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Detection limits of PCR experiments. (A) PCR with IBTSB F2/IBTSB R2 (462 bp, BoNT D) and IBTSB F4/IBTSB R4 (128 bp, BoNT D): detection limit 5 pg; (B) PCR with DS11/DS22 (497 bp, BoNT D): detection limit 5 pg; (C) PCR with P930/P932 (665 bp, 16S-rDNA): detection limit 100 pg.
Source publication
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 cont...
Contexts in source publication
Context 1
... sensitivity of PCRs was similar to those reported previously ( Takeshi et al., 1996) and 5 pg of template DNA (approximately 1650 cells) could be detected by staining their amplified products on agarose gels. Figure 5 demonstrates the sensitivity and specificity of the detection method. ...
Context 2
... little as 5 pg of template DNA from toxigenic C. botulinum strain 2301 could be detected by the BoNT/CD multiplex PCR system ( Figure 5A) and primer pair DS11/DS22 (Figure 5B) respectively. The lowest amount of template DNA of type D, which produced observable products on agarose gels with primer pair P930/P932, was 100 pg (Figure 5C). ...
Context 3
... little as 5 pg of template DNA from toxigenic C. botulinum strain 2301 could be detected by the BoNT/CD multiplex PCR system ( Figure 5A) and primer pair DS11/DS22 (Figure 5B) respectively. The lowest amount of template DNA of type D, which produced observable products on agarose gels with primer pair P930/P932, was 100 pg (Figure 5C). ...
Context 4
... little as 5 pg of template DNA from toxigenic C. botulinum strain 2301 could be detected by the BoNT/CD multiplex PCR system ( Figure 5A) and primer pair DS11/DS22 (Figure 5B) respectively. The lowest amount of template DNA of type D, which produced observable products on agarose gels with primer pair P930/P932, was 100 pg (Figure 5C). The amplification products were sequenced to proof correct amplification, because database search showed, that some regions of plants chloroplasts and mitochondria have only one mismatch in the target region of the used primer. ...
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Citations
... Clostridium species (phylum Firmicutes) are gram-positive endospore-forming anaerobic bacteria that inhabit diverse environments including animal and human gut, sewage, lake sediments, and paddy soil. Previously, several growth-enhancing Clostridium species isolated from the root and rhizosphere of rice seedlings were classified as plant growth-promoting rhizobacteria (PGPR; Febri et al., 2014;Zeiller et al., 2015); however, some caused disease in potato and kiwifruit (Shabuer et al., 2015;Spigaglia et al., 2020). In agriculture, anaerobic soil disinfestation (ASD), a preplanting soil treatment method that creates anaerobic soil conditions, was conventionally utilized to suppress the growth of soil-borne pathogens such as Ralstonia solanacearum, Fusarium oxysporum, and Verticillium spp. ...
... С другой стороны, источником повышенного количества спор могут служить органические удобрения [35], [50]. ...
Выявлено, что большинство источников экологических ри-сков контаминации спорами Clostridium botulinum меда обусловлены хозяйственной деятельностью человека. Риски контаминации спорами Clostridium botulinum меда различаются в зависимости от района сбора меда и прямо или косвенно зависят от ряда факторов окружающей среды, связанных с сельскохозяйственной деятельностью человека в районе сбора меда:-отдельные виды животноводческой деятельности и использование на-воза в качестве органических удобрений;-применение некоторых видов пестицидов;-не покрытые растительностью участки почвы. Среди природных факторов можно выделить риск вспышек распростране-ния C. botulinum, связанный с повышением температуры воздуха. Третья группа факторов ветеринарно-гигиенические аспекты работы пчело-вода. Ключевые слова: Clostridium botulinum, экологические риски, контаминация меда, сельскохозяйственное воздействие на окружающую среду. Summary. It was revealed that most sources of environmental risks of contamination of honey with Clostridium botulinum spores are caused by human economic activities. The risks of contamination of honey with Clostridium botulinum spores vary depending on the honey collection area and depend directly or indirectly on a number of environmental factors associated with human agricultural activities in the honey collection area:-certain types of livestock farming and the use of manure as organic fertilizers;-use of certain types of pesticides;-areas of soil not covered with vegetation. Natural factors include the risk of outbreaks of C. botulinum associated with rising air temperatures. The third group of factors are veterinary and hygienic aspects of the beekeeper's work. Keywords: Clostridium botulinum, environmental risks, honey contamination, agricultural impact on the environment. Введение Б отулизм можно разделить, в зависимости от пути заражения, на шесть форм. Заболевание может быть естественным образом вызвано приемом предварительно образованного токсина с пищей (пи-щевой ботулизм), инфицированием раны Clostridium botulinum, что приводит к выработке токсина in situ (ра-невой ботулизм), колонизации кишечного тракта мла-денцев (детский ботулизм) и колонизации кишечного тракта взрослых или детей старше 1 года (кишечная ток-семия, ботулизм) [21]. В отношении меда, как источника заражения, име-ются сообщения о детском ботулизме [14], [23], [24], [44], [45], а также спорные сообщения о колонизации кишеч-ного тракта взрослых [27]. Описан случай раневого зара-жения младенца при наружном применении меда [28]. Детский ботулизм, вызываемый Clostridium botulinum-редкое заболевание, связанное с особен-ностями микрофлоры ребенка возрастом до 1 года (по некоторым данным до 6 месяцев). Тем не менее, за-болевание тяжелое, и каждый его случай отдельно ре-гистрируется. С 1976 года было зарегистрировано более 1500 случаев младенческого ботулизма в более чем 15 странах мира [31]. Установленные случаи детского бо-тулизма связаны с заражением из различных объектов окружающей среды, таких как: пыль, домашние живот-ные, загрязненные продукты питания, в частности мед. Наличие спор C. Botulinum в меде, по-видимому, след-ствие не только нарушения технологии получения, но и экологических факторов, связанных с хозяйственной де-ятельностью человека [2], [6], [17]. Пчелы (Apis mellifera) собирают нектар в радиусе до 4-5 км от пасеки, соответственно факторы окружаю-щей среды в районе сбора прямо или косвенно влияют на риск загрязнения меда. В нашей работе мы рассмотрим возможные эколо-гические риски, влияющие на вероятность загрязнения спорами C. botulinum меда. Мед не является средой для развития C. botulinum, но споры в нем длительное время
... A few Clostridium species have been demonstrated to provide the beneficial effects on plants through non-symbiotic nitrogen fixation, e.g., C. butyricum and C. pasteurianum, and antimicrobial production [12], e.g., C. beijerinckii [13]. Interestingly, the effective colonization of a toxigenic strain C. botulinum 2301 and a non-toxin-producing strain C. sporogenes 1739 on clover seedlings showed enhanced plant growth [14]. ...
Objectives:
Genus Clostridium sensu stricto is generally regarded as the true Clostridium genus, which includes important human and animal pathogens and industrially relevant microorganisms. Besides, it is also a prominent member of plant-associated endophytes. However, our knowledge of endophytic Clostridium is limited.
Methods:
In this study, the endophytes were isolated under anaerobic condition from the roots of Paris polyphylla Smith var. yunnanensis. Subsequently, a polyphasic taxonomic approach was used to clarify their taxonomic positions. The fermentation products were measured in the isolates with HPLC analysis. Comparative genomics was performed on these new strains and other relatives.
Results:
In total, nine endophytic strains belonging to the genus Clostridium sensu stricto were isolated, and three of them were identified as new species. Seven of nine strains could produce acetate, propionate, and butyrate. Only two strains could produce ethanol, although genomics analysis suggested that only two of them were without genes for solventogenesis. Different from the endophytic strains, the phylogenetically closely related non-endophytic strains showed significant enrichment effects on some metabolic pathways involving environmental information processing, carbohydrate, and amino acid metabolisms, etc. It suggests that the genomes of these endophytic strains had undergone subtle changes associated with environmental adaptations.
Conclusion:
Consequently, strains YIM B02505T, YIM B02515T, and YIM B02565T are proposed to represent a new species of the genus Clostridium sensu stricto, for which the names Clostridium yunnanense sp. nov., Clostridium rhizosphaerae sp. nov., and Clostridium paridis sp. nov. are suggested.
... Clostridium is an anaerobic gram-positive bacterium belonging to the phylum Firmicutes. Clostridium was known as PGPR of rice, clover and some other crops [32,33]. Lu et al. (2006) [34] suggested that Clostridia inhabiting rice roots and the rhizosphere are probably responsible for the anaerobic decomposition of decaying root residues. ...
There has been increasing attention toward the influence of biofertilizers on the composition of microbial communities associated with crop plants. We investigated the impact of Azospirillum sp. B510, a bacterial strain with nitrogen-fixing ability, on the structure of bacterial and fungal communities within rice plant rhizospheres by amplicon sequencing at two sampling stages (the vegetative and harvest stages of rice). Principal coordinate analysis (PCoA) demonstrated a significant community shift in the bacterial microbiome when the plants were inoculated with B510 at the vegetative stage, which was very similar to the effect of chemical N-fertilizer application. This result suggested that the inoculation with B510 strongly influenced nitrogen uptake by the host plants under low nitrogen conditions. Least discriminant analysis (LDA) showed that the B510 inoculation significantly increased the N2-fixing Clostridium, Aeromonas and Bacillus populations. In contrast, there was no apparent influence of B510 on the fungal community structure. The putative functional properties of bacteria were identified through PICRUSt2, and this hinted that amino acid, sugar and vitamin production might be related to B510 inoculation. Our results indicate that B510 inoculation influenced the bacterial community structure by recruiting other N2-fixing bacteria in the absence of nitrogen fertilizer.
... Therefore, Calothrix found in the surrounding plant roots is not only remove phosphorus and nitrogen in the wastewater but also produce some important metabolites to promote plant growth and immunity to prevent plant pathogens. Previous studies also revealed that Clostridium is able to enhance the plant growth (Doni et al. 2014;Zeiller et al. 2015) as well as relate to nitrogen fixation and nitrogen utilization in wastewater (Wani et al. 2011). Clostridium might be important to plants by providing nitrogen resource. ...
Phytoremediation and bioremediation are eco-friendly methods of wastewater treatment that are widely used throughout the world to reduce anthropogenic water contamination. This study was conducted to assess the effectiveness of symbiotic bacteria in phytoremediation using two aquatic plants, Echinodorus cordifolius and Lepironia articulata, that were tested in sterilized and unsterilized groups. The results showed that unsterilized plants removed more phosphate, ammonium, nitrate and nitrite than the sterilized plants. In untreated and unsterilized E. cordifolius groups, the dominant bacterium was Calothrix (46.90 and 49.69%, respectively), which was higher than in the sterilized E. cordifolius group (38.88%). In untreated and unsterilized groups of L. articulata, Clostridium was a dominant bacterium. The proportion of Clostridium was much lower in the sterilized L. articulata group (1.31%) than in the untreated (13.71%) and unsterilized (49.02%) groups. Our results suggested that root-associated bacteria in E. cordifolius and L. articulata were effective in the removal of phosphorus and nitrogen from domestic wastewater.
... 32 Another study has demonstrated under laboratory conditions the systemic colonisation of clover (Trifolium repens) by C botulinum when the clover is grown on contaminated soil. 33 Considering these results, it may be assumed that spreading manure contaminated by C botulinum over land may be risky from an environmental, public and animal health point of view and may also lead to dissemination of the germ during this operation via the material and vehicles. ...
Background
Persistence of Clostridium botulinum in the environment is well known. Getting rid of it after animal botulism outbreaks is so tricky, especially as far as manure concerns. This study aimed at 1. describing manure management on 10 poultry farms affected by botulism and 2. assessing the persistence of C botulinum in poultry manure after the outbreak.
Methods
Each farm was visited twice at two different manure storage times (two weeks after manure removal and two months later). Fifteen samples of manure were collected on each visit and C botulinum was detected using real-time PCR.
Results
Management of manure varied among poultry farms (classical storage, addition of quicklime, bacterial flora or incineration). C botulinum was detected in the manure of all 10 farms, 56.5per cent of samples being positive. C botulinum was detected significantly more frequently at the second visit (65.8per cent vs 49.7per cent, P<0.01) and on the surface of the pile (63.1per cent vs 50per cent, P=0.025).
Conclusion
This study shows the persistence of C botulinum in poultry manure over time after a botulism outbreak and highlights manure management as a key health issue in preventing spore dissemination in the environment and recurrence of the disease.
... The study indicated that physical or chemical microclimates directly influence the differential expression of ipdC Endophytic Bacillus produces phytohormones such as abscisic acid, auxins, brassinosteroids, cytokinins, ethylene, gibberellins, jasmonates, and strigolactones, and increases nutrient (nitrogen and phosphorous) accessibility to the host (Reinhold-Hurek and Hurek 2011;Brader et al. 2014;Santoyo et al. 2016;Shahzad et al. 2016;Ek-Ramos et al. 2019). Zeiller et al. (2015) reported that C. botulinum 2301 significantly produce PGPs in a field experiment of clover. A cold-tolerant bacterial strain Exiguobacterium acetylicum 1P promotes wheat seedlings growth (Selvakumar et al. 2010), Brevibacillus brevis improve the growth of cotton crop (Nehra et al. 2016) and Bacillus spp. ...
The phyllosphere referred to the total aerial plant surfaces (above-ground portions), as habitat for microorganisms. Microorganisms establish compositionally complex communities on the leaf surface. The microbiome of phyllosphere is rich in diversity of bacteria, fungi, actinomycetes, cyanobacteria, and viruses. The diversity, dispersal, and community development on the leaf surface are based on the physiochemistry, environment, and also the immunity of the host plant. A colonization process is an important event where both the microbe and the host plant have been benefited. Microbes commonly established either epiphytic or endophytic mode of life cycle on phyllosphere environment, which helps the host plant and functional communication with the surrounding environment. To the scientific advancement, several molecular techniques like metagenomics and metaproteomics have been used to study and understand the physiology and functional relationship of microbes to the host and its environment. Based on the available information, this chapter describes the basic understanding of microbiome in leaf structure and physiology, microbial interactions, especially bacteria, fungi, and actinomycetes, and their adaptation in the phyllosphere environment. Further, the detailed information related to the importance of the microbiome in phyllosphere to the host plant and their environment has been analyzed. Besides, biopotentials of the phyllosphere microbiome have been reviewed.
... Clostridium is one of the most important genus within the Firmicutes, and members of this genus are isolated from different ecological niches (Zeiller et al., 2015), e.g., rhizosphere of pea (Pisum sativum), leaves of Miscanthus sinensis or a rice paddy field (Polyanskaya et al., 2002;Ye et al., 2005;Doni et al., 2014). ...
... Members of Clostridium genus are obligate anaerobes (some species can tolerate oxygen) motile or nonmotile, spherical or oval endospore-forming bacteria with the ability to form up to five endospores per cell (Duda et al., 1987;Zeiller et al., 2015). Members are usually Gram-positive-staining, although in some species, cells are noticed as Gram-variable or even Gram-negative-staining. ...
... Beneficial Microbes in Agro-Ecology, First Edition, 2020, 363e396 enhanced plant growth and was effective for the bacterial colonization of the rhizosphere, as well as endophytic colonization (Zeiller et al., 2015). In another study endophytic clostridia were reported to have nitrogen fixing abilities associated to gramineous plants (Minamisawa et al., 2004). ...
Among the phylum Firmicutes, members of the genus Bacillus are probably the most extensively studied as beneficial microorganisms with application in agroecology. However, members of other genera within the Phylum are equally important. This chapter highlights the beneficial role of members of the phylum Firmicutes, with the exception of Bacillus spp., which is not considered. Their importance in plant growth promotion (biofertilizers), their role in biocontrol of plant pathogens (biocontrol agent), as well as in the phytoremediation of heavy metals (metal uptake enhancers) are discussed for individual genera. All these traits are of relevance in the perspective of exploiting these organisms in sustainable agricultural systems.
... It has been reported that Clostridium spp. are able to actively colonize plants, including the roots Myamoto et al., 2004;Zeiller et al., 2015), and that they can systemically migrate from the roots to the vascular xylem, behaving as true endophytes (Zeiller et al., 2015). (Ye and Zhang, 2011;Nikaeen et al., 2015;Romanazzi et al., 2016) and is associated with biosolids. ...
... It has been reported that Clostridium spp. are able to actively colonize plants, including the roots Myamoto et al., 2004;Zeiller et al., 2015), and that they can systemically migrate from the roots to the vascular xylem, behaving as true endophytes (Zeiller et al., 2015). (Ye and Zhang, 2011;Nikaeen et al., 2015;Romanazzi et al., 2016) and is associated with biosolids. ...
Since 2012, a new pathogenic syndrome has frequently been observed in many areas of kiwifruit cultivation in Italy. Main symptoms include an initial withering of the leaves followed by a total and sudden collapse of plants, mainly occurring during summer. The withered leaves fall down and the main and secondary feeder roots appear rotten, sometimes showing a reddish‐brown discoloration. The disease, that affects both the green and yellow‐fleshed cultivars, has been called kiwifruit vine decline and it is locally known as “moria”. The syndrome has been found consistently associated with soil waterlogging, which frequently occurs either after the traditional agronomical practice to irrigate orchards through surface irrigation or after very heavy rainfall. So far, the role played by bacteria in this syndrome has not been investigated. In the present study, Clostridium spp. were isolated from both rotten roots and soils obtained from Italian kiwifruit orchards affected by the syndrome, indicating for the first time that anaerobic bacteria are able to cause damage to woody crops. C. bifermentans and C. subterminale incited symptoms to kiwifruit in both in vivo and in vitro pathogenicity tests. Soil waterlogging seems to potentially favour colonization of kiwifruit roots by anaerobic bacteria, probably because saturation of the soil can facilitate proliferation and persistence of these bacteria during long periods of the vegetative growth of the crop. The occurrence of anaerobic bacteria does not exclude the possibility that other microorganisms can play additional/synergic role(s) in causing the kiwifruit vine decline.
... This is in accordance with non-actinobacterial LAP producers such as B. amyloliquefaciens FZB42 which is associated with lettuce (Lactuca sativa) and is known to be able to promote plant growth and health by reducing disease severity (DS) caused by Rhizoctonia solani (Kröber et al. 2014). Some strains of C. botulinum are also shown to be associated with plants which are reported to produce LAPs (Zeiller et al. 2015). Goadsporin is also associated with plant as it interferes with a phytopathogen (ONAKA et al. 2001). ...
One of the most diverse groups of bioactive bacterial metabolites is the ribosomally synthesised and post-translationally modified peptides (RiPPs) with different bioactivities. The process of genome mining has made it possible to predict the presence of such clusters among the huge genomic data available today. Despite the great potential of actinobacteria in producing natural products and the myriad of completely sequenced genomes available, a comprehensive genome mining of these bacteria for RiPPs is lacking. Here, a collection of 629 complete actinobacterial genomes were analysed to explore their RiPP biosynthesis potential. Using BAGEL3 genome mining tool, the presence of 477 RiPP biosynthesis gene clusters (BGCs) was shown, including all known classes of bacterial RiPPs. RiPP-encoding potential was shown to be widespread among different members of actinobacteria especially within the plant and soil-inhabiting strains. The notable presence of LAP BGCs in plant-associating actinobacteria was also illustrated. Streptomyces, Amycolatopsis, Kitasatospora and Frankia showed greater potential in RiPP biosynthesis while lanthipeptides and lasso peptides were the most distributed RiPPs. Three cyanobactin BGCs were also detected. Generally evidence of promising ability of actinobacteria to synthesise diverse classes of RiPPs as well as information needed to rationally select appropriate taxa for rational screening of specific RiPPs are presented.
