Genetic Diversity of Pseudomonas savastanoi pv. savastanoi in California and Characterization of Epidemiological Factors for Olive Knot Development.

Olive knot, caused by Pseudomonas savastanoi pv. savastanoi, is a limiting disease in the production of table and oil olives in California. The genetic variability among 152 strains from major production areas of California was determined using BOX, ERIC, and REP primers in repetitive element sequence-based polymerase chain reaction. Overall genetic variability was low, and strains shared at least 82% similarity. Phenetic analyses identified several genotypes but most strains belonged to one of two major groups. Three copper-resistant strains had two fingerprints that were distinct from any of the sensitive strains, indicating that they may have been introduced from other production areas or hosts. In inoculations, two copper-resistant strains were mostly equally as virulent as two copper-sensitive strains. Inoculum was exuded at high levels (>108 CFU/g of knot tissue) within 10 min from hydrated olive knots, and concentrations were 2- to 3-log higher than the minimum needed to induce knot formation. Arbequina olive was significantly more susceptible to infection and developed a higher incidence of knots on leaf scar and lateral wounds (59.7 to 80.6% incidence) than Manzanillo (47.4 to 68.2% incidence). In wound-healing studies, both types of wounds were less susceptible to infection ≥10 days after injury, indicating a critical period for infection and application of bactericides during favorable environments.

Efficacy of Copper and New Bactericides for Managing Olive Knot in California.

Baseline sensitivities were established for kasugamycin and oxytetracycline for 147 strains of Pseudomonas savastanoi pv. savastanoi collected from olive knots throughout California. Minimum inhibitory concentrations for ≥95% growth inhibition ranged from 1.86 to 11.52 and 0.13 to 0.40 µg/ml for kasugamycin and oxytetracycline, respectively. In copper sensitivity evaluations, 95.3% of the strains collected grew at concentrations of metallic copper equivalent (MCE) of <20 µg/ml, 2.7% grew at MCE between 20 and 30 µg/ml (moderately sensitive), and 2% grew at MCE of 150 µg/ml (resistant). Copper resistance was never reported previously in the olive knot pathogen, and pathogenicity studies confirmed a high virulence of the copper-resistant strains. In comparative field studies, kasugamycin at 200 µg/ml performed equally to the standard copper hydroxide treatment (MCE of 1,260 µg/ml) for reducing knot development on lateral wounds of Arbequina and Manzanillo olive inoculated with a copper-sensitive strain and was better than copper using a highly copper-resistant strain. Oxytetracycline at 200 µg/ml was not as effective as copper or kasugamycin but significantly reduced the disease as compared with the untreated control. Field studies on application timings of copper, kasugamycin, and copper-kasugamycin mixtures to inoculated wounds indicated that treatments within 24 h of inoculation resulted in higher disease control than applications at later times. In greenhouse trials, copper or copper-kasugamycin applied to wounds 7 days before inoculation persisted and reduced knot incidence by >50%. Our findings indicate that kasugamycin is an effective bactericide for controlling olive knot and that the time of any bactericide application after inoculation is critical in managing the disease.

Identification of Alternaria Species Causing Heart Rot of Pomegranates in California.

Alternaria heart rot of pomegranate fruit is generally considered a minor disease. However, the current listing of the causal pathogen in California as "Alternaria sp." or as "A. alternata and other Alternaria spp." restricts trade of the crop to some major export markets where quarantines are in place in an effort to prevent the spread of unknown or undescribed pathogens. Thus, species identification of the pathogen is critical in determining whether infected fruit should be regulated by quarantines. In this study, 86 isolates of Alternaria were collected from pomegranate fruit with Alternaria heart rot symptoms from major production areas in California. An unweighted pair group method with arithmetic means analysis based on amplified fragment length polymorphisms revealed two main clusters, each with a high degree of variability. One of the clusters contained 24 isolates from pomegranate and one reference isolate of Alternaria arborescens. Reference isolates of A. alternata and A. tenuissima were found among pomegranate isolates in the other cluster, and these two species could not be separated. In maximum-parsimony analysis of ribosomal DNA internal transcribed spacer sequence data, representative pomegranate isolates all clustered with reference sequences of species in section Alternaria. Single-nucleotide differences separated A. arborescens, A. gaisen, and two pomegranate isolates from the majority of the remaining isolates. Sequence comparisons of gapdh and OPA10-2 loci indicated that none of the pomegranate isolates were identical to A. gaisen, which is a quarantine pathogen. Due to high genetic similarity and variability of morphological characteristics, revisions of the taxonomy of small-spored Alternaria spp. have been proposed by others. Based on this recent taxonomic work and work herein by us, pomegranate isolates from California can be assigned to A. alternata and A. arborescens. Reassessment of export restrictions for California pomegranate due to the previous pathogen classification is warranted. Fruit injection inoculations with conidia of 12 representative isolates 3 months before harvest caused typical symptoms of Alternaria heart rot, whereas flower inoculations did not result in fruit disease.

Quaternary Ammonium Compounds as New Sanitizers for Reducing the Spread of the Olive Knot Pathogen on Orchard Equipment.

Olive knot, caused by the wound pathogen Pseudomonas savastanoi pv. savastanoi, is a serious bacterial disease that can be disseminated by orchard equipment. Greenhouse studies confirmed that cutting tools contaminated during contact with olive knots are able to spread the pathogen to healthy olive tissue. Quaternary ammonium compounds (QACs) were assessed as sanitizing agents for contaminated equipment as a disease management strategy. In laboratory in vitro tests, QACs exhibited high toxicity against the bacterium over a broad pH range from 6 to 9 using short exposure periods (15 to 60 s) and low concentrations (5 µg/ml). QACs applied to contaminated hard surfaces in the presence of an organic load reduced bacterial recovery by ≥3.6 log10 CFU/ml. In field trials, sanitation of hedging equipment that was contaminated with the pathogen (2 × 107 CFU/ml) and used to prune olives, was successful and sometimes completely prevented new infections from occurring. Application of additional foliar spray treatments of copper or copper-kasugamycin mixtures after hedging significantly improved disease control. In laboratory and field studies, sodium hypochlorite was significantly less effective than QAC compounds in the presence of organic matter. A nonphenolic QAC formulation, however, was ineffective as a preventative treatment when applied prior to inoculation of olive wounds, whereas a copper hydroxide application was highly effective. Based on data from this research, a QAC formulation was registered for field use as a sanitizer for olive equipment in California in 2015.

Postharvest Strategies for Managing Phytophthora Brown Rot of Citrus using Potassium Phosphite in Combination with Heat Treatments.

Phytophthora brown rot, caused by several species of Phytophthora, is an economically important disease of citrus in areas with rainfall during the late stages of fruit development. Recent export restrictions of California orange fruit to China due to the presence of brown rot caused by the quarantine pathogen Phytophthora syringae have mandated more rigorous disease management. We evaluated postharvest applications with the phosphonate fungicide potassium phosphite in combination with heat treatments. In timing studies, potassium phosphite at 1,500 µg/ml was most effective when applied within 18 h after inoculation of orange fruit with P. citrophthora, reducing the incidence of decay by >96% as compared with the control. Potassium phosphite was also highly effective in inoculations with P. syringae. Heated water treatments at 60°C were consistently and highly effective in reducing the incidence of brown rot after inoculation with P. citrophthora, whereas treatments at 55 or 50°C were more variable and generally less effective. Two-stage treatments of fruit were conducted in the laboratory to simulate current packinghouse practices and to evaluate any interaction of the efficacy of potassium phosphite with treatments of two commonly used postharvest fungicides (i.e., imazalil and thiabendazole [TBZ]) or a postharvest carnauba-based fruit coating. In these studies, an aqueous imazalil-potassium phosphite (2,000 µg/ml) dip at ambient temperature that was followed by a spray treatment of imazalil and TBZ prepared in fruit coating significantly reduced the incidence of brown rot from the control. When the aqueous dip was applied at 54°C, brown rot developed in only 1% of the fruit as compared with 76% in the water control. The efficacy of potassium phosphite was also demonstrated in commercial packinghouse treatments. Based on our research, this fungicide was registered for postharvest use against brown rot of citrus and is exempt from tolerance in the United States and many other countries.

Implementation of a new tool to improve the efficacy and safety of surgical handovers.

AIMS: Compliance with European working time regulations in surgical practice has resulted in an increase in the number of clinicians caring for individual patients and subsequently an increase in the frequency of handovers. In 2007, the Royal College of Surgeons of England produced guidelines on the minimum data-set for 'safe handover'. This audit examined compliance with these guidelines before and after adopting a more detailed electronic handover 'template' with the intention of improving handover quality and patient safety. METHODS: Pre-existing surgical 'take' electronic handover sheets were reviewed daily for two weeks to assess compliance with published guidance. A new proforma was introduced, training delivered and compliance re-audited. χ2 analysis was performed to determine statistical significance. RESULTS: The handovers of 118 patients were audited before, and 114 after, the implementation of the new proforma. Name and responsible consultant were recorded in all cases. Age (52% vs. 85%, p=<0.01), location (77% vs. 95%, p=<0.01), admission date (0% vs. 39%, p=<0.01), medical history (82% vs. 94%, p=0.01), diagnosis (55% vs. 93%, p=<0.01) and management plan (81% vs. 97%, p=<0.01) showed a statistically significant improvement with the new proforma. Presenting complaint (93% vs. 98%) and investigation (90% vs. 90%) data remained good. Review frequency (5%vs.11%) and outstanding tasks (21% vs. 27%) were poorly documented. CONCLUSIONS: Significant improvement was seen in the completeness of information handed-over following the introduction of the new proforma with likely positive implications for patient safety and standard of care. Opportunity for improvement still remains however, and more specific focussed tuition for trainees is required.

Strain differences in pH-sensitive K+ channel-expressing cells in chemosensory and nonchemosensory brain stem nuclei.

The ventilatory CO2 chemoreflex is inherently low in inbred Brown Norway (BN) rats compared with other strains, including inbred Dahl salt-sensitive (SS) rats. Since the brain stem expression of various pH-sensitive ion channels may be determinants of the CO2 chemoreflex, we tested the hypothesis that there would be fewer pH-sensitive K(+) channel-expressing cells in BN relative to SS rats within brain stem sites associated with respiratory chemoreception, such as the nucleus tractus solitarius (NTS), but not within the pre-Bötzinger complex region, nucleus ambiguus or the hypoglossal motor nucleus. Medullary sections (25 µm) from adult male and female BN and SS rats were stained with primary antibodies targeting TASK-1, Kv1.4, or Kir2.3 K(+) channels, and the total (Nissl-stained) and K(+) channel immunoreactive (-ir) cells counted. For both male and female rats, the numbers of K(+) channel-ir cells within the NTS were reduced in the BN compared with SS rats (P < 0.05), despite equal numbers of total NTS cells. In contrast, we found few differences in the numbers of K(+) channel-ir cells among the strains within the nucleus ambiguus, hypoglossal motor nucleus, or pre-Bötzinger complex regions in both male and female rats. However, there were no predicted functional mutations in each of the K(+) channels studied comparing genomic sequences among these strains. Thus we conclude that the relatively selective reductions in pH-sensitive K(+) channel-expressing cells in the NTS of male and female BN rats may contribute to their severely blunted ventilatory CO2 chemoreflex.

Contributions of the Kölliker-Fuse nucleus to coordination of breathing and swallowing.

Herein we compare the effects of perturbations in the Kölliker-Fuse nucleus (KFN) and the lateral (LPBN) and medial (MPBN) parabrachial nuclei on the coordination of breathing and swallowing. Cannula was chronically implanted in goats through which ibotenic acid (IA) was injected while awake. Swallows in late expiration (E) always reset while swallows in early inspiration (I) never reset the respiratory rhythm. Before cannula implantation, all other E and I swallows did not reset the respiratory rhythm, and had small effects on E and I duration and tidal volume (VT). However, after cannula implantation in the MPBN and KFN, E and I swallows reset the respiratory rhythm and increased the effects on I and E duration and VT. Subsequent injection of IA into the KFN eliminated the respiratory phase resetting of swallows but exacerbated the effects on I and E duration and VT. We conclude that the KFN and to a lesser extent the MPBN contribute to coordination of breathing and swallowing.

Identification and Pathogenicity of Fungal Pathogens Associated with Stem-End Rot of Avocado in California.

Stem-end rot of harvested avocado fruit commonly occurs wherever the crop is cultivated. Multiple fungal species have been described as causal agents. To determine the causal pathogens of stem-end rot in California, fungal isolations were conducted from symptomatic fruit, and fungi were identified by morphological and molecular techniques. In 2010 and 2011, a total of 177 isolates were recovered from 290 avocado fruit collected from seven orchards in one of the major avocado growing areas in Southern California. The majority of isolates was identified as Neofusicoccum luteum (65%), with the remainder either as Colletotrichum gloeosporioides (33%) or Phomopsis sp. (2%). In a pathogenicity test, N. luteum caused significantly (P < 0.05) more severe stem-end rot than either C. gloeosporioides or Phomopsis sp. No significant (P > 0.05) differences in stem-end rot severity were observed between inoculations with N. luteum isolated from fruit stem-end rot and N. luteum or N. parvum isolated from branch cankers. This confirms that stem-end rot of avocado can be initiated by fungi causing branch cankers. Although low humidity and rainfall during much of the growing and harvest seasons in California are considered unfavorable conditions for the development of avocado stem-end rot, the identification of the causal pathogens is of value when decays have to be managed during outbreaks, and it stresses the importance of managing branch cankers.

QoI Resistance in Fusicladium carpophilum Populations from Almond in California and Evaluation of Molecular Resistance Mechanisms.

Disease management failures have been reported in California for almond scab caused by Fusicladium carpophilum following quinone outside inhibitor (QoI) applications. Resistance in the pathogen populations was found to be common and at high incidence in the major almond-growing regions beginning in 2003, 4 years after registration of azoxystrobin on this crop. Two levels of azoxystrobin resistance, moderate and high, were identified with 50% effective concentration (EC50) values between 0.15 and 10 µg/ml or >40 µg/ml, respectively. Reference isolates collected before resistance was detected had EC50 values <0.05 µg/ml. High-resistance was associated with a G143A mutation in the mitochondrial cytochrome b gene. For the less commonly found moderately resistant isolates, no mutations in the gene were detected between codons 122 and 212. Using primers targeting the G143A mutation or the cytochrome b gene of all F. carpophilum isolates in quantitative polymerase chain reaction (qPCR) analyses, the frequency of highly resistant isolates was accurately determined in mixtures of conidia with selected ratios of sensitive and resistant isolates. The frequency of high resistance in bulked samples of scab lesions, however, was generally underestimated compared with in vitro testing of fungicide sensitivity of fungal isolates from the same lesions. Competition experiments using conidial suspensions demonstrated stability of the highly resistant genotype in the presence of different amounts of sensitive and moderately resistant genotypes. Analysis of covariance of linear regressions of cycle threshold values on DNA concentrations derived from qPCR amplifications using two primer pairs for cytochrome b alleles with and without the G143 mutation showed that several isolates differed in their slopes and midpoints. Thus, heteroplasmy of mitochondrial-inherited QoI resistance is suggested as a likely cause for incongruence in estimating resistance frequencies using the two methods.

Distinguishing Galactomyces citri-aurantii from G. geotrichum and characterizing population structure of the two postharvest sour rot pathogens of fruit crops in California.

A growth assay in lemon juice and polymerase chain reaction amplifications using newly designed species-specific primers from endopolygalacturonase and ß-tubulin genes rapidly differentiated isolates of the morphologically similar fruit sour rot pathogens Galactomyces citriaurantii and G. geotrichum. Isolates of both species were collected from agricultural soils and decaying fruit at locations within and outside California, including worldwide locations, and were used in population genetic studies based on amplified fragment length polymorphic (AFLP) DNA markers. For all four geographically defined subpopulations (three counties of California and locations outside California) among 97 isolates of G. citri-aurantii and for the two subpopulations (origin within or outside California) among 35 isolates of G. geotrichum, the proportion of polymorphic loci and haplotypic diversity was high. In total, 82 unique haplotypes were identified for G. citri-aurantii for the four subpopulations and, of these, 80 haplotypes were unique among subpopulations. For G. geotrichum, 25 unique haplotypes were identified among the two subpopulations and no haplotype was shared. Indices of genetic differences (F(ST)) between subpopulations within each species were all low (e.g., 0.038 for G. geotrichum and 0.085 to 0.226 for G. citriaurantii), indicating a low level of genetic differentiation. Following clone correction, mating type segregation ratios for G. citri-aurantii did not significantly (P > 0.1) deviate from a 1:1 ratio for all four subpopulations or the entire population. Tests of the index of association (I(A)) and parsimony tree-length permutation tests (PTLPT) supported a random mating structure for clone-corrected data for the Kern, Tulare, and Ventura County subpopulations and the null hypothesis of random mating could not be rejected. Additionally, PTLPT also supported random mating for the "outside of California" population. For G. geotrichum, random mating was only tested using I(A) and PTLPT and the null hypothesis of random mating was not rejected (P > 0.05) using clone-corrected data. Further evidence that sexual recombination likely occurs in both species of Galactomyces was the lack of grouping consistency in the unweighted pair-group method with arithmetic mean clustering of AFLP data. A high confidence based on bootstrap values was obtained for only a few of the nodes in each of the two trees. A mixed reproduction system with an out-crossing sexual mating system and a prolific asexual phase is proposed for both species.

Efficacy and Application Strategies for Propiconazole as a New Postharvest Fungicide for Managing Sour Rot and Green Mold of Citrus Fruit.

Few postharvest treatments are available for managing sour rot of citrus caused by Galactomyces citri-aurantii and they are generally not very effective. The demethylation-inhibiting (DMI) triazole fungicides propiconazole and cyproconazole were found to be highly effective and more efficacious than other DMIs evaluated, such as metconazole and tebuconazole, in reducing postharvest sour rot of citrus. Additional studies were conducted with propiconazole as a postharvest treatment because it has favorable toxicological characteristics for food crop registration in the United States and the registrant supports a worldwide registration. Regression and covariance analyses were performed to determine optimal time of application after inoculation and fungicide rate. In laboratory studies, decay incidence increased when propiconazole applications were delayed from 8 to 24 h (lemon) or 18 to 42 h (grapefruit) after inoculation. Effective rates of the fungicide were 64 to 512 µg/ml and were dependent on inoculum concentration of the sour rot pathogen and on the type of citrus fruit. Propiconazole was found to be compatible with sodium hypochlorite at 100 µg/ml and 1 to 3% sodium bicarbonate without loss of efficacy for decay control on lemon. The addition of hydrogen peroxide/peroxyacetic acid at 80 µg/ml slightly decreased the effectiveness of propiconazole. Heated (48°C) solutions of propiconazole did not significantly improve the efficacy compared with solutions at 22°C. In experimental packing-line studies, aqueous in-line drenches applied alone or followed by applications of the fungicide in storage or packing fruit coatings were highly effective, reducing sour rot to between 0 and 1.2% compared with 83.8% decay incidence in the control when treatments were made up to 16 h after inoculation. When the fungicide was applied in either fruit coating, decay was only reduced to 49.1 to 57.1% incidence. Tank mixtures of propiconazole with the citrus postharvest fungicides fludioxonil and azoxystrobin were highly effective in reducing green mold caused by isolates of Penicillium digitatum sensitive or moderately resistant to imazalil and sour rot. Propiconazole will be an important postharvest fungicide for managing sour rot of citrus and potentially can be integrated into current management practices to reduce postharvest crop losses caused by DMI-sensitive isolates of P. digitatum.

Toxicity and Resistance Potential of Selected Fungicides to Galactomyces and Penicillium spp. Causing Postharvest Fruit Decays of Citrus and Other Crops.

A diverse collection of isolates of Galactomyces citri-aurantii and G. geotrichum, the causal pathogens of sour rots of citrus and other fruit crops, respectively, was evaluated for sensitivity to demethylation-inhibiting (DMI) fungicides of the triazole group. Propiconazole was found to be highly effective in reducing mycelial growth of both species in vitro. For 139 isolates of G. citri-aurantii, a mean effective concentration for 50% reduction of mycelial growth (EC50 value) of 0.34 µg/ml was determined; whereas, for 33 isolates of G. geotrichum, this value was 0.14 µg/ml. In a comparison of additional DMI fungicides, mean EC50 values for 60 isolates of G. citri-aurantii and 20 isolates of G. geotrichum, were 0.27 and 0.17 µg/ml for cyproconazole, 0.25 and 0.14 µg/ml for metconazole, and 1.16 and 0.73 µg/ml for tebuconazole, respectively. Propiconazole was also highly active against mycelial growth of imazalil-sensitive isolates of Penicillium digitatum, the pathogen that causes green mold of citrus, with a mean EC50 value of 0.008 µg/ml for 63 isolates. Imazalil-resistant isolates of this fungus were cross-resistant to propiconazole. When G. citri-aurantii and P. digitatum were grown at selected pH values between 3 and 9, inhibition by propiconazole occurred over the entire pH range. The fungicide was most effective at pH 5 when compared with the non-fungicide-amended control grown at the same pH. In laboratory mass platings of single-spore isolates sensitive to propiconazole onto selective media, isolates with an up to 81.6-fold decrease in sensitivity to the fungicide were recovered for P. digitatum. For G. geotrichum, isolates with an approximately twofold decrease in sensitivity were obtained. No isolates with reduced sensitivity were recovered for G. citri-aurantii. Propiconazole is currently being registered for postharvest use on citrus and other crops, and the information provided will be valuable in monitoring of fungicide resistance and in designing effective fungicide application strategies.

Effect of wetness duration and temperature on the development of anthracnose on selected almond tissues and comparison of cultivar susceptibility.

Blossoms, leaves, fruit, and woody tissues of almond can be affected by anthracnose caused by Colletotrichum acutatum. Because the disease occurs throughout rainy spring seasons, the effect of temperature and wetness duration on disease development was evaluated in controlled studies. The lowest inoculum concentration where disease developed on leaves was 10(4) conidia/ml. Longer wetness durations were needed for leaves than for blossoms and disease increased linearly with increasing wetness durations. Inoculation temperature mainly affected final disease levels. Temperature during incubation affected the rate of disease development, while final disease levels were very similar at 10, 15, or 20°C. An analysis of covariance was performed to compare regressions of the effects of wetness and temperature on disease development for several almond cultivars. For blossom inoculations at 15°C in growth-chamber studies, a common slope model was statistically sufficient to describe all four cultivars. Cultivar Nonpareil (NP) had a significantly (P<0.05) lower adjusted means at the midpoint than cultivars Carmel (CA), NePlus Ultra (NU), and Wood Colony (WC). For blossom inoculations at 20°C and for leaf inoculations at all temperatures evaluated, an unequal slope model was statistically justified for comparing regression lines. For blossoms, the slopes were significantly different (P<0.05) for pair-wise comparisons of CA-NU, NU-WC, and NP-WC. For leaves, most of the cultivars responded differently to infection at different temperatures. Two of the pair-wise comparisons demonstrated unequal slopes at all three temperatures evaluated (i.e., NU-NP and NU-WC). Overall, for blossoms and leaves, NP was the least susceptible, NU was the most susceptible, and WC and CA showed an intermediate susceptibility. In field blossom and fruit studies, a common slope model was statistically sufficient to describe all four cultivars. NP had a significantly lower midpoint (i.e., was less susceptible) than CA or WC, whereas no significant difference (P > 0.1) occurred in comparisons between CA and WC.

Postnatal ventilatory response to CO2 in awake piglets.

Abnormal ventilatory responses to increased levels of inspired CO2 during postnatal development may pose a risk for Sudden Infant Death Syndrome, primarily during periods of vulnerability. The purpose of this study was to test the hypothesis that in awake piglets the ventilatory response to hypercapnia would be attenuated between 10 and 15 days of age relative to younger and older ages. To test this hypothesis, we measured the ventilatory response to 5% inspired CO2 in piglets from postnatal (PN) days 1 through PN28. Piglets were divided into groups and exposed to 5% CO2 daily, every 3rd day or on and after PN20-21 only to avoid any plasticity that may result from repeated exposure to CO2. Room air ventilation normalized to body weight (V˙(E), ml/min/kg) declined with postnatal age in piglets from all groups. The ventilatory response to 5% inspired CO2 (expressed as % change from control) was present at birth, and we did not find an age-dependent change from PN1 to PN28 (p > 0.1). In addition, we did not find that repeated exposure (daily or every 3rd day) to 5% inspired CO2 altered the ventilatory response during this period of development. We conclude that the previously documented apparent critical period of development in piglets between 10 and 15 days of age is not associated with attenuation of the ventilatory response to 5% inspired CO2.

Effectiveness of Kasugamycin Against Erwinia amylovora and its Potential Use for Managing Fire Blight of Pear.

Comparative field studies on the management of fire blight were conducted in California on Asian and Bartlett pear using single-bactericide, mixture, and rotation treatments of selected compounds. Treatment efficacy was evaluated based on the natural occurrence of the disease or after inoculation with Erwinia amylovora. Kasugamycin at 100 mg/liter demonstrated similar or higher pre- and post-infection activity than the industry standards, streptomycin and oxytetracycline. Phytotoxicity caused by kasugamycin was observed only when five or six sequential weekly applications were done. In a six-spray rotation program including three bactericides (copper, kasugamycin, and oxytetracycline), with each being used twice, phytotoxicity was minor. Baseline sensitivity concentrations for kasugamycin were established for growth of 376 isolates of E. amylovora from California. Values for the lowest concentration where a reduction in growth on nutrient agar was observed ranged from 3.5 to 18.3 mg/liter, with a mean value of 8.7 mg/liter. Values for ≥95% inhibition of growth ranged from 6.9 to 46.7 mg/liter, with a mean value of 18.5 mg/liter. These inhibitory values for kasugamycin were higher than those for streptomycin or oxytetracycline. The in vitro activity of all three compounds was highly dependent on the agar medium used in the sensitivity assay. The activity of kasugamycin was also highly dependent on the pH of the medium and was significantly higher at pH 5.1 than pH 7.3. With the planned registration in the United States, kasugamycin represents the first new, highly effective bactericide for managing fire blight in over 40 years.

The effects of lesions in the dorsolateral pons on the coordination of swallowing and breathing in awake goats.

The purpose of this retrospective study was to gain insight into the contribution of the dorsolateral pons to the coordination of swallowing and breathing in awake goats. In 4 goats, cannulas were chronically implanted bilaterally through the lateral (LPBN) and medial (MPBN) parabrachial nuclei just dorsal to the Kölliker-Fuse nucleus (KFN). After >2weeks recovery from this surgery, the goats were studied for 5½h on a control day, and on separate days after receiving 1 and 10µl injections of ibotenic acid (IA) separated by 1week. The frequency of swallows did not change during the control and 1µl IA studies, but after injection of 10µl IA, there was a transient 65% increase in frequency of swallows (P<0.05). Under control conditions swallows occurred throughout the respiratory cycle, where late-E swallows accounted for 67.6% of swallows. The distribution of swallow occurrence throughout the respiratory cycle was unaffected by IA injections. Consistent with the concept that swallowing is dominant over breathing, we found that swallows increased inspiratory (T(I)) and expiratory (T(E)) time and decreased tidal volume (V(T)) of the breath of the swallow (n) and/or the subsequent (n+1) breath. Injections of 10µl IA attenuated the normal increases in T(I) and T(E) and further attenuated V(T) of the n breath. Additionally, E and I swallows reset respiratory rhythm, but injection of 1 or 10µl IA progressively attenuated this resetting, suggesting a decreased dominance over respiratory motor output with increasing IA injections. Post mortem histological analysis revealed about 50% fewer (P<0.05) neurons remained in the KFN, LPBN, and MPBN in lesioned compared to control goats. We conclude that dorsolateral pontine nuclei have a modulatory role in a hypothesized holarchical neural network regulating swallowing and breathing particularly contributing to the normal dominance of swallowing over breathing in both rhythm and motor pattern generation.

Anatomic changes in multiple brainstem nuclei after incremental, near-complete neurotoxic destruction of the pre-Bötzinger Complex in adult goats.

Abrupt, bilateral destruction of the pre-Bötzinger Complex (preBötC) leads to terminal apnea in unanesthetized goats and rats. In contrast, respiratory rhythm and pattern and arterial blood gases in goats during wakefulness and sleep are normal after incremental (over a month) destruction of > 90% of the preBötC. Here, we tested the hypothesis that the difference in effects between abrupt and incremental destruction of the preBötC are a result of time-dependent plasticity, which manifests as anatomic changes at sites within the respiratory network. Accordingly, we report data from histological analyses comparing the brainstems of control goats, and goats that had undergone bilateral, incremental, ibotenic acid (IA)-induced preBötC lesioning. A major focus was on the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the pontine respiratory group (PRG), which are sites thought to contribute to respiratory rhythmogenesis. We also studied the facial (FN), rostral nucleus ambiguus (NA), medullary raphé (MRN), hypoglossal (HN), and the dorsal motor vagal (DMV) nuclei. Neuronal counts, count region area (mm²), and neuronal densities were calculated using computer-assisted analyses and/or manual microscopy to compare control and preBötC-lesioned animals. We found that within the ventral and lateral medulla 2mm rostral to the caudal pole of the FN (presumed pFRG/RTN), there were 25% and 65% more (P < 0.001) neurons, respectively, in preBötC-lesioned compared to control goats. Lesioned goats also showed 14% and 13% more (P < 0.001) neurons in the HN and medial parabrachialis nucleus, but 46%, 28%, 7%, and 17% fewer (P < 0.001) neurons in the FN, NA, DMV, and Kölliker-Fuse nuclei, respectively. In the remaining sites analyzed, there were no differences between groups. We conclude that anatomic changes at multiple sites within the respiratory network may contribute to the time-dependent plasticity in breathing following incremental and near-complete destruction of the preBötC.

Determination of natural resistance frequencies in Penicillium digitatum using a new air-sampling method and characterization of fludioxonil- and pyrimethanil-resistant isolates.

ABSTRACT Fungicide resistance was identified in natural populations of Penicillium digitatum, the causal agent of green mold of citrus, to two of three new postharvest fungicides before their commercial use. Using a new air-sampling method where large populations of the pathogen in citrus packinghouses were exposed to agar plates with a continuous, wide-range fungicide concentration gradient, isolates with reduced sensitivity to fludioxonil or pyrimethanil were obtained. Resistance frequencies to fludioxonil and pyrimethanil were calculated as 9.5 x 10(-7) to 1.5 x 10(-5) and 7.3 x 10(-6) to 6.2 x 10(-5), respectively. No isolates resistant to azoxystrobin were detected. Isolates with reduced sensitivity to fludioxonil or pyrimethanil were also obtained in laboratory selection studies, where high concentrations of conidial mixtures of isolates sensitive to the three fungicides were plated onto agar amended with each fungicide at 10 microg/ml. Isolates obtained from fludioxonil selection plates in laboratory and packinghouse experiments were placed into two categories based on mycelial growth: moderately resistant isolates had 50% effective concentration (EC(50)) values of 0.1 to 0.82 microg/ml and highly resistant isolates had EC(50) values > 1.5 microg/ml. Isolates resistant to pyrimethanil all had EC(50) values >8 microg/ml. Representative isolates of the two categories with reduced sensitivity to fludioxonil varied widely in their virulence and sporulation capacity as measured by the incidence of decay and degree of sporulation on inoculated fruit, respectively, whereas pyrimethanil-resistant isolates were mostly similar to the wild-type isolate. Fungicide sensitivity characteristics for isolates from fludioxonil and pyrimethanil selection plates remained stable after passages on nonamended agar, and disease could not be controlled after treatment with the respective fungicides. Types of fungicide resistance were visualized on thiabendazole- (TBZ) and imazalil-amended selection plates that were exposed in packinghouses where resistance to these fungicides was known to occur. The qualitative, single-site resistance to the benzimidazole TBZ was visualized by two distinct subpopulations in regard to fungicide sensitivity, whereas the quantitative, multi-site resistance to the demethylation inhibitor imazalil was apparent as a continuous density gradient of colonies along the fungicide concentration gradient. Types of resistance could not be assigned to fludioxonil or pyrimethanil because a limited number of resistant colonies was obtained on each plate. Thus, with this new method, we were able to estimate fungicide resistance frequencies as well as characterize and visualize types of resistance within populations of a fungal species. This information will be used to design resistance management strategies for previous and newly registered postharvest fungicides of citrus.

Site-specific effects on respiratory rhythm and pattern of ibotenic acid injections in the pontine respiratory group of goats.

To probe further the contributions of the rostral pons to eupneic respiratory rhythm and pattern, we tested the hypothesis that ibotenic acid (IA) injections in the pontine respiratory group (PRG) would disrupt eupneic respiratory rhythm and pattern in a site- and state-specific manner. In 15 goats, cannulas were bilaterally implanted into the rostral pontine tegmental nuclei (RPTN; n = 3), the lateral (LPBN; n = 4) or medial parabrachial nuclei (MPBN; n = 4), or the Kölliker-Fuse nucleus (KFN; n = 4). After recovery from surgery, 1- and 10-microl injections (1 wk apart) of IA were made bilaterally through the implanted cannulas during the day. Over the first 5 h after the injections, there were site-specific ventilatory effects, with increased (P < 0.05) breathing frequency in RPTN-injected goats, increased (P < 0.05) pulmonary ventilation (Vi) in LPBN-injected goats, no effect (P < 0.05) in MPBN-injected goats, and a biphasic Vi response (P < 0.05) in KFN-injected goats. This biphasic response consisted of a hyperpnea for 30 min, followed by a prolonged hypopnea and hypoventilation with marked apneas, apneusis-like breathing patterns, and/or shifts in the temporal relationships between inspiratory flow and diaphragm activity. In the awake state, 10-15 h after the 1-microl injections, the number of apneas was greater (P < 0.05) than during other studies at night. However, there were no incidences of terminal apneas. Breathing rhythm and pattern were normal 22 h after the injections. Subsequent histological analysis revealed that for goats with cannulas implanted into the KFN, there were nearly 50% fewer neurons (P < 0.05) in all three PRG subnuclei than in control goats. We conclude that in awake goats, 1) IA injections into the PRG have site-specific effects on breathing, and 2) the KFN contributes to eupneic respiratory pattern generation.