Isolation of two toxins produced by Pyrenophora teres and their significance in disease development of net-spot blotch of barley

Physiological

Plant PatholoD

(1977)

10, 203-211

Isolation of two toxins produced by Pyrenophora teres and their significance in disease development of net-spot blotch of barley V.

SMEDEG~RD-PETERSEN

Department of Plant Pathology, ‘TEe Royal lhorvaldwnsvej 40, DK-1871 Copenhagen (Acceptedfor

publication

January

Veterinmy and Agricultural V, Denmark

University,

1977)

Two toxins, designated toxin A and B, were isolated from a cell-free, sterile culture filtrate of Pyrenophora term by means of extraction, gel filtration and ion-exchange chromatography. Without the presence of the pathogen each of the purified toxins incites the most important symptoms of the disease when introduced into healthy barley leaves. Toxin A is the most potent, producing symptoms in concentrations as low as 25 vggiml. The most virulent isolates produce the highest concentration of toxin in vitro. A good, although not complete, correlation was found between the host range of the pathogen and that of the toxins. Both toxins were isolated from infected but not from healthy barley leaves. The results indicate that P. teres produces two toxins which may be involved in the disease syndrome of net-spot blotch of barley. The toxins do not seem to determine pathogenic+ of P. tires but contribute to the virulence of individual isolates.

INTRODUCTION The fungus Pyrenophora teres Drechs. is the cause of net-spot blotch of barley. There exists two forms of P. teres [IO, 131, which differ only in the symptoms incited on barley. Pyrenophora teres Drechs. f. teres produces dark brown longitudinal and transverse necrotic streaks which often form a characteristic net-like pattern on leaves of infected plants. P. teres Drechs. f. maculata Smedeg. produces welldefined dark brown circular or elliptical lesions without netting. Common to both forms is that affected tissue becomes chlorotic, often with areas of water-soaked tissue. Chlorosis and water-soaking are important in the symptom complex of net-spot blotch of barley. Thus certain highly pathogenic isolates induce a blight-like reaction characterized by chlorosis and a diffuse, general water-soaking appearing 3 to 4 days after inoculation and followed by rapid necrosis of affected tissue but often with no or weak expression of the usual necrotic lesions. The symptoms incited by highly pathogenic isolates of P. teres indicated that a toxin might be operative in pathogenesis. This assumption was supported by histological investigations of barley leaves inoculated with such isolates. After hyphal penetration, chlorosis and water-soaking developed rapidly in advance of the hyphae, indicating the operation of diffusible substances. Furthermore, excised barley leaves immersed in a desalted liquid medium in which the pathogen had grown developed symptoms similar to those caused by the pathogen itself, whereas ‘7

V. Smedeghd-Petersen

204

no symptoms developed on leaves immersed in a desalted medium in which the fungus had not been grown. The present study was initiated to develop methods for production, detection and isolation of eventual toxins and determine their significance in development of the disease. An abstract including part of thii work has previously been published [II]. MATERIALS

AND

METHODS

Four monosporial isolates of Pyrenophora teresf. teres,the form of the fungus producing net lesions on barley [IO, 131, and five monosporial isolates of P. teresf. maculata, the form of the fungus producing spot lesions on barley [IO, 131, were employed in the study. The barley cultivar “Wing” which is highly susceptible to P. teres was used as the primary test plant, but also the two resistant barley cultivars “CI 9819” and “CI 9747” were used. Two bioassay procedures were employed to determine toxin activity during the purification steps. In the first procedure, five excised leaves, the first developed leaf from each plant from 12- to 14-day-old barley seedlings, were immersed in 6 ml of toxin solution in small test tubes and allowed to take up approximately 0.75 ml of solution per leaf, They were then transferred to tubes with deionized water. During the bioassay period the test tubes with the leaves were placed in the laboratory and illuminated with a 100 W Osram bulb situated 60 cm above the leaves. The leaves were evaluated for symptoms during a 5 day period using a rating scale 0 to 4: 0, no symptoms ; 1, slight chlorosis ; 2, marked chlorosis, slight necrosis; 3, marked chlorosis and necrosis; 4, extensive chlorosis and necrosis, eventually water-soaking and collapse of tissue. In a second bioassay, designated the leaf injection assay, a method of Hagborg [q was used to infiltrate barley leaves in situ with toxin. After treatment the intact plants were placed under ordinary greenhouse conditions. The reaction of infiltrated tissue to the phytotoxic solution was evaluated as indicated above. For routine toxin production, cultures were grown for 13 to 15 days in 1 1 Roux bottles containing 100 ml of Fries’ liquid medium [14]. After harvest the filtrates were pooled, filtered through a sterile Zeiss filter and stored at 5 “C for further purification. The isolation procedure involved the following steps: (1) Sterile filtrates from fungal cultures were concentrated under partial vacuum to one-tenth of the original volume. (2) After storage at 5 “C for 24 h salts and other precipitates were removed by centrifugation. (3) The clear supernatant was deproteinized by adding an equal amount of methanol. (4) The precipitate was removed by centrifugation and the supernatant concentrated for filtration through a Sephadex G-10 gel using a 2.5 x 100 cm column (bed height 93 cm, void volume 180 ml). Electric conductivity of flowing eluate was continuously measured on a Radiometer conductivity meter (type CDM 3 with recorder) by passing a flow-type measuring cell.

Isolation

and

significance

of Pyrenophora

feres

toxins

205

The effluent was collected in 5 ml fractions and each fraction determined for ninhydrin reactivity and toxic activity by means of bioassay. Solutions from ninhydrin-positive fractions were further subjected to paper chromatography on Whatman no. 1 paper with descending solvent (I-butanol-acetic acid-water, 12 : 3 : 5). (5) Toxin-containing fractions from the Sephadex column were freeze-dried, redissolved in water, applied to a column containing a strongly basic ionexchange resin in the acetate form (Dowex 1 x 8, 200/400 mesh) and eluted with a gradient of dilute acetic acid. The concentration of the acetic acid in the gradient ranged from 0.0 to 1*ON over 180 ml. In the later stages of the work, routine production of toxin was performed without the use of gel filtration. Instead, concentrated toxin solutions were desalted on a column containing a strongly acid ion-exchange resin in the hydrogen form (Dowex 50W x 8, SO/l00 mesh). After elution of the column with 2 N pyridine or 1 N aqueous ammonia all ninhydrin-reactive fractions were pooled, freeze-dried and subjected to ion-exchange using a strongly basic ion-exchange resin in the acetate form as described above. The toxins were hydrolysed according to the method of Levy & Chung [9]. One mg of toxin and 1 ml of 6 N HCl were placed in a glass tube which was then evacuated, sealed and heated in an oven at 150 “C for 4 h. The toxins were isolated from infected barley leaves following a modified procedure for isolation of free amino acids from plant material [S]. The leaves were homogenized and extracted with boiling tetrachloromethane in order to remove lipophilic compounds. After filtering through a Buchner funnel the plant residue was extracted for 15 min with boiling methanol-water [7 : 3 (v/v)]. The filtrate from the last extraction was concentrated in vacua and desalted on a column containing a strongly acid ion-exchange resin in the hydrogen form (Dowex 50W x 8, 50/100 mesh). The column was eluted with 1 N aqueous ammonia or 2 N pyridine and the eluate collected in 5 ml fractions. All ninhydrin-reacting fractions were pooled and freeze-dried. The residue was redissolved in water and applied to a column containing a strongly basic ion-exchange resin in the acetate form. The column was washed with water and eluted with a gradient of dilute acetic acid as previously described. The presence of toxins was detected on paper chromatograms by comparing with toxins isolated from culture filtrates and by bioassay. RESULTS Purification

and isolation

of the toxins

Toxin activity in the effluent from the Sephadex G-10 column as determined by bioassays on barley leaves occurred in tubes 38 to 43 and coincided with the first peak of electric conductivity and ninhydrin reaction (Figs 1 and 2). The other peaks of high electric conductivity were not related to toxic activity and only slightly related to ninhydrin reactivity. The third peak was due to elution of salts as determined by the test for chloride ions. In descending paper chromatography using I-butanol-acetic acid-water (12 : 3 : 5) as a solvent, the ninhydrin-reacting eluate fractions from the column separated into six different spots with R, values: O-05, 0.14, 0.16, O-20, O-25, O-31. The

206

V. Smedegbd-Petersen

34

36

38

40

44

42

46

48

50

52

54

56

5t

Tube number

FIG. 1. Elution pattern obtained by gel filtration of a concentrated culture teres through a Sephadex G-10 column. Electric conductivity of the effluent ously recorded and expressed in reciprocal ohms (mho) x 10”. The cross-hatched ponds to the toxin-containing fractions.

filtrate of P. was continuarea corres-

k ; I? :: .;” 8 P E m’ 1: 34

36

38

40

42

44 Tube

FIG. 2. Elution patterns obtained by gel &es through a Sephadex G-10 column. (a) evaluated on a 0 to 10 scale by spotting 5 ~1 (b) Paper chromatogram of eluate fractions.

46

48

50

52

54

56

5E

number

filtration of a concentrated culture filtrate of P. Ninhydrm reactivity of effluent fractions visually eluate from each tube on Whatman no. 1 paper. Toxin appeared in tube nos 38 to 43.

toxic effect of the fractions corresponded strictly with the occurrence of a substance with R, 0.05 (Fig. Z), whereas no activity could be detected in bioassays from fractions without this substance. Isolation of crude toxins was made from cultures grown in parallel and prepared as above; fractions were pooled in accordance with their content of material with RF 0.05. After freeze-drying the pooled fractions from tubes 38 to 43 (toxin containing) resulted in 42.3 mg light brown powder, whereas the non-toxic fraction of tubes 45 to 52 resulted in 3.4 mg dark brown, sticky material. In bioassays using a concentration of 500 pg/ml, the material from tubes 38 to 43 caused extensive chlorosis and necrosis, whereas the material from tubes 45 to 52 caused no or only traces of symptoms.

Isolation

and

significance

Pyrenophora teres toxins

of

207

By the subsequent gradient elution from a strongly basic ion-exchange resin in the acetate form, two substances with R, values of 0.09 and 0.06 were separated (Fig. 3). Fractions were pooled in accordance with the R, values and freeze-dried. Both compounds possessed toxic activity and were designated toxin A (RF O-09) and toxin B (R, O-06).

as

IS

25 I

0’

;::::=;

Tube number 46 47 44 I I o 8

37 I

48 I (j

49

50

iI

6

FIG. 3. Section of paper chromatogram showing the separation of P. teres toxin A (RF O-09) and B (Rg O-06) by ion-exchange chromatography on a strongly basic ion-exchange resin (Dowex 1 x 8, 200/400 mesh). The column was eluted with a gradient of dilute acetic acid which in concentration ranged from 0.0 to 1.0 N over 180 ml. The concentrated toxin solution which was applied to the Dowex 1 column had previously been desalted on a column containmg a strongly acid ion-exchange resin (Dowex 50W x 8, 200/400 mesh).

Chemical and physical properties of the toxins

The two toxins are very similar in all physical and chemical properties investigated. Both appear as white powders. They are readily soluble in water, NaHCO, and O-5 N HCl, but not in diethyl ether, methanol, 96% ethanol, 1-butanol and tetrachloromethane. Evidence for purity was obtained by paper chromatography and thin-layer chromatography on cellulose and silica gel plates using four different solvent systems. The R, values are listed in Table 1. In all cases only one single ninhydrin-reactive spot appeared for each toxin indicating one active component. Both toxins are fairly stable to heat and alkaline and acidic solutions. Neither autoclaving in aqueous solution at 121 “C for 15 min nor storage at room temperature for 24 h in solutions of 1 N aqueous ammonia and 1 N hydrochloric acid changed TABLE RF values

of P.

teres

toxim

Solvents I-butanol-acetic I-propanol-acetic I-butanol-pyridineter Phenol-water-cone.

acid-water acid-water

(12 : 3 : 5) (200 : 3 : 100) (1 : 1 : 1) ammonia (120 : 30 : 1)

1

A and B u&g

four dj?erent

Whatman no. 1 paper A B O-09 0.12

0.06 0.08

0.07

0.05

solvents t.l.c., cellulose A B 0.13 o-15 0.17

0.10 0.11 o-17

t.1.c., silica gel A B oa6 0.08 0.03

O-04 O-09 0.25 0.03

208

V. Smedegkd-Petersen

the RF values in the butanol-acetic acid-water system, and they retained full toxicity to barley leaves. Weak hydrolysis took place by autoclaving for 15 min in 1 N aqueous ammonia and 1 N hydrochloric acid. By hydrolysis in 6 N hydrochloric acid, toxin A separated into five ninhydrinreactive compounds on Whatman no. 1 paper [I-butanol-acetic acid-water (12 : 3 : 5)] with the following RF values: O-07, O-11, 0.17, 024, 0.35. Using the same procedure toxin B separated into six ninhydrin-reactive spots with R, values: O-04, 0.07, 0.11, 0.17, 0.24, 0.35. Both toxins are dialysable. When a solution of 10 mg of toxin A+ B in 10 ml of water was dialysed against deionized water, no toxin could be detected after 12 h, neither in bioassay nor in a ninhydrin test. The dialysability and heat stability indicate that the toxins are small molecules. An estimate of the approximate upper limit for the molecular weight was obtained by gel filtration. The stain, Blue Dextran 2000, and NaCl were added to a mixture of the toxins, applied to a Sephadex G-10 column and eluted with water. Toxins A and B were eluted simultaneously at I.1 times the void volume (V,) and NaCl at 1.6 Vs. As the exclusion limit of Sephadex G-10 is approximately 700, it is concluded that each of the toxins has a molecular weight less than 700. Neither of the toxins were found to absorb light in the ultraviolet spectral region. Studies are in progress at the Department of Organic Chemistry at this University in order to determine the chemical structure. Phytotoxicity of toxins A and B Toxin A appears to be a more powerful phytotoxin than toxin B. When excised barley leaves were immersed in a solution of 1000 pg toxin A/ml, extensive chlorosis and collapse of tissue became evident within 48 h and in 5 days the leaves became brown and entirely necrotic (Plate 1). At a concentration of 50 pg/ml, chlorosis became distinct within 3 days and necrosis within 5 days. The lowest concentration that produced symptoms within 5 days was 25 pg/ml. On control leaves in water there were no signs of chlorosis after that time. By leaf injection of toxin A in situ the first symptoms appeared within 2 to 3 days as a weak chlorotic marbling which subsequently developed into well-defined chlorotic spots surrounding brown necrotic lesions, or a general chlorosis with brown spots and browning of larger leaf areas (Plate 2). As was the case in the excised leaf assay, considerably larger toxin concentrations were required to produce water-soaking than chlorosis. Toxin B induces, qualitatively, the same symptoms as toxin A. It is, however, not as powerful and the minimum toxin concentration required to cause chlorosis was 5 to 6 times higher. Isolation of the toxinsfrom infected barley leaves Attempts were made to isolate the toxins from six infected leaf samples. Toxin A was detected in two samples infected with P. teresf. teres (the net form of the fungus) and one sample infected with P. teres f. maculata (the spot form of the fungus) as determined by paper and thin-layer chromatography. Toxin B was found in all six samples. The RF values on the chromatograms corresponded with those

C

25

50

100

200

500

1OOOpg/m

I

barley leaves of the cultivar “Wing”, 5 PLATE 1. The effect of P. teres toxin A on excised days after start of treatment. Each leaf was allowed to take up approximately 0.75 ml of toxin solutions containing from 25 to 1000 pg pure toxin A/ml, then transferred to test tubes with deionized water. Toxin-treated leaves are characterized by severe chlorosis and necrosis.

PLATE 2. The effect of P. tern toxin A on barley leaves of the cultivar “Wing” in itu, 6 days after treatment. By means of the leaf injection assay method about one-half ml of a toxin solution containing 400 pg pure toxin A/ml was injected into the tissue (two lower leaves). In control leaves deionized water was injected by the same method (upper leaf). The symptoms are characterized by chlorosis and brown necrotic spots and streaks similar to those incited by certain isolates of P. teres.

[facing pap ZOS]

Isolation

and

significance

of Pyrenophora

teres

toxins

209

of toxins A and B from culture filtrates and phytotoxic activity was demonstrated in bioassays. Although toxin B seems to be more consistently produced than toxin A, there was a great variation in the quantity found. The largest amount of toxin A+B found was about 400 kg/g leaf tissue (fresh wt). No toxin was detected in uninoculated fresh tissue. Host specificity of the toxins

Table 2 shows the reaction of twelve plant species including one susceptible and two resistant barley cultivars to fungal inoculation and to the P. teres toxins. The susceptible barley cultivar “Wing” was highly sensitive to the toxins. The two resistant cultivars “CI 9819” and “CI 9647”, which were the most resistant among a number of barley cultivars tested against Danish isolates of P. teres, were less sensitive to the toxins than “Wing”. Rye was susceptible to the pathogen and highly sensitive to the toxins. This species is not considered a host of P. teres, but in several infection experiments by the present author, rye has been infected and the fungus found to sporulate abundantly on diseased leaves. The conidia produced on rye induce typical net-spot blotch on barley leaves. Symptoms on rye after inoculation with the fungus are identical with those caused by the toxins. They do not include spots and streaks as on barley, but are characterized by heavy chlorosis and subsequent necrosis. Tomato was not infected by the pathogen but was extremely sensitive to the toxins showing total collapse and necrosis within 48 h. All other test plants were entirely or almost unaffected by the pathogen and its toxins. TABLE

of various test plants to inoculation toxins

(A+

2

zvith four isolates of B) of this pathogen Reaction Isolates N26+N175

Hordeum vulgare, “Wing” Hordeum vulgare, “CI 9819” Hwdeum vulgare, “CI 9647” Secab cereal8 Triticum aestivum Avena sativa Lolium pewme Poa prateusis Dac@s glomeratu Festuca pra.tenGs Calamagrostis canexens Lycopersicum esculentum Medicago sativa Tnifolium fnatense 0 Based on a 0 to 4 scale, with 0 indicating bility. b Ratings on a modified 0 to 4 scale which for further explanation).

P. teres and to the two purified

to inoculationa Isolates Sll+S146

4 2

4

8 0 0 0 0

do not

;: 2b 4b lb 0 0 0 0 0 0 4b 0 0

: 3b lb 0 0 0 0 8

ib lb 0 0 0

complete

Reaction to toxins=

0 0 0 resistance include

dark

and

4 complete

necrotic

netting

suscepti(see text

210

V. Smedeglrd-Petersen

DISCUSSION

One of several important criteria, which should be considered in order to evaluate the significance of a particular toxic compound in the etiology of a disease, is that the toxic compound in purified form should produce at least some of the characteristic disease symptoms when introduced into healthy plants in concentrations which could be reasonably expected in the infected tissue [Z]. The visible symptoms incited by Pyrenophora teres are characterized by necrotic net- and spot-lesions, chlorosis, water-soaking and general necrosis. Most of these symptoms are also induced by purified preparations of the toxins. One exception is that the toxins do not fully reproduce the particular net- and spot-lesions. However, there are several indications that the mechanism which is responsible for these delimited lesions is independent of the toxin production and hence different from the mechanism responsible for the remaining symptoms of net-spot blotch [12]. Thus isolates possessing a low capacity for toxin production in vitro usually do not produce chlorosis and water-soaking of the tissue but only delimited net- and spotlesions, which cause little damage to the leaves when occurring alone. Further, highly virulent isolates of P. teres may cause rapid progressing chlorosis and watersoaking of the leaf tissue without any sign of net- and spot-necrosis. On rye, virulent isolates of the pathogen cause symptoms identical with those caused by the toxins, chlorosis and cell collapse, but never net- and spot-lesions. It thus appears that chlorosis, water&oaking and the subsequent general necrosis are more important factors in the disease injury than are the particular net- and spot-lesions, despite the fact that the latter have provided the name of the disease. That the toxin-producing capacity seems to be an important factor in virulence of P. teres is further emphasized in bioassays, where increased concentrations of purified toxin cause increased symptom severity. Thus high, but still biological, concentrations of pure toxins cause a blighting reaction identical to that incited by highly virulent isolates. Several factors influence the activity of the P. teres toxins in barley leaves. Thus leaves tend to be more sensitive and the necrotic effect of the toxins higher in the winter months than in the summer. Further, the sensitivity of leaves increases with increasing age of the tissue. A similar variation has been found to exist in the sensitivity of tomato tissue to fusaric acid [7] and lycomarasmin [4]. Of the nine isolates employed in the studies, three were avirulent only producing delimited lesions without chlorosis and water-soaking. None of these three isolates produced quantities of toxins in vitro which allowed for isolation, which further indicates that there exists a correlation between virulence of individual isolates and their capacity for toxin production. The fact that toxins A and B were both isolated from barley leaves infected with P. teres but not from healthy leaves is a strong indication of their causal role in disease [15]. The highest toxin concentration found in infected barley tissue was about 4 mg toxin per g tissue (dry weight), equivalent to 400 pg/g fresh weight. According to the bioassay, this is sufficient to account for symptoms such as chlorosis, necrosis and water-soaking. The toxin concentration in infected tissue was highly variable, ranging from traces to the amount mentioned above. The variability may be due to break-down or metabolism of the toxins during disease

Isolation and significance

of Pyrenophora feres toxins

development. This has been shown to be the case with fusaric acid asmin [a, pisatin [I] and phaseollin [16]. The results indicate that toxins A and B, produced by P. teres, ficant factor in the disease syndrome of net-spot blotch of barley. do not seem to determine pathogenicity, but they contribute to individual isolates.

211

[3, 7], lycomarmay be a signiThe two toxins the virulence of

The author is indebted to Professor P. Olesen Larsen and Dr E. Bach, Department of Organic Chemistry at this University, for suggestions and valuable discussions. The investigations were supported by the Danish Agricultural and Veterinary Research Council. REFERENCES 1. DE WIT-ELSHOV~, A. (1968). Breakdown of pisatin by some fungi pathogenic to P&n sat&m. .Neth&ands Journal of Plant Pathology 74, -7. 2. DIMOND, A. E. & WAGGONER, P. E. (1953). On the nature and role of vivotoxins in plant disease. Phytopathology 43, 229-235. 3. G&JMANN, E. (1957). Fusaric acid as a wilt toxin. Phytopathology 47, 342-357. 4. G&JMANN, E. & NAEF-ROTH, S. (1953). Uber den jahreszeitlichen Gang der Welketoxin-Empfindlichkeit der Tomatenpflanzen. Phytopathalogisck