mycorrhizal fungi for sustainable agriculture-a review · 2018. 5. 15. · mycorrhiza are the rule...

AGRICULTURAL RESEARCH COMMUNICATION CENTREAgric Rev 31 (2) 93 - 1042010 wwwarccjournalscom indianjournals com

MYCORRHIZAL FUNGI FOR SUSTAINABLE AGRICULTURE-A REVIEW

Shiv Ram Singh Ummed Singh AK Chaubey and M1 Bhat

Regional Research Station Sher-e- Kashmir University of Agricultural Sciences and

Technology of Kashmir Wadura Sopore (JampK)-193 201 India

ABSTRACT

Mycorrhizal fungi are species of Glomalean fungi that intimately associated with plant

roots forming a symbiotic relationship with plant providing sugars for the fungi and the fungi

providing nutrients such as P Zn Cu etc to the plant protection from drought stress conditions

toxic effect of heavy metals and infection of some soil born diseases with increased plant growth

Further this association enhances the formation of stable soil aggregates that improves the

stability of soil structure especially under course grain soil condition and restricts soil erosion

The mycorrhizal fungi also offer a great potential for sustainable agriculture The production of

mycorrhizal fungal inocula is laborious and cost-consuming due to its obligate biotrophic nature

Spores of arbuscular mycorrhizal (AM) fungi in soil can be collected by wet sieving and decanting

method and pure culture of mycorrhizal fungi produced either by Soil Trap Culture or Single

Spore methods Thereafter spores were characterized on the basis of their shape size colours

and hyphal attachment and classified on the basis of their morphology Each genus of mycorrhizal

fungi has specific cell wall characters that may be used for identification

Key Words Mycorrhizal fungi Sustainable agriculture

One of the ecological and economically

important activities of soil fungi is the mutually

beneficial association (symbiosis) between certain

fungi and the roots of higher plants This association

is called mycorrhizae a term meaning fungi root

A mycorrhizae is formed when the appropriate

fungus invades a plant root in a process superficially

similar to infection by pathogenic fungi (Hodge

2000) However in the mycorrhizal association the

fungus and the plant have appropriately co-evolved

so that both are benefited from the relationship In

fact in natural ecosystem many plants are quite

dependent on mycorrhizal relationship and cannot

survive without them Mycorrhiza are the rule not

the exception for most plant species including the

majority of economically important plants (more

than 80 of land plant families) included in these

Emailshivramsingh22 gmail com

are many species though crops in the Brasicaceae

Chenopodiaceae Cyperaceaa Juncaceae

Urticaceae and Caryophyllaceae families generally

do not form mycorrhizal associations (Newman and

Reddell 1987) Mycorrhizal structures have been

found in fossils of plants that lived some 400 million

years ago indicating that mycorrhizal infection may

have played a role in the evolutionary adaptation of

plants to the land environment Mycorhizal fungi

derive an enormous surv ival advantage from

teaming up with plants Instead of having to compete

with all the other soil heterotrophs for decaying

organic matter the mycorrhizal fungi obtain sugars

directly from the plants root cells This represents

an energy cost to the plant which may lose as much

as 5-30 of its total photosynthates production to

its mycorrhizal fungi symbionts

94 AGRICUTURAL REVEWS

Importance of mycorrhizae 1 Mycorrhizal fungi greatly enhanced the ability

of plants to take up phosphorus and other

nutrients those are relatively immobile and exist

in low concentration in the soil solution

Arbuscular mycorrhizal fungi can playa significant role in P nutrition of crop increasing total uptake and in turn P use efficiency This might be associated with increased growth and

crop yield (Koide et aI 2004) Where

colonization by arbuscular mycorrhizal fungi is

disrupted either by cultural operation or suppressed due to high concentration of soil available P uptake of p growth and in some

cases yield can be significantly reduced even

some times crops fail to response to colonization

by native mycorrhizal fungi (Sorensen et ai 2005) Though P uptake in mainly translocated

by the arbuscular mycorrhizal association it has

become increasingly apparent that arbuscular

mycorrhizal fungi can be important in the uptake

of other nutrients by the host plant Zinc nutrition

is most commonly reported as being influenced

by the association although uptake of copper

(Cu) iron N K Ca and Mg has been reported

to be enhanced (Clark and Zeto 2000)

Arbuscular mycorrhizal fungi may also enhance

plant uptake of N from organic sources (Hodge

et ai 2001)

2 Water uptake may be improved by mycorrhizal

association making more resistant to drought

condition Several mechanisms have been

proposed to explain the effect including

increased root hydraulic conductivity improved

stomatal regulation osmotic regulation osmotic adjustment of the host and improved contact with soil particles through the binding effect of

hyphae enabling water to be extracted from

smaller pores (Auge 2004) Often both water

and nutrient uptake are higher in drought stressed mycorrhizal plants than in nonshymycorrhizal plants (Al-Karaki and Clark 1990) Increased phosphorus levels generally increase

drought resistance calculations made from

arbuscular mycorrhizal fungi indicate that the

amount of water that could travel through the

mycelia to the plant is not large enough to influence plant growth or survival However work with ectomycorhizae showed that the

fungal strands are capable of altering the water

potential of plants Seedlings were maintained

in a healthy state for at least 10 week period

when the only source of water was through

mycelia strands growing in moist peat However

arbusculpr mycorrhizal fungi can only alleviate

moderate drought stress and in more severe

drought conditions they become ineffective (Bryla and Duniway 1997)

3 Though nutrient uptake has been the focus of

much research on the mycorrhizal association

there is evidence that mycorrhizal also play a

role in the suppression of crop pest parasitic

nematodes and diseases particularly soil borne

fungal diseases by producing antibiotics

altering the root epidemics and compacting with

fungal pathogens for infection sites (Borowicz

2001) Some of the soil borne diseases which

have been experimentally suppressed using

Table 1 Selected soil borne fungal diseases controlled by AMF

Pathogen Disease Crop Reference

Aphanomces euteiches Root rot Pea (Pisum sativum) Bodker et al(2002) Fusarium oxsporum Fusarium root rot Asparagus (Asparagus ofjcinalisFrench Matsubara et al (2002)

bean (Phaseolus vulgaris) Helicobasidium mompa Violet root rot Asparagus (Asparagus officinalis Kasiamdari et al (2002) Rhizoctonia solani Root and stem rots Mung bean ( Vigna radiate) Kjoller and Rosendahl (1996) Sclerotium cepivorum White rot Onion (Allium cepa) Torres-Barragan et al (1996) Verticillium dohliae Verticilium wilt Tomato(Lycopersicon esculentum) Karagiannidis et al(2002) Pythium aphanidermatum Damping off Tomato(Lycopersicon esculentum) Jallali and Chand (1987)

95 Va 31 No 2 2010

arbuscular mycorrhizal fungi (AMF) are given

in Table 1 This reduction in the severity of

diseases occurs rather than total inhibition This

can still result in a significant increase in yield

over plants and inoculated with AMF and occurs

despite the fact that pathogen infection generally

reduces AM colonization In some cases the

apparent resistance of a plant to a pest or disease

may be simply the result of improved nutrition

(Karagiannidis et al 2002) Once colonization

of a root cell by AMF occurs the pathogens are

excluded from that cell as a result the most

effective control is achieved when colonization

by mycorrhizal fungi takes place before attack

by pathogen (Sylvia and Chellemi 2001) Other

type of pest and diseases causing organisms

which may be suppressed by arbuscular

mycorrhizal fungi include pathogenic nematodes

((Talavera et ai 2001) above ground fungal

disease and herbivores (Gange et ai 2005) Though the mechanisms involved are complex

change in nutritional status resulting in changes

to leaf defensive chemicals are likely to be

involved in above ground interactions with

herbivores (Gange and West 1994)

4 Mycorrhizal fungi have an important role in

stabilizing soil aggregates which is especially

important in an agricultural context where

cultivation trafficking and low level of soil

organic matter all tend to result in damaged soil

structure The host plant transfers as much as

20 of all fixed C to the fungal partner and in

agricultural soils AMF can produce Significant

biomass Like other soil fungi AMF can produce

significant micro-aggregates into larger macroshy

aggregates through the enmeshing effect of there

hyphae In addition AMF produce an

extracellular glycoprotein (gooey protein)

glomalin which sticks hyphae to soil and is very

effective as a cementing agent (Rilling et al 2001) Whether glomalin is important or not

general hyphae exudation and rapid hyphae

turnover provide carbon to other soil

microorganisms indirectly promoting aggregate

stability The over all effect of hyphae

enmeshment and inputs can be significant

increase in soil structural stability (Piotrowski

eta 2004)

5 The soils contaminated with high levels of metals

or salts mycorrhizal protect the plants from

excessive uptake of there potential toxins It has

been thought that the heavy metals are bound

by carbonyl groups in the pectic compounds

(hemicelluloses) of the interfacial between the

fungus and the host cells It has also been

demonstrated that plants growing in mine spoils

with heavy metals

6 As well as interacting with disease causing soil

organisms AMF also interact with a whole range

of other microorganisms in soils Bacterial

communities and specific bacterial strains

promote germination of AM fungal spores and

can increase the rate and extent of root

colonization by AM fungi (Johansson et a 2004) Once the arbuscular symbiosis has

developed AM hyphae influence the surrounding

soil which has been termed the

mycorrhizosphere resulting in the development

of distinct microbial communities relative to the

rhizosphere and bulk soil Within the

mycorrhizosphere free living N fixing bacteria

and general plant growth promoting

rhizobacteria (PGPR) (Biro et a 2000) The legume-Rhizobium symbiosis is greatly

influenced by AMF and there is some evidence

to suggest that legume nodules contain AMF

communities quite distinct from those found in

the roots of legume (Scheublin eta 2004) The

Rhizobium symbiosis is dependent on high

concentration of P and so the enhanced P

nutrition arising from the AM colonization can

result in an increase in nodulation and N2 fixation (Vazquez et al2002)

7 It can be used together with other agricultural

chemicals to some extent Mycorrhizae are

endurable to several chemical substances eg

96 AGRICUTURAL REVlEWS

I hr n notlets

Figl Ectomycorrhizal association with plant roots Fig2 Endomycorrhizal association with plant roots

Source The Nature and Properties of Soils (lOthEd) by Nyle C Brady Prentice-Hall of India Private limited New Delhi

pesticides such as endrin chlorodane methyl

parathion methyl carbofuran herbicide such

as glyphosate fuazifopbutyl chemical agents for

plant disease elimination such as captan

benomyle maneb triforine mancozed and

zineb However the effect of biocides on the

arbuscular mycorrhizal association is complex

and not easily predictable Even the effect of

fungicides is not straight forward Sreenivasa and Bagyaraj (1989) tested the effect of nine fungicides on Glomus fasciculatum in pot trails

At their recommended application rates all fungicides reduced root colonization of Rhodes grass (Chloris gayana) by 12 to 25 and spore

production by 19 to 25 but at half the recommended rate Captan (Nshy

(trichloromethylthio) cyco-hex-4-ene-l2shydicarboximide) increased all measured

mycorrhizal parameters Udaiyan et a (1990) looked at the effect of six fungicides on

arbuscrlar mycorrhizal fungal colonization and

sporolation in three types of millet (Eleusine coracana Pankum miljaceum and Paspalum scrobiculatum) under field conditions At their

recommended application rates some reduced

root colonization and sporolation while others

had no effect or increased arbuscular

mycorrhizal colonization and sporolation

depending on the species of millet involved

However when applied for control of leaf fungal

diseases at their recommended rates they had

no effect on mycorrhizal colonization Indeed

paradoxically some fungicides are regularly

shown to have no deleterious effect on arbuscular

mycorrhizal fungi and in some cases increase

mycorrhizal colonization and nutrient uptake

especially at reduced application rate (Hwang

et a 1993)

Kinds of mycorrhizae- Two types mycorrhizal

associations are of considerable practical

importance

1 Ectomycorrhiza- Ectomycorhizae covers

roots and rootlets with a thick mantel of hyphae

The fungal sheath spreads between the cortical

cells of roots Their hyphae penetrate the roots

and develop in the free space around the cells of the cortex to form a complex intercellular system

called the harting net (Harley and Smith 1983)

but do not penetrate the cortex cell wall (hence

the term ecto meaning outside) Ectomycorhizae

cause the infected root system to consist primarily

of stubby white rootlets with a characteristics Y

shape (Fig 1)These might be due to the

production of auxins that are responsible for some

of the morphological differences between

97 Vol 31 No 2 2010

mycorrhizal and non-mycorrhizal roots

Ectomycorrhizae consists of septet fungal cells

belonging to the Basidiomycetes are dependent

on carbohydrates which are supplied by the root It has been shown that C14labelled photosynthates

are rapidly translocated not only to the roots but

also into the sheath and hyphae of mycorrhizal fungi Carbohydrates mainly in the form of

sucrose obtained from the host root are rapidly

converted into typical fungal sugars such

astrehalose or mannitol In this way the organic

carbon is trapped because these sugars are only

poorly reassimilated by the piant root It has been shown that the transport of assimilates from the

aerial plant parts towards the roots is higher in

mycorrhizal infected plants than in non infected

plants The mycorrhiza is thus a sink for

photosynthates

The ectomycorrhizae group includes

hundreds of different fungal species associated

primarily with temperate or semiarid region trees

and shrubs such as pine birch hemlock beech

oak spruce and fir Further research has shown

that some of the trees and in even some tree

legumes viz Alnus Cupressus Juniperus Acacia and Casuarina has been found to associate with

both ecto and ectendomycorrhizas which have

some characteristics of both ecto and

endomycorrhiza The external mantle may be

much reduced or even absent in some mycorrhiza

on trees The Harting net is well developed but

hypha also penetrates into cells of the host The

same fungus that produces ectomycorrhiza may

on a different host plant or under different

conditions from ectendomycorrhiza Inoculation with Pisolithus tintorius and Telephora terrestrius which is most important species of ectomycorrhiza is greatly enhanced the growth of temperate forest

trees

2 Endomycorrhiza The most important members of

endomycorrhiza group are called arbuscular

mycorrhizae (AM) Formerly these were called

vesicular arbuscular mycorrhizae (VAM) but this

was shortened to AM since fungal hyphae actually

penetrate the cortical root cell wall and once inside

the plant cell form small hyphae branched structures

known as arbuscules Fungi of the endomycorrhizae

consist of aseptet hyphae are members of the

Phycomycetes and Basidiomycetes The hyphae of

these fUhgi penetrate the cells of the root cortex

forming an internal hyphae network Some hyphae

also extend into the soil For many plant species

including most agricultural crops the predominant

type of fungal infection is vesicular arbuscular

mycorrhizae (VAM) This name derives from the

occurrence of two types of structures characteristics

of the fungi belongs to the family Endogonaceae

(Fig 2) The fungus may be considered as a twoshy

phase system with a mycelium in the cortex

Table 2 Endomycorrhizal associated and non associated crops

Mycorrhizal associated crops Group Crops

Cereals Corn sorghum wheat oat barley dryland rice Legumes Soybean alfalfa lentil peas clovers greengram black gram cow pea beans pigeon pea chick pea

ground nut Lucerne Vegetables Potato tomato brinjal chilli cucumber onion garlic bitter gourd ladies finger round gourd carrot Fruits Apple plum pear cherry peach grapes citrus mulberry strawberry papaya Forest trees Maple yellow poplar red wood Plantation crops Cocoa coffee rubber Others Cotton sugarcane cassava

Mycorrhizal not associated crops Group Crops Cruciferae Cabbage mustarded canola broccoli radish turnip Chenopodiaceae Sugar beet red beet spinach

98 AGRICUTURAL REVIEWS

Classification of mycorrhizae shyClassification of mycorrhizal fungi according to Morton and Benny (1990) is given as follows

1 Order

2 Sub order

3 Family

4 Genus

connected to an external mycelium in the

rhizosphere and soil It also shows the presence of

structures at different stages in development with a

shrub like appearance called arbuscles (arbuscula

Latin=shrub) These structures are similar to

haustoria but are produced by dichotomous

branching of hyphae These structures serve to

transfer mineral nutrients from the fungi to host plants

and sugars from the plant to the fungus The second

type of structure the Vesicle (Vesicula Latin=small

bladder) may be formed by swelling of the hyphae

and may occur within or between cells and serve as

storage organs for mycorrhizae The AM fungi are

the most common and widespread group Nearby

100 identified species of fungi from these

endomycorrhizal associations in soils from the tropics to the arctic (Table 2)

The members of Glominae suborder forming

intraradical vesicles and branched haustoria called

Entrophosphora

arbuscles in mycorrhizal roots spores born

terminally intercalarily and laterally form one or

more subtending hyphal is known as vesicular

arbuscular mycorrhizae (VAM) while as members

of Gigasporinae sub order do not form vesicles and

hence are arbuscular mycorrhizal fungi (AM)

Mechanisms of nutrients and water uptake-The association between the host and fungus

is well co-ordinated Young feeding do not roots

show any sign of damage when infected Indeed

the living root is essential for the culture of these

fungi The host plant supplying the fungus with

organic compounds and the fungus assisting the

roots in exploiting the soil for water and inorganic

nutrients The relationship is of particular

importance for phosphorus nutrition since

phosphate depletion zones can readily occur around

plant roots The network of hyphae extending from

the roots into the soil enlarges the contact area

- --- -~

VeSicle extraradicat

spore hyphae

- (- ) --2- - Jmij] ----- g-WCmiddotm-middot-middot-shy - null_~_ ~ v~=--------

soil root

Fig3 Schematic picture of AM fungi colonizing root and their hyphaI extension into soil

Source Biofertilizers Manual by FNCA Forum for Nuclear Cooperation in Asia (FACAl March 2006 published by Japan Atomic Industrial Forum Tokyo Japan

between the bulk soil and the fungus-host root

association around depletion zone of soil thus create

a potential gradient against high concentration zone

from bulk of soil to the less concentrated depleted

zone by diffusion and hence facilitates a greater

uptake of phosphate It also indicates the possibility

that rate of phosphate uptake by the hyphae may

be higher than that by the root Phosphate translocation in the hyphae appears to be rapid

The phosphate is translocated in the form of small polyphosphate granules in the hyphae thus

maintaining low internal phosphate (Pi)

concentration by cytoplasmic streaming (Sorensen

et aJ 2005) Phosphate taken up by the fungus is

from the labile pool of soil phosphate and hence

from a readily available phosphate fraction

Mycorrhizal fungi may take up phosphate from less soluble phosphate which may be of particular

importance in acid tropical soils It has become

increasingly apparent that arbuscular mycorrhizae

fungi can be important in the uptake of other

nutrients by the host plant Zn Cu Fe N K Ca and Mg nutrition is most commonly reported as being

influenced by the mycorrhizal associations (Clark

and Zeto 2000)

Observation of arbuscular mycorrhizal fungi in roots

The structure of arbuscular mycorrhizal

fungus in roots is usually not observed without

appropriate staining Freshly collected root samples

should be washed gently and be free from soil

particles Roots are treated with 10 KOH solution for 30 minute to 1-2 hour in a hot bath depending on thickness of root structure Treated roots are

washed with water and treated with 2 HCI

solution Acidified root samples are stained with

0 05 trypan blue (or acid fuchsin) in lactic acid

for 10-15 minutes in a hot bath or for a few hours without heating (Phillip and Hayman 1970) The

roots are destained with lactic acid or lacto-glycerol

and are now ready for microscopic with transmitted

observation The stained roots may be observed first

under a dissecting microscope with transmitted

illumination and then observed under a compound microscope Fungal structures are stained and can

be easily recognized

100 AGRICUTURALREVIEWS

Table 3 Morphological character of AM or VAM fungal spores

Morphology Characteristics

Shape ( ie globular spher cal Irregular) Size Globular diameter (minimum-average-maximum) Irregular shape length x width (minimumshy

average-maximum) Hyphal attachment (ie sporiferous saccule bulbous suspensor etc) sporiferous saccule= Acaulospora

Entrophosphora bulbous suspensor = Gigaspora Scutellospora Auxiliary cell (presence = Gigaspora Scutellospora none ) Sporocarp (presence none) Germination shield (presence = Scutellospora absent) Surface ornamentation (ie smooth rough reticulate etc) Vesicle (Presence or absence in mycorrhizal roQts)

These characters should be recorded with careful observation of many spores

Glomus [Jiversispora Paraglomus

Acaulospora

Enlrophospora

-~~~

Gigaspora ( )gtgt

- ~ ~lbOUS suspensor

Sculeflospora

~gerrnination shield

fig 4 Morphology of representative genera of arbuscular mycorrhizal fungi

Source Biofertilizers Manual by FNCA Forum for Nuclear Cooperation in Asia (FACA) March 2006 published by Japan Atomic Industrial Forum Tokyo Japan

Isolation of spores from soils and their observation for identification

Spores of AM fu ngi in soil can be collected by the wet sieving and decanting method The gravity of spores is little than that of soil particles Successive decantation of soil suspension followed by sieving and with fine mesh can concentrate the spores from soil Since the spores are globular or sub-globular in 50-500 pm in diameter they in sievings can be recognized under a dissecting microscope

Equipments 1 Sieve Sieves with various mesh sizes At least

the following mesh sizes are required 1 mm 100

pm and 50 pm other size such as 500 pm and

250pm are preferable Stainless steel sieves are

commercially available However it is possible to make a plastic sieve with PVP tubes and

nylone mesh by yourself

2 Fine glass pipettes Tip of disposal glass Pasteur

pipette (1 ml) is softened with flame of a gas

burner and is sharpened Various sizes of tips

fitting to sizes of spores can be prepared

3 Forceps light fine tweezers is preferable

4 Dissecting microscope Stereoscopic zoom

microscope with bifurcated illuminator of fiber

arm is advisable Transmitted il lumination system is also needed

5 Compound microscope Biological compound

microscope is needed

Procedure 50 g of freshly collected soil sample is put

into 1 to 2 litres of plastic beakers Usually

rhizosphere soils are rich in arbuscular mycorrhizal

fungal spores Beaker size can be changed

depending on the soil sample size Soil is suspended

with about 500 ml to 1 litre of tap water Soil macroshy

aggregates should be crushed with hand after 10shy

30 seconds of setting down of soil particles the upper

layer of soil suspension is poured into the sieving

The procedure should be repeated until the upper

layer of soil suspension is transparent (Gerdemann

and Nicolson 1963) The sieving on the fine mesh

is collected into a small beaker and dispersed with

ultra sonication Weak sonication (ie 30 W 30 sec)

101 Vol 31 No 2 2010

is enough and strong sonication may destroy fungal spores Then the dispersed sample is again passed though the sieve Depending on the toughness of soil aggregate the sonication process can be

repeated UsuaUy AM fungal spores are collected

on 100um some small spores are on 50um To collect large spores such as Gigaspora margarita 250um sieve is efficient

Morphological observation of spores for identification

Morphology of spores is a basis for identification of arbuscular mycorrhizal fungi because the hyphae and the organs such as arbuscules and vesicles are not specific to species Spores collected from the soil often deteriorate so that they may be used only for tentative identification at genus level For detailed observation culturing the target AM fungus is required and the spores from pot culture should be used At least 30-50 spores from the same morphological spores type should be observed and more observation is required

1 Observation of intact spores under dissecting

microscope Spore collected from soil or

Rhizosphere Plant soil

1 (w ith roo ts)

Pl ant trap culture Sieving

Soil trap culture ~ I

-- r

1 i-~II (- soi l

~ I Sores

Fig 5 Methods tor trapping AM tungi

culturing medium is put in a watch glass or a

small Petri dish and their shape colour and the attachment to spores are observed Spores should be classified into each spore type based

upon morphology For each spore type detailed

observation is required

For colour description standard colour chart

such as soil colour chart or colour chart of

glomalean fungi (INVAM colour chart) should be

used The colour chart should be under the same

illumination as used for spore observation because

the colour itself is greatly affected by the

characteristics of illumination It should reminded

that attachment such as soporiferous saccule for

Acaulosporaceae and subtenting hyphae for

Gigasporaceae are often lost during collection of

spores from soil

2 Observation of spores mounted on slide glass

under a compound microscope Spores are

mounted with polyvinyle lactoglycerol (PVLG)

on a slide glass Several slides should be made

Intact and crushed spores mounted with PVLG

Polyvinyle alcohol (polymerization 1000-1500)

Fig 6 Single spore isolation

Source Biofertilizers Manual by FNCA Forum fo r Nuclear Cooperation in Asia (FACA) March 2006 published by Japan Atomic Industrial Forum Tokyo Japan

102 AGRICtITURAL REVIEWS

166 g is dissolved in 10 ml deionized water

Complete dissolution may need 6 hours at 80 dc The dissolved polyvinyle alcohol is mixed

with 10 ml of lactic acid and 1 ml of glycerol it

can be used more than a day after preparation

The Important morphological characters of AM

fungi is indicated in Tabie 3 and Fig 4

Culturing of AM fungi AM fungi need the symbiotic association

with plants for proliferation Therefore culturing AM

fungi is to indicate AM fungi to host plant and to

grow the inoculated plant For the AM fungal

inoculum spores collected from soil can be used

However spores in soil are not always active in

colonizing plants Therefore trapping culture is often

employed Soil of sieving of soil is used as medium

(Soil Trap Culture) To isolate AM fungi colonizing

roots mycorrhizal plants collected from field can be

transplanted to potting medium as Plant Trap Culture

(Fig 5) (Murakoshi et ai 1998)

1 Host plant Various mycotrophic plants can be

used viz leguminous species (Trifolium spp Medicago spp Lotus japonicus) (ie Lolium spp Paspalum notatum) and other herbaceous

species (ie Plantago spp) Onion and leek

(Alliumspp ) and maize sorghum are also good

hosts

2 Growth condition Any conditions which support good growth of host plant plants are acceptable To avoid contamination a growth chamber is preferred If green house is used it should be kept clean It should be kept clean It should be remanded that cross-contamination or contamination from dust is inevitable under open-air conditions even in growth chamber To prevent cross-contamination from other pot culture in the same chamber use of plastic bag is advisable (Walker and Vestberg 1994)

3 Procedure A Plant Trap Culture

i Rhizosphere soil is collected with shoots of

trap plant cut at the crown and roots are

finely chopped and mixed with the soils using

a sharp chopper

ii The chopped roots and soil are mixed l1 (vv )

with autoclav ed coarse sand in a

mechanical mixer or massaged well in a jurable plastic bag

iii The soil mix is then transferred to a 15 cm plastic

pot

Iv Plants seeds of suitable trap plants such tropical signal grass into the pot

as

v The pot cultures are maintained in a green house

for at least 3 months and check sporulation

from time to time By the fourth month AMF

sporulation may be at the peak Sanitary tests may also be carried out to ensure no

contamination from parasitic fungi occurs

vi Keep fertilizer application to a minimum to

encourage AMF proliferation

vii Trap culture pots are later left to dry under shade

for up to two weeks

viiiHarvest the spores using the sieving and

decanting techniques or the denSity-gradient

centrifugation technique

ix The mono specification spores are ready for

inoculation onto seedlings of the desired crops

B Single spore isolation To purify an isolated

fungus single spore isolation is needed

Even if the spores are morphologically

identical it often contains contaminants

whose morphology is very similar For single

spore isolation no specific equipment is

needed For effic ient handling two sets of

dissecting microscope are placed side by

side One microscope is for picking up

single spore from spores in a dish Another

is for inoculation of a spore on roots S

seedling placed in the pot is placed under

another microscope Under the fist

microscope single spore is picked up and

transferred under the second microscope

Under the second microscope the spore is

placed on fine roots or root tip of the

seedlings (Fig 6) If culture is successful

103 VoL 31 0 2 2(10

the detailed morphological observation is or the leaf litter under the tree is removed About required Potting medium can be dried by 10 g per plant of VA mycorrhizal biofertilizers is stopping watering to the pot After the host applied to the root hairs system and then plant wilt the dried soil containing spores covered with soil can be stored for a year at 4-5 0 C

3 For transplanting seedling simply sprinkle VA

Inoculation of Arbuscular Mycorrhizae mycorrhizal biofeliilizers adjacent to the plant roots and cover with soil l Seed inoculation- Most of cereals legume

sorghum cotton and vegetables are inoculated 4 VA mycorrhizal biofertilizers can be used in

with seed VAM culture containing infected root combination with several types of biofertilizers (Rhizobium PSB PGPR) bits viable AM-fungal propagules (including

spores) containing 8-10 viable sporesg VAM Problems and potential for VAM inoculum

culture 2 kglha is used production and utilization

2 Soil application- The AM or VAM culture should l Situation where effective indigenous VAM be applied to the open furrows 15 kglha using population is low

field soil to bulk the carrier This method is 2 Inoculation is best for transplanted crops where suitable for fruit crops and plantation crops For soil disturbances have reduced VAM

huit trees soil under the plant canopy is trenched inoculation potential

REFERENCES Al-Karaki GN and Clark RB (1990) Plant Nutr 22 1775-1784

Auge RM (2004) Canadian J Soil Sci 84 373-381

Bryla D Rand Duniway J M (1997) Plant Sai1197 85-108

Biro B etal (2000) Applied Sail Ecol 15 159-168

Borowicz VA (2001) Ecology82 3057-3068

Clark R B and Zeto S K (2000) J Plant Nutr 23 867-902

Gange A C eta (2005) Ecolagy86 603-611

Gange A C and West H M (1994) New Phytal 128 79-87

Gerdemann Jw and Nicolson T H (1963) Trans British MvcolagicaISac 46 235-244

Harley J L and Smith S E (1983) Mycorrhizal Symbiosis Academic Press London

Hwang S E etal (1993) Plant Dis 77 1093-1098

Hodge A (2000) FEMS Microbial Eco 32 91-96

Hodge A eta (2001) Nature 413 297-299

Jallali BL and Chand H (1987) in Proceedings of iSI Asian conference on Mycorrhizae at Madras (Mahadevan et

al Eds)p 209-214

Johansson J eta (2004) FEMS Microbial Eca 48 1-13

Karagiannidis N eta (2002) Sci Hart 94 145-156

Kasiamdari R S et al (2002) Plant and Soil 238 235-244

KjolIer R and Rosendahl S (1996) Mycorrhizae6 487-491

Koide R T eta (2000) NewPhytologyl48 163-168

Matsubara Y et al (2002) J Japan Soc Hod Sci 71 370-374

Morton J B and Benny G L (1990) Mycatoxan 37 471-491


Newman EI and Reddell P (1987) New Phytology 106 745-751

Phillip J M and Hayman D S (1970) Trans 01 British Mycological Soc 55 158-166

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Sreenivasa M N And Bagyaraj D J (1989) Plant and Soil 119127-132

Sorensen N eta (2005) PlantandSOI273 101-114

Sylvia D M and Chellemi D O (2001) Adv Agron 73 1-33

Talavera M etal (2001) Appl Entomol 200136 387-392

Torres-Barragan A etal (1996) Mycorrhizae6 253-257

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Walker C and Vestberg M (1994) Agric Sci Anland3 233-240

94 AGRICUTURAL REVEWS

Importance of mycorrhizae 1 Mycorrhizal fungi greatly enhanced the ability

of plants to take up phosphorus and other

nutrients those are relatively immobile and exist

in low concentration in the soil solution

Arbuscular mycorrhizal fungi can playa significant role in P nutrition of crop increasing total uptake and in turn P use efficiency This might be associated with increased growth and

crop yield (Koide et aI 2004) Where

colonization by arbuscular mycorrhizal fungi is

disrupted either by cultural operation or suppressed due to high concentration of soil available P uptake of p growth and in some

cases yield can be significantly reduced even

some times crops fail to response to colonization

by native mycorrhizal fungi (Sorensen et ai 2005) Though P uptake in mainly translocated

by the arbuscular mycorrhizal association it has

become increasingly apparent that arbuscular

mycorrhizal fungi can be important in the uptake

of other nutrients by the host plant Zinc nutrition

is most commonly reported as being influenced

by the association although uptake of copper

(Cu) iron N K Ca and Mg has been reported

to be enhanced (Clark and Zeto 2000)

Arbuscular mycorrhizal fungi may also enhance

plant uptake of N from organic sources (Hodge

et ai 2001)

2 Water uptake may be improved by mycorrhizal

association making more resistant to drought

condition Several mechanisms have been

proposed to explain the effect including

increased root hydraulic conductivity improved

stomatal regulation osmotic regulation osmotic adjustment of the host and improved contact with soil particles through the binding effect of

hyphae enabling water to be extracted from

smaller pores (Auge 2004) Often both water

and nutrient uptake are higher in drought stressed mycorrhizal plants than in nonshymycorrhizal plants (Al-Karaki and Clark 1990) Increased phosphorus levels generally increase

drought resistance calculations made from

arbuscular mycorrhizal fungi indicate that the

amount of water that could travel through the

mycelia to the plant is not large enough to influence plant growth or survival However work with ectomycorhizae showed that the

fungal strands are capable of altering the water

potential of plants Seedlings were maintained

in a healthy state for at least 10 week period

when the only source of water was through

mycelia strands growing in moist peat However

arbusculpr mycorrhizal fungi can only alleviate

moderate drought stress and in more severe

drought conditions they become ineffective (Bryla and Duniway 1997)

3 Though nutrient uptake has been the focus of

much research on the mycorrhizal association

there is evidence that mycorrhizal also play a

role in the suppression of crop pest parasitic

nematodes and diseases particularly soil borne

fungal diseases by producing antibiotics

altering the root epidemics and compacting with

fungal pathogens for infection sites (Borowicz

2001) Some of the soil borne diseases which

have been experimentally suppressed using

Table 1 Selected soil borne fungal diseases controlled by AMF

Pathogen Disease Crop Reference

Aphanomces euteiches Root rot Pea (Pisum sativum) Bodker et al(2002) Fusarium oxsporum Fusarium root rot Asparagus (Asparagus ofjcinalisFrench Matsubara et al (2002)

bean (Phaseolus vulgaris) Helicobasidium mompa Violet root rot Asparagus (Asparagus officinalis Kasiamdari et al (2002) Rhizoctonia solani Root and stem rots Mung bean ( Vigna radiate) Kjoller and Rosendahl (1996) Sclerotium cepivorum White rot Onion (Allium cepa) Torres-Barragan et al (1996) Verticillium dohliae Verticilium wilt Tomato(Lycopersicon esculentum) Karagiannidis et al(2002) Pythium aphanidermatum Damping off Tomato(Lycopersicon esculentum) Jallali and Chand (1987)

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mycorrhizal fungi for sustainable agriculture-a review · 2018. 5. 15. · mycorrhiza are the rule...

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