Description of Nothotylenchus savadkoohensis n. sp. (Rhabditida, Anguinidae) from Iran based on morphological and molecular data.

Nothotylenchus savadkoohensis n. sp. was recovered from rotten wood samples of an unidentified forest tree in the Mazandaran province and described herein. It is mainly characterized by an elongated conoid tail ending in a sharply pointed tip and four lines in the lateral field. Females of the new species have 379-662 µm long bodies with 5.8-6.9 µm long stylets ending in fine posteriorly sloping knobs, the metacorpus not valvate, the pharyngeal bulb slightly overlapping the intestine, and the vulva at 76.5-84.0% of body length. Males are also common and have 13.0-14.5 µm long spicules and bursa cloacal. By having an elongated conoid tail and four lines in the lateral field, the new species comes close to four known species, namely N. acris, N. acutus, N. antricolus, and N. truncatus. The morphological differences between the new species and the abovementioned species are discussed. The new species was sequenced for its D2-D3 segment of LSU and ITS rDNA regions. In the LSU phylogenetic tree, the currently available LSU sequences of the genus Nothotylenchus occupied distant placements from each other and the LSU sequence of the new species formed clade with a sequence assigned to Neotylenchus sp. In ITS phylogeny, the newly generated sequence of the new species formed a clade with a clade that includes sequences of Ditylenchus sp. and Neomisticius platypi and N. variabilis.

Reaction of Commercial Cultivars of Kiwifruit to Infection by Root-knot Nematode and Its Biocontrol Using Endophytic Bacteria.

Root-knot nematodes (RKN) cause considerable economic losses to kiwifruit production annually. Screening of resistant cultivars has been one of the long-standing methods to manage root-knot nematodes. Here, the reaction of the four most common commercial cultivars of kiwifruit, namely, Actinidia chinensis var. deliciosa cv. Hayward, A. chinensis var. deliciosa cv. Abbott, A. chinensis var. deliciosa cv. Bruno, and A. chinensis var. chinensis cv. Haegeum (commonly known as 'Golden' kiwifruit) to infection by the RKN, Meloidogyne incognita, was evaluated. Among examined cultivars 'Golden' was the most susceptible, having on average 52.8 galls, 56.1 egg masses per gram of root, and 642 J2 population per 200 gram of soil. 'Bruno' showed the highest resistance, with 3.3 galls, 4.1 egg masses per gram of root, and 79 J2 in 200 g of soil. Then, two potential biological control agents, namely Priestia megaterium 31.en and Agrobacterium tumefaciens 19.en were used on 'Hayward' seedlings against M. incognita and showed a significant reduction in the number of galls and egg masses on roots, juvenile population in the soil, and increased the growth parameters of the plants compared to non-treated seedlings. We demonstrated that integrated management using resistant cultivars and biological control can provide a safe and economic method to control RKN, and these resistant cultivars can be used in breeding programs.

Morphological and Molecular Characterization of Filenchus multistriatus n. sp. (Tylenchomorpha: Tylenchidae) and Data on a Known Species of the Genus from Bushehr Province, Southern Iran.

During a nematological survey in southern Iran, a population belonging to the family Tylenchidae was recovered from a tomato field in Bushehr province. The recovered population belongs to the genus Filenchus, was described and illustrated herein as F. multistriatus n. sp. It is mainly characterized by having a wide and low annulated lip region continuous with adjacent body; amphidial openings confined to the labial plate; four lines in lateral fields forming three bands, with the two outer bands broken by transverse, and the inner one broken by both transverse and longitudinal lines; and median bulb oval with visible valve and elongate-conoid tail uniformly and gradually narrowing toward the distal region, ending in a widely rounded tip. Its morphological and morphometric differences with three closely similar species were discussed. The phylogenetic relationships of the new species with other relevant genera and species were reconstructed using partial sequences of small, and large subunit ribosomal DNA (SSU and LSU rDNA) sequences. Morphometric and morphological data were also provided for an Iranian population of F. sandneri recovered from Bushehr province. Both populations were characterised using SEM data.

Dicer-like 3a mediates intergenerational resistance against root-knot nematodes in rice via hormone responses.

In a continuously changing and challenging environment, passing down the memory of encountered stress factors to offspring could provide an evolutionary advantage. In this study, we demonstrate the existence of "intergenerational acquired resistance" in the progeny of rice (Oryza sativa) plants attacked by the belowground parasitic nematode Meloidogyne graminicola. Transcriptome analyses revealed that genes involved in defense pathways are generally downregulated in progeny of nematode-infected plants under uninfected conditions but show a stronger induction upon nematode infection. This phenomenon was termed "spring loading" and depends on initial downregulation by the 24-nucleotide (nt) siRNA biogenesis gene dicer-like 3a (dcl3a) involved in the RNA-directed DNA methylation pathway. Knockdown of dcl3a led to increased nematode susceptibility and abolished intergenerational acquired resistance, as well as jasmonic acid/ethylene spring loading in the offspring of infected plants. The importance of ethylene signaling in intergenerational resistance was confirmed by experiments on a knockdown line of ethylene insensitive 2 (ein2b), which lacks intergenerational acquired resistance. Taken together, these data indicate a role for DCL3a in regulating plant defense pathways during both within-generation and intergenerational resistance against nematodes in rice.

Erratum to: Morphological and Molecular Characterization of Filenchus multistriatus n. sp. (Tylenchomorpha: Tylenchidae) and Data on a Known Species of the Genus from Bushehr Province, Southern Iran.

[This corrects the article DOI: 10.2478/jofnem-2023-0008.].

Description of Boleodorus Bushehrensis n. Sp. (Rhabditida: Tylenchidae) from Southern Iran, and Observations on a Commonly Known Species.

A new species of the genus Boleodorus, recovered from southern Iran, is described and illustrated based upon morphological and molecular data. B. bushehrensis n. sp. is mainly characterized by having a wide and low cephalic region (which is continuous with the adjacent body), the oral aperture in a depression in side view under a light microscope, four lines in the lateral field, weak metacorpus with a vestigial-to-invisible valve, and short (26-38 mm long) hooked tail with rounded tip. The females are 334-464 mm long and have a spherical spermatheca filled with spheroid sperm; males have 11.5- to 12.0-mm-long spicules and weakly developed bursa. The new species has an annulated low cephalic region, four large cephalic papillae, and small crescent-shaped amphidial openings when observed by scanning electron microscopy (SEM). Its morphological and morphometric differences with seven known species are discussed. The phylogenetic relationships of the new species with other relevant genera and species have been studied using partial sequences of small and large subunit ribosomal DNA (SSU and LSU rDNA). In both the SSU and LSU phylogenies, the sequences of B. bushehrensis n. sp. and other Boleodorus spp. formed a clade. A second species, B. thylactus, when studied under SEM, has a raised, smooth cephalic region, four large cephalic papillae, and oblique amphidial slits, with the oral opening in a depression.

Non-coding RNAs in the interaction between rice and Meloidogyne graminicola.

BACKGROUND: Root knot nematodes (RKN) are plant parasitic nematodes causing major yield losses of widely consumed food crops such as rice (Oryza sativa). Because non-coding RNAs, including small interfering RNAs (siRNA), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are key regulators of various plant processes, elucidating their regulation during this interaction may lead to new strategies to improve crop protection. In this study, we aimed to identify and characterize rice siRNAs, miRNAs and lncRNAs responsive to early infection with RKN Meloidogyne graminicola (Mg), based on sequencing of small RNA, degradome and total RNA libraries from rice gall tissues compared with uninfected root tissues. RESULTS: We found 425 lncRNAs, 3739 siRNAs and 16 miRNAs to be differentially expressed between both tissues, of which a subset was independently validated with RT-qPCR. Functional prediction of the lncRNAs indicates that a large part of their potential target genes code for serine/threonine protein kinases and transcription factors. Differentially expressed siRNAs have a predominant size of 24 nts, suggesting a role in DNA methylation. Differentially expressed miRNAs are generally downregulated and target transcription factors, which show reduced degradation according to the degradome data. CONCLUSIONS: To our knowledge, this work is the first to focus on small and long non-coding RNAs in the interaction between rice and Mg, and provides an overview of rice non-coding RNAs with the potential to be used as a resource for the development of new crop protection strategies.

Description of Longidorella (Saevadorella) caspica n. sp. (Dorylaimida: Nordiidae) from north Iran.

This contribution provides the morphological and molecular identification of a new species of the genus Longidorella (Saevadorella). L. (S.) caspica n. sp., was recovered from the rhizospheric soil of grasses in Mazandaran province in the seashore of the Caspian Sea. It is characterized by females with a length of 788 to 874 µm and a cephalic region with prominent papillae; and separated from the rest of the body by a remarkable constriction, an odontostyle of 32 to 33 µm, vulva at 52.5 to 59.0%, and a tail of 33 to 38 µm with a rounded tip. Males have 32 to 35 µm long spicules of dorylaimoid form and five to seven ventral supplements ending at 48 to 55 µm distance from cloacal pair. The new species was morphologically compared with seven nominal species viz. L. (S.) arenicola, L. (S.) cuspidata, L. (S.) magna, L. (S.) perplexa, L. (S.) saadi, L. (S.) saevae, and L. (S.) tharensis. It was further compared with similar species under the subgenus Longidorella (Enchodorella) viz. L. (E.) deliblatica and L. (E.) murithi. Besides morphological studies, molecular phylogenetic studies using partial sequences of D2 to D3 expansion fragments of 28S rDNA were performed for the new species and its phylogenetic relationships with other species and genera were discussed.

Genome-wide shifts in histone modifications at early stage of rice infection with Meloidogyne graminicola.

Epigenetic processes play a crucial role in the regulation of plant stress responses, but their role in plant-pathogen interactions remains poorly understood. Although histone-modifying enzymes have been observed to be deregulated in galls induced by root-knot nematodes (RKN, Meloidogyne graminicola) in rice, their influence on plant defence and their genome-wide impact has not been comprehensively investigated. First, the role of histone modifications in plant-nematode interactions was confirmed by pharmacological inhibition of histone-modifying enzymes, which all significantly affected rice susceptibility to RKN. For a more specific view, three histone marks, H3K9ac, H3K9me2, and H3K27me3, were subsequently studied by chromatin-immunoprecipitation-sequencing on RKN-induced galls at 3 days postinoculation. While levels of H3K9ac and H3K27me3 were strongly enriched, H3K9me2 was generally depleted in galls versus control root tips. Differential histone peaks were generally associated with plant defence-related genes. Transcriptome analysis using RNA-Seq and RT-qPCR-based validation revealed that genes marked with H3K9ac or H3K9me2 showed the expected activation or repression gene expression pattern, but this was not the case for H3K27me3 marks. Our results indicate that histone modifications respond dynamically to RKN infection, and that posttranslational modifications mainly at H3K9 specifically target plant defence-related genes.

Genome-wide DNA hypomethylation shapes nematode pattern-triggered immunity in plants.

A role for DNA hypomethylation has recently been suggested in the interaction between bacteria and plants; it is unclear whether this phenomenon reflects a conserved response. Treatment of plants of monocot rice and dicot tomato with nematode-associated molecular patterns from different nematode species or bacterial pathogen-associated molecular pattern flg22 revealed global DNA hypomethylation. A similar hypomethylation response was observed during early gall induction by Meloidogyne graminicola in rice. Evidence for the causal impact of hypomethylation on immunity was revealed by a significantly reduced plant susceptibility upon treatment with DNA methylation inhibitor 5-azacytidine. Whole-genome bisulphite sequencing of young galls revealed massive hypomethylation in the CHH context, while not for CG or CHG nucleotide contexts. Further, CHH hypomethylated regions were predominantly associated with gene promoter regions, which was not correlated with activated gene expression at the same time point but, rather, was correlated with a delayed transcriptional gene activation. Finally, the relevance of CHH hypomethylation in plant defence was confirmed in rice mutants of the RNA-directed DNA methylation pathway and DECREASED DNA METHYLATION 1. We demonstrated that DNA hypomethylation is associated with reduced susceptibility in rice towards root-parasitic nematodes and is likely to be part of the basal pattern-triggered immunity response in plants.

Discopersicus n. gen., a New Member of the Family Tylenchidae Örley, 1880 with Detailed SEM Study on Two Known Species of the Genus Discotylenchus Siddiqi, 1980 (Nematoda; Tylenchidae) from Iran.

Discopersicus iranicus n. gen., n. comb., previously described from Iran as a new species under the genus Discotylenchus, is illustrated using light microscope and scanning electron microscope (SEM) observations and further studied using molecular characters. SEM studies revealed the newly proposed genus has oblique amphidial apertures on the lateral sides of the lip region. SEM images are also provided for two species of Discotylenchus, namely D. discretus and D. brevicaudatus, as the first SEM study of the genus. These results confirmed longitudinal amphidial aperture type on lateral sides of the lip region in genus Discotylenchus, as noted by Siddiqi while erecting the genus with D. discretus as the type species. Molecular phylogenetic analyses using partial small subunit (SSU) and large subunit (LSU) rDNA sequences revealed the affinity of the genus Discopersicus n. gen. with members of the subfamily Boleodorinae, as supported by morphological characters (mainly, the oblique amphidial opening).

Rotylenchus sardashtensis n. sp., a monosexual species from Iran, with molecular identification and detailed morphological observations on an Iranian population of Rotylenchus cypriensis Antoniou, 1980 (Nematoda: Rhabditida: Hoplolaimidae).

Rotylenchus sardashtensis n. sp., a new monosexual species is described and illustrated based on morphological, morphometric and molecular studies. Rotylenchus sardashtensis n. sp. appears close morphologically and molecularly to eight known species of the genus, i.e. R. buxophilus Golden, 1956, R. eximius Siddiqi, 1964, R. breviglans Sher, 1965, R. cypriensis Antoniou, 1980, R. pakistanensis Maqbool & Shahina, 1986, R. vitis Cantalapiedra-Navarrete, Liébanas, Archidona-Yuste, Palomares-Rius & Castillo, 2012, R. paravitis Cantalapiedra-Navarrete, Navas-Cortés, Liébanas, Vovlas, Subbotin, Palomares-Rius & Castillo, 2013 and R. dalikhaniensis Aliramaji, Pourjam, Álvarez-Ortega, Pedram & Atighi, 2015 from which the morphological differences are discussed. The new species is characterised mainly in having a hemispherical and well set off lip region with 3-4 annuli under light microscopy and irregular cob-like appearance under scanning electron microscopy (SEM), a stylet 26-30 µm long, a vulva located at 61-77% of body length with double epiptygma, rounded tail with 2-4 annuli and ventral mucron at its tip. Morphologically, R. sardashtensis n. sp. could be distinguished from all similar species within the genus by its matrix code as follows: A3, B1, C1, D4, E1, F2, G2, H5, I2, J2, K1. Rotylenchus cypriensis collected from the rhizosphere of Prunus sp. in Gilangharb region is characterised with its morphological and molecular data. Phylogenetic analyses based on sequences of the D2-D3 expansion region of the 28S rRNA gene and the ITS1 fragment revealed the relationships of both species examined in present study and other species of the genus Rotylenchus Filipjev, 1936 and the family Hoplolaimidae Filipjev, 1934 included in analyses.

Molecular and morphological characterization of Veleshkinema iranicum n. gen., n. sp. (Nematoda: Hexatylina, Sphaerularioidea) from Iran.

Veleshkinema  iranicum n. gen., n. sp. is described and illustrated based on morphological, morphometric and molecular characters. The new genus is characterized by having slender females and males, stylet with asymmetrical knobs, dorsal gland orifice just posterior to subventral knob, lip region with flattened apex and eight sectors, pharynx with a non-muscular and non-valvular median bulb, pharyngeal glands slightly overlapping intestine dorsally, visible cellular cardia, female with a single gonad having a quadricolumellate crustaformeria with 8-10 cells in each column, no postvulval uterine sac and rounded and offset spermatheca containing spheroid sperm cells, males with arcuate tylenchoid spicules and subterminal bursa. The new genus is morphologically compared with four genera: Abursanema, Deladenus, Prothallonema and Sphaerularia. Molecular phylogenetic studies of the new genus using 808 bp partial sequences of SSU ribosomal RNA gene placed the new genus in a clade with Sphaerularia spp. In phylogenetic analyses using 756 bp partial sequences of the 28S ribosomal RNA gene (D2-D3 segments), the new genus formed a monophyletic group with Abursanema iranicum and Sphaerularia spp.

Further Studies on Soil Nematode Fauna in North Western Iran with the Description of One New Species.

Heterodorus youbertghostai n. sp. is described and illustrated based on morphological, morphometric, and molecular data. The new species was found in two geographically distant points in northwestern Iran and is characterized by having angular lip region, separated from the rest body by a constriction, body length of 1,432.5 to 1,751.3 µm, odontostyle length of 24 to 28 µm, rod-like odontophore, 37.0 to 42.5 µm long, lacking flanges at base, double guiding ring at 14 to 16 µm distance from anterior end, pharyngeal bulb comprising 40% to 48% of pharynx, intestine usually containing green material, female reproductive system amphidelphic with less divided short uterus, specific structure of pars distalis vaginae, bluntly conical tail, dorsally convex and ventrally flat, with rounded tip and saccate bodies in ventral side. The new species comes more close to H. conicaudatus and H. irregularis by its morphology and morphometric characters. Compared to former, it has remarkable difference in vulva position and tail characters, and compared to the latter, it could be separated by shorter body, posteriorly located vulva, wider lip region, and longer tail. In phylogenetic analyses using partial sequences of 28S rDNA D2-D3, the new species formed a fully supported clade with several isolates of H. brevidentatus, prevalent in Iran. The other nordiid taxon, Enchodorus dolichurus, already reported from Iran, was also sequenced for the same genomic region and included in phylogenetic analyses.

Rotylenchus castilloi n. sp. (Nematoda: Hoplolaimidae), a new species with long stylet from northern Iran.

Rotylenchus castilloi n. sp., a new bisexual species is described and illustrated based on morphological, morphometric and molecular data. The new species is characterised by having a hemispherical, continuous lip region with an irregular corncob-like appearance under SEM, very long stylet (62-68 µm), vulva located at 49.7-62.2% of body length from anterior end, with a protruding double epiptygma, a rounded to convex-conoid (rarely bi-lobed) tail with 8-12 annuli and specific sequences of D2-D3 segments of 28S and ITS1-rRNA genes. Differences between the new species and four other species of the genus (R. mesorobustus, R. cazorlaensis, R. magnus and R. jaeni) are discussed. Morphologically, the new species can be separated from these species mostly by its body length, lip region characters, stylet length and location of phasmid. Phylogenetic analyses using 721 bp partial sequences of D2-D3 expansion segments of the 28S and 590 bp ITS1-rRNA genes revealed the new species forming a clade with two isolates of R. eximius and two isolates of R. unisexus, two morphologically unrelated species.

Description of Ektaphelenchoides fuchsi n. sp. (Nematoda: Ektaphelenchinae) from western Iran.

Ektaphelenchoides fuchsi n. sp., recovered from a soil sample around the rhizosphere of Cucurbita maxima in western Iran, is described and illustrated based on morphological and molecular characters. The new species is characterized by its body length of 529-712 µm, continuous lip region, lateral fields with three incisures, total stylet length of 14-18 µm with rounded basal knobs, excretory pore 70-84 µm and hemizonid 87-96 µm from the anterior end, post-uterine sac short, 6-9 µm long in female and tail conoid with long filiform terminus in male. Based on morphological and molecular characters, the new species is close to E. kelardashtensis, E. attenuata, and E. musae. It differs from the closest species E. kelardashtensis by its longer sylet (14-18 vs 13-16 µm) and stylet with basal knobs vs not, longer post uterine sac (6-9 vs 3-6 µm), more posterior position of excretory pore and hemizonid (70-84 vs 55-66 and 87-96 vs 67-78 µm, respectively), and longer spicules (12-13 vs 8-10 µm). Comparisons with other species of Ektaphelenchoides are also discussed. Molecular analyses were performed based on 631 bp of the partial ribosomal RNA large subunit gene (D2/D3 of LSU) and showed that E. fuchsi n. sp. is unique when compared with other species of the genus for which sequences of that region are available. 

Description of Abursanema iranicum n. gen., n. sp. (Nematoda: Hexatylina, Sphaerularioidea) from Iran and its phylogenetic relationships.

Abursanema iranicum n. gen., n. sp. is described and illustrated based on morphological, morphometric and molecular characters. The new genus is characterized by its smooth outer and annulated inner cuticle, having two incisures in lateral field, lacking stylet knobs, having pyriform terminal bulb with stem-like extension projecting into the intestine and lacking of bursa in male. The new genus belongs to the family Paurodontidae. It most closely resembles the genera Paurodontoides and Paurodontus, but has affinities based on male characters with Gymnotylenchus of the family Neotylenchidae too. From Paurodontoides, it differs by the absence of stylet knobs and having six sectors in the head framework. Compared to Paurodontus, the new genus differs by the absence of stylet knobs, structure of the pharynx and absence of a bursa in male. It differs from Gymnotylenchus mainly by lacking of stylet knobs, presence of a basal pharyngeal bulb with an extension into the intestine and in the structure of the spicules. Molecular phylogenetic studies of the new genus using 706 bp partial sequences of the 28S rDNA D2/D3 segment revealed it forming a clade with two species of Sphaerularia in both Bayesian Inference (BI) and maximum likelihood (ML) analyses with 1.00 Bayesian posterior probability (BPP) and 0.96 bootstrap support values (BS). Using 942 bp partial sequences of 18S ribosomal RNA gene, the new genus formed a clade with a species of Deladenus with 0.86 BPP and 0.62 BS in BI and ML methods, respectively. With both BI and ML methods, this clade forms a larger highly supported clade with two species of Sphaerularia.