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.

Characterization of Thiobacillus thioparus isolated from an activated sludge bioreactor used for hydrogen sulfide treatment.

AIMS: To compare Thiobacillus thioparus population dynamics in a control and a test activated sludge (AS) bioreactor, used for hydrogen sulfide (H(2)S) degradation. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to confirm the presence of T. thioparus, and real-time PCR was used to quantify the level of this bacterium in the AS samples. The DGGE analysis showed a band for T. thioparus in all samples, with the band being more prominent in the test sample with H(2)S diffusion. It also showed that although a change occurred in the diversity of the microbial population in the test sludge after 6 weeks of H(2)S diffusion, the microbial community structure of the test and control was still similar. Thiobacillus thioparus-specific PCR primers confirmed that 50% of the isolates from both the test and control bioreactors were T. thioparus. The thiobacilli population became more efficient at degrading the diffused H(2)S. This increase in efficiency was confirmed by a significant increase in the number of isolates from the test sludge compared with those from the control sludge, when they were grown in a thiosulfate-rich liquid medium. CONCLUSIONS: It was concluded that the use of AS process for H(2)S removal encourages the population of T. thioparus to increase even at times when the total biomass concentration shows a decrease. SIGNIFICANCE AND IMPACT OF THE STUDY: The research results give an insight into the dynamics of the microbial population in an AS pilot plant used in a dual role, to treat the wastewater and H(2)S.

Impact of the nematophagous fungus Pochonia chlamydosporia on nematode and microbial populations.

The microbial and nematode populations associated with two plants (tomato and cabbage) inoculated with the nematophagous fungus, Pochonia chlamydosporia var. chlamydosporia or root knot nematode (Meloidogyne incognita), or both, were compared with those in unplanted controls. The dominant factor affecting culturable microbial populations was found to be the presence or absence of tomato plants. Generally microbial colony counts were lowest in unplanted soil, small increases were associated with cabbage and significantly greater numbers with tomato plants. Differences in microbial diversity (estimated from community profiles of carbon substrate utlisation, using Biolog) were observed between planted and unplanted soils, however, there were few differences between soils with either of the two plants. The presence of P. chlamydosporia was associated with a reduction in the numbers of plant parasitic nematodes (51%-78%) including the migratory ectoparasites, whereas free-living nematodes, culturable bacteria and bacterial populations assessed by Biolog were unaffected by the application of fungus.

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

Giant cell arteritis-a systemic spectrum including temporal arteritis and polymyalgia rheumatica.

Polymyalgia rheumatica and giant cell arteritis are closely associated disorders that may pose serious threats to the elderly. While the etiological factors remain unknown, these clinical entities may represent different expressions of a common disorder. Recent evidence suggests that autoimmunological involvement of the internal elastic lamina of the large and medium-sized arteries might play an importent role in pathogenesis. Genetic and environmental factors may also contribute. Polymyalgia rheumatica and giant cell arteritis have subtle and protean manifestations and formes frustes occur, contributing to delays in diagnosis unless the clinician holds a high index of suspicion while treating the elderly. The sequelae, including generalized disability, blindness, myocardial and cerebrovascular catastrophies can probably be avoided by timely and prudent use of corticosteroids. These are not uncommon disorders are among the most amenable to treatment of the rheumatological diseases. Family physicians must be diligent in their efforts to diagnose these disorders since early generalized and focal signs and symptoms frequently precede the catastrophic events.