Interaction Between Meloidogyne incognita, Pseudomonas fluorescens and Bacillus subtilis and its Effect on Plant Growth of Black Gram (Vigna mungo L.)
Meloidogyne incognita infection produced adverse effects on the growth of black gram (Vigna mungo L.). Inoculation of the plants with the second-stage juveniles of M. incognita, prior to bacterial inoculation, resulted in reduction of plant growth, when compared with the plants in which bacterial application was followed by nematode inoculation. Plant length, fresh and dry weights, nodules weight, leghaemoglobin content and the number of nodules per plant were found decreased in nematode infected than in infected plants. Application of bacteria Pseudomonas fluorescens and Bacillus subtilis increased the growth parameters and the number of nodules. Highest number of galls per plant were recorded on the plants infected with the nematode and not treated with bacteria. Gall number was found decreased on the plants inoculated with the nematode and treated with the bacteria than the plants not treated with bacteria. Meloidogyne incognita on infecting black gram (Vigna mungo L.) in absence of bacteria caused the formation of a number of galls on the roots, decreased plant length, plant weight, number of nodules per plant and amount of leghaemoglobin. Incorporation of bacteria into the soil after 10 days of nematode inoculation resulting in an increase in all the growth parameters considered and decreased root-knot number.
Received: January 11, 2012;
Accepted: April 03, 2012;
Published: June 05, 2012
Pulses occupy an indispensable position in the dietary habit of vast majority
of the people of India s well as abroad. These are not only the protein source
but these also contribute in restoration of soil fertility (Korlovich
and Repyev, 1995).
Black gram is an excellent source of easily digestible protein having low flatulence
property. It is a short duration and widely cultivated leguminous pulse crop
contributing substantially to the annual production of pulses. The production
and yield of black gram is declined if the crop field is infested with the nematodes
(Ali, 1994). The root-knot nematodes (Meloidogyne
sp.) are sedentary endoparasites and are among the most damaging agricultural
pests, attacking a wide range of crops including black gram (Sikora
and Greco, 1993). The infection starts with root penetration of second-stage
juveniles hatched in soil from eggs encapsulated in egg masses laid by the females
residing in the infected roots (Barker et al., 1985).
Available literature shows that Meloidogyne sp. adversely affect nodulation
and nitrogen fixation in pulse crops (Haung, 1987; Taha,
Bacteria are the most abundant microorganisms of which about 2 to 5% are rhizobacteria
which exert a beneficial effects on plant growth and therefore, are termed as
Plant Growth Promoting Rhizobacteria (PGPR) (Kloepper and
Schorth, 1978). Early studies on PGPR were more focused on biological control
of plant diseases than on growth promotion as a result of inoculants of fluorescent
pseudomonads and Bacillus subtilis, antagonistic to soil borne plant
pathogens (Kloepper et al., 1989). The PGPR-nematode
interactions have been extensively studied with the aim to manage plant-parasitic
The objectives of this study were to investigate the effect of sequential inoculation
of M. incognita, P. fluorescens and B. subtilis on plant
growth, nodulation, fresh and dry biomass in black gram cv. Pant U-30.
MATERIALS AND METHODS
Seeds of Pant U-30 black gram were sown in 15 cm diameter earthen pots filled with autoclaved soil and mixed compost (3:1). Prior to sowing, the seeds were disinfected by immersion on 1% of sodium hypochlorite (NaOCl). After germination, the seedlings were thinned to one per pot.
Culturing of nematodes: To get a large number of second-stage juveniles of M. incognita pure culture was maintained on tomato plants grown in field. The required number of freshly hatched second-stage juveniles were obtained from as per requirement.
Extraction of nematodes: For isolation of second-stage juveniles (J2), tomato plants infected with M. incognita were collected and washed properly in running tap water. Egg masses were carefully removed from the roots with the help of forceps and placed over a double layer of tissue paper spread on 3 cm diameter sieve. The sieves were placed on petri dishes containing sufficient water so that their lower part remained partially submerged in water. To avoid evaporation of water the petri dishes were kept covered with their lids. After 24 h onwards second- stage juveniles were collected in the form of suspension and stored for later use.
Culturing of bacteria: P. fluorescens and B. subtilis
were cultured on Kings B and nutrient broth media, respectively, containing
measured amount of nutrients for their growth and kept at 33°C for best
growth for 48 h. Bacterial colonies of Pseudomonas sp. fluoresced under
UV light at 366 nm were purified on Kings B agar medium (King
et al., 1954) and identified according to Krieg
and Holt (1984). The bacterial population reached >109 cells
mL-1 within 7-10 days.
Inoculation: The 10 days old seedlings were inoculated by adding required
amount of inocula through four narrow holes made around each plant. Each plant
was inoculated with M. incognita (1000 J) and bacterial culture as per
treatment (10 and 20 mL) sequentially with the interval of 10 days:
||M. incognita prior to Pseudomonas fluorescens
||M. incognita prior to Pseudomonas fluorescens (20 mL)
||M. incognita prior to Bacillus subtilis (10 mL)
||M. incognita prior to Bacillus subtilis (20 mL)
||M. incognita prior to Pseudomonas fluorescens (10 mL),
Bacillus subtilis (10 mL)
||M. incognita prior to Pseudomonas fluorescens (20 mL), Bacillus
subtilis (20 mL)
P. Fluorescens and B. Subtilis were also used as per treatment
and their detail is follows:
||Pseudomonas fluorescens (10 mL) prior to M. incognita
||Pseudomonas fluorescens (20 mL) prior to M. incognita
||Bacillus subtilis (10 mL) prior to M. incognita
||Bacillus subtilis (20 mL) prior to M. incognita
||Pseudomonas fluorescens (10 mL), Bacillus subtilis (10 mL)
prior to M. incognita
||Pseudomonas fluorescens (20 mL), Bacillus subtilis (20 mL)
prior to M. incognita
All treatments were replicated thrice. The plants were lightly watered after
inoculation and thereafter, whenever required. The pots were arranged in a randomized
block design. The experiment was terminated 60 days after inoculation and different
parameters were determined (Southey, 1986; Oostenbrink,
The data on the parameters of black gram var. PU-30 bacteria and Meloidogyne
incognita have been presented in Table 1-4.
The data recorded revealed that the shoot length increased significantly on
increasing the dose of bacterium inoculums as is evident from F1 to F6 (Table
3). when compared with control. The shortest length of the shoot was recorded
(19.4 cm) in T3 treatment (Table 1). However, maximum length
(30.7 cm) of the shoot was recorded in the treatment F6 (Table
3) but the values were significantly lower than the control (33.4 cm). Reduction
in shoot length in black gram due to Meloidogyne incognita infection
has earlier been reported by Singh (1972), Gupta
et al. (1987), Mohanty et al. (1989),
Kalita and Phukan (1993) and Haider
et al. (2003).
The root length of the plants infected with the nematode was found to be increased
significantly on increasing the amount of bacterial inoculum (Table
1 and 3). Maximum root length (14.8 cm) was recorded in
F6 plants (Table 3) which was significantly less than the
control (Table 1). Maximum and significant reduction in the
root length (5.8 cm) occurred in the plants of the treatment T3 (Table
Significant variations were observed in shoot and root weights in both the
experiments. Highest and significant reductions in the fresh and the dry shoot
weights (7.85 and 1.74 g) were noticed in T3, on comparing with the control
|| Sequential effect of M. incognita, Pseudomonas
fluorescens and Bacillus subtilis on Vigna mungo L.
|*Sequential inoculation = after an interval of 10 days
||Sequential effect of M. incognita, P. fluorescens
and B. subtilis on Leghaemoglobin, nitrogen content and dry weight
of nodules in Vigna mungo L.
|| Sequential effect of Pseudomonas fluorescens, Bacillus
subtilis and M. incognita on Vigna mungo L.
|*Sequential inoculation = after an interval of 10 days
||Sequential effect of P. fluorescens, B. subtilis
and M. incognita on Leghaemoglobin, nitrogen content and dry weight
of nodules in Vigna mungo L.
Increase in the fresh and the dry shoot weights (14.84 and 8.74 g) (Table
3) was maximum in F6 treatment which was, however, lower than the ontrol
(15.75 and 9.53). The values of the fresh and the dry weights of the roots (1.70
and 0.68 mg) were maximum when M. incognita was inoculated into the soil
prior to bacterial inoculation as is evident from T3 treatment. Maximum increase
in the fresh and the dry weights of the roots (9.63 and 6.80 g) was recorded
in F6 treatment where bacterial inoculum was introduced prior to ematode inoculation
(Table 3). The number of galls were highest in T1, where the
plants were inoculated with M. incognita prior to bacterial inoculation
(Table 1). Lowest number of galls was observed in the treatment
F6 where higher doses of both the bacteria were applied before nematode inoculation.
The highest number of effective nodules per plant (35.33) was recorded in F6
(Table 3), in which the bacteria were inoculated prior to
nematode inoculation, the values were at par with the control (36.66). However,
minimum number of root nodules was recorded in T3 treatment (Table
1). When the plants were inoculated with the nematode, Meloidogyne incognita,
significant reductions in nodule weights were observed. Maximum dry weights
of the nodules (23.87 mg) was recorded in F6 treatment where bacterial inoculum
was introduced prior to nematode inoculation (Table 4). Leghaemoglobin
content of Vigna mungo L. nodules was significantly differed at different
inoculation levels (Table 2 and 4). F6 treatment
showed a significantly maximum leghaemoglobin content compared to rest of the
treatments but less than control (4.8). Rhizosphere inoculation of bacteria
significantly increased the nitrogen content in leaves less than control. The
lowest nitrogen content were recorded in T1 treatment (0.09) (Table
Plant growth promoting bacteria play a significant role in the growth and the
development of all the leguminous crops. The presence of Pseudomonas fluorescens
and Bacillus subtilis in the roots of black gram usually produces beneficial
effects on the plant growth. Occurrence of rhizobacteria in the soil plays a
protective role against the nematodes. From the following experiment it is evident
that the damage caused to the plant was significantly lower, except in the treatment
where bacterial applications followed nematode inoculation. Presence of the
nematodes in the soil and their interaction with the plants, in absence of the
PGPR, are more damaging for the plants. The results of the experiment revealed
that inoculation of the plants with the nematodes prior to bacterial inoculation
caused reduction in plant growth as well as nodulation. From the experiment
it was found that development of the nodule was suppressed as a result of prior
nematode inoculation. The similar findings were reported by Bopaiah
et al. (1976).
Bacterial application prior to nematode inoculation resulted in an increase
in plant growth and reduced the extent of damage to the nematode inoculated
plants. From the following study it was inferred that Pseudomonas and
Bacillus both produced beneficial effects on the plant growth even in
the presence of the nematode. Similar trend was also reported by Siddiqui
and Husain (1992). Bacterial application prior to nematode inoculation resulted
in immediate establishment of the bacteria inside the root tissues; on the contrary,
earlier establishment of nematode carried out mandatory changes and caused damage
to the plant (Varshney, 1982). Increase in amount of
bacterial inoculum resulted in increase in plant growth as well as in increase
in nodulation. The usual site of nodule formation is the root tissue but nodules
were occasionally found on the galls as was reported by Hussey
and Barker (1976). Suppressed nodulation is related to the smaller size
of the root system that confirms the findings of Taha and
Raski (1969). Growth parameters, as shoot and root lengths, fresh and dry
weights of the shoot and the root showed more growth in prior pplication of
bacteria (Table 1) as compared to nematode (Table
2). The values of these parameters were found to be increased in both the
treatments on increasing the dose of bacteria.
Increase in plant growth bacterial inoculated plants, even in the presence
of the nematode was mainly due to enhancement of mineralization process, specifically
nitrogen uptake and assimilation (Griffiths, 1994). These
bacteria have been characterized for production of hydrolytic enzymes, HCN,
phenol oxidation and anti fungal activity (Insunza et
al., 2002). Nematode, when infects a plant, interferes in plant and
bacteria relationship. Colonization of the roots by pathogenic and beneficial
organisms is influenced by the nematode parasitism; but a natural antagonism
between nematode and organism, specially microorganisms suppresses the deleterious
effect of nematode (Kerry, 2000). Root knot nematodes
and rhizobacteria occupy similar niches in the soil and roots suggesting the
possibility for genetic exchange (Bird et al., 2003).
Pseudomonas fluorescens produces IAA which is helpful to promote physiological
effects on plants. Rhizosphere bacteria promote plant growth by improving the
availability of nutrients, suppressing the growth of plant pathogens or by production
of hormones such as auxins (Jangu and Sindhu, 2011).
Pseudomonas sp. possessed considerable insecticide tolerance and IAA,
siderophores (salicylic acid and 2, 3-dihydroxy benzoic acid), exo-polysaccharides,
HCN and ammonia producing traits (Ahemad and Khan, 2011).
PGPR may induce plant growth by the production of stimulatory volatiles and
phytohormones, lowering of the ethylene level in plant, improvement of the plant
nutrient status and stimulation of disease resistance mechanisms (Beauchamp,
1993). Pseudomonas fluorescens and Bacillus subtilis increased
growth of root-knot nematode infected plants of black gram. Higher doses of
bacterial were proved more beneficial than lower doses. Application of bacteria
prior to nematode inoculation was found more promising. Bacterial inoculated
plants without nematodes had conspicuously large and pink coloured nodules where
as nodules on nematode infested plants were brownish in colour. The number of
nodules per plants and their weight were significantly reduced by nematode infection
(Table 1 and 2) which was proved by Hussaini
and Seshadari (1975), Sharma and Sethi (1976) and
Chahal et al. (1985). Root-knot nematode juveniles
directly interfere with the establishment of bacteria and the secretion of hydrolytic
enzymes or growth regulators produced by nematodes may play a determinative
role in nodule development (Barker et al., 1972;
Ali et al., 1981). Leghaemoglobin content of
nodules was significantly reduced by nematode infection as compared to control
and increased on increasing the bacterial dose. Invading nematodes are considered
to disturb the functioning of nodules by altering host nutrition (Doney
et al., 1970). As leghaemoglobin regulates the supply of oxygen and
bacteroids contain nitrogenase enzyme required for the reduction of atmospheric
nitrogen to ammonia, then a decrease in these due to nematode infection would
lead to a decrease in fixation of nitrogen.
From the results of experiment, it may be concluded that M. incognita affects symbiotic nitrogen fixation not only by reducing the number of nodules but also by disturbing the functioning of nodules due to decrease in the photosynthate supply and leghaemoglobin content of nodules.
Ahemad, M. and M.S. Khan, 2011.
Assessment of plant growth promoting activities of rhizobacterium Pseudomonas putida
under insecticide-stress. Microbiol. J., 1: 54-64.CrossRef | Direct Link |
Ali, M., 1994.
Agronomy. In: 25 Years of Research on Pulses in India, Ali, M., A.N. Asthana and S.L. Mehta (Eds.). Souvenir, New Delhi, India, pp: 19CrossRef |
Ali, M.A., L.Y. Trabulsi and M.E. Abd-Elsamea, 1981.
Antagonistic interaction between Meloidogyne incognita
and Rhizobium leguminosarum
on cowpea. Plant Dis., 65: 432-435.CrossRef | Direct Link |
Barker, K.R., D. Huising and S.A. Johnston, 1972.
Antagonistic interaction between Heterodera glycines
and Rhizobium japonicum
on soybean. Phytopathology, 62: 1201-1205.CrossRef | Direct Link |
Beauchamp, C.J., 1993.
Mode of action of plant growth promoting rhizobacteria and their potential use as biological control agents. Phytoprotection, 71: 19-27.
Bird, D.M., C.H. Opperman and K.G. Davies, 2003.
Interactions between bacteria and plant-parasitic nematodes: Now and then. Int. J. Parasitol., 33: 1269-1276.CrossRef | PubMed | Direct Link |
Bopaiah, B.M., R.B. Patil and D.D.R. Reddy, 1976.
Effect of Meloidogyne javanica
on nodulation and symbiotic nitrogen fixing in mung (Vigna radiata
). Indian J. Nematol., 6: 124-130.Direct Link |
Chahal, P.P.K., I. Singh and J.K. Chhabra, 1985.
Effect of Meloidogyne incognita
on growth of mungbean. J. Res. Pb. Agric. Univ., 22: 181-183.
Doney, D.L., J.M. Fife and E.D. Whitney, 1970.
The effect of the sugarbeet nematode Heterodera schachtii
on the free amino acids in resistant and susceptible beta species. Phytopathology, 60: 1727-1729.CrossRef | Direct Link |
Griffiths, B.S., 1994.
Soil Nutrient Flow. In: Soil Protozoa, Darbyshire, J.F. (Eds.). CAB International, Willingford, UK., pp: 65-91
Gupta, D.C., K.K. Verma and I.J. Puruthi, 1987.
Studies on Meloidogyne javanica
on black gram (Vigna mungo
). Indian J. Nematol., 17: 177-179.
Haider, M.G., L.K. Dev and R.P. Nath, 2003.
Comparative pathogenicity of root-knot nematode Meloidogyne incognita
on different pulse crops. Indian J. Nematol., 33: 152-155.Direct Link |
Haung, I.S., 1987.
Intertactions of Nematode with Rhizobia. In: Vistas on Nematology, Veech, I.A. and D.W. Dickson (Eds.). Society of Nematologist Inc., Byattsville, USA., pp: 301-306
Hussaini, S.S. and A.R. Seshadari, 1975.
Interrelationship between Meloidogyne incognita
spp. on mung bean (Phaseolus aureus
). Indian J. Nematol., 5: 189-199.
Hussey, R.S. and K.R. Barker, 1976.
Influence of nematodes and light sources on growth and nodulation of soybean. Indian J. Nematol., 8: 48-52.Direct Link |
Insunza, V., S. Alstrom and K.B. Eriksson, 2002.
Root bacteria from nematicidal plants and their biocontrol potential against trichodorid nematodes in potato. Plant Soil, 241: 271-278.Direct Link |
Jangu, O.P. and S.S. Sindhu, 2011.
Differential response of inoculation with indole acetic acid producing Pseudomonas
sp. in green gram (Vigna radiata
L.) and black gram (Vigna mungo
L.). Microbiol. J., 1: 159-173.CrossRef | Direct Link |
Kalita, D.N. and P.N. Phukan, 1993.
Pathogenicity of Meloidogyne incognita
on blackgram. Indian J. Nematol., 23: 105-109.Direct Link |
Kerry, B.R., 2000.
Rhizosphere interactions and the exploitation of microbial agents for the biological control of plant-parasitic nematodes. Ann. Rev. Phytopathol., 38: 423-441.CrossRef | PubMed | Direct Link |
King, E.O., M.K. Ward and D.E. Raney, 1954.
Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med., 44: 301-307.PubMed | Direct Link |
Kloepper, J.W. and M.N. Schorth, 1978.
Plant growth promoting rhizobacteria radishes. Proceedings of the 4th International Conference on Plant Pathogenic Bacteria, August 27-September 2, 1978, Angers, France, pp: 879-882
Kloepper, J.W., R. Lifshitz and R.M. Zablotowicz, 1989.
Free-living bacterial inocula for enhancing crop productivity. Trends Biotechnol., 7: 39-44.CrossRef | Direct Link |
Korlovich, B.S. and S.I. Repyev, 1995.
Theoretical Basis of Plant Breeding. In: The Gene Bank and Breeding of Grain Legumes (Lupine, Vetch, Soya and Bean), Korlovich, B.S. and S.I. Repyev (Eds.). N.I. Vavilov Institute of Plant Industry, St Petersburg, Russia, pp: 438
Krieg, N.R. and J.G. Holt, 1984.
Bergey's Mannual of Systematic Bacteriology. Wiliam and Wilkins, Baltimore, Pages: 964
Mohanty, K.C., B.N. Routaray and S.N. Das, 1989.
Growth characteristic of black gram as affected by Meloidogyne incognita
. Indian J. Nematol., 19: 273-275.
Oostenbrink, M., 1966.
Major characteristic of the relation between nematodes and plants. Mededelingen Landbouwhogeschool Wageningen, 66: 3-46.
Sharma, N.K. and C.L. Sethi, 1976.
Interrelationship between Meloidogyne incognita
, Heterodera cajaini
sp. On cowpea (Vigna sinensis
L. Savi). Indian J. Nematol., 6: 117-123.Direct Link |
Siddiqui, Z.A. and S.I. Husain, 1992.
Interaction between Meloidogyne incognita
race-3, Macrophomina phaseolina
sp. in the root knot disease complex of chickpea (Cicer arietinum
). Fundam. Applied Nematol., 15: 491-494.Direct Link |
Sikora, R.A. and N. Greco, 1993.
Nematodes Parasites of Food Legumes. In: Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, Luc, M. and R.A. Sikora (Eds.). CAB International Institute of Parasitology, Wallingford, UK., pp: 629
Singh, R.N., 1972.
Root knot disease of urd and mung in India. Indian J. Mycol. Plant Pathol., 2: 87-87.
Southey, J.F., 1986.
Laboratory Methods for Work with Plant and Soil Nematodes. Her Majesty's Stationery Office, London, UK., ISBN-13: 978-0112427544, Pages: 202
Taha, A.H.Y., 1993.
Nematode Interactions with Root Nodule Bacteria. In: Nematode Interactions, Khan, M.W. (Eds.). Chapman and Hall, London, UK., pp: 202
Taha, A.J. and D.J. Raski, 1969.
Interrelationships between root-nodule bacteria, plant-parasitic nematodes and their leguminous host. J. Nematol., 1: 201-211.Direct Link |
Varshney, V.P., 1982.
Changes in plant growth, nematode population and nodule index as a result inoculation of cowpea (Vigna unguiculata
) with Meloidogyne incognita
and Rhizoctonia solani
. Ph.D. Thesis, Aligarh Muslim University, Aligarh, India
Barker, K.R., C.C. Carter and J.N. Sasser, 1985.
Advance Treatise on Meloidogyne. Vol. I. Biology and Control. North Carolina State University, Raleigh, USA