Diurnal effects on sporangium and zoospore production by Phytophthora ramorum on Rhododendron 'Cunningham's White'.

We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.

The Effect of Leaf Wetness on Phytophthora ramorum Zoospore Infection of Rhododendron 'Cunningham's White' and Viburnum tinus.

We performed studies using zoospore inoculum combined from nine isolates of Phytophthora ramorum and determined the effect of leaf wetness on infection of whole plants of Rhododendron 'Cunningham's White' and Viburnum tinus. The mean percentage of infected leaves for both host species increased gradually across a dew chamber moisture period of 1 to 6 h, reaching approximately 80% infection by 6 h. We also evaluated the effect of a postinoculation drying period on infectivity of the two host species with zoospore inoculum. With a 30-min postinoculation drying period, Rhododendron 'Cunningham's White' sustained less than 40% infected leaves, whereas V. tinus had an infection rate of almost 75% infected leaves. Disease percentages for both host species declined sharply with drying periods longer than 30 min. Knowledge of infectivity parameters for P. ramorum will provide a better understanding of epidemic development and lead to improved recommendations for control.

Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases.

Coniothyrium glycines, the causal agent of soybean red leaf blotch, is a USDA APHIS-listed Plant Pathogen Select Agent and potential threat to US agriculture. Sequencing of the C. glycines mt genome revealed a circular 98,533-bp molecule with a mean GC content of 29.01%. It contains twelve of the mitochondrial genes typically involved in oxidative phosphorylation (atp6, cob, cox1-3, nad1-6, and nad4L), one for a ribosomal protein (rps3), four for hypothetical proteins, one for each of the small and large subunit ribosomal RNAs (rns and rnl) and a set of 30 tRNAs. Genes were encoded on both DNA strands with cox1 and cox2 occurring as adjacent genes having no intergenic spacers. Likewise, nad2 and nad3 are adjacent with no intergenic spacers and nad5 is immediately followed by nad4L with an overlap of one base. Thirty-two introns, comprising 54.1% of the total mt genome, were identified within eight protein-coding genes and the rnl. Eighteen of the introns contained putative intronic ORFs with either LAGLIDADG or GIY-YIG homing endonuclease motifs, and an additional eleven introns showed evidence of truncated or degenerate endonuclease motifs. One intron possessed a degenerate N-acetyl-transferase domain. C. glycines shares some conservation of gene order with other members of the Pleosporales, most notably nad6-rnl-atp6 and associated conserved tRNA clusters. Phylogenetic analysis of the twelve shared protein coding genes agrees with commonly accepted fungal taxonomy. C. glycines represents the second largest mt genome from a member of the Pleosporales sequenced to date. This research provides the first genomic information on C. glycines, which may provide targets for rapid diagnostic assays and population studies.

Sporangia Production Over Time by Phytophthora ramorum on Rhododendron 'Cunningham's White' After Placement at Different Relative Humidities.

We examined the impact of relative humidity (RH) on Phytophthora ramorum sporangia production on Rhododendron 'Cunningham's White'. When diseased plants were maintained under continuous moisture in a mist tent, sporangia were collected from some plants for 22 weeks. More than 3,000 sporangia/leaf/week were collected over the first 3 weeks but levels declined to <100 sporangia/leaf/week after 7 weeks. We also examined the impact of drying on P. ramorum sporangia production. Diseased, detached leaves were maintained in humidity chambers (100, 96.2, 84.5, 74.9, and 56.2% RH) for up to 9 weeks and removed weekly to assess sporulation. For comparison, diseased leaves were harvested from plants maintained with dry foliage or subjected to 10 h of simulated dew nightly. All leaves supported sporulation following 5 weeks at 100% RH, 3 weeks at 96.2% RH, and 1 week at 84.5% RH. All leaves collected from plants subjected to nightly dew supported sporulation for 3 weeks; however, only 66.7% of leaves collected from plants with dry foliage supported sporulation after 1 week. Knowledge of the effects of RH levels on P. ramorum sporulation capacity will prove useful in terms of disease management recommendations and for development of predictive models and pest risk assessments.

Development of an Inoculation Technique and the Evaluation of Soybean Genotypes for Resistance to Coniothyrium glycines.

Red leaf blotch, caused by Coniothyrium glycines, is a serious disease affecting soybean in several African countries but has not yet appeared in the United States. The fungal pathogen is listed as a Select Agent by the Federal Select Agent Program because of potential consequence to U.S. agriculture if introduced. Sources of resistance to red leaf blotch have not yet been identified in spite of extensive field testing in Zambia and Zimbabwe. A seedling inoculation protocol was used to investigate the effects of inoculum density, temperature (20 and 25°C), and dew chamber incubation period (2 to 5 days) on disease development in 'Williams 82' soybean. Inoculum density and incubation period had significant effects on disease severity but no significant difference was observed at 20 and 25°C. Highest levels of disease were obtained using 1.94 cm3 of mycelial inoculum per 10 ml of water and incubating plants for 5 days at 100% relative humidity. Significant differences were not observed between isolates from Zambia and Zimbabwe at 25°C. In total, 23 soybean genotypes that represent nearly 90% of the genes present in U.S. soybean were evaluated and found to be susceptible, which is consistent with previous field evaluations. This method could be useful in identifying new sources of resistance to red leaf blotch.

Pyracantha 'Mohave' Fruit Infection by Phytophthora ramorum and Transmission of the Pathogen from Infected Fruit to Roots of Viburnum tinus.

Colonization of the fleshy fruit of Cornus florida, C. kousa, Laurus nobilis, Malus hupehensis, and Pyracantha 'Mohave' was observed following inoculation with sporangia of Phytophthora ramorum. However, abundant production of chlamydospores was only observed in the fruit of Pyracantha 'Mohave'. Pyracantha 'Mohave' fruit that had been inoculated with a P. ramorum sporangia suspension were placed in pots containing rooted cuttings of Viburnum tinus in a misting tent or in water-filled trays in a climate-controlled greenhouse. Runoff was collected for 24 to 30 days, and roots were plated after the final collection. Mean percent recovery from infected roots was not significantly different (P = 0.05, Tukey's test) between bottom-watered treatments in trays and misted treatments, averaging 58% for bottom-watered and 54% for mist treatments. The number of CFU collected in runoff from bottom-watered plants was consistently lower than that obtained from plants held under mist, likely due to desiccation of the fruit. The results show that root infection of V. tinus can occur by P. ramorum via infected fruit of Pyracantha 'Mohave'. This phenomenon represents a pathway of infection for P. ramorum not previously reported, which may play a role in disease epidemiology.

The effect of temperature on germination of chlamydospores of Phytophthora ramorum.

Mycelium-free chlamydospores of 12 isolates of P. ramorum representing three clonal lineages were produced with a method involving incubation in nonsterile sand at 20 C in darkness for 30 d. Chlamydospores were incubated on selective agar medium at 5, 10, 15, 20, 25 and 30 C and germination assessed after 1, 2, 4, 6 and 8 d incubation. The optimal temperature for germination based on 8 d incubation was 20 C for all three clonal lineages tested (NA1, NA2, EU1). Mean germination rates were 2, 21, 44, 67, 32 and 0 percent at 5, 10, 15, 20, 25 and 30 C respectively for all isolates combined. The highest mean germination rate was scored by isolates of the EU1 clonal lineage at 20 C (85%) after 8 d incubation However, substantial variation was observed among isolates within each clonal lineage. Overall temperatures and days of incubation on which germination was assessed isolates of the NA1 clonal lineage had the lowest mean germination, even though one isolate had the highest germination of any isolate in any lineage. The results indicate that 20 C is the optimal germination temperature for P. ramorum chlamydospores and that a great disparity in germination percentage can exist within isolates, even within a single clonal lineage.

Isolation of nine Phytophthora capsici pectin methylesterase genes which are differentially expressed in various plant species.

Phytophthora capsici causes damage on many plants species, and secretes various pectin methylesterases during all stages of infection. We identified nine Pme genes (Pcpme 1-9) from a genomic library of highly virulent P. capsici strain SD33 and further analyzed the expression pattern of nine genes on three hosts: pepper, tomato, and cucumber using qRT-PCR during all stages of infection. All nine genes were found to be differentially expressed in three host species in the course of P. capsici interaction. The expression levels of the respective genes increased from 1 to 7 dpi in pepper, while most genes presented a decreasing trend of expression from 1 to 5 dpi in tomato fruits. However, in both fruits peaks were reached at 7 dpi. In cucumber fruits, each gene showed minor expression levels from 1 to 3 dpi, exhibited definite peaks at 5 dpi, and then decreased from 5 to 7 dpi. Thus, evidence from our studies of Pcpme gene expression in different plants at a rang of time points suggests that the late stages of infection may represent the most critical time for P. capsici to successfully express or/and secret PMEs.

Effect of temperature and moisture period on infection of Rhododendron 'Cunningham's White' by Phytophthora ramorum.

We investigated the temperature and moisture conditions that allow Phytophthora ramorum to infect Rhododendron 'Cunningham's White'. Most experiments were performed with a single P. ramorum isolate from the NA1 clonal lineage. For whole plants incubated in dew chambers at 10 to 31 degrees C, the greatest proportion of diseased leaves, 77.5%, occurred at the optimum temperature of 20.5 degrees C. Disease occurred over the entire range of temperatures tested, although amounts of disease were minor at the temperature extremes. For whole plants exposed to varying dew periods at 20 degrees C and then incubated at 20 degrees C for 7 days, a dew period as short as 1 h resulted in a small amount of disease; however, at least 4 h of dew were required for >10% of the leaves to become diseased. Moisture periods of 24 and 48 h resulted in the greatest number of diseased leaves. In detached-leaf, temperature-gradient-plate experiments, incubation at 22 degrees C resulted in the greatest disease severity, followed by 18 degrees C and then 14 degrees C. In detached-leaf, moisture-tent experiments, a 1-h moisture period was sufficient to cause disease on 67 to 73% of leaves incubated for 7 days at 20 degrees C. A statistical model for disease development that combined the effects of temperature and moisture period was generated using nonlinear regression. Our results define temperature and moisture conditions which allow infection by P. ramorum on Cunningham's White rhododendron, and show that P. ramorum is able to infect this host over a wide range of temperatures and moisture levels. The results indicate that P. ramorum has the potential to become established in parts of the United States that are outside its current range.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Susceptibility to Phytophthora ramorum and Inoculum Production Potential of Some Common Eastern Forest Understory Plant Species.

Twenty-five plant species (21 genera, 14 families), which comprise a portion of the understory in forests of the Eastern United States, were evaluated for susceptibility to infection by Phytophthora ramorum. The degree to which P. ramorum is able to form sporangia and chlamydospores was also assessed on these hosts. Seedlings were spray-inoculated with a mixture (4,000 sporangia/ml) of four P. ramorum isolates followed by incubation in a dew chamber at 20°C in darkness for 5 days. Percent infection on individual leaves/leaflets was assessed visually. Mean percent leaf area infected ranged from 0.7% for Smilax rotundifolia to 93.8% for Kalmia latifolia. Eight plant species tested developed significantly larger lesion areas than those found on susceptible control Rhododendron 'Cunningham's White'. Fourteen species in addition to the susceptible control exhibited infection of over 90% of their leaves. Sporangia production by P. ramorum varied considerably among plant species, ranging from 36 per cm2 lesion area on Myrica pennsylvannica to 2,001 per cm2 lesion area on Robinia pseudoacacia. Numbers of chlamydospores produced per 6-mm-diameter leaf disk incubated in a P. ramorum sporangia suspension ranged from 25 on Ilex verticillata to 493 on Rhus typhina. The results indicate that many common understory species in Eastern U.S. forests are susceptible to P. ramorum and capable of providing ample sources of inoculum (sporangia and chlamydospores) for forest epidemics should the pathogen be introduced and should temperature and moisture conditions exist that are conducive to disease development.

Survival of Phytophthora ramorum hyphae after exposure to temperature extremes and various humidities.

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.

Recovery of Phytophthora ramorum Following Exposure to Temperature Extremes.

We examined the impact of exposure to high and low temperature extremes on recovery of Phytophthora ramorum both as free chlamydospores and within infected rhododendron tissue over a 7-day period. Chlamydospores held in moistened sand were incubated at 30, 35, 40, 0, -10, and -20°C for up to 7 days. Infected Rhododendron 'Cunningham's White' leaf disks held in sandy loam, loam, or sand at two different soil moisture levels also were subjected to these temperatures for up to 7 days, and to a variable temperature regimen for 12 weeks. Recovery was characterized by growth of P. ramorum on selective agar medium following exposures to temperature treatments. Chlamydospores held in moistened sand showed a high rate of recovery at 30°C, steadily declining recovery at 35°C, and no recovery at 40°C over the 7-day period. Chlamydospores were recovered from 0°C after 7 days, with little or no recovery observed at -10 or -20°C. In infected rhododendron tissue, P. ramorum was recovered at 20 and 30°C after 7 days but, at 35°C, the pathogen showed a decline within 2 days and no recovery by 4 days. A 40°C treatment allowed no recovery of P. ramorum from infected tissue after 2 days. For cold treatments, P. ramorum was recovered from infected leaf disks at 0 and -10°C after 7 days. At -20°C, recovery declined rapidly after 1 to 3 days and no recovery was obtained after 4 days. P. ramorum showed nearly 100% recovery from leaf disks subjected to a 12-week variable temperature treatment based on ambient summer temperatures in Lewisburg, TN. The results suggest that P. ramorum is capable of surviving some highly adverse temperature conditions for at least 7 days both as free chlamydospores in sand and within infected host tissue. Thus, P. ramorum present as free chlamydospores or within tissue of infected plants shipped to the eastern United States has the potential to survive some of the adverse conditions encountered in summer and winter in many eastern states.

Susceptibility of Some Eastern Forest Species to Phytophthora ramorum.

We tested some common Eastern forest species for their reaction to stem and leaf inoculation with Phytophthora ramorum, the pathogen that causes sudden oak death. Stem lesions were produced on inoculated seedlings of 12 Eastern forest species following 72 to 76 days of incubation in a containment greenhouse cubicle at 20°C. Chestnut oak (Quercus prinus) and white oak (Q. alba) were followed by northern red oak (Q. rubra) in susceptibility to stem inoculation. Sugar maple (Acer saccharum) and black walnut (Juglans nigra) were more resistant to stem inoculation than most of the oak species tested. P. ramorum was isolated on selective medium at distances of up to 8 mm from stem lesion margins. Foliar lesions were observed within 7 days when seedlings were inoculated with 5,000 sporangia/ml followed by incubation at 20°C in a dew chamber. Chestnut oak was followed by tanoak (Lithocarpus densiflorus) and coast live oak (Q. agrifolia) in susceptibility to P. ramorum by foliar inoculation. Chestnut oak, an ecologically important species in parts of the Eastern United States, emerged as the most susceptible host tested in both stem and foliar inoculations. The results indicate that, under controlled conditions, P. ramorum is able to infect some important Eastern forest species and cause lesions on stems and foliage. It remains to be determined whether infection of these species by P. ramorum would occur under natural conditions.

Growth and sporulation of Phytophthora ramorum in vitro in response to temperature and light.

Phytophthora ramorum, recently found in the US, is causing concern for hardwood forests and the nursery industry. In an effort to identify some of the environmental limitations to growth and sporulation we undertook a laboratory study of four US and three European (EU) isolates. On V8 media, isolates grew when incubated at 2-28 C and produced chlamydospores at 8-28 C. Sporangia were produced at all temperatures tested: 10-30 C for US isolates and 6-26 C for EU isolates. Optimal temperatures were 16-26 C for growth, 14-26 C for chlamydospore production and 16-22 C for sporangia production. US isolates grew less and produced fewer spores when exposed to increasing doses of near-UV radiation (50-300 microW/cm(2)) and visible radiation (250-1500 microW/cm(2)). EU isolates were exposed to 300 microW/cm(2) near-UV only, which significantly reduced growth of one of three isolates and had no significant effect on spore production. In our studies P. ramorum tolerated a broad range of temperature and light conditions, which suggests that it is capable of establishment in a wide geographic area.

Cultural characteristics, morphology, and variation within Claviceps africana and C. sorghi from India.

Sorghum ergot in India is caused by Claviceps africana and C. sorghi. The distributions of these two species in India is not known. Eighty-nine sorghum ergot isolates were cultured from young sphacelia obtained from male sterile sorghum plants artificially inoculated using inoculum collected in the field. Based on cultural characteristics, the isolates were separated into two groups which differed distinctly in the morphology of their sphacelia, conidia, and sclerotia. Marked differences also were observed in rates of secondary conidial production and disease spread between the groups. In combination with molecular evidence, our results confirm that the isolates placed in Group I represent C. africana and Group II isolates represent C. sorghi. C. africana was found to be widely distributed in all sorghum growing areas of India. The species first described as occuring in India, C. sorghi, appears to be restricted to a few locations in the states of Maharashtra, Andhra Pradesh, and Karnataka.

Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions.

ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

NEP1 orthologs encoding necrosis and ethylene inducing proteins exist as a multigene family in Phytophthora megakarya, causal agent of black pod disease on cacao.

Phvytophthora megakarya is a devastating oomycete pathogen that causes black pod disease in cacao. Phytophthora species produce a protein that has a similar sequence to the necrosis and ethylene inducing protein (Nep1) of Fusarium oxysporum. Multiple copies of NEP1 orthologs (PmegNEP) have been identified in P. megakarya and four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora, and P. sojae). Genome database searches confirmed the existence of multiple copies of NEP1 orthologs in P. sojae and P. ramorum. In this study, nine different PmegNEP orthologs from P. megakarya strain Mk-1 were identified and analyzed. Of these nine orthologs, six were expressed in mycelium and in P. megakarya zoospore-infected cacao leaf tissue. The remaining two clones are either regulated differently, or are nonfunctional genes. Sequence analysis revealed that six PmegNEP orthologs were organized in two clusters of three orthologs each in the P. megakarya genome. Evidence is presented for the instability in the P. megakarya genome resulting from duplications, inversions, and fused genes resulting in multiple NEP1 orthologs. Traits characteristic of the Phytophthora genome, such as the clustering of NEP1 orthologs, the lack of CATT and TATA boxes, the lack of introns, and the short distance between ORFs were also observed.

Identification of phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction-amplified region of mitochondrial DNA.

ABSTRACT Polymerase chain reaction primers spanning the mitochondrially encoded coxI and II genes have been identified that were capable of amplifying target DNA from all 152 isolates of 31 species in the genus Phytophthora that were tested. Digestion of the amplicons with restriction enzymes generated species-specific restriction fragment length polymorphism banding profiles that were effective for isolate classification to a species level. Of the 24 species in which multiple isolates were examined, intraspecific polymorphisms were not observed for 16 species, while 5 species exhibited limited intraspecific polymorphism that could be explained by the addition/loss of a single restriction site. Intraspecific polymorphisms were observed for P. megakarya, P. megasperma, and P. syringae; however, these differences may be a reflection of the variation that exists in these species as reported in the literature. Although digestion with AluI alone could differentiate most species tested, single digests with a total of four restriction enzymes were used in this investigation to enhance the accuracy of the technique and minimize the effect of intraspecific variability on correct isolate identification. The use of the computer program BioNumerics simplified data analysis and identification of isolates. Successful template amplification was obtained with DNA recovered from hyphae using a boiling miniprep procedure, thereby reducing the time and materials needed for conducting this analysis.