Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems.

Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9-SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.

First Report of Diplodia Shoot Blight and Canker Disease caused by Diplodia sapinea on Ponderosa Pine in Wyoming, USA.

Diplodia sapinea is a common fungal pathogen that causes shoot blight and canker of naturally occurring and planted pines and other conifers throughout the world. Damage can be severe severe, affectingand affect needles, new shoots, branches, and main stems, often leading to mortality. In August 2018, aerial surveys revealed 1,659 ha of ponderosa pine (Pinus ponderosa) displaying varying degrees of crown necrosis in Crook County, Wyoming (WY). In May 2019, ground surveys of this area, which was previously affected by hail the previous spring, identified typical Diplodia shoot blight and canker signs and symptoms (e.g., pycnidia, crooked shoots, discolored needles, resinous cankers). Symptoms were observed in thousands of seedlings, saplings, and mature ponderosa pine trees over the large area. Because D. sapinea had not been previously reported in WY, this study was conducted to confirm the presence and aggressiveness of D. sapinea isolates from WY on this commercially and ecologically important host tree. Pycnidia and conidia (size 34 to 39 µm × 12 to 13 µm) consistent with D. sapinea were confirmed in 19 trees from three locations in the county. Three isolates were confirmed as D. sapinea using species-specific PCR (Smith and Stanosz 2006) and are retained at the Rocky Mountain Research Station, Moscow, ID and Colorado State University, Fort Collins, CO. To confirm isolate aggressiveness, 3-year-old potted ponderosa pine seedlings were inoculated in greenhouses from March to April 2020 at the Charles E. Bessey Nursery, Halsey, NE using similar methods as previously described (Blodgett and Stanosz 1997). Wounding was conducted using a scalpel to excise a single needle fascicle from a recently expanded shoot. Cuts were made flush to the stem at 2 to 2.5 cm below an apical bud-tip. A 1.5% water-agar plug with cultured mycelia of one of the three DNA-confirmed isolates was placed over the stem wound, or sterile water-agar plugs were used as negative controls. Parafilm® (4-cm wide) was wrapped around the inoculated stems, centered at the wound for 4 weeks. Two independent trials were conducted consecutively, 3 hrs apart, in different greenhouses with five seedlings per isolate or control. Symptoms first appeared 5 days post-inoculation. All seedlings inoculated with the three isolates developed typical D. sapinea symptoms, including cankers (mean length 5.98.7 ± 1.13.0 cm standard error and 5.5 ± 0.8 cm standard error, trial 1 and 2 respectively) at 4-weeks post-inoculation, while mock-inoculated seedlings developed no symptoms. Stem segments (2-cm long) were excised, centered at canker margins (or centered 3 cm below wounds for controls), and surface disinfested for 30 sec in 70% ethanol and 5 min in 1.05% sodium hypochlorite, then placed in Petri plates containing tannic acid agar (Blodgett et al. 2003). After 3 weeks, isolates were subcultured from colony margins to Petri plates containing 1.5% water agar and autoclaved ponderosa pine needles. Pycnidia and conidia consistent with D. sapinea were confirmed after another month from all seedlings inoculated with each isolate, but not from control seedlings. This report confirms that D. sapinea is present in WY, and WY isolates can be aggressive pathogens of ponderosa pine. Reducing host water stress (Blodgett et al. 1997a, Blodgett et al. 1997b) may be the best option to manage Diplodia shoot blight and canker disease in forested sites. This can be accomplished by stand thinning and/or managing competing vegetation. Favoring non-host species might be an alternative management option in areas with severe disease. References: J. T. Blodgett et al. 2003. For. Pathology 33:395. J. T. Blodgett and G. R. Stanosz. 1997. Plant Dis. 81:143. J. T. Blodgett et al. 1997a. Phytopathology 87:429. J. T. Blodgett et al. 1997b. Phytopathology 87:422. D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006.

Gene Expression Analysis of Three Putative Copper-Transporting ATPases in Copper-Tolerant Fibroporia radiculosa.

Copper tolerance of brown-rot basidiomycete decay fungi can lessen the efficacy of copper-containing wood preservatives for wood products in-service. The purpose of this study was to evaluate wood mass loss and differential expression of three genes that have putative annotations for copper-transporting ATPase pumps (FIBRA_00974, FIBRA_04716, and FIBRA_01430). Untreated southern pine (SP) and SP treated with three concentrations of ammoniacal copper citrate (CC, 0.6, 1.2, and 2.4%) were exposed to two copper-tolerant Fibroporia radiculosa isolates (FP-90848-T and L-9414-SP) and copper-sensitive Gloeophyllum trabeum isolate (MAD 617) in a 4-week-long standard decay test (AWPA E10-19). Decay of copper-treated wood was inhibited by G. trabeum (p = 0.001); however, there was no inhibition of decay with increasing copper concentrations by both F. radiculosa isolates. Initially, G. trabeum and one F. radiculosa isolate (L-9414-SP) highly upregulated FIBRA_00974 and FIBRA_04716 on copper-treated wood at week 1 (p = 0.005), but subsequent expression was either not detected or was similar to expression on untreated wood (p = 0.471). The other F. radiculosa isolate (FP-90848-T) downregulated FIBRA_00974 (p = 0.301) and FIBRA_04716 (p = 0.004) on copper-treated wood. FIBRA_01430 expression by G. trabeum was not detected, but was upregulated by both F. radiculosa FP-90848-T (p = 0.481) and L-9414-SP (p = 0.392). Results from this study suggest that all three test fungi utilized different mechanisms when decaying copper-treated wood. Additionally, results from this study do not provide support for the involvement of these putative gene annotations for copper-transporting ATPase pumps in the mechanism of copper-tolerance.

A Nonwounding Greenhouse Screening Protocol for Prediction of Field Resistance of Hybrid Poplar to Septoria Canker.

Populus spp. and their hybrids are short-rotation woody crops which supply fiber to a diversity of industries in North America. The potential of hybrid poplar trees has been limited by the fungal pathogen Septoria musiva, the cause of leaf spot and stem canker of Populus spp. An inoculation protocol that does not rely on stem wounding to achieve infection was recently developed to screen poplar clones for resistance to Septoria canker. Prior to this study, the relationship between results obtained using this inoculation protocol and long-term field resistance of clones was unknown. Young ramets of 14 clones of hybrid poplar that were previously assigned to long-term canker damage categories (low, intermediate, and high) were inoculated with a conidial suspension of three isolates of S. musiva under greenhouse conditions. Three weeks post inoculation, lesion number, lesions per centimeter of stem length, area of stem that was necrotic, and proportion of stem area that was necrotic were measured. Logistic regression with lesion number and proportion necrotic area correctly predicted long-term disease damage categories for 11 of 14 clones tested, including the most resistant (NM6) and the most susceptible (NC11505) clones, demonstrating that this screening protocol is a promising method for prediction of long-term disease impact of the most resistant clones.

Physiological performance of an Alaskan shrub (Alnus fruticosa) in response to disease (Valsa melanodiscus) and water stress.

• Following the decades-long warming and drying trend in Alaska, there is mounting evidence that temperature-induced drought stress is associated with disease outbreaks in the boreal forest. Recent evidence of this trend is an outbreak of Cytospora canker disease (fungal pathogen Valsa melanodiscus (anamorph = Cytospora umbrina)) on Alnus species. • For Alnus fruticosa, we examined the effects of water stress on disease predisposition, and the effects of disease and water stress on host physiology. In two trials, we conducted a full-factorial experiment that crossed two levels of water stress with three types of inoculum (two isolates of V. melanodiscus, one control isolate). • Water stress was not required for disease predisposition. However, the effects of water stress and disease on host physiology were greatest near the peak phenological stage of the host and during hot, dry conditions. During this time, water stress and disease reduced light-saturated photosynthesis (-30%), light saturation point (-60%) and stomatal conductance (-40%). • Our results depended on the timing of water stress and disease in relation to host phenology and the environment. These factors should not be overlooked in attempts to generalize predictions about the role of temperature-induced drought stress in this pathosystem.

Quantification of Conidia of Diplodia spp. Extracted from Red and Jack Pine Cones.

Frequency of detection and inoculum production by the conifer shoot blight and canker pathogens Diplodia pinea and D. scrobiculata on cones of red pine (Pinus resinosa) and jack pine (P. banksiana) were studied. Cones were collected from the ground and from canopies of red and jack pine trees in mixed stands at three sites in each of two different locations during two consecutive summers in Wisconsin. Conidia were extracted in water, quantified, germination tested, and the Diplodia species present was determined using molecular methods. At least one pathogen was detected from each tree at each site in both years. Overall, more conidia were extracted from cones from canopies than cones from the ground and from red pine cones than jack pine cones. Both total numbers of conidia extracted and proportions of cones yielding D. pinea or D. scrobiculata varied by location and pine species. Cones from either the ground or canopies can be used for surveys to detect Diplodia spp. at a given site but cones from canopies may be more useful to determine the relative abundance of potentially available inoculum of these pathogens.

The Histology of Hybrid Poplar Clones Inoculated with Septoria musiva.

Septoria musiva causes stem cankers that severely limit production of susceptible hybrid poplars in eastern North America. A field experiment was conducted with resistant clone DN34 and susceptible clone NC11505 in order to (i) identify tissues colonized by the pathogen, (ii) describe tissue responses to S. musiva, and (iii) determine whether tissue responses to S. musiva differed between hybrid poplar clones. Branches of each clone were inoculated by removing the fourth or fifth fully expanded leaf and placing an agar plug colonized by an aggressive isolate of S. musiva over the wound. Seven weeks after inoculation, branches were harvested and prepared for histology. Data from nonwounded control, wounded control, and wounded and inoculated stems were collected and analyzed for effects of clone and treatment. In general, fungal colonization was more extensive in NC11505 and exophylactic and necrophylactic periderms (NPs) of clone DN34 were significantly thicker than those of NC11505, regardless of treatment. The number of NPs produced and the distance from the epidermis to the outermost layer of phellem were significantly affected by the pathogen. Inoculated stems of clone DN34 developed a single NP that formed closer to the wound surface than in wounded controls. In contrast, inoculated stems of NC11505 developed successive NPs and the first NP formed further from the wound surface than in wounded controls. These two host responses to inoculation, as well as measures of exophylactic and necrophylactic periderm thickness, may be useful as markers for the selection of poplar resistant to damage by S. musiva.

Species diversity of polyporoid and corticioid fungi in northern hardwood forests with differing management histories.

Effects of forest management on fungal diversity were investigated by sampling fruit bodies of polyporoid and corticioid fungi in forest stands that have different management histories. Fruit bodies were sampled in 15 northern hardwood stands in northern Wisconsin and the upper peninsula of Michigan. Sampling was conducted in five old-growth stands, five uneven-age stands, three even-age unthinned stands and two even-age thinned stands. Plots 100 m x 60 m were established and 3000 m2 within each plot was sampled during the summers of 1996 and 1997. A total of 255 polyporoid and corticioid morphological species were identified, 46 (18%) of which could not be assigned to a described species. Species accumulation curves for sites and management classes differed from straight lines, although variability from year to year suggests that more than 2 y of sampling are needed to characterize annual variation. Mean species richness and diversity index values did not vary significantly by management class, although mean richness on large diameter wood (> or = 15 cm diam) varied with moderate significance. Richness values on small diameter debris varied significantly by year, indicating that a large part of year-to-year variability in total species richness is due to small diameter debris. Ten species had abundance levels that varied by management class. Two of these species. Changes in the diversity and species composition of the wood-inhabiting fungal community could have significant implications for the diversity, health and productivity of forest ecosystems.

A Species-Specific PCR Assay for Detection of Diplodia pinea and D. scrobiculata in Dead Red and Jack Pines with Collar Rot Symptoms.

A polymerase chain reaction (PCR)-based assay was developed for the specific detection of the fungal pathogens Diplodia pinea and D. scrobiculata from pine host tissues. Variation among mitochondrial small subunit ribosome gene (mt SSU rDNA) sequences of Botryosphaeria species and related anamorphic fungi was exploited to design primer pairs. Forward primer DpF and forward primer DsF, each when used with the nonspecific reverse primer BotR, amplified DNA of D. pinea or D. scrobiculata, respectively. Specificity was confirmed using multiple isolates of each of these two species and those of closely related fungi including Botryosphaeria obtusa. The detection limits for DNA of each pathogen in red and jack pine bark were 50 to 100 pg µl-1 and 1 pg µl-1 in red and jack pine wood. The assay was tested using naturally occurring red and jack pine seedlings and saplings exhibiting symptoms of Diplodia collar rot. Samples from lower stems/root collars of 10 dead trees of each species from each of three sites at each of two locations were tested. Results were positive for D. pinea or D. scrobiculata for the large majorities of symptomatic bark and wood samples from both locations. For positive samples, however, there were effects of location and host species on detection of D. pinea (more frequent on red pine) and D. scrobiculata (more frequent on jack pine) (P < 0.01 in both cases). These results indicate that these new primers are potentially useful for studies in areas or hosts in which both pathogens may be present.

Specific and sensitive PCR-based detection of Septoria musiva, S. populicola and S. populi, the causes of leaf spot and stem canker on poplars.

The development of a PCR assay for the detection of the poplar pathogenic fungi Septoria musiva (teleomorph Mycosphaerella populorum), S. populicola (M. populicola) and S. populi (M. populi) is described. Three pairs of species-specific PCR primers were designed using interspecific polymorphisms in the internal transcribed spacer (ITS) of nuclear ribosomal RNA gene (rDNA) repeats. The specificity of the three primer pairs was successfully tested on a collection of 40 S. musiva, 39 S. populicola and six S. populi isolates. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assay was further confirmed with DNA extracted from 12 additional Septoria species and 17 other fungal species obtained from stems or leaves of poplars. Specific amplification of the fragments for S. musiva and S. populicola was sensitive relatively to the technique used, detecting as low as 1 pg template DNA, and 10 pg of DNA of the target species in a background of 1 ng of DNA of the other species. Moreover, using DNA purified directly from disrupted conidia, it was possible to detect with a probability of 90%, using one unique PCR assay, the DNA equivalent of 166 conidia per microl of S. musiva and 156 conidia per microl of S. populicola. The procedures developed in this work can thus be applied for rapid and accurate detection and identification of Septoria species from poplars.

Genetic Structure of Mycosphaerella populorum (Anamorph Septoria musiva) Populations in North-Central and Northeastern North America.

ABSTRACT In order to characterize the genetic variation of the poplar pathogen Mycosphaerella populorum (anamorph Septoria musiva), we have studied seven North American populations using the polymerase chain reaction random amplified polymorphic DNA (RAPD) technique. The fungal populations were sampled in 2001 and 2002 by obtaining 352 isolates from cankers and leaf spots in hybrid poplar plantations and adjacent eastern cottonwood (Populus deltoides). A total of 21 polymorphic RAPD markers were obtained with the six RAPD primers used. A fine-level scale analysis of the genetic structure within the populations revealed that subpopulations sampled on P. deltoides and on hybrid trees were not significantly differentiated. In contrast, analyses performed on the entire data set showed high levels of haplotypic diversity and moderate to high genetic differentiation, with 20% of the expected genetic diversity found at the interpopulation level. Moreover, a high and significant correlation between genetic and geographic distances among populations was found, suggesting isolation by distance of the sampled populations. Although the occurrence of the sexual stage of this fungus remained unclear in field populations, five of the six populations were at gametic equilibrium for RAPD loci, suggesting the occurrence of recombination episodes in Septoria musiva populations. Overall, S. musiva appears to consist of differentiated subpopulations, with both asexual and sexual recombination contributing to the local level of genetic structure.

Prediction of Long-Term Canker Disease Damage from the Responses of Juvenile Poplar Clones to Inoculation with Septoria musiva.

Stem cankers caused by Septoria musiva severely limit production of susceptible hybrid poplars in eastern North America. A field experiment was conducted to determine whether short-term responses of poplar stems to inoculation with S. musiva were predictive of long-term canker disease damage. Stems of 27 poplar clones were inoculated during their first season of growth by removing the fourth or fifth fully expanded leaf and placing an agar plug colonized by an aggressive isolate of S. musiva over the resulting wound. Four months after inoculation, incidence of cankers, canker length, and percent of stem circumference affected (girdle) were recorded. Clones varied greatly in canker incidence (12 to 98%), mean canker length (10 to 53 mm), and mean girdle (14 to 94%). Logistic regression analysis was used to compare these inoculation responses with canker disease damage categories assigned on the basis of information from longer-term field studies. Incidence, canker length, and girdle data were all informative, but girdle data from this field experiment correctly predicted assigned canker disease damage categories most frequently (24 of 27 clones). Responses of 15 of these clones also were evaluated in a similar greenhouse experiment. Although responses usually were predictive of long-term damage categories, the probability of correct prediction was lower than that obtained in the field experiment for most clones. These results demonstrate the feasibility and potential benefit of screening juvenile poplar clones for responses to inoculation with S. musiva before undertaking field trials for productivity and subsequent release to growers.

Differentiation of a Fusicoccum sp. Causing Panicle and Shoot Blight on California Pistachio Trees from Botryosphaeria dothidea.

A panicle and shoot blight disease of pistachio trees in California is caused by a fungus previously identified as the anamorph of Botryosphaeria dothidea. We have compared random amplified polymorphic DNA (RAPD) markers, nuclear rDNA internal transcribed spacer (ITS) region sequences, and conidium morphology of 15 isolates of the pistachio Fusicoccum to those of well-characterized isolates of B. dothidea, B. ribis, and F. luteum. Cluster analysis of RAPD markers separated the pistachio Fusicoccum isolates from B. dothidea, as did parsimony analysis of the ITS region sequences. Conidium size and shape were similar to those of B. ribis (Fusicoccum sp.) and F. luteum, but distinguishable from those of F. aesculi (the anamorph of B. dothidea). We conclude that the fungus causing panicle and shoot blight of pistachio is distinguishable from B. dothidea and is part of a complex containing B. ribis, F. luteum, and other fungi with Fusicoccum anamorphs.