Persistence of the bacterium Erwinia carnegieana in soil and its relationship to the establishment and survival of saguaro (Carnegiea gigantea) cacti

Transcription

1 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, Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 29/04/ :15:00 Link to Item

2 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

3 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment 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 acknowledgment 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 interests 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: STANLEY M. ALCORN Professor of Plant Pathology >> /96C, Date

4 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

5 TABLE OF CONTENTS... Page LIST OF TABLES vi LIST OF ILLUSTRATIONS ix ABSTRACT.... x INTRODUCTION AND LITERATURE REVIEW GENERAL MATERIALS AND METHODS PROCEDURES AND RESULTS A. Determination of Bacterial Cell, Concentrations In Water Suspensions... 8 B. Gram Stains of Erwinia carnegieana R e s u l t s C The Use of Antibiotics to Selectively Isolate Erwinia species Antibiotics Dissolved in Water Assay Disc T e s t s... l4 3 Effect of Autoclaving on Antibiotic A c t i v i t y... l6 4. Antibiotic Effect on Filamentous G r o w t h D. 5 R e s u l t s Inoculum Potential Studies Root Dip Inoculation Soil D r e n c h Needle Inoculation a. Experiment b. Experiment c. Experiment d Experiment e. Experiment R e s u l t s a. Root Inoculation b. Needle Inoculation Relationship of Temperature to Inoculum Pot e n t i a l a. Experiment b. Experiment c. Experiment R e s u l t s iv

6 V TABLE OF CONTENTS Continued Page E. The Survival of JB. carnegieana in Sterile Soil Subjected to Various Temperatures and Two Moisture C o n d i t i o n s F. 1 Results Effects of E. carnegieana on Newly 43 Germinated Saguaro Seedlings Needle Inoculation. * 46 2 Germination of Saguaro Seeds on EX carnegieana-infested Soil G. 3 R e s u l t s... Effects of EX carnegieana Suspended in Dust 49 and Sterile Water on Saguaro Blossoms and Wounded Saguaro Seedlings Infestation of Saguaro Blossoms Isolation From Blossoms 54 3 Infestation of Wounded Saguaro S e e d l i n g s a. Experiment 1 54 b. Experiment Results H. Isolation of EX carnegieana From Naturally Infested Soils Soil Collections Bacterial Isolations l a. Results DISCUSSION... 6? LITERATURE CITED l

7 LIST OF TABLES Table Page 1. Per cent transmittance of serial dilutions of 3 E.» carnegieana isolates suspended in sterile distilled water Survival of Erwinia carnegieana and 2 fungi in an aqueous solution containing actidione, griseofulvin, and erythromycin * Disc assay determinations of the inhibitory effects of erythromycin, actidione, and griseofulvin alone and in combination on the growth of certain fungi and bacteria Effect of autoclaved and non-autoelaved antibiotic agar on 6 bacterial species and the germination of spores from 3 species of fungi Effect of filter-sterilized antibiotic agar on the mycelial growth from mass transfers of Alternaria, Helminthosporium, and Fusarium spp Relationship of method of inoculation and inoculum concentration to the susceptibility of saguaro seedlings to Erwinia carnegieana Relationship of inoculum coneentration to the susceptibility of saguaro seedlings to E. carnegieana Effects of temperature and/or suspending medium on the number of cells of 15. carnegieana required to cause soft rot in saguaro seedlings Effect of temperature and inoculum concentration on the susceptibility of saguaro seedlings to ID. carnegieana vi

8 vii LIST OF TABLES Continued Ik Effect of pre- and post-inoculation temperatures on the susceptibility of saguaro seedlings to E. carnegieana. Readings were made 96 hr after inoculation. Effect of pre- and post-inoculation temperatures on the susceptibility of saguaro seedlings to 15. carnegieana. Readings were made 7 days after inoculation. Comparative effects of C and 55 C on saguaro seedlings inoculated with varying concentrations of JE. carnegieana... Effect of duration of exposure to varying temperatures on the survival and pathogenicity of JE. carnegieana stored in broth culture... Effects of duration of exposure to varying temperatures on the survival of 15. carnegieana in artificially infested sterile soil maintained at 2 moisture levels The effects of a concentrated 15. carnegieana suspension on wounded, newly germinated saguaro seedlings maintained at C... The effect of soil infested with varying cone ent rat ions of 15. carnegieana on the survival of newly germinated saguaro seedlings... Effect of 15. carnegieana infested dust and water suspensions on saguaro blossoms in the greenhouse... Effect of 15. carnegieana infested dust and water suspensions on wounded saguaro plants in the greenhouse (27 C )..... Effect of 5. carnegieana infested dust and water suspensions and humidity on saguaro plants kept in the greenhouse for 11 days after inoculation... Page Ilk

9 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

10 LIST OF ILLUSTRATIONS Figure Page 1. Needle inoculation of a saguaro seedling Concentration of 15. carnegieana cells in water suspensions as determined by per cent transmittance of light of a wavelength of 4l0 mu Saguaro seedlings showing "heat-kill" symptoms after 2k hr storage at 55 C ix

11 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 C in soil but not in broth culture. While the inoculum potential of the pathogen increases with temperatures to 50 C, 2 year old saguaro seedlings were heatkilled 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 concentrations 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

12 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.

13 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*101s 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 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

14 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 pollinators. 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

15 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 initiating infections in blossoms, seedlings, and mature saguaros.

16 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 hr and fungal cultures at 25 C for 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 homogeneous, "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

17 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 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 D r. 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.

18 Fig. 1--Needle inoculation of a saguaro seedling.

19 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.

20 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 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 ) 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 concentration of bacterial cells in 1 ml of that dilution 8

21 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 T 5 x 10"} 30.0 IQ-? _ l! 10 ^ is=5 T T l + l 5 xis:* None 1 5 x 10-1 io-i 5 x i : io: T None T 5 x 10~{ io~l 68 + io X i o, ± l io" Avg. % Trans mittance : Avg. cell/ml at 10-7 dilution = l6 b Calculated celis/ml^ No dilution=l.2 x 1 0 q 5 x 10-1 =6.3 x 10% 10~2 =1.2 x x 10 p =6.3 X 10% 10 =1.2 x 10 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.

22 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

23 Percent transmittance at 410m p 5x x x10 10' Cells of E. carnegieana per ml of suspension 5 xio Fig. 2 Concentration of 12. carnogieana cells in water suspensions as determined by per cent transmittance of light of a wavelength of 4l0 mu. Each point with its maximum and minimum represents the average number of colonies on 24 h dilution plates. - H

24 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. ) ppm. The proper weights of these were first dissolved in 20 ml of dimethylformamide (DMF) for each

25 13 liter of solution to be prepared. In the following experiments 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 precipitate 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 solutions were prepared and filter sterilized using a preautoclaved 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

26 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 h r, 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

27 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 c > Control *4* Alternaria sp 1 4* *f* 4* Control M Erwinia carnegieana isolate 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.

28 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 concentrations 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).

29 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 N N N N N Staphylococcus aureus N ^.6 N N N N Erwinia carotovora N N N N N N N N N E. aroideae N N N N N N N N N E. atroseptica N N N N N N N N N E. carnegieana N N N N N N N N N Penicillium sp. N N N 1.8 ^ N N Helminthosporium sp. N N N N N Alternaria sp. N N N 10.8_ N N Fusarium oxysporum f vasinfectum N N N N N 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.

30 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 E A 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 Escherichia coli Erwinia carotovora , E. aroideae E. atroseptica E. carnegieana t Helminthosporium sp Alternaria sp Penicillium sp Fusarium oxysporum f vasinf ectum 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.

31 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 concentrations. 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 grampositive Staphylococcus aureus were strongly inhibited by actidione and erythromycin, respectively. The effects of the antibiotics remained approximately 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

32 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 b Helminthosporium sp Fusarium oxvsporurn f. vasinfectum 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

33 21 minimum exposure period required for the inhibition of fungal spore germination in water solutions of the antibiotics (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 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

34 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 chloropicrinfumigated 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 nonwounded 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

35 Table 6. Relationship of method of inoculation and inoculum concentration to the susceptibility of saguaro seedlings to Erwinia carnegieana. Plants infected Soil drench3 Root dip,. b Inoculum Concentration (cells/ml) Injured0 roots (%) Non-injured roots (%) Injured0 roots (%) Non-injured roots (%) "very concentrated" 30d ^ (SDW) ^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.

36 "very concentrated," 10, 10^, 10, 106, , 10^, lo^, 103, 102, or 24 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 greenhouse 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 and 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,1 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

37 Table 7- Relationship of inoculum concentration to the susceptibility of saguaro seedlings to E- carnegieana. Number of cells injecteda Experiment E. carnegieana isolate "Very concentrated" (%)b 5 x (%) x 106 (%) =; 4 5 x 10^ 5 x 10 0 (SOW) ( /o) (%) (%) # # o ^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

38 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. a check inoculum. Sterile distilled water was used as 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. checks. Sterile physiological saline and SDW were used as 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 ml of the inoculum. The number of rotted plants was recorded after 7 days (Table 8).

39 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 Experiment isolate Treatment after injection "Very cone." (%)* <%) (%) (%) 105 (%) 0 (%) "Very cone." (%) (%> (%) (%) (%) 0 ( /-) # Left in laboratory (23 c) Left in laboratory (23 c) # Left in laboratory (23 O Left in greenhouse (27 c) Per cent of plants infected of 5 injected with 0.25 ml of the appropriate inoculum* recorded 7 days after inoculation. Results were

40 28 e. Experiment 3 Twenty-four hr cultures of isolate 1-12 which had just been passed through 3 saguaros, and of isolate obtained from a lyophil culture tube prepared 9 months earlier were used to make up 2 separate series of suspensions 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 ( C ) and 5 in the greenhouse (approximately 2 7 C) were injected with 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

41 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 Treatment after injection Inoculum source "Very cone. 11 (%) (%) ( /o) ( /=) (%) lo4 <%) (%) (%) (%) 0 (%) Left in green Aa ioob house ( 2 7 C ) BC Left in labora A tory (23 c) B ^Culture of 15. carnegieana isolate from 1 1 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 which was passed through sag uaro plants 3 consecutive times.

42 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 difference 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, Eight hundred ten 5 cm saguaro seedlings growing in $ x $ x 6.5 cm plastic pots were

43 31 divided into 3 equal groups (A, B, C ). For 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 5 5 C III. IV. 35 C for 6 0 hr followed by 45 C for 48 hr 3 5 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 carnegieana isolate containing 1 0, 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

44 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» 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, The results of the previous experiment show that saguaros injected with SOW developed

45 33 Table 10. Effect of pre- and post-inoculation temperatures on the s usceptibility of saguaro seedlings to IS. carnegieana. Readings were made 9 6 hr after inoculation. Number' of cells of E. carnegieana injected Plant pre- 5x xi0 6 5x10 5 5x10 5x x x Storage inoculation ( /o) Viability* of 2 temperature C treatment R b sc R s R S R s R s R S R s R S R s broth cultures 5 Ad B C A B C A B C A B C o A B ko C 40 4o : A B 0 0 4o C 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 hr before inoculation, then placed at test temperature. C = Plants acclimated to post-inoculation temperature before inocul ation. See text for details.

46 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 5X 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 B C A B C A B 6o 100 6o C A o B C 6o A B C o A B o C Room temperature A 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.

47 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 noninoculated, SDW-inoculated, and EX carnegieana-inoculated plants held at 5 5 C with similarly treated plants maintained 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 reisolations of EX carnegieana attempted (Table 1 2 ) Results The results in Table 10 further indicate that the inoculum potential of E carnegieana increases with

48 36 Table 12. Comparative effects of C and 55 C on saguaro seedlings inoculated with varying concentrations of E. carnegieana. No. of cells of E. carnegieana injected Plant pre 5 x x xl0 5 5x10 4 5x10 3 5x x Non-inoculated inoculation Storage (%) treatment temperature Ra Pb R p R P R p R P R P R p R p R p R P Group AC 55 C Group Room temp. (23-25 c) Group Ce 55 C Room temp. (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 hr before inoculation. Plants brought in from greenhouse, inoculated, and placed at storage temperature.

49 37 temperature. Below 35 C few plants rotted A distinct difference was noticed among the plants in the 3 preinoculation treatments. Those brought in from the greenhouse 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 ( %) 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 temperature 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: Group A- -5xio7 at 15 C, 5xl06 at 25 c, 5xl02 at 45 C; Group B--5xl07 at 15 C, 4 5x10 at 35 C, and 4 5x10 at 25 C, 5xl05 at 35 C, and 5xl03 at 45 C ; Group C 5xl05 at 35 C

50 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 ( 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. these plants. ID. carnegieana was not recovered from any of 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 phenomenon 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

51 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.