Development of PCR-based markers for the identification and detection of Lophodermella needle cast pathogens on Pinus contorta var. latifolia and P. flexilis.

Morphological similarities and fastidious development of increasingly emerging fungal needle pathogens impede accurate disease diagnosis and early detection. This study analyzed the specificity and sensitivity of polymerase chain reaction (PCR)-based markers developed for emerging needle cast pathogens Lophodermella concolor and L. montivaga co-occurring on Pinus contorta var. latifolia, and Bifusella linearis and L. arcuata on P. flexilis. To design primers, we utilized sequences of the internal transcribed spacer (ITS) region and single-copy gene (RH_2175) of the TCP-1/cpn60 chaperonin family searched through genomes of related species. In addition to the DNA of target and non-target fungal species that were used for primer assays, environmental samples with next generation sequencing data were used to evaluate primer sensitivity. Direct amplification using ITS primer pairs generated 248-260 bp amplicons and successfully differentiated the needle pathogens used in this study. Nested amplification of single-copy gene RH_2175 primer pairs which produced 409-527 bp amplicons detected Rhytismataceae species and discriminated both Lophodermella pathogens on P. contorta var. latifolia, respectively. While ITS-based primers had higher sensitivity than the 2175-based primers, both primer sets for L. concolor and L. montivaga detected their respective pathogens in asymptomatic and symptomatic needles. These molecular tools can help monitor and assess needle diseases for forest management and phytosanitary regimes.

Molecular characterization and phylogenetic analyses of Lophodermella needle pathogens (Rhytismataceae) on Pinus species in the USA and Europe.

Increasing prevalence of conifer needle pathogens globally have prompted further studies on pathogen identification and a better understanding of phylogenetic relationships among needle pathogens. Several Lophodermella species can be aggressive pathogens causing needle cast in natural pine forests in the USA and Europe. However, their relationships with other Rhytismataceae species have historically been based on similarities of only limited phenotypic characters. Currently, no molecular studies have been completed to elucidate their relationships with other Lophodermella needle pathogens. This study collected and sequenced three gene loci, namely: internal transcribed spacer, large ribosomal subunit, and translation elongation factor 1-alpha, from five Lophodermella needle pathogens from North America (L. arcuata, L. concolor, L. montivaga) and Europe (L. conjuncta and L. sulcigena) to distinguish phylogeny within Rhytismatacaeae, including Lophophacidium dooksii. Phylogenetic analyses of the three loci revealed that all but L. conjuncta that were sampled in this study consistently clustered in a well-supported clade within Rhytismataceae. The multi-gene phylogeny also confirmed consistent nesting of L. dooksii, a needle pathogen of Pinus strobus, within the clade. Potential synapomorphic characters such as ascomata position and ascospore shape for the distinct clade were also explored. Further, a rhytismataceous species on P. flexilis that was morphologically identified as L. arcuata was found to be unique based on the sequences at the three loci. This study suggests a potential wider range of host species within the genus and the need for genetic characterization of other Lophodermella and Lophophacidium species to provide a higher phylogenetic resolution.

Heterobasidion annosum and H. parviporum in the Southern Rocky Mountains and Adjoining States.

Collections of Heterobasidion spp. from Nebraska, Colorado, Arizona, and New Mexico were identified based on the sequence of the internal transcribed spacer region of the ribosomal DNA. The North American variant of Heterobasidion annosum sensu stricto was found on Pinus ponderosa and Juniperus virginiana in central Nebraska, southern Colorado, central Arizona, and southern New Mexico. The North American variant of H. parviporum was found on Abies concolor and Picea engelmannii in southern Colorado and central New Mexico. The pathogens were not found in a survey of conifer forests in Wyoming and the Black Hills of South Dakota. Historical records of annosus root disease are reviewed by host group to gain more insight into the potential distributions of the respective pathogens. An apparent lack of overlap in host range suggests that substitution of tree species may be a useful management approach in some cases.

Dieback and Mortality of Alnus in the Southern Rocky Mountains, USA.

Dieback and mortality of Alnus incana subsp. tenuifolia in the Southern Rocky Mountains apparently began by the late 1980s and have become a concern to land managers. A survey of alder including 68 transects from southern Wyoming to northern New Mexico indicated that, of 6,503 standing stems, 37% were dead, 29% had dieback, and 34% were healthy. Transects intercepted 1,479 m of live and 1,177 m of dead alder canopy. A second, more localized survey with 32 transects in the upper Gunnison River watershed of Colorado yielded similar results. Abundance of live sprouts was inversely related to amount of dieback and mortality in a genet, suggesting that affected genets are dying and not replacing themselves. Damage did not vary substantially by geographic area and was not related to elevation, animal browsing, or distance to nearest road. Distance to nearest stream was weakly, inversely related to severity of dieback and mortality. Symptoms were not consistent with disease of alder caused by Phytophthora alni in Europe, and isolations for Phytophthora species were negative. Cytospora canker, caused by Valsa melanodiscus (anamorph Cytospora umbrina), is the proximate cause of the dieback and mortality.