NOTES

Populations of the spinach wilt pathogen, Fusariurn oxysporum f . sp. spinaciae, in the root tissues, rhizosphere, and soil in the field

ANDRES A. REYES Ag1.ic.ri1111r.c~ C r i ~ ~ o t l o , V i r r r lo~~t l Sltrtio~l, 0111.. Crr~rtitlti LOR ?LO

Accepted October 26, 1978

REYES. A. A. 1979. Populations of the spinach wilt pathogen, Fii.sciriio~~ ~ . V . ~ . \ / I O I ' ~ I I , I f. sp. .spi~rtic~itir. in the ~-oot tissues. 1,hizosphere. ant1 soil in the field. Can. J . Microbiol. 25: 227-229.

Populations o f Fii.so~.ii~~rr o.vy.spoi.r~~~~ f. sp. .spirccic.ior in root tissues and rhizosphere soil o f tliseased spinach plants were higher than in the root tissues and rhizosphere soil o f he;rlthy plants. Populations in soil rhizosphere were higher than in nonrhizosphere soil. The fungus populations were very low in the root tissuesof the nonsusceptible strawberry, broccoli, chinese cabbage, and m ~ ~ s t a r d grown in the infestetl field. The popul;~tions were low at the beginning of the season. inc~.eased. and rcm;~inetl high during the surnmer. then dropped in the fall. The f u n g ~ ~ s populations r;~ngetl from 1600 to 2600 propag~~les/g in the lop 10cm o f soil, declined sharply between I I and 20cm. ilntl we]-e nondclectable between 41 and 60cm.

R~rl'tis. A. A. 1979. Populations of the spinach wilt pathogen, F/r.suriiol~ o.~.v.sporri~~r f. sp. .~pilrc~c.itrc,. in the root tissues, rhizosphere. and soil in the field. Cali. J. Microbiol. 25: 227-229.

Les pop~~lut ions de Fi~scrrii i~~r o.vy.cporlri~l f. sp. .sl~i~~rrc.itrc, 5ont plus elevees dans les t i ~sus des I-acines et clans les ~.hizosphk~.es LILI sol de plants tl'Cpinard mal~ides qLre dans le.; tissusdcs racincb et dans Ies rhizosphkrcs tlu sol de plants snins. Dans les sols B rhizospheres, les populations de cet organisme sont plus elevees que tlans les sols depourvus de rl1izosphe1.e~. Les populations fongiq~~es sont trcs faibles tlans les tissus de\ racines Je plantes non susceptibles, comme le fl-nisier. le brocoli, le cho~r chinois et la mout:u.de, cultivees dans des champs infestis. Les populations sont fi~ibies :ILI debut de In saison tle croissance, augmentent et demeurent elevees :IU

cour, tie I'ete et declinent :I I'nutomne. Les populz~tions fongiques varient de 1600 h 2600 propagules/g dans les pl.ernic~.s IOcm de sol. tlirninuent ~xpidernent cntre I I et 20cn1 el sont non decelablcs entre 41 et 60cm.

[ T ~ x d u i t par le journal)

FIISIII.~III)I O.II\'S~OI.III)I f. sp. .spi/z(ici(le (Sherb.) Snyd. & Hans. race 1, hereafter refel-red to as f. sp. spi/loc,irie, causes the wilt disease of spinach (Spitlocio oletacea L.) in Ontario (Reyes 1977). I t attacks spinach plants at all stages of growth aftel- the seedlings emerge from the soil but does not cause preemel-gence damping-off. The disease is severe on spinach seeded early in August but has little effect on plants seeded 3 weeks later. Disease symptonis include wilting and chlorosis of the leaves, stunting of the plant, and rotting of the roots. Spinach, lychnis, and beet are theonly plants known to be susceptible to this fungus (Reyes 1977).

in 1975 because such information was not available for f. sp. spi~lacirre.

A 25-ha ( 1 ha = 10000rn2) spinach field near Toronto, Ontario, served as the experimental site.

Root samples were taken fl-on1 plots of 5- to 6- week-old spinach, 8-week-old broccoli (B/a.s.sic,o olci.ncen L. var. itrrlicn Plenck). 6-week-old chinese cabbage (B. compcst/.i.s L . Val-. c./~it~e/lsis (L.) Makino). 6-week-old mustard (B. c~a/?lpCstt~is L.), and 3-year-old strawberry (Ft.ngarili ci/lnnos.su Duchesne). With each crop25 roots were carefully dug at random from the field. Soil adhering to the ~.oot was designated as rhizosphere soil; soil 30cni from the I-oot (believed to be free of root influence)

Considel-able knowledge has been gained on the was designated nonrhizosphere soil. The two kinds ecology of pathogenic fusaria in soils on which of soil were collected separately in 4-kg poly- susceptible and nonsusceptible crops have been ethylene bags and stored at 5OC until used. The grown (Abawi and Lorbeel- 1972; Banihashenii and roots were washed in running tap water and stored deZeeuw 1975; PI-asad and Sinha 1972; Reyes and in polyethylene bags as above. Mitchell 1962). The present study was undertaken On August 5 and 6, plant and soil samples were

WOX-4166/79/020227-03S0 1 .00/0 a 1979 National Research Council o f CanadaIConseil national de recherches du Canada

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7-28 C A N . J . MICROBIOL. VOL. 25. 1979

collected from diseased ancl healthy plots of spinach as well as from the plots of bl-occoli, chinese cabbage, rn~tst~~l-d. and stl.awbel.l.y. Ran- dom soil s:~niples were taken between rows biweekly from May 13 to June 24, every 4 weeks ~lntil September 16, and biweekly again until Oc- tober 14 to determine seasonal fluctuations in fun- gus populations. Soil samples fi-om the top 10-crn layel were also collected from three randomly selected sites from the part of the field being pre- pared for planting on September 26 to determine the range of variation in the fungus population. In addition, soil samples were collected from five other depths (10-cm intervals) at each site to deter- mine the vertical distribution of the fungus. Sub- samples flum each depth were combined to repre- sent that soil layer.

The population of f . sp. .spirltrc,itre in each sample was determined by dilution plating on modified Martin's rose bengal medium (Abawi and Lol-beer 1971). Each sample (5g) was aseptically homog- enized in 195 mL water in a Waring Blendor 3 times for 30 s each at I°C. Five dilutions (w/v) (1: 100, 1:500, 1:1000, 1:5000, 1:10000) were made from each suspension. With each dilution five replicate cultur-e plates (100 x 15 mm) were inoculated by flooding with I m L of suspension. Five replicate plates flooded with dilutions from a soil culture of f. sp. spintrcitre served as contt.ols.

The plates were placed on a labol-atol-y bench overnight (24°C) with the lid of each plate slightly raised to allow evapol.ation of excess moisture. After 5 days in an incubator at 23OC colonies o f f . sp. .spirrtrc.itre were differentiated from those of other fungi present. Based on colony size, color, and gl-owth characteristics, colonies that appeared similar to those from the soil cultut.e of f. sp. spintrc,ine on the control plates were counted. A total of 1385 representative colonies were transfer- red to potato dextrose agar (PDA) test t~lbe slants and later tested for pathogenicity on spinach seed- lings in steamed and artificially infested soil. Sixty-five percent of the colonies proved to be f. sp. sp inc~c i t r~ . This value was used throughout this study as a correction factol- in calculating the populations of the wilt fung~ls (number of colonies expressed as propagules/g, ppg, of sample). Data were analyzed statistically (Duncan 1955).

The pop~llations of f. sp. spintrcirrc. in the root tissue (73 600 ppg) and soil rhizosphere (19900ppg) of diseased plants were higher than those of roots (8800 ppg) and rhizosphere (2600 ppg) of apparently healthy plants. The fungus populations in the rhizosphere of diseased and healthy plants were significantly higher than that of nonrhizosphere soil (1920 ppg).

Root tissues of diseased plants with high popula- tions o f f . sp. spir7trc.ioc~ had numerous, coalescing, necrotic lesions. Micl.oscopic examination of the necrotic tissues stained with 1% acid fuchsin re- vealed the p~-esence of abundant myceli~ini, single-celled and multicellu1:u conidia, and thick- walled chlarnydospores (10.5-17.5 pm diam, av. 12.7 prn). The chlarnydospol'es were obsel.ved in the soil either free 01- associated with organic niat- ter. Presumably, they were released into the soil by disintegration of diseased roots. In conttxst, the root tiss~les of healthy plants contained a few le- sions which were restricted primarily to the late[-al roots. Mycelium, conidia, and chlamydospores were rarely observecl in non-necrotic roots.

These results agree with those obtained by others fou F. osjlspor.iir?i f. sp. nielonis and F. 0 . v ~ -

.spor/ir)r f. sp. ceprrc. (Abawi and Lorbeer 1972; Banihashemi and deZeeuw 1975; Wensley and McKeen 1963).

The pop~ilations o f f . sp. spiritrcioc in the root tissues of nonsusceptible cl-ops (strawberry, broc- coli, chinese cabbage, and niustnrd) were low ( 160-700 ppg) compared to that (80 000 ppg) de- tected in diseased spinach roots collected the same day. The fungus was not detected in the vascular root tissues of nonsusceptible crops but conidia and chlamydospores wese observed in the sloughed off cortical tissues. Presumably, the populations mea- sured in this study originated from the cot-tical tis- sues present at the time of honiogenization. These results show that nonsusceptible plants can harbor f. sp. spirrtrc,it/e without becoming diseased. Other fusarin have also been isolated from root tissues of symptomless, nonsusceptible plants (Armstl-ong and Armstrong 1948, Banihashenii and deZeeuw 1975).

The fungus populations in the field were low at the beginning of the season (200 ppg in early May), increased in late May (3000ppg), remained high throughout summer ( 1 300-3800 ppg), and declined in the fall (600 ppg in niid-October). These popula- tions were directly related to disease incidence and ail. temperature in the field (unpublished data). Furthet-more, these results agree with those of McKeen and Wensley (1962), who showed that populations of F . osysporunr f. sp. melonis in the field were low just before planting melons in the spring and high at the end of the growing season. Similarly, Prasad and Sinha (1972) f o ~ ~ n d that populations o f F . 0.1-ysporrir?~ f . sp. Itrtlryri in the soil were high during the hot period of the year and low during the cool, rainy season.

The f. sp. spinacicre populations in the field on September 26 ranged from 1600 to 2600 ppg in the top 10cm of soil. These populations were higher

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NOTES 229

than that (920ppg) between 1 1 and 20cm deep, - . 1972. Sever~tI aspecls o f the ecology and p:ttho~ogy of

while the lowest populations (21- 140 ppg) occurr& F ~ ~ s u t ' i u t ~ ~ o.v.v.spcn~~~t~r f. sp. c'c~xrt,. Phytopatho10;y. 62: 870-876. between 21 'lnd 40cm deep i n the s o i l ' The funglls AK~~IS.I~ROUG, G, M.. and J. K . ARMS-I.RONG. 1948. Non\~~scep-

was not detected at the 41- to 60-cni layer of soil. tible hosts as carrier5 o f wilt fir sari:^. Phptop;~thology, 38: Most of the suinach I-oots were found in the tor, 808-826.

20cn1 of soil indicating that the fungus populations were closely I-elated to the root zone. Burke (1969) found, however, that field bean roots infected with F. solrrni f. sp. pllrr~eoli were confined to the plowecl soil layer whereas in a noninfected field, the I-oots penetrated to depths of lOOcm 01. more. Perhaps. therefore. roots of noninfestecl spinach plants would have penetrateci much deeper than 20cm. It seems reasonable to suggest that depth of penetration is directly related to vigor of the plant which in turn is inversely related to severity of the disease.

Acknowledgment I thank H. W. Neufeld for technical assistance.

A e ~ w l . G S . and J. W LORBL~R 1971. Popul,ltlon\ of F r ~ . c ( ~ t t t r ~ t ~ o\v.\porrl~tr f \p tepor in otganic \ o ~ l \ In New Yo1 k Phytopathology. 61' 1042-1048

BANIHASHEMI. Z.. and D . J . DEZEEUW. 1975. The behaviourof Frr.strt.irr~rl 0.1-y.~l?orrrt)r f. sp. trlr1otli.s in the presence and ;~b- sence o f host plants. Phytopathology, 65: 1212- 1217.

BURKE. D. W. 1969. Significance o f the plowed soil I:lyel. in Frrs(rrirr~tr root rot o f beans. Phytop;tthology. 59: 1020.

DUNCAN. D. B. 1955. Multiple range and niultiple F tcsrs. Biometries. 11: 1-42.

MCKEEN. C. D.. ;~nd R. N . WENSLI'.).. 1962. Populations of Frtsot~irr~tr (1.1-y.sporrrrtr f. tt~r1otli.s in wilt infested soils. Phytopathology. 52: 21.

PKASAII. T.. and S. SINHA. 1972. Studies on the surviv:tl of Frr.strt.ir)r o.~ysporrrt~r f. Itr/lr?.~.i causing wilt of khes211.i (Ltr/lry~~rr.s .cori~~rr.s) in soil. Indian Phytop:tthol. 25: 423-427.

REYES. A. A . 1977. Spinach wilt in Ont~il-io. Plant Dis. Rep. 61: 1067- 1070.

REYES. A. A , . anti J. E. MI.I.CHEL.L. 1962. Growth response of several isol:ttes o f Frr.strt.irrt)r in rhizosphel-es of host ant1 nonhost plLtnts. Phytopathology. 52: 1196- 1200.

WEKSLEY. R. N., i ~ n d C. D . MCKEEN. 1963. Populations o f frrstrr~irrtir o.\:\'.s/)orrrt)1 f. IIICI~II~J and their relation to the wilt potential o f two soils. Can. J. Microbiol. 9: 237-249.

Characterization of an enzyme from Rhizoctorzia praticola which polymerizes phenolic compounds

Lohot.tr/o,:\. c(f'Soi1 i\.li~t.ohiolo,qy, Urprrt.rt~~etl/ c!fA,qro/ror)r> Tlrc P r t l t ~ . s~~ l i ~o r~ io Srore Utri~.pr.siry,

Utri~.o..siry Prrrk. PA. U.S.A . 16K02

Accepted October 12. 1978

Bo1.1 ..A G. J.-M.. K . D. SJOBL.AD. ant1 S.-Y. 1.1~. 1979. Characterization o f an enzynie from Rlri:oc,rotlitr ~)rcrric.oltt which polymerizes phenolic compountls. Can. J. Microbiol. 25: 229-233.

An extr:~cellul;tr phenol oxidase from the fungus Rlri;oc.rotlio prtrric,olo which polymerizes various xenobiotic phenols w;rs isolitted and ch:~racterized. The enzyme ul:ls puritietl by DEAE- cellulose and Sephadex G-200 chrorn;ttogl.aphy followed by pi.eparative polyacrylamide gel electrophoresis. Atomic ;thsorption end EPR spectroscopy indicated the presence ofcopper, and SDS gel electrophol-esis reveziled a molecular weight o f 78000. With 2.6-dimethoxyphend 21s substrate, the enzynie showed a p H optimum o f 6.7-6.9. and 21 tempel-atul-e optinit~rn o f 40°C. According to these ant1 ~tdditional chal.acteristics it appears that the enzyme belongs to the class o f l;tccases.

BOI.L,\(;. J.-M.. R. D. SJOUL,Z~I et S.-Y. LIU. 1979. Characterizarion of an enzyme from Rl~ i roc- /otritr pt.o/ic,oltr which polymel-izes phenolic compounds. Can. J . Microbiol. 25: 229-233.

A partir de Rlri;oc./otritr prerric.oltr on a isole et caracterise une phenol oxytlase ext~.;tcellulaire qui polymerise divers phenols xenohiotiques. L'enzynie a ere purifie par chroniatogri~phie SLII.

DE4E-cell~rlose et Sephadex G-200 suivi d'electrophorese pl.&pa~.ative en gel poly:tcrylamide. L'. ,I b . so~pt ion . atoniique et la spectroscopie EPR niont~,ent la presence de cuivre et I'elec-

'PI-esent address: Division o f Applied Sciences. Hztrvard University, C;trnhridge, MA. U.S.A.

0008-4 166/79/020229-05x0 I .00/0 0 1979 Narional Research Council o f CnnadaIConseil national de recherches du Canada

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