- Email: [email protected]
Septoria cirsii, a potential biocontrol agent of Canada thistle and its phytotoxin—ß-nitropropionic acid
plan cience I I S t \ I t : R S( II N 1 1 t I ( I ' ( B I I'qtll RY, I R f I \ N D
Plant Science 94 (1993) 227-234
Septoria cirsii, a potential biocontrol agent of Canada thistle and its phytotoxin - - fl-nitropropionic acid Joseph Hershenhorn a, Maurizio Vurro b, Maria Chiara Zonno b, Andrea Stierle a, Gary Strobel* a aDepartment of Plant Pathology, Montana State University, Bo-ernan. Montana 59717, USA blstituto tossine d micotossine da parassiti vegetali, Consiglio Nazionale delle Ricerche, viale Einaudi 51, 70126 Bari. Italy (Received 3 May 1993; revision received 29 June 1993; accepted 2 July 1993)
Abstract
Septoria cirsii causes leaf spot of Canada thistle (Cirsium arvense L.) and no other plant tested was susceptible to this pathogen. Because this pathogen possesses host specificity, and because it is so devastating to Canada thistle plants in the field, it is proposed for consideration as a biological control agent. In culture, S. cirsii produced copious amounts of a known phytotoxin which was identified as/3-nitropropionic acid. The toxin is inhibitory to seed germination, root elongation and causes the typical symptoms of chlorosis and necrosis on the leaves of Canada thistle which is one of the most toxin-sensitive plants of all of those tested. Key words: Phytotoxin; Biological control; Weeds; Plant pathogen
I. Introduction
Canada thistle (Cirsium arvense L.) is one of the most important weedy plants growing in virtually all temperate areas of the world and having its center of origin in the Mediterranean region [1]. Most of the serious problems with this plant are in areas receiving 450-900 m m of rainfall/year [1] and it is especially serious in pastures, rangelands, roadsides, and ditch banks [1]. It is also a weed invading fields of barley, wheat, flax, millet, oats, rye and sorghum. It is fairly common in vineyards of * Corresponding author.
Southern Europe and orchards in Canada. The plant reproduces by wind-blown plumed achenes and by an extensive rhizome system [2]. Tillage, herbicides and crop rotations are the commonly used methods for controlling this troublesome pest. In order to reduce the costs of herbicides and reduce the environmental impact involved in these practices, biological control measures are currently being explored [3]. For this reason, we sought potential pathogens that may be considered as candidates for the biological control of Canada thistle in its native region of the world, the Mediterranean area [4]. Canada thistle plants showing severe leaf blight were found in Italy and the
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03701-V
228
causal agent of the disease was Septoria cirsii [5]. This report describes the necrotic leaf spot of Canada thistle and shows that the phytotoxin, 3nitropropionic acid is produced by this fungus and that it may be one of several phytotoxins involved in symptom production. The report also gives some rationale for further testing of this important pathogen as a biological control agent. 2. Materials and methods
2.1. Septoria cirsii: isolation and identification This fungal pathogen was isolated from necrotic lesions on naturally infected Canada thistle (Cirsium arvense L) leaves near Bari in the south of Italy [5]. Infected leaves were washed thoroughly with running tap water, dried, and then placed in a moist chamber at 24°C. Two to three days later, pycnidia appeared on the majority of the necrotic portions of the leaves. Each pycnidium produces numerous pycnidiospores and several single spore cultures of this fungus were obtained for further biological testing. Ultimately, a culture of the fungus was identified as Septoria cirsii by Dr. E. Punithalingam of the Commonwealth Mycological Institute, England. 2.2. Septoria culture The fungus was maintained on Petri plates containing a medium composed of 18% (v/v) tomato juice, 0.1% (w/v) CaCO3 and 1.5% (w/v) agar. The plates were inoculated with a conidial suspension (1 ml), incubated for 1 week at 25°C in a regimen of 12 h dark/12 h light (near UV to blue at - 3 0 0 tzW/cm2). Under these conditions the fungus sporulated abundantly on these plates ( - 106 conidia/cm2). For inoculation of culture flasks used for toxin production, we placed 1 ml of a spore suspension (105-106 spores/ml) obtained from these plates into 200 ml of culture medium in a 1-1 Erlenmyer flask. Agar blocks transferred from the plates containing fungal mycelia were not effective in producing adequate fungal growth. The base medium was M-1-D [6] plus 2 g of finely cut leaves of various plants in order to study the influence of plant products on fungal growth and metabolite production. The culture flasks were shaken at 140
,L Hershenhorn et al./ Plant Sci. 94 (1993) 227-234
rev./min at room temperature (24-24°C) for 8 days prior to harvest. 2.3. Pathogenicity tests Pathogenicity tests were performed on 2-monthold plants of a number of species grown under standard greenhouse conditions. The leaves were inoculated by sprays of an aqueous suspension of S. cirsii conidia containing 105-106 spores/ml. The spore suspension was obtained by placing several ml of H20 on the surface of a maintenance plate of S. cirsii and gently scraping the surface to dislodge the conidia from pycnidia. The spore suspension was sprayed on the leaves of the test plant until they were wet. The inoculated plants were placed in a moist chamber (100% rel. humidity) for 24 h and transferred to a greenhouse maintained under standard operating conditions [7]. Symptom expression was observed and measured weekly for 4 weeks. 2.4. Toxin extraction, bioassay and chemical characterization Culture medium, as previously described [6], was filtered through eight layers of cheesecloth and exhaustively extracted three times with equal volumes of ethyl acetate. The three extracts were combined and reduced to dryness by rotary evaporation at 35°C. The residue was placed on an LH-20 column (2.5 x 120 cm) and eluted with CHC13/MeOH 1:1 (v/v). Although phytotoxic activity was expressed at several regions eluting from columns, the greatest phytotoxic activity was detected between 255-324 ml. After standing overnight at room temperature, crystals formed in some of the collection vials. The crystals were bioactive. This solution was taken to dryness by rotary evaporation and the residue dissolved in 10 ml of H20. The solution was successively extracted with equal volumes of CHCI 3 and then hexane. The bioactivity remained in the aqueous fraction. After rotary evaporation of the aqueous fraction, the residue was taken up in 1 ml of H20 and the solution placed on a mini-column of Cl8 reverse phase (Bakerbond, 40 #m, 60 .& mesh) adsorbent (in a 10-ml syringe). The column was tins-
J. Hershenhorn et a l . / Plant Sci. 94 (1993) 227-234
ed successively with 5 ml of H20, 5 ml of MeOH and 25 ml of CHCI 3. All bioactivity remained in the aqueous fraction. Finally, this aqueous fraction was placed on an LH-20 column (2.5 x 120 cm) and eluted with MeOH/H20 4:1 v/v. The bioactive fraction was collected, subjected to rotary evaporation, and crystallized from MeOH. 2.5. Bioassays
A simple leaf puncture bioassay test was used to guide us in the isolation of the phytotoxic compound(s) [7]. Unless otherwise noted, the toxin samples were adjusted to pH 5.0 with aqueous dilute NaOH. The test solution (15 #1) was placed over a puncture wound made in a leaf blade with a Hamilton syringe. The test leaves were placed in a sealed moist chamber and the effects of the toxin observed after 24 h of incubation at 25°C. The test leaves most commonly used were those of roselle (Hibiscus sabdariffa L.) since we have found that this genus is especially sensitive to many phytotoxins. The toxin was dissolved in 5% ethanol and 5 /A of solution was applied to the leaf. We also used leaves (young or old) of Cirsium arvense. Otherwise, a wide host range test was conducted with a variety of plant species to assess the specificity and general effectiveness of the phytotoxin from S. cirsii over a course of 24 h. The toxin was also studied for its effectiveness as a germination inhibitor, and an inhibitor of root elongation. Seeds of Canada thistle were surface treated with sodium chlorite (1% (v/v) for 10 min), rinsed, and placed on Petri plates (10 per plate). Each plate contained 2 ml of test solution and root lengths were measured after 5 days. In seed germination tests, 25 seeds were placed in 1 ml of test solution, and after 40 h the seeds were rinsed in distilled H20, placed on damp filter papers and the % germination recorded after 5 days. A selected crystal of the phytotoxin was subjected to X-ray analysis by Ms. Julie Lee and Dr. Jon Clardy, Cornell University. Nuclear magnetic resonance spectroscopy (NMR) was performed on a 300 mHz Brucker instrument with the sample dissolved in D20. Authentic 13-nitropropionic acid for spectral and biological comparisons was obtained from Sigma Chemical Co., USA.
229
3. Results and discussion 3.1. Septoria cirsii as a pathogen Although S. cirsii (Fig. 1) had previously been
recorded as an organism recoverable from Canada thistle, it has only recently been proposed and studied as a potential biocontrol agent [5,9]. We carried out an artificial inoculation of Canada thistle plants, as well as an assortment of other crop plants, with a spore suspension Of C. cirsii (5 plants per test species). Only Can~ada thistle developed any symptoms; necrotic lesions surrounded by chlorotic zones initially appeared on the leaves after 1 week (Fig. 2). Eventually, comparable lesions appeared on the stems and after
Fig. 1. The conidiospores (pycnidiospores) of Septoria cirsii. These spores range in size from 1-3 t~m in diameter to 40-60 p.m in length.
230
~L Hershenhorn et al./ Plant Sci, 94 (1993) 227-234
~v
Fig. 3. Diagram of a healthy plant of Cirsium arvense (Canada thistle) (right) and one that had been inoculated 3 weeks before with Septoria cirsii (left).
Fig. 2. A leaf of Cirsium arvense (Canada thistle) which had been inoculated with Septoria cirsii. Note the numerous sunken necrotic lesions. Conditions of inoculation and incubation are described in Materials and methods. The leaf is - 12 cm long.
3-4 weeks the plants were completely destroyed (Fig. 3). All other plants tested including Lycopersicon esculentum Mill. (tomato), Cucumis sativus L. (cucumber), Phaseolus vulgaris L. (garden bean), and Triticum aestivum L. (wheat) showed no symptoms, whatever. S. cirsii does possess host selectivity. The organism was successively reisolated in pure culture from infected Canada thistle plants. Subsequently it was successfully retested for pathogenicity on Canada thistle which fulfilled Koch's postulates and confirmed that this organism is a bona fide pathogen of Canada thistle. 3.2. Phytotoxic activity in S. cirsii Leaves infected with S. cirsii have symptoms
that are reminiscent of those associated with one or more phytotoxins (Fig. 2) [10]. The fungus induces necrotic spots surrounded by areas of chlorosis. It has been our experience that toxinproducing phytopathogenic fungi commonly require one or more 'activators' or biochemical cues from the host plant or another plant source in order to optimally produce one or more phytotox-
Table 1 Growth and yield of ethyl acetate extractable substances per liter of medium from Septoria cirsii grown on different plant extracts Medium
Dry weight mycelium (rag/l)
EtAc extractable a material (mg/l)
Nutrient broth (Difco) + 2 g/l Canada thistle + 2 g/l Purple nutsedge + 2 g/l Spotted knapweed
2.4
85.7
1.4 1.5 2.5
29.0 627.06 62.0
aEach of these extracts were tested in the leaf puncture bioassay test and they were phytotoxic. The uninoculated media extracted in the same manner did not possess phytotoxicity in the leaf bioassay test.
231
J. Hershenhorn et al./Plant Sci. 94 (1993) 227-234
SEPTORIA TOXIN
,
I
,
5.0
I
,
4.8
I
~
4.6
I
,
4.4
I
,
4.2
I
,
I
4.0
,
3.8
I
,
3.6
I
,
3.4
I
,
3.2
I
,
3.0
I
,
2.8
PPM
- NITROPROPIONIC ACID
,
I 5.0
,
I 4.8
,
I 4.6
,
I 4.4
,
I 4.2
,
I
,
4.0
I 3.8
t
I 3.6
,
I 3.4
J
I 3.2
,
I 3.0
,
I
,
2.8
PPM
Fig. 4. The NMR spectrum of the toxin from Septoria cirsii (A) and the NMR spectrum of ~-nitropropionic acid (B). The samples were run in 99.6% D20. The deuterated water (HDO) peak is at - 4 . 9 ppm on each spectrum.
232
ic substances [6,11]. To this end, we grew S. cirsii under standard conditions for 8 days in the M-1-D medium supplemented with 2 g/1 of finely chopped fresh leaves of Canada thistle (Cirsium arvense), spotted knapweed Centurea maculosa, or purple nutsedge (Cyperus rotundus). There was positive phytotoxic activity in the extracts of each of these cultures, however, the culture containing purple nutsedge yielded the greatest weight of material in the ethyl acetate extract, but the lowest yield of mycelial dry weight (Table 1). Ultimately, this same medium was the one from which the greatest amount of the phytotoxin was recovered, - 2 0 0 mg/1. The ethyl acetate extract made of each medium not supporting fungal growth did not possess phytotoxicity. It is apparent from these results that one or more phytotoxins is produced by S. cirsii and that the purple nutsedge leaf extract enhances the dry weight of ethyl acetate extractable substances. A crystalline, pure compound was obtained from the separatory and chromatography techniques applied to the S. cirsii culture extract obtained from the medium containing leaves of purple nutsedge (Table 1). This represents about 1/3 of the total dry weight of the ethyl acetate extract. This compound was found in each of the other cultures but to a lesser extent as judged by nuclear magnetic resonance spectroscopy (NMR). A single crystal of this phytotoxin was subjected to X-ray diffraction analysis. The techniques employed were comparable to those previously used for other phytotoxins [8]. The data unequivocally showed that the phytotoxic compound was flnitropropionic acid (also having the names hiptagenic acid, and bovinocidin) (Fig. 4). The proton NMR of the phytotoxin from S. cirsii compared with that of standard fl-propionic acid were identical (Fig. 5). Furthermore, the melting points of both of the fungal derived compound, and the standard were identical i.e., 65-67°C. Although fl-nitropropionic acid was described from the higher plant Indigofera endecaphylla [12] and a few others it is also known from a number of fungi including Aspergillus flavus, A. oryzae, A. wentii, and Penicillium atrovenetum, [ 13,14]. It has also been described from an unidentified filamentous fungus that attacks zinnia and its toxicity was
J. Hershenhorn et al. / P & n t Sci. 94 (1993) 2 2 7 - 2 3 4
O II HO
IC
/CH2 CH 2
N NO 2
Fig. 5. The structure of fl-nitropropionicacid.
implicated in the plant disease process [15]. However, to our knowledge it has never been described as a phytotoxin from a weed pathogen.
3.3. fl-Nitropropionic acid bioassays At 10 -4 M, 2 x 10 -5 M, 10 -5 M and 2 x 10 -6 M fl-nitropropionic acid there was a 28, 9, 5 and 2"/o reduction, respectively, in average root length development in the root bioassay test system as compared to the controls. However, only at 10 -4 M were the data significantly different at P < 0.05. Correspondingly, fl-nitropropionic acid was also a potent inhibitor of seed germination especially at 10 -3 and 10 -4 M at 40 h. There was 0% germination at the former concentration and 32% at the latter with 48% germination of the control. The seed germination tests were repeated three times with essentially the same results. Although a leaf bioassay test was used to guide toxin purification, a more general bioassay test employing a wide range of plant species was used to examine the specific effects of fl-nitropropionic acid on plants. In this test we employed various concentrations of the toxin dissolved in weak aqueous NaOH to a pH of 5.0 (to avoid effects due to pH). In addition, authentic fl-nitropropionic acid (pH 5.0) was also simultaneously examined and the results of the bioassay test with each test plant was identical to the reaction with the toxin isolated from S. cirsii (Table 2). One day after treatment, the leaves of Canada thistle appeared to be the most sensitive (in the necrotic reaction) to the toxin, although after 2-3 days the leaves of most species showed some effect of the toxin. Lesions formed on Canada thistle leaves (not shown) were comparable to those forming on leaves infected with S. cirsii (Fig. 2).
233
J. H e r s h e n h o r n et a l . / P l a n t Sci. 94 ( 1 9 9 3 ) 2 2 7 - 2 3 4
Table 2 The effects of a solution of fl-nitropropionic acid (pH 5.0) on a range of various plant species (leaf blades), 24 h after treatment
Table 2
(Continued)
Amount of toxin applied to a leaf puncture wound a 5 ttg
Amount of toxin applied to a leaf puncture wound a
2.5 ~tg
1 #g
Tomato 5/~g
2.5/~g
1 #g
Hibiscus subduriffa
+++
++
+
++++
+++
+
+
-
-
-
-
-
Canada thistle Circium arvense
Apple Malus sylvestris
Wheat Triticum aestivum
Cassava Manihot esculenta
-
-
-
Sunflower ~cbtyl&lon)
+++
+
-
++
+
-
+
-
-
++
+
+
Helianthus annuus
Tobacco
Lycopersicon esculentum
Roselle
Nicotiana tabacum
+++
Witloof chicory Cichorium intybus
+++
aA leaf puncture assay was used with a solution of 3nitropropionic acid adjusted to pH 5.0 with NaOH. Each sample (15 #1) was applied to a puncture wound in the leaf blade and the leaves incubated in a moist chamber at 25°C for 24 h at which time measurements were made. Scale: -, no symptoms expressed; +, lesion 0-1 mm diameter; ++, lesion I-2 mm in diameter; +++, lesion 2-4 mm in diameter; ++++, lesion 5-7 mm diameter. After 3 days the leaves of all varieties showed symptoms, fl-Nitropropionic acid from the fungus caused identical reactions on each of the above plants tested. bin this case we observed a massive chlorosis.
Potato Solanum tuberosum
Minneola tangerine Citrus r e t i c u l a t a
Annual mercury Mercurialis annua
+++
Artichoke Cynara scolymus
++
-
-
-
-
-
-
-
-
++++
+++
++
-
-
-
-
-
-
-
-
-
+++
-
Bean P h a s e o l u s vulgaris
++
Chick pea Ci c e r a r i e t i n u m
+
Common sowthistle Sonchus oleraceus
Common mallow M a l v a sp.
Green cabbage b Brassica oleracea
Lemon Citrus l e m o n
Mandarin Citrus nobilis
Parsley Petroselinum crispum
+
Pea Pisum sativum
+
Red pepper Capsicum annuum
+
Spurge E u p h o r b i a sp.
Future
prospects
T h e h o s t selectivity o f S . c i r s i i to C a n a d a thistle c o m b i n e d w i t h its ability to c a u s e r a p i d l y e x p a n d i n g l e a f lesions m a k e s it a p o t e n t i a l c a n d i d a t e for the b i o l o g i c a l c o n t r o l o f this i m p o r t a n t w e e d y plant. A l t h o u g h C a n a d a thistle is w i d e l y f o u n d in its n a t i v e M e d i t e r r a n e a n r a n g e , in s o m e locales it d o e s n o t a p p e a r to be the serious p r o b l e m it is in N o r t h A m e r i c a . T h i s m a y be d u e to the p r e s e n c e o f S . c i r s i i a n d a series o f e n v i r o n m e n t a l f a c t o r s t h a t f a v o r the d e v e l o p m e n t o f S e p t o r i a l e a f s p o t disease w h i c h s u p p r e s s e s the thistle p o p u l a t i o n . f l - N i t r o p r o p i o n i c a c i d is p r o d u c e d in s u b s t a n t i a l q u a n t i t i e s by S e p t o r i a c i r s i i . A l t h o u g h it is p h y t o toxic, its role in S e p t o r i a l e a f s p o t disease has yet to be d e t e r m i n e d . It is also the case t h a t o n e o r m o r e o t h e r p h y t o t o x i n s m a y be p r o d u c e d by S. c i r s i i since p h y t o t o x i c a c t i v i t y besides t h a t o f 3n i t r o p r o p i o n i c a c i d was n o t e d d u r i n g the p u r i f i c a t i o n process. Since C a n a d a thistle is so sensitive to the c o m p o u n d , d e r i v a t i v e s o f it s h o u l d be e x p l o r e d for their p o t e n t i a l use as selective herbicides.
+
Strawberry F r a g a r i a vesca
3.4.
4. Acknowledgements
Swiss chard B e t a vulgaris
T h e a u t h o r s wish to t h a n k the M o n t a n a W e e d
234
J. Hershenhorn et al /Plant Sci. 94 (1993) 227-234
Trust Fund and the Montana Agricultural Experiment Station for financial support. Suzan Strobel kindly provided the drawings of healthy and diseased Canada thistle plants.
8
5. References
9
1
L.G. Holm, D.L. Plucknett, J.V. Pancho and J.P. Herberger, The World's Worst Weeds. University Press of Hawaii, 1977. 2 R.J. Moore, The biology of Canadian weeds, Cirsium arvense (L.) Scop. Can. J. Plant Sci., 55 (1975) 1033-1048. 3 D.S. Broston and D.C. Sands, Field trials of Sclerotinia sc!erotiorium to control Canada thistle (Cirsium arvense). Weed Sci., 34 (1986) 377-380. 4 G.A. Strobel, Biological control of weeds. Sci. Am., 265 (1991) 72-78. 5 M. Vurro, M.C. Zonno and A. Bottalico, Sul potenziale impiego di Septoria cirsii come micoerbicida contro Cirsium arvense. Atti Giornate Fitopatologiche, 3 (1992) 189-198. 6 F. Pinkerton and G.A. Strobel, Serinol as an activator of toxin production in attenuated cultures of Helminthosporium sacchari. Proc. Natl. Acad. Sci. USA, 73 (1976} 4007-4011. 7 J. Hershenhorn, S.H. Park, A. Stierle and G.A. Strobel, Fusariurn avenaceum as a novel pathogen of spotted
l0 It
12
13
14
15
knapweed and its phytotoxins, acetamido-butenolide and enniatin B. Plant Sci., 86 (1992) 155-160. F. Sugawara, G. Strobel, L.E. Fisher, G D . Van Duyne and J. Clardy, Bipolaroxin, a selective phytotoxin produced by Bipolaris cynodontis. Proc. Natl. Acad. Sci. USA, 82 (1985) 8291-8294. Index of Plant Diseases in the United States, Agriculture Handbook no. 165, Agric. Res. Sci. USDA, Washington, DC, 1960. G.A. Strobel, Phytotoxins. Annu. Rev. Biochem., 51 (1982) 309-333. D.J. Robeson and G.A. Strobel, Influence of plant extracts on phytotoxin production and growth rate of Alternaria helianthi. J. Phytopathol., l l7 (1986) 265-269. M.P. Morris, C. Pagan and H.E. Warmlee, Hiptagenic acid, a toxicomponent of lndigofora endecaphylla. Science, l l9 (1961) 322-323. J.W. Hylin and H. Matsumoto, The biosynthesis of 3nitropropanoic acid by Penicillium atrovenetum. Arch. Biochem. Biophys., 93 (1960) 542-545. A. Evidente, P. Capretti, F. Giordano and G. Surico, Identification and phytotoxicity of 3-nitropropionic acid produced in vitro by Melanconis thelebola. Experientia, 48 (1992) 1169-1172. T. Kamikawa, F. Higuchi, M. Taniguchi, and Y. Asaka, Toxic metabolites of an unidentified filamentous fungus isolated from zinnia leaves. Agric. Chem. Biol., 44 (1980) 691-692.
Plant Science 94 (1993) 227-234
Septoria cirsii, a potential biocontrol agent of Canada thistle and its phytotoxin - - fl-nitropropionic acid Joseph Hershenhorn a, Maurizio Vurro b, Maria Chiara Zonno b, Andrea Stierle a, Gary Strobel* a aDepartment of Plant Pathology, Montana State University, Bo-ernan. Montana 59717, USA blstituto tossine d micotossine da parassiti vegetali, Consiglio Nazionale delle Ricerche, viale Einaudi 51, 70126 Bari. Italy (Received 3 May 1993; revision received 29 June 1993; accepted 2 July 1993)
Abstract
Septoria cirsii causes leaf spot of Canada thistle (Cirsium arvense L.) and no other plant tested was susceptible to this pathogen. Because this pathogen possesses host specificity, and because it is so devastating to Canada thistle plants in the field, it is proposed for consideration as a biological control agent. In culture, S. cirsii produced copious amounts of a known phytotoxin which was identified as/3-nitropropionic acid. The toxin is inhibitory to seed germination, root elongation and causes the typical symptoms of chlorosis and necrosis on the leaves of Canada thistle which is one of the most toxin-sensitive plants of all of those tested. Key words: Phytotoxin; Biological control; Weeds; Plant pathogen
I. Introduction
Canada thistle (Cirsium arvense L.) is one of the most important weedy plants growing in virtually all temperate areas of the world and having its center of origin in the Mediterranean region [1]. Most of the serious problems with this plant are in areas receiving 450-900 m m of rainfall/year [1] and it is especially serious in pastures, rangelands, roadsides, and ditch banks [1]. It is also a weed invading fields of barley, wheat, flax, millet, oats, rye and sorghum. It is fairly common in vineyards of * Corresponding author.
Southern Europe and orchards in Canada. The plant reproduces by wind-blown plumed achenes and by an extensive rhizome system [2]. Tillage, herbicides and crop rotations are the commonly used methods for controlling this troublesome pest. In order to reduce the costs of herbicides and reduce the environmental impact involved in these practices, biological control measures are currently being explored [3]. For this reason, we sought potential pathogens that may be considered as candidates for the biological control of Canada thistle in its native region of the world, the Mediterranean area [4]. Canada thistle plants showing severe leaf blight were found in Italy and the
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03701-V
228
causal agent of the disease was Septoria cirsii [5]. This report describes the necrotic leaf spot of Canada thistle and shows that the phytotoxin, 3nitropropionic acid is produced by this fungus and that it may be one of several phytotoxins involved in symptom production. The report also gives some rationale for further testing of this important pathogen as a biological control agent. 2. Materials and methods
2.1. Septoria cirsii: isolation and identification This fungal pathogen was isolated from necrotic lesions on naturally infected Canada thistle (Cirsium arvense L) leaves near Bari in the south of Italy [5]. Infected leaves were washed thoroughly with running tap water, dried, and then placed in a moist chamber at 24°C. Two to three days later, pycnidia appeared on the majority of the necrotic portions of the leaves. Each pycnidium produces numerous pycnidiospores and several single spore cultures of this fungus were obtained for further biological testing. Ultimately, a culture of the fungus was identified as Septoria cirsii by Dr. E. Punithalingam of the Commonwealth Mycological Institute, England. 2.2. Septoria culture The fungus was maintained on Petri plates containing a medium composed of 18% (v/v) tomato juice, 0.1% (w/v) CaCO3 and 1.5% (w/v) agar. The plates were inoculated with a conidial suspension (1 ml), incubated for 1 week at 25°C in a regimen of 12 h dark/12 h light (near UV to blue at - 3 0 0 tzW/cm2). Under these conditions the fungus sporulated abundantly on these plates ( - 106 conidia/cm2). For inoculation of culture flasks used for toxin production, we placed 1 ml of a spore suspension (105-106 spores/ml) obtained from these plates into 200 ml of culture medium in a 1-1 Erlenmyer flask. Agar blocks transferred from the plates containing fungal mycelia were not effective in producing adequate fungal growth. The base medium was M-1-D [6] plus 2 g of finely cut leaves of various plants in order to study the influence of plant products on fungal growth and metabolite production. The culture flasks were shaken at 140
,L Hershenhorn et al./ Plant Sci. 94 (1993) 227-234
rev./min at room temperature (24-24°C) for 8 days prior to harvest. 2.3. Pathogenicity tests Pathogenicity tests were performed on 2-monthold plants of a number of species grown under standard greenhouse conditions. The leaves were inoculated by sprays of an aqueous suspension of S. cirsii conidia containing 105-106 spores/ml. The spore suspension was obtained by placing several ml of H20 on the surface of a maintenance plate of S. cirsii and gently scraping the surface to dislodge the conidia from pycnidia. The spore suspension was sprayed on the leaves of the test plant until they were wet. The inoculated plants were placed in a moist chamber (100% rel. humidity) for 24 h and transferred to a greenhouse maintained under standard operating conditions [7]. Symptom expression was observed and measured weekly for 4 weeks. 2.4. Toxin extraction, bioassay and chemical characterization Culture medium, as previously described [6], was filtered through eight layers of cheesecloth and exhaustively extracted three times with equal volumes of ethyl acetate. The three extracts were combined and reduced to dryness by rotary evaporation at 35°C. The residue was placed on an LH-20 column (2.5 x 120 cm) and eluted with CHC13/MeOH 1:1 (v/v). Although phytotoxic activity was expressed at several regions eluting from columns, the greatest phytotoxic activity was detected between 255-324 ml. After standing overnight at room temperature, crystals formed in some of the collection vials. The crystals were bioactive. This solution was taken to dryness by rotary evaporation and the residue dissolved in 10 ml of H20. The solution was successively extracted with equal volumes of CHCI 3 and then hexane. The bioactivity remained in the aqueous fraction. After rotary evaporation of the aqueous fraction, the residue was taken up in 1 ml of H20 and the solution placed on a mini-column of Cl8 reverse phase (Bakerbond, 40 #m, 60 .& mesh) adsorbent (in a 10-ml syringe). The column was tins-
J. Hershenhorn et a l . / Plant Sci. 94 (1993) 227-234
ed successively with 5 ml of H20, 5 ml of MeOH and 25 ml of CHCI 3. All bioactivity remained in the aqueous fraction. Finally, this aqueous fraction was placed on an LH-20 column (2.5 x 120 cm) and eluted with MeOH/H20 4:1 v/v. The bioactive fraction was collected, subjected to rotary evaporation, and crystallized from MeOH. 2.5. Bioassays
A simple leaf puncture bioassay test was used to guide us in the isolation of the phytotoxic compound(s) [7]. Unless otherwise noted, the toxin samples were adjusted to pH 5.0 with aqueous dilute NaOH. The test solution (15 #1) was placed over a puncture wound made in a leaf blade with a Hamilton syringe. The test leaves were placed in a sealed moist chamber and the effects of the toxin observed after 24 h of incubation at 25°C. The test leaves most commonly used were those of roselle (Hibiscus sabdariffa L.) since we have found that this genus is especially sensitive to many phytotoxins. The toxin was dissolved in 5% ethanol and 5 /A of solution was applied to the leaf. We also used leaves (young or old) of Cirsium arvense. Otherwise, a wide host range test was conducted with a variety of plant species to assess the specificity and general effectiveness of the phytotoxin from S. cirsii over a course of 24 h. The toxin was also studied for its effectiveness as a germination inhibitor, and an inhibitor of root elongation. Seeds of Canada thistle were surface treated with sodium chlorite (1% (v/v) for 10 min), rinsed, and placed on Petri plates (10 per plate). Each plate contained 2 ml of test solution and root lengths were measured after 5 days. In seed germination tests, 25 seeds were placed in 1 ml of test solution, and after 40 h the seeds were rinsed in distilled H20, placed on damp filter papers and the % germination recorded after 5 days. A selected crystal of the phytotoxin was subjected to X-ray analysis by Ms. Julie Lee and Dr. Jon Clardy, Cornell University. Nuclear magnetic resonance spectroscopy (NMR) was performed on a 300 mHz Brucker instrument with the sample dissolved in D20. Authentic 13-nitropropionic acid for spectral and biological comparisons was obtained from Sigma Chemical Co., USA.
229
3. Results and discussion 3.1. Septoria cirsii as a pathogen Although S. cirsii (Fig. 1) had previously been
recorded as an organism recoverable from Canada thistle, it has only recently been proposed and studied as a potential biocontrol agent [5,9]. We carried out an artificial inoculation of Canada thistle plants, as well as an assortment of other crop plants, with a spore suspension Of C. cirsii (5 plants per test species). Only Can~ada thistle developed any symptoms; necrotic lesions surrounded by chlorotic zones initially appeared on the leaves after 1 week (Fig. 2). Eventually, comparable lesions appeared on the stems and after
Fig. 1. The conidiospores (pycnidiospores) of Septoria cirsii. These spores range in size from 1-3 t~m in diameter to 40-60 p.m in length.
230
~L Hershenhorn et al./ Plant Sci, 94 (1993) 227-234
~v
Fig. 3. Diagram of a healthy plant of Cirsium arvense (Canada thistle) (right) and one that had been inoculated 3 weeks before with Septoria cirsii (left).
Fig. 2. A leaf of Cirsium arvense (Canada thistle) which had been inoculated with Septoria cirsii. Note the numerous sunken necrotic lesions. Conditions of inoculation and incubation are described in Materials and methods. The leaf is - 12 cm long.
3-4 weeks the plants were completely destroyed (Fig. 3). All other plants tested including Lycopersicon esculentum Mill. (tomato), Cucumis sativus L. (cucumber), Phaseolus vulgaris L. (garden bean), and Triticum aestivum L. (wheat) showed no symptoms, whatever. S. cirsii does possess host selectivity. The organism was successively reisolated in pure culture from infected Canada thistle plants. Subsequently it was successfully retested for pathogenicity on Canada thistle which fulfilled Koch's postulates and confirmed that this organism is a bona fide pathogen of Canada thistle. 3.2. Phytotoxic activity in S. cirsii Leaves infected with S. cirsii have symptoms
that are reminiscent of those associated with one or more phytotoxins (Fig. 2) [10]. The fungus induces necrotic spots surrounded by areas of chlorosis. It has been our experience that toxinproducing phytopathogenic fungi commonly require one or more 'activators' or biochemical cues from the host plant or another plant source in order to optimally produce one or more phytotox-
Table 1 Growth and yield of ethyl acetate extractable substances per liter of medium from Septoria cirsii grown on different plant extracts Medium
Dry weight mycelium (rag/l)
EtAc extractable a material (mg/l)
Nutrient broth (Difco) + 2 g/l Canada thistle + 2 g/l Purple nutsedge + 2 g/l Spotted knapweed
2.4
85.7
1.4 1.5 2.5
29.0 627.06 62.0
aEach of these extracts were tested in the leaf puncture bioassay test and they were phytotoxic. The uninoculated media extracted in the same manner did not possess phytotoxicity in the leaf bioassay test.
231
J. Hershenhorn et al./Plant Sci. 94 (1993) 227-234
SEPTORIA TOXIN
,
I
,
5.0
I
,
4.8
I
~
4.6
I
,
4.4
I
,
4.2
I
,
I
4.0
,
3.8
I
,
3.6
I
,
3.4
I
,
3.2
I
,
3.0
I
,
2.8
PPM
- NITROPROPIONIC ACID
,
I 5.0
,
I 4.8
,
I 4.6
,
I 4.4
,
I 4.2
,
I
,
4.0
I 3.8
t
I 3.6
,
I 3.4
J
I 3.2
,
I 3.0
,
I
,
2.8
PPM
Fig. 4. The NMR spectrum of the toxin from Septoria cirsii (A) and the NMR spectrum of ~-nitropropionic acid (B). The samples were run in 99.6% D20. The deuterated water (HDO) peak is at - 4 . 9 ppm on each spectrum.
232
ic substances [6,11]. To this end, we grew S. cirsii under standard conditions for 8 days in the M-1-D medium supplemented with 2 g/1 of finely chopped fresh leaves of Canada thistle (Cirsium arvense), spotted knapweed Centurea maculosa, or purple nutsedge (Cyperus rotundus). There was positive phytotoxic activity in the extracts of each of these cultures, however, the culture containing purple nutsedge yielded the greatest weight of material in the ethyl acetate extract, but the lowest yield of mycelial dry weight (Table 1). Ultimately, this same medium was the one from which the greatest amount of the phytotoxin was recovered, - 2 0 0 mg/1. The ethyl acetate extract made of each medium not supporting fungal growth did not possess phytotoxicity. It is apparent from these results that one or more phytotoxins is produced by S. cirsii and that the purple nutsedge leaf extract enhances the dry weight of ethyl acetate extractable substances. A crystalline, pure compound was obtained from the separatory and chromatography techniques applied to the S. cirsii culture extract obtained from the medium containing leaves of purple nutsedge (Table 1). This represents about 1/3 of the total dry weight of the ethyl acetate extract. This compound was found in each of the other cultures but to a lesser extent as judged by nuclear magnetic resonance spectroscopy (NMR). A single crystal of this phytotoxin was subjected to X-ray diffraction analysis. The techniques employed were comparable to those previously used for other phytotoxins [8]. The data unequivocally showed that the phytotoxic compound was flnitropropionic acid (also having the names hiptagenic acid, and bovinocidin) (Fig. 4). The proton NMR of the phytotoxin from S. cirsii compared with that of standard fl-propionic acid were identical (Fig. 5). Furthermore, the melting points of both of the fungal derived compound, and the standard were identical i.e., 65-67°C. Although fl-nitropropionic acid was described from the higher plant Indigofera endecaphylla [12] and a few others it is also known from a number of fungi including Aspergillus flavus, A. oryzae, A. wentii, and Penicillium atrovenetum, [ 13,14]. It has also been described from an unidentified filamentous fungus that attacks zinnia and its toxicity was
J. Hershenhorn et al. / P & n t Sci. 94 (1993) 2 2 7 - 2 3 4
O II HO
IC
/CH2 CH 2
N NO 2
Fig. 5. The structure of fl-nitropropionicacid.
implicated in the plant disease process [15]. However, to our knowledge it has never been described as a phytotoxin from a weed pathogen.
3.3. fl-Nitropropionic acid bioassays At 10 -4 M, 2 x 10 -5 M, 10 -5 M and 2 x 10 -6 M fl-nitropropionic acid there was a 28, 9, 5 and 2"/o reduction, respectively, in average root length development in the root bioassay test system as compared to the controls. However, only at 10 -4 M were the data significantly different at P < 0.05. Correspondingly, fl-nitropropionic acid was also a potent inhibitor of seed germination especially at 10 -3 and 10 -4 M at 40 h. There was 0% germination at the former concentration and 32% at the latter with 48% germination of the control. The seed germination tests were repeated three times with essentially the same results. Although a leaf bioassay test was used to guide toxin purification, a more general bioassay test employing a wide range of plant species was used to examine the specific effects of fl-nitropropionic acid on plants. In this test we employed various concentrations of the toxin dissolved in weak aqueous NaOH to a pH of 5.0 (to avoid effects due to pH). In addition, authentic fl-nitropropionic acid (pH 5.0) was also simultaneously examined and the results of the bioassay test with each test plant was identical to the reaction with the toxin isolated from S. cirsii (Table 2). One day after treatment, the leaves of Canada thistle appeared to be the most sensitive (in the necrotic reaction) to the toxin, although after 2-3 days the leaves of most species showed some effect of the toxin. Lesions formed on Canada thistle leaves (not shown) were comparable to those forming on leaves infected with S. cirsii (Fig. 2).
233
J. H e r s h e n h o r n et a l . / P l a n t Sci. 94 ( 1 9 9 3 ) 2 2 7 - 2 3 4
Table 2 The effects of a solution of fl-nitropropionic acid (pH 5.0) on a range of various plant species (leaf blades), 24 h after treatment
Table 2
(Continued)
Amount of toxin applied to a leaf puncture wound a 5 ttg
Amount of toxin applied to a leaf puncture wound a
2.5 ~tg
1 #g
Tomato 5/~g
2.5/~g
1 #g
Hibiscus subduriffa
+++
++
+
++++
+++
+
+
-
-
-
-
-
Canada thistle Circium arvense
Apple Malus sylvestris
Wheat Triticum aestivum
Cassava Manihot esculenta
-
-
-
Sunflower ~cbtyl&lon)
+++
+
-
++
+
-
+
-
-
++
+
+
Helianthus annuus
Tobacco
Lycopersicon esculentum
Roselle
Nicotiana tabacum
+++
Witloof chicory Cichorium intybus
+++
aA leaf puncture assay was used with a solution of 3nitropropionic acid adjusted to pH 5.0 with NaOH. Each sample (15 #1) was applied to a puncture wound in the leaf blade and the leaves incubated in a moist chamber at 25°C for 24 h at which time measurements were made. Scale: -, no symptoms expressed; +, lesion 0-1 mm diameter; ++, lesion I-2 mm in diameter; +++, lesion 2-4 mm in diameter; ++++, lesion 5-7 mm diameter. After 3 days the leaves of all varieties showed symptoms, fl-Nitropropionic acid from the fungus caused identical reactions on each of the above plants tested. bin this case we observed a massive chlorosis.
Potato Solanum tuberosum
Minneola tangerine Citrus r e t i c u l a t a
Annual mercury Mercurialis annua
+++
Artichoke Cynara scolymus
++
-
-
-
-
-
-
-
-
++++
+++
++
-
-
-
-
-
-
-
-
-
+++
-
Bean P h a s e o l u s vulgaris
++
Chick pea Ci c e r a r i e t i n u m
+
Common sowthistle Sonchus oleraceus
Common mallow M a l v a sp.
Green cabbage b Brassica oleracea
Lemon Citrus l e m o n
Mandarin Citrus nobilis
Parsley Petroselinum crispum
+
Pea Pisum sativum
+
Red pepper Capsicum annuum
+
Spurge E u p h o r b i a sp.
Future
prospects
T h e h o s t selectivity o f S . c i r s i i to C a n a d a thistle c o m b i n e d w i t h its ability to c a u s e r a p i d l y e x p a n d i n g l e a f lesions m a k e s it a p o t e n t i a l c a n d i d a t e for the b i o l o g i c a l c o n t r o l o f this i m p o r t a n t w e e d y plant. A l t h o u g h C a n a d a thistle is w i d e l y f o u n d in its n a t i v e M e d i t e r r a n e a n r a n g e , in s o m e locales it d o e s n o t a p p e a r to be the serious p r o b l e m it is in N o r t h A m e r i c a . T h i s m a y be d u e to the p r e s e n c e o f S . c i r s i i a n d a series o f e n v i r o n m e n t a l f a c t o r s t h a t f a v o r the d e v e l o p m e n t o f S e p t o r i a l e a f s p o t disease w h i c h s u p p r e s s e s the thistle p o p u l a t i o n . f l - N i t r o p r o p i o n i c a c i d is p r o d u c e d in s u b s t a n t i a l q u a n t i t i e s by S e p t o r i a c i r s i i . A l t h o u g h it is p h y t o toxic, its role in S e p t o r i a l e a f s p o t disease has yet to be d e t e r m i n e d . It is also the case t h a t o n e o r m o r e o t h e r p h y t o t o x i n s m a y be p r o d u c e d by S. c i r s i i since p h y t o t o x i c a c t i v i t y besides t h a t o f 3n i t r o p r o p i o n i c a c i d was n o t e d d u r i n g the p u r i f i c a t i o n process. Since C a n a d a thistle is so sensitive to the c o m p o u n d , d e r i v a t i v e s o f it s h o u l d be e x p l o r e d for their p o t e n t i a l use as selective herbicides.
+
Strawberry F r a g a r i a vesca
3.4.
4. Acknowledgements
Swiss chard B e t a vulgaris
T h e a u t h o r s wish to t h a n k the M o n t a n a W e e d
234
J. Hershenhorn et al /Plant Sci. 94 (1993) 227-234
Trust Fund and the Montana Agricultural Experiment Station for financial support. Suzan Strobel kindly provided the drawings of healthy and diseased Canada thistle plants.
8
5. References
9
1
L.G. Holm, D.L. Plucknett, J.V. Pancho and J.P. Herberger, The World's Worst Weeds. University Press of Hawaii, 1977. 2 R.J. Moore, The biology of Canadian weeds, Cirsium arvense (L.) Scop. Can. J. Plant Sci., 55 (1975) 1033-1048. 3 D.S. Broston and D.C. Sands, Field trials of Sclerotinia sc!erotiorium to control Canada thistle (Cirsium arvense). Weed Sci., 34 (1986) 377-380. 4 G.A. Strobel, Biological control of weeds. Sci. Am., 265 (1991) 72-78. 5 M. Vurro, M.C. Zonno and A. Bottalico, Sul potenziale impiego di Septoria cirsii come micoerbicida contro Cirsium arvense. Atti Giornate Fitopatologiche, 3 (1992) 189-198. 6 F. Pinkerton and G.A. Strobel, Serinol as an activator of toxin production in attenuated cultures of Helminthosporium sacchari. Proc. Natl. Acad. Sci. USA, 73 (1976} 4007-4011. 7 J. Hershenhorn, S.H. Park, A. Stierle and G.A. Strobel, Fusariurn avenaceum as a novel pathogen of spotted
l0 It
12
13
14
15
knapweed and its phytotoxins, acetamido-butenolide and enniatin B. Plant Sci., 86 (1992) 155-160. F. Sugawara, G. Strobel, L.E. Fisher, G D . Van Duyne and J. Clardy, Bipolaroxin, a selective phytotoxin produced by Bipolaris cynodontis. Proc. Natl. Acad. Sci. USA, 82 (1985) 8291-8294. Index of Plant Diseases in the United States, Agriculture Handbook no. 165, Agric. Res. Sci. USDA, Washington, DC, 1960. G.A. Strobel, Phytotoxins. Annu. Rev. Biochem., 51 (1982) 309-333. D.J. Robeson and G.A. Strobel, Influence of plant extracts on phytotoxin production and growth rate of Alternaria helianthi. J. Phytopathol., l l7 (1986) 265-269. M.P. Morris, C. Pagan and H.E. Warmlee, Hiptagenic acid, a toxicomponent of lndigofora endecaphylla. Science, l l9 (1961) 322-323. J.W. Hylin and H. Matsumoto, The biosynthesis of 3nitropropanoic acid by Penicillium atrovenetum. Arch. Biochem. Biophys., 93 (1960) 542-545. A. Evidente, P. Capretti, F. Giordano and G. Surico, Identification and phytotoxicity of 3-nitropropionic acid produced in vitro by Melanconis thelebola. Experientia, 48 (1992) 1169-1172. T. Kamikawa, F. Higuchi, M. Taniguchi, and Y. Asaka, Toxic metabolites of an unidentified filamentous fungus isolated from zinnia leaves. Agric. Chem. Biol., 44 (1980) 691-692.