EFFECT OF CERTAIN SOIL FACTORS ON NODULATION IN COWPEA (Vigna sinensis)

AND LENTIL (Lens esculenta)

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

Walter of $3FulogopJjp IN

AGRICULTURE (MIGROBIOfcOGY)

BY

AbMAS ZAIDI

UNDER THE SUPERVISION OF

PROF. S. K. SAXENA

AGRICULTURE CENTRE ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 1994

- - - - T //

• ' ' ^ " ^ M M ^ R - ' ^ ; • > " ''

V FEBW)

CBECKED-2D02 £ ^ *\ CoKipuwf

DS2811

P r o f . S.K. SAXENA DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202002

C E R T I F I C A T E

This is to certify that the dissertation entitled,

"Effect of Certain Soil Factors on Nodulation in Cowpea

and Lentil" submitted in partial fulfilment of the degree

of M.Phil. (Ag.) in Microbiology of Agricultural Centre,

A.M.U., Aligarh is a bonafide record of the research n . ••

1 --• - -.

work carried out by Almas zaldi under my guidance and

supervision. No part of the dissertation has been sub­

mitted for any other degree or diploma.

The assistance and help rendered during the

courses of this investigation and sources of literature

are duly acknowledged.

(prof. S.K. SAXENA)

A C K N O W L E D G E M E N T

I am very grateful to my supervisor, prof. S.K.

Saxena, Department of Botany, Aligarh Muslim University,

Aligarh, whose suggestion and guidance gave me inspira­

tion in completion of these studies.

I am also grateful to prof. Shamim Jairajpuri,

Coordinator, Agriculture Centre and prof. Wazahat

Hussain, Chairman, Department of Botany, Aligarh Muslim

University, Aligarh, who kindly provided me all the

necessary facilities to carry out this work.

(ALMAS ZAIDI)

C O N T E N T S

PAGE NO,

1 . INTRODUCTION . . . 1 - U

2 . REVIEW OF LITERATURE . . . 1 2 - 2 8

3 . MATERIALS AND METHODS . . . 2 9 - 3 3

4 . BIBLIOGRAPHY . . . 3 4 - 4 6

* * * *

I N T R O D U C T I O N

I N T R O D U C T I O N

Pulse crops are one of the most important crops of

the world belonging to the family Leguminosease. Because

of their multidimensional uses they have been cultivated

for centuries all over the world, in India the area under

pulse cultivation is nearly 22-23 million hectares and

the annual production is about 13 million tonnes

(Anonymous, 1977).

The most important use of pulses is in food. In

developing countries pulses are main source of dietary

proteins because they are comparatively cheaper and

easily available. They constitute an important part of

the people's diet specifically of vegetarian population

in India. Pulses are also source of carbohydrates, fats,

vitamins and minerals (Anonymous, 1991).

Different pulses have varied amount of proteins,

being the highest in soybean (45%) and lowest in bengal

gram (17.1%), The protein content of the important pulses

is as follows :

Common name Botanical name protein (%)

Bengal gram Clcer arietlnum L. 17.1

Pigeon pea Cajanus cajan L. 22.7

Green gram Fhaseolus aureus L. 24 90

2

Peas Plsum sativum Roxb. 19.7

Soybean Glycine max L. 45.0

Cow pea Vlgna sinensis L. 24.6

Horse gram Dollchos blflorus L. 22.0

Some of the legumes are important fodder crops

such as cowpea, horse gram etc.

Besides their nutritional value pulses have the

unique property of maintaining and restoring soil ferti­

lity and, therefore, they are used as green manure. Cow-

pea and egyptian clover (berseem) are being used as green

manures and as rotation crops with wheat,rice and other

non-leguminous crops. Fields in which legumes are grown

maintain high population of rhizobia for several years

after harvest of the legumes (Kucey and Hynes, 1989).

Legumes possess, in their roots, nodules containing nit­

rogen fixing bacteria which capture atmospheric nitrogen

and fix it. Legumes are thus not dependent on synthetic

nitrogenous fertilizers, on the other hand, they add

about 30 kg of nitrogen per hectare to the soil and

improve its fertility e.g. clover (Trlfolium spp.) fixes

about 130 kg/hectare and cowpea about 60 to 128 kg/ha.

In legume, root-nodule-symbiosis the Rhizoblum

spp. are mlcrosymblont and legumes are macrosymbiont.

The capability of nitrogen fixation in legumes is depen­

dent on nodulation which is associated with Rhizoblum.

3

There is always speciticity in the Rhizobium with host.

Kosenko et al. (1987) reported that the phenomenon of

specific recognition between rhizobia and their host is

attributed to the plant lectins (proteins), which speci­

fically bind to the polysaccharide receptors on the

rhizobial cells. In the process of nodulation in leguminous

plants, the first step is infection of roots with the rhi­

zobia. Malek e_t al. (1989) reported that legume root exu­

dates contain chemoattractants as sugars, phenols, amino

acids or flavones etc., resulting in the colonization of

rhizobia around root hairs of legumes. Luteolin has been

found a chemoattractant produced from alfalfa for Rhizobium

meliloti (Dharmatilake and Wolfgang, 1992). Smit e£ ajL.

(1989) worked out that the attachment of bacteria to the

roots is a calcium aided process. Bhowmick f_t al. (1987)

reported that rhizobia produce indole-acetic acid (IAA).

The entry of the rhizobia into root cells occurs between

epidermal cells of the root (De Faria f_t al. 1988).

Chalifour et_ al, (1990) suggested that the infection

thread is guided by the root cell nucleus. He also pointed

out that a hydrolytic enzyme is involved in the sequence

of events from infection thread formation through rhizo­

bial release in the host cell cytoplasm. The enzyme has

been found to be of rhizobial origin. The contents of

infection thread are then released into a tetraploid cell

of root cortex. After this release of rhizobia the dis~

appearance of infection thread is attributed to its

degradation within large vacuoles from the fusion of

4

smaller vacuoles (Arya e£ al« 1985). This resulted In

increased merlstematic activity of cortical cells and

formation of the nodules*

Factors affecting nodulation

The process of nodulation occurs in nature in

soil. Therefore, soil factors influence the development

of nodules. The soil factors affecting nodulation can be

classified into physical and biotic. These factors affect

nodulation in two ways % one directly on rhizobia and

the other indirectly on plants.

Physical factors

Soil temperature :

It is an important factor that controls not only

the microbial activity but all the processes involved in

the growth of plants. The most congenial range of tempera­

ture for nodulation is 20-30°C. The low root temperature

is not only responsible for less number of nodules but

also less weight of nodules (Rice and Olsens 1988).

Sharma et aJL. (1989) pointed out that the high tempera­

ture in the semi-arid zones of Haryana could be respon­

sible for the low nodulation of pigeon pea because the

o plasmid carrying the nodulation genes is cured at 40-50 C,

giving rise to non-nodulating mutants. Elsheikh et al»

(1989) reported that the tolerance of salt to rhizobia

is dependent upon temperature, pH and carbon source.

5

Soil moisture i

No life is possible without an optimum soil mois­

ture. It plays an important role in controlling the nodu~

1ation in plants, it also controls other microbial

activities, nutrient availability and plant root growth*

Osa-Afiana et al. (1982) pointed out that the percentage

of cells of cowpea rhizobia and Rhizoblum japonlcum

surviving were directly related to the quantity of water

in the soil following dessication and that was also

influenced by relative humidity. Rhizobia are readily

transported by an advancing wetting frontc This indicates

that non-saturated flow of soil moisture contributes to

the dispersal of rhizobial inoculum in soil (Breitenbeck

et al., 1988). Postma et a_l. (1989) reported that the

rhizobial cells survived better in soil with lower initial

moisture than in soil with a higher initial moisture con­

tent. Dudeja et al. (1989) reported that after the onset

of rain, there was an Increase in the number of resident

rhizobia in the soil but after drying of soil the number

of rhizobia decreased.

Soil texture :

Texture of soil depends on percentage of sande silt

and clay and is an important soil factor for the growth

and survival of bacteria in the soil. It also affects

various soil properties such as nutrient, water holding

capacity etc., which directly play an important role in

6

growth and nodulation of legumes. Cowpea rhizobia sur­

vived better in bauxite silt-loam than in clay-loam soil

(Aarons and Ahmad, 1987). Heynen et al.. (1988) pointed

out that protozoa are partly responsible for the decline

in rhizobial number in loamy-sand soil. But bentonite

clay confers partial protection of induced rhizobial

cells against predation of rhizobia by protozoa.

El-Mokadem (1989) suggested that concentration of nitrogen

in chick pea tissues is higher in plants grown in loamy

sand than in sandy soils because of the better survival

of rhizobia in the former. Hoeflich et al. (1990) repor­

ted that the persistence of rhizobia of legume seeds

could be increased after seed pelleting with clay minerals

like kaoline and bentonite.

Hydrogen ion concentration of soil :

Soil pH also plays an important role in growth

and nodulation of leguminous plants. In winged bean

the symbiotic performance of effective strains of rhi­

zobia is best at pH 5.5 (iruthayathas and Vlassak, 1986).

Coll (1989) reported that the yield of clover inoculated

with rhizobia is found less due to the low pH of soil.

Salinity and Alkalinity :

Siddique e£ al. (1986) suggested that increased

salinity reduced the number and weight of the nodules

in pea. Whereas desallnlzation increased the production

7

and growth of nodules. El-Shlnnawi (1989) pointed out

that formation of effective nodules la reduced in saline

soils, Mirza and Tariq (1993) reported that maximum

nodulation occured in soil upto 0.4% sodium chloride.

However, the nodule mass decreased with increasing sali­

nity levels. Elsheikh and Wood (1989) found that the salt

stress in soil was more severe at an alkaline pH. Nair

et al. (1993) pointed out that tolerant rhizobia were

less efficient in symbiotic nitrogen fixation under non-

saline growth conditions of legume host, possibly due to

depression in their nitrogenous activity during salt

stress. Nodule growth was significantly reduced in treat­

ments involving salt-acclimated rhizobia.

Mineral Nutrition :

Pandey e* al. (1986) reported that the rate of

nodulation in terms of nodule number and dry weight of

Vlgna mungo was slightly inhibited at lower concentrations

and severely inhibited at high concentrations of urea and

ammonium sulphate. Nitrogenase activity was also inhibi­

ted with both types of fertilizers. Rhizobial population

size was stimulated by ammonium phosphate but was mar­

kedly inhibited with urea. Helemish and (Semell (1987)

pointed out that magnesium salts in lower concentrations

are beneficial for rhizobia. In subterranean clover,

addition of cobalt to sand culture at the rate of 0.006

to 0.06 ppm resulted not only in the formation of bigger

8

nodules but also stimulated the nitrogen fixation. Cobalt

was found predominantly in the bacteroids of nodules of

subterranean clover (Troistskaya and Severova, 1987).

Potalia (1987) reported that zinc and molybdenum dep­

ressed nitrogen stress vrhen combined with rhizobial

inoculant. Almendras and peter (1987) observed that

application of KH2P04, which did not change the soil pH,

increased significantly the percent nodule occupancy in

leguminous plants. Calcium is known to stimulate the

nodulation when present as chloride• It helps in the

attachment of rhizobia to the root hairs (Smit et_ al.

1989). Simon et al. (1990) observed that titanium in the

form of titanium ascorbate enhanced the growth rate of

rhizobia. Cadisch ejt al, (1992) suggested that phosphorus

limited the growth and nitrogen fixation to a greater

extent than potassium. Fotassium and phosphorus supply

increased the dry matter production. Phosphorus deficiency

resulted in the reduction of nodule weight.

Soil amendments with pesticides :

Surieja and Dogra (1985) found that the treatment

of soil with aldrin and lindane resulted in a reduction

in nodule number, yield and nitrogen content in bengal

gram inoculated with chickpea rhizobia, Torralba ejt ajL

(1986) pointed out that the doses of atrazlne used,

reduced the number of nodules on legume roots. Ruiz

and Bellogin (1986) observed that the fungicides

9

HcaptanH and "captafol" inhibited nodulation by Rhlzoblum

trifolii on Trifolium alexandlnum. Mogallan et a_l. (1986)

reported that the herbicide amitrol in low concentration

had a favourable effect on the growth of rhizobia.

Agarwal (1986) reported that pesticides, belonging to

the carbamate group, differed in their capacity to etfect

nodulation and nitrogen fixation. Gupta e_t al. (1988)

found that the highest number of nodules per plant and

highest grain yield were recorded with treatment of soil

with bavistin 0.1% followed by rhizobial inoculation in

chickpea. Martensson e£ al_. (1989) reported the inhibitory

action of herbicide chlorsulphuran on the nodulation and

nitrogen fixation in red clover. Torralba et al_. (1987)

observed that the herbicide 2-4-D had a very light inhi­

bitory action on the growth of rhizobia, however, it had

a clearly contrary effect on the nitrogen fixation by

the nodules. The treatment with phosphamidon inhibited

rhizobial growth temporarily but stimulated nitrogenase

activity which declined with passage of time in culture

medium.

Effect of Biotic Factors :

Since the rhizobial penetration takes place in

soil, biosphere, specially the soil microflora, is bound

to affect nodulation. Its effect may be favourable or

antagonistic. The inhibitory or stimulatory effects of

10

soil microorganisms such as bacteria, fungi and actino-

mycetes on rhizobium are well known.

Furgal et al. (1988) reported that the growth of

Rhizobium legumlposarum was inhibited by culture filtrates

of saprophytic fungi like Aspergillus spp. and Fusarlum

spp. inoculation of roots with actinomycete at earlier

stages of growth of nodule bacteria had no effect on

survival but at later stages nodule bacteria were killed

due to the growth of soil antagonistic actinomycetes

(tolyanskaya, 1985).

It is clear from the foregoing that there is a

complex relationship in the symbiosis between host and

Rhizobium. When seeds pelleted with rhizobial cultures

are sown in the field, the soil biota comes in the picture

and affects the nodulation.

Although some information is available on the eftect

of soil microorganisms on the nodulation in plants but

with the complexity of soil and with changing or shifting

of soil biosphere with intensive cultivation it becomes

necessary to make continuous efforts on the interaction

going on in the symbiotic relationship between the legume

and the rhizobia in the presence of soil fungi.

Lentil and cowpea are two important crops of the

area with multifarious uses. There has been little infor~

mation on the affect of soil fungi on the nodulation in

11

these crops. It was considered desirable to study the

relationship of some of the important fungus inhabitants

with rhizobia on these crops. The detailed plan of work

is given on following pages.

R E V I E W O F L I T E R A T U R E

12

REVIEW OF LITERATURE

The information on capability of legumes to fix

atmospheric nitrogen can be traced back to the early

nineteenth century when Boussingault (1839) pointed out

that legumes can obtain nitrogen from air when grown in

non-heated soil. Later Wilforth and Hellrigal (1888) dis­

covered the functions of the nodules formed on the roots

of leguminous plants. Beljerinch, (1888) for the first time

isolated and cultured a microorganism from the nodules of

legumes but could not identify it. Considerable work has

since been done on nodules, nitrogen fixing bacteria and

factors affecting them, in leguminous plants.

Process of nodule formation :

The process of nodulation in roots of leguminous

plants initiates with the adsorption of the bacterial cells

to root surface. This process is influenced by several

factors including the host factors and soil factors. Rovira

(1969) suggested that there has been high degree of host

specificity in the nodulation by bacterium. The bacterium

strain for a particular host will cause nodulation in a

particular plant. The host factors include besides the

genetic factors the exudations coming out of the roots.

Legumes are known to excrete a range of sugars, organic

acids and amino acids into the soil adjacent to the root

system which favoured the growth of Rhizobium.

13

Aguilar et al. (1988) reported that sucrose was

found to have strongest chemotactic response among the

carbohydrates and varrillyl alcohol among the phenollcs

produced by the plants.

Maleck e_t al. (1989), however, suggested that sugars

were better chemoattractants than amino acids but legume

root substances were the best ones. This was supported by

clouds of R. melllotl cells observed at the surface of

alfalfa roots. Dharmatilake et al . (1992) pointed out that

luteolin»a flavone produced by alfalfa plants*served as a

biochemically specific chemoattractant for R_. mililotl.

The importance of cell surface constituents of rhizobia

and legume roots in the process of attachment of the

rhizobia to root surfaces was first realized by Hamblin

and Kent (1973). He reported that seed lectins (plant

proteins) combined specifically with R. phaseoll. Bohlool

et al. (1974) pointed out that there was little or no

adhesion between Rhlzobium and plants of heterologous

cross inoculation groups. Chen et al . (1976) postulated

that rhizobia that did not nodulate soybean were not bound

by the lectins. Bhuvaneswari et: aJU (1977) obtained a very

strong correlation between the nodulating and lectin bin­

ding capacities of R. japonlcum.

Kosenko et ajU (1987) pointed out that specific

recognition between the rhizobia and root surface was due

to the plant lectins which bound to carbohydrate receptors

14

on the rhizobial cells. Dazzo et ajU (1985) pointed out

that the interface between polarly attached bacteria and

the root hair cell wall contained trifolin A in clover

plants, which helps in the process of recognition. The

production of extracellular microfibrils produced by the

host cells firmly anchored the bacteria to the root hair

tips. Anolles ejt al. (1986) and Smit et al. (1989) confirmed

the role of calcium dependent adhesion in the process of

infection.

As a result of infection by rhizobia, the first

visible response of host plants appears to be curling or

controlled growth and branching of the root hairs (Ward,

1887 and Nutman, 195 9).

Perez ejt a_l. (1989) pointed out that the curling

of root hairs has been attributed to various growth promo­

ting substances like indole acetic acid (IAA).

McCoy (1932), while studying the entry of rhizobia

into the root hairs, suggested that rhizobia entered into

root hairs through the mechanical injuries. Fahraeus

(1968) pointed out that Rhlzobium strains capable of

infecting a legume release a specific polysaccharide

which induces pectolytic activity by the root and causes

loosening of the root hair wall structure thus fascili-

tating the infection thread initiation. Chandler et al..

(1982) observed that in Stylosanthes spp.,nodule formation

15

occurred only at the lateral root junctions which was the

result of direct invasion by rhizobia through spaces bet­

ween epidermal cells.

Arya eit al. (1985) observed that after the release

of rhizobia the disappearance of infection thread was

attributed to its degeneration within large vacuoles from

the fusion of small vacuoles. Chalifour e£ aJU (1989) poin­

ted out that a hydrolytic enzyme of rhizobial origin was

involved in rhizobial release from the infection thread

into the cortical cells of the host plants. Frakash et_ a^.

(1986),while studying bacteroid branching inside nodules,

found that branched and ellipsoid bacteriods in Plsum

sativum roots were induced by R. leguminosarum.

Soil factors affecting nodulatlon :

The process of nodulatlon occurs in nature in soil.

Therefore, soil factors influence the development of

nodules. The soil factors affecting nodulatlon can be

classified into physical and biological factors. These

factors affect nodulatlon in two ways, one directly on

rhizobia and the other Indirectly on plants.

Soil Temperature :

Iswaran ejt al» (1970) observed that in many

tropical soils in which surface temperature is higher

and occasionally exceeds 40°C, the number of rhizobia

declines readily. Lipsanen et al. (1986),while studying

16

the adaptation of red clover rhizobia to low temperature,

reported that differences existed between the strains in

their ability to nodulate the host plant at different

temperatures. The most congenial range of temperature for

nodulation is 20-30 C. The low root temperature is not

only responsible for less number of nodules but also less

weight of nodules (Rice and Olsen, 1988). Sharma et_ al .

(1989) pointed out that the high temperature in the semi-

arid zones of Haryana could be responsible for the low

nodulation of pigeon pea because the plasmid carrying the

nodulation genes is cured at 40-45°C, giving rise to non-

nodulating mutants. Elsheikh et al. (1989) reported that

the tolerance of salt to rhizobia is dependent upon tempera­

ture, pH and carbon source.

Soil Moisture :

Soil moisture plays a very important role in sur­

vival of rhizobia in soil. Foulds (1971) reported that a

marked decline in population was revealed when soils

containing three different rhizobia were dried, although

the rate and extent of population reduction varied with

the soil and the test bacterium. Osa-Afiana et al. (1982)

pointed out that the percentage of cells of cowpea rhizobia

and R. japonicum survivings were directly related to the

quantity of water remaining, following desiccation and

were also influenced by relative humidity. Rhizobia are

readily transported by an advancing wetting front. This

17

indicates that non-saturated flow of soil moisture contri­

butes to the dispersal of rhizobial inoculum in soil

(Breitenbeck et a^., 1988). Postma ejt al. (1989) reported

that the rhizobial cells survived better in soil with lower

initial moisture than in soil with a higher initial moisture

content. Dudeja et al . (1989) reported that after the onset

of rain, there was an increase in the number of resident

rhizobia in the soil but after drying of soil the number

of rhizobia decreased. Yousef et_ al. (I989)>while studying

the interactive effects of inoculation and irrigation on

growth and yield of mung bean (Vigna radlata L.), observed

that irrigation with 80% of the evapotranspiration (Etp)

gave significantly the highest yield of seed and plant

top dry matter. Inoculation with the specific Rhizoblum

resulted in higher seed and dry matter yield than the

uninoculated plants. Venkateshwarlu and Ahlawat (1993)

suggested that irrigation at a ratio of 0.60 IN : CPE

(rrigation water depth : cumulative parr evaporation ratio)

with two irrigations produced taller plants with profuse

branching and root nodulation and had more dry matter

pots/plant and grain and straw yields compared with irri­

gation at 0.35 IW : CpE ratio and no irrigation.

Soil Texture :

Soil texture is also an important factor which

affects the growth of plants as well as their nodulation

because movement of air and water in soil is governed by

18

the texture of soil. Diatloff (1967) observed that during

wet periods in a black earth soil, aeration was the limi­

ting factor for nodulation of cowpea, soybeans, and native

legumes. He found that in black earth soil, sand content

was very low.

Aarons e£ a L. (1987), while studying the growth and

survival of cowpea rhizobia in bauxite silt loam and sandy

clay loam, reported that cowpea rhizobia survived better

in bauxitic silt loam than in clay loam soil. Heynen et al,.

(1988) suggested that the protozoa might be atleast partly

responsible for the decline of rhizobial numbers in loamy

sand, and that, the bentonite clay confirmed partial pro­

tection of introduced rhizobial cells against predation

by protozoa. El-Mokadem e£ ad. (1989) pointed out that

generally the nitrogen concentration in chickpea tissues

was higher in plants grown in loamy sand than those grown

in sandy soil. Hoeflich et aK (1990) suggested that the

persistance of rhizobia at legume and cereal seeds could

be elongated after seed pelleting or seed coating with

clay minerals like kaoline and bentonite and organic

substances. Heijnen et al. (1993), while working on

metabolic activity and population dynamics of rhizobia

introduced into unamended and bentonite amended loamy

sand, found that the presence of bentonite clay increased

the growth rate of rhizobia introduced into sterile soil.

Further the survival studies performed in non-sterile

19

bentonite amended loamy sand showed that the use of high

inoculum densities increased the final survival levels of

introduced rhizobia.

Hydrogen ion concentration

The low pH of soil influences nodulation by direct

and indirect effects on the bacteria and on the host.

Munns et_ aJU (1977) observed that the use of legume and

rhizobia that are adapted to soil acidity reduces the need

for expensive input of lime. Mengel e_t a_l. (1978) reported

that the critical pH for most of the soils was in the

range of pH 4.6 to 4.8 and that nodule number and weight

and the nitrogen content of plant tissues increased signi­

ficantly as the soil pH increased. Rai et ajU (1983) found

that the range of soil pH and associated acidity factors

in which nodulation and nitrogen fixation responded varied

depending on mutant strains in R_. leguminosarum associated

with Lens esculentum. The effect of soil pH on nodulation

and nitrogen fixation of winged bean Rhlzobium strains was

studied by Iruthayas (1986). He tested nine effective

Rhlzobium strains of winged bean for their symbiotic

performance under different soil pH levels. He found that

in general, all the strains performed best at pH 5.5.

Helemish et al_. (1987) found that R. leguminosarum TAL 271,

could grow in a wide range of pH and tolerated both moder­

ate acidity and alkalinity with optimum pH of 5.5. Coll

et al. (1989) pointed out that the growth of arrow leaf

clover was formed less on soils with reduced pH. Caetano

20

et al. (1989) reported the effect of pH and Ca on the

adsorption of R. mellloti to alfalfa roots. They found that

the adsorption of bacteria was abolished and viability

decreased at pH less than 6. Low pH appeared to affect the

stability of binding and cause desorption of previously

bound bacteria. Ducos ejt alL, (1989), while studying the

growth of R. meliloti 33 under laboratory conditions

(20 + 2°C) in 29 sterilized soil samples obtained from six

different soil profiles, found that the bacteria grew more

rapidly in soils which had mild to strongly alkaline pH

values (pH 7.4-9.5). Vargas e_t al . (1989) observed a

significant effect of host cultivar, ratio of inoculation

and pH on the percentage of nodule occupancy by different

strains of rhizobia in beans. He further suggested that at

low pH Rhlzoblum strain CIAT 899 had higher nodule occu­

pancy than strain UM 1116. Gemell et al. (1993), while

performing field evaluations, in acid soils, of R.

leguminosarum bv. trlfoll found that the most successful

strains in each year were those classified as acid sen­

sitive : they formed a higher proportion of the nodules

than those classified as acid tolerant. Further their

results suggested that in soil factors other than pH,

inorganic nutrients were important. Of these genetic back­

ground of strains was of such importance that it could

make improved nodulation in an acid soil.

21

Salinity and Alkalinity :

Siddique et a_l. (1986), while studying the effects

of salinization on nodulatlon and nitrogen fixation In pea,

found that Increasing the concentration of salt reduced

the number and weight of the nodules in pea in sand culture,

Desalinization (45 days after sowing) increased the produc­

tion and growth of the nodules. Salinity also reduced the

development of lehaeraoglobin in the nodules and brought

about an enhanced senesence whereas desalinization reversed

these effects to varying levels. Kassem ej^ a_l. (1986)

reported that in the presence of 1.5 per cent sodium chlo­

ride, young lucerne plants infected with three strains of

R. melllotl formed nodules which contained morphologically

normal bacteriods. With 1 per cent sodium chloride, the

nodules were colourless and the plants were less developed.

Nitrogenase activity measured in nodulated plants in

presence of sodium chloride (0.4 per cent) was poor at 1 per

cent but was completely Inhibited at 1,5 per cent sodium

chloride. El-Shinnawi et al, (1989) pointed out that

these salts were generally inhibitory towards plants

and bacteria. Number and characteristics of plant

root nodules, dry weight of plants, nitrogen content

of plants and bacterial colony count decreased. Chios

rides were the most inhibitory to nodulatlon in soil

and carbonates in the culture medium, while sulphates

were least inhibitory in either case. Formation of

effective nodules on roots of plants grown in the

22

salinized soils was very poor. Mlrza and Tariq (1993) had

seen the effect of salinity on growth and nodulation of

Trlfollum alexandrlnunu They compared the growth and nodu­

lation of plants at six levels (0-1.2 per cent) of sodium

chloride Dry mass of shoots and roots inc­

reased at low levels (0-0.2 per cent sodium chloride) but

decreased with higher sodium chloride concentrations.

Nodulation and nodule mass were also decreased with inc­

reasing salinity. Nalr ejt.al . (1993) pointed out that salt

tolerant rhizobia were less efficient in symbiotic nitrogen

fixation under non saline growth conditions of legume host

possibly due to depression in their nitrogenase activity

during salt stress. Nodule growth was significantly reduced

in treatments Involving salt acclimated rhizobia.

Effect of Nutrition :

Andrew and Nooris (1961) reported that nodule for­

mation was calcium sensitive in both tropical and temperate

legumes. The survival of tropical legumes on very poor

soil was due to their ability to extract from the soil

more calcium than temperate legumes. Freire (1976)

reported that growth and activity of Rhizobium as well as

effective nodulation requires an adequate supply of

calcium. Fandey et al^ (1986) found that rate of nodulation

in terms of nodule number and dry weight of Vlgna mungo

was slightly inhibited at higher concentrations of urea

and ammonium sulphate. Rhizobial population size was

13

stimulated with ammonium sulphate, but was markedly inhi­

bited with urea. Almendras et_ aK (1987) had shown that the

nodulation of subterranean clover by two of the sero-

groups 6 and 36 of R. trlfolll was differentially influenced

by an application of calcium carbonate which raised the pH

of the soil from 5 to 6.5. Liming the soil with either

calcium carbonate, calcium hydroxide, magnesium oxide,

significantly increased the percent nodule occupancy.

Habte et_ al. (1988) suggested that the adverse

effect of erosion of soil leading to the loss of calcium

affects the survival of rhizobium in the soil. This can be

overcome by supplimenting the soil with calcium supply.

Cadisch et_ aJU (1992) investigated the effects of potassium

on nodule formation in Centroserna acutifollum. He found

that potassium supply enhanced dry matter production by

85 per cent.

Sanoria et al . (1987) found that application of

phosphate to soil promoted greater nodulation and at the

same time Increased accumulation of nitrogen in soil.

Idris ejt al_. (1988), while studying the effect of Rhizobium

inoculation and application of phosphate in soil growing

soybean, found that application of phosphate at 60, 90,

120 kg/ha to the Bragg variety of soybean, inoculated with

a mixture of three strains of R. japonlcum in silty loam

soil, significantly improved the number of nodules per

plant and increased production of shoots and roots.

24

Cadisch et_ aK (1992) investigated the effect of phosphorus

on nodule formation. He pointed out that severe phosphorus

deficiency quickly led to a strong reduction in nodule

weight. Phosphorus was considered to have direct effect on

nitrogen fixation and nodule formation,

Mowad et al. (1987), while studying the response

of faba beans to rhizobial inoculation and fertilization

with micronutrients in sandy soils* showed that rhizobial

inoculation and micronutrient fertilization either alone or

in combination, improved seed yield, total plant dry weight,

nodule weight and nitrogen content of the leguminous plants.

They also pointed out that the deficiency of Molybdenum

or iron showed negative effect on nitrogen fixation and

plant growth as compared to Manganese or zinc deficiencies.

Potalia e_t aJL« (1987) reported that application of zinc

and Molybdenum depressed nitrogen stress in soil when

combined with rhizobial inoculant.

Troitskaya et: al. (1987) studied the influence of

R. japonicum and cobalt fertilizers on the content of

vitamin B-2 in soybean root nodules. He reported that

vitamin B-2 was found predominantly in bacteroids. Cobalt

fertilizers increased the amount of vitamin B-2 in the

bacteroids.

Simon ejt al, (1990), while studying the effects of

titanium in the form of titanium ascorbate on Rhlzoblum

japonicum, found that 1 UM and 2 uM. Titanium ascorbate

25

enhanced-the growth rate and dry matter of these strains.

But inorganic forms of titanium had no significant effect.

Effect of pesticides :

Kakralia ejt al, (1981) reported that ceresan

inhibited growth of R. leguminosarum at 1000 ppm while

brassicol did not. Brassicol or ceresan at 2 and 20 per cent

had no adverse effect on number but decreased fresh weight

and volume of nodules in pea plants. Daramola e_t ajU (1981),

while studying the effects of 3 herbicide formulations, i.e.,

preforan (2,4, dinitro-4-trifluoromethyl diphenyl ether),

Dacthal (dimethyl-2,3,5,6-tetrachlorotetraphalate) and

Dual (2-ethyl-6-methyl-N-2 raethoxyethyl or chloro-aceta-

nilide) on Rhizobium legume symbiosis, found that the highest

rate of dacthal and preforan merely decreased nodulation.

But the highest rate of dual completely killed the plants

within 14 days after planting.

Suneja et a_l. (1985) found that the treatment of

soil with aldrin and lindane resulted in a reduction of

nodule number, yield and nitrogen content in bengal gram

inoculated with chickpea rhizobia, Torralba et al, (1986)

pointed out that the closes of atrazine used, reduced the

number of nodules on legume roots. Ruiz and Bellogin

(1986) observed that the fungicides viz., captan and captafol

inhibited nodulation by R. trlfoliion Trlfollum alexandrinum

Mogollon et al, (1986) reported that herbicide amitrol

26

in low concentration had a favourable effect on the growth

of rhizobia. Agarwai et al. (1986) suggested that pesti­

cides belonging to the carbamate groups differed in their

capacity to effect nodulation and nitrogen fixation. Gupta

et al. (1988) found that the highest number of nodules per

plant and highest grain yield were recorded with treatment

with bavistin 0.1 per cent followed by rhizobial inoculation

in chickpea.

Martensson et al . (1989) reported the inhibitory

action of herbicide chlorsulphuran on the nodulation and

nitrogen fixation in red clover, Torralba elt al. (1987)

observed that the herbicide 2-4-D had a very light inhibi­

tory action on the growth of rhizobiaJ however, it had

a clear contrary effect on the nitrogen fixation by the

nodules. Mathur ej: al^ (1989) found that the treatment with

phosphamidon inhibited rhizobial growth temporarily but

stimulated nitrogenase activity which declined with passage

o£ time in culture medium. Abd-Alla et al. (1993) observed

that all doses of herbicide brominal significantly reduced

the nodulation, plant growth and nitrogen fixation in

Vlcla faba.

Biological Factors :

Mosse (1976) found that vesicular arbuscular

mycorrhizae formed a network of fungal hyphae around

infected roots which increase the absorbing capacity of

phosphate in different legumes. He further pointed out

27

that growth and nodulation of various species of legumes

were stimulated by mycorrhizal infection. Gabor e_t al.

(1987) suggested that vesicular arbuscular mycorrhizal

fungus of Glomus spp. stimulated nodule development and

activity under drought stress in soybean,

Foi eit al. (1989) studied the response of chickpea

to combined inoculation of rhizobia, phosphobacteria and

mycorrhizal fungi. They found a significantly greater

response in yield by the simultaneous inoculation with all

the three test organisms.

Angle et al. (1981) examined antagonistic effects

of mycotoxins on the growth of R. japonlcum and on the

nodulation and growth of soybean. Several fungi were found

antagonistic towards rhizobia because of their mycotoxins

produced in the sorroundings. Trichoderma viride, Rhlzppus

nigricans and Mucor vesiculosis were found consistently

antagonistic to R. japonlcum and were responsible for

reduced nodulation in soybean. Furgal et al. (1988)

reported that the growth of R. 1egumlnosarum was inhibited

by culture filtrates of saprophytic fungi like Aspergillus

nlger, Fusarlum solanl, Peniclllium notatum etc. in pea

plants. Prasad (1990) observed the inhibitory effects

of extracts of Aspergillus nlger on the length of radicle

and plumule of Dollchos lablab. The total sugar and amino

acids were found to be less in the cotyledonary leaves

but peroxidase and Indole acetic acid (IAA) activity

were stimulated.

2fl

Polyanskaya et_ al. (1985), while performing his

studies on stimulation and elimination of nodule bacteria

in soil into which the actinomycete and chltln were added*

found that with a mixed liquid culture (R. legumlnosarum

and Streptomyces olivoclnereus) the nodule bacteria sur­

vived better using the products of chitin hydrolysis

produced by actinomycete in early stages of growth but

at later stages nodule bacteria died off due to the

growth of soil antagonistic actinomycete.

Young ejt aJU (1986) reported that VA mycorrhiza

can improve the absorption of mineral phosphorus from

soil and also that the efficiency to utilize various forms

of mineral phosphorus by mycorrhizal plants depends on and

the species of mycorrhizal fungus/on the type of soil.

Li et al_. (1988) reported that the coinoculation

of legumes with antibiotic producing bacteria (Fseudomonas

spp.) and root nodule bacteria resistant to these anti­

biotics was a promising means of promoting nodulation and

possibly nitrogen fixation.

M A T E R I A L S A N D M E T H O D S

29

MATERIALS AND METHODS

Seeds of cowpea and lentil cultlvars recommended for and lentil

this area and rhlzobial culture for cowpea/wlll be obtained

ffom District Agriculture office Aligarh. The seeds will

be surface sterilized in 1:1000 parts mercuric chloride

and rinsed three times with sterilized distilled water.

The bacterization of seeds will be done by placing the

seeds in a slurry of 25 gm of rhizoblal culture and 25 gm

of sugar in 100 ml of sterilized distilled water. The seeds

will be dried at room temperature before sowing. Throughout

the studies seeds so treated will be sown in previously

autoclaved soil. Seedlings when 15 days old, will be used

for inoculation.

isolation of fungi

Fungi will be isolated from soil. The rhizospheric

soil contained in micro-spatula will be plated on steri­

lized, molten and cooled potato dextrose agar medium.

Before the agar solidifies, the sterilized dishes will be

rotated to distribute the soil in the agar evenly and

incubated at 25°C. The plates will be examined regularly.

The fungi appearing on surface of agar will be isolated

and purified by making single spore isolation (Hildebrand*

1938). The fungal cultures will be maintained in agar

slants and stored at 4°C.

30

Inoculation of plants with fungi

The test fungus will be grown on Richard's solution1*

for 15 days, in 150 ml Erlenmeyer's flask at 25°C. After

15 days the fungus will be filtered through Whatman No. 1

filter paper. The mycelial mal will be used for inocu­

lation. The fungus (5 gm) will be collected in flasks

containing 10 ml of sterile distilled water. It will be

homogenized in waring blender. The suspension containing

the fungus will be poured around the plant roots by

removing the soil and covering it with soil after inocu­

lation.

Maintaining culture of root-Knot nematode and renlfona

nematode

Seeds of tomato cv Pusa Ruby will be surface steri­

lized and sown in sterilized soil. Each seedling will be

inoculated with single eggmass of root knot nematode

collected from field. The plants so inoculated will

develop galls in a few days. The Inoculum from the galls

will be used for raizing the nematode culture on tomato

cv. Pusa Ruby. For reniform nematode, larvae will be

•Composition of Richard's solution

KNO 3 10.00 g

KH2PO4 5.oo g

MgS04.7H20 2.50 g

FeCl3.6H20 0.02 g

Sucrose 50.00 g

Agar 15.00 g

Distilled water 1000 ml.

31

isolated from soil around the caster roots. These larvae

will be placed in sterile distilled water at room tempera­

ture for development of immature females. The tomato

seedlings will be inoculated with the immature females

for raizing inoculum.

inoculation of plants with nematode

For inoculation of root-knot nematode, eggmass

from pure culture of the nematode will be treated with

sodium hypochloride (1%). The eggs will be liberated out.

The eggs so obtained will be used as inoculum. The imma­

ture females of reniform nematodes as obtained above will

be used as inoculum. A suspension of about 1000 second

stage larvae of root-knot nematode and immature females

of reniform nematode separately will be made in 5 ml of

sterile distilled water. The entire suspension will be

poured around the root of seedlings by making 4 holes.

The holes will be covered by soil after Inoculation.

Effect of living organisms on the development of

nodules.

The seedlings developed from bacterized seeds will be

inoculated as follows :

1. Root-knot nematode (Meloidogyne incognita)

2. Reniform nematode (Rotylenchulus renlformis)

3. Root-knot nematode + Aspergillus niger

32

4. Root-knot nematode + Fusarlum oxysporum

5. Root-knot nematode + Trlchoderma vlrldl

6. Root-knot nematode + renlcllllum spp.

7. Root-knot nematode + Trlchotheclum roseum

8. Reniform nematode + Aspergillus nlger

9. Reniform nematode + Fusarlum oxysporum

10. Reniform nematode + Trichoderma vlrldl

11. Reniform nematode + Eencillium spp.

12. Reniform nematode + Trlchotheclum roseum

Observations

Throughout the studies observations with regard to

growth of the plant and nodulation will be made after 45

days of sowing. Flants will be uprooted, length of root ana

sBoot, fresh and dry weight of the plant will be determined.

Plants will be washed in running water and placed on blo­

tting paper so that extra water is soaked. Length will be

determined In cm and ram and fresh weight on the weighing

pan. The plants will be dried at 60 C in oven and after

having cooled at room temperature dry weight will be deter­

mined. There will be 10 replicates for each treatment and

each study will be repeated thrice. Nodulation will be

determined by counting the number of nodules and size of

nodules. Data will be subjected to statistical analysis

to determine the significance.

33

Estimation of Leghaemoglobln from nodules

Benzidine hydrogen peroxide method (proctor, 1963)

will be used for the estimation of leghaemoglobln.

Nodules will be picked up from the roots and washed

thoroughly with double distilled water. Fresh nodular

tissue weighing 500 mg will be crushed and homogenized in

9 ml of tris-acetic acid buffer (0.1 M acetic acid plus

0.2 M tris(hydroxylmethyl) amino methane to obtain a pH

of 4.0|. The homogenate will be kept overnight in a deep

freezer to get complete extraction of Leghaemoglobln. The

homogenate will be shaken from time to time to facilitate

extraction. The volume of homogenate will be made up to

10 ml by adding tris acetic acid buffer and centrifuged

at 3000 rpm for 20 minutes. The supernatant will be collected

in a test tube. An aliquot of 0.5 ml of supernatant will

be diluted to a final volume of 4.0 ml with tris-acetic

acid buffer. To this, 2 ml of freshly prepared benzidine

reagent (100 mg benzidine with 0.5 ml of hydrogen peroxide

of 100 percent by volume added to 50 ml absolute alcohol)

will be added. The colour density will be measured at 660

nm on spectrophotometer. The results will be expressed in

bovine haemoglobin equivalents.

B I B L I O G R A P H Y

34

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