Table 1.
Isolate numbers and genome sequence information used for species specific multiplex primer design.
Table 2.
List of designed primers along with detailed information regarding their target plant species, primer sequences, expected band sizes, optimized annealing temperature (°C), and their concentration (μM) within multiplex primer mixes.
Table 3.
List of target and non-target plant and insect species used to test for individual primer specificity evaluations using singleplex PCR.
Table 4.
The result of in-silico NCBI Primer-BLAST, indicating the number of matches and number of mismatches with the intended target and non-target hits, along with sensitivity of the specific primers both individually and when used in the mix estimated by Spectra Max Gemini XPS microplate reader.
Fig 1.
The amplification of the plant target DNA using two working multiplex primer mixes (primer mix 1 & 2, panel a & b) containing several species-specific primers and their associated amplicon sizes (bp). The PCR products were visualized on a QIAxcel Advanced system, and Qiagen alignment markers were employed to separate amplicon sizes ranging from 15bp to 3000bp. The plant species were ordered by expected base pair sizes, to indicate the expected ranges of band sizes using the designed primers. Panel a) consisted of the result of multiplex PCR on a single target species from left to the right A1-A9: Cucurbita pepo, Allium cepa, Glycine max, Ipomoea batatas, Phaseolus vulgaris, Gossypium arboretum, Citrullus lanatus, Solanum lycopersicum and Arachis hypogaea, respectively, followed by A10) their mixed target species DNA sample. Panel b) consisted of the result of multiplex PCR from left to right B1-B5 including Zea mays, Solanum melongena, Solanum tuberosum, Sorghum bicolor and Cucumis sativus, respectively, followed by B6) their mixed target species DNA sample.
Fig 2.
The proportion of Melanoplus ponderosus positives for cotton DNA at the intervals of 0, 12, 24, 36, 48, 72 and 96 h post-feeding, using the multiplex primer mix.
The solid curve line represents the fitted probit model accompanied it’s of 95% confidence intervals (dotted lines). The solid vertical line is the time at which 50% of individuals are expected to test positive for plant DNA in their gut (half-life). R statistical software was used to create this figure.
Fig 3.
The concentration of remaining plant DNA ± SE in the gut of Melanoplus ponderosus after feeding at intervals of 0, 12, 24, 36, 48, 72- and 96-hours post-feeding, using the multiplex primer mix.
R statistical software was used to create this figure.
Fig 4.
The frequency of feeding on 14 different crop plant species by Melanoplus ponderosus in South Georgia agricultural fields.
The percentages of feeding on each plant species were calculated by dividing the total number of positives for each plant by the total number of tested individuals (94 individuals), multiplied by 100. The Microsoft PowerPoint program was used to create this figure.