Effects of Pseudomonas cepacia and cultural factors on the nodulation of Alnus rubra roots b y Frankia
SUSAN KNOWLTON' A N D JEFFREY 0. DAWSON Foresrr~ Depar~nerlr, Urziversiry qf Illirlois, Urbarlcr, IL. U.S.A. 61801
Received Deccmber I . 1982
KNOWLTON, S., and J . 0. DAWSON. 1983. Effects of Pserrclornorlc~s cepacia and cultural factors on the nodulation of AInlrs rltbra roots by Frankia. Can. J . Bot. 61: 2877-2882.
Infection of Alrllrs rrrbra Bong. roots by Frarlkia isolates was consistently promoted by Pserrdorr~orias cepacia "helper" bacteria under a variety of cultural conditions. Pseuclorr~or~ns cepaccia, while helpful, was ncver necessary in securing nodulation of aseptic A. rubrc~ by Frcrrlkia. Live P. cepacia cells were added with a Frankin isolate to aseptically grown A. rirbr~ secdlings on Hoagland's agar slants. This doubled, on averagc, the number of nodules formed by Frar~kia alonc when Frarikia isolate, age of Frankia inoculum, Frarlkia medium, calcium concentration of seedling substrate, phosphate concentration of seedling substrate, and pH of seedling substrate were varied. There was an apparent interaction between seedling substrate pH and P. cepacia in the promotion of nodulation. At pH levels from 4.0 to 6 .0 the infectivity of Frnnkia isolate Ar13 alone was greatly depressed compared with infectivity at the optimal pH for the growth of ArI3, around 7.0. However, at pH levels of 5 .0 .5 .5 , and 6.0 the ability of P. cepacia to promote nodulation of A. rubra seedlings by ArI3 was greater than at pH 7.0. This interaction may be due to the ability of P. cepcicia to neutralize and grow more rapidly in mildly acidic solutions in our experimental system, combined with the ability of P. cepacia to cause root-hair deformation, which has becn associated with the actinorhizal infection process.
KNOWLTON, S., et J . 0. DAWSON. 1983. Effects of Pseirdon1or~as cepacia and cultural factors on the nodulation of Alrlirs rirbra roots by Frankia. Can. J . Bot. 61: 2877-2882.
L'infection des racines de 1'Alnus rubra Bong. par des isolats de Frankia est systCmatiquement favorisCe par la bactCrie Pseudomonas cepacia, dans diverses conditions de culture. M&me s'il est utile, le P. cepacia n'est jamais nCcessaire pour assurer la nodulation de 1'A. rubra par Frankia dans des conditions aseptiques. Des cellules vivantes du P. cepacia ont CtC ajoutCes, avec un isolat de Frankia, i d e s plantules de I ' A . rirbra croissant de mani&re aseptiquedans des tubes inclines contenant de la gClose de Hoagland. En moyenne, ce traitement double le nombre de nodules formCs par le Frankia seul, lorsqu'on fait varier les facteurs suivants: l'isolat de Frarzkia, l ' ige de I'inoculum de Frarlkia, le milieu de culture du Frankia, la concentration en calcium et en phosphate du substrat de la plantule et le pH du substrat de la plantule. 11 semble y avoir une interaction entre le P. cepacia et le pH du substrat de la plantule dans l'augmentation de la nodulation. Aux pH compris entre 4,O et 6,0, l'isolat Ar13 de Frarlkia, inoculC seul, a une infectivitk beaucoup plus faible qu'au pH optimal pour la croissance de cet isolat, soit environ 7,O. Cependant, aux pH de 5,0, 5,5 et 6,0, le pouvoir du P. cepacia de favoriser la nodulation de plantules de 1'A. ritbra par l'isolat ArI3 est plus grand qu'h pH 7,O. Cette interaction pourrait &tre due h la capacitC du P. cepacia de neutraliser les solutions ICgkrement acides et de croitre plus rapidement dans ces solutions dans notre systkme experimental, de concert avec son pouvoir de provoquer des dCfomations des poils racinaires, lesquelles sont associCes au processus d'infection actinorhizienne.
[Traduit par le journal]
Introduction 7.0. It has been suggested that Frarzkia is more inhibited A number of environmental factors have been shown
to influence nodule initiation by Frankia, including pH, combined nitrogen, and presence of rhizosphere micro- organisms. Even though most actinorhizal plants are able to grow in a wide pH range, there is some indication that nodule formation is inhibited by low pH. Bond et a l . (1954) found that successful nodulation was achieved in only half of the inoculated Alnus and Myrica plants grown in water cultures maintained at pH 4.2. At the same pH, Hippophae plants did not nodulate, even though growth of nonnodulated plants supplied with nitrogen was unaffected by the acidic conditions. Nodule formation in these plants was maximum at pH
'present address: E. I. DuPont de Nemours & Co., Central Research and Development Department, Experimental Station, Wilmington, DE, U.S.A. 19898. .
at low pH than some host plants (Stewart 1966; Caiiizo et al . 1978).
In addition to the pH of the medium, high levels of combined nitrogen (10, 50, 100, 150mg NH4-N per litre) can suppress nodule formation in Alnus glutinosa (MacConnell and Bond 1957; Stewart and Bond 1971). Calcium and phosphate have been found to influence nodulation in leguminous plants (Lie 1974; Andrew 1978), although such influences have not been estab- lished for the actinorhizal association.
Work by Knowlton e t a l . (1980) indicated that microorganisms in the rhizosphere play a significant role in the infection of actinorhizal plants by Frankia. Under most conditions, aseptic Alrzus rubra seedlings inocu- lated with Frankia did not become nodulated unless other microorganisms were added to the seedling environment. Pseudomonas cepacia strains, in addition
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2878 CAN. J. BOT. VOL. 61. 1983
to several rhizosphere isolates, promoted nodulation and elicited massive root-hair deformation typical of that observed in early stages of infection. It was suggested that "helpers" promote infection by causing root-hair deformation which is associated with Frarzkia infection. Such deformation could entrap the nonmotile actino- mycete ensuring intimate contact with the root hair and, thus, promote infection.
Although infection is promoted by Pseuclornonas strains, Frarzkia is, by itself, able to infect aseptic host plants. However, infection of aseptic plants in the past has sometimes been difficult to achieve (Knowlton et al . 1980). It has been suggested that cultural conditions under which the host plant (Lalonde et al. 1980) or the Frarzkia isolate (Knowlton et al . 1980) is grown may affect the infectivity of Frankia in the presence or absence of "helpers."
The objective of this research was to determine how a variety of cultural conditions influenced the ability of Pse~idomonas cepacia strain 85 to promote the infection of aseptic Alrzus rubra Bong. seedlings by Frankia isolates. By varying the cultural conditions, we hoped to elucidate specific ways in which "helpers" promote nodule formation in our experimental system.
Materials and methods Preparntiorl of rnicroorgnr~isrns
Pure cultures of the Frnnkin isolate ArI3 (Berry and Torrey 1979) were used to inoculate most test seedlings. In one experiment, Frnrlkia isolates CpIl (Callaham et nl. 1978), AvcI 1 (Baker et nl. 1979), and AcN 1 AG (Lalonde and Calvert 1979) were tested to determine whether differences occur among Frarzkia strains with respect to the effect of "helper" bacteria.
All Frnrlkia cultures were grown in the dark in 10 mL of a liquid medium in 18-mm-diameter culture tubes. For routine maintenance, Frnnkin was grown in a yeast extract medium described by Baker and Torrey (1979). When specified, Frnnkin was grown for experimental purposes in Q-Mod (Lalonde and Calvert 1979), a Tween 80 - casamino acids medium (Blom et al. 1980), or a defined succinate medium (Tjepkema etnl. 1980). In most cases, the cultures were grown for 30 days and then used to inoculate aseptic seedlings. In some experiments, various ages of ArI3 were tested to determine their infective capacity. These included cultures of ages 2, 4, and 6 weeks.
To prepare inocula, Frankin cultures were centrifuged at 2000rpm for 15 min and the pellet was washed twice and resuspended in sterile distilled water. The bacterial suspension was applied at a concentration of 0.005 mL packed cell volume per plant.
Growth rates of ArI3 were determined by measuring packed cell volumes using Kimax-brand, Bauer-Schenck centrifuge tubes (VWR Scientific Inc., Cat. No. 21074-005) calibrated in units of 0.004 mL. Each mean growth determination was made by centrifuging four samples separately for 15 min at 2000 rpm.
One-half of all experimental plants were also inoculated with Pseudomor~as cepacia strain 85 (PC85) described by
Stanier et al. (1966). The bacteria were grown in 10 mL of a liquid medium which contained (in grams per litre) yeast extract, 5.0; dextrose, 10.0: and casamino acids (acid hydrolyzed), 5.0. The cultures were grown in shaken culture for 16 hat room temperature and then centrifuged for 15 min at 2000rpm. The pellet was washed twice and resuspended in sterile distilled water. Two drops of a visibly turbid suspension were applied to each seedling root with a sterile Pasteur pipette. For those seedlings which did not receive the Pseudomor~as inoculum, two drops of sterile distilled water were applied instead.
Prepamtior1 of seedlings Seeds of Alnus rubrn were soaked in distilled water for
several hours and surface sterilized for 30 min in 30% hydro- gen peroxide with Ivory liquid detergent added as a surfactant. They were then rinsed five times in sterile distilled water and planted aseptically in Petri dishes which contained approxi-