PM 7/158 (1) Meloidogyne graminicola

Specific scope: This Standard describes a diagnostic protocol for Meloidogyne graminicola.1

Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology.2

This Standard should be used in conjunction with PM 7/76 Use of EPPO Diagnostic Standards.

Specific approval and amendment: Approved in 2024–08. Authors and contributors are given in the Acknowledgements section.

Meloidogyne graminicola (rice root-knot nematode) is a major plant-parasitic nematode on rice and is recognized as an important constraint to rice production in Asia, where it is present in most countries in South, South-East and East Asia. It received special attention in the last few decades when yield losses due to this nematode increased, probably because of changes in agronomic practices in rice culturing, shifting to reduced water usage. However, damage can be very high in all types of rice cultivation systems (Bridge & Page, 1982; De Waele et al., 2013; Mantelin et al., 2017; Padgham et al., 2004; Peng et al., 2018; Win et al., 2016).

The genus Meloidogyne comprises over 100 species, of which several are important agricultural pests (Subbotin et al., 2021). They are obligate sedentary endoparasites and have a high reproduction rate. Meloidogyne graminicola is a facultative meiotic parthenogenetic species and sexual crosses are rare (Triantaphyllou, 1969). Meloidogyne spp. cause galling of plant roots and have a wide host range, which is another key factor for their successful survival. In addition, M. graminicola has the capacity to survive inundated (i.e. low oxygen) conditions for months (Bridge & Page, 1982).

Although widely distributed in Asia, M. graminicola was initially described from Louisiana (USA) (Golden & Birchfield, 1965). The nematode was also detected in other parts of the world, including Southern Africa (South Africa, Madagascar), South America (Brazil, Colombia, Ecuador) and Europe, where it was found in 2016 and 2018 in Italian rice fields (Fanelli et al., 2017, 2022). Phylogenetic studies showed that the Italian M. graminicola populations found in rice fields of Piedmont and Lombardy in 2016 and 2018, respectively, have genetic differences which suggests they could have been introduced independently (Fanelli et al., 2022).

Besides its main host, Asian rice (Oryza sativa), M. graminicola can multiply on several other Poaceae, including cereals such as oat (Avena sativa), pearl millet (Pennisetum glaucum), wheat (Triticum aestivum), barley (Hordeum vulgare), but also sugar cane (Saccharum officinarum), corn (Zea mays) and weeds such as barnyard grass (Echinochloa crus-galli). African rice (O. glaberrima) genotypes examined to date were less susceptible than Asian rice, however this host species can still allow multiplication of M. graminicola under dry conditions and exhibit damage (Cabasan et al., 2018). Moreover, common vegetables such as onion (Allium cepa), pea (Pisum sativum) and tomato (Solanum lycopersicum), and over 100 other plants are reported to be hosts for this root-knot nematode species. The ability to reproduce seems to vary a lot between plant varieties and M. graminicola biotypes, sometimes resulting in contradicting reports of host status.

Updated information on hosts and geographical distribution of M. graminicola can be viewed in EPPO Global Database (EPPO, 2023).

In rice, egg masses are produced within the root tissue and remain within the cortex. Hatched second-stage juveniles (J2) persist within the same root, by either migrating through the cortex and establishing a new feeding site in the same root, or by staying within the maternal gall (Bridge & Page, 1982). Due to this, flooded conditions do not interfere with the nematode life cycle. Egressing juveniles cannot re-enter the same root and consequently search for new root tips to infect plants. Flooded conditions limit the ability of J2 to reach new roots and to infect new plants. Hence, rice roots are invaded when soils are drained, and new infections are rare when soils are inundated. Once inside roots, M. graminicola can multiply rapidly (between 19 and 51 days; Bridge & Page, 1982; Fernandez et al., 2014; Rao & Israel, 1973). The nematode survives in soil as egg masses and as juveniles for at least 4 to 5 months (Bridge & Page, 1982). M. graminicola can be spread with infected rice seedlings or other plant hosts, contaminated soil, irrigation and run-off water, but not through seeds.

A flow diagram for the detection and identification of Meloidogyne graminicola is given in Figure 1.

Name: Meloidogyne graminicola Golden & Birchfield, 1965.

Other scientific name: Meloidogyne hainanensis Liao & Feng, 1995.

Taxonomic position: Nematoda: Tylenchida,3 Meloidogynidae.

EPPO Code: MELGGC.

Phytosanitary categorization: EPPO A2 no. 455; EU temporary measures 2022.4

Meloidogyne graminicola can be found in roots of infected plants, in soil, and in drainage water from inundated fields. Roots can contain all stages, however in soil and water, mainly second-stage juveniles will be present, predominantly during dry periods in rain-fed rice systems. In rice fields with regular flooding (e.g. irrigated, rain-fed lowland systems), M. graminicola may not always be present in soil and water, despite the nematode being present in the field. Juveniles emerge into the soil only when the soil is drained, thus their presence in soil is very much dependent on the watering system of the rice culture. In flooded conditions nematodes remain inside the root system. Hence, it is important to inspect rice roots for detection of M. graminicola, as they can contain many more nematodes than soil. Juveniles in soil can survive for several weeks, even when soil is flooded, however they will not infect roots until the water is removed (Bridge & Page, 1982).

Identification of M. graminicola to species level should be based on morphological and molecular analyses, using several specimens for both types of analysis. Morphological characters of M. graminicola are similar to those of other Meloidogyne species, especially those belonging to the ‘graminis’ group. In Brazil, M. graminicola was found with M. javanica, and M. oryzae in root samples from rice fields within the same county (Mattos et al., 2018; Negretti et al., 2017). Therefore, careful observations are required when performing morphological identification. All morphologically similar species, which are listed in Table 1, have rice as a host except for M. trifoliophila (Bernard & Eisenback, 1997). Considerable morphological variability among populations of M. graminicola make morphological identification difficult (Bellafiore et al., 2015; Jepson, 1983; Pokharel et al., 2010; Salalia et al., 2017). Therefore, molecular tests should be performed to achieve a reliable identification (see Section 4.3). The high intraspecific variability within M. graminicola populations (Bellafiore et al., 2015; Fanelli et al., 2017; Mattos et al., 2019; Soares et al., 2020) and the phylogenetic closeness to other Meloidogyne species infecting rice, such as M. oryzae, (Besnard et al., 2019; Mattos et al., 2018), complicates the development of molecular tests for M. graminicola identification. However, when used in combination, molecular tests included in Figure 1 result in reliable identification.

Reference material can be obtained from EURL for Plant Parasitic Nematodes and NIVIP (Netherlands Institute for Vectors, Invasive plants and Plant health), Wageningen (NL). Sequences are available in EPPO-Q-bank (https://qbank.eppo.int/).

A culture of M. graminicola can be established on rice, wheat and tomato, but also on the easily grown Echinochloa crus-galli (barnyard grass), a common weed. General guidelines for the management of nematode collections used for the production and maintenance of reference material are available in PM 7/148 (EPPO, 2023).

Guidelines on reporting and documentation are given in EPPO Standard PM 7/77 Documentation and reporting on a diagnosis.

When performance characteristics are available, these are provided with the description of the test. Validation data are also available in the EPPO Database on Diagnostic Expertise (http://dc.eppo.int), and it is recommended to consult this database as additional information may be available there (e.g. more detailed information on analytical specificity, full validation reports, etc.).

Further information on this organism can be obtained from A Troccoli (CNR-Institute for Sustainable Plant Protection (IPSP) Bari, IT), M Grossi de Sá (EURL for Plant Parasitic Nematodes, ANSES – Plant Health Laboratory, Nematology Unit, Rennes, FR), N Viaene and N Damme (EURL for Plant Parasitic Nematodes, ILVO, BE).

If you have any feedback concerning this Diagnostic Standard, or any of the tests included, or if you can provide additional validation data for tests included in this protocol that you wish to share please contact [email protected].

A regular review process is in place to identify the need for revision of diagnostic protocols. Protocols identified as needing revision are marked as such on the EPPO website.

When errata and corrigenda are in press, this will also be marked on the website.