Effects of defoliation on blackcurrant infection by Armillaria species

Field inoculation trials and laboratory studies were used to investigate the effects of defoliation stress on potted black currant plants and the infection by English and African isolates of Armillaria. Defoliation has varying effects on the carbohydrate, fatty acids and amino acids contents of roots. All isolates of Armillaria tested infected black currant plants irrespective of stress treatment; with two of the test isolates, more of the infected plants were killed with defoliation treatment. Media supplemented with water extract from defoliated roots stimulated growth of isolates compared to media supplemented with extract from non-defoliated control root tissues. The differences observed in the pathogenic behaviour of isolates, may be of importance in the epidemiology of Armillaria infections.

The effect of carrier substrate, dose rate and time of application on biocontrol efficacy of fungal antagonists against Armillaria root rot of strawberry plants.

In a glasshouse experiment using potted strawberry plants (cv. Cambridge Favourite) as hosts, the effect of selected fungal antagonists grown on 25 or 50 g of mushroom compost containing autoclaved mycelia of Agaricus bisporus, or wheat bran was evaluated against Armillaria mellea. Another glasshouse experiment tested the effect of application time of the antagonists in relation to inoculations with the pathogen. A significant interaction was found between the antagonists, substrates and dose rates. All the plants treated with Chaetomium olivaceum isolate Co on 50 g wheat bran survived until the end of the experiment which lasted 482 days, while none of them survived when this antagonist was added to the roots of the plants on 25 g wheat bran or 25 or 50 g mushroom compost. Dactylium dendroides isolate SP had a similar effect, although with a lower host survival rate of 33.3%. Trichoderma hamatum isolate Tham 1 and T. harzianum isolate Th23 protected 33.3% of the plants when added on 50 g and none when added on 25 g of either substrate, while 66.7% of the plants treated with T. harzianum isolate Th2 on 25 g, or T viride isolate TO on 50 g wheat bran, survived. Application of the antagonists on mushroom compost initially resulted in development of more leaves and healthier plants, but this effect was not sustained. Eventually, plants treated with the antagonists on wheat bran had significantly more leaves and higher health scores. The plants treated with isolate Th2 and inoculated with Armillaria at the same time had a survival rate of 66.7% for the duration of the experiment (475 days), while none of them survived that long when the antagonist and pathogen were applied with an interval of 85 days in either sequence. C. olivaceum isolate Co showed a protective effect only, as 66.7% of the plants survived when they were treated with the antagonist 85 days before inoculation with the pathogen, while none of them survived when the antagonist and pathogen were applied together or the infection preceded protection.

Multiple domestications and their taxonomic consequences: the example of Phaseolus vulgaris

The evolutionary history of P. vulgaris is important to those working on its genetic resources, but is not reflected in its infraspecific taxonomy. Genetic isolation of wild populations between and also within Middle and South America has resulted in morphological and molecular differentiation. Populations from northern and southern ends of the range are assigned to different gene pools, though intermediates occur in intervening areas. Chloroplast haplotypes suggest three distinct lineages of wild beans and several intercontinental dispersals. The species was domesticated independently in both Middle and South America, probably several times in Middle America. This, together with further differentiation under human selection, has produced distinct races among domesticated beans. The informal categories of wild versus domesticated, gene pool, and race convey the evolutionary picture more clearly than the formal categories provided by the Codes of Nomenclature for wild or cultivated plants.

Peroxyl radical-scavenging activity of coffee brews

The ORAC(FL) assay was used in non-automated mode to evaluate the specific peroxyl radical scavenging properties of the aqueous soluble components of green and roasted Arabica and Robusta coffee samples. A relationship between ORAC(FL) and the concentration of CQAs (caffeoyl quinic acids) was found for the extracts from green coffee beans. Aqueous extracts from roasted coffee beans possessed equal or stronger scavenging power than that obtained for the green coffee beans extracts and the scavenging activity depended on the variety of coffee and the roasting conditions. Brews from Robusta coffee beans showed the highest ORAC(FL). The best scavenging properties for the brews from Arabica coffee beans were detected in samples prepared from coffee beans roasted under light conditions. The data indicate that, during roasting, a complex network of reactions takes place leading to the formation of a wide number of compounds possessing specific scavenging properties. Under mild roasting conditions, caffeoyl quinic acids appear to be the main components responsible for the free radical scavenging power of coffee brews. In contrast, Maillard reaction products may be the principal components with free radical scavenging activity in more severely (medium and dark) roasted coffees.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Proof of concept of automated mapping of weeds in arable fields

The project investigated whether it would be possible to remove the main technical hindrance to precision application of herbicides to arable crops in the UK, namely creating geo-referenced weed maps for each field. The ultimate goal is an information system so that agronomists and farmers can plan precision weed control and create spraying maps. The project focussed on black-grass in wheat, but research was also carried out on barley and beans and on wild-oats, barren brome, rye-grass, cleavers and thistles which form stable patches in arable fields. Farmers may also make special efforts to control them. Using cameras mounted on farm machinery, the project explored the feasibility of automating the process of mapping black-grass in fields. Geo-referenced images were captured from June to December 2009, using sprayers, a tractor, combine harvesters and on foot. Cameras were mounted on the sprayer boom, on windows or on top of tractor and combine cabs and images were captured with a range of vibration levels and at speeds up to 20 km h-1. For acceptability to farmers, it was important that every image containing black-grass was classified as containing black-grass; false negatives are highly undesirable. The software algorithms recorded no false negatives in sample images analysed to date, although some black-grass heads were unclassified and there were also false positives. The density of black-grass heads per unit area estimated by machine vision increased as a linear function of the actual density with a mean detection rate of 47% of black-grass heads in sample images at T3 within a density range of 13 to 1230 heads m-2. A final part of the project was to create geo-referenced weed maps using software written in previous HGCA-funded projects and two examples show that geo-location by machine vision compares well with manually-mapped weed patches. The consortium therefore demonstrated for the first time the feasibility of using a GPS-linked computer-controlled camera system mounted on farm machinery (tractor, sprayer or combine) to geo-reference black-grass in winter wheat between black-grass head emergence and seed shedding.

Pseudomonas syringae pv. coryli, the causal agent of bacterial twig dieback of Corylus avellana

Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.

Evaluating the effects of tannins on the extent and rate of in vitro measured gas and methane production using the Automated Pressure Evaluation System (APES)

An in vitro study was conducted to investigate the effect of tannins on the extent and rate of gas and methane production, using an automated pressure evaluation system (APES). In this study three condensed tannins (CT; quebracho, grape seed and green tea tannins) and four hydrolysable tannins (HT; tara, valonea, myrabolan and chestnut tannins) were evaluated, with lucerne as a control substrate. CT and HT were characterised by matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS). Tannins were added to the substrate at an effective concentration of 100 g/kg either with or without polyethylene glycol (PEG6000), and incubated for 72 h in pooled, buffered rumen liquid from four lactating dairy cows. After inoculation, fermentation bottles were immediately connected to the APES to measure total cumulative gas production (GP). During the incubation, 11 gas samples were collected from each bottle at 0, 1, 4, 7, 11, 15, 23, 30, 46, 52 and 72 h of incubation and analysed for methane. A modified Michaelis-Menten model was fitted to the methane concentration patterns and model estimates were used to calculate the total cumulative methane production (GPCH4). GP and GPCH4 curves were fitted using a modified monophasic Michaelis-Menten model. Addition of quebracho reduced GP (P=0.002), whilst the other tannins did not affect GP. Addition of PEG increased GP for quebracho (P=0.003), valonea (P=0.058) and grape seed tannins (P=0.071), suggesting that these tannins either inhibited or tended to inhibit fermentation. Addition of quebracho and grape seed tannins also reduced (P≤0.012) the maximum rate of gas production, indicating that microbial activity was affected. Quebracho, valonea, myrabolan and grape seed decreased (P≤0.003) GPCH4 and the maximum rate (0.001≤ P≤ 0.102) of CH4 production. Addition of chestnut, green tea and tara tannins did not affect total gas nor methane production. Valonea and myrabolan tannins have most promise for reducing methane production as they had only a minor impact on gas production.

Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle.

The role of phase separation and feed cycle length in leach beds coupled to methanogenic reactors for digestion of a solid substrate (Part 2): Hydrolysis, acidification and methanogenesis in a two-phase system

The effect of phase separation and batch duration on the trophic stages of anaerobic digestion was assessed for the first time in leach beds coupled to methanogenic reactors digesting maize (Zea mays). The system was operated for consecutive batches of 7, 14 and 28 days for ~120 days. Hydrolysis rate was higher the shorter the batch, reaching 8.5 gTSdestroyed d-1 in the 7-day system. Phase separation did not affect acidification but methanogenesis was enhanced in the short feed cycle leach beds. Phase separation was inefficient on the 7-day system, where ~89% of methane was produced in the leach bed. Methane production rate increased with shortening the feed cycle, reaching 3.523 l d-1 average in the 7-day system. Low strength leachate from the leach beds decreased methanogenic activity of methanogenic reactors’ sludges. Enumeration of cellulolytic and methanogenic microorganisms indicated a constant inoculation of leach beds and methanogenic reactors through leachate recirculation.

Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

Genetic variability in partitioning to the yield component of cacao (Theobroma cacao L.)

Biomass partitioning of cacao (Theobroma cacao L.) was studied in seven clones and five hybrids in a replicated experiment in Bahia, Brazil. Over an eighteen month period, a seven- fold difference in dry bean yield was demonstrated between genotypes, ranging from the equivalent of 200 to 1389 kg.ha-1. During the same interval, the increase in trunk cross-sectional area ranged from 11.1 cm2 for clone EEG-29 to 27.6 cm2 for hybrid PA-150 * MA-15. Yield efficiency increment (the ratio of cumulative yield to the increase in trunk circumference), which indicated partitioning between the vegetative and reproductive components, ranged from 0.008 kg.cm-2 for clone CP-82 to 0.08 kg.cm-2 for clone EEG-29. An examination of biomass partitioning within the pod of the seven clones revealed that the beans accounted for between 32.0% (CP-82) and 44.5% (ICS-9) of the pod biomass. The study demonstrated the potential for yield improvement in cacao by selectively breeding for more efficient partitioning to the yield component.

Non-curliation of Escherichia coli O78 : K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when groan on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E, coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Contribution of fimbriae and flagella of Salmonella enteritidis to colonization and invasion of chicks

Isogenic mutants of Salmonella enteritidis defective for the elaboration of fimbrial types SEF14, SEF17, SEF21 and flagella were used to study the contribution these organelles made to colonization, invasion and lateral transfer in young chicks. The caecum, liver and spleen were colonized within 24 h following oral inoculation of 1-day-old chicks with 10(5) wild-type S. enteritidis strain LA5. However, for some mutants, the numbers of organisms recovered from internal organs was reduced significantly, particularly at 24 h post-inoculum, which supported the hypothesis that the organelles contribute to invasion and dissemination to internal organs. Specifically, mutations affecting SEF17, SEF21 and flagella contributed to a delay in colonization of the spleen, and those affecting SEF21 and flagella delayed colonization of the liver. Lower numbers of bacteria were recovered from the caecum with mutants deficient in elaboration of SEF21. Sentinel birds were colonized by LA5 or EAV40 (14(-), 17(-), 21(-), fla(-)) directly from the environment within 2 days, although a consistent slight delay was observed with the multiple mutant. Overall, our data suggest a collective role for SEF17, SEF21 and flagella, but not SEF14, in the early stages of colonization and invasion of young chicks by S. enteritidis, but these surface appendages appear unnecessary for colonization of birds from their immediate environment.

Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degreesC for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degreesC, or 250 cells per egg when eggs were stored at 30 degreesC, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs. (C) 2001 Elsevier Science B.V. All rights reserved.