Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by DGGE analysis of 16S rDNA
Konstantinov, S.R. ; Zhu Wei-Yun, ; Williams, B.A. ; Tamminga, S. ; Vos, W.M. de; Akkermans, A.D.L. - \ 2003
FEMS microbiology ecology 43 (2003)2. - ISSN 0168-6496 - p. 225 - 235.
gastrointestinal-tract - microbial ecology - rna - populations - diversity - pcr - dietary - swine - genes - identification
The effect of fermentable carbohydrates (sugar beet pulp and fructooligosaccharides) on the faecal bacterial communities of weaning piglets was analysed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in piglet faecal bacterial community structure were determined based on the Dice coefficients for pairwise comparison of the DGGE fingerprints and revealed significant changes in the faecal microbiota immediately after weaning. Piglets fed with fermentable carbohydrates showed a higher bacterial diversity and a more rapid stabilisation of the bacterial community compared with that of the animals fed with the control diet. Thirteen dominant DGGE bands were matched with sequences that showed 91-97% similarity to those derived from the Clostridium coccoides group and the Clostridium leptum subgroup. Amplicons related to Ruminococcus-like species were found in all DGGE fingerprints derived from pigs on the diet containing sugar beet pulp and fructooligosaccharides, but not in pigs on the control diet. These results indicate that these bacteria may play a role in the utilisation of dietary fibres. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions
Ramirez-Saad, H.C. - \ 1999
Agricultural University. Promotor(en): W.M. de Vos; A.D.L. Akkermans. - S.l. : Ramirez Saad - ISBN 9789058080660 - 119
bodembacteriën - frankia - microbiële ecologie - benzoaten - soil bacteria - frankia - microbial ecology - benzoates
Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.
In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .
Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).
The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).
The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .
The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.
The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.
To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).
The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).
In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.
Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.
The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.
The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.
In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.
In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.
|Detectie en ecologie van de bruinrot-bacterie: Recente resultaten van het onderzoek naar de detectie en ecologie van Ralstonia (Pseudomonas) solanacearum (ras 3), de veroorzaker van bruinrot in aardappel
Kastelein, P. ; Wolf, J.M. van der; Vuurde, J.W.L. van; Griep, R.A. ; Schots, A. ; Elsas, J.D. van - \ 1998
Gewasbescherming 29 (1998)2. - ISSN 0166-6495 - p. 39 - 41.
plantenziekten - plantenziekteverwekkende bacteriën - solanum tuberosum - aardappelen - microbiële ecologie - oogstschade - diagnostische technieken - pseudomonas - nederland - plant diseases - plant pathogenic bacteria - solanum tuberosum - potatoes - microbial ecology - crop damage - diagnostic techniques - pseudomonas - netherlands
|Ecologie en beheersing van bitterzoet in relatie tot bruinrot
Kempenaar, C. ; Groeneveld, R.M.W. ; Lotz, L.A.P. ; Wenneker, M. ; Janse, J.D. - \ 1998
Gewasbescherming 29 (1998)4. - ISSN 0166-6495 - p. 119 - 123.
plantenziekten - plantenziekteverwekkende bacteriën - solanum tuberosum - aardappelen - microbiële ecologie - oogstschade - diagnostische technieken - pseudomonas - nederland - plant diseases - plant pathogenic bacteria - potatoes - microbial ecology - crop damage - diagnostic techniques - netherlands
Bitterzoet wordt beschouwd als een belangrijke factor in de overleving van de bruinrotbacterie, veroorzaker van bruinrot in aardappel, in oppervlaktewater in Nederland. Onderzoek heeft geleid tot verbeterde inzichten in de ecologie en beheersmogelijkheden van bitterzoet langs watergangen
|Microorganisms in Foods 6: Microbial Ecology of Food Commodities.
Schothorst, M. van - \ 1997
London : Blackie Academic & Professional - ISBN 9780751404302 - 615
voedselindustrie - voedseltechnologie - micro-organismen - bacteriën - classificatie - taxonomie - dierlijke producten - bacteriologie - kiemgetal - voedselinspectie - microbiologie - voedingsmiddelen - voedselbewaring - groenteproducten - opslag - plantaardige producten - microbiële ecologie - methodologie - biologische technieken - experimenten - uitrusting - voedselmicrobiologie - voedselproducten - landbouwproducten - voedselbesmetting - toxische stoffen - xenobiotica - haccp - food industry - food technology - microorganisms - bacteria - classification - taxonomy - animal products - bacteriology - bacterial count - food inspection - microbiology - foods - food preservation - vegetable products - storage - plant products - microbial ecology - methodology - biological techniques - experiments - equipment - food microbiology - food products - agricultural products - food contamination - toxic substances - xenobiotics - haccp
|HACCP in de luchtvaartcatering.
Beumer, R.R. ; Vrouwenvelder, Th. ; Brinkman, E. - \ 1994
Voedingsmiddelentechnologie 27 (1994)10. - ISSN 0042-7934 - p. 13 - 16.
luchttransport - luchtvaartuig - dierlijke producten - kiemgetal - bacteriologie - controle - gemaksvoedsel - engineering - voedselinspectie - voedselmicrobiologie - voedselbewaring - voedingsmiddelen - bedrijfsvoering - microbiële ecologie - monitoring - voorgekookt voedsel - voorbereide voedingsmiddelen - ruimtevlucht - statistiek - kwaliteitscontroles - queuing theory - haccp - air transport - aircraft - animal products - bacterial count - bacteriology - control - convenience foods - engineering - food inspection - food microbiology - food preservation - foods - management - microbial ecology - monitoring - precooked foods - prepared foods - space flight - statistics - quality controls - queuing theory - haccp
16S rRNA as molecular marker in ecology of Frankia
Hahn, D. - \ 1990
Agricultural University. Promotor(en): A.J.B. Zehnder; A.D.L. Akkermans. - S.l. : Hahn - 126
frankia - stikstofbindende bacteriën - symbiose - rhizobium - betulaceae - microbiële ecologie - frankia - nitrogen fixing bacteria - symbiosis - rhizobium - betulaceae - microbial ecology
The research described in this thesis focusses on the role of biotic factors encountered with the establishment of the symbiosis between black alder plants ( Alnus glutinosa ) and introduced Frankia strains. A selection of plant clones and Frankia strains that gave optimal nodulation and nitrogen fixation in forestry was made. For this reason nodulation tests with increasing complexity were set up. An attempt was made to investigate whether introduced strains behaved differently on plants grown under axenic and non-axenic conditions. Since Frankia strains were difficult to identify by conventional techniques, special attention was given to the development of new molecular techniques for identification of the strains at the nucleic acid level.
Initially, plant material of two physiologically different ecotypes of Alnus glutinosa, the forest ecotype "Bentheim" and the pioneer ecotype "Weerribben", respectively, was selected. Using tissue culture techniques plant material of both ecotypes was cloned in order to obtain genetically identical plants (Chapter 2). These micropropagated plants were used to set up a standardized inoculation system under axenic conditions in order to study the genetically determined nodulation ability and nitrogen-fixing capacity of Frankia strains and to select superior Frankia strains as source of inoculum (Chapter 3). The usefulness of selected Frankia strains as inoculum was further tested under more practical conditions, in perlite as model environment for nitrogen-limited conditions and in two soils, representing natural environments with different nutritional factors and different microbial populations. The results of the inoculation tests under axenic conditions were confirmed by studies under greenhouse conditions.
The performance of the symbiosis was effected by many variables, e.g. the plant genotype, the Frankia strain and environmental conditions. The influence of the environmental conditions became more pronounced when plants were grown on either a sandy loam ("Bentheim") or a peat ("Weerribben") soil and inoculated with Frankia strains. Plant growth was positively influenced, e.g. by mycorrhizal fungi in "Bentheim" soil, or negatively influenced, e.g. by oomycetes in "Weerribben" soil. The effects of inoculation with Frankia on plant growth remained minimal. The establishment of the introduced Frankia strain was also dependent on the soil conditions. The introduced spore(-) Frankia strain was only able to compete with the natural spore(+) population of the "Weerribben" soil. Introduction of this strain to "Bentheim" soil did not show any establishment of the introduced strain. In contrast to the sandy loam of "Bentheim" which was rich in nutrients, the peat of "Weerribben" was a representative of poor soils. It could therefore be used for feasability studies in inoculation programmes. The use of pure cultures of Frankia as inoculum instead of soil or crushed nodules, has the advantage to prevent the contamination of the plant with root pathogens. Pure cultures did not result in a better symbiosis.
Atypical Frankia strains
Screening of several isolates obtained from nodules of both alder ecotypes indicated the existence of atypical, ineffective Frankia strains. The alder clones used showed variable resistance against infection of the ineffective strains (Chapter 4). When compared with growth after the addition of a single strain dual inoculation of typical, effective Frankia strains and an ineffective Frankia strain to both alder clones showed growth increment of the plants (Chapter 5). The growth enhancing effect of the ineffective Frankia strain was not paralleled by increased number of nodules. Nothing is known yet about the growth stimulation by atypical Frankia strains. The results indicate that simultaneous inoculation of different Frankia strains to Alnus plants can be profitable for the host plant.
Because the ineffective Frankia strains lacked morphological and physiological characteristics of typical Frankia strains and because nodule formation on actinorhizal plants might be reduced or even absent, detection of the ineffective strains and studies on their competitive abilities were quite difficult. Reliable markers which could be used to detect both types of Frankia in nodules and in soil without reisolation had not been available at that moment. An attempt was made to find specific markers in a molecule which was commonly used to unravel evolutionary relationships: the 16S ribosomal RNA. New sequencing techniques allowed the rapid determination of total or almost total 16S rRNA sequences. Total 16S rRNA sequences indicated the presence of conserved and variable regions. Conserved regions had been used to investigate quantitative evolutionary relationships amoung bacteria. The conserved regions of the total 16S rRNA sequence of the effective Frankia strain Ag45/Mut15 were compared with aligned sequences of other actinomycetes and used to determine the position of the family Frankiaceae in the phylogenetic tree of the actinomycetes (Chapter 6).
Analyses of variable regions of 16S rRNA of closely related organisms indicated sufficient variation, despite the fact that DNA/DNA homology studies suggested these two species might actually be one and the same. Large differences in DNA/DNA homology studies of Frankia which were also obtained between strains of one compatibility group suggested chances on large variation within the variable regions of different strains. Sequence analyses of variable regions of 16S rRNA of two ineffective Frankia strains (i.e. AgB1.9 and AgW1.1) and the effective strain Ag45/Mut1 5, all belonging to the Alnus -compatibility group, showed large differences in base composition. These sequences were used to design complementary synthetic oligonucleotides that could act as specific probes in hybridization experiments. The specificity of these probes was shown in hybridization experiments against immobilized rRNA from 23 Frankia strains belonging to different compatibiliy groups and of several related soil actinomycetes. The probes were able to distinguish between Nif +and Nif -strains, between several Nif -strains and between several Alnus compatible Nif +strains and strain AgKG'84/4 also belonging to the Alnus -compatibility group (Chapter 7). Strong strain specific sequences, however, were not obtained. The design of oligonucleotide probes opens up the possibility to investigate competitive abilities of selected strains under defined conditions, e.g. in model systems with perlite and defined Frankia strains. The question whether competition studies under these controlled conditions are ecologically relevant needs further investigations because little basic knowledge on Frankia population dynamics is yet available. The application of probes to identify introduced strains in soil remains restricted, due to the low specificity for strains. Up to now we are not able to design reliable strain specific probes that can be used to follow the establishment of introduced Frankia strains in natural environments. A much more . promising application of probes towards rRNA is concerned with the development of a genus-specific oligonucleoticle probe against Frankia (Chapter 9) that theoretically enables quantitative detection of total Frankia populations.
The application of oligonucleoticle probes in the detection of specific Frankia strains does not only depend on specificity of the probes but also on the development of a reliable isolation method for target sequences. Ribosomal RNA is preferable to DNA as target because of its relative abundance in large amounts in metabolically active cells. Actinorhizal nodules represent enrichments of Frankia, which are metabolically highly active and consequently contain large amounts of Frankia RNA. Our investigations resulted in the development of a rapid RNA extraction method that was sensitive enough to investigate strain composition also from very small nodules or lobes (Chapter 8). The detection of target sequences, however, remained limited by the design of specific probes and the ratios of different target sequences in one sample. For reliable signal expression in hybridization experiments quite similar amounts of target sequences per sample were needed.
So far, the usefulness of rRNA sequences as targets for oligonucleoticle probes was only shown in combination with pure cultures of Frankia (Chapter 7) or in metabolically highly active enrichments, e.g. nodules (Chapter 8). Terrestrial environments like soil contain populations of many different microorganisms. These populations normally grow under suboptimal nutrition conditions. Bacteria adapt to these conditions by forming special starvation cells, which are metabolically inactive and contain only low amounts of rRNA. The starvation respons often results in viable, but non-culturable populations. The recalcitrant character of Frankia, which are difficult to isolate, makes it a useful model microorganism of soil bacteria. The application of oligonucleoticle probes for detection of Frankia in soil depends on the development of an extraction method for RNA. RNA directly isolated from soil as target for Frankia specific oligonucleoticle probes was useful in detection of Frankia (Chapter 9). Quantification of the obtained signals, however, is still unreliable because Frankia is a hyphae forming organism. It is also quite difficult to correlate cell numbers (theoretical estimation) to the amount of RNA. The concentration of these molecules in an organism is a function of the activity of the individual cell. Quantification of hybridization signals therefore depends on the availability of basic information of the metabolic activity of Frankia cells in soil. This information, however, is very difficult to obtain for recalcitrant microorganisms like Frankia. It is much easier for other microorganisms, e.g. for Streptomyces . Streptomyces spores are quite easy to isolate from soil and the establishment of Streptomyces cells, i.e. as spores or as mycelium in soil, is well studied. Quantification based on hybridization signals must be possible when this basic knowledge is available. In case of Frankia methods that enable quantification must still be developed. Similar to Streptomyces these quantification methods for Frankia can be of direct character, e.g. quantitative extraction of spores, or of indirect character, e.g. determination of mycelium by phage counts.
The development of rapid and sensitive methods to detect Frankia on the basis of rRNA sequences opens up new ways to study other recalcitrant microorganisms in the environment. This molecular approach in microbial ecology can definitely further be explored when the advantages of rRNA as stable target and the rapid extraction of RNA from soil can be combined with in vitro amplification methods commonly used with DNA or mRNA. Promising approaches can also be expected in in situ studies using hybridization signal intensity of fluorescent dye labelled oligonucleotides and the amount of rRNA as criterium for bacterial activity.