A review of issues related to measuring colonization of plant roots by bacteria

 Canadian Journal of Microbiology, 1992, 38(12): 1219-1232, https://doi.org/10.1139/m92-202

ABSTRACT :. We consider true root colonists to be those bacteria that colonize roots in competitive conditions, i.e., natural field soils. Different methods of processing root samples are required if one is measuring external root colonization alone, internal colonization alone, or both. Given that most beneficial bacterial strains currently under investigation as root colonists are members of taxa naturally found in soils, a marking system is required to differentiate the introduced strain from members of the indigenous rhizosphere community. Spontaneous antibiotic resistance, immunological approaches, and foreign DNA sequences are among the marking systems that have been used and each has some possible advantages and disadvantages. More research is needed in the development and comparison of marking systems. The design of experiments to measure root colonization should take into account several statistical issues. One must decide what constitutes the sample unit for each replication of a given treatment, e.g., whole root systems or root segments. Consideration should also be given to how best to express the estimated population of root colonists (e.g., cfu/g fresh or dry weight root, cfu/cm root, or cfu/surface area root). Statistical analysis by standard analysis of variance tests should be used whenever possible to separate treatment means of colonization levels; however, one must determine that the underlying assumptions of these tests are correct for each experiment. Finally, in quantification of populations on roots, one will almost certainly encounter replications with no bacteria, i.e., zeros. 

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

Citation: Thomas P, Sekhar AC. 2014. Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria. AoB PLANTS 6: plu002; doi:10.1093/aobpla/plu002

Abstract. 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 mm) 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. PDF enclosed for further reading..

Living inside plants: bacterial endophytes

.https://doi.org/10.1016/j.pbi.2011.04.0

Abstrcat; As current research activities have focused on symbiotic or parasitic plant–microbe interactions, other types of associations between plants and microorganisms are often overlooked. Endophytic bacteria colonize inner host tissues, sometimes in high numbers, without damaging the host or eliciting strong defense responses. Unlike endosymbionts they are not residing in living plant cells or surrounded by a membrane compartment. The molecular basis of endophytic interactions is still not well understood. Several traits involved in the establishment of endophytes have been elucidated. Culture-independent methods for community analysis and functional genomic as well as comparative genomic analyses will provide a better understanding of community dynamics, signaling, and functions in endophyte–plant associations...PDF enclosed for further reading...

Plant-microbe interaction studies, including plant colonization by microbes, have benefitted from the development of high-throughput molecular methods, such as metagenomics and metatranscriptomics

CLSM images are presented as either maximum projections or single optical slides. Therefore, it is necessary to know the thickness of the confocal stack as well as the Z-step dimension, in the case of maximum projections, for interpretating the images. In particular this is critical for studying endophytism, physical interactions and spatial arrangement of microbial populations. In the case of single optical slices, the thickness of sections should also be mentioned. If colonization is scant, low resolution may contribute to a misunderstanding of signals in the images unless critical interpretation confirms bacterial signal. Size and shape characters help to distinguish bacterial cells from autofluorescent objects in the same emission range. Conspecific microbial colonies are then recognized by discernible single cells with shared phenotype.

Visualization tools are available for sophisticated analyses and improved interpretation of image data. As an example, the colonization pattern and the endophytism of the PGPR Burkholderia terricola ZR2-12  in the root system of sugar beet are impossible to assess in the maximum projection but become apparent only in the volume rendering three-dimensional modeling, and its cutting plane Such operations are possible with freeware Image .

Article Introduction to Plant-Microbe Interactions

Chapter Principles of Plant-Microbe Interactions