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Comparison of protein-lipopolysaccharide complexes produced by pathogenic and non-pathogenic strains of Verticillium dahliae Kleb. from potato
Physiological
Plant PatholoD
(1982)
20,2
13-22 1
Comparison of protein-lipopolysaccharide complexes produced by pathogenic and non-pathogenic strains of Verticillium dahliae Kleb. from potatot NACHMIAS$,
ABRAHAM $Dizkion $Department
of Plant Pathology,
VIRGINIA Agricultural
BUCHNER
9 and JAMES
Research Organization,
of Organic
Chemistry,
Weizmann
(Accepted for publication
November
1981)
Institute
Gilat
KRIKUN~ Expekent
Station, Negev,
Israel
of Science, Rehovot,Israel
Phytotoxic protein-lipopolysaccharide (PLP) complexes were isolated from dialyzed culture fluids of a pathogenic strain of Verticillium dahliae; PLP complexes which lack phytotoxic activity were isolated from a mutant non-pathogenic strain of the fungus. A comparison of profiles of the wild-type and mutant PLP eluting from an Agarose A-5 m column revealed quantitative differences between the 2 major protein peaks of each strain. The corresponding peaks of the 2 strains had similar molecular weights and in gel immunodiffusion were antigenically indistinguishable, but differences between them were seen in their chemical composition when analysed for protein, lipid and carbohydrate content. Antiserum prepared againt the components of peak 1 from the pathogenic strain reacted with an antigen in extracts of Vertisillium-infected potato plant tissue which was apparently identical to a moiety produced by the pathogen in culture.
INTRODUCTION
Certain Verticillium species produce large amounts of extracellular materials which have been related to the production of wilt symptoms. The possibility that toxins may be produced in liquid culture by tomato isolates V. dahliae and V. albo-atrum was first reported by Bewley [I]. Subsequently, other evidence was presented for the presence of toxic substances in culture filtrates of Verticillium spp. isolated from tomato [I, 51, tobacco [17], lucerne [18] and cotton [12, 13, IS, 191. Phytotoxic metabolites elaborated by these fungi in culture include low molecular weight compounds [8] and high molecular weight compounds [24] such as enzymes [IO, II], proteins [19] and polysaccharides [23]. Malysheva & Zel’tser [16] described a toxic protein-lipopolysaccharide complex (PLP) produced by a V. albo-atrum isolated from cotton plants and this was confirmed by Keen et al. [12, 131, who found a correlation between the susceptibility of numerous varieties of cotton to Verticillium wilt and their sensitivity to the PLP in a cotton leaf bioassay. From these results they concluded that the PLP may be implicated in the development of disease symptoms in cotton. Verticillium wilt is a major limitingfactorin potato production throughout the world, especially in arid regions under irrigation [14]. V. dahliae is the major pathogenic species in hot, dry climates [14]. In an attempt to develop a bioassay which could tNo.
183E
0048459/82/0202
1981 series 13 +
10 $03.00/O
@ 1982 Academic
Press Inc.
(London)
Limited
214
A. Nachmias,
V. Buchner
and J. Krikun
be used for the screening of potato cultivars for susceptibility to V. dahlias, we investigated the use of phytotoxic PLP complexes obtained from liquid cultures of an isolate of V. dahlia which was pathogenic to potato. Some plants can respond non-specifically to high molecular weight compounds and so it was necessary to ensure that compounds were not present in the extracts which mimicked the effects of the PLP complex and so caused positive reactions in resistant plant varieties. This problem might be overcome using, as a control, similar PLP complexes isolated from a non-pathogenic mutant strain of the fungus and the objective of the present study was to compare the chemical and immunological properties of PLP complexes obtained from a pathogenic potato isolate of V. dahliae with those obtained from cultures of a non-pathogenic mutant derived from it. MATERIALS
AND
METHODS
Culture methods
A pathogenic isolate of V. dahlias was isolated from an infected potato plant. A single spore isolate (Gl) was maintained on potato-dextrose agar (Difco). A spontaneous mutant (V297), arising from the single spore isolate of Gl, was selected on the same medium. Isolates of V. dahliae were tested for pathogenicity by artificial inoculation into soil, by injection into stems, or by dipping rooted cuttings into a spore suspension, Gl, which produces microsclerotia, was pathogenic by all 3 criteria; V297 did not produce microsclerotia and was not pathogenic. For production of the PLP complex, the fungi were transferred to Roux bottles, each containing 100 ml of a medium containing per litre of distilled water: glucose 20 g, asparagine 2 g, KH,PO, 1.5 g, MgSOI. 7H,O 1 g, Z&O,. 7H,O 20 mg, FeSO,. 5H,O 10 mg, CaSO, 6 mg, thiamine HCl 10 mg and pyridoxine 5 mg. The pH was adjusted to 6.7 with KOH. The cultures were incubated for 21 days at 26 “C in the dark. Bioassay
Culture fluids, after dialysis (against distilled water 1 : lO*OOO,v/v for 10 h), and PLP complexes were bioassayed for symptom-producing activity using detached leaves. The first true leaves of 3- to 6-week-old plants (potato cv. Spunta, tomato, eggplant and cotton) were excised under water and placed in vials containing 20 ml water. One hundred micrograms of the test solution in 0.2 ml was injected into the intercellular spaces of the leaves. The concentration of the test solution to be used was determined by a dilution end-point test on leaves. The highest dilution showing a clearly detectable difference between susceptible and resistant plants was employed. After injection the leaves were placed under continuous illumination (c. 7000 lx) and observed for symptoms of chlorosis and necrosis after 18 to 36 h; the degree of necrosis was determined after 72 h. Purifkation
procedure
The mycelium was removed from 21-day-old cultures by filtration and the filtrate was concentrated under vacuum at 50 “C. The concentrated filtrate was treated
Comparison
of protein-lipopolysaccharide
complexes
215
with 4 volumes of acetone at - 18 “C and allowed to stand overnight. The precipitate was collected by centrifugation at 5000 g for 15 min at 5 “C, dried and dissolved in distilled water (l/50 volume of the initial volume of the culture fluid). The solution was then loaded on to an Agarose A-5 m (Bio Rad) column (3 x 70 cm), which was eluted with distilled water at room temperature. The flow rate was 7 ml h-1 and 2-ml fractions were collected. The column was calibrated using tobacco mosaic virus (5 x lo6 daltons) as void volume marker. Peak fractions giving phytotoxic activity were pooled and concentrated as above. Analytical
methods
Protein was determined by absorbance measurement at 280 nm and by the method of Lowry et al. [15], using bovine albumin as standard. Polysaccharide was determined by the anthrone method [7], using D-&COSe as standard. Lipid was determined gravimetrically following hydrolysis of the complex in 1 N HCl for 60 min at 100 “C and extraction into diethyl ether. Identification of the carbohydrate components of the complex was carried out by gas-liquid chromatography (g.1.c.) according to Clamp et al. [4]. Electrophoresis
Disc gel electrophoresis was performed on cylindrical gels (10 cm) of 5% polyacrylamide with 0.2 M Tris-glycine buffer, pH 8.3, 4 mA per tube until the bromophenol blue marker reached the end of the tube [21]. The gels were stained with Coomassie blue R 250 for protein [S] or with Schiff’s base reagent for glycoproteins [26]. Isoelectric focusing was performed using the method of Wrigley [Zq. Antiserum production
and immunodajiiion
technique
The PLP complexes were each dissolved in 1.5 ml of 0.15 M phosphate saline buffer, pH 6.8, and emulsified by sonication for 5 min with 1.5 ml of Freund’s complete adjuvant. The antigens were injected subcutaneously into the flanks of New Zealand rabbits. A booster injection of the same material in Freund’s incomplete adjuvant was made 3 weeks later and bleeding was performed from the marginal ear vein 3 weeks after the booster injection. Immunodiffusion analysis was performed using the Ouchterlony double diffusion technique [22] in a medium consisting of O*8o/o agarose gel, 0.15 M NaCl in 60 mM barbital buffer, pH 8.6, and 0.1% sodium azide as preservative. RESULTS
Cell-free, dialysed culture filtrates from the pathogenic strain, Gl, when injected into potato leaves, induced interveinal chlorosis followed by necrosis, similar to the symptoms of wilt observed on Verticillium-infected plant (Plate 1). Dialysed culture filtrates of the non-pathogenic mutant, V297, had no detectable effect on potato leaf tissue. The phytotoxic moiety was usually present throughout the logarithmic growth phase of Gl; but the culture could only be differentiated from that of the mutant
216
A. Nachmias,
V. Buchner
and J. Krikun
at the end of the log phase, when it began to produce microsclerotia. In order to rule out the possibility of a reverse mutation to microsclerotia formation in V297, it was necessary to harvest the cultures at the onset of microsclerotia production. Essentially, all of the phytotoxic activity was recovered from redissolved acetone precipitates of the dialysed culture filtrates. The acetone precipitates were chromatographed on a column of agarose A-5 m (Fig. 1). The column eluates were analysed
(a)
50 Elutian
100 volume
150 (ml)
FIG. 1. Chromatography of extracellular products from the (a) non-pathogenic (V297) and (b) pathogenic (Gl) isolates of Verticillium d&&e on an agarose A-5 m column. Acetone precipitates of dialysed V. duhliae culture filtrates (400 ml), from the pathogenic or the nonpathogenic strain, were dissolved in distilled water and loaded on to a column (3 x 70 cm). The column was eluted with distilled water at room temperature with a flow rate of 7 ml h-r; fractions of 2 ml were collected. Tobacco mosaic virus (TMV) served as the void volume marker. A, Protein; 0, polysaccharide.
for protein, carbohydrates and lipids and tested for activity in the potato leaf bioassay. The elution profiles showed two major protein-lipopolysaccharide (PLP) peaks (Fig. 1) ; fractionation of Gl resulted in a peak I 3 times larger than peak II, whereas that of V297 gave a relatively small peak I but a larger second peak. Gross chemical analysis showed quantitative differences between components of the corresponding peaks of the 2 strains (Table 1). Quantitative differences between
PLATE 1. Phytotoxic (0.5 mg ml-‘), obtained of the pathogenic isolate
symptoms on a potato leaf, 24 h after injection by agarose A-5 m filtration of acetone-precipitated of Verticillium dahliae.
with
200 ~1 peak I culture filtrates
[facing page 2161
Comparison
of protein-lipopolysaccharide
complexes
217
them were also seen when analysed by g.1.c. for carbohydrates (Table 1). The same elution pattern as shown for Gl was observed for 6 different isolates of V. dahliae, obtained from eggplant, avocado, watermelon, olive, cotton and tomato. Filtration of the 2 major peaks on a calibrated column of agarose A-5 m indicated an estimated molecular weight of 2 x IO6 daltons for peak I and 8 x lo4 daltons for the second peak in both isolates (Fig. 2). Analysis of the peaks by polyacrylamide TABLE
Co@osition
(percentage
of dry
wt) of Verticillium pur$ed by gel filtration Pathogenic Gl Peak I
Protein Lipid Polysaccharide Galactose” Glucose” ’ Percentage
1
dahliae protei~li@opolysaccharide chromatography
isolate Peak
27 6.73 64 70 29.4 of polysaccharide,
42.7 8.83 44.2 9 69
II
Non-pathogenic isolate V297 Peak I Peak 14 18.87 60 61 37.4 determined
complexes
II
17 5.47 63 0 70 by g.1.c.
FIG. 2. Molecular weight determinations of K dahliae PLP complexes by chromatography on a column of agarose A-5 m. The column was calibrated with blue dcxtran 2000 (2 x 10s daltons), thyroglobulin, catalase, aldolase and ovalbumin.
gel electrophoresis detected 1 major band in each in both strains, and at least 5 to 7 minor bands reacting with Coomassie blue. Some of the bands gave a positive reaction with Schiff’s base stain. Isoelectric focusing of the complexes also detected several bands in each, with the major band of peak I at pH 3.8 and that of peak II at pH 4.0. Both peaks of Gl were phytotoxic to leaves of potato and certain other host plants, but not to leaves of several non-host plants (Table 2). While neither peak of the mutant was toxic for potato, each fraction was occasionally toxic for other
A. Nachmias, V. Buchner and J. Krikun
218 Phyfotoxidy of Verticillium
dahliae
TABLE 2 ~otein-lipoporysaccharidc Pathogenic
isolate Gl
Peak Potato cultivar spunta Tomato (Hosen Avocado Eggplant Olive Pepper citrus Lemon Sour orange Volkamariana Sudangrass
I
Peak
II
com#exes
on various
Non-pathogenic isolate V297 Peak I Peak
T +
+ + + + -
+ -
-
+
+
-
-
+
-
-
+
Eilon)
The bioassay was carried out by injecting ml-l). +, Symptom production, chlorosis treatment; -, no reaction.
200 ~1 liquid and necrosis,
plants
II
solution (0.5 mg PLP complex was recorded 18 to 36 h after
(b)
FIG. 3. Diagrams of the immunodiffusion patterns obtained when partially purified complexes and potato plant extracts were reacted with antisera to peak I or peak II of Gl. The inner wells contain: 1, anti-peak I; or 2, anti-peak II. The outer wells contain: (a) A,peakIofGl;B,peakIofV297;(b)A,peakIofG1;B,peakIIofG1; (c)A,peakIIof Gl ; B, peak II of V297; (d) A, peak I of Gl ; B, peak II of Gl : (e) A, peak I of Gl ; B, extract of Veium dkhliae-Meted potato plant tissue; C, extract of healthy potato plant tissue.
plants. A plant species was considered sensitive in the bioassay only if the nonpathogenic PLP gave a concomitant negative reaction. The phytotoxic activity was resistant to most attempts at inactivation, which included enzymatic treatment with protease, papain, a-arnylase and lipase; chemical treatment with potassium periodate or mild base; and heating to 98 “C for 20 min. The biological activity was destroyed only by digestion with 1 N HCI for 60 min at 110 “C.
Comparison
of protein-lipopolysaccharide
complexes
219
Antisera prepared in rabbits against peak I or peak II of Cl, reacted equally well with both peaks (Fig. 3). However, when peak I and peak II were reacted with antiserum of peak II, spurs formed between the precipitin bands, showing that each peak had some determinants lacking in the other [Fig. 3(d)]. Peaks I of Gl and V297 showed serological identity [Fig. 3(a)] when reacted with antipeak I while peaks II of the 2 strains gave a reaction of identity with anti-peak II [Fig. 3(c)]. Wh en antiserum to peak I was reacted with a water extract of V. dahliaeinfected potato tissue, a precipitin band formed which fused with that of peak I of Cl, suggesting that a substance present in V. dahlias-infected potato plants is similar to a moiety produced by the fungus in culture. DISCUSSION
The data presented here confirm the observations of others [12, 13, 161 that phytotoxic, heat-stable PLP complexes can be isolated from culture fluids of Verticillium species. Although we employed a different purification procedure, the acetoneprecipitable complexes obtained were similar to the PLP described by Keen & Long [12]. Whereas Keen et al. [13] studied only the properties of peak I, we have investigated both fractions, since both peaks of the pathogenic strain were toxic to leaves of potato and other host plants of V. dahlias. Both peaks of Gl showed specificity in the leaf bioassay because only susceptible plants were sensitive to the material in the absence of a positive reaction by the mutant PLP. between In double immunodiffusion analysis, the reaction of non-identity peaks I and II of Gl ruled out the possibility that peak I was an aggregate of components of peak II. The cross-reactivity of the antisera to the 2 fractions showed that the column-purified peaks were not homogenous and that 1 may have been contaminated with the other. This observation was confirmed by the analysis of the fractions by polyacrylamide gel electrophoresis. Although the PLP complexes of peaks I and II of V297 were serologically identical to the corresponding peaks of Cl and were of similar molecular size, there appeared to be differences between them in their chemical composition. While all the fractions were predominantly carbohydrate, the mutant peaks contained less galactose and significantly lower amounts of protein. These differences in composition may reflect qualitative differences between the PLPs of the two strains, or, alternatively, each peak may represent an unresolved mixture of the same PLP complex with a quantitative increase or decrease in one or more of the components of the mixture. Peak I was almost absent from the complexes of the mutant strain but it is unlikely that the increased amount of peak II resulted from dissociation of peak I, because the two fractions are serologically unrelated. Although gel filtration indicated a high molecular weight, peak I was resolved in 5% polyacrylamide gels, which excludes molecules of molecular weight greater than 3 x 10s daltons. This suggests that the complex can be broken down into smaller molecular weight compounds in the absence of dissociating conditions. This is in agreement with the observation of Keen et al. [12, 131 that PLP tends to form low molecular weight products in stationary phase cultures at high ionic strength. Furthermore, it is unlikely that the intact PLP complex is required for
220
A. Nachmias,
V. Buchner
and J. Krikun
biological activity because phytotoxicity was not inactivated by heat nor by enzymatic or chemical treatments which hydrolyse proteins, lipids and polysaccharides. It is possible that the PLP complex may be synthesized as a precursor molecule which is cleaved into smaller components; such a system has been described for tumorassociated antigens of several viral infected tumors in mice [3]. Keen et al. [13] presented evidence for the involvement of the PLP in symptom production by Verticillium in cotton, but were unable to unequivocally demonstrate its presence in diseased plant tissue. In the present work, antiserum to peak I detected the antigen in extracts of V. dahliae-infected potato tissue, but not in homogenates of healthy tissue, a result which supports the hypothesis that PLP may be involved in symptom production [13]. Whether the antigenic material and the phytotoxic moiety are related remains an open question. The data presented here suggest that V. dahlia produces at least two different phytotoxic materials in culture and that these compounds are not produced or are produced in altered form by the mutant. A more detailed examination of the relationship between the loss of pathogenicity and the alteration of PLP in the mutant, and of the importance of PLP to symptom production by the wild type will require biochemical purification of the active molecules. We wish to thank Mrs Janet Orenstein for her technical assistance. REFERENCES of tomato. Annals of Applied Biology 9, 116-134. S. (1977). Amino acid absorption along the intestinal tract of chicksfed heated and raw soybeanmeal. 3ournal of Nutrition 107, 1775-l 778. BUCHNER, V. (1979). Isolation and partial purification of a non-oncornaviral tumor specific transplantation antigen from TA3 tumor cells. Ph.D. thesis. Tel Aviv University, Ramat Aviv, Israel. CLAMP, J. R., Bmrrr, T. & C~AMEIERS, R. E. GLC technique as O-tðyl silyl ester (TMS). In Methods of Biochemistry, vol. 19, Ed. by G. Cluck, pp. 316-335. CRONSHAW, D. K. & PEGG, G. F. (1976). Ethylene as a toxin synergist in VerttiCCium wilt of tomato. Physiologial Plant Pathology 9,23-N FAZEKAS, G., WEB~R, S. & DATYNER, R. C. (1963). Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochemia et Biophysics Acta 71, 377-391. FIJNO, F., SCHEFFER, F. L. & Kmx, P. K. (1953). The ultramicro-determination of glycogen in liver. A comparison of anthrone and reducing sugars methods. Archives of Biochemistry and Biophyssics 45,319326. GREEN, R. J. (1954). A preliminary investigation of toxins produced in vitro by VerticiCCium alboatrum. Phytopathology k&433-437. HODGSON, R., PETERSON, W. H. & RIKER, A. J. (1949). The toxicity of polysaccharides and other large molecules to tomato cuttings. Phytopathology 39, 47-62. HOWELL, C. R. (1976). Use of an enzyme-deficient mutant of VerticiCCium dahCiue to assess the importance of pectolytic enzymes in symptom expression of Verticillium wilt of cotton. Physiological Plant Pathology 9,279-283. KAMAL, M. & WOOD, R. S. K. (1956). Pectic enxymes secreted by Verticillium dahliae and their role in the development of the wilt disease of cottom. Annals of Applied Biology 44, 322-340. KEEN, N. T. & LONG, M. (1972). Isolation of a protein-lipopolysaccharide complex from VerticilCium albo-atrum. PhysioCogicaC Plant PathoCogy 2, 307-315. KEEN, N. T., LONG, M. & ERWIN, D. C. (1972). Possible involvement of a pathogen-produced protein-lipopolysaccharide complex in Verticillium wilt of cotton. PhyszXogicaC Plant PathoCogv 2,317-331. JSRIKIJN, J. & ORION, D. (1979). Vertkillium wilt of potato: importance and control. Phytoparasitica 7, 107-l 16.
1. BE~LEY, W. F. (1922). Sleepy disease 2. BIELORAI, H., TAMYIR, M. & Humvrrz, 3.
4. 5. 6. 7.
8. 9. 10.
11. 12. 13.
14.
Comparison
of protein-lipopolysaccharide
complexes
221
15. LOWRY, 0. H., ROSEBROIJOH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. 3ownal of Biological Chemistry 193, 265-275. 16. MALYSHEVA, K. M. & ZEL'TSER, S. SA. (1968). Protein-lipid-polysaccharide complex from the culture and mycelium of the fungus Verticillium akhliae. Proceedings of the Academy of Sciences of the USSR (Kleb.) 179,231-234. 17. MCJLEOD, A. G. & SM.ITH, H. Cl. (1960). Verticillium wilt of Tobacco (III). The effect of culture filtrates of Verticillium dahliae Kleb. on tobacco. New ~ealand3ournal of Agricultural Research 4, 123-128. 18. MXCHAIL, S. H. & CARR, A. J. H. (1966). Use of culture filtrates as a rapid technique for screening Transactions of the British Mycological Society lucerne for resistance to Verticillium albo-atrum. 49, 133-138. 19. MUSELL, H. W. (1972). Toxic proteins secreted by cotton isolates of Verticillium albo-atrum. In Phytotoxins in Plant Diseases, Ed. by R. S. K. Wood, A. Ballio & A. Graniti, pp. 443445. Academic Press, London. 20. NAOHMIAS, A., BARASH, I., BUCHNER, V., SOLEL, Z. & STROBEL, G. A. (1979). Isolation of a phytotoxic glycopeptide from lemon trees infected with Phoma tracheiphila. Physiological Plant Pathology 14, 135-140. 2 1. ORNSTEIN, L. & DAVIS, B. J. (1962). Disc Electrophoresis. Distillation Products Industries, Rochester, New York. 22. OUCHTERLONY, 0. (1962). Diffusion-in-gel methods for immunological analysis. Progress in Allergy 6, 30-154. 23. PORTER, C. L. & GREEN, R. J. (1952). Production of exotoxin in the genus Verticillium. Phytopathology 42, 472 (Abstr.) . 24. STODDART, J. L. & CARR, A. H. J. (1966). Properties of wilt-toxins produced by Verticillium alboatrum. Reinke & Berth. Annals of Applied Biology !i8,81-92. 25. WRIQLEY, C. W. (1968). Analytical fractionation of plant and animal proteins by gel electrofocus. Journal of Chromatography 36, 362-365. 26. ZACHARIUS, R. M. (1969). Glycoprotein staining following electrophoresis on acrylamid gels. Ana~tical Biochemistry 30, 148-152.
Plant PatholoD
(1982)
20,2
13-22 1
Comparison of protein-lipopolysaccharide complexes produced by pathogenic and non-pathogenic strains of Verticillium dahliae Kleb. from potatot NACHMIAS$,
ABRAHAM $Dizkion $Department
of Plant Pathology,
VIRGINIA Agricultural
BUCHNER
9 and JAMES
Research Organization,
of Organic
Chemistry,
Weizmann
(Accepted for publication
November
1981)
Institute
Gilat
KRIKUN~ Expekent
Station, Negev,
Israel
of Science, Rehovot,Israel
Phytotoxic protein-lipopolysaccharide (PLP) complexes were isolated from dialyzed culture fluids of a pathogenic strain of Verticillium dahliae; PLP complexes which lack phytotoxic activity were isolated from a mutant non-pathogenic strain of the fungus. A comparison of profiles of the wild-type and mutant PLP eluting from an Agarose A-5 m column revealed quantitative differences between the 2 major protein peaks of each strain. The corresponding peaks of the 2 strains had similar molecular weights and in gel immunodiffusion were antigenically indistinguishable, but differences between them were seen in their chemical composition when analysed for protein, lipid and carbohydrate content. Antiserum prepared againt the components of peak 1 from the pathogenic strain reacted with an antigen in extracts of Vertisillium-infected potato plant tissue which was apparently identical to a moiety produced by the pathogen in culture.
INTRODUCTION
Certain Verticillium species produce large amounts of extracellular materials which have been related to the production of wilt symptoms. The possibility that toxins may be produced in liquid culture by tomato isolates V. dahliae and V. albo-atrum was first reported by Bewley [I]. Subsequently, other evidence was presented for the presence of toxic substances in culture filtrates of Verticillium spp. isolated from tomato [I, 51, tobacco [17], lucerne [18] and cotton [12, 13, IS, 191. Phytotoxic metabolites elaborated by these fungi in culture include low molecular weight compounds [8] and high molecular weight compounds [24] such as enzymes [IO, II], proteins [19] and polysaccharides [23]. Malysheva & Zel’tser [16] described a toxic protein-lipopolysaccharide complex (PLP) produced by a V. albo-atrum isolated from cotton plants and this was confirmed by Keen et al. [12, 131, who found a correlation between the susceptibility of numerous varieties of cotton to Verticillium wilt and their sensitivity to the PLP in a cotton leaf bioassay. From these results they concluded that the PLP may be implicated in the development of disease symptoms in cotton. Verticillium wilt is a major limitingfactorin potato production throughout the world, especially in arid regions under irrigation [14]. V. dahliae is the major pathogenic species in hot, dry climates [14]. In an attempt to develop a bioassay which could tNo.
183E
0048459/82/0202
1981 series 13 +
10 $03.00/O
@ 1982 Academic
Press Inc.
(London)
Limited
214
A. Nachmias,
V. Buchner
and J. Krikun
be used for the screening of potato cultivars for susceptibility to V. dahlias, we investigated the use of phytotoxic PLP complexes obtained from liquid cultures of an isolate of V. dahlia which was pathogenic to potato. Some plants can respond non-specifically to high molecular weight compounds and so it was necessary to ensure that compounds were not present in the extracts which mimicked the effects of the PLP complex and so caused positive reactions in resistant plant varieties. This problem might be overcome using, as a control, similar PLP complexes isolated from a non-pathogenic mutant strain of the fungus and the objective of the present study was to compare the chemical and immunological properties of PLP complexes obtained from a pathogenic potato isolate of V. dahliae with those obtained from cultures of a non-pathogenic mutant derived from it. MATERIALS
AND
METHODS
Culture methods
A pathogenic isolate of V. dahlias was isolated from an infected potato plant. A single spore isolate (Gl) was maintained on potato-dextrose agar (Difco). A spontaneous mutant (V297), arising from the single spore isolate of Gl, was selected on the same medium. Isolates of V. dahliae were tested for pathogenicity by artificial inoculation into soil, by injection into stems, or by dipping rooted cuttings into a spore suspension, Gl, which produces microsclerotia, was pathogenic by all 3 criteria; V297 did not produce microsclerotia and was not pathogenic. For production of the PLP complex, the fungi were transferred to Roux bottles, each containing 100 ml of a medium containing per litre of distilled water: glucose 20 g, asparagine 2 g, KH,PO, 1.5 g, MgSOI. 7H,O 1 g, Z&O,. 7H,O 20 mg, FeSO,. 5H,O 10 mg, CaSO, 6 mg, thiamine HCl 10 mg and pyridoxine 5 mg. The pH was adjusted to 6.7 with KOH. The cultures were incubated for 21 days at 26 “C in the dark. Bioassay
Culture fluids, after dialysis (against distilled water 1 : lO*OOO,v/v for 10 h), and PLP complexes were bioassayed for symptom-producing activity using detached leaves. The first true leaves of 3- to 6-week-old plants (potato cv. Spunta, tomato, eggplant and cotton) were excised under water and placed in vials containing 20 ml water. One hundred micrograms of the test solution in 0.2 ml was injected into the intercellular spaces of the leaves. The concentration of the test solution to be used was determined by a dilution end-point test on leaves. The highest dilution showing a clearly detectable difference between susceptible and resistant plants was employed. After injection the leaves were placed under continuous illumination (c. 7000 lx) and observed for symptoms of chlorosis and necrosis after 18 to 36 h; the degree of necrosis was determined after 72 h. Purifkation
procedure
The mycelium was removed from 21-day-old cultures by filtration and the filtrate was concentrated under vacuum at 50 “C. The concentrated filtrate was treated
Comparison
of protein-lipopolysaccharide
complexes
215
with 4 volumes of acetone at - 18 “C and allowed to stand overnight. The precipitate was collected by centrifugation at 5000 g for 15 min at 5 “C, dried and dissolved in distilled water (l/50 volume of the initial volume of the culture fluid). The solution was then loaded on to an Agarose A-5 m (Bio Rad) column (3 x 70 cm), which was eluted with distilled water at room temperature. The flow rate was 7 ml h-1 and 2-ml fractions were collected. The column was calibrated using tobacco mosaic virus (5 x lo6 daltons) as void volume marker. Peak fractions giving phytotoxic activity were pooled and concentrated as above. Analytical
methods
Protein was determined by absorbance measurement at 280 nm and by the method of Lowry et al. [15], using bovine albumin as standard. Polysaccharide was determined by the anthrone method [7], using D-&COSe as standard. Lipid was determined gravimetrically following hydrolysis of the complex in 1 N HCl for 60 min at 100 “C and extraction into diethyl ether. Identification of the carbohydrate components of the complex was carried out by gas-liquid chromatography (g.1.c.) according to Clamp et al. [4]. Electrophoresis
Disc gel electrophoresis was performed on cylindrical gels (10 cm) of 5% polyacrylamide with 0.2 M Tris-glycine buffer, pH 8.3, 4 mA per tube until the bromophenol blue marker reached the end of the tube [21]. The gels were stained with Coomassie blue R 250 for protein [S] or with Schiff’s base reagent for glycoproteins [26]. Isoelectric focusing was performed using the method of Wrigley [Zq. Antiserum production
and immunodajiiion
technique
The PLP complexes were each dissolved in 1.5 ml of 0.15 M phosphate saline buffer, pH 6.8, and emulsified by sonication for 5 min with 1.5 ml of Freund’s complete adjuvant. The antigens were injected subcutaneously into the flanks of New Zealand rabbits. A booster injection of the same material in Freund’s incomplete adjuvant was made 3 weeks later and bleeding was performed from the marginal ear vein 3 weeks after the booster injection. Immunodiffusion analysis was performed using the Ouchterlony double diffusion technique [22] in a medium consisting of O*8o/o agarose gel, 0.15 M NaCl in 60 mM barbital buffer, pH 8.6, and 0.1% sodium azide as preservative. RESULTS
Cell-free, dialysed culture filtrates from the pathogenic strain, Gl, when injected into potato leaves, induced interveinal chlorosis followed by necrosis, similar to the symptoms of wilt observed on Verticillium-infected plant (Plate 1). Dialysed culture filtrates of the non-pathogenic mutant, V297, had no detectable effect on potato leaf tissue. The phytotoxic moiety was usually present throughout the logarithmic growth phase of Gl; but the culture could only be differentiated from that of the mutant
216
A. Nachmias,
V. Buchner
and J. Krikun
at the end of the log phase, when it began to produce microsclerotia. In order to rule out the possibility of a reverse mutation to microsclerotia formation in V297, it was necessary to harvest the cultures at the onset of microsclerotia production. Essentially, all of the phytotoxic activity was recovered from redissolved acetone precipitates of the dialysed culture filtrates. The acetone precipitates were chromatographed on a column of agarose A-5 m (Fig. 1). The column eluates were analysed
(a)
50 Elutian
100 volume
150 (ml)
FIG. 1. Chromatography of extracellular products from the (a) non-pathogenic (V297) and (b) pathogenic (Gl) isolates of Verticillium d&&e on an agarose A-5 m column. Acetone precipitates of dialysed V. duhliae culture filtrates (400 ml), from the pathogenic or the nonpathogenic strain, were dissolved in distilled water and loaded on to a column (3 x 70 cm). The column was eluted with distilled water at room temperature with a flow rate of 7 ml h-r; fractions of 2 ml were collected. Tobacco mosaic virus (TMV) served as the void volume marker. A, Protein; 0, polysaccharide.
for protein, carbohydrates and lipids and tested for activity in the potato leaf bioassay. The elution profiles showed two major protein-lipopolysaccharide (PLP) peaks (Fig. 1) ; fractionation of Gl resulted in a peak I 3 times larger than peak II, whereas that of V297 gave a relatively small peak I but a larger second peak. Gross chemical analysis showed quantitative differences between components of the corresponding peaks of the 2 strains (Table 1). Quantitative differences between
PLATE 1. Phytotoxic (0.5 mg ml-‘), obtained of the pathogenic isolate
symptoms on a potato leaf, 24 h after injection by agarose A-5 m filtration of acetone-precipitated of Verticillium dahliae.
with
200 ~1 peak I culture filtrates
[facing page 2161
Comparison
of protein-lipopolysaccharide
complexes
217
them were also seen when analysed by g.1.c. for carbohydrates (Table 1). The same elution pattern as shown for Gl was observed for 6 different isolates of V. dahliae, obtained from eggplant, avocado, watermelon, olive, cotton and tomato. Filtration of the 2 major peaks on a calibrated column of agarose A-5 m indicated an estimated molecular weight of 2 x IO6 daltons for peak I and 8 x lo4 daltons for the second peak in both isolates (Fig. 2). Analysis of the peaks by polyacrylamide TABLE
Co@osition
(percentage
of dry
wt) of Verticillium pur$ed by gel filtration Pathogenic Gl Peak I
Protein Lipid Polysaccharide Galactose” Glucose” ’ Percentage
1
dahliae protei~li@opolysaccharide chromatography
isolate Peak
27 6.73 64 70 29.4 of polysaccharide,
42.7 8.83 44.2 9 69
II
Non-pathogenic isolate V297 Peak I Peak 14 18.87 60 61 37.4 determined
complexes
II
17 5.47 63 0 70 by g.1.c.
FIG. 2. Molecular weight determinations of K dahliae PLP complexes by chromatography on a column of agarose A-5 m. The column was calibrated with blue dcxtran 2000 (2 x 10s daltons), thyroglobulin, catalase, aldolase and ovalbumin.
gel electrophoresis detected 1 major band in each in both strains, and at least 5 to 7 minor bands reacting with Coomassie blue. Some of the bands gave a positive reaction with Schiff’s base stain. Isoelectric focusing of the complexes also detected several bands in each, with the major band of peak I at pH 3.8 and that of peak II at pH 4.0. Both peaks of Gl were phytotoxic to leaves of potato and certain other host plants, but not to leaves of several non-host plants (Table 2). While neither peak of the mutant was toxic for potato, each fraction was occasionally toxic for other
A. Nachmias, V. Buchner and J. Krikun
218 Phyfotoxidy of Verticillium
dahliae
TABLE 2 ~otein-lipoporysaccharidc Pathogenic
isolate Gl
Peak Potato cultivar spunta Tomato (Hosen Avocado Eggplant Olive Pepper citrus Lemon Sour orange Volkamariana Sudangrass
I
Peak
II
com#exes
on various
Non-pathogenic isolate V297 Peak I Peak
T +
+ + + + -
+ -
-
+
+
-
-
+
-
-
+
Eilon)
The bioassay was carried out by injecting ml-l). +, Symptom production, chlorosis treatment; -, no reaction.
200 ~1 liquid and necrosis,
plants
II
solution (0.5 mg PLP complex was recorded 18 to 36 h after
(b)
FIG. 3. Diagrams of the immunodiffusion patterns obtained when partially purified complexes and potato plant extracts were reacted with antisera to peak I or peak II of Gl. The inner wells contain: 1, anti-peak I; or 2, anti-peak II. The outer wells contain: (a) A,peakIofGl;B,peakIofV297;(b)A,peakIofG1;B,peakIIofG1; (c)A,peakIIof Gl ; B, peak II of V297; (d) A, peak I of Gl ; B, peak II of Gl : (e) A, peak I of Gl ; B, extract of Veium dkhliae-Meted potato plant tissue; C, extract of healthy potato plant tissue.
plants. A plant species was considered sensitive in the bioassay only if the nonpathogenic PLP gave a concomitant negative reaction. The phytotoxic activity was resistant to most attempts at inactivation, which included enzymatic treatment with protease, papain, a-arnylase and lipase; chemical treatment with potassium periodate or mild base; and heating to 98 “C for 20 min. The biological activity was destroyed only by digestion with 1 N HCI for 60 min at 110 “C.
Comparison
of protein-lipopolysaccharide
complexes
219
Antisera prepared in rabbits against peak I or peak II of Cl, reacted equally well with both peaks (Fig. 3). However, when peak I and peak II were reacted with antiserum of peak II, spurs formed between the precipitin bands, showing that each peak had some determinants lacking in the other [Fig. 3(d)]. Peaks I of Gl and V297 showed serological identity [Fig. 3(a)] when reacted with antipeak I while peaks II of the 2 strains gave a reaction of identity with anti-peak II [Fig. 3(c)]. Wh en antiserum to peak I was reacted with a water extract of V. dahliaeinfected potato tissue, a precipitin band formed which fused with that of peak I of Cl, suggesting that a substance present in V. dahlias-infected potato plants is similar to a moiety produced by the fungus in culture. DISCUSSION
The data presented here confirm the observations of others [12, 13, 161 that phytotoxic, heat-stable PLP complexes can be isolated from culture fluids of Verticillium species. Although we employed a different purification procedure, the acetoneprecipitable complexes obtained were similar to the PLP described by Keen & Long [12]. Whereas Keen et al. [13] studied only the properties of peak I, we have investigated both fractions, since both peaks of the pathogenic strain were toxic to leaves of potato and other host plants of V. dahlias. Both peaks of Gl showed specificity in the leaf bioassay because only susceptible plants were sensitive to the material in the absence of a positive reaction by the mutant PLP. between In double immunodiffusion analysis, the reaction of non-identity peaks I and II of Gl ruled out the possibility that peak I was an aggregate of components of peak II. The cross-reactivity of the antisera to the 2 fractions showed that the column-purified peaks were not homogenous and that 1 may have been contaminated with the other. This observation was confirmed by the analysis of the fractions by polyacrylamide gel electrophoresis. Although the PLP complexes of peaks I and II of V297 were serologically identical to the corresponding peaks of Cl and were of similar molecular size, there appeared to be differences between them in their chemical composition. While all the fractions were predominantly carbohydrate, the mutant peaks contained less galactose and significantly lower amounts of protein. These differences in composition may reflect qualitative differences between the PLPs of the two strains, or, alternatively, each peak may represent an unresolved mixture of the same PLP complex with a quantitative increase or decrease in one or more of the components of the mixture. Peak I was almost absent from the complexes of the mutant strain but it is unlikely that the increased amount of peak II resulted from dissociation of peak I, because the two fractions are serologically unrelated. Although gel filtration indicated a high molecular weight, peak I was resolved in 5% polyacrylamide gels, which excludes molecules of molecular weight greater than 3 x 10s daltons. This suggests that the complex can be broken down into smaller molecular weight compounds in the absence of dissociating conditions. This is in agreement with the observation of Keen et al. [12, 131 that PLP tends to form low molecular weight products in stationary phase cultures at high ionic strength. Furthermore, it is unlikely that the intact PLP complex is required for
220
A. Nachmias,
V. Buchner
and J. Krikun
biological activity because phytotoxicity was not inactivated by heat nor by enzymatic or chemical treatments which hydrolyse proteins, lipids and polysaccharides. It is possible that the PLP complex may be synthesized as a precursor molecule which is cleaved into smaller components; such a system has been described for tumorassociated antigens of several viral infected tumors in mice [3]. Keen et al. [13] presented evidence for the involvement of the PLP in symptom production by Verticillium in cotton, but were unable to unequivocally demonstrate its presence in diseased plant tissue. In the present work, antiserum to peak I detected the antigen in extracts of V. dahliae-infected potato tissue, but not in homogenates of healthy tissue, a result which supports the hypothesis that PLP may be involved in symptom production [13]. Whether the antigenic material and the phytotoxic moiety are related remains an open question. The data presented here suggest that V. dahlia produces at least two different phytotoxic materials in culture and that these compounds are not produced or are produced in altered form by the mutant. A more detailed examination of the relationship between the loss of pathogenicity and the alteration of PLP in the mutant, and of the importance of PLP to symptom production by the wild type will require biochemical purification of the active molecules. We wish to thank Mrs Janet Orenstein for her technical assistance. REFERENCES of tomato. Annals of Applied Biology 9, 116-134. S. (1977). Amino acid absorption along the intestinal tract of chicksfed heated and raw soybeanmeal. 3ournal of Nutrition 107, 1775-l 778. BUCHNER, V. (1979). Isolation and partial purification of a non-oncornaviral tumor specific transplantation antigen from TA3 tumor cells. Ph.D. thesis. Tel Aviv University, Ramat Aviv, Israel. CLAMP, J. R., Bmrrr, T. & C~AMEIERS, R. E. GLC technique as O-tðyl silyl ester (TMS). In Methods of Biochemistry, vol. 19, Ed. by G. Cluck, pp. 316-335. CRONSHAW, D. K. & PEGG, G. F. (1976). Ethylene as a toxin synergist in VerttiCCium wilt of tomato. Physiologial Plant Pathology 9,23-N FAZEKAS, G., WEB~R, S. & DATYNER, R. C. (1963). Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochemia et Biophysics Acta 71, 377-391. FIJNO, F., SCHEFFER, F. L. & Kmx, P. K. (1953). The ultramicro-determination of glycogen in liver. A comparison of anthrone and reducing sugars methods. Archives of Biochemistry and Biophyssics 45,319326. GREEN, R. J. (1954). A preliminary investigation of toxins produced in vitro by VerticiCCium alboatrum. Phytopathology k&433-437. HODGSON, R., PETERSON, W. H. & RIKER, A. J. (1949). The toxicity of polysaccharides and other large molecules to tomato cuttings. Phytopathology 39, 47-62. HOWELL, C. R. (1976). Use of an enzyme-deficient mutant of VerticiCCium dahCiue to assess the importance of pectolytic enzymes in symptom expression of Verticillium wilt of cotton. Physiological Plant Pathology 9,279-283. KAMAL, M. & WOOD, R. S. K. (1956). Pectic enxymes secreted by Verticillium dahliae and their role in the development of the wilt disease of cottom. Annals of Applied Biology 44, 322-340. KEEN, N. T. & LONG, M. (1972). Isolation of a protein-lipopolysaccharide complex from VerticilCium albo-atrum. PhysioCogicaC Plant PathoCogy 2, 307-315. KEEN, N. T., LONG, M. & ERWIN, D. C. (1972). Possible involvement of a pathogen-produced protein-lipopolysaccharide complex in Verticillium wilt of cotton. PhyszXogicaC Plant PathoCogv 2,317-331. JSRIKIJN, J. & ORION, D. (1979). Vertkillium wilt of potato: importance and control. Phytoparasitica 7, 107-l 16.
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complexes
221
15. LOWRY, 0. H., ROSEBROIJOH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. 3ownal of Biological Chemistry 193, 265-275. 16. MALYSHEVA, K. M. & ZEL'TSER, S. SA. (1968). Protein-lipid-polysaccharide complex from the culture and mycelium of the fungus Verticillium akhliae. Proceedings of the Academy of Sciences of the USSR (Kleb.) 179,231-234. 17. MCJLEOD, A. G. & SM.ITH, H. Cl. (1960). Verticillium wilt of Tobacco (III). The effect of culture filtrates of Verticillium dahliae Kleb. on tobacco. New ~ealand3ournal of Agricultural Research 4, 123-128. 18. MXCHAIL, S. H. & CARR, A. J. H. (1966). Use of culture filtrates as a rapid technique for screening Transactions of the British Mycological Society lucerne for resistance to Verticillium albo-atrum. 49, 133-138. 19. MUSELL, H. W. (1972). Toxic proteins secreted by cotton isolates of Verticillium albo-atrum. In Phytotoxins in Plant Diseases, Ed. by R. S. K. Wood, A. Ballio & A. Graniti, pp. 443445. Academic Press, London. 20. NAOHMIAS, A., BARASH, I., BUCHNER, V., SOLEL, Z. & STROBEL, G. A. (1979). Isolation of a phytotoxic glycopeptide from lemon trees infected with Phoma tracheiphila. Physiological Plant Pathology 14, 135-140. 2 1. ORNSTEIN, L. & DAVIS, B. J. (1962). Disc Electrophoresis. Distillation Products Industries, Rochester, New York. 22. OUCHTERLONY, 0. (1962). Diffusion-in-gel methods for immunological analysis. Progress in Allergy 6, 30-154. 23. PORTER, C. L. & GREEN, R. J. (1952). Production of exotoxin in the genus Verticillium. Phytopathology 42, 472 (Abstr.) . 24. STODDART, J. L. & CARR, A. H. J. (1966). Properties of wilt-toxins produced by Verticillium alboatrum. Reinke & Berth. Annals of Applied Biology !i8,81-92. 25. WRIQLEY, C. W. (1968). Analytical fractionation of plant and animal proteins by gel electrofocus. Journal of Chromatography 36, 362-365. 26. ZACHARIUS, R. M. (1969). Glycoprotein staining following electrophoresis on acrylamid gels. Ana~tical Biochemistry 30, 148-152.