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.

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.

Pyrenophora teres Drechs. causes net blotch of barley, a common foliar disease in cultivation zones around the world. The disease occurs in two forms, namely a net form net blotch (NFNB) caused by P. teres f. teres and a spot form net blotch (SFNB) caused by P. teres f. maculata. As in other parts of the northern Great Plains, in the Mon-Dak area (western North Dakota and eastern Montana), NFNB is prevalent. SFNB was first reported in western Montana in 1983 (1) and more recently in eastern North Dakota in 2010 (3) but not in the Mon-Dak area. In the summer of 2011, unusual spot lesions that were surrounded by necrosis or chlorosis were observed on different barley cultivars in fields at Williston, ND, Nesson Valley, ND, and Sidney, MT areas. Diseased leaves from various barley cvs. from the three locations were transferred to water agar and incubated at room temperature for 24 h to induce sporulation. Morphological examination of conidia (45 to 169 × 15 to 21 μm) did not show significant differences from a known isolate of P. teres f. teres 0-1 (provided by Tim Friesen, ARS, Fargo, ND). For pathogenicity testing, six 14-day-old plants of barley cv. Tradition were sprayed until runoff with a 2,000 spore/ml suspension of two isolates from each location and the control P. teres f. maculata isolate DEN2.6 (provided by Tim Friesen). Plants were incubated first in a lighted humidity chamber for 24 h and then in a greenhouse for 7 days at 21°C. Regardless of inoculum source, spot lesions surrounded by necrosis or chlorosis, typical of SFNB, appeared on the inoculated leaves within 7 days. Fungi isolated from symptomatic leaves were identified as P. teres and the morphology of the conidia was undistinguishable from those of P. teres f. teres. All control plants which were sprayed with sterile distilled water were symptomless. The pathogenicity test was repeated. Rapid PCR detection and amplicon sequencing (2) of the internal transcribed spacer (ITS) region of ribosomal genes was performed on field and pathogenicity test leaf lesion samples to confirm the presence of P. teres f. maculata. DNA templates were prepared using the Extract-N-Amp Plant PCR Kits (Sigma Chemical Co., St. Louis, MO) and subjected to PCR using ITS1 and ITS4 primers. Amplicons were then purified and sequenced. The 585-bp nucleotide sequences of P. teres f. maculata from Mon-Dak area were submitted to GenBank under accession nos. PtmNES1 (JX187587), PtmSDY1 (JX187588), PtmSDY2 (JX187589), and PtmWIL1 (JX187590). The sequences from the four locations shared 100% similarity and also with P. teres f. maculata (EF452471) from GenBank while showing 10 nucleotide differences (99% similarity) with P. teres f. teres (EF452472).The results represent first report of SFNB in the Mon-Dak. Barley is one of the most important crops in the area. Resistance of the NFNB and SFNB of barley are controlled by different genes (4). Based on this report, SFNB therefore have to be considered in selection of barley cultivars for cultivation in the area. References: (1) H. E Bockelman et al. Plant Dis. 67:696, 1983. (2) R. T. Lartey et al. J. Sugar Beet Res. 40:1, 2003. (3) Z. H. Liu and T. L. Friesen. Plant Dis. 94:480, 2010. (4) O. M. Manninen et al. Genome. 46:1564, 2006.

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.

Exotic perennial Lepidium draba, native to Eurasia, is an invasive weed in dense stands in rangelands and disturbed areas in several states of the western United States and an agricultural weed in the prairie provinces of Canada. To determine strategies, such as a potential multipathogen strategy (1), for biological control of the weed, surveys that included the native range were conducted in spring 2009 to detect diseases that occur on this weed. Several stunted and chlorotic plants were found scattered throughout a stand of L. draba growing in a vacant lot near Riddes, Switzerland (46°08'22.99″N, 7°9'19.02″E): ( http://maps.google.com/maps?source=earth&ll=46.13983490,7.15503250&layer= c&cbll=46.13983490,7.15503250&cbp=1,360,,0,5 ). Affected plants had reddish brown cankers on the lower stems, usually elongated and irregular in shape and slightly sunken. Insect injury was associated with the cankers. Symptoms often occurred on plants that were also infected with Rhizoctonia solani. After surface disinfestation with 0.1% sodium hypochlorite, tissue adjacent to and including lesions were plated on acidified potato dextrose agar and incubated at 20 to 25°C for 1 week. Zonate, dark gray colonies with sparse mycelia resulted that exhibited abundant, faintly pink spore masses with numerous dense clusters of black setae. Spores were single celled, hyaline, cylindrical to oval shaped, and 13.5 to 19.5 × 4 to 5.5 μm. Setae were 1- to 3-septate and 20 to 42 × 3 to 5 μm. These morphological traits correspond to Colletotrichum higginsianum. For pathogenicity tests, three 4-month-old L. draba plants were sprayed until runoff with a 106 conidia/ml suspension of the fungus and incubated for 72 h in plastic bags at 20 to 25°C in a quarantine greenhouse. Within 4 days, water-soaked lesions appeared that coalesced, resulting in chlorosis and collapse of inoculated leaves. Such symptoms are typical of infection by C. higginsianum and similar necrotrophic species (4). Fungi isolated from inoculated leaves were identified as C. higginsianum. To assess the host range of C. higginsianum, three plants each of turnip, radish, mustard greens, kale, broccoli raab, and Chinese cabbage, all in the Brassicaceae to which L. draba belongs, were inoculated with the same conditions used for the pathogenicity tests. Control plants in pathogenicity and host range tests were sprayed with sterile distilled water and all experiments were repeated at least once. All control plants were symptomless. Leaf necrosis occurred on radish and turnip and to a lesser extent on the lower leaves of Chinese cabbage and broccoli; numerous scattered dark necrotic flecks and small grayish leaf spots occurred on kale and mustard greens, respectively. These results are similar to previous studies (2,3) involving a cultivated species as the host in the field. The ITS1, 5.8S, and ITS2 sequences of this fungus (GenBank No. HM044877) were 99% similar to sequences of multiple isolates of C. higginsianum (GenBank Nos. AB042302, AB042303, AB455253, AJ558109, and AJ558110). To our knowledge, this is the first report of C. higginsianum on a wild species of the Brassicaceae, although a Colletotrichum sp. was reported on wild radish in Australia (1). References: (1) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (2) R. O'Connell et al. Mol. Plant-Microbe Interact. 17:272, 2004. (3) R. P. Scheffer. N. C. Agric. Exp. Stn. Tech. Bull. 1950. (4) H. Sun and J. Z. Zhang. Eur. J. Plant Pathol. 125:459, 2009.

The association of Rhizoctonia spp. with insect-damaged and diseased tissue of the invasive perennial Lepidium draba was documented throughout the range of L. draba that was surveyed in Europe, including Hungary, Austria, Switzerland and France. Samples that could be both maintained under cooled conditions after collection and promptly processed consistently yielded Rhizoctonia solani, 11 isolates of which anastomosed with AG-4, one with AG-2-1 and one isolate was determined to be binucleate. Comparative virulence among the isolates was analyzed using nonparametric survival analysis for interval-censored data. This analysis indicated that there were significant differences among the isolates. These findings provide further evidence that both root-attacking insects and soilborne pathogens are associated with the lower density of a perennial invasive species in its native range. Prioritization of prospective biocontrol agents should include the ability to engage in insect/plant pathogen synergisms. The application of nonparametric maximum likelihood estimation to interval-censored data for the assessment of comparative virulence among several strains of a plant pathogen is more accessible than calculation of the area under the disease progress curve.

Cercosporin is a perylenequinone pigment produced by fungi in the genus Cercospora which under light generates reactive oxygen species causing membrane damage and mortality of living cells. Our objectives were to demonstrate that fungal laccase, a lignolytic copper-containing enzyme, can degrade cercosporin and reduce cercosporin toxicity toward living cells. Cercosporin from Cercospora beticola and Cercospora bayi was treated with laccase from basidiomycete fungi (Pleurotus ostreatus and Trametes versicolor) in the dark and under constant light. Under these conditions the absorbance of the cercosporin decreased at 220, 279 and 295-500 nm within 10 min of reaction with laccase from either R ostreatus or T versicolor, indicating that basidiomycete laccase can induce changes in UV-visible spectra of cercosporin. The LIVE/DEAD (R) Bac Light (TM) VIABILITY kit and fluorescent microscopy showed more viable E. coli cells after incubation under light with cercosporin and laccase than with cercosporin alone. Lesions were apparent on sugar beet leaves exposed to cercosporin under light after 48 h, but leaves exposed to cercosporin and laccase showed visibly less damage. These data suggest that laccase from basidiomycete fungi can decrease the toxic effect of cercosporin toward microorganisms and plant tissue.

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.

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.

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.

Diffuse knapweed (DK) plants were discovered in Mosier, Wasco County, OR (45.6842°N, 121.4021°W) with crown gall-like symptoms near the soil line. Specimens were collected on 27 July 2004 and sent to the USDA-ARS at Ft. Detrick, MD for identification of disease and pathogen. Pure culture of a bacterium was obtained on potato dextrose agar, and hyperplasia and hypertrophy developed on carrot disks and tomato stems after wound inoculation with a needle contaminated by the agar culture. The same bacterium was reisolated from the galls on DK, thus fulfilling Koch's postulates. Pathogenicity tests involving needle inoculations of stems and petioles resulted in gall formation on Acroptilon repens, Carthamus tinctorius, Centaurea solstitialis, C. maculosa, C. cyanus, Crupina vulgaris, Helianthus annuus, and Rubus armeniacus. In biochemical tests typically used for identification of Agrobacterium species (3), the DK strain grew on D1M agar but not on 2% NaCl medium, produced acid from erythritol but not from melezitose, converted malonic acid to base, and turned litmus milk alkaline. These results are characteristic of Agrobacterium rhizogenes (= Biovar 2), except for the litmus milk reaction. Using 16S rRNA cluster analysis by unweighted pair group method with arithmetic mean (UPGMA, 500 replicates) and basic local alignment search tool (BLAST), the DK strain clustered most closely with A. rubi (GenBank Accession Nos. D12787 and AM181759). The DK strain differed from A. larrymoorei (GenBank Accession No. Z30542), A. tumefaciens (GenBank Accession No. AJ389896), A. rhizogenes (GenBank Accession No. AB247607), and A. vitis (GenBank Accession No. AB247599) on the basis of 16S rRNA sequence cluster analysis. The DK strain differed from A. rubi on the basis of differential reactions with erythritol, litmus milk, and 2% NaCl medium (2,4); and the 16S rRNA sequence of the DK strain differed from that of A. rubi by 11 bp (99.2% similarity). Comparisons also were made between the DK strain and two strains (83A and 135A) of A. tumefaciens (= Biovar 1), described from New Mexico on A. repens (1), a plant species in the same tribe and subtribe of the Asteraceae as DK. Host range reported for the two A. repens strains after artificial greenhouse inoculations was similar to that of the DK strain and it included diffuse knapweed (1). However, 16S sequencing, which confirmed identification of both A. repens strains as A. tumefaciens, showed they differed from the DK strain. The DK strain belongs in the genus Agrobacterium, but it could not be assigned to any known species on the basis of data from phenotypic or 16S sequence comparisons. To our knowledge, this is the first report of crown gall on diffuse knapweed in the field. This strain has been deposited into the International Collection of Phytopathogenic Bacteria at Fort Detrick (Accession No. 60099), and the 16S rRNA sequence has been deposited into the GenBank database (Accession No. EF687663). References: (1) A. J. Caesar, Plant Dis. 78:796, 1994. (2) B. Holmes and P. Roberts, J. Appl. Bacteriol. 50:443, 1981. (3) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (4) K. Ophel and A. Kerr, Int. J. Syst. Bacteriol. 40:236, 1990.

AbstractBiological control efforts against the perennial invasive Euphorbia esula/virgata in North America have left 30-50% of all treated sites without impact after 10-15 years. Those efforts focused almost exclusively on insect releases. Much evidence is available indicating that soil biotic factors affect both invasiveness and biocontrol effectiveness. The authors have shown that soilborne bacteria and fungi are linked to biomass reductions or mortality in conjunction with insect damage. To understand factors possibly affecting synergistic interaction of the insects with plant pathogens shown to cause rapid weed mortality, predominant bacteria associated with the flea beetle Aphthona flava Guill. (Coleoptera: Chrysomelidae) released to control E. esula/virgata L. in western North America, were isolated and identified. Two Euphorbia-infested sites with widely differing levels of impact 8-10 years after insect release were sampled. From the site that exhibited rapid, sweeping declines in Euphorbia density, 6 of 12 isolates were Bacillus spp., 4 were coryneform species and 2 were Pseudomonadaceae. Bacteria isolated from the Cottonwood site included some species often associated with the biocontrol of soilborne plant pathogens. The results of tests for a range of hydrolytic enzymes showed that the two groups differed in the frequency of isolates positive for such enzymes as cellulase and xylanase. Two isolates from each location representative of predominant bacterial species and their range of traits were selected for testing on E. esula/virgata in combination with Aphthona spp. After 35-37 weeks, two isolates positive for cellulase from the Knutson Creek site caused significant (P=0.05) dry weight reductions of E. esula/virgata plants of 64% and 67%, respectively, in combination with Aphthona spp. One of the two isolates from the Cottonwood site, also positive for cellulase production, caused a 60% reduction in dry weight compared with the control.

Biological control efforts against the perennial invasive Euphorbia esula/virgata in North America have left 30-50% of all treated sites without impact after 10-15 years. Those efforts focused almost exclusively on insect releases. Much evidence is available indicating that soil biotic factors affect both invasiveness and biocontrol effectiveness. The authors have shown that soilborne bacteria and fungi are linked to biomass reductions or mortality in conjunction with insect damage. To understand factors possibly affecting synergistic interaction of the insects with plant pathogens shown to cause rapid weed mortality, predominant bacteria associated with the flea beetle Aphthona flava Guill. (Coleoptera: Chrysomelidae) released to control E. esula/virgata L. in western North America, were isolated and identified. Two Euphorbia-infested sites with widely differing levels of impact 8-10 years after insect release were sampled. From the site that exhibited rapid, sweeping declines in Euphorbia density, 6 of 12 isolates were Bacillus spp., 4 were coryneform species and 2 were Pseudomonadaceae. Bacteria isolated from the Cottonwood site included some species often associated with the biocontrol of soilborne plant pathogens. The results of tests for a range of hydrolytic enzymes showed that the two groups differed in the frequency of isolates positive for such enzymes as cellulase and xylanase. Two isolates from each location representative of predominant bacterial species and their range of traits were selected for testing on E. esula/virgata in combination with Aphthona spp. After 35-37 weeks, two isolates positive for cellulase from the Knutson Creek site caused significant (P=0.05) dry weight reductions of E. esula/virgata plants of 64% and 67%, respectively, in combination with Aphthona spp. One of the two isolates from the Cottonwood site, also positive for cellulase production, caused a 60% reduction in dry weight compared with the control.

AbstractThe invasive perennial plant of Eurasian origin, Euphorbia esula/virgata, has been successfully controlled over large areas in North America with a synergism between larvae of Aphthona spp. and soilborne plant pathogens. However, a multitude of sites is not yet under control. Studies are needed on how flea beetle root herbivory may alter the microbial ecology of the rhizosphere of E. esula/virgata and how the resulting rhizosphere community may affect the synergism. Studies were undertaken at Theodore Roosevelt National Park from 2001 to 2003 to identify the predominant culturable prokaryotic species found in the rhizospheres of E. esula/virgata. The hypothesis was that distinct rhizosphere communities of E. esula/virgata would be associated with root herbivory by the flea beetle Aphthona compared with rhizospheres of E. esula/virgata from stands without insect presence. Stands with and without resident populations of Aphthona spp. were assayed by spiral plating root washes of E. esula/virgata and selecting colonies from the most dilute portion of the spiral (deemed as predominant). Gas chromatographic analysis of fatty acid methyl ester was performed on the resulting pure cultures to identify the isolates and further characterize community structures using principal component analysis. Pseudomonas syringae van Hall, Pseudomonas cichorii (Swingle) Stapp, Erwinia chrysanthemii Burkholder, all plant pathogens, were associated exclusively with herbivory by Aphthona flea beetles. Conversely, Variovorax Willems et al. 1991 and Aquaspirillum Hylemon et al. 1973 spp. were a greater proportion of predominant species from roots without Aphthona present. There were also differences in the occurrence of the root pathogen antagonistic Pantoea agglomerans Gavini et al. 1989 and Stenotrophomonas maltophilia (Hugh 1981) Palleroni and Bradbury 1993.

Soil macroaggregates (>250μm) are easily disrupted when wetted quickly, whereas microaggregates (50–250μm) are highly stable. There is little knowledge of functional groups of bacteria from microaggregates that can stabilize soil and their diversity. We isolated the predominant bacteria from microaggregates collected from two agroecosystems: a site cropped to barley (40 years with tillage) and to alfalfa (9 years with no tillage) and assayed for their potential to aggregate soil in an in vitro assay. Soil suspensions from physically disrupted microaggregates were spiral-plated and bacterial colonies were isolated from the most dilute portion of the spiral then purified. To characterize the culturable bacteria thus isolated, fatty acid methyl ester (FAME) profiles, substrate utilization patterns (Biolog), and DNA sequence analysis using 16s rRNA amplification region were performed for bacterial identification. Principal component analysis of FAME profiles and Biolog patterns was conducted to describe patterns of community composition of bacterial species within each agroecosystem. Gram-negative bacteria in microaggregates from barley soil consisted entirely of Pseudomonas spp. whereas Stenotrophomonas, Sphingobacterium, Chryseobacterium, and Acinetobacter spp. were found in addition to the Pseudomonas in microaggregates of alfalfa soil. Artificial aggregates were generated by amending a sandy soil with pure cultures of various species identified from microaggregates of both soils and tested for their water stability and their strength. The results indicated that Stenotrophomonas and Sphingobacterium species exhibited the greatest ability to stabilize soil and to increase aggregate strength among several species tested. This study demonstrated that microaggregates harbor functional groups of bacterial species capable of aggregating soil particles in vitro, and there were a greater range of such species in microaggregates of non-disturbed soil (alfalfa) than in disturbed, yearly tilled soil (barley).