Notes and brief articles 533

POLYGALACTURONASE ISOZYME PROFILES OF VERTICILLIUM

DAHLIAE ISOLATES RACES 1 AND 2 FROM DIFFERENT GEOGRAPHICALORIGINS

BY P, K. DURRANDS, R. A. KEENE, R. M. COOPER, L. W. O'GARRO AND J. M. CLARKSON

Department of Crop Protection, School of Biological Sciences, University of Bath, Claverton Down,Bath, Avon BA2 7A Y, U,K.

Activity staining of polygalacturonase isozyrnes, produced by eleven isolates of V. dahliae inpectin salts medium and resolved by flat bed PAGE IEF revealed a degree of intraspecificvariation in isozyme pattern. Eight isolates from North America, Australia and Europeshowed a common pattern of at least five PG isozymes, However, two European isolates andone American isolate had different profiles and respectively lacked one and two isozymes,There was no apparent correlation between isozyme pattern and race, pathogenicity orgeographic origin.

Verticillium dahliae Kleb. and Verticillium albo-atrum Reinke & Berth. cause vascular wilt diseasesin a wide range of wild and cultivated plant species(Snyder & Nash-Smith, 1981). The poylgalactu-ronan degrading enzymes polygalacturonase (PG;EC 3.2,1.15) and pectin lyase (EC 4,2.2.10),have been implicated as virulence factors. Isolatesof V. alboatrum from hop and tomato (Durrands &Cooper, 1988a; Mohan & Ride, 1984) producemany PG isozymes of widely differing pI in vitro.To determine whether there are substantial inter-specific differences between the PG patterns ofthese closely related pathogens we have resolved byPAGE IEF the PG's produced by eleven V.dahliae isolates of different geographical origin.

Isolates of V. dahliae attacking tomato can bedifferentiated into two races on the basis of theirpathogenicity towards cultivars possessing the Veresistance gene (Pegg, 1974). These cultivars areresistant to V. alboatrum and race 2 V. dahliae. Inthe present study race 1 and race 2 isolates of V.dahliae from the U.S.A., Greece and Australiahave been shown to produce, in contrast to V.alboatrum, a low number of PG isozyrnes and thata degree of intraspecific variation in isozymepattern exists.

The 11 isolates of V. dahliae (Table 1) weregrown on Czapek-Dox agar at 25°C and stockswere maintained at 4° on Corn Meal Agarslopes.

Polygalacturonase was induced in 250 ml Erlen-meyer flasks containing 100 ml basal salts solutionand citrus pectin (Sigma; 0'5%, w/v) as soleavailable carbon source (Cooper & Wood, 1975).To enhance recovery of PG, cultures were grownat ca pH 5'0 with the non-metabolizable buffer 2-(N-morpholino)ethanesulphonic acid (MES) (0'05M). Flasks were inoculated with two 5 mm agarplugs, removed from 7-14 days Czapek-Dox plates,and incubated on a rotary incubator (150 rev,

min-I, 25°) for 7 days. Culture filtrates were passedthrough Whatman NO.1 filter paper and centri-fuged (4000 g, 10 min) to remove conidia andblastospores.

Ammonium sulphate was used to precipitateproteins from the culture fluid as detailed byCooper, Rankin & Wood (1978).

Isoelectric focusing was performed on an LKB-2117 Multiphor at 8° in ready-prepared LKB thin-layer (1 mm) polyacrylamide gels (PAG plates)which contain carrier ampholytes to give pH range3'5 to 9'5· 19 pJ samples containing 1-5 mg proteinrnl" were applied to the gel adjacent to the cathodewith small segments (ca 10 mm") of chroma-tography paper.

The method for PG activity staining was adaptedfrom those of Lisker & Retig (1974) and Mohan &Ride (1984). After focusing, gels were immersed inNa citrate buffer (pH 5'0, 0'1 M) for 5 min followedby incubation at 30° for 30 min in sodium poly-pectate(NAPP, Sigma; 1'2 % (w/v),pH 5'0, citrate0'1 M). After gentle washing in distilled water theundegraded adsorbed NAPP was stained withruthenium red (0'05 %, w/v) for 2 h.

The eleven isolates were resolved into 3.groupsaccording to their PG isozyme banding patterns(Fig. 1; Table 1). Group 1 included the twoAustralian isolates, T34-arg from Greece and all ofthe N. American isolates with the exception of 105.

These isolates produced 3 major isozymes (esti-mated pI's 6'2, 6'6 and 6'8) and at least 2 minorbands. Group 3, which contained the remainingtwo Greek isolates, Egp and T64, had 2 minorisozymes and 2 of the major ones but lackedisozyme pI 6'8. Isolate 105 from N. Carolina hadthe simplest banding pattern (group 2) possessingonly one major isozyme (pI 6'7), which wascommon to all of the strains tested, and 2 minorbands.

Group 1 included race 1 and race 2 isolates from

Trans, Br. mycol. Soc. 91 (3), (1988) Printed in Great Britain

534 Notes and brief articles

Table 1. Race, geographic origin, PG isozyme number and pattern group of V. dahliaeisolates (see Fig. 1)

PG Isozymes

PatternIsolate* Race Origin Source No. group j

TS-1 1 California, U.S.A. 1 S 1

TS-2A 2+ Cal ifornia, U,S.A. 1 S 1

TS-2B 2 California, U.S.A . 1 5 120B 1 N . Carolina, U.S.A. 2 S 1lOS 2 N . Carolina, U.S.A. 2 3 2RG 2 N . Carolina, U.S.A . 2 S 1Egp 2 Greece 3 4 3T34-arg 1 Greece 3 5 1T64 1 Greece 3 4 3TS8 1 Australia 4 S 177- lOC 1 Australia 4 S 1

* All isolates were from infected tomato except Egp race-z which was isolated from eggplant.t Three distinct PG isozyme patterns were identified. Each isolate could therefore be assigned to a group.:t: TS-2A is a hyaline variant of the melanized wild type TS-2B.Source of isolates : (1) W. J. Tolmsoff, National Research Laboratory, Texas, U .S.A. (2) R. G. Gardener, M ountain

Horticultural Crops Research Station, N . Carolina, U.S.A; (3) E. C. Tjamos, Phytopathological Institute, Athens,Greece; (4) R. G. O'Brien, Department of Primary Industries, Indooroopilly, Australia.

the U .S .A. in addition to race 1 isolates fromGreece and Australia. Group 3 contained race 1

and race 2 isolates suggesting an absence ofcorrelation between isozyme profile and racedesignation/pathogenicity. Similarly Mohan &

3

.. . ... ....

p I

...Ii

Ride (1984) found no consistent differences be-tween the PG profiles ofprogressive and fluctuatinghop wilt isolates of V . alboatrum, Analogously, nocorrelation was found between intraspecific differ-ences of PG patterns and isolate aggressiveness

2 3 4 5 6 7 8 9 10 II

Fig. 1. Polygalacturonase isozyme patterns after broad range PAG IEF (pH 3-9'S ) of concentrated filtratesfrom 7-day pectin cultures of 11 V. dahl iae isolates; ( 1) 20B; (2) TS1 ; (3) TS-2A; (4) TS2-B; (S) lOS ; (6) RG ;(7) Egp ; (8) T34-arg ; (9) TS8; (10) T64 ; (11) 77-1OC.

Trans. Br. mycol. S oc, 9.1 (3) , (1988) Primed in Great Britain

Notes and brief articles 535amongst Sclerotinia trifoliorum isolates (Scott &Fielding, 1985).

The existence of isolates from diverse geo-graphical origins sharing the same patterns mightsuggest that PG isozymes have been conservedduring the evolution of the fungus. Alternativelythere may have been widespread dispersal of a fewisolates possibly via superficially infected seed(Hawksworth & Talboys, 197ob). This is notsupported, however, by the finding that Australianand N. American isolates belong to differentheterokaryon incompatibility groups (J. M. Clark-son & L. O'Garro, unpubl.).

Verticillium dahliae can be distinguished fromV. alboatrum on the basis of resting structureformation, temperature optimum for infection,sensitivity to uv irradiation and conidial siie(Domsch et al., 1980; Puhalla, 1973; Typas &Heale, 1977). Studies of pectic enzyme synthesis byV. alboatrum have revealed a complex pattern ofmany (> 25 apparent bands) PG isozymes (Dur-rands & Cooper, 1988a). In contrast, the isolatesof V. dahliae tested here have shown a relativelysimple pattern of 3-5 isozymes. Further isolatesneed to be tested to determine whether there isa consistent difference between the two species.Pectinase zymograms have provided workers witha ready source of taxonomic characters. Botrytisand Sclerotinia spp. exhibit interspecific variationin PG forms in contrast to species of Monilinia,which differs in pectin lyase and a-L-arabino-furanosidase patterns only (Cruickshank, 1983a,b; Willetts, Byrde, Fielding & Wong, 1977).Monilinia laxa, which produced the least numberof isozymes, also has the narrowest host range andgeographical distribution, leading Willetts et al.(1977) to suggest that increased enzyme mul-tiplicity conferred a greater adaptive capacity onthe pathogen. Assuming the isolates tested here arerepresentative of the species, V. dahliae would notappear to be restricted by the relatively lownumber of PG isozymes it produces compared toV. alboatrum as both species have been isolated froma similar and wide range of hosts (Hawksworth &Talboys, 1970a, b).

The existence of intraspecific variation in V.dahliae with respect to PG isozymes indicates thatthis characteristic could provide natural geneticmarkers for parasexual analysis (reviewed byHastie & Heale, 1984). The inheritance of PGisozyme genes is currently being investigated inparasexual crosses between auxotrophic strains ofV. dahliae. Furthermore, isolate 105, which pro-duces only one major PG isozyme, may proveuseful for isolating PG-deficient mutants (Dur-rands & Cooper, 1988a), because the problems ofphenotypic masking by other PG isozymes will be

avoided. Isolation of similar mutants of V. albo-atrum has proved useful in determining the role ofpectinases in pathogenesis (Durrands & Cooper,1988b).

REFERENCES

COOPER, R. M., RANKIN, B. & WOOD, R. K. S. (1978).Cell wall-degrading enzymes of vascular wilt fungi. II.Properties and modes of action of polysaccharidases ofVerticillium albo-atrum and Fusarium oxysporum f. sp.lycopersici. Physiological Plant Pathology 13, 101-134.

COOPER, R. M. & WOOD, R. K. S. (1975). Regulation ofsynthesis of extracellular cell wall-degrading enzymesby Verticillium albo-atrum and Fusarium oxysporum f.sp.lycopersici. Physiological Plant Pathology 5,135-156.

CRUICKSHANK, R. H. (1983a). Distinction between Scle-rotinia species by their pectic zymograms. Transactionsof the British Mycological Society So, 117-119.

CRUICKSHANK, R. H. (1983b). Pectic zymograms andtaxonomy of Botrytis. Fourth International Congress ofPlant Pathology (Melbourne), Abstract 708.

DOMSCH, K. H., GAMS, W. & ANDERSON, T. (1980).Verticillium. In Compendium of Soil Fungi, pp. 828-845.London: Academic Press.

DURRANDS, P. K. & COOPER, R. M. (1988a). Selectionand characterization of pectinase-deficient mutants ofthe vascular wilt pathogen Verticillium albo-atrum,Physiological and Molecular Plant Pathology 32,343-362.

DURRANDS, P. K. & COOPER, R. M. (1988b). The role ofpectinases in vascular wilt disease as determined bydefined mutants of Verticillium albo-atrum. Physio-logical and Molecular Plant Pathology 32, 363-371.

HASTIE, A. C. & HEALE, J. B. (1984). Genetics ofVerticillium. Phytopathologica Mediterranea 23, 130-161 -

HAWKSWORTH, D. L. & TALBOYS, P. W. (1970a). Verti-cillium albo-atrum. C.M.I. Descriptions of PathogenicFungi and Bacteria, no. 255.

HAWKSWORTH, D. L. & TALBOYS, P. W. (1970b).Verticillium dahliae. C.M.I. Descriptions of PathogenicFungi and Bacteria, no. 256.

LISKER, N. & RETIG, N. (1974). Detection of poly-galacturonase and pectin lyase in polyacrylamide gels.Journal of Chromatography 96, 245-249.

MOHAN, S. B. & RIDE, J. P. (1984). Some biochemicaland morphological characteristics of serotypes ofVerticillium albo-atrum from hop. Journal of GeneralMicrobiology 130, 3203-3218.

PEGG, G. F. (1974). Verticillium diseases. Review of PlantPathology 53, 157-182.

PuHALLA, J. E. (1973). Differences in sensitivity ofVerticillium species to ultra-violet irradiation. Phyto-pathology 63, 1487-1492.

SCOTT, S. W. & FIELDING, A. H. (1985). Differences inpectolytic enzyme patterns induced in Sclerotiniatrifoliorum by different legume host species. Trans-actions of the British Mycological Society 84, 317-324.

SNYDER, W. E. & SMITH, S. (1981). Current status. InFungal Wilt Diseases of Plants (ed. M. E. Mace, A. A.Bell & C. H. Beckman), pp. 25-50. New York:Academic Press.

TYPAS, M. A. & HEALE, J. B. (1977). Analysis of ploidy

Trans. Br. mycol. Soc. 91 (3), (1988) Printed in Great Britain

Notes and brief articleslevels in strains of Verticillium using a Coulter counter.Journal of General M icrobiology 101, 177-1180.

WILLETTS, H . J., BYRDE, R. J. W . & FIELDING, A . H.

(1977) . The taxonomy of the brown rot fungi (M oniliniaspp.) related to their extracellu lar cell wall-degradingenzymes. Journal 0/ General Microbiology 103 , 77-83.

FUSARIUM EQUISETI PATHOGENIC TO PINE

BY Z. SOLEL*, G. B. RUNION AND R. I. BRUCK

Department of Plant Pathology, North Carolina State University, Raleigh,North Carolina 27695-7616, U .S.A.

Fusarium equiseti isolated from a dead branch of loblolly pine caused a grey lesion andepinasty of the apex when inoculated on wounded young seedlings. On older seedlings aslight lesion developed on stems, but no wilt occurred.

Shoot dieback is a common symptom of pitchcanker of loblolly pine (Pinus taeda L.) caused byFusarium moniliforme Sheldon var. subglutinansWollenw. & Reink. (syn. F. subglutinans (Woll-enw. & Reinking) Nelson, Toussoun & Marasas)(Dwinell, Barrows-Broaddus & Kuhlman, 1985;Kuhlman et al., 1978). While collecting thispathogen from 4-year-old loblolly pines growing inNorth Carolina, one shoot yielded a different speciesof Fusarium subsequently identified (P . E . Nelson,pers. comm.) as F. equiseti (Corda) Sacco On potatodextrose agar (PD A) the colony first developedwhite dense aerial mycelium, which later becamelight tan ; the underside was pigmented darker tan.Radial growth at 25 °C was rapid, t6 mm days -I,compared with 8'3 mm days:" of F. moniliformevar. subglutinans, In r-week-old cultures, singlephialides produced abundant sickle-shaped macro-conidia with distinctly foot-shaped basal cells. Inz-week-old cultures, globose, thick-walled chlamy-dospores were observed singly and in chains orclumps. F. equiseti is common in soils of temperateregions and is predominantly a root pathogen(Booth, 1971; Joffe & Palti, 1969). It has beenisolated from diseased nursery-grown pine seed-lings (Bloomberg, 1981), and from diseased pinecrowns (D eL ucca et al., 1982; Kuhlman et al.,1978 ), but was not strongly pathogenic (K uhlmanet al., 1978).

The pathogenicity of a single-conidial culture ofthe F. equiseti isolate was studied. After 10 daysgrowth on PDA at 25°, conidia were washed fromculture plates with sterile water, filtered throughcheesecloth, and the concentration of spores wasadjusted to 106 conidia ml - I. The inoculum, a drop-let of approx. lOlli, was injected through a tiny and

* Present address: Department of Plant Pathology,Agricultural Research Organization, The Volcani Center,Bet Dagan, Israel.

shallow wound into the stem of 5-month-old andi -year-old loblolly pine seedlings, half-sib family8-68 unless otherwise stated, using a hypodermicsyringe. The plants were maintained in a green-house at 20-26°. On 5-month-old seedlings, inocu-lated 4-5 cm below the apex, disease symptomswere apparent after 3 days. An 8-10 mm long greylesion was formed at the point of inoculation, andthe upper part of the stem became epinastic. Thesymptoms resemble those of pitch canker, but lackthe purple discoloration of the F. moniliforme var.subglutinans lesion, and develop in half the time.On i -year-old seedlings, inoculated in the mainstem or lateral shoots, a restricted lesion, extendingto 6-12 mm at the point of inoculation, slightlysunken and with light discoloration, became ap par-ent after 3 weeks. Hi stological examination of crosssections revealed mycelium in the cortex, xylemand pith. Some resin accumulated adjacent to thepoint of inoculation on inoculated and water controlseedlings. There was no wilt of shoots, whichfrequently occurs with infection by F . moniliformevar. subglutinans. Placing an inoculum dropletwithout wounding the stem, or inoculating withsterile water, did not cause any disease symptoms.

Pathogenicity of cultures maintained on PDA at4° was reassessed after 6 months. Aggressivenesswas considerably reduced. No infection occurredon i -year-old seedlings, and infection of y-month-old seedlings took longer, 2-3 weeks, to becomeapparent. Isolates from those stems were no moreaggressive. Inoculation of young stems of half-sibfamily 8-61 was frequently negative, thus con-firming the relative resistance of this family to F .moniliforme var. subglutinans (unp ub l.) .

Our study demonstrated that a strain of F.equiseti, when entering through a wound, ispathogenic to loblolly pine. It is, however, unlikelyto be a threat to pine stands, due to it s lowinfectivity to mature stem or branches.

Trans. Br. mycol. Soc. 91 (3), (1988) Pr im ed in Great Britain