CAPSTONE PROJECT

(BTY-449A)
Vesicular Arbuscular Mycorrhiza as a potential biofertilizer
in Aloe vera

Mr. Umesh Sharma Section:

Advisor:
F77E2
Dr.Joginder singh panwar
Roll no. 08
Associate Professor
Regd. no. 3040070046
(Biotechnology)
B.tech. Biotech. (7th Sem)

Department of Biotechnology

Lovely School of Engineering (LIE)

LPU, Phagwara, India

PROJECT REPORT
 (Project Semester August-December 2010)

Vesicular Arbuscular Mycorrhiza as a potential biofertilizer

in Aloe vera

Submitted by

UMESH SHARMA

Registration No: 3040070046

Programme: BT (BIOTECHNOLOGY)

SECTION: F77E2

Under the Guidance of

Dr. JOGINDAR SINGH PANWAR

Department of Biotechnology

Lovely Institute of Engineering (LIE-F)

Lovely Professional University, Phagwara

DECLARATION

I hereby declare that the project work entitled (“Vesicular Arbuscular Mycorrhiza as a potential
biofertilizer in Aloe vera”) is an authentic record of my own work carried out at (LOVELY
PROFESSIONAL UNIVERSITY) as requirements of Capstone project for the award of degree of
B.Tech in Biotechnology, Lovely Professional University, Phagwara, under the guidance of
(Name of Faculty coordinator), during August to December, 2010).

(Signature of student)
UMESH SHARMA
Registration no: 3040070046

Date: ___________________

Certified that the above statement made by the student is correct to the best of our knowledge and belief.

Dr.Joginder singh panwar

Associate Professor

(Biotechnology)
 ACKNOWLEDGEMENT

I express my deep sense of gratitude to Dr. JOGINDER SINGH PANWAR , Assistant professor
for Bio-technology directed me in capstone project. he has been kind enough to allow me the
benefit of inspiring advice, rich experience and learned guidance.

I am highly indebted to faculty of lovely school of science and technology, Phagwara

who provided me their valuable advices, all facilities and help of books. I am also thankful to
the library and computer laboratory for the purpose of writing this project.

I am under obligation to all the prominent authors those, whose works, I have
consulted and reffered to my project.

(Umesh)
 INTRODUCTION
Arbuscular mycorrhizal (AM) fungi are common forms of symbiotic fungi that form association
with plant roots in a host non-specific manner, colonize cortical tissues and extend hyphae into
the rhizosphere. These fungi have been reported to enhance plant growth mainly by increasing
availability of phosphorus and other nutrient, producing growth hormones, improving
rhizospheric condition and altering the physiological and biochemical characteristics of the host
plant. Phosphorus is one of the major plant nutrients required in optimum amount for proper
plant growth. About 98% of Indian soils are inadequate in available P (Gaur 1987). Native soil
phosphorus is organically bound and available in the form of phytin, or its derivatives, has low
solubility and mobility, and thus is not accessible to plants. This organic P must be hydrolysed to
inorganic P for utilization by plants which can be mediated by phosphatases secreted by AM
fungi or phosphate solubilizing bacteria (Gaur and Rana 1990). Excess inorganic fertilization
decreases the extent of fungal infection by AM fungi, so an optimum situation must exist where
optimal benefits from such fungi and inorganic fertilizers are achieved. Thus tricalcium
phosphate was added as an insoluble source of inorganic phosphate which does not increase the
P level in the rhizosphere soil.
Roots of living plants support the growth of a complex of microbes which create a special habitat
that influences growth and survival of the plants . Mycorrhiza is the mutualistic association
between soil-borne fungi with the roots of higher plants. Vesicular arbuscular mycorrhizal
(VAM) fungi are known to colonize a number of tropical plants including vegetables . These
fungi use some of the root exudates and modify root physiology thereby altering the microbial
equilibrium on the root surface. Mycorrhizal plants are known to have altered nutritional status,
increased photosynthetic rates, altered levels of growth regulating substances and altered patterns
of root exudation due to changes in membrane permeability. VAM fungi are now-a-days well
recognized as biofertilizer due to their manifold advantages provided to the host plant besides
increasing nutrient and water uptake.
 REVIEW OF LITERATURE

Study of plant roots and the diversity of soil micro biota, such as bacteria, fungi and microfauna
associated with them, is important for understanding the ecological complexities between diverse
plants, microbes, soil and climates and their role in phytoremediation of contaminated soils. The
arbuscular mycorrhizal fungi (AMF) are universal and ubiquitous rhizosphere microflora
forming symbiosis with plant roots and acting as biofertilizers, bioprotactants, and biodegraders.
In addition to AMF, soils also contain various antagonistic and beneficial bacteria such as root
pathogens, plant growth promoting rhizobacteria including free-living and symbiotic N-fixers,
and mycorrhiza helping bacteria. Their potential role in phytoremediation of heavy metal (HM)
contaminated soils and water is becoming evident although there is need to completely
understand the ecological complexities of the plant-microbe-soil interactions and their better
exploitation as consortia in remediation strategies employed for contaminated soils. The ability
of AM fungi to control soilborne diseases would be strongly related to their
capacity to specifically stimulate the establishment of rhizobacteria
unfavorable to pathogen development within the mycorrhizosphere before
root infection. These multitrophic root microbial associations deserve multi-disciplinary
investigations using molecular, biochemical, and physiological techniques. Ecosystem
restoration of heavy metal contaminated soils practices need to incorporate microbial
biotechnology research and development.
Aloe vera is a wonder plant with health benefits. it is a stemless or very short stem succulent
plant with thick and fleshy leaves. Its an efficient anti-oxidant to helping in all digestion related
problems, arthritis, stress, diabetes, cancer, aids to being an enhancer of beauty. Assotiation of
VAM (vesicular arbuscular mycorrhiza) in roots of aloe vera provide nutients like phosphorus,
nitrogen,increase its water uptake capacity and make this plant more disease and drought
resistant which ultimately increase growth and production of this plant. In return to this plant
roots provides nutrition (carbon source) to the fungal hyphae. In this way both (plant and fungi)
form a symbiotic relation and proves beneficial to each other.
INTERACTION AMONG DIFFERENT COMPONENT OF
MYCORRHIZOSPHERE
VESICULAR ARBUSCULAR MYCORRHIZA PENETRATION
 STEPS OF AM FUNGI COLONISATION

• SPORE GERMINATION

• HYPHAL DIFFERENTIATION

• APPRESSORIUM FORMATION

• ROOT PENETRATION

• INTERCELLULAR GROWTH

• ARBUSCULE FORMATION
• NUTRIENT TRANSPORT

MATERIAL REQUIRED

o Soil sample (100g), distilled water.

o 250ml,500ml,1000ml beaker.
o Stirrer, sieving apparatus, filter paper (watmann No.1).
o Syringe, needle, cover slip.
 o Dissection microscope & compound microscope(10x, 40x).
o Glycerine, Trypan blue dye.

METHOD

Wet sieving and decanting technique of Gerdemann and Nicolson

• Isolation of micorrhizal spore from soil

• Mix 100 gm soil in 1 litre distilled water

• Wait for sedimentation

• Froath and sieve.each mesh posses spores and debries

• Now mix the sieving material in ¾ filled beaker of waterand wait for settling

• Pass it into filter paper and take under dissection microscope

• Isolate the spores and pick the spore with the help of needle

• Mount on glycerine slide and view under 10x,40x Microscope

• In this way spore get seperated

IDENTIFICATION OF FUNGAL INFECTION

To check the association of vesicular arbuscular mycorrhiza in to the roots Aloe vera a dye
named trypan blue is used which give the blue color appearance in the roots. this method of
identification of fungal infection is known as Philips & Haymann method.
 RESULT & DISCUSSION

Experiments were conducted in glass house to study the effectiveness of 4 arbuscular

mycorrhizal (AM) fungi on the medicinal plant Aloe vera. Aloe vera plants were grown with AM
fungi inoculation in polybags showed an increase in plant growth (leaf, stem and root dry
biomass), root colonization, phosphorus (P), nitrogen (N), sugar and barbaloin content over those
grown without inoculation.Glomus mossae was found to be the best AM symbiont for
inoculating Aloe vera.

OBSERVATION OF SPORES
 Glomus spore

CONCLUSION

Arbuscular mycorrhizal fungi are associated with most agricultural crops and provide protection
against soil-borne diseases. The degree of reduction in diseases development by AM fungi varies
with the combination of host-AM-environmental conditions. Two groups of major soil borne
pathogens of vegetables have been described here, namely nematode and fungi. Generally
inoculation with AM fungi had a negative effect on growth of pathogens, but most of the reports
for nematode diseases are on migratory and sedentary endoparasitic nematodes. The prophylactic
action of AM may further be improved with the integration of antagonistic rhizosphere microbes
to improve plant health. The mycorrhizal efficacy also varies by alteration in soil nutrition,
mycorrhizosphere, inoculation sequences and modification of cultural practices. Number of
mechanisms are involved in controlling the pathogen by mycorrhizal roots such as exclusion of
pathogen, lignifications of cell wall, changed P nutrition, exudation of low molecular weight
compounds. The phosphorus content of root and shoot tissue remains higher in mycorrhizal
custard-apple seedlings as compared to non-mycorrhizal ones and there was an increase in the
amount of phosphorus with increase in age of the plant. The percentage of phosphorus in shoot is
always higher than the root of both mycorrhizal and non-mycorrhizal plants. The external VAM
hyphae reach beyond the depletion zone around the root hairs, absorb soil P and translocate it,
perhaps in the form of polyphosphate granules, to the arbuscules where P is transferred to the
plant cell in exchange of carbon. Glomus fasciculatum inoculated custard apple seedlings
showed greater amount of chlorophyll than uninoculated control plants and there existed a
positive correlation between the chlorophyll content and the age of the seedlings.. Benefits from
VAM seem to be confined to particular growth periods namely the seedling stage. Significant
increase in chlorophyll content, shoot and root length and total biomass of different plants was
observed following inoculation with Glomus sp. Thus mycorrhizal fungi offer an
environmentally sound biological alternative to chemical fertilizers and pesticides for
maintaining plant quality and productivity in agriculture, horticulture and forestry.
Mycorrhization helper bacteria (MHB) might be exploited to improve mycorrhization, and
arbuscular mycorrhiza fungi (AMF) to improve nodulation and stimulate plant growth promoting
rhizobacteria(PGPR). It is anticipated that future commercial biofertilizers would contain PGPR,
MHB, and AMF.

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