Calluna vulgaris root cells show increased capacity for amino acid uptake when colonized with the mycorrhizal fungus Hymenoscyphus ericae

Ericoid mycorrhizas are believed to improve N nutrition of many ericaceous plant species that typically occur in habitats with impoverished nutrient status, by releasing amino acids from organic N forms. Despite the ubiquity of mycorrhizal formation the mechanisms and regulation of nutrient transport in mycorrhizal associations are poorly understood. We used an electrophysiological approach to study how amino acid transport characteristics of Calluna vulgaris were affected by colonization with the ericoid mycorrhiza fungus Hymenoscyphus ericae. Both the V_max and K_m parameters of amino acid uptake were affected by fungal colonization in a manner consistent with an increased availability of amino acid to the plant. The ecophysiological significance of altered amino acid transport in colonized root cells of C. vulgaris is discussed. © New Phytologist (2002).

Intraspecific variation in nitrogen source utilisation by isolates of the ericoid mycorrhizal fungus Hymenoscyphus Ericae (Read) Korf and Kernan

Variation in the abilities of 35 isolates of the ericoid mycorrhizal fungal endophyte Hymenoscyphus ericae from two field sites to utilise inorganic and organic nitrogen sources in axenic culture has been investigated. While most isolates showed a preference for NH₄/^- as a sole nitrogen source, considerable variation was observed in the abilities of isolates to utilise amino acids and protein (BSA). In particular, large intraspecific variation was observed for glutamine and BSA utilisation, with some isolates thriving on these substrates while others produced little growth. The data suggest that individual isolates of H. ericae may vary considerably in their abilities to supply their host plants with nitrogen from different substrates in soil. (C) 2000 Elsevier Science Ltd.

Concentration effects of 1,2-dichlorobenzene on soil microbiology

The effect of increasing concentrations (65, 130, 325, 1,300, and 3,250 μg/g soil dry weight) of 1,2-dichlorobenzene (1,2-DCB) on the microbial biomass, metabolic potential, and diversity of culturable bacteria was investigated using soil microcosms. All doses caused a significant (p < 0.05) decrease in viable hyphal fungal length. Bacteria were more tolerant, only direct total counts in soils exposed to 3,250 μg/g were significantly (p < 0.05) lower than untreated controls, and estimates of culturable bacteria showed no response. Pseudomonads counts were stimulated by 1,2-DCB concentrations of up to 325 μg/g; above this level counts were similar to controls. Fatty acid methyl ester analysis of taxonomic bacterial composition reflected the differential response of specific genera to increasing 1,2-DCB concentrations, especially the tolerance of Bacillus to the highest concentrations. The shifts in community composition were reflected in estimates of metabolic potential assessed by carbon assimilation (Biolog) ability. Significantly fewer (p < 0.05) carbon sources were utilized by communities exposed to 1,2-DCB concentrations greater than 130 μg/g (<64 carbon sources utilized) than control soils (83); the ability to assimilate individual carbohydrates sources was especially compromised. The results of this study demonstrate that community diversity and metabolic potential can be used as effective bioindicators of pollution stress and concentration effects.

Response of soil microbial communities to single and multiple doses of an organic pollutant

The effect of 100 μg 1,2-dichlorobenzene (1,2-DCB) g^-1 dry weight (dw) of soil introduced either as a single dose or multiple (10 fortnightly) doses of 10 μg g^-1 dw, on the microbial biomass, diversity of culturable bacterial community and the rate of 1,2-DCB mineralisation, were compared. After 22 weeks exposure both application regimes significantly reduced total bacterial counts and viable fungal hyphal length. The single dose had the greatest overall inhibitory effect, although the extent of inhibition varied throughout the study. Total culturable bacterial counts, determined after 22 weeks exposure showed little response to 1,2-DCB, but pseudomonad counts in single and multiple treatments were reduced to 9.7 and 0.147%, respectively, of the numbers detected in the control soil. The effect of 1,2-DCB application on the taxonomic composition of the culturable bacteria community was determined by fatty acid methyl ester (FAME) analysis. Compared to control soils, the single dose treatment had a lower percentage of Arthrobacter and Micrococcus. Multiple applications had a significant effect upon pseudomonad abundance, which represented only 2% of the identified community, compared to 45.6% in the control. The multi-dosed soils contained a high percentage of bacilli (> 25%). The effects of 1,2-DCB applications on the metabolic potential of the soil microbial community was determined by BIOLOG profiling. The number of carbon compounds utilised by the community in the multi-dosed soils (49 positives) was significantly less (P < 0.05) than detected in the single dose treatment (76) and control (66). The rate of 1,2-DCB mineralisation, determined by ¹⁴CO₂ production from radiolabelled [UL-¹⁴C] 1,2-DCB, declined throughout the study, and after 22 weeks was slightly but significantly (P < 0.05) lower in the multiply- than the singly-dosed soils. The differential response to 1,2-DCB treatments was attributed to its reduced bioavailability in soils after a single exposure, compared to multiple applications.

Toxic interactions of metal ions (Cd2+, Pb2+, Zn2+ and Sb3- on in vitro biomass production of ectomycorrhizal fungi

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd^2-, Pb²⁺, Zn²⁺ and Sb^3-, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd²⁺ and Zn²⁺ was recorded, while Pb²⁺ and Sb^3- individually had little effect. The presence of Pb²⁺ and Sb^3- in the media did however, ameliorate Cd²⁺ and Zn²⁺ toxicity in some circumstances. Interactions between Cd²⁺ and Zn²⁺ were investigated further over a range of concentrations. Zn²⁺ was found to significantly ameliorate the toxicity of Cd²⁺ to three of the four isolates tested. The influence of Zn²⁺ varied between ECM species and with the concentrations of metals tested.

Assessment of toxicological interactions of benzene and its primary degradation products (catechol and phenol) using a lux-modified bacterial bioassay

A bacterial bioassay has been developed to assess the relative toxicities of xenobiotics commonly found in contaminated soils, rivers, waters, and ground waters. The assay utilized decline in luminescence of lux- marked Pseudomonas fluorescens on exposure to xenobiotics. Pseudomonas fluorescens is a common bacterium in the terrestrial environment, providing environmental relevance to soil, river, and ground water systems. Three principal environmental contaminants associated with benzene degradation were exposed to the luminescence-marked bacterial biosensor to assess their toxicity individually and in combination. Median effective concentration (EC50) values for decline in luminescence were determined for benzene, catechol, and phenol and were found to be 39.9, 0.77, and 458.6 mg/L, respectively. Catechol, a fungal and bacterial metabolite of benzene, was found to be significantly more toxic to the biosensor than was the parent compound benzene, showing that products of xenobiotic biodegradation may be more toxic than the parent compounds. Combinations of parent compounds and metabolites were found to be significantly more toxic to the bioassay than were the individual compounds themselves. Development of this bioassay has provided a rapid screening system suitable for assessing the toxicity of xenobiotics commonly found in contaminated soil, river, and ground-water environments. The assay can be utilized over a wide pH range and is therefore more applicable to such environmental systems than bioluminescence-based bioassays that utilize marine organisms and can only be applied over a limited pH and salinity range.

Determination of the Mycotoxin Content in Distiller's Dried Grain with Solubles Using a Multianalyte UHPLC-MS/MS Method

There are more than 300 potential mycotoxins that can contaminate food and feed and cause adverse effects in humans and animals. The data on the co-occurrence of mycotoxins in novel animal feed materials, such as distiller's dried grain with solubles (DDGS), are limited. Thus, a UHPLC-MS/MS method for the quantitation of 77 mycotoxins and other fungal metabolites was used to analyze 169 DDGS samples produced from wheat, maize, and barley and 61 grain samples. All DDGS samples analyzed were contaminated with 13-34 different mycotoxins. Fumonisins were present in all 52 maize DDGS samples (81.0-6890 μg/kg for fumonisin B1), and deoxynivalenol was present in all 99 wheat DDGS samples (39.3-1120 μg/kg). A number of co-occurring mycotoxins were also identified. Due to the high co-occurrence of mycotoxins, routine screening of the animal feed ingredients is highly recommended to allow the highlighted risks to be effectively managed.

Biocontrol agents promote growth of potato pathogens, depending on environmental conditions

There is a pressing need to understand and optimize biological control so as to avoid over-reliance on the synthetic chemical pesticides that can damage environmental and human health. This study focused on interactions between a novel biocontrol-strain, Bacillus sp. JC12GB43, and potato-pathogenic Phytophthora and Fusarium species. In assays carried out in vitro and on the potato tuber, the bacterium was capable of near-complete inhibition of pathogens. This Bacillus was sufficiently xerotolerant (water activity limit for growth = 0.928) to out-perform Phytophthora infestans (~0.960) and challenge Fusarium coeruleum (~0.847) and Fusarium sambucinum (~0.860) towards the lower limits of their growth windows. Under some conditions, however, strain JC12GB43 stimulated proliferation of the pathogens: for instance, Fusarium coeruleum growth-rate was increased under chaotropic conditions in vitro (132 mM urea) by >100% and on tubers (2-M glycerol) by up to 570%. Culture-based assays involving macromolecule-stabilizing (kosmotropic) compatible solutes provided proof-of-principle that the Bacillus may provide kosmotropic metabolites to the plant pathogen under conditions that destabilize macromolecular systems of the fungal cell. Whilst unprecedented, this finding is consistent with earlier reports that fungi can utilize metabolites derived from bacterial cells. Unless the antimicrobial activities of candidate biocontrol strains are assayed over a full range of field-relevant parameters, biocontrol agents may promote plant pathogen infections and thereby reduce crop yields. These findings indicate that biocontrol activity, therefore, ought to be regarded as a mode-of-behaviour (dependent on prevailing conditions) rather than an inherent property of a bacterial strain.

Engineered alpha-defensin peptides display antimicrobial activity against oral pathogens

Introduction: Human alpha defensins are a family of neutrophil-derived antimicrobial peptides also known as human neutrophil peptides (HNPs). The defensin family of peptides are characterised by six invariant cysteine residues forming three disulphide bridges. The formation of the correct disulphide pairs complicates the synthesis of full length human alpha defensin and limits its therapeutic potential as an antimicrobial peptide. Objectives: The aim of this study was to determine whether truncated alpha defensins displayed antimicrobial activity against a range of micro-organisms including oral pathogens. Methods: Engineered peptides were synthesised by solid-phase methods using standard Fmoc chemistry. Antibacterial assays were performed using a previously described ultra sensitive radial diffusion method. A total of five engineered defensin peptides and full length alpha defensin were tested for their sensitivity against eight micro-organisms, including Gram negative bacteria, Gram positive bacteria and fungal pathogens Results: Antimicrobial activity was identified as clear zones around peptide-containing wells. Zone diameters were used to calculate minimum inhibitory concentrations (MICs) for each peptide. There was considerable variability in the susceptibility of the micro-organisms to the truncated analogues. Bacillus subtilis and Enterococcus faecalis were sensitive to the majority of the engineered peptides whereas Staphylococcus aureus, Escherichia coli and Candida albicans displayed resistance (defined as an MIC of greater than 250 ug/ml) to the truncated defensins. Of the five engineered peptides synthesised, the 2-aminobenzoic acid (Abz)-containing analogues based on the C-terminal sequence of alpha defensin displayed MIC values closest to that of the full length defensin in 5 out of 8 micro-organisms studied. Conclusion: This study demonstrates that truncated alpha defensins display variable antimicrobial activity against a range of micro-organisms, including oral pathogens. The generation of truncated defensins without disulphide bridges simplifies their synthesis and increases their therapeutic potential.

Antifungal Activity of LL-37 and Truncated Mimetics

Background: Candida albicans is a commensal organism and a constituent of the normal oral flora. Cell concentrations of 1x102 cells/ml and below are indicative of commensal colonisation in the oral cavity, above this level C. albicans can become an opportunistic pathogen; it is the most prevalent human fungal pathogen and a causal agent of the oral infection, candidiasis. The capacity of C. albicans to cause infection arises from its ability to exist in a biofilm ecosystem. Mature C. albicans biofilms display a high level of resistance to antifungals and the need for other therapeutic options has become paramount. Objectives: The objectives of the current study were to determine the antifungal activity of LL-37 (a member of the human cathelicidin family) and two truncated peptide mimetics against C. albicans in both planktonic and biofilm form. Methods: Radial diffusion assays were used to obtain the minimum inhibitory concentration (MIC) of LL-37 and the truncated mimetics KE-18 and KR-12 against planktonic C. albicans. A 96 well microtitre plate assay was employed to study the effects of the peptides on early candida biofilm formation (up to 24 hours) compared with the antifungal drug fluconazole. Biofilm quantification was achieved using the crystal violet assay. Results: MIC values obtained: LL-37 >250µg/ml; KE-18 51µg/ml; and KR-12 11µg/ml. LL-37 significantly reduced the quantity of biofilm formed by C.albicans at both the 4 h and 24 h timepoints (p <0.0001). KE-18 showed significant biofilm reduction over 4 h and 24 h (p=0.0002, p=0.013 respectively), KR-12 showed significant reduction at the 24 h time point only (p=0.0256). Conclusions: Results suggest that LL-37 has the ability to disrupt early biofilm formation of C. albicans with its potency of action similar with that of fluconazole.

Aflatoxin Exposure and Associated Human Health Effects, a Review of Epidemiological Studies

flatoxins are fungal toxins that possess acute life threatening toxicity, carcinogenic properties and other potential chronic adverse effects. Dietary exposure to aflatoxins is considered a major public health concern, especially for subsistence farming communities in sub-Saharan Africa and South Asia, where dietary staple food crops such as groundnuts and maize are often highly contaminated with aflatoxin due to hot and humid climates and poor storage, together with low awareness of risk and lack of enforcement of regulatory limits. Biomarkers have been developed and applied in many epidemiological studies assessing aflatoxin exposure and the associated health effects in these high-risk population groups. This review discusses the recent epidemiological evidence for aflatoxin exposure, co-exposure with other mycotoxins and associated health effects in order to provide evidence on risk assessment, and highlight areas where further research is necessary. Aflatoxin exposure can occur at any stage of life and is a major risk factor for hepatocellular carcinoma, especially when hepatitis B infection is present. Recent evidence suggests that aflatoxin may be an underlying determinant of stunted child growth, and may lower cell-mediated immunity, thereby increasing disease susceptibility. However, a causal relationship between aflatoxin exposure and these latter adverse health outcomes has not been established, and the biological mechanisms for these have not been elucidated, prompting further research. Furthermore, there is a dearth of information regarding the health effects of co-exposure to aflatoxin with other mycotoxins. Recent developments of biomarkers provide opportunities for important future research in this area.

An investigation of IAQ, thermal comfort and Sick Building Syndrome symptoms in UK energy efficient homes

Purpose
– Concern of the deterioration of indoor environmental quality as a result of energy efficient building design strategies is growing. Apprehensions of the effect of airtight, super insulated envelopes, the reduction of infiltration, and the reliance on mechanical systems to provide adequate ventilation (air supply) is promoting emerging new research in this field. The purpose of this paper is to present the results of an indoor air quality (IAQ) and thermal comfort investigation in UK energy efficient homes, through a case study investigation.

Design/methodology/approach
– The case study dwellings consisted of a row of six new-build homes which utilize mechanical ventilation with heat recovery (MVHR) systems, are built to an average airtightness of 2m3/m2/hr at 50 Pascal’s, and constructed without a central heating system. Physical IAQ measurements and occupant interviews were conducted during the summer and winter months over a 24-hour period, to gain information on occupant activities, perception of the interior environment, building-related health and building use.

Findings
– The results suggest inadequate IAQ and perceived thermal comfort, insufficient use of purge ventilation, presence of fungal growth, significant variances in heating patterns, occurrence of sick building syndrome symptoms and issues with the MVHR system.

Practical implications
– The findings will provide relevant data on the applicability of airtight, mechanically ventilated homes in a UK climate, with particular reference to IAQ.

Originality/value
– IAQ data of this nature is essentially lacking, particularly in the UK context. The findings will aid the development of effective sustainable design strategies that are appropriate to localized climatic conditions and sensitive to the health of building occupants.

Bursaphelenchus xylophilus : opportunities in comparative genomics and molecular host–parasite interactions

Most Bursaphelenchus species are fungal feeding nematodes that colonize dead or dying trees. However, Bursaphelenchus xylophilus , the pine wood nematode, is also a pathogen of trees and is the causal agent of pine wilt disease. B. xylophilus is native to North America and here it causes little damage to trees. Where it is introduced to new regions it causes huge damage. The most severely affected areas are found in the Far East but more recently B. xylophilus has been introduced into Portugal and the potential for damage here is also high. As incidence and severity of pine wilt disease are linked to temperature we suggest that climate change is likely to exacerbate the problems caused by B. xylophilus and, in addition, will extend (northwards in Europe) the range in which pine wilt disease can occur. Here we review what is currently known about the interactions of B. xylophilus with its hosts, including recent developments in our understanding of the molecular biology of pathogenicity in the nematode. We also examine the potential developments that could be made by more widespread use of genomics tools to understand interactions between B. xylophilus , bacterial pathogens that have been implicated in disease and host trees.

The Pine Wood Nematode: a personal view

The first report of the disease (“pine wilt disease”) associated with the pinewood nematode, goes back to 1905, when Yano reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of he disease was sought, but without success. Because of the large number of insect species that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be one of these. However, in 1971, Kiyohara and Tokushike found a nematode of the genus Bursaphelenchus in infected trees. The nematode found was multiplied on fungal culture, inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii und P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was a named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had suddenly died after the colour of its needles changed to a reddish-brown colour (Dropkin und Foudin, 2 1979). In 1981, B. lignicolus was synonymised by Nickle et al. with B. xylophilus which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides but was recognised by Nickle, in 1970,to belong in the genus Bursaphelenchus. Its common name in the USA was the "pine wood nematode" (PWN. After its detection in Missouri, it became known that B. xylophilus was widespread throughout the USA and Canada. It occurred there on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless susceptible species were stressed eg., by high temperature. This fact was an illuminating piece of evidence that North America could be the homeland of PWN. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the long-horned beetle Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century but in the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. In Japan, there are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN. The occasional transmission by less efficient species of Monochamus or by some of the many other beetle genera in the bark or wood is of little significance. In Europe, M. galloprovincialis and M. sutor transmits the closely related species B. mucronatus. Some speculate that these two insect species are “standing by” and waiting for the arrival of B. xylophilus. In 1982, the nematode was detected and China. It was first found in dead pines near the Zhongshan Monument of Nanjing (CHENG et. al. 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been 3 reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (YANG, 2003). In 1986, the spread of the PWN to Taiwan was discovered and in 1989, the nematode was reported to be present in the Republic of Korea where it had first been detected in Pinus thunbergii and P. densiflora. It was though to have been introduced with packing material from Japan. PWN was advancing. In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the import of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries in 1986 to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognised the potential danger of B. xylophilus for European forests and imposed restrictions on imports into the Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU(92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction.

Mastites ovinas: Epidemiologia, factores de virulência e antigénios imunorrelevantes de agentes etiológicos

O presente trabalho teve como objectivo o estudo da prevalência de mastites ovinas em explorações do Alentejo e a identificação dos agentes etiológicos, seus factores de virulência e epitopos imunorrelevantes. A prevalência de mastite clínica e subclínica foi 1,7% e 32,2%, respectivamente. O agente etiológico mais prevalente foi Staphylococcus epidermidis (N=115), tendo sido também identificados Staphylococcus aureus (N=27) e Streptococcus agalactiae (N=17). A pesquisa de factores de virulência permitiu identificar os padrões de susceptibilidade (N=404) e as Concentrações Inibitórias Mínimas de princípios activos (N=130). De 109 isolados de Staphylococcus epidermidis; oito revelaram capacidade para produzir biofilme in vitro. Os isolados estudados aderiam e eram internalizados por células epiteliais mamárias (N=12). A pesquisa de cinco superantigénios resultou negativa (N=27). Foram estudados os perfis proteicos de Staphylococcus epidermidis, tendo sido identificados os epitopos imunorrelevantes, reconhecidos por imunoglobulinas séricas e mamárias. Verificou-se uma resposta imunológica local específica nos animais infectados./SUMMARY - OVINE MASTITIS: EPIDEMIOLOGY, VIRULENCE FACTORS AND IMMUNORELEVANT ANTIGENES OF AETIOLOGICAL MICRORGANISMS The present work aimed at investigating the prevaleance of ovine mastitis in farms from Aletenjo and the identification of causative microrganisms, their virulence factors and immunorelevant epitopes. The preva lence of clinical and subclinical mastitis was 1.7% and 32.2%,respect ively. The most preva lent aet iologica l agent was Staphylococcus epidermidis (N=115); Staphylococcus aureus (N=27) and Streptococcus agalactiae (N=17) were also identified. The investigation of virulence factors allowed the identification of susceptibility patterns (N=404) and drug Minimal Inhibitory Concentrations (N=130). From 109 Staphylococcus epidermidis isolates; eight showed the ability to produce biofilm in vitro. The isolates studied adhered and were internalised by mammary epithelial cells (N=12). None of the five superantigens studied was detected (N=27). The protein profile of Staphylococcus epidermidis was determined, and the immunorelevant epitopes, recognised by blood and milk immunoglobulins, were identified. It was possible to detect a specific local immune response in infected animals.