Title: Diagnosis of peach phony disease
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Title: Diagnosis of peach phony disease
Physical Description: Book
Creator: French, W. J.
Publisher: Agricultural Research Center, IFAS, University of Florida
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/; AGRICULTURAL RESEARCH CENTER MONTICELLO
Monticello, Florida

Monticello ARC Mimeo Report BB 1976-3 October 11, 1976


DIAGNOSIS OF PEACH PHONY DISEASE
William J. French1

This report describes, in outline form, a method of diagnosing
phony disease based on the presence of rickettsialike bacteria (RLB)
in the xylem of infected peach trees. The information presented herein
is based on data published elsewhere (2, 3, 4, 5, 6). To summarize the
data:

a.) RLB have been found in xylem elements of roots, stems, and. leaves
of peach and wild plum trees infected with pho yis

b.) RLB have been found in constant association 4ith trees infected with
phony; they have not been found in healthy trees. 136
L 1976
c.) RLB are released from infected material by the infusion of KOH into
the xylem tissue. .
.FA.S. Univ. of Florida
d.) RLB can be observed in KOH preparation by phase contrast or-elec-
tron microscopy.

e.) Field preparations of RLB can be preserved for;later examination
without a significant decrease in numbers.

f.) Diagnosis based on the presence of RLB has been more accurate than
the standard acid-alcohol color test or diagnosis based on recog-
nition of early symptoms.

g.) RLB have been found in roots and stems of peach trees one and two
years before phony symptoms became apparent.

The following extraction procedures should be followed until ex-
perience and confidence allows the investigator to adapt the method
for his particular need. It is advisable to select root material from
known phony trees in order to become familiar with the appearance of
the RLB. A preserved suspension of RLB can be used as a reference.
Twigs can be used instead of roots. Although this method has not
been field tested as thoroughly as the root extraction method, there has

1 Associate Professor IFAS, University of Florida, Agricultural Research
a Center, Monticello 32344.


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4 = 6 '


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been good correlation between the two methods and visual symptom devel-
opment. There are ten times fewer RLB in twigs than roots but the num-
bers have been sufficient to diagnose the disease even in symptomless
trees. The distribution of RLB in the twigs is not uniform, therefore
a minimum sample of four twigs per tree is recommended.


Procedure

1. IRoots and stems: cut pieces 1.0 1.5 X 7 10 cm. Sections should
be free of discolored or decayed areas. Cut sections shorter if
extraction process exceeds 30-60 sec per section. Twigs should be
taken from one to two year old wood from each of four quadrants of
a tree.

2. Remove a 2 cm. ring of bark from each end of section.

3. Place one end of xylem cylinder into piece of plastic tubing (Fig.l)
which is attached to a..7 X 20cm glass tube. The glass tubing is
inserted through a stopper of a vacuum flask and into a centrifuge
tube, which is contained within the flask. Attach plastic tubing
to top of xylem cylinder to form a well which will hold about 1 ml
of liquid. Connect vacuum flask to vacuum pump.

4. Add 0.5 to 1 ml of 10-1M KOH solution to well. (The KOH should be
filtered through 0.2pm membrane filter and stored in a sterile con-
tainer. Check stock periodically for sterility.)

5. Turn on vacuum pump, adjust vacuum to 10-20 in. mercury as required
for slow but steady extraction of the KOH through the tissues.
Ease off vacuum before cutting off pump. Repeat steps 2-5 and
pool all four twig samples or multiple root samples from same tree

6. Centrifuge extract at about 500 G for 5 min. to clear debris. Use
supernatant. This step may be omitted when experience in RLB
identification is gained or when debris does not interfere with
observations.

7. Place one or two drops of extract on clean slide and cover with #1
cover glass. Observe with phase-contrast microscope at 400X.

8. RLB will appear as non-motile rods about .5 X 2.5 3pm. Chains of
RLB and refractive groups of RLB in gum will also be seen in many
preparations. RLB can be distinguished from debris under dark-
fiel!d by observing the perfect cylindric shape of RLB as they are
tumbled about by Brownian movement.


L_~ ___ ____





* 9


-3-




9. RLB may be preserved for over a month by neutralizing the extract
with one drop of 10% HCL solution to 20 drops of extract, then add
one drop of 1% merthiolate. Store at room temperature. The pre-
served RLB are suitable for study by electron microscope.

The KOH method has also been used in the study of other diseases
caused by RLB, notably almond leaf scorch (8) and Pierce's disease of
grape.(1). RLB associated with plum leaf scald (7) in Argentina have
been observed in KOH extracts from roots of infected plum (French and
Bakarcic, unpublished data).


plastic tubing


root or stem sample


plastic tubing


glass tubing


vacuum pump
rubber stopper


vacuum flask


ml centrifuge


Fig. 1.
peach


Apparatus used to extract rickettsialike bacteria from
roots and stems infected with phony disease.


*-N


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* 1'


-4-



LITERATURE CITED


1. Auger, J. G. and T. A.
bodies for detection
and insect vectors.


Shalla 1975. The use of fluorescent anti-
of Pierce's disease bacteria in grapevines
Phytopathology 65:493-494.


2. French, W. J. 1974. A method for observing rickettsialike bacteria
associated with phony peach disease. Phytopathology 64:260-261

3. French, W. J. 1975. Vacuum extraction of rickettsialike bacteria
from peach trees affected with phony disease. Proc. Am. Phyto-
pathological Soc. 1:90 (Abstr.) manuscript in press.


4. French, W. J. 1976. The
trees as determined by
Florida, State Hort.


incidence of phony disease in
histochenical and microscopic
Soc. 89: (in press).


wild plum
methods.


5. French, W. J..
the presence
logical Soc.


1976. Field diagnosis of peach phony disease based on
of a rickettsialike bacterium. Proc. Am. Phytopatho-
3: (Abstr. Caribbean Div. meeting, in press).


6. Hopkins, D. L., H. H. Mollenhauer, and W. J. French
ence of a rickettsia-like bacterium in the xylem of
with phony disease. Phytopathology 63:1422-1423.


1973. Occur-
peach trees


7. Kitajima, B. W., M. Bakarcic, and M. V. Fernandez-Valiela 1975.
Association of rickettsialike.bacteria with plum leaf scald dis-
ease. Phytopathology 65:476-479.

8. Mircetich, S. M., S. K. Lowe, W. J. Moller, and G. Nyland 1976.
Etiology of almond leaf scorch disease and transmission 6f the
causal agent. Phytopathology 66:17-24.


Proc.




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