Cytochrome Oxidase Subunit II Research Articles

First Report of Phytopythium litorale Causing Root Rot on Kousa Dogwood in Tennessee and the United States.

Kousa dogwood (Cornus kousa) is an economically important woody ornamental crop that exhibits creamy, white, pointed bracts in late spring, and reddish to pink drupe fruits in late summer and fall. It bears shiny dark green leaves that become reddish-purple to scarlet in the fall. In August of 2023, 3-year-old container grown C. kousa var. chinensis plants in a commercial nursery in Warren Co., Tennessee, exhibited severe yellowing, dieback and root rot symptoms (Fig. 1a and 1b). Dark brown to black lesions were observed in the root and crown region of the plants. Disease severity was 40% to 60% of root area affected, and disease incidence was approximately 40% of 1,000 plants. Surface-sterilized (10% NaOCl: 1 min) symptomatic root tissues were plated on V8-PARPH and incubated at 25°C. Sparse aerial mycelium, showing a distinct rosette or faint radiate to chrysanthemum colony pattern, was observed within four days of incubation (Fig. 2). All isolates produced ovoid or subglose, papillate, and proliferating sporangia in grass blade water cultures (Derviş et al. 2020). Sporangia measured as 19.18 to 24.80 µm X 18.08 to 22.16 µm (n = 50) with a length/width ratio of 1.06 to 1.11. Zoospores observed were between 7.07 to 9.98 µm in diameter (n = 50). Oogonia and oospores were not produced. The ribosomal internal transcribed spacer (ITS) and large subunit (LSU), as well as mitochondrial cytochrome oxidase subunit II (COX-II) genetic markers were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), and cox2-F/cox2-RC4 (Choi et al. 2015), respectively. The ITS, LSU, and COX-II sequences of isolates FBG6343, FBG6344 (ITS: PP458373 and PP461387; LSU: PP461390 and PP461391; COXII: PP477112 and PP477113) were 100% identical to those of MN306118, HQ643386, and MN206732, respectively. Based on the morphology (Nechwatal and Mendgen 2006) and sequence data, the isolates were identified as Phytopythium litorale (Nechw.) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque. The pathogenicity test was performed on 3-year-old C. kousa var. chinensis plants grown in a 3-gal container to fulfill Koch's postulates. Kousa dogwood plants were drench inoculated (800 ml/plant) with a pathogen slurry (two plates of 7-day-old culture/liter) of isolates FBG6343 and FBG6364 (five plants per isolate). Control plants were drenched with agar slurry without the pathogen. The study was conducted in a greenhouse maintained at 21 to 23°C and 70% relative humidity with a 16-h photoperiod and irrigated twice a day for 2 min using an overhead irrigation system. Forty-five days after inoculation, plants showed dieback symptoms, and dark brown lesions developed in the roots of all inoculated plants. Isolates with morphology and sequences identical to those of FBG6343 and FBG6364 were recovered from root tissues of all inoculated plants. All control plants remained symptom-free, and P. litorale was not isolated from the root tissue. Previously, P. litorale was reported to cause disease on apple, kiwi, planatus, and rhododendron (Derviş et al. 2020; Li et al. 2021; Mert et al. 2020; Polat et al. 2023). To our knowledge, this is the first report of P. litorale causing root rot of kousa dogwood in Tennessee and the United States. Identification of this pathogen as the causal agent is crucial to developing timely management practices.

First Report of Root-knot Nematode, Meloidogyne enterolobii on Passiflora edulis in Yulin, China.

Passion fruit (Passiflora edulis), a medicinal plant, was introduced into China in the early 19th century, is mainly cultivated in southern provinces (Liang et al. 2019). During March 2023, a survey was carried out and 167 samples were taken from passion fruit cultivated area in Yulin (22.6570263°E; 110.1765019°N) apart from the planting base appeared yellow leaves, stunted growth, and distinctive galls on the roots. Within the galls, Meloidogyne sp. females and egg masses were observed. From the rhizosphere soil, second-stage juveniles (J2) were extracted, and population density was 105/500 g soil. The species was determined to be Meloidogyne enterolobii based on morphological characteristics, including female perineal pattern, and genetic analyses. Female (n = 10) perineal patterns showed oval shape, with coarse and smooth striae, dorsal arch rounded to square, and lateral lines not distinct. The male head cap was high and rounded, with the head region only slightly set off from the body, knobs large, ovoid to rounded. The measurements of males (n = 10) included body length, 1,230.7 ± 244.94 (997 to 1,569) µm; a, 38.58 ± 7.8 (33.45 to 47.05) µm; c, 113.03 ± 26.22 (80.82 to 144.23) µm; stylet, 15.68 ± 1.1 (14.5 to 17.4) µm; spicules, 31.83 ± 2.84 (28.69 to 36.1) µm; tail, 11.09 ± 1.72 (8.02 to 13.38) µm; and gubernaculum length, 8.34 ± 0.28 (8.11 to 8.98) µm. Measurements of J2 (n = 20) included body length, 455.75 ± 44.94 (381 to 512) µm; a, 26.32 ± 3.89 (18.18 to 32.70) µm; c, 8.56 ± 1.2 (6.36 to 10.80) µm; stylet, 12.44 ± 0.76 (11.2 to 13.8) µm; DGO, 3.65 ± 0.54 (2.84 to 4.68) µm; tail, 53.89 ± 6.36 (39.8 to 62.2) µm; and hyaline tail terminus, 11.77 ± 2.83 (7.14 to 16.2) µm. These morphological characteristics are similar to those reported in the original description of M. enterolobii (Yang and Eisenback 1983). The sequences of the partial ITS region was amplified with V5367 (5'-TTGATTACGTCCCTGCCCTTT-3') and 26S (5'-TTTCACTCGCCGTTACTAAGG-3') primers (Vrain et al. 1992). The region between cytochrome oxidase subunit II (COII) and the 16S rRNA mitochondrial DNA (mtDNA COII) was also amplified with the primers C2F3 (5'-GGTCAATGTTCAGAAATTTGTGG-3') (Powers and Harris 1993) and MRH106 (5'-AATTTCTAAAGACTTTTCTTAGT-3') (Stanton et al. 1997). The ITS region yielded a fragment of 757 bp (OR072957) and mtDNA COII of 706 bp (OR078415). A BLAST search indicated the sequences were 100% identical to several sequences of M. enterolobii (MT406250, MH756127 and AY831967, MN269940, respectively). To confirm pathogenicity, 20 passion fruit (P. edulis Sim. f. flavicarpa) 30-day-old seedlings were transplanted into pots with an autoclaved mixture of sand and field soil (3:1) and maintained in the glasshouse at 25 ± 2°C with 65 ± 5% relative humidity. After eight weeks, fifteen plants were inoculated with 500 J2/pot (nematode culture collected from the original field), and another five uninoculated plants served as a control. Two months later, aboveground symptoms were similar to those observed in the field. Nematode reproduction occurred and root galls were observed. The reproduction factor (nematode final population density/initial population density) was 4.8. The disease caused by M. enterolobii was severe in Yulin city of Guangxi. Guangxi is an important area for passion fruit culture, with about 2000 ha, which is responsible for two-thirds of China production (Xing et al. 2020). This is the first record of P. edulis natural infection with M. enterolobii in the Yulin City of Guangxi, China.

Disclosure of genetic diversity of mackerel fish (Scomberomorus spp.) in Indonesian waters based on the mitochondrial cytochrome oxidase subunit II (COII) gene.

Mackerel fish (Scomberomorus spp.) represents a significant marine fisheries commodity in Indonesia, characterized by its high commercial value and nutritional content. To understand the intraspecific interactions and genetic variability of Scomberomorus spp., a more extensive research of Scomberomorus spp. populations, including both cultivated and wild specimens, is required. This study aimed to explore the genetic diversity of mackerel fish in Indonesian waters, focusing on the mitochondrial DNA (mtDNA) cytochrome oxidase subunit II (COII) gene, which encodes the second subunit of cytochrome c oxidase (complex IV), is essential for aerobic respiration and energy transformation. Muscle tissue samples from 18 individual mackerel fish collected from various regions in Indonesia, including Palembang, Cilacap, Rembang, Banjarmasin, Ambon, and Fak-Fak Regencies, were utilized. The genomic DNA was isolated and amplified using specific primers: CO2TF (5'-ACCGCTCTGTCACTTTCTTC-3') and CO2TR (5'-ATGTCACTAAGGGTGGTTGG-3'). Subsequently, the obtained amplicons were subjected to sequencing. The sequence data were then analyzed using the MEGA11 and DnaSP 6 software. Our findings revealed 120 variable sites within the 691 base pairs of mtDNA COII sequences, resulting in a nucleotide diversity (Pi) of 0.07169. Furthermore, we identified eight haplotypes, demonstrating a haplotype diversity (Hd) of 0.8889. Remarkably, all mackerel samples from Palembang and Cilacap clustered into discrete haplotypes, specifically haplotype 1 and haplotype 2, respectively. Our phylogenetic analysis delineated three distinct clades. Clade I, closely related to Scomberomorus cavalla, encompassed all individuals from Ambon, Palembang, Rembang, and one from Banjarmasin. Clade II, associated with Scomberomorus niphonius, included individuals from Cilacap and two from Banjarmasin. Clade III, linked to Scomberomorus semifasciatus, exclusively consisted of individuals from Fak-Fak (Papua). In conclusion, Indonesian waters harbor diverse genetic variations within Scomberomorus spp., and population relationships based on the mtDNA COII gene exhibit notable complexities. Future research endeavors should focus on further elucidating the diversity and relationships among Scomberomorus spp. in diverse Indonesian populations.

Phylogenetic analyses of Reticulitermes (Blattodea: Rhinotermitidae) from California and other western states: multiple genes confirm undescribed species identified by cuticular hydrocarbons.

Subterranean termites in the genus Reticulitermes Holmgren 1913 are among the most economically important wood-destroying pests in the western United States. Yet, there remains uncertainty regarding the taxonomy and biology of the species in this genus. The 2 species described as having distributions in this region are the western subterranean termite, Reticulitermes hesperus Banks, and the arid land subterranean termite, Reticulitermes tibialis Banks. Taxonomic studies utilizing cuticular hydrocarbon (CHC) profiles, agonistic behavior, flight phenology, and mitochondrial DNA (mtDNA) suggested that R. hesperus is a species complex comprised of 2 or more sympatric, yet reproductively isolated species. To further delineate these taxa, we examined multiple genes from samples of Reticulitermes collected in the western United States. Alates collected after recent spring and fall mating flights, as well as previously collected workers, were subjected to CHC phenotyping and DNA sequence analyses that targeted mitochondrial cytochrome oxidase subunit II (COII), mitochondrial 16S rRNA, and nuclear Internal Transcribed Spacer 1 and 2 (ITS1 and 2). Phylogenetic analyses conducted also included published sequences of other putative western Reticulitermes species. Results suggest that at least 5 species of Reticulitermes may be present in California and that Reticulitermes in Arizona consistently group into multiple clades, including samples previously identified as R. tibialis in a sister clade. These analyses further support the species status of qualitatively different CHC phenotypes and that alates swarming in spring vs. fall are reproductively isolated species.

The New Report of Root Rot on Fatsia japonica Caused by Phytophthora nicotianae in China

As an ornamental plant, Fatsia japonica has been widely used in gardens. From April 2021 to 2022, a disease that caused the wilting and root rot of F. japonica in a large area was observed, which eventually led to the plants wilting and dying, while the leaves did not fall off. This disease greatly reduced the landscape effect of plants. An oomycete species was isolated from the roots of the infected plants. This colony morphology was slightly radial to stellate, and the aerial mycelium was flocculent. Oval sporangia with papillae, apical chlamydospores and zoospores formed in sporangia were observed. The morphological characteristics were consistent with Phytophthora. For accurate identification, the internal transcribed spacer (ITS), cytochrome oxidase subunit II (COXII) and large ribosomal subunit (LSU) genes were amplified and sequenced. The species was identified as Phytophthora nicotianae using phylogenetic analysis. Finally, the disease was reproduced by inoculating healthy F. japonica with a zoospore suspension; the symptoms were consistent with those of natural infections, and the isolate obtained from artificially infected plants had the same morphological characteristics as the inoculated isolate. The results demonstrated that P. nicotianae is the pathogenic factor of root rot. of F. japonica. This is the first report of root rot on F. japonica caused by P. nicotianae in China.

First Report of Meloidogyne enterolobii Yang & Eisenback, 1983 (Guava Root-knot Nematode) Infecting Guava (Psidium guajava) in Egypt.

Meloidogyne enterolobii Yang & Eisenback, 1983 (guava root-knot nematode) is an important disease in subtropical to tropical climate in several areas of the world (Subbotin et al., 2021). It is a highly polyphagous root-knot nematode species causing major damage to a range of economically important crops. The expansion of this species is increasing worldwide creating a potential problem to the maintenance of resistance genes to other major Meloidogyne species (Castagnone-Sereno and Castillo, 2020). Additionally, the diagnosis of M. enterolobii can be challenging due to morphological similarities with other root-knot nematode species (Castagnone-Sereno, 2012). In the African continent, it has been cited in several countries of Equatorial and South Africa (Subbotin et al., 2021), but not in North Africa. Two guava groves (at Bany Salama, Natrn vally, El Beheira governorate, 30.322043N, 30.518529E; and Izbat Al Halawijah, Monshaah Alaweyah, Abu El Matamir, El Beheira governorate 30.9398050N, 30.1484430E), in Egypt, were found with significant symptoms of tree decline and root galling damage. The presence of egg masses and females of root-knot nematodes were found inside the galls (Figure 1A, B). Nematodes were extracted from soil samples with levels of 12300 and 12600 second-stage juveniles (J2s)/250 g of soil using a modified Baerman method (Hooper, 1986), respectively. Nematode root density was 24367 eggs/g of root, using the protocol described in Hussey and Barker (1973) for Izbat Al Halawijah population. For morphological and morphometrical identification, J2s and females were fixed using a hot formalin solution (4% v/v). DNA was isolated from single J2s specimen for: i) testing multiplex specific-PCR assay for M. incognita, M. javanica and M. arenaria (Kiewnick et al., 2013), and ii) amplifying and sequencing of cytochrome oxidase subunit II (COII) and the 16S rRNA mitochondrial region using the primer pair C2F3 (5'-GGTCAATGTTCAGAAATTTGTGG-3') (Powers and Harris, 1993) and MRH106 (5'- AATTTCTAAAGACTTTTCTTAGT-3') (Stanton et al., 1997). Perineal patterns of females for Izbat Al Halawijah population were typical of the species (Fig. 1D), body size (L: 520-774 µm; W: 214-487 µm), stylet length (12.5-13.7 µm) and ratio from distance from anterior end to excretory pore and stylet length (4.2) in females (n = 18), fitting with original description and others (Subbotin et al., 2021). J2s from Izbat Al Halawijah population (n=13) (Fig. 1C, E-H) showed: body length (393.5-475 µm), stylet length (11.5-13.5), excretory pore to anterior end (89-95.5 µm), tail length (50.0-60.0 µm), tail hyaline region (12.0-21.0 µm), a ratio (24.2-32.5), b ratio (4.9-6.5), c ratio (7.3-8.6) and c' (5.0-6.4), also fitting with original description and others (Subbotin et al., 2021). Specific PCR did not amplify any band (Kiewnick et al., 2013). Four J2s individuals were sequenced for COII-16S rRNA region for each population showing M. enterolobii as unique species and without intraspecific variability. Two identical DNA fragments of 814 bp obtained for both populations (OP434400 and OP434401) were compared with those in GenBank. A BLAST search indicated the sequences were 100% identical to several sequences of M. enterolobii (MF467278 and KX823371). On the basis of these results, the root-knot nematodes isolated from these two guava groves in Egypt were confirmed as M. enterolobii. This is a well-known pathogen of guava, causing important losses in this crop (Castagnone-Sereno and Castillo, 2020) and it is regulated as quarantine nematode in the Mediterranean region (EPPO).

Intraspecific Genetic Variation of Anisakis typica in Indian Mackerel Caught from the Gulf of Thailand, Samut Songkhram Province.

Anisakis nematodes infecting Indian mackerel (Rastrelliger kanagurta) were initially discovered in Thailand in our preliminary investigation. Nevertheless, the species of Anisakis collected has not been determined nor has its genetic variation been researched. Thus, this study aimed to molecularly identify the species of Anisakis specimens using the internal transcribed spacer (ITS) region of ribosomal DNA sequences. In addition, the intraspecific genetic variation was also determined using mitochondrial cytochrome oxidase subunit II (COII) gene sequences. The phylogenetic relationships of the ITS region classified all samples into Anisakis typica; however, the genetic variation between them could not be distinguished. By contrast, the phylogenetic tree analysis of the COII region identified all samples as A. typica, with 17 different haplotypes by 66 polymorphic sites and five of the substitutions resulted in amino acid change. Additionally, the distribution pattern of the COII region can be separated into two groups between South America and Asian countries. All our haplotypes belong to Asian countries. Compared with the two genetic markers used in this investigation, COII appears to be a better candidate for studying genetic variation sensitive to environmental changes and intermediate or definitive host behavioral changes.

Cytochrome Oxidase Subunit II: Potential Marker for the Identification of Forensically Significant Species of Coleoptera—A Preliminary Study

The foremost concern in forensic entomology is the explicit identification of the species recovered from the crime scene. From the different orders of insects, Diptera is the prime focus in this field, followed by Coleoptera, whose identification can be extremely helpful for corpses in later decomposition stages. In this study, cytochrome oxidase subunit II (COII) was used to check its adequacy as a genetic marker and to create a reference database for eleven species belonging to five families of Coleoptera, namely, Silphidae, Staphylinidae, Histeridae, Dermestidae and Scarabaeidae, from two different states in India to assist in the accurate identification of imperative beetle species in medico-legal entomology. To achieve this, standard protocols of DNA isolation, amplification and sequencing were followed. We concluded that the COII gene can be used as a molecular marker for the identification of forensically relevant species, as observed from the similarities between the phylogenetic relationship constructed by COII and morphological data.

First report of Pythium schmitthenneri on olive trees and in Morocco

In a grove in north-eastern Morocco in 2020, severe root rot and crown rot symptoms were noticed on olive trees (Olea europaea). Pythium schmitthenneri was identified as the pathogen based on conidia morphology and cytochrome oxidase subunit II (COXII) gene sequence analysis. Koch's postulates were verified by re-isolation of the fungus P. schmitthenneri from artificially inoculated olive seedlings after a pathogenicity test. This is the first time P. schmitthenneri has been identified as the cause of olive tree root and crown rot in Morocco.

Phylogenetic status of some unique species representing Blapstinina Mulsant & Rey (Tenebrionidae: Blaptinae: Opatrini), and implications for continued study of the subtribe

Blapstinus Dejean is the most taxonomically challenging genus within Blapstinina Mulsant & Rey (Tenebrionidae: Opatrini). With over 120 species, it is widely distributed throughout the Americas, with representatives reaching Canada on the northern range edge, and Argentina, Chile, and Uruguay in the south. Traditionally, Blapstinus has been distinguished from other blapstinoid beetles via well-developed metathoracic wings and their lack of synapomorphies present in other genera; however, fused and tapering aedeagal parameres were recently introduced as a potential autapomorphy for the genus. This study used molecular data (nuclear ribosomal 28S, cytochrome oxidase subunit II (COII), arginine kinase (ArgK), carbomyl-phosphate synthetase domain of rudimentary (CAD), and wingless (wg)) to investigate the phylogenetic placement and taxonomic status of three Blapstinus species with distinct male genitalic morphology, i.e. Blapstinus tibialis Champion (USA), B. grandis Champion (Mexico, Nicaragua), and B. punctulatus Solier (Argentina, Bolivia, Brazil, Chile, Uruguay). Analyses highlight the phylogenetic informativeness of the aedeagal morphology within the subtribe, and support an urgent need for taxonomic studies of South American taxa. Blapstinus tibialis and B. grandis were recovered as a specific lineage within Blapstinus that can be easily distinguished from remaining congeners by having tridentate parameres. A lectotype for B. grandis is designated to fix the taxonomic status of this species. Blapstinus punctulatus was recovered outside of its current genus which, along with aedeagal morphology, supports a change of status of the species. As a result, the following synonymy and combinations are introduced: Lodinus Mulsant and Rey stat. restit. (=Austrocaribius Marcuzzi syn. nov.), Lodinus araguae (Marcuzzi) comb. et stat. nov., L. punctulatus comb. nov., L. venezuelensis (Marcuzzi) comb. nov. Lectotypes for Lodinus nigroaeneus Mulsant and Rey, L. araguae, and L. punctulatus are designated to fix the taxonomic status of these species.

Phylogenetic status of some unique species representing Blapstinina Mulsant amp; Rey (Tenebrionidae: Blaptinae: Opatrini), and implications for continued study of the subtribe.

Blapstinus Dejean is the most taxonomically challenging genus within Blapstinina Mulsant Rey (Tenebrionidae: Opatrini). With over 120 species, it is widely distributed throughout the Americas, with representatives reaching Canada on the northern range edge, and Argentina, Chile, and Uruguay in the south. Traditionally, Blapstinus has been distinguished from other blapstinoid beetles via well-developed metathoracic wings and their lack of synapomorphies present in other genera; however, fused and tapering aedeagal parameres were recently introduced as a potential autapomorphy for the genus. This study used molecular data (nuclear ribosomal 28S, cytochrome oxidase subunit II (COII), arginine kinase (ArgK), carbomyl-phosphate synthetase domain of rudimentary (CAD), and wingless (wg)) to investigate the phylogenetic placement and taxonomic status of three Blapstinus species with distinct male genitalic morphology, i.e. Blapstinus tibialis Champion (USA), B. grandis Champion (Mexico, Nicaragua), and B. punctulatus Solier (Argentina, Bolivia, Brazil, Chile, Uruguay). Analyses highlight the phylogenetic informativeness of the aedeagal morphology within the subtribe, and support an urgent need for taxonomic studies of South American taxa. Blapstinus tibialis and B. grandis were recovered as a specific lineage within Blapstinus that can be easily distinguished from remaining congeners by having tridentate parameres. A lectotype for B. grandis is designated to fix the taxonomic status of this species. Blapstinus punctulatus was recovered outside of its current genus which, along with aedeagal morphology, supports a change of status of the species. As a result, the following synonymy and combinations are introduced: Lodinus Mulsant and Rey stat. restit. (=Austrocaribius Marcuzzi syn. nov.), Lodinus araguae (Marcuzzi) comb. et stat. nov., L. punctulatus comb. nov., L. venezuelensis (Marcuzzi) comb. nov. Lectotypes for Lodinus nigroaeneus Mulsant and Rey, L. araguae, and L. punctulatus are designated to fix the taxonomic status of these species.

New species and findings of emPagastia/em Oliver (Diptera: Chironomidae: Diamesinae) from Central Asia, with DNA barcoding of known species of the genus.

Chironomids of the genus Pagastia Oliver (Diamesinae, Diamesini) from the mountains of Central Asia are revised using both morphological characters and molecular data. Illustrated descriptions of the adult male Pagastia (P.) caelestomontana sp. nov. from Kirgizstan and Tajikistan, P. (P.) hanseni sp. nov. from Tajikistan, and record of a finding apparently a new species P. (P.) aff. lanceolata (Tokunaga) from Tajikistan as well as an updated a key to the determination of the adult males of all known species ofPagastia are provided. A phylogenetic framework is reconstructed based on two mitochondrial genes cytochrome oxidase subunit I (COI) sequences of 34 samples belonging to 7 species of the genus Pagastia and cytochrome oxidase subunit II (COII) available for most samples. Phylogenetic trees of some known species of the genus Pagastia were reconstructed using the combined dataset and Bayesian inference (BI) and Maximum Likelihood (ML) methods. The interspecific K2P distances between seven Pagastia species including P. (P.) caelestomontana sp. nov., P. (P.) hanseni sp. nov. and undescribed P. (P.) aff. lanceolata (Tokunaga) are 6.3-13.2 which corresponding to species level.

First Detection of Meloidogyne luci (Nematoda: Meloidogynidae) Parasitizing Potato in the Azores, Portugal.

Potato is the third most important crop in the world after rice and wheat, with a great social and economic importance in Portugal as it is grown throughout the country, including the archipelagos of Madeira and the Azores. The tropical root-knot nematode (RKN) Meloidogyne luci is a polyphagous species with many of its host plants having economic importance and the ability to survive in temperate regions, which pose a risk to agricultural production. In 2019, M. luci was detected from soil samples collected from the council of Santo António in Pico Island (Azores). Bioassays were carried out to obtain females, egg masses, and second-stage juveniles to characterize this isolate morphologically, biochemically, and molecularly. The observed morphological features and morphometrics showed high similarity and consistency with previous descriptions. Concerning the biochemical characterization, the esterase (EST) phenotype displayed a pattern with three bands similar to the one previously described for M. luci and distinct from M. ethiopica. Regarding the molecular analysis, an 1800 bp region of the mitochondrial DNA between cytochrome oxidase subunit II (COII) and 16S rRNA genes was analyzed and the phylogenetic tree revealed that the isolate grouped with M. luci isolates (99.17%). This is the first report of M. luci parasitizing potato in the Azores islands, contributing additional information on the distribution of this plant-parasitic nematode.

Molecular insights into the phylogeny of Blapstinina (Coleoptera: Tenebrionidae: Opatrini)

AbstractThe monophyly of the North and South American endemic subtribe Blapstinina (Tenebrionidae: Opatrini) is tested through phylogenetic analyses using five molecular markers [nuclear ribosomal 28S (28S), cytochrome oxidase subunit II (COII), arginine kinase (ArgK), carbamoyl‐phosphate synthetase domain of rudimentary (CAD), wingless (wg)]. Representatives of several opatrinoid subtribes were taken into consideration, including other geographically overlapping endemic genera, namely Ammodonus, Ephalus and Pseudephalus (all previously considered representatives of Ammobiina). A comparative study of morphology was performed to assess resulting phylogenetic hypotheses. Analyses support the monophyly of Blapstinina; however, they also strongly indicate that Ammodonus should be included within the subtribe. Mecysmus is nested within Blapstinus and therefore, a new synonymy, Blapstinus (= Mecysmus syn.n.), and the following combinations are introduced: Blapstinus advena comb.n., B. angustus comb.r., B. laticollis comb.n., B. parvulus comb.n., B. tenuis comb.n. Morphological analysis showed a close affiliation between Ephalus and Pseudephalus. Based on these results, Pseudephalus is synonymized with Ephalus [Ephalus (= Pseudephalus syn.n.)], and the following combination is introduced: Ephalus brevicornis comb.n. Recovered topologies also strongly support transferring Ephalus stat.n. into Opatrina, making the distribution of Opatrina amphi‐Atlantic.

First Report of the Suginami Root-Knot Nematode, Meloidogyne suginamiensis, Infecting Maple Trees, Acer palmatum, from Japan

HomePlant DiseaseVol. 104, No. 1First Report of the Suginami Root-Knot Nematode, Meloidogyne suginamiensis, Infecting Maple Trees, Acer palmatum, from Japan PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of the Suginami Root-Knot Nematode, Meloidogyne suginamiensis, Infecting Maple Trees, Acer palmatum, from JapanJ. F. Gu, Y. W. Fang, and L. L. LiuJ. F. Gu†Corresponding author: J. F. Gu; E-mail Address: jeffgu00@qq.comhttp://orcid.org/0000-0002-8514-813XNingbo Customs Technical Centre (Ningbo Inspection and Quarantine Science Technology Academy), Ningbo 315100, Zhejiang, ChinaSearch for more papers by this author, Y. W. FangNingbo Customs Technical Centre (Ningbo Inspection and Quarantine Science Technology Academy), Ningbo 315100, Zhejiang, ChinaSearch for more papers by this author, and L. L. LiuNingbo Customs Technical Centre (Ningbo Inspection and Quarantine Science Technology Academy), Ningbo 315100, Zhejiang, ChinaSearch for more papers by this author AffiliationsAuthors and Affiliations J. F. Gu † Y. W. Fang L. L. Liu Ningbo Customs Technical Centre (Ningbo Inspection and Quarantine Science Technology Academy), Ningbo 315100, Zhejiang, China Published Online:6 Nov 2019https://doi.org/10.1094/PDIS-07-19-1386-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Thousands of maple trees (Acer palmatum Thunb.) are imported from Japan to the port of Ningbo, China, each year since 2012. In 2018, during an inspection for plant-parasitic nematodes, around 100 s-stage juveniles (J2) of root-knot nematodes were extracted from soil (200 g) surrounding the roots of one of the 18 trees using a modified Baermann funnel. The roots were washed, and small round galls occurring in groups in close proximity were observed on roots with a dissecting microscope. Female root-knot nematodes excised from galls were white, globular to pear-shaped, with projecting neck, slightly flattened to round posteriorly, and without a posterior protuberance. The perineal pattern of females was slightly square to oval, with wavy and very fine, thin striae. The body length and tail of the J2 (n = 25) were 380 to 436 μm and 20.8 to 27.3 μm, respectively. Tail length/tail diameter (c′) = 2.7 to 3.7. Tail conoid and short, deeply constricted, usually one-third of tail length from a broad terminus, and 3.2 to 5.2 μm hyaline region. These morphological characteristics were similar to those of Meloidogyne suginamiensis Toida and Yaegashi, 1984 (Toida and Yaegashi 1984). M. mali Itoh, Ohshima & Ichinohe, 1969 was also reported on maple from Japan, but it could be separated by its oval outline, smooth striae of the female perineal pattern, and longer J2 tail length (30 to 40 μm) with finely rounded almost pointed terminus. The morphological identification was confirmed by sequence analysis of the region between the cytochrome oxidase subunit II (COII) gene and the 16S ribosomal RNA gene in the mitochondrial DNA (mtDNA) with primers C2F3 (5′-GGTCAATGTTCAGAAATTTGTGG-3′) and 1108 (5′-TACCTTTGACCAATCACGCT-3′) (Powers and Harris 1993). The PCR product of mtDNA was 536 bp long and analyzed by PCR-restriction fragment length polymorphism with the following restriction enzymes: AluI, BclI, DdeI, DraI, EcoRI, HindIII, HinfI, MsoI, MseI, SspI, StyI, and VspI. The result was identical to a previous report for M. suginamiensis (Orui 1998). Soil samples were collected for a pot experiment to confirm the nematode’s ability to reproduce on maple. Three maple trees were planted in infested soil and grown for 1 year. Galls, eggs, females, and juveniles were detected in all trees and identified as M. suginamiensis. To our knowledge this is the first report of M. suginamiensis on maple from Japan. M. suginamiensis was first reported on mulberry (Morus spp.) from Japan in 1984; its hosts include woody plants like mulberries, fig (Ficus carica L.), elm (Ulmus spp.), raspberry (Rubus spp.), and cherry (Prunus spp.), and also some weeds and vegetables. This species has only been reported from Japan. The mtDNA and rDNA sequences were deposited in GenBank (accession nos.: COII, MN115432; 18S, MN101829 to MN101832; ITS, MN101821 to MN101828; 28S, MN101833 to MN101838), which will benefit identification worldwide.The author(s) declare no conflict of interest.

Study on the Genetic Differentiation of Geographic Populations of Calliptamus italicus (Orthoptera: Acrididae) in Sino-Kazakh Border Areas Based on Mitochondrial COI and COII Genes.

Calliptamus italicus L. is an important pest on the desert and semidesert steppes along the Sino-Kazakh border. To elucidate the molecular mechanism of its continuous outbreaks, we studied 11 different geographic populations of C. italicus to determine: 1) the complete sequences of the entire mitochondrial cytochrome oxidase subunit I (COI) and mitochondrial cytochrome oxidase subunit II (COII) genes, and 2) performed genetic diversity, differentiation, gene flow, and molecular variation analyses. Of the 11 populations, the Yining County (YNX) population had the highest haplotype diversity and Pi values. There are significant differences in Tajima’s D and Fu’s Fs (P < 0.05). The fixation index Fst values of the total C. italicus population were 0.03352, and its gene flow Nm values of the total C. italicus population were 15.32. Taken together, there were five main findings: 1) the current genetic differentiation of C. italicus arose within populations; 2) genetic exchange levels were high between geographical populations; 3) genetic variation level was low; 4) C. italicus populations likely expanded in recently, and 5) there was no significant correlation between genetic distance and geographic distance for any geographic population. Findings from this study indicate that frequent gene exchange between populations may enhance the adaptability of C. italicus along the Sino-Kazakh border, leading to frequent outbreaks.

Meloidogyne aegracyperi n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing yellow and purple nutsedge in New Mexico.

Meloidogyne aegracyperi n. sp. is described from roots of purple nutsedge in southern New Mexico, USA. Mature females are small (310–460 µm), pearly white, with their egg masses completely contained inside root galls. The neck is often at a 90 to 130° angle to the protruding posterior end with the perineal pattern. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet is relatively long (4.0–6.1 µm), and the excretory pore is level with the base of the stylet. The anterior portion of the rounded lumen lining of the metacorpus contains 3 to 10 small vesicles. The perineal pattern has a rounded dorsal arch with a tail terminal area that is smooth or marked with rope-like striae. Only two males were found. The body twists 90° throughout its length. The DGO to the base of the stylet is long (3.0–3.3) µm. The cephalic framework of the second-stage juvenile is weak, and the stylet is short (10.1–11.8 µm). The DGO to the base of the stylet is long (3–5 µm). The tail is very long (64–89 µm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Eggs are typical for the genus and vary in length (85.2–99.8 µm) and width (37.1–48.1 µm), having a L/W ratio of (2.1–2.6). Maximum likelihood phylogenetic analyses of the different molecular loci (partial 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA gene fragments and partial Hsp90 gene) placed this nematode on an independent branch in between M. graminicola and M. naasi and a cluster of species containing M. chitwoodi. M. fallax, and M. minor. Greenhouse tests showed that yellow and purple nutsedge were the best hosts, but perennial ryegrass, wheat, bentgrass, and barley were also hosts.

IDENTIFICATION OF GENETIC DIVERSITY CYTOCHROME OXIDASE SUBUNIT II (COII) MITOCHONDRIAL GENE AS GENETIC MARKER FOR ANISAKIS SPECIES IN Euthynnus affinis

ABSTRACT This study aimed to get specific genetic marker for Anisakis sp. identification on mackerel tuna using gene sequence cytochrome oxidase subunit II (COII) mitochondrial deoxyribonucleic acid (mtDNA) and to identify taxonomic affiliation between Anisakis sp. from Indonesia and others Anisakis sp. from GenBank database. This study started with sample collections at three fish auctions in Cilacap (Central Java), morphology classification, DNA isolation, and molecular based identification using polymerase chain reaction (PCR) and sequencing methods. Molecular based identification of Anisakis used gene amplification COII mtDNA as a cell target prior to sequence. Morphology characteristic results showed that Anisakis nematodes which infected mackerel tuna classified as type II L3 larvae. Molecular based identification showed significant result, which found 530 bp COII DNA gene fragment similar to target cell. Gene sequencing alignment results of COII Anisakis gene compared with GenBank showed 11 different nucleotide sites that can be used as genetic barcode for Indonesian Anisakis sp. This study showed that Anisakis sp. infected mackerel tuna in Java Sea is Anisakis physeteris and considered as zoonosis.

Morphological and Genetic Evidence for the Synonymy ofReticulitermesSpecies:Reticulitermes dichrousandReticulitermes guangzhouensis(Isoptera: Rhinotermitidae)

The taxonomy of Reticulitermes Holmgren (Isoptera: Rhinotermitidae) in China is problematic and in need of revision. Most Chinese Reticulitermes species were described in the 1980s and 1990s, and have never been studied since then. In this study, morphological characteristics including coloration, pilosity, shape, and morphometric characteristics of Reticulitermes dichrous Ping and Reticulitermes guangzhouensis Ping were compared. In addition, a portion of the ribosomal RNA large subunit 16S (16S rRNA) and the mitochondrial DNA cytochrome oxidase subunit II (COII) genes from different populations of the 2 species were sequenced and analyzed. Morphological comparisons revealed the similarities between the 2 species in both discrete and morphometric characteristics. In the molecular phylogenetic trees inferred from COII and 16S rRNA genes, all of the examined populations of the 2 species clustered into a common clade with a high bootstrap value. Based on the morphological comparisons and the molecular analyses, it is proposed that R. dichrous is a junior synonym of R. guangzhouensis.

The Calibrated Phylogeny of the Drosophila fasciola Subgroup (D. repleta Group Wasserman) Indicates Neogene Diversification of Its Internal Branches.

The species of the Drosophila fasciola subgroup Wasserman represent the dominant section of the Drosophila repleta group Wasserman in the American rainforests and have a broad geographical distribution in the New World. However, despite of its wide range, the D. fasciola subgroup is one of the most overlooked D. repleta subgroups. Here, we report a molecular phylogenetic analysis focused on the D. fasciola subgroup using two mitochondrial [cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII)] and two nuclear [elongation factor-1alpha F1 (EF-alphaF1) and transformer (tra)] genes. Overall, we found that this subgroup is a monophyletic taxon, subdivided into two main internal branches: named Fas1 and Fas2 clades. The diversification of these clades is estimated to have begun in the middle Miocene, around 12Ma [95% high posterior density (HPD) 9.0-15Ma], and might be associated with the colonization of South America by Central America populations after the closure of Isthmus of Panama due to the temporal congruence between these events. The terminal branches had their origins estimated to be in the Pliocene or the Plio-Pleistocene transition. For the later estimates, both the geomorphological influences and the climatic oscillations of the Pleistocene may have played a role in shaping the diversification of the D. fasciola group.