Evaluation of Air Sampling and Detection Methods to Quantify Airborne Ascospores of Sclerotinia sclerotiorum.

Detection and quantification of airborne ascospores as a component of the Sclerotinia rot of carrot (SRC) forecast model is currently accomplished using the blue plate test (BPT), which uses Sclerotinia semiselective medium (SSM). A quantitative polymerase chain reaction (qPCR) assay was developed to reduce the time to specifically quantify ascospores of Sclerotinia sclerotiorum from air samples collected using a Burkard Multi-Vial Cyclone Sampler. The qPCR assay was highly sensitive and detected DNA from 0.5 to 5 × 104 ascospores within a linear range (R2 = 0.99). The qPCR assay was used to quantify ascospores of S. sclerotiorum in air samples collected over three growing seasons. Initial SRC disease was observed 8 and 34 days following detection of 9.5 and 2 ascospores m-3 of air, respectively. Results from air samples collected using an Andersen N6 Sampler and the qPCR assay were compared with the BPT. Ascospore counts from a Burkard Sampler coupled with the qPCR assay and the BPT followed similar trends. In general, fewer ascospores were detected and bioaerosol sampling efficiency was low using an Anderson Sampler. Three days were required to confirm the number of ascospores using SSM in the BPT and with an Andersen Sampler, whereas results from a Burkard Sampler coupled with the qPCR assay can provide results within 5 h of air sampling. The choice of method will depend on the available resources.

Incidence and Severity of Cavity Spot of Carrot as Affected by Pigmentation, Temperature, and Rainfall.

Field trials to determine the effect of carrot pigmentation and weather parameters on cavity spot (CS) of carrot were conducted in the Holland/ Bradford Marsh region of Ontario between 2002 and 2009. In all, 23 colored carrot cultivars from the United States Department of Agriculture (USDA) Agricultural Research Service breeding program at the University of Wisconsin (n = 5) and commercial seed companies (n = 18) were seeded in organic soil (pH 6 to 7, 45 to 75% organic matter) in late May to early June and harvested in late October or early November. Carrot roots were assessed for CS severity midseason and postharvest. Evaluations postharvest indicated that the purple pigmented carrot from breeding line 'USDA 106-3' and cultivars 'Purple Rain' and 'Purple Haze' consistently had low CS severity. The orange-pigmented 'USDA 101-23', 'Cellobunch', 'YaYa', and 'Envy' had moderate CS; and the red-pigmented carrot breeding line 'USDA 104-3' and cultivars 'Atomic Red', 'Proline Red', 'Dragon', and an unnamed line from India had high CS. Differences in CS severity in carrot cultivars between evaluations at midseason and postharvest suggest that some carrot cultivars are more susceptible to Pythium spp. inoculum in soil (alloinfection) and others to secondary infection (autoinfection) that can be attributed to the Pythium sp. involved in CS. CS severity was positively correlated with total rainfall 2 and 3 months after seeding, and was negatively correlated with number of days with air temperature ≥30°C 3 and 4 months after seeding. Soil temperature and total rainfall were the best predictors of CS incidence and severity. These results could allow a forecast of disease incidence and severity at harvest.

Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance.

This study investigated how the timing of application of the biofungicide Serenade (Bacillus subtilis QST713) or it components (product filtrate and bacterial cell suspension) influenced infection of canola by Plasmodiophora brassicae under controlled conditions. The biofungicide and its components were applied as a soil drench at 5% concentration (vol/vol or equivalent CFU) to a planting mix infested with P. brassicae at seeding or at transplanting 7 or 14 days after seeding (DAS) to target primary and secondary zoospores of P. brassicae. Quantitative polymerase chain reaction (qPCR) was used to assess root colonization by B. subtilis as well as P. brassicae. The biofungicide was consistently more effective than the individual components in reducing infection by P. brassicae. Two applications were more effective than one, with the biofungicide suppressing infection completely and the individual components reducing clubroot severity by 62 to 83%. The biofungicide also reduced genomic DNA of P. brassicae in canola roots by 26 to 99% at 7 and 14 DAS, and the qPCR results were strongly correlated with root hair infection (%) assessed at the same time (r = 0.84 to 0.95). qPCR was also used to quantify the transcript activity of nine host-defense-related genes in inoculated plants treated with Serenade at 14 DAS for potential induced resistance. Genes encoding the jasmonic acid (BnOPR2), ethylene (BnACO), and phenylpropanoid (BnOPCL and BnCCR) pathways were upregulated by 2.2- to 23-fold in plants treated with the biofungicide relative to control plants. This induced defense response was translocated to the foliage (determined based on the inhibition of infection by Leptosphaeria maculans). It is possible that antibiosis and induced resistance are involved in clubroot suppression by Serenade. Activity against the infection from both primary and secondary zoospores of P. brassicae may be required for maximum efficacy against clubroot.

First Report of Downy Mildew Caused by Peronospora belbahrii on Basil (Ocimum spp.) in Ontario.

Basil (Ocimum spp.) is one of the most commercially significant fresh culinary herb crops worldwide. In Ontario, basil is grown both in the field and in the greenhouse. In the summer of 2011, basil plants grown in a research field at the Simcoe Research Station in Norfolk County, Ontario, Canada (44°15'N, 77°35'W), were infected with downy mildew. Infected leaves exhibited interveinal chlorotic lesions on the upper surface and clear to black sporulation on the abaxial leaf surfaces. Leaf senescence and defoliation occurred at high disease severity, which reduced marketable yield. Basil downy mildew symptoms were severe on leaves of cultivars Genovese and Sweet Basil, with 40 to 100% disease incidence. Based on morphological characteristics, the basil downy mildew causal agent was identified as Peronospora belbahrii Thines (4). Infected leaves were collected and microscopic observations of the sporulating lesions were carried out and the structures measured. Sporangiophores (n = 20) were hyaline with relatively long, straight trunks and were monopodially branched, with a length of 150 to 360 µm (average 285 µm). Sporangiophores ended with two slightly curved branchlets, the longer one measuring 15 to 27 µm (average 19 µm) and the shorter one 5 to 15 µm (average 9 µm). Sporangia (n = 50) were round, or slightly ovoid, olive to brown in color, and measured 29 × 25 µm (25 to 35 × 20 to 30 µm). Genomic DNA was extracted from 10 isolates and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with ITS4 and ITS5 primers and sequenced. The sequences of the 10 isolates were nearly identical. A BLAST search of the NCBI database with the ITS sequences (GenBank Accession No. KC756923) revealed a 98 to 100% similarity to the sequences of P. belbahrii (HQ730979, FJ436024, and HQ702191) isolated from sweet basil in Florida (3), California (1), and Hungary (2), respectively. To confirm pathogenicity, 5-week-old 'Genovese' seedlings were sprayed with a suspension of 1 × 105 sporangia/ml. Plants were kept in a growth chamber maintained at 23/18°C, 60 to 85% relative humidity, and 12/12 h light/dark. Non-inoculated plants served as controls. Basil downy mildew symptoms developed after 8 days on the inoculated plants and the pathogen was identified in association with symptoms consistent with downy mildew. The non-inoculated controls remained healthy. In North America, the occurrence of basil downy mildew has been reported since 2007 (3) and the disease has spread into several U.S. states. To our knowledge, this is the first report of downy mildew on sweet basil in Canada. References: (1) C. L. Blomquist et al. Plant Dis. 93:968, 2009. (2) G. Nagy and A. Horvath. Plant Dis. 95:1034, 2011. (3) P. D. Roberts et al. Plant Dis. 98:199, 2009. (4) M. Thines et al. Mycol. Res. 113:532, 2009.

First Report of Iris yellow spot virus on Onion in Canada.

Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) is an economically important viral pathogen of onion vectored by onion thrips (Thrips tabaci Lindeman). Rapid spread of IYSV has occurred in the western United States and Georgia, with recent reports of IYSV from New York in the northeastern United States (1). In June and mid-July of 2007, symptomatic plants were found in Ontario, Canada in onions grown from sets in a home garden in Grey County (44°27'N, 80°53'W) and on a small commercial farm in Ottawa-Carleton County (45°14'N, 75°28'W), respectively. In the home garden, bleached, elongated lesions with tapered ends occurred on middle-aged leaves of 30% of 100 plants. By August 2007, 91% of these plants were symptomatic. In Ottawa-Carleton, two lesions with green centers and yellow borders occurred on a single leaf of a single plant in a field of 1,120 plants. Symptomatic leaf tissue tested positive for IYSV by IYSV-specific antiserum from Agdia Inc. (Elkhart, IN) in a double-antibody sandwich (DAS)-ELISA. These two isolated and remote finds of IYSV in Ontario prompted a survey in early August of 2007 of the Holland Marsh (44°5'N, 79°35'W), the largest onion-producing region in Ontario. Nine onion fields separated geographically across the Holland Marsh Region were scouted and one to three samples of symptomatic tissue per field were analyzed by DAS-ELISA. IYSV was confirmed in seven of nine (78%) fields surveyed and in 13 of 16 (81%) of the individual samples. A reverse transcription (RT)-PCR assay was used to verify the presence of IYSV in one new symptomatic tissue sample per field collected from three of the fields where IYSV was confirmed by ELISA. Primers specific to the small (S) RNA of IYSV (5'-TAA AAC AAA CAT TCA AAC AA-3' and 5'-CTC TTA AAC ACA TTT AAC AAG CAC-3') were used (2). The resulting 1.2-kb amplicon, which included the 772-bp nucleocapsid (N) gene was cloned and sequenced. Sequence analysis showed that the N gene of the Ontario isolate (GenBank Accession No. EU287943) shared 92 to 98% nucleotide sequence identity with known IYSV N gene sequences. The highest nucleotide sequence identity (98%) was with Genbank Accession Nos. DQ233475 and DQ233472. To our knowledge, this is the first report of IYSV infection of onion in Ontario and Canada. This finding confirms further spread of the virus within North America and the need for research to develop more effective management options to reduce the impact of IYSV on onion production. The finding of IYSV in remote and isolated locations where onions were grown from sets implies that the spread of IYSV via infected bulbs warrants further investigation as a potentially important route of distribution of the virus. References: (1) D. H. Gent et al. Plant Dis. 88:446, 2004. (2) H. R. Pappu et al. Arch. Virol. 151:1015, 2006.

First Report of Foliar and Root Infection of Carrot by Sclerotinia minor in Ontario, Canada.

In 2000 and 2001, commercial carrots (Daucus carota L.) cv. Cellobunch grown in organic soils in Ontario, Canada, developed water-soaked, dark olive-green lesions on leaves that were in contact with soil. Lesions spread rapidly to petioles and adjacent leaves when prolonged moist conditions occurred within the canopy and persisted through harvest. A soft rot lesion was observed on the crown of one carrot root in the field, but no disease symptoms were detected on carrot samples in cold storage. Symptoms on leaves and roots of carrots were similar to sclerotinia rot caused by Sclerotinia sclerotiorum, a common disease in the area, except for the signs of sparser mycelia and smaller sclerotia. White mycelium and irregular, black sclerotia (0.5 to 2 mm) that formed on the surface of diseased leaf and root tissues were plated on potato dextrose agar (PDA) and incubated at 20 ± 1°C. Within 3 to 4 days, numerous mycelial clumps developed throughout the surface of white colonies and within 5 to 7 days these clumps developed into sclerotia. The pathogen was identified as S. minor Jagger (1). In the growth-room, young and senescing leaves of eight carrot (cv. Cellobunch) plants and 20 surface-disinfested roots were inoculated with 5-mm mycelial disks from the margins of colonies of one isolate of S. minor or S. sclerotiorum grown on PDA. A control treatment of an equal set of test units inoculated with sterile PDA plugs was also included. Inoculated plants and roots were incubated at 21 ± 1°C and ≈100% relative humidity for 21 days. Plants inoculated with S. minor, lesions, mycelial clumps, and sclerotia developed on the surface of leaves and petioles 1, 2, and 10 days postinoculation, respectively and were similar to those observed in the field. Lesions progressed faster on senescing leaves and reached the base of the petiole within 7 days, however, infection of the rosette or crown did not occur under these test conditions. In roots, lesions, mycelia, and sclerotia were observed 2, 4 and 10 days postinoculation, respectively, and sclerotia sometimes adhered in large aggregate crusts. S. minor was reisolated from infected tissues, completing Koch's postulates. Carrots inoculated with S. sclerotiorum yielded similar symptoms, but the white mycelium grew into cotton-like, dense mats and produced large (5 to 10 mm) individual sclerotia. Carrot has been previously reported as a host of S. minor (2), but to our knowledge, this is the first report demonstrating the occurrence of sclerotinia rot of carrot caused by S. minor in Ontario. S. minor may not cause damage to carrots in storage, but may affect yield by weakening the tops needed for efficient mechanical harvest. Carrot crops where S. minor was identified are typically rotated with onions (Allium cepa L.), which is not a host of S. minor (2). However, this 1-year rotation is unlikely to suffice for reducing the accumulation of sclerotial inoculum in soil. The knowledge of the occurrence and prevalence of S. minor in carrot crops is a valuable consideration when planning to incorporate carrots in rotation with other hosts of S. minor. References: (1) L. M. Kohn. Mycotaxon 9:365, 1979. (2) M. S. Melzer et al. Can. J. Plant Pathol. 19:272, 1997.

Pulsed-accelerated-flow spectrometer with position-resolved observation.

The title instrument (PAF-PRO) permits the progress of rapid reactions to be monitored at 128 positions along a 2 cm observation cell as reactants flow down the cell. A decelerated push is used to give velocity changes from 21 to 2 m s(-1) during a time interval of 384 ms. Variation of flow velocity allows reaction rate constants to be resolved from the physical mixing process. The flow system brings the reacting solutions together in a 10-jet radial mixer 0.32 cm before the mixture enters the observation tube (0.203 cm width, 1.945 cm length). A masked charge-coupled device (CCD) is the array detector used to obtain transmittance data as a function of position and velocity. The masked portion of the CCD serves as a dynamic memory buffer for fast data acquisition. Pseudo-first-order rate constants are measured from 200 to 12 000 s(-1). The instrument also is calibrated for second-order reactions (equal concentrations) with initial half-lives of 0.3-1.5 ms. Applications of the PAF-PRO system for the study of fast multistep reactions are presented.

Renal lesions induced by ochratoxin A exposure in the F344 rat.

Groups of 80 male and female F344 rats were exposed by gavage to ochratoxin A, a naturally occurring mycotoxin, at levels of 21, 70, and 210 micrograms/kg body weight for up to 2 years. Ochratoxin A induced non-neoplastic renal tubular epithelial changes consisting of cytoplasmic alteration, karyomegaly, degeneration, and cysts. Exposure-related renal tubular proliferative lesions included focal hyperplasia, tubular cell adenoma, and tubular cell carcinoma. Renal tubular cell adenoma occurred as early as 9 months in 1 high-dose male rat, and both adenomas and carcinomas were seen in males by 15 months. At the terminal sacrifice, renal tubular cell tumors were found in both male and female rats, but the response was more pronounced in the males. The incidence of renal tumors in the high-dose rats was the highest of any National Toxicology Program (NTP) study completed to date. In the high-dose males approximately one-third of the renal carcinomas developed metastases. This study demonstrates that ochratoxin A is a potent renal carcinogen in the F344 rat and suggests that contamination of feedstuff by this mycotoxin may pose a potential hazard to domestic animals and man.