Glen R. Stanosz

Primers for amplification of mt SSU rDNA, and a phylogenetic study of Botryosphaeria and associated anamorphic fungi

Two PCR primers (MSU1 and MSU7) were designed to amplify part of the mt SSU rDNA gene from filamentous ascomycetes. The MSU1-MSU7 and MSU1-NMS2 primer pairs were tested with 32 isolates of 23 species in 10 genera. Minimum evolution, parsimony, and likelihood methods were used for the phylogenetic analyses of mt SSU rDNA sequences from Botryosphaeria species and related taxa. These analyses indicated that: Botryosphaeria is not monophyletic; the separation of Botryosphaeria from Guignardia is supported; except for B. vaccinii , other Botryosphaeria species and related taxa form a monophyletic group with strong jackknife support; Diplodia pinea f. sp. cupressi is different from RAPD marker groups A and B of Sphaeropsis sapinea ; the anamorph genera Sphaeropsis and Diplodia are not indicative of close relationships among different species; and the name B. stevensii may have been applied to more than one species. Botryosphaeria needs additional study and substantial taxonomic revision.

Differentiation of Botryosphaeria species and related anamorphic fungi using Inter Simple or Short Sequence Repeat (ISSR) fingerprinting

Inter simple or short sequence repeat (ISSR) fingerprinting was used to differentiate Botryosphaeria species and related anamorphic fungi. Fifty-one isolates representing 10 different Botryosphaeria species and related taxa, including two RAPD marker groups and one special form of Sphaeropsis sapinea were investigated using five ISSR primers. A total of 171 fingerprint fragments were generated from these isolates, and sizes of fragments were mostly between 200 bp and 1650 bp. Both cluster and principal coordinate analyses were used to examine separately the fingerprint data from the two previously proposed sections Hyala and Brunnea of Botryosphaeria. The results of each analysis indicated that B. dothidea and B. ribis are distinct; B. ribis and B. parva are closely related, but separable; B. mamane is distinct from other Botryosphaeria species with similar morphology or ITS sequences; the A and B RAPD marker groups of S. sapinea and S. sapinea f. sp. cupressi are probably three distinct species; B. obtusa and S. sapinea B group are closely related but distinct; and the name B. stevensii may have been applied to more than one species. This study has shown that ISSR fingerprinting can be a powerful tool for differentiating closely related filamentous fungi with very similar morphologies or ITS sequences, dissecting species complexes, and justifying newly described species.

RAPD marker and isozyme characterization of Sphaeropsis sapinea from diverse coniferous hosts and locations

Two distinct groups (A and B) of the conifer shoot blight and canker pathogen Sphaeropsis sapinea have been recognized on pine hosts in the northcentral United States. Their occurrence in a collection of isolates from much more diverse hosts and locations was studied by analysis of RAPD markers and isozymes. Seventy-nine isolates were used for RAPD marker analysis, and 37 for isozyme analysis, with 33 isolates in common. Hosts included 19 species in Pinus and species of Cedrus, Larix, Picea, Pseudotsuga , located in Africa, Australasia, Europe, and North America. Relationships among these isolates were determined using cluster analyses of presence or absence data for each amplification product or isozyme. Analyses of either RAPD marker or isozyme data placed most isolates in a group that contained previously characterized RAPD marker group A isolates. There was no strong indication of either host or geographic clustering within this highly similar, widely distributed group. Analysis of RAPD marker data placed several other isolates in a separate group that contained previously characterized RAPD marker group B isolates. This is the first report of isolates from hosts other than Pinus banksiana or P. resinosa , and from outside Michigan, Minnesota, or Wisconsin, that have been characterized as members of the RAPD marker group B.

Sphaeropsis sapinea and Water Stress in a Red Pine Plantation in Central Wisconsin

ABSTRACT A study was conducted to determine the effects of water stress resulting from competing vegetation on disease development of Sphaeropsis sapinea in red pine plantations. A 9-year-old plantation was selected in 1992 and experiments were conducted for three consecutive years. Four treatments were assigned at random to individual trees: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. Two isolates of each S. sapinea morphotype (A and B) were used to inoculate wounded lateral shoots. Disease development was measured as the maximum distance below the inoculation site at which necrotic needles were observed. Nonwatered trees with competing vegetation (nontreated condition) had significantly lower predawn needle water potentials (more water stress) and more severe disease development than trees that received the herbicide, water, or combined herbicide and water treatments. The most severe disease occurred in the driest year and the least in the wettest year. Competing vegetation indirectly affected disease development by inducing water stress, even in relatively moist years, on trees previously considered well established. Isolates of morphotype A were more aggressive than isolates of morphotype B. Conclusions from this research have implications for sustainable management of the regions conifer forests.

Effects of Moderate Water Stress on Disease Development by Sphaeropsis sapinea on Red Pine

ABSTRACT The aggressiveness of Sphaeropsis sapinea isolates was compared on water-stressed and nonstressed 3-year-old red pines (Pinus resinosa) in greenhouse and growth chamber experiments. Water was withheld from stressed seedlings to achieve mean predawn needle water potentials (psi(PD)) above -1.9 MPa. The lowest mean psi(PD) of well-watered seedlings was maintained at or above -0.8 MPa. Young shoots were inoculated by placing colonized agar plugs on wounds made by removing a needle fascicle. Two isolates of each recognized morphotype (A and B) were used in the greenhouse experiment and two isolates of morphotype A were used in the growth chamber experiment. After 4 weeks, isolates of morphotype A caused more severe symptoms and could be recovered farther from the inoculation site on water-stressed than on nonstressed trees in both experiments. In the greenhouse experiment, isolates of mor-photype A also caused more severe symptoms and could be recovered farther from the inoculation site than isolates of morphotype B, regardless of watering regime. These results indicate that water stress at levels observed typically in the field can result in increased disease development by isolates of S. sapinea morphotype A on red pine. The reduction of water stress of red pines in the field may reduce losses due to Sphaeropsis shoot blight.

Effects of polyethylene mulch in a short-rotation, poplar plantation vary with weed-control strategies, site quality and clone

Abstract The utility of mulching in various forestry applications remains unclear due to mixed results in field trials. Additionally, few studies have attempted to assess the utility of mulching across a range of conditions, to determine the circumstances that maximize the degree and longevity of mulching-related enhancements. The objective of this study was to gauge the effect of black polyethylene mulch (poly mulch) across a range of site conditions, weed-control treatments and genotypes in a short-rotation, poplar plantation, to describe the circumstances that maximize the benefits of mulching on early growth and survival. Poplar plantations composed of one native clone ( Populus deltoides Bartr. [D105]) and one hybrid clone ( P. nigra L .×P. maximowiczii A. Henry [NM6]) were established in a randomized complete-block design ( 2.4 m ×3 m spacing) during May, 1999, under intensive and minimal weed-control strategies at two sites of different soil quality. Nested within each weed-control treatment was an assessment of tree performance using poly mulch vs. no mulch. Stem volume increments (SVI, dm 3 ) were measured on trees in all treatments during the 1999 and 2000 growing seasons. During establishment (1999), poly mulch enhanced SVI in all treatment combinations. In the second year, the benefit of mulching was restricted to conditions of higher vegetative competition and lower site quality. The relative benefit of poly mulch (i.e., the ratio of SVI under mulched vs. non-mulched conditions) increased in the second year in conditions of high vegetative competition at both sites, while it decreased in the intensively managed plots at both the sites. Tree survival exceeded 90% for both the clones in all treatment combinations, except under the greatest vegetative competition (i.e., high-quality site with minimal weed control), where survival for D105 fell below 40% in both mulched and non-mulched conditions. While poly mulch may improve early growth in short-rotation, woody crop plantations under a wide range of conditions, its practical utility appears to be restricted to specific applications. At the high-quality site in this study, mulching showed little potential as an economically feasible tool under either intensive weed control (due to the rapid attrition of mulching benefits) or minimal weed control (due to low survival and slow growth). Conversely, on marginal sites poly mulch may provide a more attractive management option in both intensive and minimal weed-control applications, particularly for certain clones.

Characterization of Sphaeropsis sapinea from the West Central United States by means of random amplified polymorphic DNA marker analysis

Two morphotypes (A and B) of the conifer pathogen Sphaeropsis sapinea recently have been confirmed as distinct populations by analyses of random amplified polymorphic DNA (RAPD) markers. Because much of the research on Sphaeropsis shoot blight and canker has been conducted in the west central United States, a study was undertaken to determine the morphotype(s) of S. sapinea encountered in this region. RAPD markers were obtained for 42 isolates of S. sapinea collected in Iowa, Nebraska, Oklahoma, and South Dakota from Picea pungens, Pinus contorta, P. nigra, P. ponderosa, P. resinosa, P. sylvestris, and Pseudotsuga menziesii. Relationships among these isolates, and eight other previously characterized isolates, were determined by cluster analysis. All 42 west central region isolates were placed in a single group with the previously characterized A morphotype isolates. This result facilitates interpretation of past research in that region and extrapolation to other areas where the A morphotype of S. sapinea is present.

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.

In vitro inhibition of Sphaeropsis sapinea by natural stilbenes.

The effects of pinosylvin, pinosylvin monomethyl ether, pinosylvin dimethyl ether, and resveratrol on the fungal shoot blight and canker pathogen of conifers Sphaeropsis sapinea were examined in vitro. Effects of compounds, isolates, and concentrations on both conidial germination and mycelial growth were significant (values of P < 0.001), indicating inhibitory activity of these compounds.

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.