Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera.

Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

Introduction

Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products [1-3]. Various treatments have been developed and can be applied to eliminate organisms present in wood [3]. The two most widely used methods are heat treatment and fumigation of timber and wood products performed according to international phytosanitary standards ISPM 15 and ISPM 39 [4]. These methods are efficient for the elimination of insects but their efficacy in eliminating microorganisms can vary and is not always well documented. To evaluate the efficacy of these treatments the presence of microorganisms must be assessed following treatment of the wood products, generally using approaches based on the isolation and cultures of the microorganisms. However, culture-based methods have limitations as it is now estimated that only a small fraction of the microorganisms present in natural environments can be grown on artificial media [5,6]. In addition, some fungi grow slowly and rely on complex nutrient requirements. This is the case of certain fungi that are outcompeted by fast-growing saprophytic species [7]. These short-comings could generate false negatives, i.e. the failure to detect microorganisms that are still viable following a treatment that is inefficient. An additional challenge is that the paucity and sometimes inadequacy of distinguishing morphological traits complicates the identification of microorganisms [8].

The use of molecular methods, in particular amplification of conserved genes from genomic DNA (gDNA), followed by amplicon sequencing and DNA barcoding (matching the unknown sequence by homology in public sequence databases to provide identity), have become the standard in identification of fungi and oomycetes [9,10]. Quantitative real-time PCR (qPCR), a method that uses fluorescent dyes for detection of target DNA molecules/nucleic acids during the amplification process has increasingly replaced conventional PCR [11,12]. However, molecular detection methods are generally based on the detection of pathogen gDNA and therefore aim at detecting the presence, but not the viability, of the organism. Since DNA is stable and does not rapidly degrade following cell death, these assays are not useful to assess the viability of the targeted organisms and thus cannot help in determining the efficacy of a phytosanitary treatment. In contrast, messenger RNA (mRNA) degrades more rapidly after cell death[13-15], and is only produced by metabolically active cells, making it suitable to specifically detect living microorganisms [16].

To develop qPCR assays that can detect and quantify pathogen mRNA as an indicator of viability, it is necessary to produce complementary DNA (cDNA), which is the double-stranded DNA synthesized from a single stranded RNA (e.g., mRNA template in a reaction catalyzed by the reverse transcriptase enzyme). Complementary DNA amplified by PCR can then be used to measure the expression of mRNA and serve as a marker of cell viability in eukaryotes. Because introns are spliced out in mature mRNA, transcripts can be distinguished from gDNA. It is therefore possible to design assays utilizing a probe or primer that spans the exon-exon junction so that it can anneal to mRNA but not gDNA. This allows detection and quantification of mRNA without cross-amplification of the gDNA [17,18].

Herein, we report the design and validation of assays that amplify and provide a relative quantification of the mRNA over gDNA of two eukaryotic microorganisms that can colonize wood cambium: the oomycete Phytophthora ramorum, causal agent of sudden oak death and sudden larch death [19,20], and the blue stain ascomycete fungus Grosmannia clavigera, an important symbiont of the mountain pine beetle [21,22]. Since these two organisms belong to different kingdoms we are using them as a case-study for the development of a method that will be used to assess the viability of infectious microorganisms following wood treatments and evaluate the efficacy of such treatments.

Materials and methods

Design of assays

The program ‘‘PHYLORPH” (PHYLogenetic markers for ORPHans) was used to reconstruct the gene alignments with intron/exons junctions for P. ramorum (Pr-102 [ATCC MYA-2949];[23]) and other closely related species [24]. Two hundred and twenty eight conserved proteins for P. ramorum were identified by performing a BLAST (Basic Local Alignment Search Tool) search with the CEGMA (Core Eukaryotic Genes Mapping Approach) protein database [25] against the genome sequences of P. lateralis (GCA_000500205.2), P. hibernalis, P. foliorum, P. syringuae and P. brassicae [26] (Brett Tyler, Oregon State University, Personal communication). Based on their putative function related to basic metabolic processes and their high expression levels in transcriptome analyses [27-30], these genes are expected to be expressed under various conditions in living organisms, an important criterion in developing assays to assess viability. A total of 43 candidate gene alignments were obtained from which seven primer pairs targeting P. ramorum were designed, on two adjacent exons separated by a short intron sequence (<95 bp in length.), using Geneious (v8.1.6). Primer pairs were screened for specificity using PCR with electrophoresis on gDNA extracted from cultures of 10 Phytophthora species from clade 8 (P. ramorum, P. lateralis, P. brassicae, P. cryptogea, P. drechsleri, P. foliorum, P. hibernalis, P. porri, P. primulae and P. syringae) which are all closely related [31].

The same procedure was performed on the G. clavigera genome (isolate kw1407;[27]) using the FUNYBASE protein database [32] against the genomes of Leptographium longiclavatum, Neurospora crassa (GCA_000182925), Ophiostoma montium, O. piceae (GCA_000410735), O. novo-ulmi (GCA_000317715) and Sporothrix schenkii (GCA_000474925) [33-35]. The search returned 158 candidate alignments from which primer pairs targeting G. clavigera were designed and tested on gDNA from cultures of G. clavigera and its the sister species L. longiclavatum, L. terebrantis, L. wingfieldi and O. montium.

For candidate alignments that successfully passed PCR specificity testing, two real-time TaqMan PCR probes were designed as follows: a probe used for the detection of gDNA was designed within the intron sequence located between the two primer pairs, whereas a probe targeting the cDNA was designed to span the exon-exon junction (Fig 1A and 1B). Primers were then optimized as recommended in Feau et al. [11]. To determine the real-time PCR efficiency for each primer pair and probe a five-point standard curve was developed over a range of 10-fold dilutions from 10ng/μl to 1pg/μl of gDNA.

Fig 1

Real-time PCR detection assays targeting Phytophthora ramorum (A) and Grosmannia clavigera (B) gDNA and cDNA developed in this study.

Wood inoculation

Eight isolates of P. ramorum (two from each phylogenetic lineage i.e. EU1, EU2, NA1 and NA2, [36]; Appendix 1) and one G. clavigera isolate were used for the wood inoculations. They were obtained from long term storage, plated on carrot agar [37] and 2% malt extract agar (MEA), and sub-cultured to fresh plates 10 days prior to inoculation. In order to simulate live infection on the host, living trees were freshly felled and prepared for the artificial inoculation. Three tree species were used for the P. ramorum inoculations: Douglas fir (Pseudotsuga menziesii), Japanese larch (Larix kaempferi) and Western hemlock (Tsuga heterophylla). Lodgepole pine (Pinus contorta) was used for G. clavigera inoculation. Logs (~12cm or greater in dbh) were brushed to get rid of excess debris, rinsed with water, cut into 0.5 m long bolts and inoculated with P. ramorum, G. clavigera or a blank agar plug used as a negative control as described in [36]. Inoculated bolts were misted with water and placed in plastic bags for 28 days.

Heat treatment to determine mRNA stability

Pure cultures of P. ramorum and G. clavigera were subjected to two heat treatments: 1) a simulated spruce-pine-fir (SPF) kiln-drying schedule (from 15°C to 70°C for 7 hours) used to treat wood under the standards approved by the Canadian Food Inspection Agency (CFIA) [38]; and 2), exposure of the pathogens to 70°C for 1 hour [38] (Fig 2). Phytophthora ramorum isolate PFC-5073 [lineage NA2] and G. clavigera isolate KW1407 were grown on cellophane on V8 [39] and 2% MEA media (three replicates per time point). For the SPF kiln-drying schedule, mycelium was transferred from petri plates into 0.1mL PCR strip tubes and placed into a thermocycler. In parallel, one 1.5ml Eppendorf tube containing 30 mg of mycelia was immediately frozen to serve as a no-heat treatment control. The thermocycler was used to conduct a long heat treatment that simulated the kiln-drying schedule. Post-heat-treated samples were maintained at room temperature until its designated collection time point. The mycelium was sampled at 8 time points: 0, 6, 12, 24, 48, 96, 168 and 240 hours after the treatment. At each time point two mycelial samples were collected: one was transferred into a 1.5 ml microtube, submerged in liquid nitrogen and stored at -80°C for subsequent DNA and RNA extractions; the other one was plated on three clarified V8 (P. ramorum) or MEA (G. clavigera) agar petri dishes and incubated in a dark growth chamber at room temperature for 28 days. For the short heat treatment, a laboratory oven was preheated to 70°C to incubate 24 replicate plates of the P. ramorum and G. clavigera isolates for 1 hour. After this treatment, all plates were removed from the oven and placed at room temperature before collection at the time-points mentioned above. Mycelial samples were collected and stored as previously described for the SPF kiln-drying schedule.

Fig 2

Work flow diagram of the short (left) and long kiln (right) heat treatments. Orange plates represent Phytophthora ramorum on V8 agar, while yellow plates are Grosmannia clavigera on malt extract agar (MEA).

Chlamydospores collection

For the P. ramorum chlamydospore testing qPCR of gDNA (100 ng/μl) and RT-qPCR of cDNA (55ng/μl) were carried out for two different P. ramorum isolates (Pr-05-015 and CBS101329, both isolated from Rhododendron sp.) in biological duplicates and sample triplicates. Chlamydospores were harvested as described in Tsao [40] with the following modifications. Non-blended fungal mats were transferred onto a 75μm cell filter in a 50ml conical plastic centrifuge tube. Using sterile dH2O, these mats were rinsed while being patted with a rubber policeman until the volume of water in the falcon tubes reached approximately 10–20 ml. This was repeated twice. The tubes were then centrifuged at 10,000 x g for 2 min, and the supernatant was removed. The chlamydospore suspensions were then aliquoted into several 1.7 ml micro-tubes and centrifuged at 8000 x g for 5 min removing the supernatant. The pellets were then resuspended, combined and centrifuged at 6,000 x g for 5 min and the supernatant discarded. Final concentration of the spores was adjusted to 1x105 chlamydospore/ml using a hemocytometer. The obtained spore suspension was centrifuged at 6000 x g for 5 min, discarding the supernatant. The pellets were then suspended to a minimum volume (approximately 50μl). The chlamydospore suspension was transferred to a Lysing Matrix C grinding tube (MP Biomedicals, Santa Ana, California, USA) followed by RNA/DNA extraction as it was done for mycelia from pure cultures.

RNA and DNA extractions

Fifty to 80 mg of wood scrapings were collected at each inoculation point from eight logs of four conifer species and used for the simultaneous extractions of gDNA and mRNA. Wood samples inoculated with G. clavigera were placed in 15 ml vials with two 10 mm stainless steel balls and were submerged in liquid nitrogen to keep the samples frozen. Vials were then placed in the Geno/Grinder (SPEX SamplePrep 2010, Metuchen, New Jersey, USA) at 15,000 rpm for 30 seconds. Wood samples inoculated with P. ramorum were hand-ground using a mortar and pestle. Mycelial samples were placed in Lysing Matrix C. All samples were flash-frozen in liquid nitrogen, ground in a FastPrep-24 homogenizer (MPBiomedicals) at 5.5 rpm for 30 seconds and re-submerged in liquid nitrogen. For G. clavigera, samples were removed from the freezer and submerged in liquid nitrogen to inhibit RNA degradation. Samples were individually placed in a mortar, immersed in liquid nitrogen and ground up into fine powder.

Simultaneous extraction of gDNA and RNA was performed using the AllPrep DNA/RNA Micro kit (QIAGEN Inc., Valencia, CA) following manufacturer instructions. Three extractions were performed for each as replicates. Genomic DNA concentration was measured using the Qubit fluorometer and all culture samples were diluted down to 1ng/μl using nuclease-free water. The concentration of RNA samples was measured using the NanoDrop 1000 spectrophotometer before diluting down to 10ng/ul. RNA integrity was assessed by band fluorescence using an agarose gel stained with ethidium bromide (EtBr).

cDNA synthesis, qPCR and RT-qPCR

Using the diluted RNA, cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (QIAGEN). Two μl of gDNA Wipeout buffer (7x), 10 ng of template RNA and a variable volume of RNase-free water for a total of 14 μl was used in the first step. Then 1 μl of Quantiscript Reverse Transcriptase, 4 μl of Quantiscript RT Buffer (5x) and 1 μl of RT primer mix was added to the gDNA eliminated solution.

Two TaqMan reactions per time point were performed for the measure of mRNA stability post heat treatment. This allowed differentiation of either gDNA or cDNA with their corresponding probes. Real-time PCR mix included 0.5X Quantifast Multiplex PCR MasterMix (QIAGEN), 400 mM of each forward and reverse primer, 20 mM of TaqMan probe and 2.2 ng of template (gDNA or cDNA) for a final volume of 10 μl. Thermal cycling parameters used were 5 minutes at 95°C for enzyme activation, followed by 40 cycles of denaturation at 95°C for 30 seconds and 60 seconds of annealing/extensions at 60°C. The threshold was automatically set and generated with the Applied Biosystems StepOneTM software; qPCR efficiency was also calculated automatically through the standard curves tab.

Statistical analysis

The proportion of viable pathogen was estimated by using the ratio of mRNA over gDNA quantity i.e. quantification cycle (Cq) ratio of the real-time PCR probe targeting cDNA over Cq value of the probe targeting gDNA. This ratio was the unit of measurement used to compare the efficacy of heat treatment. Using statistical analysis software (SAS 9.4), the significance of the treatment values were tested using a two-factor ANOVA split plot, where factor A is heat treatment and factor B is sampling time points after treatments.

Results

PCR assay design and performance

Five out of the six primer pairs targeting P. ramorum were eliminated because of the presence of detectable amplification products on the agarose gel when tested with P. lateralis DNA. The primer pair that was selected for the P. ramorum assay (PH178) targets portions of a gene (Protein ID 74159; [23]) encoding for a predicted SNARE associated Golgi protein (Fig 1A) and yielded amplification products only with gDNA of P. ramorum. This gene was highly expressed in two mRNA profiling experiments with mycelial colonies of P. ramorum growing on agar media [28,29].

All 11 primer pairs designed to target G. clavigera amplified DNA from at least one non-target species. The primer pair targeting gene MS359 yielded a PCR product with all G. clavigera isolates tested and the closely related species L. longiclavatum (99.7% similarity with G. clavigera in the ribosomal internal transcribed spacer and 97.5% similarity at the genome level). Since these sister species occupy a similar niche (mountain pine beetle galleries) and have similar biology [30], we selected the assay targeting this gene to develop the mRNA assay (Fig 1B). MS359 (= G. clavigera GLEAN_5973; [27]) encodes for a putative NAD-dependent methylenetetrahydrafolate dehydrogenase that is involved in the glyoxylate and dicarboxylate metabolism pathway. This gene is known to be expressed in growing mycelium and non-germinated spores and showed stable expression levels in RNAseq experiments at 12 and 36 h post-inoculation on media supplemented with host-defense metabolites and untreated control media [27,41]. Both testing genes were chosen based on their assumed functions related to basic metabolic processes and their high expression levels in transcriptome analyses [27-30].

We verified the presence of an intron in the gDNA samples by comparing the size of the PCR products obtained by amplification of the gDNA and cDNA reverse-transcribed from mRNA of the same samples. A smaller amplification product was obtained in the PCR of the cDNA (84 bp) than the gDNA (157 bp) of gene PH178, confirming the presence of the intron in the gDNA of P. ramorum (Fig 1A). Similarly, we observed a difference in amplicon product sizes between cDNA (149 bp) and gDNA (199 bp) in MS359 of G. clavigera (Fig 1B).

TaqMan probes were added to the two selected primer pairs to design real-time PCR assays that were tested for amplification efficiency on serial dilutions of gDNA. For the assay PH178 targeting P. ramorum, the standard curve yielded a regression coefficient of 0.998 indicating low variability between independent DNA isolations and an amplification efficiency of 92.3% (Fig 3A). Efficiency of the MS359 assay targeting G. clavigera was higher (100.3%), and variability slightly lower with a regression coefficient of 0.981 (Fig 3B).

Fig 3

Log-transformed standard curve assessed with gDNA serial dilution (1:10) of (A) Phytophthora ramorum for the TaqMan probe PH178_EX (R2 = 0.992 Eff% = 99.330) and (B) Grosmannia clavigera for the TaqMan probe MS359_EX. (R2 = 0.981 Eff% = 100.303).

Assessing the performance of the detection assays in infected wood and chlamydospores

For each combination of isolate x tree species tested a necrotic lesion was observed around the point where the microorganism was inoculated, confirming the growth of these pathogens in wood. No similar necrotic lesion was observed in the controls (Fig 4). Genomic-DNA and cDNA obtained from these inoculated wood samples was successfully detected by their respective real-time PCR assay with Cq values ranging from 24.13 (G. clavigera inoculated on P. contorta) to 29.4 (P. ramorum inoculated on L. kampferi) for gDNAs (mean = 26.6 ±2.21). The Cq values obtained for the cDNAs were slightly higher, ranging from 27.4 (G. clavigera x P. contorta) to 33.4 (P. ramorum x L. kampferi) with an average of 31.2 (±2.77), indicating later detections than for gDNA (Fig 4).

Fig 4

Wood inoculation with Phytophthora ramorum (EU2 isolate P2111) and Grosmannia clavigera (isolate KW140) and corresponding real-time PCR amplification plots.

For each wood inoculation sample, gDNA and cDNA synthetized from mRNA extracted from a lesion 28 days post inoculation was tested in real-time PCR with either the PH178 assay (targeting P. ramorum) or MS356 (G. clavigera). Quantification cycle (Cq) values for gDNA (blue) and cDNA (red) are reported on each graph. Shaded area around the average line represents ±SD.

We tested the efficiency of the PH178 assay on nucleic acids extracted from P. ramorum chlamydospores. The mean Cq was 25.45 for gDNA (± 0.055) and 29.05 for cDNA (±0.038) (data not shown). No amplification was observed with gDNA and cDNA from non-inoculated wood samples as-well-as the no-template controls.

Use of molecular assays to assess viability of pathogens following heat treatment

Ratio of mRNA over gDNA quantity as measured by Cq values for cDNA and gDNA were compared following two heat treatments (SPF Kiln-drying and short heat treatment) applied to mycelium of P. ramorum and G. clavigera. For both pathogens, we found a highly significant effect of the heat treatment (F = 72.4, P < 0.0001 and F = 25.2, P < 0.0001 for P. ramorum and G. clavigera, respectively) and an interaction between treatment and time point at which the gDNA and mRNA samples were collected (F = 4.2, P < 0.001 for P. ramorum and F = 2.7, P < 0.01 for G. clavigera). This likely resulted from the difference in cDNA detection as for both species gDNA was amplified after each treatment with Cq values similar or higher than those obtained with the no-treatment control (e.g. Cq distribution ranging from 24.0 to 34.0 for the two heat treatments versus 24.0 for the controls; Fig 5A, 5B, 5E and 5F). cDNA was detected for either pathogen at the end of SPF kiln-drying schedule treatment (Cq value equal or above 40.0; Fig 5D and 5H), suggesting that this treatment efficiently killed the two pathogens. In contrast, the short heat treatment at 70°C for 1 hour seemed to be less efficient than the longer kiln treatment, with clear evidence that the mRNA degraded at different rates for the two organisms following treatment. For P. ramorum, cDNA of the targeted gene (PH178) was detected after up to 24 hours post-treatment (mean Cq = 35.8 ±4.82 for 0 to 24 hours post-treatment; Fig 5C). Similarly, G. clavigera cDNA of gene MS359 was detected until 96 hours after the short heat treatment (mean Cq = 34.2 ±3.75), suggesting incomplete degradation of mRNA and/or non-lethality of this treatment during this time (Fig 5G).

Fig 5

Efficacy of the short heat and SPF kiln-drying treatment of Phytophthora ramorum (green) and Grosmania clavigera (blue). Each graph represents distributions of cycle-threshold (Ct) values obtained by real-time PCR with the PH178 assays (targeting P. ramorum) or the MS356 assays (G. clavigera) for gDNA or cDNA extracted from cultures sampled from 0 to 240 hours after treatment. Boxplot means sharing a letter are not significantly different (p > 0.05) according to a Tukey’s HSD test.

Assessment of pathogen viability after heat treatments

Small samples of mycelia were collected at time point zero just after each heat treatment and plated onto a sterile clarified-V8/MEA agar Petri dish and grown for 28 days. No growth was observed from any of the cultures, suggesting that the two heat treatments had been lethal.

Discussion

Quantitative real-time PCR is increasingly becoming the method of choice for molecular detection of microorganisms. Conventional methods used for assessing the efficacy of treatment of wood products typically involve culturing the microorganisms. These methods have the advantage of simultaneously assessing the presence of microorganisms and their viability, but require days from initiation to result and can produce a high rate of false negatives. The present study was undertaken to evaluate the feasibility of developing a molecular method for assessing the presence and viability of wood-colonizing pathogens. Using qPCR and RTqPCR with TaqMan probes that overlap the exon-intron and exon-exon junctions, it was possible to distinguish the mRNA and the corresponding genomic gene copy (gDNA). This allowed comparison of the kinetics of degradation of both molecules following heat treatments and provided a measure of the presence and absence of microorganisms as well as an assessment of their viability.

Accurate detection of invasive pathogens is a hallmark of efficient prevention and integrated pest management programs [42,43]. In some instance assessing pathogen viability may even be more crucial. This is particularly relevant in the context of plants, plant parts and wood-material trade between countries [1-3]. RNA-based molecular assays have proven to be successful in detecting a number of different pathogens and assess their viability. For example, early studies have used levels of ribosomal RNA (rRNA) as a proxy of bacteria viability involved in periprosthetic joint infections [44], human endophthalmitis [45] and clinical infectious diseases [46]. Although all these studies reported correlation between rRNA detection signal and cell death, rRNA half-life and inconsistent retention after cell death makes it somewhat less accurate, particularly for short term experimentation [46]. Because of its relatively short half-life, mRNA has been used successfully as a viability indicator for a number of prokaryote [46-48] and eukaryote pathogens [49,50]. Another drawback of rRNA for assessing cell viability is that it does not have spliced introns and therefore it cannot be used to differentiate between RNA and gDNA templates, raising the possibility of false positive amplification from accidental gDNA contamination. In this case, a complete elimination of DNA is required prior performing RT-qPCR to ensure reliable use of rRNA as proxy of cell death. As an alternative method to differentiate between RNA and gDNA templates, Menzel et al. designed an end-primer which spans on the exon-exon junction in mRNA, so that efficient primer-annealing and PCR-amplification is only possible after splicing the intron of the targeted gene during the DNA-translation process [51]. Similarly, Leal et al. developed several reverse transcription real time PCR assay to detect living the pinewood nematode Bursaphelenchus xylophilus in wood by targeting the presence of mRNA [17,52]. These studies showed the potential of mRNA for routine detection of living pinewood nematode in commercially manufactured wood-pellets for living pinewood nematode [53].

Phytophthora ramorum causes sudden oak death in North-America and sudden larch death in UK. Despite the regulations that are in place to prevent movement of this pathogen in North America, Europe and Asia P. ramorum has spread into new areas through nursery stock exchange [54]. Accurate and rapid detection methods are crucial for preventing new introductions of this pathogen and several molecular assays have been developed to improve its detection and monitoring [31,55-57]. Phytophthora ramorum is also considered as a wood-colonizing pathogen as it can grow in xylem vessels underlying phloem lesions on several woody-perennial species [58,59]. In this context, the ability to assess its viability in plants, plant-parts and in wood-derived products following treatment may be necessary. Chimento et al. [60] developed real-time PCR primers targeting the cytochrome oxidase subunit I (COX 1) gene to investigate the viability of P. ramorum mycelial cultures after different treatments [60]. As COX1 is a mitochondrial gene that does not contain introns in Phytophthora spp. [61], their real-time PCR assay does not discriminate the genomic of the transcribed gene copies. In this study the specificity of the assay to detect living P. ramorum was increased by amplifying exclusively cDNA, ruling out potential false positives generated by gDNA contamination of the RNA sample.

The second assay we developed targeted G. clavigera, one of the mountain pine beetle fungal associate. This wood-colonizing organism was selected as a second case-study to demonstrate the efficiency of our approach in assessing the viability of an infectious fungus. Due to the high sequence similarity between G. clavigera and the closely related species L. longiclavatum [62,63], the assay developed amplified mRNA and gDNA of both species. This could be a positive attribute of this assay since both species are symbionts of the mountain pine beetle and share a very similar ecological niche in pine trees [22]; in fact it is possible that these fungi hybridize and the species limits are not clear (R. C. Hamelin and A. Capron, unpublished). These fungi cause only a wood discoloration symptom (“blue stain”) and no structural damage to the infected wood. The only issue with regard to the trade of wood products is related to the risk of long distance spread and introduction of these fungi in areas where they do not occur [3]. Since these fungi require bark beetles for dissemination, they pose a limited threat.

Our aim was to use our gDNA and mRNA-targeted real-time PCR detection assays to assess the viability of two wood-infecting microorganisms following lethal treatments to test their efficacy. The underlying hypothesis tested was that viability is related to the expression of specific genes. Therefore, monitoring specific P. ramorum and G. clavigera gene transcripts by real-time PCR was expected to provide a simple proxy for viability of these two organisms. The choice of transcript was an important consideration, not only for sensitivity but also for its expression under a variety of conditions. Genes selected to assess viability should be constitutively expressed regardless of the environmental conditions and the micro-organism life-stage as opposed to those induced following a specific environment signal [64]. This ensures that the lack of expression of a constitutive gene is due to the death of the organism instead of the gene being turned “off” by a specific environmental factor. Therefore, in order to develop markers as indicators of cell viability, it was important to identify transcripts that are present and expressed at different stage of the organism’s life cycle and under different environmental conditions. The transcripts retained for this study were chosen based on their putative function related to basic metabolic processes and their high expression levels in transcriptome analyses [27-29,41]. In addition, we validated their expression in pure cultures and for mycelium growing in wood-logs. For P. ramorum we successfully conducted the viability assay on chlamydospores, the asexual reproductive structures that are generally involved in long-term survival under adverse conditions. Although there are no reports of chlamydospores surviving in wood, they could be present in other tissues such as the bark, roots or soil associated with roots or leaves [65].

Several studies showed that the ISPM No. 15 guidelines for treatment of wood packaging material [4] were not always lethal for wood-colonizing pathogens. For example, the protocol requiring exposure for 30 minutes at a temperature of 56°C was lethal for P. cinnamomi and some other species, but not all the wood colonizing species tested [66]. No mycelial growth was observed for P. ramorum in tanoak disks and boards after a heat treatment of 60°C for at least one hour [67]. However, Chimento et al. demonstrated that a 60°C treatment for one hour did not kill the pathogen but instead delayed its growth [60]. Based on these discrepancies and since most kiln and heat treatments generate core temperatures in wood products exceeding ISPM15 requirements (e.g. for SPF products, drying temperatures are usually in the range of 70°C to 80°C; [68]), we applied on mycelial cultures a treatment of 70°C for a minimum of one hour and the kiln-drying treatment that requires temperatures from 15 to 70°C for 7 hours approved as a standard to treat SPF approved by the CFIA [38]. Accurate detection of the cell viability is therefore crucial in investigating the efficacy of these heat treatments. We used two different methods to assess cell viability. As each of these methods is based on criteria that reflect different levels of cellular integrity or functionality their outcome in declaring cells alive or dead can be different [48,69]. A classical view is to consider that viable cells still have the potential to multiply under suitable condition [48]. This was assessed in our study through a culture-based method. Though this method has a high rate of false negatives [70,71] and several studies have demonstrated that cells that have lost their “culturability” following a lethal-treatment may still retain some of their physical integrity and functionality [69,72,73]. As a second method, we assumed that the rapid degradation and synthesis inhibition of mRNA might be a useful indicator of cell mortality [48]. However, because mRNA was still detected by reverse transcriptase real-time PCR in P. ramorum and G. clavigera dead cells (as indicated by the non-recovery of living cultures) several hours following a short heat treatment (70°C for a minimum of one hour), we couldn’t conclude that mRNA provided an absolute indicator of viability according to this assumption. Instead, we can consider that the kinetics of mRNA disappearance is related to loss of cell viability: we observed that passed a certain time-point after the heat treatment (i.e. 48 hours for P. ramorum and 168 hours for G. clavigera), mRNA was no longer detected. Non-detection of mRNA just after the SPF kiln-drying schedule treatment supports this hypothesis. Possibly, the longer time of exposure to lethal temperatures in this schedule (7 hours) contributed to achieve full mRNA degradation for that treatment.

The approach presented in this study could be improved by technical improvements before envisaging operational use. For each combination of pathogen x tree tested, detection of the cDNA was always delayed comparatively to gDNA; in few cases we observed a substantial Cq value difference between the gDNA and cDNA (e.g. P. menziesii and T. heterophylla; Fig 4). Extraction of RNA from plant tissues is notoriously challenging. Particularly, yield and quality of the RNA purified from wood, in particular from conifers, is limited by the high content in polyphenols, polysaccharides and other secondary metabolites [74,75]. These compounds tend to co-precipitate with the RNA in the presence of alcohols, thereby remaining in the final extract, resulting in RNA instability and interfering with downstream enzymatic reactions such as reverse transcription and cDNA synthesis [76,77]. Optimization of RNA-extraction protocol should help improve cDNA synthesis and therefore sensitivity of the cDNA-assays.

In addition, a Two-step RT-qPCR was used for these experiments, as we thought this approach would be more convenient for a proof-of-concept study with cDNA detection. Although this approach provides more flexibility and control than a one-step RT-qPCR, it takes more time to implement and can introduce more errors. A one-step RT-qPCR involves the reverse transcription and the PCR reaction within one single tube and would be more ideal for high throughput screening. However, this method also requires careful evaluation of the conditions for both the cDNA and PCR steps that may not be optimal for either reaction. Downstream optimization of the current assays using a one-step approach should be considered but was beyond the focus of the current experiment. Another consideration would be the use of the DNA-binding photoreactive dye Propodium monoazide (PMA) that enables to distinguish between dead and living cells [78]. However, despite the ease and success in use with some organisms, the greatest concern is its limitation in technical use on environmental samples [79].

The Food and Agriculture Organization (FAO) often revises the list of suggested treatments for wood packaging material, emphasizing the importance of reducing the risk of quarantine pests associated with wood exports [4]. The present research acts as a proof-of-concept to enhance future molecular detection methods of invasive and native pathogens of phytosanitary concern. Our mRNA/gDNA detection method differentiated between dead and alive pathogens using reverse transcriptase and real-time PCR assays and was validated in pathogen-infected wood samples. These assays should provide a novel way to evaluate and compare the efficacy of wood treatments to eliminate microorganisms that are difficult to detect. Further investigation of the timing and pattern of mRNA degradation in wood and other plant tissues such as leaves and bark should be conducted using this method.

30 Aug 2019

PONE-D-19-22766

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

PLOS ONE

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Dr. Richard Hamelin

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Bonjour Richard,

Your manuscript Number PONE-D-19-22766:

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera, was critically reviewed by 5 external reviewers. Based on these reviews and my own assessment, I recommend publishing your manuscript after "Major Revision".

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Reviewer #1: This manuscript describes the design of molecular tools for the specific detection of genomic DNA and messenger RNA from two plant pathogens, i.e. the oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The novelty brought by this work is the design of species-specific tools on regions covering exon-intro junctions; in such a way that only cDNA retrotranscripted from mRNA is made amplifiable by PCR. The authors compared the amplification of coding regions of gDNA and corresponding mRNA in real-time PCR in several experimental trials, and showed that the level of mRNA decreased after mycelium treatment (e.g. heating) and could be used as a proxy for assessment of fungal or oomycete viability.

The manuscript is well written, very easy to follow and overall well organized (although I suggest minor changes, see below). The discussion section is exhaustive and interesting, although more light is shed on P. ramorum.

The experiments are adequate and compelling for most of the work, but I noticed a major discrepancy between what is claimed to be done and what is reported in the mat&met and results sections. In this respect, I recommend a major revision.

Indeed, the abstract states that “a stability analysis was conducted by comparing the ratio of mRNA to gDNA overtime following heat treatment of wood infected by the oomycete..”. Also P12L244, it is written: “Ratio of ….applied to WOOD-LOGS infected with P. ramorum and G. clavigera”. However, in the experiments reported in the manuscript, only mRNA and gDNA from MYCELIAL cultures of the pathogen were heat-treated, not from WOOD infected by the latter (see §Heat treatment to determine mRNA stability”, P6-7; in Figure 4 caption “… for gDNA or cDNA extracted from CULTURES sampled from 0 to 240 hours after treatment.”), and in discussion section L285-287, L382.

Likewise, all the discussion parts related to the appropriateness of wood treatments deserve mitigation since the experiments carried out in this work only dealt with in vitro-cultivated mycelium and not in vivo growing mycelium. The efficiency of heat treatment procedures are dependent of wood thickness, relative humidity, heat flow, etc.

I therefore suggest clarifying what was really assessed in this work. It seems that artificially infected wood were indeed tested after 28 days of incubation, but not after the two different heat treatments.

Specific comments:

L46: Ref 8 and 9 do not seem to be the most relevant ones to support the statement of the sentence.

L50: again here, ref 11 does not seem to be appropriate to the support the statement.

L69: ref 21 and 22 are more appropriate here, ref 20 a bit less supporting. Consider removing it.

L79 and 90: please could you explain why distinct DB were used for the selection of genes?

L82: on which basis the genes were selected? It is written “genes are expected to be expressed”, but how was that anticipated? The discussion section contains important information pertaining to the rationale followed for the selection, and this information should be moved to the mat&met section.

L133: number, reference of isolates and number of replicates should better be indicated here, rather than in a later paragraph (L177-179).

L140-141: the sentence sounds a bit odd.

L147: please specify what the wood samples were collected from.

L177: how was the qPCR done with these solutions as templates? 2.2 ng of template DNA are indicated a few lines above. Is it still the case?

Figure 1 caption indicates that gDNA and cDNA were used, but it is in conflict with what is written L215-216.

L201: is there one single sister species (L. longiclavatum) or several involved here? Not clear what “these sister species” refer to.

Figure 3 should remind how many replicates were included, and also explain how the curves should be interpreted (shaded areas, min and max?). Also, it should be interesting to discuss the striking lower magnitude of fluorescence yielded with cDNA extracted from P. ramorum-infected Tsuga, whereas the gDNA curves look “normal”. I noticed that there was a change of y-axis scale compared to the other plots, probably in order to better fit the gDNA fluorescence magnitude. But still, the gDNA/cDNA difference with the other plots seems awkward.

L239: I do not think that it is relevant to compare the mean Ct values obtained with chlamydospores to those obtained with infected wood samples. Chlamydospores quantity was arbitrarily set, we do not know if is close to reality in infected tissue or soil for instance.

Table 1: consider removing it, as it is a piece of arid stats. Or maybe replace “treatment” with what it stands for.

L285: heat treatment may just not degrade mRNA, it may also inhibit its synthesis.

L367-L372: I fully agree with this caveat. The authors should therefore recommend a practical procedure to assess the kinetics of mRNA.

Figure 4: The authors should discuss some questioning variations observed in cDNA quantity, e.g. the high variation in cDNA amplification between Ctrl/ 0 and 24 hrs post treatment for P. ramorum/short heat treatment. Although it seems obvious that after a certain treatment duration, no mRNA is produced, what would explain this high variation before that time? One may also wonder why gDNA and cDNA quantities (inferred from Ct values) in the control are sometimes significantly superior to the 0 tpt values? In two occasions, no mRNA is detected at all for 0 tpt, whereas the ctrl value is “normal”. Is it because 0 means that the treatment already started? If yes, 0 is propably not appropriate term.

More general comments:

The discussion section is a lot about P. ramorum, and unfortunately G. clavigera is a bit set aside. Although G. clavigera is less “famous” than P. ramorum, a more balanced discussion would be nice.

Reviewer #2: This manurscript is very appropriate for PLOS ONE. The manuscript is well written and present a new molecular assay using mRNA and gDNA to detect and show viability of Phytophthora ramorum and Grosmania clavigera. The method is well described and present new material. The 2 assays seem to work very well and could serve as base for ID and evaluation of viability of forest pathogens. The genomic was used to ID markers and selection of junction of intron/exon was good choice and well presented. The discussion could have more information comparing other methods and talk about the limitation of the assays, by example the detection of mRNA is very at the Ct value of limit of detection. What about other assays published. Other missing or clarifying information need to be add.

Specific comments:

P4L58 did you consider to add other method information to look at viability, I believe the PMA Propodium monoazide is a good method and do not need to play with RNA. Should add info and pro and cons in discussion?

P4L69 I was wondering about if the P. ramorum was infecting in wood or surface but was more clear later in manuscript. Could be presented more here. P5L82, it is really a communication or more a resource? (Bret Tyler sequences)

P6L104 for G. clavigera, only one isolate was use, how do we know it would work with multiple genotypes?

P6L109 any reason on use of only conifers, what about deciduous tree for P. ramorum?

P6L115 any specific reference for the SPF kiln drying schedule?

P8L144 "Matric" should be "Matrix" Correct in line 154.

P9L178 "586 and 101329) info on specimens, it is voucher isolate, CBS culture??? need more info on you isolates used in the manuscript?

P9 Just wondering it is better ration Ct or Ration concentration calculated, Ct,Log10?

P12L232, 33.4 seem to be very low limit? not so high you may miss lower concentration of pathogens. also should explain the range in figure 3, blurry red?? Blurry blue, same Ct but range blurry?? not clear what it mean?

P12L247 and Table 1, not so clear what the F, Fvalue is the stat need to be better explained and significance in the manuscript?

Table 1 no footnotes, what the "*" means? By? Tyope III SS?

Discussion, RNA extraction and cDNA is additional step, add info how the RNA extraction is easy or difficult and cDNA prep, risk of contamination, compare to PMA should be discuss.

What is the Ct level limit for cDNA-mRNA in other publication using similar approach?

Reviewer #3: In this MS, development of a PCR-based viability assay of tree pathogens is described. The authors showed that both genomic DNA and mRNA extracted from tree tissues under the bark were amenable for qPCR. They also showed that heat treatments could effectively kill the pathogens. Effectiveness of the heat treatments on the pathogens growing in the wood trunk was, however, not evaluated. The author needs to address this point. Below are my specific comments.

Line 18: RNA represents ... therefore only be produced by living organisms.

RNA, as well as DNA, are only produced by living organisms.

Line 164: cDNA synthesis

The authors used two-step RT-qPCR for all samples. The two-step method, which uses a stock cDNA, may be advantageous for the screening of primer pairs. However, for high-throughput assay, one-step RT-qPCR is more advantageous and give less experimental variation.

Line 231: Cycle-threshold values obtained for the cDNA were slightly higher.

For both P. ramorum and G. clavigera, the differences of Ct values between gDNA and cDNA were over 3, which were equivalent to an 8-fold difference in copy number of templates. This is a huge difference and needs to be discussed.

Line 242: No amplification was observed in the no-template control.

The vast quantity of genomic DNA and cDNA from inoculated wood samples were of the host plant origin. What was the level of background amplification when a DNA template from a non-inoculated plant tissue was used?

Line 342: indicating that the target gene is still expressed during this life-stage.

Most likely, the target gene is not expressed in the resting spores, but its transcript is stored in them.

Reviewer #4: This is an interesting manuscript describing a novel approach to the development of real-time PCR assays for viable pathogens in wood, providing significant improvements to current methods used in screening wood products. In particular the focus on only detecting viable pathogens (linked to the decay of mRNA) is valuable and will likely be applied in plant diagnostics. The manuscript is very well written, relatively free of grammatical and spelling errors (see short list below), and the statistical analyses appear sound.

The real time PCR data depicted in Fig 3 is however a bit troubling, with regard to the Ct values. The authors should mention the instrument used in the assays in the Methods section, L 170-176, and how the thresholds were established for Ct calculations. A more precise estimation of the Ct could improve upon the degree of error in their data (e.g. Fig. 4). Were instrument default parameters used in Ct calls, or did the authors adjust these parameters? Again, please state how this was set on the instrument in the Methods section. The cDNA reactions and Ct values illustrated in Fig 3 appear to be calling the Ct rather inconsistently between the samples, and this would affect the ratio and perhaps improve the error estimations. The authors should consider that Ct values over 32-33 are considered to be late amplification and may be non-specific, or at least represent an extremely low target cDNA copy number. The authors could confirm that the high Ct values are not artefactual with e.g. information from melt curves. The primers and reactions perform well using cDNA generated from pathogen RNA, but the amplifications in wood-extracted samples appears to be very weak.

Paragraph #5 of the Discussion L 345-376 should be re-written for clarity and consistency. The paragraph is very long, and wanders a bit from the first sentence (L345-346), a rather simplistic declaration, to later discussion of the reliance of the technique on the kinetics of mRNA stability (L 371 to 376). The paragraph could be reduced in length and the paragraph structure improved for clarity of the points made.

In addition, a few minor edits to the manuscript are recommended:

L63 insert “in eukaryotes”

L 134-136 “falcon” filters and tubes- Falcon is a brand not a type of tube- e.g. describe as “50 mL conical plastic centrifuge tube” or some such.

L 161 “Qubit fluorometer” list source

L 218 How was amplification efficiency calculated from the regression curves?

L236 synthesized (sp)

L 239 nucleic acids (no hyphen)

L255 ...efficiently killed... (reverse order of words)

Reviewer #5: The authors report new cDNA-based reverse transcription quantitative real time PCR (RT-qPCR) assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera. The authors test the ability of the assay to discriminate between living and dead fungal samples after two heat treatments. Additionally, the assay is used to detect the pathogens in artificially inoculated wood bolts.

The main merit of the publication is in developing molecular assays that can distinguish between dead and living mycelium based on cDNA/gDNA ratios. Assays that can evaluate pathogen viability have potential to improve phytosanitary measures. The P. ramorum assay is promising as a diagnostic tool, as it only amplifies P. ramorum DNA, and not other closely related species from Phytophthora clade 8c. However, the G. clavigera assay is not fully specific as it amplifies a closely related species: L. longiclavatum. This is problematic in terms of specific pathogen identification for diagnostic purposes. The impact of the work would be higher if the assay was specific for G. glavicera. Alternatively, the authors could develop an assay that robustly detects several closely related Leptographium/Grossmannia species.

The experimental design that was used to test the assay has some flaws and inconsistencies:

- The data for the heat treatments may not be fully comparable. Mycelial samples were processed differently for the two heat treatments: in kiln-drying, the authors used 0.1 ml tubes in a heat block, whereas in short heat treatment, they used agar plates in an oven. This might affect fungal viability and thus RNA degradation. The authors should provide data that shows this has no effect on the Cq values. Now, the ANOVA cannot distinguish if this affects the cDNA/gDNA ratios.

- No control mycelium samples for the different time points. As this is a time-point assay, the authors should include a non-treated control mycelium sample for each time point, and store them in identical conditions as the treated samples. This would tell if the expression of the gene target is actually stable in the used conditions, and would help to evaluate the suitability of this assay as a diagnostic tool.

- Wood samples were not plated to reisolate the pathogen. This would provide a concrete reference point to discuss the ability of the assay to detect the pathogens, either viable or dead.

- Wood samples were not compared for the heat treatments. This would have been an excellent simulation for the relevance of the assay as a diagnostic tool. Now the work falls short of demonstrating the applicability of this assay in phytosanitary screening.

- Imbalanced sample sizes for the two pathogens: 8 P. ramorum isolates, but only 1 G. clavigera isolate. In my opinion, the authors should have used more G. glavigera isolates.

Additionally, there are some concerns related to RNA sample quality control. The authors only used Nanodrop to quantify the RNA samples before DNAse treatment, which can distort the downstream RNA amounts added to the reverse transcription reactions. They used a kit that selects for RNA, but still, fluorescent RNA-binding dyes are the preferred method to get accurate RNA concentrations. Additionally, the authors do not report any method that was used to estimate RNA integrity (Nanodrop is not sufficient).

The manuscript has potential, but I cannot recommend the acceptance of this manuscript in its current form. The authors would need to make complementary experiments and provide more data to improve the manuscript. Especially I would like to see data for the effect of the heat treatment on the wood samples and pathogen viability, and how the assay performs as a diagnostic tool. Alternatively, the authors should re-analyze the data and re-write it as a short communication and tone down the role of the heat treatment comparison. This would in my opinion require excluding/re-developing the G. clavigera assay, as it is not species specific, but it is not a broad-spectrum assay either.

Comments and revisions

See PDF for language revisions

Please read Bustin et al. 2009 (10.1373/clinchem.2008.112797) for recommended terminology and experimental guidelines for quantitative PCR experiments

Correct real-time PCR to quantitative real-time PCR (qPCR)

Correct reverse transcription real-time PCR (RT-PCR) to reverse transcription quantitative PCR (RT-qPCR)

Correct Threshold cycle/Cycle threshold (Ct) to Quantification cycle (Cq) throughout the manuscript

Resolution of all figures is poor. Make sure you provide figure files with at least sufficient resolution (TIFF files with 300-600 DPI) and export graphs from R-studio/statistical software into EPS format to get 300 DPI resolution (see journal guidelines for preparing the images).

L47: What do you mean by universal genes? Conserved genes?

L78: How many conserved proteins, how many sequences?

L85-88: Rephrase to list P. ramorum as the primers were also tested on P. ramorum.

L94: Rephrase to list G. clavicera as the primers were also tested on G. clavicera.

L98-99: Provide citation to manuals/protocols/guidelines that were used to optimize the assays.

L113-131: The experimental setting is unclear. Please make an illustration showing the workflow, with heat treatments, what samples were used for what, differences in methods, replicates, controls, plating etc.

L103-104: Why did you decide to use 8 isolates of P. ramorum and only one G. clavicera isolate? This is a rather unbalanced experimental setting.

L103-112: How many bolts per each tree species-pathogen-time point combination?

L102-112: Why did you not plate any of the wood tissue? This would provide a reference point to evaluate what the re-isolation success is in controls vs. after heat treatment, and if the pathogens remain viable after the treatments. The manuscript would be improved if the authors provided results for re-isolation from inoculated wood samples.

L102-112: Why you did not use any of the wood samples for the heat treatments? This would be the closest to a real life simulation to evaluate the efficacy of the heat treatments. Now the results have very little connection to using the assay in a real-life diagnostic context.

L105: Provide ingredients or citation for carrot and MEA media

L118: Provide ingredients or citation for V8 and MEA media

L114-123: At what temperature were the samples stored after the SPF kiln-drying treatment before sample collection?

L120-121: Was the frozen mycelium sample in 1.5 ml tubes the only control? Did you have untreated controls stored in 0.1 ml strip tubes that were stored in same conditions as the heat treated samples, and sampled at the same time points? Flash freezing preserves mRNA better compared to a situation where a small non-heat-treated sample of mycelium is stored at room temperature. Now it is not clear how much RNA degradation occurs due to storage at room temperature, and how much is due to heat treatment. Additionally, knowing the expression of the gene over several time points would help to evaluate the suitability of this gene as a diagnostic marker. Please provide data for cDNA/gDNA ratios for control mycelial samples that are stored and sampled similarly to the heat-treated samples.

L127-129: For the short heat treatment, did you put agar plates directly to the oven? This might be a problem in terms of comparing the heat treatments. The mycelium on the plates might be heated differently than in the SPF kiln-drying treatment, where 0.1 ml Eppendorf tubes were used for heating and storing the samples after the treatment. Can you provide data that would indicate this won’t affect the degradation rate of mRNA? I would repeat this experiment by placing the mycelium into 0.1 ml Eppendorf tubes, perform the short heat treatment, and sample similarly as in the SPF kiln-drying experiment. With the current methods, I am not convinced that the data from the two heat treatments is comparable. The ANOVA won’t be able to tell if the differences in cDNA/gDNA ratios are caused by using 0.1 ml tubes vs. plates, or due to different temperature treatment, as the use of tubes or plates is nested within the heat treatment.

L159-163: Why did the authors decide to use two different methods for quantification of nucleic acids, Qubit for DNA and Nanodrop for RNA? Per MIQE guidelines, the preferred method for quantifying RNA uses fluorescent RNA-binding dyes, e.g. Qubit assay. As the RNA is not pure mRNA and the concentrations were measured before DNAse treatment, it is likely that Nanodrop readings overestimate the concentrations. This can affect the amount of RNA that is added to each RT reaction. Please address this issue and provide data that shows you get the similar Cq results from RNA samples measured with Qubit.

L159-163: What method did you use to evaluate RNA integrity? The absorbance values from Nanodrop only indicate RNA sample purity, not integrity.

L170-176: How many biological and technical replicates?

L204-208: Based on previous data, does the gene have stable constitutive expression? For diagnostic assays like the authors are developing, the gene targets should have stable expression no matter the condition. Add eg. text and references from lines 337-339.

L221-223: Figure 2 caption: Revise so that you provide the same information for both targets and indicate clearly which gene target is for which pathogen.

L234-238: Figure 3 caption: Indicate what the shaded areas are.

L250-261: Report results from statistical testing, as they are not indicated in Figure 4 or elsewhere.

L255-257: Less efficient compared to what: control, the other treatment? Be more specific.

L267-268: Table 1 indicates that replicate has a significant impact on G. clavicera cDNA/gDNA ratios. Discuss why this is observed for G. clavicera but not for P. ramorum? How was the quality of the RNA samples? How about lesion lengths, any variation in that?

L272-275: Summarize the results in 1-2 sentences in appropriate context. This is not re-isolation because what you describe is plating mycelium samples prepared from the same known pure culture. Rather you are assessing viability of mycelium after heat treatment.

L272-275 & L345-376: If I understand correctly, you did not make any re-isolations from the bolts. Discuss/Explain why not? Discuss if plating only the heat-treated mycelium samples provides a reliable reference for the viability of the pathogen in biological samples. How do the Cq values for mRNA in colonized bolts and mycelial samples compare? Based on the Cq values, what is the extent of mycelial colonization in the wood samples?

L312: At least in the US, by definition the pathogen is not regulated, but the interstate movement of certain articles from quarantined counties is regulated. See for details: https://www.federalregister.gov/documents/2007/02/27/07-892/phytophthora-ramorum-quarantine-and-regulations

L367-376: Be a bit critical and discuss if this could be resolved by plating infected wood samples after the heat treatments. There might be a time window after the treatment when the pathogen would still be able to grow, if suitable conditions (e.g. nutrients, temperature, moisture) were available. In what conditions are heat-treated/kiln-dried samples stored in industrial facilities? Is there a risk that the pathogen would still remain infectious? Does this create a risk for false-negatives? When should the diagnostic testing be done? Is this feasible?

Discussion and criticism lacks entirely for the non-specific G. clavigera assay. Discuss what diagnostic implications this might have, if you are not willing to expand the work and develop an assay that is specific for this pathogen. Are all Grosmannia/Leptographium species equally harmful? Could this assay be a more generic assay that detects several of these species?

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Submitted filename: PONE-D-19-22766_reviewer_comments_redacted.pdf

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27 Nov 2019

RESPONSE TO REVIEWER’S COMMENTS (A Word copy is also attached, with color code to make it easier to follow the comments and answers).

Reviewer #1:

The experiments are adequate and compelling for most of the work, but I noticed a major discrepancy between what is claimed to be done and what is reported in the mat&met and results sections. In this respect, I recommend a major revision.

Indeed, the abstract states that “a stability analysis was conducted by comparing the ratio of mRNA to gDNA overtime following heat treatment of wood infected by the oomycete..”.

Corrected in manuscript. “wood infected with” was deleted L.26.

Also P12L244, it is written: “Ratio of ....applied to WOOD-LOGS infected with P. ramorum and G. clavigera”. However, in the experiments reported in the manuscript, only mRNA and gDNA from MYCELIAL cultures of the pathogen were heat-treated, not from WOOD infected by the latter (see §Heat treatment to determine mRNA stability”,

P6-7; in Figure 4 caption “... for gDNA or cDNA extracted from CULTURES sampled from 0 to 240 hours after treatment.”), and in discussion section L285-287, L382.

Likewise, all the discussion parts related to the appropriateness of wood treatments deserve mitigation since the experiments carried out in this work only dealt with in vitro-cultivated mycelium and not in vivo growing mycelium. The efficiency of heat treatment procedures are dependent of wood thickness, relative humidity, heat flow, etc.

I therefore suggest clarifying what was really assessed in this work. It seems that artificially infected wood were indeed tested after 28 days of incubation, but not after the two different heat treatments.

We clarified what was done in our work i.e. all statements talking about heat treatment on infected wood have been removed and the discussion section on this point has been toned down. See modifications on L.26, L.284, L.367...

Specific comments:

L46: Ref 8 and 9 do not seem to be the most relevant ones to support the statement of the sentence.

We changed these two references for : Taylor et al. 2000 Phylogenetic Species Recognition and Species Concepts in Fungi. Fungal Genetics and Biology, 31:21-32

L50: again here, ref 11 does not seem to be appropriate to the support the statement.

Right. We removed ref. 11 from L.56.

L69: ref 21 and 22 are more appropriate here, ref 20 a bit less supporting. Consider removing it.

Done. We removed ref. 20.

L79 and 90: please could you explain why distinct DB were used for the selection of genes?

As Grosmannia is a fungus, it was searched against a fungal database i.e. FUNYBASE. Unfortunately, searching against this db couldn’t work for Phytophthora as these organisms are not fungi. For this reason, we searched Phytophthora against a more generalistic db i.e. the CEGMA eukaryotic db.

L82: on which basis the genes were selected? It is written “genes are expected to be expressed”, but how was that anticipated? The discussion section contains important information pertaining to the rationale followed for the selection, and this information should be moved to the mat&met section.

Done. See L.90-91 in M&M section.

L133: number, reference of isolates and number of replicates should better be indicated here, rather than in a later paragraph (L177-179).

This paragraph was moved up to the beginning of the M&M section entitled “Chlamydospore separation” (L.146-148).

L140-141: the sentence sounds a bit odd.

We changed the sentence for “Final concentration of the spores was adjusted to 1x105 chlamydospore/ml using a hemocytometer”. L.156-157.

L147: please specify what the wood samples were collected from.

Done. See L.163-164.

L177: how was the qPCR done with these solutions as templates? 2.2 ng of template DNA are indicated a few lines above. Is it still the case?

Yes, for all TaqMan reaction DNA templates were adjusted to 2.2ng (L.191).

Figure 1 caption indicates that gDNA and cDNA were used, but it is in conflict with what is written L215-216.

L215-216 (L.237 in the new version with track changes) was referring to Figure 2. We are not sure about what Rev. #1 is referring to with this point.

L201: is there one single sister species (L. longiclavatum) or several involved here? Not clear what “these sister species” refer to.

No, four of them but the closest one is L. longiclavatum. We change “its sister species” for “the closely related species” to discard any ambiguity (L.217).

Figure 3 should remind how many replicates were included, and also explain how the curves should be interpreted (shaded areas, min and max?). Also, it should be interesting to discuss the striking lower magnitude of fluorescence yielded with cDNA extracted from P. ramorum-infected Tsuga, whereas the gDNA curves look “normal”. I noticed that there was a change of y-axis scale compared to the other plots, probably in order to better fit the gDNA fluorescence magnitude. But still, the gDNA/cDNA difference with the other plots seems awkward.

We re-wrote Fig. 3 (NOW FIG. 4) caption as follows (L.645): Figure 4. Wood inoculation with Phytophthora ramorum (EU2 isolate P2111) and Grosmannia clavigera (isolate KW140) and corresponding real-time PCR amplification plots. For each wood inoculation sample, gDNA and cDNA synthetized from mRNA extracted from a lesion at 28 day post inoculation was tested in real-time PCR with either the PH178 assay (targeting P. ramorum) or MS356 (G. clavigera). Quantification cycle (Cq) values for gDNA (blue) and cDNA (red) are reported on each graph. Shaded area around the average represents ±SD.

This difference for gDNA vs. cDNA with Tsuga is probably the result of an inhibition of the cDNA synthesis. Unfortunately, we did not keep a record of the RNA quality after purification from wood samples and cannot verify this hypothesis.

L239: I do not think that it is relevant to compare the mean Ct values obtained with chlamydospores to those obtained with infected wood samples. Chlamydospores quantity was arbitrarily set, we do not know if is close to reality in infected tissue or soil for instance.

Corrected in manuscript, comparison sentence removed. L.251-252.

Table 1: consider removing it, as it is a piece of arid stats. Or maybe replace “treatment” with what it stands for.

Table 1 has been removed.

L285: heat treatment may just not degrade mRNA, it may also inhibit its synthesis.

We considered this important point and change the original sentence for “As a second method, we assumed that the rapid degradation and synthesis inhibition of mRNA might be a useful indicator of cell mortality [47]”. L.387.

L367-L372: I fully agree with this caveat. The authors should therefore recommend a practical procedure to assess the kinetics of mRNA.

There are two ways of addressing this issue: one if to determine the threshold after which mRNA is not detectable after lethal treatments so that testing can be done at the appropriate time. The second way would be to conduct for each targeted species an mRNA kinetics experiment to determine the rate of degradation. However, this last solution could be complicated by the fact that mRNA kinetics could be affected by the substrate treated.

Figure 4: The authors should discuss some questioning variations observed in cDNA quantity, e.g. the high variation in cDNA amplification between Ctrl/ 0 and 24 hrs post treatment for P. ramorum/short heat treatment. Although it seems obvious that after a certain treatment duration, no mRNA is produced, what would explain this high variation before that time? One may also wonder why gDNA and cDNA quantities (inferred from Ct values) in the control are sometimes significantly superior to the 0 tpt values? In two occasions, no mRNA is detected at all for 0 tpt, whereas the ctrl value is “normal”. Is it because 0 means that the treatment already started? If yes, 0 is probably not appropriate term.

The time points refer to the time after treatment, therefore, the “0” time point actually refers to when the pathogen has just been removed from their corresponding heat treatments. The x-axis for this figure was labeled to indicate that the time points 0 to 240 are the number of hours after treatment.

High variation in Ct values could be due to the difference in yield of mRNA obtained after purification (see Discussion on L.402-408) and the instability of RNA (i.e. RNA degradation).

More general comments:

The discussion section is a lot about P. ramorum, and unfortunately G. clavigera is a bit set aside. Although G. clavigera is less “famous” than P. ramorum, a more balanced discussion would be nice.

We tried to add a few more statements on G. clavigera. However, this pathogen didn’t receive the focus that P. ramorum got in terms of tool development for monitoring and surveillance, making it very difficult to comment/discuss on G. clavigera.

Reviewer #2:

The discussion could have more information comparing other methods and talk about the limitation of the assays, by example the detection of mRNA is very at the Ct value of limit of detection. What about other assays published. Other missing or clarifying information need to be add.

Yes, further discussion of the limitations to this assay was added (Paragraph starting L.398). The detection of some of the samples were at a very low Ct, but still within our cut off of 35, eliminating the possibility of false positives.

Specific comments:

P4L58 did you consider to add other method information to look at viability, I believe the PMA Propodium monoazide is a good method and do not need to play with RNA. Should add info and pro and cons in discussion?

Additional information has been added to the manuscript to address this point. Paragraph starting on L.398.

P4L69 I was wondering about if the P. ramorum was infecting in wood or surface but was more clear later in manuscript. Could be presented more here. P5L82, it is really a communication or more a resource? (Bret Tyler sequences)

Sentence has been changed to : “...two microorganisms that can colonize wood cambium”. L.75-76.

P6L104 for G. clavigera, only one isolate was use, how do we know it would work with multiple genotypes?

Yes, this is one limitation of our study. The isolate was chosen because this was the isolate that was used for genome sequencing. However, G. clavigera is highly clonal, meaning that sequence polymorphism is limited in conserved genes and we made sure that the primers developed were matching on other G. clavigera sequenced.

P6L109 any reason on use of only conifers, what about deciduous tree for P. ramorum?

Canadian forest sector mainly focus on conifers and the industry predominantly deals with the trading of such (e.g. coniferous forests account for 72% of timber volume in Canada).

P6L115 any specific reference for the SPF kiln drying schedule?

We added the following reference on L. 129:

Cai, Liping, and Luiz C. Oliveira. Evaluating the use of humidification systems during heat treatment of MPB lumber. Drying Technology 29, no. 7 (2011): 729-734.

P8L144 "Matric" should be "Matrix" Correct in line 154.

Corrected in manuscript. L.160 and L.160.

P9L178 "586 and 101329) info on specimens, it is voucher isolate, CBS culture??? need more info on you isolates used in the manuscript?

Pr-05-015 (CSL2268, P1578) was isolated from Rhododendron grandiflora from a nursery in the UK in 2002 and is Clonal lineage EU1.

CBS101329 was purchased from CBS-KNAW Fungal Biodiversity Centre (now known as Westerdijk Fungal Biodiversity Institute). It was collected from Lisse, Netherlands on Rhododendron and is of the EU1 strain. We added this information on L.147.

P9 Just wondering it is better ration Ct or Ration concentration calculated, Ct,Log10?

We are not sure we understand this question. Does Rev. #2 suggest to log-transform Ct-ratios?

However, we think it’s better to use the ratio of the Ct-values obtain for cDNA and gDNA (in theory, cDNA Ct-value should increase with loss of viability whereas gDNA Ct-value shouldn’t change).

P12L232, 33.4 seem to be very low limit? not so high you may miss lower concentration of pathogens. also should explain the range in figure 3, blurry red??

Blurry blue, same Ct but range blurry?? not clear what it mean?

Redefinition of figure 3 was done (NOW FIG. 4) - please refer to comment from Rev. #1.

P12L247 and Table 1, not so clear what the F, Fvalue is the stat need to be better explained and significance in the manuscript?

Table 1 no footnotes, what the "*" means? By? Type III SS?

Yes, Table 1 is deleted as suggested by Rev. #1.

Discussion, RNA extraction and cDNA is additional step, add info how the RNA extraction is easy or difficult and cDNA prep, risk of contamination, compare to PMA should be discuss.

Additional information has been added to the discussion that the RT-qPCR conducted here was a two-step process. Paragraph starting on L.398.

What is the Ct level limit for cDNA-mRNA in other publication using similar approach?

Similar papers (e.g. Chimento et al 2012, Leal et al 2013) used a Ct cut-off value later than 35.0 as their positive cut off.

Reviewer #3:

Effectiveness of the heat treatments on the pathogens growing in the wood trunk was, however, not evaluated. The author needs to address this point. Below are my specific comments.

Same point than Rev. #1. As stated above, we clarified that the effectiveness of the treatment was tested only on cultures.

Line 18: RNA represents ... therefore only be produced by living organisms. RNA, as well as DNA, are only produced by living organisms.

Right. We fixed that by changing the sentence: “RNA represents the transcription of genes and can therefore become rapidly unstable after cell death…”. L.20-21.

Line 164: cDNA synthesis

The authors used two-step RT-qPCR for all samples. The two-step method, which uses a stock cDNA, may be advantageous for the screening of primer pairs. However, for high-throughput assay, one-step RT-qPCR is more advantageous and give less experimental variation.

A new section has been included in the discussion to address this point. But overall, using the one-step method is less sensitive and requires to start with more RNA and though could be considered for future improvement. Paragraph starting on L.398.

Line 231: Cycle-threshold values obtained for the cDNA were slightly higher.

For both P. ramorum and G. clavigera, the differences of Ct values between gDNA and cDNA were over 3, which were equivalent to an 8-fold difference in copy number of templates. This is a huge difference and needs to be discussed.

We added some elements of discussion (+ references) about this point in a new paragraph starting on L.398. Our main hypothesis is related to the low purity/yield of the RNA extracted from conifer wood and subsequent inhibition of downstream enzymatic reaction such as reverse transcription during cDNA synthesis.

Line 242: No amplification was observed in the no-template control.

The vast quantity of genomic DNA and cDNA from inoculated wood samples were of the host plant origin. What was the level of background amplification when a DNA template from a non-inoculated plant tissue was used?

Yes, gDNA and cDNA samples from non-inoculated wood were included in the qPCR but no expression was observed. This point is now included in manuscript L.253-254.

Line 342: indicating that the target gene is still expressed during this life-stage. Most likely, the target gene is not expressed in the resting spores, but its transcript is stored in them.

I’m not sure we could have some evidence for transcript storage in resting spores. Statement has been removed.

Reviewer #4:

The real time PCR data depicted in Fig 3 is however a bit troubling, with regard to the Ct values. The authors should mention the instrument used in the assays in the Methods section, L 170-176,

Done. Applied Biosystems StepOne™ software was used for the qPCR. L.194.

and how the thresholds were established for Ct calculations.

The qPCR thresholds were generated through the StepOne™ default parameters. The parameters calculate the threshold, under “automatic Ct”. This is an analysis setting in which the software calculates the baseline start and end values and the threshold in the amplification plot. The software uses the baseline and threshold to calculate the threshold cycle (CT). A sentence was included in manuscript for clarification. L.194-195.

A more precise estimation of the Ct could improve upon the degree of error in their data (e.g. Fig. 4). Were instrument default parameters used in Ct calls, or did the authors adjust these parameters?

Default parameters of the StepOne™ software were used for the Ct threshold calculations.

Again, please state how this was set on the instrument in the Methods section. The cDNA reactions and Ct values illustrated in Fig 3 appear to be calling the Ct rather inconsistently between the samples, and this would affect the ratio and perhaps improve the error estimations. The authors should consider that Ct values over 32-33 are considered to be late amplification and may be non-specific, or at least represent an extremely low target cDNA copy number.

Details of the qPCR instrument has been included in the manuscript. L.194-195.

Though a Ct value of 32-33 is considered low as compared to gDNA samples. It is still within other similar studies’ positive amplification cut off (Chimento et al 2012, Leal et al 2013).

The authors could confirm that the high Ct values are not artifactual with e.g. information from melt curves. The primers and reactions perform well using cDNA generated from pathogen RNA, but the amplifications in wood-extracted samples appears to be very weak.

The optimization of wood extraction should help in improving the sensitivity of the assays. Due to the high amount of high quality of secondary metabolites polyphenols, tannins, terpenoids and protein inhibitors (see L.402-408).

Unfortunately, the experiments were run under the standard curve settings on the StepOne qPCR machine (not under the melting curve settings), meaning that the size of the PCR fragments cannot be confirmed with the melting curves.

Paragraph #5 of the Discussion L 345-376 should be re-written for clarity and consistency. The paragraph is very long, and wanders a bit from the first sentence (L345-346), a rather simplistic declaration, to later discussion of the reliance of the technique on the kinetics of mRNA stability (L 371 to 376). The paragraph could be reduced in length and the paragraph structure improved for clarity of the points made.

The two first sentences of paragraph #5 were removed. L.363-366.

In addition, a few minor edits to the manuscript are recommended:

L63 insert “in eukaryotes”

Done.

L 134-136 “falcon” filters and tubes- Falcon is a brand not a type of tube- e.g. describe as “50 mL conical plastic centrifuge tube” or some such.

Corrected in manuscript. L.151.

L 161 “Qubit fluorometer” list source

L 218 How was amplification efficiency calculated from the regression curves? L236 synthesized (sp)

Efficiency was an output from the StepOne qPCR machine.

L 239 nucleic acids (no hyphen)

Corrected in manuscript. L.250

L255 ...efficiently killed... (reverse order of words)

Corrected in manuscript. L.267

Reviewer #5:

The authors report new cDNA-based reverse transcription quantitative real time PCR (RT-qPCR) assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera. The authors test the ability of the assay to discriminate between living and dead fungal samples after two heat treatments. Additionally, the assay is used to detect the pathogens in artificially inoculated wood bolts.

The main merit of the publication is in developing molecular assays that can distinguish between dead and living mycelium based on cDNA/gDNA ratios. Assays that can evaluate pathogen viability have potential to improve phytosanitary measures. The P. ramorum assay is promising as a diagnostic tool, as it only amplifies P. ramorum DNA, and not other closely related species from Phytophthora clade 8c. However, the G. clavigera assay is not fully specific as it amplifies a closely related species: L. longiclavatum. This is problematic in terms of specific pathogen identification for diagnostic purposes. The impact of the work would be higher if the assay was specific for G. glavicera. Alternatively, the authors could develop an assay that robustly detects several closely related Leptographium/Grossmannia species.

Leptographium longiclavatum and G. clavigera are closely related species (i.e. two SNPs over a 710nt alignment of the ITS). They share similar morphological characteristics and evolutionary history (see Lee et al. 2005 Mycol. Res. 109:1162; Lims et al. 2004 FEMS Microbiology Letters, 237, 89). This made it very complicated to find a good tradeoff between finding a conserved enough gene in the FUNYBASE db. to anchor primers arround a intron-exon junction and enough fixed polymorphisms to be able to differentiate between L. longiclavatum and G. clavigera. As stated in the ms on L.211-214 our assay now targets both species. We added this to the Discussion (L.330-342): ‘Due to the high sequence similarity between G. clavigera and the closely related species L. longiclavatum [62,63], the assay developed amplified mRNA and gDNA of both species. This could be a positive attribute of this assay since both species are symbionts of the mountain pine beetle and share a very similar ecological niche in pine trees [22]; in fact it is possible that these fungi hybridize and the species limits are not clear (R. C. Hamelin and A. Capron, unpublished).’

The experimental design that was used to test the assay has some flaws and inconsistencies:

- The data for the heat treatments may not be fully comparable. Mycelial samples were processed differently for the two heat treatments: in kiln-drying, the authors used 0.1 ml tubes in a heat block, whereas in short heat treatment, they used agar plates in an oven. This might affect fungal viability and thus RNA degradation. The authors should provide data that shows this has no effect on the Cq values. Now, the ANOVA cannot distinguish if this affects the cDNA/gDNA ratios.

Yes, this is true. However, in both cases, the targeted temperature for the core is reached in both experiments; as long as this temperature is reached, we think that we can assume that the temperature requirements of the experiment were fulfilled.

In addition, the point of the experiment was not to compare both experiments. Instead, the aim was to use our assays to assess if some temperature/schedule used to treat wood against pests are efficient.

- No control mycelium samples for the different time points. As this is a time-point assay, the authors should include a non-treated control mycelium sample for each time point, and store them in identical conditions as the treated samples. This would tell if the expression of the gene target is actually stable in the used conditions, and would help to evaluate the suitability of this assay as a diagnostic tool.

Right, there was no non-treated control for each of the different sampling time points after heat treatment and we didn’t assess the stability of the expression of the targeted genes in non-treated conditions. However, there was a non-treated control mycelium that was frozen at the end of experiment and tested with the gDNA and cDNA assays. For both species it resulted in detectable Ct-values for the cDNA meaning that the targeted gene was expressed during the experiment if the culture was not treated.

- Wood samples were not plated to reisolate the pathogen. This would provide a concrete reference point to discuss the ability of the assay to detect the pathogens, either viable or dead.

Right, they were not plated for this experiment. However, the aim of this experiment was very “technical”. We wanted to test if gDNA and mRNA of P. ramorum and G. clavigera could be 1 - purified from inoculated wood from different conifers and 2 - detected by qPCR with our assays.

- Wood samples were not compared for the heat treatments. This would have been an excellent simulation for the relevance of the assay as a diagnostic tool. Now the work falls short of demonstrating the applicability of this assay in phytosanitary screening.

Similarly to what was discussed above in response to Rev. #1, we clarified what was really done in our work i.e. all statements talking about heat treatment on infected wood have been removed and the discussion section on this point has been toned down. Though heat treated wood samples were not tested on using the developed assays, the ability of the assays on infected woody shows promise in field trials.

- Imbalanced sample sizes for the two pathogens: 8 P. ramorum isolates, but only 1 G. clavigera isolate. In my opinion, the authors should have used more G. glavigera isolates.

Already answered above. See Rev. #2

Additionally, there are some concerns related to RNA sample quality control. The authors only used Nanodrop to quantify the RNA samples before DNAse treatment, which can distort the downstream RNA amounts added to the reverse transcription reactions. They used a kit that selects for RNA, but still, fluorescent RNA-binding dyes are the preferred method to get accurate RNA concentrations. Additionally, the authors do not report any method that was used to estimate RNA integrity (Nanodrop is not sufficient).

An additional sentence was included in manuscript regarding RNA integrity (L.180-181) and discussion on the use of RNA binding dyes as a consideration in future testing (L.416-420).

The authors would need to make complementary experiments and provide more data to improve the manuscript. Especially I would like to see data for the effect of the heat treatment on the wood samples and pathogen viability, and how the assay performs as a diagnostic tool. Alternatively, the authors should re-analyze the data and re-write it as a short communication and tone down the role of the heat treatment comparison. This would in my opinion require excluding/re-developing the G. clavigera assay, as it is not species specific, but it is not a broad-spectrum assay either.

As explained above this study is a proof of concept to demonstrate that we can target and differentiate by qPCR gDNA and mRNA (cDNA) in two wood-infecting pathogens. At the same time, we used these assays to look at the efficacy of heat-treatment in killing these pathogens. We agree that some additional experiments would be required to assess the efficacy of the wood treatment on infected wood sample. Unfortunately, we didn’t have the facility to conduct such an experiment on wood logs and/or lumber with a regulated pathogen like P. ramorum.

Please read Bustin et al. 2009 (10.1373/clinchem.2008.112797) for recommended terminology and experimental guidelines for quantitative PCR experiments Correct real-time PCR to quantitative real-time PCR (qPCR)

Done

Correct reverse transcription real-time PCR (RT-PCR) to reverse transcription quantitative PCR (RT-qPCR)

Done

Correct Threshold cycle/Cycle threshold (Ct) to Quantification cycle (Cq) throughout the manuscript

Done

Resolution of all figures is poor. Make sure you provide figure files with at least sufficient resolution (TIFF files with 300-600 DPI) and export graphs from R- studio/statistical software into EPS format to get 300 DPI resolution (see journal guidelines for preparing the images).

All our figures were 600dpi. The conversion of our files in pdf format for review purpose might have reduced the resolution of the figures?

L47: What do you mean by universal genes? Conserved genes?

We meant “conserved” genes that are found in all eukaryotic species. We change it for “conserved”. L. 52.

L78: How many conserved proteins, how many sequences?

228. See L.86.

L85-88: Rephrase to list P. ramorum as the primers were also tested on P. ramorum.

Done. L.97-98.

L94: Rephrase to list G. clavicera as the primers were also tested on G. clavicera.

Done. L.104.

L98-99: Provide citation to manuals/protocols/guidelines that were used to optimize the assays.

There was a confusion with the sentence “All assays were then optimized for use in identifying mRNA stability.” We apologize for this mistake.

Due to the constraint of having the qPCR probe located on an intron-exon junction, the assays were not optimized after their design and first testing. The only optimization done was for the primers, by following the recommendations made in Feau et al. 2018 PeerJ 6:e4392.

L113-131: The experimental setting is unclear. Please make an illustration showing the workflow, with heat treatments, what samples were used for what, differences in methods, replicates, controls, plating etc.

Let me know what you think and suggestion for improvement!

Figure caption: Work flow diagram of the short (left) and long kiln (right) heat treatments. Orange plates represents P. ramorum on V8 agar, while yellow plates as G. clavigera on malt extract agar (MEA).

Below is the draft workflow - this figure was added to the ms as a new Figure (Fig. 2; L. 129):

L103-104: Why did you decide to use 8 isolates of P. ramorum and only one G. clavicera isolate? This is a rather unbalanced experimental setting.

Our study is a proof-of-concept that the inoculated pathogen could be detected in wood samples. To do so, we only needed one individual of G. clavigera (and we picked the one that was used for genome sequencing). In addition, G. clavigera is highly clonal, and therefore our assay should also work on other genotypes.

L103-112: How many bolts per each tree species-pathogen-time point combination?

Two bolts were used for each tree species-pathogen-time point combination.

L102-112: Why did you not plate any of the wood tissue? This would provide a reference point to evaluate what the re-isolation success is in controls vs. after heat treatment, and if the pathogens remain viable after the treatments. The manuscript would be improved if the authors provided results for re-isolation from inoculated wood samples.

Same point than the fourth comment made by Rev. #5 above. Right, they were not plated for this experiment. However, the objective of the wood-inoculation exp. was to test if gDNA and mRNA of P. ramorum and G. clavigera could be 1 - purified from inoculated wood from different conifers and 2 - detected by qPCR with our assays. There was no need to re-isolate the pathogens from wood for validating that.

L102-112: Why you did not use any of the wood samples for the heat treatments? This would be the closest to a real life simulation to evaluate the efficacy of the heat treatments. Now the results have very little connection to using the assay in a real-life diagnostic context.

Yes, this is true. However, we did not have the facility & material to be able to reproduce the two heat treatment protocols with inoculated wood logs. As this is a proof-of-concept study we limited it to 1 - testing if we were able to amplify by PCR gDNA and mRNA (cDNA) extracted from inoculated wood and 2 - using our assay to determine if heat treatment protocols are accurate enough to kill mycelial cultures of G. clavigera and P. ramorum. Testing the same protocols on inoculated material will be the next step once we will be able to manage heat treatment protocols on infected wood products (logs or lumber).

L105: Provide ingredients or citation for carrot and MEA media L118: Provide ingredients or citation for V8 and MEA media

Done. L.116.

L114-123: At what temperature were the samples stored after the SPF kiln-drying treatment before sample collection?

Samples were left at room temperature and a sentence has been added to clarify this point in the manuscript. L.135.

L120-121: Was the frozen mycelium sample in 1.5 ml tubes the only control? Did you have untreated controls stored in 0.1 ml strip tubes that were stored in same conditions as the heat treated samples, and sampled at the same time points?

Flash freezing preserves mRNA better compared to a situation where a small non- heat-treated sample of mycelium is stored at room temperature. Now it is not clear how much RNA degradation occurs due to storage at room temperature, and how much is due to heat treatment.

Additionally, knowing the expression of the gene over several time points would help to evaluate the suitability of this gene as a diagnostic marker. Please provide data for cDNA/gDNA ratios for control mycelial samples that are stored and sampled similarly to the heat-treated samples.

As stated above, the frozen mycelium was the only untreated control sample for this experiment. For both species it resulted in detectable Ct-values for the cDNA meaning that the targeted gene was expressed during the experiment if the culture was not treated.

L127-129: For the short heat treatment, did you put agar plates directly to the oven? This might be a problem in terms of comparing the heat treatments. The mycelium on the plates might be heated differently than in the SPF kiln-drying treatment, where 0.1 ml Eppendorf tubes were used for heating and storing the samples after the treatment. Can you provide data that would indicate this wonʼt affect the degradation rate of mRNA?

I would repeat this experiment by placing the mycelium into 0.1 ml Eppendorf tubes, perform the short heat treatment, and sample similarly as in the SPF kiln-drying experiment. With the current methods, I am not convinced that the data from the two heat treatments is comparable. The ANOVA wonʼt be able to tell if the differences in cDNA/gDNA ratios are caused by using 0.1 ml tubes vs. plates, or due to different temperature treatment, as the use of tubes or plates is nested within the heat treatment.

Answered above. The aim of the study was not to compare the two treatments. However, when the dataset was separated by treatments and compared with the untreated control (thus eliminating the affect or tubes vs plates), there is still a significant influence of treatment. Though it may not be statistically comparable, we can see that both heat treatments showed promise in eliminating the target pathogens.

L159-163: Why did the authors decide to use two different methods for quantification of nucleic acids, Qubit for DNA and Nanodrop for RNA? Per MIQE guidelines, the preferred method for quantifying RNA uses fluorescent RNA- binding dyes, e.g. Qubit assay. As the RNA is not pure mRNA and the concentrations were measured before DNAse treatment, it is likely that Nanodrop readings overestimate the concentrations. This can affect the amount of RNA that is added to each RT reaction. Please address this issue and provide data that shows you get the similar Cq results from RNA samples measured with Qubit.

DNA and RNA were extracted in two different labs (UBC for DNA and Canadian forest service (Victoria, BC) for RNA). At the time the RNA was extracted the CFS lab only had a NanoDrop instrument. We still do not believe that this affected the outcome of our experiments.

L159-163: What method did you use to evaluate RNA integrity? The absorbance values from Nanodrop only indicate RNA sample purity, not integrity.

Extracted RNA samples’ integrity was assessed using an agarose gel for visualization of the band fluorescence. An additional sentence was included in the manuscript. L.180-181.

L170-176: How many biological and technical replicates?

Additional sentence included for clarity. L176-177.

L204-208: Based on previous data, does the gene have stable constitutive expression? For diagnostic assays like the authors are developing, the gene targets should have stable expression no matter the condition. Add eg. text and references from lines 337-339.

Additional sentence included for clarity. L.225-227.

L221-223: Figure 2 caption: Revise so that you provide the same information for both targets and indicate clearly which gene target is for which pathogen.

Corrected. Figure 2 is now Figure 3. Its caption reads as follows:

“ Log-transformed standard curve assessed with gDNA serial serial dilution (1:10) of (A) Phytophthora ramorum for the TaqMan probe PH178_EX (A) (R2= 0.992 Eff%= 99.33) and (B) Grosmannia clavigera for the TaqMan probe MS359_EX (B) for the TaqMan probe PH178_EX and MS359_EX. (R2= 0.981 Eff%= 100.303). ”

L234-238: Figure 3 caption: Indicate what the shaded areas are.

Figure caption rewrote. See answer to Rev. #1

L250-261: Report results from statistical testing, as they are not indicated in Figure 4 or elsewhere.

Tukey HSD test were done to compare means in each experiment. The results of these tests have been added to fig. 4 (Now, Fig. 5).

L255-257: Less efficient compared to what: control, the other treatment? Be more specific.

Corrected in manuscript.

L267-268: Table 1 indicates that replicate has a significant impact on G. clavicera cDNA/gDNA ratios. Discuss why this is observed for G. clavicera but not for P. ramorum? How was the quality of the RNA samples? How about lesion lengths, any variation in that?

Table 1 has been removed as suggested by Rev. #1.

L272-275: Summarize the results in 1-2 sentences in appropriate context. This is not re-isolation because what you describe is plating mycelium samples prepared from the same known pure culture. Rather you are assessing viability of mycelium after heat treatment.

Corrected and section heading redefined.

L272-275 & L345-376: If I understand correctly, you did not make any re- isolations from the bolts. Discuss/Explain why not? Discuss if plating only the heat-treated mycelium samples provides a reliable reference for the viability of the pathogen in biological samples.

Yes, the attempt to re-isolate from woody samples will provide more insight on the difficulties in “culturability” from environmental samples. That being said, the re-isolation from the bolts was not a part of the scope of the study, as the bolts were not heat treated.

How do the Cq values for mRNA in colonized bolts and mycelial samples compare?

Based on the Cq values, what is the extent of mycelial colonization in the wood samples?

It is difficult to compare the mRNA Cq values of the wood and mycelial samples as they were not of the same concentration and it is more difficult to extract woody tissue. Optimization of the woody extrataction, followed by dilution to the same concentration would be required before we can make the comparison of the Cq values. However, we can expect there to be a lower level of detection as it is usually difficult within environmental samples, especially that of wood with high levels of secondary metabolic polyphenols tannins, terpenoids and protein inhibitors. See the new paragraph about “limitations” of our study starting L. 398.

L312: At least in the US, by definition the pathogen is not regulated, but the interstate movement of certain articles from quarantined counties is regulated. See for details: https://www.federalregister.gov/documents/2007/02/27/07-892/ phytophthora-ramorum-quarantine-and-regulations

Thank you for this information. We clarified our statements accordingly on L.315-319.

L367-376: Be a bit critical and discuss if this could be resolved by plating infected wood samples after the heat treatments. There might be a time window after the treatment when the pathogen would still be able to grow, if suitable conditions (e.g. nutrients, temperature, moisture) were available. In what conditions are heat- treated/kiln-dried samples stored in industrial facilities? Is there a risk that the pathogen would still remain infectious? Does this create a risk for false-negatives?

The experiment Rev. 5 is talking about here was about testing two heat treatments on mycelial cultures of G. clavigera and P. ramorum, not on infected wood. Mycelium samples were plated right after each heat treatment (at T = 0 post treatment) and no growth was observed for each of the cultures (L.277-278). This suggests that there’s no time window after the treatment when the pathogen is still able to grow, even if his cells could still be partly functional (this point was specifically Discussed on L.382-388).

Even if these pathogens were able to grow after these treatments, industrial samples after heat/kiln-dried treatment are usually wrapped with plastic branding then kept outdoors. As such, cooler temperatures are probably not optimal for both pathogens (P. ramorum minimal growth temperature is 9°C with optima between 15 and 21°C; optimum growth temperature for G. clavigera is around 20°C). The mycelia samples in this experiment was kept at room temperature, which was more optimal for growth and no growth was obtained.

When should the diagnostic testing be done? Is this feasible?

Ideally, the diagnostic testing should be done immediately after treatment, as this will be the best to eliminate the potential of false positives. From our experiment, we saw that the kiln treated samples showed no amplification of RNA, suggesting that this temperature coupled with the long exposure rapidly degrades the RNA. Therefore, if we detect amplification of RNA in these samples, it could be the presence of viable pathogens.

Discussion and criticism lacks entirely for the non-specific G. clavigera assay. Discuss what diagnostic implications this might have, if you are not willing to expand the work and develop an assay that is specific for this pathogen. Are all Grosmannia/Leptographium species equally harmful? Could this assay be a more generic assay that detects several of these species?

Grosmannia clavigera and Leptographium longiclavatum are both equally “harmful” as they are fungal symbionts to Mountain pine beetle and do not cause structural damage to the infected woody tissue but instead just a discolouration. We added a paragraph about G. clavigera in the Discussion section L.330-342.

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3 Dec 2019

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

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Dr. Richard Hamelin

Professor of Forest Pathology

University of British Columbia

Department of Forest Sciences

Vancouver, BC, Canada

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Reviewers' comments:

15 Jan 2020

PONE-D-19-22766R1

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

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