Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Clonostachys rosea 'omics profiling: identification of putative metabolite-gene associations mediating its in vitro antagonism against Fusarium graminearum.

BACKGROUND: Clonostachys rosea is an established biocontrol agent. Selected strains have either mycoparasitic activity against known pathogens (e.g. Fusarium species) and/or plant growth promoting activity on various crops. Here we report outcomes from a comparative 'omics analysis leveraging a temporal variation in the in vitro antagonistic activities of C. rosea strains ACM941 and 88-710, toward understanding the molecular mechanisms underpinning mycoparasitism. RESULTS: Transcriptomic data highlighted specialized metabolism and membrane transport related genes as being significantly upregulated in ACM941 compared to 88-710 at a time point when the ACM941 strain had higher in vitro antagonistic activity than 88-710. In addition, high molecular weight specialized metabolites were differentially secreted by ACM941, with accumulation patterns of some metabolites matching the growth inhibition differences displayed by the exometabolites of the two strains. In an attempt to identify statistically relevant relationships between upregulated genes and differentially secreted metabolites, transcript and metabolomic abundance data were associated using IntLIM (Integration through Linear Modeling). Of several testable candidate associations, a putative C. rosea epidithiodiketopiperazine (ETP) gene cluster was identified as a prime candidate based on both co-regulation analysis and transcriptomic-metabolomic data association. CONCLUSIONS: Although remaining to be validated functionally, these results suggest that a data integration approach may be useful for identification of potential biomarkers underlying functional divergence in C. rosea strains.

Draft Genome Resources for Plant-Beneficial Fungi Clonostachys rosea Strains ACM941 and 88-710.

Clonostachys rosea strains ACM941 and 88-710 are beneficial microbes recognized for their plant disease control and growth promotion properties, respectively, when applied to economically important crops. In addition to their geographical and functional overlap, the two strains also share a high degree of genetic similarity. In an effort to identify the subtleties that underlie their strain-specific applications, their genomic sequence is reported here. The genome size of ACM941 was estimated to be 56.9 Mb, encoding 17,585 putative genes, while strain 88-710 was estimated to have a 55.5 Mb genome size, containing 17,188 predicted genes. Overall, ACM941 and 88-710 share >96% of their encoded genomes, such that their strain-specific characteristics are likely encoded in either the remaining variable 4% or differentially regulated shared genes or both. These genomic sequences form a foundation for future studies aimed at identifying the genomic and metabolic machinery driving their respective beneficial properties.

Barley "uzu" and Wheat "uzu-like" Brassinosteroid Receptor BRI1 Kinase Domain Variations Modify Phosphorylation Activity In Vitro.

Brassinosteroid insensitive1 (BRI1), a leucine-rich repeat receptor kinase, is responsible for the perception of the brassinosteroid (BR) phytohormone in plants. While recent evidence has implicated a naturally occurring Hordeum vulgare V. (barley) HvBRI1 kinase domain (KD) variant (H857R; "uzu" variation) in increased fungal disease resistance, the impact of the variation on receptor function and thus the mechanism by which disease resistance might be imparted remain enigmatic. Here, the functional implications of the uzu variation as well as the effects of newly identified naturally occurring Triticum aestivum L. (wheat) TaBRI1-KD variants are investigated. Recombinantly produced KDs of wild-type (WT) and uzu HvBRI1 were assessed for phosphorylation activity in vitro, yielding WT KM and VMAX values similar to those of other reports, but the uzu variation delayed saturation and reduced turnover levels. In silico modeling of the H857R variation showed it to be surface-exposed and distal from the catalytic site. Further evaluation of three naturally occurring wheat TaBRI1 variants, A907T, A970V, and G1019R (barley numbering) identified in the A, B, and D subgenomic genes, respectively, highlighted a significant loss of activity for A907T. A907T is located on the same surface as the H857R variation and a negative regulatory phosphorylation site (T982) in Arabidopsis thaliana BRI1. A fourth variation, T1031A (barley numbering), unique to both subgenomic A proteins and localized to the BKI1 binding site, also decreased activity. The outcomes are discussed with respect to the predicted structural contexts of the variations and their implications with respect to mechanisms of action.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

Sensor-mediated granular sludge reactor for nitrogen removal and reduced aeration demand using a dilute wastewater.

A sensor-mediated strategy was applied to a laboratory-scale granular sludge reactor (GSR) to demonstrate that energy-efficient inorganic nitrogen removal is possible with a dilute mainstream wastewater. The GSR was fed a dilute wastewater designed to simulate an A-stage mainstream anaerobic treatment process. DO, pH, and ammonia/nitrate sensors measured water quality as part of a real-time control strategy that resulted in low-energy nitrogen removal. At a low COD (0.2 kg m-3  day-1 ) and ammonia (0.1 kg-N m-3  day-1 ) load, the average degree of ammonia oxidation was 86.2 ± 3.2% and total inorganic nitrogen removal was 56.7 ± 2.9% over the entire reactor operation. Aeration was controlled using a DO setpoint, with and without residual ammonia control. Under both strategies, maintaining a low bulk oxygen level (0.5 mg/L) and alternating aerobic/anoxic cycles resulted in a higher level of nitrite accumulation and supported shortcut inorganic nitrogen removal by suppressing nitrite oxidizing bacteria. Furthermore, coupling a DO setpoint aeration strategy with residual ammonia control resulted in more stable nitritation and improved aeration efficiency. The results show that sensor-mediated controls, especially coupled with a DO setpoint and residual ammonia controls, are beneficial for maintaining stable aerobic granular sludge. PRACTITIONER POINTS: Tight sensor-mediated aeration control is need for better PN/A. Low DO intermittent aeration with minimum ammonium residual results in a stable N removal. Low DO aeration results in a stable NOB suppression. Using sensor-mediated aeration control in a granular sludge reactor reduces aeration cost.

A Universally Primed-Polymerase Chain Reaction (UP-PCR) Marker to Discriminate Clonostachys rosea ACM941 from Related Strains.

Clonostachys rosea strain ACM941 is an effective biocontrol agent against several crop diseases including Fusarium head blight. In anticipation of its increased relevance going forward, the development of a reliable DNA-based molecular marker to track it is essential. Universally primed-PCR (UP-PCR) has been used successfully to differentiate other C. rosea strains. Herein, the development of a UP-PCR marker for ACM941 is described. A combination of two primers (AS15 and L45) produced a ~450 bp fragment that was unique to ACM941 compared to other commercial biocontrol agents. Primers subsequently designed based on the obtained fragment also produced a similarly unique band from ACM941 alone. BLAST analysis of the amplified sequence did not yield any homologous sequence in available online databases or within the closely related C. rosea IK726 and CBS125111 strains' genomes. The specificity of this marker for ACM941 was validated against ten additional C. rosea strains isolated from Canada, with ACM941 producing the brightest band. Taken together, these results imply that the UP-PCR primers AS15 and L45 and the amplified fragment can be used to detect and monitor the ACM941 strain after its release into the environment.

A lavender ABC transporter confers resistance to monoterpene toxicity in yeast.

MAIN CONCLUSION: Functional expression of a multidrug resistance-type ABC transporter from Lavandulaangustifolia improved yeast resistance to geraniol, a monoterpene constituent of lavender essential oil. Plant ATP-binding cassette (ABC) transporters are a large family of membrane proteins involved in active and selective transport of structurally diverse compounds. In this study, we functionally evaluated LaABCB1, a multidrug resistance (MDR)-type ABC transporter strongly expressed in the secretory cells of lavender glandular trichomes, where monoterpene essential oil constituents are synthesized and secreted. We used LaABCB1 to complement a yeast knockout mutant in which 16 ABC transporters were deleted. Expression of LaABCB1 enhanced tolerance of yeast mutants to geraniol, a key constituent of essential oils in lavenders and numerous other plants. Our findings suggest a role for the MDR-type ABC transporters in the toxicity tolerance of at least certain essential oil constituents in lavender oil glands.

Profiling of the Transcriptomic Responses of Clonostachys rosea Upon Treatment With Fusarium graminearum Secretome.

Clonostachys rosea strain ACM941 is a fungal bio-control agent patented against the causative agent of Fusarium Head Blight, Fusarium graminearum. Although the molecular details remain enigmatic, previous studies have suggested that C. rosea may secrete F. graminearum growth inhibitors. Further toward this, experiments described herein show that induction of C. rosea cultures by the addition of an aliquot of F. graminearum(Fg)-spent media (including macroconidia), yield C. rosea (Cr)-spent media that elicited higher anti-F. graminearum activity than either control or deoxynivalenol (DON)-induced Cr-spent media. To gain additional insight into the genetic and metabolic factors modulating this interaction, transcriptomic (RNAseq) profiles of C. rosea in response to DON and Fg-spent media treatment, were developed. This analysis revealed 24,112 C. rosea unigenes, of which 5,605 and 6,285 were differentially regulated by DON and F-spent media, respectively. More than half of these unigenes were up-regulated, with annotations, most notably in the Fg-spent media treatment data, suggesting enhancement of polyketide (PK) and non-ribosomal peptide (NRP) secondary metabolite precursor synthesis, and PK/NRP-like synthases. Four ABC transporters were also up-regulated in response to Fg-spent media. Further analysis showed that the PK and NRP-like synthases belong to three gene clusters that also include ABC transporters, and other genes known to tailor secondary metabolite biosynthesis. The RNAseq data was further validated using quantitative RT-qPCR. Taken together, these results show that C. rosea responds to the presence of Fg-spent media (and to a lesser extent, DON-alone) by up-regulating unique aspects of its secondary metabolism-related genetic repertoire. The identities and roles of C. rosea secondary metabolites produced by the targeted gene clusters are now under investigation.

Identification, validation and cross-species transferability of novel Lavandula EST-SSRs.

MAIN CONCLUSION: We identified and characterized EST-SSRs with strong discrimination power against Lavandula angustifolia and Lavandula x intermedia . The markers also showed considerable cross-species transferability rate into six related Lavandula species. Lavenders (Lavandula) are important economical crops grown around the globe for essential oil production. In an attempt to develop genetic markers for these plants, we analyzed over 13,000 unigenes developed from L. angustifolia and L. x intermedia EST databases, and identified 3,459 simple sequence repeats (SSR), which were dominated by trinucleotides (41.2 %) and dinucleotides (31.45 %). Approximately, 19 % of the unigenes contained at least one SSR marker, over 60 % of which were localized in the UTRs. Only 252 EST-SSRs were 18 bp or longer from which 31 loci were validated, and 24 amplified discrete fragments with 85 % polymorphism in L. x intermedia and L. angustifolia. The average number of alleles in L. x intermedia and L. angustifolia were 3.42 and 3.71 per marker with average PIC values of 0.47 and 0.52, respectively. These values suggest a moderate to strong level of informativeness for the markers, with some loci producing unique fingerprints. The cross-species transferability rate of the markers ranges 50-100 % across eight species. The utility of these markers was assessed in eight Lavandula species and 15 L. angustifolia and L. x intermedia cultivars, and the dendrogram deduced from their similarity indexes successfully delineated the species into their respective sections and the cultivars into their respective species. These markers have potential for application in fingerprinting, diversity studies and marker-assisted breeding of Lavandula.

Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes.

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 µm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 µM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering.

Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula.

Several members of the genus Lavandula produce valuable essential oils (EOs) that are primarily constituted of the low molecular weight isoprenoids, particularly monoterpenes. We isolated over 8,000 ESTs from the glandular trichomes of L. x intermedia flowers (where bulk of the EO is synthesized) to facilitate the discovery of genes that control the biosynthesis of EO constituents. The expression profile of these ESTs in L. x intermedia and its parents L. angustifolia and L. latifolia was established using microarrays. The resulting data highlighted a differentially expressed, previously uncharacterized cDNA with strong homology to known 1,8-cineole synthase (CINS) genes. The ORF, excluding the transit peptide, of this cDNA was expressed in E. coli, purified by Ni-NTA agarose affinity chromatography and functionally characterized in vitro. The ca. 63 kDa bacterially produced recombinant protein, designated L. x intermedia CINS (LiCINS), converted geranyl diphosphate (the linear monoterpene precursor) primarily to 1,8-cineole with K ( m ) and k ( cat ) values of 5.75 µM and 8.8 × 10(-3) s(-1), respectively. The genomic DNA of CINS in the studied Lavandula species had identical exon-intron architecture and coding sequences, except for a single polymorphic nucleotide in the L. angustifolia ortholog which did not alter protein function. Additional nucleotide variations restricted to L. angustifolia introns were also observed, suggesting that LiCINS was most likely inherited from L. latifolia. The LiCINS mRNA levels paralleled the 1,8-cineole content in mature flowers of the three lavender species, and in developmental stages of L. x intermedia inflorescence indicating that the production of 1,8 cineole in Lavandula is most likely controlled through transcriptional regulation of LiCINS.

Cloning and functional characterization of ß-phellandrene synthase from Lavandula angustifolia.

En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of ß-phellandrene synthase, LaßPHLS. The ORF--excluding the transit peptide--for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni-NTA agarose affinity chromatography. While the recombinant LaßPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into ß-phellandrene as the major product, with K (m) and k (cat) of 6.55 µM and 1.75 × 10(-2) s(-1), respectively, for GPP. The LaßPHLS transcripts were highly abundant in young leaves where ß-phellandrene is produced, but were barely detectable in flowers and older leaves, where ß-phellandrene is not synthesized in significant quantities. This data indicate that ß-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.

Salmonella serovars and antimicrobial resistance profiles in beef cattle, slaughterhouse personnel and slaughterhouse environment in ethiopia.

The present study was undertaken to determine the occurrence, distribution and antimicrobial resistance profiles of Salmonella serovars in slaughter beef cattle, slaughterhouse environment and personnel engaged in flaying and evisceration during slaughtering process. A total of 800 samples (each sample type, n = 100) consisting of swabs from hides, slaughterhouse personnel hands at flaying and evisceration, rumen and caecal contents, mesenteric lymph nodes, carcasses and holding pens were collected. Of the total 100 beef cattle examined, 14% were Salmonella positive in caecal content and/or mesenteric lymph nodes. Of the various samples analysed, Salmonella was detected in 31% of hides, 19% of rumen contents, 8% of mesenteric lymph nodes, 6% of caecal contents, 2% of carcass swabs, 9% of palm swabs taken from the hands of personnel in the slaughterhouse during flaying (7%) and evisceration (2%), and in 12% of holding pen swabs. The Salmonella isolates (n = 87) belonged to eight different serovars of which S. Anatum (n = 54) and S. Newport (19) were the major serovars and both serovars were detected in all sample sources except in carcass swabs. Eighteen of the 87 (20.7%) Salmonella serovars consisting of Newport (n = 14), Anatum (n = 3) and Eastbourne (n = 1) were resistant to one or more antimicrobials. Among the antimicrobial resistant Salmonella serovars, S. Newport was multidrug resistant (15.6%) and exhibited resistance to streptomycin, sulphisoxazole and tetracycline.

Participatory investigation of Contagious Caprine Pleuropneumonia (CCPP) in goats in the Hammer and Benna-Tsemay districts of southern Ethiopia.

The study was conducted in two selected districts of Southern Omo zones of Ethiopia, namely Hammer and Benna-Tsemay, during November 2004 and May 2005 to determine the status of contagious caprine pleuropneumonia (CCPP). Participatory disease investigation was conducted in the goat flocks owned by pastoralists of the districts. Participatory methods such as proportionate piling and matrix scoring of diseases were used to characterise major diseases of goats. Clinical and post-mortem examinations and isolation of the causative agent of CCPP were done. Serological tests were conducted using CFT. CCPP (locally termed Sompo) ranked as the first important disease of goats in the study area. Local perception of causes and signs of CCPP were described. Matrix scoring between groups revealed that disease signs and causes showed weak, moderate and good agreement by Kendall's coefficient concordance (W = 0.21-0.99). The overall sero-prevalence of CCPP was 15.5%. The causative agent was isolated from sick animals in the lab. The characteristic clinical signs, gross lesions, bacteriological isolation of the causative agent supported by participatory epidemiological disease investigation revealed that CCPP is a major disease of goats in the study districts. Participatory epidemiology using indigenous knowledge could efficiently be used to generate sufficient information with minimum cost, local materials and within reasonably short period of time, assisting the designing of feasible disease control programme in developing countries.

Bovine mastitis and its association with selected risk factors in smallholder dairy farms in and around Bahir Dar, Ethiopia.

Three hundred fifty one (195 local zebu and 156 Holstein x local zebu crosses) lactating cows of smallholder farms in Bahir Dar 'milk shed' were examined from September 2003 to March 2004 to determine mastitis prevalence, isolate pathogens and identify the role of some potential risk factors. Clinical prevalence was determined through examination of abnormalities of milk, udder or cow. California mastitis test (CMT) was used for determination of subclinical mastitis prevalence. Clinical prevalence at cow level was 3.9% in crossbreds and none in local zebu breeds. Subclinical mastitis at cow level based on CMT was high (34.4%) in crossbreds compared to indigenous zebu (17.9%) (p < 0.05). Quarter subclinical prevalence based on CMT was 17.9% and 4.9% in crossbreds and local zebu, respectively. The pathogens isolated from mastitic milk (CMT positive milk) were coagulase negative staphylococci (CNS), S. aureus, Str. agalactiae, Str. dysgalactiae, Str. uberis, Micrococcus species, C. bovis, A. pyogens, B. cereus, and S. intermedius. Among these, the most frequent isolates were CNS (50%), S. aureus (19%), Str. agalactiae (8%) and Str. dysgalactiae (7%). Among potential risk factors considered, stage of lactation, parity and breed were found to affect the occurrence of mastitis significantly (p < 0.05).

Diversity of Mannheimia haemolytica and pasteurella trehalosi serotypes from apparently healthy sheep and abattoir specimens in the highlands of Wollo, North East Ethiopia.

The prevalence and serotypic diversity of Mannheimia [Pasteurella] haemolytica and Pasteurella trehalosi from nasal swabs, sera and abattoir specimens from sheep in the highlands of Wollo, North East Ethiopia was investigated. Prevalence rates of 83% and 75% of these microorganisms were found in the serum samples and nasal swabs, respectively, from apparently healthy sheep. In a local abattoir, 205 lungs were investigated, 34% of which showed pneumonia, from which samples were collected from 51 lungs and the same number of corresponding tonsils. Mannheimia and Pasteurella species were isolated from 59% of these pneumonic lungs and 69% of the respective tonsils. M. haemolytica serotypes accounted for 41 (59%) and P. trehalosi for 11 (32%) of the isolates from the abattoir specimens. The majority (67%) of isolates from nasal swabs were P. trehalosi, M. haemolytica being isolated f rom 4 (13%) of the swabs. M. glucosida was isolated only from the tonsils. The predominant serotypes of the isolates from both the nasal swabs and the abattoir specimens were M. haemolytica A1 (17%) and P. trehalosi T4 (16%) and T3 (13%). P. trehalosi T15 was less commonly encountered, while M. haemolytica A9 and A13 were not isolated. Studies on sera from 100 sheep indicated that antibodies against M. haemolytica serotype A1 (14%) were most common, followed by A5 and A8 (each 10%) and A9 and P. trehalosi T3 (each 9%) and T4 (8%). Antibodies against M. glucosida or serotype All occurred in 2% of the sera. Multiple serotypes were common in all types of samples. The importance of including in vaccines the most prevalent serotypes involved in the pneumonia of sheep in the area is discussed.

Influence of elevated CO2 and mycorrhizae on nitrogen acquisition: contrasting responses in Pinus taeda and Liquidambar styraciflua.

An understanding of root system capacity to acquire nitrogen (N) is critical in assessing the long-term growth impact of rising atmospheric CO2 concentration ([CO2]) on trees and forest ecosystems. We examined the effects of mycorrhizal inoculation and elevated [CO2] on root ammonium (NH4+) and nitrate (NO3-) uptake capacity in sweetgum (Liquidambar styraciflua L.) and loblolly pine (Pinus taeda L.). Mycorrhizal treatments included inoculation of seedlings with the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith in sweetgum and the ectomycorrhizal (EM) fungus Laccaria bicolor (Maire) Orton in loblolly pine. These plants were then equally divided between ambient and elevated [CO2] treatments. After 6 months of treatment, root systems of both species exhibited a greater uptake capacity for NH4+ than for NO3-. In both species, mycorrhizal inoculation significantly increased uptake capacity for NO3-, but not for NH4+. In sweetgum, the mycorrhizal effect on NO3- and NH4+ uptake capacity depended on growth [C02]. Similarly, in loblolly pine, the mycorrhizal effect on NO3- uptake capacity depended on growth [CO2], but the effect on NH4+ uptake capacity did not. Mycorrhizal inoculation significantly enhanced root nitrate reductase activity (NRA) in both species, but elevated [CO2] increased root NRA only in sweetgum. Leaf NRA in sweetgum did not change significantly with mycorrhizal inoculation, but increased in response to [CO2]. Leaf NRA in loblolly pine was unaffected by either treatment. The results indicate that the mycorrhizal effect on specific root N uptake in these species depends on both the form of inorganic N and the mycorrhizal type. However, our data show that in addressing N status of plants under high [CO2], reliable prediction is possible only when information about other root system adjustments (e.g., biomass allocation to fine roots) is simultaneously considered.