Research Progress HIGHLIGHTS

Activity 2 – Disease resistance

Activity 2.1 Dothistroma and lodgepole pine

Activity 2.1.1 Host gene expression in lodgepole pine

To study gene expression in response to infection we compared the responses in the progeny of controlled crosses of parents that are highly resistant (resistant x resistant) to those that are highly susceptible (susceptible x susceptible) to the fungus causing Dothistroma needle blight. Two different fungal isolates (one from BC and one from AB) with different genomic profiles were used. The inoculation experiment conducted in summer 2017 followed the procedures developed in the pilot study (described below). The inoculation results matched the expected family phenotype with resistant crosses showing less signs of infection. Total RNA was extracted from the needles at three time points and sent for sequencing. RNAseq will be used to characterize differential gene expression, identify candidate genes for resistance, and to search for SNPs to be used in the gene wide association study (Activity 2.1.2).

For more information, contact Nicolas Feau and Richard Hamelin.

Inoculation of two lodgepole pine families with Dothistroma septosporum (completed fall 2017)

We inoculated the descendants (two-year old seedlings) of two lodgepole pine families with two D. septosporum isolates. The parents of these families had been evaluated in a field test for their resistance to the DNB. One family was the result of a cross of two “DNB-resistant” parents (3055×3061), the other one with from two “DNB-susceptible” parents (3078×3108). Twelve weeks after inoculation, DNB severity was evaluated by assessing the percentage of needle showing symptoms. We found a significant effect of the tree family on DNB severity. We found a perfect match between field observations and our controlled inoculations: Descendants from the “resistant” cross (3055×3061) showed less symptoms to DNB than those from the susceptible cross.

Seedlings from this experiment were sampled for a transcriptomic study of the DNB-lodgepole pine interaction.

 

First growing season – germination (December 2016) and budset (February 2017) in lodgepole pine controlled crosses.

Second growing season – Infection levels in two lodgepole pine families at 12 weeks (September 27) after a controlled inoculation with two DNB isolates. Green: DNB-resistant family from cross of P. contorta genotypes 3055 x 3061; Blue: DNB-susceptible family from cross 3078 x 3108.

 


Inoculation pilot study (completed spring 2017)

The pilot study designed to develop an effective Dothistroma septosporum (an ascomycete fungus) inoculation procedure for lodgepole pine was successfully completed and used to determine the timing and environmental conditions for the test inoculations. The conidia (the asexual spores of the fungus) were cultivated in the lab on a sporulating medium and sprayed on the needles at a concentration of 3 million conidia per ml, achieving a germination success of 95% after 24h incubation. The seedlings experienced 20°C days (16 hours long) and 12°C nights with constant moisture on their needles. The first signs of disease appeared 5 weeks after inoculation and the symptoms (DNB red bands, necrosis and fruiting bodies) after 8 weeks. The timing of sampling was also effective, with sampling points at four days, 8 weeks, and 12 weeks post-inoculation.

For more information, contact Nicolas Feau.

Dothistroma fungus in vitro – fungus on sporulating medium (left) and the conidia (right). 

Dothistroma innoculation (left and inset) and incubation under moist conditions (right).

Signs of Dothistroma infection appeared at 5 weeks (left) and symptoms at 8 weeks (right) after inoculation.


Host gene expression in lodgepole pine (update Dec 2019)

We found a dramatic increase in gene expression in a Dothistroma septosporum-tolerant genotype of lodgepole pine, with 519 genes turned on, but only in response to one of the two Dothistroma septosporum strains that we tested. The other strain elicited very little response in gene expression, suggesting the trees may be adapted differently to different pathogen strains. The turned-on genes have functions related to plant pathogen defense. Our results provide resources to understand the genetic mechanism underlying pathogen response of lodgepole pine, which can be used to develop future management guideline.

Numbers of differentially expressed genes under different treatment. T – tolerant genotype of lodgepole pine seedlings; S ¾ susceptible genotype of lodgepole pine seedlings; D1 – D. septosporum isolate 1; D2 – D. septosporum isolate 2; 4d – 4 days post inoculation; 8w – 8 weeks post inoculation; 12w – 12 weeks post inoculation.


Activity 2.1.2 Genome-wide association study of Dothistroma resistance

The genome-wide association study designed to identify candidate genes and population variation for disease resistance or tolerance to Dothistroma is well underway. The populations selected for this study include pure lodgepole, pure jack pine and several hybrids (lodgepole x jack pine) from BC and Alberta. The 100, two-year old seedlings from each of 40 wild-collected pine populations (4000 in total) were inoculated in 2018. We used the same general inoculation procedure (Activity 2.1.1 – Pilot Study) except the inoculation was performed 3 times, allowing more time for the fungal mycelia to penetrate the stomata. After inoculation with Dothistroma, phenotypic resistance was assessed in all individuals at 15 weeks instead of 12 weeks. The 10 least and 10 most resistant seedlings in each population will be sequenced. The data will be analyzed using the SNPs generated in Activity 2.1.1 (Host Gene Expression) and used to generate a list of genes with a strong signature of association to resistance phenotypes.

Location of the pine populations sampled in British Columbia and Alberta.

 

Seed preparation and sowing (February 2017) and germination (March 2017).

 

Seedlings near the end of their first season (May 2017) and blackout chamber used to initiate budset (June 2017).


Inoculation of two-year old seedlings (completed 2018)

We used the same two DNB isolates used in the transcriptomics experiment (Activity 2.1.1) to inoculate the 40 wild collect populations: 28 populations of P. contorta, five of P. banksiana and seven of P. contorta x P. banksiana.

DNB infection severity was assessed at 15 weeks post inoculation and seedlings were rated on a scale of 1 to 5 depending on disease severity.

We found some significant differences in the response of the tree species to DNB. Overall, Jack pines (P. banksiana) were more susceptible than lodegpole pines (P. contorta) and the interspecific hybrids P. contorta x P. banksiana.

Infection severity in P. contorta, P. banksiana and P. contorta x P. banksiana populations at 15 weeks after a controlled inoculation with two DNB isolates. Boxplot means sharing a letter are not significantly different (p > 0.05) according to a Tukey’s multiple comparison test.

 

We also found some strong differences in DNB severity between the population and their regions of origin. We identified a latitudinal gradient in severity as the susceptibility of P. contorta populations was negatively correlated with the latitude (r2 = 0.51, p < 0.001); the same trend was observed for P. banksiana and the hybrid P. contorta x P. banksiana, but the relationship was not significant likely due to the limited number of populations assessed for this species (n = 5 and 7, respectively).

DNB severity in P. contorta is negatively correlated with latitude. A, Geographical location and DNB severity of the 40 Pine populations tested. B, correlation between DNB severity and latitude in P. contorta; correlation coefficient and p-value is also indicated for P. banksiana and P. contorta x P. banksiana.

 

In each pine population, we retained the ten most resistant and the ten least resistant seedlings for DNA extraction and sequencing. These data are used to generate a list of gene with strong signature of association to resistance phenotype.

 


Activity 2.2 Swiss needle cast and Douglas-fir

Activity 2.2.1 Host gene expression in Douglas-fir

We are assessing differences in gene expression between resistant and susceptible genotypes. Individuals representing the largest symptom severity differences (identified in Activity 2.2.2 see below) were selected for controlled inoculations with Swiss Needle Cast (Nothophaeocryptopus gaeumannii).

After testing different inoculation experiments, pairs of susceptible and tolerant families were prevented from infection through bagging. In comparison with naturally infected controls, differences in gene expression can be revealed. Samples have been harvested and are being prepared for sequencing.

The expression data will be analyzed to identify the Douglas-fir genes responsive to infection and the genes that differ in resistant and susceptible individuals. Fungal gene expression responses to host genotype and infection status will be determined, feeding into Activity 3 aimed at deciphering pathogenicity gene candidates.

For more information contact Juergen Ehlting.


Activity 2.2.2 Disease expression and phenotyping

Natural infection of Swiss Needle Cast (SNC) has been accessed on two sister sites (Jordan River and Lake Cowichan) in BC which are planted with Class A seed material currently used in reforestation. The sites differ in terms of climate and historic rates of infection but are geographically close to each other. The Jordan River site has a milder, wetter climate and is representative of the future high-risk climates predicted for SNC expansion.

At each site, 32 families were selected to quantify disease expression at the whole tree, branch and needle level. A total of 960 trees were sampled for branch and needle assessment. Branches were removed at mid-crown and scored for needle retention. Slide mounted needles have been visually assessed to determine levels of infection (the % of stomata occluded by the Nothophaeocryptopus gaeumannii fruiting body formation). Needle samples were also collected for molecular phenotyping and fungal load assessment. These samples have been processed as well.

Using phenotyping from both sites and known relationship among families we observe genetic variation across families and identified families that with high degrees of tolerance.

For more information contact David Montwé.

 

Visual quantification of stomatal occlusion on Douglas-fir needles. Swiss needle cast spores enter the stoma, colonize internally, and in the following spring produce fruiting bodies (pseudothecia). These fruiting bodies occlude (physically block) the stoma and limit gas exchange, causing chlorosis (yellowing) and premature abscission (needle drop).

 

 

Family means for needle retention of three year old needles. The wetter, milder site is indicated in blue, and the drier site is indicated in red. The wetter site was found to have lower needle retention than the drier site, indicating substantial differences in disease expression.

 

 

Molecular phenotyping and fungal load assessment. Real-time qPCR is being used to quantify the amount of fungal DNA relative to plant DNA. In this procedure plant and fungal DNA is extracted, amplified and run on a gel to separate and quantify the amount of DNA present.