Results

Experiment 1 – Testing of unmined leaves in the field

During the 2018 trials, L. sativae was diagnosed in 1 out of 10 of the unmined leaves, with a low DNA concentration found (1 pg DNA in 2 of 3 replicate qPCR tests). DNA concentrations from the ‘positive control’ samples averaged 123 pg across 71 samples tested (Figure 1).
During the 2019 trials, L. sativae was diagnosed in 2 out of 45 unmined leaves, with low DNA concentrations detected (<2 pg DNA in all three replicate qPCR tests). The two leaves with a low (but positive) diagnosisd, were collected from areas with known L. sativae activity, FGG and GHF. DNA of L. sativae was not amplified in any of the INJ unmined leaves, where L. sativae has never before been recorded.

Experiment 2 - eDNA persistence trial

Temperatures recorded over the period during which the leaf mines were ageing ranged from 20.1 °C to 37.6 °C (averaging 29.5 °C in the daytime and 26.7 °C overnight) and relative humidity levels ranged from 33.8% to 98.7% (averaging 64.3% in the daytime and 73.6% overnight). While the amount of DNA amplified varied widely with age of leaf mines (Figure 1), DNA was still diagnosable within leaf mines after 28 days, and the average success rate for diagnosis from an empty leaf mine (a successful diagnosis being the case where at least one of three replicate qPCR tests for an individual leaf mine yielded positive results) for mines between 0 and 28 days old was 65.1% (Figure 2). There was no effect of age of the leaf mine (F = 2.5, df = 1, p = 0.12) on the concentration of DNA amplified from the leaf material.

Experiment 3 – eDNA sensitivity under field conditions

Of the 184 leaf mine samples from M. atropurpureum directly preserved in ethanol, 82.1% yielded a positive diagnosis of L. sativae (on at least one of three replicate qPCR tests) and 17.9% yielded no diagnosis. Of the 104 leaf mines preserved onto FTA cards, 58.7% yielded a positive diagnosis of L. sativae (on at least one of three replicate qPCR tests) and 41.3% yielded no diagnosis.
There was no effect of age of the leaf mine (F = 0.5, df = 2, p = 0.60) or leaf mine length (F = 2.5, df = 2, p = 0.09) on the concentration of DNA amplified from the leaf material. However, there was a significant effect of larval remains on the concentration of DNA amplified within a leaf mine (F = 3.5, df = 8, p < 0.001). For mines preserved in ethanol, those containing the remains of a fly larva amplified on average 488.5 (+ 116) pg of DNA compared with 36.4 (+ 22) pg from mines that did not contain larval remains. For mines preserved onto FTA cards, those containing larval remains amplified on average 10.5 (+ 10) pg of DNA compared with 0.09 (+ 0.03) pg for those mines that did not contain larval remains. For leaf mines preserved in ethanol that did not contain larval remains, 89.4 % yielded a positive diagnosis.
Using the multilevel detection probability model incorporating uncertainty at the eDNA and qPCR stages, it was estimated that the probability of L. sativae diagnosis increased with the number of mined leaves and technical replicates tested as shown by the positive coefficients in Table 2 (with the exception of FTA storage). If DNA was present in a leaf mine, each qPCR replicate had an estimated 80% chance of confirming presence (with three technical replicates providing over 99% chance of confirmation). Of the covariates tested, the presence of larval remains and the preservation method were found to have significant effects on the probability of L. sativae diagnosis (Table 2). The presence of larval remains increased the probability of diagnosis by 9 times, while the use of FTA cards for preservation decreased the probability by 5 times compared with preservation of empty leafmines in ethanol (Table 2).
Assuming three technical qPCR replicates and samples are preserved in ethanol, testing of only two empty leaf mines is estimated to provide a 90% probability of L. sativae diagnosis, even if there are no larval remains present, the leaf itself is not fresh, and the mine is 50 mm (or less) in length (Figure 3). Three empty leaf mines provided a greater than 90% probability (as indicated by the 95% credible interval in Figure 3)

Experiment 4 - Field applications to delimit geographic range and host range

The empty mine eDNA method was applied to several plant samples collected within, and outside of the known range of L. sativae in Australia. Of the mines collected from known host plants of L. sativae on Thursday Island, all collected mines from S. lycopersicum and a single mine from V. unguiculata were confirmed to be L. sativae (Table 3). Of the mines collected from plants which were not known to be a host L. sativae in the Torres Strait, namely Capsicum sp., P. edulis , O. basilicum and S. jamaicensis , at least one mine per host plant was confirmed to be L. sativae . This constitutes the first formal record of mining activity of L. sativae in these plants in Australia.
Of the mines collected outside the known range of L. sativae , five (of the seven) leaf mines from M. atropurpureum collected on Zuna Island were confirmed as L. sativae , while the leaf mine collected from S. melongena in Cairns was not found to beL. sativae (Table 3)