First Report of Diplodia Shoot Blight and Canker Disease Caused by on Ponderosa Pine in Colorado, USA.

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 in 70% ethanol and 5 mins in 1.05% sodium hypochlorite. The symptomatic stem segments were then placed in petri dishes containing tannic acid agar (Blodgett et al., 2003). After 1 week on the tannic acid agar, isolates were sub-cultured on ½ PDA and identified based on colony and spore morphologically and PCR of the ITS region. This report confirms that D. sapinea is present in Colorado and that CO isolates cause disease. While the isolates collected from Colorado were not as aggressive as those previously reported for Wyoming, D. sapinea should be further studied and closely monitored. Diplodia sapinea is known to live in its host as a latent pathogen and can switch to an opportunistic pathogen under certain host-stressing conditions like drought, hail damage, and high temperatures (Ghosh et al., 2022, Stanosz et al. 2001, Terhonen et al., 2021). In 2010, a fire burned in the area near where the isolates were collected, killing approximately 50% of the trees. Prior to the fire, DSB was not observed. This suggests a stress factor, such as fire, may trigger D. sapinea to switch to its pathogenic form. Much of Colorado's montane region consists of host species for D. sapinea, including ponderosa pine. With stress factors increasing in Colorado and across western states, host trees are becoming more susceptible which may result in outbreaks of DSB.