First Report of Meloidogyne graminicola (Nematoda: Meloidogynidae) on Barley (Hordeum vulgare) in Brazil

Abstract

Winter barley (Hordeum vulgare L.) has become a growing addition to cereal crops in Brazil and, until recently, the root-knot nematode (Meloidogyne sp.) has not been reported to infect barley in this country. Meloidogyne graminicola is a major pathogen of rice (Oryza sativa L.) and causes significant yield losses in both upland and flooded fields in Rio Grande do Sul, Brazil (Negretti et al. 2017). In September 2017, many nematode galls were observed on the roots of barley plants (cv. BRS Brau), and samples were taken from areas in Santa Maria county (29°45′S; 53°38′W), Rio Grande do Sul State, Brazil. Meloidogyne sp. was identified using esterase phenotypes (n = 40), perineal patterns (n = 40), morphological measurements of second-stage juveniles (J2) (n = 50), and amplification and sequencing of the ITS1-5.8S-ITS2 rRNA region and the D2–D3 fragment of the 28S ribosomal RNA gene. The barley root systems were also processed to determine the number of J2s and eggs of Meloidogyne sp. The nematode population density observed in the samples was 2,585 eggs plus J2s per gram of barley root. The J2s had the following morphometric characteristics: length (L) = 458.2 ± 38.0 (390.0 to 476.0) µm, a = 26.6 ± 1.5 (24.9 to 29.4), c = 6.15 ± 0.4 (5.1 to 7.6), stylet = 14.2 ± 0.5 (13.4 to 15.0) µm, dorsal esophageal gland orifice (DGO) = 3.9 ± 0.4 (3.4 to 4.6) µm, tail length = 70.5 ± 4.7 (63.1 to 75.3) µm, and hyaline tail terminus = 19.5 ± 1.8 (14.5 to 25.5) µm. The female’s perineal patterns were oval with a low dorsal arch without a lateral line. The cuticular striations were smooth and thick in the dorsal region of the vulva, and the phasmids were close together (13.1 to 17.9 μm). These morphological characteristics matched the original description of M. graminicola (Golden and Birchfield 1968). The polymorphisms of esterase bands seen using electrophoresis revealed the phenotype VS-1 (Rm = 0.71) typical of M. graminicola. The sequence homologies were determined by comparing them with those from GenBank. PCR amplicons of D2–D3 of the 28S and ITS1-5.8S-ITS2 rRNA regions were 531 bp (GenBank MH707259) and 440 bp (MH703577), respectively. The sequences of both markers exhibited 99 and 100% identity, respectively, with sequences corresponding to M. graminicola isolates (D2–D3: MH843665 and KY660544; ITS: MH842692 and KY346887) from Brazil and China. Identification of the nematode species was confirmed by PCR using the species-specific sequence-characterized amplified region (SCAR) with primer sets GRAJ17-1F and GRAJ17-1R. PCR amplification using SCAR produced a specific fragment of the expected size (∼230 bp) in M. graminicola (Mattos et al. 2019). In greenhouse tests, barley plantlets from cv. BRS Brau that were maintained in pots with sterilized soil were inoculated with 5,000 eggs plus J2s of the original population of M. graminicola using 10 replicates; a noninoculated control was included in the test. After 60 days, the growth of all the inoculated plants was reduced compared with that of the control. Galling symptoms on the roots were similar to those in the field, with a nematode reproduction factor (final population/initial population) of 28.5. There were no galls on the roots of noninoculated plants, and plant development was not affected. The morphological and molecular characteristics of the reisolated root-knot nematode were identical to those of M. graminicola. M. graminicola has been reported on barley in India (Vaish et al. 2012). To our knowledge, this is the first report on M. graminicola parasitizing barley in Brazil. This finding is of great importance for Brazilian barley production because this nematode can severely damage barley plants and become a major problem for the cultivation of this crop.