Dataset Information

DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis.

ABSTRACT: Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.

SUBMITTER: Powers T

PROVIDER: S-EPMC6929643 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis.

Powers Thomas T Skantar Andrea A Harris Tim T Higgins Rebecca R Mullin Peter P Hafez Saad S Handoo Zafar Z Todd Tim T Powers Kirsten K

Journal of nematology 20190101

Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairw ...[more]

PMID: 31088028

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Project description:The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 +/- 0.5) x 10(5) bacteria (mean +/- standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and STREPTOMYCES: A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.

Project description:Soil samples collected during a survey for plant-parasitic nematodes in Tift County GA in summer 2017 were submitted for routine diagnosis of nematodes to the Extension Nematology Lab at the Department of Plant Pathology, University of Georgia, Athens, Georgia. Cyst nematodes recovered by centrifugal flotation technique were discovered in the samples from two research sites in a field with a history of tobacco and vegetable production. Cyst nematodes from tobacco (10 cysts/100?cm 3 of soil) and vegetable (2 cysts/100?cm 3 of soil) sites had similar morphological features. Morphology and morphometric measurements of the cysts and J2 ( Fig. 1A-C ) were in agreement with those of Heterodera cyperi ( Golden et al., 1962 ; Romero and López-Llorca, 1996 ). Measurements of J2 ( n = 12) included the length (range = 443-494??m, mean = 467.4??m) and width (18.3-24.4??m, 20.6??m) of body, stylet (19.1-20.8??m, 20.3??m), tail (61.6.0-66.4??m, 64.2??m), body width at anus (11.9-14.1??m, 12.8??m), and hyaline tail terminus (22.7-29.2??m, 26.3??m). The lateral field of J2 had three lines. Cysts ( n = 10; Fig. 1C ) were lemon-shaped, light to dark brown in color with protruding neck and vulval cone. The cysts had ambifenestrated vulval cone and no bullae was present. Morphometrics included body length excluding neck (370.5-714.4??m, 555.7??m); body width (165.6-411.1??m, 310.9??m); neck length (36.5-66.3??m, 49.8??m); fenestra length (26.3-42.5??m, 35.8??m), and fenestra width (19.1-31.5??m, 23.8??m). DNA was extracted from single cysts ( n = 3) and internal transcribed spacer (ITS) of rRNA and partial cytochrome oxidase I ( COI ) genes were amplified with primers TW81/AB28 and Het-coxiF/Het-coxiR, respectively ( Subbotin et al., 2001 ; Subbotin, 2015 ) and sequenced. The resulting sequences were deposited into the GenBank database (Accession no. MG825344 and MG857126) and also subjected to BLAST searches in the database. ITS sequence of H. cyperi showed 100% similarity (100% coverage) with that of a H. cyperi population from Spain (AF274388). COI sequence of H. cyperi showed 89% similarity (98% coverage) with that of H. guangdongensis (MF425735), and 88% similarity (83% coverage) with that of H. elachista (KC618473). The pathogenicity of H. cyperi was examined under greenhouse conditions using tobacco cv. K340, tomato cv. Tribute, cucumber cv. Thunder, and yellow nutsedge ( Cyperus esculentus L.). 3-wk-old seedlings of the test plants were transferred into Deepot D25L cell containers (5-cm-diam. × 25.4-cm deep) filled with sterilized sand: sand: soil mixture (1:2) and then inoculated with 1,000 eggs and J2 of H. cyperi . The plants were grown for 90?d in a greenhouse before examination of roots and extraction of cysts from the soil. Results showed that the nematode failed to reproduce on tobacco, tomato, and cucumber whereas white females and mature cysts of H. cyperi were observed on yellow nutsedge roots ( Fig. 1E ). The results confirmed that yellow nutsedeg was a host for the nematode, and tobacco, tomato, or cucumber were non-hosts. In the United States, H. cyperi was reported from Florida, North Carolina, and Arkansas ( Subbotin et al., 2010 ) infecting Cyperus spp. Yellow nutsedge is considered a serious weed problem in many cropping systems including peanut, cotton, tobacco, and vegetable crops in the Southern United States. To our knowledge, this is the first report of H. cyperi infecting yellow nutsedge in Georgia. Figure 1Photomicrographs of Heterodera cyperi from yellow nutsedge in Georgia. Whole body (A), the anterior region (B), and the posterior region (C) of J2. Cysts (D) recovered from the soil and the vulval cone of cyst with the ambifenestrate fenestra (E). A mature cyst (F) on the surface of yellow nutsedge root infected with the nematode.

Dataset Information

DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis.

Publications

DNA barcoding evidence for the North American presence of alfalfa cyst nematode, <i>Heterodera medicaginis</i>.

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