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.

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.