Rice-specific toxins produced by Bipolaris zeicola, race 3; evidence for role as pathogenicity factors for rice and maize plants

67

Physiological and Afolecular Plant Pathology (1991) 38, 67-82

Rice-specific toxins produced by Bipolaris zeicola, race 3; evidence for role as pathogenicity factors for rice and maize plants j . z. XIAO, T. Tsuo~, N.

DOKE, S. NAKATSUKA~-,

M. TSUDA+ and S.

NISHIMURA§

Plant Pathology Laboratory, Faculty of Agriculture, Nagoya Unirersity, .A'ago)'a46t, ~,Tapan; t Departmentof Applied Bioorganic Chemistry, Gifu Unirersity, Gifu 501, Japan; t Pesticide ResearchInstitute, Kyoto Unirersity, Kyoto 606,

yapan

(Acceptedfor publication December1990)

Bipolaris zeicola race 3, originally reported as a pathogen causing a leaf spot disease of maize, is also highly pathogenic to rice plants. Its spore-germination fluids (SGFs) were specifically phytotoxic to rice leaves but induced susceptibility to infection by nonpathogens on leaves of both rice and maize. Effective compounds were partially purified from the SGFs which caused leaf chlorosis only on rice and enabled spores of nonpathogenic Alternaria alternata and B. vlcloriae to infect leaf tissues of rice and maize, as judged by the formation of disease symptoms on the leaves. The partially purified compounds were further purified by HPLC and found to consist ofat least three separate components. The host-specific phytotoxicity to rice and the susceptibility-inducing activity of the components with rice and maize were evident only when combined, but not when used singly. HPLC analysis of extracts from SGFs and culture filtrates ofseveral isolates of Bipolaris and Helminthosporiumdemonstrated that the components were produced only by B. zeicola race 3. We conclude that the complex of the components may be a pathogenicity factor ofB. zeicola race 3 for rice and maize plants.

INTRODUCTION

At least 14 fungal plant pathogens have been defined to produce host-specific (or hostseiective) toxins (HSTs) clearly responsible for the determination of host-specific pathogenicity [7]. Most of the reported HSTs are produced by species of Alternaria and Hehninthosporium [8, 9, 14]. Much effort has been made to detect and identify such hostspecific factors from other pathogens, and a few have succeeded [2, 14]. Since rice production is important worldwide, host-parasite interactions of rice diseases are among the most intensively studied. Previous studies on rice blast caused by Pyricularia oryzae and rice brown leafsp0t caused by Bipolaris oryzae suggested the existence of such pathogenicity factors [1, 20,'23]. However, further efforts are necessary to critically evaluate the pathological roles, and chemical characterization of the factors is needed. Bipolaris zeicola Shoemaker (syn Helminthosporium carbonurn Ullstrup, the anamorph of Gochliobolus carbonum Nelson) is the pathogen of leaf spot disease of maize [19]. Three §Professor S. Nishimura passed away on 27 May 1990. Abbreviations used in text: HST, host-specific toxin; SGF, spore germination fluids. 0885-5765/91/010067+ 16 $03.00/0 © 1991 Academic Press Limiied 5-2

68

J.Z. Xiao et al.

pathogenic races of the fungus have been differentiated according to the symptoms expressed on different maize genotypes. Race 1 and race 2 were first recognized in 1938 and 1939, respectively [18]. Race 1 produces a host-specific toxin (HC-toxin) to which inbred lines of maize homozygous for the recessive alleles for susceptibility are highly sensitive [13]. On the leaves of these lines, race 1 causes large, oval lesions. However, on the leaves of maize lines that are not homozygous for susceptibility, race 1 causes small, necrotic lesions or flecks [5, 18]. Race 2 genetically lacks the ability to produce HC-toxin [5, 15]. The symptoms caused by race 2 on leaves of maize lines with or without the gene for HC-toxin sensitivity are similar to those caused by race I on nonsensitive lines of maize [5]. Race 3 was first described in 1973 in Pennsylvania [4]. It causes long, narrow, linear lesions on most maize genotypes and lacks host genotype specificity compared with other Bipolaris species that are pathogens of maize. In 1981, the occurrence of a leaf spot disease on maize caused by Bipolaris sp. was reported in Japan [6]. This Bipolaris sp. was later identified as race 3 ofBipolaris zeicola by using several differential host genotypes [16, 17]. In recent years, the incidence of this disease increased markedly, especially in the northern part of Japan [16]. In our preliminary study on the comparison of pathogenicity among different fungi belonging to the genus Bipolaris and Helminthosporium [22], we found that B. zeicola race 3 also caused severe symptoms on rice leaves by artificial inoculation. The spore-germination fluids (SGFs) of race 3 showed susceptibility-inducing activity and phytotoxicity on rice leaves, suggesting that the pathogen releases compound(s) responsible for the pathogenicity during spore germination. Therefore, an attempt was made to investigate the pathogenicity of race 3 to maize as well as to rice plants. We report here the partial purification and characterization of compounds which show susceptibility-inducing activity and phytotoxicity in a host-selective manner from SGFs of the pathogen, along with some attempts to observe the biological effects on the host plants.

MATERIALS AND METHODS

Plants Rice (Oryza saliva L.) cvs Sekiguchi-Asahi, Akibare and Nipponbare; maize (yea mays L.) cvs Bsss, WF-9, Kuromochi and X-125; wheat (Triticum aestivum L.) cv. C44-81 ; cucumber (Cucumis sativus L.) cv. Chibari-kyuri; eggplant (Solarium melongena L.) cv. Senryo; soybean (Glycine max Merr.) cv. Okushiro; strawberry (Fragaria grandiflora Ehrh.) cv. Morioka-16; tobacco (jVicotiana tabacum L.) cv. Samsum NN; and tomato (Lycopersicun sculentum Mill.) cv. Saturn were employed in the present study. All plants were grown in pots in a glasshouse during tile spring, summer and autumn and in phytotron (25 °C day and 20 °C night) during the winter. Young leaves of plants were used throughout all experiments.

Culture of fimgi Isolates ofB. zeicola race 2 and race 3 were obtained from diseased lesions of naturally infected maize plants in Japan. An isolate JH22 of race 3 was used in experiments where not indicated otherwise. Other isolates (listed in Tables I, 4 and 5) were

Rice-specific toxins produced by B. zeicola

69

TABLE 1

Pathogenidty of spores and susceptibility-inducing actirity and phytotoxlcity of spore-germination fluids of Bipolaris spp. and H e l m i n t h o s p o r i u m sp. to rice leaved' Susceptibility inducing activity c

Phytotoxicitya

107 1 10 125 ! 11 2 1 15 2

+ --+ + -----

5

17

--

4 17 9 1 3

25 0 3 1 n.d. e

----n.d.

Fungus

Isolate

Pathogenicityb

B. B. B. B.

KU-13 BZrl BZI101 JH22 JHI cyn A KK2 KK1 NWI

233 0 8 254 245 11 12 10 0

oryzae zeicola race 1 zeffola race 2 zeicola race 3

B. 9~odontis B. panici-miliacei B. kusanoi B. hawaiiensis B. victoriae H. nakataea Alternaria alternata

B 1-1 BI-2 HV-2 KK2-1 KK3 0-94

aThis e x p e r i m e n t was repeated twice a n d the results shown are from a single representative experiment. b N u m b e r of lesions per 20 cm 2 l e a f of rice cv. Seklguchi-Asahi 48 h after i n o c u l a t i o n w i t h spore suspension (5 x l 0 s spores m1-1) of each fungus. e N u m b e r of lesions per 20 cm 2 leaf of rice cv. Sekiguchi-Asahi 48 h after i n o c u l a t i o n M t h spore suspension (10 s spores ml -a) ofA. alternata in the presence o f s p o r e - g e r m i n a t i o n fluids of each fungus c o n c e n t r a t e d 25-fold. a L e a f chlorosls caused in a leaf p u n c t u r e assay 48 h after t r e a t m e n t by s p o r e - g e r m l n a t i o n fluids c o n c e n t r a t e d 25-fold. --, n o n e ; + , chlorosls. en.d., N o t d e t e r m i n e d .

laboratory stock cultures. The fungi were maintained on potato dextrose agar at 4 °C. Cultures were grown in l 1 Roux bottles containing 400 ml of potato dextrose broth at 28 °C. After 2 weeks of still culture, culture filtrates and mycelial mats were separated by sequential filtration through two layers of gauze and then through filter paper. Both the mycelial mats and culture filtrates were used to check for toxic substances.

Preparation of spores and spore-germinationfluids (SGFs) Spores (conidia) and SGFs were prepared by methods previously described [23]. Spores were formed abundantly when the fungus was grown on an oatmeal-V8 juice agar at 28 °Cl for 5 days in the dark. A spore suspension (5 x l0 s spores m1-1) was uniformly sprinkled onto paper iowels which had been well-washed by water and then by distilled water, and the spores were allowed to germinate 6n the towels for 24 h at 24 °C. The SGFs were harvested by squeezing the towels.

Assay for pathogenicity The pathogenicity of different isolates to rice plants was tested by using either detached leaves or intact plants. In the detached leaf test, 1 ml of spore suspension (5 x 105

J. Z. Xiao et aL

70 TABLE

2

Host-specificity of Bipolaris zeicola race 3 and susceptibility-inducing activity of the toxins from sporegermlnation fluids ofBipolaris zeicola race 3 ~ No. of lesions/20 cm ~ leaf b

Bipolaris zeicola race 3

Nipponbare Akibare Sekiguchi-Asahi Bsss

33 a 172a 213 e 17¥

0 0 0 0

44 a 64 a 94 e 0

WF-9

215f

0

0

47 t 0 0 0 0

0 0 0 0 0

0 0 0 0 0

Plant

Cultivar

Rice

Corn Wheat Eggplant Soybean Strawberry Tobacco

A. alternata +

Alternada alternata

(344-81 Senryo Okushiro Morioka-16 Samsum NN

toxinsc

aThe values indicated are a representative result from one of three experiments. bNumber oflesions per 20 cm ~ leaf48 h after inoculation ~"ith spore suspensions orB. zeicola race 3 (5 x l0 s ml-1), or spore suspensions ofA. alternata (106 ml -l) with or without toxins. CToxins partially purified with Sephadex LH-20 column chromatography at concentration of 8 units. dSmall lesions approximately 0"2 x 0"1 ram. eLarge lesions approximately 2 x 0-3 mm. fSmall water-soaked lesions.

TABLE 3

Host-spedfic

phytotoxidty to leaf tissues of the toxins from Bipolaris zelcola race 3 ~

spore-germination fluids of

Concentration of toxinsb (units) Plant

Cultivar

64

32

Rice Corn

Nipponbare Sekiguchi-Asahi Bsss

Wheat

WF-9 C44-81

. .

. .

. .

. .

. .

. .

m

Cucumber Tobacco Tomato

Chibari Samsum NN Saturn

. . .

. . .

. . .

. . .

. . .

. . .

m

+ +c + + ++ + + + . . .

16

8

4

2

+ + ++ .

+ + .

+ +

+ --

1

0

.

aThis experiment was repeated twice. bPartially purified toxins xs4th Sephadex LH-20 column chromatography. CLear chlorosis determined by leaf puncture assay and observed after a 2-day incubation. --, none; + , chlorosis ~ithin the wound; + , chlorosls up to 2 mm; + + , chlornsis up to 6 mm; + + + , chlorosis more than 1 cm in diameter.

Rice-specific toxins produced by B.

zeicola

71

TABLE 4 Pathogenicity and toxin production in spore-germination fluids (SGFs) of severalfungi

Fungus

Isolate

Pathogenicitya

Susceptibilityinducing activity of SGFsb

Bipolaris zeicola race 3

JH22 N1848 BZrl BZ1101 KK2-1

1596 1241 120 97 12

1539 2153 10 0 25

B. zeieola race 1 B. zeicola race 2 Helminthosporiura nakataea

Toxin contente 37"0 40"5 n.d.d n.d. n.d.

aNumber of lesions per 20 cm~ leaf of rice cv. Sekiguchi-Asahi 48 h after inoculation with spore suspensions (5 × l0 s ml -a) of each isolate. bNumber of lesions per 20 cm2 leaf of rice cv. Sekiguchi-Asahi 48 h after inoculation with spore suspension of a nonpathogenic Alternaria alternata isolate 0--94 in the concomitant presence of SGFs of each isolate concentrated 100-fold. eAmount of toxin component II (ng m1-1) in the SGFs determined by HPLC analysis as described in Fig. 6. aNnt detected.

TABLE 5 Toxin production in culture by Bipolaris zeicola race 3 and several otherfungi ~ Fungus

Isolate

Toxin

B. zeicola race 3

B. zeieola race 1 B. zeicota race 2

JH22 JHI JH12 JH2 SH! SH2 SM1 BZI207 BZrl BZ1101

+b + + + + + + + ---

Fungus B. blcolor B. victoriae B. or)'zae B. maydis B. sorghicola B. o~odontis B. panici-miliacci B. kusanoi B. hawaiiensis Helminthosporium nakataea

Isolate

Toxin

KS1 HV-2 KU-13 HSI-1 YK-I cyn A KK2 NWI BI-1

----------

KK2-I

--

aEach fungus was cultured in PDB medium for 2 weeks and contents ofthe toxins in culture filtrates and mycelial mats were determined by HPLC analysis. +, with toxin production; --, without toxin production. bAmount of toxin II in the mycelial mats ofisolateJH22 was approximately 009 pg mg -a dry weight, while the amount was less than 0"01 lag m1-1 in the culture filtrates.

s p o r e s m1-1) c o n t a i n i n g 0"05 % T w e e n 80 w e r e s p r a y e d o n t h r e e d e t a c h e d leaves w i t h a n a t o m i z e r . T h e i n o c u l a t e d leaves w e r e i n c u b a t e d in a m o i s t c h a m b e r a t 24 ° C a n d t h e n u m b e r o f lesions a p p e a r i n g o n t h e leaves w a s c o u n t e d 48 h a f t e r i n o c u l a t i o n . I n t h e i n t a c t p l a n t test, p o t - g r o w n p l a n t s a t t h e 4 - 6 l e a f s t a g e w e r e i n o c u l a t e d as d e s c r i b e d a b o v e . T h e i n o c u l a t e d p l a n t s w e r e k e p t in h i g h h u m i d i t y u n d e r a v i n y l b a g for 24 h a n d t h e n t r a n s f e r r e d to t h e p h y t o t r o n . P a t h o g e n i c i t y o f t h e i n o c u l a t e d s p o r e s w a s s c o r e d 72 h a f t e r i n o c u l a t i o n as t h e n u m b e r o f d i s e a s e lesions a p p e a r e d o n t h e leaves.

72

J.Z. Xiao et al.

Assay for susceptibility-inducing activity Unless otherwise stated, spores o f a nonpathogenic isolate ofAlternaria alternata (0-94), which is a stock culture in our laboratory that had been obtained from air-borne fungi, were used as a nonpathogen in most of the assay for susceptibility-inducing activity. T h e spores were prepared as described above and were suspended at a concentration of 10 ~ spores m1-1 in a test sample solution containing 0"05 % Tween 80. Inoculation was performed either by spraying or drop application. In the spraying method, 1 ml of the suspension was sprayed on three detached leaves with an atomizer. For the drop application, a droplet of spore suspension(15 ~tl) was placed on the leaf surface and covered with a small piece of lens paper (6 × 6 mm). The spore suspensions without the sample and the sample solution without the spores were used as controls. T h e inoculated leaves were incubated in a moist chamber at 24 °C and disease lesions that appeared on the leaves were scored at 48 h after inoculation.

Leaf puncture assay for phytotoxicity T h e middle portion of the blades of detached leaves was nicked with a needle, and the damaged leaf surface was immediately covered with 20 lal of test sample solution. The leaves were incubated at 24 °C in a moist chamber and the development of chlorosis a n d / o r necrosis around the nicked portion was observed 48 h after inoculation.

Root growth assay for phytotoxicity Seeds of rice and other plants were surface-sterilized with a fungicide (Topsin water solution, 1000 X, Nipponsoda Co., Ltd.) and pre-gcrminated in distilled water for 2 days at 24 °C. Seeds which had just germinated (root length less than 1 mm) were selected and transferred onto a filter paper (2 cm in diameter) in a Petri dish (2"5 cm in diameter) containing 1 ml ofsample solution to be tested. T h e seeds were incubated at 24 °(3. Length of the seedling root was measured 72 h after incubation in the test solution. Root-inhibiting activity was measured as percentage inhibition of root elongation as compared with a water control. RESULTS

Pathogenicity to rice plants and toxin production during spore germination of different fungi To test the pathogenicity of 14 isolates belonging to the genus Cochliobolus to rice plants, spore suspensions of each isolate were inoculated on detached rice leaves of cv. Sekiguchi-Asahi. Three isolates, one from B. oryzae and two from B. zeicola race 3, which are known as the pathogens of rice leaf brown spot disease and maize leaf spot disease, respectively, exhibited strong pathogenicity as judged by the appearance of abundant lesions on the leaves 2 days after inoculation (Table 1). The pathogenicity of these Bipolaris isolates were also tested by using intact rice plants of cvs SekiguchiAsahi and Nipponbare. B. zeicola race 3 caused large lesions on cv. Sekiguch~-Asahi and small lesions on cv. Nipponbare. T h e lesions were comparable in appearance to those caused by B. oryzae. T o test toxin production, the SGFs squeezed from paper towels on which spores of each of the 14 isolates had germinated were assayed for susceptibility-inducing activity

Rice-specific toxins produced by B. zeicola 73 and phytotoxicity in rice leaves. Spores of nonpathogenic A. alternata were suspended in each of the SGFs concentrated 25-fold and inoculated on rice leaves ofcv. SekiguchiAsahi. Significant susceptibility-inducing activity was found in the SGFs only of B. oryzae and B. zeicola race 3 among the isolates used (Table I), and the number oflesions appeared in a SGF concentration dependent manner on the leaves (data not shown). When phytotoxicity of the SGFs ofeach isolate was assayed by the leaf puncture assay, the SGFs of the pathogenic isolates also caused leafchlorosis on the rice leaves (Table 1). The SGFs of B. zeicola race 3 isolate JH22 were concentrated and tested for phytotoxicity by the root growth assay. Fifty percent inhibition of root elongation was detected with SGFs concentrated l-6-fold, while SGFs concentrated 50-fold almost completely inhibited root growth (Fig. 1). Neither susceptibility-inducing activity nor I00

g o= "6 "F,

'64 d8 ~ ~.i ¢,is ~'.5 21 go Concentration factor

Fro. 1. Inhibitory effect of spore-germination fluids (SGFs) ofBipolaris zeicola race 3 on the root elongation of rice seedlings. SGFs were concentrated by the factors shown. Five seeds (cv. Sekiguchi-Asahi) were employed for each concentration. Each value represents the mean ofthree replicates and the vertical bar indicates the standard deviation.

phytotoxicity was detected in the paper towel extracts and the SGFs of the other isolates which failed to show pathogenicity to rice plants. These results suggested that the isolates pathogenic to rice plants release compound(s) possessing susceptibilityinducing activity and phytotoxicity during spore germination. Investigation of the activity of the SGFs of B. oryzae has been reported [23]. Partial purification of toxins showing susceptibility-inducing activity and phytotoxicity from • SGFs @B. zeicola race 3 Unless stated otherwise, SGFs ofisolate JH22 and toxins from this isolate were used in the present study. For each experiment, 20 1 of the SGFs were used and bioassays of susceptibility-inducing activity and phytotoxicity to rice plants were employed to assay the activity during the purification procedures. The SGFs were concentrated to 10 % of the original volume at 45 °C under reduced pressure. The concentrated SGFs were extracted three times with an equal volume of ethyl acetate and then the extracts were evaporated in vacuo. Susceptibility-induclng activity and phytotoxicity were recovered in the ethyl acetate extracts. The residue (approx. 40 mg) was dissolved in 0"5-1 ml of chloroform and subjected to silica gel (Wako gel C-200, Wako Pure Chemical Industries, Ltd.) column chromatography (column: 2"5 cm in diameter packed with gel of 8 cm in height). The column was eluted stepwise With each of 100 ml of chloroform, chloroform/acetone (9:1, v/v), chloroform/acetone (4:1, v/v) and 6

M P P 38

J.Z. Xiao et al.

74

acetone. The susceptibility-inducing activity and phytotoxicity was found only in the 4:1 eluate. This eluate was evaporated in vacuo and then the residue (approx. 4 mg) was dissolved in 1 ml of methanol and further subjected to a Sephadex LH-20 (Pharmacia Fine Chemicals) column chromatography eluted with 100 % methanol. Five ml eluate fractions were collected and each fraction was assayed for susceptibility-inducing activity and phytotoxicity. The fractions eluted at 190 to 230 ml showed strong susceptibility-inducing activity and root-inhibiting activity to rice plants (Fig. 2). 100

500

~

8

*,,T-.

8

50

z5o 2

A

8"6

g aE; mC

0 -20

I

5O

I

200 150 Elution volume (ml)

a;o

FIc. 2. Sephadex LH-20 column ( l l 5 x 1"8 cm) elutlon profiles of susceptibility-inducing activity and root-inhlbitlng activity in spore-germination fluids of Bipolaris zeicola race 3. The column was eluted with methanol, and 5 ml eluate fractions were collected. Susceptibilityinducing activity (A) is indicated by the number of lesions appeared per 20 cm = leaf of rice cv. Sekiguchi-Asahi 48 h after inoculation with spore suspensions of a nonpathogenic Alternaria alternata isolate 0-94 in the concomitant presence of each fraction. Root-inhibiting activity (O) is shown by the percentage of inhibition of seedling root elongation of rice cv. Sekiguchi-Asahi.

These fractions were pooled and concentrated in vacuo, and tentatively called partially purified toxins.

Host specificity of susceptibility-inducing activity of the partially purified toxins The partially purified toxins at various concentrations were tested for susceptibilityinducing activity on rice leaves (Fig. 3). In order to compare the biological activity of samples, the concentration of toxins required to inhibit root elongation of rice 50 % was designated as one unit. When the toxins were mixed with a spore suspension of A. alternata and sprayed on detached leaves of rice cv. Sekiguchi-Asahi, necrotic lesions appeared 2 days after inoculation at concentrations higher than one unit. The number of lesions on the leaves increased in a concentration-dependent manner. Spraying of toxins alone also caused necrotic lesions on the leaves at concentrations higher than eight units. At lower concentrations ( < 8 units), necrotic lesions were only induced in the presence of spores ofA. altemata. At higher concentrations ( > 8 units), more lesions were produced in the presence of spores than by toxins alone. Spraying ofA. alternata spores suspended in water without toxins caused no visible symptom on the rice leaves. The susceptibility-inducing activity of the partially purified toxins for A. alternata was rice-specific (Table 2). The pathogen possesses a host range within the gramineous plants when artificially inoculated in the laboratory. Spraying with a spore suspension

Rice-specific toxins produced by B. zeicola

75

/

iooo

o

500

//P

4 e l

z

•

e/e

eJ °

•

ot

.o. /

00250-5 I 2 4 8 16 52 Concentration of toxins (units)

Fla. 3. Effect of the partially purified toxins from spore-germlnatlon fluids of Bipolaris zeicola race 3 on the infection of rice leaves by a non-pathogenlc Alternaria alternata isolate 0-94. Rice leaves (cv. Sekiguchi-Asahi) were sprayed with spore suspensions (106 ml -~) in the presence of the toxins at the designated concentrations ( O ) , or sprayed with toxin solution alone (O). Inoculation ofA. alternata spores alone on the leaves caused no lesions. T h e values indicated are a representative result from one of three experiments.

of race 3 on detached leaves caused large lesions on rice cv. Sekiguchi-Asahi, small lesions on rice cvs Nipponbare and Akibare, small water-soaked lesions on maize and wheat, and no symptoms on the other plants. In the presence of the toxins at a concentration of eight units, the A. alternata spores caused lesions on the leaves of rice cvs Niponbare, Akibare and Sekiguchi-Asahi, but never on those of the other plants including maize.

Host-specific phytotoxicity of the partially purified toxins The partially purified toxins were assayed for host-specific phytotoxicity by the leaf puncture method. Leaf chlorosis was induced only on rice leaves at concentrations higher than four units (Table 3). On the leaves of the other plants, no visible change was observed (Table 3). The partially purified toxins also host-specifically inhibited seedling root growth (Fig. 4). Root growth of rice was completely suppressed at 64 units. The toxins only weakly) inhibited seedling root elongation of maize and wheat: 5 0 % inhibition was obtained at a concentration of approximately 16 units. The seedling root growth of the two nongramineous plants, tomato and cucumber, were not significantly affected by the toxins at the tested concentrations. Pathogenicity orB. zeicola race 3 to maize and effect of the partially purified toxins on maize leaves Inoculation on leaves of intact plants of maize with spore suspensions orB. zeicola race 3 caused long, linear lesions typical of race 3 as previously described [4], while that with race 1 and race 2 caused small necrotic lesions. Similar results were obtained on the four cultivars of maize tested (cvs Bsss, WF-9, Kuromochi and X-125). Thus, all cultivars employed in the present study were susceptible to B. zeicola race 3. Tile susceptibility-inducing activity when assayed using A. alternata and leaf chlorosis-inducing activity of the partially purified toxins were not detected on maize leaves. Since maize has never been reported to be a host of Alternaria pathogens which suggests that A. alternata may not be suitable for experiments with maize, B. victoriae 6-2

76

J. Z, Xiao et aL I00

g

}

"6 o

50

"6

g

0

0

0"5

I

2

4

8

16

52

64

Concentration of toxins (units)

Fro. 4. Effect of the toxins from spore-germination fluids ofBipolaris zeicola race 3 on the seedling root elongation of rice (cv. Sekiguchi-Asahi, O), maize (cv. Bsss, A ) , wheat (cv. CA4-81, /X), cucumber (cv. Chibafi-kyuri, II) and tomato (cv. Saturn, I"1). The toxins used were partially purified with Sephadex LH-20 column chromatography. Assay conditions are given in ,Materials and Methods except that eight seeds of each plant were immersed in a 9 cm Petri dish containing 5 ml toxin solution. Each bar represents the mean of three determinatlons and the vertlcal bar shows the standard deviation.

which is related to B. zeicola and is a pathogen of oats but not of maize, was employed in the assay for susceptibility-inducing activity of the toxins. When the toxins were mixed with the spore suspension orB. victoriae and sprayed on detached maize leaves, small water-soaked lesions appeared 36 h after inoculation. T h e symptom was similar to that produced by B. zeicola race 3 on detached maize leaves. Spraying B. victoriae on without the toxins or the toxins without the spores induced no visible symptoms on maize leaves. The susceptibility-inducing activity of the partially purified toxins to B. victoriae was also detected on leaves of rice and wheat as well as other maize cultivars, but not on the leaves of eggplant, soybean, strawberry or tobacco. Since the spray application produced only small water-soaked lesions on the detached maize leaves which were difficult to score, the susceptibility-inducing activity of the partially purified toxins to B. victoriae on maize leaves was further confirmed by a drop assay. In the presence of the toxins, the nonpathogen produced large, developing chlorosis around the inoculated portion of maize leaves (Fig. 5). Neither the nonpathogen nor the toxins alone caused any symptom on the maize leaves. When the A. alternata spores were employed in the same drop assay, only slight symptoms were observed on the maize leaves (data not shown).

Further purification oft he toxins The partially purified toxins obtained by Sephadex LH-20 column chromatography were subjected to H P L C on a Develosil-packed column ( a D S - 5 , 4"6 x 250 mm, Nomura Chemical Co., Ltd.) with a mobile phase of acetonitrile/water (75:25, v/v) at a flow rate of I ml rain -x, and monitored at 204 nm by a U V detector. The eluates were first separated into two fractions, O and T (Fig. 6), then assayed for their susceptibilityinducing activity on rice leaves. The total activity of tile partially purified toxins was recovered in fraction T (data not shown). No activity was found in fraction O. Fraction T contained three major peak s (peaks I, II and I I I ) (Fig. 6). Each peak was

"i'f

Rice-specific toxins produced by B. A

I ;

pw

77

zeicola

C

B

D

~! ~"~

,

I~.' ~

Fro. 5. Effect ofthe partially purified toxin from Bipolaris zeicola race 3 on the infection ofmaize leaves by an isolate of B. rictoriae non-pathogenlc to maize plants. Drops of spore suspension (5 x 105 spores m1-1) orB. zeicola race 3 (A), B. rictoriae (B), B. rictoriae plus toxins (30 units) (C) and of toxin solution alone (D) were placed'on detached maize leaves. Photographs were taken after 48 h incubation.

II

o=

0

5

I0

15

20

Retention time (min)

F1o. 6. High performance liquid chromatotography (HPLC) of toxin fraction from sporegermination fluids of Bipolaris zeicola race 3. H P L C was conducted by using a reverse column (ODS-5) with a mobile phase of acetonitrile/water (75:25, v/v) at a flow rate of I ml min -t.

recovered separately and subjected to assay for the susceptibility-inducing activity. Unexpectedly, none of the individual fractions showed any activity (Fig. 7). The experiment was repeated several times, giving the same results. However, when the fractions were mixed again and subjected to the same bioassay, the activity was

3. Z. Xiao et al.

78 No. of leslons/20 cm2 leaf I4

I

7

0

"IT

"fir

400

I

-I- "It

271

I

+ "m"

196

"n- + -m"

978

I+]I+T~"

Single or mixed loxins

Fro. 7. Susceptlbility-induclng activity of the toxin fractions obtained by H P L C of the spore-germlnation fluids of Bipolaris zeicola race 3. Assay for susceptibility-inducing actMty was performed as described in Fig. 2. Each fraction at a concentration equivalent to the partially purified toxins at approximately 64 units was assayed individually, or recombined for assay. Photograph was taken 48 h after inoculation.

restored. Combinations ofpeak I and II, II and III, or I and III, exhibited significant activity, and the combination of I, II and III showed the strongest activity (Fig. 7). The three peak fractions separated by HPLC were also assayed for phytotoxicity. No single peak showed any toxicity. Their combinations induced leaf chlorosis on rice leaves and inhibited root growth ofrice seedlings (data not shown). Thus, the collective effect of the three peaks was required for tile phytotoxicity and susceptlbility-inducing activity; the components of peak I, II and III were called toxin component I, II and III, respectively, of the toxin complex.

Time course of toxin production during spore germination The spores ofB. zeicola race 3 (isolateJH22) rapidly started to germinate on paper towels so that more than 90 % ofspores had germinated within 6 h. The susceptibilityinducing activity of the germination fluids (50-fold concentrated) was first detected 12 h after incubation and then gradually increased with incubation time. The toxins in the SGFs were quantified by HPLC analysis. The amounts of the major toxin component, component II, is indicated in Fig. 8. Component II was first detectable 6 h after incubation, and then linearly increased in amount with incubation in parallel with the susceptibility-inducing activity of the SGFs (Fig. 8). Production of the toxins by different isolates Five fungal isolates were checked for toxin production (Table 4). The pathogenicity of each isolate was determined by inoculating spore suspensions at 10s spores m1-1 on detached rice leaves ofcv. Sekiguchi-Asahi. The toxins in the SGFs were quantified by HPLC analysis and the amounts of the major toxin component, component II, are indicated in Table 4. Two isolates of B. zeicola race 3 (.JH22 from Japan; N1848 from the United States) were highly pathogenic to rice plants and their SGFs showed strong susceptibility-inducing activity and contained the toxins. The other isolates including B. zeicola race 1 and race 2, and Hehninthosporium nakataea (a pathogen of millet) did not

79

Rice-specific t o x i n s p r o d u c e d by B. zeicola I00

. ~ . _

g

. ~ ,

,

I000

,

-~_ 50

-g 500

cn

~. O

I0

-

5

:9 --

.

.

.

.

6 12 18 Incubotion time (h)

0

24

O

FIn. 8. Time course study of susceptibility-inducing activity and toxin production in sporegermination fluids of Bipolaris zeicola race 3. Spore germination (m) was observed with a microscope. Susceptibility-inducing activity (Ak) is indicated by the number of lesions, as described in Fig. 2. Toxin content (0) is shown by the amount of toxin component II which was determined by tIPLC analysis on the basis of its extinction coefficient at 204 nm which had been obtained in the present study. This experiment was repeated three times, and the values shown are from a single representative experiment.

exhibit pathogenicity to rice plants, and their SGFs did not contain the toxins or show significant susceptibility-inducing activity even when concentrated 100-fold.

Detection of the toxins in culture (2ulture filtrates of each isolate were extracted with ethyl acetate. Mycelial mats dried at room temperature were immersed in 100% methanol at 25 °(2 for 48 h, and the methanol extracts were evaporated to dryness in vacuo. T h e residue was suspended in a small a m o u n t of water and extracted with ethyl acetate. T h e ethyl acetate extracts from both the culture filtrates and mycelial mats were further subjected to a toxin purification procedure by the method described above. T h e toxins were detected in the culture filtrates and mycelial mats only of the isolates ofB. zeicola race 3 (Table 5). T h e mycelial mats contained more than ten times the a m o u n t of toxins than culture filtrates did. DISCUSSION

Several lines of evidence p r o v i d e d in the present report suggest that B. zeicola race 3 produces host-specific toxins which determine its pathogenicity to rice and maize plants. These include: (i) the toxins induced leaf chlorosis only on rice plants and strongly inhibited the root growth of rice but showed only weak root-inhibiting activity to maize and wheat; (ii) the toxins enabled nonpathogens to infect host tissues of the toxin producer; (iii) the toxins were produced and released during spore germination, suggesting the importance for the initial events in the infection process; (iv) the toxins were produced specifically only by B. zeicola race 3 a m o n g the Bipolaris and Helminthosporium species tested. T h e results also indicate that the present toxins are different from those with other so-called host-specific (or host-selective) toxins (HSTs). T h e other H S T s have been known to be host-selective for certain genotypes of the hosts. B. zeicola race 3 has been found to lack a host genotype selectivity compared with other H S T producers: it exhibits a similar pathogenicity to most genotypes of maize.

80

J.Z. Xiao et aL

The present toxins were selective probably at the host species level: no cultivar or genotype selectivity were found for either the phytotoxicity or susceptibility-inducing activity on both rice and maize. The toxins induced leaf chlorosis on rice, but not on maize, even though the toxin producer was equally pathogenic to both of them. This may be due to the nature of maize plants which are known to develop little leaf necrosis or chlorosis in some cases. However, in the presence of the toxins, a nonpathogen of maize, B. victoriae was able to infect maize leaves, producing a symptom similar to that caused by pathogenic fungi. This indicates that the toxins are involved in suppression of the host resistance so that host cells become accessible or susceptible to possible invasion. T w o mechanisms can be proposed for the symptom development. One is that symptoms are caused by toxic metabolites produced by the penetrating nonpathogenic fungi; another and the more probable one is that symptoms result from the host reaction to fungal invasion. Nevertheless, the above result suggests that the toxins act also as pathogenicity factors to maize plants. All the known HSTs are found as highly toxic substances from not only culture filtrates but also from SGFs. However, the pathogenicity factors of plant pathogens are not necessarily produced in culture or are phytotoxic [8, 11, 12]. From a phytopathological point of view, metabolites released from germinating spores are more closely responsible for the initial events in the infection process than those from cultures in nutrient medium [8]. Such metabolites are involved in the suppression of host resistance, leading the producer to successful infection [3, 8, 24]. In our previous report [21], another metabolite which showed susceptibility-inducing activity to rice plants was detected and isolated from culture filtrates of B. zeicola race 3. However, the pathological role of the susceptibility-inducing factor is not yet known because the factor was not detected from the spore-germination fluids of race 3. The present toxins were produced specifically by B. zeicola race 3 during spore germination. No biologically active constituent other than the toxins were detected in the SGFs. These results strongly indicate the role of the toxins as pathogenicity factors, which was supported by the fact that the toxins were also produced in culture specifically by race 3. T h e present host-selective toxins orB. zeicola race 3 did not cause visible damage on the maize leaves, which may be one reason why previous studies failed to identify such a pathogenicity factor for maize plants. The success in the identification of toxins orB. zeicola race 3 in the present report may be due largely to the assay methods ofsurveying SGFs and assaying for susceptibility-inducing activity. Most of the known HSTs are found to consist of chemical isomers. Some isomers are biologically active and some are not. T h e partially purified toxins of B. zeicola race 3 were found to consist of at least three constituents. However, each constituent showed no phytotoxicity or susceptibility-inducing activity and complete activity was found only when combinations of them were applied. Some biological activities to animal and insects are expressed only when several complicated components are mixed [10]. T o our knowledge, tile nature of the present toxins may be a rare case where the biological activity is exhibited to higher plants by blending several components. The amount of toxin production in the SGFs is very low. From 100 1 of SGFs, the yield of the major toxin component (component II) was less than I mg. This suggests that the toxins may exhibit activities at very low concentrations. Because of the low

Rice-specific toxins produced by B. zeicola

81

yield a n d complexity of the toxin components, the toxin c o n c e n t r a t i o n tested was not directly indicated in the bioassays of activities. Large a m o u n t of toxins are required to investigate further the biological a n d chemical n a t u r e of the toxins. B. zeicola race 3 was first reported in 1973 in eastern A m e r i c a [4], a n d a b o u t ten years later, it was reported in the n o r t h e r n part of J a p a n a n d is n o w prevalent in the other parts of J a p a n [6, 16, 17]. T h e present study makes it clear that the pathogen possesses a potential ability to attack rice plants. However, the possible effect of B. zeicola race 3 u n d e r field conditions remains to be investigated. W e t h a n k D r Tsukiboshi, J a p a n N a t i o n a l Grassland Research Institute, for his gift of spore-germination fluids a n d his cooperation in the pathogenicity tests orB. zeicola race 3 isolate N1848. This research was supported in part b y G r a n t s - i n Aid for Cooperative Research Nos. 62304015 (1987, 1988) a n d 01304014 (1989, 1990) from the Ministry of Education, Science a n d C u l t u r e of J a p a n .

REFERENCES 1. ARASE,S., TANAKA,E. & NISHISIURA,S. (1989). Production of susceptibility-inducingfactor(s) in sporegermination fluids of Pyricularia oryzae. In Host-Specific Toxins: Recognition and SpecificityFactors in Plant Disease, Ed. by K. Kohmoto & R. D. Durbin, pp. 59-73. Tottori University, Japan. 2. BALLANCE,G. M., LAStARI,L. & BERNIER,(3. (3. (1989). Purification and characterization of a hostselective necrosis toxin from Pyrenophora tritici-repentis. Physiological and Molecular Plant Pathology 35, 203-213. 3. COMSrOeK,J. (3. & SCHEFFER,R. P. (1973). Role of host-selective toxin in colonizationofcorn leaves by Helminthosporium carbonum. Phytopathology 63, 24-29. 4. NELSON, R. R., BLO.~tco, M., DXAMACIO,S. & MOORE,B. S. (1973). A new race of Helminthosporium carbonum on corn. Plant Disease Reporter 57, 822423. 5. NELSON,R. R. & ULLSTRtJP,A.J. (1961). The inheritance of pathogenicity in Coehliobolas carbonum. Phytopathology 51, 1288-1291. 6. NXSmHARA,N. (1981). New disease of sweetcorn caused by Bipolaris sp. Annals of the Phytopathologieal Society offfapan 47, 367 (abstract in Japanese). 7. NXSmMURA,S. (1987). Recent development of host-specific toxin research in Japan and its agriculture use. In Molecular Determinants of Plant Diseases, Ed. by S. Nishimura, (3. P. Vance & N. Doke, pp. 11-26. Japan Scientific Societies Press, Tokyo. 8. NlStIIMURA,S. & KOHMOTO,K. (1983). Host-specific toxins and chemical structures from Allernaria species. Annual Review of Phytopathology 21, 87-I 16. 9. NXSmMURA,S. & NAKATSUKA,S. (1989). Trends in host-specific toxin research in Japan. In Host-Spedfic Toxins: Recognition and Specificity Factors in Plant Disease, Ed. by K. Kohmoto & R. D. Durbln, pp. 19-31. Tottori University,Japan. 10. OHSCGI,T., NISHmA,R. & FVKAMZ,H. (1985). Oviposition stimulant of Papilla xuthus, a citrus-feeding swallowtail butterfly. Agricultural and Biological Chemistry 87, 4968-4970. 11. OKu, H. (1980). Determination for pathogenicity without apparent phytotoxicity in plant disease. Proceeding of Japan Academy 56B, 367-371. 12. OKU,H., SHIRAISm,T. & Oucm, S. (1987). Role of specificsuppressersin pathogenicity of Myeosphaerella species. In Molecular Determinants of Plant Diseases, Ed. by S. Nishimura, (3. P. Vance & N. Doke, pp. 145-156. Japan Scientific Societies Press, Tokyo. 13. PRXNGLE,R. B. & SCHEFFER,R. P. (1967). Isolation of the host-specific toxin and a related substance with nonspecifictoxicity from Hdminthosporium earbonum. Phytopathology 57, 1169-I 172. 14. SCHEFFER,R. P. (1989). Host-specifictoxins in phytopathology: origins and evolution ofthe concept. In ttost-Specific Toxins: Recognition and Specificity Factors in Plant Diseases, Ed. by K. Kohmoto & R.D. Durbin, pp. !-17. Tottori University,Japan. 15. SCHEFFER, R. P., NELSON, R.R. & ULLSTRUP,A.J. (1967). Inheritance of toxin production and pathogenicity in Cochliobolus carbonum and C. rietoriae. Phytopathology 57, 1288-1291.

82

J. Z, Xiao et M.

16. "rSUKIBOSHI,T., KIMIGAFUKURO, T. & SATO,T. (1987). Identification ofraces ofBipolaris zdco[a, the causal fungus of Helminthosporium leaf spot on corn in Japan. Annals of the Ph.)'topalhologicalSociety or japan 53, 647-649. 17. TsuKmosm, T., SATO, T. & KBnGAFUKURO,T. (1986). Helminthosporlum leaf spot of corn caused by Bipolaris Zeicola in Japan. Annals of the PhytopathologicalSociety of.Japan 52, 492-495. ! 8. ULLSTRUP,A.J. (1941). Two physiological races ofHelminthosporiummaydisin corn belt. Phytopathology3 I, 508-521. 19. ULLSTRUP,A.J. (1944). Further studies on a species of Helminthosporiumparasitizing corn. Phytopathology 34, 214-222. 20. VIDaYASEKAV.AN, P., BORROMEO, E. & MEW, T.W. (1986). Host-specific toxin production by Helminthosporium oryzae. Phytopathology76, 261-266. 21. XIAO,J. Z., NAKATSUKA,S., TSUDA,M., DOKE, N. & NISmMURA,S. (1990). Isolation and characterization of a susceptibility-inducing factor from Bipolaris zeicola race 3. Annals of the PhytopathologlcalSociety of .,Tapan 57s (In press). 22. XrAO, J. z., NISmMURA, S. & TSUDA, M. (1988). Disease determinants produced by Bipolaris zeicda. Annals of the PhytopathologicalSociety of.Japan 54, 97 (abstract in Japanese). 23. XtAo, J. Z., TstrDA, M., DOKE, N. & NISm.XtURA,S. (1990). Phytotoxins produced by germinating spores of Bipolaris oryzae. Phytopathology80, (In press). 24. YODER, O. C. & SCHEFFER, R. P. (1969). Role of toxin in the early interaction ofIIelminthosporium t'ictoriae with susceptible and resistant oat tissues. Phytopatholog)'59, 1954-1959.