First Report of the Apple Root-Knot Nematode, Meloidogyne mali, on Maple Trees in Republic of Korea

Meloidogyne mali was originally described in Japan on roots of an apple rootstock (Malus prunifolia) (Itoh et al. 1969) and found on elm trees in Italy, Netherlands, Belgium, France and United Kingdom, and euonymus in the USA (EPPO 2018; Prior et al. 2019). In Italy, the nematode was initially described as a new species, Meloidogyne ulmi, but was later synonymized with M. mali (Ahmad et al., 2013). During the study of Meloidogyne species in Republic of Korea, galled roots were found on Acer palmatum collected in Naejangsan National Park, Republic of Korea located at 35°29'29.1"N, 126°55'42.7"E, altitude 147.8 m. Morphologically, the perineal patterns of the females was very similar to M. mali due to rounded dorsal arch and smooth, finely-spaced, indistinct striae. Lateral field shallow, narrow, and faint. Phasmids large, very distinct. Head region of second–stage juveniles flattened anteriorly to hemispherical, slightly set-off from body, without annulations, low head cap. Stylet slender, sharply pointed cone, cylindrical shaft with rounded knob sloping posteriorly. Tail conoid with irregular, and rounded end. Rectum undilated. Several micrographs were made from 25 J2s and females for mean, standard deviation and range. J2s were measured with a body length: 408.2 ± 25.1 (366-449) µm, maximum body width: 15.9 ± 1.0 (14.1-17.9) µm, stylet length: 14.1 ± 0.5 (13.1-15.3) µm, hyaline tail terminus: 10.0 ± 0.9 (8.3-11.0) µm and tail length: 31.7 ± 3.0 (26.0-36.1) µm. Females (n=25) were characterized by a body length: 656.7 ± 102.7 (516-947) µm, a stylet length: 16.4 ± 2.2 (13.9-19.0) µm, a vulval slit length: 22.2 ± 1.8 (19.8-25.7) µm, and a vulva-anal distance: 20.2 ± 2.4 (17.1-25.4) µm. Morphological measurements and configuration of perineal patterns (Fig. 1S) were comparable to M. mali (Itoh et al. 1969; Ahmed et al. 2013; Gu et al. 2020). To confirm pathogenicity, a modified version of Koch’s postulates was conducted in the greenhouse by inoculating 300 eggs from a single egg mass onto each of three, two-year-old A. palmatum plants, grown in sterilized sandy soil. After about one year, symptoms developed on the maple tree roots, with numerous galls containing females and egg masses by visual inspection. In addition, PCR was performed for the 28S rDNA D2-D3 segment and ITS region using the primers D2A, D3B, TW81 and AB28. The resulting sequences (MW522548, MW522549, MW523004 and MW523005) were at least 99% identical to other 28S rDNA D2-D3 segment and ITS region sequences on Genbank (MT406757 and JX978229). The molecular phylogenetic relationships of this species strongly supports M. mali (Fig. 2S). To the best of our knowledge, this is the first report of M. mali in Republic of Korea. The host range of M. mali includes many species which are of economic importance in fruit trees (e.g. apple, chestnut, fig, mulberry), forestry trees (e.g. elm, maple, oak, Yew), and vegetable crops (e.g. cabbage, carrot, cucumber, eggplant, soybean, watermelon). The potential danger to these economically important plants caused M. mali to be added the EPPO Alert List and also the Quarantine List of the Korean Animal and Plant Quarantine Agency. Additionally, in our survey around the Naejangsan National Park, M. mali was not found on other economically important host crops, such as grapes. Although this nematode was not detected other crops, it requires regular monitoring because it poses a serious threat to the future production of these crops.

Redescription of Ektaphelenchoides compsi Baujard, 1984 (Tylenchina: Aphelenchoididae) isolated from Pinus massoniana in Fujian Province, China

Summary Ektaphelenchoides compsi is redescribed morphologically with new molecular characterisation. It was isolated from a dead Pinus massoniana tree in Ningde City, Fujian Province, China. Detailed morphology of the spicule, female gonad, hemizonid position, arrangement of male caudal papillae and female tail terminus shape are documented. It is characterised by a lateral field with three lines (forming two bands), tripartite stylet 17.8 (17.0-19.4) μm long without basal thickenings, metacorpus rectangular with anterior 40% granular and posterior part weakly muscular, metacorpal valve slightly posterior to middle of metacorpus, excretory pore at level of nerve ring, vagina with thickened walls and strongly developed muscular bundles, vulval lips slightly protuberant, vulval flap absent, distal region of post-vulval uterine sac appearing as a weakly developed oogonia, anus and rectum indistinct, female posterior part (‘tail’) dorsally convex, conical, terminal region contracted into a bluntly pointed tip. The spicules are arcuate, 15.6 (14.3-16.3) μm along the chord, lamina smoothly curved to distal end, capitulum slightly concave, condylus well-developed with broadly rounded tip and slightly depressed at dorsal end, rostrum triangular with finely rounded tip, cucullus absent, and with seven caudal papillae present. The near full length 18S and 28S D2-D3 regions of rRNA genes sequences were characterised. The phylogenetic analyses revealed that the Fujian population of E. compsi grouped with the Zhejiang population of E. compsi, both being morphologically identical.

Symmetric Excitons in an (001)-Based InAs/GaAs Quantum Dot Near Si Dopant for Photon-Pair Entanglement

The sacrificed-QD-layer method can well control the indium deposition amount to grow InAs quantum dots (QDs) with isotropic geometry. Individual Si dopant above an (001)-based InAs QD proves a new method to build a local electric field to reduce fine structure splitting (FSS = X1−X2) and show D3h symmetric excitons. The lowest FSS obtained is 3.9 μeV with the lowest energy X state (LX) anticlockwise rotate from [1−10] (i.e., zero FSS will be crossed in a proper field). The lateral field projection induces a large eh separation and various FSS, LX, and emission intensity polarization. The lateral field along [1−10] breaks the X1–X2 wavefunction degeneracy for independent HH and VV cascade emissions with robust polarization correlation. With FSS ~4 μeV and T1 ~0.3 ns fastened in a distributed Bragg reflector cavity, polarization-resolved XX–X cross-correlations show fidelity ~0.55 to a maximal entangled state |HH> + |VV>. A higher fidelity and zero FSS will be obtained in the hybrid QD structure with a junction field integrated to tune the FSS and a sub-bandgap excitation to avoid influences from electrons in the barrier.

First report of southern root-knot nematode, Meloidogyne incognita, infecting Isatis indigotica Fortune in Anhui Province, China

Isatis indigotica Fortune, widely cultivated in China, is an important Chinese herbal medicine, mainly used to treat cold and fever. In October 2020, galls (Fig. 1), as many as 65 per root, were observed on the roots of I. indigotica in Taihe, Anhui Province, China (117°21'19.5"N, 32°57'59.5"E), and samples were taken. The infected plants were weak, and the leaves are wilting. Second-stage juveniles (J2s) were dissected from the egg masses released by females. Excretory pores of females were located nearby median bulb (Fig. 2A). The dorsal arch of the perineal pattern (n = 10) of the female was elliptical, and the dorsal arch was relatively high with smooth to wavy lines (Fig. 2B). Morphometrics of females (n=10): body length (L) = 595.5 ± 24.0 (570.0-620.5) μm, body width (W)= 350.5 ± 30.0 (320.0-390.5) μm, stylet length = 13.6 ± 0.7 (12.1-15.4) μm (Fig. 2A), and the distance from dorsal esophageal gland orifice to base of stylet (DGO) = 3.5 ± 0.2 (2.8-4.0) μm (Fig. 2B). J2s (n = 20) had the following characteristics: L = 383.2 ± 12.5 (337-430) μm (Fig. 2C), a = 22.0 ± 1.1 (20.3-24.4) μm, c = 8.4 ± 0.5 (7.5-10.5) μm, stylet length = 12.4 ± 1.5 (10.1-14.6) μm, DGO = 2.9 ± 0.6 (2.0-3.6) μm (Fig. 2D), tail length = 39.5 ±3.4 (32.0-48.5) μm and hyaline tail terminus = 10.5 ± 0.5 (9.5-11.2) μm (Fig.1E). There were four lines on the lateral field of J2s (Fig. 2F). Females and J2s obtained from galls had uniform morphological and molecular characteristics were confirmed to be Meloidogyne incognita. Live J2s were detected in all soil samples with a mean of 120 ± 15 J2s/100 ml of soil. Five 4-week-old I. indigotica plantlets, grown in pots (500cm3) with sterilized soil were inoculated with 1000 J2s from egg masses per pot and5 non-inoculated pots were used as control. Plants were well maintained under 25 ± 3°C in the greenhouse. Three plants were gently removed from the pots 30 days after inoculation, and an average of 50 galls per root was observed on the roots, and the resulting nematode reproduction factors (RF = final egg density ÷ 1,000, initial egg density) of 3.2, suggested that I. indigotica is a good host for M. incognita (Mojtahedi, 1988). There were no significant differences in main measurements and morphological characteristics between the Taihe population of M. incognita and that represented in "CIH descriptions of plant-parasitic nematodes" (Orton Williams, 1973). DNA was extracted from 5 single J2s, and ITS and 18S rDNA gene was amplified using the primer pair 18S/26S and 18s1.2a/18sr2b (Bernard et al. 2010; Vrain et al. 1992). The sequence of 18S rDNA (MW875892) was submitted to GenBank. Comparisons showed a sequence identity of greater than 99.8% for Meloidogyne incognita (MF177719.1). The rDNA sequences of M. incognita, M. hapla, M. javanica and M. arenaria are so homologous that rDNA-based differentiation is difficult. The SCAR primers can successfully distinguish M. incognita, M. hapla, M. javanica and M. arenaria. Five species-specific primer sets (Finc/Rinc; MORF-F/MTHIS-R; Jmv-F/Jmv-R; Far/Rar and Fjav/Rjav, Stanton et al. 1997; Wishart et al. 2002; Zijlstra et al. 2000) were used to species-specifically distinguish within the genus. The results (＋, ＋, －, －, －) proved that the Taihe population belonging to M. incognita. To our knowledge, this is the first report of M. incognita parasitizing I. indigotica. This finding may be important to medicinal plant industry, since M. incognita is one of the most harmful nematode pests in the world and would cause severe damage to I. indigotica.

Ottolenchus sinipersici n. sp. (Rhabditida: Tylenchidae) from the Persian Gulf mangrove forests, Iran

Summary A new species of Tylenchidae from the rhizosphere of mangrove trees in Hormozgan and Khuzestan provinces, Iran, is described based on morphological and molecular data. Ottolenchus sinipersici n. sp., is characterised by a slightly fusiform body 560-665 μm long, lateral field in the form of a narrow band with two faint incisures that are not visible in fatter females, indistinct transverse annuli under the light microscope, cephalic region continuous with the body contour, smooth and flattened dorsoventrally, longitudinal and narrow sigmoid amphidial slits, stylet delicate, 10.1-11.2 μm long, with small rounded to slightly posteriorly sloping knobs, well-developed median bulb, offset and pyriform pharyngeal basal bulb, vulva located at 66.9-69.6% of body length, offset spermatheca, short post-vulval uterine sac, spicules 18.5-20.5 μm long with highly curved blades, and a 113-135 μm long filiform tail with a hook-like or coiled terminus. In Bayesian inference phylogenetic trees based on the partial small subunit ribosomal DNA (SSU rDNA) and D2-D3 expansion segment of large subunit ribosomal DNA (D2-D3 LSU rDNA) genes, the new species clustered together with O. facultativus (KJ869310) in SSU, and forms a clade with three isolates of O. discrepans in LSU phylogeny. Ottolenchus fungivorus n. comb. (= Filenchus fungivorus) is proposed.

Description of Ruehmaphelenchus taedae n. sp. (Nematoda: Aphelenchoididae) found in Loblolly pine logs from the USA

Summary Ruehmaphelenchus taedae n. sp., isolated from Loblolly pine logs (Pinus taedae L.) from the USA, is described and figured. It is characterised by a relatively slim body (a = 42 and 43 for males and females, respectively), three lines in the lateral field, male spicules relatively small (12-18 μm) with high and dorsally bent condylus and weakly developed rostrum, bursal flap absent, short tail possessing a long terminal spike ending in a bluntly rounded tip and 8.7-13.3 μm long, vulva positioned at ca 83% of body length, vulval flap absent, vulval lips slightly protruding, post-vulval uterine branch extending for less than half of vulva to anus distance, and female tail conoid, ca 3-4 anal body diam. long, with 13.7-18.5 μm terminal projection. The new species can be separated from all other species of the genus by the male tail possessing a long terminal spike and the more anterior excretory pore. Detailed phylogenetic analysis based on 28S D2-D3 region sequences confirmed the status of this nematode as a new species.

Description of Ditylenchus acantholimonis n. sp. (Rhabditida: Anguinidae) from Iran, a morphological and molecular phylogenetic study

Summary Ditylenchus acantholimonis n. sp. is described based on morphological, morphometric and molecular characters. It was isolated from the rhizosphere soil of Acantholimon sp. in Golestan province, Iran, and is mainly characterised by having four lines in the lateral field, a pyriform to bottle-shaped offset pharyngeal bulb, post-vulval uterine sac 36.6-56.1% of the vulva to anus distance long, and a subcylindrical to conical tail with widely rounded tip. It is further characterised by short to medium-sized females, 480-617 μm long, with a fine stylet having small rounded knobs, V = 80.8-83.6, c = 11.0-13.8, c′ = 3.3-4.6, and males with 16.0-17.0 μm long spicules. The new species was morphologically compared with six species having four lines in their lateral field, rounded tail tip and comparable morphometric data namely: D. dipsacoideus, D. emus, D. exilis, D. paraparvus, D. sturhani, and D. solani. It was also compared with two species, D. ferepolitor and D. angustus, forming a maximally supported clade in the 18S tree. The phylogenetic analyses using the maximal number of Anguinidae and several Sphaerularioidea genera based upon partial 18S and 28S rDNA D2-D3 sequences revealed that Ditylenchus is polyphyletic. In the 18S tree, the new species formed a clade with D. ferepolitor (KJ636374) and D. angustus (AJ966483); in the 28S tree it formed a poorly supported clade with D. phyllobios (KT192618) and Ditylenchus sp. (MG865719).

First report of Meloidogyne arenaria infecting Maidong (Ophiopogon japonicus) in China

Maidong (Ophiopogon japonicus) is a perennial evergreen plant of the Asparagaceae, occurring mainly in China, Japan, Vietnam, and India. It grows in the damp place on the hillside below 2000 meters above sea level, under the forest or beside the stream;It has been widely cultivated in the Sichuan ofhina for medicinal uses; and it is included in the Chinese Pharmacopoeia. During April 2019, Maidong plants exhibiting symptoms of stunting, leaf wilting, and multiple galls in the roots associated with root-knot nematode (Meloidogyne sp.) were detected in a commercial field in near the city of Mianyang (N105°42′, E30°93′), Sichuan, China. The second-stage juveniles (J2) were collected from the soil in the root zone, and adult females were dissected from roots. Population densities of J2 ranged from 190 to 255 per 100 cm3. Subsequently, individual females (n=20) were extracted from root samples and submitted to Meloidogyne species identification by perineal pattern morphological analysis (n=20), and morphometric measurements of second stage juveniles (J2) (n = 20). The J2 showed the following morphometric characters：body length = 475.5 ± 24.2 µm, tail length = 55.2 ± 6.43µm, stylet length = 12.4 ± 1.56 µm and distance from dorsal esophageal gland opening to the stylet knot (DGO) = 2.97 ± 0.44 μm; perineal patterns of females showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between anus and tail terminus were observed. These morphological characteristics are consistent with Meloidogyne arenaria (Neves et al. 2016). In addition, to confirm species identification, DNA was extracted from females (Blok, et al. 1997) and D2/D3 fragments of the 28S rRNA was amplified using the universal primers D2A/D3B. The DNA fragment obtained showed a 754 bp length (GenBank accession no. MW965614) that was sequenced and analyzed, sequences were 99.8% identical to the MH359158, KX151138 and EU364889 M. arenaria sequences. Furthermore, species-specific SCAR primers Far/Rar were used as described by Zijlstra et al. 2000. The PCR produced approximately 420 bp sequences, which was identical to that previously reported for M. arenaria (Zijlstra et al. 2000). Morphological and molecular characterization supports the identification of the isolate found on Ophiopogon japonicus as M. arenaria. To verify the nematode pathogenicity on Maidong plants, Maidong seed were planted in 20-cm diameter, 10-cm deep plastic pots containing 1000 cm3 sterilized soil and infested with 2000 M. arenaria J2 per seedling, using a sterilized micropipette. Plants were maintained at 20-25°C in a greenhouse. Control plants received sterile water, and the pathogenicity test was repeated three times. After 60 days, all inoculated plants showed reduced growth compared with control. The symptoms were similar to those observed in the field, a large number of galls (38.5 ± 2.4) and egg masses (18.5 ± 0.2) were found on each root system. Maidong was considered a good host for M. arenaria in Mianyang. M. arenaria is one of the most important plant parasitic nematode with a wide geographic distribution and causes great losses in many crops around the world (Perry et al. 2009). Through investigation, this is the first report worldwide of M. arenaria infecting Ophiopogon japonicus.

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

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

Regenerable ZnO/GaAs Bulk Acoustic Wave Biosensor for Detection of Escherichia coli in “Complex” Biological Medium

A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of Escherichia coli in liquid media. The geometry of the biosensor consists of a GaAs membrane coated with a thin film of piezoelectric ZnO on its top surface. A pair of electrodes deposited on the ZnO film allows the generation of BAWs by lateral field excitation. The back surface of the membrane is functionalized with alkanethiol self-assembled monolayers and antibodies against E. coli. The antibody immobilization was investigated as a function of the concentration of antibody suspensions, their pH and incubation time, designed to optimize the immunocapture of bacteria. The performance of the biosensor was evaluated by detection tests in different environments for bacterial suspensions ranging between 103 and 108 CFU/mL. A linear dependence between the frequency response and the logarithm of E. coli concentration was observed for suspensions ranging between 103 and 107 CFU/mL, with the limit of detection of the biosensor estimated at 103 CFU/mL. The 5-fold regeneration and excellent selectivity towards E. coli detected at 104 CFU/mL in a suspension tinted with Bacillus subtilis at 106 CFU/mL illustrate the biosensor potential for the attractive operation in complex biological media.