Nighttime Applications of Germicidal Ultraviolet Light (UV-C) to Suppress Cercospora Leaf Spot in Table Beet.

Cercospora leaf spot (CLS), caused by the hemibiotrophic fungus Cercospora beticola, is a destructive disease affecting table beet. Multiple applications of fungicides are needed to reduce epidemic progress to maintain foliar health and enable mechanized harvest. The sustainability of CLS control is threatened by the rapid development of fungicide resistance, the need to grow commercially acceptable yet CLS-susceptible cultivars, and the inability to manipulate agronomic conditions to mitigate disease risk. Nighttime applications of germicidal ultraviolet light (UV-C) have recently been used to suppress several plant diseases, notably those caused by ectoparasitic biotrophs such as powdery mildews. We evaluated the efficacy of nighttime applications of UV-C for suppression of CLS in table beet. In vitro lethality of UV-C to germinating conidia increased with increasing dose, with complete suppression at 1,000 J/m2. Greenhouse-grown table beet tolerated relatively high doses of UV-C without lethal effects despite some bronzing on the leaf blade. A UV-C dose >1,500 J/m2 resulted in phytotoxicity severities greater than 50%. UV-C exposure to ≤750 J/m2 resulted in negligible phytotoxicity. Older (6-week-old) greenhouse-grown plants were more susceptible to UV-C damage than younger (2- and 4-week-old) plants. Suppression of CLS by UV-C was greater when applied within 6 days of C. beticola inoculation than if delayed until 13 days after infection in greenhouse-grown plants. In field trials, there were significant linear relationships between UV-C dose and CLS control and phytotoxicity severity, and a significant negative linear relationship between phytotoxicity and CLS severity at the final assessment. Significant differences between UV-C doses on the severity of CLS and phytotoxicity indicated an efficacious dose near 800 J/m2. Collectively, these findings illustrate significant and substantial suppression by nighttime applications of UV-C for CLS control on table beet, with potential for incorporation in both conventional and organic table beet broadacre production systems.

Sampling, a New iOS Application for Assessment of Damage by Diseases and Insect Pests Using Sequential Sampling Plans.

Regular scouting for plant diseases and insect pests by growers, crop consultants, extension educators, and researchers (herein defined as stakeholders) is the cornerstone of integrated pest management practices. Sequential sampling plans have the potential to save time and labor in field scouting and reduce the frequency of errors surrounding decision-making. The incorporation of the algorithms behind sequential sampling plans into mobile devices can make scouting for diseases and insect pests more straightforward, practical, and enjoyable. Here, we introduce an iOS application called Sampling. The application was designed for stakeholders to use on a mobile device for assessing disease and insect pest incidence in the field using sequential sampling plans. The application allows users to select a disease or insect pest from a prepopulated list and specify the objective of sampling: Estimation or classification. Conducting sequential sampling depends upon different precision levels and action thresholds within each objective. Detailed instructions for each sequential sampling plan are available as a guide. When sampling begins, users enter the number of diseased individuals at each sampling unit. The specific algorithm developed for the disease or insect pest will inform the user when to stop sampling for the desired goal and return the final incidence and precision or threshold achieved. Results are automatically saved in the application, and the user can inspect and share results by exporting them to a range of compatible programs. The initial version of Sampling (1.1) was released with the sequential sampling plans for Cercospora leaf spot of table beet. Sequential sampling plans for additional diseases or pests will be added to Sampling in subsequent versions. Sampling is available as a free download from the Apple Store (https://apple.co/3pUiYKy) and is compatible with iOS 14.0 or greater on the iPhone or iPad.

Survival of Sclerotinia sclerotiorum Sclerotia in Central New York.

White mold caused by Sclerotinia sclerotiorum is a serious disease affecting many field and specialty crops in New York (NY). The primary inoculum for white mold is sclerotia that are hardened masses of mycelia that survive adverse environmental conditions and periods of non-hosts. However, NY crop guidelines lack rotation and residue management recommendations based on local knowledge of sclerotial survival. A field trial was established in October 2020 by deploying S. sclerotiorum sclerotia in mesh bags on the soil surface or shallowly buried (placed at 3 cm depth in the soil) at Geneva, NY. Bags were periodically collected from 67 to 769 days. At each time, sclerotial retrieval (number of sclerotia) was assessed by counted and viability evaluated through myceliogenic germination. Sclerotial retrieval was significantly affected by soil depth and was higher in those on the surface than buried. Time also affected the retrieval of sclerotia which was significantly reduced after 250 days. The interaction between burial and time had a significant effect on sclerotial viability. Approximately 15% of sclerotia placed on the surface were still viable after 769 days. After 433 days, viability of buried sclerotia was also significantly reduced compared to those on the surface. After 670 days, none of the buried sclerotia were viable. These findings suggest a rotation of at least two years between susceptible crops is required to reduce primary inoculum. However, given that low inoculum densities are sufficient to initiate a white mold outbreak, a longer rotation may be beneficial. In a cultivated system, timely tillage of crop residue to bury sclerotia after harvest to promote degradation is encouraged.

Control of Phoma Leaf Spot and Root Decay of Table Beet in New York.

Disease caused by Neocamarosporium betae (syn. Phoma betae, Pleospora betae) results in reductions in plant populations, foliar disease (Phoma leaf spot [PLS]), and root disease and decay in table beet. Disease caused by N. betae has reemerged as prevalent in organic table beet production in New York. The disease can also cause substantial issues in conventional table beet production. To evaluate in-field control options for conventional and organic table beet production, small-plot, replicated trials were conducted in each of two years (2019 and 2021). The fungicides, propiconazole and difenoconazole, and premixtures, pydiflumetofen + fludioxonil or pydiflumetofen + difenoconazole, provided excellent PLS and root decay control. Azoxystrobin provided excellent (69.9%) control of PLS in 2019 and lesser (40%) control in 2021. Field trial results complemented in vitro sensitivity testing of 30 New York N. betae isolates that were all highly sensitive to azoxystrobin (mean effective concentration to reduce mycelial growth by 50%, EC50 = 0.0205 µg/ml) and propiconazole (mean EC50 = 0.0638 µg/ml). Copper octanoate and microbial biopesticides containing either Bacillus amyloliquefaciens D747 or B. mycoides strain J provided moderate (68.5 to 74.6%) PLS control as reflected in epidemic progress. The Gompertz model provided the best fit to PLS epidemics reflecting a polycyclic epidemic. Reductions in PLS severity were associated with significant decreases in Phoma root decay and increases in canopy health and the time-to-death of leaves compared with nontreated control plots. Prolonging leaf survival is critical for mechanical harvest of roots. These findings underpin the design of programs for foliar disease control in conventional and organic table beet production. Assessment of PLS severity in the field will better inform postharvest management decisions.

Development of a Sequential Sampling Plan using Spatial Attributes of Cercospora Leaf Spot Epidemics of Table Beet in New York.

Sampling strategies that effectively assess disease intensity in the field are important to underpin management decisions. To develop a sequential sampling plan for the incidence of Cercospora leaf spot (CLS), caused by Cercospora beticola, 31 table beet fields were assessed in the state of New York. Assessments of CLS incidence were performed in six leaves arbitrarily selected in 51 sampling locations along each of three to six linear transects per field. Spatial pattern analyses were performed, and results were used to develop sequential sampling estimation and classification models. CLS incidence (p) ranged from 0.13 to 0.92 with a median of 0.31, and beta-binomial distribution, which is reflective of aggregation, best described the spatial patterns observed. Aggregation was commonly detected (>95%) by methods using the point-process approach, runs analyses, and autocorrelation up to the fourth spatial lag. For Spatial Analysis by Distance Indices, or SADIE, 45% of the datasets were classified as a random pattern. In the sequential sampling estimation and classification models, disease units are sampled until a prespecified target is achieved. For estimation, the goal was sampling CLS incidence with a preselected coefficient of variation (C). Achieving the C = 0.1 was challenging with <51 sampling units, and only observed on datasets with incidence >0.3. Reducing the level of precision, i.e., increasing C to 0.2, allowed the preselected C to be achieved with a lower number of sampling units and with an estimated incidence ([Formula: see text]) close to the true value of p. For classification, the goal was to classify the datasets above or below prespecified thresholds (pt) used for CLS management. The average sample number, or ASN, was determined by Monte Carlo simulations, and was between 20 and 45 at disease incidence values close to pt, and approximately 11 when far from pt. Correct decisions occurred in >76% of the validation datasets. Results indicated these sequential sampling plans can be used to effectively assess CLS incidence in table beet fields.

Optimizing Cercospora Leaf Spot Control in Table Beet Using Action Thresholds and Disease Forecasting.

Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the dominant foliar disease affecting table-beet production in New York. CLS epidemics occur annually and, if uncontrolled, will rapidly lead to defoliation. In broad-acre production, season-long maintenance of healthy leaves is important to facilitate harvest by top-pulling. Fungicides are the dominant means of CLS control and applications are initiated at an action threshold of 1 CLS lesion/leaf. Regular fungicide application occurs thereafter without regard for scheduling based on weather-based risk. The current action threshold was evaluated with selected fungicides in two replicated field trials. Copper oxychloride + copper hydroxide and propiconazole significantly improved CLS control if initiated prior to infection. Pydiflumetofen + difenoconazole significantly reduced area under the disease progress stairs compared with other fungicides tested and was most efficacious when applications began at 1 CLS lesion/leaf. Six replicated field trials also evaluated the utility of scheduling fungicides on weather-based risk rather than a calendar approach. Two risk thresholds (moderate and high) integrating the accumulation of daily infection values based on temperature and relative humidity from a forecaster for CLS in sugar beet were evaluated. Applications of pydiflumetofen + difenoconazole were reduced from three to two by using the forecaster at either risk threshold compared with calendar applications without affecting CLS control. For propiconazole, the moderate risk threshold provided CLS control equivalent to calendar applications and saved one spray per season. Thus, there was substantial scope to reduce spray frequency by scheduling based on weather-based risk rather than calendar applications. The optimal risk thresholds for pydiflumetofen + difenoconazole and propiconazole were high and moderate, respectively. In these trials, periods of high risk occurred less frequently than moderate risk, increasing the reapplication intervals and, hence, represented a less conservative approach to disease management.

Genetic Diversity and Structure in Regional Cercospora beticola Populations from Beta vulgaris subsp. vulgaris Suggest Two Clusters of Separate Origin.

Cercospora leaf spot, caused by Cercospora beticola, is a highly destructive disease of Beta vulgaris subsp. vulgaris worldwide. C. beticola populations are usually characterized by high genetic diversity, but little is known of the relationships among populations from different production regions around the world. This information would be informative of population origin and potential pathways for pathogen movement. For the current study, the genetic diversity, differentiation, and relationships among 948 C. beticola isolates in 28 populations across eight geographic regions were investigated using 12 microsatellite markers. Genotypic diversity, as measured by Simpson's complement index, ranged from 0.18 to 1.00, while pairwise index of differentiation values ranged from 0.02 to 0.42, with the greatest differentiation detected between two New York populations. In these populations, evidence for recent expansion was detected. Assessment of population structure identified two major clusters: the first associated with New York, and the second with Canada, Chile, Eurasia, Hawaii, Michigan, North Dakota, and one population from New York. Inferences of gene flow among these regions suggested that the source for one cluster likely is Eurasia, whereas the source for the other cluster is not known. These results suggest a shared origin of C. beticola populations across regions, except for part of New York, where population divergence has occurred. These findings support the hypothesis that dispersal of C. beticola occurs over long distances.

Alternative Hosts of Cercospora beticola in Field Surveys and Inoculation Trials.

Cercospora beticola, the cause of Cercospora leaf spot (CLS) of sugar beet and table beet, has a broad range of potential alternative hosts. The role of these hosts as inoculum sources in the field is unclear and has had limited investigation since the advent of DNA-based pathogen identification. The presence of C. beticola on alternative hosts associated with table beet fields of New York was assessed in field surveys during 2016. Lesions were collected, and 71 cercosporoid conidia were isolated for phylogenetic comparison. C. beticola was identified from Solanum ptycanthum (n = 4), Chenopodium album (n = 2), and Spinacia oleracea (n = 1), whereas C. chenopodii was identified on Chenopodium album (n = 51). Artificial inoculation of 21 plants species demonstrated that C. beticola was pathogenic to Brassica kaber, Chenopodium album, Carthamus tinctorius, Rumex obtusifolius, and Spinacia oleracea. These results indicate that although C. beticola may be pathogenic to a range of plant species, the role of symptomatic tissue for inoculum production on alternative hosts in the field appears limited. Observations of C. beticola on necrotic and naturally senescent tissue suggest saprophytic survival on plant debris of a range of species, which has implications for CLS epidemics and disease management.

Spatiotemporal Attributes and Crop Loss Associated with Tan Spot Epidemics in Baby Lima Bean in New York.

Tan spot, caused by the pycnidial fungi Didymella americana and Boeremia exigua var. exigua, is a foliar disease affecting processing baby lima bean production in New York. Tan spot epidemics are prevalent, occur annually, and may result in substantial defoliation. The disease is controlled by the prophylactic application of fungicides to maximize green leaf area. Information on yield losses due to tan spot on baby lima bean yield and the benefits of fungicide applications is needed to justify investments in disease management. Four small-plot, replicated trials were conducted over 2 years in commercial baby lima bean fields to evaluate the efficacy of fungicides for tan spot control at Piffard and Leicester, NY. Applications of pyraclostrobin or boscalid significantly reduced tan spot incidence and severity compared with nontreated plots, and increased the number of leaves per stem. In 2016, the increase in green leaf area associated with fungicide application was also documented in canopy reflectance values at 830 nm. Despite the decrease in tan spot incidence and corresponding increase in crop health obtained from fungicides, this effect did not translate into significant increases in pod yield. This finding suggested that the relationship between green leaf area and yield is highly variable in baby lima bean. The spatial and spatiotemporal patterns of naturally occurring tan spot epidemics were also characterized in baby lima bean fields across western New York using disease incidence data collected in transects and grids. The spatial pattern of data collected in transects was analyzed using median runs analysis. Disease incidence data collected in two-dimensional grids were analyzed to quantify spatial pattern using spatial analysis by distance indices (SADIE). The association function of SADIE was used to quantify the spatiotemporal patterns of tan spot epidemics after crop emergence and at harvest. These findings suggested that tan spot is likely to initiate at relatively frequent, randomly positioned foci, and that subsequent, limited spread results in significant local aggregation. Hypotheses for inoculum sources and recommendations for tan spot control in baby lima bean fields in New York are discussed.

Temporal Genetic Differentiation of Cercospora beticola Populations in New York Table Beet Fields.

Annual epidemics of Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, can result in substantial defoliation in table beet fields in New York. High allelic and genotypic diversity have been described within C. beticola populations; however, information on the temporal stability of populations is lacking. C. beticola isolates were obtained from symptomatic leaves in three table beet fields in successive years. Two of the fields were organic mixed-cropping farms and the third was managed conventionally in a broad-acre cropping system. C. beticola isolates (n = 304) were genotyped using 12 microsatellite markers. Genotypic diversity (Simpson's complement index = 0.178 to 0.990), allele frequencies, and indices of differentiation between years varied. Pairwise index of differentiation values ranged from 0.02 to 0.25 for clone-corrected data, and indicated significant genetic differentiation at Farm 2. No multilocus genotype was shared between years. The shift in multilocus genotypes between years questions the role of clonally reproducing primary inoculum. Collectively, these results suggest that a dominant inoculum source for initiating annual CLS epidemics is external to the field of interest. These findings have implications for CLS disease management in conventional and organic table beet production.

Management of Cercospora Leaf Spot in Conventional and Organic Table Beet Production.

Cercospora leaf spot (CLS; Cercospora beticola) is the most important foliar disease affecting table beet. Epidemics occur annually and fungicides extend the survival of foliage to enable mechanized harvest. However, a high frequency of strobilurin-resistant C. beticola isolates necessitates the identification of fungicides with different modes of action for tactical rotation. There is also substantial demand for organically produced table beet, for which synthetic fungicides are prohibited. Five small-plot, replicated field trials were conducted over two years to evaluate conventional and Organic Materials Review Institute (OMRI)-listed products for CLS control in table beet cv. Ruby Queen at Geneva and Ithaca, New York. Benzovindiflupyr + difenoconazole significantly reduced temporal disease progress (measured by the area under the disease progress stairs; AUDPS) by 86.7 to 97.3% compared with nontreated plots, and mean survival time of leaves was significantly extended. The demethylation inhibitor, propiconazole, also provided significant disease control in two trials in 2016. Disease severity in plots treated with succinate dehydrogenase inhibitors (boscalid, fluxapyroxad + pyraclostrobin, and penthiopyrad) was significantly decreased compared with nontreated plots but less than other fungicides. Efficacious fungicides significantly increased the dry weight of foliage but did not significantly affect the dry weight of roots, and root shoulder diameter. The enhanced longevity of leaves and increased dry weight of foliage may extend opportunities for mechanized harvesting without deleteriously affecting root yield parameters which are strictly regulated for the processing markets. In two trials, copper octanoate + Bacillus amyloliquefaciens strain D747 (as Cueva + Double Nickel LC) resulted in significantly improved disease control in comparison with application of either product alone and provided comparable and reproducible disease control equivalent to conventional fungicides at both locations. The implications of these findings for CLS control in conventional and organic table beet production systems are discussed.

Genotypic Diversity and Resistance to Azoxystrobin of Cercospora beticola on Processing Table Beet in New York.

Cercospora leaf spot (CLS), caused by Cercospora beticola, is one of the major diseases affecting productivity and profitability of beet production worldwide. Fungicides are critical for the control of this disease and one of the most commonly used products is the quinone outside inhibitor (QOI) azoxystrobin. In total, 150 C. beticola isolates were collected from two commercial processing table beet fields in Batavia, NY in 2014. The mating types of the entire population were determined, and genetic diversity of a subset of samples (n = 48) was assessed using five microsatellite loci. Sensitivity to azoxystrobin was tested using a spore germination assay. The cytochrome b gene was sequenced to check for the presence of point mutations known to confer QOI resistance in fungi. High allelic diversity (He = 0.50) and genotypic diversity (D* = 0.96), gametic equilibrium of the microsatellite loci, and equal ratios of mating types were suggestive of a mixed mode of reproduction for C. beticola. Resistance to azoxystrobin was prevalent because 41% of the isolates had values for effective concentrations reducing spore germination by 50% (EC50) > 0.2 µg/ml. The G143A mutation, known to cause QOI resistance in C. beticola, was found in isolates with EC50 values between 0.207 and 19.397 µg/ml. A single isolate with an EC50 of 0.272 µg/ml carried the F129L mutation, known to be associated with low levels of QOI resistance in fungi. This is the first report of the F129L mutation in C. beticola. The implications of these findings for the epidemiology and control of CLS in table beet fields in New York are discussed.

Swede midge (Diptera: Cecidomyiidae), ten years of invasion of crucifer crops in North America.

The Swede midge, Contarinia nasturtii Kieffer (Diptera: Cecidomyiidae), a common insect pest in Europe, is a newly invasive pest in North America that constitutes a major threat to cruciferous vegetable and field crops. Since its first identification in Ontario, Canada, in 2000, it has rapidly spread to 65 counties in the provinces of Ontario and Quebec and has recently been found in canola (one of two cultivars of rapeseed, Brassica napus L. and Brassica campestris L.) in the central Prairie region where the majority of Canada's 6.5 million ha (16 million acres) of canola is grown. The first detection of Swede midge in the United States was in 2004 in New York cabbage (Brassica oleracea L.), but it has now been found in four additional states. Here, we review the biology of Swede midge, its host plant range, distribution, economic impact, pest status, and management strategies. We provide insight into this insect's future potential to become an endemic pest of brassica crops in North America. We also proposed research needed to develop tactics for handling this invasive pest in brassica crops.

Cryptic diversity, pathogenicity, and evolutionary species boundaries in Cercospora populations associated with Cercospora leaf spot of Beta vulgaris.

The taxonomy and evolutionary species boundaries in a global collection of Cercospora isolates from Beta vulgaris was investigated based on sequences of six loci. Species boundaries were assessed using concatenated multi-locus phylogenies, Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Processes (PTP), and Bayes factor delimitation (BFD) framework. Cercospora beticola was confirmed as the primary cause of Cercospora leaf spot (CLS) on B. vulgaris. Cercospora apii, C. cf. flagellaris, Cercospora sp. G, and C. zebrina were also identified in association with CLS on B. vulgaris. Cercospora apii and C. cf. flagellaris were pathogenic to table beet but Cercospora sp. G and C. zebrina did not cause disease. Genealogical concordance phylogenetic species recognition, GMYC and PTP methods failed to differentiate C. apii and C. beticola as separate species. On the other hand, multi-species coalescent analysis based on BFD supported separation of C. apii and C. beticola into distinct species; and provided evidence of evolutionary independent lineages within C. beticola. Extensive intra- and intergenic recombination, incomplete lineage sorting and dominance of clonal reproduction complicate evolutionary species recognition in the genus Cercospora. The results warrant morphological and phylogenetic studies to disentangle cryptic speciation within C. beticola.

IMA Genome-F 9: Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata.

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.

Genetic structure of Cercospora beticola populations on Beta vulgaris in New York and Hawaii.

Cercospora leaf spot (CLS), caused by Cercospora beticola, is a major disease of Beta vulgaris worldwide. No sexual stage is known for C. beticola but in its asexual form it overwinters on infected plant debris as pseudostromata, and travels short distances by rain splash-dispersed conidiospores. Cercospora beticola infects a broad range of host species and may be seedborne. The relative contribution of these inoculum sources to CLS epidemics on table beet is not well understood. Pathogen isolates collected from table beet, Swiss chard and common lambsquarters in mixed-cropping farms and monoculture fields in New York and Hawaii, USA, were genotyped (n = 600) using 12 microsatellite markers. All isolates from CLS symptoms on lambsquarters were identified as C. chenopodii. Sympatric populations of C. beticola derived from Swiss chard and table beet were not genetically differentiated. Results suggested that local (within field) inoculum sources may be responsible for the initiation of CLS epidemics in mixed-cropping farms, whereas external sources of inoculum may be contributing to CLS epidemics in the monoculture fields in New York. New multiplex PCR assays were developed for mating-type determination for C. beticola. Implications of these findings for disease management are discussed.

Global genotype flow in Cercospora beticola populations confirmed through genotyping-by-sequencing.

Genotyping-by-sequencing (GBS) was conducted on 333 Cercospora isolates collected from Beta vulgaris (sugar beet, table beet and swiss chard) in the USA and Europe. Cercospora beticola was confirmed as the species predominantly isolated from leaves with Cercospora leaf spot (CLS) symptoms. However, C. cf. flagellaris also was detected at a frequency of 3% in two table beet fields in New York. Resolution of the spatial structure and identification of clonal lineages in C. beticola populations using genome-wide single nucleotide polymorphisms (SNPs) obtained from GBS was compared to genotyping using microsatellites. Varying distance thresholds (bitwise distance = 0, 1.854599 × 10-4, and 1.298 × 10-3) were used for delineation of clonal lineages in C. beticola populations. Results supported previous reports of long distance dispersal of C. beticola through genotype flow. The GBS-SNP data set provided higher resolution in discriminating clonal lineages; however, genotype identification was impacted by filtering parameters and the distance threshold at which the multi-locus genotypes (MLGs) were contracted to multi-locus lineages. The type of marker or different filtering strategies did not impact estimates of population differentiation and structure. Results emphasize the importance of robust filtering strategies and designation of distance thresholds for delineating clonal lineages in population genomics analyses that depend on individual assignment and identification of clonal lineages. Detection of recurrent clonal lineages shared between the USA and Europe, even in the relaxed-filtered SNP data set and with a conservative distance threshold for contraction of MLGs, provided strong evidence for global genotype flow in C. beticola populations. The implications of intercontinental migration in C. beticola populations for CLS management are discussed.

Detection of Contarinia nasturtii (Diptera: Cecidomyiidae) in New York, a new pest of cruciferous plants in the United States.

The midge Contarinia nasturtii Kieffer (Diptera: Cecidomyiidae) was first confirmed in North America in Ontario, Canada, in 2000. The insect is now distributed throughout many counties in the provinces of Ontario and Québec. Nearly 1,200 farms in the northeastern United States that grow cruciferous vegetables are at risk for C. nasturtii infestation if this insect were to spread to that region. Over a period of 3 yr (2002-2004), approximately 3,000 ha of crops on 94 farms in western New York State was scouted for C. nasturtii, but none were found. In 2004, 42 experimental pheromone traps were placed in fields of cruciferous vegetables in eight counties. C. nasturtii males were captured at low levels (1-50 per trap / 8 wk) on four farms in Niagara County, but not at any other site. C. nasturtii larvae were found in plant tissue at one of the four farms. Insect specimens were identified by morphological methods, molecular methods, or both. This is the first confirmation of C. nasturtii in the United States, which we believe was made possible by the combined use of pheromone traps, morphological characters of trapped adults, and molecular methods. The early detection in New York presents an opportunity to implement measures to limit the spread and establishment of C. nasturtii across the state and into other regions of the United States.

Stable transformation of plant cells by particle bombardment/biolistics.

Particle bombardment, or biolistics, is a commonly used method for genetic transformation of plants and other organisms. Millions of DNA-coated metal particles are shot at target cells or tissues using a biolistic device or gene gun. The DNA elutes off the particles that lodge inside the cells, and a portion may be stably incorporated in the host chromosomes. A protocol for the generation of transgenic grapevines via biolistic transformation of embryogenic cell suspension cultures is detailed in this chapter. In a typical experiment, transient gene expression averaged nearly 8000 "hits" per bombarded plate. Five months after bombardment, there were nearly five putative transgenic embryos per bombarded plate. About half of the embryos were regenerated into confirmed transgenic plants. The basic bombardment procedures described are applicable to a wide range of plant genotypes, especially those for which embryogenic cell cultures are available. All users of particle bombardment technology will find numerous useful tips to maximize the success of transformation.

In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen.

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4 degrees C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at -20 degrees C or -80 degrees C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4 degrees C for a couple of weeks and then at -80 degrees C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).