VACCINIUM

Flow Cytometry determination using DAPI with internal standard = Vinca minor; performed by Plant Cytometry Services, The Netherlands, 30 June 2012. This project was a collaborative effort from the USDA ARS and the University of Florida.

Flow Cytometry determination using DAPI with internal standard = Vinca minor; performed by Plant Cytometry Services, The Netherlands, 18 May 2012.

Leaves were collected from 299 actively growing plants representing 143 accessions that are preserved in greenhouses, screenhouses, or field collections at the USDA-ARS, NCGR, Corvallis, OR. Leaf samples were also obtained for an additional 68 plants from two nurseries (9 from N1; 11 from N2), and breeders across the US including Oregon (Chad Finn, 2, OR), Minnesota (Jim Luby, 1, MN), Mississippi (Stephen Stringer, 6, MS), North Carolina (Hamid Ashrafi, 8, NC), and New Jersey (Mark Ehlenfeldt, 28, NJ). DNA was extracted from young leaves of these 363 plants using a modified Qiagen protocol described in detail by Gilmore et al. (2011). DNA amplification with the 5-SSR or the 10-SSR multiplex was performed with the Type-it Multiplex PCR mix (Qiagen Inc., Valencia, CA, USA) as detailed by Bidani et al., 2017. DNA was amplified in an Eppendorf Gradient thermocycler (Eppendorf, Westbury, NY, USA) or an MJ Research Tetrad thermocycler (BioRad, Hercules, CA, USA). The PCR protocol consisted of a “touchdown” program with an initial denaturation cycle at 94 ºC for 3 min followed by ten cycles of 40 sec at 94 °C; 45 sec at 62 °C, decreasing 1 °C each cycle; and 45 sec at 72 °C. PCR continued for an additional 28 cycles of 40 sec at 94 °C; 45 sec at 52 °C; and 45 sec at 72 °C; followed by a final extension at 72 °C for 30 min. Once amplification success was verified by 2% agarose gel electrophoresis, PCR products were pooled and separated by capillary electrophoresis with a Beckman CEQ 8000 (Beckman Coulter, Inc.). In all cases, allele sizing and visualization were performed using the fragment analysis module of the CEQ 8000 software. Individuals were scored by grouping PCR fragment sizes (alleles) into bins of less than one base pair.

Abstract
Blueberry shock virus (BlShV) is a serious problem in blueberry production in the Pacific Northwest (PNW) region of North America. Infection occurs during bloom and the virus moves into other parts of the plant in an uneven but steady manner and may take several years to become fully systemic in mature bushes. In the year after infection, emerging flower and leaf tissues die rapidly at full bloom followed by regrowth. Once symptoms have been exhibited in all parts of the plant, the plant remains asymptomatic in subsequent years. Infection in young plants of some cultivars can stunt the plant for the remainder of its life, however, more typically production is lost for one year. While there appears to be no immunity as tested by grafting, it has been recognized that the rate of infection varies among genotypes. Plants in the HCRU breeding program and at the NCGR, have been tested regularly by ELISA for BlShV, in some cases for nearly 20 years, and germplasm that is particularly slow to become infected has been identified. At Oregon State University, North Willamette Research and Extension Center (NWREC), ‘Legacy’, ‘Toro’, ‘Bluecrop’, ‘Baby Blues’, ‘Darrow’ and all rabbiteye cultivars have tested negative for BlShV for over 10 years while growing among many known positive plants. On the other extreme, ‘Berkeley’, ‘Bluegold’, ‘Brigitta Blue’, ‘Nui’ and ‘Spartan’ tested positive in the year following their 1st bloom. At the NCGR, ‘Bladen’, ‘Harding’, ‘Lateblue’, ‘Legacy’, ‘Razz’, US 612, US 693, US 845 and US 847 all tested negative after 20 years in the field. ‘Toro’ and ‘Bluecrop’, which had tested negative after 20+ years at NWREC, were positive at the NCGR, as were many rabbiteye cultivars. Crosses have been made among genotypes that are slow to test positive for BlShV to try to develop this trait in cultivars.

Methods
Leaf samples were taken from all plots in the USDA-ARS, HCRU trials (Corvallis, OR and at Oregon State University - North Willamette Research and Extension Center [NWREC], Aurora, OR) in spring of all years beginning the first year the plants had flowers (typically two years after planting). For plots that tested BlShV+, samples from individual plants were taken and reanalyzed to determine whether all plants in the plot were BlShV+. Samples were similarly taken in multiple years at the USDA-ARS, NCGR (Corvallis, OR), however, the results from the 2015 are the only ones examined. All fields contain many genotypes that have tested BlShV+ for many years providing plenty of inoculum for infecting newly established plants. The leaf samples were homogenized in blueberry buffer and the standard procedures for testing for BlShV by enzyme-linked immunosorbent assay (ELISA) testing were followed as described by Bristow and Martin (1999).

pollen germination procedured...

Abstract:
Fruits from 107 genotypes of Vaccinium L., Rubus L., and Ribes L., were analyzed for total anthocyanins (ACY), total phenolics (TPH), and antioxidant capacities as determined by oxygen radical absorbing capacity (ORAC) and ferric reducing antioxidant power (FRAP). Fruit size was highly correlated (r ) 0.84) with ACY within Vaccinium corymbosum L., but was not correlated to ACY across eight other Vaccinium species, or within 27 blackberry hybrids. Certain Vaccinium and Ribes fruits with pigmented flesh were lower in ACY, TPH, ORAC, and FRAP compared to those values in berries with nonpigmented flesh. ORAC values ranged from 19 to 131 ímol Trolox equivalents/g in Vaccinium, from 13 to 146 in Rubus, and from 17 to 116 in Ribes. Though ACY may indicate TPH, the range observed in ACY/TPH ratios precludes prediction of ACY from TPH and vice versa for a single genotype. In general, TPH was more highly correlated to antioxidant capacity than ACY was. This study demonstrates the wide diversity of phytochemical levels and antioxidant capacities within and across three genera of small fruit.

Safety. There is an explosion hazard as liquid nitrogen becomes gaseous. For proper venting procedures during liquid nitrogen milling of frozen materials, refer to Rodriguez-Saona and Wrolstad (15).

Sampling Procedures. Ripe fruit samples, as judged by flavor and color, were harvested during summer 2000 from two Willamette Valley sites: Oregon State University North Willamette Experiment Station (Aurora, OR) and the U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository (Corvallis, OR). Approximately 60 g of fruit was collected from 1 to 4 clones of each genotype. The highbush blueberry, V. corymbosum L. cv. Summit, was picked very early and very late in its season. Fruit was placed immediately on ice in the field and frozen at -10 C later that same day. Care was taken to avoid unripe, damaged, or overripe fruit. Samples were prepared according to Rodriguez-Saona and Wrolstad (15). About 40 g of berries were counted as each sample was weighed to determine average berry size. The frozen fruits were further cooled in liquid nitrogen; then they were cryogenically milled in a stainless Waring blender jar containing a lid modified with a chimney. Chilled tubes were filled with milled fruit powder and weighed, and then the powder was extracted with acetone, followed by two additional extractions with 70:30 acetone/water. The pooled supernatants were partitioned with two volumes of chloroform. The nonpolar phase was discarded, and the aqueous extracts were stored at -10 C or at -70 C if for antioxidant analysis.

Determination of Total Anthocyanins (ACY). Anthocyanin quantitation was performed by the pH differential method of Giusti and Wrolstad (16). Samples were diluted 1:150 in pH 1.0 and pH 4.5 buffers, then measured at 520 and 700 nm in a Shimadzu 300 UVVisible spectrophotometer. ACY was based on a cyanidin 3-glucoside molar extinction coefficient of 26,900 and a molecular weight of 449.2. Resultant values were expressed in terms of mg of anthocyanin/100 g of fresh-frozen fruit.

Determination of Total Phenolics (TPH). The Folin-Ciocalteu method (17) was used to determine total soluble phenolics (TPH). Extracts were diluted 1:500 or 1:1000 before incubation at 40 C. Absorption was measured at 755 nm. TPH was expressed as mg of gallic acid/100 g of fresh-frozen fruit.

Determination of Antioxidant Capacity. Antioxidant capacity was determined by ORAC and FRAP assays at the Linus Pauling Institute, Oregon State University. The ORAC assay was performed as described by Cao et al. (18) and adapted for use in a 96-well microplate fluorometer (model Cytofluor 4000, PerSeptive Biosystems, Framingham, MA). ORAC values, derived from triplicate analyses, are expressed as ímol Trolox equivalents (TE) per g of fresh-frozen fruit. Trolox is a water-soluble tocopherol analogue used as a reference compound for antioxidant capacity. The FRAP assay (19) was adapted for use in a 96-well microplate spectrophotometer (ThermoMax, Molecular Devices, Foster City, CA). FRAP values, derived from triplicate analyses, are expressed as ímol of ferric iron reduced per g of fresh-frozen fruit.

Statistical Analysis. Correlation and regression analyses were performed using Microsoft Excel Data Analysis. Differences at p > 0.05 were considered significant.

Ethnobotanical knowledge gathered by Pricilla Russell Kari from Athabascan Elders. Published by US National Park Service in 1995

Basic ecology of host blueberry species and genotypes.
The genus Vaccinium L. (blueberries, cranberries, bilberries) in the Ericaceae (Heath family) includes upwards of 450 species, most inhabiting cooler tropical microclimates (e.g., forest understory and higher elevations). The remaining species occur throughout subtropical, temperate, and boreal regions of the northern hemisphere including regions native and exotic to D. suzukii. Insular species that we tested include V. reticulatum, V. calycinum, V. cylindraceum and V. padifolium, species hypothesized to be highly susceptible to attack. Blueberries and cranberries are currently the most economically important of North American Vaccinium. Cultivated highbush-type blueberries are polyploid, more specifically, tetraploid like northern and southern highbush blueberries (V. corymbosum) and hexaploid like rabbiteye blueberry (V. virgatum syn. ashei, Hummer et al. 2015, Kron et al. 2002). Of the 29 species tested, 4 Asian blueberry species, V. myrtoides, V. bracteatum, V. oldhami, and V. smallii, probably shared a long evolutionary history with D. suzukii and, hence, could have evolved some level of resistance.

Greenhouse growing conditions and germplasm handling.
The USDA National Clonal Germplasm Repository in Corvallis, Oregon conserves wild relatives, cultivars and hybrids of Vaccinium. Non-hardy types are maintained as containerized plants in greenhouses, whereas cold hardy types grow in the field. Fruit for these species ripen between June and August. For this study, samples representing 29 species including cultivars and hybrids (Table 1), were harvested at early to peak ripeness, and were shipped overnight, for analysis at the USDA-ARS facilities in Poplarville, Mississippi. Fruit of rabbiteye and southern highbush blueberry were collected inhouse from greenhouse-grown fruit at the Poplarville Location. About 30 chilled fresh fruit were sent for each of the 140 genotypes. As the 2016 season progressed and ripening fruit became available, six separate packages of berry samples were sent on 1 June, 21 June, 12 July, 20 July, 25 July, and 3 August. Information on the 29 species used for this study are shown in Table 1. Tables 2 through 5 also provide the genotypes that provided enough intact berries for resistance screening.

Fly cultures and handling.
Drosophila suzukii adults used in these tests originated from inbred cultures reared from flies originally collected in 2014 from rabbiteye blueberry (Vaccinium virgatum syn. ashei) fruit harvested at an experimental farm near Old Creek and Indian Camp Branch, Perkinston MS (30°47014″N, 88°59026″W). Fly cultures were maintained in 68 ml plastic vials. Our D. suzukii diet included the four ingredients: 3.75 g of standard Drosophila instant media (formula 4-24, Carolina Biological Supply, Burlington, NC), an intact sterile blueberry or blackberry fruit, 20 ml water, and 0.8 mg Fleishmann’s dry baker’s yeast added as a supplemental protein source (Sampson et al. 2016). Cultures developed under ambient indoor lighting at 21°C under 60% relative humidity.

Bioassay (Screening) Protocols.
We assessed resistance (antibiosis) using detached fruit bioassays. We recorded the resulting reproductive parameters of 12.0 ± 0.4 females caged with an equal number of males (11 ± 0.5 males, Mean ± SE, n = 229 vials). Flies within each vial had access to ~10 fruits or 15g of intact unblemished fruit containing their pedicels. Pedicels were important to retain because any open wound or incision on a fruit could permit flies to circumvent resistant surfaces and accelerate oviposition 2- to 15-fold (Ioriatti et al. 2015, Rezazadeh et al. 2018). Flies and fruit from randomly selected cultures were confined to randomly-chosen (68 ml) plastic Drosophila rearing vials solely containing fruit of a known berry species and genotype. Vials remained horizontal to give flies uniform access to berries for 3 days. Our bioassay should provide adequate pest pressure to reveal antibiosis because of a mean density of 1.2 females and 1.1 males per host berry. Flies were given no supplemental food because an ability to puncture fruit for feeding and laying eggs was considered an element of resistance. Initially, fly mortality went unassessed because adults were expected to survive a 3-day confinement period without food or water. However, when we discovered that adults must feed from fruit punctures to survive, we began assessing fly mortality on subsequent fruit from 46 other host genotypes, including genotypes designated as our non-host standard. After 3 days, dead flies of both sexes were counted and remaining live flies euthanized. Fruit were then analysed for mass (g), density (berries per vial), and brix or the percentage dissolved solids, a measure of sugar content or ripeness determined using a handheld refractometer. We simultaneously measured the following fly life history parameters: (p1) subsurface eggs (viable eggs) per fruit, (p2) surface eggs (unviable eggs) per fruit, (p3) larvae per fruit, (p4) adult females per vial, (p5) adult males per vial, (p6) adult mortality (dead flies / total flies *100), (p7) viable brood per female [(p1+p3)/p4], (p8) reproductive rate over 3 d = (p7 / fruit density). A resistance index was calculated as the sum of the number of fly reproductive parameters not significant (indicated by **) from the host (Vaccinium) standard)) + 1 (for significance from non-host standard) or +2 (for non-significance from non-host standard). The minimal resistance value is 1, the maximum is 5.

The non-host resistance standard was conceived ad hoc and included fly reproductive responses to fruit collected locally from known or probable non-hosts such as Vitis rotundifolia, Ilex, Citrus, and Rhaphiolepsis. The host (Vaccinium) resistance standard was developed post hoc and included fly reproductive responses to highly-resistant host fruit collected from Vaccinium consanguineum, V. cylindraceum, V. ovatum, V. myrtoides, V. corymbosum cvs. CVAC1312 and ‘Brigitta Blue’, V. virgatum FLW84-52, V. amoenum CVAC1155.

Experimental design and analysis.
Berries of available species and genotypes were isolated with flies in 2 – 3 separate plastic vials arranged in a fully randomized design according to a no-choice bioassay, i.e., only berries of one genotype were placed in each individual vial. Berries were dissected individually and were treated as experimental replicates. Effects of genotype, ploidy level, berry weight and brix on fly reproductive parameters (i.e., eggs, larval density, female brood output, reproductive rate, and adult mortality) were analysed with correlation analysis (Proc CORR in SAS 9.4) and 1-way or 2-way multivariate analysis of covariance (ANCOVA) with berry weight and brix serving as covariates (Proc GLM in SAS 9.4). Resistance indices were compared among species using one-way analysis of variance (1-way ANOVA, Proc GLM in SAS 9.4) with mean separations accomplished with Tukey’s HSD tests at an α = 0.05. Likewise, the effect of adult mortality on oviposition rate was assessed using 1-way ANOVA after arcsine-square-root transforming adult mortality rate. Cluster analysis (PROC ACECLUS in SAS 9.4) followed by phylogenetic analysis using the first two canonical variables (PROC TREE in SAS 9.4) grouped species and associated genotypes by clutch size (eggs per berry), larvae per berry, female reproductive output, and resistance index. We adjusted female reproductive responses for variation in berry size (i.e., weight). Non-linear relationships between adult mortality and egg count, resistance index and adult mortality, ploidy and brood output, ploidy and fruit weight, ploidy and antibiosis, fruit weight and reproductive rate, fruit weight and brood output, and fruit weight versus brix were modelled using PROC NLIN in SAS 9.4.

Mineral soil adaptation indices:
A mineral soil adaptation index (0 – 10) for each genotype was calculated as follows: mineral soil adaptation score = Ʃ mean ratings for vegetative vigor (1995 and 1996), yield balance (1995 and 1996) and nutrient deficiency symptoms (1996) divided by 5. This index was used in conjunction with growth and fruiting data to evaluate genotypic response to the mineral soil environment. The mineral soil adaptation indices varied continuously within the population ranging from 2.8 to 8.9. Individuals displaying indices > or = 7.5 exhibited mineral soil adaptation.

Stringer, S.J., Sampson, B.J. and Hummer, K.E. (2017). Screening small fruit germplasm for resistance to southern populations of invasive spotted wing drosophila, SWD (Diptera: Drosophilidae). Acta Hortic. (ISHS) 1180:45-52 https://doi.org/10.17660/ActaHortic.2017.1180.7

Bioassays targeted Drosophila suzukii (Spotted-Wing Drosophila abbreviated SWD), a pest currently infesting a blueberry selection and field evaluation site at Stone County, Mississippi. From these, un-infested blueberry fruit samples were harvested from numerous genotypes and then evaluated in the laboratory for possible antixenosis against mass-reared SWD. One hundred berries were sampled from each of the 88 rabbiteye and southern highbush cultivars and selections and examined for the presence or absence of SWD feeding damage or egg laying. Berry samples sometimes had to be refrigerated and, therefore, the effects of cold storage on SWD infestation and OSI were additionally tested. In laboratory bioassays using no-choice arenas, clutch size for SWD (number of eggs per fruit) as well as total brood per fruit were assessed after 3 days and these counts correlated with berry quality attributes, which were independently recorded for each variety [berry weight, percentage soluble solids (Brixo), and harvest seasonality (early-, mid-, and late-season harvest)].

Berry samples of these same cultivars and selections were utilized in a ten replicate laboratory assay where berries were placed in 3 x 6 arrays (arenas) of specially constructed plastic bioassay chambers along with one or two fecundated female spotted-wing Drosophila flies. Partitioning analysis of variance will examine variation in SWD egg output attributable to within and between assay runs, as fruit storage and plant growing conditions may influence bioassay results. We chose 10 replicates per run and a total of 20 replicates for testing repeatability between 2 separate runs, provided there is sufficient time and material to complete 20 replications per genotype.

Plant material.

In total, 25 rabbiteye blueberry cultivars were used for this study. Notes on the origins and unusual germplasm compositions are given in Table 1. Shoots came from pairs of mature plants in experimental plantings at the P.E. Marucci Center for Blueberry and Cranberry Research and Extension (Rutgers University) in Chatsworth, N.J. Detached shoots were collected on 8 Jan. 2004 and on 10 Jan. 2005 to determine floral-bud cold hardiness. Previous studies of highbush and rabbiteye have shown this time period to be one at which plants have achieved maximum cold hardiness (Muthalif and Rowland, 1994). All plants used for shoot collection were at least 4 to 5 years old, and were selected for as much uniformity as possible. Most samples were from terminal shoots taken from upper portions of the bush.

Determination of flower bud cold hardiness.

For the freeze-thaw protocol, three 5- to 6-cm-long shoots, each with three to eight flower buds, were used for each treatment as described previously by Arora et al. (2004). Treatment temperatures chosen for the fully cold-acclimated buds covered a range from -10 to -28 C (the lowest temperature that the glycol freezing bath would consistently reach) in 2 C increments, to cover a potential range of 0 to 100 percent injury to blueberry buds for most genotypes (Arora et al., 1997). Shoots were removed from the freezing bath at their respective selected treatment temperatures, thawed overnight at 4 C, then incubated at 20 C for 24 hours. The three most apical buds were then dissected and observed for injury (visual browning of the ovaries in individual flowers) (Arora et al., 2000; Flinn and Ashworth, 1994). Each bud was rated for percentage of injured ovaries and bud cold hardiness was defined as the temperature causing 50 percent injury overall (LT50).

Statistical analysis of cold hardiness data.

Bootstrap estimates (Manly, 1997) of LT50 values and their 95 percent fiducial confidence intervals were calculated using Proc Probit (SAS Institute, 1999) for each cultivar x year combination. The nine observed data points (three proximal buds on each of the three shoots) for each temperature were resampled (n = 9 with replacement) 10,000 times. A sigmoidal (i.e., logistic) regression model was fit to percentage of injury (browning) vs. treatment temperature for each of the 10,000 sets of resampled data, and the 10,000 resulting values of LT50 and their lower and upper confidence limits were averaged to obtain a bootstrap estimate for each cultivar x year. A one-way ANOVA was conducted on these 50 LT50 estimates to compare the 25 cultivars using the duplicate year data. Cultivar x year (i.e., error) variability was partitioned into 5 sizes of variance groups so that the appropriate size of error variation was associated with each cultivar in the means comparison. The cultivar means comparison used the Sidak adjustment to ensure alpha = 0.05.

Bud size evaluations.

On 11 Jan. 2005, the cultivars in were evaluated for uniformity of flower bud size. Ratings were done on a scale of 1 to 5 with 1 = uniformly sized buds and 5 = a substantial difference between the smallest and largest buds on the cultivar.

Curatorial selection of core accessions

Study Name: Vaccinium Evaluation Data 1990 Experiment Type: Field FIELD Study Year: 1990 Year started: 01/01/1990 Year ended: 12/31/1990 Experiment length: 1 year

Study Name: Cranberry Cultivar Evaluation, 1988 Experiment Type: Field FIELD Study Year: 1988 Exp. Location: Du Bay Plots

Study Name: Resistance of Vaccinium spp. to the Leafhopper S. magdalensi Study Year: 1988