James E. Rahe

Phylogenetic relationships among Neofabraea species causing tree cankers and bull's-eye rot of apple based on DNA sequencing of ITS nuclear rDNA, mitochondrial rDNA, and the β-tubulin gene

Three fungal species responsible for anthracnose canker, perennial canker, and bulls-eye rot of apple have been considered members of the genus Pezicula for a number of years. Recent studies, however, have provided evidence to (re-)classify these species as Neofabraea . There has been a long historical debate regarding the taxonomy of two of these fungi. In Europe, both Neofabraea malicorticis and N. perennans have generally been considered N. malicorticis , while in North America a species distinction has been maintained. Phylogenetic analyses of Neofabraea isolates were based on DNA sequences of the internal transcribed spacer region of nuclear rDNA (38 isolates), the mitochondrial rDNA small subunit (partial; 48 isolates), the β-tubulin gene (partial; 25 isolates), and a combined data set (21 isolates). Our work provides evidence for the existence of four distinct Neofabraea apple pathogens including N. malicorticis , N. perennans , N. alba , and a putative new Neofabraea species that was isolated in both Europe and eastern North America. Our results indicate that the primary Neofabraea species causing tree cankers and bulls-eye rot in North America are N. malicorticis and N. perennans in the west, and N. alba in eastern Canada. N. perennans , N. alba , and the undescribed Neofabraea species were found in Europe but the presence of N. malicorticis was not confirmed by our limited sampling. Inclusion of Rosa spp. in the host range of N. malicorticis is merited.

Glyphosate, hypersensitivity and phytoalexin accumulation in the incompatible bean anthracnose host-parasite interaction*

The herbicide glyphosate, an inhibitor of the shikimic acid pathway and aromatic amino acid biosynthesis in plants, reduced the potential of Phaseolus vulgaris to defend itself effectively against races of Colletotrichum lindemuthianum without changing the nature of the interaction phenotype. Glyphosate did not block or diminish the occurrence of the hypersensitive reaction (HR) per se which is characteristic of the incompatible P. vulgaris-C. lindemuthianum host-pathogen interaction. The accumulation of phytoalexins which accompanies the HR was partially suppressed. Glyphosate at its sites of application sometimes enabled infection hyphase to grow from hypersensitive cells. Consequently, a spreading lesion formed which resulted in the collapse of the plant. The occurrence of glyphosate-induced spreading lesions was increased by treatments (e.g. light exposure, removal of cotyledons) that presumably competed for or depleted phenylalanine reserves in the plant. Spreading lesions developed at high frequency at wound sites on inoculated, glyphosate-treated plants. The increased frequency of spreading lesions caused by wounding cannot be accounted for solely by the presumed effect of glyphosate on accumulation of phytoalexins. The data indicate that infection hyphase of C. lindemuthianum remain viable at HR sites on P. vulgaris , and additionally, that the HR itself is not responsible for containment of the pathogen. The results support the conclusion that phytoalexin accumulation is a major feature of HR and a determinant of resistance in this host-parasite system.

Glyphosate treatment of bean seedlings causes short-term increases in Pythium populations and damping off potential in soils

Abstract The effect of bean root residues on populations of known isolates and unidentified Pythium species in soils was assessed, with special regard to herbicide treatment of bean seedlings. The general Pythium population in a muck soil was significantly increased 6 days after foliar treatment of bean seedlings with glyphosate, and by soil amendment with heat-killed bean roots, but not by amendment with roots of healthy bean seedlings. The enhanced populations returned to near initial levels 4 days later. Isolated populations of Pythium ultimum (a glyphosate-synergistic isolate) and Pythium coloratum (a non-synergistic isolate) in a sandy loam soil were enhanced approximately 10-fold over control by roots of healthy bean seedlings and by roots of seedlings killed with either glyphosate or paraquat. Distinct peaks in the population responses of P. ultimum and P. coloratum occurred at 9 and 18 days after treatment, respectively. Strong positive correlations were observed between the population estimates obtained by dilution plating and damping off of sunflower for both P. ultimum and P. coloratum. These results suggests that herbicide treatment of plants can cause temporary increases in both Pythium populations and damping off potential of soils.

Comparative efficacy of induced resistance for selected diseases of greenhouse cucumber.

(1990). Comparative efficacy of induced resistance for selected diseases of greenhouse cucumber. Canadian Journal of Plant Pathology: Vol. 12, No. 1, pp. 16-24.

Fungal colonization of glyphosate-treated seedlings using a new root plating technique

A new technique for assessing the number and location of fungal colonizers of entire root systems was developed, and the effect of the herbicide glyphosate on fungal colonization of roots evaluated. Fungal colonization of roots of wheat and green bush bean seedlings grown at a 25:18 °C day:night regime took place less than 48 h after treatment of the plants with glyphosate. For both plant species grown under each of four environmental conditions (a combination of two temperatures, 17° and 25°, and two soil matric potentials, −6 and −100 kPa), Pythium spp. were the most frequent colonizers of glyphosate-treated seedlings and Fusarium spp. were the second-most frequent colonizers. Colonization of control seedlings by Pythium spp. was only observed in beans grown at 17°. In glyphosate-treated wheat seedlings, less colonization by Pythium spp. occurred at 17° than at 25°, but soil water content had no significant effect. Under low soil water content, colonization by Fusarium spp. was always higher in glyphosate-treated bean or wheat seedlings than in control seedlings, whereas this differential effect was observed only for wheat grown at 25° under higher soil moisture.

Pathogenicity and DNA restriction fragment length polymorphisms of isolates of Pythium spp. from glyphosatetreated seedlings

Sixty Pythium isolates from glyphosate-treated wheat or green bush bean seedlings grown in mineral or organic soils were examined with regard to possible host and site (soil type) specificity. Pythium ultimum dominated the Pythium population isolated from glyphosate-treated root systems planted in organic soil (20 out of 27 isolates), whereas P. sylvaticum was slightly predominant in mineral soil (18 out of 33 isolates). Isolates were compared on the basis of differences in DNA sequences detected by size separation after restriction endonuclease digestion of total DNA. A computational technique to calculate distance matrix from RFLP data was developed. Intraspecific DNA variation did not correlate with host from which the isolates were obtained and there was a correlation between the DNA-based groupings of P. ultimum isolates and the soil type in which the colonized glyphosate-treated seedlings were grown. Two isolates from each fungal species were selected for evaluation of in vivo pathogenicity and host specificity. All four isolates reduced seedling emergence in wheat but only P. ultimum caused a reduction of emergence in beans. There was no indication of host specialization among the isolates tested, i.e. P. sylvaticum or P. ultimum isolates obtained from wheat did not cause a higher incidence of seedling blight of wheat than of beans and vice versa.

Species specific identification of the Neofabraea pathogen complex associated with pome fruits using PCR and multiplex DNA amplification

Five species of pathogenic fungi belong to Neofabraea. One of these, N. krawtzewii (syn. N. populi), is responsible for bark lesions on poplar (Populus) trees. The other four species cause post-harvest bulls eye rot of pome fruits, and at least two of these also cause bark cankers on pome fruit trees. Morphological variation among these species is slight, and overlap in geographic range sometimes occurs. As a consequence, identification based on conventional criteria can be tenuous. PCR primers with putative species specificity were developed following genetic analysis of the beta-tubulin gene for isolates of each of the five species of Neofabraea. PCR conditions required to achieve specificity of the primer sets were determined, and a multiplex PCR protocol was developed to optimize their diagnostic utility on apple fruits. A protocol with higher annealing temperatures in the initial PCR cycles followed by lower temperatures in later cycles gave complete species-specificity when the primer sets were used individually and in multiplex, resulting in successful detection of the pathogens from axenic culture and infected apple fruits.

Neofabraea species associated with bull's-eye rot and cankers of apple and pear in the Pacific Northwest

Species-specific primers for four species of Neofabraea associated with apple and pear fruit diseases were used in multiplex PCR assays to identify 29 putative Neofabraea isolates, primarily isolated from bulls-eye rot on pears from packing houses in Washington and Oregon. Apples were inoculated with these isolates, and tentative identifications based on morphology of conidia forming on wound-inoculated fruits were compared with identifications made using species-specific primers for different Neofabraea species. PCR-based identifications were successful for 26 of 29 isolates, and in all cases were consistent with tentative identifications based on spore morphologies. The results revealed that in addition to the two species of Neofabraea already known to occur in the Pacific Northwest, N. malicorticis and N. perennans, a third species, N. alba, is also prevalent.

Identification and role of Pythium species as glyphosate synergists on bean (Phaseolus vulgaris) grown in different soils

Five Pythium species, P. ultimum, P. sylvaticum, P. irregulare, P. coloratum , and Pythium ‘HS’ group, were identified using morphological characteristics out of 65 isolates obtained from roots of glyphosate-treated bean seedlings grown in five different soils. Various genotypes within the Pythium species were determined from RFLP patterns of total DNA. There were six RFLP types represented in P. sylvaticum , three in P. ultimum , and two each for P. irregulare, P. coloratum and P. ‘HS’ group. The potential of a representative isolate from each RFLP group to enhance the herbicidal action of glyphosate was quantified by estimating glyphosate LD 50 values on bean seedlings growing in sterilized soils amended with each isolate separately. The LD 50 values were computed by logistic regression using plant mortality data gathered 4 wk after treatment of 2-wk-old seedlings with different doses of glyphosate. Twelve of the 15 isolates of Pythium tested were glyphosate synergists on beans. The efficacy of the different isolates as glyphosate synergists varied both between species and among different RFLP types within the same species. The pathogenicity of the representative isolates to beans without glyphosate treatment was also determined. All Pythium species tested were pathogenic to varying degrees on germinating bean seeds and on 2-wk-old bean seedlings. The results indicate that several Pythium species can function as glyphosate synergists and that five different soils all yielded glyphosate synergistic Pythium isolates.