Persistence of the bacterium Erwinia carnegieana in soil and its relationship to the establishment and survival of saguaro (Carnegiea gigantea) cacti
Item Type text; Thesis-Reproduction (electronic)
Authors Takacs, Donald James, 1941-
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/554046 PERSISTENCE OF THE BACTERIUM ERWINIA CARNEGIEANA IN SOIL AND i ITS RELATIONSHIP TO THE ESTABLISHMENT AND SURVIVAL OF
SAGUARO (CARNEGIEA GIGANTEA) CACTI
by Donald James Takacs
A Thesis Submitted to the Faculty of the DEPARTMENT OF PLANT PATHOLOGY
In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 6 7 STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfill ment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowl edgment of source is made . Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the inter ests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED:
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
>> /96C, STANLEY M. ALCORN Date Professor of Plant Pathology ACKNOWLEDGMENTS
The author wishes to acknowledge his gratitude to Dr. Stanley M . Alcorn, for his advice, guidance, and informative discussions during the course of this study, and to Dr. Alice Boyle and Dr. Thomas H . McIntosh for their assistance, advice, and comments in planning and conducting the investigations reported herein and in reviewing this manuscript.
The writer also wishes to express his gratitude to the Agricultural Research Service of the United States
Department of Agriculture for the financial support which helped to make this study possible.
iii TABLE OF CONTENTS
...... Page LIST OF TABLES ...... vi
LIST OF ILLUSTRATIONS ...... ix
ABSTRACT...... x
INTRODUCTION AND LITERATURE REVIEW ...... 1
GENERAL MATERIALS AND METHODS ...... 4
PROCEDURES AND RESULTS ...... 8
A. Determination of Bacterial Cell , Concentrations In Water Suspensions . . . 8 B . Gram Stains of Erwinia carnegieana ..... 10 1. R e s u l t s ...... 12 C • The Use of Antibiotics to Selectively Isolate Erwinia species ...... 12 1. Antibiotics Dissolved in Water ..... 13 2 • Assay Disc T e s t s ...... l4 3• Effect of Autoclaving on Antibiotic A c tivity ...... l6 4. Antibiotic Effect on Filamentous G r o w t h ...... 19 5 • R e s u l t s ...... 19 D. Inoculum Potential Studies ...... 21 1. Root Dip Inoculation...... 21 2. Soil D r e n c h ...... 22 3 • Needle Inoculation...... 24 a. Experiment 1 ...... 24 b . Experiment 2 ...... 24 c. Experiment 3 ...... 26 d • Experiment 4 ...... 26 e. Experiment 5 ...... 28 4. R e s u l t s ...... 28 a. Root Inoculation...... 28 b . Needle Inoculation ...... 28 5• Relationship of Temperature to Inoculum Pot en t i a l ...... 30 a. Experiment 1 . 30 b. Experiment 2 ...... 32 c . Experiment 3 ...... 32 6 • Results ...... 35
iv V
TABLE OF CONTENTS— Continued
Page E . The Survival of JB. carnegieana in Sterile Soil Subjected to Various Temperatures and Two Moisture Conditions ...... 40 1 • Results ...... 43 F. Effects of E. carnegieana on Newly Germinated Saguaro Seedlings 46 1 . Needle Inoculation .••••••••• • * 46 2• Germination of Saguaro Seeds on EX carnegieana-Infested Soil ...... 48 3 • Results ...... 49 G. Effects of EX carnegieana Suspended in Dust and Sterile Water on Saguaro Blossoms and Wounded Saguaro Seedlings . . . . . 51 1. Infestation of Saguaro Blossoms .... 52 2 . Isolation From Blossoms • • 54 3• Infestation of Wounded Saguaro S e e d l i n g s ...... 54 a. Experiment 1 ••••••••••• • 54 b . Experiment 2 ...... 56 4. Results ...... 58 H . Isolation of EX carnegieana From Naturally Infested Soils . 60 1. Soil Collections ...... 60 2• Bacterial Isolations • • ...... 6l a. Results • . • • ...... 63 DISCUSSION...... 6?
LITERATURE CITED ...... 8l LIST OF TABLES
Table Page
1. Per cent transmittance of serial dilutions of 3 E.» carnegieana isolates suspended in sterile distilled water ...... 9
2. Survival of Erwinia carnegieana and 2 fungi in an aqueous solution containing actidione, griseofulvin, and erythromycin . . 15
3 * Disc assay determinations of the inhibitory effects of erythromycin, actidione, and griseofulvin alone and in combination on the growth of certain fungi and bacteria . . 17
4. Effect of autoclaved and non-autoelaved antibiotic agar on 6 bacterial species and the germination of spores from 3 species of fungi ...... 18
5• Effect of filter-sterilized antibiotic agar on the mycelial growth from mass transfers of Alternaria, Helminthosporium, and Fusarium spp...... 20
6. Relationship of method of inoculation and inoculum concentration to the susceptibility of saguaro seedlings to Erwinia carnegieana . 23
7• Relationship of inoculum coneentration to the susceptibility of saguaro seedlings to E . carnegieana...... 25 8. Effects of temperature and/or suspending medium on the number of cells of 15. carnegieana required to cause soft rot in saguaro seedlings ...... 27 9• Effect of temperature and inoculum concentration on the susceptibility of saguaro seedlings to ID. carnegieana...... 29
vi vii
LIST OF TABLES— Continued
Page
10. Effect of pre- and post-inoculation temperatures on the susceptibility of saguaro seedlings to E . carnegieana. Readings were made 96 hr after inoculation . 33
1 1 . Effect of pre- and post-inoculation temperatures on the susceptibility of saguaro seedlings to 15. carnegieana. Readings were made 7 days after inoculation . 34
12. Comparative effects of 23-25 C and 55 C on saguaro seedlings inoculated with varying concentrations of JE. carnegieana ...... 36
13- Effect of duration of exposure to varying temperatures on the survival and pathogenicity of JE. carnegieana stored in broth culture ...... 42
Ik. Effects of duration of exposure to varying temperatures on the survival of 15. carnegieana in artificially infested sterile soil maintained at 2 moisture levels ...... Ilk
15- The effects of a concentrated 15. carnegieana suspension on wounded, newly germinated saguaro seedlings maintained at 23-25 C . . . 47 16. The effect of soil infested with varying cone ent rat ions of 15. carnegieana on the survival of newly germinated saguaro seedlings ...... 50
17. Effect of 15. carnegieana infested dust and water suspensions on saguaro blossoms in the greenhouse ...... 55 18. Effect of 15. carnegieana infested dust and water suspensions on wounded saguaro plants in the greenhouse (27 C ) ...... 57 19- Effect of £5. carnegieana infested dust and water suspensions and humidity on saguaro plants kept in the greenhouse for 11 days after inoculation ...... 59 viii LIST OF TABLES— Continued
Page
Distribution of soft rot and pectolytic bacteria in soils collected at various sites and depths at k time intervals . . . . 64 LIST OF ILLUSTRATIONS
Figure Page 1. Needle inoculation of a saguaro seedling . • 6
2. Concentration of 15. carnegieana cells in water suspensions as determined by per cent transmittance of light of a wavelength of 4l0 mu ...... 11
3• Saguaro seedlings showing "heat-kill" symptoms after 2k hr storage at 55 C . . . . 39
ix ABSTRACT
This study concerns the ability of Erwinia carnegieana to survive in soil and the effects of such infested soil on saguaro survival and repopulation.
Actidione and erythromycin, incorporated into agar
or liquid substrates, facilitated the isolation of Erwinia
species from soil and plant material.
The optimum temperature for the survival of J£.
carnegieana in air dry or water-saturated, sterilized soil was 25 C . However, the bacterium could briefly survive at
55-60 C in soil but not in broth culture. While the
inoculum potential of the pathogen increases with tempera-
tures to 50 C, 2 year old saguaro seedlings were heat- killed at 55 C.
Newly germinated seedlings rotted when needle
inoculated with JE. carnegieana and some plants became
diseased when germinated on soil infested with high concen
trations of the bacterium. However, wounds were necessary
for root infection to occur.
In a few instances wounded saguaro seedlings, but
not blossoms, were soft rotted when insufflated with
infested dust. Water suspensions of the bacterium infected
both blossoms and wounded seedlings.
x Erwinia carnegieana was isolated from naturally infested field soils collected at the base of a rotted saguaro and between healthy saguaros but not from soil located several miles from any saguaros. The number of pathogenic isolates obtained varied with soil depth and time of year. INTRODUCTION AND LITERATURE REVIEW
The tourist "industry" is one of Arizona’s largest and most important and the giant cactus, Carnegiea gigantea
Britt. & Rose, is assumed to be one of the major tourist attractions. Since the saguaro is indigenous to Arizona and parts of California only, it is of botanical value and well worth protecting.
In the early 19*101 s a soft rot disease of the giant cactus was described by Lightle et al. (30) who named the bacterial pathogen Erwinia carnegieana Standring. At this time it was observed that both old and young saguaros were being killed. Boyle (9)> and others, further noted that in some areas within the Saguaro National Monument, near
Tucson, there were very few small, young plants to replace those that were dying. These observations were verified by annual surveys of a stand of saguaros at Saguaro National
Monument between 19^2 and 1 9 6 1 . The compiled data show a rate of decline of the older plants caused by bacterial necrosis and also a lack of natural repopulation which, if continued, would cause the complete loss of this forest by approximately 1998 (6).
The larvae of the lepidopteran moth, Cactobrosis fernaldialis Hulst, direct contact between healthy and diseased roots or branches, and infection of healthy roots
1 2 exposed to IS. Carnegieana-Infested soil were suggested as the three major means of disease spread from plant to plant
(9).
Boyle (9 ) has postulated that the requisite seed crops may be reduced by blossom infection by IS. carnegieana or attack by Cactobrosis larvae. Further, the lack of young plants may be partially accounted for by the feeding of
desert rodents (3) which have been named as the probable principal hazard to small saguaros (35)• Small seedlings also require shade for survival and are attacked by insects
(3). Alcorn (4) suggested that IS. carnegieana-infested
soil may be a means of air-dispersal of the pathogen and may play a role in seedling infection. Conceivably,
infested soil could be blown into fresh wounds and an
infection initiated or into open blossoms which could then
serve as an inoculum source for further spread by pol
linators .
According to Menzies (34) no known plant pathogenic
bacteria are obligate parasites. This is supported by the
fact that these organisms can be grown on culture media.
However, Voronkevitch (5 0 ) reported that the soft rot
causing species of Erwinia are not capable of long survival
in soil and should not be considered semi-saprophytic.
Contrarily, other workers (34, 3 8 ) describe soft-rotting
Erwinia species as being well adapted to live in soil• 3 Further, Boyle (9) demonstrated that EX carne^ieana survived in both moist and dry naturally infested soil exposed to Tucson summer temperatures for as long as 46
days. This work did not include, however, isolations from
soils collected at various depths and in various seasons nor were there any investigations of the infectivity of
Erwinia-infested soil injected into saguaros.
The isolation of specific microorganisms is often made difficult by the growth of contaminants on isolation
plates. In the past several years the selective isolation
of certain types of microorganisms has been facilitated by
incorporating into the isolation medium antibiotics and/or
other chemicals which inhibit the growth of many undesirable
microorganisms (e.g., 7 , 1 3 , 1 5 , 1 9 , 2 1 , 2 2 , 3 1 , 3 2 , 3 6 ,
4l, 44, 4?, 49)• However, a review of the literature did
not indicate that such selective media were available to
aid in isolating Erwina species.
The work reported here relates to the development
of methods to facilitate the isolation of EX carnegieana
from soil, studies on the survival of the bacterium in soil,
and studies of the role that such soil may play in initi
ating infections in blossoms, seedlings, and mature saguaros. GENERAL MATERIALS AND METHODS
Potato dextrose peptone agar (PDP) has been found through experience to be very good for culturing Erwinia species and was thus used in the following experiments. To make 1 liter of PDP, ko g of potato dextrose agar (PDA) and
5 g of Bacto agar were brought to a boil in 500 ml of distilled water. Ten g of Bacto peptone dissolved in
500 ml of distilled water were added to the boiling agar, mixed well, and the pH adjusted to 7•6. The agar was then
autoclaved for 20 min at 250 C and poured after cooling to
about 45 C.
Potato dextrose agar (PDA) was employed in the
culture of fungi. Bacterial plates and slant cultures were
incubated at 52 C for 18-24 hr and fungal cultures at 25 C
for 48-96 hr.
Bacterial suspensions were prepared by aseptically
transferring the growth from one 24 hr slant culture with a
cooled flamed loop to a tube containing 9 ml of sterile
distilled water (SDW). The tube was then vibrated for 1
min on a "Vortex" type test tube mixer to make a homoge
neous , "milky" suspension. Such suspensions, termed "very
concentrated," were used to prepare dilution series by
pipetting 1 ml of the concentrated suspension into 9 ml of
SDW, mixing, and continuing this process through a series
4 5 of dilutions. Thus, a given tube contained 10 ^ as many cells as the previous tube in the series. Sterile, disposable 2.5 ml plastic syringes with 23 gage needles were used to inject the various inocula into tomato fruits and cacti (Fig. 1).
The saguaro seedlings used in this work were 2 to 3 years old and ranged from 2.5 to 7 •5 cm in height.
Fungal spore suspensions were prepared by adding
9 ml of SOW to 4 day old PDA slant cultures of the fungus.
The tubes were then shaken for approximately 1 min on the test tube mixer. A drop of the resulting suspension in
each tube was subsequently examined under the compound microscope to verify the presence of spores.
Erwinia carnegieana isolates were from stock
cultures maintained by the Department of Plant Pathology.
Isolates 1-12, 1 0 6 , 67-3, and 65-46 had been obtained from
diseased saguaros and 62-Op-4l-2 from a diseased prickly
pear cactus, Opuntia engelmanii Salm Dyck. Cultures of
Escherichia coli Castellani and Chalmers and Staphylococcus
aureus Rosenbach were acquired from the Department of
Microbiology. The late Dr. Bernard Friedman, Market
Pathology Laboratory of the U.S.D.A. in New York City,
supplied Erwinia carotovora (Jones) Holland, while 12.
atroseptica (van Hall) Jennison and 12. aroideae (Townsend)
Holland were obtained from Dr. Robert Dickey, Department of
Plant Pathology, Cornell University. Fig. 1--Needle inoculation of a saguaro seedling. 7 Cultures of an A11 ernaria sp . and a Pend.ciIlium sp • were isolated from contaminated agar plates while a
Helminthosporium sp. and Fusarium oxysporum f. vasinfectum
Snyder and Hansen were obtained from the Plant Pathology Department stock collection.
When attempts were made to isolate EX carnegieana from a given source, PDP plates streaked with EX carnegieana isolate 1-12 and incubated for 24 hr were used to compare colony morphology. Under these conditions, the colonies are entire, smooth, convex, opaquish, and opalescent. PROCEDURES AND RESULTS
A . Determination of Bacterial Cell Concentrations In Water Suspensions
Sterile distilled water suspensions of IS. carnegieana isolates 1 0 6 , 62-0p-4l-2, and 6 7 -3 were made and each of the 3 suspensions was serially diluted 1:10 eight times. Samples from each dilution tube were read on the "Spectronic 20" colorimeter set at a wavelength of
4l0 mu (this wavelength was found to be most completely transmitted through SDW) and the per cent transmittance recorded. Sterile distilled water was used as a blank.
After the spectrophotometrie readings eight 1 ml aliquots were taken from each dilution tube and added to separate PDF plates. The plates were then swirled to distribute the inoculum evenly over the agar surface, inverted, and incubated. The suspensions were plated out within 30 min after the colorimeter readings were taken.
After incubation the colonies on each plate were
counted (only when less than 5 0 0 ) on a bacterial counter
and averaged. The approximate concentration of bacterial cells at the various dilutions were then calculated
(Table 1). A graph was made by plotting the per cent
transmittance of a dilution against the calculated con
centration of bacterial cells in 1 ml of that dilution
8 Table 1. Per cent transmittance of serial dilutions of 3 E,• carne.gieana isolates suspended in sterile distilled water.
Cell counts Colorimeter readings E. carnegieana Dilution No. colonies3 % Transmittance isolate factor (cells/ml) Dilution factor at 4l0 mu
62 -0p-4l-2 io:| T None 13-5 10 ° T 5 x 10"} 30.0 IQ-? 161 + 14 10-1 75-0 15 + 6 86.0 2 +_ 0 5 x iS:* 9 6 .0 106 T None 1 9.0 T 5 x 10-1 22.0 149 + 24 io-i 68.0 l ! 13 + 1 8 2 .0 10 ^ l + l 5 x i°:2 9 6 .0 6 7 -3 io:| T None 1 1 3 .0 10 7 T 5 x 10~{ 2 9 .0 io~l 68 + io 10-1 74.0 7 + 3 5 X i o , 84.0 iS=5 1 ± l io"2 9 6 .0 Avg. c ell/ml at 10-7 b Avg. % Trans dilution = 126 + l6 Calculated celIs/ml^ mittance : No dilution=l.2 x 1 0 q 1 1 .8 + 1 .9 5 x 10-1 =6 .3 x 10% 2 7 .0 + 3-3 10~2 =1 .2 x 1 0 ° 7 2 .3 + 2 .9 5 x 10 p =6 .3 X 10% 84.0 + 1 .3 10 =1 .2 x 10 9 6 .0 + 0 .0 kAverage number of colonies on 8 plates; T = too many colonies to read. Calculations based on results of plate counts of aliquots of the 10~7 dilution. 10
(Fig, 2). This procedure made it possible to prepare suspensions of approximate known concentrations of EX carnegieana for other experiments.
B . Gram Stains of Erwinia carnegieana
The bacterium EX carnegieana has been reported to be gram-positive (8, 9? 3 0 ) and gram-negative (11, l4).
The following experiments were conducted to determine if
EX carnegieana changes its gram-stain reaction after several broth transfers or passages through saguaros.
A broth culture of EX carnegieana isolate 1-12 was prepared from a stock culture 3 months after it had been lyophilized• After l8 hr of incubation the gram reaction was determined by the method of Pavlovich and Yall (39)• A second broth tube was then inoculated by transferring a loopful of the first broth culture to the second tube•
After the second culture had incubated, a third culture was begun and a gram stain made of the second. This procedure was carried out for 10 consecutive transfers. On each gram
stain slide EX coli was used as a gram-negative check and
S. aureus as a gram-positive check•
Three saguaro seedlings were each injected with
0.5 ml of a concentrated suspension of EX carnegieana
isolate 1-12 obtained from the first broth culture. The
following day, the bacterium was isolated from the rotting
plants, increased on PDF slants, gram stained, and injected i. —Cnetain f1. angen cls nwtr upnin a dtrie by determined as suspensions water 12. in of cells carnogieana Concentration 2— Fig. iuin lts - H - plates. dilution t mxmm n iiu ersns h aeaenme o oois n 24 on colonies of with number point Each average the represents minimum 4l0mu. and of maximum itsawavelength of light of transmittance cent per Percent transmittance at 410m p x0 0 x0 0 1 10' 5 x10 10 5x10 10 5x10 el o E creiaa e m o suspension of ml per carnegieana E. of Cells 5 xIO h 12 into 3 new plants. This procedure was repeated 3 times.
Gram-positive and gram-negative checks were again used on the gram stain slides.
1. Results Whether repeatedly transferred in broth culture or passed through saguaro seedlings, the cactus bacterium was gram-negative. In all cases the ID. coli and Sy aureus were gram-negative and gram-positive respectively.
C • The Use of Antibiotics to Selectively Isolate Erwinia species
In an attempt to formulate a method to selectively isolate Erwinia species, the following tests were performed.
The 10 organisms used in these tests were ID. carnegieana (isolates 67-3, 106, 62-Op-4l-2, and 1-12), ID. aroideae, ID. atroseptica, ID. carotovora, Sy aureus, ID. coli, Helminthosporium sp., Alternaria sp., Penicillium sp., and Fusarium oxysporum f . vasinfectum. Various isolates of the 4 Erwinia species were injected into
saguaro cactus seedlings and shown to be pathogenic before being used in these experiments.
The antibiotics and their concentrations used were
actidione (Upjohn Co.)--700 ppm, griseofulvin (Ayerst
Laboratories, Inc. ) — 500 ppm, and erythromycin (Eli Lilly
and Co . ) - - 5 0 0 ppm. The proper weights of these were first
dissolved in 20 ml of dimethylformamide (DMF) for each 13 liter of solution to be prepared. In the following experi ments the antibiotics were tested separately and in various combinations with each other for their effects upon the test organisms• Sterile distilled water and 2% DMF solution were used as controls.
When distilled water was added to griseofulvin dissolved in DMF the solution turned milky but rapidly cleared again. However, several moments.later a white precipitate, thought to be griseofulvin, began to precipi tate out of solution. Since this was possibly due to a pH factor (the pH of the laboratory distilled water varied from time to time) an experiment was performed in which griseofulvin dissolved in DMF was added to volumes of distilled water adjusted to pH 4.8, 5.7, 6.0, 6.5) 7•0,
7*9) 9*0, and 11.5 respectively by the addition of 1.0 N NaOH or 0.1 N HCL. The final concentration of griseofulvin in each solution was 500 ppm. After 15 min each solution was inspected for the presence of precipitate. 1
1. Antibiotics Dissolved in Water
One liter volumes of antibiotic and control solu tions were prepared and filter sterilized using a pre autoclaved Kruger filter holder fitted with a 60mm, 0.1 micron filter. Sterile distilled water suspensions of the
JE. carnegieana isolates were prepared along with spore suspensions of the Alternaria and Fusarium species. The 4 bacterial suspensions were adjusted to 10 cells per ml.
For each organism, 3 tubes containing 9 ml of sterile antibiotic solution and 1 tube containing 9 ml of
2% DMF solution were inoculated with 1 ml of the spore or bacterial suspension and shaken for 30 sec. The tubes were then stored at room temperature (approximately 24 C).
Samples were taken from the tubes 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, and 24 hr after inoculation. A tube was shaken for a few seconds after which 0.5 nil was removed and placed on a PDF plate (in the case of 12. carnegieana) or a PDA plate (in the case of the fungi) resulting in 3 plates per exposure period per organism.
The plates were swirled and incubated. After incubation, the amount of growth on each plate was recorded (Table 2).
2. Assay Disc Tests
Assay discs (Schleicher and Schuell #740-E, 12.7 mm) were prepared by dipping individual discs into the appropriate antibiotic solution. Discs were then drained of excess fluid by touching them to the side of the flask.
Subsequently these were dried in partly covered Petri dishes for 24 hr at 32 C.
Appropriate agar plates were seeded with 1 ml of a
SDW suspension of either a bacterium or the spores of the fungus to be tested, after which the plates were swirled to Table 2. Survival of Erwinia carnegieana and 2 fungi in an aqueous solution containing actidione, griseofulvin, and erythromycin.
Length of exposure to chemical solution3
Test organism Replicate 15 min 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr
Fusarium sp. i + + b + + 0 0 0 0 2 + + + + + + 0 0 0 0 3 c > 0 0 0 0 0 0 0 Control + + + ++ + +++ + + + 4* 4* 4- +++ +++ ++ +
Alternaria sp• 1 4* + 4- 0 0 0 0 0 2 + *f* 4* 0 0 0 0 0 3 + + ++ 4- 0 0 0 0 0 Control + + + 4--M- 4 . 4 . 4 . + + + +++ +++ +++ +++
Erwinia carnegieana isolate 1-12 106 76-3 6 2 —Op —^tl —2 aChemical solution contained 700 ppm actidione, 500 ppm griseofulvin, and 500 ppm erythromycin dissolved in 20 ml of dimethyl formamide (DMF) and subsequently k diluted to 1 liter. 0 = no growth; + = sparse scattered growth; ++ = moderate growth; +++ = dense growth. jTwo per cent DMF solution used as control solution. Growth on all ID. carnegieana plates was too dense for colony counts to be made. i6 evenly distribute the inoculum. Two discs treated with a given antibiotic were placed 3*75-5*0 cm apart on each of 3 plates for each organism tested. The plates were inverted and those seeded with bacteria were incubated for 24 hr at 32 C while the fungal plates were incubated for 72 hr at
25 C. After incubation, the diameters of inhibition zones were recorded (Table 3)»
3. Effect of Autoclaving on Antibiotic Activity
Two separate groups of the antibiotics dissolved in
DMF were prepared. One group of solutions was filter- sterilized and aseptically added to the proper volumes of newly made autoclaved POP agar to give the desired concen trations of antibiotics.
Each of the antibiotic solutions in the second group was added to a flask of freshly made non-autoclaved
PDP, adjusted to the proper volume to give the desired concentrations. The flasks were then autoclaved for 20 min at 250 C.
Sterile distilled water suspensions were made for each test organism. Two plates each of autoclaved and non- autoclaved antibiotic agar were seeded with 1 ml of a bacterial or spore suspension. The plates were swirled, inverted, and incubated for 24 hr for bacteria and 48 hr for fungi. After incubation the amount of bacterial and fungal growth on each plate was recorded (Table 4). Table 3* Disc assay determinations of the inhibitory effects of erythromycin, actidione, and griseofulvin alone and in combination on the growth of certain fungi and bacteria.
Treatments3
Test organism E A E+A G E+G A+G E+A+G DMF SDW
Escherichia coli Nb N N N NN N N N
Staphylococcus aureus 1 6 +1 .3 N 1 8 .1+^.6 N 14.9+.6 N 2 9 .0+1 NN
Erwinia carotovora NNNNNN NNN
E. aroideae N N NN N N NNN
E . atroseptica NNNNN N NNN
E . carnegieana NNNN N N NNN
Penicillium sp. N 1.6+1.6 1.4+1.9 N N 1 . 8 ^ 2 .3 .9+1.5 NN
Helminthosporium sp. N 14.9+1.3 11.9+1.2 N N 16 .9+. 8 11.2+3.1 N N
Alternaria sp. N 12.1+1.5 11.4+1.5 N N 10 . 8_+1 .6 11.1+1.7 N N Fusarium oxysporum f • vasinFectum N 8.6 + 1.2 8.4+2.5 N N 12 . 6 +1 .0 6.8+1.8 NN aE = 500 ppm erythromycin; A = 700 ppm actidione; G = $00 ppm griseofulvin; DMF = k 2% dimethyl formamide solution; SDW = sterile distilled water. Average diameter in mm of inhibition zones on each of 3 plates, 2 discs per plate ; N = no inhibition. 18
Table k . Effect of autoclaved and non-autoclaved antibiotic agar on 6 bacterial species and the germination of spores from 3 species of fungi.
Treatments* Autoclaved Non-autoclaved
Organism Plate EA G E+A E+G A+G EAG DMF SDW E A G E+A E+G A+G EAG DMF SDW
Staphylococcus aureus 1 oc + + + 4-4-4- 0 0 + + + 0 4-4-4- 4- + + 0 + + + +++ 0 0 + + + 0 + + + + + + 2 0 + + + + + + 0 0 + + + 0 + + + + 4 -4 - 0 + + + + + + 0 0 + + + 0 + + + + + + Escherichia coli 1 + + + + + + + + + 4-4-4- 4-4-4- + + + 4-4-4- +++ + + + +++ + + + +++ + + + +++ ++ + +++ + + + + + + 2 + + + + + + + + + + + + 4-4-4- + + + ++ + ++ + + + + + + + + + + +++ ++ + ++ + + + + + + + + + + + + + Erwinia carotovora 1 + + + + + + + + + + + + + + + + + + + + + 4-4-4- + + + ++ + + + + + + + + + + +++ + + + + + + + + + + + + 2 + + + 4-4-4- ++ + + + + + + + + + + 4-4-4- + + + + + + + + + + + + + + + + + + ,+++ +++ +++ + + + + + + E. aroideae 1 + + + + + + + + + + + + + + + 4-4-4- + + + + + + + + + + + + + + + +++ + + + + + + + + + + + + + + + + + + 2 + 4-4- + + + + + + + + + + + + + + + + + + + + + + + + +++ + + + ++ + + + + +++ +++ + + + + + + + + + E. atroseptica 1 4-4-4- + + + ++ + 4-4-4- + + + + + + 4- + + + + + + + + +++ + + + + + + +++ +++ + + + +++ + + + + + + 2 4-4-4- + + + 4-4-4- + + + + + + + + + + + + 4-4-4- + + + +++ + + + +++ +++ +++ +++ +++ + + + + + + E. carnegieana 1 4-4-4- 4-4-4- + + + + + + + + + + + + 4-4-4- + + + + + + +++ + + + +t+ +++ +++ +++ +++ + + + + + + 2 4-4-4- + + + + + + + + + + + + + + + + + + + + + + + + +++ + + + +++ + + + + + + + + + + + + + + + + + + Helminthosporium sp. 1 4-4-4- 0 + 0 4- 0 0 + + + + + + +++ 0 + 0 + 0 0 + + + + + + 2 4-4-4- 0 + 0 + 0 0 + + + + + + +++ 0 + 0 + 0 0 + + + + + + Alternaria sp. 1 4-4-4- 0 + + 0 + + 0 0 + + + + + + +++ 0 ++ 0 ++ 0 0 + + + + + + 2 4-4-4- 0 + + 0 + + 0 0 + + + + + + +++ 0 ++ 0 + + 0 0 + + + + + + Penicillium sp. 1 4-4-4- 0 + + + 0 + + + 0 0 + + + + + + +++ 0 +++ 0 + + + 0 0 + + + + + + 2 4-4-4- 0 + + + 0 4-4-4- 0 0 + + + + + + +++ 0 +++ 0 ++ + 0 0 + + + + + + Fusarium oxysporum f. 1 + + + 0 + + 0 + 0 0 + + + + + + +++ 0 + + 0 + 0 0 + + + + + + vasinf ectum 2 + + + 0 4-4- 0 + 0 0 4-4-4- + + + +++ 0 ++ 0 + 0 0 + + + + + + aE = 500 ppm erythromycin; A = 700 ppm actidione; G = 500 ppm griseofulvin; DMF = 2% dimethylformamide solution; k SDW = sterile distilled water. One ml of bacterial or fungal spore suspension was added to each pi ate • 0 = no growth; + = sparse growth; ++ = moderate growth over agar surface; +++ = dense growth. 19 4. Antibiotic Effect on Filamentous Growth Antibiotics (either singly or in combination) were filter-sterilized and added to flasks containing the proper volumes of freshly made PDA to give the desired concentra tions . From 5-day-old PDA slant cultures of Fusarium oxysporum f. vasinfecturn, Alternaria sp., and Helmintho- sporium sp. one-half cm squares of agar containing mycelial growth were cut. Three plates of each antibiotic agar were inoculated by placing an agar square from a slant culture in the center of the plate. This was repeated for each fungus. The plates were inverted and incubated for 5 days, after which the length of the mycelial growth radiating from the inoculum block on each plate was recorded (Table
5).
5. Results
The 3 antibiotics actidione, erythromycin, and griseofulvin had no marked inhibitory effect on any of the
Erwinia species or Escherichia coli. The fungi and gram positive Staphylococcus aureus were strongly inhibited by
actidione and erythromycin, respectively.
The effects of the antibiotics remained approxi mately the same whether they were in water solution, assay
discs, or incorporated into autoclaved or non-autoclaved
agar. Both spore germination and mycelial growth were
inhibited by actidione• Four hours were found to be the Table 5* Effect of filter-sterilized antibiotic agar on the mycelial growth from mass transfers of Alternaria, Helminthosporium, and Fusarium spp.
Treatments^
Fungus E A G E+A E+G A+G E+A+G DMF SDW
Alternaria sp. 7.5+-5b 1.5+-5 4.0+1.0 .5+. 5 0 0 0 7.51-5 1 9 .0 +1 .0
Helminthosporium sp. 6.51-5 0 6 .0 1 0 0 0 0 0 10.51-5 11-51-5 Fusarium oxvsporurn f . vasinfectum 1 6 .5 + .5 0 11.5+.- 5 0 0 0 0 1 5 -0+0 21.0+1.0 aE = 500 ppm erythromycin; A = 700 ppm actidione; G = $00 ppm griseofulvin; DMF = k 2% dimehtylformamide; SDW = sterile distilled water. Average diameter of growth in mm from 0.$ cm blocks of inoculum on each of 3 plates.
to O 21 minimum exposure period required for the inhibition of fungal spore germination in water solutions of the anti biotics (Table 2).
The griseofulvin showed a considerable amount of fungicidal activity either alone or when in combination with the other antibiotics but did not approach the almost total inhibition produced by actidione. Tests show that a precipitate (believed to be griseofulvin) formed when griseofulvin was added to distilled water adjusted to pH values ranging from 4.8 to 11.5* Griseofulvin therefore was deleted from the antibiotic solutions employed in the isolation of Erwinia in the remainder of the experiments.
D . Inoculum Potential Studies
The following experiments were performed to determine the number of cells of EX carnegieana required to cause bacterial necrosis in saguaro seedlings inoculated by various methods.
1. Root Dip Inoculation
Two hundred 2.5-3•5 cm high saguaro plants were left unplanted in the greenhouse (28 C) for 8 days to allow any injured roots to heal. One hundred of these were then wounded by cutting off the root system 2.5 cm from the
crown with a flamed pair of scissors. Ten injured and 10 non-injured seedlings were placed in each of 10 sterile
glass Petri dishes in such a way that the roots were in the 22 bottom of the dish while the above ground portion of the plants rested on the edge of the dish. The roots were not surface sterilized before treatment.
One hundred ml of SDW suspensions containing "very concentrated," 108 , 107 , 106 , 105 , l o\ 103 , 102 , and 101 cells/ml of a 24 hr culture of J£. carnegieana (isolate
1-12) were respectively added to each dish• Sterile distilled water was used as a check.
After 3 hr the saguaros were removed from the inoculum and planted in pots containing a chloropicrin- fumigated soil mixture composed of sand, silt, and peat moss (1:1:1). These pots were kept in the greenhouse and watered every 4-5 days. The number of rotted plants per treatment was recorded 24 days after inoculation (Table 6).
No isolations were made from the plants.
2. Soil Drench
. The roots of 100 saguaro seedlings were wounded (as in the root dip treatments) and planted 2 or 3 per pot in
5x6.5 cm in pots containing the same chloropicrin fumigated soil mixture. One hundred saguaros with non- wounded roots were similarly planted. The pots were separated into 10 groups, each of which included 10 non- wounded plants and 10 root-wounded plants.
Each member of the group was inoculated with one of the following concentrations of 12. carnegieana isolate 1-12 Table 6. Relationship of method of inoculation and inoculum concentration to the susceptibility of saguaro seedlings to Erwinia carnegieana.
Plants infected ,. b Soil drench3 Root dip
Injured0 Non-injured Injured0 Non-injured Inoculum Concentration roots roots roots roots (cells/ml) (%) (%) (%) (%) "very concentrated" 30d 0 0 0 108 20 0 20 0 107 10 0 10 0 106 10 0 10 0 105 20 0 0 0 10^ 0 0 0 0 103 0 0 0 0 102 10 0 0 0 101 0 0 0 0 0 (SDW) 0 0 0 0
^Twenty ml of inoculum added to each pot. Plant roots dipped into 100 ml of inoculum for 3 hr. ^Roots injured by cutting off lower portion with flamed scissors. Per cent of 10 treated plants which rotted; seedlings planted in 4 pots and stored in the greenhouse. Readings made 24 days after inoculation. Greenhouse temperature was approximately 2 7 C . 24
"very concentrated," 10®, 10^, 10®,106 , 103105 , 10^,lO^, 103, 102 , or 10^ cells/ml. Twenty ml of a given suspension were poured on the soil of each of the pots in a group. Check plants were treated with SDW. The plants were left in the green house and the number of rotted plants in each treatment recorded after 24 days (Table 6). No isolations were made.
3. Needle Inoculation
JE. carnegieana isolate 1-12 was injected into healthy saguaro seedlings and reisolated from the rotted plants 3 consecutive times in order to induce a highly virulent culture. Non-pretreated isolates 1 0 6 and 65-46 were also used in the injection experiments. a. Experiment 1
Sterile distilled water suspensions of a 24 hr culture of 1-12 were made up as, Mvery concentrated,11 10^,
107, and 10^ cells/ml. One-half ml of a given bacterial suspension or SDW was injected into each of 5, 5 cm high saguaro seedlings. The plants were left in the laboratory
(23-25 C ) and the results recorded after 7 days (Table 7)• No reisolations were made. b . Experiment 2
Separate SDW suspensions of isolates 106 and 1-12 were made up as, "very concentrated," 10^, 107 , 10^, and 5 10 cells/ml. Again, each of 5 young saguaros was injected Table 7- Relationship of inoculum concentration to the susceptibility of saguaro seedlings to E-• carnegieana.
Number of cells injecteda
"Very =; 4 E . carnegieana concentrated" 5 x 107 5 x 106 5 x 10^ 5 x 10 0 (SOW) Experiment isolate (%)b (%) (%) (°/o) (%) (%)
#1 1-12 100 100 20 0 0 0
#2 1-12 100 4o 20 0 0 0
1 0 6 100 8 0 20 0 0 0
^Seedlings were injected with 0.5 nil of the appropriate inoculum and then left at k laboratory temperature (23-25 C) for 7 days• Per cent of plants infected of 5 inoculated.
to vi 26 with 0.5 ml of an inoculum, or with SDW. The plants were left in the laboratory for 7 days and the number of rotted plants recorded (Table 7)• c . Experiment 3
Separate SDW suspensions of isolates 6^-46 and 1-12 were made up as, "very concentrated," 4x10^, 4x10^, 4x10^, and 4xl05 cells/ml. Sterile distilled water was used as a check inoculum. For each inoculum, 5 young saguaros were each injected with 0.25 ml of the inoculum. The plants were kept in the laboratory for 7 days and the number of rotted plants recorded (Table 8). d . Experiment 4
Two sets of 1-12 suspensions were made up as "very concentrated," 4x10^, 4x10^, 4x10^, and 4x10^ cells/ml.
Sterile distilled water was used as the suspending medium for 1 set and physiological saline (.8 5 % NaCl) (2 9 ) for the other. Sterile physiological saline and SDW were used as checks. The bacterial cells remained in the suspending medium for approximately 1 hr before inoculation. For each inoculum, 5 saguaro seedlings in the laboratory and 5 in
the greenhouse were each injected with 0 . 2 5 ml of the inoculum. The number of rotted plants was recorded after 7 days (Table 8). 27 Table 8. Effects of temperature and/or suspending medium on the number of cells of IS. carnegieana required to cause soft rot in saguaro seedlings.
No. of cells of IS. carnegie ana injected
Suspended in sterile water Suspended in .8 5 % NaCl
"Very "Very Treatment after cone . " 1 0 8 1 0 7 1 0 6 105 0 cone." 1 0 8 1 0 7 1 0 6 1 0 5 0 Experiment isolate injection (%)* <%) (%) (%) (%) (%) (%) (%> (%) (%) (%) (”/-)
#3 1 - 1 2 Left in 1 0 0 1 0 0 8 0 0 0 0 laboratory (23 c)
65-46 Left in 1 0 0 80 20 0 0 0 - laboratory (23 c)
#4 1 - 1 2 Left in 1 0 0 1 0 0 2 0 0 0 0 6 0 8 0 0 0 0 0 laboratory (23 O
1 —12 Left in 1 0 0 8 0 40 0 0 0 1 0 0 6 0 0 0 0 0 greenhouse (27 c)
3.Per cent of plants infected of 5 injected with 0.25 ml of the appropriate inoculum* Results were recorded 7 days after inoculation. 28 e . Experiment 3
Twenty-four hr cultures of isolate 1-12 which had just been passed through 3 saguaros, and of isolate 1 - 1 2 obtained from a lyophil culture tube prepared 9 months earlier were used to make up 2 separate series of suspen sions containing, "very concentrated," 4x10^, 4x10^, 4x10^,
4x10^, 4x10'*, 4xl02 , and 4x10'*" cells/ml. Sterile distilled water was used as a control inoculum. For each inoculum,
5 saguaro plants in the laboratory (2 3 - 2 5 C ) and 5 in the greenhouse (approximately 2 7 C) were injected with 0 . 2 5 ml of the inoculum. After 7 days the numbers of rotted plants was recorded (Table 9)•
4. Results a . Root Inoculation
The results in Table 6 demonstrate that wounds are necessary for root infection to occur; no plants with uninjured roots became diseased. Saguaros in the soil drench treatment became diseased over a wider range of inoculum coneentrations ("very concentrated"— 1 0 ^ cells/ml o and at 1 0 cells/ml). b . Needle Inoculation
The results of needle injection experiments 1-4
(Tables 7 and 8 ) show that a minimum of 5 x1 0 ^ cells of
E.* carnegieana are necessary for soft rot of saguaro 29 Table 9• Effect of temperature and inoculum concentration on the susceptibility of saguaro seedlings to 15. carnegieana.
Number of cells of IE. carnegie ana in j ured
"Very Treatment after Inoculum cone . 11 1 0 8 1 0 7 1 0 6 1 0 5 lO4 1 0 3 1 0 2 1 0 1 0 injection source (%) (%) (°/o) (°/=) (%) <%) (%) (%) (%) (%)
Left in green Aa ioob 1 0 0 6 0 20 2 0 0 0 0 0 0 house ( 2 7 C ) BC 1 0 0 1 0 0 6 0 40 0 0 0 0 0 0
Left in labora A 1 0 0 8 0 0 0 0 0 0 0 0 0 tory (23 c) B 1 0 0 2 0 2 0 0 0 0 0 0 0 0
^Culture of 15. carnegieana isolate 1 - 1 2 from 11 month old lyophilized sample. Per cent of plants infected of 5 inoculated with 0.25 ml of the appropriate c inoculum each. Culture of 15. carnegieana isolate 1 - 1 2 which was passed through sag uaro plants 3 consecutive times. 30 seedlings to occur in the laboratory (23-25 C )• However, the results of the fifth needle injection experiment (Table
9) show that when plants were inoculated and stored in the greenhouse (2? C) some became diseased after receiving as few as 105 or 10^ cells while a minimum of 10^ cells or 10^ cells was necessary to produce soft rot in plants stored in the laboratory (23-25 C ) after injection. Physiological saline, when used as a suspending medium, reduced the inoculum potential (the ability of a given number of cells to cause disease) of the bacterium. There was little dif ference between the inoculum potential of the various JE. carnegieana isolates used in these tests .
5• Relationship of Temperature to Inoculum Potential
The results of injection experiment 5 (Table 9) indicated that the number of cells of EX carnegieana necessary to cause soft rot was de creased when seedlings were stored in the warmer temperatures of the greenhouse.
Therefore, the following experiments were performed in order to determine the interrelationship of varying
inoculum coneentrations and post-inoculation temperatures
on the susceptibility of seedling saguaros to EX
carnegieana.
a. Experiment 1
October 8, 1 9 6 5 . Eight hundred ten 5 cm saguaro
seedlings growing in $ x $ x 6.5 cm plastic pots were 31
divided into 3 equal groups (A, B , C ). For 1 0 8 hr before inoculation, group A was left in the greenhouse and group B
was kept in the dark at room temperature (23-25 C ). Group
0 was subdivided into 6 (I-VI) sets of 4$ plants each.
These were acclimated to varying temperatures as follows:
I. 25 C for 108 hr
II. 35 C for 6 0 hr followed by 45 C for 24 hr and
then 24 hr at 55 C
III. 35 C for 6 0 hr followed by 45 C for 48 hr
IV. 35 C for 108 hr
V. 15 C for 60 hr followed by 5 C for 48 hr
VI. 15 C for 108 hr
All incubators were kept dark. Plants were watered
48 and 24 hr before inoculation and again 24 and 48 hr
after inoculation.
Sterile distilled water suspensions of IS. 1 2 3 4 carnegieana isolate 1 - 1 2 containing 10 , 1 0 , 1 0 , 1 0 ,
105, 10^, 10^, and 10^ cells/ml were prepared. Thirty
plants from groups A and B were each injected with 0.5 ml
of each bacterial suspension or SDW. Five plants per
inoculum per group were then placed directly at each
temperature (5 , 1 5 » 2 5 , 3 5 * 45, and 55 C ). Five acclimated plants per inoculum per treatment were also injected and
stored at the temperatures to which they had been acclimated.
These plants were removed from their respective incubators 32 only for inoculation which took about 15 min. Two 24 hr broth cultures of the bacterium were placed at each temperature as a check on its survival.
Four days after inoculation the plants were removed from the incubators and the number of rotted and/or split plants (those which split upon injection of inoculum into them) recorded for each treatment (Table 10). At this time the broth cultures were also streaked out on PDF plates to determine their viability. b . Experiment 2
February 5» 1966. The above experiment was repeated. However, in addition a group of plants was brought in from the greenhouse, inoculated, and left at room temperature in the laboratory as an additional check.
This time all plants were stored at their respective temperatures for 7 days before the results were recorded
(Table 1 1 ). Attempts were made to isolate EX carnegieana from the diseased plants in each group. The plants were surface sterilized with 2% amphyl, the spines removed by flaming, and a piece of diseased tissue removed from the rotted area and streaked on a PDF plate. The results are
also included in Table 1 1 .
c . Experiment 3
March 2 1 , 1 9 6 6 . The results of the previous
experiment show that saguaros injected with SOW developed 33 Table 10 . Effect of pre- and post-inoculation temperatures on the s usceptibility of saguaro seedlings to IS. carnegieana. Readings were made 96 hr after inoculation.
Number' of cells of E. carnegieana injected 3 2 Plant pre- 5x1 0 7 5 xi0 6 5 x10 5 5 x10 5x10 5 x1 0 5x10 1 5 0 Storage inoculation (°/o) Viability* of 2 temperature C treatment R b sc R s R S R s R s R S R s RS R s broth cultures
5 Ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +
15 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 + B 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 +
25 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +
35 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + B 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 1 0 0 0 80 0 4o 0 0 0 0 0 0 0 0 0 0 0 0 0 +
45 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + B ko 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 40 4o 2 0 0 0 0 : 2 0 0 0 0 0 0 0 0 0 0 0 0 +
55 A 0 0 0 0 60 0 0 0 0 0 0 0 0 0 0 0 0 0 B 0 0 4o 0 0 20 0 0 0 20 0 0 0 40 0 0 0 20 C 1 0 0 0 80 0 80 0 60 0 60 0 6 0 20 60 0 1 00 0 0 0 —
k+ = alive; - = dead. cPer cent of plants infected of 5 injected with 0 . 5 ml of the appropriate inoculum. ^Per cent of plants split of 5 inoculated. A - Plants in greenhouse until inoculated, then placed at storage temperature. B = Plants at 25 C in dark for 1 0 8 hr before inoculation, then placed at test temperature. C = Plants acclimated to post-inoculation temperature before inocul ation. See text for details. 34
Table 1 1 . Effect of pre- and post-inoculation temperatures on the s usceptibility of saguaro seedlings to IS. carnegieana. Readings were made 7 days after inoculation.
Number of cells of IS. carnegieana injected 4 Plant pre- 5xl07 5xi06 5xl05 5x10 5xio3 5xio2 5X101 5 0 Storage inoculation (%) Viability3 of 2 temperature C treatment Rb PC R p R p R p R p R p R p R p . R p broth cultures
5 Ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +
15 A 20 100 0 0 0 0 0 0 0 0 0 0 20 100 0 0 0 0 + B 20 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +
25 A 40 100 20 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + B 6o 100 6o 100 20 100 20 100 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +
35 A 80 100 80 100 0 0 4o 100 0 0 0 0 0 0 0 0 0 0 + B 80 100 20 100 20 100 0 0 0 0 0 0 0 0 0 0 0 0 C 6o 100 100 100 20 100 0 0 0 0 0 0 0 0 0 0 0 0 +
45 A 0 0 20 100 20 100 0 0 20 100 20 100 0 0 0 0 0 0 + B 20 100 0 0 0 0 0 0 20 100 0 0 0 0 0 0 0 0 C 100 100 0 0 4o 100 0 0 0 0 0 0 0 0 0 0 0 0 +
55 A 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 B 100 0 100 0 100 0 100 0 100 0 100 0 100 0 6o 0 100 0 C 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 - Room temperature A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + k+ = alive; - = dead. ^Per cent of plants infected of 5 inoculated with 0 . 5 ml of the appropriate inoculum concentration. ^Per cent of infected plants from which IS. carnegieana was recovered . A = Plants in greenhouse until inoculated, then placed at storage temperature. B = Plants at 25 C in dark for 108 hr before inoculation, then placed at storage temperature. C = Plants acclimated to post-inoculation temperature before inoculation• See text for details. 35 a soft rot when stored at 55 C for several days. The fact that no pathogenic organisms were isolated from these plants suggested that they may have been heat-killed.
This experiment was performed to compare non- inoculated, SDW-inoculated, and EX carnegieana-inoculated plants held at 55 C with similarly treated plants main tained at room temperature.
Two groups of 50 saguaro seedlings were acclimated to 55 C (Group A ) or stored at room temperature (Group B ) as in experiments 1 and 2 . A third group (C) of 100 seedlings was brought in from the greenhouse. Five plants from groups A and B and 10 from group C were each injected with 0.5 ml of SDW, 0.5 ml of an EX carnegieana suspension
(108 - 10**" cells/ml) , or left uninoculated. Immediately after inoculation the plants from group A were stored at
55 C and those from group B at room temperature. One-half of the plants in group C were placed at each of the 2 temperatures. All plants, except those at room temperature were stored in the dark and watered every 24 hr and all were observed after 24 hr and 48 hr. At 7 days the number of rotted plants in each group were recorded and reisola tions of EX carnegieana attempted (Table 1 2 ). 6
6. Results
The results in Table 10 further indicate that the inoculum potential of E • carnegieana increases with 36
Table 12. Comparative effects of 23-25 C and 55 C on saguaro seedlings inoculated with varying concentrations of E . carnegieana.
No. of cells of E . carnegieana injected 7 6 4 2 1 Plant pre 5 x1 0 5 x1 0 5 xl0 5 5 x10 5 x10 3 5x1 0 5 x1 0 5 0 Non-inoculated inoculation Storage (%) treatment temperature Ra Pb R p R P R p R P R PR p R p R p R P
Group AC 55 C 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 100 0 100 0 100 0
Group Room temp. 6 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (23-25 c)
Group Ce 55 C 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0
Room temp. 1 0 0 8 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (23-25 c)
^Per cent of plants soft rotted of 5 inoculated with 0 . 5 ml of the a ppropriate inoculum concentration. cPer cent of rotted plants from which IS. carnegieana was recovered. ^Plants acclimated to 55 C before inoculation. See text for details . ^Plants stored in dark at room temperature (23-25 C) for 1 0 8 hr before inoculation. Plants brought in from greenhouse, inoculated, and placed at storage temperature. 37 temperature. Below 35 C few plants rotted• A distinct difference was noticed among the plants in the 3 pre inoculation treatments. Those brought in from the green house or stored at room temperature before inoculation had a lower incidence of disease at any temperature (20-40%), and then only when injected with the more concentrated inocula (1 0 ^ - 10^ cells/ml). The saguaros which had been acclimated to higher post-inoculation storage temperatures showed a greater disease incidence (6 0 -1 0 0 %) when injected 5 4 with a minimum of 5x10 cells at 35 C, 5x10 cells at 45 C, and 5X101 cells at 55 C.
Broth cultures survived the 96 hr test period at
all but the 55 C storage temperature. Thus, although the pathogen apparently survived and caused disease in plants
at this high temperature, it was not able to survive in broth•
When the experiment was repeated and the plants
stored for 3 additional days ( 7 days) after inoculation, there was a general increase in the number of rotted
plants at each temperature (Table 1 1 ). Also, the tempera
ture range at which disease occurred included 2 5 and 1 5 C •
The temperatures and minimum numbers of cells required to
produce soft rot in the various plant treatments were: 4 Group A- -5xio7 at 15 C, 5xl06 at 25 c, 5x10 at 35 C , and 4 5xl02 at 45 C; Group B--5xl07 at 15 C, 5x10 at 25 C,
5xl05 at 35 C, and 5xl03 at 45 C; Group C— 5xl05 at 35 C 38 and at 43 C . Erwinia carnegieana was recovered from all diseased piants inoculated with the pathogen in the second test.
Most of the plants, including all of those injected with SOW, became black and had soft rot symptoms after 7 days storage at 55 C. However, the pathogen was not recovered from any of these plants.
All broth cultures except those stored at 55 C survived the test period. None of the plants stored at room temperature (2 3 - 2 5 C ) became diseased.
The results of the third experiment (Table 12) demonstrate that plants stored at 55 C may be heat-killed.
Within 7 days after inoculation, 100% of the plants stored at this high temperature (131 F) became black and soft whether inoculated with ID. carnegieana, SOW, or left uninoculated. ID. carnegieana was not recovered from any of these plants. When the saguaros in experiment 3 were observed 24 hr after inoculation it was noticed that the ridges on the upper portions of some of the plants had turned black (Fig. 3 )] after several more hours entire plants became black, soft, and watery. This same phenome non was observed on some of the plants during their period of acclimation in the 55 C incubator. Such plants were identical in appearance with those rotted by ID. carnegieana.
Plants stored at room temperature only became diseased (if at all) when injected with at least 5x10 or Fig. 3--Saguaro seedlings showing "heat-kill" symptoms after 24 hr storage at 55 C . Note the apical blackening on the plant in the left pot. 4o more cells/ml. The pathogen was recovered from most of these plants.
E . The Survival of E. carnegieana in Sterile Soil Subjected to Various Temperatures and Two Moisture Conditions
A soil mix composed of 3 parts of fine sand and 1 part clay soil was autoclaved for 12 hr at 250 C and allowed to cool to room temperature• One hundred ml of the sterile soil was placed in each of 240 cups (Dixie #120,
12 oz). Each cup was covered with a tight-fitting, trans- parent plastic lid and stored at room temperature until used. To each of 210 of the cups was added 10 ml of a 4 SDW suspension containing 10 cells/ml of a 24 hr culture of ID. carnegieana isolate 1-12 resulting in a concentration of 10^ cells/ml of soil. At this time 3 POP plates were also streaked with this suspension to confirm the viability of the bacterium.
Thirty ml of SDW was also added to 105 of these
cups. This was sufficient to soak the soil to the point where free water was visible on the surface. Additional
SDW was poured on the soil in these cups during the experi ment in order to maintain this high moisture level.
Fifteen SDW saturated and 15 non-saturated
bacterial infested cups of soil were placed at each of the
7 temperaturesi 5» 15) 25) 35) 45, 55, and 60 C. The 30 remaining uninfested cups of soil were left at room temperature as checks • Fifteen of these also were kept saturated with SOW.
As an additional check on the effect of these temperatures on the viability of jE. carnegieana, 24 tubes of nutrient broth were inoculated with the bacterium and incubated. After 24 hr the viability and purity of the organism in each broth tube was determined by streaking
loopfuls of the suspensions onto PDF plates, which were
then incubated. Immediately after the plates were streaked
the Morton closures on the tubes were sealed with masking
tape to prevent loss of moisture. The tubes were then
divided into 8 groups of 3 ? one group was placed at each
of the 7 temperatures and at room temperature.
Since JE. carnegieana has a thermal death point of .
59 C (9)* 3 PDP plates were streaked from the broth cul
tures in the 60 C incubator 24 hr after being placed there•
The plates were incubated and observed for growth. The
viability and pathogenicity of IS. carnegieana stored in
broth at the various temperatures was determined weekly (Table 13)•
At 1 week intervals through the fifth week, attempts
were made to determine the approximate numbers of cells of
JB. carnegieana which survived in 3 cups in each soil treat
ment. In addition, 3 wet and 3 dry non-infested control
cups were checked for bacterial growth each week. Table 1 3 . Effect of duration of exposure to varying temperatures on the survival and pathogenicity of IS. carnegieana stored in broth culture.a
Storage period
Temperature 24 hr 1 week 2 weeks 3 weeks 4 weeks 5 weeks C Vb pc V P V P V P V P V P
5 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y
15 3+ 3Y 3 + 3Y 3+ 3Y 3+ 3Y 3+ 3Y
25 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y
35 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y
45 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y 1 + IN
55 3- 3N 3- 3N 3- 3N 3- 3N 3- 3N
6 0 3- 3N 3- 3N 3- 3N 3- 3N 3- 3N 3- 3N Room temp. (23-25 C) 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y 3+ 3Y aThree broth cultures stored at each temperature. Cultures were 2 ^fc hr old at k start of test. V = viability; + = viable; - = non-viable. CP = pathogenicity; Y = pathogenic; N = non-pathogenic.
f- to To determine this, a cup of soil was shaken by hand for approximately 30 sec with the lid still on and then 1 0 ml were aseptically removed with a sterile beaker and placed in 90 ml of SOW in an erlenmeyer flask• After shaking the flask for a few sec, 5 ml of the suspension were pipetted into 5 ml of antibiotic solution. This mixture was shaken for 1 min on the test tube mixer and left to stand. After 4 hr (the minimum time for antibiotic
activity; Table 2) the tube was shaken again and 1 ml of the soil suspension pipetted into each of 3 PDF plates.
The plates were swirled, inverted, and incubated. After
24 hr the number of colonies of J£. carnegieana on the
plates were counted.
Representative colonies were isolated from each of
3 plates in each treatment. These colonies were increased
on PDF plants and tested for pathogenicity to young saguaro
seedlings• The colony counts of a treatment were con
sidered to be valid only when sample colonies from that
treatment proved to be pathogenic to the saguaros (Table l4) . 1
1 . Results All cultures of IS. carnegieana used in this experi- ment were found to be viable and pure.
The broth cultures kept at 60 C were non-viable
after 24 hr and after 1 week those stored at 55 C were also Table l4. Effects of duration of exposure to varying temperatures o n the survival of EX carneqieana in artificially infested^ sterile soil maintained at 2 mo isture levels.
Storage period
Sterile water saturated soil Dry soil
Temperature C 1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 we ek 2 weeks 3 weeks 4 weeks 5 weeks
5 TCT TT 3 1 2 + 7 8 T T T 368+32 152+15
15 TT T T 1 6 8 + 3 8 T T T T 2 0 8 + 1 6
25 TT TT T T T T T T
35 TTT T 3 0 + 2 2 TT T 193+13 0
45 T 240+4? 8 8 + 2 5 0 0 T 173+20 81+11 0 0
55 T 1 0 2 + 1 2 0 0 0 T 73+11 0 0 0
6 0 155+48 0 0 0 0 8 5 + 2 8 0 0 0 0 Room temp. non-infested 0 0 0 0 0 0 0 0 0 0 sterile soil — - ]T ' ' ..... Ten ml of a suspension containing 10 cel Is/ml of E. carnegieana was added to 100 ml of soil in 12 oz Dixie cups resulting in approximately 1,000 cells/ml of soil. Cups were replicated 3 times ^ for each treatment. # ^Additional sterile distilled water was added to the soil when necessary• Average number of cells per ml of soil• Determinations based on readings of 3 plates from each of the 3 replicates. 0 = no colonies; T = too many colonies to count* 45 non-viable (Table 13)• All cultures stored at 5-35 C remained viable and pathogenic after 5 weeks while only 1
culture was viable but non-pathogenic at 45 C. Unfortu nately, this non-pathogenic culture was discarded so that no further studies could be made on it. It did, however,
have the same colony morphology as the IS. carnegieana on streak plates used for comparison•
Erwinia carnegieana survival in broth and soil was
reduced by higher temperatures. Further, soil populations
were generally less in dry soil (Table l4). Approximately 5 10 cells were added to each cup of soil. After the
dilution processes employed in the recovery of the
bacterium, theoretically there should be 50 cells/ml of the
soil suspension (the amount plated out). The results in
Table l4 show that after 1 week storage at 5-55 C the
number of cells in 1 ml of wet or dry soil was too plenti
ful to count (500 or more). The bacterium survived for as
long as 2 weeks at 5 5 C and 1 week at 6 0 C , the tempera
tures at which the broth cultures failed to survive 1 week
and 24 hr respectively. Conversely, IS. carnegieana failed
to survive in soil for more than 3 weeks at 45 C while the
broth cultures stored at this temperature survived for 4
weeks. The optimum temperature for the survival of IS.
carnegieana in sterile moist and dry soil appeared to be
25 C. 46
F . Effects of E. carnegieana on Newly Germinated Saguaro Seedlings
1 . Needle Inoculation
This experiment was performed to determine the
susceptibility of newly germinated saguaro seedlings to
JE. carnegieana.
Three discs of filter paper (9 cm Schliecher and Schuell #597) were placed in the bottom of each of 5 Petri
dishes. After autoclaving for 20 min at 250 C , enough SOW
(about 1 0 ml ) was added to each dish to saturate the discs.
Saguaro seeds, collected in 1964, were soaked in 1 0 %
chlorox solution for 1 0 min after which 1 0 seeds were
aseptically transferred to each dish• The seeds were kept
under continuous fluorescent light at room temperature and
germination occurred between 3 to 5 days.
A concentrated "milky" suspension of JE. carnegieana
isolate 1-12 was prepared. Eight of the seedlings in each
plate were inoculated by injecting 0 . 2 5 ml of the inoculum
into a cotyledon with a syringe• The 2 remaining plants in
each dish were inoculated with SDW in the same manner. The
plants were subsequently left in the dishes and exposed to
continuous light. Sterile distilled water was added as
needed to keep the filter paper moist. After 1 week the
number of rotting seedlings were recorded (Table 15), after
which these and the non-rotted seedlings were each dipped
into 1 0 % chlorox solution for 5 min, crushed with a sterile Table 15 • The effects of a concentrated IS. carne.g;!eana suspension on wounded, newly germinated saguaro seedlings maintained at 23-25 C.
Rotting plants Non-rotting Plants3 Plants*3 with IS. Plants^ plants with treated rotted carnegieana .not rotted E. carnegieana Treatment No. (%) (%) (%) (%)
.2 5 ml concentrated bacterial suspension 4o 50 4o 50 25
.2 5 ml sterile distilled water 1 0 0 0 TOO 0
^Seedlings were inoculated within 3 days after germination. Results recorded 7 days after inoculation. ^Plants were cultured for E^. carnegieana either when symptoms appeared or when the j experiment was terminated. Inoculation wound became brown and dried out. - forceps and streaked on PDF-antibiotic plates ( 7 0 0 ppm actidione and 500 ppm erythromycin). After incubation, colonies resembling JE. carnegieana were isolated, increased, and tested for their pathogenicity to 2 year old saguaro seedlings.
2 . Germination of Saguaro Seeds on Ey carnegieana-Infested Soil
Each of 1 0 0 Dixie cups (No. 120, 1 2 oz) were two- thirds filled with an autoclaved soil mixture composed of
3 parts fine sand and 1 part silt. Holes had previously
been punched in the bottom of each cup to permit "bottom
watering. 11
Sterile distilled water suspensions of E.
carnegieana were made up in 2 5 0 ml volumes containing "very
concentrated," 1 0 8 , 1 0 7 , 1 0 6 , 1 0 5 , l o \ 1 0 3, 1 0 2 , and 1 0 1
cells/ml. Sterile distilled water was used as the control.
Twenty ml of a given inoculum was poured on the
soil in each of 1 0 cups. Twenty saguaro seeds were dis
tributed over the soil surface in each cup. Since these
seeds require light and high humidity for germination (1 ,
24, 2 5 ), the cups were covered with tight-fitting, trans- parent plastic covers (Dixie #920 plastic sure-snap
closures). Each was placed in a dish of water in the
greenhouse (2 ? C ).
As soon as 15 seeds germinated, the remaining seeds
were removed. Over a period of 3 weeks the seedlings in 49 the cups were observed every other day and any rotted plants were brought into the laboratory in an attempt to reisolate JE. carnegieana (Table 1 6 ). The rotted plants were surface sterilized in 1 0 % chlorox for 5 min, and then streaked on PDP-antibiotic plates. After incubation, representative, typical colonies of EX carnegieana were isolated, increased on PDP slants, and tested for patho genicity in 2 year old saguaro seedlings. No attempt was made to recover EX carnegieana from the various soils and from plants with no symptoms.
3• Results Newly germinated saguaro seedlings were found to be susceptible (Table 15) to soft rot when needle- inoculated with EX carnegieana♦ However, not all seedlings inoculated with the bacterium developed typical soft rot symptoms. In some cases the inoculation wound became brown and dried. Still, the bacterium was isolated from 25% of these latter plants.
Soft rot was also found to occur, although in small percentages, in seedlings germinated on soil infested with high coneentrations of EX carnegieana. Only 6 cups out of
1 0 0 were found to contain soft rotted saguaro seedlings during the duration of this experiment (Table l6 ). Rotted plants were found in varying percentages in 3 cups treated with 2 0 ml of inoculum at a concentration of 1 0 ^ cells/ml 50
Table l6 . The effect of soil infested with varying concentrations of IS. carnegieana on the survival of newly germinated saguaro seedlings.
Cups with Rotting plants diseased Plants^ with EX Treatment^ plants diseased carnegieana (%) (%) (%i
"Very concentrated" 1 0 9.3 Gk
1 0 8 cells/ml 1 0 8 . 0 50 CO 1 0 7 cells/ml 30 H 1 0 0
1 0 8 cells/ml 1 0 1-3 1 0 0
1 0 ^ cells/ml 0 0 0 4 1 0 cells/ml 0 0 0
1 0 3 cells/ml 0 0 0
1 0 ^ cells/ml 0 0 0
1 0 "*" cells/ml 0 0 0
Sterile water 0 0 0
aTwenty ml of an inoculum was poured on the soil in each of y 1 0 cups; each cup contained ,1 5 seedlings. Per cent of plants diseased of .1 5 0 treated . 51 6 8 and 1 cup each of those treated with 1 0 , 1 0 cells/ml, and
"very concentrated." The pathogen was isolated from most of the diseased plants.
G • Effects of E. carnegieana Suspended in Dust and Sterile Water on Saguaro Blossoms and Wounded Saguaro Seedlings
Since IS. carnegieana has been shown capable of long survival in dry soil it now seems important to determine if the infested dust can act as a carrier of the pathogen to an infectible site.
Into each of two 1 liter erlenmeyer flasks were
placed 2 0 0 gm of dry silt-clay soil. The flasks were
autoclaved for 12 hr at 250 C . After the soil had cooled
to room temperature, 50 ml of a SDW suspension containing
approximately 4x10® celIs/ml of IS. carnegieana isolate 1 - 1 2
were poured on the soil in 1 flask. The mouth of the flask
was covered with a pad made of 8 layers of sterile cheese
cloth and then placed in a 45 C incubator for 3 days, by
which time the soil was completely dry. Both flasks of soil
were then stored at room temperature.
In order to determine the number of cells which
survived the drying treatment, 1 gm of the infested soil
was placed in 9 ml of SDW and a dilution plate series made.
Sterile distilled water suspensions of the non-infested
soil were also made and plated out• After incubation 52 colonies of EX carne&ieana were counted and the number of cells per gm of soil calculated.
1 . Infestation of Saguaro Blossoms
Saguaro arms bearing flower buds were collected in late May and early June, 1965> and placed upright in large containers in the greenhouse. The buds were covered with k layers of cheesecloth in order to prevent insects from entering any open blossoms before they were treated. Saguaro flowers are known to fall within a few days if not effectively pollinated (2, 33)• Since this could obscure data from the inoculation studies, each morning open blossoms were cross-pollinated with pollen from different plants with the aid of a small camel's hair brush. Sub sequently the pollinated flowers were used for the various treatments.
A dilution series of the bacterial infested dust was made by adding the appropriate weights of sterile dust to known weights of the original infested dust. In addi tion SDW suspensions of isolate 1-12 were prepared at the same concentrations in cell/ml as the dust had in cells/gm.
These water suspensions were prepared daily when needed.
Since saguaro blossoms only remain open for approximately 1 full day (3 3 )? all of each day's blossoms were treated in the morning and a tag was tied to each 53 blossom to identify the treatment which it had received and the date of treatment.
The required amount of dust or liquid inoculum was placed in a small, sterile vial. An insufflator (for dust) or atomizer (for the liquid inocula) was attached to an air pump by a piece of rubber tubing. Before use, the insuf flators and atomizers were disinfected by passing through each 10 ml of 2% amphyl followed by 10 ml of SOW. Each was then dried in an oven.
The inocula were applied starting with the sterile and working upward to the most concentrated bacterial inocula so that the applicators could be used several times in succession. The inoculum to be applied was placed at the intake tube of the insufflator or atomizer, the air pump turned on, and the inoculum applied to the flower or wounded plant.
The treatments consisted either of insufflating or spraying 1 gm or 1 ml quantities of infested dust or bacterial suspension into individual flowers. The carriers 8 6 k 2 contained 10 , 10 , 10 , or 10 cells per ml or gm. Check flowers were appropriately treated with sterile dust or water.
After treatment the blossoms were left until they either fell off the arms without fruit set taking place
(within 2-7 days after opening) or until fruit which had formed ripened and split open (30-40 days after pollina tion) .
2. Isolation From Blossoms Test tubes containing 9 nil each of sterile anti biotic solution (actidione and erythromycin) were prepared.
Dried blossoms (blossoms became hard and dry a few days after closing) were cut longitudinally in half with a
flamed scalpel and one-half was aseptically placed into a tube of the solution. The tube was left to stand for 4 hr
at room temperature, after which it was shaken for a few
sec on a "Vortex" type test tube mixer. A flamed loop was
used to streak some of the liquid and blossom tissue on a
PDF plate. After incubation colonies resembling JE •
carnegieana were isolated and tested for pathogenicity in
2-year-old saguaros (Table 17)•
3• Infestation of Wounded Saguaro Seedlings
a . Experiment 1
Dust and SDW containing the same bacterial concen
trations used in the blossom tests were also used in this
experiment. A flamed 1 cm diameter corkbore was used to
aseptically remove a vertical core of tissue approximately
1 cm long from the tip of each of 100 saguaros previously
surface sterilized with 2% amphyl• The core of tissue was
immediately replaced until the plant was ready for 55
Table 17• Effect of 15. carnegleana infested dust and water suspensions on saguaro blossoms in the greenhouse.
Blossoms which did not set fruit Blossoms which did set fruit Blossoms Blossoms^ Blossoms Blossoms with soft Blossoms Blossoms with soft treated rotted rot bacteria treated rotted rot bacteria Treatment No No % No % No No % No %
Bacterial suspensions^
108 cells/ml 24 19 79 18 75 4 0 0 2 50 106 cells/ml 12 2 l6 12 100 8 0 0 4 50
10^ cells/ml 10 0 0 9 90 7 0 0 2 28 102 cells/ml 15 0 0 10 66 4 0 0 2 50
Sterile water 5 0 0 0 0 5 0 0 0 0 Infested dustC
108 cells/gm 10 0 0 10 100 4 0 0 2 50
106 cells/gm 4 0 0 3 75 11 0 0 7 63
10^ cells/gm 3 0 0 3 100 7 0 0 2 28
102 cells/gm 10 0 0 3 33 1 0 0 O 0 Sterile dust 5 0 0 0 0 5 0 0 0 0
Isolations made from rotted blossoms 6-9 days after inoculation and from non-rotted ones k 30-40 days after inoculation. .One ml sprayed into each open blossom One gm insufflated into each open blossom. 56 treatment• The hole was made in this position to minimize the chance of any inoculum leaking out. Groups of 10 plants were subsequently respectively inoculated with:
1 gm sterile dust; 1 ml SDW or 1 ml of a bacterial suspen sion containing 10^, 10^, 10^, or 10^ cells/ml; or 1 gm dust containing 10^, 10^, 10^, or 10^ cells/gm. The procedures for inoculating were the same as those used for blossoms, except that the inoculum was directed into the fresh injuries. Immediately after treatment, the holes in one-half of the plants in each group were refilled with the plug of tissue previously removed from them. Approximately
20 min elapsed between wounding and treatment. After 1 week the number of rotted plants in each treatment was recorded (Table l8). b . Experiment 2
An alcohol-flamed corkbore (0.5 cm in diameter) was used to remove a plug of tissue approximately 1 cm long from each of 120 surface sterilized 2-year-old saguaro seedlings. The tissue plugs were discarded. The holes were made from the top of the plant downward in order to hold the inoculum. Using the same procedures employed in experiment 1, groups of 20 plants were respectively inoculated with: 1 ml of a bacterial suspension containing
108 cells/ml; 1 ml SDW; 1 gm of dust containing approxi mately 108 cells; 1 gm sterile dust; 1 gm bacterial 57
Table 18. Effect of E_. carnegieana infested dust and water suspensions on wounded saguaro plants in the greenhouse (27 C ).
Plants infected3
Plugged wound Non-plugged wound Treatments*3 (%) ( ° / o )
Water suspensions0
10^ cells/ml 100 ko 10^ cells/ml 20 0
10 k cells/ml 0 0
10^ cells/ml 20 0
sterile water 0 0 Infested dust^
10^ cells/gm 0 0
10 cells/gm 0 0 k 10 cells/gm 0 0 2 10 cells/gm 0 0
sterile dust 0 0
^Readings made 7 days after inoculation. Five plants per treatment; plants were wounded with a flamed 1 cm diameter cork borer; plugs replaced in one- half the treatments. ^One ml per hole. One gm per hole. 58 infested dust (10^ cells/gm) plus enough SD¥ to saturate the dust once it was placed in the wound; or 1 gm of sterile dust plus SDW to saturate the dust•
After inoculation one-half of the plants in each treatment were covered with plastic bags, in order to maintain a high relative humidity and placed in the green house (27 C ) . All plants were watered every 3 days for 2 weeks, after which the number of rotting ones were recorded (Table 19).
4. Results
Colony counts made from soil dilution plates of EX carnegieana-infested dust used in these experiments showed it to contain approximately 1.5x10^ cells/gm.
As a whole, relatively few of the saguaro blossoms treated with EX carnegieana soft rotted (Table !?)• How ever , of the blossoms sprayed with 1 ml of water suspen sions containing 10^ or 10^ cells/ml, 79% and l6%, respectively, became diseased. In one instance, it was observed that ooze from a rotted blossom dripped on to an unopened bud below and this bud also rotted before it fully developed•
No blossoms treated with EX carnegieana-infested dust became diseased although the bacterium was recovered from many of these (Table 17). 59
Table 19• Effect of 15. carnegieana infested dust and water suspensions and humidity on saguaro plants kept in the greenhouse for 11 days after inoculation.
Plants infected3
Covered with Without plastic bag plastic bag Treatment (%) (%) *1
1 ml sterile water 0 0
1 gm sterile dust + .5 ml sterile water 0 0
1 gm sterile dust 0 0
1 ml bacterial suspension, 108 cells/ml 90 90
1 gm dust, 10^ cells/gm 20 20
1 gm dust, 10^ cells/gm + .5 ml sterile water 0 0
aTen plants per treatment; plants wounded with .5 cm cork borer; tissue plugs not replaced. 60
Wounded saguaro seedlings became diseased when their wounds were infested with E. carnegieana suspended in water (Tables 18 and 19) and in a few instances by EX carne#ieana suspended in dust (Table 19). Plugging inoculated fresh wounds or placing inoculated wounded plants at "high humidities1 11 produced variable results. In
one experiment (Table l8), more plants became diseased when, after inoculationv their wounds were refilled with
the plug of tissue previously removed from them. In the
second experiment (Table 19), however, no increase in
disease incidence resulted when wounds containing infested
dust were soaked with sterile water or when plants were
covered with polyethylene bags in order to maintain a "high humidity."
H . Isolation of E . carnegieana From Naturally Infested Soils
1. Soil Collections
Soil collections were made on April 2 8 , May 2 8 ,
June 29, and August 11, 1 9 6 5 * The collection sites were: I. At the base of a diseased, "bleeding" saguaro.
Ooze which dripped on the soil was included in
the collection• A sample of ooze from the plant
was also collected in a sterile tube. 6i
II. An open area between healthy saguaros; the
nearest saguaro was approximately 20 ft from the
collection site.
Ill. At the base of an old, dead, rotted saguaro. Rotted plant debris made up the top 4-6 in of the
soil profile.
IV, Beneath a decayed Opuntia clump. Rotted plant
debris made up the top 2-3 in of the soil profile.
The above collection sites were located in the
Saguaro National Monument near Tucson, Arizona.
V. An area in which no saguaros were growing. Plant
growth at the site included grasses and chollas.
The site was located several miles from the
nearest stand of saguaros.
At each site approximately 500 ml of soil was collected at the surface, 3 in, 6 in, 12 in, and l8 in levels and placed in separate, new, appropriately tagged plastic bags. The collection shovel was washed with 2%
Amphyl solution between each soil level and collection site .
2. Bacterial Isolations
Five 125 ml erlenmeyer flasks containing 20 ml
each of filter-sterilized antibiotic solution (erythromycin
and actidione) and five 250 ml erlenmeyer flasks containing 62
90 ml of SOW per flask were prepared for each soil sample collected• A 10 ml portion of soil from a bag was put into each of the 5 flasks containing the antibiotics. The flasks were swirled by hand for 1 min, covered with aluminum foil, and left to stand for 4 hr at room tempera ture . After again being swirled the soil suspension from each was added to a separate flask of SOW. After the diluted soil suspensions had been shaken for 1 min, 3 one ml aliquots were taken from each flask and pipetted onto separate PDF plates. Preliminary tests indicated that this dilution process was necessary to insure sufficient separation of bacterial colonies on the plates necessary for single colony isolation.
Laboratory tests demonstrated that jE. carnegieana, when grown on agar containing tetrazolium (triphenyl tetrazolium chloride), was able to reduce it to the highly colored insoluble formazan (2 8 ), resulting in colonies with dark pink centers. Since this property made it easier to identify IE. carnegieana colonies, the PDF used for isola tion from soils collected in June and August contained 5 ml/l of a 1% solution of tetrazolium.
After incubation, the plates were observed under a dissecting microscope. Using as checks, plates inocu lated with IS. carnegieana, colonies which morphologically resembled IS. carnegieana were isolated from the soil dilution plates and increased on PDF slants. Sterile 63 distilled water suspensions of the isolates from the April and May soil collections were injected into saguaro seed lings to test for pathogenicity. The June and August isolates were also inoculated into tomato fruits in addition to saguaros and tested for their ability to hydrolyze pectate (3).
The ooze collected from the "bleeding11 plants was also streaked on PDF plates and likely colonies isolated and similarly tested for their pathogenicity (Table 20).
a . Results
The numbers of bacterial isolates pathogenic to
saguaro seedlings and tomato fruits differed with time of year, collection site, and soil depth (Table 20).
No pathogenic bacteria were isolated from soil
collected in April and only 1 pathogenic isolate (site III,
18 in deep) was obtained in the May sampling. The June and
August collections yielded several soft rotting bacteria,
most of which (5 pathogenic to saguaros and 22 pathogenic
to tomato fruit) were isolated from soil from the surface
to 18 in deep at the base of an old decayed saguaro (site
III). It was observed that not all of the "soft rotters"
hydrolyzed pectate gel medium. Pathogenic bacteria were
isolated in smaller numbers from various depths in soil
collected at sites I, II, and IV but none at site V
(several miles from any saguaros). Several pectolytic Table 20 Distribution of soft rot and pectolytic bacteria in soils collected at various sites and depths at 4 time intervals.
Collection site3
I II III IV V Collection date*3 Soil depth, inches sc T p s T p S T P s T p s T p
April 28, 196 5 Ooze 0 0 0 0 0 0 0 3 0 0 0 0 0 6 0 0 0 0 0 12 0 0 0 0 0 18 0 0 0 0 0
May 2 8 , 1965 Ooze 0 0 0 0 0 0 0 3 0 0 0 0 0 6 0 0 0 0 0 12 0 0 0 0 0 18 0 0 1 0 0
June 29, 1965 Ooze 0 0 0 0 0 0 0 0 0 0 0 3l 4 0 0 0 0 0 0 3 0 0 0 1 1 1 1 51 10 0 0 0 0 0 1 6 1 1 k 0 0 0 0 1 4 0 1 2 0 0 1 12 0 2 3 0 1 1 2 C1 2 1 0 2 0 0 1 18 0 0 0 1 0 1 1 c1 4 0 0 0 0 0 0
August 1 1 , 1965 Ooze 0 0 0 0 0 1 0 0 0 0 1 k 1 0 0 0 0 0 5 3 0 1 1 0 0 0 0 1 1 0 0 0 0 0 0 Table 20.--Continued
6 0 1 2 0 0 0 0 0 1001000 12 0 0 0 0 0 0 0 5 1 0 1 0 0 0 0 18 0 0000003 0 0 0 0 0 0 0 aX = area at base of diseased "bleeding" saguaro; II = open area between healthy saguaros; III = area at base of old decayed saguaro; IV = area beneath rotted b Opuntia clump; V = area several miles from any saguaros. Isolates obtained from April and May soil collections tested for pathogenicity to saguaros only, all others also tested for pathogenicity to ripe tomatoes and hydrolysis of pectate gel. S = number of isolates which rotted saguaros; T = number of isolates which rotted ripe tomatoes, both at 25 C; P = number of isolates which hydrolyzed pectate gel at 32 C.
ON VI 66 bacteria were isolated from site V, however. Neither soft rot nor pectolytic bacteria were isolated from the ooze of the freshly diseased saguaros. DISCUSSION
There has been some disagreement as to the gram- staining reaction of Erwinia carneqieana. The bacterium was first reported to be gram-positive (9, 3 0 ) and Boyle
(8) demonstrated its inhibition by penicillin, a gram positive bacteria inhibitor (kO). Burkholder later described it as gram-negative (11, Ik). Boyle (9) has reported that after several months in culture EX carnegieana was more gram-negative than positive.
In recent years it has been found that the gram- stain reaction of a bacterial cell is dependent upon its ability or failure to prevent the decolorizing agent to pass through the cell wall and leach out the dye complex.
The high lipid content (easily dissolved by acetone and alcohol) of the cell walls of gram-negative bacteria fails to prevent the removal of the crystal violet dye from its cytoplasm (46). A change in the gram-stain reaction of a bacterium would thus seem to be a result of cell wall changes and would presumably be of mutational origin.
The author has recovered EX carnegieana from soil as well as diseased saguaros and all but 1 of these isolates were gram-negative. Boyle (10) has suggested the possibility of the existence of gram-negative and gram positive strains of E. carnegieana.
6? 68
Presently, work is being conducted to elucidate the presence or absence of such strains (45)• In work already done 26 bacterial isolates, all pathogenic to saguaro seedlings, have been isolated from diseased tissue of saguaros, organ pipe Lemaireocereus Thurberi (Engelm.)
Britt, and Rose, and prickly pear cacti, and 1 from ocotillo Fouquieria splendens Engelm. The isolations were made with and without the use of erythromycin (gram positive bacteria inhibitor {hOj) and all were gram negative.
In tests reported here E_. carnegieana remained gram-negative after several broth culture transfers and passages through saguaro seedlings. Although the evidence indicates that nearly all isolates of jE. carnegieana are gram-negative, no assumption can be made until far more isolations are made.
In addition to the fact that the author has isolated 1 gram-positive bacterium pathogenic to saguaros,
Boyle (9) mentioned that in her tests ID. carnegieana was inhibited by penicillin while gram-negative E. carotovora was not. Thus, the existence of the gram-positive strain of the pathogen seems certain but it does not appear to be widespread. It is important that serological and bio chemical comparisons be made between the gram-positive and gram-negative types already present and any that are 69 isolated in the future to determine just how closely related these organisms really are.
Antibiotics and various other chemicals have been used extensively in the isolation of specific types of organisms (7, 13, 15, 19, 2 1 , 2 2 , 3 1 , 3 2 , 3 6 , 4i, 43, 44,
4?, 49)• The choice of materials to be incorporated into the selective isolation medium is dependent upon the types of organisms to be eliminated and most important, the resistance of the desirable organisms to these chemicals.
In some instances (13, 15, 43, 44) media have been developed for the isolation of certain classes and genera of fungi and bacteria.
In order to make a study of the survival of 15. carnegieana in soil, it was necessary to develop a method for the consistent and selective isolation of this bacterium from soil. Actidione (cycloheximide) has been widely employed as a fungal inhibitor in isolation media
(13, 15, 2 1 , 4l, 44). Jeffers (2 1 ) reported that
Agrobacterium tumifaciens, Erwinia carotovora, Pseudomonas solanacearum, and Xanthomonas campestris made good growth on agar containing as much as 1 0 0 0 ppm of actidione while all fungi tested were greatly reduced in growth. It was suggested that actidione might be useful in the isolation
as well as the culture of phytopathogenic bacteria.
Erythromycin, a gram-positive bacteria inhibitor
(40), was employed in the isolation medium because all but 70
1 of the EX carneRieana isolates obtained in the past several years have been gram-negative. Four soft rotting Erwinia species, including 15. carnegieana # grew profusely on PDF agar containing actidione and erythromycin while various fungi and the gram positive Staphylococcus aureus were greatly inhibited by the respective antibiotics. In addition the results show that autoclaving caused no reduction in the anti-fungal activity of actidione or the effectiveness of erythromycin against Sm. aureus. This is contrary to the report by
Jeffers (2 1 ) that the activity of actidione is greatly reduced by autoclaving.
Although both can be used successfully, the anti biotics in agar have definite advantages over their water solutions in the isolation of 15. carnegieana and other soft rotting Erwinia species. With agar it is merely necessary to streak infested material onto plates and incubate them while water solutions require the material to be left in them for several hours before plating out. There is also the advantage that the material containing bacteria and fungi is continuously exposed to the activity of anti biotics when streaked on agar containing the antibiotics.
The term inoculum potential has been defined in
several ways (20) but essentially describes the ability of
inoculum (material capable of producing infection) to cause
disease• In the following discussion inoculum potential 71 will refer to the ability of a given number of cells of
EX carnegieana to cause bacterial necrosis of saguaro seedlings.
It was found that injuries must be present in order for infection to occur when saguaro roots were either dipped in a bacterial suspension or planted in soil infested with EX carnegieana. In the soil drench group, disease occurred in soil containing 2x10^ cells (20 ml of inoculum at a concentration of 10 cells/ml) while a minimum of 10^ cells/ml was required to cause infection in the root dip group. This may be explained by the fact that the roots in the infested soil were continuously exposed to the inoculum, which probably increased in the soil (2 3 , and our own results) while the root-dip group had only the 3 hr exposure to the inoculum.
More concentrated suspensions of EX carnegieana take longer at lower temperatures to cause disease than at higher temperatures• However, there was no disease at 5 or
55 C . Generally, acclimated or darkened plants are more susceptible than non-acclimated light exposed plants although the results were variable (Tables 10 and 11).
However, saguaros stored at 55 C were actually heat-killed.
The symptoms present in heat-killed saguaros are identical in appearance to those in plants rotted by EX carneqieana except that the blackened tissues of the heat-killed plants 72 are somewhat firmer. Also, the first signs of heat-kill appeared as blackened areas at the apex of the plant.
Heat-kill may play an important role in the failure of saguaro seedlings to survive and become established in the field. Since seedlings grow on the soil surface they might be exposed to temperatures of 55 C and higher during the summer months in southern Arizona when Tucson air temperatures range from 21-38 C (l?).
When considering the effect of temperature on the inoculum potential of JB. carnegieana, it becomes necessary to determine whether the effect of temperature is on the pathogen, the host, or both. Studies concerned with the effects of temperature on the biochemistry of both the saguaro and the bacterium may be helpful• It may be that at higher temperatures the pathogen produces larger quantities of enzymes used in tissue breakdown•
The formation of cork layers around wounded areas, infection pockets, and insect tunnels are known to be a defense mechanism of the saguaro (9, l8 ). Little is known
about the effect of temperature on cork formation in the
saguaro and it may prove to be an important factor in the
capability of these plants to survive invasion by ID.
carnegieana. It may be that cork formation in the saguaro
is slowed down by higher temperatures thus rendering the
plant more susceptible to invasion, colonization, and
necrosis by JB. carnegieana. 73 The survival of a bacterial cell refers to its ability to retain its faculties for reproduction in spite of, or because of prevailing conditions (48). A con siderable amount of work has been conducted to elucidate the factors in the soil which would affect the reproduction capabilities of its bacterial populations. It was shown by
Patrick (3 8 ) that the plant pathogenic bacteria used in his studies were all inhibited by some soil microorganisms, the number varying with the species of pathogen.
The food supply and the associative effects of other organisms are considered by Menzies (34) to be the 2 chief factors which influence bacterial survival in soil.
The effective role of a lack of food is not so much a lethal starvation as it is a failure to permit cell divi sion to replace those cells killed by other soil agents.
Survival in sterile soil would presumably be influenced by nutrient supply.
It is thought that most plant pathogenic bacteria survive in soil through the food supply and protection afforded by the remains of the host plant (34), although this material may also serve as a food supply for organisms antagonistic to the bacteria (42).
The bacterial plant pathogens can be divided into
3 groups: (1 ) those whose populations are almost exclu sively developed within a plant host and the soil phase is a rapidly declining one; (2 ) those which build up 74 populations within their host and these decline gradually in the soil; and (3 ) those populations which are largely produced in the soil and whose relation to plant disease is erratic and transitory (12).
When several species of bacteria, actinomycetes, and fungi were inoculated into sterile soil, all of the organisms, except 1 bacterium, increased in numbers to a maximum and then gradually decreased (23)• The presence of a gelatinous capsule is recognized as an excellent means of protecting the bacterial cell against adverse circumstances, especially dessication (l6). The cells of E_. carnegieana are enveloped by capsules (ll, l4).
It was shown by Rangaswami (42) that the numbers of
EX carotovora added to sterile soil at 27 C increased at first and then gradually decreased. Similar results with various microorganisms were obtained by Katznelson (23)•
It was found that jE • carnegieana also increased in numbers after being added to sterile soil and then gradually decreased, except at 25 C , at various rates over a 5 week period depending on the storage temperatures• The nutrients required for such multiplication may have been released from the soil during the autoclaving process (2 3 ).
The ability of IS. carnegieana to survive for 1 week in soil at temperatures of 55 C and 60 C but not in broth culture (the thermal death point of E. carnegieana is 59 C
/%/) indicates that both moist and dry soil protect the 75 bacterium from the effects of these high temperatures.
However, the results indicate that the higher the soil temperature the shorter the time interval that the bacterium remains viable. The optimum soil temperature appears to be about 25 C .
Various soft rot species of Erwinia have been shown to overwinter in both sterile and non-sterile soil (2 7 *
37)• The desert situation, however, requires the emphasis to be placed on the ability of the organism to survive through the summer• Boyle (9) has shown IE. carnegieana to be capable of surviving for 46 days in both wet and dry naturally infested soil exposed to Tucson summer air temperatures which normally range from 21-38 C (17)• The moisture level appeared to have little effect on the ability of the organism to survive in sterile and non- sterile soil which suggested that rainfall was of little importance in the natural situation.
Many antibiotic actinomycetes isolated from
Canadian soils were found to inhibit E . carotovora (2 6 ).
It seems that similar studies should be made to determine the distribution of J£. carnegieana inhibitors in soils of saguaro populated areas.
In the study involving the isolation of E_. carneqieana from soils collected at various sites, depths, and seasons, it was found that the pathogen was present in soil near and beneath saguaros but not several miles from 76 them. The greatest number of isolations was made as deep as 18 in in the soil at the base of a decayed saguaro which appeared to have been killed by bacterial necrosis. The bacterium may have been carried into the soil with the ooze when the plant was freshly diseased and then leached deeper into the soil by rain water. The recovery of the bacterium from soil located in an open area between healthy saguaros with no diseased ones nearby demonstrated that the pathogen may be moved about on soil or persists long after the host plant has disappeared. The bacterium was isolated from soil beneath a freshly diseased oozing saguaro on only one occasion and no isolations were made from the ooze itself.
The small number of samplings and/or the sampling technique may have been responsible since the ooze is supposedly a means by which soil becomes infested (9) , it seems that IS.
carnegieana should have been recovered more frequently.
With the aid of the antibiotic agar, this work should be
repeated and extended to include the fall and winter
months. It may prove profitable to study the extent of
the spread of infested soil by wind by exposing antibiotic
agar plates to the air in saguaro populated areas.
Since IS. carnegieana can survive for long periods
in soil exposed to high temperatures, it becomes necessary
to consider the role which infested soil (and dust) plays
in the survival and establishment of saguaro seedlings as
was suggested by Alcorn (4). 77
Boyle (9 ) concluded that saguaro roots growing in soil infested by ooze from a "bleeding" plant may become infected through injuries in the root cortex. Similar findings were obtained in the present studies with saguaro seedlings. When such plants had their roots dipped into a suspension of EX carnegieana or were planted in infested soil, only those with injured roots became diseased. In the field, roots may be wounded by the action of weather, rodents, insects (9 )1 or possibly nematodes. In order to determine the importance of these findings, it would seem profitable to transplant seedlings with wounded and non- wounded roots into soil which has been soaked with ooze from a diseased plant. This may be helpful in elucidating the factors affecting saguaro repopulation and in deciding where and how to plant saguaros in any future repopulation attempt.
The second aspect of infested soil to be considered is its effect on seedlings which germinate on it. The fruits and seeds of saguaros fall to the ground at the base of the plant and, under favorable conditions (1, 24), the seeds germinate. Tests show that saguaro seedlings may become diseased when germinated on soil infested with high coneentrations of J2. carnegieana. Because diseased plants were found only in a total of 6 cups of 90 inoculated cups, and because the pathogen was not recovered from all of the diseased plants, this work should be repeated. The 78 addition of inoculum to the soil at various times after the seeds have germinated might give some idea as to when the seedlings are most susceptible. Since wounds are necessary for root infection, wounding of the roots of the infected seedlings in this test probably occurred previous to infection. Sterile soil was used in this experiment and it can be assumed that wounds were probably made as the seed lings emerged from their seed coats or as the primary roots pushed their way into the soil. This type of infection may partially account for the lack of repopulation reported by Boyle (9).
The spread of the pathogen by dust particles must also be considered. Since the soil on the upper.surface is most likely to be moved by wind gusts and since this is the soil which is infested by ooze or debris from infected plants, the spread of the pathogen by wind seems quite feasible.
In order for ID. carnegieana carried on dust to cause an infection, some wound or natural opening in the plant is required. Holes made by woodpeckers are common in saguaro forests and may be an avenue of infection. Saguaro seedlings which had wounds in them were found to become diseased by ID. carnegieana carried on dust or water demonstrating that such infection is possible. Far more plants were infected by water suspensions, probably because the bacterium was free to move about on the film of moisture 1
79 covering the exposed saguaro tissue, which was thus exposed
to greater numbers of the pathogen. This would also allow
better chances to move into tissue through microwounds and
cracks. Why the addition of water to infested dust in wounds did not cause any increase in infection, is not
certain• The importance of increasing temperatures on the
inoculum potential of E_. carnegieana carried on dust should
be determined to compare it with the results of the water
suspension injection experiments. The blossoms on mature saguaro plants may also have been considered as entry sites
for ID. carnegieana. These have been reported to rot after
being sprayed with water suspensions of the pathogen (9)?
the present tests confirm this finding. However, in no case
did the infection in blossoms progress into the plants
themselves. Infested dust was not capable of producing
obvious infections in blossoms although the bacterium
survived in the blossoms for several weeks. Therefore,
even though infested dust may not cause disease in the
blossoms, these may become a source of inoculum to be
carried off by the various animals which visit the blossoms
(5) 33)• The nectar present in the base of the flower may
also be a medium in which the bacterium can multiply. It
seems necessary, therefore, to determine how well ID.
carnegieana survives and multiplies in this nectar and
whether this may be a source of inoculum for further spread
of the disease. One further aspect of infested soil should be considered. Alcorn (4) has shown that tomato and squash fruits as well as honeydew melons can be rotted by JD. carnegieana. This finding presents the possibility of future problems if these or other susceptible hosts were to be grown on E . carnegieana infested soil. LITERATURE CITED
1. Alcorn, S. M . and E. B . Kurtz, Jr. 1959• Some factors affecting the germination of seed of the saguaro cactus (Carnegiea gigantea). Am. J . Botany 46:526-529. 2. Alcorn, S. M ., S. E . McGregor, G . D . Butler, Jr., and E. B . Kurtz, Jr. 1959• Pollination requirements of the saguaro (Carnegiea gigantea)♦ Cactus and Succ. Jour. Amer. 31:39-41.
3. Alcorn, S . M . 1959 « Natural history of the saguaro. Arid Lands Colloquia. 1 9 5 9 -6 0 . The University of Arizona. pp. 23-29•
4 . Alcorn, S . M . 1 9 6 1 . Some host's of Erwinia carnegieana . Plant Dis. Reptr. 45:587-590.
5* Alcorn, S. M ., S. E. McGregor, and George Olin. 1 9 6 1 . Pollination of saguaro cactus by doves, nectar feeding bats, and honey bees. Science 133:1594- 1595.
6 . Alcorn, S . M . and Curtis May. 1 9 6 2 . Attrition of a saguaro forest. Plant Dis. Reptr. 4 5 :1 5 6 -1 5 8 .
7« Bakerspiegel, A. and J. J. Miller. 1953 • Comparison of oxgall, crystal violet, streptomycin, and penicillin as bacterial growth inhibitors in platings of soil fungi. Soil Sci. 47:123-126.
8 . Boyle, Alice M . and J . G . Brown. 1944. Effect of penicillin on a plant pathogen. Phytopathology 34:760.
9 * Boyle, Alice M . 1949 • Further studies of the bacterial necrosis of the giant cactus. Phyto pathology 3 9 :1 0 3 0 -1 0 5 2 .
10• Boyle, Alice M. 1 9 6 5 • Personal communication. University of Arizona, Tucson, Arizona.
11. Breed, R. S ., E. G . D . Murray, and N. R . Smith. 1957 * Bergey’s Manual of determinative bacteriology. Seventh edition. Williams and Wilkins Co., Baltimore. 109^ p •
81 82
12. Buddenhagen, I. W. 1 9 6 5 . The relation of plant- pathogenic bacteria to the soil. p. 269-284 In K. F. Baker and W. C . Snyder. Ecology of soil- borne plant pathogens. Univ. of California Press, Berkeley.
13• Dickey, R . S. 1 9 6 1 . Relation of edaphic factors to Agrobacterium tumifaciens. Phytopathology 31: 607-6l4.
14. Elliot, C. 1951. Manual of bacterial plant pathogens. Chronica Botanica Co., Waltham, Mass. 186 pp.
15• Georg, L. K., Libero Ajello, and M. A. Gordon. 1951« A selective medium for the isolation of Coccidioidea immitis. Science 114:3 8 7 -3 8 9 • l6 . Giltner, W . and H . V. Langworthy. 1916 . Some factors influencing the longevity of soil microorganisms subjected to dessication, with special reference to soil solution. J . Agr. Res. 5s 927-942.
17» Green, G . R . 1 9 6 2 . Arizona Climate— Supplement No. 1 . Univ. of Arizona Press, Tucson.
l8 . Hemenway, A. F . 1934. An anatomical study of traumatic and other abnormal tissues in Carnegiea gigantea. Am. J . Botany 2 1 :513-518• 19• Hine, R . B . and E . E . Butler. 1957 * Use of novo biocin for isolation of fungi from soil. Phytopathology 47:524.
20. Horsefall, J. G. and A. E. Dimond. i9 6 0 . Inoculum and the diseased population. p . 1-22 In J . G . Horsefall and A. E. Dimond. Plant Pathology, Vol. Ill. Academic Press, New York.
2 1 . Jeffers, W . F . 1954. Use of actidione in the culture of piant-parasitic bacteria. Phytopathology 44: 144.
2 2 . Johnson, L . F . 1957• Effect of antibiotics on the numbers of bacteria and fungi isolated from soil by the dilution plate method. Phytopathology 47:630- 6 3 1 .
23• Katznelson, H . 1940. Survival of microorganisms inoculated into sterile soil. Soil Sci. 49:211- 217. 83
24. Kurtz, E • B . and S . M . Alcorn• i9 6 0 . Some germina- tion requirements of saguaro cactus seeds. Cactus and Succ• Jour. Amer. 32:72-74.
25• Kurtz, E • B . and S. M. Alcorn. 1 9 6 3 • Big saguaros from little seeds grow. Cactus and Succulent Soc. J. 35:43-44.
26. Landerkin, G . B . and A. G. Lockhead. 1948. A comparative study of the activity of fifty anti biotic actinomycetes against a variety of soil bacteria. Can. J . Res. 2 6 :5 0 1 -5 0 6 .
27• Leach, J . G . 1930. Potato blackleg: The survival of the pathogen in the soil and some factors influ encing infection. Phytopathology, 2 0 :2 1 5 -2 2 8 .
2 8 • Lederberg, J . 1948• Detection of fermentative variants with tetrazolium. J . Bacteriol. 56:695*
29• Levine, M . and II. W . Schoenlein. 1930• A compilation of culture media for the cultivation of micro organisms . The Williams and Wilkins Co., Baltimore. 969 PP* 30. Lightle, P. C ., E. T. Standring, and J . G . Brown. 1942. A bacterial necrosis of the giant cactus. Phytopathology 3 2 :303-313• 31• Littman, M . L . 1947• A culture medium for the primary isolation of fungi. Science 1 0 6 :1 0 9 -1 1 1 .
3 2 . Martin, J . P . 1950. Use of acid, rose bengal, and streptomycin in the plate method for estimating soil fungi. Soil Sci. 6 9 :2 1 5 -2 3 2 .
33 * McGregor, S. E •, S . M . Alcorn, and G. Olin. 1 9 6 2 . Pollination and pollinating agents of the saguaro. Ecol. 43:259-267 *
34. Menzies, J • D • 1 9 6 3 * Survival of plant pathogens in soil. Botan. Rev. 29:79-122.
35• Niering, W . A., R. H • Whittaker, C • H • Lowe• 1 9 6 3• The saguaro: a population in relation to environ ment . Science 142:15-23*
36. Papavizas, G. and C. Davey. 1959. Evaluation of various media and antimicrobial agents for the isolation of soil fungi. Soil Sci. 8 8 :1 1 2 -1 1 7 . 84
37• Patel, M . K. 1929* Viability of certain plant pathogens in soils. Phytopathology 19i295•
3 8 . Patrick, Z . A . 1954. The antibiotic activity of soil microorganisms as related to plant pathogens. Can. J . Botany 32 : 705-735 •.
39* Pavlivich, M. S. and I. Tail. 1 9 6 1 . General micro- biology laboratory manual. Technical Publications, Desert Laboratories, Inc., Tucson, Arizona. 149 pp.
40. Pelczar, M . J . and R . D . Reid. 1958. Microbiology. McGraw-Hill Book Co., Inc., New York. 564 pp.
41. Phillips, G . B . and E. Hanel Jr. 1950. Control of mold contaminants on solid media by the use of actidione. J . Bacteriol. 6 0 :104-105•
42. Rangaswami, G . and M . Ramalingam. 1 9 6 1 . Studies on the survival of plant pathogenic bacteria in sterilized and unsterilized soil. Indian J . Microbiol. 1 :1 5 3 -1 5 6 .
43. Russell, P . 1956. A selective medium for the isola tion of Basidiomycetes. Nature 17721038-1039• 44. Schroth, M . N ., J . P . Thompson, and D . C . Hildebrand. 1 965 • Isolation of Agrobacterium tumif aciens— A_. radiobacter group from soil. Phytopathology 55 - 645-647.
45. Schuyler, M. and S. M. Alcorn. 1 9 6 6 . Personal communication.
46. Stanier, R . Y ., M . Doudoroff, and E . A . Adelberg. 1963 * The microbial world. S econd edition. Prentice-Hall, Inc., Englewood Cliffs. 753 pp•
47. Steiner, G. W. and R. D . Watson. 1 9 6 5 . Use of surfactants in the soil dilution plate count method. Phytopathology 5 5 :7 2 8 -7 3 0 .
48. Sykes, G. 1 9 6 3 * The phenomenon of bacterial survival. J. Appl. Bacteriol. 2 6 :287-294. 85
49. Vaartja, 0 . i9 6 0 . Selectivity of fungicidal materials in agar cultures. Phytopathology 50: 870-873.
50. Voronkevitch, X. V. 1 9 6 1 . The hardiness in soil of bacteria of the genus Ervrinia--organisms which cause soft rot in plants. Rev. Appl. Mycol. 40: 201.