New occurrence of Meloidogyne graminicola (Nematoda: Meloidogyninae) from rice fields in Italy: Variability and phylogenetic relationships

Abstract Since the first detection of Meloidogyne graminicola in Piedmont, North Italy, in 2016, further inspections for the presence of the rice root‐knot nematode were carried out in rice fields of neighboring regions, in accordance with the Italian NPPO (National Plant Protection Organization) to support the official phytosanitary measures, to enable the early detection of the rice pest, and to prevent its spread within the national territory. In 2018, surveys of rice fields in Lombardy region revealed a new occurrence of M. graminicola. In the present study, we confirmed the identification of the rice nematode in Lombardy using the ribosomal ITS region and the mitochondrial COI and COII genes. The sequences and phylogenetic analyses revealed that Lombardy M. graminicola population grouped in all trees in the main cluster containing Meloidogyne species belonging to graminis group, but always in a different subgroup compared to the Piedmont population of M. graminicola. These results clearly suggest that the two Italian populations have been recently and independently introduced and confirm that the geographic origin is not the main factor leading to M. graminicola population variability.


| INTRODUC TI ON
Root-knot nematodes are widely distributed causing tremendous economic losses estimated about 80 billion dollars annually (Nicol et al., 2011;Jones et al., 2013). Among Meloidogyne species, the root-knot nematode Meloidogyne graminicola Golden and Birchfield (1965) is the most important rice pest worldwide causing yield losses up to 87% (Dutta et al., 2012). Meloidogyne graminicola is a nematode species well adapted to attack different rice agrosystems, from upland to lowland, and irrigated to deep-water fields, along with more than 98 host plants including cereals and grasses (Pokharel et al., 2010). Khan (2015) reported that rice can be attacked by more than 35 plant parasitic nematodes, but M. graminicola together with few other Meloidogyne spp. is one of the most adapted species to the flooded rice systems, worldwide. Recently, during a survey in Brazil, M. graminicola was detected in rice fields along with other Meloidogyne spp. belonging to the graminis group Mattos et al., 2018). It is present in America, Africa, especially in Asia and recently in South Europe (EPPO, 2016(EPPO, , 2018Fanelli et al., 2017;Mantelin et al., 2017;Rusinque et al., 2021;Sacchi et al., 2021). In Europe, M. graminicola was detected for the first time in upland and lowland rice fields in the Piedmont region (Northern Italy) in 2016 and immediately added to the EPPO Alert List. In just one year (2016, EPPO Global Database, 2019, the total infected area increased by fivefold. Italy is the main rice-growing country in Europe, with 217,195 ha of rice (Ente Nazionale Risi, 2018). The most important rice-growing area is the section of the Po River Valley straddling the regions of Lombardy and Piedmont with more than 202,000 hectares representing 93% of the Italian rice surface (Ente Nazionale Risi, 2018;Fanelli et al., 2017;Sacchi et al., 2020Sacchi et al., , 2021. In Lombardy region, rice growing is highly specialized and is concentrated above all in the area enclosed between Pavia, Milano, and Lodi provinces. Rice fields constitute a typical element of this territory characterized by a large inter-annual variability related to the meteorological conditions (Zampieri et al., 2019). The Lombardy's rice fields have sandy soils thus draining faster than in Piedmont during summer, the water table is lower, and larger withdrawals from rivers and channels are needed for rice paddy fields irrigation.
Therefore, different water management evolution was observed with the increase of spread, in Lombardy (+69%), compared to a lower increase (+28%) in Piedmont, of rice cultivation in dry paddy fields (Torrini et al., 2020;Zampieri et al., 2019). Conditions such as those described (dry paddy fields and sandy soils) can potentially favor the activity of root-knot nematodes. Therefore, the discovery of M. graminicola in Piedmont has causing great concern so as to increase the surveillance by the Lombardy RPPO (Regional Plant Protection Organization) that reported the discovery of the first outbreak in 2018 (EPPO, 2018).
The best phytosanitary measure adopted by the Italian RPPO in Piedmont to control the spread of the M. graminicola population was the rice field flooding but, in Lombardy region, this practice is scarcely applicable due to the described soil structure characterized by a low water retention capacity and water shortage (Sacchi et al., 2021;Zampieri et al., 2019). Thus, rapid and accurate identification of Meloidogyne spp. associated with rice, specifically M. graminicola, as well as prevalence and distribution is important for adopting management strategies in the fields in Northern Italy. In the present study, a population of M. graminicola from Lombardy was characterized at molecular level in order to establish the phylogenetic relationships with the Piedmont population and other geographical isolates and to determine the origin of both Italian populations by sequencing the nuclear ITS containing region and the mitochondrial cytochrome oxidase I (COI) and the COII/16S rRNA genes.

| Nematode isolation
In 2018, surveys in rice fields showing symptoms of M. graminicola attacks ( Figure 1) were carried out in several areas of Pavia province (Lombardy). Soil and root samples from the rice-cultivated area at Cascina Scalina farm in Garlasco (Pavia province) were collected (GPS coordinates: 45.19568654643237, 8.893816095789944 for image capture and analysis. Ethanol-preserved second-stage juveniles were sent to IPSP-Bari Institute for molecular identification.
PCR cycling conditions used for amplification of the partial ITS were: an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 50 s, annealing at 55 °C for 50 s and extension at 72 °C for 1 min, and a final step at 72 °C for 7 min. For the COI, these conditions were: an initial denaturation at 94 °C for 3 min, followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 30 s and extension at 72 °C for 30 s, and a final step at 72 °C for 7 min. For the COII, PCR conditions were: an initial denaturation at 94 °C for 3 min, followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 30 s and extension at 60 °C for 30 s, and a final step at 72 °C for 7 min. 10 J2s of Piedmont M. graminicola from upland rice fields were also processed and amplified using ITS and COII primers to confirm the species occurrence in 2018. PCR products of two specimens for each molecular marker were purified after amplification using NucleoSpin (Macherey-Nagel), quantified using a Nanodrop spectrophotometer (Nanodrop Technologies) and used for cloning in pGEM-T easy vector (Promega). Eight COI, 5 COII, and 1 ITS clones of M. graminicola from Lombardy were sent at MWG-Eurofins genomics in Germany for sequencing in both directions. Three ITS and three COII clones from Piedmont upland and lowland rice fields were also sent for sequencing to MWG-Eurofins genomics in the present study.

A Basic Local Alignment Search Tool (BLAST) search at National
Center for Biotechnology Information (NCBI) was performed in order to confirm their nematode origins (Altschul et al., 1997). The newly obtained sequences for ITS containing region and the partial mitochondrial COI and COII were aligned using MAFFT V.7.450 (Katoh et al., 2019). BioEdit program V. 7.2.5 (Hall, 1999) was used for sequence alignments visualization and edited in order to improve the multialignment. Outgroup taxa, M. incognita, M. hapla, M. javanica, and M. arenaria, for each dataset were chosen according to the results of previously published data (Fanelli et al., 2017;Mattos et al., 2018;Soares et al., 2020). Phylogenetic trees, obtained for ITS, COI, and COII dataset, were performed with Maximum Likelihood (ML) method using MEGA version 7 software (Kumar et al., 2016). The phylograms were bootstrapped 1000 times to assess the degree of support for the phylogenetic branching indicated by the optimal tree for each method.
The newly obtained sequences were submitted to GenBank with the following accession numbers: for ITS OM809713-OM809716; for COI OM810293-OM810300; for COII OP024528-OP024535.

| Estimations of evolutionary divergence between sequences
The pairwise distances within the COI and COII sequences of M. graminicola belonging to the graminis-group were done in MEGA7 F I G U R E 1 (a) Uninfected rice field; (b) Rice field sowing few patchy areas (arrows); (c) Rice field severely infested by Meloidogyne graminicola; (d) Poorly growth rice plantlets showing root tip thickenings caused by massive attacks of Meloidogyne graminicola; (e) Open galled rice root showing numerous root-knot nematode females; (f) Second and third juvenile stages (top), and females (bottom) of Meloidogyne graminicola extracted from infected rice roots; (g) Secondstage juveniles came out from dissected rice roots; (h) Second-stage juveniles of Meloydogyne graminicola from Lombardy.
software package (Kumar et al., 2016). All positions with gaps and missing data were excluded. The COI and COII analyses involved 28 and 25 nucleotide sequences, respectively. Guinea, and Southeast Asia (LR215847 and MK507948; mitochondrial complete genomes) 6-8 different nucleotides (Table 2).

| DISCUSS ION
In the present study, we report on the second occurrence of M.
graminicola population in rice fields in Lombardy (North Italy), after the first occurrence in lowland and upland rice fields in Piedmont in    These findings suggest that geographical distance is not the main factor leading to M. graminicola population differentiation.
As the Italian isolates always grouped in two different subgroups with geographical distant isolates sharing the same haplotypes, this observation seems to confirm that the two Italian M. graminicola populations may have been recently and independently introduced.
Furthermore, in the current study, some M. oryzae isolates grouped within M. graminicola cluster confirming their close relationships due to recent evolution or hybrid origin of these specie well adapted to irrigated rice (Besnard et al., 2019).
In conclusion, the present study clearly demonstrates that Italian M. graminicola populations show the same genetic profiles of those from Asia and America suggesting that this species prefers asexual reproduction and is well adapted to different rice fields. In this context, it can be understood that appropriate control measures are needed to manage this pest. Rice field flooding seems to be an efficient control technique for M. graminicola in Piedmont but not in Lombardy due to the soil structure. Thus, in Lombardy, researchers are testing several trap cropping strategies to maintain a low nematode population in infested fields and also balancing water shortage due to climate change and dry-seeding practices. Regarding the entry of M. graminicola in Italy, it is not well understood yet. It seems through the movement of infested host plants, soil, waterbirds, acquatic plants or weeds acting as reservoirs for this nematode. writing -original draft (lead); writing -review and editing (lead).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
DNA sequences obtained in the present study are available at Genbank accession numbers: for ITS OM809713-OM809716; for COI OM810293-OM810300; for COII OP024528-OP024535.