Characterisation of a Meloidogyne species complex parasitising rice in southern Brazil

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1 Nematology 00 (2017) 1-10 brill.com/nemy Characterisation of a Meloidogyne species complex parasitising rice in southern Brazil Rafael R.R.D. NEGRETTI 1, Cesar B. GOMES 2, Vanessa S. MATTOS 3,LúciaSOMAVILLA 1, Roberta MANICA-BERTO 1, Dirceu AGOSTINETTO 1, Philippe CASTAGNONE-SERENO 4 and Regina M.D.G. CARNEIRO 3, 1 PPGFS/Fac. Agronomia, Universidade Federal de Pelotas, C.P. 354, Pelotas-RS, Brazil 2 Embrapa Clima Temperado, C.P. 403, Pelotas-RS, Brazil 3 Embrapa Recursos Genéticos e Biotecnologia, C.P , Brasília-DF, Brazil 4 INRA, Univ. Nice Sophia Antipolis, CNRS, UMR , Institut Sophia Agrobiotech, Sophia Antipolis, France Received: 11 November 2016; revised: 2 February 2017 Accepted for publication: 2 February 2017; available online:??? Summary Root-knot nematodes (RKN) are important plant pathogens affecting rice in South-East Asia and southern Brazil in irrigated rice fields. In order to investigate the specific diversity of RKN associated with irrigated rice in southern Brazil, Meloidogyne spp. from Rio Grande do Sul (RS) and Santa Catarina (SC) States were characterised biochemically by esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Fifty-six Meloidogyne spp. populations were detected in 48% of rice samples, and a total of five esterase phenotypes were identified, four of which presented as drawn-out bands in different positions. In RS State, M. graminicola (Est VS1), Meloidogyne sp. 2 (Est R2) and Meloidogyne sp. 3 (Est R3) were identified, which corresponded to ca 80, 40 and 10% of samples, respectively. In SC State, M. graminicola, M. javanica (Est J3), Meloidogyne sp. 1 (Est R1), Meloidogyne sp. 2 and Meloidogyne sp. 3 accounted for ca 93.75, 12.50, 62.50, and 6.25% of samples, respectively. The esterase phenotypes R1, R2 and R3 are new, never having been detected on rice before. Meloidogyne javanica showed a N1 Mdh phenotype (Rm: 1.0), while four other populations exhibited a N1a (Rm: 1.4) phenotype. All populations were tested with two SCAR markers specific to M. graminicola, which confirmed that, but no specificity was obtained with both markers in relation to the atypical populations analysed. Sequencing and phylogenetic analyses of internal transcribed spacer-rrna (ITS) were performed to infer the phylogenetic relationship of these atypical Meloidogyne spp. populations. Meloidogyne sp. 1 grouped with the mitotic parthenogenetic species, while the two others (Meloidogyne sp. 2 and sp. 3) clustered with M. graminicola and other meiotic parthenogenetic species. Taken together, these data highlight the unprecedented specific diversity of RKN associated with irrigated rice in southern Brazil. Further morphological and phylogenetic studies involving these atypical isolates will be carried out to identify this complex of species. Keywords esterase phenotypes, ITS, Meloidogyne graminicola, Meloidogyne javanica, Meloidogyne spp., Oryza sativa, phylogeny, root-knot nematode, SCAR markers. Rice (Oryza sativa L) is one of the most important foods for human nutrition. This crop is the staple food of more than three billion people and responsible for supplying 20% of the calories consumed in the world, thus playing a strategic role in solving food security issues. With an annual production of million tonnes of rice in recent harvests, Brazil is responsible for 79.3% of the production in Mercosur (Southern Common Market), which represents more than 2.5% of the total produced by the bloc. Rio Grande do Sul (RS) and Santa Catarina (SC) States stand out as the largest national producers, accounting for over 61 and 8% of grain production in the country, respectively. Therefore, this large volume produced in both southern States is considered a stabiliser for the Brazilian market and ensures the supply of this grain to the Brazilian population (SOSBAI, 2014). Approximately 29 species of nematodes are associated with losses in irrigated rice. Among these pathogens, eight species of root-knot nematode (RKN) Meloidogyne spp. affect the physiological processes involved in the devel- Corresponding author, regina.carneiro@embrapa.br Koninklijke Brill NV, Leiden, 2017 DOI /

2 R.R.R.D. Negretti et al. opment of attacked plants, resulting in a reduction in plant growth and yield of rice. However, Meloidogyne graminicola Golden & Birchfield, 1965 is considered to be the most damaging species to this crop in different regions of the world (Bridge et al., 2005). The damage caused by M. graminicola in rice is estimated by Asian producers at 11-80% (Soriano & Reversat, 2003; De Waele & Elsen, 2007). These pathogens affect the development of plants due to their harmful action on roots, altering the absorption and translocation of water and nutrients and predisposing them to environmental stresses (Whitehead, 1998). Considering that M. graminicola is an organism well adapted to surviving and multiplying in flooded areas (Prot & Matias, 1995), its occurrence has been widely reported in irrigated rice fields in several Asian countries (Soriano & Reversat, 2003; Padgham et al., 2004), as well as in the Americas (Bridge et al., 2005; Gilces et al., 2016). RKN have been reported in irrigated rice in Brazil since the 1980s (Ribeiro et al., 1984). However, the first survey for RKN in this crop was performed only 30 years later (Steffen et al., 2007). In this study, the authors detected a widespread occurrence of M. graminicola (Est.VS1) in the central depression region of RS State. Despite reports of RKN occurrence in irrigated rice in southern Brazil (Sperandio & Monteiro, 1991; Steffen et al., 2007), there has been little information so far on the diversity of Meloidogyne species in irrigated rice in this important rice-producing region. Therefore, the aim of this study was to investigate the occurrence and distribution of Meloidogyne spp. in irrigated rice fields in RS and SC States, using biochemical and molecular characterisation tools. Materials and methods SURVEY AND MORPHOLOGICAL AND BIOCHEMICAL IDENTIFICATION OF Meloidogyne SPP. In order to study the occurrence and distribution of RKN species in irrigated rice in southern Brazil, a survey was carried out in RS and SC States. Seventy-five root samples were collected in rice fields from counties located in SC (Guaramirim, Camboríu and Ilhota) and RS (Uruguaiana, Itaqui, Dom Pedrito, Bagé, Cachoeira do Sul, Rio Pardo, Camaquã, Guaíba, Viamão, Mostardas, Pelotas, Santa Vitória do Palmar and Capão do Leão). The roots of each rice sample were first selected for RKN occurrence, and population densities (eggs and secondstage juveniles, J2) were extracted and evaluated in 10 g of roots (Hussey & Barker, 1973), using a blender instead of manual agitation and 1% NaOCl. In samples where Meloidogyne sp. was detected, 40 individual females were collected from each root sample for biochemical characterisation of the species using esterase (Est) and malate dehydrogenase (Mdh) isozymes in a horizontal electrophoresis system, according to Carneiro & Almeida (2001). Enzyme phenotypes were designated with letter(s) suggestive of the species name and the number of bands (Esbenshade & Triantaphyllou, 1985, 1990; Carneiro et al., 2000). Upon Est phenotyping, populations present in each sample were identified and purified (Carneiro & Almeida, 2001) and used for morphological characterisation using the female perineal patterns. Cuttings were mounted in glycerin, examined and photographed under light microscopy (Hartmann & Sasser, 1985). MOLECULAR STUDIES OF Meloidogyne SPP. POPULATIONS DNA of all purified populations was extracted according to Randig et al. (2002) and tested with specific SCAR markers developed by Bellafiore et al. (2015) and Htay et al. (2016). PCR studies targeting the ITS1-5.8S-ITS2 rrna region were carried out using the primer set developed by Schmitz et al. (1998), i.e., forward primer 5 - TTGATTACGTCCCTGCCCTTT-3 and reverse primer 5 -TCCTCCGCTAAATGATATG-3, and amplification conditions according to Subbotin et al. (2000). PCR products were cleaned using the Wizard SV Gel/PCR Clean Up System (Promega) and cloned into the pgem-t Easy Vector (Promega), following the manufacturer s instructions. Sequence alignments were performed using ClustalX version 1.83 with default parameters (Thompson et al., 1997), with sequences from one population of M. graminicola, twoofmeloidogyne sp. 1 and one each of Meloidogyne sp. 2 and Meloidogyne sp. 3, and also sequences of other Meloidogyne spp. retrieved from the NCBI database. A tree was generated using the neighbour-joining (NJ) algorithm (Saitou & Nei, 1987) in PAUP software version 4b10 (Swofford, 2003). A sequence from Pratylenchus pinguicaudatus (Rensch, 1924) Filipjev & Schuurmans Stekhoven, 1941 (KP ) was used as outgroup. To test the node support of the generated trees, 1000 bootstrap replicates were performed and only values above 50% were considered. 2 Nematology

3 Meloidogyne species complex on rice in southern Brazil Table 1. Population code, origin, esterase (Est) and malate dehydrogenase (Mdh) phenotypes and occurrence of Meloidogyne spp. (RKN) in irrigated rice fields in Santa Catarina State, Brazil. Sample Origin Rice cultivar RKN Phenotype Occurrence (%) Est Mdh 1 Guaramirim Epagri 109 M. graminicola VS1 N1a Guaramirim Epagri 109 M. graminicola VS1 N1a Guaramirim Epagri 109 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Camboriú SCS 112 M. graminicola VS1 N1a M. javanica J3 N Meloidogyne sp. 1 R1 N1a Camboriú SCS 112 M. graminicola VS1 N1a Camboriú SCS 112 M. graminicola VS1 N1a Camboriú SCS 112 M. graminicola VS1 N1a Meloidogyne sp. 3 R3 N1a Meloidogyne sp. 1 R1 N1a 8.10 Meloidogyne sp. 2 R2 N1a Camboriú SCS 112 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Camboriú SCS 112 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Camboriú SCS 114 M. javanica J3 N Camboriú SCS 114 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Ilhota Epagri 109 M. graminicola VS1 N1a Ilhota Epagri 109 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Ilhota Epagri 109 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Ilhota Epagri 109 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a Meloidogyne sp. 2 R2 N1a Ilhota Epagri 109 M. graminicola VS1 N1a Meloidogyne sp. 1 R1 N1a The occurrence was calculated from 40 individual females per sample characterised using esterase (Est) phenotypes (Carneiro & Almeida, 2001). Results RKN were detected in 48% of rice samples. However, by separately analysing the RKN distribution by state, RKN were present in 36 and 80% of the collected samples from RS and SC States, respectively (Tables 1, 2). The majority of samples from RS State were obtained from fields cultivated by conventional tillage, while in SC rice fields had been cultivated by a pre-germination system. In the samples collected in RS State where RKN were detected, populations ranged from 8 to 1182 eggs and J2(10groot) 1. However, in the local rice fields where plants exhibited some kind of stunting or yellowing, the nematode populations ranged from 79 to 4271 eggs and J2 (10 g root) 1. In SC State, the population levels ranged from 4.7 to 771 eggs and J2 (10 g root) 1. In the rice fields from SC where the plants showed some kind of symptoms, the RKN populations ranged from 14 to 2479 eggs and J2 (10 g root) 1. Fifty-six Meloidogyne populations were characterised biochemically, of which 26 populations were from SC State and 30 from RS State (Tables 1, 2). According to the results shown, two known phenotypes corresponded to M. graminicola (Est phenotype VS1, Rm: 0.70 drawn- Vol. 00(0),

4 R.R.R.D. Negretti et al. Table 2. Population code, origin, esterase (Est) and malate dehydrogenase (Mdh) phenotypes and occurrence (%) of Meloidogyne spp. (RKN) in irrigated rice fields in Rio Grande do Sul State, Brazil. Sample Origin Rice cultivar RKN Phenotype Occurrence (%) Est Mdh 22 Uruguaiana IRGA 424 M. graminicola VS1 N1a 5.56 Meloidogyne sp. 2 R2 N1a Uruguaiana IRGA 424 Meloidogyne sp. 3 R3 N1a Uruguaiana BRS Taim Meloidogyne sp. 2 R2 N1a Uruguaiana Puitá INTA CL Meloidogyne sp. 3 R3 N1a Uruguaiana IRGA 409 M. graminicola VS1 N1a Uruguaiana IRGA 409 M. graminicola VS1 N1a Dom Pedrito IRGA 424 Meloidogyne sp. 2 R2 N1a Bagé INIA Olimar M. graminicola VS1 N1a 8.70 Meloidogyne sp. 2 R2 N1a Bagé INIA Olimar M. graminicola VS1 N1a Meloidogyne sp. 2 R2 N1a Capão Leão Puitá INTA CL M. graminicola VS1 N1a Capão Leão Puitá INTA CL M. graminicola VS1 N1a Santa Vitória do Palmar Avaxi CL M. graminicola VS1 N1a Camaquã Puitá INTA CL M. graminicola VS1 N1a Guaíba IRGA 417 M. graminicola VS1 N1a Guaíba Epagri 108 M. graminicola VS1 N1a Viamão Epagri 114 M. graminicola VS1 N1a Meloidogyne sp. 2 R2 N1a Viamão Puitá INTA CL M. graminicola VS1 N1a Meloidogyne sp. 2 R2 N1a Mostardas Puitá INTA CL M. graminicola VS1 N1a Meloidogyne sp. 2 R2 N1a Cachoeira do Sul M. graminicola VS1 N1a Cachoeira do Sul M. graminicola VS1 N1a The occurrence was calculated from 40 individual females per sample characterised using esterase (Est) phenotypes (Carneiro & Almeida, 2001). out and extending from Rm: ) and M. javanica (Est phenotype J3, Rm: 1.00, 1.20, 1.35); M. graminicola was by far the predominant species in both States. Three atypical esterase phenotypes presenting large drawn-out bands and high enzymatic activity were also detected in the RKN rice populations sampled in this study: Meloidogyne sp. 1 (Est R1, Rm 1.02, extending from 1.0 to 1.04), Meloidogyne sp. 2 (Est. R2, Rm: 0.85, 0.91, extending from 0.91 to 1.0) and Meloidogyne sp. 3 (Est R3, Rm: 0.74, 0.80, extending from 0.74 to 0.82) (Tables 1, 2; Fig. 1). Meloidogyne javanica populations showed a N1 Mdh phenotype (Rm: 1.0) while other populations exhibited N1a (Rm: 1.4) (Fig. 1). The typical morphology of female perineal patterns confirmed the specific identification of all the populations designated as M. javanica and M. graminicola on the basis of their isoenzymatic phenotypes. On the contrary, it was not possible to identify the atypical populations (Meloidogyne sp. 1, Meloidogyne sp. 2 and Meloidogyne sp. 3) by using this morphological character. Mixed populations of Meloidogyne spp. were detected in both States in ca 44% of sampled rice. However, in SC, a higher percentage (62.5%) compared to RS (30%) was observed (Tables 1, 2). Meloidogyne sp. 2 was predominant in all samples from RS containing mixtures of RKN populations, while M. graminicola populations 4 Nematology

5 Meloidogyne species complex on rice in southern Brazil Fig. 1. Esterase (Est) and malate dehydrogenase (Mdh) phenotypes observed in Meloidogyne spp. populations collected in irrigated rice plants from southern Brazil: M. javanica (J3N1), M. graminicola (VS1 N1a), Meloidogyne sp. 1 (R1N1a), Meloidogyne sp. 2 (R2N1a) and Meloidogyne sp. 3 (R3N1a) (top) and esterase phenotypes in polyacrylamide gel. A: Meloidogyne graminicola (EstVS1); B: Meloidogyne sp. 1 (Est R1); C: Meloidogyne sp. 2 (Est R2); D: Meloidogyne sp. 3 (Est R3). M. javanica (Est J3) was used as reference in each gel (bottom). Vol. 00(0),

6 R.R.R.D. Negretti et al. were more frequently detected in mixtures with other species in samples from SC State. Considering the frequency occurrence of different Meloidogyne spp. we can report for SC State: M. graminicola ca 93.75%, M. javanica ca 12.5%, Meloidogyne sp. 1 ca 62.5%, Meloidogyne sp. 2 ca 12.25% and Meloidogyne sp. 3 ca 6.25%; and for RS State: M. graminicola ca 80%, Meloidogyne sp. 2 ca 40% and Meloidogyne sp. 3 ca 10% (Tables 1, 2). All populations were tested with the specific SCAR markers developed for M. graminicola by Bellafiore et al. (2015) and Htay et al. (2016), although no strict specificity was obtained with both markers in relation to the populations analysed. The first marker revealed a fragment (640 bp) for all M. graminicola amplifications as well as for Meloidogyne sp. 1 and Meloidogyne sp. 3. For the second marker, an amplification (369 bp) was observed for all M. graminicola populations and also for Meloidogyne sp. 1 (data not shown). Additionally, sequencing of internal transcribed spacer rrna (ITS) of the populations confirmed them as M. graminicola (data not shown). The NJ tree showed that M. graminicola sequences from this study and those retrieved from databases form a single cluster (100% bootstrap value) with minor subclustering (Fig. 2). Interestingly, both Meloidogyne sp. 2 and Meloidogyne sp. 3 clustered together with M. graminicola, suggesting they are closely related. The two Meloidogyne sp. 1 populations clustered together (59% bootstrap values) and were more similar to other Meloidogyne spp. such as M. javanica, M. incognita and M. arenaria. Discussion The Meloidogyne graminis-group is the most defined group within the genus, with some species being morphologically extremely similar (Jepson, 1987). Among others, seven species that parasitise rice constitute this group: M. graminicola, M. graminis (Sledge & Golden, 1964), Whitehead, 1968, M. hainanensis Liao & Feng, 1995, M. lini Yang, Hu & Xu, 1988, M. oryzae Maas, Sanders & Dede, 1978, M. salasi López, 1984, and M. tritricoryzae Gaur, Saha & Khan, Some morphological and biological characters are common in these species: female body elongate, vulva situated on a posterior protuberance, association with an amphimictic or meiotic parthenogenetic mode of reproduction, semi-endoparasitic parasitism and males common; or mitotic parthenogenesis, males rare and females deeply embedded in the host. The VS1 esterase and N1a Mdh phenotypes were considered to be shared by M. graminicola, M. graminis and M. oryzae (Esbenshade & Triantaphyllou, 1985). More recently, different esterase phenotypes were detected for the latter two species (G1 and O1, respectively) (Blok & Powers, 2009). Considering the difficulty of characterising enzymatically nematodes from the graminis-group due to the large drawn-out bands and high enzymatic activity revealed on gels, it is possible that some species presented VS1 profiles in different positions, as was observed in this study. Here, three different esterase phenotypes (R1, R2, R3) were detected for the first time in Meloidogyne spp. populations from rice. In this way, our results confirm that the use of esterase phenotypes is a rapid and efficient method: i) to characterise Meloidogyne species and detect atypical forms; ii) to carry out an extensive field survey to determine the frequency and relative distribution of Meloidogyne spp.; and iii) to detect mixed species populations for purification prior to conducting studies on plant resistance or host specificity (Esbenshade & Triantaphyllou, 1990; Carneiro et al., 1996). Esterase phenotypes from M. graminicola (Est VS1) populations confirm the pattern of a drawn-out band with high enzymatic activity, similar to patterns reported by Esbenshade & Triantaphyllou (1985; 1990), Carneiro et al. (1996; 2000) and Steffen et al. (2007). The other drawn-out bands in different positions characterised for Meloidogyne sp. 1, Meloidogyne sp. 2 and Meloidogyne sp. 3 were detected for the first time in this study and may be considered new esterase phenotypes related to non-enzymatically characterised species like M. oryzae, M. triticoryzae, M salasi or other species detected on rice. Future morphological and phylogenetic studies involving these atypical isolates must be carried out to identify this complex of species. Although M. javanica has been reported in upland rice in Brazil since the late 1960s (Lordello, 1992), the occurrence of M. javanica associated with M. graminicola observed in SC State is the first record of this species in irrigated rice in the country. According to Carneiro et al. (2000), the Est phenotype of BrazilianpopulationsofM. graminicola was very close to the phenotype Est O1 of two populations identified as M. oryzae from Suriname and French Guiana (Blok & Powers, 2009). These populations were not characterised morphologically and the real status of M. oryzae was not confirmed by these authors. In Brazil, mixed populations of Meloidogyne have been reported in soybean (Castro et al., 2001), banana 6 Nematology

7 Meloidogyne species complex on rice in southern Brazil Fig. 2. Neighbour-joining tree showing phylogenetic relationships between Meloidogyne spp. from rice and other closely related species, based on the ITS1-5.8S-ITS2 rrna sequences. Numbers to the left of branches are bootstrap values (in %) for 1000 replicates. M. graminicola CL = M. graminicola Capão Leão Puitá INTA CL; M. sp. 2 = Meloidogyne sp. 2 Uruguaiana IRGA 424; M. sp. 3 = Meloidogyne sp. 3 Uruguaiana Puitá INTA CL; M. sp. 1 = Meloidogyne sp. 1 Camboriú SCS 114; M. sp. 1A = Meloidogyne sp. 1 Ilhota Epagri 109. Vol. 00(0),

8 R.R.R.D. Negretti et al. (Cofcewicz et al., 2004, 2005), coffee (Carneiro et al., 2005), kiwi (Somavilla et al., 2011) and fig (Medina et al., 2006), among other hosts. However, this is the first prospecting to be done in Brazil and the world using isozyme phenotypes to characterise Meloidogyne spp. from irrigated rice fields in which more than one esterase phenotype occurs. The occurrence of M. graminicola in ca 67% of samples where RKN were detected demonstrates the importance of this species for irrigated rice in southern Brazil. Considering previous reports of this species in rice fields (Sperandio & Monteiro, 1991; Sperandio & Amaral, 1994; Gomes et al., 1997), its widespread occurrence in a survey carried out in the central depression region of RS State (Steffen et al., 2007) and the results obtained here confirm the presence of M. graminicola in all ricegrowing regions in this State, as well as in SC, as previously reported by Gomes et al. (2009). In a recent survey, Gilces et al. (2016) found M. graminicola and Hirschmanniella oryzae (van Breda de Haan, 1902) Luc & Goodey, 1964 in rice fields in Ecuador in about 90 and 71% of the samples, respectively; however, it was not mentioned how the author characterised the species of RKN. All populations collected and purified were tested with specific SCAR markers for M. graminicola developed by Bellafiore et al. (2015) and Htay et al. (2016). No specificity was obtained with either markers in relation to the different esterase populations analysed. A positive amplification was observed for all M. graminicola populations, as well as for Meloidogyne sp. 1 and Meloidogyne sp. 3, showing that surveys conducted with these molecular markers can identify more than one species occurring in the same area as these markers are not species-specific for M. graminicola. Recent surveys conducted in Paraná (PR) State using esterase phenotypes have shown the predominance of Meloidogyne sp. 2 and Meloidogyne sp. 3 and the restricted occurrence of M. graminicola (Cesar B. Gomes, pers. comm.). In addition, preliminary sequencing of internal transcribed spacer rrna (ITS) showed that all M. graminicola populations belonged to a single cluster. Interestingly, both Meloidogyne sp. 2 and Meloidogyne sp. 3 clustered together with M. graminicola populations, suggesting that they are closely related and are probably species presenting facultative meiotic parthenogenesis in rice. (Castagnone-Sereno et al., 2013). Both Meloidogyne sp. 1 populations clustered together with obligatory mitotic parthenogenetic populations, suggesting that Meloidogyne sp. 1 can reproduce by mitotic parthenogenesis like M. oryzae, which was reported as having chromosomes (Esbenshade & Triantaphyllou, 1985). Recently, the population of M. oryzae from Surinam, studied first by Carneiro et al. (2000) and Tigano et al. (2005), showed the same number of chromosomes (ca 18) as M. graminicola (VS1) and a similar esterase phenotype, confirming the suspicion of an erroneous classification. This, indeed, agrees with the phylogenetic relationships depicted in Castagnone-Sereno et al. (2013), indicating that M. oryzae (mitotic) unexpectedly belongs to clade III, a group otherwise formed by meiotic parthenogenetic species, including M. graminicola. The highest population densities of RKN in roots were found on Epagri 109 and 108, SCS114, Avax Cl and Puitá Inta CL rice cultivars at a rate of RKN eggs and J2 (10 g root) 1. According to Bridge et al. (2005), 4000 J2 of M. graminicola/plant under glasshouse conditions can cause destruction of up to 72% of deepwater rice plants by drowning them. However, if we consider the total root fresh mass (ca 50 g), the nematode population in our results appears to be 2-5 times higher than that reported by Bridge et al. (2005). According to these authors, rice plants may be infected by M. graminicola J2 during the time rice plants are not flooded. However, once nematodes enter the root system, even under flooded conditions, J2 are able to multiply within host plant tissues, completing several generations during the rice cropping cycle. Moreover, it is important to note that flooded soil conditions prevent the occurrence of RKN and other nematode species that seriously affect upland rice crops (Bridge et al., 2005). There has been a decrease in water availability used for rice irrigation in southern Brazil. Thus, the use of irrigation systems that require less water may lead to changes in agricultural practices and the damage caused by nematodes to irrigated rice may increase considerably. RKN management using resistant rice cultivars and crop rotation is an important strategy to minimise nematode damage. However, detailed morphological, morphometric and molecular studies are needed to correctly identify nematode populations in rice fields. These studies should lead to a better design for control and management strategies for implementation within an integrated pest management scheme. 8 Nematology

9 Meloidogyne species complex on rice in southern Brazil Acknowledgements This work was supported by funds from FAPDF and EMBRAPA Macro 2. Vanessa Mattos and Regina Carneiro thank CNPq for their scholarships. References Bellafiore, S., Jougla, C., Chapuis, E., Besnard, G., Suong, M., Vu, P.N., De Whale, D., Gantet, P. & Thi, X.N. (2015). Intraspecific variability of the facultative meiotic parthenogenetic root-knot nematode (Meloidogyne graminicola) from rice fields in Vietnam. Comptes Rendus Biologies 338, DOI: /j.crvi Blok, V.C. & Powers, O. (2009). Biochemical and molecular identification. In: Perry, R.N., Moens, M. & Starr, J.L. (Eds). Root-knot nematodes. Wallingford, UK, CABI International, pp Bridge, J., Plowright, R.A. & Peng, D. (2005). Nematodes parasites of rice. In: Luc, M., Sikora, R.A. & Bridge, J. (Eds). Plant parasitic nematodes in subtropical and tropical agriculture, 2nd edition. Wallingford, UK, CABI Publishing, pp Carneiro, R.M.D.G. & Almeida, M.R.A. (2001). 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