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

Apr. 1997, p. 1570?1576

Methods for Observing Microbial Biofilms Directly on Leaf Surfaces 
and Recovering Them for	



Station de Pathologie Ve´ge´tale, INRA, Domaine St. Maurice,


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.



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...


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 

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...


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.


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 


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 

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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