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80Russian Journal of Nematology, 2020, 28 (2), 123 - 130
Molecular characterisation and diagnostics of some gall-forming nematodes of the family Anguinidae Nicoll, 1935 (Nematoda: Tylenchida)
using COI mtDNA
Tatiana V. Roubtsova1, Julie Burbridge2 and Sergei A. Subbotin2' 3
'Department of Plant Pathology, University of California, One Shields Avenue, 95616-8751, Davis, CA, USA 2Plant Pest Diagnostic Centre, California Department of Food and Agriculture, 3294 Meadowview Road, 95832-1448, Sacramento, CA, USA 3A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 119071, Moscow, Russia e-mail: sergei.a. [email protected]
Accepted for publication 9 December 2020
Summary. Several nematodes of the family Anguinidae inducing galls on grasses were characterised in this study. Sequences of partial COI gene were obtained from Afrina sp. from Astrebra pectinata and Anguina agrostis, A. danthoniae, A. graminis, A. tritici and Anguina sp. from Dactylis glomerata. Conventional PCR with species-specific primers were developed for rapid detection of Afrina sporoboliae, Anguina tritici, A. danthoniae and Anguina sp. from Dactylis glomerata. Real-time PCR with TaqMan probes were also designed for detection of specimens of Afrina sporoboliae and A. danthoniae. Real-time PCR assays were able reliably to detect DNA extracted from less than half of nematode specimens per a single tube.
Key words: Afrina sporoboliae, Anguina agrostis, Anguina danthoniae, Anguina graminis, Anguina tritici, Anguina sp., PCR, Real-time PCR.
The family Anguinidae Nicoll, 1935 includes mycophagous and plant-parasitic nematodes. The latter are obligate specialised parasites, which induce swellings and galls on plants. Presently, more than 16 valid and undescribed species of nematodes from the genera Afrina 1981, Anguina Scopoli, 1111 and Heteroanguina Chizhov, 1980, parasitising grasses and cereals are known (Subbotin & Riley, 2012; Mobasseri et al., 2011). Several species are considered as economically regulated important agricultural pests in many countries. Two species of Anguina, A. funesta Price, Fisher & Kerr, 1919 and A. tritici (Steinbuch, 1199) Chitwood, 1935, are listed as quarantine organisms of highest priority by USDA Animal and Plant Health Inspection Service. Several anguinid species associated with grasses were found in the USA: Anguina agrostis (Steinbuch, 1199) Filipjev, 1936 (Oregon, Washington, Virginia, Minnesota, Wyoming), A. danthoniae (Maggenti, Hart & Paxman, 1913) Brzeski, 1981 (California, Oregon), A. pacificae Cid del Prado Vera & Maggenti, 1984
(California), A. funesta (Oregon), A. agropyronifloris Norton, 1965 (Montana, Kansas, South Dakota), undescribed Anguina species from Dactylis glomerata (Oregon), Afrina sporoboliae Barrantes-Infante, Schroeder, Subbotin & Murray, 2018 (Idaho) and Heteroanguina graminophila (Goodey, 1933) Chizhov, 1980 (Minnesota, Oregon, Wisconsin) (Lehman, 1919; Subbotin & Riley, 2012; Barrantes-Infante et al., 2018; Roubtsova & Subbotin, 2020).
Molecular analysis using PCR-RFLP and ITS rDNA sequencing are reliable and rapid methods for identification of anguinid nematodes (Powers et al., 2001; Ma et al., 2004; Subbotin et al., 2004; Vovlas et al., 2011; Mobasseri et al., 2011; Barrantes-Infante et al., 2018; Cid Del Prado Vera et al., 2018). Conventional and Real-time PCR methods using species-specific primers were also developed by Ma et al. (2006, 2011) and Li et al. (2015) for detection of A. funesta, A. agrostis, A. pacificae and A. tritici. Recently, loop-mediated isothermal amplification technique was developed by Yu et al.
© Russian Society of Nematologists, 2020; doi: 10.24411/0869-6918-2020-10013
(2020) for identification of A. agrostis. Barrantes-Infante et al. (2018) was the first who provided COI gene sequences for Anguina agrostis and Afrina sporoboliae. Because of higher variability in COI gene comparing with rRNA gene sequences (Vovlas et al., 2011), COI marker provides a better resolution for species discrimination in nematodes. The goals of the present study were i) to characterise gall-forming nematodes using partial COI gene fragment; and ii) to develop conventional and Realtime PCR with species-specific primers for rapid diagnostics of some gall-forming nematodes using this gene fragment.
MATERIAL AND METHODS
Nematode samples. The list of nematode samples included in this study is given in Table 1. The identification of nematodes in this work has been made based on combined analyses of morphological, biological and molecular data (Subbotin et al., 2004). Using a stereomicroscope, an individual gall was opened with the aid of tweezers in a droplet of sterile water and the juveniles, eggs or adults were crushed under cover slip on a microscope slide.
DNA extraction, conventional PCR and sequencing. DNA was extracted from several specimens using a standard protocol with proteinase K as described by Subbotin et al. (2018). PCR with different primer sets were run to obtain amplifications of COI gene (Table 2). Two or three ^l of extracted DNA was transferred to a 0.2 ml Eppendorf tube containing: 10 ^l DreamTaq Green PCR Master Mix (2*) (Thermo Fisher Scientific), 10 ^l water and 0.15 ^l of each primer (1.0 ^g pi"1). The PCR amplification profile consisted of 4 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 45°C, and 1 min 30 s at 72°C, and a final extension of 72°C for 10 min. Two ^l of the PCR product were run on a 1% TAE-buffered agarose gel (100 V, 40 min). PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and directly sequenced at Quintara Biosciences (CA, USA). New sequences were submitted in the GenBank under accession numbers: MW086874-MW086880.
Phylogenetic analysis. New sequences for COI genes were aligned with published sequences (Barrantes-Infante et al., 2018) using Clustal X 1.83 with default options. Alignment was analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The best fit model of DNA evolution for BI was obtained using the program MrModeltest 2.2 with the Akaike
information criterion. BI analysis under the GTR + I + G model was initiated with a random starting tree and run with the four Metropolis-coupled Markov chain Monte Carlo (MCMC) for 106 generations. The MCMC were sampled at intervals of 100 generations. After discarding burn-in samples and evaluating convergence the remaining samples were retained for further analysis and the topologies were used to generate a 50% majority rule consensus tree.
Conventional PCR with species-specific primer. Species-specific primers for four gall-forming nematode species (Table 3) were designed using the sequence alignment of the COI gene for gall-forming nematodes. The PCR mixture of Taq PCR Core Kit (Qiagen) was prepared as described by Subbotin et al. (2018). The PCR amplification profile consisted of 4 min at 94°C, 35 cycles of 1 min at 94°C, 1 min 30 s at 55°C, and 2 min at 72°C, followed by a final step of 10 min at 72°C. Two ^l of the PCR products were run on a 1% TAE buffered agarose gel, stained and photographed. All gall-forming nematode samples were used to test the specificity of PCR with new designed species-specific primers.
DNA extraction and real-time PCR with species-specific primer-probe. Real-time PCR assays were developed for two species: Afrina sporoboliae and Anguina danthoniae. Specific primers developed and tested successfully in conventional PCR were used in these assays. Probe design (Table 3) was also based on a multiple alignment. TaqMan probes were labelled with FAM dye. The 20-^l PCR reaction mixture contained 10 ^l of 2* SensiFast Probe Lo-ROX master mix kit (Thermo Fisher Scientific), 1 ^l each of 200 ^M forward primer, reverse primer and 0.4 ^l probe, 5.6 ^l water and 2 ^l of DNA template. The two-step thermal cycling program was as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing and extension at 60°C for 60 s in Quant Studio 7 Flex (Applied Biosystems). Standard curves were constructed from real-time PCR performed with serial 5-fold dilutions of DNA template prepared from 50 second-stage juveniles of Afrina sporoboliae (DNA concentration = 110 ng ^l"1) and 10 females of Anguina danthoniae (145 ng ^l-1). Standard linear regressions (y = a + bx) of the log concentration of the template copies (x) versus the mean Ct values (y) were obtained. Several nematode samples (Table 1) were used to determine the specificity of assays. All reactions were performed at least three times and a negative control (water) was used in each experiment.
Table 1. Species and populations of gall-forming nematodes used in the present study.
Species Plant host Location Sample code COI GenBank accession number Reference or source
Afrina sporoboliae Afrina sp. Anguina agrostis Sporobolus cryptandrus Astrebra pectinata Agrostis capillaris Idaho, USA Windorah, Queensland, Australia Moscow region, Russia CD2919 CD2901 CD2924 MG321198-MG321204 MW086814 MW086815 B.K. Schroeder, T.D. Murray; Barrantes-Infante et al. (2018) Subbotin et al. (2004) V.N. Chizhov; Subbotin et al. (2004)
A. agrostis Agrostis sp. Brazil CD2910 MW086816 M. Mundo-Ocampo
A. danthoniae A. graminis Danthonia californica Festuca rubra California, USA Moscow region, Russia CD2992 CD2921 MW086880 MW086819 S.A. Subbotin V.N. Chizhov; Subbotin et al. (2004)
A. tritici Triticum sp. Iran CD2923 MW086811 M. Moens
Anguina sp. Dactylis glomerata Oregon, USA CD2914 MW086818 CDFA collection
Table 2. Primer sets used or designed for amplification of COI gene.
Primer code Sequence (5'-3') Reference
JB3 (f) JB4 (r) COI-F1 (f) COI-R2 (r) Anguina_COF (f) AnguinaR1a (r) AnguinaR1b (r) TTT TTT GGG CAT CCT GAG GTT TAT TAA AGA AAG AAC ATA ATG AAA ATG CCT ACT ATG ATT GGT GGT TTT GGT AAT TG GTA GCA GCA GTA AAA TAA GCA CG CTT CTW ATG CTM GGW GCW CCD GAT ATG CT CCA AAA AAC CAA AAT AGA TGC TG CCG AAG AAC CAG AAG AGG TGC TG Derycke et al. (2005) Kanzaki & Futai (2002) This study
Note: (f) - forward; (r) - reverse.
Table 3. Species-specific primer and probe sets developed in the present study for conventional and
Real-time PCR diagnostics.
Species Primer set (5'-3') Expected fragment length
Afrina sporoboliae COI-F1 - CCT ACT ATG ATT GGT GGT TTT GGT AAT TG COI AfSR1 - ACA CCA GCA CAG TGT AAA CCC Probe_Af - CTC TAT CCC CCT CTT AGA ACT ATT GGG C 248 bp
Anguina tritici COI-F1 - CCT ACT ATG ATT GGT GGT TTT GGT AAT TG COI_AnTriR1 - CTC TAC CTT ACC TCC AAG GGA 582 bp
A. danthoniae COI-F1 - CCT ACT ATG ATT GGT GGT TTT GGT AAT TG COI AnDanR1 - ACC ACT ACC TCC ATC AAC AAG Probe_AnDan - TCC TTA CCC CGA GTT AAT GCT CTT AGC 136 bp
Anguina sp. from Dactylis glomerata COI-F1 - CCT ACT ATG ATT GGT GGT TTT GGT AAT TG COI_AnDactR2 - TTA AAG TGA CGG TCG AAG ACA 432 bp
RESULTS AND DISCUSSION
Phylogenetic relationships. The length of the sequenced COI fragments ranged from 412 bp to 847 bp using several primer sets. Seven new COI sequences of gall-forming nematodes were obtained in the present study (Table 1). COI alignment contained 15 sequences of gall-forming nematodes and two sequences of Litylenchus crenatae as the outgroup and was 873 bp in a length. Phylogenetic relationships between species are given in Figure 1. Two major clades, Afrina and Anguina were resolved in this phylogenetic tree. Relationships between the studied species were congruent with those reconstructed based on the ITS rRNA gene sequences (Subbotin et al., 2004; Mobasseri et al., 2017; Barrantes-Infante et al., 2018; Cid Del Prado Vera et al., 2018).
Conventional PCR with species-specific assay.
Species-specific primers were developed for Afrina sporoboliae and three Anguina species from the USA based on differences in the COI gene sequences (Table 3; Fig. 2). Several primer sets were tested and the results of PCR with the species-specific primers, which gave a good amplification, are presented in Figure 3. The combination of the universal primer COI-F1 with the corresponding species-specific primers yielded a single PCR product of ca 250 bp for Afrina sporoboliae, 580 bp for Anguina tritici, 140 bp for A. danthoniae and 430 bp for Anguina sp. from Dactylis glomerata. PCR with the specific primers were tested with all gall-forming nematodes. No amplification was observed in samples of non-target nematodes and in the negative controls (without DNA).
Afrina sporoboliae (MG321198, USA, ID) Afrina sporoboliae (MG321204, USA, ID) Afrina sporoboliae (MG321199, USA, ID) Afrina sporoboliae (MG321202, USA, ID) Afrina sporoboliae (MG321203, USA, ID) Afrina sporoboliae (MG321201, USA, ID) L Afrina sporoboliae (MG321200, USA, ID) Afrina sp. (MW086874, Australia, CD2907)
Anguina agrostis (MW086875, Russia, Moscow region, CD2924) Anguina agrostis (MW086876, Brazil, CD2910) Anguina agrostis (MG321205, USA, WA) -Anguina tritici (MW086877, Iran, CD2923)
-Anguina sp. (MW086878, USA, OR, CD2914)
-Anguina graminis (MW086879, Russia, Moscow region, CD2921)
— Anguina danthoniae (MW086880, USA, CA, CD2992)
Litylenchus crenatae (MT080998) Litylenchus crenatae (MT080997)
Fig. 1. Phylogenetic relationships between some gall-forming nematodes: Bayesian 50% majority rule consensus tree as inferred from the analysis of the COI mtDNA dataset under the GTR + I + G model. Posterior probabilities are given for appropriate clades. New sequences obtained in this study are in bold font.
Fig. 2. The fragment of COI alignment with indications of primer and probe positions.
WP 53S
— i
^¡ZT 5 —
mm*
•
M 1 2 3 4 5 6 7 M M 1 2 3 4 5 6 7 M
Fig. 3. Gels with amplicons obtained from PCR with the species-specific primers. A: COI-F1 + COI_AfSR1 primer combination for diagnostics of Afrina sporoboliae; B: COI-F1 + COI_AnAgTr1 primer combination for diagnostics of Anguina tritici; C: COI-F1 + COI_AnDanR1 primer combination for diagnostics of A. danthoniae; D: COI-F1 + COI_AnDactR2 primer combination for diagnostics of Anguina sp. from Dactylis glomerata. Lanes: M: 100 bp DNA ladder; 1: Anguina sp. from Dactylis glomerata (CD2914); 2: Afrina sporoboliae (CD2919); 3: Anguina graminis (CD2921); 4: A. tritici (CD2923a); 5: A. agrostis (CD2924); 6: A. danthoniae (CD2992a), 7: negative control (no DNA).
Table 4. Detection of Afrina sporoboliae and Anguina danthoniae and other anguinids with Real-time PCR using
species-specific primer-probe sets.
Ct value, mean ± SD
Species Location Afrina sporoboliae Anguina danthoniae
primers/probe primers/probe
Afrina sporoboliae Idaho, USA 26.8 ± 0.9 37.7 ± 1.7
Anguina agrostis Moscow region, Russia Undetermined Undetermined
A. danthoniae California, USA 35.6 ± 1.3 19.8 ± 0.2
A. graminis Moscow region, Russia Undetermined Undetermined
A. tritici Iran Undetermined Undetermined
Anguina sp. Oregon, USA 37.5 ± 0.5 32.1 ± 0.4
Amplification Plot
y = -2.8835x+ 37.26
R!: = 0.9769
y = -3.2677x+ 27.99 R2 = 0.9927
o
• 10 12
Log Concentration
Fig. 4. A. Real-time PCR result shown on graphs Ct value for A. danthoniae with serial 5-fold dilutions of DNA template (1-5) and Anguina sp. (6); B: Standard curves calculated with the log starting quantity and threshold cycle of the 5-fold serially diluted DNA from Afrina sporoboliae (a) and Anguina danthoniae (b).
Real-time PCR with species-specific assay.
The specificity of primer-probe sets was tested in singleplex reactions against target and non-target nematodes. The specific primer-probe sets reacted with the lowest Ct values when tested against their intended target species (Table 4). In the experiment with the specific primer-probe set for Afrina sporoboliae, signals were also observed for samples with A. danthoniae and Anguina sp. but at Ct values more than 35. In the experiment with the specific primer-probe set for A. danthoniae, signals were recorded also for two samples with Afrina sporoboliae and Anguina sp. at Ct values, 37 and 32, respectively. No signals were observed with other gall-forming nematode samples and negative controls. Amplification plot and standard curves for both species were constructed from real-time PCR performed with serial 5-fold dilutions of DNA template are given in Figure 4. The results showed that the detection limits (specimen per a tube) were 0.02 female of Anguina danthoniae and 0.4 second-stage juvenile of Afrina sporoboliae approximately in 30 and 35 cycles, respectively. Thus, the Realtime PCR assays were sensitive enough to detect DNA extracted from a single specimen of the target species within an impure sample.
Previously published molecular methods of identification of gall-forming nematodes are mainly based on differences in the ITS rRNA gene sequences. This gene fragment does not always give good resolution to discriminate species; however, the COI gene fragment is more variable and suitable for diagnostics of these nematodes. The usefulness of COI marker in identification of other group of plant-parasitic nematodes was also already documented (Vovlas et al., 2011; Palomares-Rius et al., 2017). The present protocol of PCR with species-specific primers using COI gene fragments is an improvement step for gall-forming nematode diagnostics; however, additional studies and characterisations of other gall-forming nematode species will be needed to strengthen diagnostics of these parasites.
ACKNOWLEDGEMENTS
The authors thank Drs V.N. Chizhov, B.K. Schroeder, T.D. Murray, M. Moens and M. Mundo-Ocampo for providing nematode materials. This work was sponsored by USDA APHIS Farm Bill grants (cooperative agreement no. AP 18PPQS&T00C096/18-0346-000-FR-1).
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T.V. Roubtsova, J. Burbridge and S.A. Subbotin. Молекулярная характеристика и диагностика некоторых галлообразующих нематод семейства Anguinidae Nicoll, 1935 (Nematoda: Tylenchida) с помощью последовательностей COI mtDNA.
Резюме. В этом исследовании были охарактеризованы несколько нематод семейства Anguinidae, вызывающих галлы на травах. Последовательности частичного гена COI были получены от Afrina sp. из Astrebra pectinata и Anguina agrostis, A. danthoniae, A. graminis, A. tritici и Anguina sp. из Dactylis glomerata. ПЦР с видоспецифичными праймерами была разработана для быстрого обнаружения Afrina sporoboliae, Anguina tritici, A. danthoniae и Anguina sp. из Dactylis glomerata. ПЦР в реальном времени с TaqMan также была разработана для обнаружения нематод Afrina sporoboliae и A. danthoniae. ПЦР в реальном времени позволили надежно обнаружить ДНК нематод, выделенную менее чем из половины экземляра нематоды на пробирку.
