Plant Pathology (1994) 43, 261-268

Prevalence and pathogenicity of spinach root pathogens ofthe genus Pythium in Sweden

M. LARSSONDepartment of Plant and Porest Protection, Swedish University of Agricultural Sciences,PO Box 7044, S-750 07 Uppsala, Sweden

Several pathogenic Pythium spp, were isolated from spinach roots in a 4-year disease survey incommercial spinach fields in southern Sweden. Heterothallic and zoospore-producing isolates belongingto the filamentous, non-inflated zoosporangia (F) group of van der Plaats-Niterink were most abundant,followed by P. sylvaticum, P. ultimum var, ultimum and P. heterothallicum. The heterothallic hyphalswelling (HS) group and P. tracheiphilum were found in a few plants. Two types of pathogenicity testswere performed, to measure the effects of seedling infection and of root infection of older plants. Thesetests showed P. ultimum var, ultimum to be the most severe spinach pathogen inducing pre- and post-emergence damping off as well as root rot of older plants. Also P. heterothallicum and P. tracheiphilumdamaged both seedlings and older plants, Pythium sylvaticum, Pythium 'group HS" and Pythium 'groupF' were pathogenic only to seedlings, P. ultimum var, ultimum and Pythium group F were isolated insignificantly higher frequencies from spinach grown in the autumn season than in the spring. No clearrelationships were found between Pythium prevalence and disease severity index of surveyed field plants,between Pythium prevalence and plant developmental stage, or between prevalence of Pythium andother pathogens isolated. This is the first report of P. heterothallicum and P. sylvaticum being pathogenicon spinach.

INTRODUCTION

Damping off and root rot of spinach (Spinaciaoleracea) seriously limit production in severalcountries (Sumner et al., 1976; Naiki et ai, 1986;Larsson & Gerhardson, 1992), In addition toseveral soil-borne pathogens of the genera Apha-nomyces, Phytophthora, Pusarium, Cylindrocar-pon, and Rhizoctonia (Larsson & Olofsson, 1994),many Pythium spp. have been reported to beinvolved in this damping off and root diseasecomplex. Among these Pythium pathogens de-scribed are P. aphanidermatum (Watanabe, 1983;Bates & Stanghellini, 1984; Akashi et al., 1986;Naiki et at., 1986), P. butleri (Lee et al., 1975;Kusakari et al., 1979). P. dissotocum (Bates &Stanghellini, 1984), P. irregulare (Sumner et al.,1976), P. paroecandrum (Naiki et al, 1986), P.spinosum (Akashi et al., 1986), and P. ultimum(Tamietti & Garibaldi, 1978; Akashi et ai, 1986),

In investigations in Swedish spinach fields,partly reported by Larsson & Olofsson 1994:accompanying paper), we also isolated severalpathogenic Pythium spp., of which P. sylvaticumand P. heterothallicum have not previously been

reported as spinach pathogens. This paperfocuses on the prevalence of the Pythium patho-gens found in the fields surveyed and theirpathogenicity in greenhouse tests.

MATERIALS AND METHODS

Field surveys and fungal isolations

Plant samples were taken randomly from a totalof 28 commercial spinach fields as describedpreviously (Larsson & Olofsson. 1994), Data onsoil, soil pH, cultivars, cultivation methods andsampling procedures are given by Larsson &Gerhardson (1992), Methods for assessment ofroot rot severity (field disease severity index), theselection of plants for isolations and isolationprocedures were as described by Larsson &Olofsson (1994), Fungal isolations were alwaysdone from metalaxyl-treated plants as all seedswere treated in the commercial fields.

The semi-selective medium for isolation ofPythium spp, was denoted SMP and contained 17g Difco cornmeal agar, 5 mg benomyl (Bcnlate,

262 M. Larsson

50% a,i.). and 100 mg streptomycin sulphate perlitre of distilled water, Benomyl was added as adry powder to the agar base before autoclaving(120 C for 20 min), while the streptomycin sul-phate was added, also as a dry powder, afterautoclaving. Microscopic observations of myce-lial habit and branching pattern of the Pythiumspp, isolated were first made directly on the semi-selective medium, Mycelial tips from 80% of allPythium colonies were then transferred to freshlyprepared cornmeal agar (CMA) after incubationfor 2-5 d. Growth rates per 24 h were recorded onCMA at 25 C, Cultures were then stored at 4 Con CMA for use in pathogenicity tests later. Theisolates remained viable at this low temperaturefor more than 1 year and were transferred to newplates once per year.

Pathogenicity tests

The Pythium isolates were roughly grouped, butnot rigorously identified, before they were testedin greenhouse pathogenicity tests. Greenhouseconditions, cultivars. peat-sand growing mediumand seed surface-sterilization in AgNO> were aspreviously described (Larsson & Gerhardson.1990), About 50"o of the cultured isolates, ie,approximately 300 isolates, were tested in twotypes of tests, (i) An inoculum-soil mixing testinvestigated the ability to cause damping off bycomminuting agar and mycelium from 2-week-old cultures with water for 30 s in a blender andthen mixing this with the soil. One-half of a Petridish (9-cm diameter) with the Pythium isolate tobe tested and 25 ml water was used per pot( 7 x 7 x 7 cm). Immediately after mixing the fun-gal suspension with the soil, nine spinach seedswere planted per pot. Blended CMA without thegrowth of a fungus was used as a control, (ii) Aninoculum layer test investigated the ability of theisolates to cause root rot (Schmitthenner & Hilty,1962). The isolates were grown on V8-juice agarfor 2 weeks and then one-half of the agar andmycelium from one Petri dish was placed 7 cmbelow the seeds prior to seeding in plastic pots(9 X 9 X 10 cm) with 12 seeds per pot. One-half ofthe agar from a Petri dish without the fungus wasplaced at the same level in control pots,

Kor some isolates three additional methods forpathogenicity testing were used, (i) Zoospores ofeach of five /oosporc-producing isolates (pro-duced on grass leaves in water as described belowand counted in a hacmocytometer) were appliedto newly watered 7 x 7 • 7 cm plastic pots with

nine 6-day-old seedlings per pot, and 1 x 10'zoospores were gently spread onto the soil sur-faces, (ii) Seventeen isolates of different Pythiumspp, were tested by the method described byBruckart & Lorbeer (1982), by growing thePythium spp. on autoclaved oat seeds for 8 daysand then placing these inoculated oat seeds in thesoil at the same level as the spinach seeds, (iii) Sixisolates were tested on spinach seedlings on wetfilter paper in Petri dishes by placing CMA fungaldisks directly on the hypocotyls of the seedlings(Blok, 1970),

In all the pathogenicity tests, two replicates perisolate were used. All test plants were harvested 4weeks after inoculation. The roots were pulled upand washed and the severity of root rot (rangingfrom 0 to 100) was examined according toLarsson & Gerhardson (1990).

Fungal identification

All isolates giving a disease severity index of > 15in pathogenicity tests (approximately 65% of allisolates) were cultured on CMA and potato-carrot agar (PCA) (van der Plaats-Niterink, 1981)for identification. The isolates were groupedaccording to daily growth rate, occurrence ofsexual and asexual reproduction, mycelial habitand branching pattern. To induce sporangiaformation, a small piece of an agar culture wasplaced in a Petri dish with sterilized pond waterand distilled water together with boiled grassleaves at 17 C (van der Plaats-Niterink, 1981), orboiled grass leaves were placed directly on thePythium culture and then transferred 1 day laterto the water and placed at 17 C, Pythium col-onized the grass leaves, and development ofzoosporangia and zoospores was studied at theirmargins between 15 h and a few days afterwards.

All heterothallic isolates with a daily grow-thrate above 20 mm were crossed on PCA withcompatible male and female strains of P. syhati-cum obtained from Dr Sardar Kadir. SwedishUniversity of Agricultural Sciences, Uppsala,Sweden, Other heterothallic isolates with dailygrowth rates of 15-20 mm were first crossed bychance with each other on PCA. After findingcompatible partners among these isolates, theywere crossed again with the two new compatiblestrains. Shapes and sizes of anthcridia, oogoniaand oospores of heterothallic isolates were exam-ined in dual cultures, while the same character-istics were studied in single cultures for homothal-lic isolates.

Pathogenicity of .'spinach root pathogens (if the i;cnii\ Pythium

F i g , 1 , C o n t a c t l i n e s h e t u e e n e o n i p a t i h l e i s n l a l e s c f l u n h e l e r m h i i l l i L / ' i i l i i u i n s p c i i e s B , i l l i p h , i t , ' , j i j p h ,

l e m a l c s t r a i n t o t h e l e t t , t h e m a l e s t r a i n U ) t h e n g h l a n d . i n I h e s e e . i s e s . m . i l e l e ^ l s i i a i i i s i n I h c i i i i , | , ! ' i L i . i | i

(al, F whalicuniAh). /' hclcrmhalluuiu

Table I, Prevalcnee ot dilTerent f^vthmm speeies in nine f i e l d ' s , w h e r e n i ' T c i l u n i n u p l J | l ; ^ ^ ' .^ r , , ' ^ u l

l n>m w l i ieh l u i i ' j i

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Numher of planlssur\e>ed

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s e v e r i t N i n d e v l

'" P e r e e n t a g e s a r e m e a n \ a l u e s l o r a l l o l t h e 2 , N l i e l d s i i U L s i i L M l e J

RESULT.S

Pythium spp. isolated from spinach roots

All homiithallic, fast-growiny isolates (about '̂ t»tnm 24 h) without ascxu.il reproduction wereidentical and were identified as I' iiltinumi sarultimum A fev. hiimothalln, isolates tirevv, moresUiiAly (< 2(1 mm 24 h | They produeed /oosporesin y;U)bose sporany:ia and the eharaeteristies eor-rcsponded with the eleseriptum ol / ' tnn liti/'/ii-lum A similar isolate from spinaeh isolated in1986 was identified as / ' truclicirliilitni .it theCommonweal th Myeolofiieal Institute, Kew,

T K T h e - h e l e i o i l K i l l K . l , i s i - u i , i \ u n L ' l ^ , i l , l ^ • ^

W I t h o i l l s p o r M U L ' l , ! p i i ' d l l e l l ' M l l . ' l l l K i l 1 1 1 n i , , s |

e a s e s , o o i j o l l l . i i n e o l l L l e l U l l l l I I K k M , , \ i [ l / '

\ \ l i i i i t t n n i i n , l i e , i ! u l k " n i , i k ' ^ i i . i t i i s . i i i , i w ^ u

e o i i s e e | i i e n l l \ K l e i i l i l i e i l , i s / > ; , > , , ; , , . , ' ) . • I I K I I I I ^

o l e i M i t a e l o t ^ . > i i i p , i i i h l e l , . l | , 1 l e ^ \ \ , i s I \ ) S K . ! U \

r . i t h e v s l i . t i p l o I h e s i v i e > > l l i i c i v , , i l e i s o l . i i e . i i u !

d i l U i s e t o I h e s i d e I I I t h e l e n i . i k I s , ' l . i i c ( I i r I i i , . s

I s a i s t t d e s e i i l - ^ e d h \ \ . i i i t l e i 1 * 1 i . i I s - \ I U M i i k

j j i o w l h I , l i e s o l , I S ^ 1 1 m i l l I . H I I I O I , , . ' - ' , M I I I H I

t l l i . i l e u l l i i i e s \ M l l i u M i i p . i i i h l c i s i ' l . i i e s I I K l i n e , ' I

i i o e o i i i . i ( I I L ' I h i w , i s \ e i \ s h , i i p l e s e i i i M i i U ' I l i . i i

l o i / ' h i i < i < ' i l u i l l u u n i s \ \ ^ : \ \ \ \ h \ \ , i i i J e i l ' l , i , U s

264 M. Larsson

Fig. 2. Average disease severity indices induced inspinach by six Pythium spp. or groups. Two types ofpathogenicity tests were performed: (i) • the inoculum-soil mixing method (seed inoculation) and (ii) • theinoculum layer method (emerged plant inoculation).The number of isolates tested were for Fythium group F54(A). P. sylvalicum 50(B). Fythium group HS S(C), P.heterothallicum I3(D), P. ultimum var uilimum 50(E).and P. tracheiphilum 6(F). Each bar indicates thestandard error of a mean.

Niterink (1981). Other characteristics, such asforms and sizes of oogonia, oospores, antheridiaand hyphal swellings, together with the absenceof sporangia and zoospore production, also fitwith the description of P. heterothallicum (van derPlaats-Niterink, 1981). The few heterothallic fast-growing isolates, with spherical hyphal swellingswhich were neither P. sytvaticum nor P. heteroth-allicum were classified as Pythium group HS, Themost commonly recovered Pythium isolates wereheterothallic with a daily growth rate c. 20 mmand submerged colonies showing a rosette pat-tern on CMA, Filamentous, non-inflated zoo-sporangia and vesicles containing c. 30 zoosporeseach were readily produced. As no positive resultswere obtained when crossing these isolates it wasnot possible to identify them to species level andthey were classified as Pythium group F

The method for producing zoospores by plac-ing grass leaves directly on the fungus culture in aPetri dish and I day later transferring them towater was considered to be better than placing apiece of fungus culture together with grass leavesin the water. The fungus colonized the leavesfaster in the first method and consequently thezoospores were produced about I day earlier.

Field survey results

Pvthium group I was found in 687o of the fieldsinvestigated, P. svlvatuum in 61"... P. ultimum

20

15

10

020

15

^ 10

I =z

020

I 15

(a)

..dliH(b)

(c)

0 10 20 30 40 50 60 70 80 90 100Disease severity index

Fig. 3. Distribution in pathogenicity on spinach (diseaseseverity index) of three Pythium spp, where 50 or moreisolates were tested by using the inoculum-soil mixingmethod (seed inoculation), (a) P. ultimum var, ultimwn;(b) Pythium group F; (c) P. sytvaticum.

var. ultimum in 50%, and P. heterothallicum in21 % of all fields surveyed, Pythium group HS wasfound in three of the fields and P. tracheiphilum inonly one field. When calculated in percentages ofall plants investigated, Pythium group F was themost common species, being isolated from 13%of the plants (Table I), Group F isolates wereespecially abundant in one field (No, 9, Table 1);this field was also exceptional in having highfrequencies of Phytophlhora cryptogea (Larsson& Olofsson, 1994). The other Pythium spp,occurred in approximately the same frequenciesin the different fields (Table I). The four mostcommon groups of Pythium were all also isolatedin the smaller survey with surface-sterilization ofroots described previously (Larsson ft Olofsson.1994), Pythium spp, often occurred with otherpathogens such as Aphantmtyce\ cladogamus, P.crvptogea, and Fusarium spp. on the same plantroot.

Pathogenicity of spinach root pathogens of the genti.s Pythium 265

Fig. 4 . S y m p t o m s c a u s e d by t h r e e Pyihium s p p . o n s p m a c h r o o t s l a ) P uliimuni \ a r ii/iiDiiini, t h ) P M / I UIU i,m.

(c) P heterothatticum P l a n t s w i t h s y m p t o m s of P ••ykiiiuum a r e I r o m a p o t t r e a t e d b \ m e a n s c>f t he i n o e u l u m - s o i l

mi,xing m e t h o d ( seed i n o e u l a t i o n ) , w h i l e t h e p l a n t s w i t h s \ m p t o m s oi P hcicrnihalluum a n d Z' ulimmm \ a r ulnrnum

a r e f r o m p o t s t r e a t e d by t h e i n o e u l u m l a y e r m e t h o d ( e m e r g e d p l a n t i n o e u l a i i o n )

Pythium group F and P ultimum \ar. ultimumwere isolated in significantly higher frequeneies(/"^OOI) from spinach grown in the autumnseason than in the spring Pythium group F wasisolated, on average, from 3 8',, of all plants inspring crops and 16 9", in autumn erops. and P.ultimum var, ultimum was isolated from 0-9 \, ofthe plants in spring erops and 58 ' , , in autumnerops. Neither Pvthium group F nor P. ultimiiinvar, ulttmum was isolated during Ma\ P syhati-cum and P. hcteruthalluum were isolated in thesame frequeneies from May until November

In using all the data eollected and analysingrelationships between parameters b\ linearregression, there was no clear relation betweenPythium prevalenee and field disease severityindex, between Pvthium prevalenee and plantdevelopmental stage, or between prevalence ofthe vanous pathogens isolated. The eoefficient oldetermination. R\ was in most eases <() 2 andthe highest (0 3) was found when testing relation-ships between prevalence of Pythium group I- andP. crvptogcu. which often oceurred together Itwas possible to isolate all the described Pvthiumspp, from roots without any disease symptoms,i,e, with severity index =0, In addition, they eouldall be isolated ft-om small plants with the first pairof true leaves developing (even though the seedswere treated with metalaxyl) and at all plantstages up to 6-week-old plants at harvest

Pathogenicity and symptoms induced in spinach

P. ultimum var. uiiimum was the most pathogenic

of the i so la ted Pi7/ ; ; i / / " s p p ( F m s 2 a n d ,^i W h e n

mixed wi th the soil at the t ime of s o w i n g . It c a u s e d

b o t h p r e - e m e r g e n c e a n d p o s t - e m e r g e n c e d a m p -

ing oft. Of ten the ln l ec t ion spreaei u p to the

h y p o c o t y l a n d e \ c n to the c o t y l e d o n s , so the

p l a n t s rapidly d ied T h e i n o e u l u m layer m e t h o d

d id n o t resul t in damping-o tV, hu t in a r o o t ro t ol

o l d e r p l a n t s , y ie ld ing an a v e r a g e d i sease s e \ e r i t \

index of 33 (F ig . 2) .Almost the en t i r e r o o t sys t em

t u r n e d b r o w n (F ig . 4a )

P wluiliinni i so la tes \ a r i e d m p a t h o g e n i c i t y

f rom 13 t o MM) w h e n tes ted with the i n o c u l u m

soil m i x i n g m e t h o d (h )g ,^), w h e r e h o t h tcnvi lo

a n d m a l e i so la tes i n d u c e d a v e r a g e d i sease s e \ e r i t \

ind ices a r o u n d 40 They were n o n - p a t h o g e n i e

w h e n tes ted by the i n o c u l u m layer m e t h o d ( F i g

2). They s o m e t i m e s c a u s e d d a m p i n g otV, hut in

o t h e r cases e>nly a b r o w n i s h d i s c o l o r a t i o n ol the

h y p o c o t y l o c c u r r e d In o lde r p l a n t s , the c o r t e x i>l

the h y p o c o t y l o t t e n c r a c k e d a n d the i n l ec t ed

t issue w a s s l o u g h e d olT( Fig 4 h i , w hich , ippe , i red

s imi la r to s y m p t o m s i n d u c e d h \ ( , A;,/,"..',;/(/((v

( L a r s s o n & O l o f s s o n , r ' ' U )

T h e P /htcrdtliiilli, mil i so la tes tes ted \ \ e r e ,ilso

p a t h o g e n i c to s p i n a c h F e m a l e isol. i tes mduee t l ,:

d i sease s even ty index ol >s .ind ni.ile isi<|,ites g.iv e

a n index of 4tl w h e n the idocuUui i soil m i x i n g

m e t h o d w a s used , h o w e v e r , this d i f lc ie iKe iii

p a t h o g e n i c i t y w a s not s i gn i lKan i I h i s p , i i h o g e n

a l so c a u s e d a roo t rol vvhcn the moLuli i in l,i\ei

m e t h o d was used (F ig 2). the Icm.ile ,iiui ni,ile

i so la tes i n d u c i n g the s.mic disc.ise seven lv

ind ices S y m p t o m s . ippe . i red .is h i o w n vtiscv^K*-

r a t i o n s o v e r the en t i r e r o o t svstciii (I lu 4i. . ()ii

266 M. Larsson

the hypocotyl, symptoms sometimes appearedsimilar to those induced by P. sylvaticum andA. cladogamus.

The Pythium group F isolates were only slightlypathogenic on spinach (Figs 2 and 3). The methodwhere zoospore inoculation of Pythium group Fwas used induced the same disease severity(16 ± I -3) as when the inoculum-soil mixing meth-od (15 4+1 5) was used. Roots infected withthese isolates showed a light-brown discolorationand/or brown spots on the hypocotyls, or some-times over the whole root system. These isolatesalso induced typical deformations of cotyledons(bent leaves).

The few isolates of P. tracheiphilum obtainedand tested caused damage on both seedlings andolder plants, while the Pythium group HS werepathogenic only to seedlings (Fig, 2), The symp-toms caused by P. tracheiphilum included wiltedleaves, reddish coloured stems and brownishroots. Symptoms caused by Pythium group HSappeared similar to those of P. sylvaticum.

When infected oat seeds were used as inocu-lum, the isolates and species tested gave diseaseseverity indices similar to those obtained with theinoculum-soil mixing method. However, byusing the method with fungal disks placed on thespinach seedlings, the damage was somewhatgreater than that achieved by other methods.With this fungal disk method, a root lengthgrowth reduction of 10% was obtained by anisolate which was non-pathogenic when othermethods were used; a growth reduction of about50% was found with Pvthium group F isolates,and a 90% growth reduction was measured for aPythium group HS isolate, which gave a severityindex of 65 when using the inoculum-soil mixingmethod. In all pathogenicity tests, the controlplants remained healthy with disease severityindices ranging from 0 to 5,

DISCUSSION

The isolation frequency of Pythium spp, in thisinvestigation is broadly in accordance with thevalues obtained in other countries. In Japan.Pythium spp, were isolated from c. 10% of thediseased spinach plants investigated (Naiki &Kanoh, 1978; Akashi & Abe, 1985), and in theUnited States from 19% (Sumner et al., 1976),However, the pathogenic species of the genusPythium listed in this investigation differ invarious respects from those reported from othercountries. Differences in isolation techniques andagar media used may, to some extent, explain

these discrepancies. Our semi-selective Pythium-isolation medium contained streptomycin,known to inhibit certain Pythium spp., e.g. P.paroecandrum (Voros, 1965), which is reported toinfect spinach in Japan (Naiki et al., 1986). P.aphanidermatum, frequently reported as a spi-nach pathogen (Bates & Stanghellini, 1984;Akashi et al., 1986; Naiki et al., 1986) is, on theother hand, not sensitive to streptomycin (Hine &Luna, 1963) and appears to be isolated easily.This species, considered synonymous with P.butleri, is a typical plant parasite of warm regions(van der Plaats-Niterink, 1981), and in Japan wasdetected only in soil samples taken from fieldswhere the average annual air temperature was^ we (Watanabe, 1983), Thus, the lack of P.aphanidermatum in this investigation may mirrorclimatic differences between Japan and Sweden.

P. heterothallicum and P. sylvaticum have notpreviously been described as spinach pathogens,even though they are reported to be very commonin various soils. Van der Plaats-Niterink (1975)stated that P. sylvaticum is the most commonPythium species in cultivated soils in the Nether-lands, It was likewise the most common Pythiumpathogen in soils under cereal cultivation inSweden (S, Kadir, Uppsala, unpublished data,1992) and, together with P. heterothallicum, wasamong the most frequent and abundant Pythiumspp, in orchard soils in the United Kingdom(Sewell et ai, 1983), P. heterothallicum was alsoone of the most commonly isolated Pythium spp,from wheat roots in the United States whereagricultural crops including wheat, barley, lentilsand p>eas were grown in various rotations (Ingram& Cook, 1990), This frequent occurrence, and thefact that both species are heterothallic and haveto be identified by crossing in dual cultures (whichhas not been performed in other investigationswith spinach), suggest that they may be includedin the spinach damping off and root rot complexfound in other spinach-producing areas, fromwhich they have not so far been reported.

The results obtained with the two main patho-genicity tests performed, the inoculum-soil mix-ing method and the inoculum layer method, allowa division of the tested species in three distinctgroups: (i) non-pathogenic species that canreadily be isolated from spinach roots: (ii) speciesthat are pathogenic only when the inoculum is incontact with very young seedlings; and (iii)species that damage spinach plants independentof the test method used. To what extent thesefindings reflect that the pathogenicit\ tests wereperformed under only one set of environmental

Pathogenicity of spinach root pathogens of the genus Pythium 267

conditions is not known, but taking into accountthat conditions were in no way extreme, thisshould be of minor importance. Therefore, inconsidering the large heterogenicity found in thisstudy, it seems misleading to refer to the Pythiumspp. as one group of pathogens, as is often done ininvestigations where a complex of pathogens isstudied. It also seems incorrect to refer to allPythium species found on roots as pathogenicmainly on young plants or as damping offpathogens. Some clearly are non-pathogenic rootinhabitants and several are able to cause diseaseeven on older plants (Fig. 2). These studies yieldfurther evidence that the Pythium pathogens, as agroup, are very versatile.

The lack of relationships found between plantdevelopmental stage and prevalence on roots ofthe Pythium spp. that are pathogenic only toseedlings, may be somewhat remarkable. How-ever, even if most Pythium spp, are pathogensassociated with seedling diseases, they also infectfeeder roots at later stages (Hendrix & Campbell,1973), and can thus be isolated from older plants.Another obvious explanation of the lack ofrelationships is that all spinach field plants sur-veyed in this study were grown from seeds treatedwith metalaxyl, which inhibits the development ofPythium spp. on young seedlings. The metalaxyltreatment may also contribute to differences inthe species found in this study in comparison withother studies, particularly regarding the Pythiumspp, occurring during the early developmentalstages. However, as the isolations were done untilharvest time, the metalaxyl treatment is consid-ered to affect the results only slightly. Despite themetalaxyl seed treatment, all the described Pyth-ium spp, were isolated from plants where the firstpair of true leaves had just started to develop. Inseveral cases it was also possible to isolatePythium group F on the Aphanomyces-st\ecii\eagar medium containing melalaxyl (Larsson &Olofsson, 1994). These findings together indicatea possible failure of metalaxyl to control Pythiumdiseases in the field.

The isolation of P. ultimum var. ultimum andPythium group F in significantly higher frequen-cies from autumn than from spring crops agreeswith other findings that Pythium populationsgradually build up with the recurrence of wetconditions during the autumn (Schmitthenner,1970). The observation that Pythium group Foften occurs together with P. cryptogea is prob-ably also a consequence of environment; bothproduce zoospores and multiply well under wetconditions. This may be the explanation why they

thrived in field No. 9 (Table 1), which had thehighest clay content of the fields surveyed (Lars-son & Olofsson, 1994) and where the wettest soilconditions in the autumn were observed.

The findings that P. ultimum var. ultimumcauses root rot on older plants (Fig. 2) togetherwith its higher prevalence compared with theother root rot inducing Pythium pathogens (P.heterothallicum and P. tracheiphilum) indicatethat P. ultimum is the most serious threat amongthe Pythium spp, in the pathogen complex caus-ing spinach root rot. The importance of the otherPythium spp, found is probably also low incomparison with the other major root pathogenssuch as A. cladogamus and P. cryptogea (Larsson& Olofsson, 1994). The frequent isolation of thesespecies and Pythium spp. together opens up thequestion whether some kind of synergism occurs.However, in this investigation, no attempts weremade to inoculate the plants with two or morepathogens simultaneously, and thus more experi-mentation is necessary to provide conclusiveevidence in a matter of this complexity,

ACKNOWLEDGEMENTSI thank Dr S. Kadir for very valuable help inidentifying the fungal isolates and for supplyingcompatible isolates of P. sylvaticum. Professor B,Gerhardson for fruitful discussions and criticalreading of the manuscript, Dr J, Olofsson forsupport and advice, and Ms Helene Jonnerhagand Ms Ulrika Carlsson for valuable technicalassistance. The work was in part supported byNordreco AB, Bjuv, Sweden,

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