RESUMEN

To establish the association between microbial indicators and the presence of foodborne pathogens in irrigation water, Escherichia coli was enumerated using two quantification methods (plate counts and PMA-qPCR) and presence/absence of pathogenic microorganisms, including five strains from the Shiga toxigenic E. coli (O157:H7, O26, O103, O111 and O145) and Salmonella spp. were evaluated. The results confirmed that surface water can be considered a microbial hazard when used for irrigation. The levels of viable E. coli were very similar to those of cultivable E. coli, except for irrigation water obtained from water reservoirs. Comparison between the E. coli counts in samples positive and negative for the presence of pathogenic bacteria for the evaluated water sources identified E. coli level of 2.35 log cfu/100 mL as a cut-off able to correctly predict positive and negative samples with 93% sensitivity and 66% specificity, respectively. Thus, for the samples with levels of E. coli under 2.35 log cfu/100 mL (e.g., 2.24 log cfu/100 mL) there was a 90% probability that the samples were not contaminated with pathogenic microorganism in locations with similar prevalence. E. coli levels in irrigation water were affected by the ambient temperature confirming that water source and climate conditions should be taken into account by growers when designing a sampling program and the frequency of the monitoring to make a better and more efficient use of their resources.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.