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The herbaceous perennial Lepidium draba L. is an invasive weed of rangelands and riparian areas in North America and Australia. As of 2002, it had infested 40,500 ha of rangeland in Oregon and large areas in Wyoming and Utah. Little is known of plant pathogens occurring on L. draba, especially in the United States, that could be useful for biological control of the weed. Leaf spots were first noted on a stand of L. draba near Shepherd, MT in 1997. The spots were mostly circular but sometimes irregularly shaped and whitish to pale yellow. The pathogen was erroneously assumed to be Cercospora beticola since its morphological traits closely resembled that species and the area had large fields of sugar beet with heavy Cercospora leaf spot incidence. Diseased leaves of L. draba were collected in 1997 and 2007. Conidia, borne singly on dark gray, unbranched conidiophores produced on dark stromata late in the season, were elongate, hyaline, multiseptate, 38 to 120 × 2 to 6 μm (mostly 38 to 50 × 2 to 5 μm) and had bluntly rounded tips and wider, truncate bases. These characteristics were consistent with the description of C. bizzozeriana Saccardo & Berlese (2). To isolate the fungus, spores were picked from fascicles of conidiophores with a fine-tipped glass rod, suspended in sterile water, and spread on plates of water agar. Germinated spores were transferred to potato dextrose agar (PDA). The ITS1, 5.8S, and ITS2 sequences of this fungus (GenBank Accession No. EU887131) were identical to sequences of an isolate of C. bizzozeriana from Tunisia (GenBank Accession No. DQ370428). However, these sequences were also identical to those of a number of Cercospora spp. in GenBank, including C. beticola. We also compared the actin gene sequences of the Montana isolate of C. bizzozeriana (GenBank Accession No. FJ205397) and an isolate of C. beticola from Montana (GenBank Accession No. AF443281); the sequences were 94.6% similar, an appreciable difference. For pathogenicity tests, cultures were grown on carrot leaf decoction agar. Aqueous suspensions of 104 spores per ml from cultures were sprayed on 6-week-old L. draba plants. Plants were covered with plastic bags and placed on the greenhouse bench at 20 to 25°C for 96 h. Koch's postulates were completed by reisolating the fungus from the circular leaf spots that appeared within 10 days, usually on lower leaves. Spores of C. bizzozeriana were also sprayed on seedlings of sugar beet, collard, mustard, radish, cabbage, and kale under conditions identical to those above. No symptoms occurred. After the discovery of the disease in 1997, plants of L. draba in eastern Montana, Wyoming, and Utah were surveyed from 1998 to 2003 for similar symptoms and signs, but none were found. This, to our knowledge, is the first report of C. bizzozeriana in the United States. The initial report of the fungus in North America was from Manitoba in 1938 (1). It has recently been reported as occurring on L. draba in Tunisia (4) and Russia (3) and is reported as common in Europe (2). A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI No. 878750A). References: (1) G. R. Bisby. The Fungi of Manitoba and Saskatchewan. Natl. Res. Council of Canada, Ottawa, 1938. (2) C. Chupp. A Monograph of the Fungus Genus Cercospora. C. Chupp, Ithaca, NY, 1953. (3) Z. Mukhina et al. Plant Dis. 92:316, 2008. (4) T. Souissi et al. Plant Dis. 89:206, 2005.

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

Safflower is an oilseed crop adapted to the small-grain production areas of the western Great Plains, including the Northern Plains Area (NPA). In the NPA, safflower production is being evaluated for potential rotation with sugar beet. Safflower is susceptible to Cercospora carthami, whereas sugar beet is susceptible to C. beticola C. carthami has not been observed on safflower in the NPA but C. beticola is ubiquitous on sugar beet. Observation of unusual leaf spots on irrigated safflower cv. Centennial at Sidney, MT prompted this investigation of safflower as a potential alternate host of C. beticola. Safflower plants were inoculated with four isolates of C. beticola (C1, C2, Sid1, and Sid2) and incubated in growth chambers; leaf spot symptoms appeared between 3 and 4 weeks later. Polymerase chain reaction (PCR) amplification of extracts from lesion leaf tissue with C. beticola-specific primers produced fragments comparable with amplified fragments from purified cultures of control C. beticola. PCR assay of cultures of single spores from diseased safflower leaf lesions also produced fragments comparable with fragments from C. beticola cultures. Antibody that was raised from isolate C2 also bound to antigens from the single-spore cultures of the four C. beticola isolates. Inoculum from single-spore cultures from infected safflower also infected sugar beet and produced typical Cercospora leaf spot symptoms. Assay of these leaf lesions by PCR resulted in amplification of target fragments with the C. beticola-specific primers. Our results demonstrate that safflower is a new host of C. beticola.

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.

We recently isolated turnip vein-clearing virus (TVCV), a tobamovirus which causes vein clearing in Brassica rapa (turnip) and a mosaic in Nicotiana tabacum (tobacco). We present an electron microscopic and molecular characterization of TVCV. Viral particles from lower epidermis peel contained rod-shaped viral particles, typical of tobamoviruses. Viral RNA extracted from infected turnip leaves was used as template for cDNA synthesis prior to cloning in a plasmid vector. Inserts of selected cDNA clones were sequenced to obtain the nucleotide sequence of the 126 K replicase component. The nucleotide and predicted amino acid sequences were 56 to 59% identical to those of most other sequenced tobamoviruses. The least related sequence, that of cucumber green mottle mosaic virus, was more related to the TVCV lineage than it was to those of the other sequenced tobamoviruses. UV spectroscopy suggested a tryptophan content characteristic of the ribgrass mosaic virus (RMV) group. Fragmentation of the TVCV coat protein by cyanogen bromide treatment produced a profile of fragments indistinguishable from those generated from the coat protein of RMV. Thus, while symptoms of TVCV infection on Nicotiana tabacum cv. Samsun and Nicotiana clevelandii differ from those reported for RMV, TVCV appears to be closely related to RMV.

Simultaneous infection of a plant by two viruses can cause more severe disease than is caused by infection with either virus alone. Such synergy may be due to effects on the replication of one virus by the second virus or to other causes. The tobamovirus turnip vein-clearing virus (TVCV), itself causing almost imperceptible symptoms in infected turnips, exacerbated symptoms of infection of turnip by the Cabbage S isolate of the caulimovirus cauliflower mosaic virus (CaMV). The synergy in symptom production was most evident in a reduced size of leaves, providing an objective measure of synergy. In contrast, synergy did not occur when the CM4-184 isolate of CaMV was used in combination with TVCV. Both isolates of CaMV increased the level of TVCV accumulated in leaves. TVCV did not increase the level of the Cabbage S CaMV isolate. The use of Cabbage S-CM4-184 chimeras revealed that a region critical for isolate synergy in stunting was within the coat protein gene and/or the 5' one third of the reverse transcriptase gene. We conclude that the disease symptom synergy between TVCV and Cabbage S CaMV is not caused by altered levels of accumulation of the viruses, but instead reflects subtle genetic interactions mapping to the ORF IV-ORF V region of CaMV DNA.

The exotic, invasive perennial rangeland weed Lepidium draba spreads rapidly and reduces native species diversity. The extensive root system of L. draba constitutes 76% of its biomass (4). Thus, searches have been done for biocontrol agents that target root tissue or that may interact with a weevil, Ceutorhynchus assimilis, that causes galls in the crown area of L. draba. An association of Rhizoctonia spp. with root tissue of plants galled by the weevil has been documented in Europe (3). The possible presence of soilborne pathogens similar to those found in the native range has been the subject of L. draba surveys in the United States. One such survey in 2008 detected a few plants with reddened and chlorotic foliage in a stand near Shepherd, MT. Such symptoms typically indicate the occurrence of soilborne diseases on L. draba in the native range of the weed (2). The site had shown a gradual increase in the range of detectable pathogens beginning with foliar pathogens in 1997. In 2010, at the Shepherd site, L. draba plants with similar (but more severe) symptoms to those seen in 2008 were noted in a different area of the stand. Excavation of the roots in both years revealed brown, sunken crown and root cankers. Pieces of root tissue were excised from the lesions and plated on acidified PDA and Ko and Hora medium. A non-sporulating fungus was isolated from three plants. Colonies of the isolates on PDA were typical of known Rhizoctonia spp. The 2010 isolates were determined to be multinucleate using DAPI and were paired with 14 tester (including subgroups) isolates of AG-1 to AG-4 on water agar. Anastomosis was observed between the multinucleate isolates and the AG-2-1 tester isolate. Sequence analysis of ITS of the rDNA of a multinucleate isolate (GenBank KJ545577) indicated 99% similarity with an accession of R. solani AG 2-1 (AB547381). The 2008 isolates were binucleate. A binucleate isolate, KJ545578, had 100% similarity with an isolate of Rhizoctonia spp. AG-A (AY927356). Pathogenicity tests consisted of planting 6-week-old seedlings of L. draba, one per pot, in ten 85-cm-diameter pots of pasteurized soil mix infested with Rhizoctonia-colonized barley grain that had been dried and milled. An inoculum level of ~8 CFU/g (1) of air-dried soil was established by most probable number calculations from fourfold dilutions of infested soil. Controls were the same number of plants in pasteurized potting mix. Results were recorded after 3 months in a greenhouse at 20–25°C. The test was repeated. Typically, R. solani caused mortality of six to eight plants, from which it was re-isolated, whereas binuclate isolates caused stunting and lower dry weight of L. draba. Control plants remained asymptomatic. This is the first report of R. solani and binucleate Rhizoctonia spp. on L. draba in North America. References: (1) A. J. Caesar et al. Plant Dis. 93:1350, 2009. (2) A. J. Caesar et al. Biol. Control 52:140, 2010. (3) A. J. Caesar et al. Plant Dis. 96:145, 2011. (4) R. F. Miller et al. Agronomy J. 86:487, 1994.

Turnip vein-clearing virus (TVCV) and tobacco mosaic virus (TMV) represent subgroups of tobamoviruses infecting cruciferous and solanaceous plants, respectively. To identify adaptations that may have been necessary in the evolution of the TVCV subgroup from a TMV-like ancestor, the infection of turnip plants by TMV and by chimeras between TMV and TVCV was explored. TMV accumulated at spatially limited sites on inoculated turnip leaves as determined by leaf skeleton hybridization. A plasmid DNA containing a complete TVCV cDNA, when transcribed in vitro, produced RNA that was infectious to tobacco and turnip plants. TVCV-TMV chimeric genomes with junctions within coding regions were not infectious to tobacco, though the movement protein (MP) chimera was infectious to tobacco with a TMV MP transgene. Reciprocal chimeras with junctions between genes were infectious to tobacco. TVCV with a TMV MP gene infected turnips. The other tested chimeras were not detected in non-inoculated leaves, but were found in the inoculated leaves. Thus, the TMV MP is not responsible for the limitation of TMV spread in turnips.

Hieracium caespitosum (meadow hawkweed) is an exotic invasive weed belonging to a complex of hawkweed species infesting nearly 500,000 hectares of pasture and wildlands in North America, primarily in the Pacific Northwest (1). Economic losses can be up to $222 per hectare (2). Despite prolonged effort, no promising insects have been found as agents of biological control of H. caespitosum. This has led to a greater emphasis on seeking plant pathogens such as rusts and smuts as potential biological control agents. Searches for plant pathogens have included Europe, which contains a portion of the native range of the weed. In the small stands of H. caespitosum that occur in northern France, plants were found with chlorosis and significant stunting. Typically, 10 to 30% of plants in affected stands exhibited such symptoms. Excavation and examination of roots revealed discrete necrotic lesions along the length of roots and decayed root tips, roughly resembling symptoms of corky root. Ten roots with these symptoms were thoroughly washed, cut into ~0.25- to 0.5-cm-long pieces and plated on potato dextrose agar (PDA) and acidified PDA. On these media, colonies were light-salmon-beige, woolly, zonate, with older mycelium sparser with the dense occurrence of dark, hemispherical conidiomata. The conidiomata exuded pink spore masses. The spores were hyaline and falcate with acute apices, 6.2 × 1.6 μm (n = 200). These traits match published descriptions of Pilidium concavum, described as a pathogen of numerous plants usually causing leaf spots and stem necrosis (3). To corroborate this identification, the internal transcribed spacer region of rDNA was amplified by PCR using primers ITS1 and ITS4 and sequenced. BLAST analysis of the 575-bp fragment (GenBank Accession No. JX047867) showed 100% homology with the sequences of six isolates of P. concavum in GenBank, including Accession No. AY487094. To confirm pathogenicity, five each of 30-day-old H. caespitosum plants were either sprayed with, or their roots soaked for 1 hr in a 1 × 106 per ml suspension of conidia prepared from 10-day-old cultures of the fungus grown on V8 agar. An equal number of control plants were either sprayed with sterile distilled water (SDW) or their roots were soaked for an hour in SDW. To plant soaked roots, a hole was made in the pre-moistened pasteurized potting mix in each pot and inoculum or SDW was poured around them before they were covered with soil. The experiments were repeated twice. Following leaf inoculations, plants were covered with a plastic bag and placed in the greenhouse in partial shade at 20 to 25°C for 72 to 96 h. Sprayed plants were then uncovered and assessed for the appearance of lesions over the next 7 to 10 days. No foliar symptoms occurred. Plants inoculated by soaking roots showed chlorosis and stunting by 4 months post-inoculation. P. concavum was isolated from root lesions on all inoculated, symptomatic plants, confirming Koch's postulates. Although reported to cause root deterioration of strawberry (4), to our knowledge, this the first report of a root disease of H. caespitosum caused by P. concavum. References: (1) C. A. Duncan et al. Weed Technol. 18:1411, 2004. (2) L. Frid. Economic Impacts of Invasive Plants in BC Final Project Report. British Columbia: Invasive Plant Council, 2009. (3) J. A. von Arx. Plant Pathogenic Fungi. J. Cramer, 1987. (4) J. L. Maas. Compendium of Strawberry Diseases. The American Phytopathological Society, St. Paul, MN, 1998.

The exotic rangeland perennial Lepidium draba occurs as a noxious weed in 22 states, mostly in the western United States. Because chemical control measures against this invasive perennial, a member of the Brassicaceae, have not achieved adequate results, biological control is being pursued. While inventories of arthropods that feed on L. draba have been established, little is known of soilborne pathogens for possible use as biological control agents. To address this deficiency, we have surveyed for diseases of L. draba in the United States and Eurasia to identify and test potential biocontrol agents. In intensive surveys for soilborne diseases in a single infestation that is >20 years old in a cattle pasture in south-central Montana, several chlorotic, stunted plants were noted. Roots of chlorotic plants that exhibited elongated fissures from which other soilborne fungi were isolated also had numerous prominent pycnidia embedded in the crown tissue above the lesions. Examination with a dissecting microscope revealed large ostioles made evident by the wide concave inversions in the short necks of the pycnidia. Culture of root tissue on potato dextrose agar resulted in whitish, becoming pale gray colonies, with a dull peach-to-reddish tinge at the margins, with abundant single pycnidia. Conidia in vitro were mainly unicellular, variable shape, subglobose to ellipsoidal, with several guttules averaging 6 × 2.5 μm. These morphological traits are characteristic of Phoma macrostoma, which is regarded as a weak or wound pathogen. The internal transcribed spacer region of rDNA was amplified using primers ITS1 and ITS4 and sequenced. BLAST analysis of the 575-bp fragment showed a 100% homology with the sequence of an isolate of P. macrostoma that has been investigated extensively for commercialization as a biological control agent of various agricultural weeds (1), including wild mustard (GenBank No. DQ474091). The nucleotide sequence has been assigned GenBank No. HM755951. Pathogenicity tests consisted of making four 1.4-mm-diameter holes in five NaOCl (0.1%)-sterilized root sections of L. draba and pipetting ~50 to 100 μl of a 106 CFU/ml conidial suspension into the incisions, incubating the inoculated roots at 20 to 25°C overnight and planting the root sections, one per pot, in an artificial greenhouse potting mix and placing the pots in the greenhouse at 20 to 25°C. Controls were five root sections that were treated similarly except that sterile water was injected. The experiment was repeated. After 10 days, shoots that grew from inoculated roots were chlorotic and shorter than those produced from control roots. P. macrostoma was isolated from tissue of inoculated roots that became blackened distal to the inoculation points. To examine the host range of P. macrostoma on other brassica species, crowns of 2-week-old seedlings of radish, broccoli, cauliflower, broccoli raab, turnip, kohlrabi, cabbage, Chinese cabbage, mustard greens, and canola were injected with 0.5 ml of a 106 CFU/ml conidial suspension. Plants were grown in the greenhouse at 20 to 25°C for 4 weeks after inoculation and examined for symptoms. The experiment was repeated twice. Blackened root tissue with slight chlorosis occurred only on roots of radish and crowns of broccoli, from which P. macrostoma was reisolated. To our knowledge, this the first report of a disease of L. draba caused by P. macrostoma. Reference: (1) K. L. Bailey et al. U.S. Patent Application Serial No. 60/294,475, Filed May 20, 2001.

Spotted knapweed (SKW), Centaurea stoebe L., is a nonindigenous species that is invasive over large areas in the United States, especially in the west. It has been estimated that infestations of SKW cause $42 million in direct and indirect economic losses annually (2), and the weed could potentially invade 13.6 million ha of rangeland in Montana alone. Extensive efforts toward the control of SKW have included the release of 12 insects for biological control, four of which attack the crowns and roots of this short-lived perennial. To focus efforts to select potential soilborne pathogens, which could be applied in combination with insects, we conducted a survey for plant pathogens in the native range of SKW associated with damage caused by any root-attacking insects. Stunted and chlorotic SKW plants, which were colonized by larvae of Cyphocleonus spp., were found in June 1994 near the Novomar'evskaya Botanical Sanctuary (45°08′49.87″N, 41°51′02.05″E) in the Caucasus Region of Russia. A nonsporulating multinucleate fungus was isolated from the lower stem, crown, and upper root tissue of one such plant. Colonies growing on potato dextrose agar and Ko and Hora media were examined microscopically and identified as Rhizoctonia solani by the occurrence of robust, thick-walled, golden hyphae with right-angled branching and constrictions at the branch points. The anastomosis grouping of the one isolate was determined to be AG 2-2 IIIB after pairing it on water agar with 11 AG tester isolates representing all subgroups of AG 1 to AG 5. The hyphal diameter at the obvious point of anastomosis was reduced and cell death of adjacent cells was observed. In 2007, pathogenicity was determined by planting 12-week-old seedlings of SKW, one per pot, into 20 15-cm-diameter pots of a steamed greenhouse soil mix composed of sphagnum peat, sand, and Bozeman silt loam (1:1:1, vol/vol), pH 6.6, infested with R. solani-colonized barley grain that had been dried and milled. An inoculum level of 8 CFU/g of air-dried soil was determined by most probable number calculations from fourfold dilutions of infested soil. Controls were planted into noninfested soil. In both greenhouse tests, the isolate caused either mortality or a 93% mean fresh weight reduction of surviving plants, relative to the controls, after 8 months. R. solani was reisolated from necrotic root and crown tissue of dead and stunted plants but not from the controls. To our knowledge, this is the first report of R. solani occurring on SKW in Europe. The characterization and pathogenicity of Fusarium spp. isolated from insect-colonized roots of SKW in Europe was reported previously (1). References: (1) A. J. Caesar et al. BioControl 47:217. (2) S. A. Hirsch and J. A. Leitch, North Dakota Agricultural Economics Report No. 355. NDSU, Fargo. 1996.

The exotic, rangeland weed Lepidium draba L., a brassicaceous perennial, is widely distributed in the United States. For example, Oregon contains 100,000 ha of land infested with L. draba (2). Because it is capable of aggressive spread and has the potential to reduce the value of wheat-growing land (4), it is the target of biological control research. The application of multiple pathogens has been advocated for control of other brassicaceous weeds, including the simultaneous application of biotrophic and necrotrophic pathogens (3). In pursuit of this approach, in 2007, we discovered the occurrence of leaf spots on approximately 90% of L. draba plants near Shepherd, MT, which were distinct from leaf lesions caused by Cercospora bizzozeriana (1). The lesions were initially tiny, black spots enlarging over time to become circular to irregular and cream-colored around the initial black spots and sometimes with dark brown borders or chlorotic halos. Conidia from the lesions were light brown, elongate and obclavate, produced singly from short conidia, with 8 to 12 transverse septa, and 2 to 6 longitudinal septa. The spore body measured 25 to 35 × 200 to 250 μm with a beak cell 42 to 100 μm long. On the basis of conidial and cultural characteristics, the fungus was identified as Alternaria brassicae (Berk.) Sacc. Leaf tissues bordering lesions were plated on acidified potato dextrose agar. Colonies on V8 and alfalfa seed agar were black with concentric rings, eventually appearing uniformly black after 10 to 14 days. The internal transcribed spacer region of rDNA was amplified using primers ITS1 and ITS4 and sequenced. BLAST analysis of the 575-bp fragment showed a 100% homology with a sequence of A. brassicae Strain B from mustard (GenBank Accession No. DQ156344). The nucleotide sequence has been assigned GenBank Accession No. FJ869872. For pathogenicity tests, aqueous spore suspensions approximately 105/ml were prepared from cultures grown at 20 to 25°C for 10 to 14 days on V8 agar and sprayed on leaves of three L. draba plants. Inoculated plants were enclosed in plastic bags and incubated at 20 to 22°C for 72 to 80 h. In addition, three plants of the following reported hosts of A. brassicae were inoculated: broccoli, canola, Chinese cabbage, collards, broccoli raab, kale, mustard greens, radish, rape kale, and turnip. Within 10 days, leaf spots similar to those described above developed on plants of radish, canola, Chinese cabbage, and turnip and A. brassicae was reisolated and identified. Control plants sprayed with distilled water remained symptomless. These inoculations were repeated and results were the same. To our knowledge, this is the first report of a leaf spot disease caused by A. brassicae on L. draba in North America. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI No. 878750A). References: (1) A. J. Caesar et al. Plant Dis. 93:108, 2009. (2) G. L. Kiemnec and M. L. McInnis. Weed Technol. 16:231, 2002. (3) A. Maxwell and J. K. Scott. Adv. Bot. Res. 43:143, 2005. (4) G. A. Mulligan and J. N. Findlay. Can. J. Plant Sci. 54:149, 1974.

Hypertrophy and hyperplasia resembling crown galls were found on roots of Euphorbia esula/virgata at a single site in east-central Hungary in 2005. E. esula/virgata, known as leafy spurge in North America, is an invasive species causing substantial economic losses to the value of grazing lands in the Northern Great Plains of the United States and is the target of biological control. E. esula/virgata is widely distributed throughout Eurasia and is found on ditch banks, along roadsides, and in other noncultivated areas in its native range. Large galls on roots resembling crown gall were first noted in 1992 on plants collected for phylogenetic studies from three locations in east-central Hungary. One of these sites was relocated during a 2005 survey and galls were collected from infected plants. Galls were diced and incubated overnight in tubes containing 3 ml of sterile water at room temperature (20 to 25°C). The supernatant was streaked onto plates of potato dextrose agar (PDA), medium 1A, medium 2E, and Roy/Sasser medium. After 7 days, colonies were picked and streaked and subsequently purified on PDA. Of 104 isolates used to inoculate three sunflower plants each (by puncturing roots just below the soil line with a sterile dissecting needle holding a drop of fluid matrix containing bacterial cells), 35 caused galls. Thirty-three isolates were randomly selected from the 104 and used to inoculate three tomato plants each at the soil line. Seventeen caused galls, including two isolates that did not cause galls on sunflower. Finally, none of 20 randomly selected isolates caused galls on kalanchoe plants (Kalanchoe blossfeldiana). Three isolates, which formed the largest galls on sunflower, were used to inoculate five plants of E. esula/virgata growing in a 1:1:1 (peat/sand/Bozeman silt loam) potting mix. The tests were repeated. Galls were visible on inoculated plants within 6 weeks. Diagnostic biochemical tests done prior to and after reisolation indicated that the causal agent was Agrobacterium tumefaciens, which differed from A. rhizogenes in the production of alkali from litmus milk, a positive reaction for the ferric ammonium citrate and 3-ketolactose tests, and negative reactions for tests to detect the production of acid from erythritol and alkali from malonic, l-tartaric, and mucic acid. The three isolates of A. tumefaciens from E. esula/virgata had identical sequences and clustered most closely (99.8 to 99.9% similarity) with five isolates of A. tumefaciens from Tibet and Japan on the basis of cluster analysis using 16S rRNA sequences. Crown gall of E. esula/virgata has also been found in Montana and western North Dakota, and isolates were identified as A. tumefaciens biovars 1 and 2 (1) (the latter is now known as A. rhizogenes). To our knowledge, this the first report of crown gall on E. esula/virgata in Europe. Reference: (1) A. J. Caesar. Plant Dis. 78:796, 1994.