Molecular Diagnostics of pea and corn cyst nematodes

Molecular Diagnostics of Corn Cyst, Heterodera zeae and Pea Cyst, Heterodera goettingiana Nematodes
abridged from: Szalanski, A.L., D.D. Sui, T.S. Harris, and T.O. Powers. 1997. Identification of cyst nematodes of agronomic and regulatory concern with PCR-RFLP of ITS1. Journal of Nematology 29: 255-267.

INTRODUCTION Expanded international trade will require increased awareness of nematodes of regulatory concern. Cyst nematode species in particular have a notorious history of global dispersal (Barker, 1985; Baldwin and Mundo-Ocampo, 1991). Their ability to withstand desiccation in the protective cyst stage greatly enhances their dispersal capabilities. Among the cyst species suspected to have been recently introduced into the United States are the corn cyst nematode, Heterodera zeae, and the pea cyst nematode H. goettingiana. H. zeae was first described from India by Koshy et al. (1971) where it was subsequently found to be widely distributed. It was later reported from Pakistan (Maqbool 1981), the Nile Valley, Egypt, and Kent County, Maryland, USA (Ringer et al., 1987; Sardanelli et al., 1981). A second population in the U. S. has been identified in Virginia (Eisenback et al., 1993). This nematode had been considered a quarantinable species under the USDA Federal Domestic Quarantines: corn cyst nematode (CCN) 7CFR 301.90, which regulates the movement of soil, machinery, and root crops in the effected area (Anonymous 1995). On 10 October 1996 U.S. Federal H. zeae regulations were removed on the basis that it appears that the infestation is contained by the two states affected. H. goettingiana (Di Vito and Greco, 1986) occurs throughout the world and is widespread in Europe (Di Vitro and Greco 1986). Occasionally it has been reported in the U.S. (Thorne, 1961) and Stone and Course (1986) suggest that U.S. populations are chance introductions. In 1992 an established field population was discovered on pea roots in western Washington (Handoo et al. 1994). Additional findings in western Washington indicate that the pea cyst is not confined to a single field (Inglis, pers. comm.). MATERIALS AND METHODS : Nematode samples used in this study were either collected from the field, sent live as dry cysts or as cysts preserved in 95% ethanol. Many soybean and sugar beet cyst samples were processed through the University of Nebraska Plant Disease Diagnostic Clinic. Pea cyst samples were obtained from Lewis and Skagit Counties, Washington, U.S.A. and from the Department of Agriculture of Northern Ireland.

India corn cyst samples in ethanol were obtained from Hisar, Sonepat and Ambla. U.S.A. corn cyst samples in ethanol were obtained from Maryland and Virginia.

DNA Isolation, Amplication, and Sequencing: DNA was extraction from individual cysts using a phenol/choloroform extraction technique similar to Taylor et al. (1996). Ethanol precipitated DNA was resuspended in 50 ml TE (pH 7.5) and stored at -20°C until use. Single juveniles or eggs were processed for PCR by placing them in a 15µl drop of distilled water on a glass cover slip and manually disrupting them as described (Powers et al. 1993). The two amplification primers, rDNA2 and rDNA1.58S are 21 and 20 nucleotides in length respectively. The rDNA2 primer ( 5’-TTGATTACGTCCCTGCCCTTT - 3’) has been described by Vrain et al (1992) and rDNA1.58s (3’ -GCCACCTAGTGAGCCGAGCA- 5’) was designed by comparative sequence alignments of various nematode species. These primers amplify a 3’ portion of the 18S gene, entire ITS region and a 5’ section of the 5.8S gene. Primers were synthesized by the University of Nebraska-Lincoln primer synthesis lab (Lincoln, NE).

PCR reactions were conducted in a reaction mixture consisting of 1.0 ml of target DNA (from the 50 ml isolation), 5.0 ml of reaction buffer, 4.0 ml of dNTP mix (10 mM each of dATP, dTTP, dCTP and dGTP), 1.0 ml (20 mM) of each primer, 2.5 units of Taq polymerase and deionized water to a total volume of 50.0 ml. Reaction buffer, dNTP and Taq were obtained from a GeneAmp PCR reagent kit (Perkin Elmer Cetus, Norwalk, CT). The PCR amplification profile consisted of 40 cycles of 94°C for 45 sec, 55°C for one min, and 72°C for 2.0 min. The product was stored at 4°C. DNA was amplified from ten individual cysts from each population.

Amplified DNA was purified using Geneclean II (Bio 101, Inc. Vista, CA) following manufacturer protocols and resuspended in 30 ml of TE (pH 7.5). DNA was blunt ended using New England Biolabs (Beverly, MA) reagents and ligated using Stratagene (La Jolla, CA) reagents into pBluescript sk+ plasmid (Stratagene, La Jolla, CA) following Sambrook et al. (1989). Plasmid transformation and screening of clones were conducted following Sambrook et al. (1989). Positive colonies were verified by picking colonies with a sterile toothpick, replating the colony on a new LB ampicillin/tetracycline plate (Sambrook et al. 1989) and mixing the toothpick into a 50 ml PCR reaction mixture. PCR reaction was performed as before but for only 20 cycles. Five ml of PCR product was run on an agarose gel to determine if the insert was present. Single stranded product was obtained from positive clones and sequenced using a LI-COR Model 4000 DNA Sequencer (LI-COR Inc., Lincoln, NE) by the University of Nebraska-Lincoln DNA Sequencing Lab (Lincoln, NE). Two primers, T3 promotor and T7 promotor (Gibco BRL, Gaitherburg, MD), were used for sequencing in both directions. Sequences were aligned using GCG (Genetics Computer Group) PILEUP program (with a gapweight of 5.0 and a gaplengthweight of 0.3).

PCR-RFLP: Restriction sites were predicted from sequence data using the program Webcutter. Sample DNA was amplified as previously described. Predicted diagnostic markers were checked with restriction enzymes from New England Biolabs (Beverly, MA). Amplified ITS1 DNA was digested according to manufacturer’s recommendations (New England Biolabs, Beverly, MA). The digestion reaction included 7.0 µl sterile double distilled H2O, 10.0 µl amplified DNA, 2.0 µl 10x restriction enzyme buffer, and 1.0 µl of restriction enzyme. Reactions were incubated at 37 °C for 6 - 24 hours. For electrophoresis of digested DNA, the digestion mixture was added to 1.6 µl of gel dye and loaded on a 2.5% MetaPhor (FMC, Rockland, ME) agarose gel in 0.5x Tris:Borate:EDTA (TBE) buffer on a model H5 horizontal gel apparatus (BRL, Gaithersburg, MD). Ethidium bromide (0.015 mg) was added to the gel and electrophoresis buffer. Electrophoresis was at a constant voltage of 100 V for 4 hours. Gels were photographed under UV light using Polaroid film. Prints were scanned using a HPIIcx flatbed scanner and Aldus photostyler software.

RESULTS

The ITS1 primer set amplified a single fragment from both cyst DNA extractions and single egg or juvenile squashes. The ITS1 amplification product was 798 bp in length (including primers) for H. zeae, and 811 bp in length for H. goettingiana as determined by nucleotide sequencing. The restriction enzymes Alu I, Hha I, and Hinf I readily distinguished pea cyst nematode from corn cyst nematode (Fig. 1). Corn cyst nematode displayed several intraspecific restriction site differences when all five isolates were compared. For example, AvaI digestions of the U.S.A isolates produce a three fragment pattern compared to two fragment pattern observed among isolates from India. (Fig. 2). The AluI restriction pattern of the U.S.A. isolates display a 530 bp fragment not observed in the India isolates, and HaeIII digestion also produces two fragments, 424 and 118 bp, not seen in the Indian isolate (Fig. 2).