Microbiome contributes to phenotypic plasticity in saffron crocus.

Saffron crocus is a sterile plant species that propagates vegetatively, and consequently, narrow genetic variation is detected in this species. Besides the narrow genetic variation, there is significant phenotypic variation in different traits in this plant. Here we tested this hypothesis that plant microbiome is a major contributor to the phenotypic variation. We focused our analysis on culturable bacteria that were dominant in saffron fields with high stigma yield compared to the fields with low stigma yield. Following this strategy, four rhizospheric (Cupriavidus metallidurans, Bacillus sp., Solibacillus sp., and Planococcus sp.) and two endophytic bacteria (Serratia oryzae and S. odorifera) were identified. The effects of the bacteria on the growth and development of the model plant Arabidopsis were assessed both in agar plate and pot assays. Results showed that these bacteria influence the vegetative growth and flowering time of Arabidopsis. In the next step, corms of saffron were inoculated with these bacteria and the growth and development of the saffron plants were monitored for five months. Remarkably, inoculation of the bacteria had significant influence on vegetative growth, flowering time, and stigma yield of saffron crocus. Furthermore, one of the bacteria, C. metallidurans, is reported here for the first time as a naturally occurring plant-associated bacteria. Altogether our results suggest that plant microbiome is an important factor in phenotypic variation in saffron crocus.

Site and Organ-Specific Culture-Dependent Endophytic Diversity of Crocus sativus L. (Saffron) in Kashmir Himalaya, India.

The endophytic diversity of a host plant has been shown to vary across various spatio-temporal scales and between different organs of a plant, but no such study has been carried out on the economically important plant Crocus sativus (saffron). To fill this knowledge gap, the present study was undertaken to document the diversity of culturable bacterial, actinomycete and fungal endophytes at multiple sites from vegetative and reproductive organs of C. sativus. A total of 1170 culturable endophytic isolates were recovered from 6480 tissue segments of C. sativus collected from six different study sites in Pampore region of Kashmir valley in India. These isolates were identified using 16S and ITS (internal transcribed spacer) rDNA barcode sequence analysis and were classified into 84 operational taxonomic units (OTUs), including 52 bacterial OTUs, 7 actinomycete OTUs and 25 fungal OTUs. The phylogenetic analysis of sequences separated them into four phyla, namely, Firmicutes (46%), Ascomycota (30%), Proteobacteria (16%) and Actinobacteria (8%). Significant differences were observed in the diversity of endophytic assemblages across various study sites and different plant organs (P ≤ 0.001). Species richness was highest at the Baroosa site and lowest at the Chandhar site while the Shannon index was highest at the Baroosa site and lowest at the Letpur site. Among organs, species richness was highest in stigma and lowest in leaf. Similarly, Shannon index was highest for root and lowest for leaf. Further, 15 culturable endophytic OTUs showed organ specificity. The present study is the first comprehensive report that not only brings out differences in the diversity of endophytes associated with different organs and at different sites but also highlights the complexity of host-endophyte interaction at multiple scales.

Insights into the Endophytic Bacterial Microbiome of Crocus sativus: Functional Characterization Leads to Potential Agents that Enhance the Plant Growth, Productivity, and Key Metabolite Content.

The study was undertaken to unravel the culturable endophytic bacterial microbiome of Crocus sativus L. (saffron crocus) and consequently obtain potential leads to develop plant growth-promoting and biocontrol agents for increased productivity and sustainable cultivation. The endophytes formed 47 different operational taxonomic units (OTUs), spanning over 28 genera. The host was preferentially colonized by the genus Bacillus, followed by Burkholderia and Pantoea, respectively. Several endophytes possessed potential plant growth-promoting properties and inhibitory activities against the specific fungal pathogens of saffron. The endophytes, except for Microbacterium oxydans, did not cause any disease symptoms in the pot experiments. The selected cultures, Burkholderia gladioli, Streptomyces achromogenes, and three species of Bacillus, enhanced the host plant growth significantly. Based on the pot experiment results, two isolates, Bacillus mojavensis CS4EB32 and Burkholderia gladioli E39CS3, were selected for the field experiments. We obtained an increase of 67.5%, 69.8%, and 68.3% in the production of flowers with the individual and collective treatments, respectively. The treatments also enhanced the biomass of the plant and the length and weight of stigmas significantly. The endophyte treatments induced the expression of the pathway genes, resulting in a marked increase in the concentration of apocarotenoids. The study indicates that the dominant endophytes support plant growth and development in nature and present an opportunity for developing microbial formulations for the sustainability of saffron cultivation.

Field evaluation of PGP Bacillus sp. strain D5 native to Crocus sativus, in traditional and non traditional areas, and mining of PGP genes from its genome.

Native Bacillus sp. strain D5 coded as (Bar D5) has been isolated from the saffron corm that showed plant growth promotion (PGP) properties and also inhibits the growth of corm rot causing Fusarium oxysporum R1 (Fox R1) in-vitro. Bar D5 was more efficient PGP bacterium in comparison to earlier reported native bio-formulations by our group. Pot assays and field evaluation of Bar D5 confirmed its in-vivo efficacy for PGP traits and biocontrol activity as well. Pot trials were followed by field trials at traditional (Kishtwar) and non-traditional (R.S Pura) saffron cultivation areas in Jammu and Kashmir. At both places, Bar D5 bio-formulation treatment led to the increase in root number & length, shoot number & length, flower number and number & weight of daughter corms. Additionally, it also decreased the corm rot disease incidence significantly. Priming of corms with bio-formulation resulted in the reduction of pathogenic fungal load by three fold at the depth of corm sowing from ground level. The shelf life/viability of Bar D5 based bio-formulation was found to be 52% (viable spores) for one year at room temperature. Draft genome sequence of Bar D5 revealed the presence of genes necessary for PGP and biocontrol activity. Further, confirmation of gene sequences and annotation was done by amplification, re-sequencing and mapping of PGP and biocontrol genes on draft genome. Bar D5 based bio-formulation can be provided to companies/researchers interested in saffron cultivation or bio-formulation production for commercial exploitation, since saffron is grown as revenue crop across continents. The present study bridges the gap between genomics and its field application.

Antibacterial potential of Juglomycin A isolated from Streptomyces achromogenes, an endophyte of Crocus sativus Linn.

AIM: To evaluate the antimicrobial potential of Juglomycins isolated from Streptomyces achromogenes E91CS4, an endophyte of Crocus sativus Linn. METHODS AND RESULTS: The extract from E91CS4 displayed significant antimicrobial activity against several pathogens. The endophyte was identified as S. achromogenes on by 16S ribosomal gene analysis. Chemical investigation of the extract led to the isolation of two naphthoquinone antibiotics, Juglomycin A and B. Juglomycin A inhibited several pathogens, with an MIC value of 13·7µg ml-1 , whereas it was most potent against Escherichia coli, Bacillus thuringiensis and Xanthobacter flavus with MIC values of 6·8, 3·4 and 6·8 µg ml-1 respectively. It was found to reduce the biofilm formation in E. coli through inhibition of swimming and swarming motilities and downregulation of fimH gene. The α-haemolysin-related gene (hlyA) was also downregulated indicating that the compound is also reducing the virulence in E. coli. In vitro time kill kinetics showed efficient bactericidal activity of this compound. Furthermore, Juglomycin A inhibited bacterial transcription/translation in vitro, while also inducing postantibiotic effect in E. coli. CONCLUSIONS: Juglomycin A is a potential antimicrobial compound against several bacterial pathogens, particularly, E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed the promising potential of Juglomycin A as an antimicrobial agent. Efforts should be made to scale up the production of this compound and conduct further studies to explore its efficacy as an antibiotic, using in vivo models.

Plant growth promoting bacteria associated with corm of Crocus sativus during three growth stages.

Corm, being a unit of reproduction and storage, plays an indispensable role in the life cycle of Crocus sativus (saffron). This study deals with the screening of bacteria isolated from cormosphere (area surrounding the corm sheath) of C. sativus during three growth stages for biofertilizer and biocontrol property. Out of 1000 protease producing bacteria isolated from the cormosphere (soil adhering corm sheath in analogy to rhizosphere), three plant growth promoting (PGP) Bacilli namely Bacillus thuringiensis DC1, B. megaterium VC3 and B. amyloliquefaciens DC8 were selected based on multiple and maximum PGP traits along with corm rot pathogen antagonism. To evaluate in vivo efficacy of the consortia, pot trials were conducted and a significant increase in number and length of both, shoot and root were observed. Further, the disease incidence in test corms was reduced by threefold as compared to control and the number of daughter corms produced in pot trials was also increased. Since corm is a unit of reproduction/propagation, increase in its number is of major significance. This is the first report on isolation and characterization of Plant growth promoting bacteria from any cormosphere.

Cold low pressure O2 plasma treatment of Crocus sativus: An efficient way to eliminate toxicogenic fungi with minor effect on molecular and cellular properties of saffron.

In this study cold low pressure radiofrequency oxygen plasma was used for the first time to inactivate toxicogenic fungi proliferation on saffron. Varieties of plasma produced reactive oxygen species which were investigated by optical emission spectroscopy. The data were indicative of the absence of UV radiation. Effects of plasma treatment on antioxidant activity, metabolic content, colour, odour and flavour parameters and physical impact on saffron were investigated. A range of plasma powers and exposure times were assayed in suppression of fungal growth. Amongst which power of 60 W for 15 min was used to eradicate Aspergillus and other microorganisms. The ferric reducing antioxidant power was changed from 1778.21 to 1674.25 mM/g dry weight following plasma treatment. Moreover, crocin ester, picrocrocin and safranal metabolites reduced insignificantly. Additionally, plasma had no significant impact on colour, odour and flavour of saffron.

Isolation and characterization of microorganisms and volatiles associated with Moroccan saffron during different processing treatments.

Saffron may be spoiled by a variety of microorganisms during cultivation, harvesting, and post harvesting. As saffron can be dried and stored in different ways, this preliminary study explored the natural microbiota present in Moroccan saffron when subjected to different drying techniques. An analysis of the carotenoid-derived volatiles present in the saffron was also carried out. The culturable microbiota of the saffron samples dried using different methods, namely in the shade (also called natural), in the sun, or in the oven, were studied using classical and molecular approaches. The effect of the drying methods on head-space chemical volatiles was also determined. Eighty-two isolates grown in the different culture media were chosen from the colonies, and genotype analysis grouped the microorganisms into 58 clusters, revealing a wide diversity. Out of the 82 isolates, 75 belonged to the Bacillaceae family. The other isolates were distributed within the Dietziaceae, Paenibacillaceae and Carnobacteriaceae families. The dominant species was Bacillus simplex, which was detected in all samples, regardless of the drying method used. Lysinibacillus macroides was dominant in the sun-dried saffron. No pathogens were isolated, but an isolate belonging to Dietzia maris, a potential human pathogenic species, was detected. The biodiversity indexes were linked to the drying method and generally decreased as the intensity of the treatment increased. The results of this preliminary work show that the different drying methods strongly influenced the microbiota and affect the saffron volatile profile. Further analysis will be needed to determine possible effects of selected microbiota on saffron volatiles.

Porostereum sp., Associated with Saffron (Crocus sativus L.), is a Latent Pathogen Capable of Producing Phytotoxic Chlorinated Aromatic Compounds.

Saffron (Crocus sativus L.) is one of the most expensive spices in the world due to its medicinal and aromatic value. However, saffron production is severely affected by the corm rot disease throughout the saffron producing countries. In this study, we report a basidiomycetous latent pathogen of saffron, designated as CSE26, capable of producing phytotoxic compounds. CSE26 is a highly odorous basidiomycete with monomitic hyphal system. Molecular phylogeny of ITS and 28S ribosomal gene sequence of CSE26 assigned it as Porostereum spadiceum. It was found to produce corm rot in C. sativus under in vivo and field conditions, with a disease severity index of 0.7 and 0.5, respectively. CSE26 was found to produce chlorinated aromatic compounds (CAMs) having phytotoxic activity against Arabidopsis plants. Therefore, these compounds may be acting as pathogenic determinants of CSE26. However, there is a need to study the level of production of these CAMs by this fungus in the natural environment and their effects on plant health.

Mortierella alpina CS10E4, an oleaginous fungal endophyte of Crocus sativus L. enhances apocarotenoid biosynthesis and stress tolerance in the host plant.

Crocus sativus is the only plant species which produces apocarotenoids like crocin, picrocrocin and safranal in significant amounts. These compounds impart organoleptic properties to saffron (dried stigmas of Crocus flower) making it world's costliest spice. Crocus apocarotenoids have tremendous medicinal properties as well. Effect of endophytes on Crocus apocarotenoid production and the molecular mechanism involved has not been reported so far. Here we studied the effect of an oleaginous fungal endophyte, Mortierella alpina CS10E4 on Crocus growth, apocarotenoid metabolism and tolerance to corm rot disease. The results demonstrated that there was a significant improvement in many morphological and physiological traits in endophyte treated Crocus plants including total biomass and size of corms, stigma biomass, number of apical sprouting buds, and number of adventitious roots. The endophyte also shifted metabolic flux towards enhanced production of apocarotenoids by modulating the expression of key pathway genes. Further, M. alpina CS10E4 enhanced tolerance to corm rot disease by releasing arachidonic acid which acts as conserved defense signal and induces jasmonic acid production in endophyte treated Crocus corms. This is first report on effect of a fungal endophyte on Crocus apocarotenoid metabolism and stress tolerance.

Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn.

A total of 294 fungal endophytes were isolated from the corms of Crocus sativus at two stages of crocus life cycle collected from 14 different saffron growing sites in Jammu and Kashmir (J & K) State, India. Molecular phylogeny assigned them into 36 distinct internal transcribed spacer (ITS) genotypes which spread over 19 genera. The diversity of endophytes was higher at the dormant than at the vegetative stage. The Saffron microbiome was dominated by Phialophora mustea and Cadophora malorum, both are dark septate endophytes (DSEs). Some endophytes were found to possess antimicrobial properties that could be helpful for the host in evading the pathogens. These endophytes generally produced significant quantities of indole acetic acid (IAA) as well. However, thirteen of the endophytic taxa were found to cause corm rot in the host with different levels of severity under in vitro as well as in vivo conditions. This is the first report of community structure and biological properties of fungal endophytes associated with C. sativus, which may eventually help us to develop agro-technologies, based on plant-endophyte interactions for sustainable cultivation of saffron. The endophytes preserved ex situ, in this study, may also yield bioactive natural products for pharmacological and industrial applications.

Challenges of climate change: omics-based biology of saffron plants and organic agricultural biotechnology for sustainable saffron production.

Kashmir Valley is a major saffron (Crocus sativus Kashmirianus) growing area of the world, second only to Iran in terms of production. In Kashmir, saffron is grown on uplands (termed in the local language as "Karewas"), which are lacustrine deposits located at an altitude of 1585 to 1677 m above mean sea level (amsl), under temperate climatic conditions. Kashmir, despite being one of the oldest historical saffron-producing areas, faces a rapid decline of saffron industry. Among many other factors responsible for decline of saffron industry the preponderance of erratic rainfalls and drought-like situation have become major challenges imposed by climate change. Saffron has a limited coverage area as it is grown as a 'niche crop' and is a recognized "geographical indication," growing under a narrow microclimatic condition. As such it has become a victim of climate change effects, which has the potential of jeopardizing the livelihood of thousands of farmers and traders associated with it. The paper discusses the potential and actual impact of climate change process on saffron cultivation in Kashmir; and the biotechnological measures to address these issues.

Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.

Plant growth promoting bacteria from Crocus sativus rhizosphere.

Present study deals with the isolation of rhizobacteria and selection of plant growth promoting bacteria from Crocus sativus (Saffron) rhizosphere during its flowering period (October-November). Bacterial load was compared between rhizosphere and bulk soil by counting CFU/gm of roots and soil respectively, and was found to be ~40 times more in rhizosphere. In total 100 bacterial isolates were selected randomly from rhizosphere and bulk soil (50 each) and screened for in-vitro and in vivo plant growth promoting properties. The randomly isolated bacteria were identified by microscopy, biochemical tests and sequence homology of V1-V3 region of 16S rRNA gene. Polyphasic identification categorized Saffron rhizobacteria and bulk soil bacteria into sixteen different bacterial species with Bacillus aryabhattai (WRF5-rhizosphere; WBF3, WBF4A and WBF4B-bulk soil) common to both rhizosphere as well as bulk soil. Pseudomonas sp. in rhizosphere and Bacillus and Brevibacterium sp. in the bulk soil were the predominant genera respectively. The isolated rhizobacteria were screened for plant growth promotion activity like phosphate solubilization, siderophore and indole acetic acid production. 50 % produced siderophore and 33 % were able to solubilize phosphate whereas all the rhizobacterial isolates produced indole acetic acid. The six potential PGPR showing in vitro activities were used in pot trial to check their efficacy in vivo. These bacteria consortia demonstrated in vivo PGP activity and can be used as PGPR in Saffron as biofertilizers.This is the first report on the isolation of rhizobacteria from the Saffron rhizosphere, screening for plant growth promoting bacteria and their effect on the growth of Saffron plant.

Identification of a quinazoline alkaloid produced by Penicillium vinaceum, an endophytic fungus from Crocus sativus.

CONTEXT: Endophytic fungi are microorganisms living within the tissues of host plants, and have proven to be rich sources of biologically active secondary metabolites and therefore have attracted increasing attention in recent years. OBJECTIVE: To isolate and characterize bioactive constituents from the endophytic fungus cultures of Crocus sativus Linn. (Iridaceae). MATERIALS AND METHODS: Endophytes were isolated from the corm of C. sativus. Endophytic fungus cultures were subjected to repeated column chromatography. Chemical structure was elucidated based on extensive spectroscopic methods and X-ray diffraction analysis. Several pathogenic fungi isolates and tumor cell lines were employed to evaluate the antifungal and cytotoxic activities of the isolated compound. RESULTS: An isolate of Penicillium vinaceum (strain no. X17) was obtained from the corm of C. sativus. Chemical investigations of the endophyte culture broth afforded an unique quinazoline alkaloid (1), identified as (-)-(1R,4R)-1,4-(2,3)-indolmethane-1-methyl-2,4-dihydro-1H-pyrazino-[2,1-b]-quinazoline-3,6-dione, which showed cytotoxic (IC(50) range 40.55-76.83 µg/mL) and antifungal (MIC(80) range 16-64 µg/mL) activities. DISCUSSION AND CONCLUSIONS: Endophytes in C. sativus can be a rich source of novel bioactive compounds, which prompts us to expand the medicinal resource of this valuable plant in another way. Compound 1 exhibited potential cytotoxic and antifungal activities and may be considered a lead compound for promising antifungal and anticariogenic agent.

Molecular cloning and characterisation of a pathogenesis-related protein CsPR10 from Crocus sativus.

Plants have developed many mechanisms to protect themselves against most potential microbial pathogens and diseases. Among these mechanisms, pathogenesis-related proteins are produced as part of the active defence to prevent attack. In this study, a full-length cDNA encoding the CsPR10 protein was identified in fresh saffron stigmas (Crocus sativus). The deduced amino acid sequence from the nucleotide sequence of the coding region showed homology with PR10 proteins. The clone expressed as a protein in fusion with a GST tag produced a 47-kDa protein in E. coli. CsPR10 had ribonuclease activity, with features common to class II-type ribonucleases; its specific activity was quantified as 68.8 U·mg(-1) protein, thus falling within the range of most PR10 proteins exhibiting RNase activity. Antifungal activity of CsPR10 was assayed against Verticillium dahliae, Penicillium sp. and Fusarium oxysporum. CsPR10 inhibited only F. oxysporum growth, and antifungal potency was reflected in a IC(50) of 8.3 µm. Expression analysis showed the presence of high transcript levels in anther and tepal tissues, low levels in stigmas and roots, and no signal detected in leaves. This protein seems to be involved in the active defence response through activation of the jasmonic acid pathway.

Microbiological quality of saffron from the main producer countries.

A microbiological study of saffron spice was undertaken in the context of a European research project (Methodologies for Implementing International Standards for Saffron Purity and Quality, the acronym for which is SAFFIC), analyzing 79 samples obtained from the main producer countries, namely Greece, Iran, Italy, Morocco, and Spain. Current microbiological quality criteria are the same as for other spices, but saffron is added in minute quantities during the cooking process, so the health risk associated with microbial contamination might be lower. We did not detect Salmonella either by culture or by PCR methods in any sample, and Escherichia coli was only found in five samples. Enterobacteriaceae were frequently found (70.9% of the samples), but most of them belonged to species of probable environmental origin. Aerobic sporulated bacteria were also common, but only three samples contained Bacillus cereus at low levels (<200 CFU g(-1)). Clostridium perfringens counts were also very low, with only one sample reaching >100 CFU g(-1), an acceptable value. Overall, microbial contamination in saffron was markedly lower than it was in other spices.

Isolation of a new fungi and wound-induced chitinase class in corms of Crocus sativus.

In plants, various chitinases have been identified and categorized into several groups based on the analysis of their sequences and domains. We have isolated SafchiA, a novel class of chitinase from saffron (Crocus sativus L.). The cDNA encoding SafchiA is mainly expressed in roots and corms, and its expression is induced by elicitor treatment, methyl jasmonate, wounding, and by the fungi Fusarium oxysporum, Beauveria and Phoma sp., suggesting a defence role of the protein. Furthermore, in vitro assays with the recombinant native protein showed chitinolytic, and antifungal activity. The deduced protein shares high similarity with chitinases belonging to family 19 of glycosyl-hydrolases, although some changes in the enzyme active site are present. To explore the properties of SafchiA we have expressed recombinant SafchiA in Escherichia coli and generated four different mutants affected in residues involved in the catalytic activity. One glutamic acid essential for family 19 chitinases activity is not present in C. sativus chitinase suggesting that only one acidic residue is necessary for the enzyme activity, in a similar manner as family 18 glycosyl-hydrolases.

Bacillus subtilis FZB24 affects flower quantity and quality of saffron (Crocus sativus).

The effect of Bacillus subtilis FZB24 on saffron ( Crocus sativus L.) was studied using saffron corms from Spain and the powdered form of B. SUBTILIS FZB24(R). Corms were soaked in water or in B. subtilis FZB24 spore solution for 15 min before sowing. Some corms were further soil drenched with the spore solution 6, 10 or 14 weeks after sowing. Growth and saffron stigma chemical composition were measured. Compared to untreated controls, application of B. subtilis FZB24 significantly increased leaf length, flowers per corm, weight of the first flower stigma, total stigma biomass; microbe addition also significantly decreased the time required for corms to sprout and the number of shoot sprouts. Compared to the controls, picrocrocin, crocetin and safranal compounds were significantly increased when the plants were soil drenched with the spore solution 14 weeks after sowing; in contrast crocin was highest in untreated controls. Results of this study suggest that application of B. subtilis FZB24 may provide some benefit to saffron growers by speeding corm growth (earlier shoot emergence) and increasing stigma biomass yield by 12 %. While some treatment conditions also increased saffron chemical composition, these were generally not the same treatments that simultaneously improved growth yields and thus, more study is required.

Bacterial endophytes of the wildflower Crocus albiflorus analyzed by characterization of isolates and by a cultivation-independent approach.

The presence and taxonomy of endophytic bacteria of the entire aerial parts of crocus (Crocus albiflorus), a wildflower native in the Alps, were investigated. A combination of plating of plant macerates, isolation and sequence identification of isolates, and direct 16S rDNA PCR amplification followed by whole-community fingerprinting (T-RFLP) and by construction of a bacterial clone library was used. The results clearly indicated that a wide range of bacteria from diverse phylogenetic affiliation, mainly gamma-Proteobacteria and Firmicutes, live in association with plants of C. albiflorus. The community composition of the culturable component of the microflora was remarkably different from that of the clone library. Only three bacterial divisions were found in the culture collection, which represented 17 phylotypes, whereas six divisions were identified in the clonal analysis comprising 38 phylotypes. The predominant group in the culture collection was the low G+C Gram-positive group, whereas in the clone library, the gamma-Proteobacteria predominated. Interestingly, the most prominent bacterium within the uncultured bacterial community was a pseudo monad closely related to a cold-tolerant Pseudomonas marginalis strain. The results suggest that Crocus supports a diverse bacterial microflora resembling the microbial communities that have been described for other plants and containing species that have not been described in association with plants.