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Cytospora canker is one of the most important diseases affecting peach production in Colorado, yet previous efforts to characterize Cytospora species diversity in Colorado have relied exclusively on morphological traits. Recently, several new Cytospora species were described from peach orchards within the United States using molecular and morphological data, prompting the need to reexamine Cytospora spp. present on peach trees in Colorado. A total of 137 isolates of Cytospora spp. were collected from eight orchards in western Colorado. Isolates were sequenced at the internal transcribed spacer region and elongation factor I-a and assessed with reference sequences in phylogenetic analyses. All isolates from western Colorado peach trees resolved with the newly described Cytospora plurivora. In addition to molecular characterization, temperature growth and virulence assays were conducted to assess phenotypic variation among the isolates from western Colorado. Variation across isolates was found both in growth at different temperatures and in virulence. Ancestral state reconstruction analyses resolved the most virulent (and most often collected) haplotypes together in a well-supported clade from which a single monophyletic origin of high virulence can be inferred. Finally, a droplet digital PCR assay was developed for use in ongoing and future studies to detect and quantify C. plurivora from field and laboratory samples. [ABSTRACT FROM AUTHOR]

The article presents a study that aims to determine which species of Phytophthora is involved in root rot of raspberry and to investigate if pathogenicity, fungicide resistance, and genetic variation exists among Phytophthora rubi isolates collected from fields in Washington, Oregon, and California. In this study amplified fragment length polymorphism of isolates from Phytophthora were carried out. The study showed that isolates from the three U.S. states represent one mixed population.

Most Alternaria spp. are considered asexual but recent molecular evolution analyses of Alternaria mating-type genes show that the mating locus is under strong purifying selection, indicating a possible role in sexual reproduction. The objective of this study was to determine the mode of reproduction of an Alternaria alternata sensu lato population causing citrus brown spot in central Florida. Mating type of each isolate was determined, and isolates were sequenced at six putatively unlinked loci. Three genetically distinct subpopulations (SHI, SH4A, and SH4B) were identified using network and Bayesian population structure analyses. Results demonstrate that most subpopulations of A. alternata associated with citrus are clonal but some have the ability to extensively recombine through a cryptic sexual cycle or parasexual cycle. Although isolates were sampled in close physical proximity (=2,500-m² area), we were able to reject a random mating model using multilocus gametic disequilibrium tests for two subpopulations, SHI and SH4B, suggesting that these subpopulations were predominantly asexual. However, three recombination events were identified in SHI and SH4B and localized to individuals of opposite mating type, possibly indicating meiotic recombination. In contrast, in the third subpopulation (SH4A), where only one mating type was present, extensive reticulation was evident in network analyses, and multilocus gametic disequilibrium tests were consistent with recombination. Recombination among isolates of the same mating type suggests that a nonmeiotic mechanism of recombination such as the parasexual cycle may be operating in this subpopulation. The level of gene flow detected among subpopulations does not appear to be sufficient to prevent differentiation, and perhaps future speciation, of these A. alternata subpopulations. [ABSTRACT FROM AUTHOR]

Fusarium species can cause severe root disease and damping-off in conifer nurseries. Fusarium inoculum is commonly found in most container and bareroot nurseries on healthy and diseased seedlings, in nursery soils, and on conifer seeds. Isolates of Fusarium spp. can differ in virulence; however, virulence and colony morphology are not correlated. Forty-one isolates of Fusarium spp., morphologically indistinguishable from F. oxysporum, were collected from nursery samples (soils, healthy seedlings, and diseased seedlings). These isolates were characterized by amplified fragment length polymorphism (AFLP) and DNA sequencing of nuclear rDNA (internal transcribed spacer including 5.8S rDNA), mitochondrial rDNA (small subunit [mtSSU]), and nuclear translation elongation factor 1-alpha. Each isolate had a unique AFLP phenotype. Out of 121 loci, 111(92%) were polymorphic; 30 alleles were unique to only highly virulent isolates and 33 alleles were unique to only isolates nonpathogenic on conifers. Maximum parsimony and Bayesian analyses of DNA sequences from all three regions and the combined data set showed that all highly virulent isolates clearly separated into a common clade that contained F. commune, which was recently distinguished from its sister taxon, F. oxysporum. Interestingly, all but one of the nonpathogenic isolates grouped into a common clade and were genetically similar to F. oxysporum. The AFLP cladograms had similar topologies when compared with the DNA-based phylograms. Although all tested isolates were morphologically indistinguishable from F. oxysporum based on currently available monographs, some morphological traits can be plastic and unreliable for identification of Fusarium spp. We consider the highly virulent isolates to be F. commune based on strong genetic evidence. To our knowledge, this is the first reported evidence that shows F. commune is a cause of Fusarium disease (root rot and damping-off) on Douglas-fir seedlings. Furthermore, several AFLP genetic markers and mtSSU sequences offer potential for development of molecular markers that could be used to detect and distinguish isolates of F. oxysporum nonpathogenic to conifers and highly virulent isolates of F. commune in forest nurseries. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Geosmithia morbida is well documented as the causal agent of thousand cankers disease of black walnut trees. However, it is not well understood how G. morbida strains differ in virulence and how their interactions with co-occurring pathogens contribute to disease severity. In this study, we systematically investigated virulence of genetically distinct G. morbida strains. Overall, we found varying degrees of virulence, although differences were not related to genetic groupings. Furthermore, the pathogen Fusarium solani is also commonly isolated from thousand canker-diseased trees. The degree of disease contribution from F. solani is unknown, along with interactions it may have with G. morbida. This research shows that coinoculation with these pathogens does not yield a synergistic response. [ABSTRACT FROM AUTHOR]

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.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius . The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius -unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non- Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius , six tree species naturally infected by P. noxius , rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius , which is useful for long-term monitoring of BRRD status., Competing Interests: The author(s) declare no conflict of interest.

Brown root rot disease (BRRD), caused by Phellinus noxius , is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P . noxius , this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood ( Bischofia javanica ) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control., Competing Interests: The author(s) declare no conflict of interest.

The focus of investigation in this study was to consider the potential of arthropods in the dissemination of the bacterium involved in drippy blight disease, Lonsdalea quercina . Arthropod specimens were collected and tested for the presence of the bacterium with molecular markers. The bacterium L. quercina was confirmed on 12 different insect samples from three orders (Coleoptera, Hemiptera, and Hymenoptera) and eight families (Buprestidae, Coccinellidae, Dermestidae, Coreidae, Pentatomidae and/or Miridae, Apidae, Formicidae, and Vespidae). Approximately half of the insects found to carry the bacterium were in the order Hymenoptera. Estimates of the insects that are contaminated with the bacterium, and likely carry it between trees, is conservative because the documented insects represent only a subset of the insect orders that were observed feeding on the bacterium or present on diseased trees yet were not able to be tested. The insects contaminated with L. quercina exhibited diverse life histories, where some had a facultative relationship with the bacterium and others sought it out as a food source. These findings demonstrate that a diverse set of insects naturally occur on diseased trees and may disseminate L. quercina .