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© 2012 Plant Management Network.
Accepted for publication 14 September 2012. Published 24 October 2012.


Identification of Anguina funesta from Annual Ryegrass Seed Lots in Oregon


Shawn Meng, Commodity Inspection Division, Oregon Department of Agriculture, 635 Capitol Street NE, Salem, OR 97301; Steve Alderman, USDA-ARS National Forage Seed Production Research Center, 3450 SW Campus Way, Corvallis, OR 97331; Cindy Fraley and Robin Ludy, Commodity Inspection Division, Oregon Department of Agriculture, 635 Capitol Street NE, Salem, OR 97301; Fengjie Sun, School of Science and Technology, Georgia Gwinnett College, 1000 University Center Lane, Lawrenceville, GA 30043; and Nancy Osterbauer, Commodity Inspection Division, Oregon Department of Agriculture, 635 Capitol Street NE, Salem, OR 97301


Corresponding author: Shawn Meng. smeng@oda.state.or.us


Meng, S., Alderman, S., Fraley, C., Ludy, R., Sun, F., and Osterbauer, N. 2012. Identification of Anguina funesta from annual ryegrass seed lots in Oregon. Online. Plant Health Progress doi:10.1094/PHP-2012-1024-01-RS.


Abstract

In 2010, seed galls containing Anguina sp. were isolated from 14 annual ryegrass (Lolium multiflorum) seed lots submitted for phytosanitary testing. To identify the species present, the ITS1 region of the ribosomal DNA of the nematodes from the seed lots was analyzed using a PCR-RFLP method. All nematodes produced a single 540-bp DNA amplicon, which was digested with three restriction enzymes, HaeIII, HinfI, and TaqI. The resulting RFLP patterns matched those of A. funesta. To confirm these results, 525 bp of the DNA amplicon was analyzed by DNA sequencing and BLAST analysis, which verified the sequence was identical to A. funesta (Genbank Accession nos. AF363095, AF363096, and AF363104). Because of the association of A. funesta with Rathayibacter toxicus, a second PCR test was conducted to determine if the bacterium was present in the seed lots. A 200-bp DNA amplicon was amplified from two seed galls, sequenced, and subjected to BLAST analysis. Analysis of the entire DNA sequence failed to identify the bacterium present, although testing by USDA-APHIS verified the bacterium was not R. toxicus. This is the first report of A. funesta in the US; R. toxicus was not found with this detection.


Introduction

Annual and perennial ryegrasses (Lolium multiflorum Lam. and L. perenne L., respectively) are economically important crops grown in Oregon. In 2010, ryegrass seed production in Oregon ranked first and accounted for 69% of the total production in the US (7). Annual ryegrass accounted for 55% of all ryegrass production in Oregon in 2010, with a farmgate value of $50.5 million (7). The majority of annual ryegrass produced in Oregon is exported as seed to other countries.

The seed gall nematodes, Anguina species, are internationally regulated plant pests quarantined by many countries. These nematodes cause galls on seed heads of Lolium and other grass species. Anguina funesta Price, Fisher & Kerr 1979 is of particular concern in some countries because of its association with Raythayibacter toxicus (Riley & Ophel 1992) Sasaki, Chijimatsu & Suzuki 1998, the causal agent of a bacterial gummosis of seed heads, referred to as yellow slime disease in Australia (9,15). Countries require that annual ryegrass seed be tested in the laboratory to verify freedom from A. funesta prior to importation. Anguina funesta also poses a great threat to annual ryegrass and livestock production in the US because of its association with R. toxicus (12).

Rathyibacter toxicus produces a corynetoxin, which can be fatal to livestock and causes annual ryegrass toxicity (ARGT) in Australia (1,23). ARGT was first recorded in Australia in 1956 (15) and caused significant plant and animal losses (13). Since then, ARGT has been reported in South Africa in 1981 and Japan in 1997, likely associated with the importation of contaminated ryegrass seed and hay, respectively, from Australia (10,16). Although there were reports from the 1940s and 1960s of ARGT-like poisoning of livestock in Oregon, the cause was never identified (2,14,17). To date, R. toxicus is known to occur only in Australia.

Here we report the positive identification of A. funesta from Oregon annual ryegrass seed lots and discuss molecular and morphological identification of this nematode to species. We also describe challenges we encountered with a molecular protocol used for identification of potential R. toxicus contamination of seed galls.


Nematode Isolation and Morphological Identification

Official seed samples were collected from annual ryegrass seed lots intended for export according to the Association of American Seed Control Officials Handbook on Seed Sampling. From those official seed samples, Anguina juveniles were isolated using the following protocol. Approximately 100 ml of annual ryegrass seeds were soaked in 500 ml of tap water in a 600-ml beaker for 48 h. The seeds were ground at high speed for 30 sec in a Waring Commercial Laboratory Blender and then poured through 20-mesh and 500-mesh sieves nested together. The juveniles were caught on the 500-mesh sieve and the contents backwashed into the original beaker with <100-ml tap water. These washes were then poured into a plastic grid tray and examined for nematodes with a stereo microscope. Nematodes were also examined at 100× to 1000× magnification using an Olympus BX50 compound microscope and pictures were taken with a Olympus E-10 digital camera mounted on the microscope. Identification of juveniles was based on their morphological characteristics described in the literature (6,8,18,19,20,21) and compared with a positive control A. funesta provided by Dr. Ian Riley (South Australia Research and Development Institute, Adelaide, South Australia). All measurements were taken microscopically using an ocular micrometer calibrated against a stage micrometer.

Anguina funesta juveniles were isolated from 14 annual ryegrass seed lots in 2010, ten seed lots in 2011, and four seed lots in 2012. All A. funesta-infested annual ryegrass seed lots were produced in Linn Co., OR. The juveniles were initially identified as Anguina sp. based on their body length (~1.0 mm), short and thin stylet (<10 µm) with small and rounded knobs, and tail shape (6,8,18,19,20,21). Morphometric measurements of 20 A. funesta juveniles were compared to those for 20 A. agrostis juveniles collected from bentgrass (Agrostis sp.) and 20 A. agrostis juveniles collected from orchardgrass (Dactylis sp.) (Table 1, Fig. 1). Measurements were taken with specimens heat relaxed and mounted in water.


Table 1. Morphometric measurements of Anguina funesta juveniles from annual ryegrass, A. agrostis from bentgrass, and A. agrostis from orchardgrass from commercial seed production fields in Oregon’s Willamette Valley.

Parameter Mean ± standard deviation (range) (µm)
A. funesta
(annual
ryegrass)
A. agrostis
(bentgrass)
A. agrostis
(orchardgrass)
Body length 836.2 ± 14.6
(815.9 - 865.7)
795.0 ± 33.8
(726.4 - 875.6)
739.8 ± 20.0
(726.4 - 796.0)
Genital primodium
to end of tail
396.7 ± 31.1
(351.5 - 480.2)
354.9 ± 25.0
(311.9 - 415.8)
350.0 ± 21.6
(297.0 - 381.2)
Tail length 63.3 ± 3.3
(55.9 - 68.0)
57.7 ± 2.1
(53.5 - 60.8)
61.5 ± 4.2
(55.9 - 70.5)
Anterior to
excretory pore
122.5 ± 2.5
(119.1 - 128.8)
122.2 ± 2.1
(116.6 - 126.4)
122.5 ± 2.5
(119.1 - 128.8)
Esophagus length 183.0 ± 7.1
(172.5 - 194.4)
186.9 ± 10.5
(158.0 - 199.3)
183.7 ± 8.8
(167.7 - 194.4)
Genital primordium
length
20.4 ± 1.5
(18.0 - 23.0)
16.5 ± 1.5
(13.5 - 19.0)
18.1 ± 1.8
(15.0 - 23.0)
Genital primordium
width
8.8 ± 1.2
(6.0 - 11.0)
7.5 ± 0.9
(6.0 - 10.0)
8.1 ± 0.7
(7.0 - 10.00
Body width 16.6 ± 0.7
(15.0 - 18.0)
14.0 ± 0.5
(13.0 - 15.0)
14.6 ± 0.6
(14.0 - 15.0)
Medium bulb length 17.0 ± 1.1
(15.0 - 19.0)
16.2 ± 1.6
(12.0 - 19.0)
15.9 ± 1.0
(15.0 - 17.0)
Medium bulb width 8.5 ± 0.5
(8.0 - 9.0)
8.7 ± 0.8
(7.5 - 10.0)
7.9 ± 0.7
(7.0 - 9.0)
Stylet length 8.0 ± 1.0
(7.0 - 10.0)
8.0 ± 1.0
(7.0 - 10.0)
8.0 ± 0.7
(7.0 - 10.0)
Stylet base to
anterior end
10.1 ± 0.3
(10.0 - 11.0)
10.1 ± 0.5
(8.0 - 11.0)
10.5 ± 0.4
(10.0 - 11.0)
Stylet base to
esophageal gland orifice
0.9 ± 0.4
(0.5 - 1.5)
1.2 ± 0.4
(0.5 - 1.50
1.0 ± 0.3
(0.5 - 1.5)
a ratio (body length/
body width)
50.4 ± 1.8
(48.0 - 52.9)
56.9 ± 3.7
(49.1 - 62.8)
50.6 ± 2.1
(45.4 - 54.7)
b ratio (body length/
esophagus length)
4.6 ± 0.2
(4.2 - 4.6)
4.3 ± 0.2
(4.0 - 4.7)
4.0 ± 0.2
(3.8 - 4.4)
c ratio (body length/
tail length)
13.2 ± 0.8
(12.3 - 15.1)
13.8 ± 0.6
(12.8 - 15.0)
12.1 ± 1.0
(11.0 - 15.4)
Anterior to excretory
pore as % of length
14.6 ± 0.3
(14.4 - 16.0)
15.4 ± 0.6
(14.2 - 16.7)
16.6 ± 0.5
(15.3 - 17.1)
Genital primordium
to tail as % of length
47.4 ± 3.6
(43.2 - 56.8)
44.7 ± 2.7
(36.2 - 49.7)
47.3 ± 3.0
(39.8 - 51.8)


 

Fig. 1. Morphological characteristics of Anguina funesta from annual ryegrass, A. agrostis from bentgrass, and A. agrostis from orchardgrass seeds in Oregon. Bar = 10 µm, s = stylet, ep = excretory pore, gp = genital primordium, a = anus.

 

Morphometric values for Anguina species from ryegrass, orchardgrass, and bentgrass were similar, with overlapping ranges of values (Table 1). Stylet length was shorter than previously described (20,21). The shape of the tails of A. funesta were more uniformly conical than A. agrostis from bentgrass or orchardgrass (Fig. 1). The tail morphological feature may eventually be used to differentiate the two species, but more samples would need to be collected to confirm if this is a potential differentiating characteristic.


Nematode Identification with ITS PCR-RFLP and DNA Sequencing

Under a stereo microscope, either one or five Anguina juveniles were removed using a fine needle and placed in a 1.5-ml microcentrifuge tube containing 15 µl of worm lysis buffer (50 mM KCl, 0.05% Difco Bacto gelatin, 10 mM Tris pH 8.2, 0.45% Tween 20, 60 µg/ml proteinase K, and 2.5 mM MgCl2) from NemATOL, (http://nematol.unh.edu/Method-Protocol/prot/1.htm). After 15 µl of sterile mineral oil was added, the tubes were placed in a -80°C freezer for at least 1 h. The tubes were incubated at 60°C for at least 1 h and then heated at 95°C for 15 min. The nematode lysates were used for PCR immediately or stored at -20°C for future use. A total of 35 juveniles were treated for each seed lot.

The internal transcribed spacer (ITS) 1 region of the ribosomal DNA (rDNA) was amplified from the Anguina juveniles according to Powers et al. (11) with the following modifications. PCR was conducted in a MJ Research DNA Engine in a 50-µl reaction volume containing 1× GoTaq Flexi Buffer, 2 units of GoTaq DNA polymerase (Promega, Madison, WI), 2 µl of MgCl2 (25 mM), 4 µl of dNTPs (25 mM of each), 1 µl each of primers rDNA1.58S (10 µM) and rDNA2 (10 uM), and 15 µl of the treated Anguina juvenile sample. The PCR cycle conditions included an initial denaturation at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. After PCR, 5 µl of PCR product was analyzed electrophoretically on a 1.5% agarose gel in 1× TAE (Tris-Acetate-EDTA) buffer at 100 V for 1 h, and 6 µl of BenchTop 100-bp DNA Ladder (Promega, Madison, WI) was used as a molecular marker. The gel was stained with ethidium bromide and photographed using a KODAK Gel Logic 200 Imaging System. For restriction fragment length polymorphism (RFLP) analysis, 5 µl of the PCR amplicon was digested with three restriction enzymes, HaeIII, HinfI, and TaqI, following the manufacturer’s instructions (Promega, Madison, WI). After digestion, the DNA fragments were separated by gel electrophoresis and photographed as described above, and the RFLP banding patterns were compared with the positive control (11).

A single 540-bp DNA amplicon was amplified from Anguina juveniles recovered from 28 annual ryegrass seed lots that were officially sampled from 2010 to March 2012 (representative results are shown in Fig. 2). Digestion of the 540-bp amplicon with the restriction enzymes HaeIII, HinfI, and TaqI resulted in one (no digestion, 540 bp), two (448 bp and 100 bp), and two (490 bp and 58 bp) DNA fragments, respectively. The RFLP fragment patterns of the Anguina juveniles from these 28 annual ryegrass seed lots were the same as those from the A. funesta positive control (representative results are shown in Fig. 3) and matched the HaeIII, HinfI, and TaqI RFLP banding patterns for A. funesta described by Powers et al. (11).


 

Fig. 2. A single 540-bp amplicon of the ITS1 region of the rDNA was amplified from Anguina juveniles recovered from 15 annual ryegrass seed lots produced in Oregon (showing only partial results) (Lane 1 = DNA ladder; Lane 2 = positive control; Lanes 3 to 17 = Anguina sp. juveniles from annual ryegrass seed lot sample numbers 10-4380, 10-5099, 10-6320, 10-7005, 10-7009, 11-44, 11-2234, 11-4321, 11-4450, 11-4500, 11-4543, 12-301, 12-302, 11-303, and 11-581, respectively).


 

Fig. 3. Restriction fragment length polymorphism (RFLP) of the ITS1 region of the ribosomal DNA of Anguina juveniles from Oregon annual ryegrass seed lots as compared to an A. funesta positive control (showing only partial results) (Lane1 = DNA Ladder; Lanes 2-7 = RFLP with HaeIII; Lanes 8-13 = RFLP with HinfI; Lanes 14-19 = RFLP with TaqI; Lanes 2, 8, and 14 = Anguina funesta; Lanes 3, 9, and 15 = annual ryegrass seed lot sample #10-5099; Lanes 4, 10, and 16 = annual ryegrass seed lot sample #10-7005; Lanes 5, 11, and 17 = annual ryegrass seed lot sample # 11-4321; Lanes 6, 12, and 18 = annual ryegrass seed lot sample #11-4500; Lanes 7, 13, and 19 = annual ryegrass seed lot sample #12-302).


DNA sequencing was performed on the 540-bp PCR amplicon from one sample identified as A. funesta by PCR-RFLP. After PCR and gel electrophoresis as described above, the 540-bp amplicon was excised from the gel using a scalpel and purified using a QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA) per the manufacturer’s instructions. DNA sequencing was conducted at the Center for Genome Research and Biocomputing Laboratory (Oregon State University, Corvallis, OR). Approximately 525 bp of the amplicon sequence was determined to be suitable for phylogenetic analysis. This DNA sequence was submitted to GenBank (Accession Number JQ809339) and a NCBI BLAST analysis conducted to search for highly similar sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi). One HinfI and one TaqI restriction site was identified within the sequence, although no HaeIII sites were found; this was consistent with the RFLP results. NCBI BLAST analysis showed the sequence was: identical to A. funesta (AF363095, AF363096, and AF363097); 97% similar to A. agrostis (AF363094); 96% similar to Anguina sp.-Holcus (AF363104); and 95% similar to A. agropyronifloris Norton 1965 (AF33093), A. graminis (Hardy 1850) Filipjev 1936 (AF363098), A. pacificae Cid del Prado Vera & Maggenti 1984 (AF363100), Anguina sp.-Dactylis (AF363103), and Anguina sp.-Polypogon (AF363105). For phylogenetic analysis, DNA sequences of the different Anguina species were selected from GenBank and aligned with the sample sequence using Clustal X with Ditylenchus dipsaci (Kuhn 1857) Filipjev 1936 included as the outgroup (3). An unweighted pair group method with arithmetic mean (UPGMA) dendrogram was generated using PAUP 4.0 (22); the dendogram depicts the genetic relatedness of the sequences (Fig. 4). As expected, the A. funesta DNA sequence from the annual ryegrass seed lot clustered with the other published A. funesta DNA sequences in GenBank.


 

Fig. 4. A dendrogram depicting relatedness among selected ITS1 sequences of different Anguina species deposited in GenBank and the ITS1 sequence of a putative A. funesta from Oregon annual ryegrass seed lot sample #10-188 using the unweighted pair group method with arithmetic mean (UPGMA).

 

PCR Detection of Rathyibacter toxicus

During this study, two seed galls found within the 28 A. funesta-infested seed lots were visually identified as suspicious for the presence of R. toxicus based on coloration of the seed galls and were subjected to further molecular analysis using the method of Kowalski et al. (5). Sixteen other bacteria (obtained from American Type Culture Collection, Manassas, VA) were also included to verify the specificity of the PCR test: Clavibacter michiganensis subsp. insidiosus (Smith 1990) Davis, Gillaspie, Vidaver & Harris 1984; C. michiganensis subsp. michiganensis (Smith 1990) Davis, Gillaspie, Vidaver & Harris 1984; C. michiganensis subsp. nebraskensis (Smith 1990) Davis, Gillaspie, Vidaver & Harris 1984; C. michiganensis subsp. sepedonicus (Smith 1990) Davis, Gillaspie, Vidaver & Harris 1984; Agrobacterium rubi (Hidebrand 1940) Starr & Weiss 1943; A. tumefaciens (Smith & Townsend 1907) Conn 1942; A. vitis Ophel & Kerr 1990; Pantoea stewartii (Smith 1898) Mergaert, Verdonck & Kersters 1993; Pseudomonas syringae pv. coronafaciens (Elliott 1920) Young, Dye & Wilkie 1978; P. syringae pv. syringae van Hall 1902; P. syringae pv. striafaciens (Elliott 1927) Young, Dye & Wilkie 1978; P. viridiflava (Burkholder 1930) Downson 1939; Xanthomonas alfalfae (ex Riker et al. 1935) Schaad et al. 2007, sp. nov., nom. Rev; X. campestris pv. campestris (Pammel 1895) Dowson 1939; X. c. pv. phaseoli (Smith 1897) Dye 1978b; and X. translucens pv. translucens (Jones, Johnson & Reddy 1917) Vauterin, Hoste, Kersters & Swings 1995. Bacterial cells were streaked onto 523 medium plates (4) and incubated at 28°C for 3 days. One loop of bacterial cells was suspended in 100 µl of sterilized deionized H2O in a 1.5-ml microcentrifuge tube. The cell suspension was boiled at 100°C for 10 min and 4 µl was used for subsequent PCR analysis using the protocol of Kowalski et al. (5). Agarose gel electrophoresis, gel imaging, amplicon purification, and DNA sequence analysis were performed as described previously.


 

Fig. 5. PCR detection of putative Rathayibacter toxicus contamination of seed galls collected from Oregon annual ryegrass seed lots (Lane 1: 100-bp BenchTop DNA Ladder, Promega, madison, WI; Lane 2: negative control H2O; Lanes 3-20: annual ryegrass seed lot sample #10-5099 seed gall 1, annual ryegrass seed lot sample #10-5099 gall 2, Clavibacter michiganensis subsp. insidiosus, C. m. subsp. michiganensis, C. m. subsp. nebraskensis, C. m. subsp. sepedonicus, Agrobacterium rubi, A. tumefaciens, A. vitis, Pantoea stewartii, Pseudomonas syringae pv. coronafaciens, P. s. pv. syringae, P. s. pv. striafaciens, Pseudomonas viridiflava, Xanthomonas alfalfae, X. campestris pv. campestris, X. c. pv. phaseoli, X. translucens pv. translucens, respectively).


A single 200-bp DNA amplicon was detected from the two suspicious seed galls as would be expected if R. toxicus was present (5). However, a similarly sized amplicon was also amplified from several of the other bacteria tested, including all four subspecies of C. michiganensis, P. syringae pv. syringae, and X. campestris pv. phaseoli (Fig. 5). The 200-bp amplicons from the seed galls were sequenced and, when the DNA sequences were analyzed using NCBI BLAST, were found to be: identical to Rathayibacter tritici (ex Hutchinson 1917) Zgurskaya, Evtushenko, Akimov & Kalakoutskii 1993 (AM237343, AM410685, EU379299, FJ932658, and NR026158); Rathayibacter festucae Dorofeeva et al. 2002, sp. nov. (AM237343); C. michiganensis ssp. insidiosus (AM410696, GQ332306 to 332310); C. michiganensis ssp. michiganensis (HM189671, HQ144228 to 144242); C. michiganensis ssp. nebraskensis (NR037015); C. michiganensis ssp. sepedonicus (AM849034); and C. michiganensis ssp. tessellarius (Carlson & Vidaver 1982) Davis, Gillaspie, Vidaver & Harris 1984. The sequences were 99% similar to R. iranicus (FJ607310), R. rathayi (D45062, NR026159), and R. toxicus (NR037136). Subsequent testing by the USDA-APHIS-PPQ National Identifier using a different, unpublished PCR protocol confirmed the seed galls were free of R. toxicus (J. Floyd, USDA-APHIS-PPQ, Riverdale, MD, personal communication). This indicates the primer pair we used was not specific for R. toxicus and cross-amplifies with other bacteria. The Kowalski et al. (5) protocol should only be used in concert with another testing method to ensure accurate identification of R. toxicus.


Summary

This is the first report of A. funesta in the US. Anguina funesta can be rapidly and accurately identified using the PCR-RFLP method of Powers et al. (11). For laboratories without immediate access to PCR equipment, it may be difficult to differentiate the species based only on morphology of the juveniles. In our study, tails of A. funesta were more conical than those of A. agrostis, but more samples would need to be collected to confirm if tail morphology is a means to differentiate the two species. We found no evidence of R. toxicus in Oregon annual ryegrass seed lots. However, the PCR protocol (5) used to identify seed galls potentially contaminated by R. toxicus did not prove to be species-specific. Instead, this protocol cross-amplified several other bacteria found in Oregon, indicating that this protocol should not be used by itself to diagnose R. toxicus contamination.


Acknowledgments

This project was partially supported by USDA-APHIS Cooperative Agreement #11-8584-0260-CA. We thank Dr. Ian Riley for providing Anguina funesta as a positive control.


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