Meloidogyne luci, a new infecting nematode species on common bean fields at Paraná State, Brazil

Common bean (Phaseolus vulgaris L.) is one of the most important crops in Brazil and worldwide (FAO, 2015). Brazil is the second largest producer of the world and the main producer of beans in the Americas, with a total production of 3,435,370 t in approximately 3,152.917 ha (IBGE, 2013).

There are numerous limiting factors to common bean production, among them the phytonematodes occurrence. This plant species is usually infected by many nematode species, but Meloidogyne spp. (i.e. M. incognita, M. javanica and M. arenaria) are responsible for great damage worldwide (Sikora et al., 2005). These nematodes reduce the quality of vegetables and cause yield losses around 50 to 80 % per annum worldwide (Moens et al 2009; Siddiqi, 2000). Beyond these most common root-knot species, currently, other species have been cited damaging P. vulgaris around the world, as M. enterolobii, M. chitwood, M. hapla and M. brasiliensis (Brito et al 2003a,b; Hafez & Sundararaj, 1999; Charchar & Eisenback, 2002). Meloidogyne luci was detected on P. vulgaris in Distrito Federal, Brazil, but no damages were reported (Carneiro et al., 2008). Common bean plants collected on the municipality of Araucária, Paraná State, Brazil, showed symptoms of decline and stunting.

Roots showed clearly visible galls and egg masses. Therefore, we aimed to identify the infecting nematode species with the integration of morphological, biochemical and molecular approaches.

During a survey of nematode species on common bean fields in Paraná State, Brazil, galled root samples of cultivarTuiuiú (Fig.1A) were sent, in June 2012, to the Nematology Laboratory from IA-PAR, Instituto Agronômico do Paraná, collected in the municipality of Araucária (25°35'34"S, 49°24'36"W). Roots were washed with tap water and adult females were extracted from dissected roots; after, the extraction of nematodes was carried out according to Boneti and Ferraz (1981). Then, nematode population was estimated.

Fig.1

The specimens were identified through perineal patterns (Hartman & Sasser, 1985) and esterase phenotypes of 20 adult females extracted from dissected roots. Esterase phenotypes were determined using protein extract from one young egg-laying female for each reaction. For this purpose, females were placed in a hema-tocrit containing 5μl of extraction solution (Carneiro et al., 2000), macerated and transferred to 7 % polyacrylamide gel slabs. Homogenates of the isolate IPR 81 of M. javanica (Mj) (J3; Rm 1.0, 1.3 and 1.4) was our reference. Electrophoresis was performed according to Brito et al. (2004) using a Omniphor (Biosystems) equipment, at 4 °C, under constant voltage of 100 V for 15 min and 200 V for 30 min. Gel was stained for esterase activity using the α-naphtyl acetate substrate.

Scanning electron microscopy (SEM) analysis was also performed on females partially dissected from the roots. After dissection step, samples were transferred to glass vials containing 2 mL of Karno-vsky fixative solution [2.5 % v v-¹ glutaraldehyde and 2.5 % v v-¹ paraformaldehyde in 0.05 M sodium cacodylate buffer + 0.001 M calcium chloride, pH 7.0] and stored at 4 °C. Samples were post-fixed in osmium tetroxide 1 % for 2h at 25 °C, and then dehydrated in a graded acetone series (30, 50, 70, 90 and 100 %) and dried to the critical point in CO₂ (Bal-tec CPD 030; Balzers, Germany). Root tissues were mounted on aluminium stubs, sputter coated with gold (Bal-tec SCD 050; Balzers, Germany) and examined in SEM (LEO-435 VP, Cambridge, England) operating at 20 kV with a working distance ranging from 10 to 30 mm.

Genomic DNA was obtained according to NaOH method (Stan-ton et al., 1998). Amplification of the 18S-ITS1-28S region of ri-bosomal DNA was performed using the Kit Taq PCR Master Mix (Promega) and the nematode universal primers rDNA2 (5'-TT-GATTACGTCCCTGCCCTTT-3') and rDNA1.58S (5'-ACGAGC-CGAGTGATCCACCG-3'). In a microcentrifuge tube were added 25μl of the Kit Taq PCR Master Mix, 1.5μl (0.3 microM) from each primer, 18μl water mili-Q and 4μl total DNA. The DNA was subjected to a PCR with the following specifications: 94 °C (2 min); followed by 40 cycles at 94 °C (1 min), 57 °C (1 min) and 72 °C (2 min) (Cherry et al., 1997).

DNA sequences were analyzed using BLASTn megablast (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi) and deposited in GenBank (KP165404 and KP165405). The ITS1 sequences were aligned using CLUSTAL W (Tompson et al., 1997). Dendrogram was obtained by MEGA6.06 (Tamura etal 2013). Model with lowest BIC (Bayesian Information Criterion) scores was considered to describe the substitution pattern. Then, a phylogenetic tree was constructed using Maximum Likelihood with Kimura 2-parameter model (Gamma distribution) and complete deletion (1,000 replicates). Meloidogyne hapla (KJ572385.1) was used as outgroup taxa.

Fig.2

The population density in the samples was 82 nematodes per gram of roots. Characters observed on both perineal patterns and biochemical analysis were consistent with those described for M. luci. Females showed an oval to squarish perineal pattern with a low to moderately high dorsal arc and without shoulders (Fig.1B) (Carneiro etal 2014). Biochemically, we obtained L3 (Rm 1.05, 1.10, 1.25) esterase phenotypes, unique trait for M. luci (Fig.1C) (Carneiro et al., 2014).

In relation to ITS1 sequences, amplicons of 376 and 417 pb in length obtained showed 97 % and 99 % identity with known sequences of M. luci (accession number KF482363.1 and KF482364.1). Phylogenetic analysis with maximum likelihood of those sequences placed our Meloidogyne isolate in a clade (90 % bootstrap support) which included only M. luci sequences available in the GenBank database, thus confirming its identity (Fig.2). To our knowledge, this is the first report of M. luci parasitizing common bean in Paraná State; previously, it was associated with bean in Braslândia, Distrito Federal, Brazil (Carneiro et al., 2013). In general way, common bean is a good host for the major species of Meloidogyne; now, this species is included as a new concern for growers and technicians. Additional studies should be conducted in order to determine its distribution and estimates of damage.