Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria

Aims:  The ability of lactic acid bacteria (LAB) to bind fumonisins B₁ and B₂ (FB₁, FB₂) in fermented foods and feeds and in the gastrointestinal tract could contribute to decrease their bioavailability and toxic effects on farm animals and humans. The aim of this work was to identify the bacterial cell wall component(s) and the functional group(s) of FB involved in the LAB–FB interaction.
Methods and Results:  The effect of physicochemical, enzymatic and genetic treatments of bacteria and the removal/inactivation of the functional groups of FB on toxin binding were evaluated. Treatments affecting the bacterial wall polysaccharides, lipids and proteins increased binding, while those degrading peptidoglycan (PG) partially decreased it. In addition, purified PG from Gram-positive bacteria bound FB in a manner analogue to that of intact LAB. For FB, tricarballylic acid (TCA) chains play a significant role in binding as hydrolysed FB had less affinity for LAB.
Conclusions:  Peptidoglycan and TCA are important components of LAB and FB, respectively, involved in the binding interaction.
Significance and Impact of the Study:  Lactic acid bacteria binding efficiency seems related to the peptide moiety structure of the PG. This information can be used to select probiotics with increased FB binding efficiency.     

Degradation of linear alkylbenzene sulfonate by a bacterial consortium isolated from the aquatic environment of Argentina

Aims:  To isolate and identify linear alkylbenzene sulfonate (LAS)-degrading bacteria from Río de la Plata and adjacent waters, and to assay their degradation capability as a consortium and as single organisms.
Methods and Results:  A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO₂ release (mineralization) than individual cultures, and degraded 86% of LAS (20 mg l⁻¹), whereas pure strains degraded between 21% and 60%. Bacterial desulfonation from LAS was evidenced in the consortium and A. caviae strains. A complete disappearance of LAS (10 mg l⁻¹) was accomplished, and LAS levels of 50 and 100 mg l⁻¹ led to a pronounced decrease in the biodegradation extent and inhibition of culture growth.
Conclusions:  A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study:  The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system

Aims:  To evaluate whether slime-exopolysaccharides (EPS) or capsular-polysaccharide (CPS) production could protect the polymer-producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied.
Methods and Results:  An in vitro GS model containing human saliva and gastric juice was standardized. Polymer functionality on the cell viability and metabolic activity of the EPS-producing strains in the GS acidic conditions was evaluated. Two isogenic EPS/CPS deficient mutants were used for comparison. EPS or CPS conferred no significant protection on the cell viability of the studied strains after passage through the GS conditions. However, the phospho- and β-galactosidase activities of the EPS⁺ strains were higher than those of the EPS⁻. Cytoplasmic alterations in the wild-type and mutant strains and partial degradation of both EPS were detected.
Conclusions:  The presence of EPS/CPS protected the metabolic activity of the assayed LAB strains, but had no effect on survival at low pH.
Significance and Impact of the Study:  The presence of EPS/CPS as well as polymer resistance to the harsh conditions of the human GS could impact positively in probiotic strains to exert their properties in the host.     

Maturing dynamics of surface microflora in Fontina PDO cheese studied by culture-dependent and -independent methods

Aims:  To study the evolution of rind microbial communities in Fontina PDO cheese.
Methods and Results:  Four batches were examined for their surface microflora during ripening, carried out in two different maturing caves, at Ollomont and Pré-Saint-Didier, Aosta Valley region, Northwest of Italy. Culture-dependent methodologies were combined with culture-independent analysis (PCR-DGGE). Yeasts were found to increase from 10³ to 10⁶ CFU cm⁻² in 28 days, with consequent rise of surface pH, which allowed the growth of salt-tolerant bacteria, in particular coryneforms which reached 10⁹ CFU cm⁻² at the end of 3 months. Coagulase-negative cocci and lactic acid bacteria reached 10⁷ CFU cm⁻² in the same period. Debaryomyces hansenii and Candida sake were the species more constantly present throughout the whole maturing process. As early as after 1 day since manufacture, Lactococcus lactis subsp. lactis and Streptococcus thermophilus were detected on cheese rinds. Arthrobacter nicotianae , Brevibacterium casei and Corynebacterium glutamicum were found after 7–28 days .
Conclusions:  According to cluster analysis of DGGE profiles, the maturing environment seemed to influence the dynamics of microbial groups on Fontina surfaces.
Significance and Impact of the Study:  These results represent a first picture of micro-organisms colonizing Fontina PDO rinds. Further studies are in progress to better understand the origin of this surface microflora and to formulate surface starters.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Breakdown of tussock grass in streams along a gradient of agricultural development in New Zealand

Summary 1. We measured the breakdown rate of tussock grass in 12 New Zealand streams in catchments that provided a gradient of agricultural development. We also examined the microbial and invertebrate communities associated with decomposing tussock litter.
2. Pristine streams in the study had low concentrations of dissolved inorganic nitrogen (<10  μ g L⁻¹) and dissolved reactive phosphate (<3  μ g L⁻¹), whereas streams in the most developed catchments had high concentrations of nitrate (>2500  μ g L⁻¹) and phosphate (35  μ g L⁻¹), as well as greater amounts of suspended sediment and fine sediment covering the streambed.
3. Breakdown rate and microbial respiration were significantly related across the sites, and both were positively related to concentrations of nitrate and phosphate. Fungal biomass, measured as ergosterol, was positively related to microbial respiration and was also higher at sites with higher concentrations of nutrients. Total and shredding invertebrates were most abundant at the sites with high nutrient concentrations, but abundance of shredding invertebrates was not significantly related to breakdown rate. Amphipods were the most common shredding invertebrate at most sites, but probably did not contribute greatly to high rates of breakdown in streams in agricultural catchments.
4. With the exception of one site, nutrients from agricultural development appeared to have larger positive effects on litter breakdown than negative effects from sedimentation. Litter breakdown can serve as a functional measure of ecosystem health in streams, but caution should be exercised when a stress, such as land use, can have both positive (nutrients) and negative (sedimentation) effects.     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Selection and characterization of aerobic bacteria capable of degrading commercial mixtures of low-ethoxylated nonylphenols

Aims:  Isolation and characterization of new bacterial strains capable of degrading nonylphenol ethoxylates (NP n EO) with a low ethoxylation degree, which are particularly recalcitrant to biodegradation.
Methods and Results:  Seven aerobic bacterial strains were isolated from activated sludges derived from an Italian plant receiving NP n EO-contaminated wastewaters after enrichment with a low-ethoxylated NP n EO mixture. On the basis of 16S rDNA sequence, the strains were positioned into five genera: Ochrobactrum , Castellaniella , Variovorax , Pseudomonas and Psychrobacter . Their degradation capabilities have been evaluated on two commercial mixtures, i.e. Igepal CO-210 and Igepal CO-520, the former rich in low ethoxylated congeners and the latter containing a broader spectrum of NP n EO, and on 4- n -nonylphenol (NP). The strains degraded Igepal CO-210, Igepal CO-520 and 4- n -NP all applied at the initial concentration of 100 mg l⁻¹, by 35–75%, 35–90% and 15–25%, respectively, after 25 days of incubation.
Conclusions:  Some of the isolated strains, in particular the Pseudomonas strains BCb12/1 and BCb12/3, showed interesting degradation capabilities towards low ethoxylated NP n EO congeners maintaining high cell vitality.
Significance and Impact of the Study:  Increased knowledge of bacteria involved in NP n EO degradation and the possibility of using the isolated strains in tailored process for a tertiary biological treatment of effluents of wastewater treatment plants.     

13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation

The influence of microbial degradation on the ¹³C/¹²C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors αC for toluene degradation by Pseudomonas putida (1.0026 ± 0.00017), Thauera aromatica (1.0017 ± 0.00015), Geobacter metallireducens (1.0018 ± 0.00029) and the sulphate-reducing strain TRM1 (1.0017 ± 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar ¹³C/¹²C isotope fractionation factor (αC = 1.0015 ± 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil–hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a ¹³C/¹²C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

Enhanced biotransformation of TCE using plant terpenoids in contaminated groundwater

Aims:  To examine plant terpenoids as inducers of TCE (trichloroethylene) biotransformation by an indigenous microbial community originating from a plume of TCE-contaminated groundwater.
Methods and Results:  One-litre microcosms of groundwater were spiked with 100 μmol 1⁻¹ of TCE and amended weekly for 16 weeks with 20 μl 1⁻¹ of the following plant monoterpenes: linalool, pulegone, R-(+) carvone, S-(−) carvone, farnesol, cumene. Yeast extract-amended and unamended control treatments were also prepared. The addition of R-carvone and S-carvone, linalool and cumene resulted in the biotransformation of upwards of 88% of the TCE, significantly more than the unamendment control (61%). The aforementioned group of terpenes also significantly ( P  < 0·05) allowed more TCE to be degraded than the remaining two terpenes (farnesol and pulegone), and the yeast extract treatment which biotransformed 74–75% of the TCE. The microbial community profile was monitored by denaturing gradient gel electrophoresis and demonstrated much greater similarities between the microbial communities in terpene-amended treatments than in the yeast extract or unamended controls.
Conclusions:  TCE biotransformation can be significantly enhanced through the addition of selected plant terpenoids.
Significance and Impact of the Study:  Plant terpenoid and nutrient supplementation to groundwater might provide an environmentally benign means of enhancing the rate of in situ TCE bioremediation.     

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

Populations of heavy fuel oil-degrading marine microbial community in presence of oil sorbent materials

Aims:  To investigate the feasibility of applying sorbent material X-Oil^® in marine oil spill mitigation and to survey the interactions of oil, bacteria and sorbent.
Methods and Results:  In a series of microcosms, 25 different treatments including nutrient amendment, bioaugmentation with Alcanivorax borkumensis and application of sorbent were tested. Microbial community dynamics were analysed by DNA fingerprinting methods, RISA and DGGE. Results of this study showed that the microbial communities in microcosms with highly active biodegradation were strongly selected in favour of A. borkumensis . Oxygen consumption measurements in microcosms and gas chromatography of oil samples indicated the fast and intense depletion of linear alkanes as well as high oxygen consumption within 1 week followed by consequent slower degradation of branched and polyaromatic hydrocarbons.
Conclusion:  Under given conditions, A. borkumensis was an essential organism for biodegradation, dominating the biofilm microbial community formation and was the reason of emulsification.
Significance and Impact of the Study:  This study strongly emphasizes the pivotal importance of A. borkumensis as an essential organism in the initial steps of marine hydrocarbon degradation. Interaction with the sorbent material X-Oil^® proved to be neutral to beneficial for biodegradation and also promoted the growth of yet unknown micro-organisms.     

Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos

Aim:  To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos.
Methods and Results:  A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0·01 g l⁻¹). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0·2 g l⁻¹, was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0·01 g l⁻¹) by ChlD strain. The best degradation efficiency was observed at 0·1 g l⁻¹ supplement of biosurfactant, as validated by GC and HPLC studies.
Conclusion:  The addition of biosurfactant at 0·1 g l⁻¹ resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation.
Significance and Impact of the Study:  This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.     

Efficient method to isolate and purify viruses of bacteria from marine environments

Aim:  To isolate viruses of specific heterotrophic bacterial strains from marine environments using a host addition/virus amplification protocol (HAVAP) for use in phage/host systems.
Methods and Results:  Bacteria-free seawater samples containing natural viruses assemblages were inoculated with a single laboratory grown bacterial host of interest in a nutrient-enriched [peptone, Fe(III) and yeast extract] seawater suspension. These conditions enhanced the replication of only those virus(s) capable of infecting the host bacterium. After incubation, free viruses were recovered at concentrations ranging 10⁵–10¹⁰ infectious virus particles per ml of seawater. Using this approach, 15 viruses were isolated and represented 12 unique phage/host systems. Two of the hosts tested were infected by more than one virus.
Conclusions:  Isolation of high concentrations of specific viruses is possible even if their initial concentrations in native waters are low. This approach allows the recovery of phage/host systems that may not be numerically dominant.
Significance and Impact of the Study:  This host enrichment protocol for virus detection and isolation is well-suited for aquatic viral ecology studies that require phage/host systems.     

Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres

Aims:  To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes.
Methods and Results:  DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS^®-PLUS; DNeasy^®; FTA^® cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA^® cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs.
Conclusions:  ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study.
Significance and Impact of the Study:  This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi.