Biocontrol potential of endophytic fungi against phytopathogenic nematodes on potato (Solanum tuberosum L.).

Root-knot nematodes (RKNs) are a vital pest that causes significant yield losses and economic damage to potato plants. The use of chemical pesticides to control these nematodes has led to environmental concerns and the development of resistance in the nematode populations. Endophytic fungi offer an eco-friendly alternative to control these pests and produce secondary metabolites that have nematicidal activity against RKNs. The objective of this study is to assess the efficacy of Aspergillus flavus (ON146363), an entophyte fungus isolated from Trigonella foenum-graecum seeds, against Meloidogyne incognita in filtered culture broth using GC-MS analysis. Among them, various nematicidal secondary metabolites were produced: Gadoleic acid, Oleic acid di-ethanolamide, Oleic acid, and Palmitic acid. In addition, biochemical compounds such as Gallic acid, Catechin, Protocatechuic acid, Esculatin, Vanillic acid, Pyrocatechol, Coumarine, Cinnamic acid, 4, 3-indol butyl acetic acid and Naphthyl acetic acid by HPLC. The fungus was identified through morphological and molecular analysis, including ITS 1-4 regions of ribosomal DNA. In vitro experiments showed that culture filtrate of A. flavus had a variable effect on reducing the number of egg hatchings and larval mortality, with higher concentrations showing greater efficacy than Abamectin. The fungus inhibited the development and multiplication of M. incognita in potato plants, reducing the number of galls and eggs by 90% and 89%, respectively. A. flavus increased the activity of defense-related enzymes Chitinas, Catalyse, and Peroxidase after 15, 45, and 60 days. Leaching of the concentrated culture significantly reduced the second juveniles' stage to 97% /250 g soil and decreased the penetration of nematodes into the roots. A. flavus cultural filtrates via soil spraying improved seedling growth and reduced nematode propagation, resulting in systemic resistance to nematode infection. Therefore, A. flavus can be an effective biological control agent for root-knot nematodes in potato plants. This approach provides a sustainable solution for farmers and minimizes the environmental impact.

Salt-tolerant PGPR strain Priestia endophytica SK1 promotes fenugreek growth under salt stress by inducing nitrogen assimilation and secondary metabolites.

AIMS: Soil salinity is a huge obstacle in crop production worldwide. Saline soil can reduce active chemical contents in medicinal plants of the Leguminosae family through crippled normal nodule function. Intensive efforts are underway to improve yield and medicinal value of leguminous herbs under salt stress condition by using benign microbes. Here, an attempt was made to explore the salt-tolerant bacteria associated with rhizosphere of fenugreek plant (Trigonella foenum-graecum L.) and to evaluate their impact on host plant growth and metabolite of pharmaceutical importance. METHODS AND RESULTS: A salt-tolerant plant growth promoting rhizobacterial (PGPR) strain Priestia endophytica SK1 isolated from fenugreek rhizospheric soil, which increased biomass and metabolite content in plants grown under saline stress. SK1 bacterial application induced nodule formation and enhanced nitrogen and phosphorus content under salt (100 mM NaCl) stress as compared to control plants. H2 O2 production and lipid peroxidation as a measure of stress were observed high in control plants, while a reduction in these parameters was observed in plants inoculated with SK1. In addition, a significant effect was found on the phenolic compounds and trigonelline content in fenugreek plant inoculated with SK1 bacterium. An increased trigonelline content of about 54% over uninoculated control was recorded under salt stress. CONCLUSION: The results of this study revealed that the application of salt-tolerant PGPR strain P. endophytica SK1 induced nitrogen fixation machinery that leads to alleviate salt stress and improved the biosynthesis of trigonelline content in fenugreek. SIGNIFICANCE OF THE STUDY: This study extends our understanding on the significance of rhizosphere microbiome and their beneficial role in plant health under environmental stress to promote agro-eco-farming practices.

Phylogenetic diversity and plant growth-promoting activities of rhizobia nodulating fenugreek (Trigonella foenum-graecum Linn.) cultivated in different agroclimatic regions of India.

Fenugreek (Trigonella foenum-graecum Linn.), is an extensively cultivated legume crop used as a herb, spice, and traditional medicine in India. The symbiotic efficiency and plant growth-promoting potential of fenugreek rhizobia depend on the symbiont strain and environmental factors. We isolated 176 root-nodulating bacteria from fenugreek cultivated in different agroclimatic regions of India. MALDI-TOF MS-based identification and phylogenetic analyses based on 16S rRNA and five housekeeping genes classified the fenugreek-rhizobia as Ensifer (Sinorhizobium) meliloti. However, the strains represent separate sub-lineages of E. meliloti, distinct from all reported sub-lineages across the globe. We also observed the spatial distribution of fenugreek rhizobia, as the three sub-lineages of E. meliloti recorded during this study were specific to their respective agroclimatic regions. According to the symbiotic gene (nodC and nifH) phylogenies, all three sub-lineages of E. meliloti harboured symbiotic genes similar to symbiovar meliloti; as with the housekeeping genes, these also revealed a spatial distribution for different clades of sv. meliloti. The strains could nodulate fenugreek plants and they showed plant growth-promoting potential. Significant differences were found in the plant growth parameters in response to inoculation with the various strains, suggesting strain-level differences. This study demonstrates that fenugreek rhizobia in India are diverse and spatially distributed in different agro-climatic regions.

Response of intercropped barley and fenugreek to mono- and co-inoculation with Sinorhizobium meliloti F42 and Variovorax paradoxus F310 under contrasting agroclimatic regions.

In the present research, we aimed to select efficient rhizobia and plant growth-promoting rhizobacteria (PGPR) from fenugreek nodules and assess their performance as bio-inoculum for intercropped fenugreek and barley. Inoculation effects with selected bacteria were investigated firstly on fenugreek plants under greenhouse experiment and secondly on intercropped fenugreek and barley under three different agro-environmental conditions for two consecutive years. Sinorhizobium meliloti F42 was selected due to its ability to nodulate fenugreek and effectively improve plant growth. Among non-nodulating endophytic bacteria, Variovorax paradoxus F310 strain was selected regarding its plant growth-promoting traits showed in vitro and confirmed in vivo under greenhouse experiment. Field inoculation trials revealed a significant improvement in fenugreek nodulation (up to + 97%) as well as in soil enzymes activities (up to + 209%), shoot N content (up to + 18%), shoot dry weight (up to + 40%), photosynthetic assimilation (up to + 34%) and chlorophyll content of both intercropped plants in response to the mono-inoculation with Sinorhizobium meliloti F42, compared to the un-inoculated treatment at the SBR and JBS sites. Variovorax paradoxus F310 inoculation significantly increased shoot P content of both intercropped plants at the three experimental sites compared to the un-inoculated treatment (up to + 48%). It was shown that bacterial inoculation was more efficient at the low-rainfall region than the high-rainfall region. The co-inoculation with Sinorhizobium meliloti F42 and Variovorax paradoxus F310 resulted in a significant reduction in fenugreek nodulation and shoot N content. This survey showed the benefits of rhizobial and PGPR inoculation as efficient bio-inoculums to promote the cereal-legume intercropping system and highlights the influence of site-specific environmental factors on Rhizobium-PGPR-plant interactions.

Effect of Trigonella foenum Extract and ZiO2 Nanoparticles on Some Pathogenic Fungi and Bacteria

Aim: The current venture, were made to evaluate the inhibitory effect of Trigonella foenum seed Extract and ZiO2 Nanoparticles on some selected species of Fungi and Bacteria. Materials and Methods: two bacterial species included Pseudomonas aeruginosa and Staphylococcus aureus and three fungal species which is Cryptococcus neoformans, Candidda albicans and Chaetomium were used to evaluate the antibacterial activity of Trigonella foenum Extract and ZiO2 Nanoparticles. Results: This study showed that the Zirconium Oxide (ZiO2) nanoparticles have antifungal and antibacterial activities on the isolates of Cryptococcus neoformans, Candida alicans and Staphylococcus aureus, respectively. While the antimicrobial activity of Zirconium Oxide nanoparticles on the Chaetomium and Pseudomonas aeruginosa was negative. All tested fungi and bacterial isolates were found to be sensitive to Trigonella foenum seed extract, the results of the compination of the ZiO2 Nanoparticle and the Trigonella foenum seed extract were poisitive for all tested fungi isolates and bacterial isolates. The XRD analysis was done for Zirconium Oxide nanoparticles and the result showed that the biocrystallization on the surface of the Zirconium Oxide manoparticles. The average partides size was about (29.8) nm. Conclusions: This investigation conclude that the use of Trigonella foenum seed Extract has the effect of killing all bacteria and fungi under study, result indicate the Trigonella foenun seed Extract best antibacterial efficacy than the ZiO2 together (AU)

Influence of Plant Species, Tissue Type, and Temperature on the Capacity of Shiga-Toxigenic Escherichia coli To Colonize, Grow, and Be Internalized by Plants.

Contamination of fresh produce with pathogenic Escherichia coli, including Shiga-toxigenic E. coli (STEC), represents a serious risk to human health. Colonization is governed by multiple bacterial and plant factors that can impact the probability and suitability of bacterial growth. Thus, we aimed to determine whether the growth potential of STEC for plants associated with foodborne outbreaks (two leafy vegetables and two sprouted seed species) is predictive of the colonization of living plants, as assessed from growth kinetics and biofilm formation in plant extracts. The fitness of STEC isolates was compared to that of environmental E. coli isolates at temperatures relevant to plant growth. Growth kinetics in plant extracts varied in a plant-dependent and isolate-dependent manner for all isolates, with spinach leaf lysates supporting the highest rates of growth. Spinach extracts also supported the highest levels of biofilm formation. Saccharides were identified to be the major driver of bacterial growth, although no single metabolite could be correlated with growth kinetics. The highest level of in planta colonization occurred on alfalfa sprouts, though internalization was 10 times more prevalent in the leafy vegetables than in sprouted seeds. Marked differences in in planta growth meant that the growth potential of STEC could be inferred only for sprouted seeds. In contrast, biofilm formation in extracts related to spinach colonization. Overall, the capacity of E. coli to colonize, grow, and be internalized within plants or plant-derived matrices was influenced by the isolate type, plant species, plant tissue type, and temperature, complicating any straightforward relationship between in vitro and in planta behaviors.IMPORTANCE Fresh produce is an important vehicle for STEC transmission, and experimental evidence shows that STEC can colonize plants as secondary hosts, but differences in the capacity to colonize occur between different plant species and tissues. Therefore, an understanding of the impact that these plant factors have on the ability of STEC to grow and establish is required for food safety considerations and risk assessment. Here, we determined whether growth and the ability of STEC to form biofilms in plant extracts could be related to specific plant metabolites or could predict the ability of the bacteria to colonize living plants. Growth rates for sprouted seeds (alfalfa and fenugreek) but not those for leafy vegetables (lettuce and spinach) exhibited a positive relationship between plant extracts and living plants. Therefore, the detailed variations at the level of the bacterial isolate, plant species, and tissue type all need to be considered in risk assessment.

Factors in the emergence of serious human infections associated with highly pathogenic strains of shiga toxin-producing Escherichia coli.

The appearance of highly pathogenic strains of Shiga toxin (Stx)-producingEscherichia. coli (STEC) has owed largely to the acquisition of Stx-encoding prophages by strains of E. coli that have pre-existing potential as enteric pathogens, such as atypical enteropathogenic E. coli (aEPEC) and enteroaggregative E. coli (EAEC). However, while high pathogenic potential is necessary, it is not sufficient for such strains to have a serious public health impact (i.e., large outbreaks, many cases of HUS, or both). To do so requires susceptible hosts and additional elements related to transmission, such as, socio-economic, societal, and lifestyle, factors. Two examples are discussed to illustrate this. The factors involved in the emergence of serious disease associated with E. coli O157:H7 in the 1980s probably included a massive increase in population exposure to this pathogen, likely as a result of the introduction of factory farming of cattle in the 1960s, and the development and wide patronage of fast food hamburger restaurants, and, potentially, waning immunity to intimin as a result of the reduction of incidence of enteropathogenic E. coli (EPEC) infection. In the devastating outbreak of Stx2-positiveEAEC O104:H4 in 2011, the wide distribution of the proposed vehicle of transmission, imported fenugreek seeds, was decisive in the exposure of a large population in Central Europe to this pathogen. Contributing factors likely included a preference for eating raw sprouts as a healthy food choice by the affected cases, many of whom were women. Low population levels of immunity to Stx2 probably contributed to the severe clinical outcome. A better understanding of the factors responsible for the emergence of potentially dangerous STEC pathogens as well as of extensive and serious disease associated with them can enhance public health strategies to respond to them.

Biogenic synthesis of copper nanoparticles by natural polysaccharides and Pleurotus ostreatus fermented fenugreek using gamma rays with antioxidant and antimicrobial potential towards some wound pathogens.

Mono-dispersed copper nanoparticles (CuNPs) were constructed using cheap polysaccharides (citrus pectin, chitosan, and sodium alginate), and by appropriating aqueous fermented fenugreek powder (FFP) under the action of Pleurotus ostreatus (as reducing and preserving means), through the influence of gamma irradiation. The synthesized CuNPs are described by UV-Vis. spectroscopy TEM, DLS, XRD, and FT-IR. XRD study of the CuNPs confirmed the generation of metallic CuNPs. The nucleation and the production mechanism of CuNPs are moreover explained. TEM unveiled that, the ordinary diameter of CuNPs incorporated by various polysaccharides, and FFP taken in the range of 31.0 and 36.0 nm respectively. CuNPs size is influenced by many parameters such as the variety of stabilizer, pH within the organization and applied gamma dose. Evaluation of the antioxidant and antimicrobial activities of CuNPs was performed against some selected wound pathogens. The results showed that, CuNPs were a strong antimicrobial agents against microbes caused burn skin infection such as Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans (16.0, 15.0, and 15.0 mm ZOI, respectively). Additionally, CuNPs have a strong antioxidant with 70% scavenging activity against DPPH. So, due to unique characteristics of CuNPs (cost-effective with continued-term stabilization and effective features), they can recover reasonable potential in biomedical, industrial, agricultural, cosmetics, dermal products and pharmaceutical purposes.

Fate of Salmonella enterica and Enterohemorrhagic Escherichia coli Cells Artificially Internalized into Vegetable Seeds during Germination.

Vegetable seeds contaminated with bacterial pathogens have been linked to fresh-produce-associated outbreaks of gastrointestinal infections. This study was undertaken to observe the physiological behavior of Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) cells artificially internalized into vegetable seeds during the germination process. Surface-decontaminated seeds of alfalfa, fenugreek, lettuce, and tomato were vacuum-infiltrated with four individual strains of Salmonella or EHEC. Contaminated seeds were germinated at 25°C for 9 days, and different sprout/seedling tissues were microbiologically analyzed every other day. The internalization of Salmonella and EHEC cells into vegetable seeds was confirmed by the absence of pathogens in seed-rinsing water and the presence of pathogens in seed homogenates after postinternalization seed surface decontamination. Results show that 317 (62%) and 343 (67%) of the 512 collected sprout/seedling tissue samples were positive for Salmonella and EHEC, respectively. The average Salmonella populations were significantly larger (P < 0.05) than the EHEC populations. Significantly larger Salmonella populations were recovered from the cotyledon and seed coat tissues, followed by the root tissues, but the mean EHEC populations from all sampled tissue sections were statistically similar, except in pregerminated seeds. Three Salmonella and two EHEC strains had significantly larger cell populations on sprout/seedling tissues than other strains used in the study. Salmonella and EHEC populations from fenugreek and alfalfa tissues were significantly larger than those from tomato and lettuce tissues. The study showed the fate of internalized human pathogens on germinating vegetable seeds and sprout/seedling tissues and emphasized the importance of using pathogen-free seeds for sprout production.IMPORTANCE The internalization of microorganisms into vegetable seeds could occur naturally and represents a possible pathway of vegetable seed contamination by human pathogens. The present study investigated the ability of two important bacterial pathogens, Salmonella and enterohemorrhagic Escherichia coli (EHEC), when artificially internalized into vegetable seeds, to grow and disseminate along vegetable sprouts/seedlings during germination. The data from the study revealed that the pathogen cells artificially internalized into vegetable seeds caused the contamination of different tissues of sprouts/seedlings and that pathogen growth on germinating seeds is bacterial species and vegetable seed-type dependent. These results further stress the necessity of using pathogen-free vegetable seeds for edible sprout production.

Biogeography of a Novel Ensifer meliloti Clade Associated with the Australian Legume Trigonella suavissima.

Here, we describe a novel clade within Ensifer meliloti and consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genus Ensifer are best known for their ability to form nitrogen-fixing symbioses with forage legumes of three related genera, Medicago L., Melilotus Mill., and Trigonella L., which are members of the tribe Trifolieae. These legumes have a natural distribution extending from the Mediterranean Basin through western Asia, where there is an unsurpassed number of species belonging to these genera. Trigonella suavissima L. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compared the genetic diversity and taxonomic placement of rhizobia nodulating T. suavissima with those of members of an Ensifer reference collection. Our goal was to determine if the T. suavissima rhizobial strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56 T. suavissima symbionts to 28 Ensifer reference strains. Sequence data were partitioned according to the replicons in which the loci are located. The results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees, the Australian strains formed a distinct clade within E. meliloti The strains also shared few alleles with Ensifer reference strains from other continents. Carbon source utilization assays revealed that the strains are also unusual in their ability to utilize 2-oxoglutarate as a sole carbon source. A strategy was outlined for locating similar strains elsewhere.IMPORTANCE In this study, we employed a biogeographical approach to investigate the origins of a symbiotic relationship between an Australian legume and its nitrogen-fixing rhizobia. The question of the ancestral origins of these symbionts is based on the observation that the legume host is not closely related to other native Australian legumes. Previous research has shown that the legume host Trigonella suavissima is instead closely related to legumes native to the Mediterranean Basin and western Asia, suggesting that it may have been introduced in Australia from those regions. This led to the question of whether its rhizobia may have been introduced as well. In this study, we were unable to find persuasive evidence supporting this hypothesis. Instead, our results suggest either that the T. suavissima rhizobia are native to Australia or that our methods for locating their close relatives elsewhere are inadequate. A strategy to investigate the latter alternative is proposed.

Differential Attachment of Salmonella enterica and Enterohemorrhagic Escherichia coli to Alfalfa, Fenugreek, Lettuce, and Tomato Seeds.

Vegetable seeds have the potential to disseminate and transmit foodborne bacterial pathogens. This study was undertaken to assess the abilities of selected Salmonella and enterohemorrhagic Escherichia coli (EHEC) strains to attach to fungicide-treated versus untreated, and intact versus mechanically damaged, seeds of alfalfa, fenugreek, lettuce, and tomato. Surface-sanitized seeds (2 g) were exposed to four individual strains of Salmonella or EHEC at 20°C for 5 h. Contaminated seeds were rinsed twice, each with 10 ml of sterilized water, before being soaked overnight in 5 ml of phosphate-buffered saline at 4°C. The seeds were then vortexed vigorously for 1 min, and pathogen populations in seed rinse water and soaking buffer were determined using a standard plate count assay. In general, the Salmonella cells had higher attachment ratios than the EHEC cells. Lettuce seeds by unit weight had the highest numbers of attached Salmonella or EHEC cells, followed by tomato, alfalfa, and fenugreek seeds. In contrast, individual fenugreek seeds had more attached pathogen cells, followed by lettuce, alfalfa, and tomato seeds. Significantly more Salmonella and EHEC cells attached to mechanically damaged seeds than to intact seeds (P < 0.05). Although, on average, significantly more Salmonella and EHEC cells were recovered from untreated than fungicide-treated seeds (P < 0.05), fungicide treatment did not significantly affect the attachment of individual bacterial strains to vegetable seeds (P > 0.05), with a few exceptions. This study fills gaps in the current body of literature and helps explain bacterial interactions with vegetable seeds with differing surface characteristics.IMPORTANCE Vegetable seeds, specifically sprout seeds, have the potential to disseminate and transmit foodborne bacterial pathogens. This study investigated the interaction between two important bacterial pathogens, i.e., Salmonella and EHEC, and vegetable seeds with differing surface characteristics. This research helps understand whether seed surface structure, integrity, and fungicide treatment affect the interaction between bacterial cells and vegetable seeds.

Long-term survival of the Shiga toxin-producing Escherichia coli O104:H4 outbreak strain on fenugreek seeds.

A major outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 occurred in Germany in 2011. The epidemiological investigation revealed that a contaminated batch of fenugreek seeds (Trigonella foenum-graecum) was the most probable source of the pathogen. It was suggested that the most probable point of contamination was prior to leaving the importer, meaning that the seed contamination with STEC O104:H4 should have happened more than one year before the seeds were used for sprout production. Here, we investigated the capacity of STEC O104:H4 and closely related pathogenic as well as non-pathogenic Escherichia coli strains for long-term survival on dry fenugreek seeds. We did not observe a superior survival capacity of STEC O104:H4 on dry seeds. For none of the strains tested cultivatable cells were found without enrichment on contaminated seeds after more than 24 weeks of storage. Our findings suggest that contamination previous to the distribution from the importer may be less likely than previously assumed. We show that seeds contaminated with E. coli in extremely high numbers can be completely sterilized by a short treatment with bleach. This simple and cheap procedure does not affect the germination capacity of the seeds and could significantly improve safety in sprout production.

Performance of fenugreek bioinoculated with Rhizobium meliloti strains under semi-arid condition.

Rhizobium meliloti strains were isolated from the fields of S.D. Agricultural University (Gujarat, India) and were maintained in the Congo Red Yeast Extract Mannitol Agar medium. These strains were tested for their effectiveness for fenugreek crop grown under semi-arid condition. Among the six Rhizobium strains, FRS-7 strain showed best plant growth parameters like shoot length, shoot dry weight, shoot total nitrogen, root length, root dry weight, root total nitrogen, seed yield, 1000 grain weight, number of root nodules, and nodules fresh and dry weight. The performance of this strain was better as compared to 20 kgN ha(-1) treatment through urea and was even far better over control plot. Seed yields obtained with FRS-7 during two years were 10.14 and 9.66 q ha(-1); which was about 36.8% and 45.9% high over control. This strain resulted in saving of about 20 kgN ha(-1) accompanied with better crop yield and soil health. Results of the present experiments can be utilized in integrated nutrient management for cultivation of fenugreek in semi-arid areas to provide sustainability to agricultural productivity in such regions.

A gaseous acetic acid treatment to disinfect fenugreek seeds and black pepper inoculated with pathogenic and spoilage bacteria.

Contamination of spices by pathogenic and/or spoilage bacteria can be deleterious to consumer's health and cause deterioration of foods, and inactivation of such bacteria is necessary for the food industry. The present study examined the effect of gaseous acetic acid treatment in reducing Escherichia coli O157:H7, Salmonella Enteritidis and Bacillus subtilis populations inoculated on fenugreek seeds and black pepper. Treatment with gaseous acetic acid at 0.3 mmol/L, 0.6 mmol/L and 4.7 mmol/L for 1-3 h significantly reduced the populations of E. coli O157:H7 and Salmonella Enteritidis on black pepper and fenugreek seeds at 55 °C (p < 0.05). The gas treatments at 4.7 mmol/L were more effective in inactivating the pathogens than the treatment at 0.3 mmol/L. An approximately 5.0 log reduction was obtained after 3 h of treatment with 4.7 mmol/L acetic acid. No significant reductions in the population of B. subtilis spores inoculated on fenugreek seeds and black pepper were obtained after the gas treatments at 0.3 mmol/L or 0.6 mmol/L (p > 0.05). However, the gas treatment at 4.7 mmol/L significantly reduced B. subtilis spores (p < 0.05), and 4.0 log CFU/g and 3.5 log CFU/g reductions on fenugreek seeds and black pepper, respectively, were obtained after 3 h of treatment.

Results of surveillance for infections with Shiga toxin-producing Escherichia coli (STEC) of serotype O104:H4 after the large outbreak in Germany, July to December 2011.

After the massive outbreak of infections with Shiga toxin-producing Escherichia coli (STEC) of serotype O104:H4 in Germany in the summer of 2011, post-outbreak surveillance for further infections with this type of STEC was maintained until the end of 2011. This surveillance was based on national mandatory reporting of STEC infections and the associated complication of haemolytic uraemic syndrome (HUS), as well as on data obtained from a questionnaire. Between the outbreak's end (5 July) and 31 December 2011, a total of 33 post-outbreak cases were recorded. Post-outbreak cases occurred with diminishing frequency towards the year's end and resembled the outbreak cases in many respects, however the proportion of HUS among all post-outbreak cases was smaller than during the outbreak. Two thirds of the post-outbreak cases were likely infected by contact with known outbreak cases. Both laboratory and nosocomial spread was noted in this period. No post-outbreak case recalled sprout consumption as a potential source of infection. The scarcity of information conveyed by the nonculture tests routinely used in Germany to diagnose STEC made linkage of post-outbreak cases to the outbreak difficult. Though post-outbreak surveillance demonstrated the outbreak strain's potential for lengthy chains of transmission aided by prolonged shedding, our results and continued routine surveillance until the end of 2013 do not support the notion, that the outbreak strain has been able to establish itself in the German environment.

Rhizobium tibeticum activated with a mixture of flavonoids alleviates nickel toxicity in symbiosis with fenugreek (Trigonella foenum graecum L.).

The objective of this study is to explore the response of an activated Rhizobium tibeticum inoculum with a mixture of hesperetin (H) and apigenin (A) to improve the growth, nodulation, and nitrogen fixation of fenugreek (Trigonella foenum graecum L.) grown under nickel (Ni) stress. Three different sets of fenugreek seed treatments were conducted, in order to investigate the activated R. tibeticum pre-incubation effects on nodulation, nitrogen fixation and growth of fenugreek under Ni stress. Group (I): uninoculated seeds with R. tibeticum, group (II): inoculated seeds with uninduced R. tibeticum group (III): inoculated seeds with induced R. tibeticum. The present study revealed that Ni induced deleterious effects on rhizobial growth, nod gene expression, nodulation, phenylalanine ammonia-lyase (PAL) and glutamine synthetase activities, total flavonoids content and nitrogen fixation, while the inoculation with an activated R. tibeticum significantly improved these values compared with plants inoculated with uninduced R. tibeticum. PAL activity of roots plants inoculated with induced R. tibeticum and grown hydroponically at 75 and 100 mg L(-1) Ni and was significantly increased compared with plants receiving uninduced R. tibeticum. The total number and fresh mass of nodules, nitrogenase activity of plants inoculated with induced cells grown in soil treated up to 200 mg kg(-1) Ni were significantly increased compared with plants inoculated with uninduced cells. Plants inoculated with induced R. tibeticum dispalyed a significant increase in the dry mass compared with those treated with uninduced R. tibeticum. Activation of R. tibeticum inoculum with a mixture of hesperetin and apigenin has been proven to be practically important in enhancing nodule formation, nitrogen fixation and growth of fenugreek grown in Ni contaminated soils.

Activation of Rhizobium tibeticum with flavonoids enhances nodulation, nitrogen fixation, and growth of fenugreek (Trigonella foenum-graecum L.) grown in cobalt-polluted soil.

The goal of this study was to investigate the response of activation of Rhizobium tibeticum with mixture of hesperetin and apigenin to improve growth, nodulation, and nitrogen fixation of fenugreek grown under cobalt (Co) stress. The current study showed that high concentrations of Co-induced noxious effects on rhizobial growth, nod gene expression, nodulation, phenylalanine ammonia-lyase (PAL) and glutamine synthetase (GS) activities, total flavonoid content, and nitrogen fixation. Addition of a mixture of hesperetin and apigenin to growth medium supplemented with different concentrations of Co significantly increased bacterial growth. PAL activity of roots grown hydroponically at 100 mg kg(-1) Co and inoculated with induced R. tibeticum was significantly increased compared with plants receiving uninduced R. tibeticum. Total flavonoid content of root exudates of plants inoculated with activated R. tibeticum was significantly increased compared with inoculated plants with unactivated R. tibeticum or uninoculated plants at variant Co dosages. Application of 50 mg kg(-1) Co significantly increased nodulation, GS, nitrogenase activity, and biomass of plants inoculated with either or uninduced R. tibeticum. The total number and fresh mass of nodules, nitrogenase activity, and biomass of plants inoculated with induced cells grown in soil treated with 100 and 200 mg kg(-1) Co were significantly increased compared with plants inoculated with uninduced cells. Induced R. tibeticum with flavonoids significantly alleviates the adverse effect of Co on nod gene expression and therefore enhances nitrogen fixation. Induction of R. tibeticum with compatible flavonoids could be of practical importance in augmenting growth and nitrogen fixation of fenugreek grown in a Co-contaminated agroecosystem.

Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants.

An experiment was conducted to evaluate the influence of Glomus intraradices colonization on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX), and glutathione reductase (GR)] and the accumulation of nonenzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in roots and leaves of fenugreek plants subjected to varying degrees of salinity (0, 50, 100, and 200 mM NaCl) at two time intervals (1 and 14 days after saline treatment, DAT). The antioxidative capacity was correlated with oxidative damage in the same tissue. Under salt stress, lipid peroxidation and H2O2 concentration increased with increasing severity and duration of salt stress (DoS). However, the extent of oxidative damage in mycorrhizal plants was less compared to nonmycorrhizal plants. The study reveals that mycorrhiza-mediated attenuation of oxidative stress in fenugreek plants is due to enhanced activity of antioxidant enzymes and higher concentrations of antioxidant molecules. However, the significant effect of G. intraradices colonization on individual antioxidant molecules and enzymes varied with plant tissue, salinity level, and DoS. The significant effect of G. intraradices colonization on antioxidative enzymes was more evident at 1DAT in both leaves and roots, while the concentrations of antioxidant molecules were significantly influenced at 14DAT. It is proposed that AM symbiosis can improve antioxidative defense systems of plants through higher SOD activity in M plants, facilitating rapid dismutation of O2 (-) to H2O2, and subsequent prevention of H2O2 build-up by higher activities of CAT, APX, and PX. The potential of G. intraradices to ameliorate oxidative stress generated in fenugreek plants by salinity was more evident at higher intensities of salt stress.

[ITS-polymorphism of salt-tolerant and salt-sensitive native isolates of Sinorhizoblum meliloti--symbionts of alfalfa, clover and fenugreek plants].

Polymorphism of rrs-rrl sequence of ribosomal operons (intergenic sequence, ITS) was studied among 81 isolates of Sinorhizobium meliloti (AK001-AK210) derived from the collection of alfalfa nodulating bacteria of the Laboratory of genetics of ARRIAM, by using species-specific primers FGPS 1490/FGPL132VM. Isolates were obtained from nodules of different species of wild host plants from Medicago, Melilotus and Trigonella genera grown in salinized North-Western region of Kazakhstan. The typical structure of ITS, similar to that of test strain Rm1021, was dominant in native rhizobia population, while in one third of the isolates (33.3%) this sequence was divergent. Among the latter, the ITS type of strain AK83 (RCAM00182) was dominant. Here, we show for the first time that isolates with reduced level of salt-tolerance had more diverse intergenic sequences of rrn-operons. No phylogenetic separation was observed between isolates grouped on the basis of their tolerance or sensitivity towards 0.6 M NaCl. However, the frequency of divergent ITS types within the two groups of rhizobia depended on the host symbiotic preference observed in natural environment, allowing to speculate about the existence of a chromosome types specific for S. meliloti isolates with differential salt tolerance. In conclusion, we propose that in the area subjected to secondary salinization, which are also the centre of introgressive hybridization of alfalfa, micro-evolutionary processes, affecting rrn-operons and associated with salt adaptation, are also occurring in symbiotic root nodule bacteria populations.

The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers.

Rhizobia are Gram-negative Alpha- and Betaproteobacteria living in the underground which have the ability to associate with legumes for the establishment of nitrogen-fixing symbioses. Sinorhizobium meliloti in particular-the symbiont of Medicago, Melilotus, and Trigonella spp.-has for the past decades served as a model organism for investigating, at the molecular level, the biology, biochemistry, and genetics of a free-living and symbiotic soil bacterium of agricultural relevance. To date, the genomes of seven different S. meliloti strains have been fully sequenced and annotated, and several other draft genomic sequences are also available. The vast amount of plasmid DNA that S. meliloti frequently bears (up to 45% of its total genome), the conjugative ability of some of those plasmids, and the extent of the plasmid diversity has provided researchers with an extraordinary system to investigate functional and structural plasmid molecular biology within the evolutionary context surrounding a plant-associated model bacterium. Current evidence indicates that the plasmid mobilome in S. meliloti is composed of replicons varying greatly in size and having diverse conjugative systems and properties along with different evolutionary stabilities and biological roles. While plasmids carrying symbiotic functions (pSyms) are known to have high structural stability (approaching that of chromosomes), the remaining plasmid mobilome (referred to as the non-pSym, functionally cryptic, or accessory compartment) has been shown to possess remarkable diversity and to be highly active in conjugation. In light of the modern genomic and current biochemical data on the plasmids of S. meliloti, the current article revises their main structural components, their transfer and regulatory mechanisms, and their potential as vehicles in shaping the evolution of the rhizobial genome.