Louisiana State University
LSU Digital Commons
LSU Historical Dissertations and Theses
Graduate School
1985
Detection of Clavibacter Xyli Subsp. Xyli, the
Ratoon Stunting Disease Bacterium, in Sugarcane
by Dot Blot Hybridization.
Jimmy Michael Kamso-pratt
Louisiana State University and Agricultural & Mechanical College
Follow this and additional works at: http://digitalcommons.lsu.edu/gradschool_disstheses
Recommended Citation
Kamso-pratt, Jimmy Michael, "Detection of Clavibacter Xyli Subsp. Xyli, the Ratoon Stunting Disease Bacterium, in Sugarcane by Dot
Blot Hybridization." (1985). LSU Historical Dissertations and Theses. 4135.
http://digitalcommons.lsu.edu/gradschool_disstheses/4135
This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in
LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact
[email protected].
INFORMATION TO USERS
This reproduction was made from a copy of a m anuscript sent to us for publication
and m icro film in g . W h ile th e m ost advanced technology has been used to pho­
tograph and reproduce this m anuscript, the qu ality of the reproduction is heavily
dependent upon the quality o f the m aterial subm itted. Pages in any m anuscript
may have in d istin c t p rin t. In a ll cases the best available copy has been film ed.
The following explanation of techniques is provided to help clarify notations w hich
may appear on this reproduction.
1. M anuscripts m ay not always be complete. W hen it is not possible to obtain
m issing pages, a note appears to indicate this.
2. W hen copyrighted m aterials are removed from the m anuscript, a note ap­
pears to indicate this.
3. Oversize m aterials (m aps, draw ings, and charts) are photographed by sec­
tio n in g the o rig in al, b eg in n in g at the upper left hand com er and co ntinu ­
ing from left to rig h t in equal sections w ith sm all overlaps. Each oversize
page is also film ed as one exposure and is available, fo r an a d d itio n a l
charge, as a standard 35m m slide o r in black and w hite paper fo rm at.*
4. M ost photographs reproduce acceptably on positive m icrofilm or m icro­
fiche b u t lack clarity on xerographic copies made from the m icrofilm . For
an a d d itio n a l charge, all p hotographs are available in black and w h ite
standard 35m m slide form at. *
*For more inform ation about black and white slides or enlarged paper reproductions,
please contact the Dissertations Customer Services Department.
M
‘IJmversily
Microfilms
, International
8610645
Kamso-Pratt, Jimmy Michael
DETECTION OF CLAVIBACTER XYLI SUBSP. XYLI, THE RATOON STUNTING
DISEASE BACTERIUM, IN SUGARCANE BY DOT BLOT HYBRIDIZATION
The Louisiana State University and Agricultural and Mechanical Col.
University
Microfilms
International
300 N. Zeeb Road, Ann Arbor, Ml 48106
Ph.D.
1985
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy.
Problems encountered with this document have been identified here with a check mark V
^
1.
Glossy photographs or pages
2.
Colored illustrations, paper or print______
3.
Photographs with dark background
4.
Illustrations are poor copy______
5.
Pages with black marks, not original copy_____
6.
Print shows through as there is text on both sides of page_______
7.
Indistinct, broken or small print on several pages
8.
Print exceeds margin requirements_____
9.
Tightly bound copy with print lost in spine______
V
/
10.
Computer printout pages with indistinct print______
11.
Page(s)___________ lacking when material received, and not available from school or
author.
12. Page(s)
13.
.
__________seem to be missing in numbering only as text follows.
Two pages numbered
. Text follows.
14. Curling and wrinkled pages_____
15. Dissertation contains pages with print at a slant, filmed as received________
16. Other
______
University
Microfilms
International
DETECTION OF
CLAVIBA CT ER XYLI SUBSP. XYLI,
THE R ATO ON ST UNTING DISEASE BACTERIUM,
IN SUGARCANE
BY DOT BLOT HYBRIDIZATION
A Dissertation
Submit te d to the Graduate Facul ty of the
Lo ui sia na State Un iversity and
A gri cu l tu ra l and Mechanical College
in partial fulfillment of the
req ui rements for the degree of
D oc tor of Philosophy
in
The De partment of Plant Pathology and Crop Physiology
by
Jimmy Michael Kamso-Pratt
B.
S., Cumberland College,
M. S., M cN ees e State University,
December, 1985
1975
1982
ACKNOWLEDGEMENTS
I would like to thank my major professor, Dr. K.
E. Damann Jr., for his help throughout the course of my
research and for his assistance in the preparation of
this dissertation.
I would also like to express my
thanks and gratitude to Drs. J. Larkin, G. Holcomb, J.
Hoy and D. R. Mackenzie for giving me assistance and
serving as members of my Dissertation Committee.
Dr.
Wray Birchfield has been a constant source of
encouragement and kindness to me.
For this I am very
grateful.
I also give my thanks to the late Dean Prentiss
Schilling, College of Agriculture, for providing my
assistantship.
Finally, I give my special thanks to my family for
their support and patience.
ii
TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS...................................
ii
LIST OF FIGURES......
iv
ABSTRACT..............................
v
INTRODUCTION........................................
1
MATERIALS AND METHODS..............................
6
RESULTS.............................................
17
DISCUSSION..........................................
28
LITERATURE CITED...................................
30
VITA................................................
34
iii
LIST OF FIGURES
FIGURE
1.
2.
3.
4.
5.
PAGE
Miniscreening of transformed Escherichia
coli JM 83 clones by plasmid isolation
and determination of Clavibacter xyli
subsp. xyli DNA inserts in
recombinant plasmids...... ..............
19
Hybridization of Clavibacter xyli
subsp. xyli DNA, recombinant plasmids
pUC 19 DNA, Bacillus subtilis DNA with
32P-labelled C. xyli subsp. xyli
chromosomal DNA..............................
20
A comparison of Clavibacter xyli
subsp. xyli cell detection by cloned
32P-labelled C. xyli subsp.
xyli chromosomal DN A .........................
21
Detection of Clavibacter xyli subsp.
xyli cells with 32P-labelled
C. xyli subsp. xyli chromosomal
DNA ...........................................
24
Determination of the limits of detection
of C. xyli subsp. xyli sequences in
dilutions of infected sugarcane sap.........
25
Sampling of sugarcane tissues for C. xyli
subsp. xyli DNA sequences with
32P-labelled C. xyli subsp. xyli
chromosomal DNA..............................
27
iv
ABSTRACT
Recombinant DNA technology was used to produce a
probe for detection of Clavibacter xyli subsp. xyli,
the sugarcane ratoon stunting disease agent.
C. xyli
subsp. xyli chromosomal DNA was digested with the
restriction enzyme Pst 1, inserted into the plasmid pUC
19, and transformed into Escherichia coli JM 83.
Recombinant clones containing six different sizes of C.
xyli subsp. xyli DNA sequences were obtained.
Plasmid
K3-5, with an insert of 4.9Kb, and C. xyli subsp. xyli
chromosomal DNA, were radiolabelled by nick translation
using dCTP or dATP (alpha-^P).
The labelled DNAs were
used to detect homologous DNA sequences present in DNA
extracts from C. xyli subsp. xyli cells, C. xyli subsp.
xyli cells and sap from C. xyli subsp. xyli infected
sugarcane by dot blot hybridization.
The technique
proved to be rapid and sensitive for detecting C. xyli
subsp. xyli in sugarcane sap.
sensitivity varied,
The degree of
with the labelled C. xyli subsp.
xyli chromosomal DNA probe being more sensitive than
the cloned C. xyli subsp. xyli chromosomal DNA probe.
Diluted sap from infected sugarcane plants gave
positive hybridization signals whereas sap from
uninfected sugarcane plants failed to give
hybridization signals.
vi
INTRODUCTION
Clavibacter xyli subsp. xyli Davis et al. 1984,
the sugarcane ratoon stunting disease bacterium occurs
worldwide and can cause significant yield losses
especially under conditions of drought stress
(20,30).
Yield losses can range from 10 - 30% for susceptible
cultivars while resistant cultivars suffer little or no
yield loss (20).
All sugarcane cultivars are
susceptible to infection by the bacterium.
The bacterium is mechanically transmitted, and the
cutting knife is considered to be the principal means
of transmission.
Sap from infected plants can cause
disease even after dilutions of 10 J to 10
.
For this
reason, and because no causal organism was found
associated with the disease, researchers originally
assumed that the disease agent was a virus
(13, 29).
A small bacterium was found associated with the
disease in 1973 by Teakle
(31) and Gillaspie (14) and
confirmed by others (5, 17).
The bacterium was
observed in the xylem vessels of host plants with the
aid of light and electron microscopy (7, 18).
Diseased
canes were also shown to contain vascular gels which
act as plugs in the xylem vessels.
These plugs contain
microcolonies of C. xyli subsp. xyli (18).
Axenic cultures of the bacterium were obtained in
1980 by Davis et al. (8 ) and Koch's postulates were
fulfilled.
C . xyli subsp. xyli bacteria are coryneform
and can be straight or curved.
They often appear to
undergo septate division and they contain mesosomes,
which are intrusions of the cytoplasmic membrane common
in gram positive and less common in gram negative
bacteria.
The bacteria are gram positive, non-motile,
non-sporeforming, non-acidfast, oxidase negative and
catalase positive.
A relationship to some
phytopathogenic corynebacteria was suggested based on
the composition of the cell wall which includes amino
acids such as 2 , 4 diaminobutyric acid, glycine,
alanine and glutamic acid and sugars such as fucose and
rhamnose (10).
(2. xyli subsp. xyli can be
differentiated from other phytopathogenic
corynebacteria on the basis of its protein band
patterns in polyacrylamide gels, guanine and cytosine
content of its DNA, its capacity to induce ratoon
stunting disease in sugarcane and by the failure of the
phytopathogenic corynebacteria to react with antisera
to C. xyli subsp. xyli (9).
Because these bacteria
have characteristics different from those of any
existing genera, a new genus, Clavibacter was proposed
for
C. xyli subsp. xyli and for those bacteria similar
The external and general symptoms of ratoon
stunting disease are reduced height of the canes, less
weight and smaller diameter per stalk (10, 20 , 30).
Other disease symptoms are reduced vigor of infected
plants and abnormal rolling of leaves.
These symptoms
are more pronounced in times of drought stress (30 ).
Internal symptoms consist of an orange to red
discoloration of the vascular bundles at the nodes of
mature plants and a general pinkish salmon
discoloration just below the meristematic area of
immature stalks (10).
However, these symptoms are not
unique to C.. xyli subsp. xyli infection.
Environmental
conditions, nutritional imbalances and other biotic
factors can cause similar symptoms.
The inconsistent
expression of symptoms even in the severely infected
plants has caused problems in studying ratoon stunting
disease.
Methods that have been used to assay sugarcane for
the presence of the bacteria include visual symptoms
(10), light and phase contrast microscopy (7, 14),
electron microscopy (6 ,30), fluorescent antibody
staining (2 7 ) and enzyme linked immunosorbent assay
(ELISA) (15,27).
These methods are not always as
reliable or as sensitive as desired.
Fluorescent
antibody staining has been the most reliable method
used for diagnosis of the disease, but it is limited by
its fairly high detection limit (10^ cells/ml).
So
far, ELISA has not proven sensitive and consistent
enough to be of use in C. xyli subsp. xyli detection
(15, 27).
A rapid and sensitive method of detecting C.
xyli subsp. xyli in sugarcane would facilitate study of
the disease in epidemiological investigations, in
screening of sugarcane cultivars for resistance, and in
establishing control measures.
Dot blot hybridization is a part of recombinant
DNA technology that has been used for the detection of
infectious disease agents including plant viroids (26),
plant viruses (12), and plant mycoplasmas (K. S.
Derrick, unpublished observation).
The technique is
derived from colony hybridization, which has been used
to screen bacterial clones for specific DNA sequences
(25).
DNA sequences can be made into probes with a
very high degree of specificity.
These probes can be
used to search for homologous DNA sequences in host
samples.
With molecular cloning it is possible to
prepare almost unlimited amounts of nucleic acid to use
as a probe to detect a certain microorganism.
The
strong affinity of complimentary nucleic acids strands
for each other permits a highly sensitive hybridization
assay.
The technique is relatively simple,
inexpensive, and reproducible.
The major objective of this study was to clone C^.
xyli subsp. xyli chromosomal DNA fragments and to
compare the cloned and uncloned £. xyli subsp. xyli
chromosomal DNA probes for the ability to detect
xyli subsp. xyli in sugarcane samples by dot blot
hybridization.
C.
MATERIALS AND METHODS
Bacteria
Clavibacter xyli subsp. xyli was isolated from
infected sugarcane cultivar L62-96 as described below.
Corynebacterium michiganense was a gift from Dr. Rey
Cody, Auburn University, Auburn, Alabama.
Escherichia
coli JM 83, with and without pUC 19, was a gift from K.
Narva, Department of Microbiology, Louisiana State
University.
Bacillus subtilis was a gift from Dr. E.
Achberger, Department of Microbiology, Louisiana State
University.
Agrobacterium tumefaciens was a gift from
Dr. G. E. Holcomb, Department of Plant Pathology and
Crop Physiology, Louisiana State University.
Enzymes
Proteinase K, ribonuclease A, lysozymne, T4 DNA
ligase and the restriction endonuclease Pst 1 were from
Sigma Chemicals, St. Louis, MO.
The nick translation
kit was from Bethesda Research Laboratories, Bethesda,
MD.
6
Other reagents
Nitrocellulose paper (BA85/21, 82 mm circles) was
from Schleicher and Schuell, Keene, N.H.
Isolation and culture of C. xyli subsp. xyli
C. xyli subsp. xyli was isolated from infected
sugarcane vascular extracts
(31).
Single node cuttings
from mature sugarcane stalks were placed separately in
50 ml conical bottom centrifuge tubes and centrifuged
at 2000 rpm for 2 min.
Ten microliters of the vascular
extract together with a thin section of the cutting
were examined under the microscope to verify the
presence of the bacteria.
treated with 0.1 M Tris pH
The thin sections were
10 and then e xamined with a
Leitz Ortholux II microscope with a ploemopak
flourescence illuminator.
This treatment causes thin
sections of diseased sugarcane to exhibit a distinct
metaxylem autoflourescence (7).
The 10 microliter
aliquot of sugarcane sap was placed on a microscope
slide covered with a coverslip and viewed with
darkfield optics at 500X to detect the bacteria.
The
presence of the bacteria thus verified, the vascular
extract (sap) of node cuttings from the same stalk were
combined, passed through a 0.45 urn membrane filter
(Nalgene,
(27).
Rochester,
N.Y.) and inoculated into RSD broth
RSD contains 1000 ml of distilled water,
1 g
yeast extract, 1 g (NH^j^HPC^, 0.2 g MgS04 .7H20, 15
8
ml of 0.1% (wt/vol) hemin chloride stock in 0.05 M
NaOH, 1 g cysteine (free base), 0.2 g KC1, 10 ml of
filter sterilized 20% (wt/vol) bovine serum albumin
fraction V and 10 ml of filter sterilized 50%
glucose solution.
(wt/vol)
The pH of the medium was adjusted to
6.6 and cooled to 50C before adding the sterilized
ingredients.
Preparation of C. xyli subsp. xyli chromosomal DNA
C.
xyli subsp. xyli was grown in RSD broth as
static suspension cultures for 10 days.
The cells were
washed twice in TAB buffer (40 mM Tris, 20 mM sodium
acetate, 1 mM ethylene diaminotetraacetic acid (EDTA)
pH 7.8), followed by centrifugation at 2000 rpm for 15
min.
The pellet was resuspended in 25 ml of lysozyme
solution (22) (2 mg/ml) in Tris buffer (25 mM Tris-HCl,
pH 8.0, 1 mM EDTA, 100 mM NaCl plus 15% sucrose) per
liter of original culture.
a concentration of 50 mg/ml.
incubated on ice for 1 hr.
sulfate
(SDS) solution
Ribonuclease A was added to
The suspension was
Alkaline sodium dodecyl
(0.2 N NaOH and 2% SDS) was
added at 50 ml per liter of original culture followed
by agitation and incubation at 37C for 30 min.
(Personal communication from Dr. J. Jaynes,
Department
of Biochemistry, Louisiana State University).
DNA was extracted with a 25% volume of
phenol-chloroform
(1 :1 ,v/v) and centrifuged at 10,000
rpm for 10 min.
The aqueous layer was re-extracted
with chloroform-isoamyl alcohol
(24:1,v/v) and
centrifuged at 10,000 rpm for 10 min.
Two volumes of
95% ethanol-0.3 M sodium acetate were added, followed
by precipation at -20C overnight.
The DNA was pelleted
by centrifugation, washed twice with cold 70% ethanol
and centrifuged at 10,000 rpm for 5 min after each
wash.
The pellet was dried and dissolved in an
appropriate volume of TE buffer (10 mM Tris-HCl, 1 mM
EDTA pH 8.0)
(24).
Isolation of plasmid pUC 19 DNA
A colony of Escherichia coli JM 83 containing
p la smid pUC 19 was inoculated into 10 ml of L-Broth (10
g/1 bacto-tryptone, 5 g/1 yeast extract, 10 g/1 NaCl
plus 40 mg/1 ampicillin) and was incubated at 37C for
16 hr with shaking at 20,000 rpm.
The overnight
culture was used to inoculate 1 liter of L-Broth with
50 mg/1 ampicillin and grown to an optical density
(O.D.) of 0.4 at 600 nm on a Spectronic 20 (Bausch
Lomb, Rochester, N.Y.).
The plasmid was amplified with
270 mg/1 chloramphenicol and grown overnight.
The
bacteria were centrifuged at 5000 rpm for 10 min and
the bacterial pellet was suspended in 25 ml of lysozyme
solution and incubated on ice for 1 hr.
Fifty ml of
alkaline SDS was added followed by agitation and
incubation on ice for 10 min.
Fifteen ml of 3 M sodium
10
acetate, pH 4.8, was added followed by agitation and
incubation on ice for 90 min.
The DNA was extracted as
decribed above.
The DNA pellet was dissolved in 10 ml TE buffer
for each 2 liters of original culture.
Cesium chloride
(10.23 g) was added to each 10 ml dissolved DNA (to a
density of 1.02 g/ml) and 1 ml of ethidium bromide
solution (5 mg/ml ethidium bromide in distilled water)
was added.
The DNA was centrifuged at 65,000 rpm on a
Beckman 75 Ti rotor (Palo Alto, CA) for 16 hr.
The
plasmid (lower band) was removed, dialyzed overnight
against TE buffer, and extracted with water saturated
butanol six times
(discarding the top butanol layer).
The DNA was extracted with 2 ml phenol-chloroform (1:1)
and centrifuged at 10,000 rpm for 5 min.
The aqueous
phase was re-extracted with chloroform-isoamyl alcohol
(24:1) and precipitated with 2 volumes of 95% ethanol 0.3 M sodium acetate.
The DNA was washed twice with
70% ethanol, dried and dissolved in TE buffer.
(Personal
communication from Dr. J. Jaynes, Department of
Biochemestry, Louisiana State University).
Restriction digestion of C. xyli and plasmid DNA
Purified C. xyli subsp. xyli chromosomal DNA and
pUC 19 plasmid DNA were separately digested with the
restriction endonuclease Pst 1 using 20 units of enzyme
per 0.5 ug DNA with medium strength restriction enzyme
11
buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1 mM
MgCl2f 1 mM dithiothreitol) and incubated at 37C
overnight.
The digested DNA fragments were analyzed
for completeness of digestion by electrophoresis on 1%
agarose gels in 40 mM Tris-acetate, 2 mM EDTA (pH 7.8)
buffer
(24).
Formation of recombinant DNA molecules
The Pst 1 digested C. xyli subsp. xyli chromosomal
DNA and plasmid pUC 19 DNA were each extracted with
phenol-chloroform
(1 :1), chloroform-isoamyl-alcohol
(24:1), precipitated with 95% ethanol-0.3 M sodium
acetate, washed twice with cold 70% ethanol, dried and
dissolved in an appropriate volume of TE buffer.
Three
ug of Pst 1 digested C. xyli subsp. xyli chromosomal
DNA and lug of plasm id pUC 19 DNA were mixed together.
The mixture was precipitated with 95% ethanol-0.3 M
sodium acetate, washed twice with 70% ethanol and
dried.
The DNA was dissolved in 12 ml TE buffer.
Two
ml of ligation buffer (0.05 M Tris pH 7.6, 0.1 M M g C ^ /
0.1 M spermidine,
1 mg/ml bovine serum albumin) 2 ml
0.1 M d i t h r o t h r e i t o l , 2 ml 0.01 M ATP, and 1.4 ml T^
DNA ligase,
(at 100 units/ml) was added.
This was
followed by overnight incubation at 14C.
The ligated
DNA was extracted with phenol-chloroform
(1:1),
chloroform-isoamyl alcohol
(24:1), precipitated with
12
95% ethanol-0.3 M sodium acetate, washed twice with 70%
ethanol and dissolved in 20 ml TE buffer
(24).
Transformation of E. coli JM 83
E. coli JM 83 cells were transformed by a
modification of the Hanahan procedure (16).
Ten ml of
a fresh overnight culture of E. coli JM 83 without
plasmids was diluted 1:100 in L-Broth in a flask and
incubated with shaking for 3 hr.
The flask was placed
on ice as soon as the O.D.gQQ reached 0.7.
The culture
was transferred to chilled glass tubes and centrifuged
at 2,500 rpm for 15 min.
The pellet was suspended in
one-third volume of TFB buffer
(10 mM K-MES
[2[N-morpholino ethane sulfonic acid] pH 6.2, 10 mM
KC1, 45 mM M n C l 2 , 10 m M C a C l 2 , 3 m M h examine C0 C I 3) and
incubated for 30 min on ice.
The mixture was
centrifuged at 2,500 rpm and the pellet resuspended in
o ne -t we n ti et h volume of TFB buffer, and 7 ul of
dimethyl sulfoxide (DMSO) per 200 ul suspension was
added, then the mixture was swirled and place on ice.
Dithiothreitol
(DTT) was added at 7 ul per 200 ul of
suspension, swirled and incubated for 10 min on ice.
Seven ul of DMS O per 200 ul suspension was added,
followed by swirling and 5 min incubation on ice. The
mixture was pipetted into chilled Eppendorf tubes at
100 ul per tube.
Ten ul of previously ligated DNA was
added to each Eppendorf tube.
The mixture was swirled
13
and placed on ice for 30 min.
The mixture was heat
pulsed at 42C for 90 sec and placed on ice for 1-2
min.
One hundred ul aliquots of the suspension were
plated on MacConkey agar containing 25 mg/ml
ampicillin, followed by an overnight incubation at 37C.
Ampicillin resistant colonies were transferred to fresh
plates for miniscreening.
Miniscreening for plasmid analysis
Each ampicillin resistant colony was grown
overnight on 10 ml L-Broth with 50 mg/1 ampicillin.
One ml of the overnight culture was used to inoculate
10 ml of L-Broth with 50 mg/1 ampicillin.
The cells
were grown to an O.D. of 0.4 at 600 nm, amplified with
170 mg/1 chloramphenicol and grown overnight.
DNA was prepared as described above.
Plasmid
The plasmid DNAs
were digested overnight with the restriction
endonuclease Pst 1, run on a 1% agarose gel in 40 mM
TAE buffer and analyzed for the size of the inserted
DNA (24).
Nick translation of cloned C. xyli subsp. xyli
chromosomal DNA
Nick translation was done according to the
instructions on the nick translation kit.
deoxyribonucleotide solution
Five ul of
(containing all the
deoxyribonucleotides except the one which was to be
14
radiolabelled), 1 ug of DNA, 13 ul of dCTP
(alpha-^2p),
and sterile deionized water to 45 ul were added.
The
mixture was mixed briefly and 5 ul of a mixture of DNA
polymerase 1 and DNase 1 was added.
The mixture was
mixed thoroughly and incubated at 15C for 1 hr.
The
reaction was stopped by the addition of 5 ul of 0.5 m
EDTA.
The labelled DNA was separated from unlabelled
deoxyribonucleotides by G-50 Sephadex spun column
chromatography (25) or by precipitation in cold ethanol
in 7.5 M ammonium acetate
(24).
Preparation of nitrocellulose filters
C. xyli subsp. xyli cells were washed in TAE
buffer and dilutions were made in the same solution.
Replicate 5 ul samples were applied to a 1 cm square on
a nitrocellulose filter.
Five ul sugarcane sap samples
from C. xyli subsp. xyli infected and healthy
sugarcanes were also spotted on the filter.
Five ul of
a Bacillus subtilis culture was spotted on the filter
as negative control.
The filter was air dried and
incubated at room temperature for 1 hr.
The filter was
soaked in 10% SDS for 3 min and transferred with the
spotted side up to a layer of W h a t m a n # 3 filter paper
saturated with denaturing solution (0.5 M NaOH,
NaCl) for 5 min.
1.5 M
The filter was again transferred to a
Whatman # 3 filter paper saturated with neutralizing
solution (1.5 M NaCl, 0.5 M Tris-HCl pH 8.0).
The
15
filter was transferred one more time to a Whatman # 3
filter paper saturated with 2X
SSPE (20X
SSPE = 3.6 M
N a C l , 20 mM N a H 2P 0 4 (pH 7.4), 20 m M EDTA (pH 7.4))
(24).
The filter was air dried and baked
80C (25).
overnight at
After baking the filter was incubated
overnight in 3X SSC (20X SSC = NaCl 175.3g, sodium
citrate 88.2g, H 20 to 1 liter) pH 7.4, 0.1% SDS.
Hybridization
The baked filter was i m m e r s e d in 6X SSC (24) for 2
min. then placed inside a heat sealable plastic bag.
Prewarmed
SDS,
(68C) prehybridization fluid (6X SSC, 0.5%
5x Denhardt's solutioin
(Ficoll 5g,
polyvinylpyrrolidone 5g, bovine serum albumin fraction
V 5g, H 20 to 500 ml), 100 ug/ml denatured salmon sperm
DNA) was added at 0.2 ml/cm2 of filter.
The plastic
bag was sealed and incubated in a water bath at 68C for
2 hr.
At the end of the incubation period, the bag was
opened at one end and the prehybridization fluid was
squeezed out.
Hybridization solution
EDTA, 5X Denhardt's solution, 0.5% SDS,
(6X SSC, 0.01 M
100 ug/ml
denatured salmon sperm DNA) was added at 50 ul/cm"6
filter.
Then the alpha ^2P-labelled denatured probe
DNA was added and the bag was incubated in a water bath
at 68C overnight.
The filter was removed from the bag
at the end of the incubtion period and submerged in a
tray containing a solution of 2X SSC and 0.5% SDS at
16
room temperature for 5 min.
The filter was transferred
to 2X SSC and 0.1% SDS and incubated for 15 min at
room temperature with gentle agitation.
The filter was
transferred to a tray containing 0.1X SSC and 0.5% SDS
at 68C for 2 hr.
At the end of the 2 hr, the buffer
was changed and incubation was continued for 30 min
(24) .
Autoradiography
The filter was dried at room temperature on a
sheet of W h a t m a n # 3 filter paper.
The filter was
wrapped in Saran wrap and autoradiography was done at
-70C using Kodak-o-mat (AR) X-ray film and Dupont
Cronex lightning-plus intensifying screens
(24).
The
film was exposed to the filter for 24-48 hr and
developed with Kodak liquid X-ray developer (Rochester,
NY).
Hybridization was checked by the appearance of
exposed dots on the X-ray film (24).
RESULTS
DNA isolation
C. xyli subsp. xyli DNA was isolated by a
modification of the method of Birnboim and Doly (2),
which involves alkaline lysis, phenol extraction and
cesium chloride centrifugation.
This procedure gave a
DNA yield that was ten fold higher than the published
method (10) hitherto used to isolate
xyli DNA.
C. xyli subsp.
Plasmid pUC 19 DNA was also isolated as
described in Materials and Methods.
Cloning of C. xyli subsp. xyli DNA
Pst 1 digested C. xyli subsp. xyli DNA was
inserted into the Pst 1 site of the plasmid pUC 19.
The plasmid was used to transform E. coli JM 83.
The
transformed E. coli JM 83 was plated on MacConkey agar
(11) with 25 ug/ml ampicillin.
One hundred colonies
were observed on the agar plates after an overnight
incubation.
About 90% of these colonies showed a loss
of B-galactosidase activity, indicated by white instead
of purple colonies on MacConkey agar.
This loss of
activity showed that DNA was inserted into the plasmid
vector pUC 19.
Ten clones were randomly selected and
17
18
subjected to miniscreening by plasmid isolation at Pst
1 digestion.
The digests were analyzed on 1% agarose
gels (Figure 1).
Six classes of inserts of sizes 1.3Kb
(Lanes 2,3,4), 0.7Kb (Lane 5), 1.6Kb (Lane 6), 1.3Kb
and 1.0Kb (Lane 7), 0.6Kb (Lane 9) and 4.9Kb (Lane 11)
were observed
(Figure 1).
The cloned probe K3-5 and
32P-labelled C. xyli subsp. xyli chromosomal DNA were
chosen .or use as probes in the dot blot hybridization
studies.
The isolated plasmids with and without DNA
inserts were probed with radiolabelled C. xyli subsp.
xyli DNA.
The six plasmids harboring DNA fragments
hybridized with the probe as did C. xyli subsp. xyli
chromosomal DNA.
Plasmid pUC 19 without an insert did
not hybridize with the probe (Figure 2).
This
confirmed that the DNA inserts in the 6 recombinant
plasmids were from the C. xyli subsp. xyli genome.
Hybridization is indicated by dark spots on the
autoradiogram and lack of hybridization is shown by the
absence of these spots.
C. xyli subsp. xyli DNA detection
In a preliminary experiment, serial two-fold
dilutions of a suspension of C. xyli subsp. xyli
bacteria (5 x 10^ cells) were spotted on nitrocellulose
filters and probed with radiolabelled cloned and
32P-labelled C. xyli subsp. xyli chromosomal DNA.
As
shown in Figure 3, signals given by the J -labelled C.
19
Figure 1.
Miniscreening of transformed Escherichia coli JM 83
clones by plasmid isolation and determination of
Clavibacter xyli subsp. xyli DNA inserts in recombinant
plasmids.
The plasmids were prepared as described in
Materials and Methods and were digested with Pst 1 and
electrophoresed on a 1$ agrose gel (Lanes 2-11).
The
gel was stained with ethidium bromide (1 ug/ml in
water) for 15 minutes.
Lane 1: Hind III digest of
Lambda DNA (23, 9.4, 4.4, 2.3, 2.0, and 0.56 Kb).
Lane
2: K1-2 (1.3Kb).
Lane 3: K1-3 (1.3Kb).
Lane 4: K1-5
(1.3Kb).
Lane 5: K2-1 (0.7Kb).
Lane 6: K2-3 (1.6Kb).
Lane 7: K2-4 (1.3, 1.1Kb).
Lane 8: (pUC 19 with no
insert).
Lane 9: K3-1 (0.6Kb).
Lane 10: (pUC 19 with
no insert).
Lane 11: K3-5 (4.9Kb).
Hybridization of C. xyli subsp. xyli DNA, recombinant
plasmids, pUC 19 DNA, and Bacillus subtilis DNA with
J2P-labelled C. xyli subsp. xyli chromosomal DNA.
Autoradiogram of the hybridization of ^2P-labelled C.
xyli subsp. xyli DNA with:
l =2ug, 2=lug, 3 = 0.5ug, and
4 = 0.25ug of A) C. xyli subsp. xyli DNA, B) Kl-5 DNA,
C) K2-1 DNA, D) K2-3 DNA, E) K2-4 DNA, F) K3-1 DNA,
G) K3-5 DNA, H) pUC 19 DNA, and I) B. subtilis DNA.
21
Figure 3.
A comparison of the detection of Clavibacter xyli
subsp. xyli cells by cloned and 32P-labelled C. xyli
subsp. xyli chromosomal DNA. Five ul of a two-fold
dilution of C. xyli subsp. xyli cells were spotted on a
nitrocellulose filter (A). Dilutions of Agrobacterium
tumefaciens which were used as a negative control were
also spotted on the nitrocellulose filter (B). The
number of cells spotted was: 1 = 5 x 10 , 2 = 5 x 10 ,
3 = 1.25 x 109 , 4 = 6.2 x 108 , 5 = 3.1 x 108 , and
6 = 1.5 x 108 .
Autoradiogram of nitrocellulose filter hybridized with
32P-labelled C. xyli subsp. xyli chromosomal DNA.
Autoradiogram of a nitrocellulose filter hybridized
with cloned (K3-5) 32P-labelled C. xyli subsp. xyli
DNA.
22
xyli subsp. xyli chromosomal DNA probe were stronger
than those given by the cloned probe.
Agrobacterium
tumefaciens which was used as a negative control was
not recognized by the cloned or the J-‘P-labelled C.
xyli subsp. xyli chromosomal DNA probe.
The
O O
J*P-labelled C. xyli subsp. xyli DNA probe was then
tested to see how few C. xyli subsp. xyli bacteria it
could detect.
detect 5 x 10
As Figure 4 shows, the probe was able to
C. xyli subsp. xyli cells.
Bacillus
subtilis which was used as a negative control was not
recognized by the probe.
In addition,
another
experiment was done probing C. xyli subsp. xyli
chromosomal DNA and B. subtilis DNA with radiolabelled
C. xyli subsp. xyli chromosomal DNA.
subsp. xyli DNA
Only the C. xyli
hybridized with the C.
chromosomal DNA probe.
hybridize with the probe
xyli subsp. xyli
B. subtilis DNA did not
(Figure 2).
Determination of the limits of detection
The limit of detection of C. xyli subsp. xyli DNA
sequences in infected sugarcane sap was determined with
the cloned probe K305.
Two-fold dilutions of infected
and uninfected sap from sugarcane cultivar L 62-96 were
spotted on a nitrocellulose filter, and hybridized with
the probe.
The
dilution of the
probe hybridized to an eight fold
infected sugarcane sap.
The probe did
not hybridize with dilutions of uninfected sugarcane
sap (Figure 5).
24
Figure 4.
Detection of Clavibacter xyli subsp. xyli cells with
32P-labelled C. xyli subsp. xyli chromosomal DNA.
Dilutions of C. xyli subsp. xyli cells were made in
distilled water and 5 ul aliquots were spotted on a
nitrocellulose filter (A). Dilutions of Bacillus
subtilis which was as a negative control were also made
and spotted on the nitrocellulose filter (B). The
number of cells spotted were: 1 = 5 jc 10 , 2 = 5 x 10 ,
3 = 5 x 104 , 4 = 5 x 103 , 5 = 5 x 102 , and 6 = 50.
25
Figure 5.
Determination of the limits of detection of Clavibacter
xyli subsp. xyli sequences in dilutions of sugarcane
sap of infected iA) and healthy (B) cultivars L62-96
hybridized with ^ P - l a b e l l e d K3-5 DNA.
1 = undiluted,
2 = diluted 1:2, 3 = diluted 1:4, 4 = diluted 1:8,
5 = diluted 1:16, 6 = diluted 1:32.
26
Sampling of sugarcane tissues
In order to determine if bacteria could be
detected in other tissues of infected plants, assays
were done using samples from leaves, sheaths, spindles
and stalks from known infected and healthy sugarcane
plants were assayed.
These tissues were individually
homogenized and spotted on a nitrocellulose filter.
The filter was then hybridized with the ^P-labelled C.
xyli subsp. xyli chromosomal DNA probe.
The probe
recognized C. xyli subsp. xyli sequences in the leaves,
sheaths, spindles and stalks of infected but not
healthy sugarcane
(Figure 6).
27
Figure 6 .
Sampling tef sugarcane cultivar L62-96 tissues for
Clavibacter xyli subsp. xyli DNA sequences with a
32P-labelled C. xyli subsp. xyli chromos om al DNA.
Leaves (1), sheaths (2), spindles (3), and stalks (4),
from infected (A) and non-infected (B) sugarcanes were
individually homogenized and 5 ul aliquots were spotted
on a nitrocellulose filter.
DISCUSSION
This research demonstrated that dot blot
hybridization has the potential to be an effective tool
to detect Clavibacter xyli subsp. xyli/ the ratoon
stunting disease bacterium,
in infected sugarcane.
Five thousand C. xyli subsp. xyli cells were detected,
which is several orders of magnitude lower than
previous limits of detection reported for ELISA (26)
and fluorescent antibody staining (26).
This increased
sensitivity means that leaves or stalks can be sampled
without destruction of the whole plant as has been the
case in current sampling procedures.
The limits of detection could possibly be lowered
by a modification of the bacterial lysing and
hybridization steps (24).
The use of the 541 Whatman
filter paper as the solid support (23), instead of
nitrocellulose paper would allow more vigorous and may
result in more efficient lysing procedures to release
the bacterial nucleic acid without concern for the
integrity of the solid support which is a problem with
the brittle nitrocellulose paper.
With the advent of biotin-labelled probes (21), it
may be possible to detect C. xyli subsp. xyli from
28
29
infected plants in as little as two hr after
hybridization, rather than the overnight to twenty-four
hr period required for autoradiography.
In addition,
the dander inherent in the use and handling of
radioisotopes and the problem with the short shelf life
of ^2p will be eliminated.
The ^P-labelled C. xyli subsp. xyli chromosomal
DNA probe was more sensitive than the cloned probes for
the detection of C. xyli subsp. xyli however the cloned
probe should be used for future studies.
This is
because of the difficulties associated with culturing
C. xyli subsp. xyli and obtaining only a small amount
of DNA, whereas unlimited amounts of cloned DNA may be
obtained from the easily cultured E. coli.
The value of the dot blot hybridization could be
its use in quantitating bacterial populations among
sugarcane varieties as an aid in determining their
relative resistance or susceptibility to ratoon
stunting disease.
The other potential uses would be to
detect potential vectors, determine the host range and
to determine if C. xyli subsp. xyli has a soil borne
phase.
LITERATURE CITED
1.
Andiman, W. L., Gradoville, C., Heston, R.,
Neydorf, M. E., Savage, G. , Kitchingman, D. Shedd
and Miller, G.
1983.
Use of Cloned Probes to
Detect Epstein-Barr Viral DNA in Tissues of
Patients with Neoplastic and Lymphoproliferative
Diseases.
J. Infect. Dis.
148: 967-977.
2.
Birnboim, H. C. and Doly, J.
1979.
A Rapid
Alkaline Extraction Procedure for Screening
Recombinant Plasmid DNA.
Nuc. Acid. Res.
7:
1513-1522.
3.
Brandsma, J. and Miller, G.
1980.
Nucleic Acid
Spot Hybridization:
Rapid Quantitative Screening
of Lymphoid Cell Lines for Epstein-Barr Viral DNA.
Proc. Natl. Acad. Sci. U.S.A.
77: 6831-6856.
4.
Chou, S. and Merigan, T. C.
1982.
Rapid
Detection and Quantitation of Human
Cytomegalovirus in Urine through DNA
Hybridization.
N. Eng. J. Med.
308: 921-925.
5.
Damann, K. E. Jr. and Derrick, K. S.
1976.
Bacterium Associated with Ratoon Stunting Disease
in Louisiana.
Sugarcane Pathol. Newsl.
15/16:
20-22.
6.
Damann, K. E. Jr., Tejada, J. and Derrick, K. S.
1978.
Serologically Specific Microscopy of the
Bacterium Associated with Ratoon Stunting Disease
of Sugarcane.
pp. 317-320.
In Proceedings of 4th
International Conference of Plant Pathogenic
Bacteria.
Edited by Station de Pathologie
Vegetale et Phytobacteriologie. vol 1. Anger.
P. 339.
7.
Damann, K. E. Jr., Ogunwolu, E. 0. and Reagan, T.
E.
1983/84.
Ratoon Stunting Disease in Louisiana
Sugarcane:
A Preliminary Survey. La.
Agriculture.
27(2):
6-7.
8.
Davis, M. J., Gillaspie, A. G . , Jr., Harris, R. W.
and Lawson, R. H.
1980.
Ratoon Stunting Disease
of Sugarcane:
Isolation of the Causal Bacterium.
30
31
Science 210:
9.
1365-1367.
Davis, M. J., Lawson, A. G. Jr., and Harris, R. W.
1983.
Properties and Relationship of Two Xylem
Limited Bacteria and a Mycoplasmalike Organism
Infecting Bermuda Grass.
Phytopathology 73:
341-346.
10.
Davis, M. J., Gillaspie, A. G. Jr., Vidaver, A. K.
and Harris, R. W.
1984.
Clavibacter: A New Genus
Containing Some Phytopathogenic Coryneform
Bacteria including Clavibacter xyli Subsp. xyli.
sp Nov., Subsp. Nov and Clavibacter xyli Subsp
cynodontis Subsp. Nov., Pathogens that cause
Ratoon Stunting Disease.
Int. J. Syst. Bacteriol.
32: 315-326.
11.
Difco Laboratories Inc.
1967. Difco Manual of
Dehydrated Culture Media and Reagents for
Microbiology and Clinical Laboratory Procedures.
9th Edition.
Pages 131-132.
12.
French, R. C. 1983. Molecular Cloning of
Sugarcane Mosaic Virus cDNA: Use as a Probe for
the Detection of Virus Infection and Viral RNAs.
Ph.D. Dissertation.
Louisiana State University,
p. 47.
13.
Gillaspie, A. G. Jr., Irvine, J. E. and Steere, R.
C.
1966.
Ratoon Stunting Virus:
Assay Technique
and Partial Purification.
Phytopathology 56:
1426-1427.
14.
Gillaspie, A. G. Jr., Davis R. E. and Worley, J.
F.
1973. Diagnosis of Ratoon Stunting Disease
Based on the Presence of a Specific Microorganism.
Plant Dis.
Rep. 57: 987-990.
15.
Gillaspie, A. G. Jr., and Harris, R. W.
1979.
Limitations of ELISA for the Detection of RSD
Associated Bacteria in Sugarcane and Sudan Grass.
Sugarcane. Pathol. Newsl.
22: 25-28.
16.
Hanahan, D. 1983.
Studieson Transformation of
E_.
coli with Plasmids.
J. Mol. Biol.
166: 557-580.
17.
Kamiunten, H. and Wakimoto, S.
1976. Coryneform
Bacteria found in Xylem of the Ratoon Stunting
Diseased Sugarcane.
Ann. Phytopath. Soc. Japan.
42: 500-503.
18.
Kao, J. and Damann, K. E. Jr.
1978.
Microcolonies of Bacterium Associated with Ratoon
Stunting Disease found in Sugarcane Xylem Matrix.
32
Phytopathology.
6 8: 545-551.
19.
Kao, J. and Damann, K. E. Jr.
1980.
In Situ
Localization and Morphology of the Bacterium
Associated with Ratoon Stunting Disease of
Sugarcane.
Can. J. Botany.
58: 310-315.
20.
Koike, H., Gillaspie, A. G. Jr., and Benda, G. T.
A.
1982.
Cane Yield Response to Ratoon Stunting
Disease.
Int. Sugar. J. (84) 100: 131-133.
21.
Leary, J. J., Brigati, D. J. and Ward, D. C.
1983. Rapid and Sensitive Colorimetric Method for
Visualizing Biotin-Labelled DNA Probes Hybridized
to DNA or RNA Immobilized on Nitrocellulose
Bio-blots.
Proc. Natl. Acad. Sci. 80: 4405-4409.
22.
Lereclus, M. M . , Lecodet, J. and Dedonder, R.
1982. Molecular Relationships Among Plasmids of
Bacillus thuringiensis: Conserved Sequences
through 11 Crystaliferous Strains.
Mol. Gen.
Genet.
166: 391-398.
23.
Maas, R.
1983.
An Improved Colony Hybridization
Method with Significantly Increased Sensitivity
for Detection of Single Genes.
Plasmid 10:
296-298.
24.
Maniatis, T. E., Fritsch, E. F. and Sambrook, J.
1982. Molecular Cloning:
A Laboratory Manual.
Cold Spring Harbor laboratory, Cold Spring Harbor.
N.Y. p. 545.
25.
Moseley, S. L., Huq, I., Alim, A. R. M., So, M.,
Samed, P. M. and Felkow, S.
1980. Detection of
Enterotoxigenic Escherichia coli by DNA Colony
Hybridization.
J. Infect. Disease.
142: 892-898.
26.
Owens, R. A. and T. 0. Diener.
1981.
Sensitive
and Rapid Diagnosis of Potato Spindle Tuber Viroid
Disease by Nucleic Acid Hybridization.
Science
213: 670-672.
27.
Plata, M. I. 1983. Quantitative Immunological
Detection of the Ratoon Stunting Disease
Bacterium.
M.S. Thesis.
University of Florida,
p. 545.
28.
Rigsby, P. W., Dieckmann, J . , Rhodes, C. and Berg,
P.
1977.
Labelling Deoxyribonucleic Acid to High
Specific Activity in Vitro by Nick Translation
with DNA Polymerase 1. J. Mol Biol.
113: 237-251.
33
29.
Steib, R. and Forbes, I. C.
1957.
Johnson Grass
and Corn as Carriers of the Virus of Ratoon
Stunting Disease of Sugarcane.
Sugar. Bull.
(35)
23: 375-379.
30.
Steindl, D. R. L.
1951.
Stunting Disease: In
Sugarcane diseases of the World,
pp. 432-439.
Vol 1. J. P. Martin, E.V. Abbott, C. G. Hughes.
Eds. Elsevier Publishing Co. Amsterdam,
p. 542.
31.
Teakle, D. S., Smith, D. M. and Steindl, D. R. L.
1973. Association of a Small Coryneform Bacterium
with Ratoon Stunting Disease of Sugarcane.
Aust.
J. Agri. Research.
24: 869-874.
VI T A
Jimmy Michael Kamso-Pratt was born in Freetown, Sierra
Leone on February 15, 1951.
He graduated from St.
Edward’s Secondary School, Freetown, Sierra Leone in
1969.
He attended Cumberland College, Williamsburg,
Kentucky and graduated in 1975 with a Bachelor of
Science degree in Biology.
He attended Iowa State
University from 1979 to 1980.
From 1980 to 1982 he
attended the McNeese State University, Lake Charles,
Louisiana.
He received a Master of Science degree in
Microbiology from McNeese State University in May,
1982.
He entered the Department of Plant Pathology and
Crop Physiology at Louisiana State University in 1982
where he is currently a candidate for a Doctor of
Philosphy degree in Plant Pathology.
34
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
Jimmy M ic h a e l K a m s o -P ra tt
M ajor Field:
P la n t H e a lth
Title of Dissertation:
D e t e c t io n o f C l a v i b a c t e r x y l i Subsp. x y l i , t h e R a to o n S t u n t in g
D is e a s e B a c t e r iu m , i n S u g a rc a n e by D o t B l o t H y b r i d i z a t i o n
Approved:
Major Professor and iChairm
Dean of the Graduatee Sjchool
E X A M IN IN G C O M M IT T E E :
<?*y. ti
’
CUa f
OW-
Date of Examination:
Novem ber 2 7 ,
198 5