2.1. Growth and Maintenance of Non-Hosts and Weeds
The non-host plants chosen for this study: capsicum (Capsicum annum), okra (Abelmoschus esculentus), sweetcorn (Zea mays), sorghum (Sorghum bicolor), snake bean (Vigna unguiculata ssp. Sesquipedalis), and peanut (Arachis hypogaea), were supplied by a commercial nursery (PlantSmith Nursery, Darwin, NT, Australia) for all field and screenhouse trials. Weeds and weed seeds (Amaranth (Amaranthus viridis), pigweed (Portulaca oleracea), black nightshade (Solanum nigrum), wild gooseberry (Physalis angulata), fat hen (Chenopodium album), and Sabi grass (Urochloa mosambicensis)) were collected opportunistically, allowing the production of more seeds through fruiting bodies. All plants and weeds were tested for CGMMV using the methods stated in this method, prior to the trials commencing, ensuring that positive detections occurred only through manual inoculation.
2.2. Non-Host Field Site Preparation
A secure and fully enclosed fenced paddock field trial was set up at the Berrimah Farm Science Precinct at the Department of Industry, Tourism and Trade, Northern Territory, with field beds, irrigation tape, and mulch film installed. Prior to the irrigation tape and mulch film being laid, CGMMV-infected material was tilled into the soil to increase the chance of inoculum contacting the roots of the crops. Roughly 80 sweetcorn, capsicum, and sorghum seedlings were planted into the field beds, while 80 peanuts were sown.
2.4. Screenhouse Trials
The secure screenhouse at Berrimah Farm Science Precinct was outfitted with automatic watering, with 80 drippers installed per bench, and five benches in total. The following crops were tested for susceptibility to CGMMV:
S. bicolor,
A. hypogaea,
A. esculentus,
C. annum,
Z. mays, and
V. unguiculata ssp. Sesquipedalis. The following weeds were also selected for susceptibility to CGMMV:
A. viridis,
P. oleracea,
C. album,
U. mosambicensis,
S. nigrum, and
P. angulata (
Figure 1).
In 100 mm pots, potting mix produced at Berrimah Farm Science Precinct, Coir chip/coir fines/fine pine bark (50%/35%/15%), was added to 80 pots per crop. One crop per pot was then planted into the potting mix with a total of 80 pots per crop, and the crops were inoculated with CGMMV two days later. Briefly, positive material was dried and roughly chopped. A phosphate-buffered saline (PBS) solution and a small amount of silica carbide (Sigma-Aldrich, St Louis, MO, USA) was added to the material. The inoculum was then roughly rubbed onto the leaves, allowing for a small amount of damage to increase the chances of the plants becoming infected. Inoculum (~750 µL) was added directly to the soil. Inoculated leaves were tagged with white marking tags on light string to ensure these were not sampled later.
One week later, 80 cucurbit plants were placed into pots of the same size with potting mix and manually inoculated with CGMMV as a positive control, and placed on a separate bench with automatic watering. Another 80 cucurbit plants used as negative controls were potted and placed on a bench and hand watered.
Plants were left to grow for 8 weeks. Prior to sampling, material was disposed of and the screenhouse was decontaminated. A sample size of 80 was chosen, as this number of replicates could be undertaken in both a field and screenhouse setting in the space provided, with enough space between plants to prevent cross contamination. The number of samples selected would also statistically allow for greater confidence in the results, with presence/absence being tested for.
2.5. Isolation of Viral RNA, PCR Primers, and Conditions
Bulk samples (1 leaf per plant, 10 plants in total) were collected from each sample and roughly chopped, and a subsample of the material was placed into 12 cm × 15 cm Bioreba® (ThermoFisher Pty Ltd., Waltham, MA, USA) extraction bags. Total RNA was then extracted using the Isolate II Plant RNA Kit (Bioline Pty Ltd., Taunton, MA, USA) according to the manufacturer’s instructions, with the following modification made: 700 µL of RLY Buffer and 7 µL of β-mercaptoethanol were added to the extraction bag, and the material was then ground before applying the liquid to the first column.
Conventional RT-PCRs were performed using a Veriti Thermal Cycler (ThermoFisher Scientific, Waltham, MA, USA), utilising Superscript III One Step with Platinum
Taq (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturers guidelines. The conditions used in this study can be found in a recent publication from the author [
14], and the primers used were: Coat Protein (CP) (496 bp) [
15] Forward primer, 5′-GATGGCTTACAATCCGATCAC-3′ and Reverse primer, 5′-CCCTCGAAACTAAGCTTTCG-3′; Movement Protein (MP) (809 bp) [
16] Forward primer, 5′-TAAGTTTGCTAGGTGTGATC-3′ and Reverse primer, 5′-ACATAGATGTCTCTAAGTAAG-3′; CGMMV RNA helicase subunit (1053 bp) [
17] Forward primer, 5′-ATGGCAAACATTAATGAACAAAT-3′ and Reverse primer, 5′-AACCACACAGAAAACGTGGC-3′.
Visualisation of the conventional PCR products was performed using a 1% agarose gel, with amplified PCR products cleaned up with an Isolate II PCR and Gel Kit (Bioline Pty Ltd., Taunton, MA, USA). The purified PCR products were sequenced in both directions at the Australian Genome Research Facility Ltd. (Brisbane, QLD, Australia
www.agrf.org.au (accessed on 2 October 2018)). Analysis was performed using Geneious
® (Biomatters Ltd, Auckland, New Zealand). (accessed on 5 October 2018)) and compared to submitted sequences on BLAST (
https://blast.ncbi.nlm.nih.gov (accessed on 5 October 2018)). RT-qPCR was performed on a Rotor-Gene 6000 (Qiagen, Hilden, Germany) with SensiFAST
TM SYBR
® No-ROX One-Step Kit (Bioline Pty Ltd., Taunton, MA, USA) following the manufacturer’s conditions. The conditions used in this study can be found in a recent publication from the author [
14]; the primers used were: Forward primer, 5′-GTGGTTTCTGGTGTATGGAACGTA-3′, Reverse primer, 5′-CGGGAGCTGAAAATTTGCATATAGT-3′, and probe (RZ_CGMMVmp-03), 5′-[FAM]CACCCCTACAGGATTC[NFQMGB]-3′ [
18].
An internal plant control (NADH dehydrogenase ND2 subunit) [
19] was undertaken to ensure quality assurance of sample extractions.