Ptant Pathotogy (1995) 44, 250-260

Control of Verticillium wilt of cotton by means of soil solarizationand tolerant cultivars in southern Spain

J. M. MELERO-VARA, M. A. BLANCO-LOPEZ, J. BEJARANO-ALCAZAR^ andR. M. JIMENEZ-DIAZInstituto de Agricuttura Sostenibte, CSIC, Apdo. 4084, 14080 Cordoba", Departamento de Agronomia,ETSIAM, Universidad de Cordoba, and ^Departamento de Proteccion Vegetal, CIDA Cordoba, Apdo.4240, 14080 Cordoba, Spain

Eight field experiments (I-VIII) were conducted in clay soils naturally infested with a cotton-defoliating pathotype of Verticillium dahliae in the lower Guadalquivir Valley of Andalucia, southernSpain, during the period 1986-90. Experiments I-VI aimed to determine the eflBcacy of soil solarizationin reducing populations of the pathogen in soil and eventually contributing to the control ofVerticittium wilt of cotton. The population of V. dahliae in the 0- to 40-cm soil layer was reduced toundetectable or very low levels after solarization for 6 to 10 weeks. The final incidence of Verticilliumwilt in the cotton crop following solarization was reduced to 13% or less in solarized plots, compared to55-90-5% in unsolarized controls. The onset of disease incidence in the solarized plots was delayed by2-7 weeks, increased at a lower rate, and had a smaller area under the disease progress ctirve, comparedto that in unsolarized plots. Seed cotton yields in solarized plots increased by 11-3-130-9% dependingupon experiments, cultivars and years. Experiments VII and VIII aimed to determine the use of thehighly wilt-tolerant cotton cv. Acala GC 510 for control of the disease that had been cropped tosusceptible cotton cultivars the year before in solarized soils. Solarized or unsolarized plots were firstsown to susceptible cotton cvs Acala SJ2 and Coker 310, and the following year were sown to cv. AcalaGC 510. The inoculum density of V. dahliae at the time of sowing cv. Acala GC 510 in previotislysolarized plots had increased to moderate levels, but remained considerably lower than that inunsolarized plots. The final disease incidence in cv. Acala GC 510 grown in unsolarized plots was lowerthan that in susceptible cultivars grown in the same plots the year before. Furthermore, the diseaseincidence in cv. Acala GC 510 grown in solarized plots was as high as that in susceptible cultivars grownthe year before with much less initial inoculum.

INTRODUCTION

Approximately 70 000-90 000 ha of cotton(Gossypium hirsutum) are grown annually inSpain. About 90-95% is planted in the Guadal-quivir Valley of Andalucia in the southern partof the country, of which 35 000-45 000 ha aregrown intensively in the clay, fertile soils of theLower Guadalquivir Valley (Anonymous, 1992).

I'l'rticiltium wilt, caused by Verticillium dah-liae, is the most important disease of cotton \nSpain. The disease occurs in 74 82° o of the fieldsthroughout the Guadalquivir Valley, with adisease incidence range of 15 26°o (Bianco-Lopez et al, 1989). Disease incidence andseverity are highest in the Lower Guadalquivir

Valley, and occurrence is associated with highinoculum densities of a cotton-defoliatingpathotyp)e of V. dahliae (Blanco-Lopez et al.,1989). This defoliating pathotype, which wasfirst identified in the Lower Valley in 1983(Blanco-Lopez et al, 1986), has since spreadthroughout this area. The occurrence of cotton-defoliating pathotypes of V. dahliae has beenshown in Mexico, Peru and the USA, but thenature of the disease symptoms observed incotton crops in other regions suggests that theymight also be present elsewhere (Mathre et al.,1966; Schnathorst, 1969; Kannan & Srinivasan,1984).

Verticilliutn wilt of cotton can be controlled byreducing inoculum density and or the etticacs of

Control 0/Verticillium wilt of cotton 251

the initial inoculum. Several control measureshave been shown to be partially effective, butonly integrated management can effectively con-trol the disease (El-Zik, 1985). Crop rotation withnon-host crops is unlikely to reduce high levels ofinoculum density in the soil to a level sufficient fordisease control, because of the length of rotationrequired (Huisman & Ashworth, 1976) and foreconomic reasons. Furthermore, the residualinoculum of the defoliating pathotype, even atlow levels, can cause severe Verticillium wilt insusceptible cotton cultivars (Bianco-Lopez et ai,1989). Tolerant cotton cultivars are known towithstand a lower rate of development of V.dahliae within infected plants, but they seem toexert positive selection for highly virulent isolatesof the pathogen (Ashworth, 1983). V. dahliae insoil can be controlled by soil fumigation andsolarization. Soil solarization has successfullycontrolled V. dahliae in soil at different depths inseveral areas (Katan et ai, 1976; Pullman et ai,1981a; Ben-Yephet et ai, 1988; Davis, 1990). InAndaluda, soil solarization is economicallyfeasible and it has been practised successfully bygrowers for control of Fusarium wilt of water-melon (Gonzalez-Torres et ai, 1993) and Verti-cillium wilt of cotton (Melero-Vara et ai,unpublished data; Alvarado & Duran, 1992).Soil solarization is an ecologically sound controlmeasure that reduces recolonization risks, and hasbeneficial effects on soil properties and plantgrowth beyond the control of plant pathogens,weeds and insects. The principles and achieve-ments of soil solarization have been thoroughlysummarized (see, for example, Katan, 1981,1987;Stapleton et ai, 1985; Stapleton & DeVay, 1986).Tolerance of Verticillium wilt has been reported insome Upland cotton cultivars, derived mostlyfrom G. barbadense and G. darwinii (El Zik, 1985).However, most Acala cultivars that are tolerant ofmildly virulent pathotypes of V. dahliae are notuseful at the high inoculum levels of the defoliatingpathotype that occur in the Lower GuadalquivirValley of Andalucia (Blanco-Lopez et ai, 1989;Bejarano-Alcazar, 1990). Recently, new cottoncultivars have been developed, such as AcalaGC510, which are highly tolerant of the disease infields with a high inocultim density of unspecifiedpathotypes of V. dahliae (Paplomatas et ai, 1992).These cultivars might be useful for control ofVerticillium wilt of cotton under the conditionsthat prevail for cotton crops in the LowerGuadalquivir Valley.

The objective of̂ this work was to determinethe effectiveness of soil solarization, alone and in

combination with tolerant cultivars, for controlof Verticillium wilt of cotton in two consecutivecotton crops following solarization in the LowerGuadalquivir Valley of southern Spain. Preli-minary reports of part of this work have beenpublished elsewhere (Jimenez-Diaz et ai, 1991;Bianco-Lopez et al, 1992).

MATERIALS AND METHODS

Eight field experiments (I-VIII) were carried outin vertic soils (c. 61% clay, 0 9 - 1 4%> organicmatter, pH 8 3-8 8) in the Lower GuadalquivirValley, southern Spain, during the period 1986-90. The soil in these fields was naturally infestedwith a defoliating pathotype of V. dahliae, asindicated by the disease reaction of cotton PI 70-110 and CVS Acala SJ5 and Acala SJC-1 toartificial inoculation with a sample of mono-conidial isolates of the pathogen obtained fromaffected cotton, and/or the severe defoliation anddeath of infected cotton plants observed inprevious years (Bianco-Lopez et ai, 1989;Bejarano-Alcazar, 1990). Seedbed preparation,fertilization and irrigation were performed inaccordance with farmers' practices.

Effect of soil solarization on Verticillium wilt ina cotton eultivar highly susceptible to the disease

All experiments consisted of two treatments (soilsolarized or unsolarized) and were conductedaccording to a randomized complete block design.Experiments I and II were carried out during theperiod 1986-87 and consisted of six replicate plots10m long and 6 or 4 5m wide, respectively. Twoadditional experiments (III and IV) were conductedduring the period 1987-88 in order to confirm theresults of the previous experiments and to obtaininformation about the practicality and efficacy ofsoil solarization by cotton growers. Therefore, tworeplications of larger plots (15 - 30 m long and 6 5mwide) were used.

In all cases, the soil was thoroughly diskedafter harvest of the previous wheat crop, thenrotovated and furrow irrigated to field capacityin the upper 30-40 cm layer. Plots to be solarizedwere covered, 1-2 days after irrigation, withtransparent 6 5-m-wide, 37 5-^m-thick (experi-ments I, HI and IV) or 25-/jm-thick (experimentII) polyethylene sheets. Plots were solarized from17 July to 31 August in 1986. In 1987 solarizationwas extended from 17 July to 30 Septemberbecause the weather had been cloudy and rainy,with lower temperatures than usual during theearly solarization period.

252 J. M. Melero-Vara et al.

After solarization was complete, the polyethy-lene sheets were removed and plots remained inplace until early spring of the year following thatof solarization. Plots were then disked lengthwiseto reduce mixing of soil and sown with cotton cv,Coker 310, highly susceptible to V. dahtiae, inlate March 1987 (experiments I and II) and earlyApril 1988 (experiments III and IV), Plotsconsisted of four (experiment II) or six (experi-ments I, III and IV) rows 0-95 m apart with plantstand density adjusted to ca, 120000 plants/ha.In all experiments the increase in diseaseincidence, expressed as the percentage of plantsshowing foliar symptoms characteristic of Verti-cillium wilt (Schnathorst, 1981; Bianco-Lopez etal, 1989), was determined over time in the two(experiment II) or four (experiment I, III and IV)central rows of each plot at approximately 2-week intervals from June to September, Finalpercentage values were transformed into arcsin(y/100)''^ for analyses of variance. Plots wereharvested by hand-picking and seed cotton yieldswere determined for experiments II-IV. Ana-lyses of variance were made and treatment meanswere compared using Fisher's protected leastsignificant difference (LSD) at P = 0-05,

Effect of soil solarization on epidemiccomponents of Verticillium wilt and yield insusceptible cotton cultivars

Two experiments (V and VI) were conductedduring the period 1988-89 with four treatments,as follows: (1) solarized soil sown to cv. Coker310; (2) solarized soil sown to moderatelysusceptible cv, Acala SJ2 (Ashworth, 1983); (3)unsolarized soil sown to cv. Coker 310; and (4)unsolarized soil sown to cv. Acala SJ2. Theexperiments were conducted according to a split-plot treatment design within randomized com-plete blocks, with soil treatment and cultivars asthe main plot and subplot treatments, respect-ively. There were three or four blocks, respect-ively, for experiments VI and V. Soilpreparation, irrigation and plastic tarping wereperformed as for the previous experiments I-IVusing 37-5-/im-thick transparent polyethylenesheets. The main plots in experiments V(6 X 100 m) and VI (6x85m) were solarizedfrom 12 July to 2 September, and from 26 Julyto 16 September 1988, respectively. Aftersolarization was complete, the polyethylenesheets were removed and plots remained inplace until March 1989, The main plots werethen divided into two halves across their longest

side so that two subplots (6 x 50 m for experimentV and 6 X 42-5 m for experiment VI) were obtaitiedfrom each main plot, atid cultivars were randomlyallocated to them. Plots were sown in mid-March1989, and consisted of six rows 0-95 m apart andeither 50 m (experiment V) or 42-5 m (experimentVI) long, with a stand adjusted to 88 x 10̂ and156 X 10̂ plants/ha, respectively.

For both experiments the increase in diseaseincidence, expressed as the percentage of plantsshowing foliar symptoms characteristic of Verti-cillium wilt, was determined over time in the twocentral rows of each plot at 2-week intervals fromJune to September 1989, and the area under thedisease progress curve (AUDPC) was calculated.Yields were determined by combine-harvestingthe four central rows of each plot. Afterwards, 50plants per plot were randomly selected and cut4-6 cm above the soil level to determine theincidence (percentage) of plants showing vascu-lar discoloration. Disease incidence values overtime for each treatment were transformed by In[ l / ( l -y ) ] (VanderPlank, 1963), Incidence valuesof 0 or 100% disease were not included in thesetransformations. Linear regression analyses wereperformed with transformed and non-trans-formed data. The coefiJcients of determination{^) and distribution patterns of residualsagainst expected values were used to indicateappropriateness and to select the best regressionmodel (Daniels & Wood, 1980), Comparisons oflinear regressions were made in order to detectthe infiuence of soil treatment and cultivar usedon the increase in disease incidence. The finalincidence of foliar symptoms was transformedinto arcsin (K/lOO)'̂ ^ and, together with theAUDPC and yield data, was subjected toanalysis of variance. Treatment means werecompared using Fisher's protected LSD atP = 0-05,

Combined effect of soil solarization and cultivaron Verticillium wilt of cotton

At the end of March 1990, plou from experi-ments V and VI, which had remained untreatedsince harvest in September 1989, were sown tocotton cv, Acala GC510, which is highly tolerantof (erticiltium wilt (Paplomatas etal.,\ 992>, at astand of 157x10^ and 215x10^ plants/ha,respectively. Thus plots from experiments Vand VI became plots for experiments VII atidVlll, respectively. The final incidence of foliarsymptoms was determined in the two centralrows of each plot at the end of September 1990

Control o/Verticillium wilt of cotton 253

Table 1. The effect of soil solarization on the population of Verticillium dahtiae in soil, development of Verticittiumwilt and seed cotton yield of cotton cv Coker 310 during the period 1986-87

Soil treatment'

Experiment IUnsolarizedSolarized

Experiment IIUnsolarizedSolarized

Inoculum density (propagules/g dry soil)'

Before treatment

mean

233273

2418

CV(%)

879683

852854

After treatment

mean

16100

2803

CV(%)

1255

9641673

After sowing

mean

513NA'

6101

CV(%)

356NA

47-22449

Final incidenceof foliarsymptoms'

(%)

90 5 a50 b

60-1 a4-5 b

Seed cottonyield (kg/ha)"

NANA

2093 a4833 b

'Soil was solarized from 17 July to 31 August 1986.•" Determined by means of an Andersen sampler in four 500-mg aliquots of soil collected from the 0-20-cm soil layerof each of six replicate plots immediately before and after solarization. Samples of soil the year after solarizationwere collected in early May, about 1 month after sowing. CV = coefficient of variation.'Determined from plants in the four (experiment I) and two (experiment 11) central rows of each plot that wereaffected by symptoms characteristic of Verticittium wilt at the end of September, 23 weeks after sowing. For eachexperiment, values followed by the same letter are not significantly different (P = 0-05) according to LSD test.^ Determined by hand-picking all plants in the four central rows of each plot. Average of six replicate plots. Datawere not available for experiment I.'Not determined.

for both experiments. Plots were harvested as inthe previous year, and seed cotton yields weredetermined. Afterwards, 50 consecutive plantsper plot were selected to determine the incidence(percentage) of plants showing vascular disco-loration, as described for experiments V and VI.Analysis of variance and mean comparisons wereperformed on the data.

Estimation of Inoeulum density of V. dahliae inexperimental plots

The populations of V. dahliae in solarized andunsolarized soil of experiments I-VIII wereassessed immediately before and after solariza-tion, and about 1 month afler sowing either 1 year(experiments I-VI) or 2 years (experiments VII andVIII) foUowing that of solarization. Dependingupon the plot size, seven or 12 soil samples (200 g)were collected with an Edelman sampler (Eijkelk-amp, Giesbeek, The Netherlands) to a depth of 0 -20 cm (experiments I-fV, and experiments VII andVIII) or 0-20 and 20-40 cm (experiments V andVI) at equally spaced sites in the plot. The soilsamples fVom each plot were bulked, thoroughlymixed and air dried at room temperature for 4-6weeks. The soil was then crumbled by hand andwith a wooden hammer, and milled in a rotatingdrum with steel cylinders for 20 min. Four aliquots

of 500 mg each were processed per plot. Eachaliquot was divided into five 100-mg subsamplesand each subsample was plated on to a Na-polypectate semi-selective medium (Butterworth& DeVay, 1977) by means of an Andersen sampler(Andersen Sampler Inc., Atlanta, GA, USA).Plates were incubated at 25 C in the dark for 12-14 days, and were then washed free from soil withtap water. The ftingal colotiies growing on the semi-selective medium were observed with a stereoscopeat X 15 magnification. Colonies of K dahliae wereidentified on the basis of microsclerotia formed inthe medium (Butterfield & DeVay, 1977). Thenumber of colonies of V. dahliae formed from each500-mg aliquot was determined. Estimates of V.dahliae populations in solarized and unsolarizedsoils showed high variability and consequently werenot subjected to analyses of variance; instead, thestandard deviation and coefficient of variation ofestimates were calculated.

RESULTS

Effect of soil solarization on Verticillium wilt ina cotton cultivar highly susceptible to the disease

Maximum and minimum daily air temperatures

254 J. M, Melero-Vara et al.Table 2, The effect of soil solarization on the population of Verticillium dahliae in soil, development of Verticilliumwilt and seed cotton yield of cotton cv. Coker 310 during the period 1987-88

Soil treatment"

Experiment IIIUnsolarizedSolarized

Experiment IVUnsolarizedSolarized

before

mean

20-820-0

3-03-5

Inoculum density (propagules/g dry soil)*"

treatment

CV(%)

42-610-6

23-660-6

after treatment

mean CV(%)

68 3 46-10-0 —

34-3 108-40-0 —

after sowing

mean

28-81-5

1530-3

CV(%)

20-994-3

118-2141-4

Mean final incidenceof foliar

symptoms(%)''

87-7 a13-5 a

55-0 a1-8 a

Mean seedcotton yield

(kg/ha)"

1562 a3279 b

3425 a4575 b

"Soil was solarized from 17 July to 30 September 1987." Determined by means of an Andersen sampler in four 500-mg aliquots of soil collected from the 0-20-cm soil layerof each of two replicate plots immediately before and after solarization. Samples of soil the year after solarizationwere collected in early May, about 1 month after sowing. CV = coefficient of variation.' Determined from plants in the four central rows of each plot that were affected by symptoms characteristic ofI erticillium wilt at the end of September, 23 weeks after sowing. For each experiment, values followed by the sameletter are not significantly different {P = 0-05) according to LSD test.'' Determined by hand-picking all plants in the four central rows of each plot. Average of two replicate plots. Foreach experiment, values followed by the same letter are not significantly different according to LSd test.

at the experimental sites during the solarizationperiod were in the ranges 26-39°C and 13-20 C,respectively, in 1986, and 24-42 C and 14-21°Cin 1987.

The estimates of the population level of V.dahliae in soil by mid-July, before solarization,indicated that plots of experiments I and III, andthose of experiments II and IV, were heavily(>20 propagules/g dry soil) and slightly (<5propagules/g dry soil) infested with the patho-gen, respectively (Tables 1 and 2). Soil solariza-tion of these plots over a period of 6 weeks in1986 and 10 weeks in 1987 brought about areduction in the V. dahliae population toundetectable levels in the 0 20 cm soil layer inexperiments 1-IV (Tables 1 and 2). By contrast,the inoculum density of '̂. dahliae in unsolarizedsoil had either increased or remained at the samelevel during the period of solarization (Tables 1and 2). Although some soil mixing might haveoccurred during the 6-month period that elapsedbetween removal of the polyethylene sheets andsowing, the inoculum density of V. dahtiae in theMill of solarized plots was still very low at thetime of seedling emergence (Tables 1 and 2).

The reduction in the V. dahtiae population insolarized soil was associated with a highlysignificant degree of control of the disease. The

final incidence of disease in solarized plots was5% or less, except for experiment III (13-5%), incontrast to the high disease incidence occtirringin unsolarized plots, which ranged from 55 to90-5% (Tables 1 and 2). Although the meanvalues of final disease incidence in solarized andunsolarized plots for experiments III and IV werequite different (Table 2), the di£ference was notstatistically sigtuficant as revealed by analysis ofvariance. This was possibly due to the lownumber of replicates (two) in those experi-ments. The levels of disease control achieved hysoil solarization were reflected by significantincreases in the seed cotton yield of cv. Coker310, dependent upon year and experiment(Tables I and 2). Thus, while the seed cottonyield in solarized plots was increased by 131V>and 110% of that in unsolarized plou inexperiments II and III, respectively, there wasonly a 34% increase in experiment IV for whichthe yield in unsolahzed plots was 1-6-2 2 timesthat in experiments II and III.

Effect of soil solarization on epkleniiccomponents of VerticilKtun wilt and yield insusceptible cotton cultivars

Maximum and minimum daily air temperatures

Control o/Verticillium wilt of cotton 255

Table 3. The effect of soil solarization on inoculum of Verticillium dahliae in soil during the period 1988-89

Soil treatment'

Experiment VUnsolarized

Solarized

Experiment VIUnsolarized

Solarized

Soil layer(cm)

0-2020-40

0-2020-40

0-2020-40

0-2020-40

Inoculum density (propagules/g dry soil)"

before treatment

mean

39 519-4

42-138-3

64-022-0

90227-8

CV(%)^

57-937-1

304414

8-937-8

28-032-2

after treatment

mean

71-647-9

0-00-5

79-9106-0

0-00-2

CV(%)

59-951-3

200-0

18-620-4

173-2

after

mean

39-015-2

2-01-7

34-05-7

120-8

sowing

CV(%)

32-721-9

79-130-0

78-841-3

89-247-1

'Soil was solarized from 12 July to 2 September and from 26 July to 16 September 1988. for experiments V and VI.respectively."Determined by means of an Andersen sampler in four 500-mg aliquots of soil collected from each of four(experiment V) or three (experiment VI) replicate plots immediately before and after solarization. Samples of soil theyear after solarization were collected in mid-April 1989, about 1 month after sowing.'̂ CV = coefficient of variation.

at experimental sites during the solarizationperiod were in the ranges 27-42 C and 1222 C, respectively, in 1988.

The estimates of inoculum density of V.dahliae in the 0-20-cm soil layer prior tosolarization were much higher in experiments Vand VI than the estimates for experiments I-IV,and were in the range 40-90 propagules/g drysoil (Table 3). Furthermore, for the two experi-ments, the population of V. dahliae in the 20-40-cm soil layer was consistently lower than that inthe upper soil layer (Table 3). As for the previousexperiments, in experiments V and VI soilsolarization reduced V. dahliae populations toundetectabte levels both in the 0-20-cm and inthe 20-40-cm soil layers. Also, in these twoexperiments there was a marked increase ininoculum density in the two layers of unsolar-ized soils during the solarization pwriod(Table 3). V. dahliae populations in solarizedsoils remained at a low level in the upper andlower soil layers sampled, regardless of the timeperiod that elapsed between solarization andsowing. Inoculum densities in plots sown to cvsAcala SJ2 and Coker 310 were similar withineach soil treatment and experiment (e.g. 37-1 and40-9 propagules/g soil in the 0- 20-cm soil layer in

unsolarized plots, and 1-9 and 2-0 propagules/gsoil at the same depth in solarized plots, for plotssown to cvs Acala SJ2 and Coker 310 ofexperiment V, respectively). Therefore, valuesfor the two cultivars were combined (Table 3).

The marked decrease in inoculum density of thepathogen in solarized soil had a significant effecton Verticittium wilt epidemics. The linear fit forcurves of disease incidence increase over time in1989 was better for non-transformed than fortransformed data, as indicated by the higher R'values and the lack of a discernible pattern in thedistribution of residuals plotted against expectedvalues. Therefore, only linear regressions withnon-transformed incidence values are presented(Table 4). Epidemics in solarized plots weredelayed, increased at a significantly lower rate,and had a significantly lower AL DPC. comparedto epidemics in unsolarized plots (Table 4)Epidemic onset was delayed by 2 weeks mexperiment V and by 6-7 weeks in experimentVI. For each of cvs Acala SJ2 and Coker 310. therate of incidence increase was significantly lower(P<0 001) in solarized compared to unsolarizedsoils, but there were no differences ( /*=0 05)between cultivars within soil treatments. Similarly,no significant (P = 0-05) differences in AUDPC

256 J. M. Melero-Vara et al.

1eo

o

Control <?/Verticilliutn wilt of cotton 257

Table 5, Long-term effect of soil solarization on the population of Verticillium dahtiae in soil, development ofVerticittium wilt and seed cotton yield of cotton wilt-tolerant cv. Acala GC 510 grown in 1990 after a crop ofsusceptible cotton cvs Acala SJ2 and Coker 310 in 1989"

Soil treatment

Experiment VIIUnsolarizedSolarized

Experiment VIIIUnsolarizedSolarized

Inoculum density aftersowing (propagules/g

dry soil)*"

mean CV(%)

84-0 33-413-0 36-0

85-5 37-77-5 89-3

Final

foliarsymptoms

25-8 a12-4 b

14-9 a4-7 b

incidence {"/of

vasculardiscoloration

71-3 A53 5 B

64-7 a41-7 b

Seed cottonyield (kg/ha)*"

2555 A2867 B

2375 a2684 b

* Soil solarized from 12 July to 2 September (experiment V) and from 26 July to 16 September (experiment VI), 1988,was sown to Verticittium wilt-susceptible cotton cvs Acala SJ2 and Coker 310 in mid-March 1989. Plots wereharvested in September 1989 and sown to cv. Acala GC 510 at the end of March 1990 (experiments VII and VIII).'' E>etennined by means of an Andersen sampler in four 500-mg aliquots of soil collected from the 0-20-cm soil layerof each of four (experiment VII) or three (experiment VIII) replicate plots at the end of April 1990. CV = coefficientof variation.'Determined from plants in the two central rows of each plot that were affected by symptoms characteristic ofVerticiltium wilt at the end of September, 24 weeks after sowing. Vascular discoloration in the stem was determinedin 50 consecutive plants. For each experiment, values followed by the same letter are not significantly different atP = 0-05 (lower case) or /> = 0-1 (upper case) according to LSD test.'' Determined by combine harvesting. Average of four (experiment VII) or three (experiment VIII) replicate plots.For each experiment, values followed by the same letter are not significantly different at /" = 0-05 (lower case) or? = O-l (upper case) according to LSD test.

occuned for cultivars or cultivar x soil treatmentinteractions (Table 4), Furthermore, there was ahighly significant reduction in final diseaseincidence, which was in the range 3 3-12-9% insolarized plots compared with 74-6-85% inunsolarized plots (Table 4), However, neithercultivar nor soil treatment x cultivar interactionhad statistically significant effects (P<0-05). Thefinal incidence of plants showing vasculardiscoloration at harvest was 12-19-3% higherthan that of foliar symptoms, regardless ofcultivar and soil treatment, except in solarizedplots of experiment V, for which the incidence offoliar symptoms was similar to that of vasculardiscoloration (data not shown).

Soil solarization determined a significant(P<0-05) increase in seed cotton yield, whichaverated 40-5 and 131% in experiments V andVI, respectively (Table 4), Although the effects ofcultivar and cultivar x soil treatment interactionon teed cotton yield were not statisticallysignificant, there was a trend towards slightly

higher yields for cv, Coker 310 compared withAcala SJ2,

Combined long-term effect of soil solarizationand cultivar tolerance on Verticillium wilt ofcotton

The estimates of inoculum density of r. dahtiaein soil in May 1990, about 1 month after sowingthe second consecutive crop, were much higher(Table 5) than those made for the same plots theprevious year (Table 3). Populations of V,dahtiae in plots sown to cvs Acala SJ2 andCoker 310 in 1989 were very similar (e.g. 83-9 and84'3 propagules/g soil in unsolarized plots, and13-6 and 12-3 propagules/g soil in solarized plots,for plots sown to cvs Acala SJ2 and Coker 310 ofexperiment VII, respectively, regardless of thesoil treatment and experiment. Therefore, meanvalues for the two cultivars were combined(Table 5), These estimates indicated thatV, dahtiae populations in soil unsolarized or

258 J. M. Melero-Vara et al.

solarized in 1988 had increased by about 21-2-5-or 6-3-6-5-fold, respectively, after a susceptiblecotton crop in 1989 (Tables 3 and 5). When thehighly wilt-tolerant cv. Acala GC510 was grownin these plots in 1990, the final disease incidencein unsolarized plots was very low compared tothat in susceptible cvs Acala SJ2 and Coker 310grown the year before. By contrast, the finaldisease incidence in solarized plots was as high asthat observed in those susceptible cultivars withmuch less initial inoculum (Tables 4 and 5).However, the incidence of plants showingvascular discoloration was much higher thanwhen assessed by foliar symptoms, regardless ofthe experiment, soil treatment and cultivar(Table 5). Seed cotton yield was increased inplots that were solarized in 1988 compared tothat in unsolarized plots, for both experimentVII (12-2%) and experiment VIII (13%)(Table 5).

DISCUSSION

Soil solarization has been shown to control anumber of diseases caused by soilbome fungi,including Verticillium wilt (Katan, 1980; Pullmanet ai, 1981a; Ben-Yephet et ai, 1988; Davis,1990). The increased temperature in humid soilscaused by solarization affects pathogen propa-gules directly, and may also result in enhancedactivity of microbial antagonists in the soil(Katan et ai, 1976; Tjamos & Paplomatas, 1988).

Soil temperattires were not recorded in ourexperiments. Although air temperatures wereavailable during the experiments, they could notbe used for Mahrer's forecasting system toestimate soil temperatures because solar irradia-tion data were lacking. A daily maximumtemperature of 35-39'C was recorded at a depthof 25 cm in another location of the GuadalquivirValley, at a distance of 5 km from the experi-mental site, in 1989 and 1990 (J. M. M.-V. et ai,unpublished data), which is lower than thetemperatures reported from several locations inCalifornia (Pullman et ai, 1981a), but within therange of those reported in a cooler climate in Idaho(Davis & Sorensen, 1986). Similariy, comparison ofthe maximum daily air temperatures during theperiods of solarization with those reported fromsome areas of Israel and California indicate that theenvironment of the experiments reported here wasusually less extreme in relation to thermal inactiva-tion of fungal pathogens (Katan (7 ai, 1976;Pullman etai., 1981a). Nevertheless, the presentresult!, show that soil solarization successfully

reduced the population of the defoliatingpathotype of V. dahliae within at least 40 cmdepth of heavily infested clay soils. This effectwas consistent over a range of inoculum levels ofthe pathogen in soil, years and locations. Theeffectiveness of soil solarization under ourconditions agrees with the results obtained byBen-Yephet et ai (1988), who suggested that V.dahliae is highly sensitive to suboptimal tem-peratures during solarization. SinMlarly, thepresent results relate to those of Davis &Sorensen (1986), who effectively controlledVerticillium wilt of potato in a cool climate,even though V. dahliae was not significantlyreduced within a soil depth of 15-30 cm. As thenon-defoliating pathotype of I'. dahliae is moretemjjerature-sensitive at 37°C than the defoliat-ing pathotype (Pullman et ai, 1981b), soilsolarization should be a useful control measurefor reducing \'. dahliae in soils of the Guadal-quivir Valley, which are highly infested mainlywith either the defoliating or the non-defoliatingpathotype (Bianco-Lopez et ai, 1989; Bejarano-Alcazar, 1990).

The effect of solarization in reducing V.dahliae populations in soil resulted in a corre-sponding highly significant control of the diseasein Ff/-f/c////«w-wilt-susceptible cotton ctiltivarsgrown the next season, regardless of differencesin the envirormient conditions during the period1987-89. Control was achieved by delaying thetime of epidemic onset as well as by reducing therate of disease progress, which brought about alarge decrease in the final incidence of diseaseand in AUDPC values. Because of the strongeffect of early infections by I'. dahliae in reducingcotton yield (Pullman & DeVay, 1982b), the 2- to7-week delay of epidemic onset in solarized soilsobserved in these experiments is of majorsignificance in terms of reducing yield losses. Inthis study, no attempts were made to establish aquantitative relationship between initial inocu-lum density of ('. dahliae and final I'erticilliumwilt incidence, or seed cotton yield. A significantnon-linear correlation was found between initialinoculum density and final disease incidence insusceptible cotton cultivars (Pullman & DeVay,1982a; Paplomaias et ai, 1992).

Two other beneficial effects, tn addition tocontrol of Verticillium wilt, were observed in thecourse of this study, including a good control ofweeds (other than Portulaca oleracea andSotanum spp.) and of Rhizoctomia damping-off.Since Rhizoctonia damping-off occurs in mostcotton-growing areas of the worid, including

Control o/Verticilliutn wilt of cotton 259

Spain (Melero-Vara & Jimenez-Diaz, 1990), thepotential of soil solarization for its controlshould also be taken into consideration.

The overall effect of soil solarization in ourexperiments was a significant increase in seedcotton yield compared to that in unsolarizedcontrols, which is in agreement with previousrestilts (Pullman et al, 1981a). However, theextent of this increase varied with differencesbetween experiments in the various factors thatinfluence Verticillium wilt development (El-Zik,1985), including differences in the amount ofinitial inoculum (experiments III and IV),tolerance of cultivars (experiments V and VIwith susceptible cvs Acala SJ2 and Coker 310 in1989, as compared to experiments VII and VIIIwith highly wilt-tolerant cv. Acala GC510 in1990), and favourableness of weather conditionsfor disease development (severe disease with lowinoculum in experiment II associated with mildsummer temperatures). Nevertheless, in four outof five experiments (Tables 1, 2 and 4) soilsolarization resulted in a seed cotton yield of cv.Coker 310 above 4-5 t/ha, which is about 0-5-0-8 t/ha greater than the average seed cotton yieldof this cultivar in the Lower Guadalquivir Valley(Guerrero, 1984).

The results reported here indicate that soilsolarization in the Lower Guadalquivir Valley ofsouthern Spain provides satisfactory control ofVerticillium wilt caused by the defoliatingpathotype of V. datitiae. However, this controlmeasure does not seem to have a long-term effectunder these experimental conditions. When asusceptible cotton cultivar was grown in solar-ized soils, the inoculum level of the pathogen forthe next crop increased to an extent that wouldcause severe disease in susceptible cultivars.Although highly wilt-tolerant cultivars such asAcala GC510 would not be severely affected bysuch a high inoculum, they would allow for atwofold increase in the inoculum left for thefollowing crop. Therefore, the use of soilsolarization should be integrated with that oftolerant cotton cultivars and rotation with non-host crops in order to avoid a sustained increasein inoculum level in solarized soils,

A C K N O W L E D G E M E N T S

This research was supported by grants INIA7637 and DGA, J A 3621 from Instituto Nacionalde Investigaciones Agrarias and Direccion Gen-eral de Agricultura, Ganaderia y Montes, Juntade Andalueia, respectively. The authors wish to

thank J, Cobos, F, Martinez, M, Riera, J,Romero and L, Saavedra for technical assis-tance and Cooperative Algodoneras Trajano andTrobal at Marismas for allowing us to use theirfield plots and facilities.

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