Protocol for PCR diagnostics on individual root knot nematodes (RKNs)

Adapted from Holterman et al., 2009

U.S. Vegetable Laboratory-Nematology

November 19, 2019

(2X) nematode lysis buffer: recipe for 100mL solution (will remain stable at room temperature)  

• 2mL, Tris-HCl (1M stock, pH 8.0):  Final [20mM]

• 0.4mL, EDTA (0.5M stock, pH 8.0):   Final [2mM]

• 1mL Tween 20:  Final [1% v/v]

• Qs to 100mL with DNase free ddH20  

Protocol for 10 rxns:

1. Transfer 150 μL of 2X lysis buffer to a 0.5 mL snap cap tube and add 1 μL of Proteinase K [800 U/mL]. Mix by vortexing.

2. Dispense 15 μL Aliquots into 0.2 mL PCR tubes.

3. On a clean section of a glass microscope slide pipette a 15 μL droplet of nuclease free water.

4. Transfer Nematode(s) to water droplet (1 female, 1 male, or 3-5 stage 2 juveniles per tube seem to work best in our hands)

5. Thoroughly crush/cut nematodes with pipette tip or clean scalpel blade in droplet.

6. Use pipette to transfer as much nematode debris as possible to one of the lysis tubes, and mix thoroughly by pipetting and then vortexing.

7. Repeat steps 3-6 for all tubes.

8. Place tubes in a thermal cycler for incubation (37C for 30 min. followed by 85C for 2 min). Use immediately, or freeze at -20C for future use (Stopping point, freeze tubes for later use)

9. Dilute the lysate from each tube 1:100 with TE buffer or nuclease free water to reduce PCR inhibitor concentrations. (Stopping point, freeze tubes for later use)

10. Use 3.0 μL of the diluted template in a 25 μL PCR reaction (see below).

Notes on discriminating Meloidogyne enterolobii via PCR: We are currently relying heavily on two sets of mitochondrial primers to quickly discriminate M. e. from the other major tropical Meloidogyne species. The first primer set we currently use is TRNAH + MRH106 (see primer table), and use the SCAR primers MeF+MeR to confirm any positive M. e. speciation’s from the first set.

25 μL PCR reaction using Apex Taq 2X RED master mix (Genesee Scientific):

• ddH2O (nuclease free)               7.0 μL

• Apex Taq RED master mix(2X)     12.5 μL

• TRNAH primer [10 μM]              1.25 μL

• MHA106 primer [10 μM]           1.25 μL

• Diluted Template DNA               3.0 μL

Thermal cycler program “LO-EX” for mitochondrial primer sets:

Step 1: 94°C – 3 min

Step 2: 94°C – 30 sec

Step 3: XX°C – 30 sec (see table for correct annealing temperatures)

Step 4: 68°C –  1 min

Step 5: GO TO Step 2 - 39X

Step 6: 68°C –  10 min

Step 7: 12°C –  infinite  

The gel picture below is from a screen of 6 individual females we excised from different sweetpotatoes, I’m confident females 1-3 are all M. enterolobii, females 4 and 6 are likely some other species from the incognita group clade I, and female 5 was likely an old female, too far gone for a good extraction. This primer set produces a size shift instead of a presence/absence. M. i., M. j., and M. a. will all produce a band ~557 bp in length, while M. enterolobii will produce a ~720bp amplicon due to a duplication in this region specific to M. e. (Pagan et al. 2014; Baidoo et al. 2016). We confirm the positive M.e. samples using our secondary SCAR primers of choice MeF and MeR (Hu, Zhuo, and Liao 2011)  which produces a ~250bp amplicon for M. e. .  We have been sequencing additional fragments amplified from the Cytochrome c Oxidase subunits and NADH dehydrogenase (see primer table) to facilitate phylogenetic analysis (Janssen et al. 2016).

Notes on sequencing of amplicons:

In order to facilitate robust sequencing across the entire length of each amplicon we have been TA-cloning them into the pGEMt vector and then sequencing with the T7 and SP6 primers.

Primer Table

References

Baidoo, R., S. Joseph, T. M. Mengistu, J. A. Brito, R. McSorley, R. H. Stamps, and W. T. Crow. 2016. 'Mitochondrial Haplotype-based Identification of Root-knot Nematodes (Meloidogyne spp.) on Cut Foliage Crops in Florida', J Nematol, 48: 193-202.

Holterman, M., G. Karssen, S. van den Elsen, H. van Megen, J. Bakker, and J. Helder. 2009. 'Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding', Phytopathology, 99: 227-35.

Hu, MX, K Zhuo, and JL Liao. 2011. 'Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls', Phytopathology, 101: 1270-77.

Janssen, T., G. Karssen, M. Verhaeven, D. Coyne, and W. Bert. 2016. 'Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution', Sci Rep, 6: 22591.

Pagan, Chris, Danny Coyne, Regina Carneiro, George Kariuki, Nessie Luambano, Antoine Affokpon, and Valerie M. Williamson. 2014. 'Mitochondrial Haplotype-Based Identification of Ethanol-Preserved Root-Knot Nematodes from Africa', Phytopathology, 105: 350-57.