Denise R. Smith

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.

Molecular and morphological differentiation of Botryosphaeria dothidea (anawmorph Fusicoccum aesculi) from some other fungi with Fusicoccum anamorphs

Differentiation of Botryosphaeria species with Fusicoccum anamorphs has depended on recog- nition of the anamorphs. This has resulted in some confusion in the taxonomy and our understanding of the pathology of these species because these ana- morphs are very similar morphologically and the characters used for differentiation can be influenced by the substrate. We have examined 89 isolates iden- tified by the collectors as B. dothidea (F aesculi), B. ribis (Fusicoccum sp.), B. parva (F. parvum), or F lu- teum. Cluster analysis of random amplified polymor- phic DNA (RAPD) marker data identified three dis- tinct groups, which we have designated Bd (probable B. dothidea), Br (probable B. ribis, but including iso- lates of B. parva), and Fl (probable E luteum). Anal- ysis of DNA sequence of the nuclear rDNA ITS re- gion from a subset of isolates from each group sup- ports these relationships. Measurement of conidia from a subset of isolates revealed statistically signifi- cant differences in shape that can be used to differ- entiate between conidia of the Bd group isolates and those of the other groups. Conidia of the Br and Fl group isolates, however, were similar. We conclude that B. dothidea can be differentiated from these oth- er fungi with Fusicoccum anamorphs by all three tech- niques.

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.

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.

Response of Alnus tenuifolia to inoculation with Valsa melanodiscus

Abstract Valsa melanodiscus (anamorph Cytospora umbrina) has been associated with cankers, dieback, and death of alder (Alnus) stems in western North America. To determine the ability of this fungus to induce these symptoms, the responses of thinleaf alder (Alnus tenuifolia) stems to inoculation with V. melanodiscus were studied in field locations in Alaska and on plants in a greenhouse. In the field, woody stems were wounded to expose both inner bark and sapwood and inoculated in early May 2007 or September 2007 by placing a colonized agar plug over the wound. Sunken, elongated cankers that developed on inoculated stems in the field closely resembled those attributed to natural infection of thinleaf alders by V. melanodiscus. In contrast, wounded control stems exhibited strong callus production and wound closure. In the greenhouse, actively growing lateral shoots were inoculated by placing a colonized agar plug over a fresh leaf scar. Inoculation in the greenhouse resulted in development of cankers, and severity of symptoms was affected by the maturity of the shoot at the point of inoculation. These results support the conclusion that V. melanodiscus is a cause of alder dieback in western North America.

Primeiro relato da ocorrência de Septoria musiva em álamo no Brasil

O fungo Septoria musiva foi isolado de plantas de alamo com sintomas de mancha foliar e cancro no caule, nos estados do Parana e Santa Catarina, Brasil, em 2004. Testes de patogenicidade e subsequente reisolamento de Septoria musiva confirmaram a hipotese de que este fungo era o agente causal da doenca. Este e o primeiro relato de Septoria musiva em alamo no Brasil.

Detection of the Diplodia shoot blight and canker pathogens from red and jack pine seeds using cultural methods

Abstract The shoot blight and canker pathogens Diplodia pinea and D. scrobiculata commonly and abundantly sporulate on seed cones of red pine (Pinus resinosa) and jack pine (P. banksiana) collected from Wisconsin and Minnesota forests. Cultural methods were used to investigate the incidence of these fungi in seed lots obtained from government nurseries in these states. In each of three replicate trials, seeds of each lot were assigned to four treatments before incubation on semi-selective medium: (1) not surface-disinfested; (2) surface-disinfested; (3) surface-disinfested, then inoculated with D. pinea conidia; or (4) not surface-disinfested, then inoculated with D. pinea conidia. For red pine seeds, the mean percentage positive was 2.7% for treatment 1 and 1.3% for treatment 2. Jack pine seeds were less frequently positive than red pine seeds for both treatments 1 and 2. The Diplodia species cultured was identified as D. pinea in almost every case. Diplodia pinea was much less frequently recovered from seeds that were not surface-disinfested and then inoculated (treatment 4), when compared with seeds that were inoculated with D. pinea after surface-disinfestation (treatment 3). Results confirm the potential for dissemination of D. pinea on red pine and jack pine seeds, and caution is warranted before concluding absence of the pathogen based on results using cultural methods with relatively small numbers of seeds. Although the frequency of pathogen-positive seeds was low, the large numbers of seeds planted in nurseries suggest that seeds may be a potentially important route of entry of D. pinea into nursery beds.