Colletotrichum fioriniae infecting invasive Japanese hop (Humulus scandens) in the United States.

Japanese hop (Humulus scandens) is a non-native, invasive plant that colonizes disturbed riparian areas throughout the eastern United States and Canada, forming dense, monocultural stands that displace native plant communities due to a high reproductive rate, rapid growth, climbing bines, and dense shading (Balogh and Dancza 2008). It is capable of serving as a reservoir for agronomically important plant pathogens, such as the Tomato spotted wilt virus and powdery mildew species that infect commercial hemp and hop fields (Yoon et al. 2018; Weldon et al. 2020). In the spring of 2016, diseased populations of H. scandens were observed along the Monocacy River in Frederick County, Maryland with severe chlorotic and necrotic leaf lesions. Symptomatic leaves were surface sterilized and placed in moist chambers at 25°C for sporulation. Sporulating acervuli, lacking setae, developed on irregular, tan necrotic leaf lesions following 7 to 12 days in a moist chamber (Figure 1). Conidia were hyaline, aseptate, smooth-walled, fusiform to cylindrical with both ends acute (Figure 1B). Conidia measured (n = 100) [L x W; Average (+ Std. Err), range]: 12.42 µm (± 0.10), 8.41 - 14.48 µm; x 3.91 µm (±0.03), 3.03 - 4.91 µm. Monoconidial fungal cultures were obtained by transferring conidia with a sterile glass needle to acidified potato dextrose agar and incubated at 25°C for 2 to 3 days. Based on phenotypic characteristics and conidial morphology and size, the pathogen appeared to belong to the Colletotrichum acutatum complex (Damm et al. 2012). Therefore, six loci (ITS, GADPH, CHS1, HIS3, ACT, and TUB2) were amplified and sequenced from a representative isolate, 16-008, for species characterization (GenBank accessions MW023070 to MW023075) (Damm et al. 2012). For the ITS region and ACT, GADPH, and CHS1 loci, isolate 16-008 was 100% identical to C. fioriniae and shared 99% similarity to TUB2 and HIS3 for multiple accessions of C. fioriniae in GenBank. Gene sequences were aligned, trimmed, concatenated, and analyzed against 32 reference strains, within the C. acutatum complex (Damm et al. 2012). Concatenated loci were used to generate a maximum likelihood phylogeny using W-IQ-TREE (Trifinopoulos et al. 2016). Results from the phylogenetic analysis demonstrated that isolate 16-008 was most genetically similar to C. fioriniae with a bootstrap support of 100% (Figure 2). Based on phenotypic and sequence analyses, isolate 16-008 was identified as C. fioriniae. Humulus scandens seedlings from Maryland (n = 3) were inoculated with a conidia suspension (107 conidia mL-1) with 0.125% Tween 20® and applied with an atomizer until runoff. Inoculated plants were placed in a dew chamber at 25°C for 2 days. Experimental plants were distributed in a mist tent at 25°C with 14 h of light and monitored for 2 weeks. Negative control plants (n = 2) were sprayed with a sterile 0.125% Tween 20® water solution. All inoculated plants were symptomatic by 12 days post inoculation. No symptoms were observed on the mock-inoculated plants. Symptoms were identical to disease field samples. Inoculations were repeated with the same results. Colletotrichum fioriniae was reisolated and confirmed from excised leaf lesions via ITS and ACT sequencing. To our knowledge, this is the first report of C. fioriniae naturally infecting H. scandens within the United States (Farr and Rossman 2020). Future studies will evaluate the host range of this isolate due to the species broad host range and the weed's extensive distribution.

Uromyces rebeccae, sp. nov., a newly described rust on the federally endangered plant, California sea-blite (Suaeda californica).

Rust disease was observed on populations of Suaeda californica near Morro Bay, California. The pathogen was identified as a species of Uromyces based on teliospore and urediniospore morphology and nuc 28S rDNA sequence analysis. The isolate was compared with previously described species of Uromyces that infect members of Chenopodiaceae, prompting a taxonomic reevaluation of Uromyces species on Suaeda. Herein, Uromyces rebeccae is described. It can be differentiated from the closely related U. chenopodii (syn.: Aecidium chenopodii-fruticosi; U. giganteus) based on host range, teliospore morphology, and 28S sequence data. The new combination, Uromyces chenopodii-fruticosi, is made for Aecidium chenopodii-fruticosi, the oldest name for Eurasian Suaeda rust. Finally, it was determined that U. giganteus likely does not occur in the United States and that the rust of S. taxifolia in the United States likely comprises a third, yet unnamed taxon, different from both U. rebeccae and U. chenopodii-fruticosi. This is the first record of a rust fungus on S. californica. An identification key for Uromyces species reported on Chenopodiaceae is provided.

Synchytrium solstitiale: reclassification based on the function and role of resting spores.

Studies were made about resting spores of Synchytrium solstitiale, a chytrid that causes false rust disease of yellow starthistle (YST). During evaluation of this fungus for biological control of YST, a protocol for resting spore germination was developed. Details of resting spore germination and study of long-term survival of the fungus were documented. Resting spores from dried leaves germinated after incubating them on water agar at least 7 d at 10-15 C. Resting spores were viable after storage in air-dried leaves more than 2 y at room temperature, suggesting they have a role in off-season and long-term survival of the fungus. Each resting spore produced a single sorus that contained a single sporangium, which on germination released zoospores through a pore. YST inoculated with germinated resting spores developed symptoms typical of false rust disease. All spore forms of S. solstitiale have been found to be functional, and the life cycle of S. solstitiale has been completed under controlled laboratory and greenhouse conditions. Resting spore galls differ from sporangial galls both morphologically and biologically, and in comparison, each sporangial gall cleaves into several sori and each sorus produces 5-25 sporangia that rupture during release of zoospores. For this reason S. solstitiale should be reclassified as diheterogallic sensu Karling (Am J Bot 42:540-545). Because resting spores function as prosori and produce an external sorus, S. solstitiale is best placed in into the subgenus Exosynchytrium.

Life cycle of Puccinia acroptili on Rhaponticum (= Acroptilon) repens.

Russian knapweed (Rhaponticum repens) is a rangeland weed pest in the western United States. One candidate fungus for biological control of R. repens is Puccinia acroptili, which causes a rust disease. Understanding the life cycle of candidate rust fungi for weed biological control is an essential component in risk assessments and evaluations, and for P. acroptili such was unknown. For this reason greenhouse studies were undertaken to clarify the life cycle of P. acroptili under artificial conditions. Spermogonia with spermatia developed on R. repens following plant inoculation with teliospores. Artificial transfer of spermatia between spermogonia resulted in the development of aecia with uredinioid aeciospores. Inoculation with aeciospores or urediniospores resulted in uredinia containing urediniospores and occasional amphispores. Telia with teliospores and occasional mesospores developed later. Teliospores produced typical basidia with four basidiospores. These results suggest that the life cycle of P. acroptili is macrocyclic and autoecious. Inoculation with teliospores also frequently resulted in production of sori that were morphologically similar to aecia but which were not associated with spermogonia or the classical transfer of spermatia. The ontology of these sori is unknown. This is the first description of spermogonia and the first report and description of basidiospores, aecia, aeciospores, amphispores and mesospores of P. acroptili.

Factors Affecting Germination of Puccinia jaceae var. solstitialis Teliospores from Yellow Starthistle.

ABSTRACT The rust fungus Puccinia jaceae var. solstitialis is a candidate for biological control of yellow starthistle (YST). Part of the risk assessment includes determining if safflower seedlings are susceptible to infection by teliospores of P. jaceae. A protocol for germination of P. jaceae teliospores is needed to verify that teliospores used in comparative studies are viable. The protocol developed from this research has two steps: first, priming teliospores on water agar at 4 degrees C in the dark, and second, incubating them at warmer temperatures for 1 week in the presence of an exogenous stimulator. Priming longer than 4 weeks resulted in significantly greater germination than priming for shorter periods. Sources of effective stimulator included seeds and seedlings of YST or safflower. The greatest germination occurred during incubation at 18 degrees C in the dark. Teliospore germination was reduced after incubation with a 12- or 14-h photoperiod. A low percentage (<20%) of teliospores of two isolates germinated after 44 and 96 weeks of dry storage at room temperature; samples of each isolate tested after that did not germinate. Data indicate teliospores of several isolates of P. jaceae are viable, and the protocol will be used to prepare teliospores of P. jaceae for comparative studies with P. carthami on safflower seedlings.

A Simple Quantitative Procedure for Inoculation of Safflower with Teliospores of the Rust Fungus, Puccinia carthami.

A procedure is needed to determine susceptibility of safflower seedlings to teliospore inoculations of foreign accessions of Puccinia jaceae, a candidate agent for biological control of Centaurea solstitialis (yellow starthistle). The indigenous safflower rust pathogen, P. carthami, served as both model and basis for a future comparative study of these two rust fungi on safflower. The procedure involves inoculation of 2-day-old safflower seedlings on the hypocotyl near the base of the cotyledons with a known number of teliospores suspended in a 1-µl drop of a 0.1% water agar and wetting agent carrier. Advantages of this procedure include (i) quantification, by spore type, of inoculum applied to each seedling, (ii) precise placement of inoculum on test plants, and (iii) conservation of inoculum. Results were verified in studies with two safflower cultivars inoculated with 0, 15, 60, or 240 teliospores of either of two isolates of P. carthami. Plant height and dry weight data collected 6 weeks after inoculation revealed significant interactions for cultivar × inoculum concentration and for isolate × inoculum concentration. Similar results were achieved when safflower seeds were rolled in dry teliospores of P. carthami but not when seedlings grew through teliospore-infested sand. The latter two approaches (dry teliospore and infested sand inoculation) require much more inoculum and are only semiquantitative. The microliter drop procedure has potential usefulness in host range and quantitative studies involving other pathogens, including bacteria, fungi, and nematodes.