isolation and characterization of mutants of pseudomonas maltophila pm4 altered in chitinolytic...

64 Indian Journal of Microbiology (March 2007) 47:64–71

ORIGINAL ARTICLE

Isolation and characterization of mutants of Pseudomonas

maltophila PM-4 altered in chitinolytic activity and

antagonistic activity against root rot pathogens of clusterbean

(Cyamopsis tetragonoloba)

E. Yadav . D. V. Pathak . S. K. Sharma . M. Kumar . P. K. Sharma

Received: 8 March 2006 / Final revision: 15 February 2007 / Accepted 22 February 2007

Indian Journal of Microbiology (March 2007) 47:64–71

Abstract Pseudomonas maltophila PM-4, an antagonist

of pathogenic fungi including Rhizoctonia bataticola,

R. solani, Fusarium oxysporum and Sclerotinia sclero-

tiorum associated with root rot of clusterbean (Cyamopsis

tetragonoloba) was mutagenized with Tn5. Hyperchitinase

producing mutants showing large zone of colloidal chitin

dissolution were identifi ed on medium containing calcofl or

dye as an indicator. A mutant P-48 producing 137% higher

chitinase activity than the parent strain PM-4 was identifi ed.

Seed bacterization of clusterbean (Cyamopsis tetragonolo-

ba) with P-48 controlled the root rot upto 40.8% in the

presence of conglomerate of all the four fungal pathogens

Rhizoctonia bataticola, R. solani, F. oxysporum and Sclero-

tinia sclerotiorum.

Key words . Biocontrol . bacterial antagonist . root rot .

Tn5 mutagenesis . chitinase . clusterbean

Introduction

Clusterbean, Cyamopsis tetragonoloba is commonly grown

as a summer legume under arid zone in India for animal

feed, fodder and green vegetable purposes. Seed gum, a

rich source of lipopolysaccharide and polyhydroxybutyrate

(PHB), is used in various industries such as textiles, cos-

metics, explosives, paper and food processing1.

Root rot is the major disease in clusterbean under arid

zone during rainy season and may result up to 21.6%

plant loss at pre-emergence and post-emergence stages.

Fungal pathogens such as Rhizoctonia bataticola, R.

solani, Fusarium oxysporum and Sclerotinia sclerotiorum

are associated with the root rot disease2. Attempts have

been made in recent past to develop biological control

agents (BCAs) against soil borne pathogens as a potential

alternative to the use of chemical fungicides. However, their

use as biocontrol agents has not been widely adapted due to

inconsistencies in their performance3. Such inconsistencies

often result due to lack of understanding of their mode of

action and due to biotic and abiotic stresses under fi eld

conditions.

Chitin, a homopolymer of N-acetyl-D-glucosamine (Glc

- NAc) residues linked by β1-4 linkages, is a structural

component in the exoskeletons of insects, in the shells

of crustaceans, in the cell walls of many fungi and algae,

and in nematodes4. Chitinases hydrolyze chitin to soluble

oligosaccharides, mainly chitobiose, which are further

hydrolyzed to GlcNAc by GlcNAcases which is then taken

up by the cells as a carbon and nitrogen source5. Chitinases

are produced by a number of microorganisms such as

Bacillus, Pseudomonas, Serratia, Stenotrophomonas,

Streptomyces and Vibrio; and is one of the mechanism

E. Yadav . D. V. Pathak . S. K. Sharma . M. Kumar .

P. K. Sharma (�)

Department of Microbiology,

CCS Haryana Agricultural University,

Hisar - 125 004

e-mail: [email protected]

Tel: +91 / 1662 / 289292 (O), +91 / 1662 / 249338 (R)

Indian Journal of Microbiology (March 2007) 47:64–71 65

that has been implicated in biocontrol of fungal diseases6.

Other probable mechanism includes production of

antibiotics viz: 2-4-diacetylphloroglucinol, pyrrolnitrin,

siderophores, HCN and induced systemic resistance

(ISR)7,8,9,10,11. Bacterial strains with altered chitinolytic

activity have been produced by mutagenesis in Serratia

plymuthica or by introduction of chitinase gene into

heterologous strains12, 13.

A strain of Pseudomonas, isolated from sunfl ower

rhizosphere, was identifi ed as a biocontrol agent against

root rot pathogens of sunfl ower due to production of

chitinase activity. This strain completely degraded the

fungal mycelium (89% reduction in fungal biomass) of

root rot pathogens in 144 h. This bacterium was identifi ed

as Pseudomonas maltophila by Institute of Microbial

Technology, Chandigarh. Chitinase in P. maltophila PM-4

was reported to be induced by the addition of chitin containing

substrates14. Earlier Stenotrophomonas maltophila (syn.

P. maltophila ) strain C3 has been identifi ed as biocontrol

agent due to production of chitinase and loss of chitinase

activity in strain C3 through spontaneous mutation was

associated with loss of antagonistic activity 15. The objective

in this study was to mutagenize P. maltophila strain PM-4

possessing chitinase activity and identify mutants altered

in chitinase production. The mutants altered in chitinase

production were characterized to know the interrelationship

between chitinase production and biocontrol activity

against root rot pathogens in clusterbean

Materials and Methods

Organisms and growth media: Pseudomonas maltophila

PM-4, used in the present studies was collected from

Department of Microbiology, CCS, HAU, Hisar. E. coli

strain S17-1 carrying pSUP1021, a suicidal plasmid resis-

tant to kanamycin (km) was kindly supplied by Dr. J.Prell,

RWTH, Aachen, Germany. Fungal cultures were obtained

from Department of Plant Pathology, CCS HAU, Hisar

and maintained on liquid or solid potato dextrose media

(1.5% wv–1 agar). Luria broth and agar (LB/A), Tryptone

Yeast Extract (TY) media, Succinate Broth and Nutrient

Agar were used as growth media for bacterial cultivation.

To induce chitinolytic activity, bacterial culture was grown

in minimal media containing acid swollen chitin (0.5%).

Colloidal chitin was prepared as described by Lingappa and

Lockhood 16.

Mutagenesis of P. maltophila: Random Tn5 mutagenesis

of P. maltophila PM-4 was carried out using E. coli

S17-1(pSUP1021) following the procedure as described

earlier17,18.

Screening of Tn5 mutants of P. maltophila for chitinase

assay: About 2154 mutants with Tn5 insertion were

screened for chitin dissolution on growth medium

containing 0.5% acid swollen chitin (colloidal chitin) and

chitin binding fl uorescent Calcofl uor white M2R at 0.001%

(w/v) as described by Vaidya et al.19. Plates were incubated

at 30°C and examined after 72 h for formation of dark halos

against fl uorescent background under UV transilluminator

and clear zone colloidal chitin dissolution under visible

light. On the basis of size of zone formation twenty nine

mutants were picked up for further studies. These mutants

were examined for their antagonistic activity against

Rhizoctonia bataticola on PDA solid medium. Specifi c

chitinase activity was assayed as described by Morrissey

et al.20 and was expressed as μ moles of N acetylglucosamine

(NAG) ml–1 mg–1 of cell protein.

Antagonistic activity: Eight mutants with altered chitinase

activity were tested for antagonistic activity against root

rot pathogens namely R. bataticola, R. solani, Fusarium

oxysporum and Sclerotinia sclerotiorum in solid and liquid

PD medium. Mutants of P. maltophila were grown in 100

ml LB broth for 48 h and 50 μl of the culture was loaded

in center of potato dextrose agar plate. Three discs of test

pathogen (5.0 mm diameter) were placed at equidistance

from the center. Each treatment (including control without

bacteria) was conducted in triplicates. Plates were incu-

bated at 30ºC for 7 d and diameter of inhibition zone was

measured. Disk (5mm) of each fungal pathogen separately

was inoculated into 25 ml of potato dextrose broth (PDB)

in 100 ml Erlenmeyer’s fl asks. The fl asks were incubated

at 30ºC on rotary shaker at 160 rpm for 24 h. Bacterial sus-

pension (500μl containing about 105 cells) were added after

24 h of fungal growth. The fl asks were further incubated on

rotary shaker at 30ºC for 7 d. Fungal culture without bac-

terial suspension was included as control treatment. Each

treatment was replicated thrice and whole experiment was

repeated for confi rmation of results. After 7 d, the mycelium

was fi ltered through pre-weighed Whatman No. 1 fi lter pa-

per and fungal biomass was dried at 70ºC to constant weight

and then weighed.

Production of secondary metabolites: The parent wild type

strain PM-4 along with two mutants P-7 and P-48 were

grown in 30 ml succinate broth and siderophore and HCN

production was determined21, 22.

Inhibition of root rot disease in clusterbean: Two mutants

P-7 and P-48 along with the reference strain PM-4 were

tested for fungal antagonism towards the test pathogens

on clusterbean seedling in pot house experiment. Fungal

cultures were grown separately in potato dextrose broth

(PDB) at 30ºC for one week. Mycelial mats thus obtained

were harvested, cleaned with sterile water and macerated in


homogenizer. Mycelia were added at the rate of 50 mg/kg

of soil 5d before sowing. The weight of mycelium was suf-

fi cient for 50% seedling mortality. Charcoal based bacte-

rial inoculants were prepared by adding 40 ml bacterial

culture (containing109 cells/ml) to 100 g wood charcoal.

Seeds were coated with different inoculants using carboxy

methyl cellulose (CMC) as adhesive and eight seeds were

planted per pot. Seeds treated with wood charcoal powder

alone served as a negative control. Each treatment includ-

ing control was conducted in triplicates. The experiment

was conducted in the screen house under normal growing

season of the crop. Data was recorded in terms of healthy

seedlings, pre emergence and post emergence mortality. Pre

and post-emergence mortality was observed up to 7 and 45

days, respectively. Percent disease incidence and percent

disease control were calculated using following formulae-

% Disease incidence (DI) = Total no of diseased plants

x 100 Total no. of plants

% Disease control (DC) = 100 – DI in treatment

x 100

DI in control

Test for statistical signifi cance: The statistical signifi cance

of the difference between the mean of different treatments

was analyzed using one-way and two-way analysis.

Results

Tn5 mutagenesis and screening of Tn5 mutants: About

3067 mutants with Tn5 insertion (KmrNxr) were obtained

by random transposon mutagenesis of P. maltophila using

E. coli S17-1 (pSUP1021). Out of these 2154 mutants were

screened for chitin dissolution using Calcofl uor M2R as in-

dicator dye. The mutants varied in their ability to degrade

chitin as indicated by different dissolution zone in different

mutants. Some of the mutants did not solubilize chitin and

were assumed chitinase negative mutants. These mutants

were not included further in biocontrol studies. Twenty-

nine mutants showing better zone of chitin dissolution

(dark halo zone formation under UV light and clear zone

formation under visible light) were selected and tested for

chitinase activity (Fig 1).

Chitinase activity: Chitinolytic activity of selected mu-

tants was estimated in liquid media containing colloidal

chitin as sole carbon source. The different mutants showed

wide variation in chitinase activity over the parent PM-4.

Mutants P-48 and P-49 possessed higher chitinase activity

while mutants P-10 and P-11 possessed lower chitinolytic

activity in comparison to parent strain PM-4. The mutants

P-48 and P-49 showed 137.2 and 111.6 % increase in the

chitinase activity over parent PM-4 respectively; and while

there was 42.3 and 39.9, % decrease in the chitinase activ-

ity in mutants P-10 and P-11 over parent PM-4 respectively

(Table 1). Out of twenty nine mutants, eighteen isolates

showed signifi cant higher chitinolytic activity over the ref-

erence strain PM-4.

Antagonistic activity: Eight mutants selected on the ba-

sis of inhibition zone formation against R. bataticola were

screened against three other root rot pathogens namely R.

solani, F. oxysporum and S. sclerotiorum in liquid as well as

on solid PDA medium. All the mutants showed antagonistic

activity against R. bataticola, R. solani and S. sclerotiorum,

but the antagonistic activity against F. oxysporum in liquid

broth was not signifi cant in all the mutants tested except P-7

and P-48. Mutants- P-7 (having moderate chitinase activ-

Fig. 1 Screening of Tn5 mutants of P.maltophila on medium

containing colloidal chitin and calcofl or.


ity) and P-48 (hyperchitinase mutant) were statistically su-

perior to the parent PM-4 against all the four test pathogens

(Fig 2 & Table 2). Maximum inhibition zone was recorded

against R. bataticola (38.6 mm). S. Sclerotiorum and R.

solani showed 32.7 and 29.7 mm inhibition zone respec-

tively (Table 3). Ttransconjugants P-7 and P-48 performed

signifi cantly better than the parent strain PM-4 and resulted

in maximum percent inhibition against all the test patho-

gens, however both were statistically similar. The percent

inhibition was more pronounced against R. bataticola

(63.8); followed by R. solani (61.7). Minimum inhibition

was recorded against F. oxysporum (14.5 %).

Biochemical characterization: The mutants P-7 and P-

48 showed better inhibition against test pathogens and pro-

duced different levels of chitinase enzyme. These mutants

produced HCN, siderophores and IAA (Table 4).

Effect of seed bacterization on root rot control in cluster

bean: Seed inoculation of PM-4 (parent), P-7 (moderate

chitinase) and P-48 (hyperchitinase) mutants on cluster

bean cv. PNB in the presence of fungal pathogens con-

trolled root rot disease under pot culture experiment. In pots

infected with R. bataticola alone, P. maltophila PM-4, P-7

and P-48 showed 36.5, 51.3 and 44.6 percent disease con-

trol, respectively. These values for R. solani over control

were 20.7, 27.2 and 26.8 per cent and for S. sclerotiorum

were 24.5, 40.4 and 36.8 percent, respectively. Seed treat-

ment with PM-4 was not effective against fungal pathogen

F. oxysporum, while P-7 showed 22.5 percent disease con-

trol. When pots were infected with the conglomerate of all

the four fungal pathogens, seeds treated with P. maltophila

PM-4, P-7 and P-48 showed 35.2, and 40.8 percent disease

control respectively (Fig. 4).

Discussion

Antifungal mode of action of P.maltophila strain PM-4

may be mainly based on fungal cell wall degrading en-

zyme-chitinase and pigment formation as it showed good

antagonistic activity against R. solani under in vitro condi-

tion14. The ability to produce chitinase is considered crucial

for antifungal activity of strains of Serratia marcescens23,

Erwinia agglomerans6, Stenotrophomonas maltophila24,

S. plymuthica strain HRO-5.C4825 and Stenotrophomonas

Table 1 Chitinase activity of selected Tn5 mutants of

P. maltophila PM-4.

Mutants* Inhibition against

R. bataticola

Chitinase

activity**

% change over the

parent PM-4

PM-4 + 860±15 0.00

P-1 + 967±4 +12.44

P-2 - 1250±18 +45.34

P-4 - 1200±23 +39.53

P-5 + 570±34 –33.72

P-6 + 978±37 +13.72

P-7 + 750±26 –12.79

P-8 + 1280±21 +48.83

P-9 - 1590±21 +84.88

P-10 - 496±12 –42.32

P-11 - 517±8 –39.88

P-16 - 875±16 +1.74

P-17 + 1420±15 +65.11

P-18 + 830±14 –3.48

P-20 - 950±23 +10.46

P-21 + 1000±28 +16.27

P-22 + 833±19 –3.13

P-23 + 700±13 –18.60

P-24 - 1090±21 +26.74

P-25 + 1110±9 +29.06

P-26 + 1150±15 +33.72

P-27 + 1360±18 +58.13

P-28 + 1190±7 +38.37

P-33 - 1470±17 +70.93

P-37 + 1097±26 +27.55

P-46 - 900±16 +4.65

P-47 + 1560±23 +81.39

P-48 + 2040±27 +137.20

P-49 + 1820±12 +111.60

P-50 + 846±8 –1.62

CD (5%) 52.76

*Cultures was grown in colloidal chitin media for 7 days and

enzyme activity was observed by incubating 1.0 ml of bacterial

supernatant in 1.0 ml of colloidal chitin.

**One unit = one μM of N-Acetyl glucosamine produced / μg

cell protein.

Fig. 2 Inhibition zones of P-7, P-48 and PM-4 against R.

bataticola after 7d on solid media.


maltophila26. Mutants with different chitinase activity were

identifi ed on the basis of the size of halo zones in the col-

loidal chitin medium. This method has been employed

earlier to isolate hyperchitinase producing mutants of

Alcaligenes xylosoxydans19. This method provided rapid

method to identify mutants with altered chitinase activity.

Calcofl uor white M2R is widely used as an optical bright-

ener for isolation of mutants defective in exopolysacchride

production in Rhizobium and Azospirillum 27, 28. Chitinase

activity varied from 496 to 2040 units in different mutants

indicating that it is possible to alter the chitinase activity

by mutagenesis. Hyperchitinase mutants have earlier been

Table 2 Inhibition of R. bataticola, R. solani, F. oxysporum and S. sclerotiorum on growth medium containing colloidal chitin by

selected Tn5 mutants.

Isolates Inhibition zone (mm)*

R. bataticola R. solani F. oxysporum S. sclerotiorum Mean

PM-4 35.0 32.5 ±5.0 37.0 27.4

P-7 43.3 40.0 11.0 41.7 34.0

P-17 37.5 24.0 5.0 32.5 24.8

P-21 37.5 25.0 4.0 22.6 22.3

P-22 34.5 27.5 3.0 21.4 21.6

P-23 35.0 32.5 4.0 26.4 24.5

P-37 42.5 27.5 9.0 35.5 28.6

P-48 41.0 33.0 13.0 40.3 31.9

P-49 40.5 25.0 4.0 37.2 26.7

Mean 38.6± 29.7 6.4 32.7

Factor C.D (5%)

Fungal strains 0.98

Mutants 1.47

Fungal strains x Mutants 2.94

*Size of inhibition zone was in terms of diameter (mm) and inhibition zone was measured after 7 days of growth.

Table 3 Inhibition of R. bataticola, R. solani, F. oxysporum and S. sclerotiorum growth on potato dextrose broth (PDB) medium by

selected Tn5 mutants after 7d.

Isolates/Mutant Per cent Inhibition* of fungal pathogen

R. bataticola R. solani F. oxysporum S. sclerotiorum Mean

PM-4 59.8 71.3 8.5 40.2 45.0

P-7 77.5 72.4 40.4 51.8 60.5

P-17 44.5 36.0 20.6 23.1 31.0

P-21 65.9 58.0 0.0 34.8 39.7

P-22 50.1 73.2 0.0 49.8 43.3

P-23 65.8 49.7 10.8 45.8 43.0

P-37 71.3 64.4 8.3 46.6 47.6

P-48 78.7 75.6 40.1 50.5 61.2

P-49 60.5 54.4 2.0 29.6 36.6

Mean 63.8 61.7 14.5 41.3

Factor C.D (5%)

Fungal strains 1.278

Mutants 1.917

Fungal strains x Mutants 3.835

*Percent inhibition was measured after 7 d of fungal inoculation to which 500 μl containing about 105 cells was added after 24 h of

fungal growth.


identifi ed in Serratia marcescens and S.plymuthica which

formed large clearing zones on colloidal chitin medium12,29.

The hyperchitinase mutant was assumed to have a defect

in the gene involved in the negative regulation of chitinase

gene expression.30

The P-7 and P-48 differed in chitinase production but had

comparable antagonistic activity against the root rot patho-

gens. The mutant P-7 was a moderate chitinase producer but

had good antagonistic activity against root rot pathogens.

Biocontrol activity is a cumulative effect of different sec-

ondary metabolites. It is possible that when one mechanism

is impaired the production of other metabolites is increased.

Pal et al.31 observed that Tn5 mutagenesis of Pseudomonas

sp. EM85 resulted in the production of series of mutants

with altered production of HCN, siderophores, fl uorescent

pigment and antifungal antibiotics and Tn5 insertions may

have ploetrophic effects. Antibiotics production, sidero-

phore production, lytic enzymes synthesis have synergistic

effect for inhibition of plant pathogens.8 The mutants P-7

and P-48 differed in chitinase production but did not differ

in HCN, siderophores or IAA production. The molecular

characterization of mutants P-7 and P-48 of P.maltophila

will indicate the role of other secondary metabolites in

biocontrol activity. Kamensky et al.12 observed same level

of antifungal activity with the parental chitinolytic strain

and its two mutants with mini Tn5 insertion, one was a

super producer of chitinase while the other was defi cient

in chitinolytic activity. Hence other secondary metabolites

produced by the strain IC14, which were not chitinases,

played a key role in the biocontrol activity, atleast against

Botrytis cinerea and S. sclerotiorum. However, biocontrol

action of S. marcescens against S. rolfsii and R. solani was

attributed to solely due to chitinase activity32. Comparison

of Chi A negative mutant strain C5 with the wild type strain

34S1 in Stenotrophomonas maltophila for biocontrol of

root rot infecting fungus Magnaporthe poae resulted in no

obvious differences in its growth inhibition in vitro condi-

tions. However, in vivo conditions disease severity in chi

A negative mutant treated Poa pratensis was greater than

in plants treated with strains 34S133. Hence the mutation of

Chi A in S. maltophila abolishes chitinolytic activity and

affect the ability of the strain to suppress summer patch dis-

ease, providing direct evidence for the role of this enzyme

in biocontrol activity. This suggests that chitinase activity

does play a positive role in disease control. Some factors

involved in biocontrol activity have been identifi ed from

S.maltophila and includes hydrolytic enzymes like β 1-3

glucanase, protease, lipase and antibiotic compound like

maltophilin and xanthobactin34. The higher antagonistic

activity of P-7 mutant over P-48 could be due to production

of other metabolites. Role of protease and antibiotic like

compound, xanthobactin from Stenotrophomonas sp. in

biocontrol of sugarbeet have been proved35,36.

However, molecular studies of Tn5 mutants P-7 and P-48

along with the test strain PM-4 need further investigations

in terms of expression of chitinase, antibiotic production,

quantifi cation of siderophore, synthesis of plant growth

promoting substances, their role in systemic resistance and

their establishment under fi eld conditions to prove success-

ful BCAs for biocontrol of soil borne diseases in cluster-

bean and other fi eld crops.

Acknowledgements The authors thank Dr. P.P. Gupta,

Professor, Plant Pathology, CCS, HAU, Hisar for providing

fungal cultures. Thanks are also due to Dr. J. Prell,

RWTH, Aachen Germany for gift of E. coli strain S17-1

(pSUP1021) strain.

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P-48 2050 + +

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isolation and characterization of mutants of pseudomonas maltophila pm4 altered in chitinolytic...

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