[compost_tea] Re: Observing Microbial Biofilms on Leaf Surfaces + Isolation of Culturable Microbes

From: bobkacalek <bob_at_soil-life.com>
Date: Sat, 27 Mar 2004 00:33:42 -0000

Hi Steve,
Thanks for sharing, very interesting.
The advantage of quality ACT is the abundance of diversity in Micro-
organisms delivered vs one or two specific cultures.
Are the methods described (Epifluorescence microscopy, scanning
electron microscopy, and confocal laser scanning microscopy were used
to observe microbial biofilms directly on leaf surfaces)similar to
methods used by SFI? Maybe Dr. E will respond?
Thanks Again,
bob


--- In compost_tea_at_yahoogroups.com, "Steve Diver" <steved_at_n...> wrote:
> APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
> Apr. 1997, p. 1570–1576
>
> Methods for Observing Microbial Biofilms Directly on Leaf Surfaces
> and Recovering Them for
>
> http://nature.berkeley.edu/icelab/jmm/aem97.pdf
>
> CINDY E. MORRIS, JEAN-MICHEL MONIER, AND MARIE-AGNE`S JACQUES
>
> Station de Pathologie Ve´ge´tale, INRA, Domaine St. Maurice,
> France
>
>
> ABSTRACT
>
> Epifluorescence microscopy, scanning electron microscopy, and
confocal
> laser scanning microscopy were used to observe microbial biofilms
> directly on leaf surfaces. Biofilms were observed on leaves of all
> species sampled (spinach, lettuce, Chinese cabbage, celery, leeks,
> basil, parsley, and broad-leaved endive), although the
epifluorescent
> images were clearest when pale green tissue or cuticle pieces were
> used. With these techniques, biofilms were observed that were about
> 20 mm in depth and up to 1 mm in length and that contained copious
> exopolymeric matrices, diverse morphotypes of microorganisms, and
> debris. The epifluorescence techniques described here can be used to
> rapidly determine the abundance and localization of biofilms on
> leaves. An additional technique was developed to recover individual
> biofilms or portions of single biofilms from leaves and to
> disintegrate them for isolation of the culturable microorganisms
> they contained. Nineteen biofilms from broad-leaved endive, spinach,
> parsley, and olive leaves were thus isolated and characterized to
> illustrate the applications of this technique.
>
>
> Excerpts:
>
>
> INTRODUCTION
>
> It is well documented that leaves of herbaceous and woody
> plants are colonized by numerous species of bacteria, yeasts,
> and filamentous fungi...
>
>
> In many environments, it is common to find assemblages of
> microorganisms adherent to each other and/or to a surface and
> embedded in a matrix of exopolymers (8). These assemblages
> are referred to as biofilms...
>
>
> (i) Biofilms are composed of an exopolymeric matrix and multiple
> layers of microbial cells leading to the creation of physical
> barriers and the establishment of chemical gradients. (ii) They
> generally contain multiple species of microorganisms, fostering
> metabolic and genetic exchange. (iii) Many biofilm cells are
> attached to a surface...
>
>
>
> MATERIALS AND METHODS
>
> Plant material. Leaves were sampled from commercially produced
> spinach (Spinacia oleracea), Battavian lettuce (Lactuca sativa),
> Chinese cabbage (Brassica campestris subsp. pekinensis),
> celery (Apium graveolens), leeks (Allium porrum), basil
> (Ocimum basilicum), and parsley (Petroselinum crispum) purchased
> at a local supermarket. Leaves of broad-leaved endive (Cichorium
> endivia var. latifolia, cultivar Samy) were collected from mature
> plants in experimental field plots cultivated as described
> previously (18) in Montfavet, France, or purchased at a
> supermarket...
>
>
> Preparation of plant material for microscopic observation. Leaves
> of all plants except parsley were cut into squares of approximately
> 1 by 1 cm. For some leaves of broad-leaved endive, the cuticle
> was also peeled off. Parsley leaflets were cut off the petioles
> but were not cut into pieces. For epifluorescence microscopy,
> leaflets, leaf pieces, or cuticle pieces were soaked in a solution
> of acridine orange (0.01% in acetate buffer [pH 4]) for 2 min,
> rinsed twice in distilled water, set on microscope slides, and
> observed with an Olympus BHS microscope (Olympus Optical, Co.,
> Tokyo, Japan; light source model BH.RFL.W) under UV light
> filtered through a blue filter...
>
>
>
> RESULTS
>
> In situ observation. Aggregates of microorganisms embedded
> in an exopolymeric matrix were observed on all leaf material
> stained with acridine orange and examined by epifluorescence
> light microscopy, including broad-leaved endive (Fig.
> 1), spinach, lettuce, Chinese cabbage, celery, leeks, basil,
> and parsley. Epifluorescence microscopic images of biofilms on
> broad-leaved endive were of the best resolution either when
> the cuticle was peeled from the leaf surface and mounted
> under a coverslide (Fig. 1A) or when biofilms were located on
> flat, etiolated portions of the leaf....
>
>
> Biofilms were observed on all parts of the leaves (margin,
> base, and upper and lower surfaces). In the case of broadleaved
> endive, we observed about 100 biofilms. These biofilms
> were found most often at the leaf base and were rarely on the
> margin of green leaf tissue. About 90% of them were attached
> to trichomes (Fig. 1A and C)....
>
>
> SEM examination of leaf surfaces confirmed that biofilms
> could be readily observed on naturally contaminated leaf
> surfaces. Biofilms observed by SEM were as large as 1 mm
> in length (Fig. 2) or were in networks extending for several
> millimeters and were composed of a copious exopolymer matrix
> and numerous microbial morphotypes resembling bacteria,
> filamentous fungi, and yeasts as well as debris-like particles.
>
>
>
> DISCUSSION
>
> The techniques reported here permitted us to illustrate that
> biofilms in the phyllosphere may be tens of micrometers thick,
> may have a copious exopolymeric matrix, and may form extensive
> networks several millimeters long....
>
>
> The isolation technique described in this study allows for
> collection of a wide range of bacteria and other microorganisms
> coexisting in close physical proximity...
>
>
> Using the technique presented here, we have illustrated that
> diverse microorganisms, including both gram-positive and
> gram-negative bacteria as well as yeasts and filamentous fungi,
> may inhabit the same biofilm in the phyllosphere as has been
> observed in other environments (4, 22, 28)...
>
>
> The isolation technique we describe here will allow phyllosphere
> ecologists to determine which microorganisms are frequently
> associated in biofilms. This may lead to the identification of
> saprophytic organisms that play a role in plant disease processes
> incited by phytopathogenic organisms with which they are associated
> or may help to explain mechanisms of genetic exchange observed
> on leaf surfaces...
>
>
> Characterization of the microbial components of individual
> biofilms in the phyllosphere may lead to new hypotheses about
> trophic interactions or chemical communication among these
> organisms.
>
> =========================
==========================
==========
> =========================
==========================
==========
>
> SD comment:
>
> Compost teas have significant influence on microorganism
> populations in the rhizophere and phyllosphere.
>
> While biofilms are problematic in compost tea brewers,
> they are normal and beneficial in the phyllosphere.
>
> This paper elucidates direct observation of phyllosphere
> organisms, and alludes to culture methods to isolate
> microorganisms.
>
> Isolation of soil and compost microbes for identification
> by gas chromatography, following by culturing of selected
> beneficial strains in fermentation flasks, then adding back
> into compost teas prior to field application, as a liquid
> biological injection into irrigation lines, has been done
> by Midwest BioSystems in conjunction with Agri-Energy
> Resources Lab, for example.
>
> Steve Diver



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Received on Fri Mar 26 2004 - 21:06:33 EST

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