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Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

Zeiller M, Rothballer M, Iwobi AN, Böhnel H, Gessler F, Hartmann A, Schmid M - Front Microbiol (2015)

Bottom Line: In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect.To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied.Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

View Article: PubMed Central - PubMed

Affiliation: Research Unit Microbe-Plant Interactions, Department for Environmental Sciences, German Research Center for Environmental Health - Helmholtz Zentrum München Neuherberg, Germany.

ABSTRACT
In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

No MeSH data available.


Related in: MedlinePlus

Polymerase chain reaction Detection of Clostridia in shoots of inoculated plants. (A,B) PCR with 10 ng of DNA isolated from shoot after growth in soil. (A) PCR with P930/P932 (665 bp, 16S-rDNA). (B) PCR with F2/R2 (462 bp, BoNT D) and F4/R4 (128 bp, BoNT D).
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Figure 7: Polymerase chain reaction Detection of Clostridia in shoots of inoculated plants. (A,B) PCR with 10 ng of DNA isolated from shoot after growth in soil. (A) PCR with P930/P932 (665 bp, 16S-rDNA). (B) PCR with F2/R2 (462 bp, BoNT D) and F4/R4 (128 bp, BoNT D).

Mentions: This approach allowed a qualitative but no quantitative detection. By the use of a PCR system with DNA isolated from enrichment cultures of inoculated plants, C. botulinum strain 2301 could be detected sensitively in roots (r) and in shoots (sh) of clover grown for 4 weeks in an axenic system (Figure 6), as well as in the shoots of clover after 4 weeks of growth in a soil system (Figure 7). Identical results for the detection of C. botulinum strain 2301 were achieved with both primer sets.


Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

Zeiller M, Rothballer M, Iwobi AN, Böhnel H, Gessler F, Hartmann A, Schmid M - Front Microbiol (2015)

Polymerase chain reaction Detection of Clostridia in shoots of inoculated plants. (A,B) PCR with 10 ng of DNA isolated from shoot after growth in soil. (A) PCR with P930/P932 (665 bp, 16S-rDNA). (B) PCR with F2/R2 (462 bp, BoNT D) and F4/R4 (128 bp, BoNT D).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4628109&req=5

Figure 7: Polymerase chain reaction Detection of Clostridia in shoots of inoculated plants. (A,B) PCR with 10 ng of DNA isolated from shoot after growth in soil. (A) PCR with P930/P932 (665 bp, 16S-rDNA). (B) PCR with F2/R2 (462 bp, BoNT D) and F4/R4 (128 bp, BoNT D).
Mentions: This approach allowed a qualitative but no quantitative detection. By the use of a PCR system with DNA isolated from enrichment cultures of inoculated plants, C. botulinum strain 2301 could be detected sensitively in roots (r) and in shoots (sh) of clover grown for 4 weeks in an axenic system (Figure 6), as well as in the shoots of clover after 4 weeks of growth in a soil system (Figure 7). Identical results for the detection of C. botulinum strain 2301 were achieved with both primer sets.

Bottom Line: In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect.To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied.Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

View Article: PubMed Central - PubMed

Affiliation: Research Unit Microbe-Plant Interactions, Department for Environmental Sciences, German Research Center for Environmental Health - Helmholtz Zentrum München Neuherberg, Germany.

ABSTRACT
In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

No MeSH data available.


Related in: MedlinePlus