Physiological and Molecular Plant Pathology (1992) 40, 17-22

17

Chemoattraction of zoospores of the soybean pathogen,Phytophthora sojae, by isoflavones

P. F . MORRIS and E . W. B. WARDt

Agriculture Canada Research Centre, 1400 Western Road, London, Ontario, Canada , .V6G 2V4

(Accepted for publication October 1991 ;

The main infective agents in plant diseases caused by species of the fungal genera Phytophthora andPythium are zoospores that are attracted chemotactically to plant surfaces . Here we demonstratethat the simple isoflavones daidzein and genistein, which occur in soybean root exudates, arehighly effective chemoattractants for zoospores of Phytophthora sojae, an economically importantpathogen of soybeans. When added to suspensions of actively swimming zoospores, daidzein andgenistein also cause rapid encystment and germination . The isoflavones are active at concentrationsdown to 10 nM but are inactive with zoospores of several other species of Phytophthora and Pythium,non-pathogenic on soybeans. Daidzein and genistein are also inducers of nodulation genes inBradyrhizobium japonicum, the nitrogen-fixing bacterial symbiont of soybeans . Thus, both thepathogen and the symbiont identify their host by recognizing the same chemical signals .

INTRODUCTION

Species of the fungal genera Phytophthora and Pythium, causal agents of several majorplant diseases, infect plants by means of zoospores that swim freely in water films in thesoil or on plant surfaces . There is ample evidence that zoospores are attracted to plantroots and display positive chemotaxis towards compounds such as ethanol and aminoacids that may occur at or close to root surfaces [1 . 3, 16, 27] . These compounds appearto serve as general attractants and are active in the millimolar concentration range .However, several aldehydes and fatty acids have been shown to be much more effectiveattractants for Ph . palmivora [2] and there is also evidence that zoospores may beselectively attracted to roots of susceptible host plants [4] . The present study examinesthe chemotactic response of zoospores of the soybean pathogen Ph. sojae to a numberof flavonoids and isoflavonoids that occur in, and may be released into the soil from,seeds and roots of soybeans and other plants [5, 10, 13, 25, 31] . The response ofzoospores of several species more or less related to Ph. sojae that are not pathogens ofsoybeans was assessed also .

MATERIALS AND METHODS

FungiFor species of the Phytophthora megasperma complex, we have adopted the nomenclatureof Hansen & Maxwell [12] . Phytophthora sojae Kauf. & Gerd . races 1, 4 and 6 from

±To whom correspondence should be addressed .

0885-5765/92/010017+06 $03 .00/0

C 1992 Academic Press Limited

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P. F. Morris and E . W. B . Wardsoybeans = Ph . megasperma f.sp . glycinea Kuan & Erwin, were those used in earlierstudies [29 1 . Phytophthora rnedicaginis Hansen & Maxwell from alfalfa ( = Ph. megaspermaf.sp. medicagin.is Kuan & Erwin), Ph. megasperma Drechs. BR329 from alfalfa, Ph .cryptogea Pethyb . & Laff. from alfalfa and Pythium irregulare Buism . from pea wereprovided by D . J . S . Barr, Biosystematics Research Centre, Ottawa . Phytophthorainegasperma B3A from Douglas fir was provided by E . M. Hansen, Department ofBotany and Plant Pathology, Oregon State University . Corvallis, Oregon . Phytophthoracapsid Leonian from pepper and Ph. parasitica Dastur from tomato were from our ownculture collection .

Production of zoospore suspensionsIsolates of Ph. sojae, Ph. megasperma B3A, and Ph . parasitica were grown on V8 juice agarat 25 °C and suspensions of zoospores were prepared as previously described for Ph .sojae [29] . Zoospores from Ph. medicaginis and Ph. cryptogea were produced by themethods of Miller & Maxwell [23] and Duniway [7], respectively . Isolates of Ph. capsiciwere grown on V8 juice agar supplemented with 20 mg 1 -' cholesterol [15] andzoospores were prepared as previously described [30] .

The isolate of Ph. megasperma BR329 were grown on V8 juice agar for 4 days at 25 ° C .The cultures were flooded with sterile distilled water and transferred to a 16 °Cincubator. The water was replaced four times each day and zoospores were producedafter 5 days . The procedure for P_y . irregulare was similar except that the initialincubation period at 25 °C was for 2 days. Zoospore suspensions were diluted withsterile distilled water when necessary to give concentrations of 2-4 x 10 5 ml- ' .

AssayThe procedure was based on that of Khew & Zentmyer [20] . A drop of zoosporesuspension (approx . 230 µl, 2-4 x 10 5 zoospores ml- ') was placed in an assay chamberformed by the space between two cover slips, placed in parallel on a microscope slide[20] . Capillary tubes (1 1 µl) were filled with water or a solution of a test substance inwater and one tube of each was positioned with one end immersed in the zoosporesuspension . The slides were placed in an incubator at 16 °C . After 15 min the slideswere removed and the tubes were examined under a microscope . The zoospores andcysts in the tubes were counted either directly, or after fixing by addition of a drop ofcotton blue dissolved in ethanol to the assay chamber . Threshold concentrations (µMwere recorded as the lowest concentration of a compound at which there was asignificantly greater attraction of zoospores than in controls (P < 0 . 05) . Results werebased on four to six tests with zoospore suspensions prepared on different days . Exceptfor ethanol, compounds were tested over a range of concentrations, where necessaryup to their solubility limits . Ethanol was used at 25 mm throughout as a standard toconfirm activity of zoospores and for comparative purposes .

CompoundsAmino acids, cinnamic acid, biochanin A and the flavones were from Sigma (St Louis,Missouri) ; coumestrol was from Eastman Kodak Co . (Rochester, New York) . Daidzin,genistin and genistein were isolated from soybean flour [25] and purified by HPLC[24] . Daidzein, formononetin and isoformononetin were synthesized [26], and further

Chemoattraction of zoospores by isoflavones

19purified by HPLC [24] . Isoformononetin has not been described from soybean seeds orroots but has been reported to be inducibly formed in soybean leaves [17] . Compoundswith low water solubility were weighed on a six place balance in microgram amounts,dissolved in sterile distilled water and suitably diluted .

RESULTS AND DISCUSSION

Positive chemotaxis to ethanol and three amino acids was confirmed (Table 1) [1, 20] .The amino acids were attractants for all but one of the species tested, and weregenerally effective at relatively high concentrations although glutamine and asparaginewere active at micromolar concentrations with two species . With the exception ofkaempferol, which was a chemoattractant for Ph . cryptogea, none of the flavones wereeffective chemoattractants at the highest concentrations tested . The isoflavonoids wereattractants for only two of the species examined, but were especially effective for Ph .sojac . Daidzein, isoformononetin and genistein are evidently highly specific attractantsfor zoospores of this pathogen, with activity at concentrations down to 10 nM. Fordaidzein and genistein there is evidence from in vitro studies [10] that suggests that suchconcentrations could develop rapidly in the vicinity of roots and seeds in soil .Furthermore, not only were these compounds effective chemoattractants, but theystimulated rapid encystment and germination ; < 30 min) of zoospores that enteredthe capillary tubes . Zoospores that entered control tubes continued to swim randomly,often swimming out of the tube, before encystment . Similarly, when solutions ofdaidzein or genistein were added directly to suspensions of freely swimming zoosporesthere was almost immediate encystment followed by germination . This feature mayfind useful application in the investigation of biochemical and molecular mechanismsin pathogenicity where it is desirable to synchronize development of the pathogen .

In addition to demonstrating the highly selective activity of daidzein, genistein andisoformononetin for Ph. sojae zoospores, the data also provide evidence of structuralspecificity . Thus, formononetin and biochanin A, which differ from daidzein andgenistein, respectively, only in the substitution of a methoxy group for the hydroxylgroup at the 4' position, had only about one-thirtieth of their activity (Fig . I, ; .Similarly, the functional group at the 7 position may be critical since the activity of theglucosides was substantially less than that of daidzein and genistein, and quite possiblywas due to contamination with small amounts of the aglycones. However, in the caseof isoformononetin, substitution of a methoxy group for the hydroxyl at the 7 positionin daidzein did not affect activity . Such structural requirements, together with the lowthreshold for activity, suggest that Ph . sojae has evolved specific isoflavone receptors .

In legumes, isoflavones and flavones are stored as the relatively soluble glucosides,and these are released from imbibing seeds and from roots [10, 13, 25] . The glucosidesare hydrolysed to the aglycones by fl-glucosidases present in root exudates [14] or inthe soil [8] . Daidzein and genistein released from soybeans may promote growth of thenodulating bacterium Bradyrhizobium japonicum [6], they are possibly chemoattractantsfor this organism [18, 19] and are inducers of nodulation genes [21] in a manneranalogous to the induction of nod genes by flavones in other Rhizobium spp . [22] . Theresults reported here indicate that Ph . sojae has developed mechanisms for recognitionof the same chemical signals as B. japonicum, not for the establishment of symbiosis but

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Chemoattraction of zoospores by isoflavones

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FIG . 1 . Structure of isoflavonoid compounds tested as chemoattractants . Daidzein R' = R' =R3 = H ; daidzin R 1 =glucose, R' = R 3 = H ; formononetin R' = R' = H, R 3 = CH3; isoformo-nonetin R t = CH 3 , R' = R3 = H ; genistein R' = R 3 = H, R' =-0H ; genistin R' = glucose,R' _ -OH, R 3 = H ; biochanin A R' = H, R' =-OH . R3 = CH3 .

as an aid in pathogenesis, thus presenting soybeans with conflicting evolutionaryalternatives .The taxonomic relationships of Ph. sojae to other members of the Ph. megasperma

complex has been the subject of recent debate [9, 12] . It has been concluded thatwhereas Ph. megasperma attacks a broad range of plants, Ph . sojae and Ph . medicaginishave become host-specific pathogens of soybeans and alfalfa, possibly during therelatively short period of developed agriculture in North America [11] . Part of thisspecialization in Ph. sojae evidently is the ability to use daidzein and genistein as signalsto recognize its soybean host . In this it is quite distinct from Ph. medicaginis, or indeedfrom any of the other species tested . These observations may have practical value . Ifnon-host legumes can be found or engineered that also release daidzein, isoformononetinor genistein into the soil, they could be used as decoy crops to greatly reduce soilpopulations ofPh. sojae, which are known to remain infective in the absence of soybeansfor several years [28] .

REFERENCES

1 . ALLEN, R. N . & NEWHOOK, F . J . (1973) . Chemotaxis of zoospores of Phytophthora cinnamomi to ethanol incapillaries of soil pore dimensions . Transactions of the British Mycological Society 61, 287-302 .

2 . CAMERON, J . N . & CARLILE, M. J . (1978) . Fatty acids, aldehydes and alcohols as attractants forzoospores of Phytophthora palmivora . Nature 271, 448-449 .

3 . CARLILE, M . J . (1983) . Motility, taxis and tropism in Phytophthora . In Phytophthora : Its Biology, Taxonomy,Ecology and Pathology, Ed. by D . C . Erwin, S . Bartnicki-Garcia & P . H . Tsao, pp . 95-107, AmericanPhytopathological Society, St . Paul, Minnesota .

4 . CHI, C . C . & SABO, F . E . (1978) . Chemotaxis of zoospores ofPhytophthora megasperma to primary roots ofalfalfa seedlings . Canadian Journal of Botany 56, 795-800 .

5 . D'ARCY-LAMETA, A. (1986) . Study of soybean and lentil root exudates. II . Identification of somepolyphenolic compounds, relation with plantlet physiology. Plant and Soil 92, 113-123 .

6 . D'ARCY-LAMETA, A . & JAY, M. (1987) . Study of soybean and lentil root exudates . III . Influence ofsoybean isoflavonoids on the growth of rhizobia and some rhizospheric microorganisms . Plant and Soil101, 267-272 .

7 . DUNIWAY, J . M . (1976) . Movement of zoospores of Ph_ytophthora cryptogea in soils of various textures andmatric potentials. Phytopathology 66, 877-882 .

8 . EIVAZI, F . & TABATABAI, M. A . (1990) . Factors affecting glucosidase and galactosidase activities in soils .Soil Biology and Biochemistry 22, 891-897 .

9 . FARIS, M. A ., SABO, F . E ., BARR, D . J . S . & LIN, C . S . ('1989) . The systematics of Phytophthora sojae andP. megasperma . Canadian Journal of Botany 67, 1442-1447 .

10. GRAHAM, T. (1991) . Flavonoid and isoflavonoid distribution in developing soybean seedling tissues andin seed and root exudates . Plant Physiology 95, 594-603 .

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P. F. Morris and E . W. B. Ward

11 . HANSEN 1: . M . (19895 . Speciation in plant pathogenic fungi : the influence of agricultural practiceCanadian journal of Plant Pathology 9, 403-4 10 .

12 . HANSEN, E . M . & MAXWELL. D P. 1991 ; . Species of the Phytophthora megasperma complex . M_ycologia 83,

376-381 .13 . HARTWIG, U. A ., JOSEPH, C . M . & PHILLIPS, D . A . ; 19911 . Flavonoids released naturally from alfalfa

seeds enhance growth rate of Rhizobium meliloti . Plant Physiology 95, 797-803 .

14 . HARTWIG, U . A . & PHILLIPS, D . A . !1991' . Release and modification of nod-gene-inducing flavonoids

from alfalfa seeds . Plant Physiology 95, 804-807 .15 . HENDRIX, J. W . (1967), Light-cholesterol relationships in morphogenesis of Phytophthora palmivora and P .

capsici sporangia . Mycologia 59, 1107-1111 .16 . Ho, H . H . & HICKMAN, C . J . (19671 . Factors governing zoospore responses ofPhytophthora megasperma var .

sojae to plant roots. Canadian journal of Botany 45, 1983-1994 .17 . INGHAM J . L ., KEEN N. T ., MULHEIRN, L . J ., LYNE, R. L . (19811, Inducibly-formed isoflavonoids from

leaves of soybean . Phytochemistry 20, 796-798 .18 . KAPE, R ., PARNISKE, M . & WERNER, D . (19911 . Chemotaxis and nod gene activity of Bradyrhizobium

japonicum in response to hydroxycinnamic acids and isoflavonoids . Applied and Environmental Microbiology

57, 316-319 .19. KHAN, M. M . A . & BAUER, D . (1988) . Chemotaxis of Bradyrhizobium japonicum towards flavones and

isoflavones from soybean . Plant Physiology 86, 127 .20. KHEW, K . L. & ZENTMYER, G. A . i 1973) . Chemotactic response of zoospores of five species of

Phytophthora. Phytopathology 63, 1511-1517 .

21 . KOSSLAK, R. M ., BOOKLAND, R ., BARKEI, J ., PAAREN, H. E . & APPELBAUM, E. R . 11987, . Induction ofBradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max . Proceedings of thetiational Academy of Sciences of the U.S.A .' 84, 7428-7432 .

22 . LONG, S . R . (1989) . Rhizobium-legume nodulation : life together in the underground . Cell 56, 203-214 .

23 . MILLER, S . A . AND MAXWELL, D . P. ' 1982 ; . Light microscope observations of susceptible, host resistant,and nonhost resistant interactions of alfalfa with Phytophthora megasperma . Canadian journal of Botany 62,

109--116 .24. MORRIS, P . F ., SAVARD, M . E. AND WARD, E . W . B . 11991 ; . Identification and accumulation of

isoflavonoids and isoflavone glucosides in soybean leaves and hypocotyls in resistance responses toPhytophthora megasperma f.sp . glycinea . Physiological and Molecular Plant Pathology 39, 229-244 .

25. MAIM, M., GESTETNER, B., ZILKAII, S ., BIRK, Y. & BONDI, A. 11974/ . Soybean isoflavones .Characterization, determination, and antifungal activity . journal of Agricultural Food and Chemistry 22,

806-810 .

26 . PELTER, A . & Four, S . 119761 . A new convenient synthesis of isoflavones . Synthesis- 326 .

27 . ROYLE, D . J . & HICKMAN, C . J . (1964'1 . Analysis of factors governing in vitro accumulation of zoospores

of Pythium aphanidermatum on roots . 1 . Behaviour of zoospores . Canadian journal of Microbiology 10,

151-162 .28. SCHMITTHENNER, A . F . (1985 : . Problems and progress in control of phytophthora root rot of soybean .

Plant Disease 69, 362-368 .29. WARD, E. W . B., CAHILL, D. M . & BHA'rrACHARYYA, M . K . 11989) . Early cytological differences

between compatible and incompatible interactions of soybeans with Phytophthora megasperma f.sp .

glycinea . Physiological and Molecular Plant Pathology 34, 267-283 .30. WARD, E . W . B., UNWIN, C. H . & STOESSL, A . !1974) . Postinfectional inhibitors from plants . XIII .

Fungitoxicity of the phytoalexin, capsidiol, and related sesquiterpenes . Canadian journal of Botany 52,

2481-2488 .31 . WERNER, C. & HOHL, H . R . (1990) . The effects of Ca t ' and Mg ° ' on accumulation and secretion of

isoflavonoids by soybean roots . Plant Science 72, 181-191 .