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LSU Historical Dissertations and Theses
Graduate School
1990
Ecology of Erwinia Chrysanthemi, Causal Agent of
Bacterial Stem and Root Rot on Sweet Potato and
Etiology of Souring.
Valmir Duarte
Louisiana State University and Agricultural & Mechanical College
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Duarte, Valmir, "Ecology of Erwinia Chrysanthemi, Causal Agent of Bacterial Stem and Root Rot on Sweet Potato and Etiology of
Souring." (1990). LSU Historical Dissertations and Theses. 5043.
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O rd e r N u m b e r 9123186
Ecology o f Erwinia chrysanthemiy causal agent o f bacterial stem
and root rot on sw eetp otato and etiology o f souring
Duarte, Valmir, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1990
UMI
SOON.ZeebRd.
Ann Arbor, M I 48106
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Ecology of Erwinia chrysanthemi. Causal
Agent of Bacterial Stem and Root Rot on
Sweetpotato and Etiology of Souring
A
Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillm ent of the
requirements for the degree of
Doctor of Philosophy
In
The Department of Plant Pathology and Crop Physiology
by
Valmir
B.S., Federal University of
Alegre, RS,
M.S., Federal University of
Alegre, RS,
Duarte
Rio Grande do Sul, Porto
Brazil, 1977
Rio Grande do Sul, Porto
Brazil, 1981
December
1990
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ACKNOWLEDGEMENTS
I express my
Christopher
A.
sincere appreciation to my
Clark.
Gratitude
also
is
advisor,
expressed
Dr.
to
my
committee members; Dr s. A. Biel, L. L. Black, K. E. Damann, J.
W. Hoy, R. W. Schneider, and W. J. Hudnall. I would also like
to thank Jeannie A. Wilder-Ayers, Dr. Clark's former research
associate, for her assistance.
I am also grateful
to Dr.
J.
B.
Baker,
the
faculty,
staff, and students of the Department of Plant Pathology and
Crop Physiology for their support and encouragement.
Appreciation is also extended to my colleagues
Faculdade de Agronomia, UFRGS,
in the
Porto Alegre, RS, Brazil. Of
these, I extend a special thanks to the members of "the plant
pathology group".
The
financial
support
of
the
Council
of
National
Scientific and Technological Development (CNPq), Brazil, was
vital to accomplish my Ph.D. program.
I thank my parents, Ramâo and Leocadia Duarte, for their
constant moral support. I am also deeply grateful to my wife,
Nara, and my two daughters, Amanda and Cristiane,
for their
love, encouragement, and support during my years as a graduate
student.
11
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS .......................................
Ü
LIST OF TABLES .........................................
vi
LIST OF FIGURES ........................................
vii
ABSTRACT ...............................................
X
INTRODUCTION ...................................
1
CHAPTER I
REVIEW OF LITERATURE .................................
4
A.
Persistence and dissemination of Erwinia sp.
5
B.
Quantitative studies of soft-rot erwinia
...
populations .......................................
11
C.
Characterization of Erwinia chrysanthemi ........
17
D.
Phytopathogenic pectolytic clostridia ...........
21
Literature cited ...................
26
CHAPTER II
PRESENCE
OF Erwinia
chrysanthemi. CAUSAL AGENT
OF STEM
AND ROOT ROT, ON SWEETPOTATO THROUGH THE GROWING SEASON
Abstract ..............................................
36
Introduction ..........................................
37
Materials and Methods ................................
38
Results ...............................................
44
Discussion ............................................
48
Tables and Figures ..........................
51
Literature Cited ......................................
65
111
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CHAPTER III
CHARACTERIZATION OF SWEETPOTATO STRAINS OF Erwinia
chrysanthemi. CAUSAL AGENT OF BACTERIAL STEM AND
ROOT R O T ...............................................
67
Abstract ...............................................
68
I n t r o d u c t i o n .......
69
Materials and Methods ................................
71
Results ................................................
75
Discussion ............................................
78
Tables and Figures ....................................
80
Literature Cited ......................................
92
CHAPTER IV
EFFECTS
OF
INTERACTION
OF
Erwinia
chrysanthemi
AND
Fusarium solani ON SWEETPOTATO STEM R O T .............
94
Abstract ...............................................
95
Introduction ..........................................
96
Materials and Methods ................................
97
Results ................................................
100
Discussion ............................................
102
Tables and Figures ....................................
104
Literature Cited ......................................
Ill
CHAPTER V
ASSOCIATION OF PECTOLYTIC CLOSTRIDIA WITH SOURING
OF SWEETPOTATO STORAGE
ROOTS .......................
112
Abstract .............................................
113
Introduction .........................................
114
iy
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Materials and Methods ................................ 116
Results ...............................................
121
D i s c u s s i o n .........
124
Tables and Figures ...................................
128
Literature Cited .....................................
141
V I T A ....................................................
144
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LIST OF TABLES
Table
II.
Page
PRESENCE OF ERWINIA CHRYSANTHEMI■ CAUSAL AGENT OF STEM
AND
ROOT
ROT
ON
SWEETPOTATO,
THROUGH
THE
GROWING
SEASON
1.
Number of contaminated mother roots ...............
51
2.
Number of contaminated underground stems ..........
52
3.
Plant production in beds ...........................
53
4.
Yield of storage roots .............................. 54
III.
CHARACTERIZATION
OF
SWEETPOTATO
STRAINS
OF
ERWINIA
CHRYSANTHEMI. CAUSAL AGENT OF BACTERIAL STEM AND ROOT
ROT
1.
Sources of Erwinia strains ......................... 80
2.
Comparison of strains for stem rot indices .......
81
3.
Estimate of median effective dose ............
82
4.
Tests of carbon source utilization ................
83
IV.
EFFECTS
OF
INTERACTION
OF
ERWINIA
CHRYSANTHEMI
AND
FUSARIUM SOLANI ON SWEETPOTATO STEM ROT
1.
Dry weight of plants ..............................
104
2.
Disease incidence and remaining plants ..........
105
3.
Effect on sweetpotato yield ......................
106
4.
Effect of soil matric potential ..................
107
VI
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LIST OF FIGURES
Figure
Page
II. PRESENCE OF ERWINIA CHRYSANTHEMI. CAUSAL AGENT OF STEM
AND
ROOT
ROT
ON
SWEETPOTATO,
THROUGH
THE
GROWING
SEASON
2. 1. Inoculation of mother storage roots by dipping
...
55
2. 2. Inoculation of mother storage roots by
micropipette tip ...................................
56
2. 3. Plant production bed layout .......................
57
2. 4. Diagram of experiments ............................
58
2. 5. Storage root sampling m e t h o d ...................
59
2. 6 . Isolation plating .................................
60
2 . 7. Sweetpotato underground s t e m ...................
61
2. 8 . Populations of Erwinia chrysanthemi on mother
roots ................................................
2. 9. Survival in soil at three temperatures
2.10.
III.
..........
Recovery of Erwinia chrysanthemi from crate wood
CHARACTERIZATION
OF
SWEETPOTATO
STRAINS
OF
.
62
63
64
ERWINIA
CHRYSANTHEMI. CAUSAL AGENT OF BACTERIAL STEM AND ROOT
ROT
3. 1.
Toothpick inoculation ..............................
84
3. 2.
Stem rot index .....................................
85
3. 3.
Storage root inoculation ...................
86
3. 4. Comparison of strains for sweetpotato stem
rot indices .........................................
3. 5.
Comparison of strains for tomato stem rot indices.
vii
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87
88
3. 6 . Comparison of strains for bell pepper stem
rot indices ...............................
3. 7. Comparison of strains for cabbage stem rotindices
89
90
3. 8 . Comparison of strains for storage root lesion
dimension ..........................................
IV. EFFECTS
OF
INTERACTION
OF
ERWINIA
91
CHRYSANTHEMI
AND
FUSARIUM SOLANI ON SWEETPOTATO STEM ROT
4. 1.
Vine cutting inoculation .......................... 108
4. 2.
stem rot lesions on s w e e t p o t a t o ..................
109
4. 3. Length of stem rot l e s i o n s ........................
110
V. ASSOCIATION OF PECTOLYTIC CLOSTRIDIA WITH SOURING OF
SWEETPOTATO STORAGE ROOTS
5. 1. Souring symptoms ................................... 128
5. 2. Preparation of cylinders of raw storage root
tissue
............................................
129
5. 3. Anaerobiosis using wet paper towel and plastic
wrap ................................................
130
5. 4. Effect of disinfestation of storage roots on
incidence of souring ..............................
5. 5.
131
Colonies of pectolytic clostridia on PIA medium .. 132
5. 6 . Cell morphology of pectolytic b a c t e r i a ..........
133
5. 7.
Maceration of raw tissue c y l i n d e r s ..... .........
134
5. 8 . Maceration activity on different hosts ..........
135
5. 9.
Pathogenicity on s w e e t p o t a t o .....................
136
5.10.
Pathogenicity on potato tubers ...................
137
5.11.
Pathogenicity on carrot ..........................
138
viii
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5.12. Pathogenicity on o n i o n ............................
139
5.13. Combined inoculation with Erwinia chrysanthemi
and pectolytic Clostridium ........................
IX
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140
ABSTRACT
During three seasons, mother storage roots of Beauregard
sweetpotato
were
inoculated
with
Erwinia
chrysanthemi
in
March, and the pathogen was recovered from them the day they
were
bedded,
7
days
later,
and
at
the
first
and
second
pullings of slips as well as from underground stems at harvest
in
September
and
October
of
the
first
and
second
transplantings, respectively. Little stem rot occurred in the
fields during the three seasons.
Thirteen
strains
of
E.
chrysanthemi.
8
isolated
originally from sweetpotato and 5 from other hosts, and one
strain of E. carotovora subsp. carotovora were inoculated on
sweetpotato and 12 different hosts. Strains from hosts other
than sweetpotato caused little or no disease on sweetpotato.
Of 95 carbon sources tested, sweetpotato strains used the same
25, were unable to grow on 57, and varied on 13 substrates.
Jewel
and
Beauregard
sweetpotato
stem
cuttings were
inoculated with E. chrysanthemi and/or Fusarium solani before
planting
in
greenhouse,
the
greenhouse
and
in
the
field.
In
the
stem rot lesion length was greater when the two
pathogens were inoculated together than the sum of stem lesion
length produced by both pathogens inoculated separately. The
same effect was observed on Beauregard in the field in 1989
and 1990.
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When storage roots were punctured with a flamed needle
and
incubated
plastic bags,
tests,
15%
and
for 5 days
submerged
in distilled water
85 to 100 % rotted in 2 tests.
none
of
storage
roots
in
In the same
rotted when
surface
disinfested with NaOCl. Two distinguishable pectolytic sporeforming,
anaerobic
bacteria
resembling
Clostridium
in
morphology were found in the lesions, regardless of treatment.
Inoculation
of
storage
roots
with
pure
cultures
of
these
bacteria and incubation under anaerobic conditions induced
rapid maceration and production of gas, typical of "souring",
a
disorder previously
regarded
as
solely physiological
origin.
XI
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in
INTRODUCTION
Until
recently,
sweetpotato,
bacterial
Ipomoea batatas
(L.)
stem
and
Lam.,
root
caused by
rot
of
Erwinia
chrysanthemi Burkholder, McFadden, and Dimock was unknown. In
1974, a major outbreak of this disease occurred in production
areas of Georgia, and in 1975 it threatened the existence of
the sweetpotato industry in that state (7, 79). In Louisiana,
the
disease has not been found as a stem rot but
has been
found to a limited extent in storage roots in plant beds and
the
field. However,
new
cultivars
have
shown
greater
susceptibility to E. chrysanthemi when artificially inoculated
(8 , 75). Despite the potential hazard of this disease to the
sweetpotato crop, literally nothing has been reported on it.
Therefore,
I
studied several
aspects
of
E.
chrysanthemi
ecology to develop a better insight into the epidemiology and
control of the disease.
Following this reasoning, I reviewed, in chapter I, the
current
knowledge
on (a)
persistence
Erwinia
sp., (b)
quantitative studies
and dissemination
of
populations
of
of
Erwinia sp., (c) characterization of E. chrysanthemi. and (d)
phytopathogenic pectolytic clostridia.
To
address
some of
the
epidemiological
aspects
of
bacterial stem and root rot, I conducted experiments to study
(a) the presence of E. chrysanthemi on sweetpotato through the
growing season,
(b) the transmission of the bacterium from
seed roots to stems and new plants,
(c) the effect of pulling
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2
versus cutting transplants from beds with inoculated roots on
transmission and disease occurrence, and (d) the transmission
of the pathogen to stem cuttings and/or to sprout stubs and
mother roots by contaminated knives.
The results of these
investigations are reported in chapter II.
Information on strains of any pathogen has considerable
practical interest. The selection of strain (s) for evaluation
of germplasm for resistance is a particular example.
Also,
information on antibiotic resistant mutants used in ecological
studies is essential. In the investigation mentioned above, I
employed a rifampicin-resistant strain of E. chrysanthemi to
trace this pathogen through the sweetpotato growing season.
Therefore, I compared (a) virulence of sweetpotato and other
strains of E. chrysanthemi on sweetpotato and (b) on different
hosts,
(c) utilization of carbon sources by strains, and (d)
a rifampicin-resistant strain to the wild type using these
tests. The report of the results of these investigations is in
chapter III.
Several lines of evidence suggest an interaction of E.
chrysanthemi and Fusarium solani. the latter causing Fusarium
root
and
stem
canker,
on
stem
and
root
diseases
of
sweetpotato. Therefore, I conducted experiments to examine the
effects of combined inoculation of stems and roots with E.
chrysanthemi
and
F.
solani. The
results
are
reported
in
chapter IV.
Sweetpotato growers may encounter serious economic losses
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3
when excessive rainfall results in water-saturated soils for
several days prior to harvest. It leads to the asphyxiation of
harvestable storage roots and usually to a condition known as
"souring” , a disorder previously thought to be exclusively
physiological in origin. I hypothesized the involvement of E.
chrysanthemi. a facultative anaerobic bacterium. However, this
bacterium was
not
association
of
sweetpotato
became
found
in
pectolytic
evident.
affected
tissue.
clostridia
The
with
results,
Instead,
the
souring
of
demonstrating
pathogenicity of these pectolytic clostridia, are reported in
chapter V.
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CHAPTER I
LITERATURE REVIEW
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A. PERSISTENCE AND DISSEMINATION OF ERWINIA SP.
INTRODUCTION
Accurate identification of the sources of inoculum is a
prerequisite
control
for
the
strategies
dissemination
can
determination
(81).
of
Investigation
indicate
ecological
important to the success of a pathogen.
effective
disease
of persistence
and
features
are
that
The ecology of the
soft rot erwinias in relation to potatoes has been reviewed
(63). Therefore, this review intends to highlight only some of
the major aspects of persistence and dissemination of soft rot
erwinias.
A.I.
SOIL
Evidence for survival of erwinias in soil is conflicting
(63).
Despite
reports
persist in soil
primary
(5, 71,
source
of
indicating
that
the
bacteria
could
72), the significance of soil as a
inoculum
has
been
refuted
(81).
Potato
fields in Scotland were extensively infested after harvest in
September,
early
but populations declined to very
summer
infested soil,
of
the
following
year
(62).
longevity was greater in dry
low levels by
In
artificially
(1 0 % moisture)
than in wet (2 1 % moisture) soil ai i decreased as temperatures
rose,
particularly above 25 C.
carotovora
subsp.
carotovora
Because survival of the E.
and
E.
carotovora
subsp.
atroseptica in soil is apparently restricted, their presence
in fields could be attibuted to recurrent introductions from
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6
The predominance of E. carotovora subsp. carotovora or E.
carotovora subsp. atroseptica in the soil is dependent on soil
temperature (54, 64). When inoculated tubers were planted in
cool
soils
in the
field or greenhouse
(7.0-18.5 C average
minimum and 16-26 C average maximum temperature during the
first 30 days after planting) E. carotovora subsp. atroseptica
caused significantly more disease than E. carotovora subsp.
carotovora. When soil temperatures were high at planting time
(21.4-24.0 C average minimum and 29.6-35.0 C average maximum
temperature
carotovora
carotovora
for
the
subsp.
subsp.
first
carotovora
30
days
caused
atroseptica.
At
after
more
planting),
disease
than
intermediate
E.
E.
soil
temperatures both were infective (54).
A survey of a sweetpotato field
that was being harvested
showed that between 5 and 10% of the harvested roots were
rotted even though above ground plant parts were symptomless.
Erwinia chrysanthemi was isolated from soil and soil debris
collected from a harvester, but not from field soil. However,
it was suggested that E. chrysanthemi could survive in soil in
small numbers (79) .
Erwinia chrysanthemi was isolated from blackened roots,
soft discolored rhizome tissue and rhizosphere soil, but not
from field
soil
in the
immediate neighborhood
of diseased
cardamon plants (Elletaria cardomomum) (83). In contrast, soil
was considered to be the primary source of inoculum of E.
chrysanthemi. causal agent of bacterial leaf spot of tobacco.
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7
and
secondary
spread
was
shown
to be
caused
by
rainwater
splashing from infected to healthy plants (24).
Erwinia carotovora subsp. carotovora and E. carotovora
subsp. atroseptica have been isolated from fallow field soils
(53),
nonagricultural
collected
from
soils,
streams,
as well as from water samples
lochs,
and
rivers
in
arable
and
nonarable areas (35), but no Erwinia spp. were isolated from
underground waters (52). Erwinia carotovora subsp. atroseptica
and subsp.
different
carotovora were detected
cropping
Erwinias were
histories
found in all
on
in all
three
fields,
17
farms
fields with
in
Scotland.
although they were not
found consistently and were usually in small numbers.
suggested
that
the
bacteria
could
be
endemic
in
This
soil
in
Scotland (62). In another study, E. chrysanthemi survived in
mineral soil for 18 days, but was undetected in unsterile peat
1 day after infestation (44).
Using a selective,
differential medium (PT),
soft-rot
Erwinia spp. were isolated from rhizosphere soils of many crop
plants and from some field soils containing plant residues
(5) . Erwinia
carotovora was
detected
in the root
zone
of
potato plants and also in soil samples between plants, but in
smaller numbers than in the root zone. Moreover, although few
plants showed symptoms of blackleg during the growing season,
almost all tubers were infested with E. carotovora at harvest
(13).
The presence of E. carotovora in rhizosphere soil of any
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8
O f the weeds (groundsel, pineapple weed, shepherd's purse, and
speedwell) or crops (barley, wheat, cabbage, rape, and turnip)
examined in Scotland, with the exception of brassicas, was no
more frequent than in bare soil
(13). Survival was best in
association with brassicas, moderate on grasses and cereals,
and least on potatoes and weeds (62).
The
role
of
the
rhizosphere
as
a niche
in which
E.
carotovora can extend its longevity in soil has been suggested
(42, 43). However, survival of the bacteria in both bare and
rhizosphere soil was similar and dependent on the temperature
and
to
a
lesser
extent,
on
the
soil
moisture
content.
Moreover, survival was not favored by any of the plant species
examined,
including potatoes. However, E. carotovora subsp.
carotovora survived well on brassicas (42, 62).
A.2. Water
Investigation of the presence of E. carotovora in surface
water
in
North
America
showed
that
E.
carotovora
subsp.
carotovora represented 98.8% of strains recovered from surface
water samples; E. carotovora subsp. atroseptica made up the
remainder of the isolates and E. chrysanthemi was not found
(35) .
Water in the soil after heavy rain leaches E. carotovora
from rotting tubers.
Evapotranspiration forces redistribute
both water and the bacteria in the upper part of the drill as
it dries. Tubers absorb water and the lenticels soon open or
proliferate. The bacteria in the water film eventually reach
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9
the
complementary
cells
of
the
lenticels.
As
the
soil
continues to dry, the water phase becomes discontinuous and
bacterial movement is restricted (58).
A.3. Plant Residues
Erwinia chrvsanthemi survived in artificially infested
soil and in infected carnation debris in sufficient numbers to
induce
symptoms
in carnation plants after 25 and 70 days,
respectively (26). In another study, it was not recovered from
infected plant parts that had been buried in a compost pile
for 10-12 weeks (37).
A.4. Infested planting stock
Contamination of progeny tubers by E.carotovora occurs
after the mother tubers have rotted and released bacteria into
the soil (57). No relation between blackleg incidence and the
level of contamination in the harvested tubers was found (57).
Erwinia survives in the lenticels and to a lesser extent on
the tuber surface during the storage period
(57),
but
its
population or the ability to detect it decreases substantially
with time (14).
A.5. Aerosols
When simulated raindrops fall on blackleg-infected potato
stems, an aerosol containing bacterial cells is generated. The
aerosol moves readily in airstreams.
These bacteria remain
viable for more than 1 hr in a closed system at 90-95 % r.h.
and
10.6
blackleg
C
(29).
Thus,
organism
could
airborne
occur
transport
following the
of
the
potato
generation
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of
10
aerosols by raindrop
Also,
airborne
E.
impact on infected
carotovora
subsp.
stem tissue
carotovora
(30) .
and
E.
carotovora subsp. atroseptica survive for 15 min or more to
indicate that airborne spread of viable bacteria cells could
take
place,
especially
under
cool,
humid
atmospheric
conditions (31). This information helps assess the potential
significance
of
aerosol
transmission.
Thus,
potato
crops,
raised from potato stocks free from infection with soft rot
Erwinia. could be recontaminated. It also suggests it should
be possible to estimate the separation distances between crops
that would be necessary to minimize recontamination (30).
Aerosols
containing both
erwinias have
been
found
Scotland after rain during the latter half of the year
in
(59,
74), and it is postulated that erwinias could be washed out of
the air by rain to contaminate water puddles and plant leaves
(62). Contaminated leaves later rot on the soil surface and
the bacteria
contamination
are released
would
into the soil
depend
on
the
(25).
occurrence
Hence,
of
soil
aerosols
containing erwinias coupled with the requirement for rain for
deposition (62).
REMARKS
The question of whether soft rot erwinias are endemic at
low level in soil is still unresolved. However,
it has been
generally accepted that they do not have a permanent
soil
phase.
Their occurrence in fallow and nonagricultural soils
might
occur
by
repeated
introduction
from
a
variety
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of
11
sources,
results
particularly
reported
may
air
be
and
rain
explained
(62).
at
The
least
conflicting
partially
by
differences in climate and in methodology used to detect low
numbers of the bacteria (63). In common with many other plantpathogenic bacteria, the soft-rot erwinias can overwinter in
contaminated
harvest
with
plant
residues
remaining
in
the
soil
after
(63). The bacteria also may survive in association
volunteer
plants
of
the
affected
crop
or
in
the
rhizospheres of other cultivated plants and of certain weeds,
particularly in tropical regions where plant growth is often
continuous and the vegetation diverse
(63). A comprehensive
view of the ecology of these bacteria will be obtained only
when their relationships with crops other than potatoes in
temperate regions and with all crops in other regions also
have been examined (63).
B. QUANTITATIVE STUDIES OF SOFT-ROT ERWINIA POPULATIONS
INTRODUCTION
No effort is made in this review to describe in detail
any of the various techniques that have been developed to
quantitate soft-rot Erwinia or to list all their applications.
Rather, the purpose is to give an overview of the main methods
and point out some of the benefits of using them.
B.l Selective media
A weakness in quantitative work is that selective media
may kill or inhibit not only unwanted organisms but also a
part of the desired population which may be sensitive to toxic
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12
compounds, e.g. damaged, senescent, or actively dividing cells
(65). A medium (CVP) containing NaNOg, sodium polypectate, and
crystal violet was compared with other selective media for the
isolation of soft-rot bacteria from soil. Pectolytic colonies
of
Erwinia
spp.
could
be
distinguished
from
Pseudomonas spp.
by the type of depressions
pectate
and
medium
colony
morphology.
those
formed
of
in the
Recovery
of
E.
carotovora and E. carotovora subsp. atroseptica from amended
field soil on CVP ranged from 65 to 100%, depending upon soil
type and the number of bacteria added to soil samples. When
CVP
was
used
for
detection
of
soft-rot
bacteria
in
soil
samples from cabbage, carrot, and potato fields, fluorescent
pseudomonads (not soft-rot Erwinia) were the pectolytic gramnegative bacteria most frequently isolated (1 1 ).
A
substitute
of
CVP
medium
(BCVP)
polypectate manufactured by Bulmer Ltd
Plough
improves
Lane,
Hereford
both
the
atroseptica
and
production
of
E.
rate
HR4
OLE,
of
growth
carotovora
pectolytic
UK)
of
(H.
was
E.
subsp.
pits
(16).
containing
P.
a
Bulmer Ltd,
described
carotovora
carotovora
Recently,
which
subsp.
and
the
a
new
formulation for BCVP, named MBCVP, substituted KNO 3 for NaNOg,
incorporated
0.5%
peptone,
and
altered
the
CaCl 2 -agar
concentration. These modifications, combined with the use of
cold rather than boiling water and mixing by hand rather than
using a blender, produced a smooth, firm medium suitable for
streaking, on which large colonies form distinct, deep pits
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13
after 24 hr (89).
A selective medium (PT) containing polygalacturonic acid
as the carbon source was developed to detect E. carotovora
subsp. carotovora and E. carotovora subsp. atroseptica in the
rhizosphere, plant debris, and soil. Erwinia was distinguished
by the formation of clear zones (zones of pectolysis) around
the colonies after gently flooding plates with a solution of
cetrimide. Furthermore, Erwinia could be differentiated from
other
bacteria
colonies.
To
by
the
increase
formation
the
of
white,
efficiency
of
scallop-edged
the
assay,
an
enrichment technique with PT broth also was used (5).
A method was described for identifying and quantifying E.
carotovora
subsp.
carotovora.
E.
carotovora
subsp.
atroseptica. and E. chrysanthemi directly from plant tissue
and from other sources based on the pattern of growth and
cavity
formation
different
on
CVP
temperatures
with
or
(61).
without
Erwinia
erythromycin
chrvsanthemi
at
forms
cavities on CVP at 27, 33.5, and 37 C but failed to grow at 27
C
on
CVP
with
erythromycin.
The
method
allows
the
differentiation of the three erwinias in a mixed population
and
avoids
purification,
(61).
However,
time
and
a
consuming
procedures
of
isolation,
identification using physiological
study
using
45
erwinia
strains
tests
from
14
different hosts did not confirm the efficacy of the method
(40).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
14
6.2. Enrichment and estimation procedures
Low numbers of soft rot bacteria can be detected in soil
if samples are incubated anaerobically in a pectate-basal salt
solution
(53). Enrichment media, antibiotic resistance,
and
Most Probable Number (MPN) analysis were used to enumerate as
few as 1 to 10 cells of genetically engineered E. carotovora
subsp. carotovora / 10 g soil (77). The technique was applied
in studying the fate of genetically engineered E. carotovora
after introduction into soil microcosms. The MPN technique is
recommended
to
monitor
the
persistence
of
genetically
engineered E. carotovora subsp. carotovora after its release
into the environment (77).
The
concentration
of
organisms
estimated without any direct count.
in
a
liquid
can
be
The concept of MPN is
based on an application of the theory of probability.
There
are two principal assumptions: the organisms are distributed
randomly throughout the liquid, and the sample is certain to
exhibit growth whenever it contains one or more organisms (9) .
The usefulness
of MPN was postulated
for determining
numbers of E. carotovora subsp. carotovora and E. carotovora
subsp. atroseptica in soils and plant material (55). Instead
of
replicate tubes
serial
dilutions,
medium
were
placed.
The
used
of inoculated broth with aliquots
defined
on
which
conclusion
was
areas
of
a
solid
from
semiselective
drops
from
the
dilutions
that
when
working
with
were
small
populations of E. carotovora. as often occurs in soils and on
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15
plant materials, MPN may be the best choice (55).
B.3. Environment
A method that
involves wrapping tubers
in wet tissue
paper, placing them in polyethylene bags, and incubating them
in a nitrogen atmosphere for 6 to 7 days was developed for
detection of potato tubers contaminated with
E. carotovora
subsp. carotovora and E. carotovora subsp. atroseptica (56).
Further
investigation
showed
that there was
between a nitrogen atmosphere for
no
difference
incubation and a double
layer of polyvinylidene film. Two layers of polyvinylidene
film precluded the requirement for special incubation chambers
and
a
source
of
nitrogen
(14).
Latent
infection
or
contamination can also be detected when tubers are induced to
rot under anaerobic conditions induced by maintaining a film
of water over the tuber (63).
B.4. Serology
Although serological testing has been quite successful
for various phytopathogenic bacteria, serological indexing for
E.
carotovora
strains
which
(12,
is hampered by the
78,
combines
a
84).
serological
diversity of
Fluorescent antibody staining
an
antiserum
(FAS),
globulin
fraction
of
with
fluorescein-isothiocyanate,
was used
for identification of
isolates of E. carotovora subsp. carotovora and E. carotovora
subsp. atroseptica (2, 70). Fluorescing E. carotovora subsp.
atroseptica cells were detected on prepared slides in mixed
populations containing as few as 500 cells/ml mixed with 10®
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16
cells/ml
of
E.
carotovora
subsp.
carotovora. Cells
of
E.
carotovora subsp. atroseptica also were detected in soils to
which as few as 10 cells/g had been added and on potato leaves
previously
sprayed
with
bacterial
suspensions.
The
FAS
procedure also was effective in detecting E. carotovora subsp.
atroseptica
tubers
and
in tissues
of
infected
and/or decaying potato
in artificially contaminated
adult
fruit
flies
(Drosophila melanoqaster) (2). A serologically identifiable
strain of E. carotovora subsp. atroseptica was used to monitor
this pathogen in the field (34). Potato tubers were inoculated
in the field in June and the strain was detected in November
in the remains of inoculated tubers.
B.5. DNA-DNA Hybridization
Various strategies have been used to develop genomic DNA
probes
specific
for
particular
bacterial
pathogens.
One
approach has been to screen the target bacterium plus closely
related bacteria with individual or pooled DNA probes (15). A
DNA probe was developed which was specific for E. carotovora
and
hybridized
with
all
tested
strains
in
the
subspecies
atroseptica. carotovora. and betavasculorum. regardless
serogroup
specificity.
The probe,
isolated
of
from a genomic
library of Erwinia. was selected for its ability to hybridize
to genomic DNA of Erwinia. but not to genomic DNA from the
taxonomically related Escherichia coli. A single colony of E.
carotovora could be detected against a background of about
1,000 colonies of soil bacteria on nitrocellulose filters. As
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17
few as 200 colony-forming units (cfu) of E. carotovora subsp.
atroseptica could be detected in a suspension containing 2 X
10^ cfu / ml of soil bacteria (84).
REMARKS
CVP medium,
tool
for
or its substitutes, has been a very useful
quantitating
and
detecting
soft
rot
erwinias.
Enrichment procedures can help recovering these pathogens when
the
population
is
very
low.
Combining
enrichment
media,
antibiotic resistance, and MPN analysis should be considered
in ecological studies of soft rot erwinias. Likewise, serology
and
molecular
markers
are
good
tools,
facilitating
the
detection of target bacteria. However, it is still difficult,
using current available methods, to distinguish between cases
of 'die out' (complete elimination) and 'die back' (decline of
populations below detectable levels).
C. CHARACTERIZATION OF Erwinia chrvsanthemi
INTRODUCTION
Erwinia chrvsanthemi Burkholder, McFadden & Dimock was
designated as a new species based on strains that had been
isolated
from Chrvsanthemum morifolium
host range,
used
to
physiological properties,
characterize
E.
(4).
Information on
and serology has been
chrvsanthemi.
This
review
will
highlight some of these aspects.
c.l. Host Range
Strains of E. chrvsanthemi have been tested for virulence
or pathogenicity to plants or plant parts of at least 118
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18
plant species or cultivars
Artificial
plant
(3, 4,
inoculation
6 , 18,
tests
27,
with
36,
39,
strains
50).
of
E.
chrysanthemi have shown great variability. Although this may
be attributed primarily to the inoculation methods and strains
used, it also is taken as an indication that many strains of
E. chrvsanthemi cause disease in plants other than those from
which they have been isolated (18).
C.2. Physiological Properties
Erwinia chrvsanthemi. E. carotovora subsp. carotovora and
E. carotovora subsp. atroseptica are physically alike in being
peritrichously flagellated rods and biochemically alike
being
pathogenic
anaerobes,
to
plants,
gram-negative,
in
facultative
reducing nitrate to nitrite, and fermenting many
carbohydrates to acids (28, 38).
Erwinia
pectate
acetoin,
chrvsanthemi
degradation;
and
sensitivity
acid
to
has
been
production
from
ethanol,
erythromycin;
characterized
of
based
phosphatase,
growth
producing
at
gas
36
from
indole,
to
37
lactose,
or palatinose
(21).
trehalose,
C,
glucose,
utilization of malonate; inability to grow in 5% NaCl; or
produce acid from a-methyl-d-glucoside,
on
to
maltose,
In a comparative study of the
phenotypic properties of 421 strains of Erwinia species, all
strains of E. chrvsanthemi were separated from other Erwinia
spp. primarily by three physiological characters; production
of gas from D-glucose, phosphatase production, and inability
to produce acid from D-trehalose (17).
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19
Strains of E. chrvsanthemi isolated from Musa paradisiaca
L. could be distinguished from 322 strains isolated from 22
other plant hosts by the following properties: production of
acid from D-arabinose and raffinose; utilization of sodium
tartrate;
inulin,
lack
of production
mannitol,
or
of
sorbitol;
lecithinase
and
or
inability
acid
to
from
liquefy
gelatin (2 1 ).
The rice isolates were very similar to those isolated
from corn. However, the rice strains produced top soft rot or
stalk rot on corn seedlings while the corn strains were less
virulent on rice.
This suggests that the rice isolates are
distinct strains (27).
The ability to produce the enzyme tryptophanase has been
used for differentiation among the Erwinia spp.
associated
with potato soft rot disease (41). The presence of this enzyme
results
in
accumulation
of
indole
as
a
catabolite
of
tryptophan and can be detected based on its reaction with
glutaconic aldehyde which produces a red-violet polymethine
dye (10). Although some indole-positive strains can be found,
E.
carotovora
atroseptica. E.
subsp.
carotovora.
herbicola. and
E.
E.
carotovora
rhapontici
are
subsp.
indole-
negative while E. chrvsanthemi is indole-positive (10).
C.3. Pathovar
The
phenotypic
characteristics
of
352
strains
of
E.
chrvsanthemi from 28 hosts and the separation of the strains
into six subdivisions has been reported
(17,
21,
89).
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The
20
strains were assigned to a plant reaction group (I, II, III,
IV, V, and VI) based on the reactions of five or seven plant
species (21). It was proposed that the subdivisions could be
equated
with
the
pathovar
proposal.
Thus,
pathovars
dieffenbachiae. parthenii. chrvsanthemi. zeae. dianthicola.
and paradisiaca
respectively
would correspond to subdivisions I to VI,
(17,
18,
21,
89).
However,
the
proposed
six
pathovars proved to be inadequate to designate the host range
groups because many strains of E. chrvsanthemi are not limited
to causing disease only in plants from which they have been
isolated
(18). This would avoid designation of pathovars to
strains which have few or, in some cases, no distinguishing
phenotypic characteristics (18).
The plant reaction group to which a strain belongs can
not be predicted by the phenotypic properties of the strains.
No relationship could be found among the plant reactions, the
phenotypic properties, and the original host of strains (IB).
Thus, the use of the pathovar designation for strains of E.
chrvsanthemi should be avoided. Perhaps a simple solution to
this dilemma is to indicate the original host of the strain
(e.g.,
E.
chrvsanthemi
[carnation strain],
[corn
strain],
E.
chrvsanthemi
etc.) until well defined biovars can be
established that are correlated with pathogenic properties
(18).
C.4. Serology
Serological methods have been used for the identification
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21
of
E.
chrvsanthemi. and
strains
have
been
separated
into
serovars (19, 22, 8 8 ). Antigenic heterogeneity among strains
of E.
chrvsanthemi isolated from a specific host and among
strains
from
However,
serovar
different
there
and
has
hosts
been
original
no
has
been
definite
host,
demonstrated
relationship
geographical
(2 0 ) .
between
location,
and
phenotypic characteristic of the strains (19, 20, 22, 8 8 ).
REMARKS
Despite the attempts to assign pathovars or subdivisions
to
isolates of E.
chrvsanthemi. no support was
found when
either host reaction or phenotypic characters were compared
among them. On the other hand, a great antigenic heterogeneity
among isolates of E. chrvsanthemi has been reported.
D.
PHYTOPATHOGENIC PECTOLYTIC CLOSTRIDIA
INTRODUCTION
Pectolytic
clostridia
have
been
implicated
in
post­
harvest spoilage of potatoes, in cavity spot of carrots, and
in
wetwood
conditions
in trees.
Lund
(1982)
reviewed
the
possible significance of these bacteria in plant pathology.
This
review
does
not
attempt
to
further
that
of
Lund;
primarily because there has been little subseguent literature
on
Clostridia.
background
The
main
information
purpose
is
to
bring
together
for understanding the occurrence of
pectolytic clostridia associated with souring of sweetpotato.
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22
D.l. Occurrence
Contamination by pectolytic clostridia is likely to be
widespread since these bacteria are ubiquitous in most soils
(73). Prévôt et al.
(1967)
listed 17 species of pectolytic
obligate anaerobic bacteria commonly found in soils including
six
nonpigmented
and
eight
pigmented
(usually
pink)
Clostridium spp. and three Plectridium spp. Also, pectolytic
pigmented and non-pigmented bacteria belonging to the genus
Clostridium have been
found
in sugar beet
silages causing
texture loss of the ensiled mass when they are present in
excess (82).
Cavity spot was described in 1961 on carrot and parnisp
roots (33) . The disorder affects canning quality. It had been
reported to be caused by calcium deficiency, either actual or
induced by excessive potassium levels in soil (51). However,
this
hypothesis
was
not
confirmed
(6 6 ).
In
addition,
pectolytic Clostridium spp. were found on carrot roots during
investigations into cavity spot and were shown to be capable
of inducing extensive soft rotting of roots under anaerobic
conditions (67, 69).
Despite claims for association of pectolytic Clostridium
with cavity spot of carrots, the reduction of the disease in
the field and pot experiments with metalaxyl, propamocarb, and
fosetyl-Al
disease.
(49) suggested a possible fungal component in the
Further
Pvthium violae
investigation
with
cavity
spot
showed
the
disease
association
(32).
A
range
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of
of
23
Pvthium spp. produced cavities on carrots, but the relatively
slow growing P. violae gave the highest percentage of carrots
with cavities (8 6 ). Isolation of P. violae capable of inducing
lesions on carrot roots in vitro explains the reduction in
disease
symptoms
fungicide
active
following
against
applications
members
of
of
the
metalaxyl,
a
Peronosporales.
Previous failures to associate fungal pathogens with symptoms
may have
resulted
from the apparently
limited time during
which Pvthium can be isolated (32). The isolation medium and
techniques used, however, favored the growth of this genus at
the expense of Phvtophthora spp.
cavity
spot
is
still
(8 6 ). Clearly, the cause of
the subject of
controversy
but
the
involvement of Clostridium seems to be ruled out.
In many trees, both hardwoods and conifers, parts of the
interior wood appear to be water-soaked and are often darker
than
adjacent
referred
to
pectolytic
However,
as
Interior
facultative
wood
"wetwood" (46).
clostridia
the
pectolytic
wood.
play
results
anaerobes
an
also
were
clostridia
in
There
is
condition
evidence
is
that
important
role
in wetwood.
indicate
that
pectolytic,
present in
(80).
this
Whether
numbers
or
not
similar
to
bacteria,
in
particular Clostridium spp., are a primary cause of wetwood,
it seems
clear that they are
characteristic
reports
'shake',
that
the
features
trees
of
the
suffering
latter
responsible
for some of the
disorder
from
(46).
internal
being another abnormal
Moreover,
wetwood
and
condition
in
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24
standing trees, were prevalent on poorly drained soils
showed
an association with conditions of poor soil aeration which
would
favor
infection
by
Clostridium
(46).
They
also
had
suffered injuries to the roots or root collar (85, 90).
Rudd-Jones
and
Dowson
(1950)
isolated
pectolytic
Clostridia from rotting potato tubers; however, only recently
were clostridia definitely implicated in the rotting syndrome
of
potatoes
(45,
47,
48,
65).
Despite
the
ability
of
Clostridium to cause rot alone, onset of decay is accelerated
if strains of E. carotovora were present (60).
D.2. Environmental conditions
Roots exude organic substrates which stimulate growth and
metabolic
activity of
aerobes
(1).
The combined metabolic
activity of these microbes and the plant root create a high
local demand for oxygen
(23). Diffusion of oxygen could be
severely restricted during periods of high soil-water content
and be insufficient to satisfy requirements, thus creating an
anaerobic micro-site around the root. Physical measurement of
oxygen potential at the root surface is impractical, but the
presence of persistent spores of an obligate anaerobe provides
a convenient method of demonstrating that anaerobic periods
must have occurred (6 6 ). This hypothesis is supported by the
greater
number
of
pectolytic
Clostridium
found
in
the
rhizosphere of most carrot crops than in the surrounding soil
(66).
Regarding
decay
of potatoes,
depletion
of
oxygen can
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25
occur as a consequence of inadequate control of environmental
conditions during storage, transport, and marketing, and can
result from restricted ventilation, from exposure of tubers to
temperatures of about 37 C, or from the presence of a film of
water on tubers for at least several hours (46, 65). Anaerobic
conditions cause an apparent decrease in tuber resistance to
soft rot bacteria. Increases in tuber turgidity ruptures the
suberized
layer
of
the
lenticels.
This
combination
of
conditions is favorable for rapid growth of certain pectolytic
bacteria present in lenticels and for invasion of cortical
tissue
(65).
Clostridia
would
be
expected
to
thrive
in
environments that are oxygen-free, but they are also capable
of growth in conditions that are ostensibly aerobic but in
which the growth of aerobic and facultative anaerobic bacteria
can create the reducing conditions required by the obligate
anaerobe (46).
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26
LITERATURE CITED
1.
Alexander, M. 1975. Introduction to Soil Microbiology.
2nd ed., New York, John Wiley & Sons. 473 pp.
2.
Allan, E . , and Kelman, A. 1977. Immunofluorescent
stain procedures for detection and identification of
Erwinia carotovora var. atroseptica. Phytopathology
67:1305-1312.
3.
Burkholder, W. H. 1959. The causal agents of the black
stem disease of annual larkspur. Plant Dis. Rep.
43:934-935.
4.
Burkholder, W. H . , McFadden, L. A., and Dimock, A. W.
1953.
A
bacterial
blight
of
chrysanthemum.
Phytopathology 43:522-526.
5.
Burr, T.J., and Schroth, M.N. 1977. Occurrence of
soft-rot Erwinia spp. in soil, and plant material.
Phytopathology 67:1382-1387.
6.
Campbell, W. A. 1947. A bacterial root and stem disease
of guayule. Phytopathology 37:271-277.
7.
Clark, C. A., and Moyer, J. W. 1988. Compendium of
Sweet Potato Diseases. APS Press, St. Paul, MN. 74 pp.
8.
Clark, C. A., Wilder-Ayers, J. A., and Duarte, V.1989.
Resistance of sweet potato to bacterial root and
stem rot caused by Erwinia chrvsanthemi. Plant Dis.
73:984-987.
9.
Cochran, W. G. 1950. Estimation of bacterial densities
by means of the "Most Probable Number'. Biometrics
6:105-116.
10.
Cother, E. J., and Blakeney, A. B. 1987. The specific
detection of indole production by Erwinia species and
some other enterobacteria on agar. J. Appl. Bacteriol.
63:329-334.
11.
Cuppels,
D . , and Kelman, A.
1974.
Evaluation of
selective media for isolation of soft-rot bacteria
from soil and plant tissue. Phytopathology 64:468-475.
12.
De Boer, S. H . , Copeman, R. J . , and Vruggink, H. 1979.
Serogroups
of
Erwinia
carotovora
potato
strains
determined
with
diffusible
somatic
antigens.
Phytopathology 69:316-319.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
27
13.
De Boer, S. H., Cuppels, D.A., and Kelman, A. 1978.
Pectolytic Erwinia spp. in the root zone of potato
plants in relation to infestation of daughter tubers.
Phytopathology 68;1784-1790.
14.
De Boer, S. H . , and Kelman, A. 1975. Evaluation of
procedures for detection of pectolytic Erwinia spp. on
potato tubers. Amer. Potato J. 52:117-123.
15.
Denny, T. P. Differentiation of Pseudomonas svrinoae
pv. tomato from p. s. svrinaae with DNA hybridization
probe. Phytopathology 78:1186-1193.
16.
Di, Y.B . , and Kelman, A. 1988.
Modified crystal
violet pectate (CVP) medium. Page 9 in: Laboratory
Guide for Identification of Plant Pathogenic Bacteria.
2nd ed. Schaad, N. W . , APS Press, St. Paul, MN.
17.
Dickey,
R.
S.
1979.
Erwinia
chrvsanthemi:
a
comparative study of phenotypic properties of strains
from
several
hosts
and
other
Erwinia
species.
Phytopathology 69:324-329.
18.
Dickey, R. S. 1981. Erwinia chrvsanthemi: Reaction of
eight plant species to strains from several hosts and
to strains of other Erwinia species. Phytopathology
69:324-329.
19.
Dickey, R. S., Zumoff,
C. H . , and Uyemoto, J. K. 1984.
Erwinia chrvsanthemi:
Serological relationships among
strains from several hosts. Phytopathology 74:13881394.
20. Dickey, R. S., Claflin, L. E . , and Zumoff, C. H. 1987.
Erwinia
chrvsanthemi:
Serological
comparisons
of
strains from Zea mavs and other hosts. Phytopathology
77:426-430.
21.
Dickey, R. S., and Victoria, J. I. 1980. Taxonomy and
emended description of strains of Erwinia isolated
from
Musa
paradisiaca
Linnaeus.
Int.
J.
Syst.
Bacteriol. 30:129-134.
22.
Dickey, R. S., Zumoff,
C. H . , and Uyemoto, J. K. 1984.
Erwinia chrvsanthemi:
serological relationships among
strains from several hosts. Phytopathology 74:13881394.
23.
Drew, M. C. , and Lynch, J. M. 1980. Soil anaerobiosis,
microorganisms,
and
root
function.
Annu.
Rev.
Phytopathol. 18:37-66.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
28
24.
Elias, N. A., Wajid, S. M. A., and Bhakatavatsalam, G.
1987.
Studies on bacterial leaf spot disease of
tobacco. Tropical Pest Management 33:2-4, Reviewed in
Rev. Plant Path. 1988, 67:40.
25.
Elphinstone, J. G . , and Pérombelon, M. c. M.
Contamination of progeny tubers by seed- and
borne Erwinia carotovora. Potato Res. 29:77-93.
26.
Garibaldi, A. 1972. Research on carnation bacterial
wilt
disease:
Survival
in
soil
of
Erwinia
chrvsanthemi. agent of bacterial slow wilt. Actos III
Congresso du Unia'o Fitop. Mediterr. 29:32 in Haygood,
R. A., Strider, D. L . , and Echandi, E. 1982. Survival
of
Erwinia
chrvsanthemi
in
association
with
Philodendron selloum. other greenhouse ornamentals,
and in potting media. Phytopathology 72:853-859.
27.
Goto, M. 1979. Bacterial foot rot of rice caused by a
strain of Erwinia chrvsanthemi. Phytopathology 69:213216.
28.
Graham, D. C. 1964. Taxonomy of the soft-rot coliform
bacteria. Annu. Rev. Phytopathol. 2:13-42.
29.
Graham,
D.C., and Harrison, M.D.
1975.
Potential
spread of Erwinia spp. in aerosols. Phytopathology
65:739-741.
30.
Graham, B.C., Quinn, C.E., and Bradley, L. F. 1977.
Quantitative Studies on the generation of aerosols of
Erwinia
carotovora var.
atroseptica by
simulated
raindrop impaction on blackleg-infected potato stems.
J. Appl. Bacteriol. 43:413-424.
31.
Graham, B.C., Quinn, G.E., Sells, I. A., and Harrison,
M. B. 1979. Survival of strains of soft rot coliform
bacteria on microthreads exposed in the laboratory and
in the open air. J. Appl. Bacteriol. 46:367-376.
32.
Groom, M. R . , and Perry, D. A. 1985. Induction of
•cavity spot-like* lesions on roots of Baucus carota by
Pvthium violae. Trans. Brit. Mycol. Soc. 84:755-757.
33.
Guba, E. F . , Young, R. E . , and Ui,
spot disease of carrot and parsnip.
45:102-105.
34.
Harris, R. I., and Lapwood, B. H. 1978. The spread of
Erwinia carotovora var. atroseptica (blackleg) from
degenerating seed to progeny tubers in soil. Potato
Res. 20:285-94.
1986.
leaf-
T. 1961. Cavity
Plant Bis. Rep.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
29
35.
Harrison, M.D., Franc, G.D., Maddox, D . A . , Michaud,
J.E., and McCarter-Zorner, N.J. 1987. Presence of
Erwinia
carotovora
in
surface
water
in
North
America. J. Appl. Bacteriol. 62;565-570.
36.
Hartman, J. R . , and Kelman, A. 1973. An improved
method for the inoculation of corn with Erwinia spp.
Phytopathology 63:658-663.
37.
Hoitink, H. A., Herr, L. J., and Schmitthenner, A. F.
1976.
Survival
of
some
plant
pathogens
during
composting of hardwood
tree bark.
Phytopathology
66:1369-1372.
38.
Hugh,
R . , and
Leifson,
E.
1953.
The
taxonomic
significance
of
fermentative
versus
oxidative
metabolism of carbohydrates by various Gram-negative
bacteria. J. Bacteriol. 66:24-26.
39.
Jabuonski, R. E . , Takatsu, A., and Reifschneider, F.
J. B. 1986. Evaluation of pathogenicity of Erwinia
isolated from potato, tomato, and other host plants.
Fitopatol. Bras. 11:587-597.
40.
Janse,
J.
D.,
and
Ruissen,
M.
A.
1988.
Characterization
and
classification
of
Erwinia
chrvsanthemi
strains
from
several
hosts
in
The
Netherlands. Phytopathology 78:800-808.
41.
Kelman, A., and Maher, E. A. 1985. Isolation and
identification of the Erwinia spp. that cause soft
rot. Pages 11-12, in: Report of the International
Conference on Potato Blackleg Disease, ed. Graham, D.
C . , and Harrison, M. D. Oxford: UK Potato Marketing
Board.
42.
Kikumoto, T., and Sakamoto, M. 1969. Ecological studies
on the soft-rot bacteria of vegetables. VI. Influence of
the development of various plants on the survival of
Erwinia aroideae added to soil. Ann. Phytopathol. Soc.
Japan 35:29-35.
43.
Kikumoto, T., and Sakamoto, M. 1969. Ecological studies
on the soft-rot bacteria of vegetables. VII. The
preferential stimulation of the soft-rot bacteria in the
rhizosphere of crop plants and weeds. Ann. Phytopath.
Soc. Japan 35:36-40.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
30
44.
Lim, w. H.
pineapple
Pathologie
Fourth Int.
France.
45.
Lund, B. M. 1972. Isolation of pectolytic clostridia
from potatoes. J. Appl. Bacteriol. 35:609-614 .
46.
Lund, B. M. 1982. Clostridia and plant disease: New
pathogens? Pages 263-283 in: Phytopagenic Prokaryotes,
Vol 1, Mount, M. S., and Lacy, G. H. , ed. Academic
Press, N Y .
47.
Lund, B. M., and Kelman, A. 1977. Determination of the
potential for development of bacterial soft rot of
potatoes. Am. Potato J. 54:221-225.
48.
Lund,
B. M . , and Nicholls,
J.
influencing the soft-rotting of
bacteria. Potato Res. 13:210-214.
49.
Lyshol,
A. J., Semb,
L . , and Taksdal,
G.
1984.
Reduction of cavity spot and root dieback in carrots by
fungicide applications. Plant Pathology 33:193-198.
50.
Martin, W. J., and Dukes, P. D. 1975. A bacterial soft
rot of sweet potato plants. Proc. Am. Phytopathol.
Soc. 2:57.
51.
Maynard,
D. N . , Gersten,
B . , Vlack,
E.
F . , and
Vernell,
H.
F.
1961.
The
effects
of
nutrient
concentration and calcium levels on the occurrence of
cavity spot. Proc. Amer. Soc. Hort. Sci. 78:339-342.
52.
McCarter-Zorner, N.J., Franc, G.D., Harrison, M.D.,
Michaud, J.E., Quinn, C.E., Sells, I.A., and Graham,
D.C. 1984. Soft-rot Erwinia bacteria in surface and
underground
waters
in
southern
Scotland
and
in
Colorado, United States. J. Appl. Bacteriol. 57:95105.
53.
Meneley, J.C., and Stanghellini, M.E. 1976. Isolation
of soft-rot Erwinia spp. from agricultural soils using
an enrichment technique. Phytopathology 66:367-370.
54.
Molina, J. J . , and Harrison, M. D. 1980. The role of
Erwinia carotovora in the epidemiology of potato
blackleg. II. The effect of soil temperature on disease
severity. Am. Potato J. 57:351-363.
1978. Survival of Erwinia chrvsanthemi on
surfaces.
Pages 743-746 in: Station de
Végétale Phytobacteriologie, éd.,
Proc.
Conf. on Plant Pathogenic Bacteria, Angers,
C.
1970.
Factors
potato tubers by
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
31
55.
Pérombelon, M. C. M. 1971. A quantal method for
determining
numbers
of
Erwinia
carotovora
var.
carotovora and E. carotovora var. atroseptica in soils
and plant material. J. Appl. Bacteriol. 34:793-798.
56.
Pérombelon, M. C. M. 1972. A reliable and rapid method
for detecting contamination of potato tubers by Erwinia
carotovora. Plant Dis. Rep. 56:552-554.
57.
Pérombelon, M. C. M. 1973. Sites of contamination and
numbers of Erwinia carotovora present in stored seed
potato stocks in Scotland. Ann. Appl. Biol. 74:59-65.
58.
Pérombelon, M. C. M. 1976. Effects of environmental
factors during the growing season on the level of
potato tuber contamination. Phytopath. Z. 85:97-116.
59.
Pérombelon, M. C. M . , Fox, R. A., and Lowe, R. 1979.
Dispersion of Erwinia carotovora in aerosols produced
by the pulverization of potato haulm prior to harvest.
Phytopath. Z. 94:249-260 .
60.
Pérombelon, M. C. M . , Gullings-Handley, J., and Kelman,
A. 1979. Population dynamics of Erwinia carotovora and
pectolytic clostridia in relation to decay of potatoes.
Phytopathology 69:167-173.
61.
Pérombelon, M. C. M . , and Hyman, L. J. 1986. A rapid
method for identifying soft-rot erwinias directly from
plant material based on their temperature tolerances
and sensitivity to erythromycin. J. Appl. Bacteriol.
60:61.
62.
Pérombelon, M. C. M . , and Hyman, L. J. 1989. Survival
of soft rot coliforms,
Erwinia carotovora subsp.
carotovora and E. carotovora subsp. atroseptica in
soil in Scotland. J. Appl. Bacteriol. 66:95-106.
63.
Pérombelon, M. C. M . , and Kelman, A. 1980. Ecology of
the soft-rot erwinias. Annu. Rev. Phytopathol. 18:361387.
64.
Pérombelon, M. C. M . , and Kelman, A. 1987. Blackleg
and other potato diseases caused by soft rot erwinias:
Proposal
for revision of terminology.
Plant Dis.
71:283-285.
65.
Pérombelon, M. C. M . , and Lowe, R. 1972. The effects of
bile salts media and the age of inocula in quantitative
studies of populations of Erwinia
carotovora var.
carotovora and E. carotovora var. atroseptica. J. Appl.
Bacteriol. 34:501-506.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
32
66.
Perry, D. A.
1982. Pectolytic Clostridium spp. in
soils and rhizospheres of carrot and other arable
crops in east Scotland. J. Appl. Bacteriol. 52:403408.
67.
Perry, D. A.,
and Harrison, J. G. 1977. Pectolytic
anaerobic bacteria cause symptoms of cavity spot in
carrots. Nature 269:509-510.
68.
Perry, D. A., and Harrison, J. G. 1979. Cavity spot of
carrots. I. Symptomatology and calcium involvement.
Ann. Appl. Biol. 93:101-108.
69.
Perry, D. A.,and Harrison, J. G. 1979. Cavity spot
of carrots. II. The effect of soil conditions and the
role of pectolytic anaerobic bacteria. Ann. Appl.
Biol. 93:101-108.
70.
Phillipis,
J.
A.,
and Kelman,
A.
1982.
Direct
fluorescent antibody stain procedure applied to insect
transmission of Erwinia carotovora. Phytopathology
72:898-901.
71.
Powelson, M.
L. 1980. Seasonal incidence and cause of
blackleg and a stem soft rot of potatoes in Oregon.
Am. Potato J. 57:301-306.
72.
Powelson, M.
L. 1984. Soil and seed tubers as sources
of inoculum
of Erwinia carotovora pv. carotovora for
stem soft rot of potatoes. Phytopathology 74:429-432.
73.
Prévôt, A. R . , Turpine, A., and Kaiser, P. 1967.
Les Bactéries Anaerobies. Dunod, Paris, 2188 pp. in:
Pérombelon, M. C. M . , Gullings-Handley, J . , and fëlm=n,
A. 1979. Population dynamics of Erwinia
carotovora and
pectolytic clostridia in relation to
decay of potatoes.
Phytopathology 69:167-173.
74.
Quinn, C. E . , Sells,
I. A., and Graham, D. C. 1980.
Soft rot Erwinia bacteria in the atmospheric bacterial
aerosol. J. Appl. Bacteriol. 49:175-181.
75.
Rolston, L. H . , Clark, C. A., Cannon, J. M . , Randle, W.
M . , Riley, E. G . , Wilson, P. W . , and Robbins, M. L.
1987.
'Beauregard' sweet potato. HortSci. 22:13381339.
76.
Rudd-Jones,
D . , and
Dowson,
W. J.
1950. On the
bacteria responsible for soft rot in stored potatoes,
and the reaction of the tuber to invasion by Bacterium
carotovorum (Jones) Lehmann and Neumann. Ann. Appl.
Biol. 37:563:569.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
33
77.
78.
Scanferlato, V. s . , Orvos, D. R . , Lacy, G. H., and
Cairns Jr.,
J. 1990. Enumerating low densities
of
genetically engineered Erwinia carotovora in soil.
Lett. Appl. Microbiol. 10:55-59.
Schaad, N.
W. 1979. Serological
plant pathogenic bacteria. Annu.
17:123-147.
identification
of
Rev. Phytopathol.
79.
Schaad, N.W., and Brenner, D.
1977. A bacterial wilt
and root rot of sweet potato caused by Erwinia
chrvsanthemi. Phytopathology 67:302-308.
80.
Schink, B . ,
Ward, J. C . , and Zeikus, J. G. 1981.
Microbiology
of
wetwood:
Importance
of
pectin
degradation and Clostridium species in living trees.
Appl. Environ. Microbiol. 42:526-532.
81.
Stanghellini, M. E. 1982. Soft-rotting bacteria in the
rhizosphere.
Pages
249-261
in:
Phytopagenic
Prokaryotes, Vol 1, Mount, M. S., and Lacy, G. H . , ed.
Academic Press, NY.
82.
Suzzi, G. , Papa, F . , and Grazia, L. 1987. Pectolytic
Clostridia isolated from sugar beet pulp silages in
Italy. J. Appl. Bacteriol. 63:481-485.
83.
Tomlinson, D. L . , and Cox, P. G. 1987. A new disease of
cardamom
(Elettaria cardamomum) caused by Erwinia
chrvsanthemi in Papua New Guinea. Plant Pathology
36:79-83 in Rev. Plant Path. 198 :365, Abstract.
84.
Ward, L. J . , and De Boer, S. H. 1990. A DNA probe
specific for serologically diverse strains of Erwinia
carotovora. Phytopathology 80:665-669.
85.
Ward, J. C . , Kuntz, J. E . , and McCoy, E. M. 1969.
Bacteria associated with "shake" in broadleaf trees.
(Abstr.) Phytopathology 59:1056.
86.
White, J. G.
1986. The association of Pvthium
with cavity spot and root dieback of carrots.
Appl. Biol. 108:265-273.
87.
Woodward,
E. J . , and Robinson, K. 1990. An improved
formulation and method of preparation of crystal
violet pectate medium for detection of pectolytic
erwinia. Lett. Appl. Microbiol. 10:171-173.
88.
Yakrus,
M . , and Schaad,
N. W.
1979. Serological
relationships among strains of Erwinia chrvsanthemi.
Phytopathology 69:517-522.
spp.
Ann.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
34
89.
Young,
J.
M.,
Dye,
D.
W.,
Bradbury, J.
F.,
Panagopoulos, C. G., and Robbs, C. F. 1978. A proposed
nomenclature and classification for plant pathogenic
bacteria. N. Z. J. Agric. Res. 21:153-177.
90.
Zeikus, J. G . , and Ward, J. C. 1974. Methane formation
in living trees:A microbial origin. Science
184:11811183.
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CHAPTER II
PRESENCE OF ERWINIA CHRYSANTHEMI. CAUSAL AGENT OF STEM AND
ROOT ROT ON SWEETPOTATO, THROUGH THE GROWING SEASON
35
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36
PRESENCE OF ERWINIA CHRYSANTHEMI. CAUSAL AGENT OF STEM AND
ROOT ROT, ON SWEETPOTATO THROUGH THE GROWING SEASON
ABSTRACT
Storage
roots
of
sweetpotato,
cv.
Beauregard,
were
inoculated at bedding by either dipping them without injuring
in a suspension of a rifampicin-resistant, virulent strain of
Erwinia chrysanthemi (Ech-2-rr) or by inserting a pipette tip
containing 50 ^1 of the same suspension into the root.
transplanting,
At
slips were cut or pulled and transplanted to
the field two times in succession. Ech-2-rr was recovered from
inoculated roots at 0 and 7 days after bedding,
and at the
first and second pullings. The bacterium also was present on
or in symptomless vine cuttings after transplanting to the
field, and in or on the underground stem, and daughter storage
roots at harvest. However, little stem rot was observed in the
field. Inoculation of stem cuttings at transplanting did not
affect disease occurrence. There was no difference in disease
occurrence
when
pulled
transplanting,
but
samples
pulled
from
respectively.
When
or
Ech-rr
was
and
slips
cut
detected
cut
of
slips
cv.
were
in
23%
treatments
Jewel
used
were
and
at
cut
9%
for
of
harvest,
with
a
contaminated knife, Ech-2-rr was recovered from the cut stub
of the sprout,
from transplants at the subsequent cutting,
from the surface of the mother root, and from the underground
stem at harvest.
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37
INTRODUCTION
Until
sweetpotato
recently,
bacterial
fIpomoea batatas
(L.)
stem
and
root
rot
of
Lam.), caused by Erwinia
chrysanthemi Burkholder, McFadden, and Dimock was unknown. In
1974, a major outbreak of this disease occurred in production
areas of Georgia, and in 1975 it threatened the existence of
the
sweetpotato
industry
in
that
state
(2,
14).
In
a
preliminary description of the disease, it was concluded that
the bacterium responsible was similar to E. carotovora
(9).
Eventually, it was determined that the disease of sweetpotato
is caused by E. chrysanthemi (14). In Louisiana, the disease
has not been found as a stem rot but has been found to a
limited extent in storage roots in plant beds and the field.
However, new cultivars have shown greater susceptibility to E.
chrysanthemi when artificially inoculated (3, 13). Therefore,
the objectives of this study were to determine a) the presence
of E. chrysanthemi on sweetpotato through the growing season,
b) the transmission of the bacterium from seed roots to stems
and new plants, c) the effect of pulling and cutting the stem
from beds with inoculated roots on transmission and disease
occurrence, and d) the transmission of the bacterium to stem
cuttings
and/or
to
sprout
stubs
and
mother
roots
by
contaminated knives. A preliminary report on this research has
been presented previously (7).
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38
MATERIAL AND METHODS
Inoculum.
A
rifampicin-resistant,
Erwinia chrysanthemi
originally
isolated
throughout
this
(Ech-2-rr)
from
study.
virulent
strain
of
was selected from a strain
sweetpotato
Ech-2-rr
was
(Ech-2)
and
used
pathologically
and
physiologically indistinguishable from the wild-type strain
Ech-2 (Duarte and Clark, unpublished). Cells of the bacterium
grown on nutrient broth in a rotary shaker for 24 hr at 25 C
were concentrated by centrifugation and suspended in sterile
distilled
water
(SDW).
The
bacterial
suspensions
used
throughout this study were adjusted to an optical density of
0.15 at 620 nm (approx. 10^ cfu/ml).
Bedding.
In
1988,
1989,
and
1990,
storage
roots
of
Beauregard sweetpotato were inoculated at bedding by either
dipping them without
injuring
in a suspension of Ech-2-rr
(Fig. 1) or by inserting a micropipette tip containing 50 n l
of the same suspension to a depth of about 1 cm
it in position
and leaving
(Fig. 2). There were seven randomized blocks
with single, 10-root plots per treatment per block. A separate
row
with
four
roots
per
block
of
each
treatment
was
established from which samples were taken (Fig. 3).
To
determine
the population
of
Ech-2-rr
on
the
root
surface, the mother roots were sampled at four times (Fig. 4).
Storage roots were dug and washed individually in plastic bags
containing
100 ml of SDW (Fig. 5). Aliquots of 0.01 ml of the
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39
serially diluted wash water were plated on circular areas of
CVP-RC medium
(crystal
violet pectate medium amended with
rifampicin and cycloheximide, 100 and 25 mg / 1, respectively)
cut
in agar plates with
a metallic tube
cap.
Plates were
checked for Ech-2-rr colonies after 48-hr incubation at 32 C
(Fig. 6).
Transplanting.
At transplanting,
slips were pulled or
pulled and cut from beds of each treatment of mother roots and
transplanted to the field two times in succession (first and
second pullings)
from
beds
mother
with
roots)
(Fig. 4). The six treatments (pulled or cut
control,
were
dipped
planted
complete block design.
in
or
the
pipette
field
in
tip-inoculated
a
randomized
Each treatment was replicated seven
times in 10-plant plots. Sweetpotato slips were planted 30 cm
apart with 90 cm between plots and 120 cm between rows.
After the second pulling in 1989 and 1990, mother storage
roots were dug and number of sound mother roots recorded.
To assess the presence of Ech-2-rr on stem cuttings at
transplanting, slips were harvested from different treatments
in the plant bed and transferred individually to plastic bags
with 50 ml of SDW. After at least 30 min, a 1-ml aliquot of
the suspension was added to 9 ml of enrichment medium, crystal
violet pectate broth (CVPB), which contained 4.5 g of sodium
polypectate,
1 ml of 0.075% aqueous crystal violet,
0.5 g
NaNOg, and 2.25 ml of 1 N NaOH per 500 ml of hot, distilled
water
(4,
12),
or serially diluted and plated on CVP-RC.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
40
Aliquots of inoculated enrichment medium were plated on CVP-RC
after
incubating
for 2 and 4 days at 32 C.
Colonies were
counted after incubating the plates for 48 hr at 32 C.
Sprout-cutting
inoculation.
In
another
experiment
conducted in the field in 1989 and repeated in 1990,
cuttings
from
noninoculated
storage
roots
were
sprout
pulled
or
pulled and cut and inoculated by dipping the basal end of the
slip
into
a
suspension
noninoculated
pulled
of
and
Ech-2-rr;
cut
stems.
the
Each
controls
were
treatment
was
replicated five times in 10-plant plots.
Underground stem. At harvest, two underground stems (Fig.
7)
from each plot were transferred individually to plastic
bags containing 100 ml of SDW.
The same procedure used to
assess for presence of Ech-2-rr on storage roots was followed.
Presence of the pathogen on/in daughter roots and root
rot assessment.
In 1988,
two daughter roots from each plot
were transferred individually to plastic bags containing 100
ml
of
SDW.
After
7-day
incubation
at
room
temperature,
serially diluted samples were plated on CVP-RC. Plates were
checked for Ech-2-rr colonies after 48-hr incubation at 32 C.
In 1989, an adaptation of a technique used with Irish potatoes
to determine bacterial soft rot potential was attempted with
sweetpotatoes. Two storage roots from each plot were punctured
ten times at different sites with a flamed needle, placed in
polyethylene bags, immersed in distilled water, and incubated
at room temperature (25 C ±2) for 5 days (1).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
41
Survival in storage.
In October,
1989,
four crates of
daughter roots from vines produced by mother roots inoculated
with Ech-2-rr in March and one crate from noninoculated were
stored at 18 ± 3 C from November to March,
1990.
Also,
2
crates of Beauregard sweetpotato storage roots, harvested in
a field other than those of the experiments, were inoculated
with Ech-2-rr by immersion in a cell suspension. Presence of
Ech-2-rr on storage roots was assessed in March, at bedding,
and May by peeling and transferring around 1 g of root skin to
tubes with 9 ml of CVPB.
To recover from vines in May, 5-cm
of aboveground stem was cut with a flamed scalpel, transferred
to
tubes with CVPB
in the
field,
and
incubated
at
32
C.
Aliquots of 0.01 ml were plated on CVP-RC medium after 48- and
96-hr incubation. CVP-RC plates were incubated at 32 C for 48
hr and then checked for Ech-2-rr colonies.
Knife
transmission.
sweetpotato
were
planted
Storage
in
roots
of
25-cm-diameter
Beauregard
pots
in
the
greenhouse. After sprouts reached about 30 cm long, they were
cut
with
a
knife
previously
dipped
in
a
suspension
of
Ech-2-rr. After 2 mo, the presence of Ech-2-rr on stems and
mother storage roots was assessed using enrichment medium as
described previously.
In the field, slips of Jewel sweetpotato also were cut
with a contaminated knife in 1988 and 1989. Both inoculated
and noninoculated treatments were replicated four times in
10-plant plots. At harvest, two underground stems were sampled
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
42
from each
plot,
and presence
of Ech-2-rr was
assessed
as
described previously.
Survival in soil.
Olivier silt loam soil was collected
from Burden Plantation in Baton Rouge, sieved,
and infested
with a suspension of Ech-2-rr in November, 1989. Infested soil
was then transferred to four 25-cm-diameter clay pots and kept
outdoors during the winter. At different intervals, 5-g-soil
samples were added to tubes with 9 ml of CVPB and incubated at
32 C for 48 and 96 hr. Aliquots of 0.01 ml were plated on
discs of CVP-RC medium cut in agar plates with a metallic tube
cap,
and plates
incubation
at
infestation,
checked
32
pots
C.
for Ech-2-rr colonies
In
were
April
1990,
transferred
125
to
days
the
after 48-hr
after
greenhouse
soil
and
planted with sweetpotato storage roots. After 2 mo, storage
roots
and
shoots
were
assessed
for
the
presence
of
the
bacterium as described above.
Soil from Burden Plantation, Baton Rouge, LA, was sieved
through a 2-mm sieve,
plastic
bags.
Fifty-ml
and 500-g quantities placed
volumes
of
Ech-2
or
in nine
Ech-2-rr
cell
suspensions, or sterile, distilled water were added to soil in
each of three bags, and one bag was incubated at 4, 25, or 37
C.
Presence of Ech-2 or Ech-2-rr was assessed as described
above, but including CVP without antibiotics to recover Ech-2.
Field soil was sampled monthly,
from July to October,
1989, for presence Ech-2-rr. Cores of soil, 2 cm in diameter
and 15 cm long, were taken with a soil sampler. Four cores.
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43
from about 15 cm away from the underground stems of plants in
the different treatments,
(5) . In the week
were combined to form one sample
following harvest
in 1988
and
1989,
the
presence of Ech-2-rr also was assessed in all plots of the
second transplanting;
about 500 g of soil was sampled from
each plot. Presence of Ech-2-rr in the soil was assessed as
described above.
Survival on crate wood. Wood squares (12 x 12 x 3 mm) cut
from sweetpotato storage crates were infested by submerging
them
in
a
suspension of
rotted
inoculated
with Ech-2-rr.
transferred
to
petri
After
dishes
storage
30
and
root
min,
previously
squares
maintained
at
were
room
temperature. Three squares from different petri dishes were
transferred daily to l25-ml Erlenmeyer flasks with 10 ml of
nutrient broth and shaken for 30 min. Then, 0.01-ml aliquots
were plated on discs of CVP-RC medium cut in agar plates with
a
metallic
tube cap.
Colonies
were
counted
after 48-hr
incubation at 32 C. This experiment was repeated once.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
44
RESULTS
Recovery during the growing season. During three seasons,
mother storage roots were inoculated with E. chrysanthemi in
March, and the pathogen was recovered from them the day they
were
bedded,
pullings
7
days
(Table
1)
later,
as
well
and
as
at
the
from
first
and
underground
second
stems
at
harvest of the first and second transplantings in September
and October, respectively (Table 2). The pathogen was detected
on or in 15,
15,
71,
43,
29 and 15% of symptomless vines
produced by mother roots
suspension
or
inoculated
inserting
by dipping
pipette-tips
in
a cell
containing
cell
suspension in 1988, 1989, and 1990, respectively. It was not
recovered from stems of noninoculated mother roots.
Populations
decreased
of
chrysanthemi
on/in
from March to July in plant beds
ranged from approx.
pulling
E.
in 1989,
10 to 10® cfu / cm^.
mother
(Fig.
roots
8) ; they
After the second
the number of sound mother roots was not
significantly different between plots of those inoculated by
dipping in a cell suspension
(39 roots)
and plots of those
inoculated with pipette tips
(23 roots)
but was lower than
those of noninoculated mother roots
(54 roots). Numbers of
sound mother roots were not different among treatments
in
1990.
The number of plants produced by storage roots was not
affected by
pulling
in
inoculation treatments,
1989
when production was
except
for the
significantly
second
reduced
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
45
(Table
3).
Likewise,
sweetpotato
yield
by
weight
was
not
affected, but the number of storage roots produced from plants
of
the
second
transplanting
varied
significantly
among
treatments in 1989 (Table 4). Little stem rot occurred in the
fields during the three seasons; one or two out of seventy
plants in some treatments showed symptoms.
The effect of pulling vs. cutting the transplants. Erwinia
chrysanthemi was recovered
from 23 and 9% of
underground
stems of daughter plants produced from pulled or cut sprouts
from mother roots inoculated at bedding (combined data from
Table 2).
pathogen
When stems were inoculated at transplanting, the
was
recovered
from
40,
50,
93,
and
93
%
of
underground stems of daughter plants produced from pulled or
cut transplants in 1989 and 1990, respectively. Regardless of
the time of inoculation with E. chrysanthemi. no effect of
pulling vs. cutting the transplants on stem rot incidence was
observed in either year.
Presence of the pathogen on/in daughter roots and root
rot assessment. At the second harvest in 1988, E. chrysanthemi
was
recovered
from
0,
15,
and
0,
and
15,
43,
and
29%
of
sampled daughter roots from pulled and cut sprouts from mother
roots noninoculated and inoculated at bedding by dipping into
a cell suspension or with pipette tips, respectively. Attempts
to assess root rot potential in 1989 by a polybag test failed.
The incubation conditions led to a high incidence of souring,
a disease with which clostridia have been associated (6). No
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
46
Ech-2-rr was recovered from rotting tissue.
Survival
daughter
inoculated
in
roots
storage.
from
with
March,
or
in
sprouts
Ech-2-rr
October to March,
1990.
May,
aboveground stems.
Ech-2-rr
was
produced
in March,
not
by
1989,
detected
mother
and
on
roots
stored
from
It was not recovered at bedding in
1990,
However,
or
at
harvest
from
the
5-cm
on storage roots inoculated in
October, 1989, the populations of Ech-2-rr were about 10^ cfu
/ g of root skin at the same sampling times,
although they
were not detected on the 5-cm aboveground stems.
Knife transmission.
When sweetpotato sprouts were cut
with a contaminated knife in the greenhouse, the bacteria left
on the cut stubs were recovered from the mother storage root
and new sprouts two months later. When stems were cut with a
contaminated knife in the field, Ech-2-rr was recovered from
6 and 4 out of 8 underground stems sampled at harvest in 1988
and
1989,
respectively.
It
was
not
recovered
from
noninoculated control plants in either year.
Survival in soil. Erwinia chrysanthemi was not recovered
from
soil
kept
in
clay
pots
outdoors
for
50
days
after
infestation in November, 1989, or in May, 1990, after storage
roots
were
grown
in
it
for
2
mo.
The
bacterium
was
not
recovered from storage root surfaces or sprouts either.
When soil was infested with the wild type or the mutant
strain of E. chrysanthemi and incubated at 4, 25, or 37 c,
only the mutant was recovered 21 days later from soil at 4 C.
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47
Neither
Ech-2
nor
Ech-2-rr
was
recovered
30
days
after
infestation at either incubation temperature.
Erwinia chrysanthemi was not recovered from field soil
sampled around the plants
from any treatment
from July to
October, 1989, or from soil sampled after harvesting in 1988
or 1989.
Survival on crate wood. The populations of the pathogen
on infested crate wood decreased exponentially with time and
it was not detected 9 days after infestation (Fig. 4).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
48
DISCUSSION
Erwinia chrysanthemi present on or in mother roots in
March,
at
bedding,
transplanting
field
on
or
was
in June-July,
in
symptomless
transmitted
to
sprouts.
the pathogen was
vines
and
At
taken to the
detected
on
or
in
underground stems and daughter roots at harvest in SeptemberOctober. The bacteria survived the storage period on storage
roots inoculated in October and were recovered in March,
bedding,
and
May,
at
pulling
time.
These
results
at
have
important implications for the control of the bacterial stem
and
root
rot.
Three
potential
identified: mother roots,
sources
of
inoculum
are
stem cuttings, and daughter roots
that will be mother roots in the next season.
A
crucial
issue
arises
as
a
consequence
of
virtual
absence of disease in the field over the 3-yr period of this
study. We found no evidence indicating that the mutant used in
this study is less virulent than the wild type strain of E.
chrysanthemi
Beauregard,
genotypes
(Duarte and Clark, unpublished) . The cultivar,
is
to
one
of
bacterial
the
most
root
rot
susceptible
but
is
sweetpotato
intermediate
to
bacterial stem rot (3). Consequently, factors other than the
presence
of
the
pathogen
or
relative
storage
root
susceptibility are likely to affect disease development in the
field.
This line of reasoning is supported by at least two
observations.
pipette
tip
First,
bedded
containing
a
mother
high
roots
number
of
wounded
bacterial
with
a
cells
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49
in general,
did not completely
rot.
Second,
the
number of apparently intact inoculated mother roots remaining
after the second pulling was lower than noninoculated ones
only in 1989. Coincidently, heavy rains occurred in May-July
of that year (950 m m ) .
Unlike Irish potato in which contaminated mother tubers
are
planted
in
the
field
and
are
the
main
contamination of daughter roots by E. carotovora
source
of
(10), the
transmission of E. chrysanthemi from mother roots to daughter
roots depends basically on the presence of the bacteria on or
in the transplants.
This study showed that the pathogen is
carried on or in sweetpotato transplants to the field. The
lack of disease development in the field could be attributed
to the low numbers of pulled
transplants.
Thus,
transplanting,
but
we
(23%) or cut (9%) contaminated
inoculated pulled
the
disease
and
incidence
cut
still
slips
at
remained
negligible. However, the presence of E. chrvsanthemi on or in
underground
stems
of
sweetpotato
plants
increased
substantially compared to plants produced from pulled or cut
sprouts
from mother roots
inoculated on the day they were
bedded.
The main advantage in using cut-sprouts or vine cuttings
instead of pulled sprouts is that they do not carry Fusarium
wilt, black rot, soil rot or scurf pathogens to the field (8).
Even though the percentage
recovery of E.
chrvsanthemi
at
harvest from plants produced from pulled sprouts was greater
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
50
than those from cut sprouts, the importance of survival of E.
chrvsanthemi at low populations should not be underestimated
because only a few cells can function as primary inoculum if
conducive conditions occur, as is known in Irish potato (5,
11),
Thus,
the importance of type of transplanting on the
epidemiology
of
E.
chrvsanthemi
still
needs
further
investigation.
Contaminated tools may transmit E. chrvsanthemi to the
first
and,
by
contaminating
the
cut
stubs,
subsequent
transplantings. In addition, the bacteria may be present in or
on daughter roots produced from those transplantings. Evidence
for this mode
of transmission was
provided
by
results
of
greenhouse and field experiments using knives contaminated
with E. chrvsanthemi. This revealed an aspect of epidemiology
of this disease
interest
and
for which there
requires
further
is considerable practical
investigation,
such
as
the
importance of mechanical harvesters on the transmission of the
bacteria from plant beds to the field.
Regarding
survival
of
E.
chrvsanthemi
in
soil,
our
results agreed with those previously reported (14) that this
pathogen does not survive in the soil or survive
in small
numbers difficult to recover. Either way, it does not seem to
play an important role in the survival of the pathogen (14).
However, investigation on soil conditions conducive to disease
occurrence will lead to a better insight into the epidemiology
of this disease.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
51
CHAPTER II
TABLE
1.
Number
of
contaminated mother roots.
Number
of
contaminated mother roots of Beauregard sweetpotato inoculated
with Erwinia chrvsanthemi
(Ech-2-rr)
at bedding by dipping
into a cell suspension or with pipette tips
Number of contaminated mother roots
/ total number sampled
Evaluation
time
Year
Noninoculated
Dipped
Pipette-tip
Day zero
1988
1989
1990
0 / 5
0 / 7
0 / 7
5 / 5
6 / 7
7 / 7
3 / 5
3 / 7
7 / 7
Day seven
1988
1989
1990
0 / 5
0 / 7
0 / 7
5 / 5
6 / 7
7 / 7
3 / 5
5 / 7
7 / 7
First pulling
1988
1989
1990
0 / 5
0 / 7
0 / 7
2 / 5
5 / 7
3 / 7
1 / 5
6 / 7
2 / 7
Second pulling 1988
1989
1990
0 / 7
0 / 7
2 / 7
1 / 7
3 / 7
4 / 7
3 / 7
3 / 7
4 / 7
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52
CHAPTER II
TABLE 2. Mtunber of contaminated underground stems. Number of
contaminated
underground
stems
of
Beauregard
sweetpotato
daughter plants produced from pulled or cut stems from mother
storage roots inoculated with Erwinia chrysanthemi (Ech-2-rr)
at bedding by dipping into a cell suspension or with pipette
tips
Number of contaminated underground
stems /
total number sampled
Noninoculated
Pulled Cut
Harvest-l?
Harvest-2^
y •^
Dipped
Pulled Cut
Pipette-tip
Pulled Cut
1988
0/7
0/7
4/7
1/7
4/7
3/7
1989
0/7
0/7
0/7
1/7
1/7
1/7
1990
0/7
0/7
2/7
0/7
0/7
0/7
1988
0/5
0/5
3/5
1/5
2/5
0/5
1989
0/7
0/7
1/7
0/7
1/7
0/7
1990
1/7
0/7
0/7
0/7
0/7
0/7
Harvest
of
the
first
and
second
transplantings,
respectively;
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53
CHAPTER II
TABLE 3. Plant production in beds. Plant production in beds of
sweetpotato storage roots noninoculated or inoculated with
Erwinia
chrysanthemi
at
bedding
by
dipping
into
a
cell
suspension or with a pipette tip containing inoculum
Number of plants / plot*
Treatments
1988
1989
1990
Firsty Second
First Second
First Second
Control
23
19
60
62 a*
34
30
Dipped
25
25
63
45 b
31
22
Pipette-tip
17
15
51
28 c
38
24
Weight of plants (kg) / plot*
Control
0.86
0.44
1.72
1.37 a
1.26
0.53
Dipped
0.66
0.46
1.54
1.06 ab
1.29
0.48
Pipette-tip
0.60
0.20
1.28
0.67 b
1.45
0.45
*Mean of seven 10-root plots
ypirst and second pullings
=Means with the same letter for the 2nd pulling in 1989 are
not significantly different according to Duncan's multiple
range test (P=0,05). Differences for all other pullings were
not significant (P=0.05).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
54
CHAPTER II
TABLE 4. Yield Of storage roots. Yield of storage roots from
plots planted with Beauregard sweetpotato plants produced from
mother roots dipped into a suspension of Erwinia chrysanthemi
or inoculated with inoculum in a pipette-tip at bedding and
cut or pulled at transplanting
Number of storage roots / plot*
1988
Treatments
Control
First? Second
1989
1990
First Second
First Second
Pulled
79
24
51
50 b^
43
36
Cut
64
28
58
63 a
47
30
Pulled
67
28
59
64 a
51
32
Cut
73
31
57
59 ab
37
28
Pipette- Pulled
75
18
57
60 ab
42
24
tip
65
24
61
51 b
41
23
Dipped
Cut
kg of storage roots / plot*
Control
Pulled
18.4
3.9
7.6
8.3
14.0
6.7
Cut
16.4
5.3
9.3
10.6
12.6
7.8
Pulled
16.6
4.7
8.7
9.4
15.8
6.5
Cut
20.3
5.8
8.8
9.9
11.4
6.5
Pipette- Pulled
17.7
2.5
8.5
8.6
13.3
5.0
tip
20.2
4.5
8.2
9.1
14.7
5.4
Dipped
Cut
*Mean of seven plots
ypirst and second transplantings
%Means with the same letter in the same column are not
significantly different according to Duncan's multiple range
test; differences in other tests were not significant (P =
0.05).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
55
Fig.
2.1.
Inoculation of mother storage roots by dipping.
Inoculation
of
mother
storage
roots
by
dipping
without
injuring in a suspension of Erwinia chrvsanthemi (Ech-2-rr) at
the time of bedding.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
56
Fig. 2.2. Inoculation of mother storage roots by micropipette
tip.
Inoculation
of
mother
storage
roots
by
inserting
a
micropipette tip containing 50 Ml of a suspension of Erwinia
chrvsanthemi (Ech-2-rr) at the time of bedding.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
57
Fig. 2.3. Plant production bed layout. Four and ten-root plots
bedded in two rows in March; one row to take samples (left)
and the other (right) for plant production.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
58
PART OF PLANT MONTH
SAMPLED
88-90
STAGE
AT
SAMPLING
T R E A T M E N T S
r CONTROL
MOTHER ROOTS — MARCH— BEDDING -- DIPPED
SEVEN
DAYS
AFTER
PIPETTE-TIP
INOCULATED
APRIL
ut
K
r CONTROL
ULLED
ULI
MOTHER ROOTS
1-jCUT
UlY
FIRST
--DIPPED
HARVEST
UuULLED
LI
STEM CUTTINGS
PIPETTE-TIP
INOCULATED
r-CUT
LpUULLEDl
JUNE
ut
K
r CONTROL
ULLED
ULI
:UT
MOTHER ROOTS
JULY-
SECOND --DIPPED
PULLING
HARVEST
“ClLLED
STEM CUTTINGS J
PIPETTE-TIP
INOCULATED
k:
:UT
ULLED-
AUGUST
DAUGHTER ROOTS
SEPT—
HARVEST
1st transplanting
BELOW-GROUND
STEM REGION
DAUGHTER ROOTS
OCT-
HARVEST
2nd transplanting
BELOW-GROUND
STEM REGION
Fig.
2.4.
Diagram of
experiments.
indicating part of plant sampled,
Diagram
of
experiments
month when samples were
taken, stage at sampling, and treatments.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
59
Fig. 2.5. Storage root sampling method. A mother root dug from
the bed and washed individually in a plastic bag containing
100 ml of sterile distilled water to assess for presence of
Erwinia chrvsanthemi.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
60
Fig. 2.6. Isolation plating. Colonies of Erwinia chrvsanthemi
after 48-hr incubation at 32 C on circular areas of CVP-RC
medium cut in agar plates with a metallic tube cap.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
61
Fig.
2.7.
(arrows)
Sweetpotato
underground
stem.
Underground
stem
of sweetpotato plants was sampled for presence of
Erwinia chrvsanthemi at harvest.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
62
Populations of Erwinia chrysanthemi on Mother Roots
7
■ Control
^ D ip p e d ---------□ Tip-inoculated
DAY ZERO
6
5
7 DAYS AFTER
2ND PULUNG
4
1ST PULUNG
3
2
o
D)
1
■ X
NR
0
88 89 90 88 89 90 88 89 90 88 89 90
Sampling time
Fig.
2.8. Populations of Erwinia chrvsanthemi on the day of
bedding,
seven
pullings
from
days
later,
sweetpotato
and
at
mother
the
roots
first
and
second
noninoculated
or
inoculated at bedding by dipping in a cell suspension or with
pipette; NR = not recorded.
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63
Survival in soii at three temperatures
Ech-2-rr
Ech-2-rr
Ech-2-rr
Tim e (days)
Fig.
2.9.
Recovery
of
wild
type
strain
Ech-2
of
Erwinia
chrvsanthemi and a rifampicin-resistant strain Ech-2-rr from
soil at three temperatures.
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64
Recovery of Erwinia chrvsanthemi
from crate wood
12
10
o
ay
Q
8
o
6
%
O
CL
2
NR NR
3
4
5
6
7
9
16
20
Time (days)
Fig. 2.10. Recovery of Erwinia chrvsanthemi from wood squares
infested by dipping into a suspension of rotted storage root
previously inoculated with Ech-2-rr,
a rifampicin-resistant
strain; NR = not recovered.
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of the copyright owner. Further reproduction prohibited without permission.
65
LITERATURE CITED
1.
Adams, M. J . , Hide, G. A., and Lapwood, D. H.
Sampling
potatoes
for
the
incidence
of
diseases and levels of inoculum. Ann. App.
107:189-203.
1985.
tuber
Biol.
2.
Clark, C. A., and Moyer, J. W. 1988. Compendium of
Sweet Potato Diseases. APS Press, St. Paul, MN. 74 pp.
3.
Clark, C. A., Wilder-Ayers, J. A., and Duarte, V.1989.
Resistance of sweet potato to bacterial root and
stem rot caused by Erwinia chrvsanthemi. Plant Dis.
73:984-987.
4.
Cuppels,
D . , and Kelman,
A.
1974.
Evaluation of
selective media for isolation of soft-rot bacteria
from soil and plant tissue. Phytopathology 64:468-475.
5.
De Boer, S. H . , Cuppels, D. A., and Kelman, A. 1978.
Pectolytic Erwinia spp. in the root zone of potato
plants in relation to infestation of daughter tubers.
Phytopathology 68:1784-1790.
6.
Duarte,
V.,
and
Clark,
C.
A.
1990.
Pectolytic
clostridia associated with souring of sweetpotato.
(Abstr.) Phytopathology 80:1030.
7.
Duarte, V., and Clark, C. A. 1990. Presence of Erwinia
chrvsanthemi. causal agent of bacterial stem and root
rot
on
sweetpotato,
through
the growing
season.
(Abstr.) Phytopathology 80:1030.
8.
Edmond,
J.
B . , and Ammerman,
G. R.
1971. Sweet
Potatoes: Production, Processing, Marketing, ed. The
Avi Publishing Co., Westport, Connecticut. 334 pp.
9.
Martin, W. J., and Dukes, P. D. 1975. A bacterial soft
rot of sweet potato plants. Proc. Am. Phytopathol.
Soc. 2:57.
10.
Pérombelon, M. C. M. 1974. The role of the seed tuber
in the contamination by Erwinia carotovora of potato
crops in Scotland. Potato Res. 17:187-199.
11.
Pérombelon, M. C. M . , and Kelman, A. 1980. Ecology of
the soft rot erwinias. Annu. Rev. Phytopathol. 18:361387.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
66
12.
Phillips,
J.
A.,
and
Kelman,
A.
1982.
Direct
fluorescent antibody stain procedure applied to insect
transmission of Erwinia carotovora. Phytopathology
72:898-901.
13.
Rolston, L. H . , Clark, C. A., Cannon, J. M . , Randle, W.
M . , Riley, E. G . , Wilson, P. W . , and Robbins, M. L.
1987.
'Beauregard' sweet potato. HortSci. 22:13381339.
14.
Schaad, N. W . , and Brenner, D. 1977. A bacterial wilt
and root rot of sweet potato caused by Erwinia
chrvsanthemi. Phytopathology 67:302-308.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CHAPTER III
CHARACTERIZATION OF SWEETPOTATO STRAINS OF ERWINIA
CHRYSANTHEMI. CAUSAL AGENT OF BACTERIAL STEM AND ROOT ROT
67
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68
CHARACTERIZATION OF SWEETPOTATO STRAINS OF ERWINIA
CHRYSANTHEMI. CAUSAL AGENT OF BACTERIAL STEM AND ROOT ROT
ABSTRACT
Thirteen strains of Erwinia chrvsanthemi. eight isolated
originally from sweetpotato and five from chrysanthemum, corn,
dieffenbachia, African violet, and rice, respectively, and one
strain of E. carotovora subsp.
were
inoculated
carnation,
in
cotton,
bell
rice,
carotovora from bell pepper
pepper,
squash,
corn, sweetpotato, and tomato.
cabbage,
soybean,
cantaloupe,
sugarcane,
sweet
Strains from hosts other than
sweetpotato caused little or no disease on sweetpotato roots
and stems. Based on stem rot index, most sweetpotato strains
were more virulent on tomato,
bell pepper,
and sweetpotato
plants than the other strains,
except a rice strain which
showed
bell
similar
sweetpotato
virulence
strains
tomato and bell
on
produced
pepper.
greater
stem
rot
pepper than on sweetpotato.
resistant mutant of E. chrvsanthemi
In
(Ech-2-rr)
A
general,
indices
on
rifampicinproduced 40%
smaller storage root lesions than the wild type, but lesions
were
similar to or greater in size than other sweetpotato
strains. No difference in stem rot virulence was found between
mutant and wild type. Of 95 carbon sources tested, sweetpotato
strains used the same
25,
were unable to grow on
57,
varied on 13 substrates.
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and
69
INTRODUCTION
Similarities
characteristics
have
and
been
differences
noted
for
in
strains
phenotypic
of
Erwinia
chrvsanthemi Burkholder, McFadden, and Dimock from different
hosts (6 , 12, 13) . Strains of E. chrvsanthemi have been tested
for virulence or pathogenicity to plants or plant parts of 118
plant species or cultivars (5). Strains isolated from the same
host usually were similar even though the host plants had been
collected from different locations.
The latter
observation
suggested that the hosts of E. chrvsanthemi may be attacked by
specific types of the pathogen which might be recognizable by
phenotypic characteristics (4).
When
first
detected
on
sweetpotato,
isolates
of
chrvsanthemi were studied primarily foridentification.
E.
They
were pathogenic to 13 plant species in addition to sweetpotato
(15). Another study investigated the reaction of sweetpotato
genotypes to stem and root inoculations, and concluded that
the
response
of
stem
cuttings
was
not
correlated
to
the
response of storage roots of the same genotypes to a strain
(Ech-2)
of E. chrvsanthemi
(2). An important aspect of the
bacterial stem and root rot that has not been investigated in
detail to date has to do with the characterization of isolates
of E. chrvsanthemi from sweetpotato.
Antibiotic resistance has been used in ecological studies
of
several
bacterial
plant
pathogens
resistant strain of E. chrvsanthemi
(18).
A
(Ech-2-rr)
rifampicinwas used to
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70
monitor the pathogen during the sweetpotato growing season
(7) . Bacterial resistance to rifampicin, generally mediated by
a mutation in the p
subunit of RNA polymerase
(16), has a
chromosomal nature which affords greater stability than occurs
with plasmid-borne markers and is also advantageous since the
mutation is not easily transferable (3). Although antibioticresistant
mutants
have
identical
to wild
types
been
(18),
shown
to
in one
virulence also has been reported (14).
maintain
virulence
case a reduction of
Thus, it is necessary
to thoroughly compare the mutants to the wild type.
This paper
(a) compares virulence on sweetpotato among
sweetpotato and other strains of E. chrvsanthemi. (b) tests
strains
for pathogenicity on different hosts,
(c) compares
utilization of carbon sources by strains, and (d) compares a
rifampicin-resistant
strain
to
the
wild
type
using
these
tests.
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71
MATERIALS AND METHODS
Inoculum. The host, Identification number, location, and
source of strains of E. chrvsanthemi and E. carotovora subsp.
carotovora
used
are
presented
in
Table
1.
Cells
of
the
bacteria grown on plates of yeast extract-dextrose-calcium
carbonate agar (10, 20, 20, and 15 g/L, respectively) at 32 C
for 24-48 hr were suspended in sterile distilled water. The
suspensions were adjusted to an optical density of 0.15 at 620
nm (approx. 1 0 ^ cfu / ml).
Inoculation. To test for pathogenicity, five plants per
bacterial strain, unless otherwise specified, of bell pepper
{Capsicum annuum L.)
oleracea
cantaloupe
(Dianthus
morifolium
var.
'California Wonder',
capitata
fCucumis melo
L.)
Jersey
Hemsl.),
chrysanthemum
cotton
(Brassica
Wakefield',
L.) 'Hales Best Jumbo',
carvophvllus) ,
(Ramat)
'Early
cabbage
carnation
fChrysanthemum
fGossvoium hirsutum L.)
'HG-660', rice fOrvza sativa L.)
'Lemont', squash fCucurbita
moschata
'Early
(Duschene)
Duschene)
squash), soybean (Glycine max (L.) Merr.)
fSaccharum
L.
hybrid)
L.'Golden Queen'),
'CP70-321',
sweetpotato
Golden'
(crookneck
'Davis', sugarcane
sweet
corn
(Ipomoea batatas
fZea
[L.]
mays
Lam.)
'Beauregard' and 'Jewel', and tomato fLvcopersicon esculentum
Mill.)
in
'Walter' were grown (five per 15-cm-diameter clay pot)
sand-soil-Jiffy Mix mixture
(1:1:1)
in the greenhouse.
Sugarcane and chrysanthemum rooted cuttings and bell pepper,
cabbage, cantaloupe, carnation, cotton, squash, soybean, sweet
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72
corn,
and
tomato
plants
were
15-
to
30-days-old
at
inoculation. Rice plants were grown to the tillering-booting
stages before inoculation, and pots were kept in a 5-cm layer
of water.
Terminal cuttings about 25-30 cm long were taken
from sweetpotato vines growing in the field and grown for 1 wk
in the greenhouse before inoculation. Toothpicks previously
dipped in bacterial suspension were inserted into stems of
plants and left in position (Fig. 1). Noninfested toothpicks
were inserted in control plants.
Seven
days
after
inoculation,
30 days
for rice
and
sugarcane, stems were cut open longitudinally and severity of
stem rot was recorded using the following rating system; 0 =
no
symptoms,
inoculation,
=
1
2 =
localized
and
inoculation,
4
at
the point
of
localized necrosis extending up to 1-2 cm
from point of inoculation,
inoculation
necrosis
decay
3 = stem collapsed at point of
extending
stem
2-5
decayed
cm
but
from point
=
most
of
green
remaining at apex
(Fig.
2), and 5 = plant dead
of
tissue
(2). Trials
were performed two times unless otherwise specified.
Storage root inoculations. Sweetpotato storage roots were
inoculated by inserting a micro-pipette tip containing 50 fx l
of serially diluted cell suspension of each strain to a depth
of about 1 cm and leaving it in position (9). The inoculated
roots were placed
in plastic vegetable baskets,
that were
stacked, covered with a black polyethylene bag, and incubated
at
25-28
C for
6
days.
The
roots were
then cut
in cross
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73
section through the point of inoculation
(Fig.
3), and the
depth and diameter of each lesion was measured (2 ). EDgQ, the
inoculum
concentration
that
gave
lesions
in
50%
inoculations, was estimated from plots of the log^o
each
dilution
versus
probit
response
cfu of
(converted
percentage of inoculations producing lesions)
of
from
(2). Regression
analysis was performed to determine if there was a significant
correlation
between
inoculum
concentration
and
response
probit.
Carbon source utilization. The carbon source utilization
profiles of the 13 strains of E. chrvsanthemi and 1 strain of
E. carotovora subsp.
carotovora were obtained using 95-test
microplates (GN MicroPlate ). Twenty-four-hr-oId tryptic soy
agar cultures of E. chrvsanthemi strains were transferred to
tubes containing normal
saline
(0.85% NaCl)
using sterile,
cotton-tipped swabs. The bacterial concentration was adjusted
to an optical density of 0.17 ±0.01 at 590 nm. Microplates
were inoculated by filling all wells with 150 i i l of bacterial
suspension and incubated at 27 C. Microplates were read after
4 hr and 24 hr; results are based on the 24-hr reading. The
process of matching the metabolic pattern of the strains was
performed with the aid of Biolog's computer software package,
MicroLog
(10).
Biochemical
tests.
D-trehalose
is
one
of
the
carbon
sources of the microplate system described above. Thus, the
results of testing for production of acid from that sugar was
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74
Obtained from there.
slices
(2 ),
and
Rotting of potato
phosphatase
(1 )
were
(8 ) and sweetpotato
tested
following
procedures reported elsewhere.
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75
RESULTS
Steu rot index. Based on stem rot index, most sweetpotato
strains of
chrysanthemi were more virulent on sweetpotato,
tomato, and bell pepper plants than other strains (Fig. 4-6).
In general,
sweetpotato
strains produced
greater
stem
indices on bell pepper and tomato than on sweetpotato.
sweetpotato
strains,
the
rice strain
(Ech-24)
rot
Like
killed bell
pepper plants by 7 days after inoculation in test 1. Although
the indices were lower in test 2, Ech-24 continued to perform
similarly to sweetpotato strains
(Fig.
6 ).
Two sweetpotato
strains, Ech-18 and Ech-31, caused some disease (indices > 2)
on cabbage in test 1 , and the rice strain caused significantly
more disease
(indices 2 and > 4) than all other strains in
test 1 and 2 (Fig. 7).
Stem
between
rot
indices
Ech-2-rr
sweetpotatoes
(Fig.
were
and
Ech-2
4),
not
on
significantly
Beauregard
different
and
Jewel
but they were lower in tests 2 on
tomato (Fig. 5) and bell pepper stems (Fig. 6 ).
Stem inoculations of cantaloupe, carnation, cotton, rice,
squash,
soybean,
produced
little
sugarcane,
or
no
and sweet corn with all strains
disease
(Table
2).
Two
sweetpotato
strains, Ech-37 and U-09, and the rice strain, Ech-24, induced
necrosis
extending
inoculation
in
more
sugarcane
than
stems
5
cm
from
(Table
2).
the
point
of
Inoculation
of
chrysanthemum plants induced intermediate (index = 3 )
levels
of
disease
(Table
2).
Reaction
of
rice
to low
to
stem
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76
inoculation with sweetpotato strains ranged from 0 to 2; 4
strains did not induce any reaction (index = 0)
Storage
root
inoculations.
Most
(Table 2).
sweetpotato
strains
produced larger lesions than other strains when inoculated on
storage roots (Fig. 8 ). Strain Ech-18 induced small lesions,
similar to those
sweetpotato.
caused by
strains
from hosts
other
than
Storage root lesions in Beauregard sweetpotato
produced by the rifampicin-resistant mutant were about 40%
smaller than those produced by the wild type, but lesions were
similar to or greater in size than other sweetpotato strains
(Fig. 8 ). The median effective dose was calculated only for
Ech-2 (3.18), Ech-18 (3.60), Ech-31 (3.94), Ech-19 (2.74), and
Ech-24
(3.78),
which
the
regression
significant correlation between
analysis
showed
inoculum concentration and
response probit (Table 3).
Carbon sources. Of the 95 carbon sources in the MicroLog
plates,
all sweetpotato strains of E. chrvsanthemi used the
same 25 carbon sources (N-acetyl-D-galactosamine, L-arabinose,
D-fucose, D-galactose, a-D-glucose, m-inositol, D-manitol, Dmannose,
D-melibiose,
^-methyl
glucoside,
D-raffinose,
sucrose, methyl pyruvate, mono-methyl succinate, citric acid,
D-galacturonic
acid,
D-gluconic
acid,
D-saccharic
acid,
succinic acid, L-asparagine, L-aspartic acid, glycerol, D,L-°<glycerol
phosphate,
glucose-l-phosphate,
and
glucose- 6 -
phosphate). The sweetpotato strains varied on 13 (Table 4) and
gave a negative reaction on the other 57 substrates.
Among
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77
negative
reactions,
glycogen,
Tween
40,
a-lactose,
D-
glucuronic acid, uridine, or thymidine were utilized at least
by
one
Strain
strain
other than the
Ech-18,
which
also
sweetpotato
was
less
ones
virulent
(Table
than
4).
other
sweetpotato strains, was the only one not to utilize formic
and bromo succinic acids (Table 4).
Biochemical
phosphatase
trehalose,
tests.
positive,
and unable
to
produce
acid
from
D-
important properties to separate E. chrvsanthemi
from other Erwinia spp.
isolated
All eight sweetpotato strains were
originally
(4). All
strains,
from other hosts,
including those
macerated
slices of
sweetpotato storage roots and potato tubers.
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78
DISCUSSION
Sweetpotato strains of E. chrvsanthemi were more virulent
than
strains
isolated
from other hosts when
inoculated in
storage roots or stems of sweetpotato. These results agreed
with the hypothesis that in general, strains isolated from a
specific host are often more virulent when reinoculated to the
same species than when inoculated to other host plants (1 1 ).
In addition,
the
heterogeneity
in virulence
present
among
sweetpotato strains either for sweetpotato root or stem rot
should be considered in selection of strain(s),
from one or
more locations, for evaluation of sweetpotato germplasm.
The greater virulence of sweetpotato strains on tomato
and
bell
pepper
than
on
sweetpotato
stems
increases
the
credence that many strains of E. chrvsanthemi often are not
limited to causing disease only in plants from which they have
been isolated.
It seems likely that the plant from which a
strain was isolated may not necessarily reflect the host in
which the strain originally occurred (5).
Based
on
the
results
of
infectivity
titration,
the
Diffenbachia strain, Ech-19, was the most virulent. However,
the dimension of storage root lesions produced by that strain
does not support this assertion.
The
common
substrates
by
utilization
sweetpotato
of
25
strains
and
variability
suggests
there
on
13
are
no
striking differences in utilization of carbon sources among
these strains. This is supported by similarity coefficients
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
79
with
the
stains
of
E.
chrvsanthemi
used
to
compile
the
Microlog data base.
The utilization of formic acid is dependent on an enzyme
system known as formic hydrogenlyase. When this enzyme system
is
absent,
production
the
and
fermentation
with
the
of
sugar
production
of
occurs
formic
without
acid
as
gas
an
important end product (17). Ech-18, a sweetpotato strain of E.
chrysanthemi.
and
Ecc-44,
a
bell
pepper
strain
of
E.
carotovora subsp. carotovora were the only strains which did
not utilize formic acid.
According
to
the
E.
chrvsanthemi
profile
(updated
metabolic profiles furnished by BIOLOG, Inc., October, 1990),
78% of tested strains utilized lactate as a carbon source. In
another study, 10 sweetpotato strains of E. chrvsanthemi were
reported as producing acid from lactose
(15),
although the
configuration for lactose was not mentioned. None of the eight
sweetpotato strains in this study utilized a-lactate.
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80
CHAPTER III
TABLE 1. Sources of Erwinia strains
Bacterial
species
Echf
Eccf
Original
Strain
host
Geographical
Provided
origin
by
Sweetpotato
Ech-2
NC
Moyer
Sweetpotato
Ech-2-rr
NC
Duarte
Sweetpotato
Ech-18
GA
Dickey
Sweetpotato
Ech-26
GA
Dickey
Sweetpotato
Ech-28
GA
Dickey
Sweetpotato
Ech-31
LA
Duarte
Sweetpotato
Ech-37
GA
Duarte
Sweetpotato
U-09
TX
Philley
Chrysanthemum
Ech-15
PA
Dickey
Corn
Ech-17
NC
Dickey
Dieffenbachia
Ech-19
CA
Dickey
African violet Ech-21
FL
Duarte
Rice
Ech-24
JAPAN
Dickey
Bell pepper
Ecc-44
LA
Duarte
%Ech = Erwinia chrvsanthemi; Ecc = Erwinia carotovora subsp.
carotovora
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81
CHAPTER III
TABLE 2. Comparison of strains for stem rot indices.
Host
reactions to inoculation with Erwinia chrysanthemi strains and
E. carotovora subsp. carotovora
rot inde%y »^
stem
Strain
Spx
pq X
CT
CH
CR
SC
RC
SQ
Ech-2
+
+
NT
NT
0
1
1.8
0
0
1.0 ab
Ech-2-rr
+
+
1
2
0
0
2
0
0
0.7 b
Ech-18
+
+
1
3
0
1
0
0
0
1.3 ab
Ech-26
+
+
0
1
0
0
0
0
0
NT
Ech-28
+
+
1
3
0
1
0
0
0
NT
Ech-31
+
+
1
3
0
0
0
0
0
1.3 ab
Ech-37
+
+
NT
NT
0
3
2
0
0
0.7 b
u-09
+
+
NT
NT
0
3
1.5
0
0
0
b
Ech-15
+
+
1
NT
0
NT
NT
0
0
0
b
Ech-17
+
+
2
1
0
0
0
0
0
1.3 ab
Ech-19
+
+
1
1
0
NT
NT
0
0
0.3 b
Ech-21
+
+
1.5 NT
0
NT
NT
0
0
0
Ech-24
+
+
1
1
0
3
2.5
0
0
2.3 a
Ecc-44
+
+
1
0
0
NT
NT
0
0
1.0 ab
so
CO
b
*SP = slices of storage root of Beauregard sweetpotato; PO =
slices of potato tuber (- = no soft rot; + = soft rot);
yRated on a scale of 0-5, where 0 = no symptoms and 5 = plants
dead. NT = not tested; CT = cotton, 2 plants; CH =
chrysanthemum, 1 plant; OR = carnation, 2 plants; SC =
sugarcane, 1 plant; RC = rice, 4 plants; SQ = squash, 5
plants; CO = sweet corn, 4 plants; SO = soybean
^Means in the same column followed by a common letter are not
significantly different(Duncan's multiple range test, P =
0.05). Statistical analysis was not performed with other data.
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82
CHAPTER III
TABLE 3. Estimate of median effective dose. Estimate of median
effective dose (EDgg) from the linear regression equation of
the log^o of cfu of strains versus probit responses
Strain
P > F
Ech-2
0.05
Ech-2-rr
NS=
Ech-18
0.01
Ech-26
NS
Ech-28
NS
Ech-31
0.05
Ech-37
NS
U-09
NS
Ech-15
NS
Ech-17
NS
Ech-19
0.05
Ech-21
NS
Ech-24
0.01
Ecc-44
NS
r2
Regression equation
ED 50
0.90
Y = 1.32 X + 0.79
3.18?
0.97
Y = 2.75 X - 4.90
3.60
0.89
Y = 2.30 X - 4.07
3.94
0.99
Y = 2.60 X - 2.16
2.74
0.94
Y = 1.36 X + 0.16
3.78
yiiog^o bacterial population = effective median dose
^NS = not statistically significant
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
83
CHAPTER III
TABLE 4. Tests Of carbon source utilization. Tests of carbon
source
utilization
that
varied
among
strains
of
Erwinia
chrysanthemi and g. carotovora subsp. carotovora (Ecc-44)
Carbon
D G T
e 1 w
X y e
t c e
r o n
i g
n e 4
n 0
source
C a M D P
e — a - s
1 1 1 m i
1 a t e c
D D C
- - i
r s s
a o o c o 1 o f r a
b t s i s f b c
i o e b e i i o
o s
i
n t n
s e
o
o o i
e
s 1 t
s
e
e
i
c
F
o
r
m
i
c
a
c
i
d
a
c
i
d
D D M
t a
g L 1
1 - o
u 1 n
c a i
u c c
r t
o i a
n c c
i
i
c a d
c
a i
c d
i
d
B S L
r u o c g
m c 1
o i u
n t
s i a
u c m
c
i
c a c
i c
n i a
i d c
c
i
d
a
c
i
d
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Strain?
Ech-2
Ech-2-rr
Ech-18
Ech-26
Ech-28
Ech-31
Ech-37
U-09
Ech-15
Ech-17
Ech-19
Ech-21
Ech-24
Ecc-44
- +
- - - +
- - - + - - -
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
- - + +
—
+
+
-
+
+
-
+ +
+
+ +
+ +
+ +
+ +
+ + +
+ +
- +
+ +
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + - - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
L U T G
- r h 1
s i y U
e d m C
r i i o
i n d s
n e i e
e
n e 1
+
+
+
+
+
+
+
+
+
S
t
a
t
i
s
S
i
m
i
1
.
a
r
i
t
-
s
y
P
h
U
b
o
g
s
p
h
a
t
e
r
o
u
P
+
+
+
+
+
+
+
+
+
—
+
+ +
+
- + +
+ + —
A
A
A
A
A
A
A
A
B
A
A
A
B
-
+
-
t
V
a
1
u
e
s
.84
.81
.63
.53
.71
.67
.50
.76
.70
.58
.86
.78
.87
.55
?Ech-2 to U-09 from sweetpotato; Ech-15 to Ecc-44 from other
hosts
z+ = positive reaction; - = negative reaction
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
84
Fig. 3.1. Toothpick inoculation. Sweetpotato stems inoculated
with
toothpicks
previously
dipped
in Erwinia
chrvsanthemi
suspension.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
85
Fig. 3.2. stem rot index. Sweetpotato plant with most of stem
decayed but green tissue remaining at apex; stem rot index =
4.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
86
Fig. 3.3. storage root inoculation. Sweetpotato storage roots
cut in cross section through the point of inoculation with
pipette tip containing Erwinia chrvsanthemi suspension.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
87
Comparison of strains for
sweetpotato stem rot
100
LSD - 0.57
04
P-
0.0001
4 80
C.V. - 52%
O)
R®- 0.78
40 Ü
Ech-2
Ech-18
Ech-28
Ectv37
Ech-15
Ech-19
Ech-24
Ech-2-fr
Ech-26
E cM I
IW »
Ech-17
Ech-21
Eco44
Strain
100
LSD - 0.45
04
Jewel
Sweetpotato
P ■ 0.0001
C.V. - 33%
4 80
R®« 0.87
-- L g Q
40 p
20
Ech-2
Ech-18
Ech-28
Ech-37
Ech-2-fr
Ech-26
Ech-31
Ech-15
Ech-17
Ech-24
Ecc-44
Ech-21
Strain
Fig. 3.4. Comparison of strains for sweetpotato stem rot
indices. Stem reactions of Beauregard and Jewel sweetpotatoes
to inoculation with strains of Erwinia chrvsanthemi from
sweetpotato (Ech-2 to U-09), chrysanthemum (Ech-15), corn
(Ech-17), Dieffenbachia (Ech-19), African violet (Ech-21), and
rice (Ech-24), and with a strain of E. carotovora subsp.
carotovora from bell pepper (Ecc-44). Stem rot index: 0 = no
symptoms, 5 = dead plant.
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88
LSD - 0.57
Tomalo
TMtl
P • 0.0001
C.V. -16%
R®«0.03
40 O
NT
NT
—I
--1
—
"r- "i“— r
E c h -2
E c h -1 8 E c h -2 8 E c h -3 7 E c h -1 5 E c h -1 9 E c h -2 4
E c h - 2 - r r E c h -2 6 E c h -3 1
U -0 9
E c h -1 7 E c h -2 1 E c c -4 4
Strain
5T
LSD > 0.9
Tomato
Teal 2
P - 0.0001
l<
C
C.V. - 31%
R®- 0.88
s '
+ 40 Ü
I'
E c h -2
E c h -1 8 E c h -2 8 E c h -3 7 E c h -1 5 E c h -1 9 E c h -2 4
E c h - 2 - r r E c h -2 6 E c h -3 1
U -0 9
E c h -1 7 E c h -2 1 E c c -4 4
Strain
Fig. 3.5. Comparison of strains for tomato stem rot indices.
Stem reactions of tomato to inoculation with strains of
Erwinia chrvsanthemi from sweetpotato
(Ech-2 to U-09),
chrysanthemum (Ech-15), corn (Ech-17), Dieffenbachia (Ech-19),
African violet (Ech-21), and rice (Ech-24), and with a strain
of E. carotovora subsp. carotovora from bell pepper (Ecc-44).
Stem rot index: 0 = no symptoms, 5 = dead plant.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
89
5T
Bell peppe
Testi
LSD = 0.57
C
P = 0.0001
C.V. = 10%
i ’
40 O
ë'
NT NT
0 .1- '^— - 'f— -T-— I---- 1— " r • '" f " ‘ ^ r ‘— I— "T-— I— ^
Ech-2
Ech-18
Ech-28
Edv37
Edi-15
Ech-19
Ech-24
Ech-2-rr
Ech-26
Ech-31
U-09
Ech-17
Ech-21
Eoo44
Strain
100
LSD = 1.0
Bell peppei
Test 2
P = 0.0019
C.V. = 68%
CD
R®= 0.51
Ech-2
Ech-18
Ech-28
Ech-37
Ech-15
Ech-19
Ech-24
Ech-2-rr
Ech-26
Ech-31
U-09
Ech-17
Ech-21
Eco-44
Strain
Fig. 3.6. Comparison of strains for bell pepper stem rot
indices. Stem reaction of bell pepper to inoculation with
strains of Erwinia chrvsanthemi from sweetpotato (Ech-2 to U 09), chrysanthemum (Ech-15), corn (Ech-17), Dieffenbachia
(Ech-19), African violet (Ech-21), and rice (Ech-24), and with
a strain of E. carotovora subsp. carotovora from bell pepper
(Ecc-44). Stem rot index: 0 = no symptoms, 5 = dead plant.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
90
100
LSD m 0.83
Ic
P > 0.0001
C.V. - 36%
■
-4
80
D)
R * - 0.66
■e
E
40 P
<p
35
NT
NT
Ech-2
Ech-18 Ech-28
Ech-37 Ech-15
Ech-19
Ech-24
Ech-2-rr Ech-26 Ech-31
U-09
Ech-17 Ech-21
Ecc-44
Strain
100
5
LSD « 0.5
P - 0.0001
0 4
•D
80
C
E
I
u>
3
60
2
40 P
1
0
m
Ech-2
Ech-18
Ech-28
Ech-37 Ech-15
Ech-19
Ech-24
Ech-2-rr Ech-26 Ech-31
U-09
Ech-17 Ech-21
Ecc-44
Strain
Fig. 3.7. Comparison of strains for cabbage stem rot indices,
stem reaction of cabbage to inoculation with strains o f
Erwinia chrvsanthemi from sweetpotato
(Ech-2 to U - 09),
chrysanthemum (Ech-15), corn (Ech-17), Dieffenbachia (Ech-19),
African violet (Ech-21), and rice (Ech-24), and with a strain
of E. carotovora subsp. carotovora from bell pepper (Ecc-44).
Stem rot index: 0 = no symptoms, 5 = dead plant.
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91
Comparison of strains for storage
root lesion dimension
Beauregard
Storage root
LSD = 4.2
P = 0.0001
C.V. = 90%
Ech-2
Ech-18
Ech-28
Ech-2-rr Ech-26
Ech-37
Ech-31
Ech-15
U-09
Ech-19
Ech-17
Ech-24
Ech-21
Ecc-44
Strain
Fig. 3.8. Dimension of storage root lesions produced by
inoculation with
strains
of Erwinia
chrvsanthemi
from
sweetpotato (Ech-2 to U-09), chrysanthemum (Ech-15), corn
(Ech-17), Dieffenbachia (Ech-19), African violet (Ech-21), and
rice (Ech-24), and with a strain of E. carotovora subsp.
carotovora from bell pepper (Ecc-44).
Reproduced with permission
of the copyright owner. Further reproduction prohibited without permission.
92
LITERATURE CITED
1.
Barber, M . , and Kuper, S. W. A. 1951. Identification
of Staphvlococcus pyogenes by the phosphatase reaction.
J. Pathol. Bacteriol. 63:65-66.
2.
Clark, C. A., Wilder-Ayers, J. A., and Duarte, V.
1989. Resistance of sweet potato to bacterial root and
stem rot caused by Erwinia chrvsanthemi. Plant Dis.
73:984-987.
3. Compeau, G . , Al-Achi, B. J., and Platsouka, E . , and
Levy,
S. B.
1988.
Survival of rifampin-resistant
mutants of Pseudomonas fluorescens and Pseudomonas
putida in soil systems. Appl. Environ. Microbiol.
54:2432-2438.
4.
Dickey,
R.
S.
1979.
Erwinia
chrvsanthemi:
A
comparative study of phenotypic properties of strains
from
several
hosts
and
other
Erwinia
species.
Phytopathology 69:324-329.
5.
Dickey, R. S. 1981. Erwinia chrvsanthemi: Reaction of
eight plant species to strains from several hosts and
to strains of other Erwinia species. Phytopathology
71:23-29.
6.
Dickey, R. S., Claflin, L. E . , and Zummoff, C. H.
1987. Erwinia chrvsanthemi: Serological comparisons of
strains
from
Zea
mavs
and
other
hosts.
Phytopathology 77:426-430.
7.
Duarte, V., and Clark, C. A. 1990. Presence of Erwinia
chrvsanthemi. causal agent of stem and root rot, on
sweetpotato
through
the growing
season.
(Abstr.)
Phytopathology 80:1030.
8.
Dye, D. W.
Erwinia. I.
11:590-607.
9.
Maher, E. A., and Kelman, A. 1983. Oxygen status of
potato tuber tissue in relation to maceration by
pectic enzymes of Erwinia carotovora. Phytopathology
73:536-539.
10.
Marello, T. A., and Bochner, B. R. 1989. Metabolic
reactions of Gram-negative species.
Biolog,
Inc.,
Hayward, CA. 105 pp.
1968. A taxonomic study of the genus
The 'amylovora' group. N. Z. J. Sci.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
93
11.
McIntyre, J. L., Sands, D. C . , and Taylor, G. S. 1978.
Overwintering, seed disinfestation, and pathogenicity
studies of the tobacco hollow stalk pathogen, Erwinia
carotovora var. carotovora. Phytopathology 68:435-440.
12.
Miller, H. N . , and McFadden, L. A. 1961. A bacterial
disease of philodendron. Phytopathology 51:826-831.
13.
Munnecke,
D.
E.
i960.
Bacterial
stem
Dieffenbachia. Phytopathology 50:696-700.
14.
Scanferlato, V. S., Orvos, D. R . , Cairns, J. Jr, and
Lacy,
G. H.
1989. Genetically engineered Erwinia
carotovora in aquatic microcosms: Survival and effects
on functional groups of indigenous bacteria. Appl.
Environ. Microbiol. 55:1477-1482.
15.
Schaad, N. W . , and Brenner, D. 1977. A bacterial wilt
and root rot of sweet potato caused by Erwinia
chrvsanthemi. Phytopathology 67:302-308.
16.
Sippel, A. E . , and Hartmann, G. R. 1968. Mode of
action of rifampicin on the RNA polymerase reaction.
Biochim. Biophys. Acta 157:218-219.
17.
Stanier, R. Y . , Doudoroff, M . , and Adelberg, E. A.
1963. The Microbial World. 2nd ed., Prentice-Hall,
Englewood Cliffs, NJ. 753 pp.
18.
Weller, D . M . , and Saettler, A. W. 1978. Rifampinresistant
Xanthomonas
phaseoli
var.
fuscans
and
Xanthomonas phaseoli: Tools for field study of bean
blight bacteria. Phytopathology 68:778-781.
rot
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of
CHAPTER IV
EFFECTS OF INTERACTION OF ERWINIA CHRVSANTHEMI AND FUSARIUM
SOLANI
ON SWEETPOTATO STEM ROT
94
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95
EFFECTS OF INTERACTION OF ERWINIA CHRYSANTHEMI AND FUSARIUM
SOLANI
ON SWEETPOTATO STEM ROT
ABSTRACT
Jewel
and
Beauregard
sweetpotato
stem
cuttings
were
inoculated with Erwinia chrvsanthemi and/or Fusarium solani
before planting in the greenhouse and in the field.
In the
greenhouse, stem rot lesion length was 111% and 120% greater
on Jewel sweetpotato and 29% greater on Beauregard when the
two pathogens were inoculated together than the sum of means
of stem lesion length produced by both pathogens inoculated
separately. In the field, the percentages of sampled plants of
Beauregard showing stems with rotted tissue or black streaks
were
greater
in
1989
and
1990
when
inoculated
with
both
pathogens (66 and 20) than with E. chrvsanthemi (20 and 10) or
F.
solani
(16 and 4)
separately.
When soil was kept under
three soil matric potentials (0, -0.3, and -0.5 bars)
greenhouse,
means
in the
of lesion length were greater on plants
inoculated with E. chrvsanthemi. alone or combined, than F.
solani alone regardless of soil matric potential.
When both
organisms were applied to wounded storage roots, no synergism
was observed.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
96
INTRODUCTION
There are many accounts in the literature suggesting an
association of Fusarium spp. with the potato blackleg disease
(1,
5,
1,
8,
9,
10,
11).
It
has
been
hypothesized
that
colonization of the roots and vascular system by F. solani
generally weakens the plant, making it more susceptible to the
soft rot bacteria (11).
Erwinia chrvsanthemi and F. solani cause bacterial stem
and root rot and Fusarium root and stem canker, respectively,
on sweetpotato
floomoea batatas
(L. ) Lam.)
(2, 3,
6).
Both
diseases have two phases, a stem rot and a root rot phase (3).
Similarities
regarding
occurred
suggest
may
the
plant
a possible
parts
in which
symptoms
interaction between
these
pathogens. On the other hand, inoculations of plants in field
experiments with E. chrvsanthemi and F. solani separately have
failed to induce the severity of stem rot sometimes observed
in the field. Furthermore,
several sweetpotato samples with
symptoms of both diseases have been observed. Therefore, this
study was conducted to examine the results of combined effects
of E. chrvsanthemi and F. solani on stem and root diseases of
sweetpotato.
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97
MATERIALS AND METHODS
Inoculum. Isolates Ech-2 of E. chrvsanthemi and M-10 of F.
solani both isolated from sweetpotato and obtained from J. W.
Moyer,
Department of Plant Pathology,
North Carolina State
University, were used throughout this study. Twenty-four-hrold nutrient broth
cultures
optical density of 0.15 at
of Ech-2
620 nm
were
(ca.
adjusted
lO^cfu/ml).
to
an
Spore
suspensions of isolate M-10 were prepare by washing from 5day-old
potato
dextrose
distilled water
cheesecloth.
(SDW)
agar
(PDA)
cultures
with
sterile
and filtering through four layers of
The concentration of spores was then determined
with a hemacytometer and adjusted to 10® per ml.
Stem inoculations, vine cuttings of Jewel sweetpotato in
1988 (Test 1 = 8/11-9/27 and Test 2 = 10/4-11/2) and Jewel and
Beauregard
in
1989
(Test 3 = 6/20-8/1)
were
cut
from the
field. All expanded leaves and petioles were removed and vine
cuttings were
inoculum,
immediately dipped into Ech-2
combined
inoculum,
M-IO
inocula with each organism at the same
concentration as when inoculated alone,
or distilled water
(Fig. 1). Twenty five cuttings per treatment were planted in
15-cm-diameter clay pots in the greenhouse using five plants
per pot in a completely randomized design.
Six weeks after inoculation, roots were washed free of
soil, stems were cut longitudinally, and the length of stem
rot lesions was measured (Fig. 2). Plants were then dried for
48 hr at 60 C, and roots and shoots were weighed separately.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
98
In
the
field,
sweetpotatoes
were
stem
cuttings
inoculated
of Jewel
as
and
described
Beauregard
above.
Each
treatment was replicated five times in 10-plant plots in a
randomized complete block design. Cuttings were planted 30 cm
apart with 90 cm between plots and 120 cm between rows.
Prior to harvesting, stems of five plants in 1989 and of
all ten plants in 1990 were sectioned longitudinally at the
ground level incidence of stems with rotted tissue or black
streaks was recorded. At 1990 harvest,
the total number of
plants per plot was recorded and storage roots were counted
and weighed.
Effect of soil matric potential on stem rot. Stem cuttings
of
Jewel
were
inoculated
with
E.
chrvsanthemi
and/or
F.
solani. as described above, planted in pots with soil:sand
(1:1),
and grown in the greenhouse under three soil matric
potentials; zero, -0.3, and -0.5 bars. Four replicate pots (5
plants/pot)
for each treatment were placed in a completely
randomized complete design. One tensiometer was installed in
one pot of each of the 12 treatments. The soil in all pots of
that
treatment
was
saturated
with
water
each
time
the
tensiometer indicated the respective matric potential. Six wk
after inoculation, the length of stem rot lesions and plant
dry weight were measured as previously described.
Storage root inoculations. Storage roots were wounded by
gently scraping the periderm from a spot on the median of the
root with a flamed scalpel. A 5-mm-diameter plug was removed
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
99
with a flamed cork borer from the margin of a 5-day-old PDA
culture of F. solani (M-10), and placed (mycelium side down)
on
the
wound
(4).
Inoculation
with
E.
chrvsanthemi
was
accomplished by submerging wounded storage roots in a cell
suspension
of
10^ cfu /
ml.
In the
combined
inoculation,
inoculation with F. solani followed the bacterial inoculation.
Inoculated roots were placed in plastic vegetable baskets,
which were stacked, covered with a black polyethylene bag, and
incubated
at
25
±2
C
for
6
wk.
Evaluation
was
made
by
measuring the external diameter and internal depth of each
lesion (4).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
100
RESULTS
Stem inoculations. In test 1 at the greenhouse, no disease
was recorded on Jewel plants inoculated with F. solani alone.
Lesion length of plants inoculated with E. chrvsanthemi and F.
solani combined was 111% greater than those inoculated with E.
chrvsanthemi alone (Fig. 3). In test 2, lesions were longer in
the
F.
solani-inoculated
treatments.
than
in
the
Ech-2
or
combined
The results in test 3 were similar for both Jewel
and Beauregard to results for Jewel in test 1 (Fig. 3).
Dry weights
alone
or
of plants
combined with
noninoculated
water
inoculated with
F.
solani
control
in
were
test
E.
less
1
chrvsanthemi
than
(Table
for
1).
the
No
significant differences were found in dry weight of plants
among treatments in tests 2 and 3.
Field experiments. The percentages of sampled plants of
Beauregard showing stems with rotted tissue or black streaks
were
greater
in
1989
and
1990
when
inoculated
with
both
pathogens (66 and 20) than with E. chrvsanthemi (20 and 10) or
F. solani
(16 and 4) inoculated separately. The synergistic
effect of the combined inoculation was detected on Beauregard
in both years but not on Jewel
remaining
plants
per plot
(Table 2). Neither number of
at harvest
(Table
2)
nor yield
(Table 3) were significantly different for any treatment in
either
year
(Table
3).
In
1989,
Beauregard
sweetpotato
inoculated with E. chrvsanthemi and F. solani combined (Table
3) produced more storage roots than when either was inoculated
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
101
alone.
Effect
of
soil
matric
potential
on
stem
rot.
No
interaction was observed between E. chrvsanthemi and F. solani
on lesion length when the effect of soil matric
potential was compared (Table 4). Disease level was higher on
plants inoculated with E. chrvsanthemi. alone or combined, and
very low with F. solani.
Storage
root
inoculations.
No
interaction
between
E.
chrvsanthemi and F. solani was found when storage roots were
inoculated. Erwinia chrvsanthemi alone caused no disease while
lesion dimensions on storage roots inoculated with F. solani
(19.5
mm)
or
combined
(15.5
mm)
were
not
significantly
different.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
102
DISCUSSION
A synergistic interaction between E. chrvsanthemi and F.
solani
on
length
of
stem
rot
lesion was
observed
in
two
greenhouse experiments and in two experiments conducted in the
field in 1989 and 1990. This may account for the greater stem
rot
severity
that
develops
in
the
field
under
natural
conditions than in controlled inoculated with the individual
pathogens (4).
I
cannot
account
for
the
lack
of
interaction
in
greenhouse test 2. It may be related to the fact that plant
growth was less in test 2 thant tests 1 or 3. Differences in
temperature in the greenhouse during tests 1, 2, and 3 do not
appear to account
for the differences
in the results.
The
means of maximum and minimum daily temperatures were 31, 29,
and 30 C in tests 1, 2, and 3, respectively.
Although no interaction was found when storage roots were
inoculated
with
E.
chrvsanthemi
and
F.
solani.
this
is
apparently because bacterial root rot failed to develop in any
treatment. The superficial wounding which favors Fusarium root
rot development (4) may not be suitable for bacterial root rot
development. Relative humidity was not controlled during this
study, and it is expected to affect bacteria, fungi, and their
interactions differently.
Bacterial soft rot can be induced
when pieces of surface-sterilized potato tubers are inoculated
with F. roseum cultivar
'Sambucinum', F. solani f. pisi. F.
solani f. phaseoli. and other fungi and incubated in a moist
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
103
chamber; soft rot was prevented by seed piece treatment with
Dithane M-45,
Fusarium
Polyram,
(9).
sweetpotato
Although
can
be
and benomyl
the
after
inoculation with
development
restricted
by
the
of
F.
solani
on
ring
to
vascular
superficial lesions within the cortex of the root
(4), the
presence of a soft rot bacterium such as E. chrvsanthemi and
adequate environmental conditions are expected to be conducive
to root rot disease. Furthermore, in addition to breaking the
storage root barrier, an effect of F. solani similar to that
observed
pieces
on potato
with
fungus
tuber,
in which
trigger
inoculation
bacterial
soft
of
rot,
potato
can
postulated and investigated on sweetpotato.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
be
104
CHAPTER IV
TABLE
1.
Dry
sweetpotato
weight
of
cultivars
inoculation with
plants.
Jewel
and
Dry
weight
of
Beauregard
plants
6
wk
of
after
Erwinia chrysanthemi and/or Fusarium solani
under greenhouse conditions
Dry weight of plants (g)^’^
Test 1
Test 2
Test 3
Jewel
Jewel
Jewel
Control
18.1
2.5
33.6
32.6
Ech
12.3
2.7
32.1
34.6
Fs
14.7
2.2
25.2
30.0
Ech + Fs
10.1
2.8
30.6
27.5
LSD
3.8
0.9
6.1
P
0.006
0.46
0.16
CV (%)
20
25
21
Beauregard
^Mean of five 5-plant pots. Plants were dried for 48 hr at 60
C.
^ Means of test 3 are compared within the two columnr.
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Further reproduction prohibited without permission.
105
CHAPTER IV
TABLE
2.
Disease
incidence
and
Incidence
remaining
and
remaining
plants
at
plants.
harvest
Disease
for Jewel
and
Beauregard sweetpotatoes inoculated in the field with Erwinia
chrysanthemi and/or Fusarium solani at transplanting
Plants per plot with
Remaining plants
stem rot symptoms?
per plot=
19
19
8 9
9 0
19
9 0
Beauregard Jewel Beauregard Jewel Beauregard Jewel
Control
0.4
0.0
0.0
0.0
9.0
8.0
Ech
1.0
1.0
0.5
0.0
7.7
7.6
Fs
0.8
0.8
0.2
0.0
8.4
9.0
Ech + Fs
3.3
1.4
1.0
0.2
8.5
9.2
LSD
2.0
0.4
1.5
P > F
0.01
0.04
0.79
CV (%)
131
181
19
^Mean of five 10-plant plots. Five plants per plot in 1989 and
all
ten
plants
in
1990
were
sampled.
Either
soft
rot
vascular discoloration were counted as stem rot symptoms.
^Plants counted at harvest.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
or
106
CHAPTER IV
TABLE
3.
Effect on sweetpotato yield.
Yield
of Jewel
and
Beauregard sweetpotatoes inoculated with Erwinia chrvsanthemi
and/or Fusarium solani at transplanting
Weight (kg) of storage roots/ plot?
19
8 9
Jewel
Beauregard
Control
Ech
Fs
Ech + Fs
1 9 9 0
5.6
6.1
5.1
7.4
Beauregard
13.1
9.8
13.6
10.6
3.6
3.2
4.0
3.2
Jewel
8.7
6.1
7.8
6.6
Number of storage roots / plot^’^
Control
Ech
Fs
Ech + Fs
40
54
49
74
b-d
b
be
a
33
38
39
34
38
37
44
36
d
Cd
cd
d
36
26
22
28
^Mean of five 10-plant plots
=Means within each pair of columns for each year and parameter
followed by the same letter are not significantly different
using Duncan's multiple range test; differences in other tests
were not significant (P = 0.05).
Pr > F
1990
1989
Source
Block
Cultivar
Treatment
Cv * Trt
Kg/plot
#/plot
kg/plot
#/plot
0.4685
0.0001
0.6451
0.1762
0.3085
0.0001
0.0486
0.0388
0.8034
0.7241
0.2172
0.6246
0.4976
0.0004
0.3104
0.1780
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107
CHAPTER IV
TABLE 4. Effect Of soll matric potential. Effect of soil
matric potential on interaction between Erwinia chrvsanthemi
and Fusarium solani on plant growth and stem rot lesion length
Dry weight (g)
Soil matric
potential
(bars)
Inoculation
Root
Shoot
Control
Ech
Fs
Ech+Fs
Control
Ech
Fs
Ech+Fs
Control
Ech
Fs
Ech + Fs
47.Or^
12.4
14.7
13.9
4.4
5.9
5.2
6.5
3.3
4.0
3.2
2.5
0.0
-0.3
— 0 .5
combined
combined
combined
Combined
Combined
Combined
Combined
Control
Ech
Fs
Ech + Fs
0.0
-0.3
— 0 .5
Root + Shoot
9.8
10.1
13.4
11.9
8.4
8.5
9.6
9.3
6.2
6.1
5.3
5.2
Lesion
length
(mm)
56.9
22.6
28.1
25.9
12.9
14.4
14.8
16.1
9.5
10.5
8.6
7.7
0.0
36.0
0.0
21.5
0.0
15.0
0.0
26.7
0.0
46.5
1.7
40.7
22.0 a 11.3 a
5.5 b 9.0 b
3.3 b 5.7 c
33.4a
14.6 b
9.1b
14,4
10.4
21.5
18.3
7.5
7.7
7.7
26.5
15.9
17.2
16.6
0.0
31.5
0.6
29.7
8.2
8.3
9.5
8.8
b
a
b
a
^Means within a column followed by the! same letter are not
significantly different using Duncan's multiple range test;
differences in other tests were not significant (P =: 0.05).
Combined = treatments combined
Pr > F
Source
Root
Shoot
Inoculation (A)
0.417
0.464
0.440
0.001
0.014
0.000
0.001
0.422
0.374
0.455
0.375
0.839
Soil matric pot.
A X B
2
C. V.
r
(%)
(B)
0.34
181.52
0.62
25.30
Root + Shoot
0.41
94.40
Lesion
0.39
154.60
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108
Fig. 4.1. Vine cutting Inoculation. Inoculation by dipping the
sweetpotato vine cuttings in cell and/or conidial suspension
of Erwinia chrvsanthemi and Fusarium solani. respectively.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
109
Fig.
4.2. Stem rot lesions on sweetpotato. Sweetpotato stem
cuttings showing stem rot lesions of different lengths 6 wk
after inoculation with Fusarium solani.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
110
Length of stem rot lesions
100
□ Control
□ Ech
80
□ Fs
0 Ech+Fs - 80
£
60
E.
LSD >28
LSD > 9.9
LSD -1 4 .3
•P > 0.0001
P - 0.0005
P > 0.0001
C.V. > 64%
C.V. > 53%
C.V. > 80%
f
40
QL
O)
II
20
0
60
i
20
/
/
/
1 (Jewel)
/
3 (Jewel)
2 (Jewel)
3 (Beauregard)
Test
Fig.
4.3.
Length of stem rot lesions on plants 6 wk after
inoculation in the greenhouse with Erwinia chrysanthemi (Ech)
and or Fusarium solani (Fs)
Reproduced with permission
of the copyright owner. Further reproduction prohibited without permission.
Ill
LITERATURE CITED
1.
Blodgett, E. C. 1947. Comments on black rot, a storage
disease of potatoes in Idaho. Plant Dis. Rep. 31:10-13.
2.
Clark, C. A.
1980. End rot, surface rot, and stem
lesions caused on sweet potato by Fusarium solani.
Phytopathology 70:109-112.
3.
Clark, C. A., and Moyer, J.
W. 1988. Compendium of
Sweet Potato Diseases. APS Press, St. Paul, MN. 74 pp.
4.
Clark, C. A., Randle, W. M . ,
and Pace, C. S. 1986.
Reactions of sweet potato selections to Fusarium root
and stem canker caused by Fusarium solani. Plant Dis.
70:869-871.
5.
Davis, J. R . , Sorensen,
L. H . , and Corsini, G. S.
1983. Interaction of Erwinia spp. and Fusarium roseum
'sambucinum' on the Russet Burbank potato. Amer.
Potato J. 60:409-421.
6.
Moyer, J. W . , Campbell, C. L . , and Averre, C. W. 1982.
Stem canker of sweet potato induced by Fusarium sdlmi.
Plant Dis. 66:65-66
7.
Nielsen, L. W.
1949. Fusarium seedpiece decay
of
potatoes in Idaho and its relation to blackleg. Idaho
Agric. Exp. Sta. Univ., Res. Bull. No. 15, 31 pp.
8 . Nielsen,
L. W . , and Moyer, J. W. 1979. A Fusarium root
rot of sweet potatoes. Plant Dis. Rep. 63:400-404.
9.
10.
11.
Stanghellini,
M.
E . , and
Russell,
J.
D.
1971.
Induction of bacterial seedpiece decay of potatoes by
various
soil-borne
fungi.
(Abstr.)
Phytopathology
61:1324.
Zink, R. T., and Secor, G. A. 1979. The presence
fungal wilt pathogens increases the incidence
potato blackleg. (Abstr.) Amer. Potato J. 56:487.
of
of
Zink, R. T., and Secor, G. A. 1982.
Interaction of
fungal wilt pathogens and potato blackleg. Plant Dis.
66:1053-1056.
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CHAPTER V
ASSOCIATION OF PECTOLYTIC CLOSTRIDIA WITH SOURING
OF SWEETPOTATO STORAGE ROOTS
112
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113
ASSOCIATION OF PECTOLYTIC CLOSTRIDIA WITH SOURING OF
SWEETPOTATO STORAGE ROOTS
ABSTRACT
Storage roots of Beauregard sweetpotato fIpomoea batatas)
were
either
not
treated,
disinfested
with
1%
NaOCl,
or
inoculated with Erwinia chrvsanthemi (Ech-2) and half of the
roots from each treatment were punctured with a flamed needle.
They were
then
incubated
for 5 days
at
25 C submerged
in
distilled water in plastic bags. Maceration occurred in 93 and
8 , 8 and
roots
0 , and
from
storage
the
roots,
100 and
control,
60% of punctured and non-punctured
disinfested,
respectively.
and
Erwinia
Ech-2-inoculated
chrvsanthemi
was
reisolated only from artificially inoculated storage roots.
However,
forming,
at
least
two
distinguishable
sulfate-reduction
negative,
pectolytic
gelatin
spore-
hydrolysis
positive, anaerobic bacteria resembling Clostridium spp. were
found
in
most
lesions,
regardless
of
treatment.
Clostridia were differentiated by the diameter
The
two
(1.0 and 2.5
mm) of pitting on double-layer pectate medium. Isolates of the
two
Clostridia
macerated
sweetpotato,
potato,
carrot,
and
onion, but affected tissue became watery and no slimy masses
were
formed
unlike
the
'sticky
rot'
syndrome
reported
previously on potato.
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114
INTRODUCTION
Sweetpotato,
encounter
Ipomoea
serious
economic
batatas
(L.)
losses
when
Lam., growers
excessive
may
rainfall
results in water-saturated soils for several days prior to
harvest. A yield loss of 82.5% of
'Centennial' was observed
after storage roots were submerged under water for 72 hr (22).
Typically, flood-damaged storage roots are totally decayed in
the ground or exhibit shortened storage life due to excessive
weight
loss
Genetic
and
rotting
variation
in
during
tolerance
curing
of
and
storage
sweetpotatoes
to
(13).
flood
damage was demonstrated, but this variation is inconsistent
from year to year (27).
The internal atmosphere of harvested sweetpotato roots
submerged in water was found to be almost exclusively CO 2 72
hr after submergence
(l).
Ethanol also accumulated rapidly
with increasing time of soil saturation (8 ). This asphyxiation
leads to a condition known as "souring", a disorder previously
thought
to
be
exclusively
physiological
in
origin
(6 ).
Actually, souring is a complex of symptoms of which the most
obvious are rapid maceration of tissues of the whole storage
root
(Fig.
1)
and
a
strong
smell
of
byproducts
of
fermentation.
Although many bacteria possess the ability to produce
tissue-macerating enzymes, only a few have been associated
with decay of living plant tissues.
spp.,
Bacillus
subtilis.
B.
These
meaaterium.
include Erv/inia
B.
polvmyxa.
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115
Pseudomonas marginalis. and pectolytic strains of Xanthomonas
camoestris. Pseudomonas. Clostridium spp., and Flavobacterium
spp.
(14, 21). Conclusive
data
that
certain
pectolytic
Clostridia can cause decay in tubers in the absence of Erwinia
were presented (19) , and an association between spore-forming
anaerobic bacteria and rotting in potatoes , Solanum tuberosum
L . , has been observed by several researchers (3, 16, 20, 26).
Clostridial rots are strikingly different from those caused by
E. carotovora and produce considerable slime with many gas
bubbles and a distinctive odor
(23).
Pectolytic clostridia
also have been implicated in 'wetwood', conditions in trees
(17,
24,
25,
28).
However,
nothing
has
been
reported
on
involvement of pectolytic clostridia in sweetpotato storage
root rots. Moreover, they were not detected on sweetpotato in
a survey for Clostridium in various crops in Japan (26).
The
objective
of
this
study
was
to
determine
if
pectolytic clostridia or other bacteria like E. chrvsanthemi
are
involved
in
souring
of
sweetpotato
storage
roots.
A
preliminary report of this research has been published (1 0 ).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
116
MATERIALS AND METHODS
Effect of surface disinfestation. One hundred and twenty
Beauregard sweetpotato storage roots were divided into three
groups. The first group received no treatment (control), the
second was dipped in a solution of 1% NaOCl for 10 min and
washed in tap water (surface disinfested), and the third was
dipped in a suspension (ODg2o = 0.15) of Erwinia chrvsanthemi
isolate Ech-2. Twenty roots of each group were punctured ten
times at different sites with a flamed needle. Then all roots
were
transferred
distilled water,
to
plastic
tied,
bags
(2/bag),
and incubated
flooded
for 5 days
with
in larger
plastic bags at about 25 C. This experiment was performed
twice.
Isolations.
removing
a
small
Pectolytic
bacteria
portion
tissue
of
were
at
extracted
the
margin
of
by
the
lesion, and suspending it in sterile distilled water (SDW).
Gram
negative
suspension
on
incubating
the
bacteria
crystal
plates
were
isolated
violet
pectate
for
48
hr
at
28
by
streaking
(CVP)
C
plates
(3).
the
and
Pectolytic
Clostridia were isolated by streaking the suspension on plates
of
nutrient-dextrose
hydrochloride
(NDA)
agar
amended
with
0.5%
that had previously been
cysteine
stored under
anaerobic conditions in order to be reduced (24 h r ) . After the
plates were streaked,
they were inverted and chloroform was
placed in the lid so that the vapors would eliminate nonsporeforming
bacteria.
The
NDA
plates
were
incubated
in
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
a
117
disposable environmental chamber for 48-72 hr at 32 C. The
environmental chamber is a plastic bag that contains a gas
generator/resazurin
oxygen
indicator,
consisting
of
two
tablets of potassium borohydride and sodium bicarbonate and an
ampule
of
hydrochloric
acid
(2.7
N ) , two
catalyst
cups
containing palladium catalyst, and an indicator containing an
ampule
of
resazurin
(Bio-Bag
Dickinson Microbiology Systems)
were
incubated
under
aerobic
Type
A,
Multi-Plate,
Becton
(2). Streaked NDA plates also
conditions
to
check
for the
presence of Bacillus.
Sets of 10 storage roots of Beauregard and Centennial
sweetpotato harvested in Chase, LA, and Jewel and Porto Rico
harvested in Baton Rouge, LA, were assessed for the presence
of pectolytic clostridia and E. chrvsanthemi. All roots were
punctured 10 times at different sites with a flamed needle,
transferred to plastic bags
(1 root/bag),
and flooded with
sterile distilled water. Plastic bags were tied and incubated
for 5 days in larger black plastic bags at 25 ± 2 C. The
isolation procedure is described above.
Characterization of isolates. Besides anaerobic growth,
pectolytic
clostridia
were
identified
on
the
basis
of
characteristic colony morphology and color on NDA and potato
infusion
agar
(PIA)
(16,
18),
morphology
of cells
from a
suspension of macerated tissues and in Gram stain of 10 to 16hr-old cultures, motility,
catalase test after exposing the
colonies to air for 1 hr,
and pathogenicity tests
(3,
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4).
118
Sulfate reduction was tested using a lactate medium
(yeast
extract, 1 g; sodium lactate, 4 g; NH 4CI, 0.5 g; K 2HPO 4 , 1 g;
MgS 0 4 .7 H 2 0 , 0.2
g;
CaCl 2 .2 H 2 0 ,
g;
0.1
FeS0 4 .7 H 2 0 ,
Na 2S0 4 , 0.5 g / L) in screw-capped tubes
tested
by
cysteine
inoculating
nutrient
hydrochloride
migration
and
broth
0.4%
g;
(11). Motility was
amended
agar
0.1
and
with
0.5%
observing
any
(11). Gelatin liquefaction was determined by spot
inoculation of nutrient agar amended with 0.4% gelatin. Plates
were incubated anaerobically for 3 days at 28 C and flooded
with
5 ml
of
acid mercuric
chloride
solution
(11).
Pure
cultures of clostridia were stored in SDW in screw cap tubes
(13x100 mm) at room temperature.
Pectolytic activity of ClAl and ClBl was estimated by
measuring the diameter of pits formed on double-layer pectate
medium
(DLPM)
(15) at 16,
20,
23, 28, and 37 C. Maceration
activity of ClAl and ClBl isolates was compared by weighing
the
nonmacerated
tissue
of
cylinders
of
Beauregard,
Porto
Rico, and Jewel sweetpotatoes, potato, and carrot 48 hr after
inoculation.
Inoculation
methods.
Storage
disinfested with 1% NaOCl
borer,
cylinders of raw
aseptically
(Fig.
2),
for 10 min.
storage
and
roots
were
peeled
Using a flamed cork
root tissue were
transferred
and
to
screw
obtained
cap
tubes
(13x100 mm) containing à sterile solution of 0.05 % cysteine
hydrochloride. Tubes were inoculated with cells from 24-hr-old
colonies and incubated for 72 hr at 32 C.
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119
Whole
storage
roots
disinfested as described
of
Beauregard
above,
sweetpotato
were
dipped in a suspension of
pectolytic Clostridium, punctured with a flamed needle, and
kept in distilled water in plastic bags at 25 C for 5 days.
Control storage roots received the same treatment but were not
dipped in the bacterial suspension.
Sterile
toothpicks were
colonies grown
touched
on NGA and inserted
to
typical
24-hr-old
into disinfested whole
storage roots to a depth of about 2.5 cm. The toothpicks were
left in position and storage roots were wrapped in a wet paper
towel and plastic wrap and incubated at 25 C for 5 days inside
clear plastic bags (Fig. 3). The same procedure was used to
inoculate potato tubers, carrot roots, and onion bulbs.
Storage roots also were sliced in half longitudinally and
inoculated by streaking cells of 24-hr-old colonies grown on
NGA onto the center of one of the slices. Afterwards, the two
slices were joined together, wrapped with wet paper towel and
clear plastic, and incubated at 25 C for 5 days.
Interaction.
The
reaction
of
whole
storage
roots
to
inoculation with cell suspensions of E. chrvsanthemi and/or
pectolytic clostridium (ClBl) was recorded. Micropipette tips
containing 50 ^1 of inoculum were inserted into Jewel storage
roots and
roots
were
left in position.
placed
Following inoculation,
in plastic
vegetable
baskets
storage
that
were
stacked and covered with a black polyethylene bag (6 ) . Number
of decayed roots was recorded 4 and 10 days after inoculation
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
120
in the first and second trials, respectively.
trial,
extension
and
color
of
rotted
In the second
tissues
were
recorded.
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also
121
RESULTS
Effect Of surface disinfestation. When maintained under
water
for
5 days,
5 and
10% of nondisinfested,
uninjured
storage roots rotted in tests 1 and 2, respectively.
injured,
85 and 100% of storage roots rotted.
All
When
injured
storage roots inoculated with E. chrvsanthemi rotted in both
trials while 90 and 30% of inoculated roots rotted even when
uninjured
in
the
first
and
second
tests,
respectively.
Uninjured,
disinfested storage roots did not rot when kept
under water for 5 days. When surface disinfested storage roots
were
wounded,
15%
and
none
rotted
in
tests
1
and
2,
respectively (Fig. 4).
Isolation. Pectolytic, spore-forming, sulfate-reduction
negative,
gelatin
forming white
hydrolysis
positive,
anaerobic
colonies on NGA and PIA media
(Fig.
bacteria
5)
and
resembling Clostridium spp. in cell morphology (Fig. 6 ) were
isolated from all samples of souring-affected tissues.
Two
isolates, ClAl and ClBl, were chosen and used throughout this
investigation. ClAl formed small, round isolated colonies on
PIA medium while ClBl formed large, thin spreading colonies.
Both ClAl and ClBl formed nonspreading colonies on NGA.
Souring symptoms developed in 7, 4, 6 , and 9 out of 10
storage roots of Beauregard and Centennial from Chase, LA, and
Jewel
and
Porto
respectively,
Rico
when
sweetpotatoes
injured
and
from
Baton
maintained
Rouge,
LA,
underwater.
Pectolytic bacteria with the same characteristics described
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122
above
were
isolated
from
all
souring
tissues
but
no
E.
chrvsanthemi was detected.
When cylinders of aseptically sampled, raw storage root
tissue were
inoculated with
(2.1-2.5x4.9x5 .6 f m )
ClAl,
swollen bacterial
cells
with terminal oval spores (2.0 /xm) were
consistently observed by microscopic examination
(Fig.
6 ).
Swollen cells were not found on solid media, only rod-shaped
cells. Swollen cells also were found in tissue from the edge
of decay in inoculated storage roots.
In some cases, despite
disinfestation with NaOCl, rots developed in storage roots and
a great number of swollen bacterial cells also were detected
in the tissues along with rod-shaped cells.
Inoculations.
ClAl induced rotting in tissue cylinders
kept in SDW in tightly capped tubes
(Fig.
7),
in punctured
storage roots submerged in distilled water in closed plastic
bags, and in inoculated storage roots wrapped with wet paper
towel and plastic wrap.
Pectolytic Activity.
On DLPM,
pectolytic activity was
greater at 28 (pit diameter = 2 . 9 mm) and 37 C (3.9 mm) than
at 20 (0.8 mm) or 24 C (1.3 mm). Colonies were visible at 16
C but no craters were formed.
Isolate ClBl
showed greater
maceration activity on Beauregard sweetpotato,
potato,
and
carrot than ClAl (Fig. 8 ).
Host Range. Sweetpotato isolates ClAl and ClBl macerated
sweetpotato (Fig. 9), potato (Fig. 10), carrot (Fig. 11), and
onion
(Fig.
12) when inoculated using toothpicks.
Symptoms
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123
were distinct from the sticky rot symptom reported on potato
previously (3), as no slimy mass was formed at any time.
Interaction.
storage
roots
inoculated
chrvsanthemi and pectolytic clostridium
aerobic conditions,
13).
E.
combined,
in
showed significantly greater number of
decayed roots than with E.
(Fig.
(ClBl)
with
chrvsanthemi or ClBl separately
This difference was not detected in the second
trial (Fig. 13), however, the lesion dimension (33 mm) induced
by
the
combined
inoculation
was
30%
larger
than
by
E.
chrvsanthemi alone (23 m m ) . In addition, the dark zone which
commonly
develops
at
the
border
of
the
lesion,
that
is
associated with the restriction of lesion development in some
genotypes
(7),
was
not
formed
when
both
bacteria
were
COinoculated.
Inoculation of ClBl alone produced no or very
small lesions
(1-2 mm) in both trials.
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124
DISCUSSION
This study provides the first evidence to support the
hypothesis that pectolytic anaerobes are associated with the
disorder of sweetpotato known as souring.
association
of
pectolytic
clostridia
Evidence for the
with
souring
of
sweetpotato storage roots includes; surface disinfestation of
storage roots eliminated or reduced consistently the incidence
of
souring;
chrvsanthemi
Koch's
postulates were partly
was
isolated
not
from
fulfilled;
controls;
and
E.
similar
pectolytic clostridia were isolated from storage roots of two
cultivars
from
different
locations.
In
addition,
only
pectolytic Clostridia were isolated consistently from souring
tissues.
The first requirement of Koch's postulate was met only
partially
isolate
because
there
pectolytic
naturally
was
no
opportunity
to
clostridia from storage
occurring souring
in
the
field.
attempt
roots
to
with
However,
the
isolation of similar bacteria from souring tissues of storage
roots
of
different
cultivars from different
locations
indicated that these bacteria are associated with sweetpotato
storage roots. Whether or not pectolytic clostridia are the
primary or sole cause of souring of sweetpotato,
it seems
clear that they are responsible for some of the characteristic
features of the disorder.
The
hypothesis
that
E.
chrvsanthemi
is
involved
in
souring of sweetpotato was not supported. When artificially
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125
inoculated in disinfested storage roots, E. chrvsanthemi was
recovered
from
the
margins
of
the
lesion
and
no
other
pectolytic bacteria were found. However, when non-disinfested
storage roots were stabbed with a flamed needle,
and kept
under water in a plastic bag, no Erwinia sp. were detected but
pectolytic
clostridia
were
consistently
recovered.
These
results are similar to those found with Irish potato where
pectolytic clostridia are capable of initiating soft rots in
tubers incubated anaerobically in the absence of detectable
soft-rot Erwinia (20). Symptoms caused by E. chrvsanthemi are
different from those caused by clostridia. Maceration and gas
production seem to be greater with clostridia. Comparisons of
kinds
of
pectolytic
enzymes,
speed
of
maceration,
and
particularly composition of gases emanating from macerated
tissues
should
be
conducted
with
E.
chrvsanthemi
and
pectolytic clostridia.
Combined inoculation of E. chrvsanthemi and pectolytic
Clostridium
(ClBl)
under
aerobic
conditions
resulted
in
increased numbers of rotted storage roots and larger lesions.
Thus,
the
possibility
of
an
interaction
between
E.
chrvsanthemi and clostridia deserves further investigation.
In order for Clostridium spp. to play a primary role in
attacking plant tissue, they probably require the ability to
degrade
pectic
fulfilled
this
substances
(17).
requirement by
The
showing
sweetpotato
isolates
a strong pectolytic
activity and macerating not only sweetpotato but also carrot.
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126
potato, and onion. Although pectolytic enzyme production may
be important in pathogenesis, it also has potential use by the
sweetpotato industry. Chopped carrots, potatoes, celery, and
many fruits are readily reduced to loose aggregates of cells
by pectolytic enzymes and used for the manufacture of their
concentrates
develop
(5).
Likewise,
interest
pectolytic
in
enzymes
the
utilizing
in
a
sweetpotato
these
process
industry
bacteria
that
or
requires
may
their
tissue
maceration.
The sweetpotato producing areas of Louisiana receive an
average annual rainfall of 130 to 145 cm. Evenly distributed
throughout
the year,
this
rainfall
would provide
adequate
moisture for most crops grown in the state but distribution
is
often
very
erratic
(12).
In
1990,
for
instance,
the
rainfall was zero in August and 890 mm in September, a harvest
month,
at
anaerobiosis
areas.
Burden
Plantation,
is unlikely to be
Baton
Rouge.
restricted
only to
However,
flooded
Certain locations or even microsites in soil in the
field can develop anaerobiosis (9).
No attempts were made in this study to characterize the
species of pectolytic bacteria isolated from sweetpotatoes.
However,
further
evidence
regarding
the
importance
of
pectolytic clostridia might be gained by characterization of
strains
and assessment of their occurrence
field and storage.
in rots
in the
Investigations have been carried out on
effects of wet soil on fleshy roots (13, 27) , changes in the
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127
internal gas concentrations
(l),
tolerance of cultivars to
flooding damage (27), but microbiology has been ignored.
•Souring' previously has been regarded as a physiological
disorder. The involvement of clostridia does not discount the
importance of the host and/or environment but adds another
biotic component which may help to further understand flooding
damage or related stresses in storage roots.
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128
Fig. 5.1. Souring symptoms. Sweetpotato storage root cut open
to show internal maceration that is characteristic of souring.
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129
Fig. 5.2. Preparation of cylinders of raw storage root tissue.
The storage root was peeled and disinfested with 1% NaOCl for
10 min. Using a flamed cork borer, cylinders of raw storage
root tissue were obtained aseptically.
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130
Fig. 5.3. Anaerobiosis using wet paper towel and plastic wrap.
Potato
tubers
inoculated
Clostridium using
with
an
isolate
infested toothpicks.
of
pectolytic
Tubers were wrapped
with wet paper towel and plastic wrap, and incubated inside a
plastic
bag.
Gas
formation,
indicated
by
bubbles
around
toothpicks, is a characteristic of souring.
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131
□ Non-Punctured
s Punctured
W
15
40 CO
Control
Ech
NaOCl
Treatment
□ Non-Punctured
Q Punctured
W
15
Test 2
P ) 10
0)
P)
40 (0
«
Control
Ech
NaOCl
Treatment
Fig. 5.4. Effect of disinfestation of storage roots on
incidence of souring. Number and percentage of rotted
sweetpotato storage roots with no treatment
(control),
inoculated with Erwinia chrvsanthemi (Ech ) , or disinfested
with NaOCl, and punctured or not with a flamed needle. Roots
were incubated individually for 5 days at 25 C submerged in
distilled water in plastic bags.
Reproduced with permission
of the copyright owner. Further reproduction prohibited without permission.
132
Fig.
5.5.
Clostridia,
Colonies
isolates
of
pectolytic
ClBl
(left)
clostridia.
and ClAl
Pectolytic
(right),
on PIA
medium after 48-hr anaerobic incubation at 32 C. ciBl forms
spreading colonies while C1B2 forms white colonies on this medium.
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133
Fig.
5.6.
Cell morphology of pectolytic bacteria.
bacterial cells
spores
were
(2.1-2.5 X 4.9-5 .6 f m )
found
when
either
Swollen
with terminal oval
cylinders
of
aseptically
sampled, raw storage root tissue or whole storage roots were
inoculated with pure cultures of pectolytic clostridia.
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134
Fig.
5.7.
Maceration
of
raw
tissue
cylinders.
Completely
macerated cylinders of raw storage root tissue three days
after
inoculation
with
pectolytic
clostridia
(3
tubes
left). Noninoculated tube is on the right.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
on
135
Maceration activity on different hosts
2 5 -,--------------------LSD = 0.09
P = 0.0001
C.V. = 18%
FF= 0.97
20.5
Control
-
Beauregard
Jewel
Porto Rico
Carrot
Potato
Host
Fig. 5.8. Maceration activity on different hosts. Maceration
activity of two Clostridium isolates
(ClAl and ClBl)
tissue
of
cylinders
cultivars,
potato
of
storage
tubers,
and
roots
carrot
three
roots,
on raw
sweetpotato
estimated
weighing the nonmacerated tissue 48 hr after inoculation.
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by
136
Clostr-iHi„j^ (C12A)
Fig.
5.9. Pathogenicity on sweetpotato. Sweetpotato storage
roots inoculated with toothpicks infested with an isolate of
pectolytic Clostridium (left) and noninoculated (right).
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137
Fig.
5.10.
inoculated
Pathogenicity on
with
toothpicks
potato
infested
tubers. Potato
with
an
tubers
isolate
pectolytic Clostridium (left) and noninoculated (right).
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of
138
Fig.
with
5.11.
Pathogenicity on carrot.
toothpicks
infested
with
an
Carrot root inoculated
isolate
of
pectolytic
Clostridium.
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139
Fig. 5.12. Pathogenicity on onion. Onion bulb inoculated with
toothpicks infested with an isolate of pectolytic Clostridium.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
140
□ Ech-2 + CIB1
D)
60
March
Time
Fig. 5.13. Combined inoculation with Erwinia chrvsanthemi and
pectolytic Clostridium. Percent storage roots of Beauregard
sweetpotato that rotted following inoculation with Erwinia
chrvsanthemi (Ech-2), Clostridium sp. (ClBl), or combination
(Ech-2+ClBl). Roots were incubated under aerobic conditions
for 4 days in March and 10 days in April at ± 25 C. Bars with
the same letter are not significantly different (Duncan's
multiple range, P = 0.0001).
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141
LITERATURE CITED
1.
Ahn, J. K . , Collins, W. W . , and Pharr, D. M. 1980. Gas
atmosphere
in
submerged
sweet
potato
roots.
HortScience. 15:795-796.
2.
Becton
Dickison
Microbiology
Systems.
4361216. Cockeyville, Maryland, 21030, USA.
3.
Campos,
E . , Maher,
E. A.,
and Kelman,
A.
1982.
Relationship of pectolytic clostridia and Erwinia
carotovora strains to decay of potato tubers in
storage. Plant Dis. 66:543-546.
4.
Cato, E. P., George, W. L . , and Finegold, S. M. 1986,
Genus Clostridium Prazmowski 1980. Pages 1141-1199 in:
Sergey's Manual of Systematic Bacteriology, Vol. II.
5.
Chesson, A. 1980. Maceration in relation to the post­
harvest handling and processing of plant material. J.
Appl. Bacteriol. 48:1-45.
6.
Clark, C. A., and Moyer, J. W. 1988. Compendium of
Sweet Potato Diseases. APS Press, St. Paul,MN. 74 pp.
7.
Clark, C. A., Wilder-Ayers, J. A., and Duarte, V.1989.
Resistance of sweet potato to bacterial root and stem
rot caused by Erwinia chrvsanthemi. Plant Dis. 73:984987.
8.
Corey, K. A., Collins, W. W . , and Pharr, D. M. 1982.
Effect of duration of soil saturation on ethanol
concentration and storage loss of sweet potato roots.
J. Amer. Soc. Hort. Sci. 107:195-198.
9.
Drew, M. C . , and Lynch, J. M. 1980. Soil anaerobiosis,
microorganisms,
and
root
function.
Annu.
Rev.
Phytopathol. 18:37-66.
10.
Duarte,
V.,
and
Clark,
C.
A.
1990.
Pectolytic
Clostridia associated with souring of sweetpotato.
(Abstr.) Phytopathology 80:1002.
11.
Gerhardt,
P. 1984. Manual of methods for general
bacteriology.
American
Society
for
Microbiology,
Washington, DC. 524 pp.
Cat.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
No.
142
12.
Hammet, H. L . , Constantin, R. J . , Jones,
L. G.,
Hernandez, T. P. 1982. The effect of phosphorus and
soil moisture levels on yield and processing quality of
'Centennial' sweet potatoes. J. Amer. Soc. Hort. Sci.
107:119-122.
13.
Kushman, L. J . , and Deonier, M. T. 1954. Effects of
weather, date of harvest, and curing treatments on
keeping qualities of Porto Rico sweet potatoes. Proc.
Amer. Soc. Hort. Sci. 71:369-375.
14.
Liao, C. H . , and Wells, J. M. 1987. Association of
pectolytic strains of Xanthomonas camoestris with soft
rots of fruits and vegetables at retail markets.
Phytopathology 77:418-422.
15.
Lund, B. M. 1969. Properties of some pectolytic,
yellow pigmented. Gram negative bacteria isolated from
fresh cauliflowers. J. Appl. Bacteriol. 32:60-67.
16.
Lund, B. M. 1972. Isolation of pectolytic clostridia
from potatoes. J. Appl. Bacteriol. 35:609-614.
17.
Lund, B. M. 1982. Clostridia and plant disease: New
pathogens?
Pages
263-283
in:
Phytopathogenic
Prokaryotes, Vol 1, Mount, M. S., and Lacy, G. S.,
eds. Academic Press, NY.
18.
Lund, B. M . , Brocklehurst, T. F . , and Wyatt, G. M.
1981.
Characterization of
strains
of Clostridium
puniceum
sp.
nov., a
pink-pigmented,
pectolytic
bacterium. J. Gen. Microbiol. 122:17-26.
19.
Lund,
B. M . , and Nicholls, J.
influencing the soft-rotting of
bacteria. Potato Res. 13:210-214.
20.
Pérombelon, M. C. M . , Gullings-Handley, J., and Kelman,
A. 1979. Population dynamics of Erwinia carotovora and
pectolytic Clostridium spp. in relation to decay of
potatoes. Phytopathology 69:167-173.
21.
Pérombelon, M. C. M . , and Kelman, A. 1980. Ecology of
the soft-rot erwinias. Annu. Rev. Phytopathol. 18:361387.
22.
Phills, B. R. 1972. Effect of ethephon, temperatures,
water-submerged roots and cultivars on sweetpotato
plant production. MS Thesis. La. State Univ., Baton
R ouge.
C. 1970.
Factors
potato tubers by
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
143
23.
Rudd-Jones,
D . , and Dowson,
W. J.
1950.
On the
bacteria responsible by Bacterium carotovorum (Jones)
Lechmann & Newmann. Ann. Appl. Biol. 37:563-569.
24.
Schink, B., Ward, J. C., and Zeikus,
J. G. 1981.
Microbiology
of
wetwood:
Importance
of
pectin
degradation and Clostridium species in living trees.
Appl. Environ. Microbiol. 42:526-532.
25.
Schink, B., Ward, J. C., and Zeikus,
J. G. 1981.
Microbiology of wetwood: Role of anaerobic bacterial
populations
in living trees.
J.
Gen.
Microbiol.
123:313-322.
26.
Suyama, K . , Negishi, H . , Tjahjono, B . , and Fujii, H.
1988.
Investigation
on
occurrence
of
pectolytic
Clostridia on various crops in Japan. (Abstr.) Int.
Congr. of Plant Path. 11:1-2.
27.
Ton, C. s., and Hernandez, T. P.
78. Wet soil stress
effects on sweet potatoes. J. Amer. Soc. Hort. Sci.
103:600-603.
28.
Ward, J. C . , Kuntz, J. E . , and McCoy, E. M. 1969.
Bacteria associated with "shake" in broadleaf trees.
(Abstr.) Phytopathology 59:1056.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
VITA
Valmir Duarte was borne in Porto Alegre, RS, Brazil on
October 12, 1953, and raised in Canoas, Brazil. He earned his
B.S.
and
M.Sc.
degrees
at
the
Agronomy
College,
University of Rio Grande do Sul, Porto Alegre,
Federal
in 1977 and
1981, respectively.
In March
began
working
1980,
as
before
finishing the M.Sc.
instructor
of
microbiology
course,
and
he
plant
pathology courses at the 'Pontiflcia Universidade Catolica',
Uruguaiana, RS, until December 1980, when he was hired by the
Agronomy College of the Federal University of RS. Since then,
he has been teaching microbiology and plant pathology in the
Agronomy College. He was also instructor of a plant pathology
course in the Agronomy College of the Federal University of
Santa
Catarina,
Florianopolis,
SC,
during
one
semester
in
1982. In December 1983, he was elected chairman of Fitotecny
Department, holding the position for two years. Currently, he
is maintaning the rank of associate professor in Brazil and
receiving a Brazilian scholarship from the Council of National
Scientific and Technological Development (CNPq).
Valmir Duarte has been pursuing a Ph.D.
degree in the
Department of Plant Pathology and Crop Physiology, LSU, with
a minor in microbiology, since the summer, 1987. He has been
working under the direction of Dr. C. A. Clark.
144
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
D O C TO R AL E X A M IN A T IO N AND DISSERTATION REPORT
Candidate;
V alm ir Duarte
Major Field; P la n t H e a lth
Title of Dissertation;
Ecology o f E rw in ia chrysanthem i, causal
agent o f b a c t e r ia l stem and ro o t r o t
on sweetpotato and e tio lo g y o f souring
Approved;
MMajor
ajor Profess
Professor and Chairman
Dean of the Graduate School
EXAMINING COMMITTEE.
Û/mW
0 ' ^ ' ^ ----------------
Date of Examination:
November 16, 1990
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