16S-23S rDNA Research Articles

Molecular phylogeny of Boninia (Platyhelminthes: Polycladida), with description of a new species from the Pacific coasts of Panama.

Mesopsammic polyclad members in the family Boniniidae have attracted attention in terms of their evolutionary shifts of microhabitat and their unique morphology such as a pair of pointed tentacles extending from the anterolateral margins and prostatoid organs harbouring stylets. Here, we establish a new species of this family as Boninia panamensis sp. nov. from the Pacific coasts of Panama, based on its morphological characteristics of (i) four cerebral and 61-80 marginal eyespots, (ii) two prostatoid organs located anterior and posterior to the penis papilla, and (iii) two uterine canals departing from the anterior part of the Lang's vesicle. We also report Boninia cf. uru from Hawai'i, USA, based on its morphological identity with B. uru from Okinawa, Japan, along with their genetic distances for the partial cytochrome c oxidase subunit I (COI ) sequences, which were beyond the range of intraspecific differences observed in congeners in this study. Boninia oaxaquensis is also reported from Panama as a new locality for the species. Involving the above-mentioned three species sequenced herein, we reconstructed molecular phylogenetic trees of Boninia based on the four gene markers (18S rDNA, 28S rDNA, 16S rDNA and COI ). Our phylogenetic trees indicated the synapomorphy within the genus Boninia of the small numbers of stylets (2-4) and the connection route of the uterine canals to the Lang's vesicle. The results also showed a characteristic distribution pattern in which pairs of species in distinct lineages occurred sympatrically with different microhabitats, as observed in Boninia uru and Boninia yambarensis in Okinawa and B. panamensis sp. nov. and B. oaxaquensis in Panama. In addition, we discuss possible speciation pathways in this genus based on the tree topology. ZooBank: urn:lsid:zoobank.org:pub:D414BACD-C14A-4B34-8AF9-7ACBA28F46D0.

A new species of Thoracophelia (Annelida, Opheliidae) from the Yellow Sea of South Korea.

Thoracophelia Ehlers, 1897 is a genus of Opheliidae characterised by the body divided into three distinct regions, modified parapodia in chaetiger 10 and a ventral groove restricted to the posterior half of the body. To date, 18 species have been described in the genus. Amongst them, six species have been recorded in northeast Asia. A new species, Thoracopheliafoliformis sp. nov., was discovered in the intertidal zone of the Yellow Sea, South Korea. This is the first Thoracophelia species report from the Yellow Sea. This new species is closely related to T.dillonensis (Hartman, 1938) from California and T.ezoensis Okuda, 1936 from Japan in having pectinate branchiae. However, the new species can be distinguished from the two species by the unique combination of the following characteristics: 15 pairs of wrinkled pectinate branchiae with 12-15 filaments at best development and a foliaceous mid-ventral plate in the pygidium instead of one or two thick ventral cirri. Detailed descriptions and illustrations of T.foliformis sp. nov. are provided. Sequences of the mitochondrial cytochrome c oxidase subunit I (COI), nuclear 18S ribosomal DNA (rDNA) and 28S rDNA of the new species were determined and analysed.

Plant, bacteria and fungi crosstalk: Direct and indirect biocontrol mechanisms of sugarcane rhizoplane Pseudomonas species against Fusarium wilt

In order to identify an effective rhizoplane Pseudomonas species to combat sugarcane wilt pathogen (Fusarium sacchari), twenty Pseudomonas isolates were obtained from the sugarcane rhizoplane of different genotypes. The three-tier confrontation (direct, indirect and remote) tests revealed antagonistic activity of the bacteria, bacterial non-volatile and volatile compounds. In dual culture assay, SRP19 (46.30%) and SRP20 (39.30%) have shown the highest inhibition against F. sacchari. 16S-23S rDNA sequencing identified them as Pseudomonas chlororaphis subsp. aurantiaca (SRP19) and Pseudomonas aeruginosa (SRP20). Exploration of biocontrol mechanisms revealed the potential roles for IAA production, extracellular enzyme secretion (proteases, cellulases and chitinases), HCN synthesis, siderophore production, pyocyanin or fluorescein production and phosphate solubilization. The molecular analysis revealed the presence of genes (phz, prnD, phlD and pltC) involved in the biosynthesis of antimicrobial peptides like phenazines, pyrrolnitrin, 2,4-diacetyl phloroglucinol and pyoleuteorin, involved in suppressing Fusarium. The antagonistic activity was further confirmed under greenhouse conditions by sett treatment with potential antagonistic isolates, which increased germination and reduced the pre-emergent mortality by 60% and post-emergent seedling mortality by 76% in SRP19 treated pots. The study revealed that the isolate Pseudomonas chlororaphis subsp. aurantiaca strain SRP19 can act as PGPR with both plant growth promoting and pathogen suppressing activity that can be used as an effective biocontrol agent against Fusarium wilt of sugarcane.

Integrative description of two new species of the genus Mesobiotus (Eutardigrada, Macrobiotoidea) from Russia, with an updated phylogeny of the genus

In this study, we describe two new species of Mesobiotus based on morphological data collected through light and scanning electron microscopy. Descriptions include DNA sequences of four commonly used molecular markers (18S rDNA, 28S rDNA, ITS-2, and COI). Mesobiotus efa sp. nov. was discovered in North-West Russia and belongs to the group of species with smooth cuticle, harmsworthi-type OCA, typical Mesobiotus claws IV with unindented lunules, and egg chorion with reticulated processes in form of ‘sharp wide cones’ or ‘cones with long slender endings’, egg process bases with well-developed crone of dark thickenings without finger-like projections, and egg shell surface between the processes with ridges without reticulation, areolation or semi-areolation. It can be distinguished from all know species of this group by a unique combination of morphological and morphometric characters. Mesobiotus vulpinus sp. nov. was found in the Russian Far East, and is similar to Mesobiotus mauccii by having an egg chorion with polygonal relief. The new species can be distinguished from M. mauccii by having a narrower buccal tube, by details of oral cavity armature, and by longer egg chorion processes. Furthermore, we provide results of the phylogenetic analyses of the genus Mesobiotus conducted in this study.

Cryptic invasions of Pseudopolydora (Annelida: Spionidae), with description of a new species from Queensland, Australia

During surveys worldwide, we collected adult and larval specimens of Pseudopolydora Czerniavsky, 1881 similar to P. achaeta Radashevsky & Hsieh, 2000 and P. rosebelae Radashevsky & Migotto, 2009 far from their type localities in Taiwan and Brazil, respectively. Analysis of sequence data of five gene fragments: mitochondrial COI and 16S rDNA, nuclear 18S rDNA and 28S rDNA, and Histone 3 (3114 bp in total) of individuals from Nha Trang Bay (Vietnam), São Paulo (Brazil), Florida (USA), and the Caribbean Sea (Martinique) confirmed their conspecificity with P. achaeta from Taiwan. Based on the morphology, we also report this species for the first time for the East Sea (South Korea) and provide new records for the north-western part of the Sea of Japan (Russia). The analysis also showed the conspecificity of worms from Vietnam with P. rosebelae from Brazil. Based on the morphology, we also report P. rosebelae for the first time for the Caribbean Sea (Belize), South China Sea (Thailand), and Hawaii. Developed pelagic larvae of P. achaeta and P. rosebelae from Nha Trang Bay are described and illustrated. A worm from the Great Barrier Reef (Queensland, Australia) earlier identified as P. cf. rosebelae, is referred to the new species Pseudopolydora nivea sp. nov. Following our earlier hypothesis about the origin of Pseudopolydora in the Indo-West Pacific Ocean, we explain the occurrence of these worms, including Pseudopolydora floridensis Delgado-Blas, 2008, on the Atlantic coasts of North and South America by unintentional transportation in ballast water, followed by successful invasions. We assume that the penetration of warm waters of the East China Sea through the Korea Strait and the warming of the waters of the Sea of Japan have led to the extension of the range of tropical-subtropical P. achaeta to the north-western part of the Sea of Japan.

Base-substitution rates of nuclear and mitochondrial genes for polyclad flatworms

The increase in the use of molecular methodologies in systematics has driven the necessity for a comprehensive understanding of the limitations of different genetic markers. Not every marker is optimal for all species, which has led to multiple approaches in the study of the taxonomy and phylogeny of polyclad flatworms. The present study evaluates base-substitution rates of nuclear ribosomal (18S rDNA and 28S rDNA), mitochondrial ribosomal (16S rDNA), and protein-codifying (cytb, cox1) markers for this taxonomic group, with the main objective of assessing the robustness of these different markers for phylogenetic studies. Mutation rates and Ti/Tv ratios of the other markers were assessed for the first time. We estimated substitution rates and found cytb to be the most variable, while 18S rDNA was the least variable among them. On the other hand, the transition to transversion (Ti/Tv) ratio of the different genes revealed differences between the markers, with a higher number of transitions in the nuclear gene 28S and a higher number of transversions in the mitochondrial genes. Lastly, we identified that the third codon position of the studied protein-codifying genes was highly variable and that this position was saturated in the cox1 marker but not in cytb. We conclude that it is important to assess the markers employed for different phylogenetic levels for future studies, particularly in the order Polycladida. We encourage the use of mitochondrial genes cytb and 16S for phylogenetic studies at suborder, superfamily, and family levels and species delimitation in polyclads, in addition to the well-known 28S and cox1.

Molecular detection of Class 1, 2, and 3 integrons in hypervirulent and classic Klebsiella pneumoniae isolates: A cross-sectional study.

The "hypervirulent" variant of Klebsiella pneumoniae (hvKp) is an emerging pathogen that cause life-threatening infection. The present study was conducted to identify the prevalence of hvKp and to investigate the presence class 1, 2, and 3 integrons in these isolates. A cross-sectional study was conducted at three teaching hospitals, Ahvaz, South-west of Iran, from January 1, 2019 to December 31, 2020. Samples were collected from inpatients and included only the first samples collected from each patient. K. pneumoniae strains were isolated from different specimens using biochemical test and confirmed by targeting 16S-23S rDNA internal transcribed spacer. HvKp isolates were recovered using string test and were further characterized by detection virulence-associated genes (rmpA, iucA, and magA). Antibiotic susceptibility patterns of isolates were determined using the disc diffusion method. Isolates were screened for presence the integron genes (intI, intII, and intIII) and repetitive element sequence-based polymerase chain reaction (PCR) performed to determine strain relatedness. SPSS version 22 was used for the data analysis. Seventy-one (77%) of isolates showed multidrug-resistant (MDR) phenotype. HvKP accounted for 14% (13/92) of cKp isolated from blood (46%) and urinary tract infection (38%), and the great majority of them (61.5%; 8/13) exhibited MDR phenotype. Using the PCR assay, 29 of 92 isolates (31.5%) were found to have positive results for the presence of IntI. Three of the IntI-positive strains were hvKP. Class 2 integron was present in 8/92 cKp isolates. Integron Class 2 was found to coexist with Class 1 integron in 3/8 isolates. All integron-positive isolates (IntI and/or IntII) were resistant to at least three different classes of antibiotics and showed MDR phenotype. No Class 3 integrons were detected among the isolates. The results of our study revealed that considering the role of integrons in facilitating the acquisition and dissemination of resistance genes among bacteria, monitoring the emergence of hvKp, emphasizing on the mechanism of antimicrobial resistance, can prevent from the spread of carbapenemase-producing hvKp strains.

Integrative taxonomy supports two new species of Macrobiotus (Tardigrada: Eutardigrada: Macrobiotidae) allowing further discussion on the genus phylogeny

In this study, I describe two new species of Macrobiotus based on morphological data collected through light and scanning electron microscopy. Both species are accompanied by DNA sequences from four commonly used molecular markers (18S rDNA, 28S rDNA, ITS-2, and COI). Macrobiotus ovovittatus sp. nov. was discovered in Greenland and can be distinguished from similar taxa of Macrobiotus by its continuous, solid, and clearly wrinkled egg surface, adorned with sparse, very small and irregularly spaced pores. Additionally, the terminal discs of egg processes are covered in multiple light-refracting dots, resembling crocheted napkins. Macrobiotus mileri sp. nov. was found in Israel and is characterized by unique pore arrangements in its body cuticle, expressed in two distinct animal forms: (i) forma porata with large pores arranged in five distinct patches and (ii) forma aporata with single, almost undetectable pores. It also features weakly defined convex terminal discs with smooth edges. Furthermore, the phylogenetic analyses conducted in this study offer the most updated phylogeny of superclade I within the family Macrobiotidae. This facilitates additional discussion concerning the interrelationships among species within the genus Macrobiotus and the circumscription of species groups within it.

Detection of Rickettsia raoultii in Vermipsylla alakurt-Like Fleas of Sheep in Northwestern China

IntroductionTo date, a total of 2574 validated flea species have been discovered. Vermipsyllidae is a family of fleas that comprises at least eight species. Vermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Here a novel Vermipsylla species was described, and rickettsial agent was also detected in it.MethodsA total of 128 fleas were collected directly from 260 pastured sheep in China. Of these, eight representative fleas (four males and four females) were identified by key morphological features. Meanwhile, 120 flea DNAs, including six flea samples for molecular taxonomy, were subjected to Rickettsia spp. DNA detection. The molecular identity of fleas was determined by amplification and sequenmce analysis of four genetic markers (the 28S rDNA genes, the 18S rDNA genes, the mitochondrial cytochrome c oxidase subunit I and subunit II). In addition, five Rickettsia-specific gene fragments were used to identify the species of the rickettsial agents. The amplified products were sequenced and phylogenetically analyzed.ResultsThe morphological characteristics of the flea species identified in this study were similar to Vermipsylla alakurt, but presented difference in hair number of the metepimeron, the third tergum, the genitals and the tibiae of hind leg. The 18S rDNA, 28S rDNA and COII genetic markers from fleas showed the highest identity to those of V. alakurt, shared 98.45% (954/969), 95.81% (892/931) and 85.86% (571/665) similarities, respectively. However, the COI sequence showed the highest identity to that of Dorcadia ioffi with 88.48% (576/651) similarity. Rickettsia raoutii tested positive in 14.17% (17/120) flea DNA samples.ConclusionOur study reports the detection of R. raoultii in V. alakurt-like fleas infesting sheep in China.

Molecular phylogeny and ethology of the Family Plagiostomidae (Platyhelminthes, Prolecithophora), with integrative description of a new species, Plagiostomum robusta A. Wang, sp. nov.

The taxon Prolecithophora, which is closely related to the well-known clade Tricladida, is rarely studied, particularly in terms of molecular phylogeny and behavioral characteristics. In this study, we employed an integrative analysis of molecular phylogeny, histology, and ethology to describe a new marine species, Plagiostomum robusta A.Wang, sp. nov. of the order Prolecithophora. Additionally, we obtained its nearly complete mitogenome sequence with annotations of 12 protein-coding genes, two rRNA genes and 22 tRNA genes. P. robusta is characterized by several features, including a pair of short tentacles, a terminal mouth and a terminal gonopore, a large variable pharynx, two extended front ends of the intestine, paired testes located behind the ovaries and outside the vitellaria, a muscular and movable distal sac, and a long tubular penis surrounded by a sheath. The phylogenetic analysis, based on 18S rDNA and 28S rDNA, revealed that the new species formed a clade with six Plagiostomum flatworms, indicating a close kinship with Plagiostomum. Despite contradictions between the traditional morphological classification system and the results of molecular phylogenetics, we chose to assign the new species to Plagiostomum based on comprehensive considerations. Moreover, our study has provided more insights into the behavioral features of Plagiostomidae species, as we found that P. robusta fed on other flatworms and engaged in cannibalism, and unexpectedly it mated via hypodermic inpregnation. This study represents the first comprehensive description of the reproductive behavior of Plagiostomidae species, contributing to the more in-depth understanding of the biological characteristics of this group.

Identification of Sarcocystis and Trichinella Species in Muscles of Gray Wolf (Canis lupus) from Lithuania.

Apicomplexan Sarcocystis and Trichinella nematodes are food-borne parasites whose life cycle is carried-out in various wildlife and domestic animals. The gray wolf (Canis lupus) is an apex predator acting as an ecosystem engineer. This study aimed to identify the species of Sarcocystis and Trichinella found in the muscles of gray wolves in Lithuania. During the 2017-2022 period, diaphragm, heart, and hind leg samples of 15 animals were examined. Microscopical analysis showed the presence of two types of Sarcocystis parasites in 26.7% of the analyzed muscle samples. Based on the sequencing of five loci, nuclear 18S rDNA, 28S rDNA, ITS1, mitochondrial cox1, and apicoplast rpoB, S. arctica, and S. svanai were identified. The current work presents the first report of S. svanai in gray wolf. Phylogenetically, S. svanai clustered together with S. lutrae, infecting various carnivorans, and S. arctica was most closely related to S. felis from domestic cats. Trichinella spp. were found in 12 gray wolves (80%). For the first time, Trichinella species were molecularly identified in gray wolves from Lithuania. Trichinella britovi was confirmed in all of the isolated Trichinella larvae using a multiplex PCR. Gray wolves in Lithuania may serve as a major source of zoonotic pathogens due to the presence of these parasites.

New species of Dermoergasilus Ho & Do, 1982 (Copepoda: Cyclopoida: Ergasilidae) parasitizing endemic cichlid Paretroplus polyactis (Bleeker) in Madagascar.

Dermoergasilus madagascarensis n. sp. is described from the gills of Paretroplus polyactis, an endemic cichlid fish in Madagascar, using a combined morphological (light microscopy and SEM) and molecular approach (partial 18S rDNA, 28S rDNA, and COI sequences). The new species is characterized mainly by possessing: (i) roughly pentagonal cephalosome; (ii) antennal endopodal segments covered with slightly inflated membrane; (iii) maxillule bearing 2 equally long outer setae and a minute inner seta; (iv) interpodal sternites of swimming legs ornamented with 3–4 rows of spinules; (v) genital segment and first abdominal somite both barrel-shaped; and (vi) a caudal ramus projecting into a digitiform process with inconspicuous terminal seta and bearing 3 terminal setae. The obtained DNA sequences of Malagasy species represent the first molecular data for species of Dermoergasilus. The 28S rDNA phylogeny showed the affiliation of D. madagascarensis n. sp. to Ergasilidae and its sister relationship with cosmopolitan Ergasilus sieboldi von Nordmann, 1832. The first checklist for all species of Dermoergasilus is provided.

Genetic variation of the freshwater snail Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand, inferred from 18S and 28S rDNA sequences.

Indoplanorbis exustus, a freshwater pulmonate snail, is widely distributed in tropical and subtropical zones and plays a significant role as an intermediate host for trematode parasites. Various genetic markers have been used for species identification and phylogenetic studies of this snail. However, there are limited studies about their molecular genetics based on nuclear ribosomal DNA (rDNA) genes. A genetic analysis of I. exustus in Thailand was conducted based on the nuclear 18S rDNA (339 bp) and 28S rDNA (1036 bp) genes. Indoplanorbis snails were collected from 29 localities in 21 provinces covering six regions of Thailand. Nucleotide sequences from 44 snails together with sequences from the GenBank database were examined for phylogenetic relationships and genetic diversity. All sequences of the selected nucleotide regions exhibited a high level of similarity (99%) to the sequences of I. exustus in the GenBank database. The maximum likelihood tree based on the 18S and 28S rDNA fragment sequences of I. exustus in Thailand revealed only one group with clear separation from another genus in the family Planorbidae. The I. exustus 28S rDNA sequences showed intraspecific genetic divergence ranging from 0 to 0.78% and were classified into 8 different haplotypes. Conversely, the 18S rDNA data showed lower variation than the 28S rDNA data and revealed a single haplotype and intraspecific distances of zero among all sampled individuals. The haplotype network of 28S rDNA sequences of I. exustus in Thailand revealed six unique haplotypes and two haplotypes shared by at least two regions. Overall, both markers were successful in the identification of I. exustus. However, these markers, particularly the 18S rDNA, may not be suitable for genetic analysis within the species, particularly for population genetic studies, due to their limited variation as seen in this study. In summary, this study not only enhances understanding of genetic variation in I. exustus but is also useful for the selection of molecular markers in future genetic research.

Two new species of Sabatieria de Rouville, 1903 (Nematoda: Araeolaimida: Comesomatidae) from the Southern Ocean.

Two new free-living marine nematode species of the genus Sabatieria de Rouville, 1903 are described from the Southern Ocean using morphological and molecular data. Sabatieria brevicaudata sp. nov. is characterized by body length 14761740 m, short cephalic setae, 1324% corresponding body diameter long, lateral differentiation of body cuticle with only slightly coarser punctations, spiral amphidial fovea with 3 turns, spicules arcuated and 1.31.6 cloacal body diameter long, slightly arcuated gubernacular apophyses, and twelve precloacal supplements, in the form of small pores. Sabatieria multipora sp. nov. is characterized by body length 21392324 m, short cephalic setae (47 m long, 3040% corresponding body diameter long), cuticle with lateral differentiation of distinctly coarser punctations extending from anterior edge of amphidial fovea to anterior of intestine and in the tail region, spiral amphidial fovea with 3 turns, males with spicules 1.92.0 cloacal body diameter long, seventeen fine tubular precloacal supplements with distance between adjacent supplements increasing towards anterior, and with curved gubernacular apophyses. We obtained 18S rDNA and 28S rDNA sequences from the two new species, which are both closely related to other species of Sabatieria. Our analyses also show that several genera of Comesomatidae Filipjev, 1918 do not form monophyletic clades.

On the Origin of Cultivated Roses: DNA Authentication of the Bourbon Rose Founding Pedigree

Rose flowers have been cultivated for their fragrance and their garden value since ancient times. Very ancient cultivars became famous locally for their specific use, and competitive horticultural activities progressively established, leading, with time, to landraces with limited polymorphism. The most famous examples are the oil-bearing Damask roses from Iran and the Yueyue Fen garden strain from China. In 1817, a new rose, allegedly a hybrid from the two previous lineages, was discovered at Reunion. From this plant, as early as the 1820s, a new founder group, the Bourbon roses, was developed in France, which immediately stirred up deep passions among botanists and skilled enthusiasts. Today, more than 30,000 named cultivars have been raised either as garden and landscape plants for the cut rose market or as indoor pot plants. The market handles billions of euros a year, making the rose the most economically important crop worldwide. Following the inheritance of SSR DNA markers, we here propose a reconstitution of the very early lineage of Bourbon roses, clarifying one of the major steps, if not the major one, that links these very ancient heritage roses to modern roses.

DNA Barcoding of Pomegranate (Punica granatum L.) Cultivars in Duhok Province- Kurdistan Region/ Iraq Using 18S–28S rRNA and ITS Region

Pomegranate is a tree species with a significant plant diversity, therefore molecular methods are necessary to define and verify approaches to recognize rapidly and correctly. This research looks at a genetic strategy for identifying pomegranate varieties in Duhok KRG. The approach is based on application of ITS region, PCR-RFLP, sequencing and SNP identification. For this study, 14 pomegranate accessions were taken from various regions, namely the Center of Duhok, Amedi, Akre, Zaxo, the South of Duhok, and Sulav. The PCR product of the 18S–28S rDNA intergenic spacer was 854bp, and the sequence analysis revealed a 99.94 percent similarity with other accession numbers in NCBI, demonstrating the use of the 18S–28S rDNA intergenic spacer for identifying and barcoding pomegranate cultivars. The PCR product of the ITS region was 700bp. This result was then employed for PCR-RFLP using two restriction enzymes namely RsaI GT/AC and HaeIII GG/CC which helped grouping as well as genetic similarities. This study Further involved sequencing examined genes were compared using the NCBI BLASTn tool and clustalo (Version 1.2.4) to determine gene location and SNP. According to this study, the result of PCR-RFLP revealed poor association between pomegranate physical morphology and genetic features, while SNP identification was identified between studied cultivars. Moreover, this result showed possible DNA barcoding of pomegranate cultivars under the study.

Establishment of CRISPR-Cas-based antiparasitic agents for the swimming crab parasite Mesanophrys sp.

Mesanophrys sp. represents a parasitic ciliate, significantly jeopardizing the survival of farmed swimming crabs (Portunus trituberculatus). However, certain drawbacks exist in traditional broad-spectrum antiparasitic drugs, necessitating the development of innovative antiparasitic methods. Here we report the development of a series of CRISPR-Cas-based antiparasitic engineering bacteria, termed AEB-Cas(s), capable of sequence-specific killing of Mesanophrys sp.. The AEB-Cas(s) are generated by introducing well-designed prokaryotic plasmid vectors into chassis bacteria to express CRISPR effector protein and guide RNA targeting the 18S rDNA, 28S rDNA, and DHC7 genes of ciliates. By virtue of the bacteria-feeding behavior of ciliates, a feeding-delivery (FD) strategy is established for the targeted delivery of exogenous proteins and RNAs into Mesanophrys sp.. Based on the FD strategy, AEB-Cas9 and AEB-Cas13 antiparasitic agents are feeding to the ciliates, and their antiparasitic effect on Mesanophrys sp. are evaluated, respectively. Our results demonstrate that AEB-Cas(s) exhibit significant differences in the inhibitory effect when targeting different ciliates' genes. Specifically, AEB-Cas9 and AEB-Cas13 exhibit 39.0% and 52.2% inhibition rates targeting the 18S rDNA gene, respectively. In comparison, the inhibition rates are 55.2% and 73.6% targeting the 28S rDNA gene, respectively. Notably, both exhibit the highest lethality targeting the DHC7 gene, with inhibition rates of 56.5% in AEB-Cas9 and 89.4% in AEB-Cas13, respectively. Our findings highlight the potential of AEB-Cas(s) as effective antiparasitic agents against Mesanophrys sp., and provide a promising approach for preventing and treating other aquatic ciliate diseases.

IDENTIFICATION OF LENS CULTIVARS IN MARKET BY MOLECULAR TOOLS: DNA BARCODING AND SSRs

Substitution of plant cultivars of high commercial value with a cheaper, lower quality one is a common fraud committed against consumers and producers. Since it is one of the most widely grown legumes, lentil (Lens culinaris Medik.) is suitable for such frauds. This study aimed to identify lentil cultivars which are registered and authorized in the market in Türkiye by using current molecular methods. For this purpose, 26 lentil cultivars were analyzed for 15 SSR markers and two DNA barcode regions (trnH-psbA and matK). A high allele diversity was observed by 12 scorable SSR markers, and the average number of alleles was determined to be 16. One of the important findings was the presence of “cultivar-specific alleles” that can be used to identify each cultivar in the lentil market in Türkiye. At least one “cultivar-specific allele” was obtained for each cultivar. The lentil cultivars were also analyzed by two DNA barcode regions as trnH-psbA and matK. While it was observed that the rate of the intra-species variation for the trnH-psbA region was low and 26 varieties were divided into 7 groups, higher rate was found for matK and samples were distributed into 14 groups. Nevertheless, it was observed that intra-species discrimination can be made more effective when both loci are used together and 26 species were distributed into 18 different groups. We expect that the results of this study, especially the cultivar-specific SSR alleles and DNA barcoding sequence data may be used routinely to identify production and packaged products that are commercially available in markets.

First report of 'Candidatus Phytoplasma asteris' associated with yellowing, scorching and decline of almond trees in India.

The almond, a commercially important tree nut crop worldwide, is native to the Mediterranean region. Stone fruit trees are affected by at least 14 'Candidatus Phytoplasma' species globally, among which 'Candidatus Phytoplasma asteris' is one of the most widespread phytoplasma infecting Prunus dulcis, causing aster yellows disease. Recently, almond plantations of Nauni region were consistently affected by phytoplasma, as evidenced by visible symptoms, fluorescent microscopic studies and molecular characterization. During several surveys from May to September 2020-2022, almond aster yellows phytoplasma disease showing symptoms such as chlorosis, inward rolling, reddening, scorching and decline with an incidence as high as 40%. Leaf samples were collected from symptomatic almond trees and the presence of phytoplasma was confirmed through fluorescent microscopic studies by employing DAPI (4, 6-diamino-2-phenylindole) that showed distinctive light blue flourescent phytoplasma bodies in phloem sieve tube elements. The presence of phytoplasma in symptomatic almond trees was further confirmed using nested PCR with specific primer pairs followed by amplification of 16S rDNA and 16S-23S rDNA intergenic spacer (IS) fragments. Sequencing and BLAST analysis of expected amplicon of the 16S rDNA gene confirmed that the almond phytoplasma in Himachal Pradesh was identical to the aster yellows group phytoplasma. Phylogenetic analysis of 16S rDNA almond phytoplasma also grouped 'Prunus dulcis' aster yellows phytoplasma within 16SrI-B subgroup showed 94% nucleotide identity with 'Prunus dulcis' phytoplasma PAEs3 and 'Prunus dulcis' phytoplasma PAE28 from Iran. This research presents the first host report of 'Candidatus Phytoplasma asteris' infecting almonds in India, expanding the knowledge of the diversity and distribution of phytoplasma strains affecting almond trees globally.

Comparative Cytogenetic of the 36-Chromosomes Genera of Orchidinae Subtribe (Orchidaceae) in the Mediterranean Region: A Summary and New Data.

This article provides a summary of the current knowledge on the cytogenetics of four genera, which are all composed of 36 chromosomes, within the Orchidinae subtribe (Orchidaceae). Previous classical studies have revealed differences in karyomorphology among these genera, indicating genomic diversity. The current study includes an analysis of the current knowledge with an update of the karyotype of 47 species with 36 chromosomes from the genera Anacamptis, Serapias, Himantoglossum, and Ophrys. The study discusses comparisons of karyotypes among these genera that used traditional techniques as well as karyotype asymmetry relationships with various asymmetry indices. Additionally, the study reports new findings on polyploidy in Anacamptis pyramidalis and Serapias lingua, which were observed through karyotype and meiotic metaphase analyses in EMC. Moreover, the study detected B chromosomes for the first time in A. papilionacea and A. palustris. The article also describes the use of fluorescent in situ hybridization in some specimens of A. papilionacea and A. collina to locate different sites of the 18S-5.8S-25S rDNA and 5S rDNA ribosomal complexes on chromosomes. The information derived from these cytogenetic analyses was used to refine the classification of these orchids and identify evolutionary relationships among different species and genera.