J. Phytopathology 144, 143-146 (1996)© 1996 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-1785

Department of Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden

Suppressiveness to Clubroot, Pea Root Rot and Fusarium Wilt in Swedish Soils

Y.WoRKU and B.GERHARDSON

Authors' address; Department of Plant Pathology, Swedish University of Agricultural Sciences, Box 7044. S-750 07

Uppsala, Sweden

With 2 figures

Received August 31, 1995; accepted November 15, 1995

Abstract

Natural soil samples, collected from farms in central and southernSweden, were tested for soil disease-suppressiveness. The soils weremixed with inocula of either Plasmodiophora brassicae. Aphanomyceseuteiches or Fusarium oxysporum. f.sp. spinaciae and disease symptomswere scored on Chinese cabbage, pea and spinach respectively, thatwere grown in the soil-inocula mixture under greenhouse conditions.Most field soils limited the development of disease to some degreecompared with a commercial 5/)fta^num-sand-soil mixture, and about10% were strongly suppressive to disease outbreaks caused by either P.brassicae, A. euiheiches. or F. oxysporum f.sp. spinaciae. Some testedsoils gave higher disease indices of A. euteiches compared with thecontrol growing medium and were regarded disease synergistic. Thesuppressiveness found was specific for each disease tested and in nocase was a soil sample strongly suppressive to two of the diseases tested.

Ziisammenfassung

Siippressivitat schwedischer BMen gegenOber Kohlhemie, Wurzelfaaleder Erbse und Fusarium-Welke

Bodenproben von landwirtschaftlichen Betrieben in Mittel- undSudschweden wurden auf ihre Suppressivitat gegentiher bodenbtirtigenKrankheiten getestet. Den Bdden wurde Inokulum von Plasmodiophorahrassicae. Aphanomyces euteiches bzw. Fusarium oxysporum f.sp. spina-ciae beigemischt. In den Boden-Inokulum-Mischungen wurden China-kohl-, Erbsen- bzw. Spinatpflanzen unter Gewachshausbedingungengehalten und auf Krankheitssymptome untersucht. Im Vergleich zueiner im Handel erhaltlichen Mischung von Torf, Sand und Bodenbegrenzten die meisten Feldboden die Krankheitsentwicklung inbestimmtem AusmaO. Etwa zehn Prozent der Feldboden waren starksuppressiv gegentiber Krankheitsausbruchen, die dureh P. brassieae, A.euteiches bzw. F. oxysporum f.sp. spinaciae verursacht werden. Einigeder geprflften Boden verstarkten den Befall dureh A. euteiches im Ver-gleich zum Kontroll-Wachstumsmedium und wurden als synergistischauf die Krankheit wirkend eingestuft. Die gefundene Suppressivitatwar ftir die einzelnen Krankheiten spezifisch, keine Bodenprobe wargegenQber zwei gerprOften Krankheiten stark suppressiv.

Introduction

Soil physical and chemical factors profoundly influence soil-

borne pathogens and the crop diseases they cause. As a conse-

quence, specific diseases rarely appear on soils with certain

characters, e.g. clubroot where soil pH is high (Garret, 1970)

or Fusarium wilt on heavy clay soils (Schneider, 1982). There

are also reports of soils that restrain specific diseases even where

adverse soil physical or chemical factors are not present. In

such cases, the suppressiveness often is lost when the soil is

heated or sterilized in other ways, indicating involvement of

biological agents (Alabouvette 1986). This type of disease-spec-

ific suppressiveness, reported from several locations, is either

naturally occurring or is induced by monoculture (Baker and

Cook, 1974; Schneider, 1982; Cook and Baker, 1983; Hornby

1983, 1990, Oyarzun, 1994), and may be of great agricultural

and economic interest (Alabouvette, 1986). Biological factors

as a background for soil disease suppressiveness may also be of

interest as a possible source for isolating agents for biological

disease control (Cook and Baker, 1983; Louvet et a l , 1981).

Disease-specific soil suppressiveness with a biological back-

ground has not hitherto been reported from Scandinavia and

systematic surveys have not been performed. In this paper we

report on development of test procedures and results of a pilot

survey of suppressiveness to three soil-bome pathogens in soil

samples collected from various locations in central and southern

Sweden.

Materials and Methods

Soils tested

Soils were sampled from cultivated fields that were chosen randomlyon farms in the central and southern part of Sweden. Subsamples werecollected with an auger and taken from an area of about 50 x 50 m inthe central part of the fields chosen. A sample of about 10 1 from eachfield was collected from the top 20 cm soil layer. All the subsampleswere mixed in a plastic bag, transported to the department and storedin a cool room at 4 C unti! used for testing.

PreparatioD of patbogen inoculum for soil tests

Inocula were prepared with the objective of obtaining resting structuresthat could be stored as dry preparations and could be mixed with thesoil to be tested without losing infectivity until test plants were planted.Plasmodiophora brassicae Woron inoculum was prepared by mixingpulverized decomposing ciubroot galls with heavily infested field soil.After air drying and sieving ( < 2 rrmi) the inoculum was stored at— I 8 X . Pathogenicity of the prepared inoculum was regularly testedhy mixing 1-1.5Vo (w/w) of inoculum with our control soil (Commercialplanting soil: Hasselfors P-jord, a fully fertilized mixture of sand andpeat with a pH of about 5.6) and planting 10-day-old Chinese cabbage(Brassica campestris ssp. pekinensis, cv. Granaat) seedling in this pottedmixture. After 6 v^eeks growth in the greenhouse the plants were gradedfor symptoms and given a disease index as described below.

Inocula of Aphanomyces euieiches Drechs. and Fusarium oxysporum

tJ.S Copyri6h.Ctoan«Cen.=rCc«teSla,c™«: 0 9 3 1 - 1 7 8 5 / 9 6 / 4 4 0 3 - 0 1 4 3 S 1 1.50/0

144 WoRKu and GEKHARDSON

Schlecht. f.sp. spinaciae (Sherb.) Snyd. & Hans were prepared by grow-ing the fungi in an autoclaved commeal-sand mixture (1 kg sand, 30 gcorrmieal and 130 ml of tap water) in glass jars at ca 23'C for 30 days.After air drying and sieving (< 2 mm), the inocula were stored at —4°C.Pathogenicity was tested as described for the P. brassicae inoculum,but 8-day-old pea seedlings (Pisum sativum L., cv. Bodil) were used astest plants for the A. euteiches inoculum and 11-day-old spinach (Spi-nacia oleracea L.. cv. 'Medania') seedlings for testing the F. oxysporumf.sp. spinaciae inoculum.

Before using the inocula for testing suppressiveness they were testedin control soil in a series of dilutions. The percentage (w w) of inoculumgiving a disease index of 80-95 in this test was then used for testingsuppressiveness in field soils.

Disea^ indices

For evaluation of clubroot infections, the cabbage test plants were dugup and the roots carefully washed with water and scored visually foramount of cluhroot infection on a four-degree scale (0, healthy plants(no cluhs); 1. plants with slight swellings on lateral roots; 2, plants withsmall clubs and/or a few bigger ones; and 3, plants with severe swellingon lateral and or tap roots). As disease index we used the average scoreof al! plants in a treatment multiplied by 100 to give an index rangngfrom 0 (all plants healthy) to 100 (all plants in the highest score).

A. euteiches infections were scored on pea test plants that wereremoved from the soil, washed and evaluated for rot in the roots andepicotyl on a scale ranging from 0 (no disease, roots free of symptoms) to4 (water soaking, browning, and decay involving all roots and epicotyl,cortex easily sloughed off. epicotyl shrivelled or rotted). (Sherwood andHagedom, 1958; Olofsson, 1967; Sundheim, 1972; Papavizas, 1974).Infections off. oxysporum f.sp. spinaciae were scored on spinach plantsdug up and washed which were likewise given scores from 0 to 4 (0, nodisease, no symptoms; 1, light brown lateral roots, yellow lower leaves;2, brown discolouration of most of the root system, yellow lower leaves;3, brown to black discolouration of the whole root system, beginningof wilting of shoots; and 4, the plant fully wilted and dead). Diseaseindices ranging from 0 to 100 were then calculated as described above.

Greenhouse testing procedure

The field soil samples were air dried in the laboratory from 2 to 8 weeksand in a few cases longer. They were then mixed with washed sand in aratio of 1 :1 (v.'v) to help to eliminate differences in texture and water-holding capacity. This soil-sand mixture was then thoroughly mixedwith inoculum, usually about 1% (w.'w), poured into plastic pots (13cm diameter, 10.5 cm deep; four pots per test soil sample), wateredfrequently, and incubated for 7 days at 25 C in the greenhouse to allowthe test soil to interact with the inoculum. After this incubation period,the pots were planted with the test plant seedlings used for testing thetype of inoculum applied. Each pot was planted with either five 10-day-old Chinese cabbage seedlings, four 8-day-old pea seedlings or four 11-day-old spinach seedlings, respectively. The seedlings were producedby placing seeds on sterilized greenhouse soil in plastic pots.

The planted pots used in the test were placed in a greenhouse at 20-23°C with a light period of 14 h/day. They were watered as requiredbut given no fertilizers. The Chinese cabbage plants were scored fordisease symptoms after 6 weeks and the pea and spinach plants after 4weeks.

Two controls were included in each test series: (I) control soil withoutinoculum and (2) control soil with inoculum. In addition, we also testedthe field soils without inoculum in most experiments to detect anynatural infestations.

Heating of test soilsSome of the tested soils showing disease suppressiveness were heatedto 60°C for 10 min (Olsson and Gerhardson, 1991) or autoclaved at120°C for 20 min (Rouxel et al., 1988) and then retested to evaluateinvolvement of heat sensitive factors in the suppressive reaction.

ResultsDevelopment of the test procedureBefore adopting the test procedure used, involvement of variousfactors on test results were tested in a nimiber of pilot experi-ments. Planting test plant in the soil-inoculum mixture directlyafter the mixing gave high disease indices in all soils tested andresults were not repeatable in subsequent experiments. Whenwe instead incubated the watered soil-inoculum mixture for7 days in the greenhouse before planting the test plants, cleardifferences between soils was obtained and the results also couldbe repeated by testing the same soil in consecutive experiments.

Greenhouse temperatures and water regimes also stronglyinfluenced test results and in order to be able to make com-parisons between tests, condition had to be stable from test totest.

For the field soils tested, and especially the strongly disease-suppressive soils, the amount of inoculum applied to the soildid not affect the disease index significantly, at least not untilvery high doses were applied (Fig. 1). For routine tests, pro-duction and storage of inocula were a critical factor. The vari-ous batches of inocula seldom showed the same infectivity incontrol soil, and all inocula lost infectivity after several monthsof storage.

Results of testing field soilsWe found highly varying degrees of soil suppressiveness for the142 field soils tested, as shown in Table 1. For some soils wealso obtained a higher disease index than in the control soil,indicating disease-synergistic characters. Results of one specificexperiment where such effects are obtained are shown in Fig. 2.

No correlation was found in disease indices obtained for P.brassieae and A. euteiches when they were tested in the samesoils. The correlation coefficient R" was 0.186 between the dis-ease indices obtained for these pathogens in 23 soils tested withboth.

Effect of headng on disease suppressiveness

Effects of heating or autoclaving some of the field soils sup-pressive to A. euteiches or P. brassicae varied depending onsoil and pathogen tested. Heating two soils suppressive to P.brassieae had only a small and non-significant effect on thedisease indices obtained after heating, while heating one soilthat was highly suppressive for A. euteiches dramatically influ-

I Control soil

I Suppressivefield soil

0.1 g

Inoculum/100 g soilFig. 1 Results of a typical experiment where three different doses ofinoculum of Aphanomyces euteiches were applied to control soil (agreenhouse growing medium, see text) and a disease-suppressive fieldsoil. Differences are statistically significantly different for the field soil

Soil Disease-suppressiveness in Swedish Soils 145

Table 1Results of grouping the soils testedafter indices obtained in tests withthe three pathogens

Pathogentested

Aphanomyces euteichesFusarium oxysporumPtasmodiophora bra.'isicae

Total

Hosttested

PeaSpinachChineseCabbage

Number of soils with disease index0-25

203

5

26-50

91

11

21

51-75

385

23

66

76-100

221

23

46

Totalnumbertested

717

60

138

enced the disease index, which after the heating rose from 15.6to 87.5.

DiscussionThe test method worked out here gives one type of index to asoil quality parameter that we call disease suppressiveness. Ininterpreting thts value, or index, two main questions arise; (I)to what extent has this index, obtained in greenhouse tests,relevance in a field situation and (2| to what extent is this indexdependent on the test procedure adopted? The results presentedhere do not give an answer to the first question. Farmers'experience from fields where the soil samples were collectedhave given indicatioti that the index obtained may have a clearfield relevance. However, more conclusive evidence awaitsfurther, more rigorous investigations in field trials that are nowunder way. The second question is related to the first, but isalso of interest in ttself where tests are to be standardized sothat consecutive tests can be compared.

Within reasonable limits, the amount of inoculum mixed withthe test soil is evidently not critical for the outcome of a test, asshown for one pathogen in Fig. 1. More cntical are inoculumpreparation and storage that, according to our experiences, leadto routines where all inocula are repeatedly standardized indilution series tests. Watering regime and temperature in thegreenhouse also strongly affect the index. However, these par-ameters mainly affect the value of an index and seemingly littleor not at all the rank of indices among different soils tested.Thus, the watering regime and the greenhouse temperaturechosen may be more a question of convenience and stan-dardization than of quality of the test.

The effect of mixing test soils with sand, which may have

affected the outcome of the tests, was not given a rigorousexperimental evaluation. However, it is very difficult to usefield soil without some kind of dilution, as theti possibilities ofwatering, mixing in of inoculum and also of washing roots freefrom soil would be very dependent on soil type. Type andamount of material mixed in may well have a profound influ-ence on the outcome of the test, especially in some soils and,therefore, should be given attention in test systems that are tobe used routinely. Another critical parameter affecting quahtyof the test is probably treatment of test soils after inoculum hasbeen mixed in. If test plants are planted or sown directly aftermixing in the inoculum, the soil as such apparently affects theinoculum or infection process very little. In our test procedurefinally adopted, we watered the soil sample-inoculum mixtureand left it for I week without test plant to allow the wet soil toreact with the inoculum. The time chosen here was somewhatarbitrary and for a routine test should be evaluated more care-fully. Several authors have discussed this problem (Alabouvetteet al., 1982; Linderman et al., 1983; Alabouvette, 1991), andpossibly a special regime may have to be worked out for eachspecific disease to be tested.

The results of our tests, as summarized in Table 1, show thatmost of the soils collected have an ability to suppress diseasesmore than the control soil used. This was to be expected asmost natural soils possess some ability to limit diseases, but itis somewhat surprising that we found around 10% of the soilsamples to be strongly suppressive. One explanation may bethat the samples tested were not taken randomly in a statisticalsense, as we were looking for suppressive soils. Another obviousexplanation is that there are a substantial part of strongly dis-ease-suppressive soils among those sampled. Clearly, the test

100

80

° 20 • II I • ! ! ! Ml• I I I n IM JL2 2oU

§u

Field soil No.

Fig. 2 Results of a test with Aphanomyces euteiches inoculum in 15 fields soils (numbered 1 to 15). One soil, no. 7, shows strong suppressivenessand another soil, no, 10, disease-synergistic effects Control 1 is a greenhouse growing medium without inoculum and control 2 the same mediummixed with the same amount of inoculum as the field soils

146 WoRKU and GERHARDSON

could also have been inadequate for testing practically usabledisease suppressiveness in the field, as discussed above.

The suppressiveness to P. brassicae infection found probablydoes not have a biological background as heating two P. bras-5;fof-suppressive soils at 60^C did not influence their sup-pressivene.ss. Further, as soil pH of suppressive soils was nodifferent from those in soils found to be non-suppressive, thepossibility that the suppressiveness found solely was a soil pHeffect is also unlikely. There are few reports in the literaturedealing with suppressiveness to ciubroot and so there are fewindications of other possible mechanisms involved (Hsieh andWang, 1986; Rotixel et al., 1988). From a practical view pointthe suppressiveness is still of gTeat interest. Intensive attemptsto control this disease for almost a century and a half has notso far led to any completely effective control measures (Karling1968, Rouxei et al., 1988) and, thus, the possibility of makingbetter use of suppressive soils is very appealing.

The suppressiveness to pea root rot found most probably hasa biological background as it was affected by heating and, thus,it shows similarities to suppressiveness against several othersoil-bcme diseases (Schneider, 1982). Some of the soils testedfor suppressiveness to A. euteiehes gave higher disease indicesthan the control soil (Fig. 2). This indicates synergistic effectsfrom these soils, which under field conditions might beespecially conducive to this disease. Such effects have practicalimplications, but are also of purely scientific interest in thestudy of root infections, especially if there is a truly biologicalbackground. However, few soils with this character were foundand the phenomenon should be better confirmed by furtherinvestigation.

The test of suppressiveness to P. oxysporum f.sp. spinaciae ina few soils was undertaken mainly because suppressiveness iswell documented for other forma speciales of this pathogen(Alabouvette, 1986, Cook and Baker, 1983). The chances offinding suppressive soils thus might be good and the results(Table 1) also show that suppressiveness probably present.However, few soils were tested and no strongly suppressive soilwas found.

In agreement with several other investigations (Schneider,1982; Cook and Baker, 1983; Hornby, 1983,1990; Alabouvette,1986), we found suppressiveness against the various diseasestested to be very specific. This points to mechanisms specific foreach disease and to the impossibility of designing tests to findstrong, general suppressiveness to many diseases.

Acknowledgement

We thank Agronomist Ann-Charlotte Wallenhammar for help withtaking soil samples and gratefully acknowledge financial support fromStiftelsen Lantbniks-forskning, without which this investigation wouldhave not been possible.

LiteratureAlabouvette, C. (1986): Fusarium wilt suppressive soils from Chateau-

renard region: review of a 10 year study. Agronomie 6,273—284.Alabouvette, C. (1991): Suppressive soils and practical application of

biological control of fusarium diseases. In: Bay-Petersen, J. (ed). Thebiological Control of Plant Diseases, pp. 120-129, F.F.T.C. Bookseries No. 42, Kuo Thai Color Printing Co., Ltd., Taipei, Taiwan.

Alabouvette, C, Y. Couteaudier, } . Louvet (1982): Comparaison de lareceptivite de differems sols et substrats de culture aux fusariosesvasculaires. Agronomie 2, 1-6.

Baker, K. F., R. J. Cook (1974): Biological control of plant pathogens.W. H. Freeman, San Francisco.

Cook, R. J. (1988): Management of environment for the control ofpathogens. In: Wood. R. K. S. and M. J. Way (eds). BiologicalControl of Pests, Pathogens and Weeds: Developments and Pros-pects, pp. 61-72, The Royal Society. London.

Cook R. J , K. F. Baker (1983): The Nature and Practice of BiologicalControl of Plant Pathogens, American Phytopathology Society, StPaul, MN.

Garret, S. D. (1970): Pathogenic Root-infecting Fungi Cambridge Uni-versity Press. London.

Hornby, D. (1983); Suppressive soils. Annu. Rev. Phytopathol. 21,65-85.

Hornby, D.. ed. (1990): Biologicai Control of Soil-borne Plant Patho-gens. CAB Intemationai, Walhngford, UK.

Hsieh, W. H., J. F. Wang (1986): Investigation on suppressive soils ofciubroot of crucifers in Taiwan. Plant Protect. Bull. (Taiwan) 27, 3-10.

Karling, J. S. (1968): The Plasmodiophorales, 2nd edn, Hafner Puhl.Co.. New York.

Linderman. R. G., L. W. Moor, K. F. Baker, D. .A. Cooosey (1983):Strategies for detecting and characterising systems of biological con-trol of soilbome plant pathogens. Plant Dis. 67, 1058-1064.

Louvet, J., C. Alabouvette. F. Rouxel (1981): Microbiological sup-pressiveness of some soils to Fusarium wilts. In: Nelson, P. E., T. A.Toussoum, and R. J. Cook (eds). Fusarium: Diseases, Biology, andTaxonomy, pp. 261-275. The Pennsylvania State University Press,PA.

Oiofsson, J. (1967): Root rot of canning and freezing peas in Sweden.Acta Agr. Scand. 17, 101-107.

Olsson. S., B. Gerhardson (1992): Effects of long-term barley mono-culture on plant-affecting soil microhiota. Plant Soil 143, 99-108.

Oyarzun, P. J , 1994: Root rot of peas in the Netherlands; fungalpathogens, inoculum potential and soil receptivity. Ponsen andLooijen BV, Wageningen, the Netherlands. 208 pp.

Papavizas. G. C, W. A. Ayers (1974): Aphanomyces species and theirroot diseases in peas and sugarbeet. USDA-ARS Tech. Bull. 158 pp.

Rouxel, F., M. Briard, B. Lejeune (1988): Studies of soil receptivenessto clubroot caused by Plasmodiophora brassicae: experiments onresponses of a series of vegetable soils in Brittany. In: Cavalloro.R., and C. Pelerents (eds). Progress on Pest Management in FieldVegetables, pp. 145-152, A. A. Balkema, Rotterdam, Netherlands.

Schneider, R. W., ed. (1982): Suppressive Soils and Plant Diseases,American Phytopathology Society, St. Paul, MN.

Sherwood, R. T., D. J. Hagedom (1958): Determining the commonroot rot potential of pea fields. Wise. Agric. Exp. Sta. Bull. 531, 12pp.

Sundheim, L. (1972): Physiological specialization in Aphanomvceseuteiches. Physiol. Plant Pathol. 2, 301-306.