Evaluation of Six Models to Estimate Ascospore Maturation in Venturia pyrina.

Estimates of ascospore maturity generated by models developed for Venturia pyrina in Victoria, Australia (NV and SV), Oregon, United States (OR), and Italy (IT) or for V. inaequalis in New Hampshire, United States (NH-1) or modified in Norway (NH-2) were compared with observed field ascospore release of V. pyrina from 21 site-year combinations. The models were also compared with ascospore release data from laboratory assays. In the laboratory assays, the forecasts of the NH-1 and NH-2 models provided the best fit to observed spore release. Under field conditions, the lag phases and slope coefficients of all models differed from those of observed release of ascospores. Identifying the precise time of bud break of pear to initiate degree-day accumulation was problematic at both Australian sites. This resulted in a higher deviance between bud break and first released ascospore compared with the sites in Norway and Belgium. Linear regressions of observed release against forecasted maturity generated similarly high concordance correlation coefficients. However, where differences were noted, they most often favored models that included adjustment for dry periods. The NH-2, IT, and NV models using pooled data also provided the most accurate estimates of 95% ascospore depletion, a key event in many disease management programs.

First Report of Blossom Blight and Green Fruit Rot of Sweet Cherry Caused by Sclerotinia sclerotiorum in Oregon.

During May and June of 2006, 'Lapins' sweet cherry (Prunus avium) trees were observed with white fungal growth on blossoms and young fruit in two commercial orchards in central Oregon (Wasco County). Entire blossom clusters and 30% of fruit clusters were affected. Rot on the fruit was firm, light brown, and covered the entire fruit, extending halfway down the pedicel. Affected blossoms were light brown and stuck to adjacent fruit and blossoms. Six disease samples from fruit, four samples from pedicels, and four samples from blossoms were surface sterilized and plated on potato dextrose agar acidified with lactic acid ([APDA] 1.5%). A white fungus producing sclerotia measuring 4 to 8 mm in diameter was recovered from all the samples after 10 days on APDA at 25°C. Koch's postulates were satisfied by inoculating green, pea-sized 'Lapins' cherry fruit with mycelial plugs colonized by the white fungus. Symptoms and signs similar to those seen in the orchard were produced. The same sclerotium-producing fungus was recovered from all inoculated fruit. The pathogen was identified as Sclerotinia sclerotiorum (Lib.) de Bary on the basis of the size of sclerotia and nested PCR using fungal universal primer pair ITS4/ITS5 and S. sclerotiorum-specific primer pair SSFWD/SSREV (2). A negative control, devoid of DNA templates in the reaction mix, was included in the PCR assay. S. sclerotiorum is endemic on wheat in Wasco County. The affected orchards were surrounded by wheat fields in which snow mold disease that is caused by S. sclerotiorum was particularly serious in the spring of 2006 compared with previous years. Rot on cherries was first observed and very severe in May of 2005, with symptoms and signs mainly on leaves of all trees across 45 ha. New symptoms continued to appear on cherry until mid-June. During 2006, most symptoms and signs were on fruit with some leaf spotting. Both years had unusually wet springs with 12.2 cm of rainfall received from April to June 2005 and 12.4 cm from April to June 2006, which is well above the 6.81 cm average for the previous 5 years. S. sclerotiorum causes green fruit rot of stone fruits, including almond, apricot (3), nectarine, and peach (1). To our knowledge, this is the first report of S. sclerotiorum causing blossom blight and green fruit rot on sweet cherry in Oregon. References: (1) K. M. Flint. Green fruit rot. Page 15 in: Compendium of Stone Fruit Diseases. The American Phytopathological Society. St. Paul, MN, 1995. (2) J. Freeman et al. Eur. J. Plant Pathol. 108:877, 2002. (3) R. E. Smith. Phytopathology 31:407, 1931.

Timing of Preharvest Infection of Pear Fruit by Botrytis cinerea and the Relationship to Postharvest Decay.

Botrytis cinerea causes significant levels of postharvest decay in the winter pear cultivar d'Anjou. The objectives of this study were to determine the timing of B. cinerea infection of pear stems and calyxes in the orchard during the growing season, to investigate the development of gray mold in storage, and to determine whether preharvest levels of B. cinerea in pear stems and calyxes can be used as predictors of gray mold levels observed in storage. Very low levels of B. cinerea were isolated from stem tissue prior to harvest. In a single year repeat experiment, stems sampled at harvest had higher levels of infection than those sampled earlier in the season. Little or no stem end gray mold was detected in fruit after 3 months in air-storage; however, incidence increased between 6 and 8 months. Calyx end gray mold was detected at low levels in fruit stored for up to 8 months. The mean incidence of stem end gray mold was 3.6 and 2.0%, and incidence of calyx end gray mold was 1.2 and 0.2%, in 1996 and 1997, respectively. Calyxes were susceptible to infection soon after full bloom; however, inoculation of calyxes in April or May did not result in higher levels of calyx end gray mold in storage. Therefore, preharvest level of calyx infection is a poor predictor of calyx end gray mold in storage. In addition, application of benomyl in the orchard reduced the level of B. cinerea in blossoms but had no effect on levels of calyx end gray mold of fruit in storage. Packing and shipping fruit within 3 to 6 months of harvest may mitigate economic losses due to gray mold.

Involvement of Aureobasidium pullulans and Rhodotorula glutinis in Russet of d'Anjou Pear Fruit.

d'Anjou pear, the main cultivar grown in the Mid-Columbia Region of Oregon, is subject to russeting of the fruit surface, resulting in reduced quality and value. The role of Aureobasidium pullulans and Rhodotorula glutinis in russet of pear fruit was studied. Inoculations were done at full bloom in 1998 and 1999 and petal fall in 1999 with a log range of concentrations up to 108CFU/ml. Populations of A. pullulans on floral and fruit tissue were monitored during spring 1999 and 2000 in six orchards with a history of russet. Russet of fruit in both studies was evaluated at harvest. In 1998 neither fungus increased russet. In 1999, inoculation with two strains of each fungus at 108 CFU/ml increased russet. Inoculation with 104 or 106 CFU/ml did not increase russet in either year. In commercial orchards, there was no correlation between fruit russet and the populations of A. pullulans on floral and fruit tissue. Populations were less than 103 CFU/g of tissue. We conclude that A. pullulans and R. glutinis are not major contributors to russet of d'Anjou pear fruit in the Mid-Columbia Region.

First Report of Phytophthora syringae Causing Rot on Apples in Cold Storage in the United States.

A decay of 'Granny Smith' apples (Malus domestica Borkh.) was observed in 1988, 1990, and 1991 on fruit grown in the lower Hood River Valley of Oregon and stored at 0°C. Harvested fruit were drenched with thiabendazole and stored in October in all years. In mid-November, fruit were sized, drenched with sodium hypochlorite, and returned to cold storage. Decay was observed in January when fruit were removed from cold storage, sorted, and packed. Decayed areas were light brown and firm with a slightly indefinite margin. Losses were less than 1% of fruit packed. Diseased fruit were surface-disinfested with 95% ethanol, and tissue pieces were transferred aseptically to potato dextrose agar acidified with lactic acid and incubated at approximately 22°C. The fungus consistently isolated was identified as Phytophthora syringae (Kleb.) Kleb. based on morphological characters (3). Sporangia were persistent and averaged 60 µm long (range 59 to 69) × 40 µm wide (range 37 to 43). Antheridia were paragynous, and oospores averaged 37 µm (range 31 to 46). 'Golden Delicious', 'Granny Smith', and 'Gala' apples were inoculated with mycelial plugs from a 7-day-old culture of P. syringae and incubated 12 days at 5°C and 7 to 12 days at 22°C. Twenty fruit of each cultivar were used-ten were inoculated, and ten uninoculated fruit served as controls. Lesions developed on all inoculated fruit but not on uninoculated controls. Lesions were spherical, chocolate brown, and firm with no evidence of external mycelia. Lesion morphology was similar on all cultivars. P. syringae was reisolated from lesion margins of all infected fruit. This postharvest decay of apples has not been observed in the Hood River Valley since 1991. Fruit rot of apples caused by P. syringae is known in Canada (1) and is common in the United Kingdom (2), but has not been reported previously in the United States. To our knowledge, this is the first report of postharvest decay of apples by P. syringae in the United States. References: (1) R. G. Ross and C. O. Gourley. Can. Plant Dis. Surv. 49:33, 1969. (2) A. L. Snowdon. A Color Atlas of Postharvest Diseases. CRC Press, Inc., Boca Raton, FL, 1990. (3) G. M. Waterhouse. The Genus Phytophthora. Misc. Publ. 12. The Commonwealth Mycological Institute, Kew, Surrey, England, 1956.

Detection and Quantification of Botrytis cinerea by ELISA in Pear Stems During Cold Storage.

Botrytis cinerea was detected and quantified in pear stems from six orchards in the Pacific Northwest, and changes in fungal biomass in the stems after 6 and 8 months of cold storage in regular (air) atmosphere were studied. The fungus was detected by plating stem halves on selective medium and by enzyme-linked immunosorbent assay (ELISA) using the Botrytis-specific monoclonal antibody BC-12.CA4. Both methods demonstrated that the incidence of B. cinerea increased from 6 to 8 months, but ELISA was more sensitive than standard isolation. Quantitative ELISAs on stems indicated that over 200 µg of B. cinerea biomass per gram of stem tissue was present in the stems of visibly rotted fruits, but usually less than 35 µg/g was present in stems from fruits without visible gray mold. Aureobasidium pullulans, Penicillium spp., Alternaria spp., and Cladosporium spp. were commonly isolated from stem tissue. A. pullulans was present in 86% of the stems from which B. cinerea was detected. Use of the monoclonal antibody BC-12.CA4 could help in determining the infection path of B. cinerea in pear stems and detection of latent infections, enabling the timing and method of control of stem end rot to be optimized.

Effect of Fenarimol Application Timing on Fruit Shape of Bartlett Pear.

Plots were established at Ukiah and Davis, California, and Medford and Hood River, Oregon, to evaluate the effect of fenarimol (Rubigan 1 EC), applied at various stages of bloom development, on fruit shape of Bartlett pear. At each location, 10 different treatment schedules were applied at various combinations of bud burst (BB), green cluster (GC), white bud (WB), and full bloom (FB). In schedules that included an application at FB, the fruit length:diameter (L/D) ratio was significantly reduced in at least three of four locations when compared to untreated controls. When fenarimol was applied at BB, GC, WB, and FB, the L/D ratio was reduced at all locations. Treatment at BB only did not affect L/D ratio. Schedules involving GC or WB, but not FB, resulted in reduced L/D ratios at only one or two locations. When data from all four locations were combined, L/D ratios were significantly reduced by all fenarimol treatments except BB alone, and the smallest L/D ratios resulted from treatments which included applications at both WB and FB. Fruit stem length was significantly reduced by all timings of fenarimol application except GC at Medford, and all timings except BB and GC at Ukiah. Fruit weight did not differ among treatments.

Variability in Postharvest Decay Among Apple Cultivars.

The level of resistance to decay caused by four fungal pathogens, the force required to break the epidermis, and the extent of open sinuses as measures of potential decay resistance were determined for the fruit of several apple cultivars. No single cultivar was the most resistant to each of the four pathogens in this study. In addition, each cultivar that was the most resistant to one pathogen also was the most susceptible to one of the other pathogens. However, Royal Gala was the most resistant to the wound pathogens Botrytis cinerea, Penicillium expansum, and Mucor piriformis. Fuji and Oregon Spur II were more resistant than other cultivars to Pezicula malicorticis. The epidermis (skin) of Fuji and Granny Smith were the most resistant to puncture, requiring an average of 81.5 and 87.0 Newtons, respectively, to break the epidermal layer. Fuji had the highest percentage of fruits with open sinuses, with a mean of 38%, whereas Braeburn had 0% of fruit with open sinuses. The cultivar information presented herein may be a valuable measure of decay resistance and may be of use to the apple industry.

Control of Brown Rot and Blue Mold of Sweet Cherry with Preharvest Iprodione, Postharvest Cryptococcus infirmo-miniatus, and Modified Atmosphere Packaging.

The effectiveness of preharvest iprodione and postharvest Cryptococcus infirmo-miniatus treatments alone and in combination for control of decay of sweet cherry fruit was studied. Also, the effect of a modified atmosphere on brown rot control was evaluated as a part of the iprodione-C. infirmo-miniatus combinations. A single preharvest application of iprodione at 1.13 kg a.i./ha reduced brown rot in stored sweet cherry fruit in both years of this study. Significantly better control of brown rot was obtained when cherry fruit that received a preharvest iprodione application also were treated with a postharvest dip in a suspension of C. infirmo-miniatus containing 0.5 to 1.5 × 108 CFU/ml. Brown rot was reduced by modified atmosphere packaging (MAP) alone and further reduced as a result of a C. infirmo-miniatus-MAP synergism. Incidence of brown rot was reduced from 41.5% in the control to 0.4% by combining preharvest iprodione and postharvest C. infirmo-miniatus treatments with MAP.

Effect of Dolomitic Lime on Production of Asci and Pseudothecia of Venturia inaequalis and V. pirina.

An autumn application of dolomitic lime to infected leaves on the orchard floor resulted in a decrease in the percentage of apple and pear leaves with pseudothecia, the number of pseudothecia per leaf, and the number of asci per pseudothecium of Venturia inaequalis and V. pirina. A dolomitic lime application of 5.08 metric tons per hectare reduced the ascospore dose the following spring up to 88% for pear and 92% for apple.

Enumeration of bacterial and yeast colonists of apple fruits and identification of epiphytic yeasts on pear fruits in the Pacific Northwest United States.

A procedure for the isolation of diverse culturable microflora for the estimation of the population size of yeasts and bacteria on the surface of pome fruits is described. Maximum numbers of morphologically distinct colonies of both yeasts and bacteria were recovered from apple fruit surfaces when fruits were shaken for 5 min in sterile phosphate buffer plus tween, sonicated for 5 min, and aliquots of the buffer plated onto diluted yeast malt agar and diluted nutrient broth agar, respectively. The yeast and bacterial populations on the surface of unsprayed Golden Delicious apple fruits were approximately 8.0 x 10(3) and 9.5 x 10(4) colony forming units (cfu) per cm2, respectively. The densities of yeasts on the surface of pear fruits collected from Yakima, Wa, Cascade Locks, Medford, and Hood River, OR, were approximately 7.3 x 10(3), 6.4 x 10(3), 4.1 x 10(3), and 9.9 x 10(2) cfu.cm-2, respectively. The highest number of morphologically different yeast isolates were recovered from pear fruits from Cascade Locks and Hood River, Oregon and Yakima, Washington. Aureobasidium pullulans was present on fruits in all pear orchards sampled whereas Cryptococcus albidus and Rhodotorula glutinis were isolated from 80% of the orchards. Other yeasts colonizing pear fruit surfaces in 20-60% of the orchards were Cryptococcus infirmo-miniatus, Cryptococcus laurentii, Debaryomyces hansenii, Rhodotorula aurantiaca, R. fujisanensis, R. minuta and Sporobolomyces roseus.

Characterization of the two tryptophan residues of the lactose repressor from Escherichia coli by phosphorescence and optical detection of magnetic resonance.

The native lactose repressor from Escherichia coli (Lac Rep) and two single-point mutants, W220Y and W201Y, were investigated using low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Emission from two tryptophan residues was evident in the phosphorescence spectrum of native Lac Rep at 77 K. Using the single-point mutants, the triplet-state properties of tryptophans 201 and 220 were obtained independently. Trp 220 was characterized as a partially solvent-exposed residue (0,0 band centered at 409.5 nm), while tryptophan 201 exhibited the properties of a buried residue (0,0 band centered at 413.5 nm). Both single-point mutant proteins experienced changes in tryptophan triplet-state properties as a result of binding either of two inducer sugars: isopropyl beta-D-thiogalactoside, a monosaccharide, or melibiose, a disaccharide. Putative singlet-singlet energy transfer from tryptophan 220 to tryptophan 201 was also investigated, but the quantitative results must be viewed with some caution.

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.

Needlepoint teeth for jaws of vascular surgical clamps.

A vascular surgical clamp is described which has a single row of needlepoint teeth for the jaws. In our experience, the use of fine round and pointed teeth seems to afford less trauma and greater control of the vessel.

The prediction of healing in ischemic lesions of the foot. A comparison of Doppler ultrasound and elevation reactive hyperemia.

The results of the Elevation Reactive Hyperemia Test (ERHT) and the transcutaneous Doppler ultrasound ankle blood pressure (DAP) have been compared in a series of 115 cases with ischemic lesions of the foot. The ERHT was carried out by simple elevation of the foot or elevation with the circulation temporarily obstructed by a blood pressure cuff following which the foot was gradually lowered until the reactive hyperemia was seen in the skin and the height above the right atrium was estimated. The appearance of the hyperemia at 35 cm above the right atrium will allow healing of local ischemic lesions and at the height of 45 cm or above indicates a zone at which amputation may be carried out successfully. In the same series the DAP using 60 mmHg as the cut off point showed 40% healing with a pressure less than this level and 40% failed with a pressure above this level. The DAP gave no consistent prediction of the healing of ischemic lesions or of amputations.

Distribution, population dynamics, and characteristics of ice nucleation-active bacteria in deciduous fruit tree orchards.

Deciduous fruit tree orchards located in the Pacific Northwest were surveyed over a 3-year period for the presence of ice nucleation-active (INA) bacteria. In the Yakima Valley, only about 30% of the fruit tree orchards contained INA bacteria (median population ca. 3 x 10 CFU/g [fresh weight]) in contrast to nearly 75% of the orchards in the Hood River Valley (median population ca. 5 x 10 CFU/g [fresh weight]). These INA populations ranged from less than 10 to over 10 CFU/g (fresh weight) of blossoms and, in Hood River Valley orchards, generally comprised over 10% of the total bacterial population. Populations of INA bacteria fluctuated during the year with highest levels developing on buds and flowers during the cool, wet spring, followed by a drop in populations during the warmer, drier, summer months and finally a gradual increase in the autumn. The INA bacteria persisted on dormant buds from which they again colonized young developing vegetative tissues. All INA bacteria were identified as Pseudomonas syringae. The frequency of ice nucleation at -5 degrees C for these strains ranged from nearly every cell being INA to less than 1 in 10 cells. The median frequency of ice nucleation at -5 degrees C was 10 cells per ice nucleus. The INA P. syringae strains from individual orchards were diverse with respect to bacteriocin typing and in ice nucleation frequency. The consistent absence of detectable INA bacteria or presence of low populations in most of the orchards surveyed during periods when critical temperatures (i.e., -2 to -5 degrees C) were common indicated a limited role for INA bacteria in frost susceptibility of most Pacific Northwest orchards.