Virus elimination by meristem-tip culture from a range of Alstroemeria cultivars

A technique for virus elimination by meristem culture was developed for a range ofAlstroemeria cultivars. Meristems were excised from the rhizome tips and placed on a medium with indole-3-butyric acid, the required concentration of which was cultivar dependent: Six to eight weeks after dissection a shoot had formed which was transferred to a medium without growth regulators. On filter paper bridges, in a liquid medium, root formation was better than on a solid medium. In many cases a new rhizome developed. If not, the plantlet eventually died. Transfer into soil was more successful with the plantlets rooted in liquid medium than with those rooted in solid medium. Virus elimination was cultivar dependent, but in most cultivars plants resulting negatively in serological tests could be obtained. After repeated testing and selection for horticultural properties these plants may be used to start high quality mother plots.Samenvatting Een techniek werd ontwikkeld om door middel van meristeemcultuur virus te elimineren uit een aantalAlstroemeria cultivars. De meristemen werden uit de toppen van de rhizomen geprepareerd en op een voedingsbodem met IBA als auxine geplaatst. De IBA-concentratie nodig voor scheutvorming was afhankelijk van de cultivar. Na een periode van zes tot acht weken had het meristeem zich ontwikkeld tot een scheutje, dat vervolgens werd overgebracht op een voedingsbodem zonder groeistoffen. In ongeveer twee maanden vormden zich dan wortels. Deze wortelvorming was beter op een vloeibare voedingsbodem met papieren bruggetjes dan op een vaste voedingsbodem van agar. In veel, maar niet in alle gevallen, vormde zich ook een nieuw rhizoom. Indien geen rhizoom werd gevormd stierf de plant. Gewortelde plantjes groeiden beter in grond indien de beworteling op papieren bruggetjes had plaatsgevonden. Het succes van het elimineren van hetAlstroemeria-mozaïek virus hing af van de cultivar. Na herhaalde toetsing kunnen de negatief reagerende planten worden gebruikt voor de opbouw van een partij gezonde moederplanten. Op deze manier kan de kwaliteit van het uitgangsmateriaal vanAlstroemeria worden verbeterd.     

The musk rat (Ondatra zibethicus) as intermediate host of cestodes in the Netherlands

An investigation on the presence of larval cestodes in musk rats (Ondatra zibethicus) was carried out in two regions of the Netherlands (east Groningen and south Limburg) where in a earlier study foxes with Echinococcus multilocularis were found. A total of 1726 musk rats were dissected (1200 in Groningen, 526 in Limburg). Larval stages of Taenia taeniaeformis were most frequently found (total 44.8%: Groningen 42%, Limburg 51.3%), followed by T. martis (total 6.1%: Groningen 0.7%, Limburg 18.6%). Infections with T. crassiceps (total 0.3%: Groningen 0%, Limburg 1.0%), T. polyacantha (total 0.2%; Groningen 0.3%, Limburg 0%) and E. multilocularis (0.1%: Groningen 0.1%, Limburg 0%) were rare. Infections with T. taeniaeformis were more frequent in adults (71.8%) than in juveniles (34.2%). The same was found for T. martis: adults 15.3%, juveniles 2.5%. This difference was also reflected in the relation between weight of the animals and presence of infection. Heavier animals (>1000 g) were more often infected with T. taeniaeformis (74.1%) than animals less than 1000 g (34.8%). In musk rats weighing less than 500 g (n=155) only 5.2% were infected, but above 1200 g, 82.6%. The highest number of T. taeniaeformis was 28, of T. martis 13, of T. crassiceps >1000 and of T. polyacantha 24. The E. multilocularis was in a very young stage, a few white spots in the liver. Although E. multilocularis infections were exceptional, it is expected that with a rise in the number of infected foxes in the Netherlands the number of infected musk rats will increase.     

Trend analysis of Trichinella in a red fox population from a low endemic area using a validated artificial digestion and sequential sieving technique

Freezing of fox carcasses to minimize professional hazard of infection with Echinococcus multilocularis is recommended in endemic areas, but this could influence the detection of Trichinella larvae in the same host species. A method based on artificial digestion of frozen fox muscle, combined with larva isolation by a sequential sieving method (SSM), was validated using naturally infected foxes from Latvia. The validated SSM was used to detect dead Trichinella muscle larvae (ML) in frozen muscle samples of 369 red foxes from the Netherlands, of which one fox was positive (0.067 larvae per gram). This result was compared with historical Trichinella findings in Dutch red foxes. Molecular analysis using 5S PCR showed that both T. britovi and T. nativa were present in the Latvian foxes, without mixed infections. Of 96 non-frozen T. britovi ML, 94% was successfully sequenced, whereas this was the case for only 8.3% of 72 frozen T. britovi ML. The single Trichinella sp. larva that was recovered from the positive Dutch fox did not yield PCR product, probably due to severe freeze-damage. In conclusion, the SSM presented in this study is a fast and effective method to detect dead Trichinella larvae in frozen meat. We showed that the Trichinella prevalence in Dutch red fox was 0.27% (95% CI 0.065-1.5%), in contrast to 3.9% in the same study area fifteen years ago. Moreover, this study demonstrated that the efficacy of 5S PCR for identification of Trichinella britovi single larvae from frozen meat is not more than 8.3%.     

Biological control of Rhizoctonia root rot on bean by phenazine- and cyclic lipopeptide-producing Pseudomonas CMR12a

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.     

Molecular Monitoring of SRB Community Structure and Dynamics in Batch Experiments to Examine the Applicability of in situ Precipitation of Heavy Metals for Groundwater Remediation (15 pp)

Background, Aims and Scope   Sulfate-reducing bacteria (SRB) are known for their capacity to reduce and precipitate heavy metals (HM) as metal sulfides, offering the opportunity to create an in situ reactive zone for the treatment of heavy metal-contaminated groundwater, a process called in situ metal precipitation (ISMP). The applicability of the ISMP technology first has to be investigated at a laboratory scale before going into an on site application. The evaluation and optimization of the ISMP process is facilitated when physical/chemical analysis techniques are combined with molecular tools that specifically monitor the abundance, diversity and dynamics of the indigenous sulfate reducing microbial community. In this study, batch experiments were conducted in order to investigate the feasibility of ISMP as a groundwater remediation strategy for an industrial site contaminated with elevated levels of Zn, Cd, Co and Ni. Methods   The potential of different types of carbon source/ electron donor (lactate, acetate, methanol, ethanol, Hydrogen Release Compound?, molasses) to stimulate the sulfate reduction and metal precipitation activity of the naturally present (or indigenous) SRB community was explored. In addition, the effect of amending vitamin B12 and yeast extract was evaluated. The ISMP process was monitored by combining analytical analyzes of process parameters (SO42-concentration, heavy metal concentrations, pH, Eh) with molecular tools such as SRB subgroup and genus specific PCR, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis of clone sequences, based on either the 16S rRNA or the dsr (dissimilatory sulfite reductase) gene. Results and Discussion   The efficiency of different carbon-sources to stimulate the ISMP process followed the order HRC 〉 molasses 〉 methanol 〉 lactate 〉 ethanol 〉 acetate. Within 10 weeks, the highest sulfate and metal removal efficiencies ranged from 85% to 99%. Addition of yeast extract boosted the ISMP process, whereas vitamin B12 negligibly affected SRB activity. Analysis of the sulfate reducing population by SRB subgroup and genus specific PCR demonstrated that members of the genus Desulfosporosinus dominated in all batch tests, while 16S rDNA DGGE profiles additionally revealed the presence in the microbial communities of non-sulfate reducing bacteria within the family Clostridium and the -proteobacteria. The dsrB-based DGGE profiles allowed us to assess the diversity and dynamics of the sulfate reducing community and added to a better understanding of the effects of different batch conditions on the ISMP process. Remarkably, all dsrB sequences affiliated with the dsrB gene sequence cluster found in Desulfotomaculum, which received their xenologous dsrB gene from the -proteobacteria. Conclusions   The batch experiments, which aimed at stimulating the activities of the indigenous SRB communities, demonstrated that these communities were present and that their activities could be used to obtain efficient in situ precipitation of the contaminating heavy metals. This opens the possibility to test this concept in the future as an on site demonstration as part of the groundwater strategy for the heavy metal contaminated site. Although batch setups are suitable for preliminary feasibility studies for ISMP, they do not reflect the in situ situation where sulfate and heavy metal and metalloid polluted groundwater are supplied continuously. A sulfate reducing strain JG32A was isolated from whose 16S rRNA gene affiliated with the genus Desulfosporosinus, while its dsrB gene sequence clustered with Desulfotomaculum dsrB gene sequences, which received their xenologous dsr genes from -proteobacteria. Therefore we hypothesize that the batch experiments enrich members of the Desulfosporosinus genus that possess a non-orthologous dsrB gene. Recommendation and Perspective   The next step towards an on site pilot test for ISMP will be the setup of a series of column experiments, with process conditions that are selected based on the above mentioned results. This will allow to define optimal ISMP process conditions and to test its long-term efficacy and sustainability before going into an on site bioremediation application. By applying the described molecular tools together with physical-chemical analyzes, it can be investigated whether the same SRB community is enriched and which type of C-source is most effective in promoting and sustaining its growth and sulfate-reduction activity.     

Variation in two Begonia × hiemalis clones after in vitro propagation

In order to study the variation of Begonia × hiemalis after in vitro propagation, one plant of ‘Aphrodite Pink’ and one of ‘Schwabenland Red’ were vegetatively propagated in four ways. One group of plants was obtained by propagation by means of cuttings. For the second group, the bacterial elimination system, as developed by Hakkaart and Versluijs (1983), was applied which includes an in vitro propagation step. The third and fourth groups were obtained by adding one and two cycles, respectively, of in vitro propagation after the bacterial elimination, so that one, two and three propagation cycles could be compared. One cycle, followed by bacterial elimination, gave nearly uniform offspring. However, when this was followed by one or two cycles of in vitro propagation, the variation increased. Variation also increased when the size of the plantlets obtained in vitro decreased. Thus, an important source of variation can be avoided by eliminating the smallest in vitro plantlets. It is recommended to flower and select the plants before propagation by conventional cuttings, whether or not a further in vitro propagation is applied after the bacterial elimination.     

Some factors affecting glassiness in carnation meristem tip cultures

A study was made of the effect of agar concentration on glassiness during meristem tip culture of carnation. Increasing the agar concentration from the usual 6 g/l to 12 g/l decreased glassiness, but at the same time reduced plant growth. A concentration of 8 or 10 g/l reconciled these conflicting effects. The type of closure of the tubes was also found to affect glassiness, the looser types (cotton wool, metal caps, steri stops) being better than the tighter ones (aluminium foil and parafilm). In the first days after excision of the meristem tip a period existed in which a rather high temperature of 26° C favoured the development of normal plants later. The positive response to high temperature was around 3 days after excision.