Principal Component Analysis and Molecular Characterization of Reniform Nematode Populations in Alabama.
ABSTRACT: U.S. cotton production is suffering from the yield loss caused by the reniform nematode (RN), Rotylenchulus reniformis. Management of this devastating pest is of utmost importance because, no upland cotton cultivar exhibits adequate resistance to RN. Nine populations of RN from distinct regions in Alabama and one population from Mississippi were studied and thirteen morphometric features were measured on 20 male and 20 female nematodes from each population. Highly correlated variables (positive) in female and male RN morphometric parameters were observed for body length (L) and distance of vulva from the lip region (V) (r = 0.7) and tail length (TL) and c' (r = 0.8), respectively. The first and second principal components for the female and male populations showed distinct clustering into three groups. These results show pattern of sub-groups within the RN populations in Alabama. A one-way ANOVA on female and male RN populations showed significant differences (p ≤ 0.05) among the variables. Multiple sequence alignment (MSA) of 18S rRNA sequences (421) showed lengths of 653 bp. Sites within the aligned sequences were conserved (53%), parsimony-informative (17%), singletons (28%), and indels (2%), respectively. Neighbor-Joining analysis showed intra and inter-nematodal variations within the populations as clone sequences from different nematodes irrespective of the sex of nematode isolate clustered together. Morphologically, the three groups (I, II and III) could not be distinctly associated with the molecular data from the 18S rRNA sequences. The three groups may be identified as being non-geographically contiguous.
Project description:Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. The specimens were identified as Bursaphelenchus antoniae Penas et al., 2006 based on 18S rDNA molecular sequence vs only 95% similarity with PWN B. xylophilus. Compared to the previously described Portuguese population of B. antoniae, the sequences generated for the MA population were 98.3% similar in the ITS1, 2 rDNA and 99.9% similar for 28S rDNA. There was 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae. This population has morphology consistent with that of Penas et al., 2006; however, the female tail on this MA pine population is mucronate and more attenuated than in B. antoniae from Portuguese P. pinaster found in association with Hylobius sp. Ecological associations of both populations of B. antoniae are discussed.Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. The specimens were identified as Bursaphelenchus antoniae Penas et al., 2006 based on 18S rDNA molecular sequence vs only 95% similarity with PWN B. xylophilus. Compared to the previously described Portuguese population of B. antoniae, the sequences generated for the MA population were 98.3% similar in the ITS1, 2 rDNA and 99.9% similar for 28S rDNA. There was 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae. This population has morphology consistent with that of Penas et al., 2006; however, the female tail on this MA pine population is mucronate and more attenuated than in B. antoniae from Portuguese P. pinaster found in association with Hylobius sp. Ecological associations of both populations of B. antoniae are discussed.
Project description:Longidorids are economically important plant-parasitic nematodes because several species are virus vectors. Populations of Paralongidorus sali and Longidorus jonesi , isolated from woody perennials of Hangzhou, Zhejiang, China, were characterized molecularly and morphologically. The morphometric data of the Chinese populations of both species were compared with other populations. The present study provided a first record of the occurrence of Paralongidorus in China coupled with description of the first-stage Juvenile and male of L. jonesi . Phylogenetic analysis based on 18S and D2-D3 expansion segments of 28S gene indicated that L. jonesi clustered with L. jonesi reported from Japan and P. sali grouped with P. bikanerensis from Iran. Considering the pathological and economic importance of this group of nematodes, the study emphasized the need of updated descriptions from accurately identified specimens, isolation of sufficient material for examination, and molecular and phylogenetic analysis for a better understanding and diagnostics of Longidorid nematodes.
Project description:The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.
Project description:Three populations of neotylenchid nematodes were isolated in Ningbo, P. R. China, from white pine lumber (Pinus monticola) imported from the USA. The nematodes were morphologically intermediate between Hexatylus and Deladenus. The nematodes were molecularly characterized based on sequences of the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer sequences. The phylogenetic inferences placed the nematodes with other neotylenchid nematodes, i.e., Fergusobia and Rubzovinema. Based on the morphology and phylogenetic analysis, this nematode is described herein as Delatylus andersoni n. gen., n. sp. The new genus/species is characterized by the female body habitus ranging from nonobese to semiobese and from straight to dorsally curved when heat relaxed, cephalic framework with six unequal sized lip sectors, lateral fields having 10 to 12 lines, 4 to 5 guide rings on the stylet, excretory pore posterior to the nerve ring, spermatheca diminished or absent, vulvar opening large, and relative proximity of vulva to the anus. Detailed morphological and molecular characterization of the new genus/species is presented along with the comparison of the related genera.
Project description:Little information is available on host-parasite relationships between bivalves and larval nematodes. Herein, we describe nematode larvae (likely stage 2) in the infraorder Ascaridomorpha infecting the foot, intestine, and mantle of a freshwater mussel (Alabama rainbow, Villosa nebulosa [Conrad, 1834]) and detail histopathological changes to infected tissues. A total of 43 live mussels from the South Fork of Terrapin Creek, Alabama, were collected between 2010 and 2014, with 14 sectioned for histopathology and 29 dissected. Of the 14 sectioned mussels, 5 appeared to be uninfected, and 7, 1, and 1 had histozoic infections observed in the foot and intestine, intestine only, and mantle edge and foot, respectively. Twenty-three of 29 (79%) of the mussels dissected were infected by live nematodes, and mean nematode abundance was 8.3 (CL?=?5.23-13), with 2 mussels infected with >100 nematodes each. Thus, with a total of 32 of the 43 collected mussels observed with nematodes, overall infection prevalence was 74.4% (CL?=?0.594-0.855). The 18S rDNA of this nematode was 99% similar to that of several ascaridids (species of Kathlaniidae Lane, 1914 and Quimperiidae Baylis, 1930) that mature in aquatic/semi-aquatic vertebrates; the recovered 18S phylogenetic tree indicated this nematode from V. nebulosa shares a recent common ancestor with Ichthyobronema hamulatum (Ascaridomorpha: Quimperiidae; GenBank Accession Number KY476351). Pathological changes to tissue associated with these infections comprised focal tissue damage, but a cellular response was not evident. The Alabama rainbow possibly represents an intermediate or paratenic host. Given these results, the nematode is likely not pathogenic under normal stream conditions; however, high intensity infections in the foot could inhibit pedal extension and retraction; which would have demonstrable health consequences to a freshwater mussel. Based on our review of the bivalve mollusc parasite literature, a collective biodiversity of 61 nematodes reportedly exhibit some degree of symbiosis (from commensal to parasitic) with 21 bivalves (28 nematode spp. from 17 marine bivalve spp.; 33 nematode spp. from 4 freshwater bivalve spp.); only four records exist of putatively parasitic nematodes from Unionida. The present study represents the first description of a nematode species that invades the tissues of a Unionidae species.
Project description:Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the taxonomic confusion of the X. americanum species complex.
Project description:Fragments of four candidate reference genes of Aphelenchoides besseyi, including actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin conjugating-3 enzyme (UBC) and alpha-tubulin (?-tubulin) were cloned from the transcriptome database of A. besseyi. The expression level of these four candidate reference genes and a commonly used reference gene of A. besseyi (18S rRNA) in three experimental conditions, including the four life stages (female, male, juvenile and egg) of two populations and the mixed-stage nematodes of four populations with different origins and hosts were analyzed by RT-qPCR. The expression stability of the five candidate reference genes under the three experimental conditions was analyzed by ?Ct, geNorm, NormFinder and RefFinder respectively. The analysis results of ?Ct, geNorm, NormFinder and RefFinder all indicated that UBC was the gene with the highest average ranking of stability. In conclusion, the expression stability of UBC was optimal under the three experimental conditions, indicating that UBC could be used as a suitable reference gene instead of 18S rRNA in the RT-qPCR analysis for A. besseyi.
Project description:Since the nematodes Trichuris trichiura and T. suis are morphologically indistinguishable, genetic analysis is required to assess epidemiological cross-over between people and pigs. This study aimed to clarify the transmission biology of trichuriasis in Ecuador.Adult Trichuris worms were collected during a parasitological survey of 132 people and 46 pigs in Esmeraldas Province, Ecuador. Morphometric analysis of 49 pig worms and 64 human worms revealed significant variation. In discriminant analysis morphometric characteristics correctly classified male worms according to host species. In PCR-RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS-2) and 18S DNA (59 pig worms and 82 human worms), nearly all Trichuris exhibited expected restriction patterns. However, two pig-derived worms showed a "heterozygous-type" ITS-2 pattern, with one also having a "heterozygous-type" 18S pattern. Phylogenetic analysis of the mitochondrial large ribosomal subunit partitioned worms by host species. Notably, some Ecuadorian T. suis clustered with porcine Trichuris from USA and Denmark and some with Chinese T. suis.This is the first study in Latin America to genetically analyse Trichuris parasites. Although T. trichiura does not appear to be zoonotic in Ecuador, there is evidence of genetic exchange between T. trichiura and T. suis warranting more detailed genetic sampling.
Project description:The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.
Project description:Root-knot nematodes (Meloidogyne spp.) are the most common major pathogens of many crops throughout the world, impacting both the quantity and quality of marketable yields. In this study, a total of 244 root-knot nematode populations from various hosts from 39 counties in Arkansas were tested to determine the species diversity. Molecular characterization was performed on these populations by DNA sequencing of the ribosomal DNA 18S-ITS-5.8S, 28S D2/D3 and a mitochondrial DNA fragment flanking cytochrome oxidase gene subunit II - the intergenic spacer. Five species were identified, including M. incognita (Kofoid & White, 1919) Chitwood, 1949 from soybean, cotton, corn and various vegetables (232 samples); M. hapla Chitwood, 1949 from rose (1 sample); M. haplanaria Eisenback, Bernard, Starr, Lee & Tomaszewski, 2003 from okra, tomato, peanut, Indian hawthorn, ash, willow and elm trees (7 samples); M. marylandi Jepson & Golden in Jepson, 1987 from grasses (3 samples); and M. partityla Kleynhans, 1986 from pecan (1 sample) through a combined analysis of DNA sequencing and PCR by species-specific primers. Meloidogyne incognita is the most abundant species that was identified in 95% samples and was the only species in field crops including soybean and cotton, except for one population of M. haplanaria from soybean in Logan County (TK201). Species-specific primers were used to verify M. incognita through PCR by species-specific primers. Unlike historical data, M. arenaria, M. javanica and M. graminis were not detected from any of the samples collected during this study. This result is essential for effective and sustainable management strategies against root-knot nematodes in Arkansas.