The Northern Root-Knot Nematode Meloidogyne hapla: New Host Records in Portugal

Simple Summary Damage caused by plant-parasitic nematodes (PPNs) is usually underestimated as many farmers are oblivious of their presence. Root-knot nematodes (RKNs) are an economically important group of PPNs and one of the most devastating. The species Meloidogyne arenaria, M. hapla, M. incognita and M. javanica are considered the most important, due to their worldwide distribution and polyphagia, being the northern RKN, Meloidogyne hapla the most important species occurring in cold regions. During 2019–2022, surveys were carried out in different districts of Portugal in horticultural and ornamental crops. From the samples collected, nine were identified morphologically and molecularly as M. hapla, indicating that its prevalence is increasing, and demonstrating its importance and impact in agricultural systems. Abstract Root-knot nematodes (RKNs), Meloidogyne spp., are a group of plant-parasitic nematodes (PPNs) of great economic significance worldwide. The northern root-knot nematode, Meloidogyne hapla, is one of the most important species of RKNs occurring in cold regions. In Portugal so far, M. hapla has been found parasitizing potato and fig trees. During surveys carried out in 2019–2022 in fields for horticultural and ornamental production, soil and root samples were collected. Roots were observed under a stereomicroscope to determine the presence of galls. Nematodes were extracted from the soil. Morphological features showed a high similarity and consistency with previous descriptions of the genus. For molecular analysis, total genomic DNA was isolated from single nematodes and used to amplify in multiplex reaction using the species-specific primers JMV1, JMV2 and JMVhapla, and for sequencing of the ITS region with the primers TW81/AB28. Multiplex PCR amplification produced a specific fragment of 440 bp and PCR amplification of the ITS region yielded a single fragment of 550 bp, as expected. The obtained sequences showed a similarity ranging from 99.8% to 100% with the sequences of M. hapla available in the database. The phylogenetic tree revealed that the isolates grouped with M. hapla isolates. From the 690 samples collected, M. hapla was detected in three different hosts (grapevine, eucalyptus and potato) in four districts of mainland Portugal and on Madeira Island. To our knowledge, this is the first report of M. hapla infecting the grapevine and eucalyptus in Portugal.


Introduction
Horticulture crops represent about 50% of the Portuguese agricultural added value. The most important crops are grapevines, oranges, apples, pears, cole crops, peaches, processing tomatoes and potatoes. Ornamentals (cut flowers and potted plant production) in increasing areas represent about 600 ha [1].
Plant-parasitic nematodes (PPNs) are an important constraint to agricultural production as the losses they cause have been estimated from USD 175 billion per year [2,3].
However, the full extent of nematode damage is likely to be underestimated as many growers are unaware of their presence [4].
The northern RKN, Meloidogyne hapla, was first described from a potato field in Long Island, New York (Chitwood 1949) and is one of the most important species of RKNs occurring in temperate regions, as it can withstand the cold. Eggs and juveniles can survive field temperatures below 0 • C, with some studies recording survival down to −15 • C in soil for a prolonged period of time. The optimum temperature for penetration and development of M. hapla is in the range of 20-25 • C and a mean temperature of 28 • C may prevent its development [15][16][17]. Meloidogyne hapla is a sedentary, biotrophic parasite of plants that overwinters in soil or diseased roots, and multiplies by both sexual (amphimixis) and asexual (parthenogenesis) reproduction, in contrast to the most widespread, three other RKNs [18]. Unlike many other RKN species, M. hapla eggs and juveniles can survive field temperatures below 0 • C. However, there is no evidence of its inability to survive in hot temperatures [17]. It parasitizes nearly all temperate vegetables of economic importance, reducing considerably yield and even causing total crop losses [14,19]. Surveys of vineyards and potatoes conducted in the United States found M. hapla to be the most abundant RKN present in the fields [20]. In Portugal, M. hapla was first detected in 2008 in fig trees and a year later found parasitizing the potato; since then, no other detections have been reported [7,8].
Therefore, the aim of the present study was to morphologically and molecularly identify the isolates of the RKN found in the survey of different counties and crops across mainland Portugal and Madeira Island, namely in grapevines, potato and eucalyptus.

Sampling and Nematode Isolates
During 2019-2022, surveys of horticultural and ornamental fields were carried out, resulting in a total of 690 soil and root samples collected from various districts in Portugal. Soil sampling was carried out from the rhizosphere at about 15 cm in depth for horticultural crops and 70 cm for grapevines and trees. Each sample consisted of 5 to 8 cores, sampled in the vicinity of plants that presented symptoms, and in zigzag at roughly equal intervals in asymptomatic fields. Each composite soil sample was placed in a polyethylene bag and brought for analysis. A 400-mL subsample was taken from each composite sample and the nematodes were extracted according to the protocol PM 7/119 (1) [21]. The suspension was observed under a stereomicroscope (Nikon SMZ1500, Tokyo, Japan) and suspected specimens of Meloidogyne observed using a bright-field light microscope (Olympus BX-51, Hamburg, Germany) for confirmation. When second-stage juveniles (J2) of Meloidogyne were detected in the soil suspension, bioassays were carried out by planting tomato plants cv. Oxheart in the remaining soil from the sample and maintained in a quarantine greenhouse for two months to obtain different developmental stages for further studies. Roots (from sampling and bioassays) were observed under stereomicroscope. Egg masses were handpicked from infected roots and used to establish cultures of each isolate.

Morphological Characterization
Ten second-stage juveniles (J2) from each positive sample were placed in a drop of water on a glass slide, gently heat killed for morphological characterization using a brightfield light microscope (Olympus BX-51, Hamburg, Germany) and photographed with a digital camera (Leica MC190 HD, Wetzlar, Germany).

Molecular Characterization
The species-specific primers, SCAR markers, and the ITS region were selected for molecular characterization of Meloidogyne hapla isolates found in the survey. The total DNA was extracted from a single juvenile using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The SCAR region was amplified in multiplex PCR using the primers JMV1 (5 -GGATGGCGTGCTTTCAAC-3 ), JMV2 (5 -TTTCCCCTTATGATGTTTACCC-3 ) and JMVhapla (5 AAAAATCCCCTCGAAA AATCCACC3 ), and the ITS region, using the forward primer, TW81 (5 -GTTTCCGTAGGT GAACCTGC-3 ) and the reverse AB28 (5 -ATATGCTTAAGTTCAGCGGGT-3 ) [22,23]. PCR reactions were performed in a 50-µL final volume mixture containing 25 µL of Supreme NZYTaq II 2× Green Master Mix (NZYTech, Lisboa, Portugal), 1 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 [23,24]. PCR products were resolved by electrophoresis at 5 V.cm −1 , in agarose gel (1.5%) containing 0.5 µg/mL of ethidium bromide and 0.5× of 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 & Concentrator Kit (Zymo Research Corp, Irvine, CA, USA), according to the manufacturer's instructions. Amplicons were sequenced at the INIAV (Oeiras, Portugal) on an ABI PRISM 3730xl (Applied Biosystems, Walthman, MA, USA) DNA analyzer. The newly obtained sequences were manually checked, edited and assembled. The sequences were compared to those of M. hapla 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.
Phylogenetic analyses were conducted using MEGA 11 [25], the maximum likelihood (ML) and the Kimura 2-parameter model. The robustness of the ML tree was inferred using 1000 bootstrap replicates.

Distribution
A total of 690 samples were collected from the south to the north of mainland Portugal and Madeira Island. From the total of samples collected, Meloidogyne hapla was detected in nine, corresponding to 1.5%. The detections were in four districts of the southern and northern regions and on Madeira Island. Meloidogyne hapla was not found in the central region. Three hosts were identified (potato, grapevine and eucalyptus) (Table 1) ( Figure 1).

Symptoms
The field symptoms (above-and below-ground) observed and the species of nematodes found in the soil are described in Table 2.

Symptoms
The field symptoms (above-and below-ground) observed and the species of nematodes found in the soil are described in Table 2.

Morphological Characterization
Morphological characterization from the recovered second-stage juveniles was performed and was in agreement with previous descriptions of the genus (Figure 4) [24, 26,27]. The second-stage juveniles were vermiform, slender, and clearly annulated. The head region was slightly set off from body. The stylet was delicate and narrow, and the dorsal gland orifice (DGO) long, compared to the other three most common species. The knobs were rounded and appeared set off from the shaft. The excretory pore was distinct and hemizonid anterior or adjacent to excretory pore. The tail was long and slender with a narrow, tapering terminus that had several distinct annulations; the tail delimitation was not very clear in M. hapla. The hyaline tail terminus of M. hapla is highly variable and can go from deformed with an irregular shape to regular V shapes.

Molecular Characterization
The PCR amplification of species-specific SCAR markers yielded a single fragment of 440 bp as expected ( Figure 5). The amplification of ITS (TW81/AB28) yielded a single fragment of about 550 bp. The nucleotide sequences obtained in this study were deposited into the GenBank database (NCBI) under the accession numbers, OP364010, OP364011, OP364012, OP364013, OP364014, OP364015, OP364016, OP364017 and OP364018. A BLAST search of the nucleotide sequences showed a similarity ranging from 99.8% to 100% with the sequences of M. hapla available in the database (Table 3). Morphological characterization from the recovered second-stage juveniles was performed and was in agreement with previous descriptions of the genus (Figure 4) [24, 26,27]. The second-stage juveniles were vermiform, slender, and clearly annulated. The head region was slightly set off from body. The stylet was delicate and narrow, and the dorsal gland orifice (DGO) long, compared to the other three most common species. The knobs were rounded and appeared set off from the shaft. The excretory pore was distinct and hemizonid anterior or adjacent to excretory pore. The tail was long and slender with a narrow, tapering terminus that had several distinct annulations; the tail delimitation was not very clear in M. hapla. The hyaline tail terminus of M. hapla is highly variable and can go from deformed with an irregular shape to regular V shapes.

Molecular Characterization
The PCR amplification of species-specific SCAR markers yielded a single fragment of 440 bp as expected ( Figure 5). The amplification of ITS (TW81/AB28) yielded a single fragment of about 550 bp. The nucleotide sequences obtained in this study were deposited into the GenBank database (NCBI) under the accession numbers, OP364010, OP364011, OP364012, OP364013, OP364014, OP364015, OP364016, OP364017 and OP364018. A     The molecular phylogenetic analysis is presented in Figure 6.

Discussion
Nematodes are often overlooked because of the ambiguity of the symptoms they cause, and the difficulty in detecting and identifying them. Moreover, the impact the RKN has in agricultural areas reinforces the need for accurate diagnosis at the species level. Conventionally, the identification of Meloidogyne species has been based on morphological characters of second-stage juveniles, perineal patterns of adult females and isozyme phenotypes. Isozymes are highly reliable, but have its drawbacks, as it requires a specific developmental stage (adult females) as well as considerable skills. Many different DNAbased methods have been used to identify M. hapla, among which include species-specific SCAR primers and the ITS region; these methods allow to identify the species in a timely and efficient manner. Since the eradication of nematodes is a complex task, the identification of the Meloidogyne spp. in the area of interest is of primary importance to implement

Discussion
Nematodes are often overlooked because of the ambiguity of the symptoms they cause, and the difficulty in detecting and identifying them. Moreover, the impact the RKN has in agricultural areas reinforces the need for accurate diagnosis at the species level. Conventionally, the identification of Meloidogyne species has been based on morphological characters of second-stage juveniles, perineal patterns of adult females and isozyme phenotypes. Isozymes are highly reliable, but have its drawbacks, as it requires a specific developmental stage (adult females) as well as considerable skills. Many different DNA-based methods have been used to identify M. hapla, among which include species-specific SCAR primers and the ITS region; these methods allow to identify the species in a timely and efficient manner. Since the eradication of nematodes is a complex task, the identification of the Meloidogyne spp. in the area of interest is of primary importance to implement management strategies that minimize damage. This will allow for the adoption of preventive practices such as the appropriate deployment of cultivars. The presence of M. hapla has extended throughout the country, from detections in two districts in 2009 to five in the following four years (2019-2022). Such occurrences and increased detection draw attention to its potential to adversely affect economically important horticultural and ornamental crops. These detections were in northern and southern coastal counties-places where the climate is influenced by the Atlantic Ocean. Districts located up north are cooler and rainy, while the south becomes gradually warmer and sunnier; however, the Atlantic moderates the temperature, providing adequate conditions for M. hapla development and survival, as average temperatures throughout the year vary from 5 • C to 25 • C.
To our knowledge, this is the first report of Meloidogyne hapla in the grapevine and eucalyptus in Portugal, adding valuable information to the current situation of this organism in the European Plant Protection Organization (EPPO) zone.

Conclusions
Root-knot nematodes are one of the most devastating plant parasitic nematodes, affecting the yield and quality of many crops. Their wide host range makes the management of RKNs extremely difficult; so, identifying the nematode species we are dealing with and understanding the spread, survival and damage potential, in this case of the Meloidogyne hapla, is the first step to managing it efficiently and effectively.
This study confirmed the presence of M. hapla in the country and provided evidence of its dispersion and ability to survive in different crops, contributing important data that can be used in integrated management programs.