Response of bean calli to filtrate from Pseudomonas syringae pv. phaseolicola and correlation with whole plant disease reaction

Physiological andMolecular PlantPathology (1986) 28,353-358

Response of bean calli to filtrate from Pseudomonas syringae pv. phaseolico/a and correlation with whole plant disease reactiorr] C. 1. HARTMAN, G. A. SECOR,]. R. VENETIE and D. A. ALBAUGH Department of PlantPathology, North Dakota SlateUnioersuy, Fargo, ND 58105, U.S.A. (Aceeptdfor publication November 1985)

Callus cultures of bean (Phaseolus vulgaris L.) cultivars were tested for response to toxic filtrates from liquid cultures of Pseudomonas .ryrillgae pv. phaseolicola. Callus reaction to the filtrates ranged from necrosis and reduced growth to no necrosis and normal growth. Plants of each cultivar were also inoculated in the greenhouse with suspensions of the pathogen. Charlevoix (red kidney) was the most susceptible in the greenhouse, and Charlevoix calli were most severely affected by the filtrate. Emerson (great northern) or Admiral (navy) were most resistant to the strains in the greenhouse, and the corresponding calli developed no necrosis on medium containing filtrate. Plant and calli reactions were correlated using Spearman's ranking correlation coefficient, and a highly significant r value (0'971) was obtained. Results suggest that a callus screening system can identify bean cultivars resistant to halo blight.

INTRODUCTION

Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Burkholder 1926) Young, Dye, and Wilkie 1978 (ISPP List 1980) is a serious disease of bean iPhaseolus vulgaris L.) in many bean producing areas of the world. Current methods of screening for halo blight resistance involve inoculating bean plants with the pathogen and ranking disease severity. This method is both labor and greenhouse intensive; and reactions during testing may vary with environmental conditions. Pseudomonas syringae pv. phaseolicola produces phaseolotoxin, (N-phosphosulphamyl) ornithylalanylhomoarginine [12], which causes the chlorotic halos and systemic chlorosis typical ofbean halo blight disease [11]. Bejaj & Saettler [1] found a growth inhibition response of bean callus tissue to P. syringae pv. phaseolicola filtrate in medium due to the presence of this "halo toxin". Cell culture systems have been used successfully for in vitro selection for disease resistance. Several of these systems have used a toxic filtrate and have established a correlation between toxin resistance and resistance to the pathogen (2, 7, 14, 16]. A similar system could be utilized to test dry bean genotypes for resistance to bacterial pathogens, since beans readily initiate callus in culture and the halo blight pathogen produces toxin in culture. Resistance has been reported in cultured meristems screened using phaseolotoxin [6J. The objective of this study was to determine whether a callus Abbreviations used in text: MB5, modified B5 medium; MB5-fil, MB5 medium with culture filtrate; NDB, nutrient dextrose broth. tPublished with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article number 1426. 0885-5765/86/030353 + 06 $03.00/0

© 1986 Academic Press Inc. (London) Limited

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screening system for resistance to P. syringae pv . phaseolicola in beans could be reliably used instead of greenhouse inoculations.

MATERIALS AND METHODS

Pseudomonas syringae pu. phaseolicola strains Four strains of P. syringae pv phaseolicola were used for callus and greenhouse testing. The first (Strain V) , a highly virul ent strain, was initially isolated from a diseased plant collected in eastern North Dakota. Axenic cultures were repeatedly tested on, and reisolated from red kidney cv. Charlevoix to insure pathogenicity . Strain V had th e biochemical characteristrics of the species [15]. Isolate HB-38 was obtained from A. W . Saettler, Michigan State University, isolate 83-K2 was obtained from A. K. Vidaver, University of Nebraska, and a phaseolotoxinless mutant, isolate G-50, was obtained from S. S. PatiI, University of Hawaii. Pseudomonas syringae pv . phaseolicola cultures used to produce filtrate for in vitro tests and inoculum for greenhouse inoculations were grown in 100 ml nutrient dextrose broth (N DB) for 72 h on an orbital shaker (100 r min - 1) in 250 ml flasks at 26 "C. Flask s were inoculated with I ml of bacterial suspension, tubidometrically standardized at 3 x 108 cfu ml " ! [15]. Extracts to be used for callus testing were filter sterilized by passage through a 0·22 urn nitrocellulose filter . Crude filtrate was added to the test medium after the medium had been autoclaved and cooled to 45 °C.

Callus initiationand proliferation Callus cultures were initiated from 5 mm 2 sections of newly opened trifoliate leaves of bean cultivars. Leaves were surface sterilized in 70% ethanol for 5 min, transferred to 0'5% NaGCI for 5 min and rinsed in two changes ofsterile distilled water. Leafsections were aseptically placed on modified B5 medium (M B5) [5] and incubated for one month. The MB5 con tained 2 mg 1-1 a-naphthalcneacetic acid, 2 mg 1- 1 2,4-d ichlorophenoxyacetic acid and I mg 1- 1 kinetin.

Callustesting To test for toxic components in ND13, 300 mll - 1 l"'DB was incorporated into MB5. After 1 month on MR5 medium, calli (approx. Q. I 50 g) of each of the cultivars were placed on test media (five colonies per plate). Calli on MB5 were used as the control. Plates were incubated in clear plastic boxes at 22 ± 1 °C with continuous, cool, fluorescent light. Fresh weights used to compare callus growth on MB5, MB5 with NDB and MB5 with culture filtrate (MB5-fil) were obtained from 10 plates per cultivar per treatment, and results were used to determine the influence of media type on callus growth. In the second exp eriment, calli (as above) of nine bean cultiva rs were placed on media containing 300 ml offiltrate from 48 h cultures of P. syringae pv. phaseolieola, Strain V. The purpose of this experiment was to determine ifcuitivars of varying resistan ce co P. syringae pv. phaseolicola responded differentially in vitro. After 1 month, the area of browning and percent necrosis was tallied for each culti var, Cultivars were then ranked according to severity of necrosis.

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355

In the third experiment, calli of 10 cultivars were placed on media containing 200 ml of filtrate from 48 h cultures of either Strain V, 83-K2, HB-38, or G-50. At this rate weight differences were observed, and cultures were ranked according to fresh weight. The purpose of this experiment was to determine if differences in P. syringae pv. phaseolicola pathogenicity could be detected invitro. After I month, calli from 10 plates per cultivar per treatment were weighed.

Greenhouse testing Seed from bean cultivars were planted in 6-inch pots in pasteurized soil and grown under natural light in the greenhouse at 24± 5 ac. Ten plants per cultivar were inoculated with P. syringae pv. phaseolicola within 48 h after the unifoliate leaves had flattened. Both unifoliate leaves were inoculated (one inoculation site on each side ofthe mid-vein) with a suspension of P. syringae pv. phaseolicola diluted to 3 x 10 6 cfu ml in sterile distilled water with 0·002% Tween 80 (Polyoxyethylene (80) Sorbitan Monooleate) (10). The inoculum was injected into the leaves using an artists' air brush sprayer at 1·13 kg cm- 2 air pressure [9]. Sterile water inoculations were used as controls. Three weeks after inoculation, plants were rated for disease reaction according to a greenhouse scale of increasing plan t susceptibility. 1. No symptoms. 2. Light water congestion. 3. Moderate water congestion-no chlorosis. 4. Moderate water congestion with chlorosis on upper leaf surface. 5. Heavy water congestion at main infection site with congestion spots away from main site-chlorosis or darkening of upper leaf surface. 6. Heavy water congestion over large areas oflower leaf surface including spotting away from main site. Darkening of site on upper leafsurface--discoloration of veins on lower surface and chlorotic halo around main site. 7. Darkening of main infection site upper and lower leaf surfaces. Tissue collapse beginning, but encompassing less than 50% of main infection site. 8. Tissue collapse of entire main infection, necrosis and upper leaf surface discoloration at spots away from main site.

Spearman's coefficient of rank correlation [3] was used to test the relationship between callus screening rankings and greenhouse screening rankings. A significant coefficient would indicate that susceptibility rankings from in vitro screenings could be used as a substitute for greenhouse screening. RESULTS AND DISCUSSION

Comparison of callus fresh weights from seven cultivars of beans grown on MB5, MB5-NDB, or MB5-fil showed that MB5-fil significantly reduced growth in all cases (Table I). Although the addition of nutrient dextrose broth (MB5-NDB) significantly reduced growth compared with the control MB5, this clearly was not the cause of the drastic growth reduction displayed in MB5-fil treatments (Table I). Calli reactions (Table 2) ranged from 100% necrosis in Charlevoix to 0% necrosis in Emerson. Reaction was consistent over two trials although some intermediately

C. L. Hartman et al.

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1 Comparison offiesh weights (g)" rif calli from seven cultiuats if beans grown on tissue culture medium (MB5) , MB5 wilh }fDB, and MB5 with culturefiltmle ( M B5-jiI) from Pseudomonas syringae po. phaseolicola TABLE

Media type Cultivar Charlevoix Black Turtle Olathe Admiral Cranberry Mecosta Ouray

MB5

MB5·NDB

MB5·fil

3'94-ab 4-'59a 5·73a 3·82a 3·63a 7·33a 1·96a

2·14-b 4·41a g·32b 3·04-b 2.50b 4·95b 1·I4-b

O·BOb O..H e O·71c 0·35c 0·42c O·BOe

oase

"Average fresh weight often plates. bN umbers followed by the same letter (from left to right , in the same row ) are not sign ificantl y different (P = O'OI) according to Duncan's new multiple range test.

TABLE 2 Correlation ofseedlingpathogenicity lest with callitestingofninebean cultiuars ( Pseudomonas syringae pu. phaseolicola Strain V)

Gr eenhouse seedling susceptibility ra nk

Percentage callus necrosis Class Dark red kidney Snap Cranberry Ligh t red kidney Pinto Small Re d Black turtle Pinto Great northern

Cultivar

Trial I

Trial 2

Pooled"

Trial I

Charlevoix Early Gallatin M ichicran Mec osta Resist o UI3 7 T-39 O uray Emerson

100b

100

100

10

9490 B2

91 95 79 54 56 41 33 0

93 93 BI 61 55 45 31 0

2 43 5 6 7 8 9

69 53

49 30 0

Trial 2

Pooled

2 3

2 3 S 6 5 7 B 9

4-

7 5 6 8 9

"S p earman's Ranking Co rrelati on Coefficient r=0'97 I for pooled data of two trials, significant at a~O ·OI . bAverage of I0 calli per tre atment. Filtrate rate of 300 mll- ' . e I = most susceptible, 9 = most resistant, based on average of 10 plants/cui tivar.

reacting cultivars changed by a rank of one or two. Percentage callus necrosis was significantly correlated with greenhouse seedling susceptibility rank (r= 0,9 71 , a~O ·Ol ) .

Cultiva rs rea c ted differentially to strain of bacteria used (Table 3) . Charlevoix appeared to be universally susceptible while Emerson and Admiral showed resistance

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357

3 Weights of calli derived from ten bean cultiuars grown in media with culture filtrate from four strains of Pseudomonas syringae p», phaseolicola compared togreenhouse pathogenicity Jests TABLE

Strain V Cultivar Charlevoix Cranberry Early Gallatin Olathe Resisto Ouray Black Turtle Mecosta Emerson Admiral Spearman's ranking correlation coefficient

Strain 83·K2

Filtrate' GH b 0·39 0·34 0·88 1·31 0·35 0·82 0·80 0·65 2·11 2·35

(3) (1) (7) (8) (2) (6) (5) (4) (9) (10)

0·842*

I 2 5 6 3 7 8 4 10 9

Filtrate 0·23 0·59 0·39 1·44 0·27 0·85 0·63 2·30 1·06 2·33

(1) (4) (3) (8) (2) (6) (5) (9) (7) (10)

0·648*

GH b

I 4 2 3 7 5 6 8 9 10

Strain HB-38 Filtrate 0·35 0·54 0·69 1·91 0·29 0·55 2·65

(2) (3) (4) (6)

(I) (5) (8)

HO(IO} 3·15 (9) 2·22 (7)

0·709*

GH b 1 3 2

4 6 5 7 8 9 10

Strain G-50" Filtrate 0·87 1·07 1·26 1·88 1·04 1·34 1·75 \060 2·03 1·67

GH b

(I)

I

(3) (4) (9) (2) (5) (8) (6) (10) (7)

2 3 5 4 8 6 7 9

!O

0·721 *

'Callus fresh weight in grams; filtrate rate of 200 mil-i. Numbers in parenthesis are rankings of calli. bGreenhouse rank determined by greenhouse rating scale. "Phaseclotoxinless mutant. *u<;;0·05.

to all strains tested. Mecosta, usually resistant, was susceptible to Strain V, indicating that a callus screening system could be used to detect the occurrence of new pathogenic strains of P. syringae pv. phaseolicola. Reduction of callus fresh weight was significantly correlated with greenhouse seedling susceptibility rank for all four strains (Table 3). Although strain G-50 did not produce phaseolotoxin, it was capable of infection and caused reduced growth of callus cultures. This may indicate the presence of other toxic metabolites. This suggests that crude filtrates may be better material for screening than purified phaseolotoxin. In greenhouse tests for experiments two and three, the most susceptible cultivars developed lesions with chocolate brown centers surrounded by chlorotic regions on the leaf. As the disease progressed, systemic toxemia and general chlorosis developed in the new tissue resulting in plant death of the most susceptible cultivar, Charlevoix. More resistant cultivars initially developed necrotic tissue at the lesion center, but new leaves did not express systems. The most resistant cultivars, Emerson and Admiral, appeared to completely recover from initial symptoms of the disease. Callus reactions based on either percentage necrosis (Table 2) or fresh weight (Table 3) were significantly correlated with symptom production in the greenhouse inoculated bean cultivars. Resistant and susceptible cultivars were easily distinguished using the callus screening system. The callus screening system was not as useful in separating cultivars with intermediate reactions, yet this is also a problem with greenhouse screening. The high correlation between callus reactions and whole plant disease reaction indicates that callus testing could be used for screening Phaseolus

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germplasm for halo blight resistance. Callus testing also appears to be useful in distinguishing bacterial strains. By expanding the cell culture filtrate system to include developed cultivars, the practicality of a tissue culture assa y is increased. If cell culture screening can provide a convenient, more accurate method of evaluating bean cultivars for resistance to halo light, then the breeder is provided with a helpful tool. The authors wish to thank]. Tuskan and K. Grafton for critical evaluation of this manuscript and help with statistical methods. REFERENCES I. BAJAJ, Y. P. S. & SAETTLER, A. W. (1970). Effect of halo toxin-containing filtrates of Pseudomonas phaseolicola on the growth of bean callus tissue. Phytopathology 70,1065-1067. 2. BEHNKE, M. (1980). General resistance to late blight of Solanum tuberosum plants regenerated from callus resistant to culture filtrates of Phytophthora infastens. Theoretical Applied Genetics 56, 151-152. 3. DAiIIlEL, W. W. (1978). Rank correlation and other measures of association. In Applied Nonparametric Statistics, pp. 300-306. Houghton Mifflin Company, Boston, U.S.A. 4. DURBIN, R. D. (1982). Toxin and pathogenesis. In Phytopathogenic Prokatyotes, Vol. l., Ed. by M.S. Mount & G. H. Lacy, pp. 423--441. Academic Press, New York. 5. GAMBoRG, O. L., MILLER, R. A. & OJIMA, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. ExperimentalCellResearch 50, 151-158. 6. GANTOTTI, B. V., KARTHA,K. K. & PATIL, S. S. (1985). In vitro selection of phaseo lotoxin resistant plants using mcristern culture of bean tPhoseolus vulgaris). Phytopathology 75, 1316-1317. 7. HARTMAN, C. 1., MoCov, T. J. & KNOUS, T. R. (1984). Selection of alfalfa (Mtdicago sativa) cell lines 8.

9. 10. I I. 12. 13. 14. 15.

16.

and regeneration of plants resistant to the toxin(s) produced by Fusarium oxysporum r. sp. medicaginis. Plant Science Letters34,183-194. HILDEBRAND, D. C. & SCHROTH, M. N. (J971). Identification of the fluorescent pseudomanads. In Proceedings of tile Third International Conference oj Plant Pathogenic Bacteria, pp. 281-289. Wageningen, The Netherlands. KENNEDY, B. W. & CROSS,]. E. (1966). Inoculation procedures for comparing reactions of soybeans to bacterial blight. Plant Disease Reporter SO, 560-565. LAWRENCE,J. A. & REYNOLDS, K. 1. (1984). Growth and lesion development of Xanthomonas campestris pv. phaseoli on leaves of red kidney bean plants exposed to hydrogen fluoride. Plrytopathology 74, 578-580. MITCHELL, R. E. & BIELESKI, R. 1. (1977). Involvement of phaseolotoxin in halo blight of beans. Transport and conversion to functional toxin. Plant Physiology 60, 723-729. MOORE, R. E., NIEMCZUR, W. P., Kwox, O. C. H. & PATlL, S. S. (1984). Inhibitors or ornithine carbamoyl transferase from Pseudomonas syringae pv. phaseolotoxin. Tetrahedron Letters 25, 3931-3934. MURASHlGE, T. & SKOOG, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologica Plan/arum 15, 473-497. SACRISTAN, M. D. (1982). Resistance responses to Phoma lingam of plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus. Theoretical AppliedGenetics 61, 193-200. SCHAAD, N. W. (1980). Initial identification of common genera. In Laboratory Guide for Identification of Plant Pathogenic Bacteria, Ed. by N. W. Schaad, pp. I-II. American Phytopathological Society, St. Paul, Minnesota, U.S.A. THANUTONG, P., FURUSAWA, I. & YAMAMOTO, M. (1983). Resistant tobacco plants from protoplastderived calluses selected for their resistance to Pseudomonas and Alternaria toxins. Theoretical Applied Genetics 66,209-215.