Bioinoculants in plant growth promotion and disease management

M. ALAM Microbiology & Plant Pathology Division, CIMAP, Lucknow

The crop production by conventional agriculture system could not keep pace with the pace of human population increase. To maintain the productivity sufficient for human consumption, modern agriculture system was evolved where inorganic fertilizers and highly effective chemicals are commonly used . But they are not a long-term solution to the crop, human and animal health and environment because their intensive and indiscriminate use posed many serious problems.

Deleterious effect of chemical pesticides

I)Development of resistant strains of the pathogen: ii)Build up of harmful residues in the edible plants: "Pesticides in the Diets of Infants and Children" A report of National Research Council of the National Academy of Sciences (1998) concluded that: People in this age group could be at considerable health risk from consumption of foods containing pesticide residues iii)Non-target side effect of beneficial micro flora iv)Environmental pollution : According to WHO estimate every year approx. 7.5 lakh people get sick due to pesticide poisoning14000 die in agony.

To avoid such problems, attention have been diverted towards the use of beneficial and effective microorganisms as bioinoculant for improvement in the productivity and disease management. They are a distinct possibility for the future and can be successfully exploited in modern agriculture without affecting our precious ecosystem.

Bioinoculant
Preparations containing active strains of microorganisms in sufficient numbers  PGPR & N-Fixing Bacteria  Actinomycetes  AM Fungi  Antagonistic Fungi

N2 Fixing Bacteria Rhizobium, Azotobactor Azospirillum P. Fluorescens B. subtilis

Streptomyces

Trichoderma Paecilomyces

AM Fungi

BIO INOCULANT

Functions of Bioinoculant  Improve soil quality& soil health  Enhance crop production and protection,  Conserve natural resources, and  Ultimately create a more sustainable agriculture and environment

Beneficial microorganisms
     Can fix atmospheric nitrogen, Decompose organic wastes residues and detoxify pesticides, Suppress plant diseases and soil-borne pathogens, Enhance nutrient cycling, and Produce bioactive compounds such as vitamins, hormones and enzymes that stimulate plant growth.

Bioinoculant act by
     Fixing atmospheric nitrogen, Decomposing organic wastes residues and detoxifying pesticides, Suppressing plant diseases and soil-borne pathogens, Enhancing nutrient cycling, and Producing bioactive compounds such as vitamins, hormones and enzymes that stimulate plant growth.

Mycorrhiza
By solubilizing insoluble phosphorus and makes available to the plant.Thus enhance the plant growth. Compete for nutrition with other soil microorganisms and there by suppress their growth. Occupy root surface to check infection by other pathogen. Induce resistance in the host against pathogens.

Plant Growth Promoting Rhizobacteria (PGPR) * Reducing population of the pathogen by

producing siderophore, lytic enzymes etc. Preventing the pathogen from court of infection on the plant Limiting disease development after infection

Streptomyces
Act by antibiosis through production of powerful antibiotic & lytic enzymes. Compete for the nutrition

Bioinoculant Requirements
 
Presence of suitable substrate and Optimum conditions for metabolizing their substrates Including available water, oxygen (depending on whether the microorganisms are obligate aerobes or facultative anaerobes), pH and temperature of their environment

OBJECTIVES
Isolation, identification and screening of potent bioinoculant Application and determination of their competitive ability and survival proliferation Selections and their evaluation individually or in combinations Evaluation and optimization of inoculum doses Development of methods for mass production Testing of the product for growth and disease management

Effect of G. aggregatum treatment on the growth of Java citronella

Growth Treatments
No. of tillers Herb Yield (g) Root Yield (g) Oil Content (%)

Ga (T1) 15 d Ga + Pa (T2) Sim Ga + Pa (T3) Pa (T4) Untreated Control (T5)

9 6
5.5

35.0a
27.6b 16.2c

32.2a
20.4b 8.0c

1.44a
1.35a 0.98c

2.5
8

12.6c
22.6b

5.6c
17.2b

0.92c
1.18b

anagement of chlorosis in Java citronella by slip treatment with G. aggregatum

Ga (T1)

Chlorosis of Java Citronella

% Chlorosis

100 90 80 70 60 50 40 30 20 10 0 2 weeks 4 weeks 8 weeks

15 d Ga + Pa (T2)

Sim Ga + Pa (T3)

Pa (T4)

Untreated Control (T5)

Effect of G. aggregatum and P. aphanidermatum treatment on the N, P and K contents and acid phosphatase activity of Java citronella

mg g-1 dw 10 12 14 16 0 Ga (T1) 15 d Ga + Pa (T2) Sim Ga + Pa (T3) Pa (T4) Untreated Control (T5) 2 4 6 8

mg g-1 dw 10 15 20 25 30 0 Ga (T1) 15 d Ga + Pa (T2) Sim Ga + Pa (T3) 5

mM p nitroph m-1 mg-1

Shoot Root

Pa (T4) Untreated Control (T5) Root

m g g -1 d

Shoot

10

15

20

25

0
Ga (T1) 15 d Ga + Pa (T2) SimGa + Pa (T3) Pa (T4) Untreated Control (T5)

G a (T 1 )

15 d Ga + P a (T 2 )

S im G a + P a (T 3 )

P a (T 4 )

S ho o t Root

U n tre a te d C o n tro l (T 5 )

Effect of different AM inoculation on the productivity of geranium

Treatments Av. herb yield/pot (g) 217.3 269.5 335.4 Essential oil content (%) 0.17 0.17 0.15 0.18 0.16 0.16 0.21 0.18 0.15 0.17 Av. oil yield/pot (ml.) 0.35 0.46 0.50 0.66 0.42 0.42 0.52 0.60 0.42 0.44 Root colonization (%) 75.0 60.5 Spores/g soil

Control GA GF GM AK1 AK2 AK3 GA+GM GA+GF GA+GF+GM

24 26 27 32 36 36 60 60 38

371.3
264.5 266.5 246.6 332.5

82.0
65.7 64.3 100.0 90.0

380.9
260.9

82.5
68.0

C. cassicola S. sclerotiorum

C. acutatum A potential strain of Streptomyces showing antagonistic activity against plant pathogens

Effect of Streptomyces culture filtrate on the growth of different isolates of Rhizoctonia solani
4-day-old CF Test organism CF Conc. (%) Inhibition Over control (%) 44.4 55.5 68.8 61.1 100 100 6-day-old CF Inhibition Over control (%) 66.6 77.7 83.3 69.0 83.3 100

Growth in 3days(mm)

Growth in 3days(mm)

0(control) R. solani (OP-Rh1) 5 10 20 0(control) R. solani (Pyr-Rh1) 5 10 20

90 50 40 28 90 35 Nil Nil

90 30 20 15 90 28 15 Nil

Effect of Streptomyces culture filtrate on the growth of different phytopathogenic fungi

Test organism 4-day-old CF Inhibition Over Growth in control 3days (%) (mm) 65 15 0 0 83 65 50 30 85 0 0 0 76.9 100 100 21.6 39.8 63.9 100 100 100 6-day-old CF Inhibition Growth in 3days (mm) 63 0 0 0 84 68 65 35 84 0 0 0 Over control (%) 100 100 100 19.0 22.6 58.3 100 100 100

CF Conc. (%)

0(control) C. acutatum (Ger-cola) 5 10 20 0(control) F. moniliforme (Rosm-fm) 5 10 20 0(control) C. andropogonis (Aloe-Ca) 5 10 20

Isolation of plant growth promoting rhizobacteria (PGPR) from the soil

Soil Samples Medi a Colonies/g soil

Colonies characters
small, circular with yellow center & white border small, circular, yellow small, white, circular with rough ends white, circular and little larger than that on NA 1-large, circular, light yellow center; 2-very small,white,circular 1-large,circular,light brown in center 1-large,circular, light brown in center & white outside; 2-small,white, circular 1-large,circular, light brown in center 2-small,white, circular

Geranium (3) NA PAM Geranium (5) NA PAM Geranium (11) NA

8x10-6 17x10-6 7x10-6 3x10-6 6x10-7 10x10-7 8x10-7 9x10-6 20x10-6 10x10-6 7x10-6

PAM Geranium (13) NA

PAM

Antagonistic activity of different Bacillus strains against pathogenic fungi

B1 vs A. alternata, C. acutatum F. monaliformae & R. solani

B2 vs A. alternata & C. acutatum

B2 vs C. andropogonis and R. solani

B3 vs C. acutatum & C. capsici

Different strains of Bacillus sp. and Pseudomonas sp. showing inhibition zone against plant pathogenic fungi.
Inhibition zone (in mm) against the growth of Antagonist Strain Origin Fusarium oxysporum Bacillus sp. Do B (CAG) B (Aspergill us) B2 B3 B4 Geranium
rhizosphere
Curvularia andropogonis Alternaria alternata Aspergillus niger

20

15

Chlorophytum

rhizosphere

20

20

22

Do Do Do
Pseudomonas sp.

Soil Do Geranium
rhizosphere

20 15 07

30 05 -

15 15 11

07 08

Psf1

Do

15

12

15

21

Different strains of Bacillus sp. and Pseudomonas sp. showing inhibition zone against plant pathogenic fungi.
Inhibition zone (in mm) against the growth of Antagonist Strain Origin C. capsici C. acutatum R.solani (Rosm) R. solani (Pyre.)

Bacillus sp.

B (CAG) B (Aspergill us)

Geranium
rhizosphere
Chlorophytum

15

25

06

25

Do

rhizosphere

20

32

07

Do Do Do
Pseudomonas sp.

B2 B3 B4

Soil Do Geranium
rhizosphere

15 25 10

20 12

20 07 -

10 10

05

Psf1

Do

20

30

10

Effect of different strains of Bacillus culture filtrate on the spore germination of plant pathogenic fungi.
Spore Germination (%) After 6 hrs. Inhibition over Control (%)

Antagonist

CF Conc (%)

A.alternata

C. capsici

A. alternata

C. capsici

C B (Aspergillus) 10 20 80 C 10 B4 20 80

100 69 59 06 100 70 55 9

100 68 56 03 100 50 45 05

31 41 94 30 45 91

32 44 97 50 55 95

Bacillus IBA

C
Effect of Bacillus on the growth of geranium cuttings

Effect of Bacillus treatment on rooting and survivality of Geranium

Treatment & date 20-11-01 B1 IBA Control 05-12-01 B1 IBA Control 1-1-02 B1 IBA Control Rooting initiation % survivality Two node 70.0 69.23 60.0 100 100 90.0 100 100 97.0

Normal 100 100 87.5 100 100 90 100 100 98.0

Apical 100.0 95.83 83.33 100.0 100.0 95.83 100 100 100

25 27 32 26 28 35 22 24 27

Performance of Bacillus strain B1

Early Root Initiation

Promote Plant Growth

Enhanced Root Biomass Enhanced Biomass

Geranium

Chlorophytum

Effect of Bacillus treatment on rooting and survivality of Geranium

Treatment & date 20-11-01 B1 IBA Control 05-12-01 B1 IBA Control 1-1-02 B1 IBA Control Rooting initiation % survivality Two node 70.0 69.23 60.0 100 100 90.0 100 100 97.0

Normal 100 100 87.5 100 100 90 100 100 98.0

Apical 100.0 95.83 83.33 100.0 100.0 95.83 100 100 100

25 27 32 26 28 35 22 24 27

Influence of bioinoculant on the productivity of Geranium

350 300 250 200 150 100 50 0
400 350 300 250 200 150 100 50 0

al on e

ol

Th 1

G A

Cont r ol

GA + P sf 1+ A1

GA + P sf 1 +A 1 +

GA + P sf 1 +A 1 +

C on tr

400 350 300 250 200 150 100 50 0

C on tr

+P sf

G A

G A

G A

G A

+T h1

ol

Ps f

1+Th1

Bioinoculant increases herb yield of Geranium

40 0 350 300 250
200 350 300 250

200 150 100 50 0 Control GA GM GF

150 100 50 0 Cont rol GA +GM GA +GF GA +GF+GM

40 0 350 300 250 200 150 100 50 0

Effect of different bio-inoculants on the productivity of geranium

Treatments Control Av. Herb Yield/pot (g) 153 Oil Content (%) 0.16 Oil Yield/pot 0.27 Spore population/g soil Root colonization (%) Survival (%) 100

GA
PSF1 A1 B1 TH1 GA+PSF1 GA+A1 GA+B1

299
252 154 196 250 274 271 311

0.15
0.15 0.16 0.17 0.17 0.16 0.17 0.16

0.48
0.38 0.23 0.29 0.38 0.45 0.43 0.50

24
40 30 28

46.6
55.7 32.8 53.7

Do
Do Do Do Do Do Do Do

GA+TH1 GA+PSF1+A1
GA+PSF1+A1+B1 GA+PSF1+A1+B1+TH1

334 346
321 346

0.15 0.14
0.13 0.16

0.51 0.51
0.46 0.52

24

78.5 64.0
67.5 44.2

Do

30
24 28

Do
Do Do

GA+A1+VC
GA+B1+VC

346
341

0.15
0.15

0.54
0.52

20
12

66.0
53.5

Do
Do

GA=Glomus aggregatum, A1=Streptomyces sp, B1=Bacillus sp., Th1=Trichoderma harzianum, PSF=Pseudomonas fluorescens, VC=vermicompost

GA+PSF+A1 Control Effect of bioinoculant on the growth of geranium

AM fungal infection in the roots of geranium

Bacillus Psf

GA+B1+ Th1
Influence of Bioinoculant on the growth of Safed Musli

Influence of PGPRs on the productivity of Safed Musli

Treatment Plant No.of fingers/pl fresh wt. (g) ant 9.7 10.7 13.3 7.7 14.7 13.0 11.0 8.7 9.7 8.3 45.0 48.6 49.5 49.7 52.6 52.1 76.1 42.7 48.8 47.0 Finger Fresh wt. (g/ Plant) 19.3 26.3 28.4 20.5 35.3 28.7 33.5 21.7 24.7 20.3

Control IBA Unknown(Drench B1 Psf Unknown (foliar) Unknown + B1 Unknown + Psf Gibberlic Acid GA3 + Unknown

Effect of Glomus aggregatum on the biomass, root colonization and P content of pyrethrum in presence of R. solani Shoot Biomass (g) Wilt (%)
100 80 60 40 20 0 Control 15 d pr 5 d po R. solani Sim

80 60 40 20 0
S im
Sim

C ol ontr

AM Colonization
5 4 3 2 1 0

Shoot P content (mg/g)

R. solani

R solani .

15dpr

Control

15 d pr

5 d po

5dpo

Control

Bacillus

Management of wilt disease of pyrethrum by bioinoculants and chemical treatment

Treatments Disease incidence Effectiveness

Uninoculated Control Inoculated (R. solani) Glomus aggregatum 21 days prior Simultaneous 6 days after

Absent Severe Absent Mild Severe

----Highly effective with full protection Partially effective No effect

Streptomyces sp. 21 days prior Simultaneous 6 days after

Severe Mild Severe

No Effect Partially Effective No Effect

C.

Bacillus sp. 21 days prior Simultaneous 6 days after Chemical (Ridomil mancozeb) 21 days prior Simultaneous 6 days after

Absent Mild Severe Severe Absent Mild

Highly effective with full protection Partially Effective No effect No effect Highly effective with full protection Partially effective

D.

Management of pyrethrum wilt by G. aggregatum treatment

G. Aggregatum + R. solani solani Control

Root colonization by G. aggregatum

Influence of bioinoculant on the yield of pyrethrum flowers under field conditions

Fresh flower Yield/plot (g) Treatments R1 R2 R3 Av. Kg/ha

Dry flower Yield/plot (g)

R1

R2

R3

Av.

Kg/ha

Control

513.0

475.4

530.0

506.1

843.5

135.0

118.9

154.1

136.0

226.6

G. intraradices

682.1

543.8

817.3

681.0

1135.0

246.1

167.1

283.5

232.3

387.2

Bacillus

820.0

886.6

876.0

848.3

1434.8

275.0

281.0

273.0

276.3

460.5

T. harzeanum

1104.0

1317.0

1074.0

1165.0

1941.7

340.2

381.8

324.0

348.7

581.1

THANKS