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ROLE OF VAM FUNGI IN THE MANAGEMENT OF PHYTONEMATODES ON TOMATO
DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
IN
BOTANY
" I • :•• t ' • • ' " ^ '
• • ; - - ^^ " B y • •
ZEHRAKHAN
•r'l
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2007
'\M
A % J^^ 10 ^
DS3808
dedicated To
My Loving (Parents
Irshacf MaRmoocf M.Sc. Phi).
Reader
Department of Botany Aligarh Muslim University Aligarh 202 002 India Phone: 0571-2702016(0) Mob: 09412535631
Date: August 10,2007
Certifitate
This is to certify that the dissertation entitled "Role of VAM fungi in the
management of phytonematodes on tomato" embodies original and bonafide
work carried out under my supervision by MINS Zchra Khan. It may be
submitted to the Aligarh Muslim University, Aligarh towards the fulfillment of
requirements for the Degree of Master of Philosophy in Botany.
(Dr. Irshad Mahmood) Research Supervisor
ACKNOWLEDGEMENT
/ bow to the Almighty for all that He has blessed me with and I have never, ever-felt abondoned by His Grace. I pray to Him to keep it the same way for me in this life and the life thereafter
I acknowledge, with all respect and regard to my supervisor Dr. Irshad Mahmood for the help and guidance. His invaluable help, support and teaching shall remain the guiding force for the rest of my life.
I am also thankful to Professor Ainul Haque Khan, Chairman, Department of Botany, A.M.U. Aligarh for providing necessary laboratory facilities.
I would fail in my duty if I do not acknowledge my heartfelt thanks to Prof. K.G. Mukherji and Prof. Bhatnagar, Department of Botany, Delhi University for the help. My gratitude particularly to Prof Mukherji who taught me the identification technique ofVAMis beyond the reach of my expression.
It is my privilege to mention my deep sense of reverence to Mr. Sonal Bhatnagar and Mr. Sayeed Ahmad, for his advice and valuable suggestion.
I feel pride and privilege for my loving father Mr. Mohd. Wall Khan and beloved mother Mrs. Nasreen Begum who lit the flame of learning in me. Their prayer and sacrifices helped me to gain this success.
A special note of thanks to my seniors and friends, Farha, Bushra, Humera, Sheeba, Zeba, Shweta, Sangeeta, Swam, Qaisar and Uzma for their constant help, encouragement throughout the writing of this dissertation.
I have no words to express my special feelings for my brothers and sisters Hammad, Nadeem, Wajahat, Nasir, Fatima and Shagufta who always provided me warm shoulder with affection and regards. It is their love, care and inspiration that has been instrumental in overcoming the complexities at every step of this work.
I am sincerely behold to Mr. H.K. Sharma who very painstakingly typed the manuscript despite his personal engagements.
Zehra Khan
CONTENTS
Page No.
INTRODUCTION 1
REVIEW OF LITERATURE 6
SECTION I The occurrence of arbuscular mycorrhizal fungi (AMF) Characterization of soil, spore population of AMF in soil And root colonization of some commonly cultivated crops In Pilibhit District - A survey
Introduction 27
Materials and Metliods 27
Results 29
Discussion 39
SECTION II
Comparative efficacy of different Arbuscular-mycorrhizal Fungal spp. (AMF) on tomato (Lycopersicon esculentum Mill.)
Introduction 42
Materials and Methods 43
Results 47
Discussion 55
SECTION III Effect of AM fungus, Glomus fasciculatum and root-knot nematode, Meloidogyne incognita on growth and development of tomato
Introduction 57
Materials and Methods 58
Results 70
Discussion 82
LITERATURE CITED 87
LIST OF TABLES
Table Page No.
number
Section I
1. Soil characteristics of four sites of Pilibhit district 30
2. Total spore counts at different sites in different crops 32
3. Spore counts of G. fasciculatum at different sites 32 under different crops
4. Spore counts of G/oww^ wo^^eae at different sites 32 under different crops
5. Spore counts of Glomus etunicatum at different sites 35 under different crops
6. Spore counts of G/owwi'co«6'?nc/wm at different sites 35 under different crops
7 Spore counts of ^caw/o^pora/aevw at different sites 35 under different crops
8 Spore counts of Gigaspora gigantea at different sites 38 under different crops
9 Mycorrhization at different sites under different crop 38
Section II
10 Effect of different arbuscular mycorrhizal (AM) 48 flingi on shoot, root and total lengths of tomato var. Pusa Ruby at different stages of growth
11 Effect of different arbuscular mycorrhizal (AM) 50 fungi on shoot, root and total fresh weights (g/plant)
of tomato var, Pusa Kuby at different stages of growth
Effect of different arbuscular mycorrhizal (AM) 12
13
fungi on shoot, root and total dry eights (g/plant) of tomato var. Pusa Ruby at different stages of growth
Effect of different arbuscular mycorrhizal (AM) fungi inoculation on mycorrhizal infection of tomato var. Pusa Ruby at different stages of growth
52
54
Section III
14 North Carolina differential host test reaction chart 61
15 Inoculation Schedule (Section III) 63
16 Interaction effect of spore density of mycorrhizal 71 fungus G. fasciculatum and population of nematode Meloidogyne incognita on the length of tomato plants
17 Interaction effect of spore density of mycorrhizal 73 fungus G. fasciculatum and population of nematode Meloidogyne incognita on the fresh weight of tomato plants
18 Interaction effect of spore density of mycorrhizal 74 fungus G fasciculatum and population of nematode Meloidogyne incognita on the dry weight of tomato plants
19 Interaction effect ofspore density of mycorrhizal 75 fungus G. fasciculatum and population of nematode Meloidogyne incognita on the N, P and K content of tomato plants
20 Interactioneffectof spore density of mycorrhizal 77 fungus G. fasciculatum and population of nematode Meloidogyne incognita on mycorrhization G. fasciculatum in tomato plant
21 Interaction effect of spore density of mycorrhizal 80 fungus G. fasciculatum and population of nematode Meloidogyne incogntia on the root-knot disease parameters in tomato plant
LIST OF FIGURES
Figure No.
Section I
a Showing the sHde of Glomus fasciculatum
b. Showing the sUde of Glomus mosseae
c Showing the sHde of Glomus etunicatum
d Showing the shde of Glomus constrictum
e Showing the shde of Acaulospora laevis
f Showing the shde of Gigaspora gigantea
INTRODUCTION
INTRODUCTION
Agriculture is one of the major sector of the Indian economy. 70 per
cent of the population is dependent upon it for their livelihood and contributes
over 40 per cent of the gross national production. Food production has always
remained a matter of great concern for the people all over the world. Any effort
to improve food production necessarily involve step to protect the crop against
pests and diseases. Among the pest, the plant parasitic nematodes are
recognized as potentially serious constraint to agricultural production
throughout the world.
During the last few decades plant parasitic nematodes have definitely
made their presence felt as important pest of economic crops besides insects,
bacteria, fungi, viruses, weeds etc. The prevalent pest complexity has
accentuated due to induction of new production input such as high yielding
varieties, increased irrigation, water fertilizers, pesticides etc.
Use of biocontrol agents for the management of plant parasitic nematode
or to integrate management studies are the current emphasis area. For obvious
reason many natural enemies attract nematodes in soil and reduce their
populations. Exploitation of these enemies for practical use in management of
plant parasitic nematodes at present seems to be practically demanding and the
relevant approach in the greater awareness of pollution free environment (Khan
andHussain, 1990).
In India, the importance of the nematodes as a constraint to successful
crop production was first realized with the discovery of the cyst nematodes on
wheat (Vasudeva, 1958) and potato (Jones, 1961). Since then number of
nematode problems of national importance have emerged.
Plant parasitic nematodes, cause diverse kinds of damages to crop
plants. They are of global importance as parasites of agricultural crops. Among
all the plant parasite nematode, root-knot nematodes Meloidogyne spp. being a
polyphagus in nature is a major threat in the production of various crops, all
over the world and cause enormous crop damage (Sasser, 1990).
Tomato, Lycopersicon esculentum an important vegetable crop and its
cultivation is worldwide. Fresh ripe fruits are refreshing and appetizing tomato
are consumed in the form of juice, puree, paste, ketchup, sauce, soup and
powder. Ripe tomatoes contain glucose and fructose as principal sugars.
Tomato contains a gluco-alkaloid tomatine which is used as a precipitating
agent for cholesterol. There are several constraints for the successful
cultivation of tomato, out of which root-knot nematode alone causes about
20.60% world wide yield loss (Sasser, 1989). In India several nematologists
have assessed the loss in tomato crop caused by root-knot nematode, 26.5-
73.30% by Seshadri, (1970); Bhatti and Jain (1977), observed a loss of 46% ,
whereas Reddy (1985) in his crop loss assessment study reported 39.11% yield
loss of tomato due to Meloidogyne incognita. Jain et al. (1994) reported 47.3-
71 % yield loss due to M javanica and M incognita.
Limited importance has been placed on the management of nematodes
by non chemical approaches to plant disease control. Due to excessive use of
pesticides large number of once effective chemicals have become uneffective
due to development of resistance by pathogen (Delp, 1980). Several
nematicides have been withdrawn from the market because of health and
environmental problems associated with their production and use (Thomson,
1987). As a result of increasing public concern over the use of pesticides on
food production, some pests management researches have focused efforts on
developing an alternative approach to synthetic chemicals by introducing the
use of biological agents. Weller (1988) reported that the Microorganism
present in the rhizosphere provides a front line defense for pathogens attacking
roots. In recent years much attention has been generated in minimizing the
excessive use of chemicals against the pest by using an alternative measures
such as biological methods (Baker and Cook, 1974; Cook and Baker, 1983,
Blakeman and Fokkema, 1982; Vidauer, 1982; Kerry, 1992; Dalpe and
Monreal, 2004; Pal and Gardner, 2006).
Among the natural biological processes that contribute to soil nutrients
management are symbiosis between plant and bacteria (as in nitrogen
fixation)or fungi (as in mycorrhiza). Arbuscular mycorrhiza (AM)increase the
plant ability to absorb phosphorus, minor elements, translocate certain mineral
elements and water (Hayman, 1982; Ratnnayake et al, 1978). They are also
known to reduce damage caused by pathogen (Dehne and Schoenbeck,1975;
Bagyaraj et al, 1979; Hussey and Roncadori, 1982; Sitaramaiah and Sikora,
1982 and Smith 1987). Increased flow of nutrients was observed in mycorrhizal
plants which impart mechanical strength to the host .AM fungi improved plant
growth, control root pathogens, increased nutrient uptake and hormone
production withstand water stress and are potential means of management of
waste lands.
The AMF belongs to the Zygomycetes and have recently regrouped in a
single order, the Glomales (Morton and Benny, 1990), the majority of known
3
species belong to the family Glomaceae (Pirozynski and Dalpe, 1989). VAM
are characterized by the presence of two specialized structures, vesicles and
dichotomously branched haustoria with the host cell called arbuscules.
Arbuscules are formed intracellular^ as internal mycellial structure which
penetrate mechanically and enzymatically into cortical cells in the form of
highly ramified aborescences within a few days of infection. Bifurcated
arbuscules hyphae are enveloped by host derived encasement layer and
continuously invaginate host plasmalemma and form a wide contact between
the two symbionts but are short lived and can be digested with in few days
after formation of host vesicles .The so called vesicles have now been termed
chlamydospores (Mehrotra.,1993; Mehrotra and Baijal,1994; Will et al., 1995).
A second type of structure which have swollen thick walled appearance are
also produce by internal mycelium intercellularly. They can act as infective
propagules and remain viable for long periods (Biermann and Linderman,
1983; Diop et al., 1994) and are also known to absorb and release phosphorus
in the process of transfer between the symbionts. Plants provides the ftingus
upto 80 per cent or more of the net energy fixed as sunlight to below ground
processes. Some of this energy goes into root growth; but a high proportion
may be used to feed mycorrhizal fungi. On the contrary the AM fungi living in
the hot zone greatly influence the ability of plants to establish through effects
on plants in number of ways.
1. increased nutrient uptake
2. increased disease resistance
3. enhanced water relation
4. increased soil aggregation
Use of microbial inoculants seem to be better alternative to achieve the
objective of management of root knot nematode disease and improvement of
plant growth which at present seems to be practically demanding in view of the
greater awareness of health and safe environment. Keeping in view of the
above, the present study was undertaken to explore the possibility of achieving
an effective biocontrol of nematode disease by AM fungal exploitation on
tomato plant. That resulted to decreased input cost, increased yields and
environmental benefits.
The present work consist of following aspects
1. A survey of some cultivated crops of Pilibhit district, to determine the
frequency of occurrence and distribution of AM fungi.
2. Collection of AM fungi from agricultural fields and their multiplication
by single spore inoculation technique and maintenance of pure culture
under pot condition.
3. Screening of comparative efficacy of different species of AM fungi to
determine the efficient AM inoculant in tomato in sterilized soil under
greenhouse condifion.
4. Efficient strain of AM fungus Glomus was tested alone in various
combination with Meloidogyne incognita on tomato (var. Pusa Ruby).
REVIEW OF LITERATURE
REVIEW OF LITERATURE
While various epiphytes and endophytes may contribute to biological
control. The importance of mycorrhizae deserved special consideration.
Mycorrhizae are formed as the result of mutalistic symbiosis between fungi
and plants and occur on most plant species. Because they are formed early in
the development of the plants, they nearly represent ubiquitous to root colonists
that assist plants with the uptake of nutrients and also prevent root infections by
reducing the access sites and stimulating root defense. VAM fungi have been
found to reduce the incidence of root nematode (Linderman, 1990). Numerous
reviews over the past 15 years on the efficacy of AM fungi as biological
control agent have been done (Schenck and Kellam, 1978; Schoenbeck, 1979;
Dehne, 1982; Schenck, 1987; Caron, 1989; Jalali and Jalali, 1991; Siddiqui and
Mahmood, 1995). The main objective of this review is to analyse the role of
AMF on biological control of plant diseases. However the information
regarding the interaction of these organisms with nematodes are meagre and is
completely lacking in Indian context. It is therefore a need to give a short
review on this aspect.
VAM as biofertilizers :
Much interest has been shown in the recent past on the root fungus
association and the possible beneficial role of fungal mycelia which often
mantle plant roots. This was brought about for first time in 1842 and was given
name as Mycorrhiza by Frank (1885). The importance of AMF as a tool, for
improving the growth and productivity in diverse growth of plants was
recognized only after the work of Gerdemann (1968) and Mosse (1972).
However, during last two decades a lot of information has been gathered about
the taxonomy, ecology, physiology and anatomy of AM fungi and their relation
with host especially in relation to uptake of phosphorus and also other mineral
nutrients, hormone production and root diseases (Gianinazzi et al, 1982;
Harley and Smith, 1983; Gianinazzi and Gianinazzi-Pearson, 1986; Grahm,
1988). Attempts have been made to cultivate them in artificial culture media
and use them in different plant production system (Gianinazzi et al., 1984;
Hall, 1988; Gianinazzi etal., 1990).
Wong et al. (2005) studied the effect of biofertilizer containing N, P and
K solubilizers and AM fungi on maize growth. They identified biofertilizer as
an alternative to chemical fertilizers to increase soil fertility and crop
production in sustainable farming. They concluded that the use of biofertilizer
resulted in the highest biomass and seedling height. This study also indicated
that half the amount of biofertilizer application had similar effects when
compared with organic fertilizer or chemical fertilizer treatments.
AM fungi are not host specific but can exhibit certain preferences if
screened against different plants and therefore, have a very wide host range.
They were also occurring over a wide range of agroclimatic condition (Mosse,
1972). AMF produce three types of spores, zygospores, azygospores and
chlamydospores (Trappe and Schenck, 1982; Koske et al., 1983).
The vesicular arbuscular mycorrhizal fungi (VAM) also known as
arbuscular mycorrhizal or endomycorrhizal fungi) are all members of the
Zygomycota. The order glomales of class zygomycetes which encompasses
representing 6, the genera Glomus, Sderocystis, Acaulospora, Entrophosphora,
Gigaspora, Scutellospora with around 170 described species (Verma and
Hock, 1995; Morton and Benny, 1990; Smith et ai, 1997).
AM fungi are ubiquitous and have been recovered from the soils of a
variety of habitats. In India, VAM fungi are very well distributed in cultivated
than in non-cultivated soil. Mosse and Bowen (1968), Sparling and Tinker
(1978), Schenck and Kinloch (1980). Mukerji and Kapoor (1990) reported that
more number of spores in cultivated lands than non-cultivated land.
Janardhanan et al. (1994) reported presence of VAM fungi from alkaline soil.
AM fungi develop association with nearly all cultivated plants whether
they are agricultural, horticultural or fruit crops. There are about 300,000
receptive hosts and 170 AM fungal species reported (Anonymous, 1974;
Mukerji, 1996). Barea et al. (1993) pointed out that indigenous or introduced
VAM fungi are involved in the development of different crop production
system. Important crops associated with VAM include wheat, maize, all
millets, potato, beans, soybean, tomato, oranges, grapes, sugarcane (Bagyaraj,
1991).
Seasonal variation have a remarkable influence on the spore number
(Khan, 1975; G.P. and Tinker, 1978;). The spore populations of VAM fungi
also vary at different sites in the same geographical area. Powell and
Sithamparanathan (1977) noted that spore number and type also influenced by
soil factor. Bayles (1967) observed that spores is also influenced by the soil
pH. Khan (1975, 1978) reported that Glomus mosseae is very common in near
neutral soil and very rare in acidic soils.
Schmidt and Snow (1986) found that AM fungi were present to varying
degree in the roots of at least some members of all plant communities sampled.
Medina et al. (1988) observed that legume species differed in per cent root
colonization and total spore density among locations. Mohankumar et al.
(1988) revealed that most plant harboured AM fungi were influenced by
temperature and moisture status. Kim et al. (1989) noted that out of 103 plant
species (41 families) collected from limestone sites in Korea republic, 98
species were associated with AM fungi. Hussain et al. (1995) have been
observed in the roots of 14 hydrophytes and suggested that high AM.
Colonization in plant root is due to the availability of oxygen. Venkataramanan
et al. (1990) had given qualitative and quantitative distribution of AM spores in
soil samples from Indian habitats viz., Assam, Arunachal Pradesh, Manipur,
Mizorum and Nagaland. The highest counts were made in the planes of Assam.
The most abundant species were found to be Scutellospom nigra, Sclerocystis
rubriformis and Glomus macrocarpum. They suggested that the inoculation
trials in hilly areas with AM fungi proved beneficial to crops such as chilli,
soybean and maize.
Peng and Shen (1990) observed that spore density and frequency of
occurrence of AM fungi are greater in autumn than on the spring. Payal et al.
(1994) reported that AM colonization happens to be the lowest during winter
and highest during late summer and autumn. Kuhn et al. (1991) reported that
out of 43 species of flowering plants examined, 40 are nearly associated with
VAM fungi. Root colonization by vesicular arbuscular mycorrhizae ranged
from 6 to 28 per cent and total spore density ranged from 23-256 per 100 g soil.
Level of AM colonization varies between the two crops from site to site and
not related to soil properties (Talui<;clar and Germida, 1993) and species to
species (Muthukumar et al. 1996).
VAM as growth promoter :
Interest in arbuscular mycorrhizal fungi propagation for agriculture is
increasing due to their role in the promotion of plant growth and health
(Gerdemann, 1968; Harley, 1969; Tinker, 1975; Howeler et al, 1987; Linxian
and Hao, 1988; Raju et al, 1989). The use of AMF for crop productivity
requires a selection of an efficient and appropriate fungus and it has been
assessed by many workers (Jensen, 1982; Krishna et al, 1984). There are
several reports of some species being more efficient than others (Kuo and
Huang, 1982; Mosse, 1977). Glomus tenius was more efficient than the
indigenous fungi at a particular location. The agricultural importance of AMF
is mainly due to their ability to increase phosphate uptake and other major and
minor nutrients of crop plants (Mosse et al, 1973). The main constraints in
exploiting beneficial effects of AM fungus in improving agricultural
productivity is the obligate nature of symbiont large scale production of
commercial inoculum that provide users with product of quality and efficiency
is not possible. Presently, use of AM fungi is confined to green house and pot
culture studies and to a limited extent in plant propagation nurseries
(Rangaswami, 1990). Effect of VAM fungi on various kinds of plants has been
studied by various workers (Asif er al, 1995; Muhammad and Hussain, 1995;
Kehri and Chandra, 1990; Menge et al, 1978; Plenchette et al, 1981;
Ragupathy and Mahadevan, 1995; Adjoud etal, 1996).
10
Growth yield and dry matter increase by AM inoculation had been
reported in many crops such as barley, onion, soybean and rice (Owersu and
Mosse, 1979; Bagyaraj et ai, 1979; Sanni, 1976). Koch et al. (1997) observed
that VAM inoculated garlic plants had larger and more green leaves and
increased photosynthetic rate, especially at low light intensities, and higher
fresh and dry weights than plants in uninoculated plots. Root colonization of
AM fungus Glomus species is known to increase phosphorus content in the
mycorrizal root tissue of maize plants to 35 percent by G. mosseae and 98% by
G. fasciculatum.
Almomany (1991) studied the response of beans, broadbean and
chickpea plant to inoculation with Glomus species. Eight different locally
obtained Glomus species including G. pallidum, G. mosseae, G. fasciculatum,
G. etunicatum and G. monosporum were inoculated into bean, broad bean and
chickpea plant to examine their effect on plant growth. He concluded that G.
fasciculatum and G. mosseae were the most effective in colonizing the roots of
beans, broad bean and chickpea plants, G. mosseae increased the shoot growth
of beans, broad beans and chickpea by 52, 117 and 190% respectively. Effect
of G. fasciculatum, G. mosseae, Gigaspora calospora and Acaulospora laevis
on sorghum variety C026 was seen by Prabhakaran et al. (1995) who found
increased biogrowth of both the plants by G. fasciculatum.
Edathil et al. (1996), studied the interaction of multiple VAM fungal
species on root colonization plant growth and nutrient status of tomato
seedlings. Tomato seedlings were grown in sterile, phosphorus deficient soil
and inoculated with 4 species of Glomus i.e. Glomus aggregatum, G.
11
fasciculatum, G. geosporum and G. sinuosum that resulted in a significantly
higher shoot length and biomass, than non-mycorrhizal plants. The VAM
combination, containing all endophytes promoted markedly better shoot length
and biomass than other combination.
Iqbal and Mahmood (1998) studied the effect of single and multiple
VAM inoculants on the growth parameters of tomato. G. mosseae resulted
maximum growth of plants followed by G. constrictum.
Utility of mycorrhizal application in horticultural crop production were
under evaluation in many fruit crops like citrus (Menge et ai, 1978; Onkarayya
and Sukhada, 1993), apple (Plenchette et ai, 1981; Sharma and Bhutani, 1995)
and strawberry (Hughes et ai, 1978). In all cases, beneficial effect of
inoculation has been recorded. Sivaprasad et al. (1995) reported a maximum
plant heights (12 cm) and fresh weight (4.38 kg) in jack fruit (Artocarpus
heterophylla) inoculated with G. mosseae. Different VAM fungi have been
found to have different efficiencies in increasing fruit yield and nutrient uptake.
In tomato plant (Raverkar et al, 1994; Raverkar and Bhandari, 1995).
Mamatha and Bagyaraj (2002) studied the effect of different levels of
VAM inoculum application on growth and nutrition of tomato under
greenhouse conditions. Different levels of G. fasciculatum inoculum tried (0,
0.5, 1.0, 1.5, 2.0 and 2.5 kg/m" of nursery bed). 2.0 kg inoculum per m of the
nursery bed improved the plant growth, biomass, P content, mycorrhizal root
colonization. Future increase in inoculum level did not significantly improved
seedling growth and nutrition. Again Mamatha and Bagyaraj (2003) conducted
a green house study to determine the effect of different method of VAM
12
inoculum application on growth and nutrient uptake of tomato seedlings grown
in raised nursery beds. G. mosseae application resulted an increase of plant
height, number of leaves, leaf areas, plant biomass, phosphorus content and per
cent colonization.
Increased growth responses and nutrient uptake has been reported
because of AM symbiosis in several field grown forage and seed legumes. In
case of pigeon pea, the inoculated plants had higher mycorrhizal root
colonization, shoot and root dry weight and phosphate content (Manjunath and
Bagyaraj, 1984; Ramraj and Shanmugam, 1990). Glomus leptotrichum, G.
macrocarpum, Acaulospora laevis, Gigaspora margarita and G. fasciculatum
were equally good in promoting shoot and root dry weight (Reddy and
Bagyaraj, 1990). In black gram dry weight were found to be significantly
higher on inoculation with G. fasciculatum, G. constrictum, G. versiforms and
Acaulospora species (Umadevi and Sitaramaiah, 1990). Sylvia et al. (2001)
evaluated the influence of arbuscular mycorrhzial (AM) fiangi on the
competitive pressure of bahiagrass on the growth of tomato. Tomato grown
alone was very responsive to mycorrhizal colonization, shoot dry weight of
inoculated plants was upto 243 per cent greater than that of non-inoculated
plants. Tomato grown with bahiagrass had reduced root and shoot growth
across all treatments compared with tomato grown alone but there was an
increase in shoot weight following AM fungal inoculation. They concluded that
the role of mycorrhizae in plant competition for nutrients is markedly impacted
by soil nutrient status and reduced P application.
13
Mycorrhizal fungi enhance the absorption of nutrients by increasing the
total surface area of the roots. Absorbed phosphorus is probably converted into
polyphosphate granules in the external hyphae (Callow et al, 1978) and passed
to the arbuscules for transfer to the host (White and Brown, 1979). This flow of
phosphorus occurs in the presence of acid phosphatase (Gianinazzi et al,
1979). The mycelial network in mycorrhizal plants enables them to extract
phosphorus from places beyond the zone of low concentration around the roots
and depend on the distribution and phosphate uptake of external hyphae
(Jakobson et al, 1992). AM fungi also stimulates the plant uptake of zinc,
copper, sulphur, potassium and calcium although not as markedly as
phosphorus (Copper and Tinker, 1978).
Anion (1982) reported the production of phytohormones by G. mosseae
and considered them as important for evaluation of isolates for inoculum
production.
Effect of fertilizers on host benefit by mycorrhizal fungi
Phosphorus deficiency is one of the major limiting factor for crop
growth and yield in the tropics, mycorrhiza help to conserve and use
phosphorus efficiently. Improved phosphorus nutrition when colonized with
AMF has been demonstrated for hundred of cultivated plants. By extending
past the P-depletion zone formed around the root system, the flingal soil
network is able to maintain P transports to plant for longer periods (Hodge,
2000; Lange et al, 2000; Miller et al, 2001).
Many researches have shown that under high phosphorus soil
conditions, AMF are almost of no use to the plant and symbiosis is temporarily
14
inhibited. However, very low phospiiate fertilizer addition to soil, can increase
per cent colonization of root system, possibly through direct effect on AM
fungus symbiosis efficiency (Bolan et al., 1984).
Hao et al. (1991), estimated the development of mycorrhizal infections
by assessing the percentage of VAM infection of mungbean in pot culture with
greyed alluvial soil and red soil. Most of which were deficient in available
phosphorus. Results showed that infections of indigenous VAM fungi in most
soil were low and there was usually a lag phase. But after introducing
mycorrhizal fungi into the soil lag phases of infections were decreased or
disappeared and plant growth was greatly stimulated. Phosphorus encouraged
mycorrhizal infection in most soils deficient in available phosphorus, but the
optimum amount of P fertilization was different between alluvial soil and red
soil.
The P status of the plant strongly affects AM colonization and spore
production and sporulation. This intum determines cell membrane permeability
and root exudation of carbohydrates and amino acids available metabolites to
the fungus (Grahm et al, 1981). In field addifion of P fertilizer to soil reduces
either AM fungal colonization or sporulation in a variety of crops, including
maize, corn, soybean, clover, small grains and teak stumps (Lu and Miller,
1989; Durga et al, 1995). Tawarya and Saito (1996) also found that addition of
P to host plant influenced the composition of root exudates and thereby
increase hyphal growth of AM fungi.
Susai (1991) studied the effect of phosphate applicafion on infection of
vesicular arbuscular mycorrhizal fungi in some horticultural crops. In yield
15
• tests on maize, soybean, tomato, carrot and Artium lappa, the application of
phosphorus fertihzers increased shoot dry weight, increased shoot phosphorus
content after second cropping (86 day after sowing) and decreased mycorrhizal
infection rate to varying degree. Mycorrhizal spore number on rhizosphere soil
(soybean, tomato and maize) was much higher in soil without added
phosphorus. It is concluded that VAM fungi promote phosphate uptake in low
phosphate soils during the early stages of plant growth.
Elwan (1993) observed that maize shoot and root dry weight, root and
shoot length, root surface and transpiration rate were increased with P fertilizer
inoculated with AM fungi. In another experiment Elwan (1993) determined
that the uptake of P, K, Ca, Mg, Fe, Mn, Cu and Zn were highest when plant
received P + AM in combination followed by P fertilizer alone.
Araujo et al. (1996) studied the effect of phosphate fertilizer on tomato
inoculated with Glomus etunicatum. They observed that plants received 60 mg
P/kg soil resulted in enhancing the growth and P accumulation rate while at
120 mg P/ka soil resulted a sharp decline in growth, P accumulation and P
utilization rate at the early growth stages and increased at the later stage.
Stimulatory effect on plant growth by fertilizer application were evident
from several studies (Fabig et al., 1989; Zaghloul et al., 1996; Singh, 1996).
Dhinakaran and Savithri (1997) studied the effect of different P treatments and
VAM fungal inoculation in tomato cv C03 grown in pots filled with calcareous
soil. Phosphorus was applied as single phosphate at 75 or 100 kg ^lOslha.
Phosphorus was applied as single superphosphate at 100 kg PjOs/ha that
resulted a significant increase on the yield of tomato, phosphorus contents and
16
P uptake of tomato fruits. These pattern increased more with the increase in
applied P and VAM fungal inoculation increase fertilizers.
Khaliq et al. (1997) found that VAM inoculation at three phosphorus
application levels (3, 10 and 30 mg/kg) significantly suppressed maize seedling
growth and phosphorus inhibition of mycorrhizal infection increased with
increasing rates of application.
Posta et al. (1997) reported that the positive effect of mycorrhizal
inoculation on shoot growth decreased as P rate increased. For the various soil
volumes, the effect of mycorrhizal inoculation on the shoot mass increased as
soil volume increased. In all cases mycorrhizal plants had higher P contents. P
application reduce the degree of root colonization and also the quantity of
external hyphae.
Olsen et al. (1996) studied the effect of VAM fungal inoculum, Glomus
mosseae on capsicum {Capsicum annum L.), sweet com {Zea mays L.) and
tomato {Lycopersicon esculentum) grown in a low P sandy loam and along with
different levels P (0, 10.3, 30.9, 9 to 278 mg P/kg oven dry soil; P,, P2, P3,, P4 or
P5 respectively) and 2 levels of N (50 or 200 mg N/L). At low P rates, dry
weight of sweet corn and tomato, plants colonized with VAM did not differ
from uninoculated plants, despite inoculated plants having higher P
concentrations in index tissues. For all three crops, lack of VAM response at
high P was related to a lower VAM colonization.
Asmah (2004) studied the effect of two-phosphorus sources (triple
superphosphate and Ghafsa phosphate rock), applied at rates equivalents to 44
kg ha' and 22 kg ha'', on VAM fungal infection in roots, dry matter yield and
17
nutrient content of maize grown in an oxisol and an alfisol. The application of
44 kg P ha"' resulted in root infection by VAM fungi was not significantly
different (p < 0.01) from when no P was applied. Root infection was
significantly greater when P was applied as triple, superphosphate at the rate of
22 kg ha"' phosphate rock treatment at both rates of application that resulted in
significantly greater root infection than in control or with triple superphosphate
was applied at 44 kg ha'V Plant P uptake increased in all soils with different P
treatments compared with the control.
VAM fungal in nematode control:
Plant parasitic nematodes and mycorrhizal fungi are commonly found
inhabiting the same soil and colonizing roots of their host plants. These two
groups of micro-organism exert a characteristic, but opposite effect on plant
health. The potential role of mycorrhizal fungi for the control of nematode
diseases has recently received considerable attention (Osman et al., 1990;
Ahmad and Aslayed, 1991; Sankaranaryanan and Sundarbabu, 1994; Santhi
and Sundarbabu, 1995; Price et ai, 1995; Kerry, 1996).
Arbuscular mycorrhizal fungi (AMF) variously influence a number of
plant nematode interactions (Atilano et ai, 1981; Grandison and Cooper, 1980;
Hussey and Roncadori, 1982). High populations of endoparasitic nematodes
and spores of endomycorrhizal fungi were found in a survey made by Hasan
and Jain (1987) indicating that these nematodes do not affect the VAM flingi
and vice versa. However, in some crops like gram, cowpea and pigeonpea, low
incidence of root-knot nematode in the roots having high level of VAM fungi
was observed (Hasan and Jain, 1987). Jain and Hasan (1986) in a different
18
investigation indicated that the presence of nematodes did not adversely affect
VAM sporulation and reported that, where there was only 50 per cent root
colonization, nematode number was lower and they rarely infect VAM
colonized region of roots, but on the other side, the roots infected by
endoparasitic nematode were not colonized by VAM fiingi while the damage
done by plant parasitic nematodes is compensated by mycorrhizae unless close
to the nematode feeding site. Stimulation of root growth by VAM provides
greater habitat and sometimes resulted in increase of nematode population.
Increased spore production and higher root colonization by VAM fungi in the
presence of nematodes have also been observed (Ingham, 1988). Jain and Sethi
(1989) showed that the occurrence of Heterodera cajani and VAM fungi.
Glomus fasciculatum and G. epigaeus in Vigna unguiculata were largely
independent of each other and the organism modify the effect of each other to
some extent. However, an increase in nematode inoculum invariably resulted in
reduced root infection and spore production by mycorrhizal fungi. The
presence of G. fasciculatum showed a profound adverse effect on cyst
production and multiplication of nematode, while G. epigaeus exhibited a
reverse trend.
The antagonistic effects of AM fungi on nematodes may be either
physical or physiological in nature. The nematode control may be through
improved plant vigour, physiological alteration of root exudates or through
direct role of mycorrhizae in retarding the development and reproduction of
nematodes within root tissues. Several studies report an antagonistic effect of
mycorrhizal fungi on plant parasitic nematodes (Fox and Spasoff, 1972;
Grandison and Copper, 1986; Macguidwin et al, 1985; Osman et ai, 2005).
19
Sitaramaiah and Sikora (1980) observed that Glomus mosseae increased the
resistance of tomato plants to Rotylenchulus reniformis infection. AM fungi
can alter the physiology of the root, including root exudates which were
responsible for chemotactic attraction of nematode. Sikora (1978) suggested
that attractiveness of the root system to M. incognita larvae was altered by the
presence of G. mosseae. Sitaramaiah and Sikora (1982) expressed the other
version of increasing resistance in tomato plants colonized by Glomus
fasciculatum against Rotylenchulus reniformis by delaying the nematode attack
in roots and less formation of egg/eggsac. Glomus fasciculatum adversely
affected R. reniformis during several phases of its life cycle.
Several investigations Atilano et al. (1981), Cason et al. (1983),
Roncadori and Hussey (1977), Healed et al. (1989) have suggested that
increased nutrient uptake of mycorrhizal fungi enhances plant tolerance relative
to detrimental effects on nematode. Similarly it had been found that the
presence of mycorrhizae increase the tolerance of plants to diseases (Chandra
and Kehri, 1996). The increase of giant cells formed by mycorrhizal plants
was significantly low in tomato plants, infected with root-knot nematode M.
incognita and mycorrhizal root did not prevent penetration by the nematode
larvae (Suresh et al, 1985).
Baghel et al (1990) observed that Glomus mosseae stimulated the
growth of Citrus jambhiri seedlings. The nematode Tylenchulus semipenetrans
decreased plant growth and simultaneous inoculation with AMF and nematode
partly neutralized the adverse effect of the nematode, as the fungus was
limiting the development of the nematode. Sivaprasad et al. (1990) showed that
20
the pre-inoculation of Piper nigrum cv. Panniyur cuttings with Glomus
fasciculatum or G. etunicatum reduced the root knot (Meloidogyne incognita)
index by 32.4 and 36.0 per cent respectively, reduced nematode population in
roots and surrounding soil, and significantly increased growth even in the
presence of nematode. The tolerance of kiwi plants to M javanica was
increased in presence of G. etunicatum (Verdejo et al., 1990).
Srivastava et al. (1990) found that acid phosphatase activity in leaf was
greater in Meloidogyne incognita inoculated tomato plants than Glomus
fasciculatum inoculated plants while phosphorus concentration was generally
higher in Glomus mosseae inoculated plants. Singh et al. (1990) noted that pre
occupation of tomato (Pusa Ruby) roots with Glomus fasciculatum resulted in
an increase in lignin and phenols and this might increase the resistance in
tomato plants against root knot nematode, Meloidogyne incognita. Mittal et al.
(1991) showed that tomato tissues forming galls following inoculation with
Meloidogyne incognita had no AM fungi while roots lacking nematode galls
had vesicles and arbuscules of Glomus fasciculatum, which inhibits the
formation of nematode galls. Glomus fasciculatum was found to be the better
and most effective mycorrhizal fungus when compared to G. mosseae in its
overall performance (Sharma and Trivedi, 1994).
Glomus fasciculatum application in nursery beds helped the mycorrhiza
to colour the tomato roots before transplantation to the main field, thereby
preventing the penetration and development of nematode in the infected plants
(Sundarbabu and Sankaranarayanan, 1995).
21
Mishra (1996) studied the inter-relationship oi Meloidogyne incognita,
G. fasciculatum and the three commonly used herbicides. Higher level of VAM
after 60 days of inoculation improved growth of tomato plant, whereas higher
levels of M incognita caused progressive reduction in plant growth.
Simultaneous incorporation of VAM and nematode resulted in maximum
multiplication of VAM. Pre-establishment of G. fasciculatum increased plant
growth, decreased the size and number of galls and improved NPK uptake over
plants inoculated with the nematode alone or pre-inoculated with the nematode
to VAM. Herbicidal combination with VAM and nematode showed overall
decrease in growth of tomato plants, multiplication of Glomus and uptake of
NPK.
Sundarbabu et al. (1996) observed that when Glomus fasciculatum was
inoculated 15 days prior to nematode inoculation, that resulted an enhancing
the growth of tomato cv. COS and suppress M incognita multiplication.
Simultaneous inoculation followed the similar pattern. G. fasciculatum was
unable to suppress nematode growth when the nematode was inoculated 15
days prior to fungus.
Mishra and Shukla (1997) reported that simultaneous inoculation of G.
.fasciculatum with M incognita caused greater reduction in the number and size
of the nematode induced root galls. Application of G. fasciculatum 15 days
prior to the nematode significantly decreased the number of size of the galls
and increased growth of tomato var. Pusa Ruby as compared to plant
inoculated with nematode alone or inoculation with nematode 15 days prior to
22
VAM. NPK contents of tomato plants were significantly higher with prior an
simultaneous application of the VAM and nematode.
Cofcewicz et al. (2001) under greenhouse conditions studied the
interaction of arbuscular mycorrhizal fungi Glomus etunicatum and Gigaspora
margarita and root knot nematode, Meloidogyne javanica and their effects on
the growth and mineral nutrition of tomato. The result pointed out that the
shoot dry matter yield was reduced by nematode infection and this reduction
was less pronounced in plants colonized with G. etunicatum than plant
colonized with G. margarita and non-mycorrhizal plants. The higher tolerance
of plants colonized with G. etunicatum to M. javanica appeared to be
associated with P nutrition.
Labeena et al. (2002) evaluated the ability of five arbuscular mycorrhiza
viz., Glomus fasciculatum, Glomus macrocarpum, Gigaspora margarita,
Acaulospora laevis and Sclerocystic dussi to mitigate damage caused by M.
incognita on tomato cv. Pusa Ruby. They found that Glomus fasciculatum was
the most efficient in promoting plant growth in the presence of nematode. The
developmental stages of nematodes in the roots and nematode density in soil
were suppressed by the AM fungi and the most pronounced effect was
exhibited by Glomus fasciculatum.
Kantharajan et al. (2003) carried out a survey to determine the
biodiversity of vasicular arbuscular mycorrhiza (VAM) Glomus fasciculatum in
the rhizosphere of different crops in seven district of southern Kamataka, India.
Among 108 soil collected, only 8 isolates of G. fasciculatum were selected on
the basis of spore morphology and spore load and were evaluated against root
23
knot nematode. Meloidogyne incognita race-1, on tomato. Amongst the isolates
tested, the Shimoga, areca isolate was the most effective in increasing the plant
growth parameters, the most number of chlamydospores and highest per cent
colonization. Nematode population in soil, number of galls and egg masses per
root system were lowest in Shimoga banana isolate followed by Shimoga areca
isolate.
Carling et al. (1995) studied the individual and combined effects of two
arbuscular mycorrhizal fungi (AMF). Meloidogyne arenaria and phosphorus
(P) fertilization (0, 25, 75 and 125.7 g/g soil) on peanut plant growth and yields
in green house. Best growth and yields usually occurred at 75 and 125 g P
regardless of inoculation treatment. In challenge inoculations, VAM increased
peanut plant tolerance to the nematode and offset the growth reduction caused
by M. arenaria at the two lower of P levels.
Osman et al. (2005) studied the interaction of root-knot nematode and
VAM fungi on common bean plants {Phaseolus vulgaris L.) in the greenhouse.
They concluded that the inoculation with VAM fungus caused a significant
increase in plant height and fresh weight compared with non-treated plants.
Meloidogyne infection significantly decreased plant height and dry weight.
When fungus was inoculated at 15 and 30 days prior to M incognita infection,
a significant increased in fresh weight was observed. The inoculation with
VAM fungus caused a significant increase phosphorus content. However, it
was significantly decreased in plants inoculated with nematode alone and in
plants inoculated with VAM fungus and M incognita at the same time. Gall
index and final nematode population were significantly increased when
nematodes were inoculated at the same time with fungus, although there were
24
significant decrease in nematode final population and gall index when plants
were inoculated with nematodes at 15 and 30 day after mycorrhzial infection.
A decreased in percentage of fungal colonization was observed when
nematodes were inoculated with fungus simultaneously.
Castillo et al. (2006) studied the effect of single and combined
inoculation of olive planting stocks cvs. Arbequina and Picual with the
arbuscular mycorrhizal ftmgi (AMF) Glomus intraradices, Glomus mosseae or
Glomus viscosum and the root knot nematodes M. incognita and M. javanica on
plant performance and nematode infection. They concluded that prior
inoculation of olive plants with AMF may contribute to improving the health
status and vigour of cvs Arbequina and Picual planting stocks during nursery
propagation.
In several studies AM fungi had shown an antagonistic influence on the
population of plant parasitic nematode (Bagyaraj et al, 1979; Saleh and Sikora,
1984; Cooper and Grandison, 1986; Sitaramaiah and Sikora, 1982; Carling et
al, 1989; Sivprasad et al, 1990; Rao et al, 1992; Sankaranarayanan and
Sundarbabu, 1994; Kerry, 1996). Nematode susceptible plant colonized by AM
fungi were better able to tolerate plant pathogenic nematode (Kellam and
Schenck, 1980; Sitaramaiah and Sikora, 1982; Grandison and Cooper, 1986;
Diederichs, 1987; Jain and Sethi, 1989; Osman et al, 1990; Sankaranarayanan
and Sundarbabu, 1994). As a result of interaction in general, the severity of
nematode diseases was reduced in mycorrhizal plants. Biological control is
more inconsistent although improvements in performance might be expected
from more research on individual agents and their successful application.
25
SECTION I
SECTION -1
THE OCCURRENCE OF ARBUSCULAR MYCORRHIZAL FUNGI (AMF), CHARACTERIATION OF SOIL, SPORE
POPULATION OF AMF IN SOIL AND ROOT COLONIZATION OF SOME COMMONLY CROPS IN PILIBHIT DISTRICT -
A SURVEY
INTRODUCTION
An extensive survey of agricultural fields of Pilibhit district of some
important cultivated crops was conducted for quantitative and qualitative
assessment of AM fungal spores population and per cent root colonization by
AMF at four sites under field conditions. The four sites selected were Puranpur
(Ai), Bisalpur (A2), Nyorea (A3), and Mainakote (A4), having some commonly
cultivated crops like tomato {Lycopersicon esculentum Mill.), chickpea {Cicer
arietinum Linn), turmeric {Curcuma longa Linn.), chilli {Capsicum annum
Linn.), pigeonpea {Cajanus cajan (Linn.) Millsp.), sugarcane {Saccharum
officinarum Linn.), wheat {Triticum aestivum Linn.).
The soils of the experimental sites were sandy loam and the
characteristics of the soils are given in (table 1). The temperature of the sites
ranges 21.3 to 33.4°C while relative humidity from 65.5 to 78.20% in a
calendar year. All the sites are moderately rainfed and have satisfactory
irrigational facilities with a soil moisture 5 to 9.80%.
MATERIALS AND METHODS
Soil sampling and root sample collection :
Sampling was done for each crop separately from the fields of cultivated
crops at site Ai, A2, A3 and A4 in different months i.e. tomato December, 2006;
chickpea - January, 2006; turmeric - June, 2006; chilli - May, 2006;
pigeonpea - September, 2006; sugarcane - April 2006, wheat - March, 2006.
Soil samples (soil cones of 5 cm diam.) were collected at random from
each soil with the help of soil auger upto a depth of 15 cm near the plant base.
Forty such samples were collected from each plant species and were
thoroughly mixed to make a composite sample, seven samples of lOOg soils
were used for recovery of spores.
Seven root samples for each plant species were collected at random in
order to assess root colonization of AM fungi. A representative sample of the
entire root system was obtained from five different portions of the root system
after washing by tap water.
Soil characteristics :
The soil samples were brought to laboratory, marked and packed in
polythene bags and their electric conductivity (EC) and pH were measured with
EC meter and pH meter respectively in the extract collected from 1:1 soil/water
suspension. The texture of soil in relation to particle size was determined by
hydrometer method (Allen et al, 1974); total organic carbon by the method
given by Walkley and Black (1934); total nitrogen by microkjeldahl method
(Nelson and Sommers, 1972) and total phosphorus by molybdenum blue
method (Allen et al, 1974).
Quantitative estimation of spores from the soil:
Spores of AM fungi were isolated by wet sieving and decanting method
(Gerdeman and Nicolson, 1963). For this a sample of 100 g dry soil was mixed
27
in water (1000 ml) and the heavier particles were allowed to settle for few
seconds. The liquid was poured through a coarse soil sieve to remove large
piece of organic matter the liquid passed through this sieve was collected and
again passed through a sieves of 80, 100, 150, 250 and 400 mesh. Spores
obtained on sieves were collected with water in separate beakers. The spores
were counted in 1 ml of the suspension in nematode counting dish under the
stereoscopic microscope. The final number of spores/100 g of soil was
calculated accordingly for each crop.
Assessment of colonization by AM Fungi:
Clearing and staining (Phillips and Hayman, 1970)
Roots were washed with tap water and cut it into 1 cm long segments
and then boiled in 10% KOH solution at 90°C for 45 minutes. KOH solution
was then poured off and roots were rinsed well in a beaker until no brown
colour appeared in the rinsed water. Alkaline H2O2 which was used to bleach
the roots was made by adding 3m of NH4OH to 30 ml of 10% H2O2 and 567 ml
of tap water. The roots were rinsed thoroughly at least three times using tap
water to remove the H2O2. Roots were then treated with 0.05% trypan blue (in
lactophenol) and kept for overnight for destaining in a solution, prepared with
acetic acid (laboratory grade) 875 ml, glycerine 63 ml and distilled water
63 ml.
The cellular contents were removed by this method and the AM fungal
structures get stained dark blue. This stained root segments were used for
determining the root colonization by AM flingi.
28
Percentage of root colonization and per cent arbuscules were determined
by slide method (Giovanretti and Mosse, 1980). The root segments were
selected at random from the stained sample and mounted on microscopic slides
in groups of ten. One hundred to one hundred and fifty root segments from
each samples were used for the assessment. The presence or absence of
colonization in each root segment was recorded and result was expressed as
percentage of root colonized. The root colonization (mycorrhizal infection in
the roots) was calculated as follows :
Number of mycorrhizal segments % AM association =
Total number of segments served
Isolated spores were identified with the help of keys provided by
different workers (Trappe, 1982; Hall and Fish, 1979; Bakshi, 1974; Rani and
Mukerji, 1988; Srinivas et al., 1988). The data collected during this study were
statistically analyzed in simple randomized design by the method of Panse and
Sukhatmi (1985).
RESULTS
Soil collected from the four sites were alkaline with pH ranging from 7.1
at site Ai to 8.4 at site A4 and sandy loam in texture. The electric conductivity
was low at site A3 and high at site A4 in comparison to other two sites. The
organic carbon content of the soil at site A4, is highest, followed by A2, Aj and
A3. As far as the soil nutrient is concerned, nitrogen content was highest at site
A4 while P ranged between 7.0 to 8.1 kg/ha, highest being at site Ai. The
percentage of soil samples having a particular species of AM fungi idenfified
are given in table 1.
29
i^ 00 0^ o
o
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o ^^ ©•r s a s "
_u 'a « bxi u 0
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o
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iri
in o i n
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00
00
rsi
00
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^ t ^
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30
The data provided in table 2 indicate that the total spore number per
lOOg of soil varied with the crop as well as with the site. The sugarcane crop
showed the highest number of spores, whereas chickpea show the minimum at
almost all the four sites investigated.
At site I, wheat showed the second highest number of spore followed by
pigeon pea, tomato and turmeric. The statistical analysis of the data clearly
shows that the spore number of chickpea, chilli and pigeon pea were varied
significantly from one another.
At site II, the highest and lowest spore numbers were recorded in wheat
and chickpea, respectively. The number of spore in turmeric was close to
pigeon pea. Similar trend were shown by chickpea, turmeric and chilli. The
second highest number of spores were recorded in sugarcane followed by
tomato and pigeon pea. The spore number in chickpea, chilli and sugarcane
were statistically significant than other crops while pigeon pea was at par with
sugarcane.
At site III, although the general trend was the same as in others the
highest number of spores were recorded in sugarcane and lowest in chickpea.
The spore numbers obtained from turmeric, chilli and pigeonpea were
significantly higher than other crops.
At site IV, the spore number in general was found to be poor as
compared to other sites. The highest and lowest spore number were recorded in
wheat and chilli. Turmeric show the second highest followed by crops like
tomato, chickpea, sugarcane and pigeon pea.
31
Table - 2 : Total spore counts at different sites in different crops
Crop
Tomato
Chickpea
Turmeric
Chilli
Pigeon pea
Sugarcane
Wheat
CD.
Site I
346
320
341
326
361
440
380
12
Site II
360
310
322
332
356
370
400
14
Site III
360
280
281
336
354
420
367
9
Site IV
220
270
280
187
216
260
314
7
Table -3 : Spore counts of Glomus fasciculatum at different sites under different crops.
Crop Site I Site II Site III Site IV Tomato Chickpea Turmeric Chilli Pigeon pea Sugarcane Wheat CD.
109
125
132
80
164
186
162
8
64
72
177
180
108
126
128
10
121
88
158
64
76
168
177
7
120
56
62
105
74
96
77
6
Table -4 : Spore counts of Glomus mosseae at different sites under different crops.
Crop Tomato Chickpea Turmeric Chilli Pigeon pea Sugarcane Wheat CD.
Site I Site II Site III Site IV 160
^9 95
75
55
141
136
4
107
112
36
74
80
148
194
5
167
100
64
104
130
148
130
7
48
59
73
47
36
85
130
6
32
Fig. a. Showing the slide of Glomus Fasciculatum
\ \ 4.^' * •>
fi
\
n
Fig.b. Showing the Slide of Glomus Mosseae
1:
Fig. e. Showing the Slide of Acaulospora laevis
Fig. f. Showing the Slide of Gigaspora Gigantea
Table -5 : Spore counts of Glomus etunicatum different sites under different crops.
Crop Site I Site II Site III Site IV
Tomato Chickpea
, Turmeric Chilli Pigeon pea Sugarcane Wheat CD.
31
39
50
74
40
14
37
5
0
66
35
31
104
13
%9 6
34 41
29
55
25
68
28
5
43
38
40
13
25
35
37
4
Table -6 ; Spore counts of Glomus constrictum at different sites under different crops.
Crop Site I Site II Site III Site IV
Tomato • Chickpea Turmeric Chilli Pigeon pea Sugarcane Wheat CD.
37
67
50
9
6
50
20
4
80
15
32
7
16
14
57
6
20
48
18
46
38
8
16
5
6
25
35
12
56
26
28
5
Table -7 : Spore counts of Aculospora laevis at different sites under different crops.
Crop Site I Site II Site III Site IV
Tomato
Chickpea
Turmeric
Chilli
Pigeon pea
Sugarcane
Wheat
CD.
9
0
8
44
50
37
19
3
41
0
24
24
48
66
26
5
12
0
12
65
47
28
12
3
3
48
40
10
25
0
6
2
35
Fig. c. Showing the Sh'de of Glomus etunicatum
• ^ • - ,
a>.
Jt -a* •
Fig. d. Showing the Slide of Glomus constrictum
At site IV, the highest number of spores was recorded under Tomato
cultivation which was closely followed by Turmeric, Chickpea, wheat and
sugarcane with minor variations. The lowest number of spores were recorded
from chilli field.
It was observed that the number of spores of a G. etunicatum of all the
four sites were very low as compared to G. fasciculatum and G. mosseae.
Table 6 gives the data of spore population of Glomus constrictum (Fig.
• d) in different sites and different crops.
At site I, the highest number of spores were obtained under chickpea
cultivation while turmeric and sugarcane resulted the same number of spores.
The lowest number of spores were recovered from pigeonpea followed by chilli
and are insignificant to each other.
At site II, highest number of spores were recorded under Tomato
cultivation followed by wheat and lowest under chilli cultivation. Chickpea,
chilli and sugarcane were more significant than others.
At site III, the spore number happened to be the highest under chickpea
cultivafion. The least number of spores were obtained under sugarcane while
chilli and chickpea remained almost at the same level.
At site IV the highest number of spores were recorded under Pigeon Pea
cultivation which is followed by Turmeric cultivation. The least number of
spores were obtained under tomato cultivation. Chickpea and chilli were more
significant than others.
Table 7 show spore population ofAculospora laevis (Fig. e) at different
sites and crops. At site I, the highest number spores were recorded under
36
Pigeon pea cultivation followed by chilli and sugarcane while the lowest was
obtained from Turmeric and Tomato where spore number remained at the same
level. No spores were obtained under chickpea cultivation.
At site II, the highest number of spores were recorded under sugarcane
cultivation, followed by pigeonpea and tomato and were statistically significant
to each other. No spore was recovered from chickpea field.
At site III, chilli yielded the highest number of spores while tomato,
turmeric and wheat remained at the same level and were statistically are
insignificant.
At site IV, the highest number of spores were obtained under chickpea
and lowest under Tomato cultivation. At this site tomato and chilli were
significant than others. No spore was recovered from sugarcane field.
The table 8 show, the number of spores of Gigaspora gigantea (Fig. f)
under different sites and different crops. At site I highest spores were recorded
under Pigeon pea followed by chilli and lowest were obtained under turmeric
cultivation. No. spores were obtained under, Tomato, chickpea and wheat
cuhivation.
At site II, highest number of spores was recorded under tomato
cultivation followed by chickpea, sugarcane and wheat crops resulted a very
small number of spores while no spore was recovered from pigeonpea field.
At site III, the highest spores were recorded under pigeonpea cultivation
while chilli, chickpea and wheat show a close proximity and were insignificant
to each other.
37
Table - 8 : Spore counts of Gigaspora gigantean at different sites under different crops.
Crop
Tomato
Chickpea
Turmeric
Chilli
Pigeon pea
Sugarcane
Wheat
CD.
Site I
0
0
6
44
46
12
0
3
Site II
68
45
18
16
0
3
6
3
Site III
6
3
0
2
38
0
4
2
Site IV
0
44
30
0
0
18
36
4
Table -9 : Mycorrhization at different sites under different crops.
Crop
Tomato
Chickpea
Turmeric
Chilli
Pigeon pea
Sugarcane
Wheat
CD.
Site I
87
65
76
68
89
94
93
6
Site II
85
63
65
71
83
90
91
6
Site III
78
60
61
68
70
81
77
4
Site IV
76
58
60
56
62
74
71
5
38
At site IV, the highest spores were recorded under chickpea followed by
wheat and turmeric. No spores were recorded under tomato, chilH, and
pigeonpea cultivation. The spore number were poor as compared to other
species.
Table 9 shows, the percent colonization of AM fungi in different sites
under different crops. A glance at the data clearly indicates that the percentage
colonization occurred to the maximum extent under sugarcane cultivation in all
the sites yielding an average of 84.75%. In wheat, the colonization percentage
recorded at second highest level in all the four sites with an average of 83.0%
followed by tomato with an average of 81.50%. Almost similar percentage of
colonization were recorded from chilli, turmeric and chickpea. In pigeonpea
percent colonization happened to be 76.0%. The per cent colonization in
, different crops followed the following descending order viz. sugarcane, wheat,
tomato, pigeonpea, chilli, turmeric and chickpea. It was also evident from the
data given in table 9, that the percent colonization differ widely at different
sites under all the crops surveyed depending on the soil conditions and
environmental factors.
DISCUSSION
The results of this investigation clearly indicate that most of the plants in
all the sites are mycorrhizal. The degree of AM formation varied in all crop
species. AM colonization in roots was below 84.75% at all four sites. The
average AM colonization in all crops at four site ranged between 61.50% to
84.75%. The colonization increased gradually with an increase in the spore
39
number. Due to difference in pH and soil characteristics, the number of spores
. as well as root percentage colonized varied the highest being at site Ai.
All the crops were colonized by the AM fungi extensively and there was
no definite relationship between crop and AM fungi as the spores number in
soils collected during different crops season varied at different sites. But AM
fungi preferred sugarcane and wheat crops than the others. In general the
leguminous as well as the other crop species are mycorrhizal in nature (Mosse,
1975). Multiple infection was observed in all the cultivated crops at all the four
sites in this survey and the diversity in AM fungal spore types was seemingly
very high and AM fungal chlamydospores were quite common in all the
samples. Similar results were obtained by (Khalil et ai, 1992, Mosse 1973,
Hayman and Strold '1979. Padmavathi et ai, (1991) also observed that spores
belonging to more than one species of AM fiingi are often found in agricultural
soils. Glomus was the dominant genus followed by Acaulospora and
Gigaspora. The spores of only three genera were found in the sample collected
from all the four sites in the present study, but the level of colonization varied
from site to site. Similar results were also obtained by (Sulochana et ai, 1990;
Peng and Shen 1990 and Blaszkowski 1993). Muthukumar et al, (1996)
pointed out that AM fungal colonization varied considerably between the
species., Difference in the per cent colonization and sporulation of the
symbiont in association with different crops receiving similar environment may
be attributed to the specificity of the symbiont to the crop (Mosse, 1973;
Kruckteman, 1975).
40
It has been reported that availability of the substrate in root and the
various soil factors affected the production of external hyphae. The complex
environmental factors, seasonal fluctuations, agricultural practices and there
soil microflora may also influence the development of mycorrhizal fungi
(Schwab et al, 1983; Abott and Robson, 1985).
AM fungi are wide spread in occurrence and due to their potential for
crop improvement they have been investigated extensively (Mukerji, 1995;
Mukerji and Dixon, 1992; Powell and Bagyaraj, 1984). Natural occurrence and
predominance Glomus, Acaulospora and Gigaspora in the cultivated soils at all
the four sites indicates that there is a need of maintaining them in cultivated
soils through proper management. They may be utilized as biofertilizer and
also as biocontrol agents. The bi-directional transport enhanced the plant
growth and lead to completion of fungal life cycle. For this reason mycorrhizal
symbiosis are attractive systems in agriculture.
41
SECTION II
SECTION II
COMPARATIVE EFFICACY OF DIFFERENT ARBUSCULAR-MYCORRHIZAL FUNGAL SPP. (AMF) ON TOMATO
(LYCOPERSICONESCULENTUM MILL.)
INTRODUCTION
Arbuscular mycorrhizal (AM) association is known to improve plant
grow1;h through better uptake of nutrients and increased resistance or tolerance
to drought and root pathogens in many species of leguminous and other crop
plants (Singh, 1994; Mosse, 1973). AM fungi vary in their physiological
interaction with different hosts and hence, in their effect on plant growth.
Species and strains of AM fungi have been shown to differ in the extent to
which they increase nutrient uptake and plant growth (Powell et al, 1980).
These observations have led to introduction of the term "efficient" or effective
strains (Abbot and Robson, 1981). Generally those fungi that infest and
colonize the root system more rapidly are considered to be "efficient" strain
(Mums and Mosse, 1980). The usefulness of mycorrhizae is especially
appropriate in the development of sustainable system of agriculture (Mosse,
1986), so as to produce desirable effect of improving plant growth and
inducing resistance to pathogen in given environmental conditions (Bali et al.
1987). Tomato is one of the important vegetable plants, used in different forms
viz ., juice, paste, ketchup, soup and powder. The information to select efficient
AM fungi for inoculating tomato (var. Pusa Ruby) to achieve better growth and
drought resistance is still meager. Hence, there is need to identify specific host
-endomycorrhizal association and to define conditions under which these
association function efficiently. The present study is a step in this direction to
identify the efficient AM fungi for tomato crop.
MATERIALS AND METHODS
Starter culture of AM fungus (inoculum production)
Collection of soil sample:
Morphologically different types of spores recovered from the
rhizosphere soils were collected separately. In order to collect spores of AM
fungi from each sites, fifty soil samples were collected from the crop fields in
with the help of soil auger upto a depth of 15 cm from the rhizosphere of the
plants.
Isolation of spores
Spores of AM fungi present in the soil samples were isolated by wet
sieving and decanting method described by Gerdemann and Nicolson (1963).
Samples of 100 g dry soil was taken in 1000 ml of water thoroughly shaked
and left for a minute to settle down the heavier particles. The soil solution was
passed through coarse sieve first and then decanted on to a series of sieves of
varied size i.e. 80, 150, 250 and 300 mesh. The spores obtained on sieves were
collected with water in separate beakers. The spore suspensions were
repeatedly washed by Ringers' solution (NaCl 6g 1"', KCl 0.1 g 1'' and CaCli
0.1 g r ' in disfilled water of pH 7.4) in order to remove the adhered soil
particles from the spores. The following species were found to be of common
occurrence in the agricultural fields.
Glomus fasciculatum
Glomus mosseae
Glomus etunicatum
Glomus constrlctum
Acaulospora laevis
Gigaspora glgantea
43
All the above mentioned species were evaluated for their potential as
effective AM inoculant for tomato (var. Pusa Ruby). The spores of AM fungi
were identified under a dissecting microscope with the help of the synoptic
keys suggested by Trappe (1982). The spores of AM fungal species were
separated by picking and used for pot culture. Spores were separated with a
microspatula and picked up by a Pasteur pipette fitted with a rubber bulb.
These tools were surface sterilized for 2 minutes in a solution containing
chloroamine T 20 g/1, streptomycin 300 mg/1 and Tween 80 in trace amount/1
of distilled water.
Maintenance of AM fungi culture :
Pure cultures of six AM fungi viz., Glomus fasciculatum, Glomus
mosseae, Glomus etunicatum, Glomus constrictum, Acaulospora laevis,
Gigaspora gigantea collected during the survey (Section-1) were raised on
Rhode's grass {Chloris gay ana Kunth) grown in pots under glasshouse
conditions. To raise Rhode's grass, seeds were surface sterilized with 0.1 per
cent solution of HgCl2 and sown (5 seeds per pot) in 9 cm clay pots,
containing sterilized soil (66% sand, 24% silt, 8% clay, OM 2%, pH 7.5).
Fifty spores of each AM fiingal species per pot were layered at 6 and 2 cm
depth in 50 clay pots. After emergence, seedlings were thinned and one
seedling was maintained in each pot. After 125 days, the plants were
uprooted and the spores were isolated by wet sieving and decanting method
from the pot soil and the roots were stained and examined for the AM
colonization. The spores, hyphal fragments and small plant root segments
were then used for further experiments. The population of different AM
44
fungi in tiie inoculum was assessed by the most probable number method
(Porter, 1979).
Inoculation of AM fungus :
In order to select efficient AM inoculant for tomato, the AM fungi
recovered from agricultural fields were evaluated for their efficiency in
improving the performance of the crop (tomato: var. Pusa Ruby). The
following AM fungi were used in this experiment Glomus fasciculatum, G.
mosseae, G. etunicatum, G. constrictum, Acaulospora laevis and Gigaspora
gigantea.
Seedlings of tomato (Lycopersicon esculentum Mill.) cv. Pusa Ruby
were raised in clay pots (25 cm diam.) from seeds surface sterilized with -
0.01% mercuric chloride. The surface sterilized seeds were sown in the pots
filled with autoclaved sandy loam soil (66% sand, 24% silt, 8% clay, 2%0M,
pH 7.7) and one-week - old seedling were transplanted in 15 cm diam. pots. In
each pot filled with 920 g sterilized soil; 80g soil with AM inoculum was
added later to make the amount of soil 1 kg/pot. Before transplantation of
seedlings, the mycorrhizal inoculum of different AM fungi was separately
placed below the seedling by the layering method (Menge et al, 1977). The
inoculum was spread as a layer at a depth of 3-5 cm in the pot at the time of
planting. The seedlings were recovered with a layer of soil to ensure the
development of an efficient host fungus association. The inoculum consisted of
a mixture of infected root segments and soil with extramatrical hyphae and
spores (1000 spores/pot) from cultures of different AM fiingi maintained on
Rhode's grass, as described earlier. For each treatment 15 replicates were
45
maintained. A control series was also maintained where no inoculum of AM
fungi was added to the soil. Pots were watered appropriately and maintained in
a glass house bench with air temperature ranging from 30±2°C.
The plants were examined 20, 40 and 60 days after the transplantation
for determining the plant growth and mycorrhization of the plants. The
samples of the roots and soil were processed as described earlier in Section I.
Mycorrhization was recorded in terms of mycorrhizal intensity in roots i.e.,
external colonization percentage, internal colonization percentage, per cent
arbuscules, average number of chlamydospores in 1 cm root segment and
number of spores recovered from 100 g dry rhizosphere soil. All the data
related to growth of shoots and roots, root infection, spore population were
analyzed statistically by the method of Pause and Sukhatme (1985). Minimum
difference, required for significance (CD) at 5% level was calculated by the
ANOVA model.
The performance of the crop raised with added inoculum of selected
AM fungal species was compared with that of control and the AM fungus
causing maximum improvement in the performance over control was selected
as efficient AM inoculants for the tomato var. Pusa Ruby.
Parameters studied :
During experimentation the following parameters were determined for
each treatment of the experiments at different growth periods.
Shoot and root lengths
Fresh and dry weights of shoot and root
Af>
Mycorrhization in term of:
External and internal colonization percentage, per cent arbuscules,
number of spores recovered from 100 g dry rhizosphere soil.
Plant growth parameters :
Length, fresh weight as well as dry weight of shoots and roots at
different stages of growth were recorded for each treatment. Plants of each
treatment were taken out from the pots and soil particles adhering to roots
were removed by washing in tap water and properly labelled. In the
laboratory, lengths of shoot and root were measured by measuring tape and
fresh weights of shoot and root were determined with the help of a physical
balance. For determining dry weights of root and shoot, plants from each
treatment were wrapped in blotting paper sheets, labelled and dried in a hot air
oven running at 60°C for 24-48 h till a constant weight is obtained.
Root colonization and the spore estimation :
At the termination of the experiments root colonization in term of
percentage external and internal colonization, per cent arbuscules, estimation
of spores (in 100 g rhizosphere soil) in the same soil samples were used for
assessment of the root colonization, as described in the section I.
RESULTS
Plant length :
The effect of different AM fungi at the different intervals on plant length
was examined in terms of shoot, root and total length of the tomato plants
(Table 10). Shoot length increased as the time intervals after inoculation
47
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48
increased from 20 to 60 days. Glomus fasciculatum treated plants, showed
increase in shoot at each interval, compared to the control as well as to those
inoculated with Acaulospora laevis and Gigaspora gigantea. Shoot lengths of
plants inoculated with either A. laevis or G. gigantea did not differ from
control. Inoculation of tomato plants with G. mosseae or G. constrictum
showed a better performance at 40 days interval. The root length was also
promoted by G. fasciculatum at all the growth intervals. Treatment of G.
fasciculatum improved the root length to the same extent as that of G.
constrictum at 40 and 60 days intervals but to a lesser extent at 20 days
seedling compared to the plants inoculated with G. constrictum or G. mosseae.
The growth effects due to treatment with AM fungi on total plant length
are given in Table 10. Total plant length in control, A. laevis or G. gigantea
were similar and lower compared to the ones treated with G. fasciculatum. No
significant difference in plant length observed in plants inoculated with A.
laevis or G. gigantea compared to control.
The highest (37%) growth promotion was recorded in case of G.
fasciculatum and it was closely followed by G. mosseae (36%) at 60 days
interval. However, on 40 days old plants, G. constrictum promoted the highest
level of growth (26.60%) followed by G. mosseae (24.41%) and G
fasciculatum (23.13%).
Plant fresh weight:
At all the growth intervals G. fasciculatum promoted the highest value
of shoot fresh weight, compared to those treated with the other five AM fungi
and control, but it was apparently at par with G. constrictum inoculated plants.
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50
Inoculation of G. etunicatum, G. gigantea and A. laevis however, resulted in no
significant increase in the shoot fresh weight over the control at 20 days
interval. The fresh weight of plants inoculated with G. gigantea did not differ
from control at 40 days interval but slight increase in shoot fresh weight
occurred at 60 days interval compared to control. Inoculation of plants with G.
consthctum resulted in higher increase in shoot fresh weight than those
inoculated with G. etunicatum at 20, 40 and 60 days intervals.
At 40 days intervals the plants treated with G. fasciculatum showed
higher root fresh weight than all the other AM fungi but it was almost
equivalent to those plants inoculated with G. constrictum and G. etunciatum. At
60 days interval, the root fresh weight was the highest in the plant treated with
G. fasciculatum.
Total fresh weight of tomato plants was improved by inoculation with
G. fasciculatum, plants inoculated with G. etunicatum and G. constrictum at 40
days interval were at par. At the final stage of growth A. laevis and G. gigantea
treatments resulted significantly higher plant fresh weight than control but
lower than that of G. fasciculatum (Table 11). Inoculation of G. fasciculatum
resulted in significant increase in total fresh weight percentage of the plant over
the control as well as the other five AM fungi.
Plant dry weight:
At 60 days interval, G. fasciculatum inoculation resulted in significantly
superior shoot dry weight than all others. At 40 days interval G. mosseae and
G. constrictum resulted almost the same amount of dry weight equal value of
dry weight was supported in G. fasciculatum inoculated plants at 20 days
51
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52
interval. No significant difference in siioot dry weight was recorded in plants
inoculated with A.laevis or G. gigantea compared to control at 40 days
intervals.
As in all the other cases, G. fasciculatum supported significantly high
root dry weight at 60 days interval. The root dry weight supported by G.
fasciculatum and G. mosseae did not vary to any significant level at 40 days
intei-val. Root dry weight in the control plants was found to be at par with those
inoculated with G. constrictum at 20 days interval.
The total dry weight of tomato plants inoculated with G. fasciculatum
proved to be significantly high at 60 days growth interval compared to other
treatments (Table 12). Plants inoculated with G. constrictum resulted in
significantly high dry weight over that of G. mosseae at 60 days interval. Equal
values of plant dr>' weight was recorded in plants treated with G. constrictum
and G. etunicatum at 20 days interval. The highest total plant dry weight was
supported by G. fasciculatum at all the three intervals, which was closely
followed by G. mosseae and G. constrictum at 40 days interval.
MYCORRHIZATION
The mycorrhization of AM fungi was estimated by using four
parameters as given in (Table 13).
The value for all the parameters in plants inoculated with G.
fasciculatum was higher than all the other treatments. The values of
mycorrhization in case of A. laevis and G. gigantea were lesser than the other
AM fungi. The highest percentage of outer colonization (78.2%), internal
colonization (66.2%), arbuscules (53.20%) was recorded in plants treated with
53
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54
G. fasciculatum which was followed by G. constrictum (68%), (55%) and
(46%) at 40 days interval. G. fasciculatum inoculated plants also resulted in the
highest spore population in 100 g soil/pot (300, 326,an4^820yc6iftBared to all
other AM fungi at all the three intervals. / " f ' ^ *7, p \ G
DISCUSSION ' V
In the present study, it has been found that the mycorrliigat^-^tus and
growth responses are higher in all treatments compared to the control and the
different strains of AM fungi differ in their capacity to promote plant growth.
Better plant growth in AMF infected plants could be due to enhanced nutrient
contents of the plant. Enhanced absorption and accumulation of several
nutrients such as N, P, K, Zn, Mn, Fe, Ca and S infected plants have been
reported (Bowen et al., 1975; Selvaraj et al, 1986 and Dhillion, 1992). AM
fungi also enhance the concentration of different organic compounds in roots
and can improve the productivity of the host plant (Selvaraj et al., 1995). Out
of the six different AM fungi tested, G. fasciculatum caused highest increase in
dry weight over the control. The results of the present study are due to
increased P uptake and other nutrients, resulting in better growth, yield and dry
matter. This has also been reported for many crops such as barley, onion,
soybean, rice, blackgram (Sanni, 1976; Bagyaraj et al., 1979; Owusu and
Mosse, 1979; Kuo and Haung, 1982; Luis Brown, 1986; Jeffries, 1987;
Umadevi and Sitaramiah, 1990). Sundaram and Arangarasan (1995) reported
that out of four cultures of AM fungi G. fasciculatum gives the highest fruit
yield in tomato plants. Bagyaraj et al. (1989) reported that different strains of
AM fungi have different capability to increase the nutrients uptake and plant
55
growth, and therefore there is a need for selecting the efficient ones. It has been
found in the present study that the extent of mycorrhization in soil varies with
different AM fungal species. They also vary in their preference to the host.
Root colonization has been found to facilitate more host fungal contact and
exchange of nutrients resulting in better plant growth. Similar observation has
been made by Abbott and Robson (1982) out of the six AM fungi investigated
G. fasciculatum promoted better plant growth and nutrient content of the plants
than others, G. fasciculatum inoculated plants also showed the highest
percentage of outer colonization (78.2%), and internal colonization (66.2%),
arbuscles (53.2%) and spore production (820) compared to others. A similar
kind of result has been reported by Abbott and Robson (1985). These
mycorrhizal mycelia coupled with increased nutrient uptake results in the better
performance of the mycorrhizal plants (Rhodes and Gerdemann, 1975). In the
present study it has been found that G. fasciculatum support the highest plant
growth in terms of length and biomass of the plants at maturity. Biomass
production, mycorrhizal colonization. Sporulation have been found to be
significantly high in the G. fasciculatum inoculated plants as compared to all
the other five AM fungal species. It could be, therefore, designated as the
potential AMF inoculant in sandy clay loam soil for tomato var. Pusa Ruby for
the successful plant growth and yield.
56
SECTION III
SECTION III
EFFECT OF AM FUNGUS, GLOMUS FASCICULATUM AND ROOT-KNOT NEMATODE, MELOIDOGYNE INCOGNITA ON
GROWTH AND DEVELOPMENT OF TOMATO.
INTRODUCTION
The arbuscular mycorrhizal fungi (AMF) are associated with all the
cultivated crop plants and can significantly increase the nutrient uptake
(Hayman, 1982, Mosse, 1975). AMF and plant parasitic nematodes occur
together in the rhizosphere of the plants in association with the same root tissue
for their growth and development and while doing so they exert a characteristic
but opposite effect on plants. The interaction between AM fungi and plant
parasitic nematodes has been studied by several workers (Fox and Spasoff,
1972; Sikora and Schoenbeck, 1975., Schenck and Kellam, 1978., Bagyaraj et
al, 1979., Sikora, 1981., Hussey and Roncadori, 1982; Mishra and Shukla,
1995; Edathil et al, 1996; Mamatha and Bagyaraj, 2002).
Concomitant colonization and infection of roots by mycorrhizal fungi,
pathogens and other microbes inevitably interfere with each other's activities
(Linderman, 1985). Several studies on interaction have shown that AM fungi
associations generally induce tolerance to nematode susceptible plants (Heald
et al., 1989; Cooper and Grandison, 1987; Lingaraju and Goswamia, 1993) and
mitigate the adverse effects of plant parasitic nematodes on plant growth
(Sikora, 1979; Jain and Sethi, 1987). Fox and Spasoff (1972) demonstrated that
as little as 10 spores/pot of Gigaspora gigantea suppressed the reproduction of
Heterodera solanacearum on tobacco. Pratylenchus brachyrus levels on cotton
were reduced with 250 spores/pot (Hussey and Ronchdori, 1982). Schenck and
Kellam (1978) obtained reduction in the number of galls of Meloidogyne
incognita on soybean with 25 spores of Glomus macrncarpiis var.
macrocarpus. (Similarly population of Rotylenchulus reniformis reduced by
150 chlamydospores and egg masses and the adult females of M incognita
with 300 chlamydospores of Glomus fasciculatum on tomato and cotton
respectively (Sitaramaiah and Sikora, 1982, 1996). In the present study,
glasshouse experiment was designed to find out the individual and concomitant
effects of Glomus mosseae and Meloidogyne incognita on the growth of tomato
plants and their population dynamics.
MATERIALS AND METHODS
Plant culture :
Seedlings of tomato cv. Pusa Ruby were raised in clay pots (30cm
diam.) from seeds surface sterilized with 0.01% mercuric chloride. The surface
sterilized seeds were sown in the pots filled with autoclaved sandy loam soil
(66% sand, 24% silt, 8% clay and 2% OM) of pH 7.7.
Root-knot nematode culture :
In the study root-knot nematode, Meloidogyne incognita (Kofoid and
White) Chitwood, one of the commonest root-knot nematode species in the
area, was used. Field populations of M. incognita were collected from tomato
and egg plant, (Solanum melogena L.), species of root-knot nematode, infecting
roots of tomato or egg plant in fields were tentatively identified on the basis of
the characteristics of perineal patterns of the females. Roots infected with M.
incognita were chopped and added to the pots containing sterilized field soil
and the tomato plants raised earlier as mentioned above (3 weeks-old) were
58
transplanted. After 50 days, single egg mass culture of the nematode was
established. Seedlings of tomato plants were transplanted in clay pots (30cm
diam.) containing autoclaved soil. Single egg mass of the nematode M
incognita obtained from the roots of the plants was added in each pot near the
roots of the seedlings. The pots were kept in glasshouse at 27°C (+2°C).
Subculturing was done approximately at every 3 months by inoculating new
tomato seedlings with at least 15 egg masses, each obtained from a single egg
mass culture in order to maintain sufficient inoculum for further experimental
studies.
Identification of the root-knot nematode :
The identity of the root-knot nematode was confirmed by studying the
characteristics of perineal patterns of females and conducting North Carolina
differential host test (Eisenback et al., 1981., Taylor and Sasser, 1978.,
Hartman and Sasser, 1985). Fifteen perineal patterns of females of each single
egg mass population were prepared and their characteristics were examined in
order to identify the species (Eisenback et al., 1981).
For North Carolina differential host test (Taylor and Sasser, 1978,
Hartman and Sasser, 1985) seedlings of tomato cv. Rutgers, tobacco cv. NC95,
pepper cv. California Wonder, peanut cv. Florunner, watermelon cv.
Charleston Grey and cotton cv. Deltapine-61 were grown in clay pots (one
seedling/pot) having sterilized soil in triplicate. Two additional replicates of
tomato were included to determine the time of termination of the test.
After determining the number of second stage juveniles (J2) per ml,
plants in each pot were inoculated with 5000 J2. Juveniles were added to a
<Q
depression made in the soil at the time of transplanting. Inoculated plants were
kept at glasshouse at 27-30°C). Fifty to sixty days after inoculation, roots were
harvested and thoroughly washed with tap water and examined for the presence
of galls. Roots with very high infection were stained with Phloxin B to
determine the number of egg masses. Number of galls and egg masses were
counted and GI and EMI were rated on 0-5 scale;
0 = 0
1 = 1-2
2 = 3-10
3 = 11-30
4 = 31-100 and
5 = more than 100 galls or egg masses per root system (Taylor and Sasser,
1978).
After the rating of root system, results were compared with the
differential host test reaction chart (Table 14). This confirmed the identity of
the species determined by the perineal pattern method.
Preparation of inoculum and inoculation :
Second stage juveniles (J2) of the nematode were used as inoculum in
the study. Second stage juveniles were obtained by incubating egg masses
collected from the roots of tomato plants maintaining single egg mass culture
of M incognita. Egg masses were incubated in coarse sieve fitted with double
layered tissue paper and placed on Baerman funnel containing water. The
sieves were then placed in an incubator (temp 25°C). After 72h, number of
hatched juveniles (J2) were collected in a beaker and number of juveniles per
60
V2
T5 O
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61
ml was standardised by counting the juveniles from ten, 1 ml samples.
Average number of juveniles was then calculated to represent the number of
juveniles per ml of the suspension.
For inoculation, the suspension containing J2 was taken in micropipette
controller and added near the roots of the seedlings. The holes were covered
with soil after inoculation. Nematode inoculation density consisted of uniform
quantity of suspension containing either 0, 150, 300, 600, 1200, 2400 and
4800 freshly hatched second stage juveniles (J2)/pot.
Preparation of inoculum of the AM fungus:
Glomus fasciculatum (Nicol & Gerd) Gerdemann & Trappe, inoculum
was maintained and multiplied on Rhodes grass (Chloris gayana) as in
section II.
Inoculation
For inoculation, inoculum of G. fasciculatum for each pot consisted of
uniform quantity of suspension containing 0, 50, 100, 200, 400 and 800
chlamydspores which were retrieved from the soil using wet-sieving and
decanting technique (Gerdemann and Nicolson, 1963) and were placed just
3 cm below the soil surface.
Six levels of G. fasciculatum and seven levels of Meloidogyne
incognita were used in combination for their effect on plant growth and on
each other (Table 15). The chlamydospores number as given constituted the
levels of G. fasciculatum (designed as GFQ, GFi, GF2, GF3, GF4 and
GF5). Similarly freshly hatched second stage juveniles numbering as above
62
> o a
o 02 H
u
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53
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63
constituted M. incognita levels (designated as MIQ, MIi, MI2, MI3, MI4, MI5
and MIe). The treatments were replicated five times, arranged in a
completely randomized block design and maintained in a glasshouse at 28-
35°C. The plants were harvested at 60 days after inoculation and all the
parameters studied in section II were studied along with the following
additional parameters viz., Nematode population (both in soil and root),
number of galls/root system, number of egg masses/root system, number of
eggs/egg mass (Fecundity). Data were analysed statistically using analysis of
variance in factorial design as mentioned by Fisher (1950) and CD was
calculated at P-0 .05 .
Plant growth :
Plant growth parameters and chemical parameters were studied by the
methods mentioned in section II.
Galls and egg masses :
At termination of the experiment, roots of harvested plants were
washed under the tap and examined for the presence of galls. Number of galls
per root system was counted. Roots were immersed in a aqueous solution of
Phloxin B (0.15 g/lit tap water) for 15 minutes to stain the egg masses. Egg
masses per root system were counted (Taylor and Sasser, 1978).
Fecundity :
The number of eggs per egg mass is known as fecundity. It was
measured by shaking vigorously 10 egg masses in 5.25 % NaOCl solution.
The eggs were separated from egg mass and collected over 500 mesh sieve.
64
From the sieve, eggs were transferred into a beaker and 0.35% acid fuchsin (in
25% lactic acid) was added into 20 to 25 ml of suspension with boiling for 1
minute for staining the eggs. After cooling, the eggs were counted and the
eggs per egg mass were calculated to find out the fecundity.
Nematode population :
For estimating root population of nematodes (J2 + J3 + J4, mature
females), root from each replicate was weighed and cut into 1 cm length. One
gram of root pieces was stained with acid fuchsin and lactophenol (Byrd et al.,
1983). The root pieces were placed between two slides, examined under
stereoscopic microscope and number of J2 + J3 + J4 counted. Then total number
of J2 + J3 + J4 for the whole root system of the replicate was calculated. For
counting the number of females, 1 g of root pieces were transferred in 5%
HNO3) and incubated at 25°C. After 72h root pieces were gently teased to
release the females. The number of females/g of root was counted and total
number of females for the whole root system was calculated (Hussey and
Barker, 1973). The means of replicates were then calculated.
The eggs were extracted from the roots of each treatment separately by
Chlorox method of Hussey and Barker, (1973). Roots from each treatment
were cut into l-2cm pieces. One gram of the root pieces were shaken
vigorously in 200 ml of 1.0% NaOCl solution for 1 to 4 minutes. Then NaOCl
solution was passed through a 200 mesh sieve, rested over a 500 mesh sieve to
collect free eggs. After this 500 mesh sieve with eggs was placed under a
stream of cold water to remove residual NaOCl (rinsed for several minutes).
The rinsed roots were put under water to remove additional eggs and were
65
collected by sieving. The number of eggs was then counted in counting dish
under stereoscopic microscope. The total number of eggs was calculated by
multiplying the number with the fresh weight of the root in the treatment. Soil
population (J2 + male) of the nematode was estimated by modified Cobb's
sieving and decanting technique (Southey, 1986).
Plant analysis :
Plant samples of each treatment from the glasshouse experiment were
processed for estimating phosphorus (P), nitrogen (N) and potassium (K)
contents of shoots and roots.
Estimation of nitrogen, phosphorus and potassium
For estimation of nitrogen, phosphorus and potassium in plant materials
(shoot + root) from each treatment, samples were digested in the following
way. Estimations were done from the mixed powder of plants of various
treatments, in a given experiment. Hundred mg of oven dried powder of the
shoots and roots from each treatment was transferred separately in 50 ml
Kjeldahl flask, then 2 ml of chemically pure H2SO4 was added and flask was
heated on Kjeldahl assembly for about 2 h till dense fumes were given off and
the contents turned black. Then 0.5ml of pure 30% H2O2 was added after 15
min. of cooling. Heating was continued till the colour changed to light yellow.
Extract was heated again for half an hour and cooled for 10 min. for getting it
clear and then 3-4 drops of 30% H2O2 was added drop wise followed by
heating for 15 minutes. After that, the digested material was transferred in 100
ml volumetric flasks with 3-4 washings and volume was made upto capacity.
This digested material was used for estimating N, P and K, (Linder. 1944;
Lundegardh, 1951)
66
Nitrogen
Prior to estimating nitrogen present in the digested plant material,
standard curve was prepared by the following method.
Nitrogen standard curve
Different dilutions in 10 test tubes (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8,
0.9 and 1.0 ml) were made from the solution prepared by dissolving 0.236g of
ammonium sulphate in 100 ml of distilled water. The volume was then made
upto 5 ml in each test tube by adding distilled water. A control was also run
side by side. After that 0.5ml Nesseler's reagent was added followed by 5 ml of
distilled water. Per cent transmittance was read at 525nm from spectrophoto
meter on developing yellow orange colour after half an hour. Then a curve was
drawn between concentration in X axis and O.D obtained in Y axis.
Estimation ofN in plant material
An aliquot of 10 ml was transferred to 100 ml volumetric flask to
which 2ml of 2.5N NaOH was added so as to neutralize the excess of acid.
10% sodium silicate was added to prevent turbidity. The volume was made
upto capacity. 5ml of aliquot was taken in three test tubes followed by the
addition of 0.5 ml of Nesseler's reagent with shaking. Then 10 ml volume was
made by adding distilled water. After waiting for 5 min, the per cent
transmittance was read at 525 nm. The concentration was read with the help
of 0.0. from standard curve (Linder,1944).
Phosphorus standard curve
Different dilutions of potassium dihydrogen orthophosphate (KH2PO4)
viz., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml concentrations were
67
taken into separate test tubes and final volume was made upto 5 ml by adding
required amount of distilled water. One ml ammonium molybdic acid and 0.4
ml. of I -amino 2-nepthol 4-sulphonic acid were added in each test tube and
the volume was made upto 10 ml with distilled water. After half an hour, the
percent transmittance was read at 625nm. Standard curve was drawn between
concentration and O.D. in X and Y axis respectively.
Estimation ofP in plant materials
Five ml of aliquot of digested plant material was taken in three test
tubes, to which 5 ml of distilled water was added. After that I ml of ammonium
molybdic acid was added, with shaking, followed by addition of 0.4 ml of 1-
amino 2-nepthol 4-sulphonic acid and the volume was made up to 10 ml. The
control was run side by side and per cent transmittance was read at 625 nm.
after half an hour. The P concentrations in plant materials was calculated from
the standard graph using O.D. (Fiske and Row, 1925)
Potassium standard curve
Potassium was estimated using flame photometer. Different dilutions of
potassium chloride viz., 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ppm were prepared by
dissolving 91 mg of oven dried KCl in 100 ml of distilled water and diluting 0,
1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml of the stock solution to 100 ml. The flame
photometer was calibrated to zero using distilled water and to 100 using 10
ppm KCl solution and readings for other standard solutions were taken. A
standard graph was drawn taking concentration in X axis and readings obtained
from the flame photometer in Y axis.
68
Estimation ofK in plant materials
The digested plant materials were diluted with distilled water so as to
maintain the K concentration in the final solution ranged between 2 to 8 ppm
and the reading was taken in the flame photometer and with the help of the
standard graph, the concentration of K in plant materials was calculated.
The data collected for morphometries were statistically analysed as
under to determine the degree of authenticity of results.
Standard deviation (S.D)
Standard deviation is a measure of fluctuation if. a population produced
as a result of chance factors of sampling from the same population. S.D. for
each parameter of morphometries were calculated by the following formula
(X-X!)^ + ( X - X 2 ) ^ + . . . + (X-XN)^
S.D. = ± N-1
where,
X = Meanof observations i.e. arithmetic mean
X], X2, ... XN = observations
N = Number of observations
Coefficient of variation (C.V)
This measures the relative magnitude of variation present In
observations relative to the magnitude of their arithmetic mean. It IS defined
as the ratio of S.D. to arithmetic mean expressed as percentage
SD C.V.- xlOO
X
69
Data presented in the tables of the morphometries are in the form of Mean ±
S.D. and C.V. Like other parameters, CD was calculated for morphometries
also using the mean values obtained for each replicates.
RESULTS
Plant length:
The combined effect of Meloidogyne incognita and Glomus
fasciculatum was studied at different levels of inoculum of the nematode and
the mycorrhizal fungal spores. The nematode caused adverse effect on the
growth of tomato plants, while the AM fungus G. fasciculatum promoted their
shoot and root growth. When both were applied together, the adverse effect of
the nematode was mitigated to some extent by the AM fungus (Table 16). The
addition of the AM fungal spores had reduce the adverse effect of the nematode
at all population levels studied i.e. from 150 to 4800 J2/plant which was
proportional to the AM fungal spores/plant. For instance at 150 J2 level the
shoot length was reduced by 1.64 per cent while the reduction was 37.82 at
4800 J2/plant. At the same level of nematode, the addition of spore (400/plant)
shoot length increased by 41.44 per cent over the control. Further addition of
spores 800/pot did not bring any significant increase in shoot length at 2400 J2
level of nematode population.
Similar trend was observed for root length and total length, the
maximum effect of nematode was exhibited by a population of 4800 J2/plant
resulting in a loss of 31.60 per cent in root length. This loss was completely
eliminated when the culture medium was supplied with 400 spores/plant,
resulting in a gain of 11.49 per cent instead a loss of 31.62 per cent.
70
Table 16: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the length of tomato plants.
Spore density No/Pot Cont. 50 100 200 400 800 Mean CD
Spore density No/Pot
Cont. 50 100 200
Cont 30.4 34.1 36.8 41.3 41.9 44.8 38.2
150 29.9 33.3 37.4 40.9 43.0 43.8 38.0
Glomus fasciculatum
Cont 17.4 17.7 17.7 19.1
150 16.8 17.5 17.8 18.8
{ Shoot Length (cm) Nematode inoculum density (J2)
300 26.8 31.0 36.8 41.1 43.3 42.8 36.9
= 1.53
600 22.9 28.8 34.5 39.8 42.4 43.4 35.3
1200 21.0 25.9 30.7 37.8 44.1 41.9 33.5
2400 19.8 24.8 28.3 35.9 43.0 41.8 32.2
4800 18.9 24.4 28.1 33.3 39.7 40.4 30.8
M incognita ~ 1.65 Interaction =
Root Length (cm) Nematode inoculum density (J2)
300 15.4 15.0 17.4 18.0
600 14.5 15.3 16.8 18.4
1200 14.0 13.8 14.9 17.7
2400 13.8 13.2 13.8 16.6
4800 11.9 12.5 14.2 15.4
M 24.2 28.9 33.2 38.5 42.4 42.7
-4.1
M 14.8 15.0 16.2 17.7
400 19.8 20.0 20.4 19.4 18.6 19.4 17.4 19.2 800 20.4 19.4 19.9 18.8 20.5 18.5 18.6 19.4 Mean 18.6 18.3 17.6 17.2 16.5 15.9 14.9 CD
Spore density No/Pot Cont. 50 100 200 400 800 Mean
Glomus fasciculatum
Cont 47.8 51.8 54.5 60.4
61.7
64.5 56.8
150 46.7 50.8 55.2 59.1
63.0
63.2 56.3
= 0.947 M. incognita =
Total length (cm)
1.02
Nematode inoculum density (J2) 300 42.2 46.0 54.2 59.1
63.7
62.7 54.6
600 1200 38.2 35.0 44.1 39.7
51.3 45.6 58.2 55.5
61.8 62.7
62.2 62.4 52.6 50.15
2400 33.7 38.0 42.1 52.5 62.4
60.3
48.1
Interaction
4800 31.0 36.9 42.3 48.7 57.1
59.0
45.8
= 2.4
M 39.0 43.9 49.4 56.2
61.6
62.1
CD Glomus fasciculatum = 2.1 M. incognita = 2.0 Interaction = 6.2
71
The nematode caused a loss of 35.14 per cent in the total length, the loss
was completely overcome when the fungal spores where applied at the rate of
400/.plant and gain of 30.54 per cent in total length resulted.
Plant fresh weight:
The interaction effects of M. incognita and G. fasciculatum on the fresh
weight of shoots and roots are given in Table 17. Shoot fresh weight gradually
declined with the increase in the number of nematode (J2) per plant. No
significant reduction in fresh weight of the plant occurred at 150and 3OOJ2
inoculum levels compared to control. Effect of G. fasciculatum on other hand
was found to be increasingly positive with the growing number of spores,
although it was significant at 50 levels.
In case of shoot fresh weight the maximum interaction effect (55.14 per
cent) was found at 2400 J2 level with 400 spore application per plant. Higher
level of spores i.e.800/plant did not bring out any change in shoot fresh weight,
root fresh weight as well as the total fresh weight. The root fresh weight was
reduced at marginal reduction at 150 J2 levels of M incognita and it increased
at higher nematode levels bringing about significant reduction with the
maximum (44.87), occurring at 2400 J2 level (Table 17).
Plant dry weight:
The data on shoot, root and total dry weight are given in Table 18.
Inoculation of M incognita caused decreased in the dry weight of the plant.
Significant reduction occurred when nematode number was increased above
300 J2 level. In all the three cases of weight analysis, 2400 and 4800
J2 produced the same degree of reduction. G. fasciculatum produced beneficial
72
Table 17: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the fresh weight of tomato plants.
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 21.4 23.1 27.8 31.3 33.8 36.4 28.9
150 20.9 22.6 26.3 31.2 34.7 35.4 28.4
Shoot fresh weight (g/plant) Nematod(
300 20.0 21.0 25.7 31.7 35.3 34.9 28.1
; inoculum density (J2) 600 17.2 18.9 23.8 29.2 33.7 32.8 25.9
1200 15.9 18.0 20.7 28.1 32.4 33.5 24.7
2400 14.4 16.6 18.5 26.8 33.2 32.4 23.6
4800 14.6 16.0 18.3 27.3 32.2 31.9 23.3
M 17.7 19.4 23.0 29.3 33.7 33.9
CD Glomus fasciculatum = \ .Q M. incognita = \ .2 Interaction = 2.8
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 7.8 8.7 9.8 12.0 13.3 14.0 10.9
150 7.6 8.6 10.0 11.6 13.2 13.7 10.7
Root fresh weight (g/plant) Nematode inoculum density (J2)
300 6.6 8.0 9.4 11.3 12.1 13.3
10.11
600 6.0 6.9 8.7 10.3 12.7 12.8
9.5
1200 5.3 6.1 7.9 9.2 12.0 13.4 8.9
2400 4.3 4.5 6.6 8.7 11.3 12.7 8.0
4800 4.5 4.7 6.0 8.1 11.7 12.3 7.8
M 6.01 6.7 8.3 10.2 12.3 13.1
CD Glomus fasciculatum "= 0.691 M. incognita-Q Ml Interaction = 1.3
Spore density No/Pot Cont. 50 100 200 400 800 Mean
CD
Cont 29.2 31.8 37.6
43.3 47.1
50.1 39.8
150 28.5 31.2
36.3 42.8 47.9
48.9 39.2
Glomus fasciculatum
Total fresh weight (g/plant) Nematode inoculum density (J2)
300 26.6 29.0 35.1 43.0 47.4 48.2 38.2
= 1.5
600 23.2 25.8
32.5
39.5 46.4 45.4 35.4
1200 21.2 24.1
21.6 37.3 44.4
46.7 32.5
M. incognita = 0.\1
2400 18.7 21.1 25.1 35.5
44.5 45.1
4800 19.1 20.7
24.3 35.4
43.9 44.2
31.6 31.2
Interaction =
M 20.7 26.2
30.3 39.5 45.9
46.9
3.0
73
Table 18: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the dry weight of tomato plants.
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 5.10 5.27 6.33 7.27 8.36 8.64 6.82
150 5.03 5.36 6.28 7.41 8.40 8.54 6.83
Shoot dry we ight (g/plant) Nematode inoculum density (J2)
300 4.75 4.89 6.11 7.63 8.51 8.39 6.71
600 4.10 4.88 5.79 6.91 7.62 7.54 5.99
1200 3.78 4.25 4.98 6.71 7.86 7.77 5.89
2400 3.35 3.84 4.53 6.29 7.98 7.84 5.63
4800 3.20 3.74 4.13 6.48 7.59 7.86 5.50
M 4.18 4.54 5.45 6.95 8.04 8.08
CD Glomus fasciculatum = {).Ill M. incognita-025 Interaction = 0.61
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 2.13 2.28 2.61 3.12 3.31 3.50 2.82
150 2.08 2.33 2.70 3.03 3.40 3.48 2.83
Root dry wei ght (g/plant) Nematode inoculum density (J2)
300 1.80 2.13 2.48 2.95 3.21 3.52 2.68
600 1.64 1.77 2.10 2.48 3.13 3.10 2.37
1200 1.35 1.61
1.98 2.45 3.22 3.21
2.30
2400 1.15 1.28 1.81 2.25 2.81 3.28 2.09
4800 1.12 1.41 1.65 2.13 2.85 3.13 2.04
M 1.61 1.83 2.19 2.63 3.13 3.31
CD Glomus fasciculatum = 0.07 M. incognita = 0.08 Interaction = 0.15
Spore density No/Pot Cont. 50 100 200 400 800 Mean
CD
Cont 7.23
7.55 8.94
10.39
11.67 12.14 9.65
150 7.11
7.69 8.98 10.44
11.8
12.0 9.67
Glomus fasciculatum
Total dry wei ght (g/plant) Nematode inoculum density (Jj)
300 600 6.55 5.74 7.02 6.21 8.59 7.89
10.58 9.39
11.72 10.75 11.91 10.64 9.39 8.43
1200 5.13
5.86 6.96 9.16
11.08
10.98 8.31
2400 4.50 5.12 6.34 8.54
10.76 11.12 7.56
4800 4.32
5.15 5.78
8.61 10.44
10.99 7.54
= 0.27 M. incognita = 0.30 Interaction =
M 5.79 6.37 6.51
9.58
11.17
11.39
= 0.72
74
Table 19: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the N, P and K content of tomato plants.
Spore density No/Pot Cont. 50 100 200 400 800 Mean
Cont 1.75 2.0 2.15 2.11 2.23 2.18 2.07
150 1.84 2.03 2.13 2.17 2.06 2.30 2.08
Nitrogen contents Nematode inoculum density (J2)
300 1.71 1.90 1.98 1.96 2.19 2.21 1.99
600 1.78 1.94 2.16 2.08 2.02 1.98 1.99
1200 1.64 1.83 2.01 2.16 2.07 2.22 1.98
2400 1.58 1.73 2.14 1.92 2.15 2.09 1.93
4800 1.69 1.80 1.95 2.06 2.11 2.18 1.96
M 1.71 1.89 2.07 2.06 2.11 2.16
CD Glomus fasciculatum-Q.\6 M. incognita = ]^S Interaction = NS
Spore density No/Pot "
Cont. 50 100 200 400 800 Mean
Cont 0.215 0.277 0.301 0.311 0.339 0.334 0.296
150 0.201 0.271 0.294 0.297 0.336 0.323 0.287
Phosphorus contents Nematode inoculum density (J2)
300 600 0.221 0.199 0.269 0.281 0.286 0.267 0.296 0.313 0.331 0.329 0.347 0.316 0.291 0.270
1200 0.204 0.257 0.281 0.285 0.317 0.338 0.280
2400 0.191 0.260 0.271 0.282 0.344 0.336 0.280
4800 0.205 0.269 0.256 0.273 0.327 0.331 0.276
M 0.205 0.269 0.279 0.293 0.331 0.384
CD Glomus fasciculatum = 0.02 M. incognita = NS Interaction = NS
Spore density No/Pot Cont. 50 100 200 400 800 Mean
CD (
Cont 1.45 1.54
1.75 1.85 2.01
2.06 1.77
jlomusfas
150 1.32 1.58
1.65 1.82 2.16
2.01 1.75
ciculatun
Potassium contents Nematode inoculum density (J2)
300 600 1.46 1.42 1.48 1.38
1.55 1.47 1.87 1.74 1.91 2.11
2.08 2.06 1.72 1.69
I = 0.033 M. inci
1200 1.32 1.39
1.40 1.75 1.95 1.94 1.62
2400 1.22
1.35 1.41
1.76 1.87
2.09 1.61
ognita = NS
4800 1.31 1.41
1.32
1.65 1.96
2.06 1.61
Interaction =
M 1.35 1.44
1.50
1.77 1.99 2.04
= NS
75
effect by promoting the plant dry weight. Shoot, root and total dry weight were
enhanced significantly as the spore number was increased upto 800. Significant
increase in dry weight was obtained in the plants inoculated with 800 spores
than that of 400 spores/plant. The maximum loss of dry weight caused by M
incognita amounted to 37.25, 47.41 and 40.24 per cent respectively in shoot,
root and total dry weight at the population level of 4800 J2 per plant. This loss
in all three cases was totally overcome by the addition of 800 spores of G.
fasciculatum per plant which resulted in a gain of 54.II, 46.94 and 52.00 per
cent, in case of shoot, root and total dry weight respectively.
Nutrient status :
In general M. incognita reduced the N, P and K contents of the plants at
different levels of inoculum. While Glomus fasciculatum treatments improved
the nutrient status significant (Table 19).
MYCORRHIZATION
The effect of M. incognita on the growth and development of G.
fasciculatum was studied. G. fasciculatum extensively colonized the tomato
plants when inoculated with spores. The external colonization percentage
(Table 20) increased significantly with the increasing number of spores per
plant. The colonization declined to 22.6 per cent from 27.1 per cent at the spore
level of 50 per plant when the nematode level was raised to 4800 J2 from 150
J2. With the increasing spore concentration, from 150 to 800/plant the per cent
colonization increased to 64.5 per cent at the level of 150 J2.
76
Table 20: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on mycorrhization G. fasciculatum in tomato plants.
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 0.0 26.7 35.4 48.9 60.5 62.3 38.9
150 0.0
27.1 34.8 50.1 59.5 64.5 39.3
External colonization (%)
Nematode inoculum density (J2) 300 0.0 25.1 36.8 45.5 57.6 63.9 38.15
600 0.0
21.3 33.7 39.7 54.5 66.2 35.9
1200 0.0 23.7 27.1 33.1 56.3 64.7 34.1
2400 0.0 17.3 20.3 26.4 46.5 53.2 27.2
4800 0.0 17.0 18.8 24.9 43.3 46.9 25.1
M 0.0 22.6 29.5 38.3 54.2 60.2
CD Glomus fasciculatum = 2.\ M. incognita = 2.3 In tera^rf^A6 ^^''^•-)S
1 •^ il 80%
Spore density No/Pot Cont 150
Internal colonization (%) Nematode inoculum density (J2)
300 600 1200 2400
\ \ X V
4800
^^ ---' '
. -:
M Cont. 50 100 200 400 800 Mean
CD
0.0 34.4 43.3 38.8 50.5 49.8 36.1
0.0 33.4 45.4 37.8 50.0 51.1 36.3
Glomus fasciculatum '-
0.0 30.4 31.8 30.5 40.3 41.8 29.1
-2.6
0.0 28.4 43.5 46.0 52.1 55.0 37.5
0.0 27.4 32.2 35.0 41.4 39.6 29.2
M incognita = 31
0.0 25.1 32.4 27.8 35.4 34.0 25.7
0.0 19.4 28.9 31.0 38.6 37.8 25.9
Interaction = 6.0
0.0 28.3 36.7 35.2
44.04 44.1
Spore density No/Pot Cont 150
Percent Arbuscules Nematode inoculum density (J2)
300 600 1200 2400 4800 M Cont. 50
100 200 400 800 Mean
0.0 28.8 37.3 36.0 41.3 39.8
30.5
0.0 30.0 38.7 34.1 39.5 42.0 30.7
0.0 23.0 36.9 37.8 35.8 38.0 28.7
0.0 18.8 34.6 38.4 41.3 40.8 28.9
0.0 20.8 39.1 37.4 34.4 38.4 28.3
0.0 22.0 32.4
37.3 38.8 36.4 27.8
0.0 18.4
31.1 37.1 40.3 38.4
27.5
0.0 23.1 35.7 36.8 38.7 39.1
CD Glomus fasciculatum = 2.^ M. incognita ^'HS Interaction = NS
77
Spore density No/Pot
Cont. 50 100 200 400 800 Mean CD
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 0.0 19.8 21.2 23.4 25.3 27.2 19.4
Number ol
150 0.0 20.5 21.0 22.6 24.4 26.4 19.1
Glomus fasciculatun
Cont 0.0 515 514 528 550 625 455
• chlamydospores / ( cm root Nematode inoculum density (J2)
300 0.0 18.2 20.2 21.4 24.1 25.8 18.2
2 = 2.0
Number of
150 0.0 511 504 541 551 521 545
600 0.0 16.8 18.4 20.3 19.1 21.8 16.0
1200 0.0 18.6 21.4 17.1 18.6 21.4 16.1
M incognita = NS
2400 0.0 17.1 18.6 21.8 23.1 24.4 17.5
4800 0.0 14.4 17.1 19.6 21.8 23.2 16.0
Interaction = NS
chlamydospores /100 g soil Nematode inoculum density (J2)
300 0.0 520 477 596 541 600 455
600 0.0 507 457 566 570 560 443
1200 0.0 405 411 541 550 610 419
2400 0.0 401 388 420 339 350 316
4800 0.0 200 437 255 268 275 239
M 0.0 17.9 19.7 20.8 22.3 24.3
M 0.0 437 418 492 481 519
CD Glomus fasciculatum = 50 M. incognita - 56 Interaction = 58
78
The internal colonization increased with the increasing level of spore
concentration, from 28.3 to 44.04 per cent, when the spores increased from 50-
800/plant.
The development of arbuscules followed the same trend as that of
external and internal colonization.
The number of chlamydospores per centimeter root length also depicted
a similar trend as that of external and internal colonization. The number of
chlamydospores per lOOg of soil show the maximum at 800 spore level. At
1200 and 2400 Ji levels of inoculum there was a significant decrease in the
chlamydospores.
Root-knot disease :
The effect of G. fasciculatum on the growth and development of M.
incognita has been studied in terms of root-knot disease. Population of the
nematode in soil increased significantly as the number of inoculated juveniles
increased. Nematode population showed a significant decrease compared to
control, irrespective of spore concentration (Table 21).
Root population of the nematode was suppressed by G. fasciculatum.
Significant reduction in the root population occurred. The reduction was
proportionate to the spores concentration upto 400 spore level but at 800 level,
there was no appreciable difference in the nematode population.
The number of egg masses per root system increased with increasing
level of M incognita. The number of egg masses per root system was reduced
with increasing spore concentration oiG. fasciculatum. It was observed that the
200, 400 and 800 spore levels caused the same degree of reduction.
79
Table 21: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on root-knot disease parameters in tomato plants.
Spore density No/Pot
Cont.
50 100 200 400 800 Mean
Cont
0 0 0 0 0 0 0
150 1046
1170
908 1006
974 1028
1022
Nematode population
Nematode inoculum density (J2)
300 4114
3967
3817
3966
4395
4496
4125
600 6723
6826
6266
6194
6683
6469
6526
1200
8266
9744
8667
8844
9746
9193
9076
2400
13481
12516
11216
11767
12479
11762
12203
4800
16074
14002
15744
15001
14147
14084
14842
M 7100
6889
6659
6882
6917
6718
CD Glomus fasciculatum = 400 M. incognita = 475 Interaction ==936
Spore
density
No/Pot
Cont.
50 100 200 400 800 Mean
Cont
0 0 0 0 0 0 0
150 25 18 12 6 4
11
Nematode population/root
Nematode inoculum density (J2)
300 48 38 25 10 3 5 21
^n 89 67 32 26 11 12 39
1200
146 108 55 38 8 13 61
2400
168 143 68 58 21 11 78
4800
180 156 84 60 15 13 84
M 93 75 39 28 9 8
CD Glomus fasciculatum = 7 M incognita = 9 Interaction = 15
Spore
density
No/Pot
Cont.
50 100 200 400 800 Mean
Cont
0 0 0 0 0 0 0
150 25 18 12 6 4 3 11
Nematode population/root
Nematode inoculum density (J2)
300 48 38 25 10 3 5 21
600 89 67 32 26 11 12
39
1200
146 108 55 38 8 13 61
2400
168 143 68 58. 21 11 78
4800
180 156 84 60 15 13 84
M 93 75 39 28 9 8
CD Glomus fasciculatum = 2 M. incognita - 3 Interaction = 5
80
Spore density No/Pot Cont. 50 100 200 400 800 Mean
Cont 0 0 0 0 0 0 0
150 52 44 53 35 41 44 44
Nematode of c !ggs/egg masses Nematode inoculum density (J2)
300 64 51 50 43 40 43 48
600 69 62 52 51 40 38 52
1200 81 84 80 90 78 70 80
2400 73 82 69 84 74 70 75
4800 96 94 90 79 68 65 82
M 62 59 56 54 48 47
CD Glomus fasciculatum = 5 M. incognita = 6 Interaction = 10
Spore density No/Pot
Cont. 50 100 200 400 800 Mean
Cont 0 0 0 0 0 0 0
150 85 65 47 18 12 8 39
Nematode of egg; j/egg masses Nematode inoculum density (J2)
300 76 75 42 30 14 7
40
600 116 87 48 33 16 11 51
1200 150 107 56 40 12 14 63
2400 182 124 65 44
17 12 74
4800 194 144 72 52 15 13 81
M 114 86 47 31 13 9
CD Glomus fasciculatum = 8 M. incognita - 10 Interaction = 18
81
Fecundity of the nematode increased at different inoculum levels of M.
incognita. Treatment with 1200,2400and 4800 J2/plant level had more eggs/egg
mass than at the other inoculum levels. Inoculation of G. fasciculatum at the
rate oflOO/plant and above caused significant reduction in the fecundity.
The number of galls per root system was reduced to a significant level
by different treatment of G. fasciculatum. The reduction in gall number
occurred proportional to the spore level with the maximum (90.58% per cent)
occurring with the highest level of spores (800 level).
DISCUSSION
The present study showed that Glomus fasciculatum is beneficial to the
tomato plants as it helps in plant growth by promoting shoot and root
elongation and enriching nutrient status. The improvement in plant growth
characteristics in mycorrhizal plants compared to control indicates the
existence of complex physiological and biochemical relationship between
Glomus fasciculatum and tomato. AM fungi have been reported to alter the
physiology of root and reduce the root exudation (Grahm, 1981) causing
changes in phytohormone levels (Allen et al., 1980) and photosynthetic rates
(Aliened a/., 1981).
The isolates of G. fasciculatum has been found to improve the nutrient
status of tomato plants, as the phosphorus, nitrogen and potassium contents
were enhanced greatly in the plants improved nutrient status in the mycorrhizal
plants resulted in increased biomass production and growth potential. This
supports the earlier findings that mycorrhizal infection can cause a beneficial
physiological effect on host plants by increasing uptake of soil phosphorus
82
(Gerdemann, 1968, 1975). Better growth of cotton plants with Gigaspora
calospora and G. margarita has been observed earlier by Roncadori and
Hussey (1977). Similar results have been obtained for sorghum, upland rice
and other crop plants by Singh and Tilak (1990; Asif e/ ai, 1995 and Mosse et
ai, 1973). It has been suggested by a number of workers that mycorrhizae can
influence plant water relationship by reducing plant resistance to water
transport (Safir et ai, 1972; Hardic and Layton, 1981; Allen et al., 1982) and
thus may also improve drought tolerance.
In general root-knot nematode caused adverse effect on tomato piant,
while AM fungus produced beneficial effects by promoting the growth when
both were applied together, the adverse effect of the nematode was nullified to
a great extent by the endomycorrhizal fungus. The addition of G. fasciculatum
spores reduced the adverse effect of the nematode M incognita. The nematode
caused a maximum loss amounting to 37.25, 47.41, 40.24 in shoot, root and
total dn/' weights of the host plant at 4800 J2 level and this loss was overcome
by the addition of 800 spores/plant and there was also an improvement in the
shoot, root and total dry weight by 54.11, 46.94 and 52.0 respectively,
compared to control. A vegetative correlation has been found between
mycorrhizal development and the populations of plant parasitic nematode by
Schenck and Kinloch (1974); Sitaramaiah and Sikora (1996) on cotton with G.
fasciculatum and Rotylenchulus reniformis.
Nematode population in the soil generally decreased in concomitant
inoculation. Nematode population in root, egg mass production, fecundity, root
galling and also root-knot indices and egg mass indices were found to be
83
affected in the present study in the presence of endomycorrhizal fungus.
Reproduction and development of M. incognita have been found to be affected
markably by G. fasciculotum at the rate of 400 and 800 spores/plant at all the
inoculum levels of nematode. Saleh and Sikora (1984) also recorded that
symbiont caused reduction by 59 per cent in neamtode population at a spore
level of 750/plant. In present study it has been found that M. incognita reduced
the mycorrhizal colonization percentage, as well as arbuscules and
chlamydospores number, although the values improved with the increasing
inoculum levels of G. fasciculatum.
Mycorrhizal fungi which form the symbiotic association with plant
roots, promote plant health and growth have also been suggested by others
(Gerdemann, 1968; Harley, 1969; Howeler et al, 1987; Lin-Xian and Hao,
1988; Raju et al, 1990 and Tinker, 1975). The present observations clearly
show that increasing levels of G. fasciculatum improve plant growth and
health, while M. incognita suppresses the same, and the adverse effect of the
nematode become lesser and lesser as the number of G. fasciculatum spores
increase. Mycorrhizal plants with low newmatode inoculum showed maximum
growth potential than the mycorrhizal plants with higher inoculum levels of
nematode. The number of nematodes in the roots, the number of egg
masses/root system have also been noted to be lower in the mycorrhizal plants
compared to non-mycorrhizal ones. The reduction in the development and
severity of root-knot disease may be due to complex physiological and
biochemical changes in the mycorrhizal roots and the resistance of the host
induced by the AM fungus. This is in agreement with earlier investigations
which reveal that nematode susceptible plants colonized by endomycorrhizal
84
fungus are better able to tolerate plant pathogenic nematodes by showing better
growth (Verdejo et al, 1990; Palacino and Leguizaman, 1991; Lingaraju and
Goswami, 1993 and Sikora and Sitaramaiah, 1995). In a previous study Singh
et al. (1990) found that pre-infection of tomato roots with G. Jasciculatum
made Pusa Ruby resistant to root-knot nematode by bringing about biochemical
changes in the levels of ligneous and phenolic compounds.
Mycorrhizae help to withstand the antagonistic effects of the nematode
and increase plant growth. Application of G. fasciculatum at the higher
inoculum level benefits the plant most and promotes the plant health by
significantly reducing the nematode population in root, egg mass production,
root galling and fecundity, the above results also indicate that very few
nematodes are present in severely colonized roots. It is also evident that G.
fasciculatum colonized roots are less penetrated by the nematode. Similar
results have also been obtained by Kellam and Schenck (1980) and Grandison
and Cooper (1986). The damage due to nematode disease is generally reduced
in roycorrhizal plants (Carling et al, 1989; Osman et al., 1990; Price et ah,
1995).
Improved phosphorus nutrition by VAM fungi results in root exudation
(Graham et al, 1981) and has been correlated with reduction in soil borne
diseases (Graham and Menge, 1982). Thus, it may be the reason for the
reduced damage by the neamtode in the mycorrhizal plants and may also be
nematode parasitism by AM fungi changes in root morphology,
histopathological changes, physiological and biochemical changes, changes in
the host nutrition (Siddiqui and Mahmood, 1995a,b and Siddiqui and
85
Mahmood, 1998) and competition for food and space (O'Bannon and Nemec,
1979; Saleh and Sikora, 1984) and may also be due to the production of
antifungal and antibacterial metabolities.
Application of M. incognita decreased by the mycorrhizal infection and
sporulation of G. fasciculatum through the mycorrhization increases with
increase in G. fasciculatum inoculum levels. Schenck and Kinloch (1974) and
Linderman (1985) also obtained similar results in the field observations relating
the incidence of mycorrhizal fungi to the incidence of endoparasitic nematodes.
Nematode influenced with mycorrhizal colonization G. fasciculatum on
cowpea (Lingaraju and Goswami, 1993).
86
LITERATURE CITED
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Adjoud, D., C. Plenchette, R. Halli-Hargas and F. Lapeyrie 1996. Response of
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