Mycorrhizal Technology for Reclamation of - isca.co.in 978-93-84659-47-9.pdfa variety of purposes like planning reclamation programs, rational land use planning, ... A.1 Saline soils

Mycorrhizal Technology for Reclamation of

Saline Waste Land of Indian Thar Desert

By

DR.NISHI MATHUR Head, Department of Biotechnology

Mahila P.G.Mahavidyalaya

Kamla Nehru Nagar, Jodhpur, Rajasthan,INDIA

2016

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ISBN:978-93-84659-47-9

Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert iii

DEDICATED

TO

MYBELOVED

PARENTS

Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert iv

S.NO. NAME OF CHAPTER PAGE NO.

1. INTRODUCTION 2-4

2. MATERIAL AND METHODS 5-21

3. RESULTS 22-45

4. DEVELOPMENT OF ARBUSCULAR MYCORRHIZAE 46-50

5. DISCUSSION 51-59

6. SUMMARY 60-61

7. REFERENCES 62-87

CONTENTS

Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 1

Chapter – 1

Introduction

Land is facing serious threats of deterioration due to unrelenting human pressure and

utilisation incompatible with its capacity. The information on land degradation is needed for

a variety of purposes like planning reclamation programs, rational land use planning, for

bringing additional areas into cultivation and also to improve productivity levels in degraded

lands. Land degradation has numerous environmental, economic, social and ecological

consequences. There can be rather serious effects in terms of soil erosion, loss of soil fertility

and thus reduced plant growth or crop productivity, clogging up of rivers and drainage

systems, extensive floods and water shortages. It is estimated that some forms of land

degradation constituting 75% of the earth’s usable landmass affect 4 billion people in the

world. About 15% of the world population is effected by land degradation which is likely to

worsen unless adequate and immediate measures are taken to arrest the degradation

processes. The largest category is land affected by water and wind erosion, which account for

80 percent of degraded followed by salinization / alkalization and waterlogging. Reliable time

series data are available only for salt affected land, which has grown from 7.18 million

hectares in 1987 to over 10 million in 1993 (Annon, 2002). According to NRSA / DOS project

on ’Mapping of salt affected soils of India on 1:250,000 scale’, the area under salt affected

soils in the country is 6.727 million hectares. An estimated area of 2.46 million ha land is

suffering from water logging in irrigation commands in India (Anonymous, 1991). “An area is

said to be waterlogged when the water table rises to an extent that soil pores in the root

zone of a crop become saturated, resulting in restriction of normal circulation of the air,

decline in the level of oxygen and increase in the level of carbon dioxide”. It 2 may result in

various types of soil degradation like physical degradation or chemical degradation or

salinity. Satellite data are being used regularly for mapping and monitoring of waterlogged


areas. Salt affected areas are one of the most important degraded areas where soil

productivity is reduced due to either salinization( EC > 4 dS/m) or sodicity (ESP > 15) or both.

The soils with EC more than 2 dS/m in black soils and >4 dS/m in non-black soils was

considered as saline in the present project. Soils with soil pH more than 8.5 results in increase

of exchangeable sodium percentage (ESP) in soils (> 15) and are termed as sodic. Based on

the type of problem, it has been divided into saline, sodic and saline-sodic. Under NR Cenus

project three types of salt affected soils viz., saline, sodic and saline-sodic are mapped using

three seasons satellite data, field work and analysis of soil samples under three severity

classes namely slight, moderate and strong. A.1 Saline soils These soils occurs in arid and

semi-arid regions, coastal areas, irrigated commands and peripheries of streams in peninsular

regions. The soil pH is usually less than 8.5 and EC is more than 4 dS/m. On satellite data it is

seen in light grey to white with association of poor crop growth. In severe cases, there may

not be any vegetal cover, even grass. A.2 Sodic soils Usually it occurs in the older alluvial

plains. Because of high sodium content, soils will be moist during post-monsoon season

which can be seen easily in the post-monsoon image. It appears on satellite data as grayish

white / dull white discrete patchy. It occurs as 3 contiguous patches with smooth texture on

the image. Multi temporal data set will help in delineation of affected areas and to some

extent severity classes. The soil pH values will be more than > 8.5, and EC will be < 4 dS/m

and ESP is greater than 15. A.3 Saline Sodic Soils The Saline-Sodic soils occur in arid and

semiarid regions. It appears as grayish white with red and white mottle color on the image.

Bright white tone, dominantly in Indo-Gangetic alluvial plains. In coastal plain it is creamy

white color with mottle tone. The soil pH is greater than or equal to 8.5 and EC is greater than

or equal to 4 dS/m.


Chapter – 2

Materials and Methods

MYCORRHIZAL TECHNIQUES

Collection

Isolation of Spores

Identification of Spores

Staining Procedure for Root

Percentage of Root Colonization

Mass Multiplication of Inoculum

Inoculation of Mycorrhizae in Seed Lings of Tree Species

PLANT ANALYSIS

Plant Height and Biomass Dry Weight

Phosphorus

Nitrogen

Acid / Alkaline Phosphatase

Nitrate Reductase

Total Phenol

Peroxidase & Polyohenol Oxidase

MYCORRHIZAL TECHNIQUES


Collection

During the present research investigation work rhizospheric soil of the Casuariana

spp. were collected from various localities of Western Rajasthan namely

Jodhpur Region

Pali Region

Udaipur Region

Mount Abu Region

The periodical survey of Western Rajasthan was undertaken in order to collect the

rhizospheric soil as well as root samples. Root and Rhizosphere soil samples for plant species

were collected from five individuals at different stages of growth (vegetative and

reproductive). Care was taken during collection that roots of shrubs and tree species could be

positively identified, so take them carefully without sample mixing. Root samples were

washed thoroughly free of attached soil particles and cut into several small segments and

stained within 24 hours or preserved in formalin-acetic acid alcohol upto six months before

staining. Rhizosphere soil from roots and adjacent to plants were collected. Soil samples

collected from different indivisuals of a species were mixed to form a composite sample.

These composite soil samples were used for the isolation of VAM fungal spores and for soil

chemistry.

Isolation of Spores

To Isolate Mycorrhizal spores from the soil many methods can be employed. Out of

these, techniques three has been used in present investigation.


1. Wet sieving and decanting technique: The soil samples collected were processed by

wet sieving and decanting technique of Gerdemann and Nicolson (1963) to obtain

spores. The details are as follows-

100 g soil was taken and mixed in luke warm water in a large beaker and the heavier

particles were allowed to settle down.

The suspension was then poured through a coarse sieve (710 µm) to remove large

pieces of organic matter.

The roots and organic matter on the sieve were washed with a fine jet of water from a

squeeze bottle to ensure that all the small particles have passed through. The washings

which have passed through the sieve were collected.

The particles were resuspended by stirring several times and this suspension was

decanted through 500 µm, 250 µm, 125 µm, 105 µm and 53 µm sieves respectively to

retain the desiring spores.

Each sieve was washed into separate small beakers and was examined in turn. Root

pieces retained on the 710 µm sieves were examined for attached hyphae, spores and

sporocarps under stereomicroscope.

The organic matter from 250 µm sieve was examined for sporocarps and large spores.

The 105 µm sieving yield most spores since their size range was between 100 µm and

250 µm and spores smaller than 100 µm often occurred in moths, trapped on the 105

µm sieve. Small detached spores were found on the 53 µm sieve.

Spores were picked up with the help of plastic syringe and were mounted in polyvinyl

alcohol lacto-glycerol (PVLG) (Koske and Tessier, 1983) and observed under stereo

microscope for identification. The fungal propagules were used as primary inoculum for

the pot culturing of different species.

Spore Number and variability are counted by using grid-line intersect method, whatman

filter paper No. 1(size, 11cm diameter) (By Gaur and Adholeya, 1994).


For clayey soil, which blocks the sieve by forming suspension, precipitate the particles

in 0.1M sodium pyrophosphate. If the spore number is low, “host baiting technique” or “trap

pot culturing” may be employed. A soil sample from the test soils are added to a sterilized

greenhouse potting mix and planted with a suitable trap or bait crop. A mixture of a

perennial grass and a legume is preferable. After 3 to 5 months the potting mix can be used

for enumerating AM spores.

2. Sucrose Centrifugation: Wet sieved material processed with Sucrose Centrifugation

method of Smith and Skipper, 1979. to obtain spores. The details are as follows-

Take a suspension of spores with debries collected from the sieving in a 50ml centrifuge

tube and make upto 35ml with distilled water. Centrifuge at 2000 rpm for 10 min.

Filter the supernatant

Suspend the pellet remaining after the first centrifugation in enough 2M sucrose

solution and bring the volume to 35ml

Stir it vigorously, centrifuge at 200 rpm for 10min., filter the supernatant and collect the

spores.

This technique gives a suitable and easy way to collect spores from soil sample even

they present in low quantity in soil.

3. Alternative method for Ohm’s technique: Wet sieved material processed with

Alternative method for Ohm’s technique of Menge, 1982. to obtain spores. The details

are as follows-

Transfer soil sieving to a blender and at high speed for 1 or2 min. This frees any spores

attached to the roots, or in sporocarps or in the clay particles.

Pass contents of blender through a fine sieve and wash the colloidal material

thoroughly with a strong stream of water.


Add 10 ml of 20% sucrose into a clean 50ml centrifuge tube, followed by 10ml of 40%

and then 10ml of 60% sucrose into the bottom of the tube.

Add 10-15 ml of blended sieving onto the surface of the 20% sucrose layer.

Centrifuge for 3 min. at 300 rpm.

Remove debris which gathers at the 20-40% and / or 40-60% interfaces. Often the layer

of spores is visible and can be removed without taking any of the debris which

remained in solution.

Rinse spores on a fine sieve with a strong stream of water to remove sucrose and collect

the spores.

Identification of Spores

In the present investigation, different species of AM fungi were identified with the

help of synoptic key of Trappe (1982), manual of Schenck and Perez (1987) and Marton

(1988). For that species and genera of AM fungi were identified on the basis of morphology of

their resting spores i.e. chlamydospores.

Staining Procedure for Root

VAM root infection consists of intra and intercellular hyphae and vesicals together

with finely branched Arbuscules within the host cortical tissue. The anatomical feature

characteristic of VAM infection cannot be seen unless the infected roots are suitably stained.

To observe the VAM infection, two technique used. The details are as follows-

1. Method of Philips and Hayman (1970)


Roots are washed in tap water, but not vigorously enough to detach the external

mycelium. Cut root into 2 cm segments

Then root pieces simmered at about 90 0C for 15-30 minutes (depending upon hardness

of the roots) in 10% KOH. KOH solution clears host cytoplasm and nuclei and readily

allows stain penetration.

Rinse root segments 4-5 times in tap water.

After that, acidified root segments by immersing them in 2% HCl for 5minutes. Acid is

poured off and stain is added viz. 0.05% trypan blue in lactophenol.

Root segments are kept in stain overnight (covered). Stain was poured off, lactic acid:

glycerol (1:1) was added and roots were kept over night in this liquid to destain the host

tissue.

The squashed roots were examined under the microscope. To observe hyphae,

vesicles and arbuscules under light microscope the root pieces were mounted sealed with

D.P.X. on glass slide temporarily in lactophenol or permanently in poly vinyl alcohol. The

coverslip was pressed gently to make the roots flattened and observe under microscope for

the infection.

2. Method of Brundrett et al., (1984) observation of arbuscules: For study of arbuscules in

more clear way Brundrett et al., (1984) proposed a new method, which involves staining by

chlorazol black dye. The detailed procedure of this method is as follows-

Reagents- FAA, 10% KOH, 80% Lactic acid, Glycerin, 95% ethanol, Chlorazol black E, Basic

fuchsin, Chloral hydrate.

Preparation of mounting fluid- 20 g Chloral hydrate + 20 g Gum arabic + 20 ml Glycerine + 3

ml distilled water + 10 drops of basic fuchsin (0.3 g/10 ml 95% ethanol).

Roots were washed in tap water and fixed overnight or stored in Formalin-Acetic Acid-

Alcohol (FAA).


Rinsed with several changes of tap water to remove FAA. These were transferred into

a 10% KOH solution in autoclave resistant jars.

Roots were then sterilized in autoclave for 15 minutes at 121 0C. Samples with delicate

roots may require shorter sterilizing time.

They were rinsed with several changes of tap water followed by deionised water.

Then, the roots were transferred into a staining solution consisting of equal volumes

of 80% lactic acid, glycerin and distilled water with 0.1% chlorazol black E. Stain for 1

hour or longer at approximately 90 0C. Staining solution was prepared several hours

before use and undissolved particles were allowed to settle down.

After decanting overnight in glycerin roots were mounted on slides using mounting

fluid. Roots were examined under a light microscope.

Percentage of Root Colonization

The percentage of root colonization of AM fungi in the roots was calculated by

Gridline intersect method of Giovannetti and Mosse (1980). The stained root pieces were

spread evenly on a plastic petridish.. A grid of line was marked on the bottom of the dish to

form 1 cm squares. To facilitate the observations, the roots were immersed in a solution of

glycerol and water (1:8 v/v). The glycerol increases the viscosity of the medium and prevents

excessive movements of roots. The roots were than observed under the microscope. The

petri plates were moved first horizontally and than vertically along the grid line. Two

observations were recorded simultaneously:

(a) Total number of roots intersecting grid lines.

(b) Total number of infected roots intersecting grid lines.


From this the percentage of root colonization was calculated by using the following formula-

100

% xline grid inginteresect Roots of Number Total

line grid ngintersecti roots infected of Number TotalonColonizatiRoot

When sufficient root pieces were not available, the slide method was followed. Root

pieces 1 cm long were selected at random from a stained sample and mounted on

microscopic slides in groups of ten. Presence of infection was recorded and percentage of

infection was calculated.

Mass Multiplication of Inoculum

The pot trial was conducted at the Department of Botany, JNVU, and Jodhpur during Rain

spring season of 2007-08.

PVC pots of 18 cm diameter were filled (sterilized with alcohol) with sterile sand : sandy

loam (1: 1 by volume) soil @ 3 kg/pot.

Soil sterilized by autoclaving at 15-lbs/sq inch pressure, 121 0C for 40 minutes. (It was

done twice with a day interval in between). The soil had 20 kg P2O5/ha (NH4F + HCl

extractable) with a pH of 7.2 surface sterilized seeds of Cenchrus ciliaris and Sorghum

vulgaris were grown in funnels and transplanted to pots after 30 days (Plate 2 b & c).

Seven efficient strains of VAM fungi isolated from soil of different sites were separately

placed 3 cm below the soil surface before sowing the seeds in funnel culture. Pots were

watered regularly. They were neither allowed to dry nor were flooded.

Pots were examined regularly for purity of the inoculum and were maintained through

out the course of investigation.


Since Cenchrus ciliaris and Sorghum bicolor is a perennial grass, so it was possible to

maintain pot cultures regularly by cutting the aerial part of the plant time to time.

Uninoculated plants were kept as control.

The plants received 50 ml per pot of Ruakura nutrient solution (Smith et al., 1983)

without P once in 30 days.

The plants were harvested after 90 days. After harvesting, shoot and root’s fresh weight

and dry weight were recorded. Soil of pot culture is used for spore source for inoculum

and further physiological and biochemical studies.

Inoculation of Mycorrhizae in Seed Lings of Tree Species

Inoculation of Mycorhizal spores done by three methods

1. Pellates mathods: (Menge and Timmer, 1982.)

Use inoculum of VAM fungi consisting of ground granular crudely produced pot culture

inoculums containing plant roots, mycorhizal spores and growth media such as perlite,

peat mass, vermiculate, sand or soil for field inoculation.

Air dry this inoculums to about 5-20% moisture.

Prepare mycorhizal pellets by mixing 20 parts mycorhizal inoculums, 1 part autoclaved

sedimentary clay (mean particle size, 16 um) and 1 part autoclaved tertiary sedimentary

clay (mean particle 2-6 um)

Add water until the mixture is malleable and could be rolled into pellets.

Make pellats each weighing 1.4 g and use them before 28 days of storage.


Prune the host plants to soil level. Remove the compacted soil mass from the pot and

plunge it in water to save even the very fine roots.

Chop the recovered roots and homogenize the roots and soil in a sterile blender.

Examine for the presence of plants pathogens, mycorrhizal hyperparasites, cultural

purity, spore number and spore maturation.

Air dry the soil mixture to the point at which there is no free water. After drying, pack

the culture in plastic bags and seal to prevent further drying.

Store at 5 Co.

2. Direct Inoculation from Pot culture Inoculum:

The inoculum in form of pot soils containing extrametrical chlamydospores and AMF

infected roots pieces of Cenchrus ciliaris and Sorghum vulgaris was placed 4 -5 cm below the

soil surface before sowing.

The seeds were sown and were kept in glass house under temperatures 25-35 0C. The

seedlings were regularly examined for the mycorrhizal development. The samples were

harvested on the requirement for further studies.

3. Production of alginate entrapped VAM inoculum:

Sand and soil mixture containing azygospores and infected root segments (chopped) of

Cenchrus ciliaris and Sorghum bicolor infected with VAm fungi grown for 90 days served

as the mycorhizal inoculums.

The inoculums were air dried and passed through 400 um sieve.

To an aqueous suspension of sodium alginate (2%), 10% of the sieved sand: soil

inoculums of the VAM fungus plus 2% of the carrier material (perlite, sorlite, talc,


vermiculite, kaolinite and bentonite were added separately and mixed using a magnetic

stirrer.

This mixture was passed through a sieve onto 0.1 M sterile calcium chloride solution to

form beads (Plate 4 d) After 30 minutes the beaded inoculums was rinsed with tap

water.

Aportion of the alginate entrapped wet VAM inoculums was dried to surface dryness

and stored at 4o C. This formed the carrier based alginate entrapped wet VAM

inoculums (Kropacek et al., 1989; Strllu and Plenchette, 1991). A portion of the beads

were air dried for 5 days to form dry VAM alginate inoculums packed in polythene bags

and stored at room temperature (32 ± 5o).

The pH of the carrier materials used in the study was estimated by using a digital pH

meter (substrate: water ratio = 1:10 w/v).

The moisture content of the wet VAM beads was determined after drying to a constant

weight. The number of propagules in the different carrier based alginate entrapped

VAM inoculum was determined by the MPN method using four-fold dilution (Sieverding,

1991).

The alginate beads were solubilized in 0.2 M sodium citrate solution (pH adjusted to 7.2)

prior to carrying out the MPN test.

The Propagule numbers in the dry VAM alginate beads was computed from the

propagule numbers of the wet VAM alginate beads and its moisture content.

A pot culture experiment was also conducted to know the effect of alginate entrapped

VAM inoculums on the colonization of roots and growth of wheat as the host plant. The soil

used for this study was an alfisol (Fine, Kaolinthic, isohyperthermic type, Kanhaplustalfs) of

pH 7.2 with 2.4 mg available P/g (NH4F + HCl extractable) and an indigenous VAM population

of 0.31 propagules/g of soil. Earthernware pots (18 cm deep x 18 cm diameter) were filled


with 3.5 kg of soil and inoculated with alginate entrapped wet and dry VAM inoculums (with

perlite, soilrite, talc and vermiculite as carriers) and sand : soil inoculum of Glomus mossae at

the rate of 300 prpagules per pot. Four Two seedlings were planted per pot. Each treatment

had three replicates. The pots were arranged in a glass house (temperature 29 ± 2o C) in

randomized complete block design and watered whenever necessary. Fifty ml Rkura nutrient

solution was added thrice (first application with P, 20 days after planting and the other two

later applications without P, on 40 and 60 days after planting.)

Observation on plant height, fresh weight and dry weight of shoot and bulb 90 days

after planting. The plants were harvested 90 days after planting. Plant samples were oven

dried at 60o C to a constant weight to get plant biomass. Phosphorus and potassium content

of the shoot and leaf samples were determined, by the Vanado molybdate phosphoric yellow

colour method (Jackson, 1973) and flame photometric method respectively. Mycorhizal

colonization pf the root was determined by the grid line- intersect method (Giovannetti and

Mosse, 1980) after staining the roots with trypan blue ( Phillips and Hayman, 1970). The data

obtained from the pot expermint was subjected to analysis of varience by randomized

complete block design and treatment means were seprated by Duncan’s multiple range

(DMR) test (Little and Hills, 1978).

PLANT ANALYSIS

Plant Height and Biomass Dry Weight

Plant heights were recorded in cm and plant dry weights were recorded after drying

them in hot air oven at 80 0C for 48 hours.

Phosphorus


Total phosphorus was estimated by Vanadomolybdate method of Jackson (1973).

Following reagents were prepared-

Reagents:

a. Ammonium molybdate - This solution was prepared by dissolving 6.25 g ammonium

molybdate in 250 ml of distilled water.

b. 5N Sulphuric acid - 14.1 ml of conc. sulphuric acid was taken and final volume was

made up to 100 ml by adding distilled water.

c. Stannous chloride - 250 mg of SnCl2 was dissolved in 10 ml of conc. HCl by heating up-

to boiling. Volume was made up-to 25 ml by addition of distilled water. This solution

was prepared freshly while performing experiments.

d. Standard solution - 43.8 mg of KH2PO4 was dissolved in 100 ml of distilled water. This

was 100-ppm solution. From this stock solution of 100 ppm the stock was diluted to 10

ppm by adding distilled water in the ratio of (1:9) 1 ml stock and 9 ml distilled water.

e. Triacid mixture - HNO3, perchloric acid and H2SO4 were mixed in the ratio of 10:3:1

respectively following the method of Krishna and Dart (1984).

Preparation of standard curve:

The stock solution was pippetted out in 11 test tubes ranging from 0.1 ml to 1.0 ml.

The volume was raised to 1 ml by adding respective quantities of distilled water. 0.4

ml of ammonium molybdate, 0.4 ml of H2SO4, 0.25 ml of SnCl2 (freshly prepared) were

added to each test tubes. O. D. was read at 700 nm.

Extraction of plant material:

50 mg of dried plant material was taken in digestion tube and 3 ml of triacid mixture

was added. Plant material was digested for one hour and then allowed to cool down.


Final volume of digested solution was made up to 25 ml, 0.2 or 0.3 ml of plant extracts

were raised to 1 ml by adding respective quantities of distilled water to it 0.4 ml of

H2SO4, 0.25 ml of SnCl2 (freshly prepared) and 0.4 ml ammonium molybdate solution

were added.

It was incubated for 10 minutes and then 2 ml of distilled water was added. The O. D.

was taken at 700 nm.

Nitrogen

Reagents:

a. Sodium thiosulphate

b. Sodium hydroxide pellets

c. 0.7 g of mercuric oxide

d. Potassium sulphate

e. Salicylic acid

f. 0.08 g of methyl red and 0.02 g of methylene blue in 50 ml of ehanol

g. Boric acid

h. 0.02 N NaOH

Procedure:

Weigh 2 g of sample and transfer to a Kjeldhal flask.

Add 40 ml of concentrated sulfuric acid containing 2 g of salicylic acid and mix well.

Allow standing for 1 hour with occasional shaking.

Add 5 g sodium thiosulphate, shake, let stand 5 min, and then heat until forthing

ceases.


Turn off the heat, add 0.7 g of mercuric oxide and 15 g of potassium sulphate,

Cool, add about 200 ml of water, wait until it cools to room temperature, and then

add a few zinc granules.

Tilt the flask and carefully add without agitation, 50 ml of a solution containing 500 g

of sodium hydroxide pellets and 40g of sodium thiosulphate, dissolved in 1 litre of

water.

Immediately connect the flask to the distillation system with the condenser tip

immersed in 50 ml standardized 0.1 N boric acid in a receiving flask.

Then rotate the digestion flask slowly to mix the contents, and heat to collect 200 ml

of distillate.

Acid / Alkaline Phosphatase

Reagents:

a. Acetate buffer (pH-5.0) ( For acid phosphatase)

b. 0.1M Tris-HCl buffer (pH-8.0) (For alkaline phosphatase)

c. 50 mM p-Nitrophenyl phosphate (p-NPP) (0.18 g/10 ml in d.water)

d. 0.5 M KOH

Procedure:

Take 2.5 ml soil suspension into a 10 ml test tube.

Add 0.5 ml of 0.1 M acetate buffers and 3.3 ml d.water.

Add 0.5 ml of 50 mM p-nitrophenylphosphate solutions.

Incubate ona shaker, in the dark, at 40oC for 1 hour.

Add 2.5 ml of 0.5 M KOH to termination of enzyme reaction in the above mixture.


Centrifuge at 1500 rpm for 10 min to removeal of precipitate, if any.

Collect the supernatant and measure the OD at 450nm.

Calculation:

One unit of enzyme activity is expressed as the amount of enzyme required to

liberate1umol p-nitro phenol produced g-1wet soil h-1

Enzyme activity (U/h/g wet wt) =

Molar extinction coefficient of p-nitro phenol = 18.8 M/LPath length = 1.0 cm

= 18.8 x OD at 405 x 1.0 x DF x Total volume of water added (ml)

Incubation time (h) x Initial wet wt of soil, in g

Nitrate Reductase

The assimilatory reduction of nitrate by plants is a fundamental biological process in which

highly oxidized form of inorganic nitrogen is reduced to nitrite and then to ammonia

(Plummer, 1988). Nitrate reductase is a substrate inducible enzyme of high molecular weight

containing FAD, cytochrome and molybdenum as prosthetic groups. Depending upon the

electron donor two major types of nitrate reductase occurs-

(a) Ferredoxin dependent nitrate reductase (blue green algae)

(b) Pyridine-nucleotide dependent nitrate reductase (higher plants)

For the assay of nitrate reductase in-vitro, Wray and Filner’s method (1970) was used.

500 mg of plant material was homogenized in 5 ml of extraction media containing 0.1 mol/l

phosphate buffer (pH 7.5) and 1 mM cysteine. The reaction mixture containing 0.5 ml of

enzyme extract, 0.01 ml of KNO3 (0.1 M), 0.5 ml of phosphate buffer (0.1 M pH 7.5), 0.1 ml of

NADH (1mM and 0.1 ml of double distilled water was incubated for 15 minutes at 30oC). The


reaction was terminated adding 1 ml of sulphanilamide (1% in 3 N HCl) and 1 ml of 2.02% N-

Naphthylene diamine dihydrochloride (NEDD). The reaction mixture was centrifuged to

discard proteins and absorbance was recorded at 540 nm against blank in which enzyme was

added after sulphanilamide. The standard curve was prepared by using 1 µg/ml of NaNO2.

Total Phenol

Total phenol was determined following Mahadevan’s method (1975) using folin-

ciocalteu reagent. Estimation of the total phenols with folin-ciocalteu reagent is based on the

reaction between phenols and an oxidizing agent phosphomolybdate that results in the

formation of a blue complex (Bray and Thorpe, 1954).

Fresh plant materials were extracted in 80% ethanol in soxhlet apparatus. The alcohol

extract was evaporated to dryness and was redissolved in 30% methanol. The methanolic

extract was used for calorimetric estimation of total phenols.

Suitably diluted methanolic extract of the plant material was taken in test tubes. To it

1 ml of folin-ciocalteu reagent (diluted with equal volume of double distilled water) was

added followed by 2 ml of Na2CO3 (20% W/V) solution. The test tubes were shaken gently and

then heated in a boiling water bath for exactly one minute. It was then cooled down under

running tap water. The blue colored solution was suitably diluted with distilled water and the

absorbance was measured at 650 nm in a spectrophotometer. Qualitative estimation of total

phenols was attempted with the help of standard curve prepared from different

concentrations of catechol (5 to 50 µg). The polyphenolic contents are expressed as mg/g

fresh weight. All the experiments were performed in triplicates to avoid errors. Average value

of five observations was considered for final calculations of total phenols.

Peroxidase & Polyohenol Oxidase


(a) Peroxidase and Polyphenol oxidase (PRO and PPO)

Root pieces were homogenized in 0.1 M phosphate buffer (pH 7.0), with a pre-chilled

mortar and pestle at 4 0C. The homogenate was centrifuged at 5,000 rpm for 15 minutes and

the supernatant was used for enzyme assay. Peroxidase activity was measured by incubating

the enzyme with guaiacol and hydrogen peroxide (Racusen and Foote, 1965). The arbitrary

unit of enzyme activity chosen was change in absorbance of 0.001/sec. Polyphenol oxidase

activity was measured at 420 nm, using the method of Mahadevan (1975). The activity is

presented in terms of absorbance of 100 mg/g fresh weight of tissues.


Chapter – 3

Results

It is well recognized that AM fungi helps in improving plant biomass production and

nutrient uptake under different climatic condition. In order to evaluate potentiality of

different AM fungi on biomass production and nutrient uptake in two species of Acacia s

(namely A.tortilis and A. nilotica).

After collection of plant species the studies were further carried out to find out the

effect of abiotic factors viz. soil pH, moisture organic carbon, phosphorous and nitrogen on

spore population and percentage root colonization by AM fungi.

For this purpose soil samples along with roots were collected from rhizosphere soil of the

Acacia from various localities viz.Pachpadara , Balotara , Luni and Phalodi .Respective soil of

all the four localities was sandy (Table 1, Histogram 1), and 86.5-89.0 per sent sand particles

present in sampled rhizospheric soil.

Mycorrhizal spore population at various localities varied from 90-150 spores per gram

of soil. Soil sample of Pachpadara showed minimum spore population i.e. 90 spores per g of

soil while sample from Balotara showed maximum spore population i.e. 150 spores per g of

soil.

Percentage root colonization by different AM fungi at various localities varied from

48-68 per cent. Soil sample collected from Phalodi showed minimum percentage of root

colonization i.e. 48 per cent and maximum is observed in the sample collected from Luni i.e.

68 per cent.


During the present study mycorrhizal spore population was not found to be correlated

with percentage root colonization in rhizosphere of Acacia spp., collected from various

localities of the region. This suggests that it is not the quantity of mycorrhizal spores which

affects the root colonization, but it is the potentiality of AM spore which decides the rate of

colonization.

Since the rhizosphere soil samples of the Acacia spp. at one locality were almost

similar in various physical factors only one observation of each locality is represented in the

table (Table 2a and 2b). It is clear from the observations that soil pH at various localities

varied from 8.6 –9.4, while soil moisture varied from 8.5 – 9.0 per cent, organic carbon ranged

from 0.25 –0.40, soil phosphorus was 35 – 50 k/ha and soil nitrogen 20 – 36 k/ha at different

localities.

While correlating mycorrhizal root colonization with abiotic factors, it was observed

that increase in soil pH with decrease in soil phosphorus and soil nitrogen resulted in

increased percentage root colonization as the sample of Pali showed. However soil moisture

and soil organic carbon level could not be correlated with percentage root colonization by AM

fungi during the present study. Possible reason for this might be almost similar level of soil

moisture (8.5 –9.0) and organic carbon (0.25 –0.40 per cent).

Observation further reveal that all the ten species were found distributed in all the

four localities. Among the five Acaulospora morrowae,Glomus constrictum,Gigaspora

gigantean, Sclerocystis rubiformis and Scutelospora nigra,were found in lesser number at

various localities, while Glomus deserticola,G.faciculatum, G. mossae, Gigaspora margarita

and Sc. calospora were equally distributed at all the places with maximum in number. Hence

the observation suggests that AM fungi belonging to ten species at various localities,

surveyed during the present study. However their number varies from different localities.

Selection of suitable genera and efficient strain of AM fungi:

During the first phage of study different genotypes of AM fungi were studied for

selection of suitable genera and efficient strain of AM fungi.to this region. It was observed


that plant height was ranged between 18-45 cm at 60 days, 28-57 cm at 90 days and 55-112

cm at 120 days of AM inoculation for A.tortilis and 20-38 cm at 60 days, 27-52 cm at 90 days

and 52-110 cm at 120 days for A .nilotica. Plant dry weight ranged between2.1-4.0 g at 60

days, 3.56-5.4 g at 90 days and 5.4- 12.1 g at 120 days, presented in Table 2a and 2b. The

most efficient 6 native genotypes were further studied with inoculation of different AM fungi.

It is observed that most efficient genera for each genotype isGlomusmossae and

Glomusdeserticola. These results were followed by genotype G. faciculatum, Gigaspora

margarita Sc. calospora and A.morrovaeSo for further study these six genotypes were used.

Biomass production and nutrient uptake.

Influence of different AM fungi on nutrient uptake (phosphorus and nitrogen) and

productivity of the Cowpea was presented in Table 6a and 6b and Histogram 6a and 6b. This

was studied by sowing these two cultivars with treatments of six suitable and efficient

species of AM fungi. Arbuscular mycorrhizal inoculation resulted in increased biomass

production and productivity (height and plant dry weight) in Acacia spp. irrespective of the

mycorrhizal species as compared with non-mycorrhizal plants. The height of A.tortilis ranged

from 60 cm - 112 cm of different mycorrhiza treated plants, as compared with 55 cm. of non-

mycorrhizal ones. The most efficient response was observed by Glomus mossae which

resulted in almost two fold increase in height followed by Gigaspora margarita while least

response was observed in Acaulospora morrowae treated plants. Where as the height of A.

nilotica ranged from 64 cm - 110 cm of different mycorrhiza treated plants, as compared with

52 cm. of non-mycorrhizal ones. The most efficient response was observed by Glomus

deserticola which resulted in almost two fold increase in height here too followed by G.

mossae and Gigaspora margarita while least response was observed in Acaulospora

morrowae treated plants. Similar trend in the efficacy of different AM fungi towards increase

in productivity of the Acacia spp. was also observed during the present studies. In A.tortilis

40-80 per sent root AM colonization were observed in different AM treated plants as

compared with no association present in non-mycorrhizal ones and least 34 per sent is in

Gigaspora margarita and in A. nilotica 36-55 per sent root AM colonization were observed in


different AM treated plants as compared with no association present in non-mycorrhizal ones

and least 31 per sent is in Gigaspora margarita. It is clear from the observation that

inoculation of Glomus mossae and Glomus deserticola in A.tortilis and A. nilotica respectively

resulted in 100 per cent increase in productivity followed by G. facciculatum while least

response was observed in Acaulospora morrowae treated plants as compared with non-

mycorrhizal plants.

The phosphorus uptake of the host plant ranged from 3.8-7.8 mg/g dry weight in

A.tortilis and 3.6-7.6 in A. nilotica as they are different mycorrhiza treated plants as

compared with 3.4-3.6 mg/g dry weight of non-mycorrhizal ones. The most efficient response

was observed by G. mossae and G. deserticola which resulted in more than two fold increase

in phosphorus uptake followed by G. facciculatum and Gi. margaritawhile least response was

observed in Acaulospora morrowae treated plants. Similar trend in the efficacy of different

AM fungi towards increase in nitrogen uptake of the Cowpea was also observed during the

present studies. In case of A.tortilis 4.2-8.5 mg/g dry weight and in A.nilotica 4.1-7.9 mg/g dry

weight nitrogen was observed in different AM treated plants as compared with 3.7 and 4.0

mg/g dry weight of non-mycorrhizal ones. It is clear from the observation that inoculation of

Glomus deserticola resulted in 100per cent increase in nitrogen uptake followed by G.

facciculatum and Gigaspora margarita while least response was observed in Acaulospora

morrowae treated plants as compared with non-mycorrhizal plants.

Influence of AM fungi on enzymatic changes:

AM fungi are well known to bring about physiological changes in plants via increasing

enzymatic activities i.e. acid and alkaline phosphatases, nitrate reductase, peroxidase and

polyphenol oxidase etc. Among these enzymes phosphatases are important enzymes of

phosphorus metabolism while nitrate reductase is important enzyme of nitrogen metabolism,

peroxidase and polyphenol oxidase are the two important enzymes of defense mechanism of

plants. Keeping all these facts in mind another sets of experiments were designed to evaluate

potentiality of different microorganisms towards increasing activities of these enzymes in the

Acacia plants.


The results of influence of symbiotic relationship towards acid phosphates, alkaline

phosphatase and nitrate reductase of the plant are presented in histograms. It is clear from

the observations that in Acacia tortilis inoculated by different AM fungi resulted in 0.10-0.39

(M mol-1 h-1 kg-1 Fr. wt) nitrate reductase activity as compared with 0.06 (M mol-1 h-1 kg-1 Fr.

wt) of non-inoculated ones.

In Acacia tortilis 0.08-0.38 (M mol-1 h-1 kg-1 Fr. wt) nitrate reductase activity present

when they inoculated with AM fungi, where as 0.06 (M mol-1 h-1 kg-1 Fr. wt) nitrate activity

present in non-inoculated ones. Glomus mossae and Glomus deserticola inoculation resulted

in more than two fold increases in nitrate reductase activity followed by Glomus facciculatum

while least response was observed in A. morrovae treated plants as compared with the non-

inoculated plant species. Similarly acid phosphatase activity in different AM fungi treated

plants varied from 1.31-1.82(X104 n mol PNP hydro S-1 g-1) as compare with 1.2 (X104 n mol

PNP hydro S-1 g-1) of non-inoculated plant species. The acid phosphatase activity was

increased up to more than 83 per cent due to Glomus deserticola and Glomus

mossaeinoculation as compared with non-inoculated ones.

Plant resistance and servility improvement:

When plants are exposed to open fields, there are several factors, which results in

destruction of the plant species. Among these factors soil microorganisms particularly soil

borne plant pathogens plays a vital role in managing the plants growing in natural habitats. In

order to save the plants from attacking soil borne pathogens, some technology should be

applied by which plants can develop resistance against attacking pathogens. Arbuscular

mycorrhizae are now days well recognized as biocontrol agents, some rhizobacteria are also

helpful in this respect. In view of all these facts experiments were designed to find out

potentiality of different AM fungi towards biological control of soil borne plant pathogens.

For this purpose two enzymatic estimations were done namely peroxidase and polyphenol

oxidase. Since these two enzymes are important enzymes of phenolic metabolism of the

plant, as they bring about oxidation of phenols into quinones, which are well known to be

toxic to the attacking plant pathogens, the experimental studies carried out during the


present study can be useful. The results of these experiments are presented in Table: 10a and

10b.In Acacia tortilis peroxidase activity due to inoculation of different microorganisms

ranged from 102.3-158.4 (unit mg-1 Protein) as compared with 95.5 (unit mg-1 Protein) of non-

inoculated plants. Same experiment resulted into 104.2-156.4 (unit mg-1 Protein) in AM fungi

inculated and 97.0 (unit mg-1 Protein) in non-inoculated. As observed in previous

experiments Glomus deserticola and G. mossae responded most efficiently by increasing

more than 57 per cent activity of this enzyme in thehostplantas compared with controls after

120 days of inoculation. Similarly polyphenol oxidase activity and total phenolic contents

were also increased due to different AM fungi inoculation. Polyphenol oxidase activity ranged

from 110.3-147.6 units in different microbial treated plants as compared with 102.4 of control

in A.tortilis .Glomus mossae resulted in almost 43 per cent increase in polyphenol oxidase

activity with 44 per cent increase in total phenolic contents. As per in A.senegal Polyphenol

oxidase activity ranged from 112.3-153.6 units , Glomus desrticola in different microbial

treated plants as compared with 103.4 of control.

0%

20%

40%

60%

80%

100%

So

il %

Locations of Soil Collection Luni ,Pachpadara ,Balotara , Phalodi

Histogram 1: Physical characterstic of rhizosphere soil of Various Locations

Clay (%)

Silt (%)

Fine sand (%)

Coarse sand (%)


0

20

40

60

80

100

120

Control Gi. margarita Glomusdeserticola

G. mossae

Pla

nt

Heig

ht

(in

cm

)

Treaments

Histogram 2a: Influence of different AM fungi on biomass production (plant height) of Acacia nilotica.

60 Days

90 Days

120Days

0

20

40

60

80

100

120


G. mossae

Pla

nt

Heig

ht

(in

cm

)

Treaments

Histogram 2b: Influence of different AM fungi on biomass production (plant height) of Acacia tortilis.

60 Days

90 Days

120Days


0

10

20

30

40

50

60

70

80


G. mossaePla

nt

AM

co

lon

iza

tio

n in

p

erc

en

t

Treatment

Histogram 3a: Influence of different AM fungi on plant AM colonization of Acacia nilotica.

60 Days

90Days

120Days

0

10

20

30

40

50

60

70


G. mossae

Pla

nt

AM

co

lon

iza

tio

n in

pe

rce

nt

Treatment

Histogram 3b: Influence of different AM fungi on plant AM colonization of Acacia tortilis .

60 Days

90Days

120Days


0

1

2

3

4

5

6

7

8

9


G. mossae

mg

pe

r p

lan

t

Treatment

Histogram 4a : Influence of Arbuscular Mycorrhizae on nutrient uptake in Acacia nilotica after six months of inoculation

Totalphosphorus(mg pt-1)

0

1

2

3

4

5

6

7

8

Control A. morrovaeGi. margaritaScutelospora calosporaGlomus deserticolaG. faciculatumG. mossae

mg

pe

r p

lan

t

Treatment

Histogram 4b : Influence of Arbuscular Mycorrhizae on nutrient uptake in Acacia tortilis, After six months of inoculation

Totalphosphoru…


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Control Gi. margarita Glomus deserticola G. mossae

Co

nc

en

tra

tio

n

Treatment

Histogram 5a : Changes in nitrate reductase, acid and alkaline phosphatase activities in Acacia nilotica after six months of AM inoculation

Nitratereductase (Mmol-1 h-1 kg-1 Fr. Wt.)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2


Co

nc

en

tra

tio

n

Treatment

Histogram 5b : Changes in nitrate reductase, acid and alkaline phosphatase activities in Acacia tortilis after six months of AM inoculation

Nitratereductase(M mol-1 h-1 kg-1 Fr.Wt.)


0

20

40

60

80

100

120

140

160


Co

nc

en

tra

tio

n

Treatment

Histogram 6a : Changes in total phenol, peroxidase and polyphenol oxidase activity in root of Acacia nilotica after six months of AM inoculation

Total Phenol (%dry wt.)

0

20

40

60

80

100

120

140

160


Co

nc

en

tra

tio

n

Treatment

Histogram 6b : Changes in total phenol peroxidase and polyphenol oxidase activity in root of Acacia tortilis after six months of AM inoculation

Total Phenol(% dry wt.)

PRO activity(unit mg-1Protein)


DESCRIPTION OF GENERA

Periodical survey of different parts of Western Rajasthan was undertaken to collect

and identify different AM species associated withAcacia sp. Rhizosphere soil samples

collected from various localities revealed presence of eighteen arbuscular mycorrhizal species

belonging to the five genera viz. Acaulospora, Gigaspora, Glomus, Sclerocystis and

Scutellospora associated with these plant species. The detailed description of these AM

species is as follows-

Acaulospora Gerd. & Trappe emend Berch.

Spores produced singly in soil or in sporocarp that may attain several cm in length,

spores globose, subglobose, ellipsoid with oily content; borne laterally on the subtending

hyphae of large, terminal relatively thin walled, sporogenous saccule. Spore composed of

essentially two distinct, separable wall groups.

Gigaspora Gerdemann & Trappe

Spores produced singly in soil, large, variable in shape, usually globose to subglobose,

often ovoid, obovoid, pyriform or irregular, borne on a bulbous suspensor like cell, usually

with narrow hyphae. Spore wall structure of a single wall group, lacking flexible walls. Thin

walled, echinulate auxillary cells borne in soil on straight or coiled hyphae, formed singly or in

clusters.

Glomus Tul. & Tul.

Chlamydospores borne terminally on single (rarely two) undifferentiated, non

gametangial hyphae in sporocarps or individually in soil. Spore contents at maturity

separated from attached hyphae by a septum or occluded by spore wall thickening. Spores of


most Glomus species are borne singly. A few species are known only from sporocarps.

Glomus species are very common in cultivated soils and widespread in native grasslands and

forests.

Sclerocystis Berk. & Broome

Chlamydospores form in sporocarps or single, crowded layer of erect spores that

surrounds the side and top of a spore-free, control mass of tightly interwoven hyphae. The

sporocarps may be borne singly in soil or fused together with organic debris.

Scutellospora Walker & Sanders

Spores produced singly in soil, large, variable in shape, usually globose or subglobose,

but often ovoid, obovid, pyriform or irregular especially when constrained during formation;

borne on a bulbous suspensor like cell, usually with a narrow hyphae. Spore wall structures of

at least two wall groups. Germination by means of one or more germ tubes produced near

the spore base from a germination shield formed upon or within a flexible inner wall.

DETAILED DESCRIPTION OF VARIOUS SPECIES

Acaulospora morrowae Spain & Schenck

Azygospores formed singly in the soil, borne laterally on hyphae ending in a globose

hyphal terminus (58-) 79 (-94) µm diameter with walls 0.5-1 µm thick: hyphae at the point of

spore attachment 10-12 µm wide; hyphal terminus contents subhyaline to white; distance

between the hyphal terminus and the developing azygospore 100-160 µm; terminus contents

emptying to form the spore, leaving a hyaline, thin walled, empty terminus that readily

collapses and detaches from the spore; spores rarely found with an attached terminus. Young


azygospores with light yellow walls and white contents, becoming light yellow with globular,

transparent contents in reflected light.

Spores predominately globose or subglobose, (63-) 79-92 (-120) µm diameter, but also

lacrimoid to irregular, 86-100 X 64-96 µm diameter; spore wall 2-4 (-6) µm thick consisting of

several wall layers, readily apparent on broken spores; outer wall 0.5-1 µm thick, hyaline in

water, adhering to wall two but swelling in lactophenol, separating and sometimes with

adhering debris; wall two light yellow, 1.5-3 µm thick; wall three brittle, hyaline 0.5 µm thick;

wall four membranous, 0.5 µm thick, usually adhering to wall five; wall five membranous, 0.5

µm thick forming vesicular-arbuscular mycorrhizae.

Acaulospora laevis Gerdmann & Trappe

Sporocarps unknown.Spores forms singly in soil, sessile, born laterally on a wide, thin-

walled hypha 30-40µ diameter. that terminates near by in a globose, thin walled vesicle.

Vesicle approximately the same size as the spore, developing to full size prior to spore

formation, with dense, white contents, becoming empty and shrunken at spore maturity and

then usually lost in sieving. Spores smooth, 119-300 x 119-520 µ, globose to sub-globose,

ellipsoid or occasionally reniform to irregular, dull yellow in youth, becoming deep yellow

brown to red-brown or dark olive brown at maturity. Spore wall continuous except for the

occluded opening consisting of three layers : a rigid, yellow-brown to red-brown outer wall 2-

4µ thick ,and two hyaline inner membranes ,the inner most sometimes minutely roughened:

in older specimens wall at times becoming minutely perforate and the outer surface

sloughing away. Spore contents globose to somewhat polygonal (reticulate in optical

section). Hypha below spore attachment giving rise to many slender branches 1-2.5 µ

diameter. Vesicles in vesicular-arbuscular mycorrhizae thin walled and lobed.

Acaulospora mellea Spain & Schenck

Azygospores formed singly in soil;borne laterally on hyphae tapering to a globose to

sub-globose swollen hyphal terminus 90-100 µ diam. Azygospores honey coloured to yellow

brown, globose to sub-globose,95 -105µ diameter, ellipsoidal or irregular ,96-130 x 78-92 µ


,spore wall 4-8µ thick. Hyphal terminus contents white, emptying during spore formation,

resulting in a transparent to subhyaline receptacle attached to the azygospores : hyphal

terminus remains attached to young spores: old spores in soil usually devoid of hyphal

terminus.

Gigaspora gigantea (Nicol. & Gerd.) Gerd. & Trappe

Azygospores formed singly in the soil, 353-368 X 345-398 µ, globose to ellipsoid,

greenish yellow, with a thin outer wall tightly covering an inner wall 5-7 µ thick and

continuous, except for an occluded pore at the attachment. Suspensor like cells bulbous, 42-

48µ diameters, giving rise to slender hyphae that project to the spore.Spherical to clavate

vesicles formed in soil 22-37 X 20-34 µ, in clusters of 1-16 on complex system of inter-coiled

hyphae.

Diagnostic feature:

Mature spore bright yellow with greenish tinge. Germ tubes produce directly through the

spore wall in the base region. Vesicles formed in soil have septate echinulation at apices.

Vesicles lacking in roots. Spores with two-layered wall, which is rarely 7 μm thick.

Gigaspora margarita Becker & Hall

Azygospores formed singly in the soil, dull white with a light greenish-yellow tint;

mostly spherical 143-330 µ diameter, averaging 265 µ, occasionally ellipsoidal 232-252 X 234-

250 µ; spore walls continuous, except for an occluded pore from 4-12 µ thick, with 1-6 walls;

outer wall thin, smooth, 1-2 µ thick, readily cracking under light pressure. Germ tubes

produced directly through the spore wall near the bulbous suspensor without forming an

enclosed compartment separating it from the spore contents. Azygospores attached to a

single, hyaline to yellow bulbous suspensor 24-50 µ attached to separate hyphae with hyphal

branches. Extrametrical vesicles hyaline to yellow, turbinate, obovate or clavate, 17-36 µ

diameters formed in clusters of 5-14 on coiled hyphae in the soil.

Diagnostic feature:


This is readily distinguished from other member of the genus in having white spores

with laminated wall and white clustered, varty vesicles. Spores white, having a spore wall

consisting of several laminations. Spore wall with up to 10 fused laminations; 1.5-4 μm thick,

which do not readily separate, when spores are crushed.

Gigaspora roseaNicol & Schenk

Azygospores produced signally in soil, predominantly globose, 230-305 μm diam,

occasionally sub globose, white to cream in color with a rose-pink tint on the azygospore wall

near the hyphal attachment encompassing up to half the spore. Pink coloration variable from

distinctly rose pink to barely detectable layers 1-2 μm thick.Outer wall layer smooth.

Suspensor like cell

attachment to azygospore usually spherical, occasionally sub globose, subtending

hyphae, 7-14 μm wide, hyphal walls 1-2 μm thick, septate. Soil born vesicles in clusters of 5-

12 on coiled hyphae, individual vesicles 19-32 μm wide, and echinulate with spines up to 5.0

μm long and 2.5 μm wide.forming mycorrhizae with arbuscule.

Diagnostic features:

G. rosea can be distinguished from other light spored species of Gigasporaby the rose

pink tint associated with the wall of the azygospore near the wall of the attachment. Soil

borne vesicles echinulate, with spines (5 μm long and 2.5 μm wide). Spores white to cream.

Spore wall consist of 2-5 inseparable layers.

Glomus deserticola Trappe, Bloss & Menge

Spores borne singly or in loose clusters in soil or within roots, globose to subglobose

(47-) 54-115 X (38-) 52-102 µ, shiny, smooth, reddish brown, with a single, sometimes

laminated wall (1.5-) 2-2.5 (-4) µ thick. Attached hypha 6-12 µ in diameter, cylindrical, the

walls thickened and reddish brown, especially thick adjacent to the spore but not occluding


the hypha. Interior of the spore wall at the hyphal attachment thickened at maturity to form

an inner-mounded collar, which appear to be closed by a membranous septum.

Diagnostic feature:

Interior of the spore wall at the hyphal attachment thickened at maturity to form an

inner-mounded collar, which appears to be closed by a membranous septum. Spore walls

deep reddish brown. Wall diameter 1-4 μm. Reddish brown hyphal attachment not occluded

by wall thickening.

Glomus fasciculatum (Thaxter sensu Gerd.) Gerd. & Trappe

Chlamydospores borne free in soil, in dead root lets, in loose aggregations, in small

compact clusters and in sporocarps. Sporocarps up-to 8 X 5 X 5 mm, irregularly globose or

flattened, tuberculate grayish brown. Peridium absent. Chlamydospores 35-105 µ diameter

when globose, 75-150 X 35-100 µ when subglobose to obovate ellipsoid, sublenticular,

cylindrical or irregular; smooth or seeming roughened from adherent debris. Spore walls

highly variable in thickness (3-17 µ), hyaline to yellow or yellow brown, the thicker wall often

minutely perforate with thickened inward projections. Hyphal attachments 4-15 µ diameter,

occluded at maturity. Walls of attached hypha often thickened to 1-4 µ near the spore.

Diagnostic feature:

Thicker walls of the spore often minutely perforated with thickened invert

projections. Chlamydospores borne free in soil in aggregates, in small compact clusters and in

sporocarps; peridium absent. Chlamydospores tightly packed together.

Glomus geosporum(Nicol. & Gerd.) Walker

Sporocarps unknown. chlamydospores formed singly in soil, globose to subglobose or

broadly ellipsoid, 100-290 μm, smooth and shiny or with a dull appearance, or roughened

from adherent debris; light yellow-brown and transparent to translucent when young,

becoming dark yellow-brown to dark red-brown at maturity.


Spore walls 4-18μm thick, 3 layered, with a thin, hyaline, tightly adherent outer wall

(<1μm), a yellow brown to red – brown laminated middle wall (316 μm); and a yellow to

yellow brown inner wall (< 1μm) that appears membranous and that forms a septum

separating the spore contents from the lumen of the subtending hypha. Spores with straight

to recurved, simple to slightly funnel shaped subtending hypha up to 200 μm long.Occasional

spore lacking a subtending hypha due to breakage close to the spore base.

Spore contents uniform oil droplets when young, becoming increasingly granular in

appearance with age; cut off by a thick septum that protrudes slightly into the subtending

hypha after rupture of the septum.

Diagnostic feature:

Spore cut of by a septum that protrudes slightly into the subtending hypha. Spores

red brown to opaque at maturity.

Glomus macrocarpum Tul. & Tul

Sporocaps are fragmentary, non of the pieces more than 5 mm diameter.Spores are

usually slightly longer than wide, sub-globose or globose, to irregular, (90-)120(-140) x (70-

)110(-130) μm. Spore wall is composed of two distinct layers :outer layer is thin (1-2 μm) and

hyaline when mounted in water or glycerol, usually swelling to at least twice its original

thickness in lactic acid : inner wall layer is yellow in section,6-12 μm thick ,with a series of

laminations occasionally visible or rarely appearing as two distinct layers, swelling relatively

little in lactic acid. Spores taper to the point of attachment of the single persistent hypha. The

average diameter of the hyphae at this point is 16 μm. The inner wall at maturity thickens to

occlude .The pore of the attached hyphae and the wall thickening continues into the

subtending hypha for up to 90 μm from the spore .Infrequently the pore seems to be closed

by septum that is thinner than the normal occluding wall thickening. Spores

characteristically bear a straight, long subtending hypha which may extend up to 100 μm

before branching or breaking.


Glomus mosseae (Nicol.& Gerd.)Gerd.& Trappe

Sporocarps 1-10 spored, globose to elliposoid ,up to 1mm diameter.Peridium of

loosely interwoven, irreguraly branched ,hyaline, septate hyphae 2-12 μm diameter., the

walls upto 0.5 μm thick ,frequently anastomosing to form a thin network, enclosing the

chlamydospores entirely, incompletely or with some spores unenclosed .Endocarpic and

ectocarpic spores similar. Chlamydospores yellow to brown, globose to ovoide, obovoid or

somewhat irregular, 105 -310 x 110-305 μm, with one or occasionally to funnel–shaped

bases 20-30(-15) μm diameter, divided from subtending hyphae by a curved septum ;walls 2-

7 μ thick ,with a thin often barely perceptible hyaline outer membrane ,and a thick,

brownish-yellow inner wall.

Sclerocystis rubiformis Gerd. & Trappe

Sporocarps dark brown, subglobose to ellipsoid, 180 X 180-375 X 675 µ, consisting of a

single layer of chlamydospores surrounding a central plexus of hyphae, resembling a

miniature black berry.Peridium nearly absent, individual spores at times partially enclosed in

a thin network of tightly appressed hyphae. Chlamydospores dark brown, obovoid to

ellipsoid or subglobose, 37-125 X 29-86 µ, with a small pore opening into the thick walled

subtending hypha. Spore wall laminated, 3.7-13.5 µ thick, often perforated. A variable stalk

like projection protrudes near the base of some spores.

Diagnostic feature:

Spore wall often perforated, and often with thick, perforated projections on the inner

surface. Peridium nearly absent.

Sclerocystis corieomoidesBerk. & Broome

Sporocarps 340-600 μ broad, subglobose to pulvinate, flattened at base, at times

borne on a short stalk up to 100µ broad, white when immature, becoming tan to dull brown

when fully mature, gregarious in mats containing large number of sporocarps fused together

laterally and one above the other to about 4 sporocarps thick. Peridium 20-70 μ thick, of


interwoven hyphae. Chlamydospores 50-86 (-102) X 35-52 (-82) μ, obovoid-ellipsoid to

oblong-ellipsoid, often but not always cut off from subtending hyphae by septa just below

spore base, arranged in a single layer, tightly grouped in a hemisphere around a central

plexus of hyphae. Spore absent at base of sporocarp. Chlamydospore wall up to 4 μ thick at

base and 2 μ thick at apex, brown.Forming vesicular arbuscular mycorrhizae.

Diagnostic feature:

Young sporocarps wide enclosed in a peridium 20-70 μ thick of interwoven hyphae.

Sporocarps fused together, laterally and vertically to about 4 sporocarps thick.

Sclerocystis microcarpus Iqbal and Bushra

Sporocarps dark brown, 100-420 µ in diam, globose to subglobose, minutely verrucose

from exposed tips of spores formed radially in a single, tightly packed layer around a central

plexus of hyphae; peridium lacking.Chlamydospores clavate, cylindrical clavate with a small

pore opening into the thick walled subtending hyphae. Chlamydospores walls laminate,

brown, generally thickest at apex.


Sporocarps minutely verrucose from exposed tips of spores. Chlamydospores broader at the

upper end.Sporocarp 100-420 µ in diam.

Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders

Spores formed in the soil, terminally on a bulbous suspensor like cell; translucent,

hyaline to pale greenish-yellow, globose, ellipsoidal or cylindrical, occasionally broader than

long; 114 X 285 X 110-412 µ. spore wall structure of four walls in two groups (group A and B).

Group A consisting of an inner, brittle, hyaline to pale yellow, very finely laminated wall 3-5 µ

thick that may be surrounding by a thin very closely appressed hyaline unit wall, 0.5-1 µ thick.

Group B of two hyaline membranous walls.Wall 3, 0.5-1 µ thick often wrinkling in crushed


spores.Wall 4, 1-1.5 µ thick.Suspensor like cell borne terminally on a septate subtending

hypha; 33-48 µ broad.


Spores translucent hyaline to pale greenish yellow. Smooth knobby vesicles borne

singly on coiled hyphae in the soil. The oval germination shields often with invagination along

the margin. Pore at the attachment occluded. Inner wall group consist of 2 juxtaposed

membranous walls.

Scutellospora nigra (Red head) Walker & Sanders

Azygospores formed singly in the soil, dark brown to black spherical, and 297-500 µ

diameters with an inner and outer wall. Outer wall black to dark brown, pitted with larger

pores, 7-10 µ diameter; inner wall light brown, transparent. Suspensor like hyphal

attachment light brown attached laterally, 40-60 X 80-120 µ. accessory soil borne vesicles

dark brown, globose to subglobose, smooth to knobby, in usually tight clusters of 3-12 on

coiled or twisted hyphae arising from straight hyphae 4.8-9.6 µ in width.

Diagnostic feature:

This can be readily separated by its large black, shiny spores, with pores in the outer

wall. Suspensor-like cells 40-120 μm diameter.Spores with 2 walls.

Scutellospora heterogama (Nicol. & Gerd.) Walker & Sanders

Spores borne singly in the soil, terminally, subterminally, or laterally on a bulbous

suspensor-like cell; globose to subglobose or irregular; 150-220 μm, ellipsoidal specimens up

to 210 X 230 μm; pale yellow-brown to red-brown. Spore wall structure of four walls in two

groups (A and B). Group A with an outer ornamented unit wall (1) tightly adherent to an inner

wall (2).Wall 1 brittle, pale yellow to pale brown.1-1.5μm thick, excluding the hyaline warts

(papillae). Warts very densely crowded, usually touching or less than 0.5 μm + apart at the

base, 0.5-1 μm high, 0.5-1 μm diameter. Wall 2 yellow-brown, finely laminated, 4-7μm thick.

Group B of two membranous walls (3 and 4) separated by an apparent amorphous cementing


layer. Each wall hyaline, <1 μm thick; total thickness of walls and separating material 1.5-3

μm. Suspensor like cell borne terminally on a coenocytic to sparsely septate subtending

hypha; 21 42 μm wide; yellow-brown. Wall suspensor like cell 1-2.5 μm thick distally,

thickening to 2.5-4 μm at the spore base. One or two peg-like hyphal projections present or

lacking; when present 10-16 X 5-9 μm, arising from the suspensor like cell and projecting

toward the spore.

Diagnostic features

The small, closely crowded warts on the spore surface and the wall surface

differentiate S. heterogama from other species. Wall group B of 2 membranous walls

separated from an apparent amorphous cementing layer, each wall less than 1μm thick.

Scutellospora aurigloba (Hall) Walker & Sanders

Spores ectocarpic, globose or more rarely polymorphic, 200-420x 130-420 x 130-420

μm diameter, pale yellow, transparent and shining when yellow and becoming dull at

maturity. Spore wall 2-4 layered, outer wall coloured 6-16 μ thick, inner walls approx. 1 μm

thick , colourless to yellow.Spores formed on a bulbous suspensor 40-70 μm diameter .Walls

of subtending hypha 3-10 μm thick, yellow to light brown. Subtending hypha sometimes with

a well to poorly developed lateral projections. Pore approx. 4 μm diameter without a septum

cup or dome shaped septa often form in the subtending hypha.




Chapter – 4

Development of arbuscular mycorrhizae

Arbuscular mycorrhizae are well recognized as biofertilizers now days. Since the plant-

fungus relationship in this case is of symbiotic type, both the organisms get benefited from

each other. The fungus produces external as well as internal structures to establish symbiotic

relationship with the host. The infection of the roots by an arbuscular mycorrhizal fungus and

subsequent development of the AM could be categorized into following four stages-

(a) Spore germination or initiation of hyphal growth from the infective propagules.

(b) Growth of hyphae through the soil to the host roots.

(c) Penetration and successful initiation of infection in roots and,

(d) Spread of infection development of a mycorrhizal relationship with root and spore

production.

Brundrett et al., (1985) stated that the application of VA mycorrhizal symbiosis to

agriculture and forestry requires an understanding of the events that occur during the

establishment of this association.

The present investigation was undertaken to study the sequence of events in the

colonization process and the time required for the formation of each stage in the Cowpea.

The plant roots of Cowpea collected from the fields as well as inoculated in the pots

were regularly examined. A series of squash preparation reveals the presence of different

stages in the development of arbuscular mycorrhizal infection in the roots of Cowpea.


The infective propagules of different AM fungi exist in the form of penetrating

chlamydospores in the fields. The pot experiments have shown that the infection in plant

roots was initiated either by the germ tube formed on the germination of chlamydospores or

by young juvenile hyphae. It was observed that the young developing feeder roots come in

contact with the germ tube of germinating spore on the third or fourth day of inoculation.

The infecting hyphae developed close contact and adhered strongly to the root surface. This

adherence leads to appressoria formation or direct penetration of the epidermal cells by the

rupturing of outer cell wall. The penetration was rarely without appressorium formation. The

penetration of the host was a continuous process and could be seen even at later stages,

when endophyte had already established itself in the cortical cells. This was followed by the

development and ramification of fungal hyphae, which grows, inter as well as intracellularly .

The arbuscule formation.was observed on the seventh day of inoculation. Arbuscules

development started after the penetration of the host cell wall by a lateral branch produced

from hyphae of the adjoining cell.These hyphae became the arbuscular trunk and showed

repeated dichotomous branching in the cell. The arbuscule occupy a major portion of the

host cell and can be seen in the various stages of development. The arbuscules were

ephemeral structures and remain active only for four to fifteen days. During degeneration the

finer hyphae of arbuscular were the first to collapse into a dense residual mass.

The arbuscular trunk was quite apparent in the centre of the cells and was the last

hyphal elements to collapse.

It was of interest to notice that young arbuscule was present in the cell adjacent to a

cortical cell containing collapsed arbuscule. It was observed that the endophyte remains

confined to the cortical region of the host.

The vesicle formation was noticed on the eighth day onwards, after the penetration of

the host cell. The vesicles were oval spherical or irregularly lobed. These are thick walled

structures formed terminally in the inter or intracellular spaces, with their size ranging

between 9-45 µm in diameter. The vesicles were usually multinucleate having open


connections with parent hyphae. Vesicles have been considered to function as temporary

storage organs. They also serve as propagules as such as roots decay or develop into thick

walled chlamydospores functioning as reproductive structures. Due to these properties the

so-called vesicles are now termed as chlamydospores (Mehrotra, 1997).

In addition to this extramatrical hyphae commonly developed into the soil up to some

distance around the roots. The mature extramatrical hyphae also bear the resting spores. The

chlamydospore formation was observed in the host tissue on the surface of rootlets and also

in the rhizosphere .

The process of development of mycorrhizal infection in the roots of the Cowpea plant

is consistent with observations made on other endomycorrhizae (Reddy, 1996; Kumar et. al,

1997 and Chandra and Jamaluddin, 1999). The infection of the roots was possible through the

germ tube formed on the germination of chlamydospores or through the young fungal

hyphae. The roots were penetrated either directly or was accompanied with the formation of

appressoria.

The penetration of host tissue was a continuous process and was seen at later stage

when the endophyte had already established itself inter as well as intracellularly.




Chapter – 5

Discussion

Periodical survey of four regions of Western Rajasthan namely, Jodhpur, Pali, Udaipur,

and Mount Abu revealed that nearly eighteen AM fungi were found associated with the two

Acacia species namely, Acacia tortilisand Acacia nilotica. The frequency of occurrence of

mycorrhizal spores in rhizosphere soil and root colonization of the two host plant was found

to be affected by abiotic factors like soil pH, soil phosphorus and soil nitrogen at all the above

localities. Different abiotic factors and their influence on spore population and percentage of

root colonization are presented in Tables 1 & 2.

During the present study increase in soil pH with decrease in soil phosphorus and

nitrogen was found to be correlated with increasing colonization of host root by the AM fungi

at all the localities irrespective of host plant. Edaphic factors such as soil texture, soil fertility,

soil moisture, soil temperature and soil pH may effect the composition, distribution and

efficacy of AM fungi in the natural habitat (Singh, 1999). Singh and Tewari (1999) reported

seasonal fluctuation in number of VAM spores in soils of sand dunes. Pavan Kumar et. al.,

(1999) reported that the percentage of infection was suppressed under the influence of

effluence. They observed that the spore population in the rhizosphere soil varied both with

the plant and also with the type of pollutants.

Soil pH is the major edaphic factor, which effect the establishment and efficiency of

mycorrhizal fungi in natural vegetation.

Siddu and Behl (1997) observed relative tolerance of VAM fungi to graded level of pH

(7.8 - 10.5) and there influence on P uptake in Prosopis juliflora. They showed that increase in


pH adversely effect growth, biomass and P concentration in seedlings of Prosopis.

Chlamydospore formation in the rhizosphere soil by all three VAM fungi decreased with

increase in soil pH. Soil pH and available soil nutrient have a cumulative effect on the

efficiency of VAM on different plant species (Singh, 2000). Domisch et. al., (2002) reported

effect of soil temperature on root colonization by AM fungi in Scot pine seedlings. The

observed increase in AM sporulation and rate of colonization of the two Acacia species due to

increase in soil pH was found to be correlated to the distribution of different AM species in

these arid and semi arid areas. The concentration of K, soil moisture and organic carbon could

not be correlated with AM spore population and root colonization of the two plant species in

this region. The reason for such observation could be very low soil moisture level, almost

similar quantitative occurrence of K at different localities and very low organic carbon level of

the soils of this region. AM fungi and its potentiality to establish symbiotic relationship with

the host plant is affected most severely by the nutrient status of the host plant as well as the

rhizosphere soil. Since AM fungi compensate to nutrient deficiency of the host plant, its

potentiality to colonize the root is likely to be decrease with increase in nutrient status of

both the rhizosphere soil and the host plant (Mathur and Vyas, 2000).

Arbuscular mycorrhizae are well known to be of ubiquitous occurrence. Its distribution

in Indian Thar Desert has been reported (Mathur and Vyas, 1995 I).

However, occurrence of AM fungi inassociation with Acacia tortilisand Acacia

niloticahas not been studied properly. The present study reveal occurrence of eighteen

species of AM fungi in different arid and semi arid regions of Western Rajasthan. The type of

plant species had almost no effect on sporulation of a particular AM species in this region.

There has been a phenomenal increase of interest on AM fungi in recent years leading to

numerous surveys for enumerating and accessing AM fungal species and their colonization of

host plants in different regions of this country (Muthukumar and Udaiyan, 2000). The

significance of AM fungi is based on its wild spread occurrence in natural ecosystems. Until

now, there has been a paucity of information on the mycorrhizal status of Acacia species of

this region.


The present study revealed association of eighteen species of five genera of AM fungi

with the two Acacia species. These genera and species were invariably present throughout

the region irrespective of the host plant type present at particular locality. Among the five

genera the species belonging to genera, Gigaspora, Glomus and Scutellospora was found very

common while the species belonging to genera Acaulospora and Sclerocystis were found

comparatively at lower rate in distribution.

Our results support the previous observations about frequent occurrence of Glomus in

different regions of Indian Thar Desert (Mathur and Vyas, 1995 I). Though AM fungal species

were not found to be effected by host specificity for its distribution in this region however,

some species were found to be more abundant in its occurrence as compare with the others.

All the eighteen AM fungal species were successfully cultured on Cenchrus ciliaris and

Sorghum bicolor forpreparation of pure pot cultures. The inoculum from these pot cultures

was used to inoculate the two Acacia species. Both the plant species viz. Acacia tortilisand

Acacia niloticawere successfully colonized by different AM fungal species. The symbiotic

relationship was established very well between AM fungal species and the host plant species.

All the stages of symbiotic relationship i.e. appresoria formation, hyphal penetration to the

cortical region, intra-cellular penetration and formation of Arbuscules and formation of

vesicles of different size and shape at both inter as well as intra cellular level was observed

during the present study.

Seedlings of the two Acacia species were inoculated with commonly found eight

arbuscular mycorrhizal species for the further studies. In this phase of experiment efforts

were made to exploit the potentiality of different arbuscular mycorrhizae on biomass

production and nutrient uptake in the two Acacia species namely, Acacia tortilisand Acacia

nilotica. Observations revealed that different arbuscular mycorrhizal species varied in their

efficacy to improve biomass production and nutrient uptake in the two Acacia species.

Scutellospora nigra was found to be most efficient in increasing biomass production and

nutrient uptake of Acacia tortiliswhileGigaspora gigantea was proved most efficient for

Acacia nilotica.


The improved biomass production of the two Acacia species by AM fungi during

present study could be due to improved nutrient status of the host plant provided by

efficiently root colonization by the particular AM endophytes.

Phosphorus nutrient exerts a significant influence on plant growth and

development.Arbuscular mycorrhizae acts as biofetilizer for the host plant having attachment

of their hyphal system with root s of the plant acting as extension to the root system. Thus,

external hyphae of the mycorrhizal plant more rapidly exploit a given volume of soil for

available P then roots of non-mycorrhiza plant. Under nutritional deficient conditions AM

fungi increases mobility of P (which is very less mobile under natural conditions) thereby

increase in the availability of the nutrients for the host plants. The bidirectional exchange of

nutrients is the basis of the arbuscular mycorrhizal symbiosis; in this way, the fungus interacts

with host plant roots to increase their absorption of water, phosphate, and other nutrients

from the soil. In turn, the plant provides photosynthesized sugars to the fungus, a

phenomenon that provokes many cellular, physiological, and energetic changes in the host

roots (Ramos et al. 2009).

Linderman (1999) reported that the response of plant to VAM fungi is highly variable,

being influenced by host plant physiology, genotype, environmental conditions and root

excretions. In order to have a good plant growth effective mycorrhizal symbiosis is most

essential. Hence, screening for efficient VAM fungi for a particular plant suitable to a

particular agro-climatic region is needed. Al-Karaki (2000) reported improved growth and

nutrient uptake of tomato plants under slat stress conditions. Fidelibus et. al., (2000)

reported variation in efficacy of different AM fungi to improve growth of lemon under dry soil

conditions. Bhattacharya and Bagyaraj (2002) reported variations in effectiveness of different

AM fungal isolates on coffee.

They suggested that extent of growth and nutritional status enhanced by AM fungi

varied with the isolates of AM fungi inhabiting the roots of coffee seedlings. Arbuscular

mycorrhizal (AM) fungi facilitate inorganic N (NH4 + or NO3−)uptake by plants, but their role

in N mobilization from organic sources is unclear. They hypothesized that arbuscular


mycorrhizae enhance the ability of a plant to use organic residues (ORs) as a source of N.This

was tested under controlled glasshouse conditions by burying a patch of OR in soil separated

by 20-μm nylon mesh so that only fungal hyphae can pass through it. The fate of the N

contained in the OR patch, as influenced by Glomus claroideum, Glomus clarum, or Glomus

intraradices over 24 weeks (Atul-Nayyar. et al,2009).

Nikolau et. al., (2002) reported variation in different mycorrhizal species on biomass

production and mineral uptake of Vitis venifera. Hart and Reader (2002) reported the

variation in efficacy of different AM fungi for biomass production and nutrient uptake could

be due to difference in the size of the mycelium of the AM fungi. Allison (2002) also observed

similar type of results in Achillia millefolium and suggested that the variation could be due to

difference in nutritional status of the host plant. Cavagnaro et. al., (2003) reported relative

variation in effectiveness of different AM fungi on growth and P nutrition of a Paris type

arbuscular mycorrhizal. Scagel (2004 a) reported increased nutrient uptake and biomass

production of harlequin flower due to AM fungi. Linderman and Davis (2004) reported varied

response of marigold genotypes to inoculation with different AM fungi. Jamalluddin and

Chandra (1995) reported improved growth performance of Eucalyptus by VAM fungi in the

coalmine spoils of Korba due to improved nutrient uptake. Verma and Jamalluddin (1995)

reported mycorrhiza mediated improved biomass production of Tectona grandis due to

improved nutrient uptake by the endophytes.Jamalluddin et al., (1997) reported symbiotic

relationship of VAM fungi in different bamboos. Chandra and Jamalluddin (1999) reported

variation in percentage of root colonization by VAM fungi in different plant species.

Bhattacharya et. al., (1999) reported improved biomass production and nutrient uptake in

bamboos in wasteland soils. Mathur and Vyas (2000) reported improved biomass production

and nutrient uptake of Ziziphus mauritiana by different AM fungi under drought stress

conditions.

Different species and even geographic isolates of the same species of AM fungi might

vary with respect to their ability to colonize roots and improve plant growth (Graham et. al.,

1996; Pelletier and Dionne, 2004). Relatively high water and nutrient soil inputs might,


overtime favour proliferation of species or strain of AM fungi colonizing Citrus roots (Fidelibus

et. al., 2000).

Hence, from the above discussion it is clear that different arbuscular mycorrhizal fungi

vary in their efficacy to improve biomass production and nutrient uptake of the host plant.

However, the important factors that determine the potential benefits of a particular

mycorrhizal species to a host plant are the nutritional level of the host plant, availability of

nutrient in the rhizosphere soil of the particular plant, the root system of the plant and

efficiency of the particular mycorrhizal species to compensate the nutritional requirement of

the host plant.

In order to further understand the physiology of symbiosis experiment were also

conducted to evaluated the potentiality of different AM fungi towards uptake of micro-

nutrients, Zinc (Zn), Iron (Fe), Copper (Cu) and Manganese (Mn) as well as chlorophyll, sugars,

starch, protein, carotenoids and phenolic contents in the two Acacia species. The

observations revealed considerable increase in all the parameters due to different AM fungi

in both these Acacia species. Adriaensen et. al., (2004) reported increased uptake of zinc by

pines inoculated with mycorrhizal fungi. Bi et. al., (2003 a) reported increase uptake of Zn by

red clover at early stages of arbuscular mycorrhizal development. Jamal et. al., (2002)

reported increase uptake of Zn and Nickel (Ni) form contaminated soil by soybean due to

arbuscular mycorrhizal association. Liao et. al., (2003) reported increased uptake of heavy

metals by arbuscular mycorrhizae under different soil types. Mogueira et al., (2002) reported

removal of Mn toxicity by soybean due to mycorrhizal symbiosis. Chen et. al., (2003) reported

increased Zn uptake by red clover growing in a calcareous soil by arbuscular mycorrhizae. Al-

Karaki et. al., (2000) reported increased uptake of Zn, Cu and Fe in tomato by arbuscular

mycorrhizae under salt stress conditions.

Schubert et. al., (2004) reported increase in sucrose content in roots of soybean

colonized by different AM fungi. Mathur and Vyas (1995 I) reported increased chlorophyll,

carotenoids, sugar and protein content of Ziziphus zylopyrus by different VAM species.

Joseph et. al., (1999) reported increase sugar and starch contents in Pueraria


phaseolidesinoculated with 11 different mycorrhizal species. Prasad and Bilgrami (1999)

reported increased chlorophyll and sugar content in saccharum officinarum due to VAM

inoculation. Prasad (1999) reported increased chlorophyll, sugar and protein content in

Acacia nilotica due to inoculation with indigenous fungi. Auge (2001) reported increased

chlorophyll content of different host plants due to VA mycorrhizal symbiosis. Declerck et al.,

(2002) reported increase chlorophyll and sugar contents in micropropagated bananas by in

vitro monoaxenically produced arbuscular fungi. Mathur and Vyas (2000) reported increased

biomass production, nutrient uptake, protein, chlorophyll, and sugar contents of Ziziphus

mauritiana by different VAM fungi under water stress conditions.

From the above discussions it is clear that arbuscular mycorrhizae brings about certain

physiological changes of the host plants by improving carbohydrates, protein and

photosynthetic pigments in different plant species. This beneficial effect of the endophytes

could be attributed to either improved nutrient uptake which resulted in over all change in

the metabolism of the host plant or it can be due to improved leaf surface area which

resulted in increase photosynthetic rate thereby increasing the carbohydrates contents of the

host plants.

The above physiological changes in the two Acacia species could also be due to

increasing various enzymatic activities like phosphatases, nitrate reductase, peroxidase, poly

phenol oxidase etc. In view of these facts experiments were conducted to find out application

of different AM fungi towards biochemical changes of the two Acacia species.

Observations revealed that significant increase in activities of all the four enzymes

were recorded due to different arbuscular mycorrhizal species in both Acacia species. Pearson

et. al., (1991) reported that increased phosphates activity (both acid and alkaline

phosphatases) by VAM fungi is due to presence of specific isozymes of the two enzymes in

AM colonized plants. Zhu and Smith (2001) reported increased phosphatases of wheat plant

by arbuscular mycorrhizal plant under field condition. Buscot et. al., (2000) reported changes

in various enzymatic activities by mycorrhizal symbiosis in natural ecosystem. Mathur and

Vyas (2000) reported biochemical changes in Tamarix aphylla by different VAM fungi. This


phosphatases activity (increased) due to above reasons might also be responsible for P

uptake in mycorrhizal colonized plants. Similarly increase in nitrate reductase activity in the

two Acacia species was observed irrespective of the mycorrhizal treatment. Nitrate reductase

is one of the important enzymes of nitrogen metabolism in all the plant. This increased NR

activity in roots and leaves of mycorrhizal colonized clover was attributed to improved P

nutrition provided by the mycorrhizal symbiosis. Similarly, Mathur and Vyas (1995 II)

reported increase in NR activity in Ziziphus nummularia by different VA mycorrhizal fungi.

Endomycorrhizal fungal species like Glomus macrocarpum and Glomus mossae have also

been shown to reduce nitrate ions. Mc Farlend et. al., (2002) reported increased nitrate

reductase activity of the plants of a deciduous forest ecosystem by arbuscular mycorrhizae.

Hobbi and Colpaert (2003) reported increased nitrate reductase and Glutamine synthetase

activity in different plant species by arbuscular mycorrhizae. The results in present study

suggest that with a capacity for reducing nitrate, it is likely that the symbiotic effectiveness of

the arbuscular mycorrhizal fungi is enhanced in terms of nitrogen acclimation and

translocation to the host plant. The increased peroxidase and polyphenol oxidase activities in

the two Acacia species by different AM fungi during present study can be important. These

two enzymes are of great importance in the defense mechanism of the plants against

attacking pathogens. These two enzymes bring about oxidation of phenols into quinines,

which are well to be toxic to the plant pathogens. The observe increase in peroxidase

activities during the present study is indirect effect of the mycorrhizal symbiosis. Further, it is

the P mediated effect on peroxidase activity (which was provided by the mycorrhizal

symbiosis). Lower peroxidase activity is well known in low P roots than in high P roots (Mc

Arthur and Knowels, 1992). Reduction in such activities may be indicative of a lower capacity

for the induction of a defense response from the plant. Since low P roots have lower capacity

for ethylene generation and thereby also have less peroxidase activity then high P roots

which further suggests that high P roots have high capacity for ethylene generation which

results in higher peroxidase activity hence increased P uptake by AM fungi might have

resulted in increase P activity of the two Acacia species during the present study.


A positive correlation was observed between total phenolic accumulation and

polyphenol oxidase activity in AM colonized both Acacia species in the present study.

Accumulation of phenol in mycorrhizal colonized plant has been well recognized in plant.

Mathur and Vyas (1995 III) reported changes in isozyme patterns of peroxidase and

polyphenol oxidase in roots of Ziziphus species by different VAM fungi. They reported

correlation between increase in isozyme numbers and activity of both these enzymes by the

AM fungi. Hence during present study the increase peroxidase and polyphenol oxidase

activities in the two Acacia species can also be due to mycorrhiza specific isozymes of the two

enzymes in the roots of both the Acacia species. The present study clearly reveal application

of arbuscular mycorrhizal fungi in improving status of arid and semi arid regions of western

Rajasthan in various ways i. e. by improving nutrient status and biomass production of the

Acacia species, by removing higher metals from the wastelands, by changing host plant

physiology. Pelletier and Dionne (2004) reported improved survival and establishment of turf

grass without irrigation and fertilizer inputs by inoculating with AM fungi. All these factors

collectively would contribute for development of arid and semi arid wastelands.

Chapter – 6


Summary-------------------------------------------

Salinisation of soils and ground water is a serious land degradation problem in arid and

semi arid areas, and is increasing steadily in many parts of India, causing major problems

for land productivity. On a world scale there is an area of around 380 million hectares that

is potentially usable for agriculture, but where production is severely restricted by salinity.

These areas occur predominantly in regions where evaporation exceeds precipitation. The

problem of saline soils is ever increasing, due to poor irrigation and drainage practices,

expansion of irrigated agriculture into arid zones with high evapotranspiration rates, or

land –clearing, which leads to rising saline water tables i.e. dry land salinity. Dry land

salinity is a major environmental problem in arid and semi-arid regions of India. The

impact of agricultural clearing through salinisation extends across the country, but they

are particularly severe in saline areas of Indian Thar Desert, which covers nearly 45% area

of this region. The major parts of saline habitat includes Luni, Pachpadra, Balotra,

Bikaner, Churu, Osian, Nagaur, Barmer and Jaisalmer to Kuchh of Ran. Physical,

chemical and biological constrains in soil horizons impose an additional stress on plants in

these habitats, restricting plant growth and development. Hard setting, crusting,

compaction, acidity, alkalinity, nutrient deficiency and high temperature are major factors

that cause these constrains. Productivity of many salt affected soils has declined due to

inappropriate land use practice, over grazing or removal of trees for various purposes. In

Indian Thar Desert irrigation by fresh water is not possible, due to severe scarcity of

water. Thus exploiting the possibilities of using salt stress water for irrigation, especially

drainage and underground water is of great importance. Thus, there is an urgent need to

develop new technologies to cope with these adverse climatic conditions. Mycorrhizal fungi

are well recognized as biofertilizers now a day. Due to their manifold benefits provided to

the host plant, they are being frequently used in revegetation and reclamation programme

worldwide. By improving nutrient uptake and water transport, they help the plants to

survive more efficiently under adverse climatic conditions of drought prone areas.

Mycorrhizal fungi have also been shown to reduce transpiration rate and increase water

use efficiency of plants under arid and semiarid conditions, where water is the most

important factor which determine plant growth. Under arid and semi arid conditions water


is the most important factor which determine plant growth.Arpuscular mycorrhizae

improve water economy of plants. Major constrains imposed by saline habitat are physical

, chemical and biological; like structural decline in the form of compaction and

crusting,high concentration of sodium salts, chloride ,carbonate and bi carbonate and soil

borne pathogens. Arbuscular mycorrhizal fungi by various mechanisms help the plant to

over come these constrains thereby improving their survival and establishment under

saline habitats.

AM FUNGI

BETTER EXCESS TO

NUTRITIONAL STATUS

MODIFICATION OF

PLANT PHYSIOLOGY

i.e.OSMOTIC MODIFICATION

PHOTOSYNTHESIS

PROTECTION AGAINST SALT STRESS

MYCORRHIZAL MECHANISM FOR SURVIVAL OF PLANTS UNDER SALINE HABITAT


Chapter-7

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