Pseudomonas syringae as a microbial antagonist of Ceratocystis ulmi in the apoplast of American elm

Trans. Br . mycol. Soc. 80 (3) 389-394 (1983)

Printed in Great Britain

PSEUDOMONAS SYRINGAE AS A MICROBIAL ANTAGONIST OF CERATOCYSTIS ULMI IN THE APOPLAST OF AMERICAN ELM By DONALD F . MYERS* AND GARY A. STROBEL Department of Plant Pathology , Montana State Univers ity, Bo z eman, MT 59 717, U.S.A. Isolates of Pseudomo nas syringae are antagonistic to Ceratocy stis ulmi, the causal agent ofDutch elm disease. P. syringae grows and produces antimycotics on expressed elm sap and on an extract of elm wood . When specific isolates of P . syr ingae , designated M27m and M323m, were introduced into the apoplast of greenhouse-grown elm seedlings, they established themselves without significant phytotoxicity and suppressed the development of vascular discolouration due to Dutch elm disease. Biological control strategies for plant diseases most often involve modifying the external environment of the plant or pathogen to make them unfavourable for pathogen development (Corke, 1978). If an antagonist could be established within the apoplast (the dead cells, tissues, and intercellular spaces of the stems and roots associated with water uptake) of a host plant, the development of a vascular pathogen might be inhibited. Dutch elm disease was chosen as the host-parasite complex because its causal agent, Ceratoeystis ulmi (Bu ism .) Moreau (syn . Ophiostoma ulmi (Buism .) Nannf.) is a vascular pathogen that causes an economically important disease for which traditional control strategies have been unsuccessful on a large scale (Sinclair & Campana, 1978). Pseudom onas syringae van Hall was selected as the antagonist because (i) P . syringae and its many biotypes can exist as epiphytes. While it is a pathogen with a broad host range (Stapp, 1961), P. syringae is not a reported pathogen of elm; (ii) Although it is not a vascular pathogen, P. syringae can colonize internal tissues of leaves, wood, and bark (Crosse, 1966); (iii) P. syringae produces various antibiotics including the antimycotic syringomycin (Sin den, DeVay & Backman, 1971) and Ceratocystis was one of the most sensitive genera tested in screening tests of the toxicity of this antimycotic (D eVay, Gonzales & Wakeman, 1978). Here we report on the introduction of P. syringae into the apoplast ofAmerican elm and its effectiveness as an antagonist of C. ulmi in laboratory and greenhouse tests. Preliminary reports of this work have been made (Myers & Strobel, 1981; Strobel & Lanier, 1981). ., Present address : University of Florida, AREC, P .O. Drawer A, Belle Glade, FL 33430, U.S.A.

MATERIALS AND METHODS

Isolates of P . syringae were obtained from D . C . Sands, Montana State University, Bozeman, MT 59717 and maintained in sterile distilled water at 12°C and reisolated on medium B of King et al . (1954) at 28°. The aggressive isolate of C. ulmi CU-SF was obtained from N. K . Van Alfen, Utah State Univ., Logan, Utah 84321 and maintained on potato dextrose agar (D ifco) at room temperature in the dark for 7-10 days and then under continuous fluorescent light at 24°. The term 'aggressive ' is used sensu Gibbs, Heybroek & Holmes (1972). Fifteen isolates of P . syringae were assayed for their antagonistic activity to an aggressive isolate of C. ulmi (CU - 5F) . Five pI of a cell suspension of P. sy ringae (ea 1 x 108 cfujml) grown in potato dextrose broth (D ifco) and diluted to 10 pi with distilled water was deposited aseptically on the surface of a modified mineral salts agar medium (D G A) . DGA consisted of Dye's salts (D ye, 1963), 1 % glucose, and 1'2 % Noble agar. Plates were incubated for 3-4 days at 28°. Each treatment was replicated three times and the experiment was repeated once . A spore suspension of C. ulmi (5 x 106 conidiajml) was sprayed onto the surface of each plate, and the plates were incubated at room temperature for 2 days. Antagonism was estimated by determining the area of inhibition surrounding and including the bacterial colony. Twenty-five isolates of C. ulmi, including aggressive and non-aggressive types, were bioassayed for their sensitivity to P. syringae isolates M27m and M323m and to the extracted antimycotics of isolate M323m by the same plate test except that the extract was deposited into an 18·8 mm" well in the agar . For the extraction of antimycotics , P. syringae

39°

Pseudomonas antagonising Ceratocystis ulmi

was grown on potato dextrose broth adjusted to final concentrations of 1'5 % glucose and 0"4 % casein hydrolysate (Sigma) in standing culture. The cultures were incubated and extracted according to Gross & DeVay (1977). The ability of P. syringae to produce antimycotics toxic to C, ulmi in an agar medium containing elm sap or elm wood extracts was also tested by the plate test described above. Sap was expressed from cuttings of new growth from mature elm trees with a plant water status console (Model 2005, Soilmoisture Equipment Corp" Santa Barbara, CA 93105) (Van Alfen & Turner, 1975). Fluid passing through the stems, equal to the volume of the fluid in the stem, was collected at 5-10 bars, concentrated 10 x by flash evaporation at 35°, freeze dried, and the resulting powder stored over P 205' The powder was dissolved in distilled water at 0, 0'01, 0'05, 0'1, and 1'0 % (wIv) with 1'2 % Noble agar, pH 7'0, P, syringae M27m and M323m were tested for antagonism to C. ulmi CD-5F as described previously, For preparation of wood extracts, twigs were harvested from the current year's growth of mature elms, the bark discarded, and 2-5 em twig segments homogenized with an 'Omni-Mixer' (Sorvall) for 2 min in distilled water in the proportion 1: 5 (wIv). The extract was filtered through two layers of cheesecloth and centrifuged at 4° for 20 min at 4000 g. The pellet was discarded and the supernatant liquid (11'3 mg (dry wt/ml) diluted with distilled water and incorporated at 1, 5, 8, 17, and 25% (v/v) with 1'3% Noble agar, pH 7'1. Each treatment was replicated three times. For greenhouse tests, seeds from American elms (Ulmus americana L.) were planted in sandy loam soil in 21'5 cm diam plastic pots. The seedling elms were maintained at 25-30° under natural light. When seedlings were 1-2 m high, they were pruned to single stems. At the beginning of their second growth period after germination the seedlings were treated with either P. syringae M323m, M323nm, M27m, or M27nm by a gravity flow method in mid-May. Isolates M323m and M27m were used because they had produced relatively large amounts of antimycotics in our screening tests and, because they represented different pathotypes of P. syringae, we felt that there was a higher probability of success. Non-mucoid isolates M323nm and M27nm, which did not produce antimycotics, were used as controls. The bacteria were grown for 2 days at 28° on King B medium. Cells were scraped from each plate, washed twice in distilled water, suspended in 1 % glucose (wIv) and adjusted to 5 x 108 cfu/rnl. Sixty-ml plastic bottles fitted with plastic tubing adapters and rubber tubing (3 mm inner diam) were filled with these suspensions and hung about 1 m above the soil level of each pot. A

hole, 0"44 ern diam, was drilled into an internode in the seedling about 10 em above the soil line and fitted with a plastic tubing adapter sealed to the plant with silicone rubber adhesive. The system allowed cell suspensions to flow from the reservoir into the apoplast of the seedling. Maximum uptake of the bacterial suspension occurred during the first 48 h and the total uptake was 12-60 ml (x = 36). Randomly selected groups of seedlings (8-14 seedlings/group), treated with M323m, M323nm, M27m, or M27nm, were inoculated with C. ulmi CD-5F two weeks after treatment with the bacteria. For inoculation, a flap of bark (5-10 x 20-25 mm), ca 10 em above the injection site, was peeled back and the exposed apoplast was flooded with 1 ml of a suspension of C. ulmi (1 x 104 spores/ml). The bark flap was replaced and wrapped with masking tape and aluminium foil. Four groups of seedlings, each treated with either M323m, M323nm, M27m, or M27nm, were not challenged with C. ulmi. One group of non-treated seedlings was inoculated with C. ulmi alone and one group was treated with 1 % glucose only. All plants were examined for foliar wilt and yellowing 10 weeks after the inoculation with C. ulmi and the percentage of plants showing these symptoms in a treatment was determined. Vascular discolouration was determined as the percentage of the total length of the stem apoplast discoloured per plant. To determine the viability and movement of P. syringae and C. ulmi in the seedlings, stems were cut into 2-3 em pieces and split in half longitudinally. One half was placed on a P. syringae-selective medium (Sands & Scharen, 1978) and the other half on a C. ulmi-selective medium (Miller, Sands & Strobel, 1981). RESUL TS

Of the 15 isolates of P. syringae tested for antimycotic activity against C. ulmi, M323m was the most antagonistic (Table 1). Eight lacked antimycotic activity, including M323nm and M27nm, which were non-mucoid selections made from the mucoid isolates M323m and M27m respectively. Twenty-five isolates of C. ulmi tested varied up to 75-fold in sensitivity to P. syringae M323m, 19-fold in sensitivity to P. syringae M27ffi, and i i-fold in sensitivity to crude extracts of antimycotics from P. syringae M323m (Table 2). No isolate tested was totally insensitive either to P. syringae or to its extracted antimycotics. Although most of the isolates of C. ulmi were relatively similar in sensitivity to P. syringae M323m and M27ffi, isolate TN was relatively insensitive to M323m and sensitive to M27m. The aggressiveness of an isolate of C. ulmi towards elm was apparently not related

D. F. Myers and G. A. Strobel Table 1. Antimycotic activity of isolates of Pseudomonas syringae against Ceratocystis ulmi isolate CU-SF P. syringae isolate

M3 23m M3 23nm M27m M 27nm M3 19 M30 8 M305 M307 M46 S1-a M33 1 M3 27 M3 26 M313 M30 6 M7 1-b

Natural host

Zone of inhibition (mm")

Pear Barley Pear Safflower Cucumber Lettuce Barley Apple Pear

Apple Tomato

706 ° 254 ° 3°2 254 112 112 64 5° ° ° ° ° ° °

39 1

to its sensitivity to the antimycotics produced by P . syringae. Elm wood extracts and expressed elm sap supported the growth and production of antimycotics by P. syringae as determined by the size of the inhibition zone . Isolate M323m consistently produced more antimycotic activity than isolate M27m. Antimycotic activity was detected in a wood extract concentration of 5 % (1·g mm" (isolate M27 m) and g'1 mrn" (isolate M323m», but the greatest activities were found at 25 % (g' 1 mm" (isolate M27m) and 20·6 mrn" (isolate M323m». In elm sap, the greatest antimycotic activity was produced at a concentration of 0'05 % ( r- B mm " (isolate M27m) and 16'9 rnm " (isolate M323m». Detectable activity also was found at a concentration of 0'01 % (o -S rnm" (isolate M27m) and 5'9 mm " (isolate M323m», which we estimate to be the natural solute concentration of elm sap. P. syringae could be isolated from all bacteriatreated seedlings, but not from seedlings treated with the fungus alone. In a few seedlings kept for

Table 2. Antimycotic actiuity of Pseudomonas syringae isolates M27m and M323m and culture filtrates from M323m against Ceratocystis ulmi Zone of inhibition (rnrn") C. ulmi isolate*

M3 23m

lA-55 (A) (NA) TN VA-BL (A) WI (A) IL-D (A) MO-Co (A) OH-5 (A) MA -N (N A) CO-FM (A) AL-Ad (A) MN-1 (A) (NA) IA-3 ME-41 (NA) NH-14 (NA) MA-B (NA) MT-1a ? CD-5 F (A) IA-2 (N A) NY-CI ? NY-42 (N A) ME-Or (I) OH-9 (A) (A) VT-7 MT-1d ? MO-66 ?

7 13 20 20 39 50 5° 79 95 113 133 154 201 227 227 346 380 380 415 4 15 45 2 45 2 491 49 1 531

M3 23m culture filtrates 79 7°7 95 908 go8 855 855 572 804 855 855

M27m 28 380 20

153 380

254 201 754 804 201 754 804 6 15 254 254 7°7 660 804 201 804 3 14 660 254 201 855 754 754 3 14 * Isolates were designated by an abbreviation for the state where they were isolated, by an abbreviation for the city or by a number. The aggressiveness rating (A = aggressive , I = intermediate, NA = not aggressive) was determined by standard pathogenicity tests made by the donor of the isolate.

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Pseudomonas antagonising Ceratocystis ulmi

Table 3. Symptoms of Dutch elm disease in greenhouse-grown American elm seedlings treated with Pseudomonas syringae isolates and inoculated with Ceratocystis ulmi Treatment with P. syringae Isolates producing antimycotics

Isolates not producing antimycotics

Not treated M323m M27m M323nm (a) Vascular discolouration (%)* Inoculated with C. ulmi Not inoculated with C. ulmi

54Y (11)

21x (11)

2X(8)

67 Y(10) OX

(8)

OX

(8)

(b) Percentage of trees showing wilt or yellowing

Inoculated with C. ulmi Not inoculated with C. ulmi

73

9

0

20

50

0

0

0

0

0

* Values followed by the same letter are not significantly different (P = 0'05) according to the Newman Keuls Test. The number of plants in each treatment is shown in parentheses. observation in the greenhouse, the bacteria could be isolated up to 1 year after treatment. P. syringae was found an average of 21 em (range 0-64) from the injection point in seedlings treated with isolate M323m and 8 em (range 0-12) in seedlings inoculated with M27m. No attempt was made to determine concentrations of bacteria in treated seedlings. C. ulmi was isolated from all seedlings inoculated either with C. ulmi alone or with combinations of C. ulmi and P. syringae isolates not producing antimycotics. In seedlings inoculated either with the antimycotic-producing isolates M323m or M27m and then challenged with C. ulmi, the fungus was isolated in 82 and 88 % of these seedlings respectively. In these plants, the fungus could be isolated an average of 58 ern (range 4Cr(1) from the injection point in those treated with M323m and 5 ern (range 0-10) with M27m. Vascular discolouration was observed in all greenhouse-grown elms inoculated with C. ulmi and not treated with P. syringae. Treatment of elm seedlings with either of the antimycotic-producing isolates of P. syringae, M323m or M27m, significantly reduced vascular discolouration compared to seedlings inoculated with M323nm, M27nm, or C. ulmi alone (Table 3). Isolates M27m and M27nm produced no phytotoxicity and M323m and M323nm caused a small amount of phytotoxicity as measured by vascular discolouration. The nonantimycotic-producing M323nm and M27nm failed to reduce the amount of vascular discolouration in seedlings challenged with C. ulmi. Treatment of these elms with either M323m, M323nm, or

M27m also significantly reduced the percentage of C. ulmi-inoculated seedlings showing wilt or yellowing. The amount of crown symptoms per seedling was, however, extremely variable and crown symptoms were not observed in all trees inoculated with C. ulmi, so crown symptoms were not used as a measure of effectiveness. DISCUSSION

The suppressive effects of P. syringae treatments on vascular discolouration may be significant to future attempts to develop this method as a control for Dutch elm disease. These effects were probably not due to the introduction of exogenous antimycotics with the bacteria, because the bacteria produced relatively small amounts of antimycotics on the medium used to grow them for uptake studies, and because bacterial cells were washed prior to the treatment. Since isolates of P. syringae that do not produce antimycotics had no significant effect on the development of vascular discolouration, we conclude that production of antimycotics in vivo is a possible factor in the suppression of vascular discolouration. Nevertheless, the mechanism of suppression is unknown. Because antimycotics produced by P. syringae are toxic to many isolates of C. ulmi in vitro, they may have direct toxic effects on the pathogen in vivo. Other mechanisms, however, including the induction of host resistance mechanisms by the antimycotic or the production of other toxic compounds by P. syringae may be involved.

D. F. Myers and G. A. Strobel The high percentage of C. ulm i recovered in seedlings treated with antimycotic-producing isolates of P. syringa e indicates that the fungus was not eradicated. We do not know if the population ofthe fungusin treated seedlings was reduced. Commonly, initially successful therapeutic treatments to arrest Dutch elm disease in the field fail in the subsequent growing season because the pathogen has not been eradicated (Sinclair & Campana, 1978). Nevertheless, in some bacteria-treated seedlings inoculated with C. ulmi, the fungus was eradicated in parts of the apoplast where antimycotic-producing bacteria were found and also in adjacent tissues, but not throughout the apoplast (M yers & Strobel, unpubl.). This indicates that suppressive effects may not be transmitted large distances from the bacteria. The final distribution of the bacteria in seedlings is determined both by their location immediately after introduction and their ability to move subsequently. In our experiments, P. syringae was found at varying distances from the point of introduction at the end of the experiment, but not throughout the seedlings in all cases. Methods of introduction including pressure injection techniques, quantitative changes in bacterial populations after introduction using marked strains, detection and distribution of the antimycotics in the tree, and other possible mechanisms of supp ression need to be studied. A strategy using a single isolate of P. syringae may not be the best choice for modifying apoplast microflora. Of the 25 isolates of C. ulmi tested for sensitivity to P. syringae , four isolates showed little sensitivity to M323m. Nevertheless, one of the four, isolate TN, was highly sensitive to isolate M27m. This indicates that mixtures of several antimycotic-producing isolates may be useful in further testing of this strategy. Stimulation of antimycotic production by chemical treatments of elm (My ers, Sands & Strobel, 1978) may produce temporarily higher amounts of antimycotics and increa se the suppressive effects in vivo. The long-term effects of P. syringa e on elm must be studied as well as the possible serious consequences to other crops of introducing new isolates of P. syr ingae into geographical areas where they are not found . Finally, more effective antagonists might be engineered by transferring the plasmid presumably coding for antimycotic production (Gonzales & Vida ver, 1977; Hopwood, 1978) from P. syringae into bacterial residents of the elm apoplast. Resident bacteria are common in the wetwood and apoplast of elm (Stipes & Campana, 1981). Our results can be used to demonstrate only a possibility that this strategy could be employed in disease control. While vascular discolouration is a significant internal symptom of the disease and

393

useful indicator of microbial antagonism, the primary external symptoms, wilt and crown discolouration, must also be evaluated. In our greenhouse experiments, the appearance of crown symptoms was too variable to draw any useful conclusions. Crown symptoms will, therefore, have to be evaluated in mature trees in the field. Currently, extensive field testing is underway in Canada, Holland, the United States, and Great Britain. While this strategy does not provide, at its present stage of development, a control for Dutch elm disease, it does provide a significant new direction for future research aimed at exploiting microbial antagonism for the control of diseases of perennial crops. The authors gratefully acknowledge the technical assistance of Ms M. Kenfield and Mr K. Bodily, and the many discussions with Drs D. Sands and F. Holmes. Mr R. Johnston and Dr J. Martin helped with statistical analyses. This project was supported by a DUEL grant from the Freshwater Biology Foundation, Navarre, MN . It is a contribution of the Montana Agricultural Experiment Station, paper no . 936, Journal series. During a portion of this work Gary A. Strobel was the principal investigator on NSF grant ISP-7916343. REFERENCES

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Pseudomonas antagonising Ceratocystis ulmi

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(Received for publication 19 May 1982)