improvement of seed quality and field emergence offusarium moniliforme infected sorghum seeds using...

Journal of the Science of Food and Agriculture J Sci Food Agric 79 :206–212 (1999)

Improvement of seed quality and fieldemergence ofFusarium moniliforme infectedsorghum seeds using biological agentsN S Raju,1 S R Niranjana,1 G R Janardhana,1 H S Prakas h,1 H Shekar Shetty1

and S B Mathur21 Seed Pathology Laboratory , Department of Studies in Applied Botany,Manas agangotri , Univers ity of Mys ore-570 006, Karnataka, India

2 Danis h Government Ins titute of Seed Pathology for Developing Countries , PO Box 34, Ryvangs Alle 78, DK-2900, Hellerup, Copenhagen,

Denmark

Abstract : Five diþ erent cultivars of sorghum seeds infected with a varied degree of Fusarium monili-

forme were treated with biocontrol agents. Pure cultures of Pseudomonas ýuorescens, Trichoderma

harzianum and Chaetomium globosum at the rate of 1 Â 108 cfu g—1 and talcum based formulations of

(28 Â 107 cfu g—1), (19 Â 107 cfu g—1) and (4 Â 106 cfu g—1) at the rate of 6 g kg—1 and 10 g kg—1 of seeds

were used, respectively. The treated seeds were evaluated for per cent reduction of F moniliforme,

seed germination, vigour index and üeld emergence. It was found that the pure culture of P ýuores-

cens was more eþ ective in reducing the F moniliforme infection followed by T harzianum and C

globosum than the Bavistin treated and untreated seeds. Formulations of P ýuorescens were eþ ective

in reducing the F moniliforme infection and also in increasing the seed germination, vigour index and

üeld emergence, followed by T harzianum and C globosum treatments in comparison with control.

1999 Society of Chemical Industry(

Keywords: sorghum; Fusarium moniliforme ; seed quality ; üeld emergence; biological agents

INTRODUCTION

Sorghum (Sorghum bicolor (L) Moench) is the thirdmost important cereal crop in India produced forfood in the semi-arid regions. This crop is vulnerableto 23 diþerent fungal diseases1 of which head blightand grain mould caused by F moniliforme isresponsible for signiücant crop yield losses of up to30%.2 Grain infected with F moniliforme appears tobe completely pink and black and such seeds havepoor germination capacity. Therefore they cannot beused for seed purposes.3 Although Thiram andBavistin are known to be eþective against a widerange of fungal pathogens,4 including seed-borne Fmoniliforme, their eþect on the seed quality is poorlyunderstood. Moreover, the fungicides have failed toimprove the seed quality parameters. Therefore, inthe present study, an attempt has been made to usebiological agents in place of chemical fungicides, notonly to reduce the F moniliforme incidence but also toimprove the seed qualities of sorghum.

MATERIALS AND METHODS

Source of seed sample

Sorghum seeds of üve diþerent cultivars, namely,SGMN 1018, SGMN 10115, SGMN 1044, SGMN

2012 and SGMN 2037, were collected from theInternational Crops Research Institute for SemiaridTropics (ICRISAT), Patancheru, Hyderabad, India,grown in Kharif, 1994.

Multiplication and preparation of biocontrol agents

The antagonistic strains of Pseudomonas ýuorescens,Trichoderma harzianum and Chaetomium globosumwere isolated from the native soil, maintained onnutrient medium and then used as potential bio-control agents. P ýuorescens was mass multiplied byinoculating on Kings ‘B’ medium and incubated at26^ 1¡C in a BOD incubator. After 48h of incu-bation, culture broth was centrifuged at 10000] gfor üve minutes. The pellet was suspended in steriledistilled water for the seed treatment. P ýuorescensformulation (28] 107 cfu g~1) was prepared bymixing 100ml of P ýuorescens suspension and 25g oftalcum powder under sterile conditions. Carboxylmethyl cellulose (2.5g) was also added to 250g offormulation and stored in the form of talc andpacked in polythene bags under ambient conditions.5

Fungal bioagents T harzianum and C globosumwere mass multiplied on potato dextrose agar (PDA)petri plates and incubated at 22^ 2¡C under 12/12h/h cycles of a dark and NUV light for 10 days. Pure

* Corres pondence to : H Shekar Shetty, Seed Pathology Labora-tory, Department of Studies in Applied Botany, Manas agangotri,

Univers ity of Mys ore - 570 006, Karnataka, India

(Received 19 Augus t 1997 ; revis ed vers ion received 3 March 1998;

accepted 15 April 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50 206

Biological agents for seed quality improvement

conidial mass was suspended in sterile distilled waterfor the seed treatment. Formulations were preparedby mixing the conidial mass with talcum powder(1 : 10w/w) and packed in polythene bags and storedunder ambient conditions of 23^ 2¡C and used asand when required.

Seed treatment with biocontrol agents

Sorghum seeds were treated with cell or conidial sus-pension of P ýuorescens, and conidia of T harzianumand C globosum at the rate of 1] 108 cfu g~1 bymixing 400 seeds with 5ml of cell/conidialsuspension. Formulations of P ýuorescens(28] 107 cfu g~1), T harzianum (19] 107 cfu g~1)and C globosum (4] 106 cfu g~1) in the form ofslurry treated to sorghum seeds at the rate of 6g kg~1and 10g kg~1 of seeds, respectively. After 24h oftreatment the seeds were air dried and then weresubjected to F moniliforme incidence, germinationtest, vigour index and üled emergence test.

Seed treatment with Bavistin

Commercially available fungicide Bavistin (methyl-2-benzimidazole carbamate) was used, since it is astandard fungicide recommended for seed protectionin India. Sorghum seeds were treated with Bavistinat the rate of 2g kg~1 of seed as slurry.

Screening for Fusarium moniliforme incidence in

the seeds

Seeds (400) of each cultivar were screened to recordthe per cent incidence of F moniliforme by StandardBlotter Method.6

The germination test

Treated seeds (400) were also placed between paperrolls in four replicates of 100 seeds each for germi-nation. The rolls were kept at 23^ 2¡C in a seed ger-minator. The ürst count of normal seedlings wastaken on the fourth day and the second count on theseventh day.

Vigour index (VI)

Normal seedlings were evaluated for vigour index.The root and shoot lengths of the normal seedlingswere measured and vigour index (VI) was calculatedusing the formula:7

VI \ (mean root length] mean shoot length)

] percentage germination.

Field emergence test

Fields were ploughed well and the soil was levelled,fertiliser NPK was added at the rate of80 : 60 : 40kg ha~1. Sorghum seeds treated with bio-agents, Bavistin and untreated seeds were taken inthree replicates of 100 seeds each. They were sown inthree rows (5m) which were distributed randomly,with an isolation distance of 45cm between rows and

15cm between plants, respectively. The üelds wereirrigated immediately after sowing. The üeld emer-gence was recorded seven days after sowing.

Statistical analysis

Data obtained with each cultivar was taken as repli-cate and values obtained were subjected to arcsintransformation and then analysis of variance.

RESULTS

Effect of biocontrol agents on incidence of Fusarium

moniliforme

The eþect of biocontrol agents on per cent reductionof F moniliforme incidence in treated seeds overcontrol are presented in Table 1. Pure culture of Pýuorescens at the rate of 1] 108 cfu g~1 reduced theF moniliforme incidence by 76%. On the other hand,reduced incidence of 69% and 70% was recorded forthe talcum based formulations at the rate of 6g kg~1and 10g kg~1 of seed, respectively. Pure culture of Tharzianum at the rate of 1] 108 cfu g~1 reduced theincidence of F moniliforme by 67%, whereas talcumbased formulation of the same at the rate of 6g kg~1and 10g kg~1 seeds reduced the incidence by 65%and 66%, respectively. The pure culture of C glo-bosum reduced the incidence of F moniliforme by65%. The talcum based formulation of the samereduced the incidence by 62% and 63%, respectively.The Bavistin treatment reduced the incidence by57% over control.

Effect of biological agents on seed germination

An increase in seed germination in all cultivars ofsorghum treated with biocontrol agents and Bavistinwere compared with the values of the untreated seedsand they are presented in Table 2.

Treatment of sorghum seeds with the pure cultureof P ýuorescens at the rate of 1] 108 cfu g~1increased the germination by 25% whereas thetalcum based formulations of the same increased thegermination by 19% and 14%, respectively. T har-zianum pure culture increased the germination by17%. Formulations of the same at 6g kg~1 and10g kg~1 increased the germinations by 14% and16%. Pure culture C globosum increased the germi-nation by 13%. However, the talcum based formula-tions of C globosum at the rate of 6g kg~1 and10g kg~1 seeds increased the germination by 11%and 12%, respectively. The chemical fungicideBavistin increased the germination by 5%.

Effect of biocontrol agents on seed vigour

P ýuorescens pure culture (1] 108 cfu g~1) increasedthe vigour by 59% and the talcum based formula-tions of P ýuorescens increased the seed vigour by39% and 41%, respectively. T harzianum pureculture increased the vigour by 28% and talcumbased formulation increased the vigour by 23% and28%. Pure culture of C globosum increased the seed-

J Sci Food Agric 79 :206–212 (1999) 207

NS Raju et al

Table 1. Effect of different biocontrol agents on the incidence of s eed-borneF moniliforme in five different cultivars of s orghum on

s tandard blotter method

Treatments Incidence of F. moniliforme

(%)

Cultivars Mean1

SGMN 1018 SGMN 10115 SGMN 1044 SGMN 2012 SGMN 2037

1 Control 53.31 57.73 54.76 52.12 53.31 54.24] 0.96h2 Pure culture of

Ps eudomonas 9.97 12.52 13.81 13.56 13.31 12.63^ 0.70afluores cens

(1] 108 cfu gÉ1)

3 Formulation of

P fluores cens 15.18 15.34 15.46 16.74 16.43 15.03^ 0.57b(6 g kgÉ1)

4 Formulation of

P fluores cens 12.92 14.18 15.89 15.89 15.34 14.84^ 0.48bc(10 g kgÉ1)

5 Pure culture of

Trichoderma

harzianum 16.11 16.95 18.91 18.15 17.75 17.57^ 0.53bcd(108 cfu gÉ1)

6 Formulation of

T harzianum 16.74 18.43 20.00 18.91 19.09 18.13^ 0.55bcd(6 g kgÉ1)

7 Formulation of

T harzianum 16.43 17.46 19.64 18.43 18.72 18.89^ 0.73bcd(10 g kgÉ1)

8 Pure culture of

Chaetomium 20.70 16.43 20.00 18.43 18.91 18.89^ 0.73bcdglobos um

(108 cfumlÉ1)

9 Formulation of

C globos um 22.14 18.43 21.72 20.27 20.27 20.56^ 0.65bcdef(6 g kgÉ1)

10 Formulation

C globos um 21.56 17.95 21.13 19.37 19.37 19.87^ 0.65bcde(10 g kgÉ1)

11 Bavis tin 24.88 24.58 19.37 23.17 24.88 23.37^ 1.05g(2 g kgÉ1)

1 Values given are means ^SE. Figures followed by different letters in rows differ s ignificantly when s ubjected to DMRT (PO 0.05)

ling vigour by 25% whereas formulations of C glo-bosum increased the seedling vigour by 17% and21%, respectively. As a result the Bavistin treatmentincreased the vigour by 20% (Table 3).

Effect of biocontrol agents on field emergence

Pure culture of P ýuorescens increased the üeld emer-gence by 22% and the formulations of the same by17% and 18%, respectively. Pure culture of T har-zianum at the rate of 1] 108 cfu g~1 increased theüeld emergence by 15% and the formulations of thesame increased the üeld emergence by 12% and13%, respectively. The pure culture of C globosumincreased the üeld emergence by 10% whereas thetalcum based formulations of C globosum increasedthe üeld emergence by 9% and 10%, respectively(Table 4). The Bavistin increased the üeld emer-

gence by 6%.

DISCUSSION

In the present investigation, üve diþerent sorghumcultivars having diþerent ranges of F moniliformeinfected seeds were treated, with P ýuorescens, Tharzianum and C globosum and evaluated for thereduction of F moniliforme incidence, eþect on theseed germination, seedling vigour, üeld emergenceand grain yield. All the antagonistic organismsreduced F moniliforme incidence signiücantly, butthey also increased seed germination, seedling vigourand üeld emergence, although the results varied withdiþerent biocontrol treatments.

There is hardly any literature available on theeþect of P ýuorescens, T harzianum and C globosum

208 J Sci Food Agric 79 :206–212 (1999)


Table 2. Effect of different biocontrol agents on s eed germination of s orghum cultivars

Treatments Germination

(%)

Cultivars Mean1


1 Control 57.61 57.73 57.42 58.24 58.05 57.81^ 0.14a2 Pure culture of

Ps eudomonas 73.26 72.85 72.24 73.05 71.57 72.59^ 0.30hfluores cens

(1] 108 cfu gÉ1)

3 Formulation of

P fluores cens 68.44 70.00 68.87 69.47 67.86 68.92^ 0.73cdefg(6 g kgÉ1)

4 Formulation of

P fluores cens 69.47 64.16 69.73 63.43 63.94 66.12^ 1.42cd(10 g kgÉ1)

5 Pure culture of

Trichoderma 69.12 69.47 66.42 66.25 67.21 67.69^ 0.67cdefharzianum

(108 cfu gÉ1)

6 Formulation of

T harzianum 65.65 67.78 66.82 65.12 66.42 66.35^ 0.46cd(6 g kgÉ1)

7 Formulation of

T harzianum 68.03 68.87 67.62 65.88 66.65 67.12^ 0.52cde(10 g kgÉ1)

8 Pure culture of

Chaetomium 64.90 64.90 66.03 65.12 66.03 65.39^ 0.26cdglobos um

(108 cfu gÉ1)

9 Formulation of

C globos um 64.53 65.27 64.33 63.65 64.23 64.40^ 0.26c(6 g kgÉ1)

10 Formulation

C globos um 64.97 65.73 64.97 65.05 64.97 65.13^ 0.14c(10 gmgÉ1)

11 Bavis tin 59.34 61.00 65.42 58.89 60.20 60.97^ 1.17b(2 g kgÉ1)

1 Values given are means ^SE. Figures followed by different letters s ignificantly differ when s ubjected to DMRT (P¹ 0.05)

to reduce F moniliforme in sorghum seed. F monili-forme is an important seed-borne pathogen onsorghum and it reduces the seed germination, via-bility and üeld emergence,2 as well as nutritionalqualities.8 To control F moniliforme, chemical fungi-cides such as Bavistin and Thiram are used.9,10 Pýuorescens is known to reduce the seed-borne infec-tion of fungi in radish,11 and T harzianum is knownto control many fungal diseases such as seed rot andearly stem rot in maize and sorghum caused by Fmoniliforme, Rhizoctonia solani and Pythiumultimum.12 Seed treatment formulation of Tri-choderma and Gliocladium for biological control ofMacrophomina phaseolina in sesamum has beenreported.13 T koningii has been used to control theweb blight of cowpea caused by R solani.14 Theculture ültrate of C globosum reduced downy mildew

incidence in pearl millet.15All the antagonists reduced F moniliforme inci-

dence in all the sorghum cultivars and the resultsindicated that the biological control agents are moreeþective than the recommended chemical Bavistin toreduce F moniliforme in sorghum seeds.

Among the antagonists, P ýuorescens was moreeþective in reducing seed-borne infection F monili-forme in sorghum seeds. The pure culture of all thethree antagonists used were more eþective inreducing F moniliforme infection than the talcumbased formulations. This may be due to the loss ofviability of spores in the formulation.13

All the three antagonists tried in our experimentincreased the seed germination signiücantly. Thechemical fungicide Bavistin treated seeds showedhigher seed germination than the untreated ones, and

J Sci Food Agric 79 :206–212 (1999) 209

NS Raju et al

Table 3. Effect of different biocontrol agents on vigour index (VI) of s orghum cultivars

Treatments Vigour index (VI ) Mean1

Cultivars



P fluores cens 1201.75 1316.25 1225.75 1191.00 1260.00 1238.95^ 22.65f(1] 108 cfu gÉ1)

3 Formulation of

P fluores cens 1137.00 1120.00 1021.00 1041.25 1100.00 1083.85^ 22.53bcde(6 g kgÉ1)

4 Formulation of

P fluores cens 1157.75 1227.75 1061.50 1078.25 1099.50 1124.95^ 30.41bcde(10 g kgÉ1)

5 Pure culture of

T harzianum 997.00 993.75 992.11 1022.50 984.25 997.92^ 6.49bcd(1] 108 cfu gÉ1)

6 Formulation of

T harzianum 917.75 955.00 962.00 967.50 973.75 995.20^ 9.85b(6 g kgÉ1)

7 Formulation of

T harzianum 987.25 980.00 991.75 987.00 980.00 985.20^ 2.28bc(10 g kgÉ1)

8 Pure culture of

Chaetomium 980.10 970.00 985.75 960.00 965.00 972.71^ 4.75bcglobos um

(1] 108 cfu gÉ1)

9 Formulation of

C globos um 870.50 863.25 959.50 940.50 910.25 908.80^ 18.86b(6 g kgÉ1)

10 Formulation

C globos um 888.75 905.00 982.75 987.75 931.25 939.10^ 20.03b(10 g kgÉ1)

11 Bavis tin 890.50 897.50 948.75 971.25 978.75 937.35^ 18.40b(2 g kgÉ1)

1 Values given are means ^s tandard error. Figures followed by different letters s ignificantly differ when s ubjected to DMRT (P¹ 0.05)

the antagonist treated seeds also showed still higherper cent of seed germination. This may be due to thereduction of F moniliforme incidence. P ýuorescenshave been reported to increase the germination intreated seeds of radish in commercial greenhouses.11T harzianum has shown increased seed germinationand seedling vigour of lettuce seeds.10 It has beensuggested that some Pseudomonas have the ability tosynthesise hydrogen cyanide which is known toinhibit the expression of pathogenic fungi,17 and alsothe ability to hydrolyse Fusaric acid produced bysome Fusarium spp.18 P ýuorescens is known toproduce the plant growth regulators like gibberellins,cytokinins and indole acetic acid.19 The ability of Pýuorescens to increase the üeld emergence, vigourand germination was attributed to the plant growthpromoting substances produced by the bacteria thatcould act to enhance various stages of plantgrowth.20h22

T harzianum is known to produce chemical com-pounds such as chytinolytic enzymes, glucanase and

proteases.23 The antagonistic nature of C globosumwas not studied well but the secondary metabolites ofC globosum have reduced the disease incidence inpearl millet.15 All the üve diþerent varieties ofsorghum have shown a much increased vigour whentreated with biological agents than when treated withthe chemical agent Bavistin. The reason for theincrease in vigour may be certain chemicals producedby P ýuorescens, T harzianum and C globosum whichare known to have increased growth rate as reportedby Lynch and Hobbie16 and Kimura et al.24 Asimilar type of higher üled emergence was alsoobserved in the üeld conditions in all the diþerentcultivars of sorghum. In recent years much attentionhas been given to non-chemical systems for seedtreatment as well as to protect them against seed-borne pathogens. The present study has shown thatbiological control agents like P ýuorescens, T harzia-num and C globosum, which are eco-friendly andmuch more eþective, can be used in the place ofchemical fungicides.

210 J Sci Food Agric 79 :206–212 (1999)


Table 4. Effect of different biocontrol agents on field emergence of s orghum in different cultivars

Treatments Cultivars Mean1



Ps eudomonas 72.85 70.63 70.63 73.05 73.05 72.04^ 0.57efluores cens

(1] 108 cfu gÉ1)

3 Formulation of

P fluores cens 70.36 69.30 69.30 66.03 70.00 69.99^ 0.76bcd(6 g kgÉ1)

4 Formulation of

P fluores cens 70.36 65.73 69.73 65.97 71.57 69.47^ 0.93bcd(10 g kgÉ1)

5 Pure culture of

Trichoderma 68.03 70.00 66.82 64.38 70.36 67.91^ 1.09bcdharzianum

(108 cfu gÉ1)

6 Formulation of


7 Formulation of


8 Pure culture of

Chaetomium 63.58 63.22 65.80 65.42 67.78 65.16^ 0.86bcglobos um

(108 cfu gÉ1)

9 Formulation of

C globos um 63.22 61.82 64.38 64.53 67.78 64.30^ 0.98b(6 g kgÉ1)

10 Formulation

C globos um 65.12 62.24 64.53 65.12 68.03 65.00^ 0.92bc(10 g kgÉ1)

11 Bavis tin 63.58 60.67 60.87 61.14 64.38 62.16^ 0.77b(2 g kgÉ1)

1 Values given are means ^s tandard error. Figures followed by different letters in rows differ s ignificantly when s ubjected to DMRT(P¹ 0.05)

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improvement of seed quality and field emergence offusarium moniliforme infected sorghum seeds using...

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