Molecular Profiling on Surface-Disinfected Tomato Seeds Reveals High Diversity of Cultivation-Recalcitrant Endophytic Bacteria with Low Shares of Spore-Forming Firmicutes.

Seeds are known to harbor diverse microorganisms offering protective effects on them with the prospects of quick root colonization at germination, selective recruitment as endophytes, and possible vertical transmission. The study was undertaken to assess the gross seed-internal bacterial community in tomato and to confirm if spore-forming Firmicutes constituted major seed endophytes adopting cultivation versus molecular approach on surface-sterilized seeds. Testing the initial seed wash solutions of "Arka Vikas" and "Arka Abha" cultivars showed > 1000 bacterial cfu per dry seed, largely Bacillus spp. Tissue homogenates from surface-disinfected seeds did not show any cultivable bacteria on enriched media for 1-2 weeks, while 16S rRNA V3-V4 taxonomic profiling revealed a huge bacterial diversity (10-16 phyla per cultivar). Proteobacteria formed the dominant phylum (65.7-69.6% OTUs) followed by Firmicutes, Actinobacteria, Bacteroidetes, and a notable share of Euryarchaeota (1.1-3.1%). Five more phyla appeared common to both cultivars in minor shares (Acidobacteria, Planctomycetes, Chloroflexi, Spirochaetes, Verrucomicrobia) with the ten phyla together constituting 99.6-99.9% OTUs. Class level and family level, the cultivars displayed elevated bacterial diversity, but similar taxonomic profiles. Arka Vikas and Arka Abha showed 114 and 107 genera, respectively, with 63 common genera constituting 96-97% OTUs. Psychrobacter formed the dominant genus. Bacillus and related genera constituted only negligible OTU share (0.16-0.28%). KEGG functional analysis showed metabolism as the major bacterial community role. One-month-old in vitro seedlings showed the activation of some originally uncultivable bacteria uninfluenced by the OTU share. The study reveals a high diversity of cultivation-recalcitrant endophytic bacteria prevailing in tomato seeds with possible vertical transmission and significant roles in plant biology.

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.

High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction, and unsuspected persistence.

MAIN CONCLUSION: Molecular and microscopic analyses reveal enormous non-cultivable endophytic bacteria in grapevine field shoots with functional significance. Diverse bacteria enter tissue cultures through surface-sterilized tissues and survive surreptitiously with varying taxonomic realignments. The study was envisaged to assess the extent of endophytic bacterial association with field shoot tissues of grapevine and the likelihood of introduction of such internally colonizing bacteria in vitro adopting molecular techniques targeting the non-cultivable bacterial community. PowerFood®-kit derived DNA from surface-sterilized field shoot tips of grapevine Flame Seedless was employed in a preliminary bacterial class-specific PCR screening proving positive for major prokaryotic taxa including Archaea. Taxonomic and functional diversity were analyzed through whole metagenome profiling (WMG) which revealed predominantly phylum Actinobacteria, Proteobacteria, and minor shares of Firmicutes, Bacteroidetes, and Deinococcus-Thermus with varying functional roles ascribable to the whole bacterial community. Field shoot tip tissues and callus derived from stem segments were further employed in 16S rRNA V3-V4 amplicon taxonomic profiling. This revealed elevated taxonomic diversity in field shoots over WMG, predominantly Proteobacteria succeeded by Actinobacteria, Firmicutes, Bacteroidetes, and 15 other phyla including several candidate phyla (135 families, 179 genera). Callus stocks also displayed broad bacterial diversity (16 phyla; 96 families; 141 genera) bearing resemblance to field tissues with Proteobacterial dominance but a reduction in its share, enrichment of Actinobacteria and Firmicutes, disappearance of some field-associated phyla and detection of a few additional taxonomic groups over field community. Similar results were documented during 16S V3-V4 amplicon taxonomic profiling on Thompson Seedless field shoot tip and callus tissues. Video microscopy on tissue homogenates corroborated enormous endophytic bacteria. This study elucidates a vast diversity of cultivation-recalcitrant endophytic bacteria prevailing in grapevine field shoots, their in vitro introduction, and unsuspecting sustenance with possible silent participation in tissue culture processes.

Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria.

The interior of plants constitutes a unique environment for microorganisms with various organisms inhabiting as endophytes. Unlike subterranean plant parts, aboveground parts are relatively less explored for endophytic microbial diversity. We employed a combination of cultivation and molecular approaches to study the endophytic bacterial diversity in banana shoot-tips. Cultivable bacteria from 20 sucker shoot-tips of cv. Grand Naine included 37 strains under 16 genera and three phyla (Proteobacteria, Actinobacteria, Firmicutes). 16S rRNA gene-ribotyping approach on 799f and 1492r PCR-amplicons to avoid plant organelle sequences was ineffective showing limited bacterial diversity. 16S rRNA metagene profiling targeting the V3-V4 hypervariable region after filtering out the chloroplast (74.2 %), mitochondrial (22.9 %), and unknown sequences (1.1 %) revealed enormous bacterial diversity. Proteobacteria formed the predominant phylum (64 %) succeeded by Firmicutes (12.1 %), Actinobacteria (9.5 %), Bacteroidetes (6.4 %), Planctomycetes, Cyanobacteria, and minor shares (<1 %) of 14 phyla including several candidate phyla besides the domain Euryarchaeota (0.2 %). Microbiome analysis of single shoot-tips through 16S rRNA V3 region profiling showed similar taxonomic richness and diversity and was less affected by plant sequence interferences. DNA extraction kit ominously influenced the phylogenetic diversity. The study has revealed vast diversity of normally uncultivable endophytic bacteria prevailing in banana shoot-tips (20 phyla, 46 classes) with about 2.6 % of the deciphered 269 genera and 1.5 % of the 656 observed species from the same source of shoot-tips attained through cultivation. The predominant genera included several agriculturally important bacteria. The study reveals an immense ecosystem of endophytic bacteria in banana shoot tissues endorsing the earlier documentation of intracellular "Cytobacts" and "Peribacts" with possible roles in plant holobiome and hologenome.

Use of Plant Preservative Mixture™ for establishing in vitro cultures from field plants: Experience with papaya reveals several PPM™ tolerant endophytic bacteria.

KEY MESSAGE: Prevalence of diverse PPM™-tolerant endophytic bacteria in papaya, the broad-spectrum microbicide specified for use in plant tissue cultures, capable of surviving covertly in MS-based medium, with implications in contamination management. Plant Preservative Mixture™ was employed for establishing papaya (Carica papaya) tissue cultures from field explants. Comparing three recommended practices for controlling endogenous microbial contaminants, axillary shoot tips (1.0-1.5 cm) from cv. Arka Prabhath were treated with PPM™ 5% for 4 h (T1), 50% for 10 min (T2) or 100% for 10 min (T3) and cultured in MS-based papaya establishment medium (PEM). By 4-6 weeks, all treatments proved non-rewarding with cultures succumbing either to microbial contamination (80% in T1) or phytotoxicity effect/contamination (90% in T2 and 95% in T3). Another trial adopting a multi-step surface sterilization treatment (carbendazim-cetrimide-HgCl2) followed by culturing in 0.05% PPM-supplemented PEM showed 35% obvious bacterial contamination compared with 40% in control. Single colonies from pooled bacterial growths were tested on 0.1% PPM-incorporated nutrient agar (NA) registering 60% isolates as PPM sensitive. Twenty PPM-surviving isolates were selected and identified. This showed 85% Gram-positive bacteria including 80% under phylum Firmicutes (55% spore-forming Bacillaceae and 25% Staphylococcaceae) and 5% Actinobacteria, and 15% Gram-negative Proteobacteria. About 50% isolates remained wholly non-obvious upon culturing on PEM while the rest showed slow growth with many displaying growth enhancement upon host tissue extract supplementation. Culturing the isolates on PPM-supplemented NA indicated 90-95% as tolerating 0.05-0.1% PPM and 65% overriding 0.2% PPM. The isolates, however, did not display obvious growth in PPM-supplemented PEM where the spore formers survived. The results indicate the prevalence of diverse PPM™-tolerant endophytic bacteria in papaya most of which survive covertly in MS-based medium and the need for taking this into account while using PPM™ for contamination management.

Improved recovery of Bacillus spores from nonporous surfaces with cotton swabs over foam, nylon, or polyester, and the role of hydrophilicity of cotton in governing the recovery efficiency.

Evaluating different swabbing materials for spore recovery efficiency (RE) from steel surfaces, we recorded the maximum RE (71%) of 10(7) Bacillus subtilis spores with Tulips cotton buds, followed by Johnson's cotton buds and standard Hi-Media cotton, polyester, nylon, and foam (23%) swabs. Among cotton swabs, instant water-absorbing capacity or the hydrophilicity index appeared to be the major indicator of RE, as determined by testing three more brands. Tulips swabs worked efficiently across diverse nonporous surfaces and on different Bacillus spp., registering 65 to 77% RE.

Long-term survival of Bacillus spores in alcohol and identification of 90% ethanol as relatively more spori/bactericidal.

This study was taken up with a view to generate basic information on spore hardiness to ethanol in various Bacillus species and related genera, and to assess the effectiveness of different levels of ethanol as a bacterial disinfectant. Predominantly spore-bearing cultures of five Bacillus spp. (B. pumilus, B. subtilis, B. megaterium, B. fusiformis and B. flexus) that were isolated from the spent-alcohol used during plant tissue culture work were challenged with aqueous ethanol (25, 50, 60, 70, 80 and 90% v/v) in 1 ml volumes at 10¹°â»¹¹ CFU ml⁻¹. Monitoring the spore endurance through spotting and plating revealed prolonged tolerance (>12 months) at different alcohol levels depending on the organism except in 90% where no survival was observed beyond 2-12 months. Spores of related genera like Paenibacillus and Lysinibacillus also showed long-term ethanol survival. Alcohol tolerance of spore-forming organisms depended on the extent of spores and spore hardiness, which in turn varied with the organism, strain, age of culture, growing conditions and other factors as authenticated with ATCC strains of B. pumilus and B. subtilis. Aqueous 90% ethanol caused instant inactivation of vegetative cells in different spore formers and twelve other non-sporulating Gram-positive and Gram-negative organisms tested. Taking into account both vegetative cells and spores, the appropriate concentration of ethanol as a disinfectant emerged to be 90% followed by absolute ethanol compared with the generally recommended 70-80% level.

Cytobacts: Abundant and Diverse Vertically Seed-Transmitted Cultivation-Recalcitrant Intracellular Bacteria Ubiquitous to Vascular Plants.

We have recently described 'Cytobacts' as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for 'Cytobacts' through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of 'Cytobacts' in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3-V4 amplicon profiling on shoot-tip tissues of four representative plants-tomato, watermelon, periwinkle, and maize-showed high bacterial abundance and taxonomic diversity (11-15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3-V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document 'Cytobacts' comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.

Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures.

The study was undertaken with a view to unravel the source of bacterial colony growth observed in a section of micropropagated triploid watermelon cultures that were supposedly cleansed of the associated endophytic bacteria through antibiotic treatment, and thereafter maintained under stringent sterility checks to prevent lateral intrusion of contaminants. Five different bacteria were retrieved from colony growth-displaying watermelon cultures that were previously treated with gentamycin and five isolates from cefazolin-treated stocks with the organisms showing tolerance to the respective antibiotic. These watermelon cultures were in degeneration phase (over 6 months after the previous sub-culturing), while the actively maintained counterpart stocks appeared healthy with no colony growth on different bacteriological media during tissue-screenings. The latter cultures, however, revealed abundant motile, tetrazolium-stained bacterial cells in microscopy, suggesting tissue colonization by non-culturable endophytes. PCR screening on healthy cultures endorsed tissue colonization by different bacterial phylogenic groups. A few organisms could be activated to cultivation from healthy watermelon stocks through host tissue extract supplementation, which also enhanced the growth of all the organisms. The study indicated that a fraction of antibiotic-tolerant bacteria survived intra-tissue in non-culturable form during the preceding cleansing activity, multiplied to substantial numbers thereafter, and turned cultivable in degenerating cultures contributed by tissue breakdown products. This study brings out the existence of a deep endophyte association in tissue cultures which is not easily dissociable. It also signifies the utility of in vitro system for investigations into plant-endophyte association and to bring normally non-culturable novel organisms to cultivation facilitating their future exploitation.

Vertical Transmission of Diverse Cultivation-Recalcitrant Endophytic Bacteria Elucidated Using Watermelon Seed Embryos.

Seed transmission of endophytic microorganisms is a growing research area in plant biology and microbiology. We employed cultivation versus cultivation-independent approaches on excised embryos from watermelon seeds (6-12 months in storage) and on embryo-derived in vitro seedlings (EIVS) to assess the vertical transmission of endophytic bacteria. Surface-disinfected watermelon seeds bore abundant residual bacteria in the testa and perisperm tissues, predominantly Bacillus spp. propounding the essentiality of excluding all non-embryonic tissues for vertical transmission studies. Tissue homogenates from re-disinfected seed embryos displayed no cultivable bacteria during the 1-week monitoring. Bright-field live microscopy revealed abundant bacteria in tissue homogenates and in embryo sections as intracellular motile particles. Confocal imaging on embryo sections after SYTO-9 staining and eubacterial fluorescent in situ hybridization (FISH) endorsed enormous bacterial colonization. Quantitative Insights Into Microbial Ecology (QIIME)-based 16S rRNA V3-V4 taxonomic profiling excluding the preponderant chloroplast and mitochondrial sequences revealed a high bacterial diversity in watermelon seed embryos mainly Firmicutes barring spore formers followed by Proteobacteria, Bacteroidetes, and Actinobacteria, and other minor phyla. Embryo-base (comprising the radicle plus plumule parts) and embryo-cotyledon parts differed in bacterial profiles with the abundance of Firmicutes in the former and Proteobacteria dominance in the latter. EIVS displayed a higher bacterial diversity over seed embryos indicating the activation from the dormant stage of more organisms in seedlings or their better amenability to DNA techniques. It also indicated embryo-to-seedling bacterial transmission, varying taxonomic abundances for seed embryos and seedlings, and differing phylogenic profiles for root, hypocotyl, and cotyledon/shoot-tip tissues. Investigations on different watermelon cultivars confirmed the embryo transmission of diverse cultivation recalcitrant endophytic bacteria. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes formed the core phyla across different cultivars with 80-90% similarity at genus to phylum levels. Conversely, freshly harvested seeds displayed a dominance of Proteobacteria. The findings revealed that dicot seeds such as in different watermelon cultivars come packaged with abundant and diverse vertical and seedling-transmissible cultivation recalcitrant endophytic bacteria with significant implications for plant biology.

Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures.

This study was initiated to assess whether the supposedly axenic plant cell cultures harbored any cultivation-recalcitrant endophytic bacteria (CREB). Adopting live-cell imaging with bright-field, fluorescent and confocal microscopy and bacterial 16S-rRNA gene taxonomic profiling, we report the cytoplasmic association of abundant and diverse CREBs in long-term actively maintained callus and cell suspension cultures of different plant species. Preliminary bright-field live-cell imaging on grape cell cultures showed abundant intracellular motile micro-particles resembling bacteria, which proved uncultivable on enriched media. Bacterial probing employing DNA stains, transmission electron microscopy, and Eubacterial FISH indicated abundant and diverse cytoplasmic bacteria. Observations on long-term maintained/freshly established callus stocks of different plant species-grapevine, barley, tobacco, Arabidopsis, and medicinal species-indicated intracellular bacteria as a common phenomenon apparently originating from field shoot tissues.Cultivation-independent 16S rRNA gene V3/V3-V4 amplicon profiling on 40-year-old grape cell/callus tissues revealed a high bacterial diversity (>250 genera), predominantly Proteobacteria, succeeded by Firmicutes, Actinobacteria, Bacteriodetes, Planctomycetes, and 20 other phyla, including several candidate phyla. PICRUSt analysis revealed diverse functional roles for the bacterial microbiome, majorly metabolic pathways. Thus, we unearth the widespread association of cultivation-recalcitrant intracellular bacteria "Cytobacts" inhabiting healthy plant cells, sharing a dynamic mutualistic association with cell hosts.

Endophytic bacteria associated with growing shoot tips of banana (Musa sp.) cv. Grand Naine and the affinity of endophytes to the host.

A cultivation-based assessment of endophytic bacteria present in deep-seated shoot tips of banana suckers was made with a view to generate information on the associated organisms, potential endophytic contaminants in tissue-cultured bananas and to assess if the endophytes shared a beneficial relationship with the host. Plating the tissue homogenate from the central core of suckers showed colony growth on nutrient agar from just 75% and 42% of the 12 stocks during May and November, respectively (average 58%; 6 x 10(3) colony-forming units per gram), yielding diverse organisms belonging to firmicutes (Bacillus, Brevibacillus, Paenibacillus, Virgibacillus, Staphylococcus spp.), actinobacteria (Cellulomonas, Micrococcus, Corynebacterium, Kocuria spp.), alpha-proteobacteria (Paracoccus sp.), and gamma-proteobacteria (Pseudomonas, Acinetobacter spp.). Each shoot tip showed one to three different organisms and no specific organism appeared common to different sucker tips. Tissue homogenate from shoot tips including the ones that did not yield culturable bacteria displayed abundant bacterial cells during microscopic examination suggesting that a high proportion of cells were in viable-but-nonculturable state, or their cultivation requirements were not met. Direct application of cultivation-independent approach to study endophytic bacterial community using bacterial 16S ribosomal RNA universal primers resulted in high interference from chloroplast and mitochondrial genome sequences. Dislodging the bacterial cells from shoot tips that did not show cultivable bacteria and incubating the tissue crush in dilute-nutrient broth led to the activation of four organisms (Klebsiella, Agrobacterium, Pseudacidovorax spp., and an unidentified isolate). The endophytic organisms in general showed better growth at 30-37 degrees C compared with 25 degrees C, and the growth of endophytes as well as pathogenic Erwinia carotovora were promoted with the supply of host tissue extract (HTE) while that of the isolates from nonplant sources were inhibited or unaffected by HTE, suggesting an affinity or dependence of the endophytes on the host and the prospect of an HTE-based assay for discriminating the nonendophytes from endophytes.

In Vitro Activation of Seed-Transmitted Cultivation-Recalcitrant Endophytic Bacteria in Tomato and Host⁻Endophyte Mutualism.

This study was aimed at exploring seed transmission of endophytic bacteria in tomato utilizing aseptic in vitro conditions. Cultivation-based studies were undertaken on two tomato cultivars "Arka Vikas" and "Arka Abha" employing surface sterilized seeds, aseptically germinated seeds and in vitro grown seedlings at different stages. Bacillus sp. appeared primarily as seed externally-associated bacteria. Tissue homogenate from extensively surface-sterilized seeds, day-3 germinating seeds, or 10-day in vitro seedlings did not show any cultivable bacteria on two bacteriological media. Indexing of 4-week old healthy seedlings with seed-coat removal following seed germination showed bacterial association in 50-75% seedlings yielding 106-107 cfu g-1 tissues. Four endophytic bacteria appeared common to both cultivars (Kosakonia, Ralstonia, Sphingomonas, Sphingobium spp.) with three additional species in "Arka Abha". The bacterial strains showed a manifold increase in growth with host-tissue-extract supplementation. Seed inoculations with single-isolates stimulated germination or enhanced the seedling growth coupled with the activation of additional endophytic bacteria. In vitro seedlings upon recurrent medium-indexing over eight weeks showed gradual emergence of endophytic bacteria. The study reveals the seed internal colonization by different bacterial endophytes in a cultivation-recalcitrant form, their activation to cultivable state during seedling growth and transmission to seedlings with mutualistic effects.

Effects Due to Rhizospheric Soil Application of an Antagonistic Bacterial Endophyte on Native Bacterial Community and Its Survival in Soil: A Case Study with Pseudomonas aeruginosa from Banana.

Effective translation of research findings from laboratory to agricultural fields is essential for the success of biocontrol or growth promotion trials employing beneficial microorganisms. The rhizosphere is to be viewed holistically as a dynamic ecological niche comprising of diverse microorganisms including competitors and noxious antagonists to the bio-inoculant. This study was undertaken to assess the effects due to the soil application of an endophytic bacterium with multiple pathogen antagonistic potential on native bacterial community and its sustenance in agricultural soil. Pseudomonas aeruginosa was employed as a model system considering its frequent isolation as an endophyte, wide antagonistic effects reported against different phytopathogens and soil pests, and that the species is a known human pathogen which makes its usage in agriculture precarious. Employing the strain 'GNS.13.2a' from banana, its survival in field soil and the effects upon soil inoculation were investigated by monitoring total culturable bacterial fraction as the representative indicator of soil microbial community. Serial dilution plating of uninoculated control versus P. aeruginosa inoculated soil from banana rhizosphere indicated a significant reduction in native bacterial cfu soon after inoculation compared with control soil as assessed on cetrimide- nalidixic acid selective medium against nutrient agar. Sampling on day-4 showed a significant reduction in P. aeruginosa cfu in inoculated soil and a continuous dip thereafter registering >99% reduction within 1 week while the native bacterial population resurged with cfu restoration on par with control. This was validated in contained trials with banana plants. Conversely, P. aeruginosa showed static cfu or proliferation in axenic-soil. Lateral introduction of soil microbiome in P. aeruginosa established soil under axenic conditions or its co-incubation with soil microbiota in suspension indicated significant adverse effects by native microbial community. Direct agar-plate challenge assays with individual environmental bacterial isolates displayed varying interactive or antagonistic effects. In effect, the application of P. aeruginosa in rhizospheric soil did not serve any net benefit in terms of sustained survival. Conversely, it caused a disturbance to the native soil bacterial community. The findings highlight the need for monitoring the bio-inoculant(s) in field-soil and assessing the interactive effects with native microbial community before commercial recommendation. varying interactive or antagonistic effects. In effect, the application of P. aeruginosa in rhizospheric soil did not serve any net benefit in terms of sustained survival. Conversely, it caused a disturbance to the native soil bacterial community. The findings highlight the need for monitoring the bio-inoculant(s) in field-soil and assessing the interactive effects with native microbial community before commercial recommendation.

Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects.

This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8-9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen.

Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples.

We propose a simple technique for bacterial and yeast cfu estimations from diverse samples with no prior idea of viable counts, designated as single plate-serial dilution spotting (SP-SDS) with the prime recommendation of sample anchoring (100 stocks). For pure cultures, serial dilutions were prepared from 0.1 OD (100) stock and 20 µl aliquots of six dilutions (101-106) were applied as 10-15 micro-drops in six sectors over agar-gelled medium in 9-cm plates. For liquid samples 100-105 dilutions, and for colloidal suspensions and solid samples (10% w/v), 101-106 dilutions were used. Following incubation, at least one dilution level yielded 6-60 cfu per sector comparable to the standard method involving 100 µl samples. Tested on diverse bacteria, composite samples and Saccharomyces cerevisiae, SP-SDS offered wider applicability over alternative methods like drop-plating and track-dilution for cfu estimation, single colony isolation and culture purity testing, particularly suiting low resource settings.

Significant Effects Due to Peptone in Kelman Medium on Colony Characteristics and Virulence of Ralstonia solanacearum in Tomato.

The study was taken up to assess if the media constituents played any role in governing the variable colony characteristics or pathogenicity of the bacterial wilt pathogen, Ralstonia solanacearum cultured on the widely employed Kelman medium. The effects due to the constituents 2,3,5-triphenyl tetrazolium chloride (TTC), peptone, casein hydrolysate and glucose on colony characteristics were investigated using -80°C stored culture of strain 'NH-Av01' (race 1, biovar 3) isolated from tomato. Comparing the pigment inducing TTC from two brands, its source or mode of storage/incorporation did not impart any significant effects. The source of peptone, on the other hand, displayed striking effects on the extent of colony growth, fluidity and red pigmentation depending on type, brand or batch / lot of manufacture as documented with 20 different formulations. Significant differences in the pathogenicity of isolate derived from different peptone sources in seedling-challenge assay on tomato were observed. The observations on peptone effects were endorsed with four other isolates belonging to distinct geographic locations, crops (eggplant, chilli, ginger) or races (race 1 or 4). The peptone source did not influence the pathogen-responses in biovar tests but notably altered the pattern of lawn formation and inhibition zone development during antagonistic assays. Casein hydrolysate displayed some variable effects while glucose source had no effect. This study brings to light the significant modifying effects by the peptone-constituent in Kelman medium on the physiology of R. solanacearum and the virulence of isolate and the need to consider the source of media components during culture maintenance, host-pathogen interaction studies or microbe-microbe interaction investigations.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria.

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50-100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed 'Brownian motion'-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host-endophyte interaction in vitro and in vivo.

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (approximately 1 cm) of papaya (Carica papaya L. 'Surya') planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2-4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea (P. ananatis), Enterobacter (E. cloacae), Brevundimonas (B. aurantiaca), Sphingomonas, Methylobacterium (M. rhodesianum), and Agrobacterium (A. tumefaciens) or two Gram-positive genera, Microbacterium (M. esteraromaticum) and Bacillus (B. benzoevorans) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550=0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.

Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya.

This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35-50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14-17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.