*Ficus microcarpa* **Bonsai "Tiger bark" Parasitized by the Root-Knot Nematode** *Meloidogyne javanica* **and the Spiral Nematode** *Helicotylenchus dihystera***, a New Plant Host Record for Both Species**

#### **Duarte Santos 1, Isabel Abrantes <sup>1</sup> and Carla Maleita 1,2,\***


Received: 5 July 2020; Accepted: 20 August 2020; Published: 24 August 2020

**Abstract:** In December 2017, a *Ficus microcarpa* "Tiger bark" bonsai tree was acquired in a shopping center in Coimbra, Portugal, without symptoms in the leaves, but showing small atypical galls of infection caused by root-knot nematodes (RKN), *Meloidogyne* spp. The soil nematode community was assessed and four Tylenchida genera were detected: *Helicotylenchus* (94.02%), *Tylenchus* s.l. (4.35%), *Tylenchorynchus* s.l. (1.09%) and *Meloidogyne* (0.54%). The RKN *M. javanica* was identified through analysis of esterase isoenzyme phenotype (J3), PCR-RFLP of mitochondrial DNA region between COII and 16S rRNA genes and SCAR-PCR. The *Helicotylenchus* species was identified on the basis of female morphology that showed the body being spirally curved, with up to two turns after relation with gentle heat, a key feature of *H. dihystera*, and molecular characterization, using the D2D3 expansion region of the 28S rDNA, which revealed a similarity of 99.99% with available sequences of the common spiral nematode *H. dihystera*. To our knowledge, *M. javanica* and *H. dihystera* are reported for the first time as parasitizing *F. microcarpa*. Our findings reveal that more inspections are required to detect these and other plant-parasitic nematodes, mainly with quarantine status, to prevent their spread if found.

**Keywords:** 28S ribosomal DNA; mitochondrial DNA region; pest interception; plant-parasitic nematodes; SCAR-PCR

#### **1. Introduction**

The globalization era opens up new trade routes and increases the volume and complexity of cross-border transactions of goods. The plant sector (plant products, germplasm, grafts and live plants) has been part of the general trend in increased trade. This exchange of species between distant geographical regions of the globe creates new pathways for the introduction of alien plant pests and diseases [1,2].

The introduction of a non-native organism in a new environment produces unpredictable effects. A species may have low impact in its native range, but much greater impact when introduced to new areas, putting native biodiversity and local production systems at risk [1,3,4]. For instance, the introduction of the alien pinewood nematode *Bursaphelenchus xylophilus* in Portugal has caused huge environmental and economic losses in Portuguese pine forests, while in North America, where this nematode is native, it does not cause significant mortality to native conifers [5–7]. These problems are expected to be intensified in the future as climate change is predicted to facilitate the further spread of these species, since many of these new pathogens are of tropical and subtropical origins [8,9]. Recently,

the tropical root knot nematode (RKN) *Meloidogyne luci* (Alert List of the European and Mediterranean Plant Protection Organization—EPPO) and *M. enterolobii* (A2 List of Pests EPPO) were detected in Portugal. *Meloidogyne luci* was found to be associated with potato (*Solanum tuberosum*) and tomato (*S. lycopersicum*), the ornamental plant *Cordyline australis*, and the weed *Oxalis corniculata*, whereas *M. enterolobii* was detected in the ornamental plants *Cereus hildmannianus*, *Lampranthus* sp., *Physalis peruviana* and *Callistemon* sp. Taking into account its aggressiveness and distribution, there is a high probability of spread in the Mediterranean region and also in Europe, becoming a potential threat to the agricultural economy [10,11].

The European Commission has proposed an import ban on 35 genera of plants for planting, other than seeds, in vitro material and natural or artificially dwarfed woody plants for planting from countries outside the European Union (EU). *Ficus carica*, common fig, is the only species of the genus *Ficus* included on the list. The ban was put into effect in December 2019 and aims to reduce the probability of the introduction of harmful organisms in the EU [12]. During a survey (3 years) in the Netherlands, around 20% of samples of imported plants for planting and ornamentals from 21 countries showed quarantine nematodes and 11% other important nematodes [13].

*Ficus* constitutes one of the largest genera of flowering plants (Angiosperms) that, according to the plant list version 1.1 (http://www.theplantlist.org), has 919 accepted species, being primarily found in tropical and subtropical environments throughout the world [14]. During the past few decades, plants from this genus have become quite popular as indoor house plants.

Despite the diversity of *Ficus* species, the research regarding plant-parasitic nematodes (PPN) has been focussed on the edible fig tree *F. carica* native to western Asia and introduced in the Mediterranean region. A number of PPN species have been reported as parasitizing fig trees in many countries, the most prevalent belonging to the genera *Helicotylenchus* (spiral nematodes), *Heterodera* (cyst nematodes), *Meloidogyne* (root-knot nematodes), *Paratylenchus* (pin nematodes), *Pratylenchus* (root lesion nematodes, RLN) and *Xiphinema* (dagger nematodes) [15–24]. Of these, the most common are the RKN species *M. arenaria*, *M. hapla*, *M. hispanica*, *M. incognita* and *M. javanica*, economically important species that directly target plant roots and prevent water and nutrient uptake, resulting in growth or even plant death in extreme cases, and the species *Heterodera fici*, a worldwide parasite of ornamental and cultivated *Ficus* species [24,25].

Concerning *Ficus* bonsai, besides *F. carica*, other species, such as *F. benghalensis*, *F. macrophylla*, *F. microcarpa*, *F. retusa* and *F. rubiginosa*, have been considered suitable for bonsai plants, but records of PPN on them are few and scarce. Some bonsai plants with nematode infections have been intercepted in Europe and other parts of the world [13,26,27].

The species *F. microcarpa*, the Indian laurel tree, sometimes confused with *F. nitida* and *F. retusa*, is widely distributed as an ornamental plant either outdoors or indoors and is known for its pharmacological properties: antioxidant, antibacterial, anticancer, anti-diabetic, anti-diarrhoeal, anti-inflammatory, anti-asthmatic, hepatoprotective and hypolipidemic [28]. This *Ficus* species is a host of many pests, including the Cuban laurel thrips (*Gynaikothrips ficorum*), the Ficus leaf-rolling psyllid (*Trioza brevigenae*), and the Ficus whitefly (*Singhiella simplex*), among others [29]. The PPN found associated with *F. microcarpa* include the genera *Helicotylenchus*, *Meloidogyne*, *Pratylenchus*, *Tylenchorynchus* and *Xiphinema*, and the RKN *M. enterolobii* species have been intercepted in bonsais or plants for planting imported from China or Egypt [27].

The aims of the present study were to find the nematode diversity associated to *F. mircrocarpa* bonsai plant, to characterize/identify the RKN, *Meloidogyne* sp., and the spiral nematode, *Helicotylenchus* sp., parasitizing *F. microcarpa* (Figure 1a) and to enlarge the knowledge on the phytoparasitic nematodes of this *Ficus* species.

**Figure 1.** *Meloidogyne javanica* parasitizing *Ficus microcarpa*. (**a**) *F. microcarpa*. (**b**,**c**) *F. microcarpa* infected roots. (**d**) Polyacrylamide gel stained for esterase activity. J3, *M. javanica* (*F. microcarpa* isolate); R, *M. javanica* (reference isolate). (**e**) *Hinf*I (1), *Alu*I (2) and *Dra*III (3) digestion patterns of the approximately 1800-bp amplification products from *M. javanica*, using C2F3 and MRH106 primers. M, DNA marker (HyperLadder II; Bioline). (**f**) DNA amplification product using Fjav and Rjav primers. 4, Negative control; 5, *M. javanica*; M, DNA marker (HyperLadder II; Bioline). Scale bar: 1 mm.

#### **2. Results and Discussion**

*Meloidogyne* females plus egg masses (Figure 1b,c) and *Helicotylenchus* specimens (Figure 2a) were detected in fresh and stained roots. Although *Helicotylenchus* spp. are classified as ectoparasites or semi-endoparasites, they can penetrate the roots and were already found completely embedded in the cortical tissue of the root system of sycamore (*Platanus occidentalis*) [30]. The galls were small and hard, mainly in woody roots (Figure 1b,c), which is common in woody perennial plants. The PPN detected in the soil sample (130 g) of *F. microcarpa* bonsai belonged to four genera: *Helicotylenchus* (94.02%), *Tylenchus* s.l. (4.35%), *Tylenchorynchus* s.l. (1.09%) and *Meloidogyne* J2 (0.54%). The spiral nematode *Helicotylenchus* was the most prevalent PPN, detected in very high numbers, with approximately 3000 nematodes.

For the RKN isolate, the biochemical characterization resulted in three bands of esterase (relative mobility %: 0.38; 0.45; 0.49), which is the characteristic phenotype exhibited by *M. javanica* (J3) isolates (Figure 1d). The mtDNA *COII* and 16S rRNA genes region amplified with the primer set C2F3/MRH106 yielded a single fragment of 1800 bp. When the amplified product was digested with the restriction enzyme *Hinf*I, no digestion occurred. *Alu*I and *Dra*III generated three fragments of approximately 1000, 580, and 240 bp and two fragments of approximately 1000 and 800 bp, which is in accordance with other results for this species [31] (Figure 1e). Additionally, molecular characterization of the RKN

species with the species-specific primers Fjav and Rjav produced a fragment size of 600 bp, as expected, thus confirming the presence of *M. javanica* (Figure 1f).

This RKN species is known to parasitize *F. carica* and is one of the most widely distributed species and the second highest in economic importance after *M. incognita* [22,23,32]. *Meloidogyne javanica* was first reported from Portugal on potato in Azores [33]. Since then, it has been found on several economically important crops, including *Humulus lupulus*, *Musa* sp., *Phaseolus vulgaris*, *Prunus persica*, *S. lycopersicum* and *S. tuberosum*, ornamental plants, such as *Cordyline australis* and *Dianthus plumarius*, as well as many other dicots [11,34–40].

*Helicotylenchus* females were spirally curved, with up to two turns after relation with gentle heat, a key feature of *H. dihystera* (Figure 2b), the tail dorsally convex-conoid to a narrow terminus with a slight projection (Figure 2d) and lateral field with four non-areolated incisures (Figure 2e). Males were not found [41]. Amplification of the D2D3 expansion region of the 28S rDNA gene resulted in a product of ca. 750 bp (Figure 3). Sequences (744 bp) were submitted to the GenBank database with accession numbers MT277384, MT277385 and MT277386. The three sequences of the *Helicotylenchus* sp. from *Ficus microcarpa* (Fm) were compared and nine nucleotide changes at positions 57, 77, 98, 189, 537, 541, 546, 548 and 572 in alignment were identified. The comparison of this region with the *Helicotylenchus* sequences available in the GenBank database revealed a similarity of 99.99% with *H. dihystera.* Phylogenetic analysis from the alignment of Fm *Helicotylenchus* 28S rDNA sequences with available sequences of similar *Helicotylenchus* spp. [41] revealed that this isolate and all listed *H. dihystera* sequences appeared together in a well-separated clade with 100% bootstrap support (Figure 4)*,* confirming the morphological identification. Considering a common start and end point to *Helicotylenchus* spp. (560 bp), the Fm sequences differed in several positions from at least one *H. dihystera* sequences included in the analysis; however, only three (4, 493 and 519) position changes were completely distinct from *H. dihystera* sequences (Figure S1). Fm *H. dihystera* sequences had divergences ranging from 0.2 to 2.2% when compared with *H. dihystera* sequences and 3.7 to 6.5% to the other *Helicotylenchus* species (Table S1).

**Figure 2.** *Helicotylenchus dihystera* (females) light microscope photographs. (**a**) Infected *Ficus microcarpa* root. (**b**) Whole specimen. (**c**) Anterior region in lateral view. (**d**) Posterior region in lateral view. (**e**) Lateral field with four lateral lines. Scale bars: 20 μm (**a**–**d**), and 50 μm (**e**).

**Figure 3.** DNA amplification product obtained from *Helicotylenchus dihystera* isolate identified on *Ficus microcarpa* to the D2D3 expansion region of the 28S rDNA gene (1). 2, negative control. M, DNA marker (HyperLadder II; Bioline).

**Figure 4.** Neighbor-joining tree based on analysis of alignment of D2D3 expansion region of the 28S rDNA gene sequences of the *Helicotylenchus dihystera* isolate identified on *Ficus microcarpa* (FmHe) with available sequences of close *Helicotylenchus* spp. (*H. leiocephalus*, *H. microlobus* and *H. pseudorobustus*) [41]. The percentage of replicate trees in which the associated *Helicotylenchus* spp. clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary distances were computed using the maximum composite likelihood method and all positions containing gaps and missing data were eliminated.

Although *H. dihystera* is considered as a polyphagous species with a wide distribution, reports on its pathogenicity are very few, and it is rarely recognized as an economically important PPN [30,42–44], even when high population densities are found. This is the first report of *H. dihystera* infecting *F. microcarpa;* however, it has been associated with *F. benjamina, F. carica*, *F. elastica, F. formosana* and

*F. retusa* [21,26,44–47]. In Portugal, *H. dihystera* was reported as being associated with *Begonia* sp., *Colocasia esculenta*, *Cactus* sp., *Mentha* sp., *Musa* sp., *Pelargonium* sp., Polygonaceae, beans, maize and tomato [45,48–50].

Although no specific quarantine measures are being implemented against *M. javanica* or *H. dihystera*, preventive measures are particularly important to decrease the risk of spread into a region where they do not exist. Once nematodes are established in the soil, their eradication is very difficult. Thus, the use and transportation of clean, healthy, nematode-free planting material is a prerequisite for limiting the spread of nematodes. Plant parts liable to carrying PPN in trade/transport can be bulbs/tubers/corms/rhizomes, growing medium accompanying plants, roots and micropropagated plants. During routine inspections, the detection of nematode infections can be easily overlooked or misdiagnosed, as low to moderate populations of nematodes may cause no visible aboveground symptoms, making it harder to diagnose them [51,52]. Furthermore, above-ground symptoms are non-specific and usually involve stunting, lack of vigour, leaf nutritional deficiencies and temporary wilting in periods of water stress and high temperatures. The examination of roots can reveal the presence of galls that are specific symptoms associated with the occurrence of *Meloidogyne* spp., but the symptoms caused by *Helicotylenchus* spp., when present, can be confused with the damage associated with poor nutrition or injury caused by pathogens that attack the root system (other nematodes, bacteria, fungi and/or virus).

To our knowledge, *M. javanica* and *H. dihystera* are here reported for the first time as parasitizing *F. microcarpa*. Although both PPN species are common and widely distributed, our findings emphasize the importance of inspections by governmental authorities to find out whether imported material is free of PPN. If plants are grown in infested soil and then commercialized, it increases the probability of PPN dissemination to new regions and/or other suitable hosts with potential impact on economically important crops.

#### **3. Materials and Methods**

In December 2017, a *F. microcarpa* "Tiger bark" bonsai tree with a phytosanitary certificate was acquired by the first author in a shopping center in Coimbra, Portugal, showing small galls in the protruding roots, which aroused our attention, but without symptoms in the leaves (Figure 1a,b). Consequently, a few roots and a soil sample of 130 g were collected from the pot. Roots were observed directly and stained with acid fuchsin to detect nematode-infected plant tissues [53]. Nematodes were extracted from the soil, according to the Tray Method [54], followed by microscopic examination of nematode diversity and the genera identified and quantified.

#### *3.1. Root Knot Nematode Characterization*/*Identification*

Egg masses from *F. microcarpa* galled roots were propagated on tomato, *Solanum lycopersicum*, cv. Coração-de-Boi, in a growth chamber. After two months, the infected tomato roots were gently rinsed with tap water and 5 young egg-laying females were, individually and randomly, handpicked with their respective egg masses to glass blocks with NaCl 0.9% to obtain pure cultures. Individual young egg-laying females were characterised biochemically by electrophoretic analysis of esterases. Esterase electrophoresis was performed using polyacrylamide gels following the methodology described by Pais and Abrantes [55]. The individual females were transferred to micro-haematocrit tubes containing 5 μL of extraction buffer (20% sucrose and 1% Triton X-100), macerated and stored at −20 ◦C. Before electrophoresis, the samples were centrifuged at 8905 *g*, at −5 ◦C for 15 min. Electrophoresis was performed at 6 mA/gel during the first 15 min and then at 20 mA/gel for about 45 min using the Mini-Protean Tetra System (Bio-Rad Laboratories, Hercules, CA, USA). The gels were stained for esterase activity with the substrate α-naphthyl acetate, in the dark at 37 ◦C. Protein extract from five females of *M. javanica* was included in each gel as a reference. A pure culture (designated as Fm) of RKN was established by inoculating the 5 individual egg masses onto tomato to obtain a sufficient number of second-stage juveniles for molecular characterization. Electrophoretic analysis of esterases

was repeated after two months to confirm the biochemical identification and the relative movement of each band calculated taking as reference the buffer front (Relative mobility, Rm%).

Biochemical identification was further confirmed by PCR-RFLP of mtDNA region between COII and 16S rRNA genes with C2F3 and MRH106 primers, and by SCAR-PCR with the species-specific primers Fjav and Rjav, using a pellet of second-stage juveniles (J2) obtained from egg masses of the pure isolate [31,56]. Briefly, for mtDNA region amplification, each PCR contained 1X PCR buffer, 1.8 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of each primer, 2.5 U Taq DNA polymerase (Bioline), and 50 ng DNA. Amplification was conducted using the following conditions: initial denaturation at 94 ◦C for 4 min, followed by 40 cycles of 94 ◦C for 30 s, 60 ◦C for 30 s, and 72 ◦C for 60 s, and a final extension for 10 min at 72 ◦C. After amplification, the PCR product was digested separately with 5 U *Hinf*I, *Alu*I and *Dra*III. For SCAR-PCR, the PCR reactions were the same as for the mtDNA region, except the primers (0.3 μM of each primer) and the amplification conditions (35 cycles of denaturation at 94 ◦C for 30 s, annealing at 52 ◦C for 30 s and extension at 72 ◦C for 1 min).

#### *3.2. Spiral Nematode Characterization*/*Identification*

*Helicotylenchus* specimens from soil and roots were propagated on the same tomato cultivar, in a growth chamber. Two/three months after inoculation, with approximately 3000 specimens, nematodes were extracted from roots/soil, according to the generalist Tray Method [54] and used to *Helicotylenchus* species characterization/identification and isolate maintenance, respectively. The characterization and identification of the *Helicotylenchus* species was based on the morphological characters of 10 females (body shape after relaxed with gentle heat and number of incisures) and ribosomal DNA (rDNA) sequencing.

DNA was extracted and purified from 20 spiral nematodes, extracted from tomato roots, using the DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions, and the D2D3 expansion region of the 28S rDNA gene was amplified using D2A (5 -ACA AGT ACC GTG AGG GAA AGT TG-3 ) and D3B (5 -TCG GAA GGA ACC AGC TAC TA-3 ) primers [57]. The PCR products were analysed on 1% agarose gel stained with GreenSafe (Nzytech), purified from the gel with the MiniElute Gel Extraction kit (QIAGEN, Valencia, CA, USA), quantified using the NanoDrop 2000C spectrophotometer (Thermo Scientific), cloned and sequenced. Sequences were compared with available close *Helicotylenchus* spp. sequences in GenBank [41]. Sequences were aligned using CLUSTALW multiple alignment in BIOEDIT software [58]. The evolutionary history was inferred using the neighbor-joining (NJ) and maximum likelihood (ML) methods in MEGA 7, as described in Santos et al. [10,59].

**Supplementary Materials:** The following materials are available online at http://www.mdpi.com/2223-7747/ 9/9/1085/s1, Figure S1: Multiple sequence alignment of Fm *Helicotylenchus* (FmHe1, FmHe2 and FmHe3) and available close *Helicotylenchus* spp. (*H. dihystera*—AB933469.1, MH156808.1, MH142614.1, KX822142.1, KF486503.1, KF443217.1; *H. pseudorobustus*—KM506820.1, HM014280.1, DQ328751.1; *H. microlobus*—KM506806.1, KM506805.1, KM506804.1; *H. leiocephalus*—HM014269.1, HM014268.1) sequences of D2D3 expansion region of the 28S rDNA gene (560 bp), Table S1: Pairwise sequence divergences between Fm *Helicotylenchus* and available close *Helicotylenchus* spp. sequences in GenBank of D2D3 expansion region of the 28S rDNA gene using MEGA7.

**Author Contributions:** Conceptualization, D.S. and C.M.; methodology, D.S., I.A. and C.M.; software, C.M.; validation, D.S., I.A. and C.M.; formal analysis, I.A.; investigation, D.S. and C.M.; resources, I.A. and C.M.; data curation, D.S. and C.M.; writing—original draft preparation, D.S.; writing—review and editing, I.A. and C.M.; visualization, D.S., I.A. and C.M.; supervision, I.A. and C.M.; project administration, I.A. and C.M.; funding acquisition, I.A. and C.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by CFE, Department of Life Sciences, UC, and CIEPQPF, Department of Chemical Engineering, UC, and FEDER funds through the Portugal 2020 (PT 2020) "Programa Operacional Factores de Competitividade 2020" (COMPETE 2020) and by "Fundação para a Ciência e a Tecnologia" (FCT, Portugal), under contracts UIDB/04004/2020, UIDB/00102/2020, POCI-01-0145-FEDER-031946 (Ref. PTDC/ASP-PLA/31946/2017), POCI-01-0145-FEDER-029392 (Ref. PTDC/ASP-PLA/29392/2017), Project ReNATURE—Valorization of the Natural Endogenous Resources of the Centro Region (Centro2020, Centro-01-0145-FEDER-000007) and by "Instituto do Ambiente, Tecnologia e Vida". Duarte Santos is funded by a doctoral Fellowship financed by FCT (SFRH/BD/146196/2019).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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

**Leidy Rusinque 1, Filomena Nóbrega 1, Laura Cordeiro 2, Clara Serra <sup>3</sup> and Maria L. Inácio 1,4,\***


**Abstract:** 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.

**Keywords:** identification; EST phenotype; root-knot nematodes; mtDNA

#### **1. Introduction**

Potato, *Solanum tuberosum*, is the third most important crop in the world after rice and wheat, with more than 156 countries producing it, and hundreds of millions of people depending on it for survival. According to Food and Agriculture Organization of the United Nations FAO estimates, in 2018, over 368 million metric tons of potatoes were produced worldwide, a substantial increase from 333.6 million metric tons in 2010. China is the biggest producer of potatoes worldwide, with an estimated production of 91 million metric tons, while Europe accounts for 106 million metric tons [1]. In Portugal, this crop has great social and economic importance, since it is grown throughout the country, including the archipelagos of Madeira and the Azores. On average, 430,000 metric tons of potato are produced, with the most representative production areas being Bragança, Chaves, Aveiro, Viseu, Oeste Region, and Montijo [2].

Plant-parasitic nematodes are a hampering factor in potato production and quality. Many species have been reported to be associated with potato, among which are the potato cyst nematodes *Globodera* sp., the root-knot nematodes (RKN) *Meloidogyne* sp., the lesion nematodes *Pratylenchus* sp., the potato-rot nematode *Ditylenchus destructor*, and the false root-knot nematode *Nacobbus aberrans* [3].

**Citation:** Rusinque, L.; Nóbrega, F.; Cordeiro, L.; Serra, C.; Inácio, M.L. First Detection of *Meloidogyne luci* (Nematoda: Meloidogynidae) Parasitizing Potato in the Azores, Portugal. *Plants* **2021**, *10*, 99. https://doi.org/10.3390/plants 10010099

Received: 22 November 2020 Accepted: 4 January 2021 Published: 6 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional clai-ms in published maps and institutio-nal affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

RKN are one of the oldest known parasitic nematodes of plants and considered serious pests of economically important crops [4,5]. The genus comprises more than 90 species [6] and many have been reported in Portugal: *Meloidogyne arenaria* (Neal, 1889) Chitwood, 1949; *Meloidogyne chitwoodi* Golden et al., 1980; *Meloidogyne enterolobii* Yang and Eisenback, 1983; *Meloidogyne hapla* Chitwood, 1949; *Meloidogyne hispanica* Hirschmann, 1986; *Meloidogyne incognita* (Kofoid and White, 1919) Chitwood, 1949; *Meloidogyne javanica* (Trub, 1885) Chitwood, 1949); *Meloidogyne luci* Carneiro et al., 2014; *Meloidogyne lusitanica* Abrantes and Santos, 1991; and *Meloidogyne naasi* Franklin, 1965 [7–11].

*Meloidogyne luci* was first described in 2014 on different plant species in Brazil, Chile, and Iran [12]. Due to its morphological resemblance and similar esterase (EST) phenotype to *M. ethiopica*, several populations of *M. ethiopica* in Europe were reclassified and identified as *M. luci* using biochemical and molecular analyses. In Portugal, it was detected in 2013 in a potato field near Coimbra [9] and was recently found parasitizing tomato, *Solanum lycopersicum,* the ornamental plant *Cordyline australis*, and the weed *Oxalis corniculata* [13]. Since *M. luci* is a polyphagous species with many of its host plants being of economic importance, it poses a risk to agricultural production, especially for potato. Furthermore, its detection in Europe shows that it has the potential to enter the region and survive under temperate conditions [14]. For those reasons, in 2017 *M. luci* was added to the European Plant Protection Organization (EPPO) alert list and in 2019 a national survey was implemented aiming to avoid dispersion.

The aim of the present study was morphological, morphometric, biochemical and molecularly characterize the isolate of RKN *M. luci* found in the Azores islands.

#### **2. Results and Discussion**

Morphological characterization from the recovered second-stage juveniles (J2), males and females of the isolate of *M. luci* was performed, as were morphometric studies on J2 (Table 1).

**Table 1.** Morphometric comparison of second-stage juveniles (J2) of *Meloidogyne luci* from the Azores, Portugal, with the original description (Carneiro et al., 2014). All measurements are in μm and in the format mean ± standard deviation (range).


\* length/max. body width; \*\* length/tail length.

#### *2.1. Morphological and Morphometric Characterization*

The J2 were vermiform, slender, and clearly annulated. The head region was slightly set apart from the body. The stylet was delicate, narrow, and sharply pointed; the knobs were small and oval shaped. The excretory pore was distinct and the hemizonid was anteriorly adjacent to the excretory pore. The tail was conoid with a rounded tip and the hyaline terminus was distinctive (Figure 1a–d).

**Figure 1.** *Meloidogyne luci* light microscope observations. Second-stage juvenile: (**a**) whole specimen; (**b**) anterior region, (**c**) tail region. Female: (**d**) egg-laying female, whole specimen; (**e**) anterior end; (**f**) perineal pattern (bar = 20 μm).

Females were elongated, ovoid, or pear-shaped, with a prominent neck (Figure 1d). The head was slightly set apart from the body. The stylet was robust, with knobs well developed. The stylet cone was wider near the shaft and the shaft was wider near the junction with knobs (Figure 1e). The perineal pattern was oval to squarish, with the dorsal arch high to low and rounded. The striae were smooth and wavy, widely separated, and continuous. The lateral lines were weakly demarcated and the perivulval region was free from striae (Figure 1f). The patterns were found to be highly variable.

The males were vermiform, bluntly rounded posteriorly, and with an anterior end narrowing. The body cuticle was annulated, with large annuli. The head region was not set off from the body. The stylet was robust, and the cone was larger than the shaft and increased in width near the junction with the shaft. The knobs were rounded and small, merging gradually into the shaft. The tail was short and the spicules were curved.

Morphological features are valuable tools for RKN identification due to their low cost and ease of learning the skills, with accuracy depending on the number of characteristics to be evaluated and the number of specimens. The species identification of *Meloidogyne* based on these characters is nevertheless a challenge because morphological differences between RKN species are in most cases indistinctive and measurements of individual specimens in general overlap.

The morphology and morphometrics were compared to the description of *M. luci* made by [9] and the results were consistent (Table 1). However, due to the intraspecific variability, its identification became difficult; for instance, characteristics such as the morphology of the perineal pattern were highly variable and could be found in more than one species. Therefore, morphological and biometrical diagnostic characteristics need to be supported by other studies, such as biochemical and molecular.

#### *2.2. Biochemical Characterization*

The EST phenotype from young egg-laying females exhibited three bands (relative mobility, Rm: 1, 1.10, 1.20), corresponding to the *M. luci* L3 phenotype [11]. *Meloidogyne ethiopica* also presented an EST phenotype of three bands (Rm: 0.93, 1.13, 1.24). The three EST bands

observed in *M. javanica* (Rm: 1, 1.17, 1.26) were used as a reference to determine the relative position of *M. luci* and *M. ethiopica* bands (Figure 2).

**Figure 2.** (**a**) Phenotypes of protein homogenates from one egg-laying female of the *Meloidogyne* species: C—Positive control *M. luci*; 1: L3—*M. luci* esterase (Azores), J3—*M. javanica*, and E3—*M. ethiopica*, and (**b**) relative mobility L3—*M. luci*, J3—*M. javanica*, and E3—*M. ethiopica*.

> Many studies have shown the usefulness of the nonspecific EST phenotype as the quicker, more reliable, and more stable method to identify *Meloidogyne* spp. [15,16]. The EST phenotype found in the isolate from the Azores was similar to the Portuguese isolate found parasitizing potato in Continental Portugal in 2013. The first band was located at the same level of the band of the reference *M. luci* and *M. javanica*. Since *M. ethiopica* was included for comparison, it could be clearly seen that this first band was well above. Therefore, in spite of the similarity between *M. luci* and *M. ethiopica,* the patterns have clear differences and can be consider reliable in the identification of these two species.

#### *2.3. Molecular Characterization*

The PCR amplification of mtDNA COII/16S rRNA yielded a single fragment of 1800 bp. The nucleotide sequence obtained in this study was deposited into the GenBank database (NCBI) under the accession number MW160418. A BLAST search of the nucleotide sequence showed a similarity of 99.17% with the sequences of *M. luci* available in the database.

The molecular phylogenetic analysis is presented in Figure 3. The phylogram revealed one clade, supported by a bootstrap value of 93%, that included all isolates of *M. luci* from other countries, the isolate from the Azores, and the isolates of *M. ethiopica*. The isolates of *M. javanica, M. incognita*, and *M. arenaria* formed separate major clades with bootstrap values of 81, 97, and 99%, respectively.

According to [17], the region of mtDNA COII/16S rRNA is useful in the identification of the closely related species *M. luci* and *M. ethiopica*. In this study, that region allowed us to identify the isolate from the Azores as *M. luci*. However, due to the closeness between the species, the molecular markers needed to be used in combination with biochemical analysis.

In general, the eradication of nematodes is very difficult, and it is even more so when it comes to the RKN species, especially *M. luci*. Its ability to adapt to temperate conditions and a wide variety of hosts make its management a challenge. Additionally, several nematicides have recently been strictly regulated or banned in the EU, due to the adverse impacts on the environment and human health, reducing the alternatives for control. Therefore, to define sustainable management strategies, an accurate diagnosis and knowledge of the species is required, with the combination of biochemical and molecular analysis being the best approach for RKN species identification. Furthermore, not only does the identification have constrains, but the detection does as well, as occurred in this

study. Plants may either not present any symptoms, or they can often be misdiagnosed, as symptoms may appear similar to other factors.

**Figure 3.** Phylogenetic relationships of *Meloidogyne luci* isolate collected from the Azores, Portugal, and *M. luci* isolates from other geographical regions, including other species of the *Meloidogyne* group, based on the sequence alignment of the mtDNA region between COII and 16S genes. The dendrogram was inferred by using the maximum likelihood method and the Hasegawa–Kishino–Yano model with 1000 bootstrap replication. Bootstrap values are indicated at the nodes. The analysis involved 30 nucleotide sequences and there was a total of 1596 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. \* Recently reclassified as *M. luci*, according to [16].

> Finally, due to the threats and problems presented above, to evaluate the distribution and potential impact of this nematode, a national survey was implemented after 2019 in Continental Portugal and the Azores.

> To our knowledge, this is the first report of *M. luci* in the islands of the Azores, Portugal, adding valuable information to the current location of this organism in the EPPO zone.

#### **3. Materials and Methods**

#### *3.1. Nematode Isolates*

During the 2019 National Survey in the Azores islands, soil samples were collected from the council of Santo António on Pico Island. Each consisted of 5 to 8 cores sampled at roughly equal intervals. Six composite soil samples were placed in polyethylene bags and brought for analysis. A 400 mL subsample was taken from each composite sample and the nematodes were extracted using sieving and decanting together with centrifugal technique according to protocol PM 7/119 (1) [18]. The suspension was observed under a stereomicroscope (Nikon SMZ1500, Tokyo, Japan) and suspect specimens of *Meloidogyne* were observed using a bright-field light microscope (Olympus BX-51, Hamburg, Germany) for confirmation.

For positive detections of *Meloidogyne* it was necessary to perform bioassays in order to obtain material (females, egg masses, and males) for identification. Bioassays were carried out by planting tomato plants cv. Oxheart in the remaining soil from the analyzed sample and maintained in a quarantine greenhouse for two months. Females and egg masses were handpicked from the infected tomato roots.

#### *3.2. Morphological and Morphometric Characterization*

Nematodes were placed in a drop of water on a glass slide and gently heat killed for morphological and morphometric characterization using a bright-field light microscope (Olympus BX-51, Hamburg, Germany) and photographed with a digital camera (Leica MC190 HD, Wetzlar, Germany). The measurements were taken using the Leica LAS Live. Perineal patterns of adult females were cut from live specimens in 45% lactic acid and mounted in glycerine.

#### *3.3. Biochemical Characterization*

Young egg-laying females were handpicked from infected tomato roots and transferred to micro-hematocrit capillary tubes with 5 μL of extraction buffer (20% sucrose *v*/*v* and 1% Triton X-100 *v*/*v*). The females were macerated with a pestle, frozen, and stored at −20 ◦C until use. Proteins were separated by polyacrylamide gel electrophoresis (PAGE) on thin-slab 7% separating polyacrylamide gels, in a Mini-Protean II (BioRad Laboratories, Hercules, CA, USA) according to [19]. The gels were stained for EST activity with the substrate α-naphthyl acetate. Protein extracts from young egg-laying females of *M. ethiopica* and *M. luci* were included in each gel for comparison and a protein extract of an isolate of *M. javanica* was used as a reference.

#### *3.4. Molecular Characterization*

The mtDNA COII/16S rRNA region was selected for molecular characterization of the *M. luci* isolate from the Azores islands. The total DNA was extracted from the egg masses using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The mtDNA COII/16S rRNA region was amplified using the primers C2F3 (5 -GGTCAATGTTCAGAAATTTGTGG-3 ) and 1108 5 -TACCT TTGACCAATCACGCT-3 [20]. PCR reactions were performed in a 50 μL final volume mixture containing 25 μL Supreme NZYTaq II Green Master Mix, 10 μL of isolated DNA, and 0.2 μM of each primer in a Biometra TGradient thermocycler (Biometra, Göttingen, Germany). Thermal cycling conditions were as described by [17]. PCR products were resolved by electrophoresis at 5 V.cm−<sup>1</sup> in agarose gel (1.5%) containing 0.5 μg/mL ethidium bromide and 0.5x Tris-borate-EDTA (TBE) running buffer. Amplifications were visualized using the VersaDoc Imaging System (BioRad Laboratories, Hercules, CA, USA). PCR products were purified using the DNA clean and concentrator kit (Zymo Research Corp, Irvine, CA, USA), according to the manufacturer's instructions. Amplicons were sequenced in both directions at STABVida Sequencing Laboratory (Lisbon, Portugal) on a DNA analyzer ABI PRISM 3730xl (Applied Biosystems). The newly obtained sequence was manually checked, edited, and assembled. The sequence was compared to those of *M. luci* and other relevant sequences of *Meloidogyne* spp. available in the GenBank database using the BLAST homology search. The multiple alignment of the retrieved sequences was performed using ClustalW multiple alignment in BioEdit (Figure S1).

Phylogenetic analyses were conducted using MEGA X v10.1 [21] and the maximum likelihood (ML) method based on the Hasegawa–Kishino–Yano model. The robustness of the ML tree was inferred using 1000 bootstrap replicates.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2223-7 747/10/1/99/s1, Figure S1: Alignment of *M. luci* isolate from the Azores islands and available sequences on GenBank.

**Author Contributions:** Conceptualization, L.R., F.N., and M.L.I.; methodology, L.R., F.N., C.S., L.C., and M.L.I.; software, F.N.; validation, L.R., F.N., C.S., L.C., and M.L.I.; formal analysis, M.L.I. and F.N.; investigation, L.R., F.N., and M.L.I.; resources, L.R., F.N., C.S., L.C., and M.L.I.; data curation, L.R., F.N., and M.L.I.; writing—original draft preparation, L.R.; writing—review and editing, F.N., C.S., L.C., and M.L.I.; visualization, L.R., F.N., C.S., L.C., and M.L.I.; supervision, F.N., C.S., and M.L.I.; project administration, F.N., C.S., L.C., and M.L.I.; funding acquisition, F.N., C.S., L.C., and M.L.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Funds through the FCT—Foundation for Science and Technology through the R&D Unit, UIDB/04551/2020 (GREEN-IT—Bioresources for Sustainability).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in Figure S1: Alignment of *M. luci* isolate from the Azores islands and available sequences on GenBank.

**Acknowledgments:** The authors would like to thank the staff at the Nematology Laboratory at INIAV—Nema-INIAV and the Laboratory of Molecular Genetics.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Article Pratylenchus vovlasi* **sp. Nov. (Nematoda: Pratylenchidae) on Raspberries in North Italy with a Morphometrical and Molecular Characterization †**

**Alberto Troccoli 1, Elena Fanelli 1, Pablo Castillo 2, Gracia Liébanas 3, Alba Cotroneo <sup>4</sup> and Francesca De Luca 1,\***


**Abstract:** Root-lesion nematode species rank third only to root-knot and cyst nematodes as having the greatest economic impact on crops worldwide. A survey of plant-parasitic nematodes associated with decaying raspberries (*Rubus* sp.) in northern Italy revealed that root-lesion nematodes were the most frequently occurring species among other phytonematodes. Several *Pratylenchus* species have been associated with *Rubus* sp. in Canada (Quebec, British Columbia) and USA (North Carolina, Maryland, New Jersey) including *P. penetrans* and *P. crenatus.* In the roots and rhizosphere of symptomatic raspberries, nematodes of two *Pratylenchus* spp. were detected. Detailed morphometrics of the two root-lesion nematode isolates were consistent with *Pratylenchus crenatus* and with an undescribed *Pratylenchus* species. The extracted nematodes were observed and measured as live and fixed materials and subsequently identified by integrative taxonomy (morphometrically and molecularly). The latter species is described herein as *Pratylenchus vovlasi* sp. nov., resulting morphometrically closest to *P. mediterraneus* and phylogenetically to *P. pratensis*. The molecular identification of *Pratylenchus vovlasi* sp. nov. was carried out by sequencing the ITS region, D2-D3 expansion domains of the 28S rRNA gene and a partial region of the nuclear *hsp90* gene. ITS-RFLP and sequence analyses revealed that *Pratylenchus vovlasi* sp. nov. had species-specific restriction profiles with no corresponding sequences present in the database. The phylogenetic relationships with ITS and D2-D3 sequences placed the *Pratylenchus vovlasi* sp. nov. in a clade with *P. pratensis* and *P. pseudopratensis*. This research confirms the occurrence of cryptic biodiversity within the genus *Pratylenchus* as well as the need for an integrative approach to the identification of *Pratylenchus* species.

**Keywords:** Bayesian inference; D2-D3 expansion domains of the 28S rRNA gene; *hsp90* gene; integrative taxonomy; ITS

#### **1. Introduction**

Raspberries (*Rubus* sp.) have a long history of human consumption and cultivation in Europe, with Russia being the leading producer [1]. Raspberries have been eaten fresh for thousands of years. In the last decades, there has been an increase in the demand for fruit and fruit-based products as consumers seek out healthier dietary options. In particular, berries are considered one of the best dietary sources of bioactive compounds that have

**Citation:** Troccoli, A.; Fanelli, E.; Castillo, P.; Liébanas, G.; Cotroneo, A.; De Luca, F. *Pratylenchus vovlasi* sp. Nov. (Nematoda: Pratylenchidae) on Raspberries in North Italy with a Morphometrical and Molecular Characterization . *Plants* **2021**, *10*, 1068. https://doi.org/10.3390/ plants10061068

Academic Editors: Carla Maleita, Isabel Abrantes and Ivânia Esteves

Received: 15 April 2021 Accepted: 21 May 2021 Published: 26 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

important antioxidant properties, with associated health effects such as protective effects against several cancers and cardiovascular disorders [2,3] and thus the berry market is expected to expand during the next years. Raspberries are the most productive in areas with mild winters and long, moderate summers. The production of raspberries is greatly influenced by biotic and abiotic factors. Among the biotic factors, plant-parasitic nematodes have an important role in the reduction of the raspberry yield [4,5]. Several genera of plantparasitic nematodes have been found to be associated with the raspberry [4–9]. The most frequently observed species in soil and the roots of raspberries belong to the root-lesion nematode genus *Pratylenchus*, mainly *P. crenatus* Loof, 1960, (71.7% of soil samples, 53.4% of root samples), *P. penetrans* (Cobb, 1917) Filipjev and Schuurmans Stekhoven, 1941 (51.6%, 54.2%) and *P. scribneri* Steiner in Sherbakoff and Stanley 1943 (9.2%, 10.2%; [9]). In Italy, raspberries are grown mainly in the northern areas including Trentino Alto Adige, the main production area, Verona province and Piedmont. Other Italian areas growing raspberries are in Romagna in the north and Calabria, Sicily, Campania and Basilicata in the south. Considering the economic significance of root-lesion nematodes on raspberries and the need to accurately distinguish these damaging species for their practical management in the field, we provide here the morphometrical and molecular characterization of a new root-lesion nematode, *Pratylenchus vovlasi* sp. nov. parasitizing raspberries in the Piedmont area. The specific objectives of this paper were: (i) to carry out a comprehensive identification with morphological and morphometric approaches of *P. vovlasi* sp. nov. with a differential diagnosis to closely related species; (ii) to provide a molecular characterization of *P. vovlasi* sp. nov. and estimate its phylogenetic relationships with other representatives of the *Pratylenchus* genus using three molecular markers: two ribosomal markers (the D2-D3 expansion segments of the 28S rRNA and the internal transcribed spacer region (ITS rRNA) and the nuclear region of the partial heat shock protein 90 (*hsp*90) gene.

#### **2. Results**

Two species were identified from raspberries in the Piedmont region. Detailed morphometrics and molecular analyses of the two root-lesion nematode populations were consistent with *P. crenatus* and with an undescribed *Pratylenchus* species. As *P. crenatus* is a well-known and well molecular characterized species, we concentrated our efforts on the integrative taxonomic identification of the undescribed species.

#### *2.1. Molecular Characterization*

For molecular analyses, a total of five individual specimens for each molecular marker were amplified. The D2-D3 expansion domains of *28S* rRNA, *ITS* rRNA and partial *hsp90* genes of the new species *P. vovlasi* sp. nov. yielded single fragments of ~800 bp, 700 bp and 300 bp, respectively, based on a gel electrophoresis. The D2-D3 for *P. vovlasi* sp. nov. (OA984892–OA984893) showed a very low intraspecific variability with one different nucleotide and no indels (99% similarity). The D2-D3 for *P. vovlasi* sp. nov. differed from the closest related species, *P. pseudopratensis* Seinhorst, 1968 (JX261965) by 18 nucleotides and 0 indels (97% similarity), *P. pratensis* (De Man, 1880) Filipjev, 1936 (KY828298) by 23–26 nucleotides and 0 indels (96–97% similarity) and from *P. vulnus* (JQ003993) by 72–73 nucleotides and four to ten indels (89–90% similarity).

The ITS region for *P. vovlasi* sp. nov. also showed a low intraspecific variability by 19 nucleotides and 6 indels (97% similarity). The ITS1 for *P. vovlasi* sp. nov. (OA984869– OA984870) showed a low similarity with all of the ITS sequences of *Pratylenchus* spp. deposited in NCBI including the most similar species, *P. pratensis* (=*P. lentis*) (AM933158, AM933147, AM933149) and *P. fallax* Seinhorst, 1968, (FJ719921, FJ719917), by 97–98 different nucleotides and 34–39 indels (86% similarity).

The partial *hsp*90 gene amplified products of two individual specimens were cloned and four clones for each individual specimen were sequenced (OA984937–OA984944). The sequence analyses revealed the occurrence of two different fragments for each specimen and both fragments coded for *hsp90* differed in the length of the intron (52 bp vs. 43 bp) and

nucleotide variability. The 298 bp fragment showed an 82% similarity (254/310 identities) with the 310 bp fragment at a nucleotide level. At an amino acid level, the two fragments showed 96% identities (81/84) and 98% positives (83/84). The new *hsp90* sequences for *P. vovlasi* sp. nov. showed a high intraspecific variability by 3–60 nucleotides and 0–13 indels (81–99% similarity). The *hsp90* sequences for *P. vovlasi* sp. nov. differed by 13, 16 and 61 nucleotides and 4, 4 and 24 indels from the most closely related species, *P. speijeri* De Luca, Troccoli, Duncan, Subbotin, Waeyenberge, Coyne, Brentu and Inserra, 2012 (HE601547) with a 96% similarity, *P. coffeae* (Zimmermann, 1898) Filipjev and Schuurmans Stekhoven, 1941 (HE601548) with a 95% similarity and *P. hippeastri* Inserra, Troccoli, Gozel, Bernard, Dunn and Duncan, 2007 (HE601549) with an 81% similarity, respectively.

#### *2.2. Restriction Profiles*

PCR-RFLP analyses of the ITS region allowed us to determine the species-specific patterns for the Italian population of *P. vovlasi* sp. nov. (Figure 1) that clearly identified this species.

**Figure 1.** Restriction fragments of an amplified ITS of *Pratylenchus vovlasi* sp. nov. Al: *Alu*I, Av: *Ava*II, B: *Bam*HI, D: *Dde*I, H: *Hinf*I, R: *Rsa*I and M: 100 bp ladder.

#### *2.3. Phylogenetic Relationships*

The phylogenetic relationships among *Pratylenchus* species inferred from the analyses of the D2-D3 expansion domains of 28S rRNA, ITS and the partial *hsp90* gene sequences using BI are shown in Figures 2–4, respectively. The phylogenetic trees generated with the ribosomal and nuclear markers included 54, 60 and 31 sequences with 693, 548 and 284 positions in length, respectively (Figures 2–4). The D2-D3 tree of *Pratylenchus* spp. showed a well-supported subclade (PP = 1.00) including *P. vovlasi* sp. nov., *P. pratensis* and *P. pseudopratensis* (Figure 2). All other clades followed the same pattern as previous studies on *Pratylenchus* species.

**Figure 2.** Phylogenetic relationships of *Pratylenchus vovlasi* sp. nov. within the genus *Pratylenchus*. A Bayesian 50% majority-rule consensus tree as inferred from the D2 and D3 expansion domains of the 28S rRNA sequence alignment under the general time-reversible model of the sequence evolution with a correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

The 50% majority-rule consensus ITS BI tree showed several clades that were not well-defined (Figure 2) but a well-supported subclade (PP = 1.00) including *P. vovlasi* sp. nov. and *P. pratensis* (Figure 3). This subclade was phylogenetically related to *P. vulnus* Allen and Jensen, 1951 and *P. kumamotoensis* Lal and Khan, 1990 in a moderately supported subclade (PP = 0.95) (Figure 3). Finally, the *hsp90* BI tree confirmed the occurrence of two isoforms within *P. vovlasi* sp. nov. that were clearly separated in two independent well-supported subclades (PP = 0.98 and PP = 1.00, respectively) (Figure 4).

*2.4. Morphology and Morphometry of Pratylenchus vovlasi sp. nov.*

*Pratylenchus vovlasi* **sp. nov.** (http://zoobank.org/urn:lsid:zoobank.org:act:3B91A2 A8-B809-47BA-A822-CF380EBC0245, accessed on 2 April 2021).

**Figure 3.** Phylogenetic relationships of *Pratylenchus vovlasi* sp. nov. within the genus *Pratylenchus*. A Bayesian 50% majority-rule consensus tree as inferred from the ITS sequence alignment under a transversional model with a proportion of invariable sites and a rate of variation across sites (TVM + I + G). Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are in bold letters. Scale bar = expected changes per site.

#### 2.4.1. Description

Female: the body assumes an almost straight to open C posture when heat-killed (Figure 5A). The lip region is slightly offset from body contour and bears three annuli, which narrow in diameter towards the anterior end (Figures 5D and 6D). In the en face SEM view lip region, they appear dumb-bell-shaped (Figure 7A,B,D,I) with an acute pattern (sensu Subbotin et al. [10]) fitting the group II according to the classification scheme of Corbett and Clark [11]. The stylet is relatively small and delicate with conus about 45 ± 2.5 (41–49) % of the entire stylet length (Table 1). The stylet shaft is slender ending with rounded basal knobs, which are slightly anteriorly flattened. The pharyngeal procorpus narrows just anterior to the small, oval metacorpus. The valve of the median bulb is conspicuous. The isthmus is short, encircled by a nerve ring and widening to a pharyngeal lobe with a dorsal nucleus just posterior to the cardia and ventro-sublateral nuclei near the tip of the pharyngeal lobe (Figure 5B,C) and overlapping the intestine ventro-laterally for almost a two-body diameter at the cardia level. The secretory-excretory pore is level with the cardia just posterior to the hemizonid. The body annulation is distinct; the lateral field usually with four smooth incisures and a few specimens have an additional line (Figure 6K) or more rarely a few oblique striae (Figure 6J) in the middle of the central band. The outline of outer bands becomes indented towards the tail end, posterior to phasmid, with the inner lines fusing just posterior to the phasmid. The genital tract is well developed with oocytes arranged in a single row. The spermatheca is large and spherical to oval, usually full of sperm (Figure 5F,E,G); its posterior margin is 50.0 ± 9.6 (36.5–64.5) μm from the vagina (Table 1). The vulva is slightly sunken and the vulval lips are not prominent (Figure 5E,F). The post-uterine sac is about 1.1 vulval body diameter long and is usually undifferentiated. The phasmids are located to the mid-tail, 13.2 ± 2.2 (11–17) μm from the tail tip. The tail is typically subcylindrical, tapering towards the tip with a rounded-truncate terminus in most specimens; a few specimens have broadly rounded, conical tails with coarsely pointed or indented striated termini (Figures 5H,I and 6F,H,I,L).

**Figure 4.** Phylogenetic relationships of *Pratylenchus vovlasi* sp. nov. within the genus *Pratylenchus*. A Bayesian 50% majority-rule consensus tree as inferred from the *hsp90* sequence alignment under the general time-reversible model of the sequence evolution with a correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are in bold letters. Scale bar = expected changes per site.

> Male: similar to the female except in the posterior end of the body and in a slightly smaller and usually slender body length. The lip region is usually higher and narrower than in the female (Figure 6B,C). The stylet is slightly smaller than that of female with smaller, more rounded knobs. The pharyngeal bulb is small and round; the isthmus is slender and rather short ending in a long, narrow glandular lobe. The testis is outstretched and filled with round spermatozoa in the vas deferens. The spicules are paired, weakly cephalated and ventrally arcuate. The gubernaculum is simple and slightly curved. The tail is conical and bent on the ventral side with a prominent, crenate bursa.

#### 2.4.2. Type Host and Locality

*Pratylenchus vovlasi* sp. nov. was found associated with the roots and soil of *Rubus* sp. with a population density of 650 nematodes/250 cm<sup>3</sup> of soil in the locality of Prarostino, Turin province, Piedmont Region, north Italy.

**Figure 5.** Line drawings of *Pratylenchus vovlasi* sp. nov. (**A**) entire female and male; (**B**–**C**) the female (**B**) and the male (**C**) pharyngeal regions; (**D**) detail of the female lip region; (**E**) detail of the female posterior region; (**F**) the female reproductive system with details of the lateral field; (**G**) en face view; (**H**,**I**) the female tail; (**J**) the male tail.

#### 2.4.3. Type Material

Holotype, female and male paratypes mounted on glass slides were deposited in the nematode collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), CNR, Bari, Italy (collection numbers IPSP-M-1238-1250). The additional paratypes were distributed to the United States Department of Agriculture Nematode Collection, Beltsville, MD, USA (collection number IPSP-M-1237), WaNeCo Plant Protection Service, Wageningen, The Netherlands, (collection number IPSP-M-1246) and Instituto de Agricultura Sostenible, CSIC, Córdoba, Spain (collection number IPSP-M-1245).

#### 2.4.4. Diagnosis and Relationships

*Pratylenchus vovlasi* sp. nov. is characterized by a lip region slightly offset with three annuli narrowing towards the anterior end, sub-median sectors fused with the oral disc and separated by a lateral sector to give a dumb-bell -shaped pattern; the stylet is rather small (15.0 μm long) with rounded knobs and a short pharyngeal overlap. The lateral field has four smooth incisures in most specimens, the spermatheca is round to oval and is usually full of sperm, the vulva is located in a relative anterior position and the tail is subcylindrical with a truncate, smooth terminus and with common males. The matrix code of the new species according to Castillo and Vovlas [12] is A2 B2, C2, D2, E2, F3, G1,2, H1, I2, J1, K1 (Table 2).

Related species sharing with *P. vovlasi* sp. nov. a three lip annuli, a divided face as seen with SEM (Group 2 according to Corbett and Clark [11]), a functional spermatheca and numerous males include *P. bhattii* Siddiqi, Dabur and Bajaj, 1991, *P. kralli* Ryss, 1982, *P. mediterraneus* Corbett, 1983, *P. thornei* Sher and Allen, 1953, *P. pratensis*, *P. pseudopratensis* Seinhorst, 1968, *P. penetrans*, *P. fallax* and *P. convallariae* Seinhorst, 1959.

**Figure 6.** Light photomicrographs of *Pratylenchus vovlasi* sp. nov.: (**A**) entire female and male; the female (**B**) and the male (**C**) anterior regions; (**D**) the female lip region; (**E**,**G**) detail of the vulval region showing the spermatheca; (**F**,**H**,**I**,**L**) detail of the female tail (arrow in H shows the anus); (**J**,**K**), detail of the lateral field incisures showing the oblique striae (arrowed in J) or an additional line in the central band (arrowed in K); (**M**,**N**), detail of the male tail. Scale bars: A = 100 μm; B, C, E–M = 20 μm; D = 10 μm.

**Table 1.** Morphometrics of the holotype and paratypes of *Pratylenchus vovlasi* sp. nov. All measurements are in μm and in the form: mean ± S.D. (range).



**Table 1.** *Cont.*

Abbreviations: a = body length/greatest body diameter; b = body length/distance from the anterior end to the pharyngo-intestinal junction; DGO = distance between the stylet base and the orifice of the dorsal pharyngeal gland; c = body length/tail length; c' = tail length/tail diameter at the anus or cloaca; G1 = anterior genital branch length expressed as a percentage (%) of the body length; L = overall body length; *n* = number of specimens on which measurements are based; o = distance from the stylet base to the dorsal esophageal gland outlet × 100/total stylet length; T = distance from the cloacal aperture to the anterior end of the testis expressed as a percentage (%) of the body length; V = distance from the body anterior end to the vulva expressed as a percentage (%) of the body length; PUS = Post uterine sac length.

> *P. vovlasi* sp. nov. is most closely related to *P. mediterraneus*, matching 10 out of 11 characteristics according to the matrix code [12] and differing from it just by a slightly shorter pharyngeal overlap (32 ± 6.7 (20–43) vs. 25–55 μm, code I2 vs. I3 in the tabular key), a face pattern showing transverse incisures in the middle of the sub-medial sectors (not present in *P. mediterraneus*), lateral sectors fused (vs. separated) with the oral disc, smooth lateral fields, not areolated vs. crenate (outer bands), occasionally areolated and with the middle band variously ornamented (Figure 6J,K and Figure 7K). From *P. pratensis*, *P. fallax*, *P. penetrans* and *P. vulnus*, it differs in the en face SEM pattern (belonging to Group 2 vs. Group 3, according to Corbett and Clark, [11]) and the tail tip morphology despite a certain degree of overlap (mostly truncate and smooth vs. usually oblique and annulated in *P. pratensis*, rounded or with a slightly irregular contour in *P. fallax*, generally rounded in *P. penetrans* and narrowly rounded to subacute and occasionally irregular in *P. vulnus*). Furthermore, the new species differs from *P. pratensis* by a mostly rounded vs. oval to rectangular spermatheca and the number of tail annuli (14–20 vs. 20–28); from *P. fallax* by a shorter stylet (range: 14.3–16.3 vs. 16–17 μm) and a smaller c' value 1.8 ± 0.2 (1.4–2.3) vs. 2.5 (2.0–3.0); from *P. penetrans* by a slightly shorter stylet mean length (15 vs. 16), a slightly more anterior position of the vulva (78 (74–80) vs. 78–84%) and in a fewer number of tail annuli on the ventral surface (15.6 ± 2.0 (14–20) vs. 15–27); from *P. vulnus* by a shorter post-uterine sac (1.1 vs. ca. 2.0 vulval body diameter; code F3 vs. F6 after Castillo and Vovlas, [12] (Table 2) and a more anterior position of the vulva (78 (74–80) vs. 77–82%). From *P. pseudopratensis*, it differs in the en face SEM view (not dumb-bell-shaped

in *P. pseudopratensis*), a shorter stylet (range: 14.3–16.3 vs. 16–17 μm), the shape of the spermatheca (rectangular, sometimes empty in *P. pseudopratensis*) and the tail tip (smooth vs. crenated; code H1 vs. H2 after Castillo and Vovlas, [12]).

**Figure 7.** SEM photomicrographs of *Pratylenchus vovlasi* sp. nov.: (**A**–**D**), detail of the female lip region; (**E**,**F**), detail of the male anterior region; (**I**) the female en face view; (**G**,**H**) the vulval region in the sub-lateral (**G**) and the ventral (**H**) view; (**J**–**L**), detail of the female tail; (**M**) detail of the male tail. Scale bars: (**A**,**D**,**F**–**J**)=5 μm; (**B**,**C**,**E**) = 2.5 μm; (**K**–**M**) = 10 μm.

Other *Pratylenchus* species with three lip annuli, a functional spermatheca and the presence of males and with a matrix code (sensu Castillo and Vovlas, [12]) similar to *P. vovlasi* sp. nov. have been described without examining their lip patterns. These species include *P. convallariae*, *P. kralli* and *P. bhattii*, from which the new species differs by the following characteristics: from *P. convallariae*, by a shorter stylet (15 (14.3–16.3), feature C2 in Table 2, vs. 17 (16–18) μm feature C3), a shorter PUS (1.1, feature F3 vs. more than 1.4–2 vulval body diameter, feature F6 in Table 2) and by a smooth tail tip (H1) vs. coarsely and often irregularly annulated (H2); from *P. kralli*, in the stylet knob shape (mostly rounded vs. anteriorly directed), the post-uterine sac length (1.1 vs. more than 1.5 vulval body diameter), the tail tip is truncated, smooth and more rarely pointed vs. pointed and showing a slight groove; from *P. bhattii* by a slightly longer stylet (15 (14.3–16.3) vs. 13.5 (13–14) μm) and a more posterior vulva (V = 78 vs. 73% mean value) with the vulval lips continuous with the body contour vs. raised in a prominent protuberance. Finally, the new species can be compared with *P. rwandae* Singh, Nyiragatare, Janssen, Couvreur, Decraemer and Bert, 2018, but differs from it by a labial region in the en face view showing clearly separated sub-median sectors with transverse incisures in the middle (vs. slightly separated from the lateral sectors and without transverse incisures), males present (vs. absent), females with mostly round and full spermatheca (vs. oval to rounded and empty), lateral fields with four incisures (vs. six or more at the mid-body) and a tail with a fewer number of ventral annuli (14–20) and usually a truncated tip (vs. 18–28 tail annuli and a highly variable tail tip).


**Table 2.** Polytomous key of *Pratylenchus vovlasi* sp. nov. and the morphologically most closely related species.

  present.  = 18–20; **5** = >20. **Group D: 1** = absent or reduced; **2** = rounded to spherical; **3** = oval; **4** = rectangular. **Group E: 1** = <75; **2** = 75–79.9; **3** = 80–85; **4** = >85. **Group F: 1** = <16; **2** = 16–19.9; 20–24.9; **4** = 25–29.9; **5** = 30–35; **6** = >35. **Group G: 1** = cylindrical; **2** = subcylindrical; **3** = conoid. **Group H: 1** = smooth; **2** = striated; **3** = pointed; **4** = with ventral projection. **Group I:**<30; **2** = 30–39.9; **3** = 40–50; **4** = >50. **Group J: 1** = four; **2** = five; **3** = six to eight. **Group K: 1** = smooth bands; **2** = partially or completely areolated bands. \*\* PUS = post-uterine sac length.

**3** =

 **1** =

#### 2.4.5. Etymology

The species epithet, *P. vovlasi*, is dedicated to Dr. Nikos Vovlas, an eminent Italian nematologist and taxonomist from the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy.

#### **3. Discussion**

The identification of the *Pratylenchus* species is difficult because many diagnostic characteristics overlap and also due to the increasing number of nominal species. An accurate identification is needed in order to adopt appropriate control strategies. The primary objective of this study was to identify and molecularly characterize root-lesion nematodes parasitizing raspberries cultivated in the Piedmont region. The present study reports on the occurrence of two root-lesion nematodes associated with raspberry fields in the Piedmont region, *P. crenatus*, along with an abundant species herein identified as *P. vovlasi* sp. nov. *Pratylenchus crenatus* was previously reported on raspberries in several European countries [6,9].

Our results demonstrated that the application of rRNA molecular markers integrated with morphological studies could help in the diagnosis and characterization of root-lesion nematode species. Based on the molecular characterization using the D2-D3 expansion domains of the 28S rRNA gene, ITS region and the partial hsp90 gene, the abundant species was clearly identified as *P. vovlasi* sp. nov. This species proved very similar in morphometry and morphology to *P. mediterraneus*, differing only by a shorter pharyngeal overlap, as well as with *P. thornei*, *P. penetrans*, *P. fallax* and *P. convallariae*. By blasting at NCBI the D2-D3 region, it was 96–97% similar to *P. pratensis* and *P. pseudopratensis*, respectively, while by using ITS sequences it was 85–87% similar to *P. pratensis* and *P. fallax*. These results represented an additional confirmation of the extraordinary cryptic diversity of the nematodes of the genus *Pratylenchus*, as reported in previous studies [13–17]. Phylogenetic analyses of the ITS and the D2-D3 sequences confirmed a sister relationship between *P. vovlasi* sp. nov. with *P. pratensis* and *P. pseudopratensis*. Furthermore, in both phylogenetic trees, the new species was closely related to *P. vulnus* and *P. kumamotoensis* (Figures 2 and 3). Major clades for D2-D3 and ITS phylogenetic trees were highly correlated with previous phylogenetic studies carried out by Subbotin et al. [10], Palomares-Rius et al. [14,18] and Araya et al. [17]. We agreed with De Luca et al. [13] who suggested that the ITS-containing region allowed a better discrimination among the closely related species studied because it evolved faster than the D2-D3 expansion segments of 28S rDNA and accumulated more substitution changes.

The current study confirmed the occurrence of different hsp90 isoforms in the *Pratylenchus* species as already reported by Fanelli et al. [19]. In *P. vovlasi* sp. nov. the different isoforms differed from each other in the length of the intron and the nucleotide variability grouping in two well-supported clusters. This finding suggested that the different isoforms of *P. vovlasi* sp. nov. hsp90 arose by gene duplication events relatively recently because the nucleotide variability was low and the gene structure was still conserved [20,21]. Similarly, these results confirmed that the primers for the amplification of the hsp90 gene could also amplify other paralogous genes in Pratylenchus and could be used for species delimitation within this genus [22,23]. Furthermore, the occurrence of different *hsp90* isoforms in *P. vovlasi* sp. nov. confirmed that this gene family could contribute to the adaptation to different hosts and to different environments.

In this study, *P. vovlasi* sp. nov. was isolated from raspberries in north Italy together with *P. crenatus* in the same area. Further research on its pathogenicity and the economic damage on this crop is needed.

#### **4. Materials and Methods**

#### *4.1. Nematode Isolate and Morphological Studies*

No specific permits, other than that of the farm owner, were required for the indicated fieldwork studies. The soil samples were obtained in raspberry cultivated areas in Piedmont and did not involve any endangered species or those protected in Italy, nor were the sites protected in any way.

The soil samples recovered from the rhizosphere and roots of the raspberries located in the Piedmont region were sent to the IPSP laboratory, Bari, in the autumn of 2010. The samples were collected with a shovel from the upper 50 cm of the soil of raspberries arbitrarily chosen at random. The nematodes were extracted from 500 cm3 of the soil by centrifugal flotation [24]. The specimens for light microscopy were killed by gentle heat, fixed in a solution of 4% formaldehyde + 1% propionic acid and processed to pure glycerin using Seinhorst's method [25]. The specimens were examined with a Zeiss III compound microscope with a Nomarski differential interference contrast at powers up to ×1000. The measurements and drawings were made at the camera lucida on glycerin-infiltrated specimens. All measurements were expressed in micrometers (μm) unless otherwise stated.

For the scanning electron microscope (SEM), fixed specimens were dehydrated in a gradient series of ethanol, critical-point dried, sputter-coated with gold according to the protocol of Abolafia et al. [26] and observed with a Zeiss Merlin Scanning Electron Microscope (5 kV; Zeiss, Oberkochen, Germany).

#### *4.2. DNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing*

DNA was extracted from 20 single individual root-lesion nematode specimens. The specimens were handpicked and placed singly on a glass slide in 3 μL of the lysis buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 15 mM MgCl2, 0.1% Triton × 100, 0.01% gelatin with 90 μg/mL proteinase K) and then cut into small pieces by using a sterilized syringe needle under a dissecting microscope. The samples were incubated at 65 ◦C for 1 h and then at 95 ◦C for 15 min to deactivate the proteinase K [27]. The following sets of primers were used for the amplification of the gene fragments in the present study: (i) D2-D3 expansion segments of the 28S rRNA gene using forward D2A and reverse D3B primers; (ii) ITS1- 5.8-ITS2 rRNA using forward TW81 and reverse AB28 primers [28,29]; (iii) the *hsp*90 gene using forward U831 and reverse L1110 primers [30]. New sequences were submitted to the GenBank database under the accession numbers indicated on the phylogenetic trees.

#### *4.3. PCR-RFLP*

Ten μL of each ITS-PCR product from three individual nematodes were digested with five units of the following restriction enzymes: *Alu*I, *Ava*II, *Bam*HI, *Dde*I, *Hin*fI and *Rsa*I. The digested products were separated onto a 2.5% agarose gel by electrophoresis, stained with gel red dye, visualized on a UV transilluminator and photographed with a digital system.

#### *4.4. Phylogenetic Analysis*

Sequenced genetic markers in the present study (after discarding primer sequences and ambiguously aligned regions) and several *Pratylenchus* spp. sequences obtained from GenBank were used for the phylogenetic reconstruction. The outgroup taxa for each dataset were selected based on previous published studies [17,18,23]. Multiple sequence alignments of the newly obtained and published sequences were made using the FFT-NS-2 algorithm of MAFFT v. 7.450 [31]. The sequence alignments were visualized using BioEdit [32] and edited by Gblocks ver. 0.91b [33] in the Castresana Laboratory server (http: //molevol.cmima.csic.es/castresana/Gblocks\_server.html, accessed on 2 April 2021) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences + 1; maximum number of contiguous no conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half).

The phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MRBAYES 3.2.7a [34]. The best-fit model of DNA evolution was calculated with the Akaike information criterion (AIC) of JMODELTEST v. 2.1.7 [35]. The best-fit model, the base frequency, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates in the AIC were then used in the phylogenetic analyses.

The BI analyses were performed under a general time-reversible model with a proportion of invariable sites and a rate of variation across sites (GTR + I + G) model for D2-D3 and the partial *hsp90* gene and under a transversional model with a proportion of invariable sites and a rate of variation across sites (TVM + I + G) model for the ITS region. These BI analyses were run separately per dataset with four chains for 4 × 106 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding the burn-in samples of 30% and evaluating the convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. The trees from all analyses were visualized using FigTree software version v.1.42 [36].

**Author Contributions:** Conceptualization, F.D.L., A.T. and P.C.; sampling, A.C.; methodology, A.T., E.F. and G.L.; software, F.D.L. and P.C.; data curation, E.F. and F.D.L.; writing—original draft preparation, F.D.L., A.T. and P.C.; writing—review and editing, F.D.L., A.T., E.F. and P.C.; funding acquisition, F.D.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** F.D.L. acknowledges the Piedmont region through project NEMVIR/302/2010.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** SEM pictures were obtained with the assistance of technical staff and the equipment of the Centro de Instrumentación Científico-Técnica (CICT), University of Jaén. The staff of Settore Fitosanitario e Servizi tecnico-scientifici of the Piedmont region who collected the samples are also acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Review*
