First Report of Scutellonema brachyurus (Steiner, 1938) Andrassy, 1958 and Occurrence of Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949 in Belgium

Abstract A study of plant-parasitic nematodes in the Botanical garden at Ghent University in Belgium revealed the presence of two tropical nematode species, i.e. Scutellonema brachyurus and Meloidogyne incognita. Scutellonema brachyurus was recovered, only once, for the first time in Belgium from Musa basjoo and is morphologically characterized. M. incognita, forming galls on Hedychium greenii, was recovered in all seasons over three consecutive years and is morphologically and molecularly characterized. Although no unequivocal evidence was found to indicate that these nematodes pose a current threat in Belgium, in the light of climate change, it is crucial to improve our knowledge of potential tropical nematode activity in more Northern countries.

Recently, the presence of tropical nematodes has been reported from several Mediterranean or more southern European countries (Wesemael et al., 2011;EPPO, 2019). However, tropical nematodes do not yet pose a problem in the more northern parts of Europe, including Belgium. According to Bebber et al. (2013), there is clear evidence of a general, climate change-driven, poleward migration of pests and plant pathogens, a movement that can include tropical plant-parasitic nematodes. Interestingly, our survey revealed the presence of Scutellonema brachyurus and the tropical root-knot nematode Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 for the first time, respectively, on banana (Musa basjoo Siebold & Zucc. ex Iinuma) and red ginger (Hedychium greenii W. W. Sm.) in Belgium.

Materials and methods
After removing detritus layer from the surface, soil and root samples were collected from the upper 30 cm soil layer around the rhizosphere of Musa basjoo and Hedychium greenii at the Botanical garden of Ghent University (GPS coordinates: N: 51°2′6.8″, E: 3°43′22.7″ and N: 51°2′6.7″, E: 3°43′22.4″, respectively). Several samples were taken from September 2017 to June 2019 to check for the survival of nematodes through the winter season. For morphological characterization, vermiform nematodes were extracted by the modified Baermann tray method (Whitehead and Hemming, 1965). After that, permanent slides were made following Singh et al. (2018). Mature females of M. incognita were extracted directly from the galls under a stereomicroscope, using a scalpel and forceps. Perineal patterns were cut and cleaned following Hartman and Sasser (1985) and mounted in glycerine. Microphotographs were made from permanent slides using an Olympus BX51 DIC Microscope equipped with a digital camera. Measurements were made based on the obtained pictures using Image J 1.51 (Schneider et al., 2012). For molecular characterization, primers NAD5F2/NAD5R1 were used to amplify the Nad5 mtDNA gene following the protocol of Janssen et al. (2016). Nad5 mtDNA sequences of M. incognita in Belgium were aligned with 73 reference sequences of tropical root-knot nematode species from Janssen et al. (2016) and other closely related sequences from GenBank using Muscle on Geneious R11 (www.geneious.com) to check for polymorphic nucleotide positions.

Remarks
Morphological traits and measurements of the female Belgian population of S. brachyurus is in full agreement with the type population of Steiner (1938), except for the slightly shorter stylet (26.8±1.1 (25.2-28.6) µm vs 29 µm). However, this variation is known in other populations of S. brachyurus (Table 1). Van Den Berg et al. (2013) described two types of S. brachyurus (type A and type B), and S. brachyurus type A can be differentiated from type B by the main following traits: lip region with 4 to 6 annuli vs mainly three, rarely 4 to 5 annuli; 4 to 12 blocks on basal annulus vs 8 to 20 blocks; secretory-excretory pore located opposite anterior part to mid-region of overlapping pharyngeal lobe vs from rarely opposite mid-isthmus to mostly opposite the posterior part of pharyngeal gland lobe up to its posterior border. The Belgian population of S. brachyurus belongs to S. brachyurus type B. Unfortunately, our effort to recover the Belgian population of S. brachyurus for molecular analysis was unsuccessful. Males were also not found.

Remarks
In general, the morphology of M. incognita in Belgium is in agreement with the description of Whitehead (1968) (the perineal pattern of Belgian population of M. incognita belongs to M. incognita incognita type). Only a few variations of measurements were observed such as a wider upper range of body length of juveniles (406 (374-420) µm vs 371 (337-403) µm) and a larger DGO value of the males (3.8 (3.2-4.4) vs 2.1 (1.4-2.5)). However, these variations are small and fall within the range known from other populations (Table 1). Six Nad5 sequences were obtained with a length from 544 to 599 nucleotides. The sequences of M. incognita in Belgium were identical to each other and five other reference sequences of M. incognita (specimen ID: T384, T532, M8, M20, M21) from Morocco, Egypt, and Tanzania in the study of Janssen et al. (2016). During winter time, the aerial parts of the host plant were cut down and the growing area was covered by wood chips. Meloidogyne incognita has been found at any time of sampling in all seasons over three consecutive years.

Discussion
This study reveals the presence of S. brachyurus and M. incognita in Belgium, species known to be prevalent in warm areas, especially in tropical regions (CABI, 2019). Morphologically, the Belgian population of the spiral nematode belongs to the S. brachyurus group type B according to Van Den Berg et al. (2013). However, the presence of cryptic species in the S. brachyurus group have only been molecularly unequivocally defined (Van Den Berg et al., 2013;Van Den Berg et al., 2017), and therefore, molecular data are needed to confirm the group of S. brachyurus to which the Belgian population belongs. Unfortunately, multiple attempts to recover this nematode for molecular studies failed.
The morphological identification of tropical rootknot nematodes is known to be greatly hampered by phenotypic plasticity and interspecific similarities (Hunt and Handoo, 2009), for example the variation in the number of post-labial annuli of M. incognita in Belgium (ranging from 1 to 3 annuli) confirmed the plasticity of the numbers of head annuli in the genus Meloidogyne. Therefore, we used an integrated approach including the mitochondrial barcode region Nad5 as a reliable marker to identify tropical root-knot nematodes.
The Belgian population of M. incognita was discovered for the first time on a specimen of Hedychium greenii that was planted outside several years ago   10.9 ± 0.8 (9.5-12.0) 24.7 ± 0.9 (23.9-25.8) 15.6 ± 0.7 (14-17) Conus length 14.1 ± 1.1 (12.7-15.8) 6 ± 0.5 (5-6) 13.9 ± 0.5 (13.2-14.5) 9 ± 0.9 (8-10) Shaft length 10.1 ± 0.39 (9.5-10.5) 4 ± 0.6 (3-5) 8.0 ± 0.6 (7.6-8. in the botanical garden of Ghent University, with the accompanying Belgian weather conditions (cold winter seasons). This plant is an exotic plant that was imported from the Himalayas, which in its native habitat can be found growing at altitudes up to 5000 feet (about 1666m), and it is known to tolerate temperatures as low as 15 °F ( −9°C). However, M. incognita is known as a tropical nematode and distributed in warmer climates (Wesemael et al., 2011). Most likely, this nematode originated from one of the tropical plants that were imported and planted in the botanical garden of Ghent University. The fact that the growing area was covered by wood chips during winter time may have created more suitable conditions for M. incognita. Meloidogyne incognita has already been reported in Belgium by Coolen et al. (1974) in nurseries growing multiflora tuberous begonias. However, without detailed taxonomical information, its species status can therefore not be assured.