Title: Collection of palm samples for electron microscopic examination
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Title: Collection of palm samples for electron microscopic examination
Physical Description: Book
Creator: Norris, R. C.
Publisher: Agricultural Research and Education Center, IFAS, University of Florida
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COLLECTION OF PALM SAMPLES
FOR ELECTRON MICROSCOPIC
EXAMINATION
R. C. Norris and R. E. McCoy
University of Florida, IFAS
Agricultural Research and Education Center
3205 S. W. College Avenue
Fort Lauderdale, Florida 33314 U. S. A.


[U[ME i R ARY
J.F.A.S.- Unv. of Florid
!.F.A.S.-Univ. of Florida


-REC Fort Lauderdale -Research Report FL-82-4


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Coconut and African oil palms are
subject to a variety of systemic lethal
diseases. Two different presumed
pathogens of these palms in the Carib-
bean region are mycoplasmalike
organisms (MLO) (Fig. 1) and flagel-
lated protozoa (Fig. 2). MLO are
associated with lethal yellowing
disease of coconut and an additional
30 or more species of palms. Phytomonas
staheli, a flagellated protozoan of the
family Trypanosomatidae, is associated
with both hartrot disease of coconut
palm and marchitez sorpresiva disease
of African oil palm. Despite distinct
differences in the associated micro-
organisms, lethal yellowing, hartrot,
and marchitez sorpresiva share
strikingly similar symptomatology.
They are all lethal diseases, killing
palms within four months of the onset
of symptoms. Symptoms include loss of
fruit, necrosis of inflorescences,
spear leaf collapse, and a subsequent
rot of the growing point.

Both MLO and Phytomonas may be
distributed unevenly and with highly
variable concentrations in palm tissue.
This, coupled with the large size of
mature palms and the small portion that
can be examined with an electron
microscope, limits the efficiency of
this method of examination. Never the
less, at the present time electron
microscopic examination is the only
method of confirming the presence of
suspected MLO in symptomatic palms.

Techniques for collecting palm
samples for electron microscopic exam-
ination are given in this report. These
procedures apply when collecting
samples from palms affected by the three
diseases mentioned above or diseases of
unknown etiology. However, electron
microscopy is not suitable as a rapid
diagnostic technique and should be used
only after potential problems caused by
fungi, bacteria, nematodes, and mites
are first eliminated by simpler
k diagnostic techniques.


Fig. 1. Electron micrograph
of mycoplasmalike organisms
(darts) in sieve tube element
of coconut palm. CW = cell
wall of sieve tube element.


Fig. 2. Phytomonas staheli
from plant, photographed
under phase contrast microscopy.,









TISSUES FOR COLLECTION

The plant part to be sampled for
electron microscopic examination
depends on the presumed disease organ-
ism. If the cause of disease is very
uncertain, specimens should be taken
from two or three sites on the palm.
MLO are most reliably detected in
tissues of the bud* of the palm but may
also be found in yellow tissue of the
flag leaf, roots, and inflorescences.
The bud tissues are more difficult to
collect than other tissues but are
preferred because this region is
apparently a "sink" for MLO. The best
time to collect the sample is when the
suspected MLO infection is in an
advanced stage, with most fronds yellow
to dead and collapsed, and preferably
with a secondary rot extending down
the spear leaf towards the bud.

In the early stages of hartrot
and marchitez sorpresiva, Phytomonas
can be found in bud tissues; in more
advanced stages of disease, they can
lso be found in roots and
inflorescences. Whereas MLO can only
be visualized with the electron
microscope, Phytomonas is readily
visible in the light microscope at
400X when phase contrast optics are
used, and presence of this organism
can be determined before tissue is
collected for electron microscopy.

Tissues to be collected will cut
easily with a razor blade. Woody,
desiccated, or rotted material should
not be collected. Specific
collection sites are:

Leaf. For MLO in coconut palm,
collect yellow leaflet tissue of the
flag leaf (which appears in the center
of the crown prior to development of
systemic yellowing) or below tip
necrosis of the spear leaf. MLO have
not been reported in leaves of palm

*Bud = heart = cogollo, the
Smeristematic tissue of a palm.


species other than coconut.


Phytomonas has not been reported
in palm leaf tissue.

Root. MLO may be present in lethal
yellowing coconut roots showing distal
necrosis. Collect whole pieces of
secondary roots or the central
vascular core (stele) of primary roots.

To check for Phytomonas, remove
adventitious roots from the base of a
palm with advanced symptoms of hart-
rot or marchitez sorpresiva. Strip
off the cortex, squeeze juice from the
stele, and examine with phase contrast
light optics at 400X. About ten
samples should be checked per tree.
Only samples known to contain protozoa
should be taken for electron micro-
scopic examination.

Inflorescence. A distal necrosis of
unopened flower stalks is
characteristic of palms infected by
either MLO or Phytomonas. If lethal
yellowing is suspected, collect tissue
from the base of the flower stalk,
near the point where it is attached
to the main trunk.

Using phase contrast optics,
check for Phytomonas in juice squeezed
from tissue below the necrotic tips.
If found, collect tissue for electron
microscopy.

Bud or heart. Cut down the palm,
remove mature leaves, and take the bud
back to the laboratory or to a shaded
area in the field. Use a machete to
remove leaf bases one at a time,.
starting with the oldest (Fig. 3).
Close to the growing point or bud, the
operation becomes more delicate since
the inner tissue is very fragile. At
this point it would be better to use
a knife or razor blade so as not to
damage the bud. The tissue to be
collected will be the bases of







immature, unemerged leaves within 1
to 3 cm of the growing point (Fig. 4).
This tissue will not be woody. Do not
collect tissue that is rotted or
desiccated.

For suspected MLO, collect three
leaf bases per palm (Fig. 5). In
mature coconut or oil palms, the oldest
leaf base to be sampled will be within
2 to 3 cm of the growing point, will
have a petiole diameter of not more
than 15 mm and will probably not be
more than 15 cm long. The youngest
leaf base collected will be within
1 cm of the growing point, will have a
petiole diameter of not more than 5 mm,
and will probably not be more than 3 cm
long. Choose a third leaf base between
these two sizes.

Again, confirm presence of
Phytomonas before collecting tissue
for electron microscopy. Phytomonas
may also be present in mature'tissue
of the palm crown, below the growing
point (Fig. 6).

STORAGE OF SAMPLES BEFORE FIXATION

Ultrastructural changes in plant
tissues can occur rapidly, and so for
best results fixation should begin as
soon as possible after tissue is
removed from the plant. Until
dissection can begin, keep the plant
material cool but do not freeze. If
necessary, the palm crown may be held
overnight and dissection begun the
next day. Leaf, root, or inflorescence
tissues should be dissected within
ten hours after removal from the plant,
but earlier if possible. Phytomonas
can remain viable in palm tissues for
two or three days after collection if
the plant parts are kept cool and are
not allowed to dry out. Thus the
organisms can still be detected with a
light microscope, although appearance
at the electron microscope level will
be less than optimal.


Fig. 3. Palm researcher,
Dr. F. W. Howard, removing
mature leaf bases from a coconut
palm, prior to collecting un-
emerged leaf bases for electron
microscopy.


Fig. 4. Coconut palm bud after
removal of most of the leafbases.











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Fig. 5. Coconut palm bud after removal of all leaf bases. For suspected MLO,
collect specimens from three of the five youngest leaf bases.





FIXATION


Fig. 6. Growing point or meristem
(arrow) of coconut palm. Phytomonas
may be present in mature tissue
below the meristem.


If palms are located some
distance from the laboratory, it will
be necessary to dissect tissues and
begin fixation in the field. In this
case, trim specimens to 5 mm X 5 mm
X 1 cm pieces, with the long axis
parallel to the long axis of the
plant part (Fig. 7). Immediately
drop specimens into a vial of
fixative. Keep the vials of
specimens cool, preferably in a
refrigerator, but do not freeze at
any time. Leave specimens in
fixative overnight (18 to no more
than 24 hours) if refrigerated;
reduce time to 6 to 10 hours if not
refrigerated. After the appropriate
fixation time, remove the fixative
and immediately replace with buffer.
Fill vials to the top with buffer so
that the specimens remain submerged
during transit.


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Fig. 7-8. Specimen trimming. 7. Specimens cut from a coconut leaf base in the
field. These should be put into fixative immediately. 8. Tissue cut from a
coconut leaf base in the laboratory. These should be further trimmed to embedding
size while bathing with fixative.


Upon return to the laboratory,
retrim specimens to a final embedding
size of ca 1 X 1.5 X 0.5 mm thick.
Bathe specimens in fixative while
trimming. These final specimens
should be slightly longer in the
transverse direction of the phloem so
that tissue orientation can be
recognized after embedding. Leave
the final specimens in fixative
overnight in the refrigerator before
proceeding with osmium tetroxide
post-fixation.

When initial dissection is done
in the laboratory, cut slices of tissue
(Fig. 8) while bathing in fixative,
trim to ca 1 X 1.5 X 0.5 mm as
described above, and proceed with
fixation schedule.

CHEMICALS FOR FIXATION

The following fixatives and
buffers are recommended for palm
samples. Cacodylate buffer is
preferred when fixation must begin in
the field. If fixation is begun in
the laboratory, collidine buffer is
preferred but cacodylate buffer may
be used.

All chemicals for electron
microscopy should be handled with care.
Avoid contact with skin and clothing;
do not inhale vapors. Work in a fume


hood if possible or in a well-venti-
lated area. Dispose of excess or
used chemicals by flushing with large
quantities of water.

Stock Solutions

(1) 0.2 M Sodium cacodylate buffer
Na(CH3)2As04-3H20 4.28 g
Add sufficient water to
bring volume to 100 ml(
Adjust to pH 7.4 with ca 5 ml
of 0.2 N HC1.

(2) 0.2 M s-Collidine buffer
Purchase from any commercial
electron microscope (EM) supply
house.

(3) 8% Glutaraldehyde
Purchase from any commercial EM
supply house.

(4) 8% Paraformaldehyde solution
Powdered paraformaldehyde 8 g
Add sufficient water to
bring volume to 100 ml
Stir while heating to dissolve.
Add drops of 0.1 N NaOH until
the solution becomes clear.

Fixative

8% Glutaraldehyde 10 ml
8% Paraformaldehyde in water 10 ml
0.2 M Cacodylate* or


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collidine buffer


Transport or rinse buffers

Dilute 0.2 cacodylate* or
collidine buffer to 0.1 M with
water.


*To cacodylate buffer, add 0.1 g
calcium chloride and 2 g sucrose.






SUGGESTED READING

McGhee, R. B. and A. H. McGhee. 1979.
Biology and structure of Phytomonas
staheli sp. n., a trypanosomatid
located in sieve tubes of coconut and
oil palms. J. Protozool. 26:348-351.

Norris, R. C. and R. E. McCoy. 1983.
Specialized electron microscopic
techniques for mycoplasma-like
organisms in plant tissues. In:
"Methods in Mycoplasmology" (S. Razin,
ed.), Vol. I, pp 63-69. Academic
Press, New York.

Thomas, D. L. 1979. Mycoplasmalike
bodies associated with lethal declines
of palms in Florida. Phytopathology
69:928-934.

Waters, H. 1982. Light and electron
microscopy. In: "Plant and Insect
Mycoplasma Techniques" (M. J. Daniels
and P. G. Markham, eds.), pp 101-151.
Croom Helm, London.





Fig. 3 courtesy of Jim DeFilippis.


20 ml




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