The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria     

Molecular identification and control of endophytic contamination during in vitro plantlet development of <Emphasis Type="Italic">Fagonia indica</Emphasis>

Microbial contamination is the major cause of economic losses in commercial and scientific plant tissue culture laboratories. For successful micropropagation, it is important to control contamination during in vitro cultures. The present study was designed to isolate, identify and eradicate endophytic contaminants from in vitro cultures of medicinally important plant Fagonia indica. A total of eight distinct bacterial isolates from in vitro grown plantlets of F. indica were selected based on analysis of colony morphology. The endophytic bacterial contaminants identified at the species level through 16S rRNA sequence analysis were Enterobacter xiangfangensis, Bacillus vallismortis, Bacillus tequilensis, Terribacillus halophilus, Pantoea dispersa, Serratia marcescens subsp. Sakuensis, Staphylococcus epidermidis and Bacillus atrophaeus. It was observed that almost 60% of seedlings were contaminated with Bacillus sp. and out of those, Bacillus tequelensis contributed to most infections (70% out of the Bacillus infections). The other most frequently occurring bacteria were Bacillus vallismortis, Terribacillus halophilus and Serratia marcescens subsp. sakuensis. Furthermore, the addition of antimicrobials to the media either completely inhibited or drastically decreased the growth of endophytic bacteria as compared to the control in which 92% of the plantlets were contaminated with these endophytes. Nine different antibiotics (rifampicin, teicoplanin, gentamicin, vancomycin, ciprofloxacin, tobramycin, tetracycline, doxycycline and ampicillin) were tested for their activity against the identified endophytes. Antibiotics such as ciprofloxacin and tobramycin showed a good response and inhibited the growth of all the bacterial isolates at low doses compared to the other antibiotics. Tobramycin was the most effective as it inhibited the growth of five of the bacterial isolates at a dosage as low as 4 mg/L. In case of tetracycline (16 mg/L) and doxycycline (64 mg/L), the contamination frequency in plantlets was 25.6 and 45%, respectively. It is, therefore, important to search for more endophytes, causing adverse effects during in vitro cultures and should devise a feasible anti-microbial strategy for controlling such contamination.     

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Key message

Cefotaxime (100 mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production.

Abstract

Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F₁ hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100 mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100 mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.     

Predominant culturable crude oil-degrading bacteria in the coast of Kuwait

Total of 272 crude oil-degrading bacteria were isolated from seven locations along the coast of Kuwait. The analysis of the 16S rDNA sequences of isolated bacteria revealed the predominance of six bacterial genera: Pseudomonas, Bacillus, Staphylococcus, Acinetobacter, Kocuria and Micrococcus. Investigation of the factors associated with bacterial predominance revealed that, dominant culturable crude oil-degrading bacteria were better crude oil utilizers than the less frequently occurring isolates. Bacterial predominance was also influenced by the ability of bacteria to adapt to the level of organic content available. Predominant culturable bacteria constituted 89.7–54.2% of the total crude oil-degrading bacterial communities. Using 16S-RFLP analyses to assess the diversity of the dominant crude oil-degrading bacterial genera, four phylotypes of Pseudomonas sp. and seven phylotypes of Bacillus sp. were determined. This suggested high degree of diversity of crude oil-degrading bacterial population at the strain level, but low diversity at the genus level.     

A Laboratory Case Study of Efficient Polyhydoxyalkonates Production by Bacillus cereus,a Contaminant in Saccharophagus degradans ATCC 43961 in Minimal Sea Salt Media

A contaminating bacterium growing along with the stock culture of Saccharophagus degradans ATCC 43961 (Sde 2-40) on marine agar plate was isolated and investigated for its ability to produce polyhydoxyalkonates (PHA). Preliminary screening by Sudan black B and Nile blue A staining indicated positive characteristic of the isolate to produce PHA. The isolate was able to grow and produce PHA in minimal sea salt medium broth. PHA quantification studies with gas chromatographic analyses of the dry cells derived from culture broths revealed accumulation of PHA in bacterial cells. PHA production started after 20 h and increased with cell growth and attained maximum values of 61 % of dry cell weight at 70 h of cultivation. After 70 h, a slight decrease in the level of PHA content was observed. The nature/type of PHA was found to be poly(3-hydroxybutyraye) by Fourier transform-infrared spectroscopy. Microbiological and 16S rRNA gene sequencing analyses suggested that the PHA producing bacterial isolate belongs to Bacillus genera and shows 100 % nucleotide sequence similarity with Bacillus cereus species in GenBank. This study is a first report for ability of Bacillus species to grow in marine sea salt media and produce PHA. The media used for the polymer production was novel in the context of the genus Bacillus and the production of PHA was three-fold higher than Sde 2-40 using same growth medium. This study shows that the contaminant bacteria once properly investigated can be used for advantageous characteristic of metabolites production in place of original cultures.     

Heat treatment induced bacterial changes in irrigation water and their implications for plant disease management

A new heat treatment for recycled irrigation water using 48 °C for 24 h to inactivate Phytophthora and bacterial plant pathogens is estimated to reduce fuel cost and environmental footprint by more than 50 % compared to current protocol (95 °C for 30 s). The objective of this study was to determine the impact of this new heat treatment temperature regime on bacterial community structure in water and its practical implications. Bacterial communities in irrigation water were analyzed before and after heat treatment using both culture-dependent and -independent strategies based on the 16S ribosomal DNA. A significant shift was observed in the bacterial community after heat treatment. Most importantly, bacteria with biological control potential—Bacillus and Paenibacillus, and Pseudomonas species became more abundant at both 48 and 42 °C. These findings imply that the new heat treatment procedure not only controls existing plant pathogens but also may make the heat-treated irrigation water a more antagonistic environment against plant pathogens, promoting sustainable disease management.     

Variations in leaf morphology and DNA methylation following in vitro culture of Malus xiaojinensis

Juvenile trees of Malus xiaojinensis produce leaves with three to five lobes; however, adult trees do not. Thus, M. xiaojinensis serves as a model plant for studies on rejuvenation of woody perennials. To better understand effects of in vitro culture, number of passages, and plant growth regulators on observed somaclonal variations in leaf morphology, total levels of DNA methylation and expression of associated genes, including DRM2 and MET1, of apical shoot meristems of M. xiaojinensis, were evaluated. Following 17 passages of nodal stem segment culture and four passages of callus culture, levels of DNA methylation and expression of DRM2 and MET1 were determined, and found to decrease from the 11th passage of nodal stem segment culture or adventitious bud culture and from the first passage of callus culture. Levels of DNA methylation increased with increasing 2,4-dichlorophenoxyacetic acid concentrations, but decreased significantly with increasing 6-benzyladenine (BA) concentrations. Expression of DRM2 and MET1 in regenerated plantlets were negatively correlated with BA concentrations in the culture medium. Overall, less than 11 passages of nodal stem segment or adventitious bud culture did not contribute to incidence of somaclonal variation, even in the presence of low concentrations of BA (<0.5?mg/L) in the culture medium. Moreover, when nodal stem segment cultures were maintained for up to 17 passages, complete rejuvenation was observed.?However, epigenetic changes were detected following the first passage of callus cultures.     

Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures

Endophytic bacterial contamination is a major constraint to the establishment and maintenance of aseptic Aglaonema cultures. The objectives of the present study included the identification of endophytic bacterial contaminants from micropropagated Aglaonema cultures and the investigation of effective antibiotic treatment for their control. Bacterial contaminants isolated from 181 infected stem nodal explants of six Aglaonema cultivars were identified following the amplification of 16S ribosomal RNA gene and partial sequence analysis. A total of thirteen different bacterial species were identified and these were found to be mostly associated with soil and water. The bacteria were subsequently subjected to antimicrobial susceptibility and minimum inhibitory concentration tests. Three antibiotics, including gentamicin, tetracycline and chloramphenicol, were selected for their effectiveness at low concentrations of 4?C32?mg?l^?1 to inhibit bacterial growth in most of the bacterial species found in the present study. The incorporation of these antibiotics into the culture medium was found to effectively reduce the incidence of bacterial contamination in three of the four Aglaonema cultivars tested. Therefore, sanitation of the irrigation water and growth substrate while raising the stock plants, as well as the appropriate use of antibiotics during the in vitro culture stage will be important factors governing the success of Aglaonema micropropagation in the future.     

Isolation and characterization of Bacillus sp. strain BC01 from soil displaying potent antagonistic activity against plant and fish pathogenic fungi and bacteria

Fungal and bacterial pathogens infect a diverse range of hosts including various plant and animal species. Fungal and bacterial diseases, especially of plants and aquatic animals, such as fish, lead to significant damage to crops and aquaculture, respectively, worldwide. The present study was conducted to isolate and characterize potent Bacillus strains with significant antagonistic activity against the major plant and fish pathogenic fungi and bacteria. We randomly collected 22 isolates of Bacillus from the soil, rhizosphere, and sediment from different parts of Bangladesh. Initial characterization, based on in vitro antagonistic activity on the culture plate, resulted in the selection of four gram-positive Bacillus sp. isolates. Among these, the isolate BC01, obtained from soil demonstrated the highest broad-spectrum anti-bacterial and anti-fungal activities. We confirmed the genus of BC01 to be Bacillus by morphological and biochemical tests as well as using molecular data analysis tools, including the study of 16s rDNA, phylogenetic relationship, and evolutionary divergence scores. The isolate significantly inhibited the mycelial growth of the plant pathogen, Penicillium digitatum and fish pathogen, Aphanomyces invadans in vitro. The anti-bacterial effect of the isolate was also evaluated against Pseudomonas spp. and Xanthomonas spp., the two deadliest plant pathogens, and Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus iniae, three major fish pathogens that are primarily responsible for global aquaculture loss. The results of the present study could pave the way for developing potent drugs to combat microbial infection of plants and fish.     

A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.)

We have discovered a bacterial contaminant in some cell cultures of Datura innoxia (Mill.). The bacterium was tentatively identified as a species of Hyphomicrobium on the basis of its morphology and life cycle, and was isolated and grown in pure culture on a defined medium. The contaminant was not macroscopically observable in plant cell cultures. It caused neither a reduction of plant cell growth nor a noticeable increase in culture turbidity. Furthermore, it was not readily detectable by many standard assays for culture contamination: it would not grow alone in plant culture medium or yeast extract potato dextrose medium, and grew only very slowly on nutrient agar or beef-peptone medium. Repeated treatments with a combination of streptomycin (100 g/ml) and carbenicillin (100 g/ml) eliminated the contaminant from D. innoxia cell cultures without harming the plant cells.     

Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.     

Isolated Phosphate-Solubilizing Soil Bacteria Promotes In vitro Growth of Solanum tuberosum L.

The capacity of four bacterial strains isolated from productive soil potato fields to solubilize tricalcium phosphate on Pikovskaya agar or in a liquid medium was evaluated. A bacterial strain was selected to evaluate in vitro capacity of plant-growth promotion on Solanum tuberosum L. culture. Bacterial strain A3 showed the highest value of phosphate solubilization, reaching a 20 mm-diameter halo and a concentration of 350 mg/l on agar and in a liquid medium, respectively. Bacterial strain A3 was identified by 16S rDNA analysis as Bacillus pumilus with 98% identity; therefore, it is the first report for Bacillus pumilus as phosphate solubilizer. Plant-growth promotion assayed by in vitro culture of potato microplants showed that the addition of bacterial strain A3 increased root and stems length after 28 days. It significantly increased stem length by 79.3%, and duplicated the fresh weight of control microplants. In this paper, results reported regarding phosphorus solubilization and growth promotion under in vitro conditions represent a step forward in the use of innocuous bacterial strain biofertilizer on potato field cultures.Key words: Bacillus sp., phosphorus soluble, Pikovskaya agar, potato rhizosphere, plant growth promoting rhizobacteria     

Cultivable internal bacterial flora of ticks isolated in Hungary

From six sampling sites in north-western Hungary 126 questing ticks of three species (Ixodes ricinus, Dermacentor reticulatus, Haemaphysalis concinna) were sampled. After inactivating the external bacteria on the surface of ticks, the internal bacterial flora was cultured (two kinds of agar media at three temperatures with aerobic and anaerobic conditions were applied), and 116 strains were isolated. Our results showed, that after a blood meal the bacterial contamination of ticks was much higher in Ixodes and Dermacentor, than in Haemaphysalis, indicating different host range or blood meal habits. Most (89.7 %) of the bacteria were Gram-positive, the most frequent genera were the Staphylococcus (18.1 %) and Bacillus (7.8 %). The percentage of bacteria part of the skin and mucosal flora was 21.6 %. Among the environmental bacteria 14 were found with reported medical importance. The results show, that members of some genera are able to replicate inside the ticks (Mycobacterium, Bacillus) which can increase their potential risk. Isolated bacteria/tick ratio continuously grew from larvae to adults, indicating that larvae probably are hatched sterile, but later bacterial uptake from the environment and from the hosts increases bacterial contamination. Ten anaerobic bacteria were cultured, mostly Propionibacterium acnes, a facultative skin pathogen. No significant differences were found between the isolated bacteria of the six sampling sites. Our work showed, that internal bacterial community of ticks is diverse, novel strains were isolated several with medical importance, some bacteria multiplicate inside ticks, and that ticks continuously take up bacteria from the environment. Our study first described anaerobic bacteria from ticks.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications

The study was envisaged to assess the extent of normally uncultivable endophytic bacteria in field papaya plants and in vitro established cultures adopting cultivation vs molecular analysis and microscopy. Surface‐sterilized axillary shoot‐buds of papaya ‘Arka Surya’ revealed high bacterial diversity as per 16S rRNA metagene amplicon sequencing (6 phyla, 10 classes, 21 families) with an abundance of Pseudomonas (Gammaproteobacteria), which also formed a common contaminant for in vitro cultured field explants. Molecular analysis of seedling shoot‐tip‐derived healthy proliferating cultures of three genotypes (‘Arka Surya’, ‘Arka Prabhath’, ‘Red Lady’) with regular monthly subculturing also displayed high bacterial diversity (11–16 phyla, >25 classes, >50 families, >200 genera) about 12–18 months after initial establishment. ‘Arka Surya’ and ‘Red Lady’ cultures bore predominantly Actinobacteria (75–78%) while ‘Arka Prabhath’ showed largely Alphaproteobacteria corroborating the slowly activated Methylobacterium sp. Bright‐field direct microscopy on tissue sections and tissue homogenate and epi‐fluorescence microscopy employing bacterial DNA probe SYTO‐9 revealed abundant intracellular bacteria embracing the next‐generation sequencing elucidated high taxonomic diversity. Phylogenetic investigation of communities by reconstruction of unobserved states‐ PICRUSt‐ functional annotation suggested significant operational roles for the bacterial‐biome. Metabolism, environmental information processing, and genetic information processing constituted major Kyoto Encyclopedia of Genes and Genomes KEGG attributes. Papaya stocks occasionally displayed bacterial growth on culture medium arising from the activation of originally uncultivable organisms to cultivation. The organisms included Bacillus (35%), Methylobacterium (15%), Pseudomonas (10%) and seven other genera (40%). This study reveals a hidden world of diverse and abundant conventionally uncultivable cellular‐colonizing endophytic bacteria in field shoots and micropropagating papaya stocks with high genotypic similarity and silent participation in various plant processes/pathways.     

Rhizoplane microbiota of superior wheat varieties possess enhanced plant growth-promoting abilities

BACKGROUND

Microbes affect the growth of plants. In this study, the diversity and plant growth-supporting activities of wheat rhizospheric bacteria were examined.

METHODS

Sampling was performed thrice at different phases of plant growth. Microbes associated with the rhizoplane of three wheat varieties (Seher, Lasani, and Faisalabad) were cultured and assessed for their plant growth-promoting abilities based on auxin production, hydrogen cyanide production, phosphate solubilization, and nitrogen fixation.

RESULTS

Bacterial load (CFU/mL) declined, and the succession of bacterial diversity occurred as the plants aged. Most auxin-producing bacteria and the highest concentrations of auxin (77 μg/mL) were observed during the second sampling point at the tillering stage. The Seher variety harbored the most auxin-producing as well as phosphate-solubilizing bacteria. Most of the bacteria belonged to Bacillus and Pseudomonas. Planomicrobium, Serratia, Rhizobium, Brevundimonas, Stenotrophomonas, and Exiguobacterium sp. were also found.

CONCLUSION

These results suggest that the rhizoplane microbiota associated with higher-yield plant varieties have better plant growth-promoting abilities as compared to the microbiota associated with lower-yield plant varieties.

     

Culturable bacterial diversity from a feed water of a reverse osmosis system,evaluation of biofilm formation and biocontrol using phages

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.     

Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays

Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10⁶) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.     

Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 10²–10⁵ colony forming units g⁻¹ fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR^+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 10⁵ – 4.0 × 10⁷ colony forming units g⁻¹ tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.     

Isolation of endophytic bacteria from embryogenic suspension culture of banana and assessment of their plant growth promoting properties

Endophytic microorganisms have been reported from various plants. In the current study, somatic embryogenic cultures of banana (Musa accuminata AAA cv. Grand Naine) were found to have association with endophytic bacteria which were present initially in a covert state. The presence of bacteria was detected only in suspension cultures derived from the somatic embryogenic cultures. The bacteria isolated from embryogenic cell suspension culture were identified as Ralstonia sp. and Bacillus sp. The Ralstonia sp. interestingly showed the presence of various plant growth promoting properties including indole acetic acid and siderophore production. Also the strain was found to have the potential to solubilize phosphate and produce ammonia. Growth enhancement effect of Ralstonia sp. on Vigna radiata seedlings showed promising results and the growth parameters were found to be statistically significant when compared to control. Identification and confirmation of the plant growth promoting properties of Ralstonia sp. makes the study significant with promising applications.