A single amino acid mutation in Spo0A results in sporulation deficiency of Paenibacillus polymyxa SC2.

Sporulating bacteria such as Bacillus subtilis and Paenibacillus polymyxa exhibit sporulation deficiencies during their lifetime in a laboratory environment. In this study, spontaneous mutants SC2-M1 and SC2-M2, of P. polymyxa SC2 lost the ability to form endospores. A global genetic and transcriptomic analysis of wild-type SC2 and spontaneous mutants was carried out. Genome resequencing analysis revealed 14 variants in the genome of SC2-M1, including three insertions and deletions (indels), 10 single nucleotide variations (SNVs) and one intrachromosomal translocation (ITX). There were nine variants in the genome of SC2-M2, including two indels and seven SNVs. Transcriptomic analysis revealed that 266 and 272 genes showed significant differences in expression in SC2-M1 and SC2-M2, respectively, compared with the wild-type SC2. Besides sporulation-related genes, genes related to exopolysaccharide biosynthesis (eps), antibiotic (fusaricidin) synthesis, motility (flgB) and other functions were also affected in these mutants. In SC2-M2, reversion of spo0A resulted in the complete recovery of sporulation. This is the first global analysis of mutations related to sporulation deficiency in P. polymyxa. Our results demonstrate that a SNV within spo0A caused the sporulation deficiency of SC2-M2 and provide strong evidence that an arginine residue at position 211 is essential for the function of Spo0A.

First Report of Blackleg Caused by Leptosphaeria maculans on Canola in Idaho.

Canola (Brassica napus L.) is produced in the dryland agriculture areas of eastern Washington State and northern Idaho, often in rotation with cereal cropping systems. Canola is also used as a rotation crop in irrigated circles in the Columbia Basin of Washington and southern Idaho, where potato is the main cash crop. In 2011, 7,700 ha of canola were harvested in Idaho and 4,200 ha in Washington. One of the major diseases of canola around the world is blackleg, caused by Leptosphaeria maculans (aggressive) and L. biglobosa (non-aggressive). Both Washington and Idaho have been considered blackleg-free, and production of canola in Idaho is subject to government regulations. Canola seed originating from outside of Washington and Idaho should have a phytosanitary certificate. This disease is widespread in Canada and the U.S. Northern Plains, Midwest, and South, and is the major disease of canola in these areas. In August 2011, a sample from a canola field in Bonners Ferry, Idaho, was brought for diagnosis to Washington State University. The canola stems showed the typical gray to dark grey lesions with black pycnidia. The pycnidia and conidia were examined microscopically, and found to be similar to descriptions of Phoma lingam, the anamorph of L. maculans (2). Samples were sent to the University of Manitoba for confirmation with PCR. The pathogen was cultured out of stems on V8 juice agar amended with streptomycin and 22 single pynidiospore isolates were made from the cultures. DNA was extracted from the cultures using methods described in Fernando et al. (1) and a multiplex PCR was performed with species-specific primers for L. maculans and L. biglobosa. The reaction should produce a 330-bp amplicon for L. maculans and a 440-bp amplicon for L. biglobosa. Based on this, all 22 isolates were identified as L. maculans. The susceptible cultivar Westar was inoculated with the isolates, by wound inoculating 7-day-old cotyledons with a concentration of 107 spores/ml. Plants were kept in a moist chamber at 23°C. After 14 days, plants were rated for disease with a 0 to 9 scale, where 0 = no infection and 9 = tissue collapse and appearance of pycnidiospores. Isolates with rating ≥5 are considered virulent. All isolates produced a rating of 7 to 9, indicating a high level of virulence. The source of the seed used in the infested fields is not known at this time. This disease is seedborne, and may pose a threat to the two major vegetable and oilseed brassica seed production areas of Washington: the Skagit River valley of western Washington and the Columbia Basin area of central Washington. In addition, the susceptibility of Pacific Northwest varieties of canola and other brassica oilseeds is largely unknown. References: (1) W. G. D. Fernando et al. Plant Dis. 90:1337, 2006. (2) S. Roger Rimmer et al. Compendium of Brassica Diseases, APS Press, 2007.

Population Structure, Chemotype Diversity, and Potential Chemotype Shifting of Fusarium graminearum in Wheat Fields of Manitoba.

This study was to investigate the variation of acetyl ester derivative of DON at 15-position oxygen (15ADON) and acetyl ester derivative of DON at 3-position oxygen (3ADON) chemotypes and potential chemotype shifting of Fusarium graminearum based on the population structure of this species in Manitoba. The study was conducted in 15 locations with wheat cvs. Superb and AC Barrie in Manitoba from 2004 to 2005. Percentages of chemotypes 3ADON and 15ADON of F. graminearum ranged from 0 to 95.7 and 4.3 to 100%, respectively. The 3ADON chemotype was distributed in the southern part of Manitoba and predominant in Morris and Horndean. The two chemotypes almost shared the same percentage in Portage la Prairie. The 15ADON chemotype was predominant in the other locations. Significant gene flow was found among the populations from Sanford, Portage la Prairie, Hamiota, Plumas, Rapid City, and Virden; the populations from Cartier, Rivers, Killarney, and Souris; and the populations from Morris, Kenville, and Dauphin. There were no differences between the populations from two wheat cultivars and two chemotypes. The great variation of chemotype likely resulted from the great genetic diversity of F. graminearum. Sexual recombination, population age, and cropping system could result in genetic and chemotypic diversities. Wheat seed shipment and long-distance spore transportation of F. graminearum potentially caused the genetic migration and chemotype shifting in Manitoba.

Development of a SCAR Marker to Track Canola Resistance Against Blackleg Caused by Leptosphaeria maculans Pathogenicity Group 3.

Blackleg of rapeseed and canola (Brassica napus) is caused by various pathogenicity groups (PG) of Leptosphaeria maculans. The disease occurring in the Canadian prairies for the last two decades was caused by PG2 and was controlled by host resistance. PG3 and PG4 isolates have been found recently in Canada, but there is no resistance available against these pathogenicity groups in commercial Canadian varieties. This study sought to identify canola cultivars that could be used as sources of resistance to PG3 and to develop molecular markers for marker-assisted selection. Resistance to PG3 specifically was found in B. napus 'Dunkeld' and 'Quinta', while B. juncea 'Cutlass' and 'Domo' proved to be resistant to PG2, PG3, and PG4. A set of F2 progeny of 'Westar' (susceptible) × 'Dunkeld' was used to identify genetic markers linked to PG3 resistance. These markers were physically located on a BAC clone from B. rapa subsp. pekinensis containing a homolog to a serine threonine 20 (ste20)-like kinase in Arabidopsis thaliana. Thus, we have developed a sequence characterized amplified region (SCAR) marker available for marker-assisted selection in breeding canola for resistance against blackleg caused by L. maculans PG3. This work has received a provisional patent (serial # 60/977,933 - Oct. 5, 2007).

First Report on the Presence of Phoma Blackleg Pathogenicity Group 1 (Leptosphaeria biglobosa) on Brassica napus (Canola/Rapeseed) in Iran.

Canola/rapeseed (Brassica napus L.) is a new crop in Iran, grown since 1996. In 2006, 180,000 ha were planted. During the same year, leaf and upper stem lesions (3) were observed on cv. Hyola 401 at rosette and flowering stages in the Gorgan Province in northern Iran. Field disease incidence ranged from 1 to 40%. Several isolates from stem lesions were sent to the Department of Plant Science, Blackleg Research Lab, University of Manitoba, Canada from the Agricultural and Natural Resources Research Center of Golestan Province of Iran for pathogenicity group identification. The blackleg pathogen is divided into several pathogenicity groups on the basis of phenotypic interaction (IP) of isolates on differential cvs. Westar, Glacier, and Quinta. Isolates PG2, PG3, PG4, and PGT are highly virulent, but PG1, which recently has been named Leptosphaeria biglobosa (2), is weakly virulent. Colonies of the blackleg pathogen were reisolated from their original medium, potato dextrose agar, and grown onV8 agar medium and incubated under light for 2 weeks. Pure cultures of the pathogen were then characterized by colony morphology, pycnidia, and measurement and microscopic observation of pycnidio-spores. Fungal colonies formed with concentric rings containing pycnidia with pink ooze on V8 agar. Pycnidia were globose and as much as 200 µ 200 µm. They had a prominent beak on the ascomata that was enlarged, cylindrical, central, terete, erect, and 150 to 200 × 100 µm. Pycnidiospores were cylindrical, straight, 4 to 5 × 2 µm, and hyaline (2). To identify the pathogenicity group of the Iranian isolates, pycnidiospores were harvested from single-spore cultures after 14 days of incubation under continuous cool-white fluorescent light (1). One-week-old cotyledons from the differential cvs. Westar, Glacier, and Quinta were inoculated with pycnidiospore suspension concentration of 2 × 107 spores per ml of the four Iranian isolates. Each cotyledon lobe was punctured with forceps and inoculated with a 10-µl droplet of spore suspension. Disease evaluations were made 10 to 14 days after inoculation using a 0 to 9 rating scale. Inoculations were repeated twice with identical results yielding only the PG1 type reaction. To our knowledge, this is the first report of the presence of L. biglobosa (PG1; B-type) on canola in Iran. Differential testing fulfilled Koch's postulates. L. biglobosa seems to be less damaging compared with L. maculans, but severe phoma stem lesion epidemics have been associated with the L. biglobosa in Poland (3). The importance of this weakly virulent pathogen, whenever the relative humidity increases, has been demonstrated in greenhouse conditions (A. El-Hadrami, W. G. D. Fernando, and F. Daayf, unpublished data). Since the relative humidity in northern Iran is high, an epidemic may occur if appropriate management practices are not utilized to minimize inoculum levels. References: (1) W. G. D. Fernando and Y. Chen. Plant Dis. 87:1268, 2003. (2) R. A. Shoemaker and H. Brun. Can. J. Bot. 79:412, 2003. (3) J. S.West et al. Plant Pathol. 48:161, 2001.

Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction.

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.

A GacS deficiency does not affect Pseudomonas chlororaphis PA23 fitness when growing on canola, in aged batch culture or as a biofilm.

Pseudomonas chlororaphis PA23 is a biocontrol agent that protects against the fungal pathogen Sclerotinia sclerotiorum. Employing transposon mutagenesis, we isolated a gacS mutant that no longer exhibited antifungal activity. Pseudomonas chlororaphis PA23 was previously reported to produce the nonvolatile antibiotics phenazine 1-carboxylic acid and 2-hydroxyphenazine. We report here that PA23 produces additional compounds, including protease, lipase, hydrogen cyanide, and siderophores, that may contribute to its biocontrol ability. In the gacS mutant background, generation of these products was markedly reduced or delayed with the exception of siderophores, which were elevated. Not surprisingly, this mutant was unable to protect canola from disease incited by S. sclerotiorum. The gacS mutant was able to sustain itself in the canola phyllosphere, therefore, the loss of biocontrol activity can be attributed to a reduced production of antifungal compounds and not a declining population size. Competition assays between the mutant and wild type revealed equivalent fitness in aged batch culture; consequently, the gacS mutation did not impart a growth advantage in the stationary phase phenotype. Under minimal nutrient conditions, the gacS-deficient strain produced a tenfold less biofilm than the wild type. However, no difference was observed in the ability of the mutant biofilm to protect cells from lethal antibiotic challenge.

Genetic Diversity of Gibberella zeae Isolates from Manitoba.

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight, one of the most important diseases of cereals in the Canadian prairies for the last decade. In 2002, 60 isolates of G. zeae were collected and single spored from naturally infected spikes of wheat from Carman and Winnipeg in Manitoba. These isolates were compared using vegetative compatibility analysis and polymerase chain reaction (PCR)-based sequence related amplified polymorphisms (SRAP). Sixteen vegetative compatibility groups (VCG) were found among the 50 isolates tested. Five VCGs were found in the two locations, five in Carman and six in Winnipeg. Eight SRAP primer pairs amplified 90 polymorphic DNA fragments from 60 isolates and identified 59 distinct haplotypes. Among seven pairs of isolates, each pair from a distinct spike, four had isolates with different VCGs and six comprised different SRAP haplotypes. Principal component analysis and UPGMA separated the dataset into two main groups, each with isolates from both locations. The analysis of molecular variance also revealed that 75 and 20% of the variance was associated with differences among individual isolates and varieties sampled, respectively. Geographic location was not a significant source of variation at P = 0.05 and accounted for only 4% of total variance. A low correlation between VCG and SRAP marker data was detected. This study showed that, although genetic diversity is high among G. zeae isolates, Carman and Winnipeg collections have a similar genetic makeup and are likely part of the same population. The significant proportion of variance accounted by the variety compared with the geographic origin of isolates suggests that seedborne inoculum might have contributed to the genetic diversity within the G. zeae collection under study.

First Report of Pathogenicity Group-3 of Leptosphaeria maculans on Winter Rape in Hungary.

Blackleg, caused by Leptosphaeria maculans, is an increasing threat to winter rape (Brassica napus L.) in Hungary. The winter rape acreage has been increasing, and the occurrence and severity of the disease has become widespread in all rapeseed-growing regions throughout Hungary in a very short time. The blackleg-infected rape stubbles were collected in the autumn of 2003 in Ikervár, County Vas where the disease was severe. Ascospores were obtained from pseudothecia growing on infected rape stubble (susceptible cvs. GK Helga and Aladin). Three single-spore cultures were grown on V8 agar medium at room temperature and fluorescent light. The culture characteristics fit the type culture description for L. maculans. Pycnidiospores that formed on V8 plates were flooded with 10 ml of sterile distilled water. Seeds of cvs. Westar, Glacier, and Quinta obtained from the Department of Plant Science, University of Manitoba, Canada were sown in plastic pots containing peat mix. Seedlings were maintained in a growth chamber at 24°C with 90% relative humidity and a 16-h photoperiod. Seven days after sowing, cotyledons were wound inoculated with a 10-µl droplet of pycnidiospore suspension (1.5 × 107 spores ml-1). Interaction phenotypes (IP) were scored 10 days after inoculation using a 0 to 9 scale (1). All three isolates from Ikervár were highly virulent on cvs. Westar (8.8 to 8.9) and Glacier (8.1 to 8.3) and avirulent on cv. Quinta (0.8 to 0.9). The IP ratings indicated that these isolates belonged to pathogenicity group-3 (PG-3). To our knowledge, this is the first report of the presence of L. maculans PG-3 in Hungary. At the current time, PG-3 has caused at least 30% yield losses in susceptible cultivars of winter rape. Reference: (1) A. Mengistu et al. Plant Dis. 75:1279, 1991.

Induced Resistance to Blackleg (Leptosphaeria maculans) Disease of Canola (Brassica napus) Caused by a Weakly Virulent Isolate of Leptosphaeria biglobosa.

Blackleg of canola is a disease complex of at least two fungal species: Leptosphaeria maculans and L. biglobosa. Isolates of L. biglobosa typically are weakly virulent or avirulent and are assigned to pathogenicity group 1 (PG-1). Isolates of L. maculans are highly virulent and encompass pathogenicity groups PG-2, PG-3, and PG-4. In greenhouse tests, percent lesion/leaf area (PLLA) on cotyledons of two Brassica napus cultivars, Westar and Invigor 2153, was smaller when L. biglobosa (PG-1) was either pre- or co-inoculated at 0, 12, 24, and 48 h with virulent isolates of L. maculans in PG-2, PG-3, and PG-4. On six-leaf-stage plants of Westar, the PLLA declined significantly compared with the control when the lower leaves were treated with either PG-1 or salicylic acid, then challenged with a PG-2 isolate 24 h later. In addition, the activity of four enzymes (chitinase, ß-1,3-glucanase, peroxidase, and phenylalanine ammonia lyase) was greatly enhanced at 48 and 72 h when cotyledons of Westar were inoculated first with PG-1 followed by PG-2 24 h later, compared with a water control treatment. Field experiments conducted in 2003 and 2004 showed decreased blackleg severity in plants inoculated with PG-1 alone or prior to PG-2 compared with plants inoculated with PG-2 alone or prior to PG-1.

Molecular Detection of Apiosporina morbosa, Causal Agent of Black Knot in Prunus virginiana.

A specific and sensitive polymerase chain reaction (PCR) assay was developed to detect Apiosporina morbosa, the causal agent of black knot disease on chokecherry, Prunus virginiana (including the cultivar 'Shubert Select'). A pair of A. morbosa-specific forward and reverse primers (AMF and AMR) was designed from the internal transcribed spacer (ITS) regions of A. morbosa, preamplified by universal ITS primers ITS1 and ITS4, and compared with the ITS region sequences of Fusarium, Alternaria, Phoma, and Cladosporium species associated with black knots. The primers were tested for their specificity to A. morbosa detection in the PCR assays using DNA derived from 64 pure cultures, including 42 single-spore isolates of A. morbosa and 22 isolates of other fungi, as well as healthy and diseased plant branches collected from the field. A product of ~400 bp was amplified from DNA of all isolates belonging to A. morbosa. No product was amplified from DNA of other fungal species, confirming the specificity of the newly designed primers. Within plant tissues, the pathogen was detected at further distances from the edges of knots on thicker branches bearing larger knots compared with thinner branches bearing smaller knots. The PCR assay has shown high sensitivity, needing only 100 fg of the A. morbosa DNA for a reliable PCR amplification with the AMF and AMR primers.

First Report of Pathogenicity Groups 3 and 4 of Leptosphaeria maculans on Canola in North Dakota.

Blackleg, caused by Leptosphaeria maculans (Desmaz) Ces. & de Not (anamorph = Phoma lingam), is an economically important disease of canola (Brassica napus L.) worldwide and was first detected in North Dakota in 1991 (3). L. maculans can be categorized into one of several pathogenicity groups (PGs) on the basis of the interaction phenotypes in differential canola cvs. Westar, Glacier, and Quinta by using a standard screening protocol in the greenhouse (4). With this system, PG1 strains are weakly virulent and PG2, PG3, and PG4 are highly virulent. The predominant strains of L. maculans in North Dakota are PG1 and PG2 (3). In cooperation with the Oilseed Pathology Lab in the Department of Plant Science, University of Manitoba, blackleg-infested canola stubble was collected arbitrarily from fields in North Dakota during August and September of 2003. Isolates of the pathogen were obtained by plating surface-sterilized (2% NaOCl), collected stubble on V8 agar containing 0.03% chloramphenicol at 22°C under continuous cool-white fluorescent light. Pycnidiospores were harvested from single pycnidia after 14 days of incubation with the Miracloth filtering method (2) and stored at -20°C. Each isolate was passed once through cv. Westar to maintain virulence. Isolates were confirmed as being L. maculans by the presence of characteristic pink pycnidia formed on V8 agar and the characteristic symptoms caused on inoculated cotyledons of cv. Westar. The PG test was performed using a standard screening protocol (4) and was repeated three times for each isolate. For each isolate, 12 7-day-old cotyledons of each differential cultivar were wound inoculated with 10 µl of a pycnidiospore suspension (1 × 107 per ml). Disease severity on cotyledons was assessed 12 days after inoculation with a 0 to 9 scale (0 to 2 = resistant; 3 to 6 = intermediate; and 7 to 9 = susceptible). A total of 106 isolates were obtained from the stubble collected from 47 fields. Of these isolates, three were characterized as PG1, 94 as PG2, six as PG3, and one as PG4; two isolates could not be characterized according to the PG system as described (4). PG3 isolates originated from two fields in Cavalier County and one field in Ward County. The PG4 isolate was from Cavalier County. To our knowledge, this is the first time highly virulent strains of PG3 and PG4 have been detected in North Dakota. PG3 and PG4 strains of L. maculans were found only recently in western Canada (1,2). The discovery of these PGs in North Dakota and western Canada has immense implication to canola breeding programs and blackleg control, since these PGs may cause greater levels of blackleg severity on canola cultivars that are resistant to only PG2 type isolates. References: (1) Y. Chen and W. G. D. Fernando. Plant Dis. 89:339, 2005. (2) W. G. D. Fernando and Y. Chen. Plant Dis. 87:1268, 2003. (3) H. A. Lamey and D. E. Hershman. Plant Dis. 77:1263, 1993. (4) A. Mengistu et al. Plant Dis. 75:1279, 1991.

Seasonal and Diurnal Patterns of Spore Dispersal by Leptosphaeria maculans from Canola Stubble in Relation to Environmental Conditions.

Seasonal and diurnal patterns of spore dispersal by Leptosphaeria maculans, which causes blackleg disease of canola, were studied in two consecutive field seasons using a 7-day Burkard spore sampler and rotorod impaction spore samplers. Ascospores of L. maculans were trapped from mid-June to the end of July, whereas pycnidiospores were trapped from mid-July until the end of July or early August. Ascospores and pycnidiospores were trapped between 9:00 P.M. and 4:00 A.M., when air temperatures were 13 to 18°C and relative humidity was >80%. Peak ascospore and pycnidiospore dispersal was associated with rain events. Peak ascospore dispersal was found to occur several hours after rainfall ≥2 mm, and ascospore dispersal continued for approximately 3 days after such events. Peak pycnidiospore dispersal occurred during the same hours as rain events. More ascospores and pycnidiospores were carried in the direction of prevailing winds than in other directions. To the south of the inoculated area, the gradients of disease incidence and stem disease severity were -19.2 and -0.8 m-1, respectively. Disease incidence and stem severity declined by 50% 12.5 and 5.5 m from the inoculated area, respectively. To the north of the inoculated area, the gradients of disease incidence and stem severity were -21.5 and -0.7 m-1, respectively. Disease incidence and stem severity declined by 50% 14.0 and 5.2 m from the inoculated area, respectively. In 2001, ascospores and pycnidiospores were trapped within 25 m of the inoculated area, whereas pycnidiospores were trapped up to 45 m from the inoculated area.

First Report of Canola Blackleg Caused by Pathogenicity Group 4 of Leptosphaeria maculans in Manitoba.

Leptosphaeria maculans (Desmaz.) Ces. & de Not., causal agent of blackleg of canola (Brassica napus L.), was initially placed in several pathogenicity groups (PG) on the basis of the interaction phenotypes (IP) of L. maculans isolates on the differential canola cvs. Westar (W), Glacier (G), and Quinta (Q) (4). PG1 isolates are weakly virulent and PG2, PG3, and PG4 isolates are highly virulent. In Manitoba, the L. maculans population consists mainly of PG2 isolates (virulent on W and avirulent on G and Q), a few PG1 isolates (avirulent on W, G, and Q), and PGT (virulent on W and Q, but avirulent on G) (3). Since the blackleg fungus is known to have a high level of evolutionary potential, the Oilseed Pathology Laboratory at the University of Manitoba, Winnipeg, Canada, examines the pathogenic variability of L. maculans isolates from the Canadian Prairies and North Dakota each year. During 2002, the presence of PG3 (virulent on W and G and avirulent on Q) was reported in Manitoba (1). During 2003, a canola field located at La Riviere, Manitoba, 200 km southwest of Winnipeg, was found to be severely affected by blackleg. Stubble from this field was arbitrarily collected in mid-April 2004, and 98 single-pycnidia pure cultures were obtained by isolating fungi from surface-sterilized (2% sodium hypochlorite), infested residue, cultured on V8 agar at room temperature under cool-white florescent light for 24 h. Pycnidiospores were harvested after 14 days of incubation using the Miracloth filtering method (1). PG testing was performed using the three differential cultivars in the greenhouse. Known PG2, 3, and 4 isolates, 86-12, Liffole-6, and PL30.2, respectively, were included as positive controls. For each of the 98 isolates, 12 7-day-old cotyledons of each differential cultivar grown in Metro Mix were wound-inoculated with 10 µl of a pycnidiospore suspension (1 × 107 per ml) (1). Inoculated plants were maintained in the greenhouse (16/21°C night/day and a 16-h photoperiod with cool-white florescent light). The experiment was repeated three times. Disease severity on cotyledons was assessed 12 days after inoculation with a 0 to 9 scale (0 to 2 = resistant; 3 to 6 = intermediate; and 7 to 9 = susceptible). Of the 98 isolates tested, five were PG1, 51 were PG2, 24 were PG3, 13 were PGT, and five were PG4. The isolates classified as PG4 gave IP reactions of 7 to 9, 7 to 9, and 6.6 to 8.2, on W, G, and Q, respectively. PG3 was reported one year ago, but highly virulent isolates belonging to PG4 have not been previously detected in Manitoba. To our knowledge, this is the first report of the occurrence of PG4 isolates of L. maculans, and the first report of PG4 causing canola blackleg in Manitoba. The appearance of PG4 may be evidence of pathogen population changes occurring under high-selection-pressure exerted by resistance genes in commercial cultivars (2), or through importation of PG4 isolates with canola seed. References: (1) W. G. D. Fernando and Y. Chen. Plant Dis. 87:1268, 2003. (2) B. J. Howlett. Can. J. Plant Pathol. 26:245, 2004. (3) M. Keri et al. Can. J. Plant Pathol. 23:199, 2001. (4) A. Mengistu et al. Plant Dis. 75:1279, 1991.

Virulence and Genetic Variability Among Isolates of Mycosphaerella pinodes.

Fifty-eight isolates of Mycosphaerella pinodes, collected from western Canada, New Zealand, France, Australia, the United Kingdom, and Ireland, were analyzed for pathogenic and genetic variation according to their virulence on six differential cultivars of field pea (AC Tamor, Bohatyre, Danto, Majoret, Miko, and Radley) and amplified fragment length polymorphism markers. The 56 isolates were classified into 15 pathotypes. Pathotype 1 consisted of 31 isolates that were virulent on all six pea differential cultivars. Pathotypes 14 and 15 consisted of eight isolates that were avirulent on all six differential cultivars or virulent on one of six differential cultivars. The analysis of molecular variance showed that 57.2% of the total variation was caused by differences among populations, and 42.8% was due to molecular diversity within populations. Phylogenetic analysis of molecular variation of isolates showed that most of the Canadian isolates and four Australian isolates formed two clustered groups, respectively, regardless of virulence on the six differential cultivars. Isolates from New Zealand were geographically clustered into two groups. However, the isolates from France, Ireland, and the United Kingdom were clustered with the Canadian isolates.

First Report on the Presence of Leptosphaeria maculans Pathogenicity Group-3, the Causal Agent of Blackleg of Canola in Manitoba.

Blackleg, caused by Leptosphaeria maculans (Desmaz.) Ces. & De Not. (anamorph = Phoma lingam) (Tode:Fr.) Desmaz.), is an economically important and serious disease of canola (Brassica napus L.) in Australia, Europe, and Canada. L. maculans isolates can be categorized into four pathogenicity groups (PGs) on the basis of the interaction phenotypes (IP) on the differential canola cvs. Westar, Glacier, and Quinta (1) by using a standard screening protocol in the greenhouse. PG1 isolates are weakly virulent and PG2, PG3, and PG4 isolates are highly virulent. In Manitoba, L. maculans population consists mainly of PG2 (virulent on cv. Westar; avirulent on cvs. Glacier and Quinta) and a few PG1 isolates (avirulent on all three differentials). The Oilseed Pathology Lab in the Department of Plant Science, University of Manitoba examines the pathogenic variability of blackleg isolates obtained from Manitoba each year. In 2002, the blackleg-resistant cv. Q2, was found to be severely infected in Roland, Manitoba. The canola stubble collected from a coop trial plot (Roland, Manitoba) and a farm in East Selkirk (60 km northeast of Winnipeg, Manitoba) was isolated for the blackleg fungus. Small pieces of stubble were cut from the pseudothecia forming section and surface sterilized with 1% sodium hypochlorite solution for 3 to 5 min and then rinsed in sterile distilled water. V8 agar medium containing 1% streptomycin sulphate was used to culture the isolates under continuous cool-white fluorescent light for 14 days. Pure cultures of the pathogen were isolated and characterized as L. maculans by means of colony morphology, pycnidia, and microscopic observations of pycnidiospores. Pycnidiospores that formed on V8 plates were flooded with 10 ml of sterile distilled water and then harvested by filtering through sterilized Miracloth and kept at -20°C. The isolates were passed once through cv. Westar to maintain their virulence. The PG test was performed with the three differential cultivars. Two additional cultivars, Q2 (resistant to PG2 isolates) and Defender (moderately resistant to PG2 isolates), were included for comparisons. Twelve 7-day-old cotyledons of each differential cultivar grown in Metro Mix were wound inoculated with a 10-µl droplet of pycnidiospore suspension (1 × 107 pycnidiospores per ml). Inoculated cotyledons were maintained in the greenhouse (16/21°C night/day and a 16-h photoperiod). The experiment was repeated twice. Disease severity on cotyledons was assessed 12 days postinoculation by using a 0 to 9 scale (2). All five isolates from Roland and East Selkirk were highly virulent on Glacier (6.4 to 7.7), Q2 (7.1 to 8.2), and Defender (7.2 to 8.4), but intermediately virulent on Quinta (4.5 to 5.4). This clearly indicated that these isolates were of PG3. Isolates of PG2 have been predominant in Manitoba for the past 25 years, and highly virulent isolates belonging to PG3 had not been detected previously. To our knowledge, this is the first report of the presence of PG3 in L. maculans in Manitoba. References: (1) A. Mengistu et al. Plant Dis. 75:1279, 1991. (2) P. H. Williams. Crucifer Genetics Cooperatives (CrGC) Resource Book, University of Wisconsin-Madison, 1985.

First Report of Blackleg Disease Caused by Leptosphaeria maculans on Canola in Brazil.

Canola (Brassica napus L.) is a relatively new crop in Brazil, having been grown there for approximately 8 years. In 2000, leaf lesions and stem cankers were observed in cvs. Hyola 420 and Hyola 401 in farmers' fields in the state of Rio Grande do Sul. Cankered stems were received at the University of Manitoba, Canada, from Rio Grande do Sul for disease identification. Small pieces of the stem were cut from the cankered area, and standard protocol was followed to surface sterilize the stem pieces. Stem pieces were plated on V8 agar medium and incubated under light for 12 days. Typical fungal colonies with concentric rings containing pycnidia formed on the V8 agar. The colony characteristics were typical of the blackleg pathogen, Leptosphaeria maculans (Desmaz.) Ces. & De Not. (anamorph = Phoma lingam) (Tode:Fr.) Desmaz.). Blackleg is an economically important and serious disease in many parts of the world including Australia, Canada, the United States, and Europe. L. maculans strains can be characterized in four pathogenicity groups (PG1 through PG4) based on differential testing procedures giving interaction phenotype (IP) reactions (2). Two weeks after plating on V8 media, plates were flooded with sterile distilled water, and pycnidiospores were harvested. Flats of multipots filled with Metro Mix were seeded with three cultivars (Westar, Glacier, and Quinta). One-week-old cotyledons from the three cultivars were inoculated with pycnidiospore suspensions (2 × 107 pycnidiospores per ml) of seven Brazilian isolates, numbered 7, 8, 9, 11, 15, 16, and 18, respectively. Each cotyledon leaf, punctured in the center with a needle, was inoculated with a 10-µl droplet of the inoculum. Disease evaluations were made 11 days after inoculation using a 0 to 9 rating scale (1). This screening was repeated three times from February 2001 to October 2001. After the second repeat, the isolates from Rio Grande do Sul were passed through the highly susceptible canola cv. Westar. Results from all four trials were consistent, and yielded one PG1 isolate (No. 7) and six PG3 isolates. PG1 is classified as a nonaggressive strain, whereas PG3 isolates are classified as aggressive. PG3 isolates would have an IP reaction of 7 to 9, 7 to 9, and 3 to 6 on cvs. Westar, Glacier, and Quinta, respectively. PG2 is the most commonly found aggressive strain in the Canadian prairies. PG3 is predominantly found in Australia, the United Kingdom, and the United States. To our knowledge, this is the first report of blackleg disease caused by L. maculans on canola in Brazil. Differential testing fulfilled Koch's postulates and determined the PG groups found in Brazil (PG1 and PG3). References: (1) P. A. Delwiche. Genetic aspects of blackleg (Leptosphaeria maculans) resistance in rapeseed (Brassica napus) Ph.D. thesis. University of Wisconsin-Madison, 1980. (2) A. Mengistu et al. Plant Dis. 75:1279, 1991.