Reproducibility of T1ρ and T2 quantification in a multi-vendor multi-site study.

OBJECTIVE: To evaluate the multi-vendor multi-site reproducibility of two-dimensional (2D) multi-echo spin-echo (MESE) T2 mapping (product sequences); and to evaluate the longitudinal reproducibility of three-dimensional (3D) magnetization-prepared angle-modulated partitioned k-space spoiled gradient echo snapshots (MAPSS) T1ρ and T2 mapping (research sequences), and 2D MESE T2 mapping, separated by 6 months, in a multi-vendor multi-site setting. METHODS: Phantoms and volunteers (n = 5 from each site, n = 20 in total) were scanned on four 3 T magnetic resonance (MR) systems from four sites and three vendors (Siemens, General Electric, and Phillips). Two traveling volunteers (3 knees) scanned at all 4 sites at baseline and 6-month follow-up. Data was transferred to one site for centralized processing. Coefficients of variation (CVs) were calculated to evaluate reproducibility. RESULTS: For baseline 2D MESE T2 measures, average CV were 0.37-2.45% (intra-site) and 5.96% (inter-site) for phantoms, and 3.15-8.49% (intra-site) and 14.16% (inter-site) for volunteers. For longitudinal phantom data, intra-site CVs were 1.42-3.48% for 3D MAPSS T1ρ, 1.77-3.56% for 3D MAPSS T2, and 1.02-2.54% for 2D MESE T2. For the longitudinal volunteer data, the intra-site CVs were 2.60-4.86% for 3D MAPSS T1ρ, 3.33-7.25% for 3D MAPSS T2, and 3.11-8.77% for 2D MESE T2. CONCLUSION: This study demonstrated excellent intra-site reproducibility of 2D MESE T2 imaging, while its inter-site variation was slightly higher than 3D MAPSS T2 imaging (10.06% as previously reported). This study also showed excellent reproducibility of longitudinal T1ρ and T2 cartilage quantification, in a multi-vendor multi-site setting for both product 2D MESE T2 and 3D MAPSS T1p/T2 research sequences.

Multi-vendor multi-site quantitative MRI analysis of cartilage degeneration 10 Years after anterior cruciate ligament reconstruction: MOON-MRI protocol and preliminary results.

OBJECTIVE: To describe the protocol of a multi-vendor, multi-site quantitative MRI study for knee post-traumatic osteoarthritis (PTOA), and to present preliminary results of cartilage degeneration using MR T1ρ and T2 imaging 10 years after anterior cruciate ligament reconstruction (ACLR). DESIGN: This study involves three sites and two MR platforms. The patients are from a nested cohort (termed as Onsite cohort) within the Multicenter Orthopaedic Outcomes Network (MOON) cohort 10 years after ACLR. Phantoms and controls were scanned for evaluating reproducibility. Cartilage was automatically segmented, and T1ρ and T2 were compared between operated, contralateral, and control knees. RESULTS: Sixty-eight ACL-reconstructed patients and 20 healthy controls were included. In phantoms, the intra-site coefficients of variation (CVs) of repeated scans ranged 1.8-2.1% for T1ρ and 1.3-1.7% for T2. The inter-site CVs ranged 1.6-2.1% for T1ρ and 1.1-1.4% for T2. In human subjects, the intra-site scan/rescan CVs ranged 2.2-3.5% for T1ρ and 2.6-4.9% for T2 for the six major compartments. In patients, operated knees showed significantly higher T1ρ and T2 values mainly in medial femoral condyle, medial tibia and trochlear cartilage compared with contralateral knees, and showed significantly higer T1ρ and T2 values in all six compartments compared to healthy control knees. The patient contralateral knees showed higher T1ρ and T2 values mainly in the lateral femoral condyle, lateral tibia, trochlear, and patellar cartilage compared to healthy control knees. CONCLUSION: A platform and workflow with rigorous quality control has been established for a multi-vendor multi-site quantitative MRI study in evaluating PTOA 10 years after ACLR. Our preliminary report suggests significant cartilage matrix changes in both operated and contralateral knees compared with healthy control knees.

Multi-vendor multi-site T1ρ and T2 quantification of knee cartilage.

OBJECTIVE: To develop 3D T1ρ and T2 imaging based on the same sequence structure on MR systems from multiple vendors, and to evaluate intra-site repeatability and inter-site inter-vendor reproducibility of T1ρ and T2 measurements of knee cartilage. METHODS: 3D magnetization-prepared angle-modulated partitioned k-space spoiled gradient echo snapshots (3D MAPSS) were implemented on MR systems from Siemens, GE and Philips. Phantom and human subject data were collected at four sites using 3T MR systems from the three vendors with harmonized protocols. Phantom data were collected by means of different positioning of the coil. Volunteers were scanned and rescanned after repositioning. Two traveling volunteers were scanned at all sites. Data were transferred to one site for centralized processing. RESULTS: Intra-site average coefficient of variations (CVs) ranged from 1.09% to 3.05% for T1ρ and 1.78-3.30% for T2 in phantoms, and 1.60-3.93% for T1ρ and 1.44-4.08% for T2 in volunteers. Inter-site average CVs were 5.23% and 6.45% for MAPSS T1ρ and T2, respectively in phantoms, and 8.14% and 10.06% for MAPSS T1ρ and T2, respectively, In volunteers. CONCLUSION: This study showed promising results of multi-site, multi-vendor reproducibility of T1ρ and T2 values in knee cartilage. These quantitative measures may be applied in large-scale multi-site, multi-vendor trials with controlled sequence structure and scan parameters and centralized data processing.

First Report of Rhizoctonia spp. Causing a Root Rot of the Invasive Rangeland Weed Lepidium draba in North America.

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.

First Report of Spot Form Net Blotch Caused by Pyrenophora teres f. maculata on Barley in the Mon-Dak Area of the United States.

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.

First Report of a Root and Crown Disease of the Invasive Weed Lepidium draba Caused by Phoma macrostoma.

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.

First Report of Anthracnose Stem Canker of the Invasive Perennial Weed Lepidium draba Caused by Colletotrichum higginsianum in Europe.

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.

Direct Polymerase Chain Reaction-Based Detection of Cercospora beticola in Field Soils.

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.

First Report of a Root and Crown Disease Caused by Rhizoctonia solani on Centaurea stoebe in Russia.

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.

First Report of Leaf Spot Caused by Cercospora bizzozeriana on Lepidium draba in the United States.

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.

First Report of a Leaf Spot Caused by Alternaria brassicae on the Invasive Weed Lepidium draba in North America.

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.

First Report of Crown Gall Caused by Agrobacterium tumefaciens on Euphorbia esula/virgata in Europe.

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.

Safflower: A New Host of Cercospora beticola.

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.

Isolate-specific synergy in disease symptoms between cauliflower mosaic and turnip vein-clearing viruses.

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.

Limitations to tobacco mosaic virus infection of turnip.

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.

An Arabidopsis thaliana mutant with virus-inducible phenotype.

The role of host factors in plant viral diseases is not well understood. To study this important aspect of plant-pathogen interaction, we identified an Arabidopsis thaliana mutant, designated vid1 (virus-inducible dwarf), with altered responses to viral infection. Specifically, vid1 resembled the wild-type plants when healthy but developed a severely dwarfed phenotype with a loss of apical dominance following infection by a tobamovirus. Genetic segregation showed that the vid1 phenotype is caused by a recessive mutation in a single gene. Since systemic viral infection is thought to interfere with the host plant intercellular transport, we propose that the vid1 mutation affects this transport process. Combination of the mutation and viral infection may disrupt transport of developmental regulators, such as hormones, causing formation of the vid1 phenotype. Indeed, the effect of vid1 mutation was repressed by exogenous application of a plant hormone auxin. Potentially, the vid1 mutant will help characterize the mechanism of virus-plant interaction and formation of plant viral disease symptoms.

Inhibition of plant viral systemic infection by non-toxic concentrations of cadmium.

Heavy metal, such as cadmium, have a significant impact on plant physiology. However, their potential effect on plant-pathogen interaction, an important biological process, has not been examined. This study shows that exposure of tobacco plants to non-toxic concentrations of cadmium completely blocked viral disease caused by turnip vein clearing virus. Cadmium-mediated viral protection was due to inhibition of the systemic movement of the virus, i.e. its spread from the inoculated into uninoculated leaves. Exposure of plants to cadmium had no effect on viral replication, assembly and local movement within the inoculated leaf. Analysis of the viral presence in different tissues suggested that cadmium treatment inhibited virus exit from the vascular tissue into uninoculated leaves rather than its entry into the host plant vasculature. Higher, toxic levels of cadmium did not produce this inhibitory effect on viral movement, allowing the systemic spread of the virus and development of the viral disease. These observations suggest that cadmium-induced viral protection requires a relatively healthy, unpoisoned plant in which non-toxic levels of cadmium may trigger the production of cellular factors which interfere with the viral systemic movement.

Identification of an Arabidopsis thaliana mutation (vsm1) that restricts systemic movement of tobamoviruses.

Following inoculation, many plant viruses spread locally from cell to cell until they reach the vascular system, through which they then move to other parts of the plant, resulting in systemic infection. To isolate host genes involved in systemic transport of plant viruses, ethyl methanesulfonate-mutagenized Arabidopsis thaliana plants were screened for significant delays in the systemic movement of turnip vein clearing virus (TCVC). One such mutant, designated vsm1 (virus systemic movement), was identified. Unlike the wild-type plants, vsm1 did not develop viral disease and did not allow the systemic spread of the virus. The local viral movement within the inoculated vsm1 leaves, however, was not affected. TVCV systemic movement within the vsm1 plants was likely blocked at the step of viral entry into the host plant vasculature from the infected leaf tissue. vsm1 plants also restricted the systemic movement of another tobamovirus but not of an unrelated carmovirus.

Study of the aetiologic agents of meningitis in Kumasi, Ghana, with special reference to Cryptococcal neoformans.

OBJECTIVE: To evaluate the importance of Cryptococcus neoformans, an opportunistic in meningitis, in healthy and HIV infected patients in Kumasi, Ghana. DESIGN: A prospective study; isolating the aetiologic agents of meningitis from cerebrospinal fluid (CSF) using standard methods. SETTING: Kumasi city, Ashanti region of central Ghana. SUBJECTS: One thousand five hundred and seventy patients suspected of meningitis, including 28 HIV infected and AIDS patients. MAIN OUTCOME MEASURE: The pattern and distribution of the main etiologic agents of meningitis in Kumasi, Ghana shown. RESULTS: Of the 1570 CSF samples examined, 1256 (80%) showed no abnormality. Of the 314 (20%) with abnormalities, 147 were bacterial, and 167 diagnosed aseptic. No cryptococcal cells were found. CONCLUSION: There is a paucity of cryptococcal meningitis in Kumasi, Ghana, and it is not recommended to screen routinely for the fungus in meningitis investigations.

Movement and subcellular localization of a tobamovirus in Arabidopsis.

Tobamoviruses represent a well-characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus-plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis. Here, we describe infection of Arabidopsis by a recently discovered crucifer-infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis, causing mild and fast-developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus-plant interaction.