root-knot nematodes (RKNs). To identify the species of RKNs associated with pistachio trees in Kerman and Khorasan Razavi provinces of Iran, 84 localities were investigated and 37 isolates of the predominant species, Meloidogyne javanica, were identified based on the perineal pattern of females and species-specific primers Fjav and Rjav. Eight isolates (KS1-1, KS1-10, KN7-1, KR3-1, KR8-1, KSh1-1, KhF1-4, and KhF2-1) were further characterized using morphological, morphometric, and molecular data (Small subunit [SSU] rDNA, COII-16S, and NADH dehydrogenase subunit 5 [Nad5] mtDNA sequences). The SSU sequences of these eight isolates showed over 98% identity with several sequences of tropical Meloidogyne species. The COII-16S and Nad5 sequences displayed 99.63 and 100% identity with other available M. javanica sequences, respectively. This study provided, for the first time, comprehensive morphometric, molecular, and geographical distribution data of M. javanica isolates parasitizing pistachio in two main pistachio-producing provinces of Iran. Based on the results of this research, the prevalence of M. javanica in the pistachio orchards of the studied regions was 44.04%, indicating its widespread distribution. Rafsanjan in Kerman province and Mahvelat in Khorasan Razavi province were two highly infested cities based on the present study. Therefore, regular monitoring is essential for effective management of this pest.
Criconemoides iraqicus n. sp. recovered from the rhizospheric soil of pomegranate in Misan province, Iraq, is described based on morphological and molecular data. The new species is characterized by its lip region comprised of two annuli, true submedian lobes absent, pseudolips present, body annuli smooth and with few anastomoses (R = 76-79), stylet 64.6-75.3 μm long, with anchor-shaped basal knobs, excretory pore at one to three annuli posterior to the pharynx base, vulva closed, vulval lips not projecting above body contour, and tail conical with one to three terminal lobes. Based on the number of body annuli, stylet length, smooth annuli, and shape of postvulval body region, C. iraqicus n. sp. is closely similar to C. amorphus, C. ananasi, C. geraerti, C. informis, C. neoinformis, and C. tenuiannulatus. The phylogenetic relationships of the new species with representatives of the family Criconematidae were reconstructed and discussed using partial sequences of the small subunit, D2-D3 expansion segments of the large subunit, and internal transcribed spacer regions of ribosomal DNA (SSU, LSU D2-D3, and ITS rDNA) based on Bayesian inference (BI). In phylogenetic trees, sequences of the new species formed clades with corresponding sequences of C. geraerti, C. informis, Discocriconemella parasinensis, and D. sinensis with different levels of relatedness.
The Stilbonematinae live in symbiosis with ectosymbiotic bacteria covering their cuticle, which evidently constitute their food. In different Stilbonematinae genera, two pharynx types are found, depending on the arrangement of the bacterial coat. Species descriptions show that most Stilbonematinae species with a thick multilayer of symbionts have a two-part pharynx with a predominantly muscular posterior bulb. In contrast, in cases of a thin monolayer of bacteria, the nematodes predominantly show a three-part pharynx with a distinctly swollen muscular corpus at their anterior end. This indicates a shift of the main pumping structure from the terminal bulb to the anterior corpus. Consequently, the amount of contractile filaments in the terminal bulb should decrease. Using phalloidin staining in combination with confocal laser scanning microscopy, light microscopy, and transmission electron microscopy, we measured and compared the filamentous actin (F-actin) volume in the posterior bulb in several Stilbonematinae species representing both pharynx types. Two-part pharynges had a larger relative F-actin volume in the terminal bulb than three-part pharynges. In the latter, prominent gland tissue occupied most of the space between the reduced muscles. This supports our hypothesis of two distinct feeding modes: ingestion of large amounts of food in species with a two-part pharynx ("gourmands") requiring a muscular terminal bulb vs discriminant grazing on a thin bacterial coat in species with a three-part pharynx ("gourmets").
Citrus production worldwide is severely impacted by a devastating disease known as citrus greening, or Huanglongbing (HLB). The phytopathogen causing HLB is transmitted between trees by the Asian citrus psyllid, Diaphorina citri, which acts as the primary vector. Currently, there is no cure for HLB, and management efforts primarily rely on the use of insecticides. However, there is a growing need for alternative, environmentally sustainable control methods, such as RNA interference (RNAi). We investigated the impact of gene silencing on the mortality of D. citri by targeting two genes, vacuolar-sorting protein/sucrose non-fermenting protein 7 (DcSnf7) and inhibitor of apoptosis 5 (DcIap5). Gene silencing was initially assessed by delivering synthesized double-stranded RNA (dsRNA) to D. citri through topical feeding. To further assess gene suppression in vivo, we used a virus-induced gene silencing (VIGS) approach to deliver RNAi to psyllids via citrus plants. Suppressing DcSnf7 and DcIap5 individually resulted in elevated nymph mortality; however, the combined suppression of both genes using dual dsRNA treatment did not yield an additive effect. We modified the infectious Citrus tristeza virus (CTV-T36) clone to individually and jointly carry the truncated genes, DcSnf7 and DcIap5. Over two successive generations, D. citri reared on plants inoculated with CTV-tSnf7, CTV-tIap5, or the combined construct CTV-tSnf7-tIap5 exhibited increased mortality at all life stages, as well as significantly reduced fecundity and fertility, compared to insects reared on non-infected or CTV-wt-inoculated control plants. In addition, these VIGS plants shortened the lifespan of D. citri. Notably, the dual construct CTV-tSnf7-tIap5 consistently produced the most pronounced reductions in survival, fecundity, fertility, and longevity of D. citri across all experiments, exceeding the effects observed with either single-gene construct. Our results suggest that silencing key genes of D. citri using RNAi mediated by VIGS represents a promising control strategy that could play a role in HLB management. © 2026 Society of Chemical Industry.
Coslenchus iranicus n. sp., recovered from the rhizosphere of soapwort in South Khorasan province, Eastern Iran, is described based on morphological and molecular data. The new species is characterized by its cuticle with 18 longitudinal ridges excluding lateral lines, lateral field with three incisures, lip region unstriated, stylet 9.4-12.0 µm long, spermatheca filled with sperm, post-vulval uterine sac absent, vulva with large vulval flaps, and tail mostly with a pointed terminus, though in a few specimens with a finely rounded tip. Based on general characterization, C. iranicus n. sp. is closely similar to C. areolatus, C. franklinae, C. japonicus, C. leiocephalus, and C. maritus. The phylogenetic relationships of the new species with representatives of the family Tylenchidae were reconstructed and discussed using partial sequences of the small subunit, D2-D3 expansion segments of the large subunit, and internal transcribed spacer regions of ribosomal DNA based on Bayesian inference. In the SSU rDNA and LSU rDNA phylogenetic trees, sequences of the new species formed clades with corresponding sequences of C. polonicus and C. leiocephalus with different levels of relatedness, respectively.
Nanobodies specifically recognizing atrazine were successfully obtained through immunization of alpacas and phage display libraries, and an indirect competitive immunoassay (ic-ELISA) based on nanobodies was constructed with an IC50 of 0.062 μg/mL and a minimum detection limit of 0.01 μg/mL. An Au@PtNPs with excellent colorimetric performance, high catalytic activity, and high stability was synthesized. The combination of Au@PtNPs and nanobodies led to the development of a direct competitive immunoassay (dc-ELISA) with higher specificity, convenience, lower cost and an IC50 value of 0.032 μg/mL which was lower than the ic-ELISA. Based on the Au@PtNPs and nanobodies, a novel LFIA model for qualitative and semi-quantitative detection of atrazine was constructed with a visual detection limit of 5 ng/mL. The present study is of great significance for the construction of atrazine immunoassay combining nanobodies and nanoenzymes, which provides a sensitive, specific, simple and reliable method for the detection of atrazine in the environment.
Aeonium, or tree houseleek (Aeonium arboreum), is a bushy, perennial succulent and a popular ornamental plant in regions such as California, New Zealand, Australia, Sicily, Gibraltar, and Chile. It features rosettes of soft, waxy leaves at the tips of sparsely branched or occasionally single, bare stems. It is drought-tolerant and has a variety of colors and forms, making it a popular ornamental plant. In July 2024, a diseased Aeonium plant was submitted by a home gardener from Los Angeles County, California, to the Department of Nematology at the University of California, Riverside (UCR), for diagnosis. Root galls were observed on the plant, and further examination revealed high numbers of root-knot nematodes (Meloidogyne sp.). Molecular species identification was conducted using ribosomal DNA, mitochondrial haplotyping, and species-specific primer techniques, including the TRNAH/MHR106 and MORF/MTHIS primer sets, along with Meloidogyne incognita-specific primers (MIF/MIR). Amplification and sequencing of the marker genes identified the root-knot nematode infecting Aeonium as M. incognita. To our knowledge, this study presents the first report of M. incognita infecting Aeonium worldwide.
Pristionchus pacificus is an important model organism with a well-developed suite of molecular tools and a genomic dataset. Studies that integrate population genetics, ecology, and evo-devo in P. pacificus are supported by an extensive and robust phylogeny consisting of more than 50 Pristionchus species and close to 3,000 strains. Asia by and large has emerged as the biodiversity hot spot of the genus, becoming the focus of recent sampling efforts for Pristionchus isolates. Here, we describe a new androdioecious species of Pristionchus discovered in recent samplings in the Philippines based on a combination of molecular markers, morphological and morphometric data, and mating experiments. All strains of the new species were collected from Scarabeoid beetles in the Philippines and are basal to a sub-clade of the maupasi clade. Pristionchus endotocus n. sp. exhibits constant bagging and a strong bias toward the stenostomatous morph under laboratory conditions and might therefore provide an additional reference point for life history trait studies in Pristionchus.
Currently available nematode identification techniques rely on visual microscopic examination of their morphology and limited molecular assays. These methods generally serve their purpose of enumerating nematode genera and informing management recommendations. However, when identifying variations in pathogenicity or virulence within nematode populations and species - which is crucial for specific plant-parasitic nematode management recommendations - these methods are insufficient. Here, we demonstrate that nucleotide sequence information for tens of thousands of monoclonal antibodies (mAbs) can be generated for identification purposes using a single-cell RNA-seq of mature B cells obtained from mice immunized with nematode antigens. We also provide proof of concept by synthesizing two of these mAbs in vitro and demonstrate specificity using ELISA. Since mAbs can bind to a variety of molecules, their potential use may surpass discrimination among pathotype groups and shed light on what contributes to pathogenicity or virulence of nematodes that produce, or are associated with, these antigenic molecules.
Strawberries are primarily cultivated in the Central Region of Costa Rica due to the favorable growing conditions there. However, several factors can affect the final yield and quality of strawberries, including the presence of plant-parasitic nematodes (PPN). Unfortunately, no surveys have been conducted in the country to identify the PPN affecting production. This study aimed to identify morphologically PPN genera associated with strawberry in the Central Region of Costa Rica, and to identify the Meloidogyne and Pratylenchus species using molecular techniques. A nematode survey was performed between 2018 and 2021 across four provinces: Cartago, Alajuela, Heredia, and San José. The most frequent nematodes found in both root samples (n = 55) and soil samples (n = 53) were Meloidogyne (Frequency of occurrence, FO = 78% in root and 62% in soil) and Pratylenchus (FO = 56% and 43%, respectively) (p < 0.05). Molecular techniques with species-specific primers, such as PCR-RFLP and PCR, allowed for the identification of 13 Meloidogyne populations, all confirmed to be M. hapla. DNA sequencing of the partial mitochondrial COI gene and PCR with species-specific primers found 11 Pratylenchus populations, with 10 identified as P. penetrans and one as P. hippeastri. Further studies should focus on pathogenicity assays with a diversity of strawberry cultivars to assess damage potential and develop strategies for integrated management of PPN in strawberry production.
Chrysodeixis includens (Lepidoptera: Noctuidae) is a significant soybean pest in the southern United States. As it has developed resistance to many commonly used insecticides, alternative control measures are necessary. Entomopathogenic nematodes (EPNs) may be one such alternative. Our previous study found that a surfactant Southern Ag Surfactant (SAg Surfactant), significantly increased the mortality caused by Steinernema carpocapsae on the first instars of Helicoverpa zea in corn plants. In this study, SAg Surfactant and two more adjuvants - dish soap and vegetable oil - were tested for efficacy of S. carpocapsae on C. includens larvae under laboratory and greenhouse conditions. The three adjuvant treatments tested were 0.125% dish soap (Soap), dish soap combined with 0.25% vegetable oil (Soap & Oil), and 0.066% SAg Surfactant. In laboratory conditions, insect mortality caused by S. carpocapsae 72 hr after application was significantly higher with the Soap & Oil treatment than with the no-adjuvant treatment at 1 and 4 hr of exposure, as well as with the Soap treatment at 4 hr of exposure. No significant difference was observed among the EPN with and without adjuvant treatments when the exposure times were extended to 8 and 24 hr. However, compared to the no-adjuvant treatment, insect mortality 24 hr after application was significantly higher for all EPN adjuvant treatments at 8 hr of exposure and for the Soap & Oil treatment at 4 and 24 hr of exposure. These results suggest that these adjuvants shortened the time needed for EPNs to kill C. includens larvae. In the first trial, under greenhouse conditions, insect mortality 72 hr after application was not affected by the adjuvant treatments. In the second trial, all the adjuvant treatments increased insect mortality. However, in the third trial, only the Soap & Oil treatment caused higher mortality compared to the no-adjuvant treatment. Additionally, the Soap & Oil treatment yielded the highest number of viable EPNs in most of the three trials, although this result was statistically significant only at one sampling point. Overall, our results showed that the adjuvants could enhance the efficacy of S. carpocapsae on C. includens larvae.
The distribution of economically significant plant-parasitic nematodes in pulse crops in the Canadian Prairies is relatively unknown. Reports suggested that Ditylenchus dipsaci in yellow pea export was likely the nonquarantine species D. weischeri, a Canada thistle (Cirsium arvense) parasite. To determine if D. dipsaci is found in pulse plants and understand nematode distribution in the Canadian Prairies, a survey was conducted in commercial yellow pea, lentil and chickpea fields in Alberta, Saskatchewan, and Manitoba. Samples of pulse and thistle plants (flowers or pods, stems and leaves) and soil were collected from 94 fields. Nematodes were identified by morphological features and molecular analyses (species-specific PCR, PCR-RFLP, and sequencing of the partial 18S, 28S and ITS of the rDNA gene). High densities of plant-parasitic nematodes - Pratylenchus, Paratylenchus, Helicotylenchus and Telotylenchinae - were found in several fields. Ditylenchus weischeri, a parasite of thistles and not pulse crops, was recovered from 20 fields across Alberta, Saskatchewan and Manitoba; D. dipsaci was found in pods of one yellow pea field in Manitoba. These results confirm the high prevalence of D. weischeri on creeping thistle in pulse fields and the near absence of the quarantine pest D. dipsaci.
The goal of this study is to test whether parasitic Hyalorbilia spp., isolated from sugarbeet cyst nematodes (Heterodera schachtii) in California, could also suppress soybean cyst nematodes (H. glycines). Three H. oviparasitica clade strains, DoUCR50, HsImV27, and ARF18-L, were cultured in powdered peat for use in greenhouse nematode suppression tests. Soils were amended with 500 CFU/cm3 of each fungal strain or autoclaved peat inoculum as a control, seeded with soybeans (Glycine max cv. Williams 82), and 3 weeks later, inoculated with 250 J2 of H. glycines. After 1,260 degree days, cysts and females were collected by flotation sieving and enumerated. DoUCR50, HsImV27, and ARF18-L reduced cysts and females of H. glycines by 73, 87, and 0%, respectively. Egg parasitism was tested by incubating eggs of H. schachtii and H. glycines on water agar cultures of DoUCR50 and HsImV27. Individual eggs of H. schachtii or H. glycines were parasitized by DoUCR50 at rates of 15 or 13%, respectively. Eggs placed in pairs were parasitized at 38 and 43%, while eggs placed in groups of four were parasitized at 70 and 63%, respectively. HsImV27 parasitized fewer than 10% of eggs of either nematode, regardless of egg group size. Both DoUCR50 and HsImV27 were observed to parasitize white females of H. glycines and H. schachtii in tissue culture.
During a survey on the members of the family Neotylenchidae in northern Iran, a population of Deladenus was recovered, which, based on the morphology and molecular characters, identified as a new species. Deladenus hyrcanus sp. n., was isolated from dead wood of Acer velutinum in Balaband forests, Mazandaran Province. Morphologically, the new species is characterized by the moderate body length of the mycetophagous females (718-806 μm) with c = 10.2-12.6, c' = 3.5-4.4 and V = 86-87, stylet length 7.5-10 μm with three small knobs inclined backward, the lateral field with 7-8 incisures, position of excretory pore at level of hemizonid or slightly posterior to it, lacking a post-uterine sac, elongate-conoid tail (58-72 μm), with rounded to pointed terminus and males not observed. According to similarities in general morphology, D. hyrcanus sp. n., resembles D. gilanica and D. zyzyphus; however, the new species is distinguished from them in body length, lateral field incisures, and tail length. It was furthermore compared with D. aridus, D. bonabensis and D. brevis, by similarities in the position of the excretory pore and hemizonid, posterior to the nerve ring, lack of a median chamber, and a conoid tail narrowing to a rounded to pointed terminus. Molecular phylogenetic analysis based on D2-D3 expansion domain of 28S rDNA placed the new species close to D. gilanica in a clade with 1.00 posterior probability. The measurements, line illustration, and LM photographs are provided for the new species.
The new nematode species Hoplolaimus tuberosus n. sp., isolated from potato rhizosphere in Budwale sub-county, Mbale district, Eastern Uganda, is characterized based on light and scanning electron microscopy alongside four molecular markers. Females of H. tuberosus n. sp. are moderately large (1.2-1.6 mm) and exhibit distinctive morphological features, including an offset lip region with 4-5 lip annuli, a basal lip annule divided into 10-12 irregular blocks, a robust stylet (45-50 μm), a variable lateral field, characterized by one incisure (zigzag longitudinal line formed by anastomoses) anteriorly and posteriorly, and 2-3 irregular, incomplete striae at mid-body, a secretory-excretory pore positioned anterior to the hemizonid, 6 gland nuclei, and a hemispherical to bluntly rounded tail with 8-10 annuli. Males are slightly smaller at 1.0-1.3 mm, have a basal lip annule divided into 2-4 blocks and relatively long spicules (46-58 μm). Phylogenetic analyses of COI mtDNA, ITS-rRNA, 18S-rRNA and D2D3 of 28S-rRNA demonstrated a close relation of the new species with morphologically similar species (Hoplolaimus columbus, Hoplolaimus indicus, Hoplolaimus seinhorsti, Hoplolaimus dubius and Hoplolaimus pararobustus) yet H. tuberosus n. sp. had in all analyses a distinct phylogenetic position. The population density of 50-75 H. tuberosus n. sp. per 100 ml of soil, combined with the polyphagous nature of related Hoplolaimus species, suggests that this new species could pose a significant pest threat to potato crops, warranting further pathogenicity studies.
Foliar nematodes are economically important plant pathogens affecting a wide range of hosts, including food crops, ornamentals, and forest trees. Among these, Litylenchus crenatae (family Anguinidae), the causal agent of beech leaf disease (BLD), poses a growing threat to American beech forests. Traditional nematode staining methods, such as acid fuchsin staining, have been widely used for plant-parasitic nematodes found in roots, but their application to foliar tissues remains limited due to differences in tissue structure and stain permeability. In this study, we optimized two acid fuchsin-based protocols for brightfield and fluorescence microscopy to improve the visualization of L. crenatae in symptomatic beech leaves. The improved staining methods provided high-resolution visualization of L. crenatae at the surface and within the leaf tissues using both dissected and whole-mount samples. These enhanced visualization methods offer a practical tool for investigating the spatial distribution and dynamics of foliar nematodes within distinct layers of the leaf.
A new species of free-living nematode inhabiting microbialites in Great Salt Lake, Utah, USA is described both molecularly by 18S-sequencing and morphologically with scanning electron microscopy and differential interference contrast (DIC) microscopy. Diplolaimelloides woaabi sp. nov. (family Monhysteridae, order Monhysterida) is characterized by a combination of the following characters: ocelli present; a relatively small body size (<1.5 mm); short anterior sensory setae; cryptospiral amphidial fovea; a funnel-shaped anterior buccal cavity and reduced secondary cavity; fused lips; long double spicules and conspicuous male bursa displaying four pairs of post-cloacal papillae arranged in a (2 + 2) pattern, a single mid-ventral pre-cloacal papilla, two pairs of papillae posterior to the bursa, and an additional offset mid-tail papillae pair; and a pair of sub-apical extensions on spicules. An updated key to all species of Diplolaimelloides Meyl, 1954 is given. This species is notable for its adaptation to hypersaline microbialites, positioning it as both an extremophile and a potential bioindicator of ecological change in Great Salt Lake.