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.

tubes and placed into a thermocycler. In parallel, one 1.5ml Eppendorf tube containing 30 mg of mycelia 121 was immediately frozen to serve as a no-heat treatment control. The thermocycler was used to conduct 122 a long heat treatment that simulated the kiln-drying schedule. The mycelium was sampled at 8 time 123 points: 0, 6,12,24,48,96,168 and 240 hours after the treatment. At each time point two mycelial 124 samples were collected: one was transferred into a 1.5 ml microtube, submerged in liquid nitrogen and 125 stored at -80 o C for subsequent DNA and RNA extractions; the other one was plated on three clarified V8 126 (P. ramorum) or MEA (G. clavigera) agar petri dishes and incubated in a dark growth chamber at room 127 temperature for 28 days. For the short heat treatment, a laboratory oven was preheated to 70 o C to 128 incubate 24 replicate plates of the P. ramorum and G. clavigera isolates for 1 hour. After this treatment, 129 all plates were removed from the oven and set aside at room temperature before collection at the time-130 points mentioned above. Mycelial samples were collected and stored as previously described for the SPF 131 kiln-drying schedule.

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Chlamydospores Separation 133 Chlamydospore harvesting from P. ramorum was carried out according to Tsao [36] with the 134 following modifications. Non-blended fungal mats were transferred onto a 75µm falcon filter in a 50ml 135 falcon tube. Using sterile dH 2 O, these mats were rinsed while being patted with a rubber policeman until 136 the volume of water in the falcon tubes reached approximately 10-20 ml. This was repeated twice. The 137 tubes were then centrifuged at 10,000 x g for 2 min, and the supernatant was removed. The 138 chlamydospore suspensions were then aliquoted into several 1.7 ml micro-tubes and centrifuged at 139 8000 x g for 5 min removing the supernatant. The pellets were then resuspended, combined and 140 centrifuged at 6,000 x g for 5 min and the supernatant discarded.   We verified the presence of an intron in the gDNA samples by comparing the size of the PCR 210 products obtained by amplification of the gDNA and cDNA reverse-transcribed from mRNA of the same 211 samples. A smaller amplification product was obtained in the PCR of the cDNA (84 bp) than the gDNA 212 (157 bp) of gene PH178, confirming the presence of the intron in the gDNA of P. ramorum (Fig. 1A).

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TaqMan probes were added to the two selected primer pairs to design real-time PCR assays that 216 were tested for amplification efficiency on serial dilutions of gDNA. For the assay PH178 targeting P.
217 ramorum, the standard curve yielded a regression coefficient of 0.998 indicating low variability between 218 independent DNA isolations and an amplification efficiency of 92.3% ( Fig. 2A). Efficiency of the MS359 219 assay targeting G. clavigera was higher (100.3%), and variability slightly lower with a regression 220 coefficient of 0.981 (Fig. 2B). For each combination of isolate x tree species tested a necrotic lesion was observed around the 227 point where the microorganism was inoculated, confirming the growth of these pathogens in wood. No 228 similar necrotic lesion was observed in the controls (Fig. 3). Genomic-DNA and cDNA obtained from 229 these inoculated wood samples was successfully detected by their respective real-time PCR assay with C t 230 values ranging from 24.13 (G. clavigera inoculated on P. contorta) to 29.4 (P. ramorum inoculated on L.
231 kampferi) for gDNAs (mean = 26.6 ±2.21). Cycle-threshold 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 233 of 31.2 (±2.77), indicating later detections than for gDNA (Fig. 3). Use of molecular assays to assess viability of pathogens following heat treatment 244 Ratio of mRNA over gDNA quantity as measured by C t values for cDNA and gDNA were 245 compared following two heat treatments (SPF Kiln-drying and short heat treatment) applied to 246 wood-logs infected with P. ramorum and G. clavigera. For both pathogens, we found a highly significant 247 effect of the heat treatment (F= 72.4, P < 0.0001 and F= 25.2, P < 0.0001 for P. ramorum and G. 248 clavigera, respectively) and an interaction between treatment and time point at which the gDNA and 249 mRNA samples were collected (F= 4.2, P < 0.001 for P. ramorum and F= 2.7, P < 0.01 for G. clavigera) 250 (Table 1). This likely resulted from the difference in cDNA detection as for both species gDNA was 251 amplified after each treatment with C t values similar or higher than those obtained with the no-252 treatment control (e.g. C t distribution ranging from 24.0 to 34.0 for the two heat treatments versus 24.0 253 for the controls; Fig. 4A, B, E and F). For both pathogens, no cDNA was detected at the end of SPF kiln-254 drying schedule treatment (C t value equal or above 40.0; Fig. 4D and H), suggesting that this treatment 13 255 killed efficiently the two pathogens. In contrast, the short heat treatment of 70 o C for 1 hour seemed to 256 be less efficient, with clear evidence that the mRNA degraded at different rates for the two organisms 257 following treatment. For P. ramorum, cDNA of the targeted gene (PH178) was detected after up to 24 258 hours post-treatment (mean C t = 35.8 ±4.82 for 0 to 24 hours post-treatment; Fig. 4C). Similarly, G.
259 clavigera cDNA of gene MS359 was still detected until 96 hours after the short heat treatment (mean C t 260 = 34.2 ±3.75), suggesting incomplete degradation of mRNA and/or non-lethality of this treatment during 261 this time (Fig. 4G).
262 Figure 4. Efficacy of the short heat and SPF kiln-drying treatment of Phytophthora ramorum (green) and of the assay to detect living P. ramorum was increased by amplifying exclusively cDNA, ruling out 324 potential false positives generated by gDNA contamination of the RNA sample.

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Our aim was to use our gDNA and mRNA-targeted real-time PCR detection assays to assess the 326 viability of two wood-infecting microorganisms following lethal treatments to test their efficacy. The 327 underlying hypothesis tested was that viability is related to the expression of specific genes. Therefore, 328 monitoring specific P. ramorum and G. clavigera gene transcripts by real-time PCR was expected to 329 provide a simple proxy for viability of these two organisms. The choice of transcript was an important 330 consideration, not only for sensitivity but also for its expression under a variety of conditions. Genes 331 selected to assess viability should be constitutively expressed regardless of the environmental 332 conditions and the micro-organism life-stage as opposed to those induced following a specific 333 environment signal [60]. This ensures that the lack of expression of a constitutive gene is due to the 334 death of the organism instead of the gene being turned "off" by a specific environmental factor. analyses [30,37,38,40]. In addition, we validated their expression in pure cultures and for mycelium