acibenzolar-s-methyl induces systemic resistance in cotton against black root rot caused by...

CSIRO PUBLISHING

www.publish.csiro.au/journals/app Australasian Plant Pathology, 2005, 34, 499–507

Acibenzolar-S-methyl induces systemic resistance in cotton against black rootrot caused by Thielaviopsis basicola

A. H. MondalA,C,D, D. B. NehlA,C and S. J. AllenB,C

ANSW Department of Primary Industries, Locked Bag 1000, Narrabri, NSW 2390, Australia.BCotton Seed Distributors Ltd, PO Box 117, Wee Waa, NSW 2390, Australia.

CAustralian Cotton Cooperative Research Centre, Narrabri, NSW 2390, Australia.DCorresponding author. Email: [email protected]

Abstract. Black root rot, caused by Thielaviopsis basicola, is a serious disease of cotton in Australia, causingnecrosis of the root cortex, delayed seedling development and fruit set, and yield loss when severe. We investigatedthe potential for acibenzolar-S-methyl to induce resistance in cotton against T. basicola in naturally infested soil.In pot experiments, soaking seeds in solutions of acibenzolar-S-methyl (25 or 50 μg/mL for 3 to 5 h before planting)consistently reduced disease severity on tap roots by 20–30%. In field experiments, acibenzolar-S-methyl wasapplied either as a spray over the seed during sowing (in-furrow spray), as a seed dressing, by the soaking method, oras foliar sprays over seedlings. In the field, seed-soaking reduced the severity of black root rot on tap roots by 33%.The in-furrow spray (25 μg acibenzolar-S-methyl/mL at 550 L/ha) reduced the severity of symptoms on tap rootsby 24%, increased the number of relatively healthy lateral roots by 350% and increased fruit number by 29%. Seeddressing with 3.3 mg acibenzolar-S-methyl/kg seed did not affect disease severity, whereas 6 mg/kg reduced theseverity on tap roots by 20%. The foliar sprays were ineffective. To our knowledge, this is the first report of theinduction of systemic acquired resistance against a soilborne plant pathogen in the field using a chemical seedtreatment. With appropriate rates and application methods, acibenzolar-S-methyl has potential as a component inan integrated disease management strategy for black root rot of cotton.

Additional keywords: benzothiadiazole, BTH, Gossypium hirsutum, root fungi, SAR.

IntroductionBlack root rot, a soilborne disease caused by the fungus

Thielaviopsis basicola, is pandemic in Australian cotton(Gossypium hirsutum) (Nehl et al. 2004). The pathogeninvades the roots during the first 2 to 8 weeks of thecrop, causing dark brown to black necrosis of the cortex,and reduced root and shoot development (Hillocks 1992).The fungus produces abundant, short-lived, thin-walledendoconidia and thick-walled, melanised chlamydospores onroots (Linderman and Toussoun 1967). Both types of sporeare capable of infecting cotton roots. Seedling mortality isgenerally not increased by black root rot (Nehl et al. 2004).Cotton root tips are not infected by T. basicola (Hawes andBrigham 1992) and plants in the field resume normal growthas temperatures increase. When black root rot of cotton issevere, the reduced seedling growth results in delayed cropmaturity and yield loss (Nehl et al. 2004).

Black root rot is difficult to control because ofthe persistence of chlamydospores that are produced in

abundance in cotton. Rotation with non-host crops such ascereals delays, but does not prevent, ongoing increases in theseverity and distribution of black root rot with each cottoncrop (Nehl et al. 2004). None of the currently availablecultivars of cotton have effective resistance against T. basicola(Allen 2001) and no fungicides are available in Australia forcontrol of the disease. Flooding of fields during summer isan effective control measure for black root rot (O’Niell 1997)but its deployment is severely limited by the availability ofwater and the topography of fields. There are few optionsfor control of black root rot of cotton, other than delayingplanting until soil temperatures are warmer.

Induction of systemic acquired resistance (SAR) bybiological or chemical means has been shown to protectplants against viral, bacterial, fungal and nematode pathogensamong a range of crops (Zhao and Guo 2003). The protectionafforded by SAR is frequently non-specific and long-lasting(Kessmann et al. 1994a, b). In cotton, application of either2,6-dichloroisonicotinic acid or acibenzolar-S-methyl as

© Australasian Plant Pathology Society 2005 10.1071/AP05089 0815-3191/05/040499

500 Australasian Plant Pathology A. H. Mondal et al.

foliar sprays induced SAR against Alternaria macrosporaand Verticillium dahliae (Colson-Hanks and Deverall 2000;Colson-Hanks et al. 2000). Foliar application or soil-drenching with 2,6-dichloroisonicotinic acid did not induceresistance against T. basicola (Colson 1997). However,Mondal (2001) found that treatment of the seed of cowpea(Vigna unguiculata), faba bean (Vicia faba), mung bean(Vigna radiata), soybean (Glycine max), field pea (Pisumsativum) and chickpea (Cicer arietinum) with acibenzolar-S-methyl induced resistance against T. basicola, even thoughthe compound had no fungicidal activity against the pathogen.In this paper, several methods of application of acibenzolar-S-methyl on cotton for the control of black root rot areinvestigated.

MethodsCotton seeds treated with fungicides at standard rates

(pentachloronitrobenzene 1.08 g/kg seeds; metalaxyl 150 mg/kgseeds) were used in all experiments. Acibenzolar-S-methyl (Bion500 WG, a.i. 500 g/kg) was kindly provided by Mr K. McKee,Syngenta Crop Protection Pty Ltd. The population density ofT. basicola in soil was determined in sub-samples using theselective medium TB-CEN, with methods described previously(Nehl et al. 2004).

The severity of black root rot was assessed on the basis ofdiscoloration on taproots and rated on a scale of 0–10 where 0 = nodisease, 1 = 1–10% and 10 = 91–100% discoloration as describedpreviously (Nehl et al. 2004). Discoloured or blackened roots wererandomly bioassayed on TB-CEN medium to confirm the infectionby T. basicola. When the severity of disease on taproots in the fieldexperiments was very high among the treatments, the number ofrelatively healthy lateral roots on each plant was counted. Lateral rootswere considered to be relatively healthy when <50% of their length wasdiscoloured or blackened.

Pot experiments

The pot experiments used soil collected from fields at the AustralianCotton Research Institute (ACRI) near Narrabri, NSW or at a farm nearWee Waa, NSW that were infested with T. basicola (125 and 427 cfu/gsoil, respectively). Soil was stored in the shade in unsealed bags andthen mixed thoroughly before use. Cotton was sown (five seeds/pot,unless otherwise stated) in polystyrene foam cups (700 mL) filledwith soil to 2 cm from the top, with three drain holes punched inthe base. Unless stated otherwise, plants were grown in a glasshouse(range 15–30◦C, night/day). The plants were watered periodicallyby placing pots in trays partly filled with water until the soil wasmoist. Disease severity was expressed as the mean for all seedlingsin each pot.

Rate and duration of seed soaking. Prior to sowing, cotton seeds(cv. DeltaPine 90) were soaked in solutions containing 0, 25 or 50 μgacibenzolar-S-methyl/mL distilled water for 12 h. The soaking operationin this experiment, and others described hereafter, was carried out inthe dark at room temperature (23◦C). The solutions were drained andthe seeds were allowed to dry for at least 10 min before sowing in theWee Waa soil. The treatments were repeated thrice. Disease severity wasassessed 24 days after sowing (DAS).

In a second experiment, cotton seeds (cv. Sicala V2) were soakedfor 5 h using the same concentrations of acibenzolar-S-methyl/mL anddrying as before. Seeds were sown in ACRI soil at eight seeds/pot.The treatments were repeated thrice. Disease severity, shoot height andshoot fresh mass were assessed at 19 DAS.

In a third experiment, cotton seeds (cv. Sicala V2) were soaked foreither 45 or 90 min using the same concentrations of acibenzolar-S-methyl and drying as before. The seeds were sown in Wee Waa soil.The treatments were repeated thrice. Disease severity and shoot drymass were assessed at 28 DAS.

Cotton cultivars. Seeds of cvv. Sicala V2, Sicot 189, DeltaSapphire, Siokra 1–4, Sicot 53, Delta Jewel and Delta Emeraldwere soaked in solutions containing 0 or 25 μg acibenzolar-S-methyl/mL for 3 h and sown in Wee Waa soil, with six replicates.The plants were grown in a controlled environment chamber(19/23◦C 12 h night/day cycle). Disease severity on taproot andlateral roots was assessed at 28 DAS and the shoot dry mass wasalso recorded.

Field experiments

Cotton was sown in raised beds 1 m apart using disc-planters thatcut mini-planting-furrows at a depth of 5 cm in the middle of beds. Seedwas dropped into the mini-planting-furrows, which were then closedwith press wheels ∼30 cm behind the discs. Irrigation was providedin bigger furrows between the beds and tail-water was collected withreturn drains. At least 4 weeks before sowing, fertiliser (anhydrousammonia) was injected into the sides of the beds at commercialrates (generally 180 to 200 kg N/ha). Sowing rates ranged from15 to 18 seeds/m of beds.

Foliar spray. Cotton (cv. NuCOTN 37) was sown on 3 October 1998in a commercial field near Wee Waa, NSW (from which soilwas collected for the pot experiments) in plots (two rows × 10 m,with 2 m buffer between all plots) with four treatments in a Latinsquare design. Treatments were untreated; a foliar spray withacibenzolar-S-methyl (50 μg/mL) at 16 DAS; a foliar spraywith acibenzolar-S-methyl (75 μg/mL) at 16 DAS; and foliar sprayswith acibenzolar-S-methyl (50 μg/mL) at 12 and 16 DAS. Thesesolutions of acibenzolar-S-methyl were applied to seedlings usinga hand-held, pressurised garden-sprayer until the point of incipientrunoff. Disease severity on tap roots was assessed in two lots offive plants at 26 DAS, starting ∼2 m from each end of one row ineach plot replicated four times. Shoot dry mass was also recorded.

Foliar spray and seed dressing. Cotton (cv. Sicala V2i) was sownon 16 October 1998 in a commercial field near Warren, NSW, in plots(two rows × 10 m) with four treatments in a randomised block designwith six replicates. The treatments were untreated (standard fungicidecoated), acibenzolar-S-methyl as a seed dressing, acibenzolar-S-methylas a foliar spray, and acibenzolar-S-methyl as both the seed dressingand the foliar spray. The seed dressing, acibenzolar-S-methyl solution(100 μg/mL), was applied over the existing standard fungicidal coatingof cotton seeds at the rate of 33 mL of solution/kg seed (equivalentto 3.3 mg acibenzolar-S-methyl/kg seed), by agitating the seed andsolution vigorously in a polythene bag. Seed was dried for at least 30 minbefore sowing. The foliar spray was applied at 18 DAS by sprayingthe seedlings with a solution of acibenzolar-S-methyl (50 μg/mL) asin the earlier experiment. Disease severity on tap roots was assessedon five plants in the middle of each row of each plot and plantestablishment was assessed by counting seedlings in 5 m of each rowof each plot at 26 DAS.

Seed dressing. Acibenzolar-S-methyl solution (300 μg/mL)was applied to cotton (cv. Sicot 289RR) as a seed dressing at therate of 20 mL of solution/kg seed (equivalent to 6 mg acibenzolar-S-methyl/kg seed), with agitation and drying as before. Thisprocess removed very little of the standard fungicide seed coating.Seeds with the standard coating only were used as the control.The seed from both treatments was sown on 17 October 2003 ina commercial cotton field near Wee Waa, NSW, in single-row plots15 m long, using a randomised block design with six replicates.The seedling establishment was recorded from entire row and theseverity of black root rot was assessed in ten plants/plot at 28 DAS.

Systemic resistance against Thielaviopsis basicola Australasian Plant Pathology 501

The number of true leaves/plant was assessed in 20 plants/plot at28 and 45 DAS.

Seed soaking. Cotton seeds (cv. NuPEARL) were sown on9 October 2001 in a field at the ACRI, in plots (four rows × 45 m)with two treatments in a randomised block design with eight replicates.The seed was either unsoaked, as the control, or soaked for 3 h ina solution of acibenzolar-S-methyl (25 μg/mL) as in the pot experiments,except it was kept in the shade during soaking. The mean population ofT. basicola in that part of the field was 250 cfu/g soil. The rate of sowingwas 15 seeds/m of bed. The severity of black root rot was assessed at34 DAS in five adjacent plants in each of the second and third rows ofeach plot, at 20 m from the tail-water return drain. Lateral roots werealso assessed for black root rot. The shoots of these plants were oven-dried (70◦C, 48 h) before weighing. Plant establishment was assessedby counting the number of seedlings in a 10 m interval in the first andfourth row in each plot, starting at positions 15 m from the tail-waterreturn drain.

In-furrow spray. Cotton (cv. Sicot 80) was sown on 4 October 2001in a commercial cotton field west of Moree, NSW, in plots(eight rows × 500 m) with two treatments in a randomised blockdesign with three replicates (eight rows buffer between each block).The treatments were water (550 L/ha) and acibenzolar-S-methyl (25 μgacibenzolar-S-methyl/mL, at 550 L/ha) applied as in-furrow sprays(i.e. into the planting-furrow) before closure by the press wheels.The population of T. basicola close to the tail-water drain in that fieldwas 400–500 cfu/g soil. The severity of black root rot was assessedin 30 plants (ten plants each in the second, fifth and seventh rows)at a point 20 m from the tail-water drain in each plot, at 35 DAS.The shoots of these plants were oven-dried (70◦C, 48 h) beforeweighing. Fruit production was assessed at 118 DAS by counting thetotal number of bolls (>2 cm diameter) in 2 m sections of the second,fifth and seventh rows in each plot, starting ∼1 m beyond the gapscaused by disease assessment. To provide additional replication, allthese parameters were assessed in the same manner at points 50 mfrom the tail-water drain in each plot. Seed cotton was harvestedusing a commercial cotton picker (John Deere Pty Ltd) from wholeplot (∼500 m2) across the length of the field. The whole picker wasweighed in a high capacity (∼15 tonnes) scale each time before thestarting and at the finishing of picking each plot. The differencebetween the initial and final weight was used as the weight of seedcotton from that plot. The yield was calculated as bales/ha basedon 40% ginning outturn.

Statistical analysis. Analysis of variance was conducted usingthe statistical programme Minitab (Release 9.2, Minitab Corporation).Before analysis, data were screened for normality and for homogeneityof variance among groups and transformed if appropriate. Means werecompared using Fisher’s least significance difference test. In the in-furrow spray experiment, data were analysed by two-way AOV, withdistance from the tail-water drain (20 m or 50 m) as a factor. Distancefrom the tail-water drain did not have significant effects and, therefore,the analysis for each parameter is presented using six observations ineach treatment (two observations from each plot).

Results

Pot experiments

Rate and duration of seed soaking. In the first experiment,when cotton seed, cv. DeltaPine 90, was soaked for 12 h,the severity of black root rot was decreased by 17 to31% in acibenzolar-S-methyl treatments (Fig. 1). In thesecond experiment, the severity of black root rot wasdecreased by 44 to 58% in acibenzolar-S-methyl treatments(Fig. 2). Shoot height increased significantly (P = 0.002)

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Fig. 1. Severity of black root rot in potted soil from a naturally-infested field at Wee Waa NSW following soaking of cotton seed(cv. DeltaPine 90) in acibenzolar-S-methyl for 45 min. The diseaseseverity was assessed on tap roots at 24 days after sowing ona 0–10 scale. Error bars represent s.e.m.

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Fig. 2. Severity of black root rot in potted soil from a naturally-infestedfield at the Australian Cotton Research Institute following soakingseed in acibenzolar-S-methyl for 5 h. Plant height is presented inshaded columns and the severity of black root rot of cotton(cv. Sicala V2) is presented in unshaded columns. The diseaseseverity was assessed on tap roots 19 days after sowing. Error barsrepresent s.e.m.

as the rate of acibenzolar-S-methyl increased. The shoot freshmasses for the treatments 0, 25 and 50 μg acibenzolar-S-methyl/mL were 0.58, 0.65 and 0.63 g/plant, respectively,which were not significantly different (P = 0.56). In the


third experiment, disease severity was decreased by 29%when seed (cv. Sicala V2) was soaked for either 45 min(Fig. 3) or 90 min (P < 0.001, data similar and not presented).Soaking seed for 45 or 90 min did not have any significanteffects on shoot (P = 0.55) or root (P = 0.89) growth (datanot presented).

Cotton cultivars. In all seven cultivars, soaking seed inacibenzolar-S-methyl reduced the severity of symptoms ontap roots by 15 to 28% (Fig. 4A). The number of relativelyhealthy lateral roots was increased by 43 to 152% (Fig. 4B).The greatest response to acibenzolar-S-methyl was observedin cultivars Sicot 189, Delta Jewel followed by Sicot 53,Siokra 1–4 and Delta Emerald. A reduced intensity ofdiscoloration on tap root was observed in acibenzolar-S-methyl treatment. Acibenzolar-S-methyl increased the shootbiomass of Delta Jewel by 14% (889 and 1017 mg/plantfor untreated and treated plants, respectively, P = 0.05).Significant changes in shoot growth were not observed inthe other cultivars in this experiment.

Field experiments

Foliar spray. The mean disease severity (0–10 scale) wasnot significantly (P = 0.93) different for four treatments atthe Wee Waa site in 1998 when acibenzolar-S-methyl wasapplied as foliar spray. Foliar application caused stunting ofcotton plants and shoot growth decreased by 21% comparedwith the control treatment (Fig. 5).

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Fig. 3. Severity of black root rot in potted soil from anaturally infested field at Wee Waa NSW following soakingseed of cotton (cv. Sicala V2) in acibenzolar-S-methyl for 45 min.The disease severity was assessed on tap roots at 28 days after sowing.Error bars represent s.e.m.

Foliar spray and seed dressing. In the field experimentat Warren in 1998, the seed dressing and foliar treatmentswith acibenzolar-S-methyl had no effect (P = 0.77) onseverity of black root rot (Fig. 6). A delay (2–3 days) inseedling emergence was obvious at 15 DAS in all plotswith the seed dressing treatment but was not quantified.However, at 26 DAS, seedling emergence was 55, 56, 54and 55% (P = 0.81), respectively, for treatments untreated(standard fungicide coated), acibenzolar-S-methyl asa seed dressing, acibenzolar-S-methyl as a foliar spray, andacibenzolar-S-methyl as both the seed dressing and thefoliar spray.

Seed dressing. When acibenzolar-S-methyl was appliedas a seed dressing at Wee Waa in 2003, the severityof black root rot on tap roots was decreased by 20%(Table 1). This decrease in disease severity at 28 DASwas reflected by a subsequent increase in plant growth,assessed as the number of true leaves at 45 DAS. Seedlingestablishment was only slightly reduced by treatment withacibenzolar-S-methyl.

Seed soaking. The severity of black root rot wassignificantly reduced by soaking cotton seed in acibenzolar-S-methyl treatment at the ACRI site (Table 2). A reducedintensity of discoloration on tap root was observed inacibenzolar-S-methyl treatment as observed in the potexperiment. No significant increase in shoot weight or thenumber of relatively healthy lateral roots was observed.However, the plant stand was reduced by 42% inthe acibenzolar-S-methyl treatment. This experiment wasconfounded by competition for moisture with a severeinfestation by nut grass (Cyperus rotundus) shortly afteremergence of the cotton, when lateral root growth would havebeen impeded.

In-furrow spray. When acibenzolar-S-methyl was appliedto cotton seed as an in-furrow spray, the severity of blackroot rot on tap roots was decreased by 24% and the numberof relatively healthy lateral roots was increased by 350%(Table 3). A reduced intensity of discoloration on tap rootwas observed in acibenzolar-S-methyl treatment as observedin the pot experiment and seed soaking experiment at ACRIfield. The in-furrow spray did not have any adverse effect onseedling establishment or shoot growth. Fruit production wassignificantly higher in the acibenzolar-S-methyl treatmentat 118 DAS but this was not reflected as an increase inyield (Table 3).

Discussion

The severity of black root rot of cotton, in both potted soiland in the field, was consistently reduced by a seed soakingtreatment of acibenzolar-S-methyl before planting. Diseaseseverity in the field was also reduced when acibenzolar-S-methyl was used as an in-furrow spray at planting or as a seeddressing (one out of two) before planting. Acibenzolar-S-methyl decreased the extent of infection on tap roots and


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Fig. 4. Severity of black root rot in potted soil from a naturally infested field at Wee Waa, NSW, following soaking seed of six cotton cultivars inacibenzolar-S-methyl for 3 h. The severity of black root rot was assessed on tap roots and healthy lateral roots were counted from each tap root ofcotton cultivars. Error bars represent s.e.m., n = 6.

lateral roots, reduced the intensity of discoloration of roots,and in some cases, increased shoot growth and fruit number.These effects were clearly systemic because acibenzolar-S-methyl was applied to the seed and the reductions in diseaseseverity were evident after germination, as the root systemexpanded through space and time. In vitro studies havedemonstrated that acibenzolar-S-methyl has no antifungalactivity against T. basicola (Mondal 2001), other fungalpathogens of cotton (Colson-Hanks et al. 2000) and plantpathogens in general (Zhao and Guo 2003). This lack offungicidal activity and our observations of systemic effectsare key criteria confirming that acibenzolar-S-methyl inducedSAR against black root rot in cotton.

To our knowledge, this is the first report of the inductionof SAR against a soilborne plant pathogen, in the field,using a chemical seed treatment. Acibenzolar-S-methylhas been shown to induce resistance in several plants againstroot-infecting fungi (Jensen et al. 1998; Dann et al. 1998;Benhamou and Belanger 1998a, b; McFadden et al. 2001; Aliet al. 2000) and nematodes (Owen et al. 2002). Dann et al.(1998) used acibenzolar-S-methyl in the field as a foliar spray.Jensen et al. (1998) induced SAR against Rhizoctonia solaniin cabbage seedlings by soaking the seed in acibenzolar-S-methyl before sowing in a controlled environment cabinet.None of these studies used acibenzolar-S-methyl as a seedtreatment in the field.


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Fig. 5. Severity of black root rot and growth of field grown cottonseedlings, cv. NuCOTN 37, (sown on 3 October 1998, assessed at26 days after sowing) following a foliar spray with aqueous solutions ofacibenzolar-S-methyl at 10 days after sowing in a field near Wee Waa,NSW. Error bars represent s.e.m.

T. basicola reproduces prolifically on cottonroots (Rothrock and Nehl 2000) and populations of200 to 500 cfu/g soil are frequently observed in soilin cotton fields in Australia (Nehl et al. 2004). Aninoculum level of 50 cfu/g soil is sufficient to causesubstantial disease in cotton and 100 cfu/g is effectivelya saturation level, above which almost the whole tap root

Table 1. Severity of black root rot of cotton, cv. Sicot 289RR (sown on 17 October 2003, assessed at 28 days aftersowing) following application of acibenzolar-S-methyl as a seed coating before sowing in a field near Wee Waa

Acibenzolar-S-methyl Disease severity Seedling establishment True leaves (number/plant)(mg/kg seed) (0–10 scale) (plants/m) 28 DASA 45 DAS

0 6.5 12.7 1.65 5.26 5.4 11.8 1.67 5.5

Probability (n = 6) P < 0.001 P = 0.041 NSB P < 0.001

ADAS = days after sowing.BNS = not significant.

Table 2. Severity of black root rot of cotton, cv. NuPEARL (sown on 9 October 2001, assessed at 34 days after sowing),following soaking seed in acibenzolar-S-methyl before sowing in a field at ACRI

Acibenzolar-S-methyl Disease severity Relatively healthy lateral roots Seedling establishment Shoot dry mass(μg/mL) (0–10 scale) (no./plant) (plants/m) (mg/plant)

0 6.9aA 6.6 13.0a 33925 4.6b 5.7 7.6b 375

Probability P = 0.021 NS P < 0.001 NS

AValues followed by the same letter in columns are not significantly different by pairwise comparison of means withFisher’s l.s.d. at the stated probability level (n = 8, NS = not significant).

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Fig. 6. Severity of black root rot of cotton (cv. Sicala V2, sown on16 October 1998) seedlings at Warren when acibenzolar-S-methyl wasapplied as seed dressing and/or foliar spray and the disease was assessedat 26 day after sowing. Error bars represent s.e.m.

will be infected (Holtz and Weinhold 1994; Nehl et al.2004). The inoculum levels in our experiments ranged from125 to 427 cfu/g of potted soil and from 250 to 500 cfu/gof soil in the field, and were more than adequate fortesting the potential of acibenzolar-S-methyl to induceSAR against black root rot. The temperatures in theglasshouse, controlled environment chamber and in the


Table 3. Severity of black root rot of cotton, cv. Sicot 80 (sown 4 October 2001, assessed at 35 days after sowing) following application ofacibenzolar-S-methyl as an in-furrow spray at sowing in a field near Moree, NSW

Acibenzolar-S-methyl Disease severity Relatively healthy lateral roots Seedling establishment Shoot dry mass Fruiting Yield(μg/mL) (0–10 scale) (no./plant)A (plants/m) (mg/plant) (bolls/m) (bales/ha)

0 9.6aB 1.5b 10.6 211 21b 8.025 7.3b 6.8a 11.6 224 27a 8.3

Probability P < 0.001 P < 0.001 NS NS P = 0.019 NS

ALateral roots were considered to be relatively healthy when <50% of their length was discoloured or blackened.BValues followed by the same letter in columns are not significantly different by pairwise comparison of means with Fisher’s l.s.d. at thestated probability level (n = 6, NS = not significant).

field were also conducive for disease development (Nehlet al. 2004).

In the pot experiment comparing cotton cultivars and inthe field experiment at Moree, the effects of acibenzolar-S-methyl on disease were manifest to a much greater extent onlateral roots than tap roots. Although the whole tap rootof cotton can become infected and ‘blackened’ (Nehl et al.2004) the vascular tissue is usually not invaded (Walker et al.1998). As temperatures increase, cotton seedlings resumenormal growth and slough off the dead cortical cells (Kingand Presley 1942). Since the tips of cotton roots are generallynot infected by T. basicola (Hawes and Brigham 1992), lateralroot growth may continue even when infection of the cortexof tap roots is at a saturation level, within the zone of soilused for assessment. Although disease severity in the tap rootswas reduced significantly by acibenzolar-S-methyl in the fieldexperiment at Moree, infection of the tap roots was stillsubstantial. It is likely that the greater number of relativelyhealthy lateral roots, with acibenzolar-S-methyl, was a keyfactor in the subsequent increases in plant growth and fruitingin that experiment. Pot experiments with seven cultivarsshowed a similar trend of increased number of healthy lateralroots. Although there is a clear indication of reduced diseaselevels (per cent root affected) it is possible that there was somedilution of the disease effect due to enhanced root growth asa consequence of the chemical treatment. This needs to beinvestigated in relation to understanding the mechanism ofaction of the chemical. In the field experiment at ACRI,acibenzolar-S-methyl decreased disease severity on tap rootsbut did not improve the health of lateral roots, possibly due tohigh competition of nut grass. Our assessment of lateral roothealth is a novel method to measure the severity of blackroot rot in crop plants. Although this method is more time-consuming than the conventional assessment of symptoms onthe tap root, it appears to give a better measure of the relativestatus of the disease.

Soaking cotton seed in acibenzolar-S-methyl reducedseedling establishment substantially in the field experimentat ACRI and in another field experiment in which T. basicolawas not present in the soil (unpublished data). Anecdotalobservations suggested that acibenzolar-S-methyl was toxic

to cotton at 100 μg/mL for 3 h of soaking but toxicity wasnot observed at 25 or 50 μg/mL for the same period ofsoaking (Mondal 2001). This effect was not observed inthe pot experiments or in the field experiments in whichacibenzolar-S-methyl was applied as a seed coating or in-furrow spray. Therefore, toxicity of acibenzolar-S-methyl tocotton at 25 or 50 μg/mL for 3 h of soaking was not thereason for poor establishment. During the soaking process,some of the standard fungicide seed coating was removedfrom the seed and this may have reduced the effectivenessof these fungicides against damping-off pathogens in thefield experiments. However, imbibition during the soakingincreased the size of seeds. A subsequent investigationshowed that soaking for 3 h in water reduced the numberof seeds delivered by the mechanical planter by 20%in comparison with unsoaked seeds (unpublished data).This imbibition effect on the rate of sowing appears tobe partly responsible for the reduced plant stand in thesoaking experiment at ACRI, as the control treatment wasnot soaked. Imbibition did not occur before sowing in thein-furrow spray and seed coating experiments and theseapplication methods would be preferable for commercialuse of acibenzolar-S-methyl.

Seed dressing with 3.3 mg acibenzolar-S-methyl/kg ofseed was not effective against black root rot at the Warren site.In the seed dressing experiment at Wee Waa, we used 6 mg/kgof seed, which was equivalent to the amount of acibenzolar-S-methyl imbibed in the seed soaking experiment at Narrabri(calculated using observations of the volume of waterimbibed in 3 h by cotton seed). As seed dressing with6 mg/kg, or soaking at an equivalent rate, induced SAReffectively, it appears that 3.3 mg/kg was too low to induce aneffective resistance response in cotton. Mondal (2001) alsoobserved that foliar spray of acibenzolar-S-methyl as low as6.3 mg/kg effectively induced SAR in green bean againstColletotrichum lindemuthianum.

Foliar application of acibenzolar-S-methyl at rates greaterthan 50 μg/mL suppressed cotton growth in the field.McFadden et al. (2001) also observed phytotoxic effects incotton with increasing rates of application of acibenzolar-S-methyl as a foliar spray and a soil drench. Our foliar


applications of acibenzolar-S-methyl in the field wereprobably not effective because black root rot was welldeveloped in those crops at the time of application. Anyresistance response that might have occurred was clearlytoo late to prevent infection and/or too weak to promoterecovery of the seedling.

T. basicola infects young roots soon after germination.Seed treatment is a better option than foliar treatmentto activate SAR against T. basicola because it pre-emptsinfection; however, a period of hours to days is requiredto express SAR after treatment (Kuc 1982; Kessmann et al.1994b). Seed treatment requires no added labour costs tothe farmer. In our experiments, the most effective rate forapplication of acibenzolar-S-methyl for control of black rootrot was 6 mg/kg seed (or the equivalent with seed-soaking),which was not phytotoxic. SAR induced by acibenzolar-S-methyl could be integrated with other control methods forblack root rot. The combined use of acibenzolar-S-methyl andfungicides to control fungal pathogens has been demonstratedin nursery plants (Ali et al. 2000). Furthermore, inducedresistance against Alternaria leaf spot, Verticillium wiltand bacterial blight of cotton using foliar application ofacibenzolar-S-methyl has been demonstrated (Colson-Hankset al. 2000). The potential for using acibenzolar-S-methylin an integrated disease management approach would beenhanced if seed treatment proves to be effective against otherpathogens of cotton.

The expression of the cascade of defence responsesinvolved in SAR occurs over a period of hours to days (Kuc1982; Kessmann et al. 1994b). SAR genes that are associatedwith pathogen recognition, transduction of defence signals,and expression of pathogenesis-related proteins have beenwell documented (Zhao and Guo 2003). Benhamou andBelanger (1998a, b) observed that acibenzolar-S-methylenhanced the resistance of cucumber and tomato againstPythium and Fusarium, respectively. The heightenedresistance was associated with increases in the depositionof electron-opaque material onto the inner surface of cellwalls and intercellular spaces, and increased accumulationof phenolic compounds, at the site of pathogen penetration.McFadden et al. (2001) found that inoculation of cottonwith V. dahliae increased the expression of several defencegenes. Acibenzolar-S-methyl enhanced resistance againstV. dahliae but did not affect the expression of the defencegenes examined in that study. Zeringue and Shih (2000)found that application of chitosan, methyl jasmonate ora heat soluble mycelial extract to cotton seed induced theproduction of numerous compounds in seedlings and matureplants. The defence responses induced by acibenzolar-S-methyl in cotton are clearly complex and yet to befully resolved.

In conclusion, among the different methods of applicationused for acibenzolar-S-methyl, seed soaking, in-furrowspray and seed dressing (one out of two trials) induced

SAR against black root rot in cotton. To our knowledge,this is the first successful use of a chemical seed treatmentto induce SAR against a soilborne plant pathogen in thefield. Repetitive monocultures of cotton have contributedto the current pandemic of black root rot in Australiancotton (Nehl et al. 2004). No single method gives completecontrol and resistant cultivars are not available (Allen2001). Acibenzolar-S-methyl gives only a partial controlof black root rot but could be an important componentof an integrated management approach, particularly as ithas potential to induce a SAR against a broad spectrumof pathogens.

Acknowledgements

This research was funded by the Cotton Research andDevelopment Corporation and NSW Department of PrimaryIndustries. We thank Dr Om Jhorar for his assistancewith assessment of fruiting and yield in the experimentat Moree.

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Received 6 August 2004, accepted 24 May 2005

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