Prepman ultra

We tested for Bd infection by using skin swabs and a qPCR assay [24 (link)]. The swabbing protocol was standardized by performing 45 strokes on the venter and limbs with a sterile rayon-tipped swab (MW-113, Medical Wire & Equipment, Wiltshire, United Kingdom). Genomic DNA was extracted from the swabs using the Prepman Ultra (Applied Biosystems®, Life Technologies Pty Ltd, Carlsbad, California, USA) and a bead beater to break the fungal cell walls for two minutes, and then the extract was diluted 3:47 in PCR water. Extracted DNA was then analysed using quantitative real time PCR following Boyle et al. [24 (link)]. We conducted the analysis in singlicate to maximise both cost efficiency and test accuracy [25 (link),26 ] including a positive and negative control and a series of dilution standards (to estimate infection load in zoospore equivalents, ZE).
To test for inhibition of the swab DNA, a subset of 20 samples was haphazardly selected and an internal positive control (VIC^TM IPC, Applied Biosystems®, Life Technologies Pty Ltd, Carlsbad, California, USA) was added to the qPCR reaction. No inhibition was detected in those samples. Because many samples returned high (>1000ZE) infection loads and prevalence was high, we concluded that inhibition due to high zoospore loads was unimportant. We prioritized resources to increase sample size rather than including IPC’s in every reaction.

Phytophthora cinnamomi isolates were obtained from declining avocado orchards in Tzaneen, Limpopo, South Africa. Permission to collect isolates was obtained from individual farmers and from Westfalia Technological Services. Pathogenicity of isolates was confirmed by infecting avocado plants in a pre-trial and assessing disease development. See supplementary material for details on individual infection trials. Infection was confirmed by re-isolation of the pathogen and subsequent DNA extraction using Prepman™ Ultra (Applied Biosystems, Foster City, CA). Isolates were confirmed as P. cinnamomi by use of the species specific LPV3 primers (LPV3 F 5′-GTG CAG ACT GTC GAT GTG-3′, LPV3 R 5′-GAA CCA CAA CAG GCA CGT-3′) [73] in a polymerase chain reaction (PCR).

During fieldwork in 2018, 2019 and 2021, we swabbed live frogs of the new species with a synthetic dry swab (Medical Wire & Equipment #113) to quantify infection by Batrachochytriumdendrobatidis (Bd). We stroked swabs across the skin of juveniles and adults a total of 30 times per individual: five strokes on each side of the abdominal mid-line, five strokes on the inner thighs of each hind leg and five strokes on the foot webbing of each hind leg. We used a standard quantitative Polymerase Chain Reaction (qPCR) assay using DNA extracted from swabs to quantify the level of infection (Boyle et al. 2004 (link)). Following the protocol of Boyle et al. (2004) (link) and Hyatt et al. (2007) (link), we extracted DNA from swabs using 40 µl of PrepMan Ultra (Applied Biosystems). We analysed each extract once with a QuantStudio 3 qPCR system (ThermoFisher Scientific). We calculated the number of zoospore equivalents (ZE) (i.e. the genomic equivalent for Bd zoospores) by comparing the sample results with a serial dilution of standards (gBlock synthetic standards, IDT DNA, Iowa, USA). We considered any sample with ZE > 1 to be infected or Bd-positive.

Following animal death and preservation, we used quantitative polymerase chain reaction (qPCR) to quantify Bd-infection intensity of all individuals in the Bd-exposure treatments. Additionally, we investigated Bd-infection status in two unexposed individuals per species per trial as well as every unexposed individual that died during the trials. To sample the individuals for Bd, we used a sterile, fine-tipped, dry swab (Medical Wire and Equipment, Corsham, Wiltshire, England) and swabbed the right ventral surface of individual frogs 10 times including the feet, legs, and drink patch. Individual swabs were placed into sterile screw-capped vials. Bd-DNA was extracted by adding 60 μL of Prepman Ultra (Applied Biosystems, Carlsbad, California), heating the vial for 10 min at 100° C, cooling the vials for 2 min, obtaining the supernatant, then diluting it to a 10% solution. We conducted qPCR using an ABI PRISM 7500 sequencer (Applied Biosystems) according to the methods of Boyle et al. [46 (link)]. All samples were run in triplicate and averaged. If a sample tested positive for Bd-DNA in only one replicate we reanalyzed the sample. If a second analysis was required, we re-swabbed the individual on their left side and analyzed the sample from the second swabbing.

Polymorphism analysis of the internal transcribed spacer (ITS) region of the 16S-23S ribosomal DNA sequence was performed according to the method of Daffonchio et al. [23 (link)]. The template DNA was prepared from each of the strains using Prepman Ultra (Applied Biosystems, CA, USA). PCR was done with TaKaRa ExTaq Hot Start Version (Takara Bio, Shiga, Japan), template DNA, and the primers in a reaction volume of 50 μL. The PCR conditions were initial denaturation at 94°C for 2 min, then 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, and final extension at 72°C for 2 min.

Fifty μL of either RNA-free water (Qiagen Sciences Inc., Germantown, MD) or Prepman Ultra (Applied Biosystems, Foster City, CA) were added to thawed pellets. Samples were then placed in a dry bath at 99°C for 15 minutes, allowed to cool, and centrifuged for 3 min at 13,000 rpm (15,682 × g). The supernatant was transferred to a sterile 1.7 mL micro-centrifuge tube. Assay volume for calculations ~ 50 μL.