Publications by T.D. Ramsfield

Histopathology of the endophytic system and aerial shoots of Arceuthobium americanum infected by Colletotrichum gloeosporioides

Fri, 25 Nov 2011

Infection of the endophytic system of Arceuthobium americanum parasitizing Pinus contorta var. latifolia by the fungus Colletotrichum gloeosporioides was assessed on naturally infected and inoculated localized dwarf mistletoe plants. Two different techniques were utilized to attempt to inoculate C. gloeosporioides into the endophytic system: the fungus was applied as Stabileze formulated product onto cut A. americanum shoots and as a mycelial plug to wounded A. americanum infected host branches. The fungus was cultured from infected shoots and the basal cup region of naturally infected A. americanum, but not pine tissues that included the endophytic system. Following inoculation of cut shoots, the shoots were abscised from the dwarf mistletoe plant, and the fungus was not isolated 1 year after inoculation. The fungus was recovered from the wounded region of infections that were inoculated with a mycelial plug, but not outside of the wounded area. Hyphae with morphology that matched that of C. gloeosporioide were observed microscopically in the shoots and basal cup region of naturally infected A. americanum, but not in the samples from the inoculated treatments. Thus, we were unable to confirm that C. gloeosporioides infected the endophytic system of A. americanum parasitizing P. contorta var. latifolia, although xylem continuity between the aerial and endophytic systems was observed.

The phenology and impact of Caliciopsis arceuthobii on lodgepole pine dwarf mistletoe, Arceuthobium americanum

Fri, 16 Jan 2009

Arceuthobium americanum Nuttal ex Engelmann in Gray (lodgepole pine dwarf mistletoe) causes significant losses to the timber industry. The fungus Caliciopsis arceuthobii (Peck) Barr shows high specificity to Arceuthobium spp., and therefore infection of A. americanum was monitored for 4 years to determine the effect of this fungus on fruit production. The contribution of stand-level and locally produced inoculum in the infection process was also studied to better understand the epidemiology of the pathogen. Over the period of the study, C. arceuthobii caused an average annual fruit reduction of 58%. When the contribution of local and stand-level inoculum was modelled, the results suggested that on A. americanum infections with = 4 ascospore-producing immature fruit, more than half of the new C. arceuthobii infections on the same plant are derived from locally produced ascospores. The effect of the fungus on the host suggests that it has potential as a biological control agent for A. americanum, and further studies are needed to attempt to induce inoculum production in culture.

Identification and molecular characterization of a new double-stranded RNA virus infecting Chondrostereum purpureum

Mon, 16 Mar 2009

A new double-stranded RNA (dsRNA) virus, designated Chondrostereum purpureum cryptic virus (CPCV), belonging to the family Partitiviridae, has been identified in the basidiomycete fungus Chondrostereum purpureum. The virus does not appear to be distributed widely and was detected in only one (PFC2064) of 20 C. purpureum isolates screened. Visualization by electron microscopy revealed isometric virus particles, approximately 30 nm in diameter. Analysis of purified dsRNA revealed two distinct bands that were cloned and sequenced. Double-stranded RNA1 was 1920 base pairs (bp) in size, and dsRNA2 was 1757 bp, excluding the poly(A) tails at the 3' terminus of each fragment. Each RNA contains a single open reading frame (ORF) with short 5' and 3' untranslated regions, 44 and 115 nucleotides (nt) and 90 and 227 nt, respectively. The deduced 587 amino acid (aa) protein (Mr = 68.1 kDa) encoded by the dsRNA1 ORF showed homology to the RNA-dependent RNA polymerase (RdRp) of viruses belonging to the family Partitiviridae. Eight motifs associated with RdRp were identified, including the highly conserved glycine – aspartic acid – aspartic acid sequence. The dsRNA2 ORF encodes the putative coat protein subunit (480 aa, Mr = 52.8 kDa). Basic local alignment search tool analysis of the nucleotide sequence of dsRNA2 revealed no matches in GenBank; however, low identity (16%–24%) was observed for the CPCV coat protein amino acid sequence compared with members of the family Partitiviridae. The dsRNA profile, amino acid sequence alignments, and phylogenetic analyses all indicate that CPCV is most closely related to members of the genus Alphacryptovirus within the family Partitiviridae.

Infection of Arceuthobium americanum by Colletotrichum gloeosporioides and its potential for inundative biological control

Fri, 16 Sep 2005

Inundative biological control of Arceuthobium americanum occurring on Pinus contorta var. latifolia with the fungus Colletotrichum gloeosporioides was investigated. Isolates of C. gloeosporioides were collected throughout British Columbia, Canada, and one isolate was selected for assessment based on its growth and sporulation in culture. The fungus was formulated using the 'Stabileze' method and inoculated onto A. americanum under field conditions. It became established on some replicates and there was a higher incidence of C. gloeosporioides on treated replicates than controls. In some replicates, the treatment reduced fruit production, leading to a decrease in the reproductive capacity of the dwarf mistletoe plant; however, the efficacy was highly variable and not significant.

Biological control approach for management of dwarf mistletoes

Mon, 12 Jul 2004

Dwarf mistletoes (Arceuthobium spp.) are destructive forest pathogens that parasitise commercially important conifer species. Timber losses result from growth reduction, from wood degradation, from increased predisposition to attack by bark beetles, decay, and sapstain fungi, and ultimately from plantation failure. Research and experience in North America have demonstrated the potential use of hyperparasitic fungi as biological control agents for management of dwarf mistletoes. Although much information is available on the mycobiota associated with dwarf mistletoes, significant research and development are required for these to become operational tools. The most promising biological control agents are Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. and Neonectria neomacrospora (C.Booth & Samuels) Mantiri & Samuels which attack shoots and berries, and the endophytic systems of dwarf mistletoe. The use of these two hyperparasitic fungi as potential biological control agents for management of dwarf mistletoes is under investigation. The development of an effective and efficient biological control strategy will reduce the impact of dwarf mistletoes on timber production in areas where traditional silvicultural control, such as retention silviculture or partial harvesting systems, is not practical.

Factors related to seed production by lodgepole pine dwarf mistletoe. (Abstract).

Mon, 22 Jan 2001

To establish which factors are correlated with lodgepole pine dwarf mistletoe seed production, attributes of 67 female dwarf mistletoe plants and the infected host tree were measured. Stepwise multiple regression analysis indicated that the number of dwarf mistletoe shoots, maximum shoot length, dwarf mistletoe swelling diameter and length, and distance of the dwarf mistletoe infection from the stem of the host tree were significantly correlated with fruit production. Host tree volume, branch length, number of lateral branches and the dwarf mistletoe rating were not significant. The calculated r2 of 0.2761 was low, and the number of fruit produced was found to be highly variable. These results suggest that other factors, such as dwarf mistletoe infection age, pollination success and the action of pathogens on dwarf mistletoe seeds, have a significant role in fruit production.

Fungal parasites of lodgepole pine dwarf mistletoe in British Columbia

Sat, 26 Aug 2000

Lodgepole pine dwarf mistletoe (Arceuthobium americanum) is a serious pest in British Columbia (BC) forests. In BC, the trend towards reduced cut-block sizes and partial cutting systems results in infection of the regenerating conifers by seed dispersal from infected adjacent and overstory trees. Biological control using native fungal parasites is currently being studied as an alternative method of control. During the summer of 1998, diseased dwarf mistletoe plants were collected from the interior of BC. Fungi reported to infect lodgepole pine dwarf mistletoe include Caliciopsis arceuthobii, Cylindrocarpon gillii, and Colletotrichum gloeosporioides, and all were found to infect A. americanum in BC. Colletotrichum gloeosporioides was distributed throughout the province and was isolated from infected shoots and berries. Studies will be focused on the effects of Colletotrichum gloeosporioides on the endophytic system, and shoot mortality and regeneration, as these determine dwarf mistletoe seed production over time.

Variation in the mitochondrial DNA of the potential biological control agent Chondrostereum purpureum

Sat, 26 Aug 2000

The mitochondrial DNA (mtDNA) of Chondrostereum purpureum (Pers.:Fr.) Pouzar was extracted and purified, and the size ranged from 51.8 to 66.4 kb. One isolate each from British Columbia, Alberta, Finland, the Netherlands, and New Zealand were found to have identical BamHI mtDNA restriction patterns, resulting in a mitochondrial genome of 63.8 kb. An additional isolate from British Columbia and one from Switzerland had different banding patterns, however, resulting in mitochondrial genomes of 66.4 kb and 51.8 kb, respectively. A sequence-characterized amplified region (SCAR) assay, based on a polymerase chain reaction, was developed to rapidly screen a larger population of 84 isolates from North America, Europe and New Zealand. Two SCARs, one encoding the NADH 4 gene (3 kb) and the second encoding the ATPase VI and cytochrome b genes (5.1 kb), were digested with 24 restriction enzymes. There were no polymorphisms in the NADH 4 containing SCAR, while a single polymorphism was detected by Nsi I in the ATPase VI - cytochrome b containing SCAR. Two mitochondrial haplotypes, which were distributed throughout the sample population were thus identified. The coancestry coefficient (q) for all subpopulations of the sample population was calculated to be 0.0353. The level of gene diversity in the mtDNA of C. purpureum suggested that the chance introduction of novel mitochondrial genes following biological control applications of the fungus is relatively low.

Variation in the mitochondrial DNA of Chondrostereum purpureum

Sat, 26 Aug 2000

The variation in the mitochondrial DNA (mtDNA) of Chondrostereum purpureum, a potential biological control agent for competing hardwood trees in conifer plantations and utility rights-of-way, is currently being assessed. Extraction, purification, and restriction digestion of the mtDNA indicated that it is approximately 67 kb in size, close to the 50-52 kb reported for Schizophyllum commune, a closely related basidiomycete. Restriction digestion of the mtDNA of four isolates, two from British Columbia, Canada, one from Switzerland, and one from Finland, with the restriction endonucleases BamHI and HindIII, showed a high degree of similarity. To screen a larger sample population, a 3000 bp fragment putatively containing the NADH oxidoreductase 4 gene, is being amplified by PCR and digested with restriction endonucleases. Initially, this fragment was amplified from five isolates, each with different ribosomal DNA patterns, and screened for polymorphisms with restriction endonucleases. To date, 18 restriction endonucleases have been tested and none have shown polymorphisms. The uniformity of the total mtDNA banding patterns, and the failure of 18 restriction enzymes to show polymorphisms, suggests that there is a low degree of variation in the mtDNA of Chondrostereum purpureum. Further screening for polymorphisms in this fragment is ongoing at this time, and additional sequences will be screened.

Geographic variation in the mitochondrial DNA of Chondrostereum purpureum

Sat, 26 Aug 2000

The mitochondrial DNA of the biological control agent Chondrostereum purpureum was assessed as an indicator of total genetic variation in a sample population of isolates collected from North America, Europe, and New Zealand. Extraction and purification of the mitochondrial genome allowed calculation of an approximate size of 67 kb. Restriction digestion of the total mitochondrial DNA from eight isolates of C. purpureum with the restriction endonuclease BamHI and comparison of the resultant banding patterns by UPGMA analysis, indicated that isolates from British Columbia, Alberta, Finland, the Netherlands, and New Zealand had exactly the same restriction pattern. Two other isolates had different banding patterns and had similarity coefficients of 0.917 and 0.655 respectively. To study a large sample population, a rapid PCR based assay was developed. PCR primers were designed to amplify two sequence characterized amplified regions (SCARs). The first contained the NADH 4 gene and the second the ATPase VI and cytochrome b genes. Restriction digestion of the first SCAR with 24 restriction endonucleases failed to show any polymorphisms. Restriction digestion of the second SCAR with the endonuclease Nsi I was able to differentiate two mitochondrial haplotypes. A sample population of 84 isolates was screened and both haplotypes were found in all geographic regions surveyed. The calculated value of Nei's gene differentiation statistic, GST, was 0.0935; indicating that the diversity between sub-populations was not significantly different from the diversity within sub-populations. The two degree of variation in the total mitochondrial DNA, the inability of 24 restriction endonucleases to differentiate populations in the first SCAR, and the finding that two mitochondrial haplotypes are distributed throughout the sample population suggest that the risk of introducing rare mitochondrial genes into new regions as part of biological control activities is low.

Molecular analysis of the population structure of Chondrostereum purpureum

Sat, 26 Aug 2000

Chondrostereum purpureum is a potential biological control candidate for competing hardwood vegetation in forest regeneration sites and utility rights-of-way. The population structure of C. purpureum is being assessed to determine the amount of genetic variation within the population and the possible risks involved with movement of individual isolates after deployment in the field. Restriction fragment length polymorphisms (RFLPs) of the ribosomal DNA have been characterized in a sample population of C. purpureum collected world wide. Three unique RFLP patterns were observed: the first was found to be worldwide in distribution, the second was only present in North America, and the third was found only in Europe and New Zealand. Gene flow was observed in the North American population by the near equal distribution of nuclear type patterns in the center of the continent where the distinct eastern and western populations merge. Polymorphisms in the mitochondrial DNA are currently being assessed to further clarify the population structure of this fungus and to devise appropriate biocontrol strategies.

Population structure and dynamics of the biocontrol agent Chondrostereum purpureum (Abstract)

Sat, 26 Aug 2000

The white-rot basidiomycete fungus Chondrostereum purpureum has excellent potential for development as a biological control of North American forest weed species. We are developing molecular genetic markers to study the natural population biology of C. purpureum and to monitor the environmental fate of specific isolates applied as a biological control. We have characterized restriction fragment length polymorphisms (RFLPs) in the intergenic spacer region of the ribosomal DNA (rDNA) repeat which allow differentiation of inter-continental isolates. Frequencies of rDNA nuclear types within North America suggest there have been no significant barriers to gene flow on this continent. Work is in progress to develop molecular markers that can be used to identify specific C. purpureum isolates and to study population structure and dynamics on an intra-continental scale. Several methods, including random amplification of polymorphic DNA (RAPD), sequence-characterized amplified regions (SCAR), and direct DNA sequencing, are being applied.

Assessment of the genetic variability of Chondrostereum purpureum (Abstract)

Sat, 26 Aug 2000

The genetic variation of 111 isolates of the biocontrol agent Chondrostereum purpureum, collected from Europe, New Zealand, and North America, was assessed by analysis of ribosomal DNA (rDNA). The rDNA repeat of one isolate of C. purpureum was cloned into the Lambda vector EMBL-3, and restriction mapped by probing with cloned rDNA. With PCR, it was determined that the 5S gene was transcribed in the same direction as the 26S gene. The large nontranscribed spacer region of the rDNA was amplified by PCR and analyzed by restriction fragment length polymorphisms. Three nuclear type patterns were identified using the restriction endonuclease Hae III. Nuclear type I was found in North American, European and New Zealand isolates. Nuclear type II was detected in isolates collected from North America and nuclear type III was observed in isolates from Europe and New Zealand. Geographic separation is the likely cause of the isolation of nuclear types, allowing identification of isolates from different geographic origins. Gene flow in the North American population was indicated by the observation of two nuclear type patterns, both found in the east and west. Nuclear type I was the predominant nuclear type in eastern North America indicated by a frequency of 0.66 and nuclear type II occurred with a frequency of 0.90 in western North America. The distribution of nuclear type patterns in North America indicates that gene flow is occurring across the continent but that geographic separation has caused nuclear types to be predominant in separated areas.

Development of Chondrostereum purpureum as a biocontrol agent for red alder (Alnus rubra) in utility rights-of-way (Abstract)

Sat, 26 Aug 2000

A study has been initiated to evaluate the bioherbicidal efficacy of the biocontrol agent Chondrostereum purpureum for control of red alder in utility rights-of-way near Duncan, B.C. An experiment was laid out in a randomized complete block design, with 5 blocks, 6 treatments and 20 observations (red alder trees) per treatment within each block. Trees were cut 15-20 cm above ground and assigned one of the following treatments: 1) formulated C. purpureum (PFC 2139); 2) formulated C. purpureum (PFC 2090); 3) Glyphosate (12%); 4) Carbopaste; 5) formulation control; and 6) slash brushing control. Ten months after treatment, measurements of number of live resprouts, maximum resprout height, and dieback of the treated trees were recorded and subjected to analysis of variance. The mean resprout growth for both biological and chemical formulations was significantly different from the slash control but not from the formulation control. Maximum shoot height exhibited a significant difference between the slash control and each of the chemical and biological formulations. Chemical formulation and C. purpureum (PFC 2090) were different from the formulation control. Chemical treatments significantly differed from the formulation control with respect to dieback.

Detection of double-stranded RNA and virus-like particles in Chondrostereum purpureum and their effects on virulence (Abstract)

Sat, 26 Aug 2000

Chondrostereum purpureum is a wound pathogen of hardwood trees and is also under evaluation as a mycoherbicide for unwanted brush species in Canadian conifer plantations and utility rights-of-way. Twenty isolates of C. purpureum collected from different host plants across Canada were examined for the presence of double-stranded RNA (dsRNA) elements and virus-like particles (VLPs). Using agarose gel electrophoresis and scanning electron microscopy, dsRNA and VLPs were found in only one isolate (PFC 2064). Two dsRNA fragments with 1600 and 1800 base pairs were detected on 1% agarose gel, while isometric VLPs of 30 nm in diameter were observed. Curing or eliminating dsRNA elements from isolate PFC 2064 was achieved by protoplast isolation and regeneration. Protoplast suspensions were prepared by digesting mycelial mats with lysing enzymes, and the protoplasts were regenerated in a regeneration medium osmotically stabilized with 0.6 M sucrose. When representative dsRNA-containing parent isolate (PFC 2064), two non-cured protoplast regenerated progeny isolates, and 13 dsRNA-free (cured) protoplast regenerated progeny isolates were inoculated into golden delicious apple and dormant black cottonwood cuttings, lesions produced by dsRNA-free progeny were not significantly larger than those produced by the dsRNA-containing isolates. Ongoing research is focused on screening large populations of Canadian and foreign isolates of C. purpureum to determine the prevalence of dsRNA and to elucidate their biological significance with respect to virulence and other characteristics.

Geographic variation of Chondrostereum purpureum detected by polymorphisms in the ribosomal DNA

Sat, 26 Aug 2000

Variation in the ribosomal (rDNA) repeat was analyzed for 107 isolates of the pathogenic fungus Chondrostereum purpureum, collected from Europe, New Zealand, and North America. The rDNA repeat of a representative Canadian isolate of C. purpureum was cloned into a l vector EMBL-3, and a detailed restriction map was constructed. Variation in the large non-transcribed spacer region of the rDNA was determined for the entire collection of isolates following amplification by the polymerase chain reaction (PCR) and analysis of restriction fragment length polymorphisms (RFLPs). Three distinct nuclear type patterns were identified using the restriction endonuclease HaeIII. Nuclear type I was found in North America, European, and New Zealand isolates. Nuclear type II was only detected in isolates collected from North America, and nuclear type III was observed in isolates collected from both Europe and New Zealand. Nuclear type I was the predominant nuclear type in eastern North America as indicated by a frequency of 0.78, and nuclear type II occurred with a frequency of 0.89 in western North America. Gene flow across the continent was indicated by nearly equal nuclear type distributions (nuclear type I, 0.41; nuclear type II, 0.59) in central North America, but geographic separation has led to unequal nuclear-type distributions across North America.

The differentiation of Chondrostereum purpureum isolates by cycloheximide sensitivity and L-dopa activity

Sat, 26 Aug 2000

Chondrostereum purpureum is a wound pathogen of hardwood trees, and is also under evaluation as a mycoherbicide for unwanted brush species in forestry and utility rights-of-way. A biochemical test (L-DOPA staining) was used to determine the genetic identity. There was no formation of a zone of antagonism (or black line) between the confronting margins of heterokaryon Canadian isolates in the presence of L-DOPA. European and New Zealand isolates when paired with each other or with Canadian isolates produced a black line after similar treatment. Cycloheximide sensitivity was determined by radial growth on cycloheximide-amended malt agar. There was little intraspecific variation in sensitivity among Canadian, European and New Zealand isolates (LD50 0.85-1.50). These biochemical data indicate that C. purpureum isolates share common metabolic pathways. Such genetic uniformity is crucial for the registration of C. purpureum as a mycoherbicide.