Grapevine rootstocks drive the community structure of arbuscular mycorrhizal fungi in New Zealand vineyards.

AIM: Arbuscular mycorrhizal fungi (AMF) are often regarded as non-specific symbionts, but some AMF communities show host preference in various ecosystems including vineyards. Grapevine plants are very responsive to AMF colonization. Although these fungi have potentially significant applications for sustainable agricultural ecosystems, there is a gap in knowledge regarding AMF-grapevine interactions worldwide and especially in New Zealand. This study focused on identifying AMF taxa colonizing grapevines in New Zealand vineyards and investigated the effect of grapevine rootstocks on AMF community diversity and composition. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) and trap cultures were used to characterize the AMF communities. Grapevine roots from three vineyards and nine rootstocks were analysed by DGGE and used in trap cultures for AMF recovery. Trap cultures allowed the recovery of six AMF spore morphotypes that belonged to Ambispora sp., Claroideoglomus sp., Funneliformis sp. and Glomus sp. Bands excised, reamplified and sequenced from the DGGE were assigned to Glomus sp., Rhizophagus sp. and Claroideoglomus sp. The AMF community analyses demonstrated that rootstock significantly (P < 0·05) influenced the AMF community composition in all sites. CONCLUSIONS: The study showed that for a comprehensive identification of AMF, both results from trap culture and molecular work were needed and that the rootstock cultivar was the main driver of the arbuscular mycorrhizal community colonizing the roots. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a firm foundation for future research exploring the beneficial use of AMF in enhancing grapevine production and sustainability.

Apple endophyte community is shaped by tissue type, cultivar and site and has members with biocontrol potential against Neonectria ditissima.

AIMS: This research aimed to identify factors influencing endophyte community structure in apple shoots and the bioactivity of cultured representatives against the fungal pathogen Neonectria ditissima. METHODS AND RESULTS: The endophyte community in leaves and stems of the apple cultivars 'Royal Gala' and 'Braeburn' were analysed by a cultivation-independent method (PCR-DGGE) which showed that tissue type, cultivar and site were determinant factors, with the endophyte taxa in 'Royal Gala' more variable than that in 'Braeburn', with leaf endophyte communities typically differing from stems in both cultivars. Seasonal (spring vs autumn) and regional (Nelson vs Hawke's Bay) variations were not obvious in woody stems. A collection of 783 bacterial and 87 fungal endophytes were recovered from leaves and stems of 'Royal Gala', 'Braeburn', 'Scilate' and/or 'Scifresh' from Nelson (nine sites) and Hawke's Bay (five sites) in spring and from Nelson (three sites) in autumn. A dual culture plating assay was used to test their ability to inhibit the mycelial growth of N. ditissima. Thirteen bacterial (mean of percent inhibition ≥20%) and 17 fungal isolates were antagonistic towards N. ditissima. These isolates belonged to the bacterial genera Bacillus and Pseudomonas, and fungal genera Chaetomium, Epicoccum, Biscogniauxia, Penicillium, Diaporthe, Phlyctema and two unidentified fungal isolates. CONCLUSIONS: Endophyte communities in apple shoots were determined by tissue type, cultivar and site. Endophytic bacterial and fungal isolates inhibiting N. ditissima growth in vitro were found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provided new evidence of factors influencing apple endophyte community in New Zealand. Endophytes with potential to reduce N. ditissima infection were identified, with the potential to be developed into a biocontrol strategy for European canker.

Diet Supplementation with Soy Protein Isolate, but Not the Isoflavone Genistein, Protects Against Alcohol-Induced Tumor Progression in DEN-Treated Male Mice.

Diethylnitrosamine-treated male mice were assigned to 4 groups: a casein-based 35% high fat ethanol liquid diet (EtOH), an EtOH diet made with soy protein isolate protein (EtOH/SOY), an EtOH liquid diet supplemented with genistein (EtOH/GEN) and a chow group. EtOH feeding, final concentration 5% (v/v), continued for 16 wks. EtOH increased incidence and multiplicity of basophilic lesions and adenomas compared to the chow group, (p < 0.05). The EtOH/SOY group had reduced adenoma progression when compared to the EtOH and EtOH/GEN group, (p < 0.05). Genistein supplementation had no protective effect. Soy feeding significantly reduced serum ALT concentrations (p < 0.05), decreased hepatic TNFα and CD-14 expression and decreased nuclear accumulation of NFκB protein in EtOH/SOY-treated mice compared to the EtOH group (p < 0.05). With respect to ceramides, high resolution MALDI-FTICR Imaging mass spectrometry revealed changes in the accumulation of long acyl chain ceramide species, in particular C18, in the EtOH group when compared to the EtOH/SOY group. Additionally, expression of acid ceramidase and sphingosine kinase 1 which degrade ceramide into sphingosine and convert sphingosine to sphingosine-1-phosphate (S1P) respectively and expression of S1P receptors S1PR2 and S1PR3 were all upregulated by EtOH and suppressed in the EtOH/SOY group, p < 0.05. EtOH feeding also increased hepatocyte proliferation and mRNA expression of ß-catenin targets, including cyclin D1, MMP7 and glutamine synthase, which were reduced in the EtOH/SOY group, p < 0.05. These findings suggest that soy prevents tumorigenesis by reducing inflammation and by reducing hepatocyte proliferation through inhibition of EtOH-mediated ß-catenin signaling. These mechanisms may involve blockade of sphingolipid signaling.

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues.

Glycosylated proteins account for a majority of the posttranslation modifications of cell surface, secreted, and circulating proteins. Within the tumor microenvironment, the presence of immune cells, extracellular matrix proteins, cell surface receptors, and interactions between stroma and tumor cells are all processes mediated by glycan binding and recognition reactions. Changes in glycosylation during tumorigenesis are well documented to occur and affect all of these associated adhesion and regulatory functions. A MALDI imaging mass spectrometry (MALDI-IMS) workflow for profiling N-linked glycan distributions in fresh/frozen tissues and formalin-fixed paraffin-embedded tissues has recently been developed. The key to the approach is the application of a molecular coating of peptide-N-glycosidase to tissues, an enzyme that cleaves asparagine-linked glycans from their protein carrier. The released N-linked glycans can then be analyzed by MALDI-IMS directly on tissue. Generally 40 or more individual glycan structures are routinely detected, and when combined with histopathology localizations, tumor-specific glycans are readily grouped relative to nontumor regions and other structural features. This technique is a recent development and new approach in glycobiology and mass spectrometry imaging research methodology; thus, potential uses such as tumor-specific glycan biomarker panels and other applications are discussed.

Culturing conditions affect biological control activity of Trichoderma atroviride against Rhizoctonia solani in ryegrass.

AIMS: Effects of culture conditions on productivity, germinability and bioactivity of Trichoderma atroviride LU132 conidia were assessed to identify the factors affecting conidium 'fitness' (quantity and quality) and to withstand variable environmental conditions, increase conidial productivity, and perform optimum bioactivity. METHODS AND RESULTS: The interaction effects of temperatures (20 or 30°C) vs hydrocarbon types (dextrose or sucrose in constant C : N 5 : 1) were assessed for bioactivity and colonization potential in pot experiments with ryegrass in the presence of pathogen, Rhizoctonia solani. Trichoderma atroviride produced in different culture conditions increased some growth parameters of ryegrass plant and also reduced the pathogenicity effects of R. solani. For example, Trichoderma colony produced at 20°C with sucrose increased all plant growth parameters and conidia produced at 20°C with dextrose gave the greatest bioactivity. CONCLUSION: The bimodal population cycle in T. atroviride recurred in pot experiments in a manner similar to that previously observed in agar plates but indicating that simulated natural conditions shortened the Trichoderma life cycle. Trichoderma colonized ryegrass root system and symbiotically interacted with ryegrass and greater ryegrass colonization resulted from medium production treatment with dextrose rather than sucrose. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the effects of inoculum production conditions on conidium quality of Trichoderma to colonize and to maintain populations in host rhizospheres, and also the ability to promote plant growth and suppress a soil-borne disease. The results of these experiments provide new knowledge on how manipulation of culture conditions of T. atroviride LU132 can influence conidium fitness, as a basis for optimizing commercial production of the fungus as a biological control agent.

Effects of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride.

AIMS: The goal was to determine the effect of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride LU132 against Rhizoctonia solani. METHODS AND RESULTS: The incubation temperatures of 20, 25 or 30°C were assessed on the production of T. atroviride conidia under constant light over a 25 and 50 days periods. The resulting conidia were also studied for germination and bioactivity. Conidium production was maximum at 25°C after 20 days. The second peak of conidium production occurred at 45-50 days. Incubation at 25°C after 15 days showed optimum production of T. atroviride LU132. Conidia produced at 30°C gave the greatest germination and bioactivity in comparison with incubation at 20 or 25°C. CONCLUSION: This study indicates that the temperature at which conidia of T. atroviride are produced affects germination and bioactivity. Formulations based on production of the high conidia yield may not result in optimal bioactivity and there is a trade-off between quantity and quality of T. atroviride LU132 conidia. Conidium production was shown to be a continuous process, and increased under a dark/light regime. This is the first report of bimodal conidium production in a Trichoderma biological control agent (BCA), which is likely to be on 20 days cycle, and is dependent on colony age rather than abiotic factors. Conidia produced after 15 days are likely to be the most suitable for use in commercial production of this strain as a BCA. SIGNIFICANCE AND IMPACT OF THE STUDY: Most studies on Trichoderma-based BCA have only shown the effect of culture conditions on the high conidia yield regardless of conidium quality. This study is the first report on conidium quality affected by principal culture conditions for Trichoderma biological control formulations.

Increased hyphal branching and growth of ectomycorrhizal fungus Lactarius rufus by the helper bacterium Paenibacillus sp.

Paenibacillus sp. EJP73 has been previously demonstrated as a mycorrhization helper bacterium (MHB) for the Lactarius rufus-Pinus sylvestris symbiosis in both laboratory and glasshouse experiments. In the present study, the effect of Paenibacillus sp. EJP73 metabolites on L. rufus EO3 pre-symbiotic growth was tested in two agar plate-based systems. Specifically, volatile metabolites were investigated using a dual plate system, in which the presence of strain EJP73 resulted in a significant negative effect on L. rufus EO3 hyphal radial growth but enhanced hyphal branching and reduced internode distance. Soluble metabolites produced by strain EJP73 were tested on L. rufus EO3 growth in single-agar plate assays by incorporating bacterial cell-free whole or molecular weight fraction spent broth into the agar. Whole spent broth had a negative effect on hyphal growth, whereas a low molecular weight fraction (100-1,000) promoted colony radial growth. Headspace and spent broth analysis of strain EJP73 cultures revealed 2,5-diisopropylpyrazine to be the most significant component. Synthesised 2,5-diisopropylpyrazine and elevated CO2 (2,000 ppm) were tested as specific volatile metabolites in the dual plate system, but neither produced the response shown when strain EJP73 was present. Increased pre-symbiotic hyphal branching leading to increased likelihood of plant infection may be an important MHB mechanism for strain EJP73. Although the precise signal molecules could not be identified, the work suggests a number of metabolites may work synergistically to increase L. rufus root colonisation.

First Report of Leptosphaeria biglobosa as a Stem Canker Pathogen of Brassicas in New Zealand.

Phoma black leg or stem canker, caused by Leptosphaeria maculans or L. biglobosa, is an important disease of brassicas, causing significant crop losses in areas such as Europe, Australia, and North America (1). Samples collected in 2011 from canola and forage brassica (swede, kale, and turnip) crops in the main New Zealand growing regions (Southland, Central Otago, Canterbury, Hawkes Bay, and Manawatu) to identify the causal agent(s) of the characteristic stem cankers, found many isolates of L. maculans, which has been reported previously in New Zealand (2), and three isolates identified by colony characteristics as L. biglobosa. Of the latter, two isolates were from canola (Brassica napus) stem cankers from Darfield and Lincoln, Canterbury, and one was from a kale (B. oleracea) stem canker from Lincoln. An isolate (ICMP10665) of similar morphology, from the International Collection of Microorganisms from Plants (ICMP), obtained from a basal rot lesion on a cauliflower (B. oleracea var. botrytis) plant in Levin, New Zealand in 1979, was also evaluated. The initial, incorrect identification of the latter isolate as L. maculans predates the reclassification of L. maculans group B isolates as a new species, L. biglobosa (1). These four isolates produced fluffy white mycelium and a yellow pigment on potato dextrose agar (PDA) after 5 days' growth, and abundant black-brown, globose pycnidia containing cylindrical hyaline conidia after 7 days. In contrast, L. maculans isolates had slower growth and no pigment production (4). Amplification of genomic DNA using species-specific primers LmacR, LmacF, and LbigF (1) generated a PCR product of 444 bp that is typical of L. biglobosa isolates. Sequencing of the PCR product from each of the four isolates showed they were 100% identical to a sequence of L. biglobosa 'brassicae' in GenBank (JF740198). To confirm the species identity of the isolates, the rDNA, actin, and ß-tubulin gene regions were amplified (1,3). Sequences for the rDNA (568 bp), actin (941 bp), and ß-tubulin (410 bp) gene regions were 99% identical to sequences of the same regions of isolates in GenBank for L. biglobosa 'brassicae' (AY48997, AY748949.1, and AY748997.1, respectively). The four L. biglobosa isolates were tested for pathogenicity on a canola cultivar commonly grown in New Zealand (Flash). Cotyledons of 10-day-old seedlings (n = 12 seedlings/isolate or control treatment) grown in a potting mix in pots were pricked with a sewing needle, and each wound inoculated with 10 µl of the appropriate conidial suspension (106 conidia/ml) or 10 µl sterilized distilled water for the control treatment. Leaf lesions that developed on the inoculated cotyledons were characteristic of those caused by L. biglobosa, i.e., small and dark with a distinct margin. No pycnidia were produced on the lesions. No lesions developed on the cotyledons of the non-inoculated control plants. The causal agents were confirmed as L. biglobosa by the colony morphology of isolates that grew from surface-sterilized, inoculated leaf lesions plated on PDA amended with 100 µg/ml ampicillin. The fungus was not isolated from control leaf tissue. To our knowledge, this is the first report of L. biglobosa as a pathogen of canola and kale in New Zealand. This finding shows that both causal agents of black leg are present in New Zealand's brassica cropping areas. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) H. C. Smith and B. C. Sutton. Trans. Brit. Mycol. Soc. 47:159, 1964. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) R. H. Williams and B. D. L. Fitt. Plant Pathol. 48:161, 1999.

First Report of Cylindrocladiella parva as a Grapevine Pathogen in New Zealand.

Isolates morphologically identified as Cylindrocladiella parva were isolated from characteristic black foot symptoms on a grapevine (Vitis vinifera) rooted on 101-14 rootstock from Central Otago in 2005 and 101-14 rootstocks from a nursery in the Auckland Region in 2007 and 2008. On potato dextrose agar, the isolates initially produced cottony, white mycelia that turned grayish cream or golden cream within 10 days, the initially tawny colony undersides becoming dark brown with age. Conidia (0 to 1 septate; 16.4 to 17.0 [16.7] × 2.3 to 2.6 [2.5] µm) and abundant chlamydospores were produced. To confirm identity of the isolates, genomic DNA was extracted and the ribosomal DNA (rDNA) and ß-tubulin gene were amplified and sequenced (3,4). Sequences of the PCR products were compared with sequences in GenBank. The rDNA (535 bp) and ß-tubulin (297 bp) sequences of the four isolates were 100 and 99% identical, respectively, to reported sequences of C. parva in GenBank (AY793454, grapevine isolate (4)/AY793455 for rDNA; AY793486/AY793488, grapevine isolate (4)/AY793489/HM034822 for ß-tubulin). Although C. parva was previously isolated from grapevines in New Zealand (2) and rootstocks of mature grapevines, cuttings, and graft unions of grafted young grapevines in South Africa (4), its role as a pathogen of Vitis spp. has not been confirmed (2,4). However, it has been reported as a pathogen of Eucalyptus spp. (1) and was also isolated from Telopea speciosissima and Macadamia integrifolia in New Zealand (2,4). The C. parva isolates were tested as a mixed inoculum (four isolates) for pathogenicity on roots of 10 grapevine rootstock plants each of cvs. 101-14 and Schwarzmann (Sch). The rootstocks were grown in potting mix for 4 months, after which the root systems of all vines were wounded with an asparagus knife with a sharp, square tip, driven vertically down into the soil at four equidistant locations approximately 8 cm from the trunk. Each plant was inoculated with 50 ml of the mixed-isolate conidial suspension (106/ml), or 50 ml water (controls), followed by 50 ml of water. After 7 months of growth, the plants were harvested. For C. parva-inoculated plants, internal blackening of the stem base tissue was observed. Isolations from surface-sterilized trunk bases recovered C. parva from four and nine plants of 101-14 and Sch, respectively, with C. parva infections in 25 and 48%, respectively, of the four wood pieces taken per plant. Plants inoculated with water had no blackening and no C. parva was isolated from their stem bases. Mean shoot dry weights of inoculated plants (17.9 and 15.0 g for 101-14 and Sch, respectively) were significantly lower (P = 0.035) than noninoculated controls (26.5 and 20.0 g for 101-14 and Sch, respectively). Mean root dry weights were reduced by C. parva inoculation, although not significantly (32.7 and 27.0 g for C. parva inoculated 101-14 and Sch, respectively, and 36.2 and 27.4 g for control 101-14 and Sch, respectively). To our knowledge, this is the first report of C. parva as a pathogen of grapevines (2,4) and suggests that along with Cylindrocarpon spp., C. parva is part of the pathogen complex responsible for black foot of grapevines. References: (1) P. W. Crous et al. Plant Pathol. 42:302, 1993. (2) P. D. Gadgil et al. Fungi on Trees and Shrubs in New Zealand. Fungal Diversity Press, Hong Kong, 2005. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) G. J. van Coller et al. Australas. Plant Pathol. 34:489, 2005.

First Report of Neofusicoccum macroclavatum as a Canker Pathogen of Grapevine in New Zealand.

In a 2008 survey, 120 isolates of the Botryosphaeriaceae were recovered from a representative subsample of Vitis vinifera plants and propagation materials collected in nine New Zealand grapevine nurseries. Isolates were identified by amplified ribosomal DNA restriction analysis (ARDRA) (1) as Neofusicoccum luteum (56%), N. parvum (18%), N. australe (8%), Diplodia mutila (7%), Botryosphaeria dothidea (5%), D. seriata (3%), and N. ribis (2%). One isolate (M353) from 1 cm below the graft union of a nonsymptomatic 1-year-old grafted plant from the Nelson Region was not identified by ARDRA and was morphologically distinct from all others. Mycelium produced by the novel isolate on potato dextrose agar (PDA) was initially moderately dense, flat, and white and turned olivaceous brown within 10 days. The isolate did not produce pycnidia in PDA or prune extract agar, but when grown in water agar with sterile pine needles for 8 weeks at 25°C and a 12-h light/dark regimen, small, black pycnidia covered with mycelium were produced but no conidia were observed. To identify the novel fungus, genomic DNA was extracted and the ribosomal DNA (rDNA), ß-tubulin gene, and elongation factor α-1 gene were amplified and sequenced (4). The sequences of the PCR products were compared with sequences present on GenBank. The rDNA (503 bp), ß-tubulin (371 bp), and elongation factor α-1 gene (227 bp) sequences of M353 were 100% identical to reported sequences of N. macroclavatum on GenBank (Accession No. DQ093199/198/196 for rDNA, DQ093207/206 for ß-tubulin, and DQ093219/217 for elongation factor α-1). These genes differed from the same genes in other Neofusicoccum species by at least 11, 2, and 3 base pairs, respectively. The N. macroclavatum isolate was tested for pathogenicity on wounded grapevine (Sauvignon blanc) green shoots and 1-year-old rooted canes (n = 4 per plant type) using mycelium plugs from a 4-day-old PDA culture. Sterile agar was used for the negative control. Green shoots inoculated with N. macroclavatum developed brown lesions with an average length of 40.5 mm 6 days after inoculation. Bark from inoculated 1-year-old canes was peeled off 28 days after inoculation and brown-to-black lesions on the wood, with an average length of 52 mm, were observed. Control plants produced no lesions. The pathogen was consistently reisolated from the inoculated plants while none were found in negative control plants. To our knowledge, this is the first report of N. macroclavatum as a pathogen of grapevines and the first report of its presence in New Zealand (3). N. macroclavatum was first reported as a pathogen of Eucalyptus globulus in Western Australia in 2005 and has not been reported as a pathogen of grapevines (2). References: (1) A. Alves et al. FEMS Microbiol. Lett. 245:221, 2005. (2) T. T. Burgess et al. Australas. Plant Pathol. 34:557, 2005. (3) J. Sammonds et al. N. Z. Plant Prot. 62:248, 2009. (4) B. Slippers et al. Mycologia 96:83, 2004.

The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion.

Levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) protein and neurotrophin receptor mRNA in adult sympathetic neurons were investigated following surgical removal of preganglionic input and/or in vivo administration of NGF. Expression of trkC and p75, but not trkA, was significantly decreased following a 3-week deafferentation of the superior cervical ganglion (SCG). Protein levels of NGF and NT-3 in the SCG were unchanged by deafferentation. A 2-week intracerebroventricular infusion of NGF without deafferentation resulted in enhanced mRNA levels of trkA, trkC, and p75 as well as significantly increased NGF and NT-3 protein in the SCG. When NGF infusion followed deafferentation, both trkA and p75 showed significant increases while trkC levels were similar to control values. NGF protein was not increased in the SCG when deafferentation preceded exogenous NGF, yet NT-3 was elevated and levels were similar to cases receiving NGF infusion only. These results support a role for preganglionic input in trkC and p75 expression in adult sympathetic neurons. The increased levels of NT-3 protein and trkC gene expression observed following NGF infusion suggest that NGF influences NT-3 regulation in adult sympathetic neurons. In addition, the present findings provide evidence that, when preganglionic input is removed prior to the NGF infusion, NT-3 effectively competes with NGF for trkA binding. Taken together, we propose that NT-3 may play a role in the robust sprouting of sympathetic cerebrovascular axons previously observed following NGF administration, particularly when deafferentation precedes the NGF infusion period.

Efficacy of partially hydrolyzed corn syrup solids as a replacement for lactose in manufactured liquid diets for neonatal pigs.

Feeding manufactured liquid diets to early-weaned pigs improves growth performance and reduces days to market weight compared with pigs receiving pelleted dry feed. Few alternative dietary ingredients are utilized in manufactured liquid diets other than byproducts of the dairy industry, especially for sources of carbohydrates. This experiment was designed to evaluate the efficacy of starch from partially hydrolyzed corn syrup solids (CSS), at two different levels of hydrolyzation, as a replacement for lactose in manufactured liquid diets. Forty-eight pigs were removed from sows at 1 d of age and randomly assigned to one of three treatments: 1) control with lactose as the carbohydrate source, 2) lactose replaced (gram for gram) with CSS (dextrose equivalent [DE]-20), and 3) lactose replaced with DE-42. In addition, 10 pigs were randomly removed from several litters to provide estimates of initial body composition and small intestinal variables. Twenty-four pigs were removed from the study on d 10 of treatment, and the remaining 24 pigs were removed on d 20 of treatment. Pigs averaged 9,845 +/- 191 g at d 20 of treatment regardless of dietary treatment (P > 0.20). No differences in ADG, ADFI, or feed efficiency were detected between treatment groups from d 0 to 20 (P > 0.19). Whole-body water, protein, lipid, and ash accretion rates were unaffected by dietary treatment from d 0 to 10 or from d 0 to 20 (P > 0.20). The replacement of lactose with CSS did not affect intestinal villi height or width, or crypt depth (P > 0.10). Pigs fed lactose tended to have greater lactase activity on d 10 than pigs fed CSS (P < 0.07). Also, pigs fed lactose tended to have lower oligosaccharidase activity than pigs fed the DE-20 diet on d 20 (P < 0.07). No other differences in lactase, maltase, or long oligosaccharidase specific activity on d 10 or 20 of treatment were detected (P > 0.12). Plasma urea nitrogen concentrations were unaffected by diet on d 10 and 20 of treatment. In addition, dry matter digestibility of the diets averaged approximately 85.6 +/- 0.8% and was unaffected by dietary treatment or day of treatment. These results suggest that partially hydrolyzed CSS can be used as a replacement for lactose in manufactured liquid diets for neonatal pigs.

The fungal biocontrol agent Coniothyrium minitans: production by solid-state fermentation, application and marketing.

Biological control agents (BCAs) are potential alternatives for the chemical fungicides presently used in agriculture to fight plant diseases. Coniothyrium minitans is an example of a promising fungal BCA. It is a naturally occurring parasite of the fungus Sclerotinia sclerotiorum, a wide-spread pathogen which substantially reduces the yield of many crops. This review describes, exemplified by C. minitans, the studies that need to be carried out before a fungal BCA is successfully introduced into the market. The main aspects considered are the biology of C. minitans, the development of a product by mass production of spores using solid-state fermentation technology, its biocontrol activity and marketing of the final product.

Physiological responses to supercooling and hypoxia in the hatchling painted turtle, Chrysemys picta.

We investigated physiological responses to supercooling in hatchling painted turtles (Chrysemys picta) which remain in their natal nests over winter and therefore may become exposed to subzero temperatures. These turtles are freeze tolerant but also must rely on supercooling to survive exposure to the lower temperatures occurring in nests during winter. We compared whole-body concentrations of lactate, glucose, glycerol, and ATP in turtles chilled at 0 degrees C, -4 degrees C, or -6 degrees C for 5 days, or at 6 degrees C for 19 days. In a companion experiment, we measured metabolite concentrations in turtles exposed to a hypoxic environment for 1 day, 4 days, or 8 days. Supercooling and hypoxia exposure were both associated with an increase in concentrations of lactate and glucose and a decrease in glycerol concentrations (albeit no change in the ATP pool), suggesting that supercooling induces functional hypoxia. We conclude that hypoxia tolerance may be an important pre-adaptation for surviving exposure to subzero temperatures in hatchling C. picta.

Therapeutic action: a new theory.

This article introduces a new clinical construct: "Interaction structure." Interaction structures are recurrent, mutually influencing patterns of interaction between therapist and patient. The experience, recognition, and comprehension of the meaning of such repetitive interactions are a fundamental component of therapeutic action. This bipersonal or dyadic model attempts to bridge those theories of therapeutic action that focus on insight and self-understanding and those that emphasize the patient's experience of the therapist. In this model, insight and relationship have complementary roles, since psychological knowledge of the self can develop only in the context of a relationship where the therapist endeavors to understand the mind of the patient through the medium of their interaction. The interaction-structure construct and its associated theory of therapeutic action are based on empirical research. A single-case research model is described that shows that process and outcome in psychoanalytic treatments can be studied within individual treatments in a way that is scientifically persuasive. Three case studies illustrate how each patient-therapist pair has a unique interaction pattern, and how these patterns are linked to therapy outcome. The essential place of formal, empirical study in psychoanalytic theorizing and in clinical work is demonstrated.

Luteinizing hormone depletes ascorbic acid in preovulatory follicles.

OBJECTIVE: The objective of the present studies was to determine whether luteinizing hormone (LH) depletes ascorbic acid in the preovulatory follicle. DESIGN: Controlled, prospective experimental study. SETTING: University-based research center. ANIMAL(S): Sprague-Dawley female rats. INTERVENTION(S): Follicular growth and ovulation were induced in immature rats by gonadotropin treatment. MAIN OUTCOME MEASURE(S): Analysis of ovary, follicle, and oocyte levels of ascorbic acid by colorimetric analysis and high-pressure liquid chromatography. RESULT(S): Ovarian ascorbic acid was maximally depleted (50%) within 2 h of LH treatment and was sustained for 8 h. Follicle ascorbic acid levels were unchanged 1 h after LH injection but were significantly reduced within 2 h (40%). Incubation of isolated preovulatory follicles for 3 h with hCG or with menadione (a generator of oxygen radicals) reduced ascorbic acid levels. Isolation of cumulus-enclosed or denuded oocytes depleted ascorbic acid to undetectable levels, but follicular ascorbic acid levels were only moderately depleted by isolation and incubation. Accumulation of ascorbic acid by oocytes was significantly enhanced by the presence of intact cumulus cells. CONCLUSION(S): Elevation of LH and the production of oxygen radicals deplete ascorbic acid in the preovulatory follicle.

Cornual heterotopic pregnancy: contemporary management options.

This review covers the clinical presentations, treatments, and outcomes of cornual heterotopic pregnancies reported in the literature. Infertile women with a history of ectopic pregnancy, tubal surgery, or disease are at increased risk for cornual heterotopic pregnancy when they undergo in vitro fertilization. Women who have undergone bilateral salpingectomy also seem to be predisposed to this condition when they undergo in vitro fertilization. We recommend that these patients be followed up closely after a successful in vitro fertilization cycle with monitoring of serum beta-human chorionic gonadotropin levels and serial transvaginal ultrasonography because of the high associated morbidity. Laparotomy remains the treatment of choice for rupture of a cornual heterotopic pregnancy. In the absence of cornual rupture, however, medical management is an option that eliminates the risk of surgery and anesthesia and results in outcomes similar to those associated with surgical treatment. Currently there is insufficient evidence to recommend any single treatment modality, and the decision should be based on such factors as clinical presentation, surgeon's expertise, side effects, overall cost, and the patient's preference.

Empty follicle syndrome: evidence for recurrence.

The empty follicle syndrome (EFS) is a frustrating condition in which no oocytes are retrieved in an IVF cycle. Although this is an infrequent event in IVF patients, the economic consequences as well as the emotional frustration of a cancelled cycle due to the inability to obtain oocytes are enormous. The mechanisms responsible for EFS remain obscure, though many hypotheses have been put forward ranging from dysfunctional folliculogenesis to a drug-related problem. We found that the EFS is a rare event (1.8% of oocyte retrievals) but with profound implications for counselling the couple about their future reproductive performance. The chances of recurrence of EFS increase with the age of the patient (24% recurrence rate for the 35-39 year age group, and 57% for those over 40 years). We postulate that ovarian ageing, through altered folliculogenesis, may be implicated in the aetiology of EFS and its recurrence.

Psychotherapy for depression: current and future directions in research, theory, practice and public policy.

This article is based on a symposium held at the 1998 Annual Meeting of Society for Psychotherapy Research (Snow Bird, Utah). Recognized experts addressed current and future directions in psychotherapy for depression from the perspectives of process and outcome research, basic research, theoretical models, clinical practice and training, and public policy. The specific issues discussed at the symposium included the strengths and limitations of major forms of psychotherapy; the therapeutic factors common and unique to different approaches; the future viability of current theories of depression; the role of treatment manuals in clinical practice and training; the development of new interventions based on basic research; and the priorities that should guide federal funding.

Characterization of the Menkes protein copper-binding domains and their role in copper-induced protein relocalization.

Menkes disease is a fatal X-linked disorder of copper metabolism. The gene defective in Menkes disease (ATP7A) encodes a copper transporting P-type ATPase (MNK or ATP7A) with six copper-binding domains at its N-terminus. MNK is normally localized to the trans -Golgi network in cultured cells, but relocates to the plasma membrane in the presence of elevated extracellular copper. In this study, the role of the six copper-binding domains on copper-induced redistribution is investigated. In a recombinant clone, when all the wild-type copper-binding motifs are mutated from GMXCXXC to GMXSXXS and the cells grown in medium containing elevated copper, relocalization of the recombinant protein to the plasma membrane was not observed. Using the same assay with any one of the six copper-binding domains intact, MNK moves to the plasma membrane in a way indistinguishable from the wild-type protein. Therefore, the copper-binding domains are vital for MNK trafficking and only a single domain is sufficient for this redistribution to occur.