Monitoring of Root-Knot Nematodes (Meloidogyne spp.) in Croatia (2022–2024): Occurrence, Distribution and Species Identification

Abstract

Root-knot nematodes (RKNs) of the genus Meloidogyne spp., are among the most economically important groups of plant-parasitic nematodes worldwide, causing significant economic losses through yield reduction across a wide range of crops. In Croatia, although the presence of Meloidogyne spp. has been documented for decades, data at the species level was limited. As accurate identification is crucial for implementation of effective management strategies, we attempted to fill this gap. This study presents the results of a national survey of RKNs affecting potato crops as well as an early warning programme targeting vegetable crops, conducted across Croatia between 2022 and 2024. Nematodes were identified using morphological analyses (female perineal patterns and second-stage juveniles) and molecular methods (PCR with group-specific and species-specific primers, as well as DNA sequencing). Meloidogyne spp. were detected in 61 out of 210 samples, corresponding to an infestation rate of 29%. Four species were identified: M. incognita, M. hapla, M. arenaria, and M. javanica. Notably, M. incognita and M. javanica are reported here for the first time in Croatia. These results provide updated insights into the distribution and identity of RKNs in Croatia, thereby establishing a foundation for the implementation of sustainable management strategies.

1. Introduction

The genus Meloidogyne (Gőldi, 1892) commonly referred to as root-knot nematodes (RKNs), represents one of the most damaging groups of plant-parasitic nematodes globally [1]. These obligatory, sedentary endoparasites infect a wide range of host plants, particularly horticultural crops of major economic importance, including tomato, potato, cucumber, melon and pepper where they cause significant yield and economic losses.

Their impact is particularly pronounced in warm and temperate agricultural regions, where they disrupt root systems by inducing characteristic gall formation. This physiological disturbance impairs water and nutrient uptake, ultimately reducing plant vigour, crop yield, and market quality [2,3].

Global yield losses caused by plant-parasitic nematodes (PPN), particularly species of the genus Meloidogyne, are estimated to exceed USD 157 billion annually, accounting for up to 12.3% of total production losses in susceptible crops worldwide [4,5]. These nematodes are particularly problematic in intensive agricultural systems where monoculture and limited crop rotation facilitate their proliferation [3].

The most species with the widest global distribution, including Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949, M. javanica (Trub, 1885) Chitwood, 1949, M. arenaria (Neal, 1889) Chitwood, 1949, and M. hapla (Chitwood, 1949), exhibit a broad host range and high reproductive capacities, which make management strategies challenging to implement [2,3]. Several tropical RKN species, such as M. incognita, M. arenaria, M. javanica, M. enterolobii, M. luci, and M. hispanica, are considered invasive and highly damaging agricultural pests. Their spread is facilitated by global trade, changes in agricultural practices (e.g., reduced pesticide use), and climate change [6,7,8]. Warmer soil temperatures and longer growing seasons enhance the reproductive potential of Meloidogyne spp., potentially expanding their reach to previously unsuitable areas [9,10,11]. Continuous surveillance and risk assessment are therefore critical for both protected and open-field production systems.

To prevent the introduction or to limit the spread within the European Union (EU), three RKN species (M. chitwoodi, M. fallax, and M. enterolobii) are listed as quarantine pests under Commission Implementing Regulations [12,13]. These species are subject to surveillance and regulatory control. The European and Mediterranean Plant Protection Organisation (EPPO) has also included additional Meloidogyne spp. in its A1 (M. ethiopica) and A2 lists (M. mali, M. luci, and M. graminicola) of quarantine pests due to their significant agricultural impact [14,15].

Consequently, Meloidogyne species are of considerable phytosanitary importance within the European Union (EU). Their presence in vegetable fields and greenhouses presents serious challenges, particularly in intensive cropping systems. Croatian legislation aligns with EU plant health regulations and includes regular monitoring and control measures for regulated Meloidogyne species. A national surveillance programme for M. chitwoodi and M. fallax in potato (Solanum tuberosum L.) crops has been implemented since 2019 under this legislative framework. In addition, an early warning monitoring programme for Meloidogyne species in vegetable fields and greenhouses was launched in 2022. Although M. incognita, M. javanica, M. arenaria, and M. hapla are currently not classified as quarantine pests, they are frequently encountered in Croatia. These species are monitored due to their agronomic relevance [3,11,16,17,18].

The agricultural sector plays a crucial role in Croatian rural development and food security, contributing approximately 3.4% to the national Gross Domestic Product (GDP) [19]. Despite this modest economic share, agriculture remains vital for regional employment and livelihoods. Croatia possesses approximately 1.5 million hectares of agricultural land, with vegetable production occupying 13,000–14,000 hectares, of which 10% is under protected cultivation such as greenhouses and polytunnels [20]. Despite this production base, Croatia’s self-sufficiency rate in vegetable production is only ~47%, resulting in substantial reliance on imports to meet domestic demand. Crop production is therefore strategically important for enhancing national food self-sufficiency and reducing dependence on food imports.

Croatia is divided into four statistical regions (the Adriatic, Pannonian, Northern, and the City of Zagreb) and 21 administrative counties. The country’s diverse agro-climatic zones, from continental lowlands to Mediterranean coastal areas, support a wide range of crops [21].

Economically important horticultural crops include tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.), cucumber (Cucumis sativus L.), lettuce (Lactuca sativa L.), watermelon (Citrullus lanatus L.), melon (Cucumis melo L.) and various species of the genus Brassica spp. [22]. These crops are predominantly cultivated in greenhouse production systems, where elevated soil temperatures combined with high relative humidity create favourable conditions for the proliferation of nematodes. Potato, another major crop, is grown primarily in the northern regions (e.g., Međimurje and Varaždin counties) and extensively across the Pannonian (e.g., Bjelovar-Bilogora and Osijek-Baranja counties), where favourable soil and climatic conditions are particularly suitable for its production [20].

RKNs are well-established pests in Croatia, particularly with high prevalence in the Adriatic region encompassing Istria, Zadar, Split-Dalmatia, and Dubrovnik-Neretva County. The predominance of sandy and light loamy soils in these areas, combined with a Mediterranean climate characterised by rainy winters and warm to very warm summers, creates highly favourable conditions for nematode reproduction.

Historical records indicate the first detection of Meloidogyne spp. in Croatia during the mid-1960s, primarily affecting vegetables and ornamentals. Subsequent occurrences have been documented across different agricultural areas [23,24].

To date, only three Meloidogyne species (M. arenaria, M. hapla, and M. artiellia) have been officially reported in Croatia [25,26,27]. However, more recent studies conducted in neighbouring Slovenia and Serbia have confirmed the presence of other species, such as M. luci and M. incognita, thereby highlighting the necessity for further investigation in Croatia [18,28,29,30].

Accurate identification of Meloidogyne species is essential for implementation of effective management strategies. Traditional diagnostic methods based on morphological and morphometric traits, such as female perineal patterns and features of second-stage juveniles, are often constrained by extensive intra- and interspecific variability, which complicates species-level differentiation [2]. Classical approaches, including differential host testing [31] and isozyme phenotyping [32], remain valuable but have limitations. Biochemical assays, such as electrophoretic analysis of carboxylesterase (EST) and malate dehydrogenase (MDH) isoenzymes, are cost-effective tools; however, their applicability is restricted to egg-laying females [32,33].

Over the past 25 years, molecular diagnostics techniques have become indispensable for Meloidogyne identification [33,34,35]. Among these, Sequence Characterised Amplified Region PCR (SCAR-PCR) is the most widely applied technique, alongside markers based on ribosomal and mitochondrial DNA as well as other DNA-based approaches, such as amplified fragment length polymorphism (AFLP), effectively target species-specific polymorphisms and are independent of nematode life stage, providing rapid results; nevertheless, occasional inconsistencies in diagnostic reliability have been reported [6,8,35,36,37,38,39]. Increasingly, classical and molecular tools are integrated and intensively re-examined to improve diagnostic precision [8,40,41]. Combining morphological, biochemical and molecular methodologies is considered essential for reliable species identification of RKNs [42,43].

Despite the agricultural importance of Meloidogyne spp., data on their presence in Croatia remain limited, with many infestations likely unreported. This study aims to update current knowledge on the occurrence and geographical distribution of Meloidogyne species in Croatia, thereby supporting the future effective control of RKN in Croatia.

2. Materials and Methods

2.1. Sampling and Nematode Extraction

Between 2022 and 2024, soil and root samples were collected by the Croatian Agency for Agriculture and Food, Centre for Plant Protection (CAAF-CPP) as part of the national survey targeting Meloidogyne chitwoodi and M. fallax in potato crops. Additionally, samples were collected under an early warning programme for RKNs in vegetable crops grown in both field and greenhouse conditions across Croatia.

Soil samples were collected from the potato fields, and samples composed of soil and roots were collected from vegetable cultivation sites.

Sampling was conducted across 3 regions and 15 counties covering mainland and coastal regions of Croatia (Figure 1). The selection of potato fields was based on national production data and the multi-annual EU surveillance programme for quarantine pests. Vegetable sites were chosen based on prior infestation reports or selected randomly. Sampling sites were stratified by crop type and geographical distribution to ensure representative coverage, and fields were randomly selected in accordance with Croatian and EU plant health surveillance protocols.

Potato and tomato were the main crops surveyed; however, samples from other crops such as carrot (Daucus carota subsp. sativus L.), melon, watermelon, cucumber, pepper, courgette (Cucurbita pepo L.), lettuce, beans (Phaseolus vulgaris L.), kale (Brassica oleracea var. sabauda L.), cabbage (Brassica oleracea cv. capitata L.), parsley (Petroselnum crispum L.), strawberry (Fragaria × ananassa L.), pomegranate (Punica granatum cv. Wonderful L.) and ornamental plants (Buddleja davidii L.) were included as they represent known host plants of RKN, showed suspicious symptoms of infestation, and were considered relevant for assessing the potential occurrence of these nematodes on previously unreported hosts in Croatia.

Soil around the potato roots was sampled with the auger (diameter of 2 cm) at a depth of 15–20 cm taking at least 100 subsamples per hectare, following a rectangular grid pattern with sampling intervals of 5–20 m. Approximately 1500 mL of soil was collected per site and stored in labelled polythene bags and refrigerated until analysis for a maximum of one month.

At vegetable production sites, roots were sampled in addition to collecting a soil sample. Root systems with residual soil of two symptomatic plants were collected per site and stored in labelled polythene bags and refrigerated until analysis for a maximum of ten days [44].

Out of the total 210 samples collected, 139 were soil samples obtained exclusively from potato fields, while the remaining 71 samples, derived from other crops, included 54 samples of both soil and symptomatic root material and 17 samples of only soil (

Table 1. Number of samples collected during the monitoring of RKNs on potatoes and other vegetables in Croatia from 2022 to 2024.

Type of the Monitoring	2022	2023	2024	Total (2022–2024)
National survey of RKNs on potato	37	50	52	139
Early warning programme of RKNs on vegetables	12	30	29	71
Total per year	49	80	81	210

, Figure 2).

Motile nematodes were extracted by flotation and sieving from 200 mL soil subsamples following Cobb [45] in accordance with EPPO Diagnostics Standard PM 7/119 (1) [46]. Soil was washed in water, decanted and nematodes were collected on sieves of different aperture followed by cleaning the suspension with Baermann funnel. The resulting suspensions were initially examined under a stereomicroscope (Olympus SMZ16, Hamburg, Germany).

Root samples were assessed for the presence of galls, thoroughly washed, and dissected under a stereomicroscope at 40× magnification (Olympus SMZ16, Hamburg, Germany) using a scalpel and a nematological needle.

Second-stage juveniles (J2s), females and egg masses were isolated for morphological and molecular analysis.

2.2. Morphological Analysis

Morphological identification of females was performed by examining perineal patterns following the Hartman and Sasser protocol [31]. J2s were fixed in triethanolamine–formaldehyde (TAF) and examined at 400× magnification using an Olympus BX-51 light microscope (Hamburg, Germany). Images and measurements were acquired using CellSens Dimension^® software version 5.1.2108 (Olympus CAM XC 10).

Diagnostic features included the shape of the perineal region (vulva, anus, lateral lines, striae, and phasmids) and morphological traits of J2s (body and stylet length, tail morphology, and hyaline tail tip). Identification was based on standard nematological keys [42,47,48,49,50,51].

2.3. DNA Extraction and Molecular Identification

Genomic DNA was extracted primarily from isolated egg masses, and occasionally from second-stage juveniles or males of each population using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. DNA samples were stored at −20 °C until further use.

Molecular characterisation involved PCR using group-specific, species-specific SCAR primers, and mitochondrial DNA (mtDNA) markers [33,34,35,38,52].

Group-specific primers targeted tropical Meloidogyne species and the M. ethiopica complex (M. ethiopica, M. luci, M. inornata). Species-specific SCAR primers identified M. incognita, M. javanica, M. arenaria, M. hapla, M. chitwoodi and M. fallax (

Table 2. Primers used for the molecular identification of Meloidogyne species.

Primer Name	Target Species/ Group of Species	Primer Sequences (5′-3′)	Expected Fragment Length (bp)	References
C2F3 Mt575	tropical Meloidogyne group	5′-GGTCAATGTTCAGAAATTTGTGG-3′	621	[34,38]
		5′-AGAACTTAAACTCTAAATAAC-3′
Me309 Me549R	M. ethiopica group	5′-CTAATTTGGGTGAATTT-3′	241	[38]
		5′-AATCAAAATCTTCTCCT-3′
JMV1 JMV2 JMV hapla JMV tropical	M. hapla M. chitwoodi M. fallax M. incognita	5′-GGATGGCGTGCTTTCAAC-3′	440 540 670 615	[52]
		5′-TTTCCCCTTATGATGTTTACCC-3′
		5′-AAAAATCCCCTCGAAAAATCCACC-3′
		5′-GCKGGTAATTAAGCTGTCA-3′
Finc Rinc	M. incognita	5′-CTCTGCCCAATGAGCTGTCC-3′	1200	[35]
		5′-CTCTGCCCTCACATTAGG-3′
Far Rar	M. arenaria	5′-TCGGCGATAGAGGTAAATGAC-3′	420	[35]
		5′-TCGGCGATAGACACTACAACT-3′
Fjav Rjav	M. javanica	5′-GGTGCGCGATTGAACTGAGC-3′	670	[35]
		5′-CAGGCCCTTCAGTGGAACTATAC-3′
NAD5F2 NAD5R1	Meloidogyne spp.	5′-TATTTTTTGTTTGAGATATATTAG-3′	610	[33]
		5′-CGTGAATCTTGATTTTCCATTTTT-3′
C2F3 1108	M. incognita and M. javanica M. arenaria M. hapla	5′-GGTCAATGTTCAGAAATTTGTGG-3′	1700 1100 520	[34]
		5′-TACCTTTGACCAATCACGCT-3′

).

PCR reactions (25 µL final volume) contained 12.5 µL Emerald Amp^® MAX PCR MasterMix (Takara Bio Inc., Shiga, Japan), 1 µL each of forward and reverse primers (10 µM), 8.5 µL nuclease-free water, and 2 µL of genomic DNA (~5 ng). Reactions were run on an ABI 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) under conditions specified in Supplementary Materials S1 (Tables S1–S5).

Amplicons were resolved by electrophoresis on 1% agarose gels in 1× TAE buffer (AccuGENE^®, Lonza, Switzerland) at 110 V for 45 min, and visualised using the UVIdoc HD2^® system (UVITEC Ltd., Cambridge, UK).

Initial screening used C2F3/Mt575R primers (621 bp) for tropical species, followed by Me309F/Me549R for the M. ethiopica group. Species-specific primers were used to confirmed identity: Finc/Rinc (1200 bp) for M. incognita, Far/Rar (420 bp) for M. arenaria, Fjav/Rjav (720 bp) for M. javanica. Multiplex PCR using primers JMV1, JMV2, JMVhapla, and JMVtropical enabled simultaneous detection of M. chitwoodi (540 bp), M. fallax (670 bp), M. hapla (440 bp), and M. incognita (615 bp). For additional confirmation of species identity, two mtDNA regions were amplified using primer sets C2F3/1108 developed by Powers and Harris in 1993 [34], and NAD5F2/NAD5R1 by Janssen et al. in 2016 [33] (Table 2).

PCR products were purified using the DNA Purification Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Amplicons were directly sequenced in both directions by a commercial services Macrogen Inc. (Amsterdam, The Netherlands) or Genewiz Europe (Leipzig, Germany). Raw chromatograms were edited in Geneious Prime 2025.2.1. (Biomatters Ltd., Auckland, New Zealand), and final identifications were confirmed by BLAST [53] searches against the NCBI GenBank database reference sequences (National Center for Biotechnology Information, Bethesda, Maryland, USA).

2.4. Bioassay of Meloidogyne Populations

Unidentified RKN-positive samples collected during monitoring were subjected to a bioassay. Infected soil and plant debris were placed in pots, and susceptible tomato seedlings (Solanum lycopersicum cv. ‘Novosadski rani’, Semenarna, Ljubljana, Slovenia) were planted.

Pure cultures of unidentified Meloidogyne populations were established from single egg masses and maintained on tomato under controlled growth chamber conditions. The growth chamber was set to 25 °C (day)/22 °C (night), with a 12 h photoperiod and 65–70% relative humidity. Tomato seedlings with at least six leaves were inoculated with 1000 eggs. These conditions allowed for nematode propagation and the production of sufficient inoculum for further morphological and molecular analyses.

Plants were grown for 45 days post-inoculation, after which roots were washed and inspected for galling under a stereomicroscope. Female nematodes and egg masses were dissected from infected roots; egg masses were used for DNA extraction, while freshly isolated females and J2s were used for morphological characterisation. All residual materials were sterilised post-extraction.

2.5. Statistical Analyses

Statistical analyses were performed to evaluate differences in Meloidogyne species frequency and abundance across Croatian counties. The occurrence and frequency of root-knot nematodes at both the genus and species levels were calculated using the following formulae:

Occurrence of the genus or species = Number of samples with RKN infection × 100/Total number of samples analysed

Absolute frequency = Number of samples with species × 100/Number of samples collected

Relative frequency = Frequency of occurrence of the species × 100/Sum of the frequency of all Meloidogyne spp.

3. Results

3.1. Detection of Meloidogyne spp.

During the monitoring period from 2022 to 2024, a total of 210 samples were collected and analysed from 15 counties across three Croatian regions. Of these, Meloidogyne spp. were detected in 61 samples, representing an overall infestation rate of 29%. Supplementary Materials S2 (Table S6) provides a comprehensive overview of all positive samples, including cultivation conditions, host plants, sampling localities, regions, results of PCR analyses, and identified RKN species during monitoring in Croatia from 2022 to 2024.

From the 139 soil samples collected in potato fields as part of the national survey, Meloidogyne spp. were found in 13 samples (9.4%). In the early warning programme for vegetable crops, which included a total of 71 samples, 54 samples of both soil and symptomatic roots and 17 samples of soil only, Meloidogyne spp. were detected in 48 samples (67.6%), indicating a higher level of infestation among the surveyed vegetable crops compared to potato (

Table 3. Number and percentage of samples with detected Meloidogyne spp. during the monitoring of root knot nematodes on potatoes and vegetables in Croatia from 2022 to 2024.

Type of the Monitoring	2022	2023	2024	Total Positive Detections (2022–2024)
National survey of RKNs on potato	1 (2.7%)	4 (8.0%)	8 (15.4%)	13 (9.4%)
Early warning programme of RKNs on vegetables	7 (58.3%)	24 (80.0%)	17 (58.6%)	48 (67.6%)
Total per year	8 (16.3%)	28 (35.0%)	25 (31.3%)	61 (29.0%)

).

The highest number of detections was recorded in samples from potato, cucumber, tomato, melon and pepper, all of which are the most important host crops for RKNs. In Croatia, detections were also confirmed for the first time on kale, Buddleja davidii, pomegranate, potato, melon, watermelon, courgette and bean (Supplementary Materials S2 (Table S6)).

3.2. Species Identification of Meloidogyne spp.

Species identification was conducted using a combination of morphological and molecular methods. Among the 61 positive samples, four Meloidogyne species were identified: M. incognita, M. hapla, M. arenaria, and M. javanica.

The most prevalent species was M. incognita, followed by M. hapla, while M. arenaria and M. javanica were detected in a smaller number of samples. Mixed populations were observed in several cases, including combinations of M. incognita with M. hapla, M. arenaria, and M. javanica (Supplementary Materials S2 (Table S6)).

The perineal patterns of M. incognita, observed in 43 populations, were typically rounded to oval, with a high, squarish dorsal arch and smooth to wavy striae in the perineal area. Variability in the perineal pattern was observed among the identified species M. arenaria and M. incognita. The perineal shape ranged from rounded to ovoid in both species, with characteristic lateral lines observed in M. arenaria, whereas they were absent or only weakly developed in M. incognita.

M. arenaria exhibited a generally low dorsal arch with distinct shoulders and lateral lines. The striae were coarse and varied from smooth to wavy. In some cases, the perineal patterns formed one or two “wings” extending laterally, marked by fusion of the dorsal and ventral striae. Patterns with short lateral incisures resembled those of M. javanica.

The perineal patterns of M. javanica were easily distinguishable from other species by the presence of prominent lateral lines. Patterns were generally ovoid or rounded with a low dorsal arch.

The posterior cuticular pattern of M. hapla was approximately circular, composed of closely spaced, smooth or slightly wavy striae. The dorsal arch was low (Figure 3).

Second-stage juveniles (J2s) were vermiform, slender, and annulated with the head region slightly set off from the body. The stylet was slender, delicate, and sharply pointed, with small basal knobs. The excretory pore was distinct and positioned posterior to the hemizonid. The tail was conoid with a rounded to pointed tip and a conspicuous hyaline region terminating in a narrow, long or short or irregular outline. Diagnostic characters included body and stylet length, tail morphology, and the structure of the hyaline tail tip (Figure 4).

The affiliation of the populations to the tropical RKN group and the M. ethiopica group was assessed using two PCR reactions. A total of 51 populations were identified as tropical RKNs, while the primers for M. ethiopica group did not yield amplification products in any of the tested samples.

Species identification of Meloidogyne spp. was further confirmed in 61 samples using species-specific SCAR primers. Amplification of the 1200 bp fragment with Finc/Rinc primers confirmed M. incognita in 43 samples and in 6 samples of mix populations. Far/Rar primers confirmed M. arenaria in one sample, while Fjav/Rjav primers confirmed M. javanica in one sample. Multiplex PCR with JMV primers identified M. hapla in ten samples. No amplification was observed for M. chitwoodi or M. fallax.

Further confirmation of species identity in ten samples was performed through mtDNA sequencing, comparison to sequences in the public domain with BLAST search and deposited in the GenBank under the accession numbers PX310634–PX310637 and PX317675–PX317680. Two mtDNA regions were amplified using primers targeting part of the gene for NADH dehydrogenase subunit 5 (NAD5) and the 3′ portion of the gene that codes for cytochrome oxidase subunit II (COII) through a portion of the 16S rRNA (lRNA) gene. BLAST searches revealed high similarity (≥98–100% identity) to M. incognita in four samples, M. arenaria and M. hapla in two samples and one M. javanica and M. javanica & M. arenaria (Supplementary Materials S3 (Table S7)).

3.3. Distribution of Meloidogyne Species

Between 2022 and 2024, Meloidogyne spp. were detected in 8 out of 15 surveyed counties. Of these, four counties were located in the Adriatic, three in the Pannonian and one in the Northern region.

A total of 61 RKNs populations were identified, comprising 43 populations of M. incognita (20.4%), 10 of M. hapla (4.8%), one of M. arenaria (0.5%), and one of M. javanica (0.5%). Additionally, six mixed populations were detected: three of M. incognita and M. javanica (1.4%), two of M. incognita and M. hapla (0.96%), and one of M. incognita and M. arenaria (0.5%).

M. incognita was recorded in three counties within the Adriatic region (Dubrovnik-Neretva, Zadar, and Istria) but was not found in the Pannonian or Northern regions. M. hapla was identified in two Adriatic counties (Zadar and Split-Dalmatia), three Pannonian counties (Brod-Posavina, Virovitica-Podravina, and Osijek-Baranja), and one in the Northern region (Međimurje County). Populations of M. arenaria and mixed populations of M. incognita and M. arenaria were detected in Zadar County. Two mixed populations (M. incognita and M. javanica) were found in Zadar and one in Dubrovnik-Neretva County. In Dubrovnik-Neretva County, single populations of M. javanica and two mixed populations of M. incognita + M. hapla were identified (Supplementary Materials S2 (Table S6)).

Positive detections of Meloidogyne spp. were most frequent in Dubrovnik-Neretva and Zadar Counties, while the Northern region (Međimurje County) had the lowest frequency of detection.

The greatest species diversity was observed in Zadar County, with three species and two mixed populations, followed by Dubrovnik-Neretva County with two species and two mixed populations. Only a single species was identified in each of the following counties: Split-Dalmatia, Brod-Posavina, Virovitica-Podravina, Osijek-Baranja, and Međimurje (Figure 5).

3.4. Host Plant Associations

In this survey, several host plants were analysed for the presence of root-knot nematode (RKN) species for the first time in Croatia. Meloidogyne species were found parasitizing 14 different host plants, including ten in open-field conditions and six under greenhouse cultivation (Supplementary Materials S2 (Table S6)).

M. incognita was the most prevalent species under greenhouse cultivation, detected on tomato, melon, cucumber, pepper, bean, and lettuce, in the Adriatic region (Zadar, Istria, and Dubrovnik-Neretva counties). Under field conditions, M. incognita was detected on potato, kale, pomegranate, watermelon, melon and courgette in the Adriatic region (Zadar, Istria, and Dubrovnik-Neretva counties).

M. arenaria was identified on the ornamental plant Buddleja davidii in a private garden in Zadar County, as well as in a mixed population with M. incognita on tomato.

M. hapla was detected in open fields on parsley in Međimurje County (Northern region), on carrot in Zadar County and on potato in both Split-Dalmatia County (Adriatic region) and the Pannonian region (Brod-Posavina and Virovitica-Podravina counties).

Two mixed populations of M. incognita and M. hapla were identified in Dubrovnik-Neretva County, isolated from tomato and melon. Furthermore, three mixed populations of M. incognita and M. javanica were found in Dubrovnik-Neretva and Zadar counties, isolated from melon and lettuce. A single population of M. javanica was detected on melon in Dubrovnik-Neretva County (Adriatic region).

The species with the widest host range in this study was M. incognita, recorded on 11 different plant species. M. hapla was found on four hosts, while M. arenaria and M. javanica were each identified on one host. No infestations were recorded on cabbage or strawberry in field conditions.

To the best of our knowledge, this study represents the first official report of the presence of M. incognita and M. javanica in Croatia. It also provides the first record of M. incognita in Croatia on the following host plants: pomegranate, kale, potatoes, courgette, melon and watermelon under open fields conditions as well as on bean, lettuce and melon in greenhouse production. Furthermore, M. arenaria was recorded on Buddleja davidii, M. javanica on melon, and M. hapla on potato for the first time in Croatia.

4. Discussion

This study aimed to identify Meloidogyne species and determine their distribution across Croatia. Species identification was conducted through an integrated approach that combined morphological observations with molecular techniques, primarily PCR-based methods and, where necessary, DNA sequencing. Accurate species identification is essential for the effective and sustainable management of RKNs.

Relying solely on morphological traits for species identification is unreliable due to significant interspecific variability and overlapping features among different species [54]. While morphological characteristics can support diagnostics, they are often insufficient alone and are best utilised in combination with molecular data. Consequently, molecular identification remains essential for accurate and definitive species determination [43].

For example, several RKN species (e.g., M. luci, M. paranaensis) were misidentified as M. incognita or M. arenaria based on perineal patterns typical of these species [55]. However, even molecular identification methods have their limitations. According to Bačić et al., [30], the species-specific PCR primers Finc/Rinc for M. incognita are not always entirely specific and may yield positive results with M. luci DNA. The identification of M. luci in Serbia was confirmed only after species-specific PCR for M. luci became available [40]. In the Turkish study, according to Aydınlı et al., [54], who conducted molecular PCR analyses with the Finc/Rinc primer set, did not give reproducible amplifications for some of the populations.

Biochemical methods, such as isozyme pattern analysis, offer a more reliable alternative. However, they often require a bioassay to obtain females at a specific developmental stage, which can be time-consuming [42]. For this reason, PCR analysis is more practical for routine and rapid diagnosis of RKN species.

The recognition of species relationships [33], the hybrid origin of tropical RKN species [56], and the associated taxonomic ambiguities are driving ongoing research and development of new molecular diagnostic methods for selected tropical RKN species [41].

Global warming has facilitated the migration of tropical RKN species into new areas, leading to the emergence of previously undocumented species in Europe [7,10,28,29,55,57,58,59]. Several Meloidogyne species, particularly tropical RKNs, are now recognised as invasive agricultural pests [8,11,30,54]. As climatic conditions become increasingly favourable for their development, tropical RKNs are expected to pose a growing threat in temperate regions. These species are also capable of surviving in Mediterranean and continental European climates, including Croatia, potentially causing significant economic losses.

Our results confirmed the presence of M. incognita, M. javanica, M. hapla, and M. arenaria, which is consistent with several studies on the occurrence and distribution of RKNs in the region. Before this study, three Meloidogyne species (M. arenaria, M. hapla, and M. artiellia) had been officially reported in Croatia [25,26,27]. However, M. artiellia has been reported only once, on tobacco (Nicotiana tabacum L.) by Oštrec [25], and was not detected in this survey as the study did not target field crops.

The study, which examined the effects of global warming on the distribution of tropical RKN species in France, Portugal, Serbia and Slovenia, identified M. incognita as the most frequently occurring species, followed by M. arenaria, M. javanica, M. hispanica, M. luci, and M. enterolobii [8]. Recent results from Serbia confirmed the presence of M. incognita, M. arenaria, and a mixed population of M. hapla and M. javanica, as well as the first detection of M. luci [10,30].

On the other hand, a study from Turkey shows slightly different frequencies of RKN species; in the Middle Black Sea region of Turkey, M. arenaria (42.2%) and M. luci (41.1%) were the most prevalent, while M. javanica (12.2%) and M. incognita (4.4%) were detected in fewer samples [54].

The current survey confirmed that RKNs are widely distributed across Croatian agricultural regions. The detection of M. incognita and M. javanica for the first time in Croatia, together with the identification of several new hosts, represents a significant advancement in the understanding of RKN diversity at the national level. Our results expand the known host range of RKNs in Croatia. These findings are in line with reports from neighbouring countries such as Serbia, where M. incognita, M. arenaria, M. hapla, and M. javanica are widely distributed, and from Turkey, where different frequencies but mainly overlapping species of RKN have been observed.

Aligned with studies from France and Portugal, which demonstrated the increasing spread of tropical species linked to climate change, our results confirm that Croatia is also vulnerable to further introductions and establishment of invasive RKNs.

Our findings align with projections from the ongoing Nem-Emerge project, which indicate areas of potential RKNs emergence in Croatia, as illustrated by the suitability map (Supplementary Materials S4 (Figure S1)) [41].

Croatia’s geographical position at the intersection of Mediterranean and continental climatic zones makes it particularly susceptible to both temperate and thermophilic Meloidogyne species. The predominance of sandy and light loamy soils, in combination with the Mediterranean climate of the Adriatic region, provides particularly favourable conditions for their development compared with other regions.

The confirmed presence of M. incognita, M. javanica, M. hapla, and M. arenaria, together with the risk of potential introduction of quarantine species such as M. luci and M. enterolobii, highlights the need for continued national monitoring programmes and the urgent development of effective management strategies.

5. Conclusions

This study contributes significantly to the current understanding of the distribution and diversity of Meloidogyne species across Croatian agricultural regions. The results confirm the widespread occurrence of RKNs and document the presence of previously unreported species and 14 host associations, primarily vegetables in open-field cultivation, but also ornamentals and fruit trees. Notably, M. incognita, M. javanica, M. hapla, and M. arenaria were confirmed, with M. incognita and M. javanica reported in Croatia for the first time. New host records include M. incognita on pomegranate, kale, and potato under open-field conditions, and on common bean in greenhouse production; M. arenaria on Buddleja davidii; and M. javanica on melon.

Given Croatia’s position at the intersection of Mediterranean and continental climatic zones, the country is particularly susceptible to both temperate and thermophilic Meloidogyne species. The ongoing monitoring programme proved successful in detecting two Meloidogyne species previously unrecorded in Croatia, alongside several new host associations, and should therefore be continued. Further sampling and species determinations are underway or planned for 2025 and 2026.

The findings also highlight broader phytosanitary risks linked to ongoing climate change, the intensification of greenhouse production, and increasing regional trade, all of which may accelerate the introduction and establishment of high-risk tropical and quarantine Meloidogyne species, such as M. enterolobii and M. luci.

In this context, consistent surveillance, accurate species-level diagnostics, and the implementation of integrated pest management (IPM) strategies are essential to safeguarding Croatia’s diverse and economically important cropping systems.