ARTICLE IN PRESS

0038-0717/$ - se

doi:10.1016/j.so

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Soil Biology & Biochemistry 39 (2007) 2797–2805

www.elsevier.com/locate/soilbio

Effect of sewage sludge on suppressiveness to soil-borne plant pathogens

Raquel Ghinia,�, Flavia Rodrigues Alves Patrıciob, Wagner Bettiola,Irene Maria Gatti de Almeidab, Aline de Holanda Nunes Maiaa

aEmbrapa Environment, CP 69, 13820.000, Jaguariuna, SP, BrazilbInstituto Biologico, CP 70, 13001-970, Campinas, SP, Brazil

Received 16 November 2006; received in revised form 1 June 2007; accepted 8 June 2007

Available online 2 July 2007

Abstract

The objective of this study was to evaluate the effect of sewage sludge on soil suppressiveness to the pathogens Fusarium oxysporum f.

sp. lycopersici on tomato, Sclerotium rolfsii on bean, Sclerotinia sclerotiorum on tomato, Rhizoctonia solani on radish, Pythium spp. on

cucumber, and Ralstonia solanacearum on tomato. Soil samples were collected from an experimental corn field in which sewage sludge

had been incorporated once a year, since 1999. Sludge from two sewage treatment stations in Brazil (Franca and Barueri, SP) were

applied at the rates of one (1N), two (2N), four (4N) and eight (8N) times the N recommended doses for the corn crop. Soil

suppressiveness was evaluated by methods using indicator host plants, baits and mycelial growth. There was no effect of sewage sludge

on soil suppressiveness to Fusarium oxysporum f. sp. lycopersici in tomato plants. For S. rolfsii, reduction of the disease in bean was

inversely proportional to the dose of Franca sludge. The incidence of dead plants, caused by S. sclerotiorum, was directly proportional to

sludge doses applied. For R. solani and R. solanacearum, there was a linear trend with reduction in plant death in soils treated with

increasing amounts of sludge from Franca. There was an increase in the pathogen community of Pythium spp., proportional to the

amounts of sewage applied. The effects of sewage sludge varied depending on the pathogen, methodology applied and on the time

interval between the sewage sludge incorporation and soil sampling.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Biosolids; Fusarium oxysporum; Sclerotium rolfsii; Sclerotinia sclerotiorum; Rhizoctonia solani; Pythium; Ralstonia solanacearum; Disease

suppression

1. Introduction

The disposal of sludge, or biosolids, generated by thesewage treatment process is a problem. Because this wastematerial is a rich source of organic matter and nutrients, itsuse in agriculture and forestry is an alternative to disposal.In general, Brazilian soils used in agronomic practices arelow in nutrients, organic matter, and cation exchangecapacity (CEC) and water retention capacities. Sludgeapplication can be beneficial in improving soil chemical,physical, and biological properties (Santos and Bettiol,2003; Bettiol and Camargo, 2006).

Among these, suppressiveness to soil-borne plant patho-gens is one of the most important properties. Suppressive

e front matter r 2007 Elsevier Ltd. All rights reserved.

ilbio.2007.06.002

ing author. Tel.: +5519 38678764; fax: +55 19 38678740.

ess: raquel@cnpma.embrapa.br (R. Ghini).

soils are defined as those in which disease development issuppressed even when the pathogen is introduced in thepresence of a susceptible host (Baker and Cook, 1974).There is a wealth of information on the nutritional effect ofsewage sludge to plants; however, when it comes to theeffect on plant diseases, there is a lack of data worldwide,which calls for further studies. Because of its rich organicmatter content, sewage sludge can contribute to the controlof plant diseases, particularly in view of its capacity tostimulate the soil microbiota (Santos and Bettiol, 2003).In Brazil, few studies have been conducted to evaluate

the effect of sewage sludge on soil suppressiveness to plantdiseases and they were of short-duration not allowingevaluation of impacts on agroecosystems (Bettiol, 2003;Santos and Bettiol, 2003; Leoni and Ghini, 2006).Information on such impacts will allow the selection ofcrops that could benefit from sludge applications.

ARTICLE IN PRESSR. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–28052798

A long-term interdisciplinary study was conducted toevaluate the environmental impact of the agricultural useof sewage sludge. Sludge, produced in the wastewatertreatment plants of Barueri and Franca, were appliedannually for 6 years, and corn was grown after eachapplication. Data on communities of organisms, heavymetal concentrations, nitrogen mineralization rates, andphysicochemical properties of the soils, among others, werereported by Bettiol and Camargo (2006), and Fernandeset al. (2005).

The objective of this research was to study the effect ofapplication of different doses and types of sewage sludgeon soil suppressiveness to Fusarium oxysporum f. sp.lycopersici, Sclerotium rolfsii, Sclerotinia sclerotiorum,Rhizoctonia solani, Pythium spp., and Ralstonia solanacear-

um, in soil samples collected from long-term field experi-ment conducted with corn by Bettiol and Camargo (2006),and Fernandes et al. (2005).

2. Materials and methods

2.1. Field experiment

The experiment was conducted at the EmbrapaEnvironment Experimental Field, located in Jaguariuna,Sao Paulo state (latitude 221410S, longitude 471W).The altitude of the site is 570m a.s.l. The climatetype is humid subtropical; mean annual rainfall is1335.4mm and mean annual temperature is 21.7 1C(Cepagri, 2007). The soil was a dark red distroferriclatosol (clayey texture) and the physical chemicalproperties in the 0–20 cm layer before the studyonset were: pH in water ¼ 5.8, OM ¼ 25.5 g kg�1,P ¼ 3.5mg dm�3, K ¼ 1.51mmolc dm�3, Ca ¼ 27.5mmolc dm�3, Mg ¼ 8.5mmolc dm�3, Al ¼ 1mmolc dm�3,H ¼ 35mmolc dm�3, CEC ¼ 73.5mmolc dm�3, BS% ¼50.8, clay ¼ 450 g kg�1, Cu ¼ 12.3mg kg�1, Mn ¼ 7.2mgkg�1, Ni ¼ 8.4mg kg�1, Pb ¼ 10.1mg kg�1, and Zn ¼11.6mg kg�1 (Fernandes et al., 2005; Rangel et al., 2006).

Sewage sludge was obtained from biological or second-ary wastewater treatment plants located in Barueri, SaoPaulo, which treats home and industrial sewage (Baruerisludge), and in Franca, Sao Paulo, which treats only homesewage (Franca sludge). Both plants employ activated-sludge process. The most important characteristics of thesetwo types of sludge were earlier described in Bettiol et al.(2006), and Fernandes et al. (2005).

The study treatments were: control (C); sewage sludgeapplied based on the N concentration that provides thesame amount of N as in the mineral fertilizationrecommended for maize—90 kg ha�1 (Raij et al., 1996)(1N); and two (2N), four (4N), and eight (8N) times the Nrecommended dosage for the corn crop. Calculations ofsludge rates were performed as a function of the Navailable for plants, considering the N mineralization rateas 30% (Cetesb, 1999). Supplementary K was applied fortreatments involving sewage sludge, when necessary

(Bettiol and Camargo, 2006). The wet sludge had beenincorporated annually in six applications, since 1999 (Apriland December 1999, October 2000, November 2001,November 2002 and December 2003). It was toss-distributed in the total area of the experimental plots,and incorporated to a depth of 20 cm with a rotary harrow,3–4 days before sowing. For the present work, soil sampleswere collected from March 2003 to August 2005, at a depthfrom 0 to 20 cm, in at least five sampling points per plot ofthe experiment.The field experimental was set up as a completely

randomized split blocks design with three replications.Each plot measured 10� 20m, with 12 rows per plot. Plotswere separated by hedgerows with at least 5m on each side,planted with Bracchiaria grass kept at a short height. In theassays for investigating pathogen suppressiveness, potsfilled with soil samples from field experimental plots weredefined as the experimental unit. Different levels ofpathogen infestation were then tested for each type ofsludge and dose combination. Thus, in each greenhouseassay, the factors investigated (type of sludge, sludge doseand level of pathogen infestation), were arranged in asplit–split plot design.Tomato (Lycopersicon esculentum Mill.), bean (Phaseo-

lus vulgaris L.), cucumber (Cucumis sativus L.), and radish(Raphanus sativus L.) were used as indicators of soilsuppressiveness to diseases. However, it must be pointedout that sludge application in vegetables is forbidden(Conama, 2006).

2.1.1. Soil suppressiveness to Fusarium

F. oxysporum f. sp. lycopersici isolate TO245 (race 2,collected on diseased tomato in Paty do Alferes, RJ) wascultured in potato-dextrose liquid culture medium onshaker for 25 days. Spore suspensions were incorporatedinto the soil samples collected in field experiment atconcentrations of 0, 103, 104, 105, 106, and 107 cfu g�1 drysoil. Cultivar Viradoro tomato plantlets, approximately1-month old, grown in trays containing container mediumdisinfested in a solar collector were transplanted to 1.5 lcapacity pots. One plantlet was used per pot, in threereplicates for each plot of the field, totaling nine pots pertreatment. The plants were watered by drip irrigation,allowing for equal water per pot.Soil pH was determined in water at soil/solution ratio of

1:2.5 (w:v), using a Digimed DM-20 (Digicrom AnaliticaLtd., Sao Paulo, SP). Electrical conductivity was measuredfrom soil/water suspension (1:5, w:v), using a mCA-150conductivity meter (MS Technopon Instrumentac- ao Cien-tıfica, Piracicaba, SP). Occurrence and severity of diseasewere evaluated 40 days after transplanting seedlings. Plantswere washed, their stalks cut lengthwise into two equalparts, and vascular darkening was evaluated by means of arating scale, following the methodology of Tokeshi andGalli (1966), modified as follows: 1 ¼ healthy plant;2 ¼ plant with brown vessels in the first internode region,without other visible symptoms; 3 ¼ plant with brown

ARTICLE IN PRESSR. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–2805 2799

vessels up to the height of the first leaf, with yellowing of atleast one leaflet; 4 ¼ plant showing vessel browning up tohalf of the stem length, with yellowing of two or moreleaves; 5 ¼ plant showing vessel browning until near theleader shoot, with most leaves wilted, except the leadershoot; 6 ¼ dead plant or plant showing vessel browningand wilted leaves up to the leader shoot.

Isolations were made to confirm the presence of thepathogen. Plant development was determined via drymatter mass. The assay was repeated three times, in theperiods of March 2003, and January and March 2004(Experiments 1, 2, and 3, respectively).

2.1.2. Soil suppressiveness to Sclerotium

Soil collected in field experimental plots was placed in 2 lcapacity plastic pots and cultivar carioquinha bean seedswere sown in a circle (10 seeds per pot). Thirty-five S. rolfsii

sclerotia, produced in Petri dishes containing potato-dextrose-agar medium (PDA), were buried in the centerof the pot at a distance of 1.5 cm from the seeds. Three potswere used per field plot, totaling 90 pots. The pots weremaintained in the greenhouse and moisture was providedby drip irrigation. Emergence percentage of seedling, plantheight, and severity of the disease [(determined by arating scale according to Schoonhoven and Pastor-Corrales (1987))] were evaluated. The assay was repeatedtwice, in February and April 2004 (Experiments 4 and 5,respectively).

2.1.3. Soil suppressiveness to Sclerotinia

The inoculum was prepared from sclerotia ofS. sclerotiorum transferred to Petri dishes containingPDA medium, incubated for 7 days at 25 1C. The funguswas then cultured in carrot and corn meal agar medium.

The assay (Experiment 6, conducted in January 2005)consisted of six cultivar Viradoro tomato seedlingstransplanted to pots (800ml capacity), in three replicates,totaling 90 pots containing inoculum and 12 pots withoutinoculum. The seedlings were placed around the fungusinoculum (2 g sclerotia per pot) located in the center of thepot. Water was supplied by drip irrigation. The plants weremaintained in an air-conditioned room at 20 1C, in order tofavor disease development. Total number of dead plantsand apothecia produced per pot was counted during 30days. Plant segments showing damping-off symptoms weretransferred to PDA medium for isolations of the pathogen.

2.1.4. Soil suppressiveness to Rhizoctonia

The soil of each plot from the experimental field wasdivided into two subsamples and placed in 1 l capacity pots.Only one of the subsamples received R. solani inoculum(4 g of wheat seeds colonized with the pathogen). The soilwas then moistened up to field capacity. After remainingfor 7 days in greenhouse, 50 radish seeds were sown perpot. Seedling emergence was evaluated 10 days aftersowing; fresh matter was evaluated 30 days later.

The assay was repeated twice, in February 2004 (Experi-ment 7) and August 2005 (Experiment 8).

2.1.5. Soil suppressiveness to Pythium

Cultivar caipira cucumber seeds were sown in pots (20seeds per pot) containing 1.5 l soil previously autoclaved at120 1C for 3 h. Two pots were prepared for each fieldexperimental plot. Four days after seedling emergence,200ml of soil collected in each plot treated with sewagesludge were mixed with 4 g of oatmeal to stimulate Pythium

development (Lourd et al., 1986) and distributed on the potsurface in contact with the root collars of plantlets. Thenumber of diseased plantlets was determined after 7 days.The assay was repeated twice, in February 2004 (Experi-ment 9) and April 2005 (Experiment 10).Pathogen recovery was also evaluated using potato cubes

as bait. Samples containing 500 g soil from each plot wereplaced in plastic bags, taken to the laboratory and sifted(2mm mesh) and then moistened to field capacity. Thesamples were maintained in the laboratory for 1 week andthen each sample received 50 potato cubes (4mm sides)that remained dipped for 1 h in a solution containing100 mg l�1 streptomycin sulfate and 20 mg l�1 benomyl. Thesamples were then oven-dried for 16 h at 3172 1C in openplastic bags. Upon retrieval from the soils, the potato cubeswere arranged in Petri dishes (15 cm diameter, 25 cubes perdish) containing agar-water medium added of 100 mg l�1

streptomycin sulfate and 20 mg l�1 benomyl. The number ofcubes containing mycelial growth characteristic of Pythium

spp. was annotated 24 h later (Experiment 11).

2.1.6. Soil suppressiveness to Ralstonia

Soil samples from each field experimental plot wereplaced in two 2 l capacity pots. The soil of each pot thenreceived a 200ml suspension of R. solanacearum containingapproximately 2� 106 cfuml�1. Five 30-day old tomatoseedlings, cultivar Santa Cruz VF5700, previously devel-oped on disinfested container medium, were then trans-planted into each pot. Seedling development was monitoredfor 30 days and the number of wilted plants was recorded.Re-isolations were conducted to confirm the presence of thepathogen. The assay was repeated twice, in April (Experi-ment 12) and November 2004 (Experiment 13).

2.1.7. Statistical analysis

The effect of sludge doses and infestation levels, for eachtype of sludge, was evaluated via analysis of variance andregression methods (po0.05), using the MIXED procedureof the SAS System (SAS, 1993). Linear or quadratic modelswere fitted to describe the effect of sludge dose increases onresponse variables of interest (for example: rating, drymatter, and others). In cases where adequate model fittingwas obtained (correlation coefficients significant), thecorresponding models were plotted in graphs showing theinfluence of sludge dose on the response of interest. Forthose situations where sludge influence did not followed

ARTICLE IN PRESSR. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–28052800

regular patterns, scatterplots were used to present theresults.

3. Results

In the F. oxysporum f. sp. lycopersici assays, an increasein dry matter production of tomato plants was observed assludge doses increased (F-test, po0.01), for all pathogeninfestation levels. In most cases, the relationship was linear,demonstrating that waste incorporation can be beneficialto plant development (data not presented). In general, noeffect of dose or type of sludge on disease severity wasobserved (Fig. 1).

With respect to suppressiveness to S. rolfsii, the severityof disease showed a quadratic response to the increase of

Barueri

0

2

4

6

0

2

4

6

0

2

4

6

0 3 4 5 6

No

tes

0 5 6 7

0 5 6 7

Sewage slud

0 1 2 4 8

0 1 2 4 8

0 1 2 4 8

A

C

E

Fig. 1. Effect of Franca and Barueri sewage sludge doses on the severity of Fu

6 ¼ dead plant), at different inoculum concentrations (0 ¼ control witho

5 ¼ 105 conidia ml�1 soil, 6 ¼ 106 conidia ml�1 soil, and 7 ¼ 107 conidia ml�1

1N, 2N, 4N, and 8N ¼ sludge doses required to provide zero, one, two, four,

sludge dose, with inflection points at 2N and 4N forBarueri and Franca sludge, respectively (Fig. 2, Experi-ment 4). There was a tendency for reduction in severity ofdisease in plants grown at higher sludge concentrations(Fig. 2, Experiment 5). No dose effect was observed ondisease severity for the Barueri sewage sludge in Experi-ment 5. However, plant toxicity symptoms were observedfor higher applied sludge doses, resulting in smaller plants,foliage distortion and other malformations (data notpresented).In the S. sclerotiorum assays, the incidence of dead plants

was directly proportional to the sludge doses applied(Fig. 3). The Franca sludge did not have an effect on theproduction of apothecia; however, a quadratic responsewas obtained with the application of Barueri sludge,

Franca

0

2

4

6

0

2

4

6

0

2

4

6

0 3 4 5 6

0 5 6 7

0 5 6 7

ge doses (N*)

0 1 2 4 8

0 1 2 4 8

0 1 2 4 8

B

D

F

sarium oxysporum f. sp. lycopersici in tomato (notes: 1 ¼ healthy plant to

ut inoculation, 3 ¼ 103 conidia ml�1 soil, 4 ¼ 104 conidia ml�1 soil,

soil), in Experiments 1 (A and B), 2 (C and D), and 3 (E and F). *0N,

and eight times the amount of mineral fertilization N.

ARTICLE IN PRESS

Experiment 4 Experiment 5

Note

s

Barueri

y = –0.0759x2 + 0.3383x + 4.8627

R2 = 0.5072

Franca

y = –0.0974x2 + 0.744x + 3.8477

R2 = 0.6022

Franca

y = 0.0634x2 – 0.7836x + 5.03

R2 = 0.8745

0

1

2

3

4

5

6

7

8

0 2 4 80

1

2

3

4

5

6

Sewage sludge doses (N*)

1 0 2 4 81

Fig. 2. Effect of Franca (’——) and Barueri (J– – –) sewage sludge doses on the severity of Sclerotium rolfsii in bean plants (notes: 1 ¼ no symptoms to

9 ¼ approximately 75% or more of the tissues covered by lesions, with severe reduction of the root system), in Experiments 4 and 5. *0N, 1N, 2N, 4N, and

8N ¼ sludge doses required to provide zero, one, two, four, and eight times the amount of mineral fertilization N.

Barueri

y = –0.2419x2 + 1.771x + 22.8

R2 = 0.6987

15

17

19

21

23

25

27

29

0 1 2 4 8 0 1 2 4 8

Ap

oth

ecia

pe

r p

ot

Franca

y = –0.0571x2 + 2.0208x + 15.908

R2 = 0.6538

Barueri

y = –0.0496x2 + 1.4094x + 18.815

R2 = 0.9039

0

5

10

15

20

25

30

De

ad

pla

nts

Sewage sludge doses (N*)

Fig. 3. Effect of Franca (’——) and Barueri (J– – –) sewage sludge doses on the production of Sclerotinia sclerotiorum apothecia and number of dead

tomato plants (Experiment 6). *0N, 1N, 2N, 4N, and 8N ¼ sludge doses required to provide zero, one, two, four, and eight times the amount of mineral

fertilization N.

R. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–2805 2801

differing from the result obtained when the number of deadplants was evaluated.

In Experiment 7, conducted with R. solani for soils thatreceived Franca sewage sludge, the emergence and devel-opment of radish seedlings, in the presence or absence ofthe pathogen, were proportional to the amounts of sludgeapplied, with a linear response. For Barueri sludge, therewas a quadratic response with an inflection point at 4N foremergence and fresh matter in plots containing R. solani.There was a linear response in plots without the pathogen(Fig. 4). In the second experiment (Experiment 8), nosludge application effect was observed with respect to plantemergence. Nevertheless, the results showed an increase inplant development directly proportional to the sludge doseapplied. Fresh matter responses were linear for Baruerisludge with or without the pathogen; for Franca sludge, theresponse was linear in the presence of the pathogen, andquadratic with inflection at 4N in its absence (Fig. 4).

With regard to Pythium in the bait test, there was anincrease in pathogen population directly proportional tothe sewage sludge dose applied (Fig. 5C). Populations werehigher in soils that received Franca sludge. The percentageof dead plants was directly proportional to sludge doses(Figs. 5A and B), and the responses in both assays werelinear for the Franca sludge and quadratic for the Baruerisludge, with an inflection point at the 4N dose.

An increase in the percentage of wilted plants, caused byR. solanacearum, was observed in Experiment 12 up to the4N dose for the Franca sludge. At the 8N dose, there was areduction in the percentage of wilted plants, similar to thesoil without sludge (Fig. 6). In Experiment 13, a reductionwas observed in the number of wilted plants, inverselyproportional to the sludge doses applied (Fig. 6).

4. Discussion

Introduction of sewage sludge had a pathogen species-specific effect on the soil suppressiveness. The absence ofeffects observed with F. oxysporum f. sp. lycopersici

differed from previous results obtained in the sameexperimental field from which the samples were taken.Bettiol (2003) observed that increased doses of both typesof sludge resulted in increased incidence of corn stalk rotcaused by Fusarium. In both studies, positive correlationswere observed between sludge concentrations and electricalconductivity and negative correlations were observed withpH (data not presented). The reduction in pH values in thesoil solution is due to the release of N-NH4

+ during thesludge mineralization process in the soil, and the highN-NH4

+ contents could indicate a greater release of H+ tothe medium, promoting acidification (Epstein, 2003;Evangelou, 1998). Electrical conductivity increased in

ARTICLE IN PRESS

Experiment 7 Experiment 8

Em

erg

en

ce

(%

)

Barueri

y = –1.7343x2 + 17.336x + 21.277

R2 = 0.8502

Francay = 3.5152x + 31.479

R2 = 0.76370

10

20

30

40

50

60

70

0 1 2 4 8 0 1 2 4 8

0 1 2 4 8

0 1 2 4 8

0 1 2 4 8

0 1 2 4 8

A

0

10

20

30

40

50

60

70B

Without R. solaniy = 1.4708x + 21.294

R2 = 0.5097

With R. solani

y = 1.8053x + 6.3407

R2 = 0.65160

10

20

30

40With R. solani

y = 0.8893x + 14.786

R2 = 0.6954

Without R. solani

y = –0.2738x2 + 2.6131x + 10.553

R2 = 0.9152

0

4

8

12

16

20

24

Fre

sh

ma

tte

r (g

)

With R. solani

y = –0.7926x2 + 7.3402x + 7.8695

R2 = 0.7216

Without R. solani

y = 2.855x + 21.768

R2 = 0.8005

0

10

20

30

40

50

Without R. solani

y = 0.8714x + 11.532

R2 = 0.7489

0

4

8

12

16

20

24

Sewage sludge doses (N*)

C

E

D

F

Fig. 4. Effect of Franca (’——) and Barueri (J– – –) sewage sludge doses on the emergence of radish seedlings in Experiments 7 (A) and 8 (B) and on the

fresh matter of plants grown in soils treated with different Franca (C ¼ Experiment 7 and D ¼ Experiment 8) or Barueri (E ¼ Experiment 7 and

F ¼ Experiment 8) sewage sludge doses, with (- - - -) or without (——) the addition of Rhizoctonia solani. *0N, 1N, 2N, 4N, and 8N ¼ sludge doses required

to provide zero, one, two, four, and eight times the amount of mineral fertilization N.

R. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–28052802

response to increases in the amounts of sludge incorpo-rated. The differences between these results could be due todifferences in Fusarium species involved and alterations insoil properties due to the experimental set up using fieldplots versus potted soil from field plots. Cotxarrera et al.(2002) observed that a container medium produced withsewage sludge compost and plant and animal residues wassuppressive to F. oxysporum f. sp. lycopersici. This could beexplained by the contents and quality of the organic matterin the container medium, as well as by other characteristics,considerably different from those occurring in the presentwork.

With respect to S. rolfsii, there was a tendency for reductionin severity of disease in beans grown at higher sludgeconcentrations, which is in agreement with data by Santosand Betttiol (2003) (Fig. 2, Experiment 5). The plant toxicitysymptoms observed suggested a toxic effect due to the presenceof heavy metals. In the same experimental field from which thesamples were taken, Silva et al. (2006) observed that the totalcontents of heavy metals in soil after three applications of8N dose of Barueri sludge were Cu ¼ 61.1mgkg�1,Ni ¼ 18.3mgkg�1, and Zn ¼ 89.0mgkg�1, while in controltreatment were Cu ¼ 15.7mgkg�1, Ni ¼ 7.6mgkg�1, and

Zn ¼ 14.0mgkg�1. Also Rangel et al. (2006) observed thatsuccessive applications of sewage sludge caused increases in Znand Mn contents in maize leaves: the dose 8N of Baruerisewage sludge promoted an increment of up to 270% of Zn,and 35% of Mn concentration, in relation to the controltreatment.The data obtained for S. sclerotiorum differ from those

obtained by Lumsden et al. (1983, 1986) and Millneret al.(1982), who observed that sludge induced suppres-siveness to this pathogen. Possibly, this difference in resultswas due to the methodology employed in the present study,in which sclerotia were added just a few centimeters fromthe plants. Another important aspect is that in the studieswhere suppressiveness to Sclerotinia occurred, the sludgewas composted before application to the soil and, inaddition, higher doses were used.In this study, there was considerable variation in the

influence of sewage sludge on suppressiveness to R. solani.Others have found variation depending on the methodused. Kuter et al. (1988) observed that sludge eitherdecreased or increased R. solani depending on the age ofthe container medium produced. On the other hand,Millner et al. (1982) observed that container medium

ARTICLE IN PRESS

Franca

y = 9.669x – 3.6719

R2 = 0.9733

Barueri

y = –1.5772x2 + 14.441x – 4.3383

R2 = 0.8158

0

10

20

30

40

50

60

70

80

90

0 4 8

De

ad

pla

nts

(%

)

0

10

20

30

40

50

60

70

80

90

De

ad

pla

nts

(%

)

0

10

20

30

40

50

60

70

80

90

Baits w

ith P

yth

ium

spp.

(%)

A

Franca

y = 9.33x + 0.25

R2 = 0.8975

Barueri

y = –1.3217x2 + 11.957x + 2.6671

R2 = 0.5295

B

Franca

y = 5.65x + 35.85

R2 = 0.8298

Barueri

y = 2.3493x + 19.748

R2 = 0.8586

C

Sewage sludge doses (N*)

1 2

0 4 81 2

0 4 81 2

Fig. 5. Percentage of dead cucumber seedlings by post-emergence

damping-off caused by Pythium spp. in soils treated with different Franca

(’——) or Barueri (J– – –) sewage sludge doses, in Experiments 9 (A) and

10 (B), and Pythium spp. recovery using potato cubes as baits

(C) (Experiment 11). *0N, 1N, 2N, 4N, and 8N ¼ sludge doses required

to provide zero, one, two, four, and eight times the amount of mineral

fertilization N.

R. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–2805 2803

produced with sewage sludge controlled damping-offcaused by this pathogen in bean, cotton, and radish undergreenhouse conditions. However, in the field, these authors

confirmed that sludge was effective in controlling thepathogen in pea, but not in cotton. Most of the studies thathave demonstrated the potential of sewage sludge forinducing suppressiveness to R. solani were carried out usingcontainer medium and higher doses of the residue thanthose incorporated into the soil in the present study. Theresults obtained in the present work could also be due tothe time in which the assays were installed, in relation tothe sludge applications: the first and the second wereinstalled 2 months and 8 months after application of theresidue, respectively.

R. solani suppressiveness tests were also conducted usingmycelial growth in soils contained in Petri dishes, followinga methodology similar to that used by Grunwald et al.(1997) and the bait method described by Huang andKuhlman (1991). There were no treatment effects with thebait method. In the mycelial growth test, a quadraticresponse was observed with an inflection point at 4N forthe Franca sludge. No effects on pathogen growth weredetected for the Barueri sludge (data not presented).Lumsden et al. (1983) and Tuitert et al. (1998) observed

that sewage sludge stimulated suppressiveness to R. solani.However, these authors used composted sewage sludge, inwhich the C:N ratio was relatively higher than those usedin this work (C:N of Franca sludge ¼ 6.4; Baruerisludge ¼ 7.6). This is important since Fenille and Souza(1999) demonstrated a difference in behavior betweenorganic matter sources with different C:N ratios in induc-ing suppressiveness to R. solani. Bailey and Lazarovits(2003) discussed that ammonia liberation following appli-cation of high-N organic amendments is responsible forcontrol pathogens in soil. This may explain why theseamendments with low C:N ratio (o10) are most oftenfound to suppress plant diseases.The observed increase in Pythium populations was

probably responsible for the increase in seedling damp-ing-off. Since damping-off in control plots was rarelyobserved, it is likely that sludge applied to the soilstimulated native pathogen populations and/or introducednew Pythium propagules. These propagules, if present inthe sludge, could have survived during activated-sludgeprocess. Pythium aphanidermatum presents a relativetolerance to high temperatures since its oospores cansurvive for 30min at temperatures as high as 52.5 1C(Bollen, 1985). Indeed, Lumsden et al. (1983) observed thatthe survival of P. aphanidermatum oospores was higher insoil containing sludge (87%) than in soil without thisresidue (17%), after 2 weeks of incubation. As that periodincreased (after 4 months of incubation), such differenceswere no longer observed. However, the authors observedthat the P. aphanidermatum propagules seemed to beprotected in the soil containing sludge. Also Ghini et al.(2002) did not observe any sewage sludge effect in inducingsuppressiveness to P. aphanidermatum.Although there have been few studies of the effect of

incorporation of organic residues on soil suppressiveness toR. solanacearum, a relative reduction in the incidence of

ARTICLE IN PRESS

Experiment 12 Experiment 13

Dis

eased p

lant (%

) Franca

y = –2.7581x2 + 21.629x + 65.8

R2 = 0.7882

0

20

40

60

80

100

120

1 2 4

A

Francay = –5.1595x + 84.719

R2 = 0.8426

Barueriy = –3.3748x + 86.124

R2 = 0.7079

0

20

40

60

80

100

120

B

Sewage sludge doses (N*)

0 8 1 2 40 8

Fig. 6. Percentage of cv. Santa Clara tomato plants with wilting caused by Ralstonia solanacearum in soils treated with different amounts of Franca

(’——) or Barueri (J– – –) sewage sludge, in Experiment 12 (April 2004) and Experiment 13 (November 2004). *0N, 1N, 2N, 4N, and 8N ¼ sludge doses

required to provide zero, one, two, four, and eight times the amount of mineral fertilization N.

R. Ghini et al. / Soil Biology & Biochemistry 39 (2007) 2797–28052804

bacterial wilt has been observed in sites where organicresidues (such as chicken manure and others) were added(Hartman and Elphinstone, 1994). Natural suppressivenessto R. solanacearum was observed in some soils (French,1994), associated with antagonistic rhizobacteria of thefluorescent pseudomonas group (Hayward, 1991; Trigaletet al., 1994) and other species such as Burkholderia glumae

and B. cepacia, in addition to bacteria of the generaBacillus and Erwinia (Trigalet et al., 1994). Populations ofsome of these microorganisms may have increased withtime in the soils treated with sludge, causing a reduction inthe incidence of the disease.

In the present study, the results varied depending on themethodology applied and on the time interval between thesewage sludge incorporation and soil sampling. In general,both types of sewage sludge increased suppressiveness ofthe soil to disease caused by S. rolfsii, R. solani andR. solanacearum, but decreased suppressiveness to thosecaused by S. sclerotiorum and Pythium spp., and had noeffect on disease associated with Fusarium oxysporum f. sp.lycopersici. The effects of sewage sludge on plant diseasesshould be studied further, since the production of this typeof waste, whose final disposal is mainly in agriculture, iscontinuously increasing in Brazil and worldwide.

Acknowledgments

This research was supported by CNPq (NationalCouncil for Scientific and Technological Development,Project 473385/03-1).

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