Genomic biosurveillance detects a sexual hybrid in the sudden oak death pathogen

Abstract

Invasive exotic pathogens pose a threat to trees and forest ecosystems worldwide, hampering the provision of essential ecosystem services such as carbon sequestration and water purification. Hybridization is a major evolutionary force that can drive the emergence of pathogens. Phytophthora ramorum, an emergent pathogen that causes the sudden oak and larch death, spreads as reproductively isolated divergent clonal lineages. We use a genomic biosurveillance approach by sequencing genomes of P. ramorum from survey and inspection samples and report the discovery of variants of P. ramorum that are the result of hybridization via sexual recombination between North American and European lineages. We show that these hybrids are viable, can infect a host and produce spores for long-term survival and propagation. Genome sequencing revealed genotypic combinations at 54,515 single nucleotide polymorphism loci not present in parental lineages. More than 6,000 of those genotypes are predicted to have a functional impact in genes associated with host infection, including effectors, carbohydrate-active enzymes and proteases. We also observed post-meiotic mitotic recombination that could generate additional genotypic and phenotypic variation and contribute to homoploid hybrid speciation. Our study highlights the importance of plant pathogen biosurveillance to detect variants, including hybrids, and inform management and control.

Fig. 1. Hybridization between divergent clonal lineages EU1 and NA2 of Phytophthora ramorum.

We analyzed 95 whole genomes of P. ramorum and extracted 450,656 single nucleotide polymorphisms (SNP) to characterize the populations; a Population structure analysis of P. ramorum using a principal component analysis; each dot represents the genome of a P. ramorum isolate; b Ancestry estimation using Admixture analysis of P. ramorum lineages and putative hybrids at K = 4; each bar represents the genome of a P. ramorum isolate; c Neighbor-net phylogenetic network reconstructed from a matrix of pairwise Nei’s genetic distances between isolates of P. ramorum. Samples 16-237-021 and 16-284-032 are the two putative hybrids.

Fig. 2. Haplotype phylogeny shows independent assortment of alleles at nuclear loci and uniparental inheritance of mitochondrial haplotype in the Phytophthora ramorum hybrid.

Neighbor-joining tree of haplotypes of P. ramorum indicates meiotic recombination with nuclear phased haplotypes clustering with haplotypes of one of the two parents and mitochondrial haplotypes clustering with lineage NA2. Phased haplotypes were obtained by using short genome regions with physical phasing when two or more variants co-occur on the same sequencing read. The following genomes were used: 14-1270, 15-1093 (EU1), 04-0372 (NA1), 5-0954 (NA2) and P2111 (EU2) for a Conserved hypothetical protein, b peroxisomal membrane protein and c cell 12 A endoglucanase. It is not unexpected to have more than two alleles at the conserved hypothetical protein for the EU1 lineage since two isolates were used. For the mitochondrial genomes d, the following samples were used: 03-0110, 05-7036, 08-3469, BBA-26-02, CC1048, P2599 (EU1); 04-0372, MK548, Pr1556 (NA1), 04-25165, 05-18753, 13-0781 (NA2); P2111, P2460, P2461 (EU2). The mitochondrial genome comprised 103 SNPs. For the two hybrid samples (16-237-021 and 16-284-032), the two haplotypes are indicated with h1 and h2.

Fig. 3. Phytophthora ramorum hybrid can sporulate, infect a host and has intermediate phenotypes.

a Morphology of P. ramorum hybrid isolate 16-284-032 growing on carrot agar, showing fluffy growth where sporangia are produced; b Sporangia produced by the P. ramorum hybrid in culture; c Rhododendron leaves infected by hybrid P. ramorum isolate 16-284-032; d Growth of P. ramorum lineages and hybrids on carrot agar medium; ***p < 0.0001, one-way ANOVA with multiple comparisons using Tukey test; e Growth of P. ramorum lineages and hybrids measured as necrotic area (π x lesion length x width) over time on rhododendron leaves; there was no significant difference in lesion growth among the lineages and hybrids. The lower and upper boundaries of each box in the boxplots (d, e) indicate limits of the interquantile range (IQR) between the 25th (Q1) and 75th percentile (Q3). Bars (whiskers) below and above the box indicate the minimum (Q1 – 1.5*IQR) and maximum (Q3 + 1.5*IQR) values of the distribution. The horizontal line in each box represents the median and outliers are represented with circles.