RESUMO
Stubby-root nematodes (family Trichoridae) are an economically important group of ectoparasites that feed on roots, vector tobraviruses, and cause substantial crop loss (1,2,3). In June 2013, 48 soil samples were submitted to the Nematology Laboratory at Ohio State University for nematode analysis from a field planted to corn in Wood County, Ohio. The soil texture was sandy and the field was previously planted to wheat and soybean in 2011 and 2012, respectively. Nematodes were extracted from 100 cm3 soil by decanting and sieving followed by sucrose centrifugal flotation. Phytoparasitic nematodes were identified and counted based on morphological traits to genus at 40× to 63× magnification. Nematode genera parasitic to corn recovered from these samples included Helicotylenchus, Hoplolaimus, Paratrichodorus, Pratylenchus, and Tylenchorhynchus. Stubby-root nematodes (Paratrichodorus sp.) were detected in more than 60% of the samples with a maximum count of 52 per 100 cm3 soil. Individual stubby-root nematodes were hand-picked and identified to species under a compound light microscope as Paratrichodorus allius (Jensen, 1963) Siddiqi, 1974 according to morphological and morphometric characteristics (1). Females (n = 14) were observed with the intestine not anteriorly overlapping the esophagus, posterior subventral esophageal glands overlapping the intestine, caudal pores, absence of spermatheca, and vaginal sclerotization reduced in lateral view. Body length ranged from 475.8 to 840.5 µm (mean = 652.2 µm), and onchiostylet length ranged from 37.7 to 47.4 µm (mean = 42.9 µm). DNA was extracted from single adult females (n = 4) and the 18S rRNA region was amplified with 18S (TTGATTACGTCCCTGCCCTTT) and 26S (TTTCACTCGCCGTTACTAAGG) primers (4). PCR products were purified and sequenced. The sequence was deposited in GenBank (Accession No. KF887974) and was compared with previously deposited sequences by means of BLAST search. The comparison revealed a sequence similarity of 98 to 99% with both P. allius and P. teres (AM269895, AM087124, AJ439572, FJ040484, AJ439575, and AM087125). P. allius and P. teres can be difficult to discriminate using both morphological characteristics and molecular sequencing (3). Therefore, a universal primer (BL18: 5' CCCGTCGMTACTACCGATT 3') and species-specific primers designed to produce PCR products of 432 bp (PAR2: 5'-CCGTTCAAACGCGTATATGATC-3') and 677 bp (PTR4: 5'-CCTGACAAGC'IWGCACTAGC-3') were used for P. allius and P. teres, respectively (3). DNA from individuals used for sequencing was used in PCR reactions with each species-specific primer. DNA samples yielded PCR products of 432 bp with the P. allius-specific primer set and had no reaction with the P. teres-specific primer set. Molecular results and morphological observations confirmed the presence of P. allius in the samples. P. allius is a polyphagous migratory ectoparasite and a vector for Tobacco rattle virus (TRV). The known distribution of P. allius has previously been limited to the Pacific Northwest, where it was originally described as an important pathogen in potato production (2,3). Corn and wheat have been reported as suitable hosts; although they are not susceptible to TRV, crop loss may result from direct damage to roots (2,3). Nematode management recommendations for corn and wheat will depend on the distribution of this nematode. To our knowledge, this is the first report of P. allius in Ohio. References: (1) W. Decreamer. Rev. Nematol. 3:81, 1980. (2) H. Mojtahedi and G. S. Santo. Am. J. Potato Res. 76:273, 1999. (3) E. Riga et al. Am. J. Potato Res. 84:2, 2007. (4) T. C. Vrain et al. Fundam. Appl. Nematol. 15:563, 1992.
RESUMO
Root-knot nematodes (Meloidogyne spp.) are common parasites attacking turfgrasses in the United States, but the species of these nematodes is typically unresolved unless targeted surveys are performed (3). Using morphometric analysis and an RFLP method (3), an investigation of a golf course green in Florida with a history of infestation by root-knot nematodes was conducted to identify the species present. This 'Tifdwarf' bermudagrass (Cynodon dactylon × C. transvaalensis) putting green at the University of Florida Research Unit in Citra, FL, exhibited irregular patches of declining turf. Turf roots in these symptomatic areas had galled root tips with adhering egg masses, characteristic of infection from Meloidogyne spp. Mean populations of 5,149 ± 708 Meloidogyne second stage juveniles per 100 cm3 of soil were extracted from the rhizosphere of these symptomatic plants. Morphological measurements from 20 of these juveniles were slightly less than those published previously for M. marylandi, but were still distinct enough to discriminate them from M. graminis, which commonly infects bermudagrass in Florida (3). Body length averaged 396.1 ± 4.9 (376.7 to 420.0) µm with a mean width of 16.3 ± 0.5 (13.3 to 18.3) µm, stylet lengths were 11.2 ± 0.7 (6.7 to 12.3) µm, tail lengths averaged 54.7 ± 1.9 (47.5 to 65.0) µm with the hyaline region of the tails 9.9 ± 0.7 (8.3 to 14.2) µm. Mature females extracted from symptomatic root tissue lacked a posterior cone-like protuberance of the vulva typical of M. graminis. DNA was extracted from 15 single juveniles using a NaOH digestion method (2). The mitochondrial DNA region was amplified with PCR using the primers C2F3/1108 5'-GGTCAATGTTCAGAAATTTGTGG-3' and 5'-TACCTTTGACCAATCACGCT-3' (3). This resulted in a DNA fragment 520 bp in length, which upon digestion with SspI restriction enzyme produced four bands 148, 103, 91, and 67 bp in length, similar to what has been reported for M. marylandi (3). The PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Valencia, CA) and sequenced at the University of Florida, Cancer Research and Genetics Institute. Sequences were compared with those in GenBank by means of BLAST search. The comparison showed a sequence similarity of 98% with M. marylandi (GenBank Accession No. JN241918.1). Although M. marylandi has been reported on bermudagrass in many areas of the United States and other places throughout the world (1,3,4), to our knowledge, this is the first detection of this nematode in Florida. Further studies will be conducted to determine the prevalence, incidence, severity of damage caused by M. marylandi, and determine a possible mode of dispersal on turfgrasses. References: (1) A. M. Golden. J. Nematol. 21:453, 1989. (2) J. Hübschen et al. Euro. J. Plant Pathol. 110:779, 2004. (3) M. A. McClure et al. Plant Dis. 96:635, 2012. (4) Y. Oka et al. Nematol. 5:727, 2003.
