Background: Development and application of DNA-based methods to distinguish highly virulent isolates of Fusarium oxysporum f. sp. koae [Fo koae; cause of koa wilt disease on Acacia koa (koa)] will help disease management through early detection, enhanced monitoring, and improved disease resistance-breeding programs. Results: This study presents whole genome analyses of one highly virulent Fo koae isolate and one non-pathogenic F. oxysporum (Fo) isolate. These analyses allowed for the identification of putative lineage-specific DNA and predicted genes necessary for disease development on koa. Using putative chromosomes and predicted gene comparisons, Fo koae-exclusive, virulence genes were identified. The putative lineage-specific DNA included identified genes encoding products secreted in xylem (e. g., SIX1 and SIX6) that may be necessary for disease development on koa. Unique genes from Fo koae were used to develop pathogen-specific PCR primers. These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo. Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae. Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa. Conclusions: These results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands.

Background and Objectives: Koa (Acacia koa A. Gray) is an economically, ecologically, and culturally valuable tree species endemic to Hawaii. A vascular wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. koae Gardner (FOXY) induces high rates of mortality in plantings and threatens native koa forests as well. Landowners are reluctant to consider koa for reforestation purposes in many areas due to the risk of mortality caused by FOXY. Producing seeds with genetic resistance to FOXY is vital to successful koa reforestation and restoration. The Hawaii Agriculture Research Center (HARC), with both public and private partners, operates a tree improvement program to develop wilt-resistant koa populations in Hawaii. The population genetics of koa is poorly understood and seed zones are evolving. Thus, HARC uses provisional seed zones based on genetic and biogeographic variables and has selected wilt-resistant koa populations that are locally found in Kokee, Kauai (eco-regions) of Hawaii. Materials and Methods: To make these selections, virulent FOXY isolates were used in previous seedling inoculation trials to evaluate resistance levels among koa families in greenhouse experiments, and the most resistant families were used in the field trial reported here. Results: In this trial, survival rates two years after planting varied by family, and ranged from 45% to 95%, but all resistant families had greater survival rates than the susceptible control (25%). The trial has been converted to a seed orchard. Conclusions: The higher survival rates of the families are encouraging and seeds coming from the orchard will improve the success of future restoration and reforestation efforts. Within these resistant families it was also possible to make some selections based on height, growth, diameter, and stem form. Thus, the establishment of a wilt-resistant seed orchard results in locally adapted, eco-region specific, disease-resistant koa seed that will allow for the restoration of this iconic species and provide plant material for commercial reforestation opportunities at the landscape level.

Passalora sequoiae is a foliar pathogen to conifer tree species. In this study, we conducted whole-genome and transcriptome analyses on isolates of P. sequoiae collected from symptomatic Leyland cypress leaves from a Christmas tree farm in Mississippi. The objectives for this research were to elucidate the pathogenicity mechanisms of P. sequoiae by characterizing the genome and transcriptome and possibly identify unique and shared predicted genes in comparison with non-conifer/canker and foliar pathogens in the family Mycosphaerellaceae. P. sequoiae was found to be similar to other foliar Mycosphaerellaceae pathogens and likely represents a hemibiotrophic lifestyle based on comparisons across pathogens. The genome and in planta transcriptome highlighted some unique features of P. sequoiae: the significant presence of chitin synthases and fructose-degrading carbohydrate-degrading enzymes, trans-AT PKS genes, and antibiotic gene clusters that were unique to P. sequoiae compared with the other Mycosphaerellaceae species genomes. Several transcripts that were highly expressed in planta were identified as effectors, yet the functions were not characterized. These targets provide ample resources to continue to characterize pathogen−conifer host interactions in conifer foliar pathogens. Furthermore, this research helps build genomic resources for an important plant pathogen on Leyland cypress that will further our ability to develop novel management practices that could begin with breeding for resistance.

Diplodia shoot blight and canker disease (DSB), caused by the fungal pathogen Diplodia sapinea (Fr.) Fuckel (syn=Sphaeropsis sapinea (Fr.) Dyko & Sutton), is found on 2-3 needled pines including ponderosa pine (Pinus ponderosa) and other conifers (Blodgett and Stanosz 1999). Typical symptoms of DSB are short, necrotic needles, necrosis of current-years growth that can progress into older growth, resinous branch and bole cankers, dieback, dead tops and branches, infected cones, and sapwood staining (Blumenstein et al., 2021, Caballol et al., 2022) The latent pathogen is known to persist asymptomatically within the tree allowing it to accumulate unnoticed in healthy trees. (Terhonen et al., 2021). While D. sapinea is a major issue in natural pine stands, outbreaks can occur in nurseries and seed orchards due to monocultures and other stress-inducing factors (Aragonés et al., 2021). Diplodia sapinea outbreaks have been reported in North America, Africa, and several European countries (Blumenstein et al., 2021). Since D. sapinea has never been reported in Colorado, studies were conducted to confirm the presence, pathogenicity, and potential movement of DSB from Wyoming into Colorado. In 2018, DSB symptoms were observed in ponderosa pine stands during aerial surveys in Wyoming, and were confirmed in ground surveys in 2019 (Blodgett et al., 2021). Isolates from host trees were confirmed as Diplodia sapinea using species specific PCR (Blodgett et al., 2021). In 2021, a group of approximately 90-year old ponderosa pines exhibiting DSB symptoms were observed in the northeastern foothills of Colorado. Two D. sapinea isolates were collected from branches off two symptomatic trees. Xylem and phloem tissue samples were cut from the disease margin and placed on 1/2 strength potato dextrose agar (PDA) (Hardy Diagnostics; Santa Maria, CA). Another isolate was collected from the same tree in 2023 from a symptomatic branch. Branches were surface sterilized in 10% sodium hypochlorite for 2 min, placed in sterile water for 1 min, and then placed in humid chambers. Pycnidia were observed after a week. Diplodia sapinea spores were identified morphologically and after 1 day, a single spore was transferred to ½ PDA agar (for each of the three isolates). The three isolates were confirmed as D. sapinea using colony PCR to amplify the internal transcribed spacer (ITS) and translation elongation factor 1 alpha (tef1-α). The master mix for ITS amplification contained 1 µl of the forward and reverse primers ITS1/ITS4 diluted to 10 ng/µl (White et al. 1990), 12.5 µl of GoTaq Green 1x (Promega), and 8.5 µl of MH2O. DNA was diluted to 10 ng/µl and 20 ng were used for a total reaction volume of 25 µl. The cycling parameters followed those described by White et al (1990). The reaction mixture was similar to the tef1-α amplification, however, the forward and reverse primers EF-688F and EF-1251R were used and PCR cycling parameters were completed methods by Carbone and Kohn (1999). Samples were run on a 1.5% agarose gel to visualize amplified PCR products using GelRed® and sent to Eurofins Genomics for forward and reverse sequencing of both the ITS and tef1-α . The sequences obtained were then compared to reference strains found in the NCBI database using the BLAST algorithm. The ITS sequences of isolates CO21-1 and CO21-2 resulted in a query coverage (QC) of 100% and percent identity (PI) of 99.81% for D. sapinea (PP467714 and PP860312) and for isolate CO23-1 resulted in QC=99.8% and PI=100% (PP467692). The tef1-α sequences of CO21-2 and CO23-1 resulted in a QC=93.94% and PI=99.8% and QC=83.43% and PI=100% to D. sapinea (MG015730 and MT592070), respectively. A phylogeny of the tef1-α was produced using CO21-2 and CO23-1 closely-related species obtained from GenBank. To infer the phylogenetic relationship, an alignment of the tef1-α sequence data was performed using MAFFT v7.490 and edited manually. The species used for the phylogeny were Diplodia sapinea (KF729440, KF729470, DQ458880), Diplodia intermedia (GQ923850, GQ923826), Diplodia seriata (AY573220, GU121862), Diplodia africana (EF445383), Diplodia scrobiculata (KF766399), and Diplodia coryli (EU673284). Sphaeropsis visci (MH863218) and Botryosphaeria dothidea (AY259092) were used as the outgroups in the analysis. A maximum likelihood phylogeny was produced using PhyML version 3.3.20180621 (Guidon et al. 2010) with 200 bootstraps (BS). The resulting phylogeny showed that the Colorado isolates form in a clade (BS=60%) with the reference isolates of D. sapinea. Sequences for isolates CO21-2 and CO23-1 were submitted to GenBank with accession numbers PQ583516 and PQ583515 for the ITS and PQ588602 and PQ588603 for the tef1-α, respectively. These samples are stored at Colorado State University, Fort Collins, CO. Two-year-old ponderosa pine seedlings were inoculated in greenhouses at Colorado State University, using methods previously described in Blodgett et al. (2021). A sterilized scalpel was used to remove a single needle fascicle, 2 to 2.5 cm below the apical bud. A 5 mm 1.5% water agar plug colonized by one of two D. sapinea isolates was placed on the wound and wrapped with parafilm. Sterile water plugs were used as negative controls. Inoculated trees were observed for 6 weeks. Two experiments were conducted simultaneously with five seedlings per isolate or a negative control (n=30). After 6 weeks, 25% of seedlings inoculated with CO21-1 and CO21-2 isolates developed lesions ranging from 1.85 mm to 5.55 mm in length. The experiment was repeated with isolates CO21-2 and CO23-1 which resulted in 20% of inoculated seedling developing lesions ranging from 5.86 mm to 18.21 mm in length. None of the seedlings inoculated with a control plug developed symptoms in either experiment. Stem segments from all seedlings were cut, centered at the original wound, and surface sterilized for 30 seconds...