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Diagnosis of peach phony disease

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Title:
Diagnosis of peach phony disease
Creator:
French, W. J.
Publisher:
Agricultural Research Center, IFAS, University of Florida
Language:
English

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University of Florida
Rights Management:
Copyright Board of Trustees of the University of Florida
Resource Identifier:
153990736 ( OCLC )

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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 rickettfialike 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 ,a ,_!nll-.ea,.ves.
of peach and wild plum trees infected with ph,,b"-,Yv , , P

b.) RLB have been found in constant association 4ith trees infected with
phony; they have not been found in healthy trees.
O 1976
c.) RLB are released from infected material by the InfusiOn of KOH into
the xylem tissue.
F.A.S. - Univ. of Florida~
d.) RLB can be observed in KOH preparation by phasticontrastor-elec-a.
tron mi croscopy.

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 recognition 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 experience 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
Center, Monticello 32344.





4 = 6


~been good correlation between the two methods and visual symptom development. There are ten times fewer RIB in twigs than roots but the numbers have been sufficient to diagnose the disease even in symptomless trees. The distribution of RLB in the twigs is not uniform,9 thereftrej 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.1)
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 0.2,pm membrane filter and stored in a sterile container. 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 RIB
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. RIB will appear a -s non-motile rods about .5 X 2.5 - 3jjm. Chains of
RIB and refractive groups of RIB in gum will also be seen in many
preparations. RLB can be distinguished from debris under darkfiel~d by observing the perfect'cylindric shape of RIB as they are
tumbled about by Brownian movement.





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 preserved 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 rlckettsialike bacteria from roots and stems infected with phony disease.


-N





* 1'


-4



LITERATURE CITED


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


Shalla 1975. The use of fluorescent antiof 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. Phytopathological Soc. 1:90 (Abstr.) manuscript in press.

4. French, W. J. 1976. The incidence of phony disease In wild plum
trees as determined by histochenical and microscopic methods. Proc.
Florida, State Hort. Soc. 89: (in press).


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


1976. Field diagnosis of peach phony disease based on of a rickettsialike bacterium. Proc. Am. Phytopatho3: (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. Occurpeach trees


7. Kitajima, V. 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 66:1724.




Full Text

PAGE 1

~--------~------------------,-----------. AGRICULTURAL RESEARCH CENTER MONTICELLO Monticello, Florida Monticello ARC Mimeo Report BB 1976-3 \ October 11, 1976 DIAGNOSIS OF PEACH PHONY DISEASE William J. French 1 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, , 5, 6). To st.111111arize the ~h: , a.) RLB have been found in xylem elements of roots, stems_, _ !lnd leaves ,.~ ,, .. ~ of peach and wild plum trees infE?cted with pho~dlv~si:tBRJ\HY b.) RLB have been found in constant association Jith trees infected with phony; they have not been found in hea 1 thy tri,e _ es. _ . 1 1976 , Dt-c 3 c.) RLB are released from infected material by th~ infusion . of KOH into the xylem tissue. . i _ . ~ !.F .A .S. Univ. ot Florida d.) RLB can be observed in KOH preparation by phase ~ .contras-t.. or -elec .. ,., _ .. _ _ _ tron microscopy. e.) Field preparations of RLB can be preserved for :1 ater 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 recognition 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 Center, f.bnticello 32344. / Research --. /

PAGE 2

. . . ..... .. ----------------------------------------2.been good correlation between the two methods and visual symptom development. 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 : i a minimum sample of four twigs per tree is reconrnended. Procedure l.'~ !Roots and stems: cut p-ieces 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. Rennve a 2 cm. ring of bark from each end of section. 3. Place one end of xylem cylinder into piece of plastic tubing (Fig.1) 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-lM KOH solution to well. (The KOH should be filtered through 0.2J-1ffi membrane filter and stored in a sterile container. 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.i 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 3fT1. 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 fielid by observing the perfect cylindric shape of RLB as they are tumbled about by Brownian movement / /

PAGE 3

... -39. 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 bf RLB, notably alrrond 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 plt.nn (French and Bakarcic, unpublished data). 1----plastic tubing root or stem sample plastic tubing glass tubing vacuum pump A--,!:;---rubber stopper -500 ml vacuum flask _ -15 ml centrifuge Fig. 1. Apparatus used to extract rickettsialike bacteria from peach roots and stems infected with phony disease '

PAGE 4

-4LITERATURE CITED .1. Auger, J. G. and T. A. Shalla 1975.. The use of fluorescent anti . bodies for detection of Pierce's disease bacteria in grapevines and insect vectors Phytopa tho 1 ogy 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 pathol ogical Soc. 1: 90 (Abs tr.) manuscript in press. 4. French, W. J. 1976. The incidence of phony disease in wild plum trees as determined by histochenical and microscopic methods. Proc. Florida, State Hort. Soc. 89: (in press). 5. French, W. J •. 1976. Field diagnosis of peach phony disease based on the presence of a rickettsialike bacterium. Proc. Am. Phytopathological Soc. 3: (Abstr. Caribbean Div. meeting, in press). . 6. Hopkins~ D. L., H. H. Mollenhauer, and W. J. French 1973. Occur . ence of a rickettsia-like . bacterium in the xylem of peach trees with phony disease. Phytopathology 63:1422-1423. 7. Kitajima, E. 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 of the causal agent. Phytopathology 66:17-24. . I


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