BackgroundCannabis powdery mildew, caused by the fungal pathogen Golovinomyces ambrosiae, poses a significant threat to licensed producers as its presence on marketable products can compromise product innocuity. While there is a focus in research for resistance genes within the Cannabis sativa germplasm, there is a lack of genetic information regarding the infecting agent, preventing validation of their effectiveness against different populations as the plant-pathogen interaction most likely follows a gene-for-gene relationship. In this paper, we assembled the first G. ambrosiae genome, providing insights into its genomic content and potential virulence determinants. The assembly was made using a hybrid approach, combining Oxford Nanopore Technologies long reads and Illumina short reads.ResultsThe resulting 155.2 Mb genome is composed of 73 contigs, 13 scaffolds and has a completeness score of 97.5%. Subsequent analysis highlighted the substantial transposable elements content of the pathogen, occupying 82.64% of its genomic composition. Prediction of protein-coding genes revealed 6995 highly confident gene models, including 169 candidate effector proteins. Among the latter, we highlighted 14 candidates sharing key characteristics of confirmed effectors in other powdery mildew pathosystems such as the presence of signal peptides, RALPH-like domains, and Y/F/WxC motifs.ConclusionsThe result of this study provides valuable resources for future identification of avirulence genes within the G. ambrosiae species, responsible of conferring resistance when encoded effectors are recognized by a cognate host's resistance genes. Those findings will lead to guided breeding strategies and, ultimately, the selection of cultivars adapted to the pathogen's virulence profile.

Exploitation of disease resistance genes in soybean (Glycine max (L.) Merr.), as an effective method for management of Phytophthora sojae (Kauf. & Gerd.), is on the verge of an impasse. Few of the known resistance genes are commercially exploited, and even fewer have been precisely identified. Therefore, little is known about the identities or relationships between those genes, a hindrance preventing optimal introgression of new sources of resistance into elite soybean lines. In this study, we have applied state-of-the-art nucleotide-binding and leucine-rich repeat gene capture (RenSeq) using a set of approximately 80,000 unique baits on near-isogenic lines, whole-genome resequencing, and bulked segregant analysis to uncover a resistance gene that has remained elusive for 40 years. This work highlights the reassessment of the Rps3b locus from Chr13 to Chr7 and the description of two alleles, from Turkish and Chinese landraces, of a sole candidate gene. We have identified Rps3b in four, fully resequenced, genetic backgrounds, including the original PI from 1985, in which the resistance gene was originally described. Specificity of the resistant alleles was achieved through phenotypic characterization with field isolates carrying virulent and avirulent forms of the corresponding effector, Avr3b. Surprisingly, these alleles showed extremely high synteny and sequence identity with Rps11 consistent with allelism, and conferred a resistance phenotype indistinguishable from that of the recently cloned Rps11. These results offer new sources of resistance for breeders that are effective against the current P. sojae pathotypes in the field.

Cranberry fruit rot (CFR) has been a major challenge in cranberry production affecting fruit quality, particularly in Massachusetts and New Jersey. It is known to be a disease complex associated with several diverse fungi. This study provides the first comprehensive assessment of CFR fungal population dynamics across wild, organic, and conventional cranberry production systems in southeastern Massachusetts. By employing multiplex PCR, a high-throughput molecular method for the simultaneous detection of multiple fungi, we investigated the prevalence and diversity of 11 most commonly associated fruit rot fungi in 2021 and 2022, in 32 (23 conventional, 4 organic and 5 wild) and 50 (40 conventional, 4 organic and 6 wild) cranberry bogs respectively. Significant differences in the detection of CFR fungi were observed across these ecosystems. Conventional fields showed varied fruit rot incidence, ranging from 2-42% in 2021 and 1-48% in 2022. Species richness analysis indicated that on average, wild bogs were more species-rich and diverse, with 5.5 CFR fungi detected per sample compared to 4 CFR fungi in both conventional and organic bogs. Organic bogs exhibited a significant decline in species richness from 6 fungi in 2021 to 2 fungi in 2022. Except for Phomopsis vaccinii, which was not observed in organic bogs in either year, all 11 CFR fungi were detected across the ecosystems: some in both growing seasons and some in only one. Key CFR fungi such as Coleophoma empetri, Allantophomopsis cytisporea and Botryosphaeria vaccinii were consistently found across all ecosystems and both years of study. Analysis of year-to-year variation indicates that wild bogs exhibited higher fungal carryover, with 8 out of the 11 CFR fungi persisting from one year to the next, compared to 6 fungi in conventional bogs and 4 fungi in organic bogs. Notably, C. empetri, B. vaccinii, and A. cytisporea demonstrated year-to-year persistence across all three bog types. Among these, C. empetri exhibited the greatest persistence across all three ecosystems, with 96%, 100% and 60% of the conventional, organic and wild bogs maintaining its presence across both years. Colletotrichum spp. persisted in 43% of the conventional bogs but detected only in one of the two years studied in organic and wild bogs resulting in 0% persistence from one year to the next. The study also noted a general decrease in Botryosphaeria vaccinii and an increase in Physalospora vaccinii across all bog types. This research highlights the varying persistence and complex dynamics of CFR fungi across different cranberry ecosystems, as well as the potential impact of management practices on fungal populations.

IntroductionCranberry (Vaccinium macrocarpon) is an important crop grown in the United States and Canada, with the province of Québec being the world’s largest producer of organic cranberry. However, cranberry fruit rot (CFR), caused by 12 fungal species, has become a major issue affecting yield.MethodsA molecular detection tool was used to detect the presence of the 12 CFR fungi and evaluate CFR species across three farms with different fungicide strategies in Québec. The incidence and frequency of CFR fungi were evaluated for 2020 and compared with 2018 data from the same farms in Québec. Machine-learning models were used to determine the possibility of explaining CFR disease and species based on climate, and analyze the effects of weather variables on CFR presence andprimary fungal species.ResultsThe most predominant CFR species remained the same in both years, with Godronia cassandrae and Coleophoma empetri being the two most common, but some species showed changes in relative abundance. Furthermore, this study examined the diversity variations in 2018 and 2020, with data showing an overall increase in diversity over the period. The results showed that fungicide applications impacted the species composition of CFR among the farms. Five weather variables (daily snow on the ground (cm), total daily precipitation (mm), daily atmospheric pressure (kPa), daily relative humidity (%) and daily temperature (°C)) were selected and found to contribute differently to the model with atmospheric pressure being the most important. Surprisingly, temperature and precipitations did not influence much the incidence of fungal pathogen species and each CFR species behaved differently in response to environmental factors.DiscussionOverall, this study highlights the complexity of predicting CFR disease, as caused by 12 fungi, and of developing effective disease management strategies for CFR.

Cranberry fruit rot (CFR) pathogens are widely reported in the literature, but performing large-scale analysis of their presence inside fruit has always been challenging. In this study, a new molecular diagnostic tool, capable of identifying simultaneously 12 potential fungal species causing CFR, was used to better define the impact of CFR across cranberry fields in Québec. For this purpose, 126 fields and 7,825 fruits were sampled in three cranberry farms distributed throughout the province and subjected to comparative analyses of fungal presence and abundance according to cultural practices, sampling times, and cranberry cultivars. All 12 pathogens were detected throughout the study, but as a first major finding, the analyses revealed that four species, Godronia cassandrae, Colletotrichum fructivorum, Allantophomopsis cytisporea, and Coleophoma empetri, were consistently predominant regardless of the parameters studied. Comparison of conventional and organic productions showed a significant reduction in fungal richness and relative abundance. Interestingly, Monilinia oxycocci was found almost exclusively in organic productions, indicating that fungicides had a strong and persistent effect on its population. Surprisingly, there were no significant differences in fungal relative abundance or species richness between fruit sampled at harvest or in storage, suggesting that there may not exist a clear distinction between field and storage rot, as was previously thought. Comparative analysis of fungal species found on eight different cranberry cultivars indicated that they were all infected by the same fungi but could not rule out differences in genetic resistance. This large-scale analysis allows us to draw an exhaustive picture of CFR in Québec and provides new information with respect to its management.

The use of resistance genes in elite soybean cultivars is one of the most widely used methods to manage Phytophthora sojae. This method relies on effector-triggered immunity, where a Resistant to P. sojae (Rps) gene product from the plant recognizes a specific effector from the pathogen, encoded by an avirulence (Avr) gene. Many Avr genes from P. sojae have been identified in the last decade, allowing a better exploitation of this type of resistance. The objective of the present study was to identify the Avr gene triggering immunity derived from the soybean resistance gene Rps8. The analysis of a segregating F-2 progeny coupled with a genotyping-by-sequencing approach led to the identification of a putative Avr8 locus. The investigation of this locus using whole-genome sequencing data from 31 isolates of P. sojae identified Avr3a as the likely candidate for Avr8. Long-read sequencing also revealed that P. sojae isolates can carry up to five copies of the Avr3a gene, compared to the four previously reported. Haplotype and transcriptional analyses showed that amino acid changes and absence of Avr3a transcripts from P. sojae isolates caused changes in virulence towards Rps8. Functional analyses using CRISPR/Cas9 knockout and constitutive expression demonstrated that Rps8 interacted with Avr3a. We also showed that a specific allele of Avr3a is recognized by Rps3a but not Rps8. While Rps3a and Rps8 have been previously described as closely linked, this is the first report of a clear distinction hitherto undefined between these two resistance genes.

Abstract In the last decade, more than 70 quantitative trait loci (QTL) related to soybean [Glycine max (L.) Merr.] partial resistance (PR) against Phytophthora sojae have been identified by genome‐wide association studies (GWAS). However, most of them have either a minor effect on the resistance level or are specific to a single phenotypic variable or one isolate, thereby limiting their use in breeding programs. In this study, we have used an analytical approach combining (a) the phenotypic characterization of a diverse panel of 357 soybean accessions for resistance to P. sojae captured through a single variable, corrected dry weight; (b) a new hydroponic assay allowing the inoculation of a combination of P. sojae isolates covering the spectrum of commercially relevant Rps genes; and (c) exhaustive genotyping through whole‐genome resequencing (WGS). This led to the identification of a novel P. sojae resistance QTL with a relatively major effect compared with the previously reported QTL. The QTL interval, spanning ∼500 kb on chromosome (Chr) 15, does not colocalize with previously reported QTL for P. sojae resistance. Plants carrying the favorable allele at this QTL were 60% more resistant. Eight genes were found to reside in the linkage disequilibrium (LD) block containing the peak single‐nucleotide polymorphism (SNP) including Glyma.15G217100, which encodes a major latex protein (MLP)‐like protein, with a functional annotation related to pathogen resistance. Expression analysis of Glyma.15G217100 indicated that it was nearly eight times more highly expressed in a group of plant introductions (PIs) carrying the resistant (R) allele compared with those carrying the susceptible (S) allele within a short period after inoculation. These results offer new and valuable options to develop improved soybean cultivars with broad resistance to P. sojae through marker‐assisted selection.

Group I catalytic introns are widespread in bacterial, archaeal, viral, organellar, and some eukaryotic genomes, where they are reported to provide regulatory functions. The group I introns are currently divided into five types (A-E), which are themselves distributed into several subtypes, with the exception of group I type D intron (GI-D). GI-D introns belong to the rarest group with only 17 described to date, including only one with a putative role reported in fungi, where it would interfere with an adaptive response in the cytochrome b (COB) gene to quinone outside inhibitor (QoI) fungicide resistance. Using homology search methods taking into account both conserved sequences and RNA secondary structures, we analysed the mitochondrial genomes or COB genes of 169 fungal species, including some frequently under QoI selection pressure. These analyses have led to the identification of 216 novel GI-D introns, and the definition of three distinct subtypes, one of which being linked with a functional activity. We have further uncovered a homing site for this GI-D intron type, which helps refine the accepted model of quinone outside inhibitor resistance, whereby mobility of the intron across fungal mitochondrial genomes, would influence a fungus ability to develop resistance to QoIs.

The complex etiology of cranberry fruit rot (CFR) (Oudemans et al., 1998) has made it difficult to precisely identify the fungi involved in CFR and their relative importance in North America. To remedy this situation, a multiplex PCR approach targeting the 12 most commonly reported fungi in CFR was recently developed (Conti et al., 2019). However, in surveys conducted in Eastern Canada, the molecular tool revealed the presence of an unknown fungus in more than 30% of the collected samples. Analyses were thus undertaken to identify this species. From 117 rotten fruit collected at harvest in 2017, 34 samples of the unknown fungus, all morphologically similar, were isolated but not detected using the molecular tool. Their ITS ribosomal regions were sequenced using universal primers (Vilgalys and Hester, 1990; White et al., 1990) and searched against the GenBank database using the Blastn tool (Altschul et al., 1990). The top match was obtained with Godronia cassandrae (accession number: MH855281 (Vu et al., 2019), 98-100% of identity and an E-value of 0.0), even though some isolates had minor nucleotide differences, as presented in the tree. Sequences were deposited in GenBank as accession numbers MT599989 to MT600022. Since G. cassandrae had been reported, albeit rarely, on cranberry in Michigan (Olatinwo et al., 2003), it was supposed to amplify with the molecular tool developed from the strain DAOM C216021 (AAFC, Ottawa, ON) identified in 1993 on Vaccinium angustifolium as G. cassandrae. Analysis of the sequences used to build the specific primers from this strain confirmed the DAOM strain as being Neocucurbitaria juglandicola, which was never diagnosed in our cranberry samples. To confirm this revised diagnosis, a multi-sequence alignment (MSA) was performed on the ITS regions of the isolates from rotten cranberries and sequences available for the genus Godronia in the NCBI nucleotide database (NCBI txid269064). This MSA allowed us to find discriminant regions between Godronia spp. A pair of PCR primers specific to G. cassandrae found on cranberry fruit was then designed (the forward and reverse sequences are AAT CAG TGG CGG TGC CTG TC and TAC CGC TTC ACT CGC CGT TAC, respectively), generating 196 bp amplicons, with an annealing temperature of 65°C. The diagnosis of 7,835 fruit sampled at three time points (harvest, after three and after six weeks of storage) in 2018, from four cranberry farms located in Québec (CA) and Nova Scotia (CA), detected G. cassandrae in 2350 samples (30%). To assess the pathogenicity of four specimens from 2017, Koch's postulates were completed on two healthy fruit per isolate. The fruit were wounded with a sterilized pick and individually inoculated; two fruit were used as control. Based on our observations, the fungi isolated from cranberry fruit displayed a pale lemon yellow mycelium and black pycnidia. Conidia are hyaline, cylindrical and divided by a single septum. These morphological characters are similar to the ones described in the literature for G. cassandrae (Polashock et al., 2017). Rot symptoms appear as a discoloration from the firm, red and healthy cranberry fruit to a yellowish-orange softer fruit. Molecular characterization of the re-isolated fungus confirmed the presence of G. cassandrae. We report Godronia cassandrae for the first time as a major cause of CFR in Eastern Canada. Its prevalence in cranberry fields of Québec and Nova Scotia suggests that it supplants Physalospora as the main fungus involved in CFR in Eastern Canada.

Cranberry fruit rot (CFR) is arguably one of the most limiting factors of cranberry (Vaccinium macrocarpon) production throughout its growing areas. The disease is caused by a group of closely related fungi that require identification using long and cumbersome steps of isolation and microscopic observations of structural features. The objective of this study was to develop a molecular assay to simultaneously detect and discriminate 12 of the most important fungal species reported to be pathogenic on cranberry fruit to facilitate the diagnosis of CFR. As the first approach, internal transcribed spacers and large subunit regions of all fungi were sequenced and confirmed with sequences available in the NCBI database. These data were used to develop primers able to differentiate seven of the 12 species. The five remaining species, including three in the Phacidiaceae family and two in the Glomerellaceae family, were differentiated on the basis of a more discriminant marker, the translation elongation factor 1-α. Two PCR reactions were optimized to clearly delineate the 12 species. The multiplex test was first validated using pure fungal cultures; it was subsequently validated using fruit collected in cranberry beds in eastern Canada. In the latter case, the test was rigorous enough to clearly discriminate the fungal pathogens from contaminants. Within the tested samples, Physalospora vaccinii and Coleophoma empetri were most commonly found. This molecular test offers scientists, diagnosticians, and growers a powerful tool that can rapidly and precisely identify fungi causing CFR so they can implement appropriate control methods.

Secondary contact between closely related taxa routinely occurs during postglacial migrations. After initial contact, the location of hybrid zones may shift geographically or remain spatially stable over time in response to various selective pressures or neutral processes. Studying the extent and direction of introgression using markers having contrasted levels of gene flow can help unravel the historical dynamics of hybrid zones. Thanks to their contrasted maternal and paternal inheritance, resulting in different levels of gene flow for mitochondrial and chloroplast DNA (mtDNA and cpDNA), the Pinaceae stand out as a relevant biological model for this purpose. The objective of the study was to assess whether the hybrid zone between Abies balsamea and Abies lasiocarpa (two largely distributed Pinaceae) has moved or remained stable over time by analysing the distribution of cytoplasmic DNA variation as well as published palaeobotanical data. Interspecific gene flow was higher for cpDNA than mtDNA markers; hence, the geographic distribution of mitotypes was more congruent with species distributions than chlorotypes. This genetic signature was contrary to expectations under a moving hybrid zone scenario, as well as empirical observations in other conifers. Genetic evidence for this rare instance of stable hybrid zone was corroborated by the colonization chronology derived from published fossil data, indicating that the two fir species initially came into contact in the area corresponding to the current sympatric zone 11 kyr ago. While an explanatory analysis suggested the putative influence of various environmental factors on the relative abundance of cytoplasmic genome combinations, further research appears necessary to assess the role of both demographic history and selective factors in driving the dynamics of hybrid zones.

The phylogeographic structure and postglacial history of balsam fir (Abies balsamea), a transcontinental North American boreal conifer, was inferred using mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) markers. Genetic structure among 107 populations (mtDNA data) and 75 populations (cpDNA data) was analyzed using Bayesian and genetic distance approaches. Population differentiation was high for mtDNA (dispersed by seeds only), but also for cpDNA (dispersed by seeds and pollen), indicating that pollen gene flow is more restricted in balsam fir than in other boreal conifers. Low cpDNA gene flow in balsam fir may relate to low pollen production due to the inherent biology of the species and populations being decimated by recurrent spruce budworm epidemics, and/or to low dispersal of pollen grains due to their peculiar structural properties. Accordingly, a phylogeographic structure was detected using both mtDNA and cpDNA markers and population structure analyses supported the existence of at least five genetically distinct glacial lineages in central and eastern North America. Four of these would originate from glacial refugia located south of the Laurentide ice sheet, while the last one would have persisted in the northern Labrador region. As expected due to reduced pollen-mediated gene flow, congruence between the geographic distribution of mtDNA and cpDNA lineages was higher than in other North American conifers. However, concordance was not complete, reflecting that restricted but nonetheless detectable cpDNA gene flow among glacial lineages occurred during the Holocene. As a result, new cpDNA and mtDNA genome combinations indicative of cytoplasmic genome capture were observed.