Bottor of ^liilasiopl)? · 2018. 1. 4. · dose (60.00 ppm) were 51586.90 squ and 43981.70 squ respectively. Thus the test doeses (7.5-60.0 ppm) of the heavy metals caused reductions

INFLUENCE OF CADMIUM AND LEAD ON THE PATHOGENICITY AND CONTROL OF THE ROOT-KNOT NEMATODE, MELOIDOGYNE

INCOGNITA ON TOMATO

ABSTRACT

THESIS SUBMITTED TO THE ALIGARH MUSLIM UNIVERSITY, ALIGARH

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

Bottor of ^liilasiopl)? IN

BOTANY

GHAZALA PARVEEIM

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH -202002 (INDIA)

1995

Industrialisation and heavy application of chemical

fertilisers have created many problems of contamination of

toxic materials in soils. Sewage sludge, industrial wastes

and fertiliser impurities contain many heavy metals such as

Pb, Cd, Ni, Cr, etc. All these metals are knwon to be highly

toxic to plants and animals. Growth and activity of micro

organisms including plant pathogens may be greatly

influenced by the nature and concentration of heavy metal

contamination in soil, which in turn may influence the

disease development in plants.

The focal theme of the present study is to assess

the impact of two common heavy metal pollutants, Cd and Pb

on the root-knot nematode, Meloidogyne incognita, and its

pathogenicity and control on tomato, Lycopersicum esculentum.

The results of different experiments embodying the thesis

are briefly presented as under:

Effect of heavy metals on the hathcinq of Meloidogyne incognita in vitro:

In vitro study revealed that both the heavy metals,

viz. Cd and Pb were highly inhibitory to juvenile hatching of

the root-knot nematode, Meloidogyne incognita. There was a

progressive decrease in hatching with an increase in the

concentration of the heavy metals. The inhibition in the

hatching relative to the control was in the range of 90.7-

74.4% and 100.0-77.6% respectively in different

concentrations (60.0-7.5 ppm) of Cd and Pb, indicating that

Pb was more inhibitory than Cd. In all the cases inhibition

in juvenile hatching at different concentrations of the heavy

metals tested was statistically significant at P=0.01 and

P=0.05 .

2. Effect of heavy metals on the mortality of Afeloidoq-yne incognita in vitro-.

Both the heavy metals, viz. Cd and Pb were found to be

highly toxic to the root-knot nematode, Meloidogyne

incognita. The mortality of the 2nd stage juveniles (J2)

increased with an increase in the concentration of the heavy

metals and the exposure period. There was a linear

relationship between nematode mortality and concentration of

the metals. Lead was found to be more toxic than cadmium.

With the help of regression lines Ec50 values were

calculated; for Cd it was 50.8 ppm at 96 h exposure and for

Pb it was 13.9 ppm at 96h of exposure and 53.5 ppm at 72 h.

Response of different cultivars of tomato Lvcoversicon esculentum to the-root knot nematode. Afeloidoqyne incocynita in relation to heavy metals:

The results of the experiments indicated that all

the test cultivars were susceptible to Meloidogyne incognita

to varying extent. Minimum reduction in fresh weight was

observed in tomato cv. Punjab Kesari and maximum in cv.

Damyanti. The plant growth was further reduced in all the

cultivars excepting in cv. Arka Vikas when the plants were

also treated with the heavy metals. As far as the root-knot

development was concerned, lowest root-knot index was

observed in cv. Arka Vikas and maximum in cv. Pusa Ruby. The

results also revealed that susceptibility of the cultivars

was greatly influenced by the treatment of the heavy metals.

Lead was found to be more inhibitory to the root-knot

development as well as plant growth as compared to that of

cadmium.

4. Effect of heavy metals on the penetration of second stage juveniles (J2) of the root-knot nematode, Afe.I oidoqyne incognita into the roots of tomato, Lycopersicon esculentum cv. Pusa Rxiby.-

Both the test heavy meatls, viz. Cd and Pb

significantly inhibited the penetration of second stage

guveniles (J2) of the root-knot nematode, Meloidogyne

incognita into the roots of tomato cv. Pusa Ruby when the

seedlings were kept in different concentrations of the heavy

metals (7.5 and 15.0 ppm) for different durations (12,24,

48h); Pb was more effective in causing inhibition in larval

penetration than Cd.

5. Effect of heavy metals on the root-knot development and plant growth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby:

An experiment was conducted to assess the

influence of the heavy metals, viz. Cd and Pb on root-knot

disease caused by Meloidogyne incognita and plant growth

parameters of tomato cv. Pusa Ruby. Both the nematode as

well as the heavy metal brought about significant decline in

fresh and dry weights of tomato plants as well as

chlorophyll content of leaves. There was a progressive

increase in retardation of the above parameters with an

increase in the level of the heavy metals and it was further

ameliorated significantly when plants were also inoculated

with the nematode. It was interesting to note that plant

damage due to the heavy metals in terms of percentage was

comparatively less in neamtode inoculated plants than

uninoculated ones. Lead was found to be more effective than

cadmium. A similar trend was also observed in the water

absorption efficiency of plant roots. Both the heavy metals

also had an adverse effect on the root-knot development. It

caused complete check on root-knot development at higher

concentrations, viz. 3 0.0 and 60.0 ppm of both the heavy

metals.

Effect of heavy metals on the growth of females of the root-knot nematode, Afeloidoqyne incognita:

The results of the experiment confirm the

findings of the earlier experiments with respect to the

development of root-knot disease on tomato, as the growth of

nematode female itself was significantly retarded due to

the treatment of both the heavy metals tested.

As compared with 203611.70 sq JJL average cross-

sectional area of the nematode females in the untreated

control, the lowest concentration of cadmium and lead (7.5

ppm) reduced it to 158942.40 sq^ and 144500.30 sq/a

respectively. The corresponding figures for the highest test

dose (60.00 ppm) were 51586.90 squ and 43981.70 squ

respectively. Thus the test doeses (7.5-60.0 ppm) of the

heavy metals caused reductions in the growth of the nematode

females to the extent of 21.94-74.67% in case of cadmium and

29.03-78.40% in case of lead. Not only the growth of the

females of Meloidogyne incognita was retarded due to the

treatments of the heavy metals but the treatments also caused

aberrations in their shapes.

Effect of heavy metals on protein profile of females of the root-knot nematode Meloidogyne incognita:

Effect of heavy metals, viz. cadmium and lead was

studied on the protein profile of the females of

Meloidogyne incognita. Both the heavy metals brought about

reduction in the number of polypeptides. A declining trend

was observed in the number of polypeptides with increasing

concentration of the heavy metals. As compared to 19

polypeptides encountered in the untreated control, the number

of polypeptides decreased to 13 and 11 when plants were

treated with 7.5 and 15.0 ppm of Cd in soil respectively. The

corresponding figures for Pb were 13 and 6 respectively.

These results again are in conformity with the earlier

results indicating more injurious nature of lead as compared

to cadmium.

It was interesting to note that polypeptides of

higher moelecular weights were replaced by polypeptides of

low molecular weights indicating a possible fragmentation

of polypeptides of higher molecular weights due to the

action of the heavy metals.

8. Efficacy of organic amendments against the root-knot nematode, Afeloidogyne incognita on tomato, Lycopersicon esculentum in relation to heavy metals:

Efficacy of the organic amemdments, viz. oil-seed

cakes of neem (Azadirachta indica) and castor {Ricinus

communis) and rice polish against the root-knot nematode,

Meloidogyne incognita attacking tomato cv. Pusa Ruby was

compared in presence and absence of the heavy metals, e.g.

cadmium and lead. All the organic materials tested,

significantly reduced the root-knot development and improved

plant growth. Neem cake caused greatest improvement in plant

growth parameters followed by castor cake, neem leaf, castor

leaf and rice polish.

Both the heavy metals were found to be injurious

to tomato plants and lead being more injurious than cadmium

as evident from the reductions in the growth parameters and

this reduction was more than that caused by the nematode in

all the treatments (organic amendments as well as inorganic

fertilisers). The combined effect of heavy metals and the

nematode was much higher than that caused by either of them

and in all the cases it was more than the sum total of the

reductions caused by the heavy metals and the nematode alone

excepting the application of neem and castor cakes to the

soil, indicating the beneficial effects of both the oil -seed

cakes against the heavy metals and the root-knot nematode.

Efficacy of Paecilomyces lilacinus against the root-knot nematode, Meloidoovne incognita on tomato, Lycopersicon esculentvm in relation to heavy metals:

In vitro studies revealed that both the test

metals, viz. lead an cadmium were toxic to the nematode bio-

control fungus, Paecilomyces lilacinus as evident from the

reductions in the dry mycelial weight and sporulation of the

fungus. It was found that there was a gradual reduction with

increasing doses of the heavy metals. As in earlier

experiments, lead was found to be more toxic to the fungus

than cadmium.

The test fur gus, P. lilacinus controlled the

root-knot nematode, M. incognita significantly and thus

improved the plant growth. However, its potentiality as a

bio-control agent against the nematode was impeded by the

heavy metal treatment, lead being more harmful than cadmium.

10. arrnitini.ation of heavy matals in tomato. Lycopersicon esculentum infected with the root-knot nematode, Mel oi docryne incooni ta:

Amounts of the heavy metals accumulated in the

tomato plants (shoots as well as roots) were assessed with

the help of Atomic Absorption Spectrophotometry (AAS) on

dry weight basis. It was found that there was more

accumulation of the heavy metals in roots than in shoots.

However, in both the cases, there was a tendency of more

accumulation with increasing dose of the heavy metal. It

was interesting to note that uptake of the heavy metals was

more in nematode-inoculated plants than uninoculated ones.

11. Micronutrient status of heavy metal stressed tomato, Lycoversicon esculentum infected with the root-knot nematode, Afeloidoqyne incocroita:

The uptake/accumulation of the heavy metals, as

observed in the above experiment, had a great impact on

the translocation of other micronutrients present in the

soil. Manganese (Mn) was observed in maximum amount as

compared to Cu and Zn in heavy metal-stressed plants both in

the nematode -inoculated as well as uninoculated sets. It was

quite interesting to note that the micronutrients absorbed

in the inoculated plants were in a much lesser amounts than

in the uninoculated ones, indicating an inverse relation

between the amounts of the heavy metals absorbed in the

inoculated sets and absorption of Cu, Zn and Mn.

INFLUENCE OF CADMIUM AND LEAD ON THE PATHOGENICITY AND CONTROL OF THE ROOT-KNOT NEMATODE, MELOIDOGYNE

INCOGNITA ON TOMATO

THESIS SUBMITTED TO THE ALIGARH MUSLIM UNIVERSITY, ALIGARH

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

JBottor of ^liiloisoplip IN

BOTANY

GHAZALA PARVEEN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH - 202002 (INDIA)

1995

_ i w &a > '

. •) 4 '"1 ^ ,

vi^^ N^^ l

T4776

Zhe thesis entitled " Jnfluenee of cadmium and lead on the pathogenicity and control of root-knot nematode, jMM^^lM^ //f^^mYa on tomato'\ is a faithful record of the bonafide research work carried out by Ms, Qhazala Parveen, Senior Cecturcr of botany. Women's College, AJH. U., Aligarh, as a teacher candidate under our guidance and supervision. Jier work is up-to-date and originaL Jô part of this thesis has been submitted for any other degree or diploma. She is allowed to submit her thesis to the Migarh Muslim University, Migarh for consideration of the award of the degree of Doctor of Philosophy in botany.

MJUSMWC?/^ J/:AM Pk.D..DSc., Pg êmMtal..7PSJ.7SS.7KKS

Professor of Plant A^matology. Department of botany,

Migarh Muslim University. Aligarh

(SUPSKVJSOK)

f.WKt SAM/U££M /(y/JJ\^

MSc..Pk.D.(S0il ekemistrf)

Keader. Department of Applied Chemistry. ZM. College of Engineering 4 Zeehnology.

Aligarh Muslim University. Aligarh

(eO-SUPBKVJSOK)

dirst, J offer my thanks to the Almighty Allah, for having blessed me with inspiration and persistent patience without which J could not have seen this work through.

J deem it my most pleasant duty to acknowledge my indebtedness and to convey my deep sense of profound gratitude to Prof. M. Mashkoor Alam. 'Deptt. of botany, AM.U., Aligarh under whose supervision J could venture to complete my thesis. Mis unstinted and unrelenting help and sagacious ness and proficiency in the subject enabled me to quell the problems came across during the completion of the thesis.

J also wish to extend my heartfelt thanks to Prof. Abrar M. Khan, doyen of Plant Jêmatology and Professor Emeritus in the 'Deptt. of botany with whom J have had the privilege to discuss the subject. Me has always been a great source of personal and professional inspiration, to me.

J wish to cKtend my thanks to Prof. Wazahat Musain, Chairman, Deptt. of Uotany, AMU., Aligarh, for his solicitude and helpful demeanour throughout the course of study.

My earnest thanks are due to Prof. (Mrs.) Zakia A. Siddiqui, Principal. Women's College for sparing me from the college and her constant encouragement and affection shown to me. whenever. J needed them the most.

J am also thankful to Dr Samiullah Khan. Header. Deptt. of Applied Chemistry. Z.M. College of Engineering and technology for sorting out the problems while working on Atomic Absorption spectrophotometer for an lysis of heavy metals. Zhanks are also due to Prof At Ajmal. the then Chairman. Deptt. of Applied Chemistry, Z.M. College of Engineering ^ Zechnology for allowing me generously to work in his department.

J would like to have a special mention of Prof. Wajih A- Jîzami. Deptt. of Zoology. AM. U. Aligarh and his student Dr Khalid Saifullah for their altruistic and helpful attitude and allowing me to work on Electrophoresis for analysis of proteins and analysing the data thereafter.

My thanks are also due to all my colleagues and friends who assisted me, in one way or the other, during the course of present study.

J have no words to express fully the indebtedness, benevolance and gratitude J owe to my parents and their affection, compassion and clemency which has always been a source of inspiration to me.

— Q.P.

CONTENTS

INTRODUCTION 1-5

REVIEW OF LITERATURE 6-36

MATERIALS AND METHODS 3 7-58

RESULTS 59-160

Effect of cadmium (Cd) on the hatching of the root-knot 59 nematode, Meloidogyne incognita in vitro.

Effect of lead (Pb) on the hatching of the root-knot 59 nematode, Meloidogyne incognita in vitro.

Effect of cadmium (Cd) on the mortality of second stage 64 juveniles (J2) of the root-knot nematode, Meloidogyne incognita in vitro.

Effect of lead (Pb) on the mortality of second stage 64 juveniles (J2) of the root-knot nematode, Meloidogyne incognita in vitro.

Response of some selected cultivars of tomato, 69 Lycopersicon esculentum to the root-knot nematode, Meloidogyne incognita in the presence of cadmium (Cd) .

Response of some selected cultivars of tomato, 74 Lycopersicon esculentum to the root-knot nematode, Meloidogyne incognita in the presence of lead (Pb).

Effect of cadmium (Cd) on the penetration of second 79 stage juveniles(J2) of the root-knot nematode, Meloidogyne incognita into the roots of tomato, Lycopersicon esculentum cv. Pusa Ruby.

Effect of lead (Pb) on the penetration of second stage 79 juveniles (J2) of the root-knot nematode, Meloidogyne incognita into the roots of tomato, Lycopersicon esculentum cv. Pusa Ruby.

Effect of cadmium (Cd) on the root-knot development 84 caused by the root-knot nematode, Meloidogyne incognita and growth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby.

Effect of lead (Pb) on the root-knot develoment caused 95 by the root-knot nematode, Meloidogyne incognita and growth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby.

Effect of cadmium (Cd) on the growth of the females of 107 root-knot nematode, Meloidogyne incognita on tomato, Lycopersicon esculentum cv. Pusa Ruby.

Effect of lead (Pb) on the growth of the females of 107 root-knot nematode, Meloidogyne incognita on tomato, Lycopersicon esculentum cv. Pusa Ruby.

Effect of cadmium (Cd) on the protein profile of 114 females of root-knot nematode, Meloidogyne incognita.

Effect of lead (Pb) on the protein profile of females 117 of root-knot nematode, Meloidogyne incognita.

Efficacy of organic amendments on the root-knot 120 development caused by Meloidogyne incognita and plant grwoth parameters of tomato, Lycopersicon esculentuiii cv. Pusa Ruby in the presence of cadmium ( Cd) .

Efficacy of organic amendments on the root-knot 127 development caused by Meloidogyne incognita and plant grwoth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby in the presence of lead (Pb).

In vitro growth of nematode biocontrol fungus, 135 Paecilomyces lilacinus in the presence of cadmium (Cd) .

In vitro growth of nematode biocontrol fungus, 135 Paecilomyces lilacinus in the presence of lead (Pb) .

Efficacy of Paecilomyces lilacinus on the root-knot 142 development caused by Meloidogyne incognita and plant growth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby in the presence of cadmium (Cd).

Efficacy of Paecilomyces lilacinus on the root-knot 144 development caused by Meloidogyne incognita and plant growth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby in the presence of lead (Pb).

Estimation of cadmium (Cd) by Atomic Absorption 146 Spectrophotometry (AAS) in tomato, Lycopersicon esculentum cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

Estimation of lead (Pb) by Atomic Absorption 149 Spectrophotometry (AAS) in tomato, Lycopersicon esculentum cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

Estimation of micronutrients, viz. Cu, Zn and Mn by 152 Atomic Absorption Spectrophotometry (AAS) in cadmium stressed tomato, Lycopersicon esculentum cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

Estimation of micronutrients, viz. Cu, Zn and Mn by 156 Atomic Absorption Spectrophotometry (AAS) in lead stressed tomato, Lycopersicon esculentum cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

DISCUSSION 161-181

SUMMARY 182-186

REFERENCES 187-19y

mznoDuezjo^

1. INTRODUCTION

Nematodes are multicellular invertebrates which belong to

the phylum Nematoda. These are small, elongate, tubular

animals that live on the moist surfaces,in the soil, in fresh

and marine waters, and in the interior of animals and

plants. Out of known nematode species, about 50 percent are

marine and 25 percent are free living forms. Remaining ones

are either plant parasitic (10%) or animal parasitic (15%)

forms.

Plant parasitic nematodes are one of the important

limiting factors of plant growth and productivity, as they

cause great destruction to plants singly or in collaboration

with other pathogens and parasites. There is hardly any

economic crop and for that matter any plant which is not

being parasitised by one or more nematode species at a given

time. The crop losses due to nematodes are not only in the

form of reduced plant growth and yield but are also in the

marketable quality of the produce. In a recent study based on

a world-wide survey, Sasser & Freckman (1987) have estimated

crop losses to the tune of $ 100 billion per annum.

The main sphere of activity for most of the plant

parasitic nematodes is soil. Even the aerial parasites also

have a soil-phase in their life-cycle. As one can visualise,

the soil environmental factors - biotic as well as abiotic -

greatly influence the nematode biology and behaviour,

directly or indirectly through host plants. Much information

is available with respect to the influence of physical,

chemical and biotic factors of the soil on plant parasitic

nematodes and the diseases they cause. However, it is quite

meagre in case of extraneous factors, e.g. pollutants, which

are being introduced continuously in the ecosystem.

The definition of "pollution" given by Holdgate (1979)

is widely recognised. This states that "pollution is the

introduction by man into the environment of substances or

energy liable to cause hazards to human health, harm to

living resources and ecological systems, damage to structures

or amenity or interference with legitimate uses of the

environment".

Pollutants generally affecting the soil environment, may

be broadly categorised as inorganic and organic pollutants.

Inorganic Pollutants : Inorganic pollutants are lead,

cadmium, iron, manganese, nickel, chromium, mercury, arsenic,

copper, cobalt, selenium, zinc, etc. It also comprises of

some metals and metalloids, viz. antimony, gold,

molybdenum, silver, thallium, uranium, vanadium, etc. These

inorganic pollutants are continuously added to the soil

through industrial effluents, organic wastes, refuse burning,

transport, power generation, smoke release from domestic and

industrial outlets, etc. These have been found to suppress

the plant yields at their different levels of concentrations

(Dijkshoorn et al., 1979; II'in & Stepanova, 1980; Brauns &

Anthony, 1983; Page,1974, 1981).

Organic Pollutants : Various industrial wastes, sewage

sludges and domestic wastes have been reported to contain

many types of organic compounds like alcohols, ketones,

aldehydes, amines and others. Accumulation of these chemicals

in soils and their subsequent mobilisation into the plants

have posed a serious concern ( Cole et al., 1969) .

The rapid industrialisation has not only caused

significant changes in aerial enviornment but also in soil

environment. The aerial pollutants emanating from industries

not only effect the aerial parts of the plants but they also

affect the below ground parts when they come to the soil

through rains, winds, gravity, etc. Similarly, the industrial

effluents are directly incorporated into the soil. In such

situations, not only the host plants but the whole soil

biota is greatly influenced. These pollutants most often

contain various heavy metals.

The toxic metals naturally occur in very small

quantities in the earth's crust (<1000 ppm) and hence are

called "trace metals". Theseare further arbitrarily sub

divided on the basis of their densities: those having

densities below 5g/cm^ are called "light metals" and those

with densities above 5g/cm- are designated as "heavy metals".

The metals like Hg, Pb, Cu, Cd, Zn, Ni etc. are generally

known as "toxic heavy metals".

The total metal content of a soil is the result of

inputs of metals from several sources: parent material,

atmospheric deposition, fertilisers, agrochemicals, organic

wastes and other inorganic pollutants, minus losses in metals

removed in crop material, leaching and volatilasation

(Alloway, 1990). The concentrations of heavy metals in

agricultural soils vary from place to place depending upon

the quantum of inputs. Alloway (1990) has given range of

different heavy metals in agricultural soils as under :

Element

Ag As Au Cd Co Cr Cu Hg Mn Mo

Range (mg/kg

0.01-8 0.1-50 0.001-0.02 0.01-2.4 1-40 5-1500 2-250 0.01-0.3 20-10000 0.2-5

soil) Element

Ni Pb Sb Se Sn Ti U V W Zn

Range (mg/kg soil)

2-1000 2-300 0.05-260 0.01-2 1-200 0.03-10 0.07-9 3-500 0.5-83 10-300

The above range is based on the available data (Alloway,

1990) which in all the probability may increase manifolds,

keeping in view to-days scenario of urbanisation and

pollution hazards.

Effects of gaseous and heavy metal pollutants have been

studied on various micro-organisms such as fungi,

cyanobacteria, algae, lichens, viruses, etc. (Babich &

Stotzky, 1982). There are also some reports available on the

impact of pollutants on marine and free-living nematodes.

Marine nematodes have been considered promising indicators of

pollution because of their ubiquity and taxonomic diversity

(Hodda & Nicholas, 1986). These nematodes have already been

used in bioassay studies to determine the relative toxic

effects of sediments (Howell, 1982; Tietjen & Lee, 1984), or

fractions of sediments (Samoiloff et al., 1983), or

environmental pollutants (Feldmesser & Rebois, 1966; Popham &

Webster, 1979). Some work has also been done on the effect of

pollutants especially the gaseous ones on plant parasitic

nematodes. However, not much information is available

relative to the impact of heavy metals on them. An effort

has been made to review the relevant literature on the above

aspect in the subsequent chapter.

2. REVIEW OF LITERATURE

India is basically an agriculture based country, where

agricultural technologies in practice are both traditional

and modern. However, the former one is quite predominant. As

far as the output is concerned, it is quite low as compared

to the advanced countries. The major constraints for the low

productivity of our crops are low rain fall, low fertiliser

inputs, less availability of certified seeds, traditional

agriculture systems in practice and biotic (pests, parasites,

pathogens) and abiotic (environmental stresses including

pollution problems) agencies. More recently " Pollution

Pathology" has emerged as distinct discipline where

interactive effects of pollutants and pathogenic agencies and

their impact on crops are studied (Darley & Middleton, 1966;

Heagle, 1973; Heck, 1966,1968; Kosuge, 1969; Rich, 1964).

As man continues to add pollutants to the environment,

they continue to be widely dispersed. As a result, plants,

plant pathogens and plant pathogenesis are all being

affected. At present, very little is known of the effects of

pollutants on pathogenic diseases of plants. The pollutants

may effect the pathogenesis in different ways; it may be

increased or decreased through a direct effect of the

pollutant or indirectly through pollutant - induced changes

in the host plant or through changes in other aspects of the

environment. With all pollutants, the threshold concentration

required to injure plants is affected by the resultant of

duration of exposure X cone, of pollutant and a multitude of

interacting biological and meteorological variables.

IMPACT OF POLLUTANTS ON FUNGI (Table 1 )

Sulfur dioxide:

Sulphur dioxide (SOj) is released into the atmosphere

during fuel combustion, petroleum refinement, ore smelting

and through the natural sulfur cycle. There are several

accounts of the effects of industrial emissions on the

incidence of fungal diseases in the field.

Sulf' r dioxide may act directly upon the fungi or

indirectly through some effects upon plant tissues, soil or

water. The oxidation and dissolution of SO2 in water has

significantly increased the acidity of precipitation, thereby

increasing the acidity of soils, rivers and several areas of

the world.

Decreased parasitism of the species of Cronartium ,

Melampsora, Peridermium and Puccinia was observed in the

localities where plants were injured by SO2 (Scheffer &

Hedgcock, 1955). Jancarik (1961) observed the occurrence of

sporocarps of various fungi (mostly basidiomycetes) in a

severely polluted area and in a relatively clean industrial

area. SO2 has rarely been reported to increase the incidence

of fungal diseases. There was an increase in trees injured

by SO2 and infected with Armillaria mellea (Donaubauer,

1968; Jancarik, 1961; Kudela & Novakova, 1962).

Fungal hyphae of different species vary in sensitivity

to large doses of SO2 (Mc Callan & Weedon, 1940). Growth of

Aspergillus niger, Alternaria brassicicola and Didymellina

macrospora was not affected but a Penicillium sp. was

slightly stimulated in nutrient solutions that contained the

equivalent of 90 ppm SO2 (Saunders, 1966). The spores of most

fungi appeared to be resistant to direct exposure to SO2.

Moist spores apparantly are more sensitive to SO2 than dry

spores. Spores can be affected by SO2 dissolved in water

(Saunders, 1966).

Ozone :

Ozone is the most important plant pathogenic component

of photochemical oxidant air pollution and causes extensive

plant injury in many areas of the world. The effects of O3

on higher plants are documented in several reviews

(Middleton, 1961; Rich, 1964; Darley & Middleton, 1966; Heck,

1968) .

Effects of ozone on obligate parasitism have been well

studied. Studies were made on two rust fungi (Heagle, 1970;

Heagle & Key, 1973) and two barley powdery mildews (Schuette,

1971; Heagle & Strickland, 1972). Ozone can decrease

infection, invasion and sporulation of parasitic fungi. High

doses of O3 inhibited development of disease in petals

inoculated with Botrytis gladiolorum (Magie, 1960,1963). It

decreased the disease incidence of chrysanthemum petals

caused by Botrytis spp. even though the petals were injured

(Magie, 1963) . Small doses of O3 can also inhibit obligate

parasitims (Heagle, 1970). Ozone can affect flowering plants

in many ways that could alter the processes of plant

parasitism by fungi (Darley & Middleton, 1966; Heck, 1968;

Rich, 1964) .

Ozone also effects fungal growth, sporulation and spore

germination (Hibben & Stotzky, 1969; Ingram & Haines, 1949;

Kormelink, 1967; Kuss, 1950). Ozone is more effective than

SO2 in decreasing spore germination. The sensitivity of 0^

depends upon fungus species, spore morphology, moisture and

substrate.

Ozone may adversely affect the morphology of various

micro-organisms. In Alternaria solani, it was found that

conidia germinated while they were still attached to

conidiophore (Rich & Tomlinson, 1968) . There was significant

loss of pigmentation and light refractive globules in hyphae

in Colletotrichum lindemuthianum (Treshow et al., 1969).

Suppression of aerial hyphae has been reported in

Trichoderma viridae, Botrytis allii, Penicillium egyptiacum,

P.expansum, Phytophthora cactorium, Colletotrichum

lindemuthianum, Alternaria oleracea and Helminthosporium

sativum (Watson, 1942; Hibben & Stotzky, 1969; Treshow et

al. ,1969) .

10

Fluoride;

Fluoride is widely found in soil, rocks and minerals.

It is released into the air when these materials are heated

or treated with acid. Hydrogen fluoride (HF) is generally

considered to be the most important plant pathogenic

fluoride. Major fluoride sources are the ore smelting and

phosphate fertiliser industries. The tissues of most plants

which are not exposed to fluoride pollution may contain

about 2-20 parts per million fluoride. Concentration of

fluoride ranging between 25-50 ppm may cause injury to the

sensitive plant species. Some species can accumulate several

thousand parts per million with no visible effects.

Direct effect of fluoride on parasitism has not yet

been reported. However, there are some reports available from

a few Isiboratory and greenhouse studies. Colony growth of

fungi was inhibited on agar containing sodium fluoride

(NaF) . Inhibition of Verticillium alboatrum and

Helniinthosporium sativum occurred at more NaF than it was

required to inhibit Pythium debaryanum. Growth of Botrytis

cinerea and two CoIIetotrichuin spp. was stimulated slightly

at low concentration (Treshow, 1965). Sodium fluoride in

nutrient agar decreased sugar utilisation and formation of

itaconic acid by Aspergillus terreus (Lai & Bhargava, 1962).

Fluoride concentration,in the leaves, of more than 300

ppm decreased foliar infection and invasion by two bean

pathogens, Uromyces phaseoli and Erysiphe polygoni and one

11

tomato pathogen Alternaria solani. This is somewhat greater

than is usually found in vegetaion near emission sources. It

is possible that smaller amounts of fluoride in leaf *• issue

could cause stimulation of disease similar to the

stimulation of fungal growth on agar containing NaF (Treshow,

1965).

IMPACT OF POLLUTANTS ON BACTERIA (Table 2)

Sulphur dioxide:

No effects of SO2 on plant pathogenic bateria have been

reported. However, Mrkva & Grunda (1969) reported that change

occurred in bacterial population in the area of SO2

emissions. Fewer numbers of bacteria were found in the soil

near a smelter where the soil pH, soil potassium and soil

magnesium were decreased.

Hydrogen fluoride:

Effects of fluoride have been studied on the parasitism

of bean by Pseudomonas phaseolicola (c.f., Heagle, 1973).

Fluoride may decrease disease caused by P. phaseolicola.

Nitrogen dioxide:.

Sulphur dioxide (SO2) and nitrogen dioxide are probably the

most important air pollutants in those industrialised

countries situated at higher altitudes ( Heck, 1968) . The two

gases are usually found together because the combustion of

12

sulphur containing fuels produces nitric oxide as well as

SO2. Photosynthetic microbes, located in the top layer of

soil and capable of raducing nitrate to nitrite, are

presumably another source of atmospheric nitric oxide and

nitrogen dioxide (Makarov, 1969).

NO2 has been found to inhibit active transport uptake

of glucose by Pseudomonas aeruginosa (Rowe et al., 1979). NO2

(as HNO2) was found to be mutagenic to Bacillus spp.,

Aspergillus nidulans and Neurospora crassa (Babich & Stotzky,

1974; Fishbein, 1976). The concentration of NO2 of 0.15 nl'-^

was found to be most toxic to aerosolised Rhizobium ineliloti

at high r.h. levels, i.e. 95% (Won & Ross, 1969).

IMPACT OF POLLUTANTS ON VIRUSES (Table 3)

Pollutants affecting viral diseases have only been

studied in case of tobacco mosaic virus (TMV) . It has been

found that both fluoride and ozone either predispose pinto

bean leaves to TMV infection or increase the infectivity of

the virus particles. The number of TMV lesions was greater on

inoculated bean leaves than on the controls when leaves

contained 100-300 ppm fluoride (Treshow, 1965; Treshow et

al., 1967). But when the concentration of pollutant exceeded

500 ppm the reverse was found (Treshow, 1965) . Ozone causes

increased virus multiplication in tobacco or increased

infectivity of existing virus particles or both (Brennan &

Leone, 1970) .

13

IMPACT OF POLLUTANTS ON NEMATODES

Several reports of the effects of pollutants on plant

pathogens have been published, but relatively few have

concerned nematodes (Heagle, 1973). Studies relating to the

alteration in the course of development of plant diseases

caused by nematodes are also quite meagre.

Since very little is known about the effects of air

pollution on nematodes and since industrial and urban

pollution is becoming more prevalent, plant pathologists

should be concerned with this problem not only because of the

possible effect on the biological pathogens per se in

relation to disease development, but also because of the

possible usefulness of the response of these organisms as

indicators of the source and extent of air pollution.

In the context of pollution impacts on nematodes,

preliminary studies have been made on marine/free living

forms and the work on plant parasitic nematodes is rather a

recent interest of the scientists.

FREE-LIVING/MARINE NEMATODES (TABLE 4)

Marine nematodes have been considered promising

indicators of pollution because of the ubiquity and taxonomic

diversity. Hodda & Nicholas (1986), found that polluted areas

were more diverse taxonomically than unpolluted areas. Quite

a large number of reports are available on the effects of

pollutants on marine and free living nematodes. Toxicity of

14

various metals has been studied in detail using the nematode,

Caenorhabditis elegans. The testing was performed with

soluble forms of Ag, Hg, Cu, Be, Al, Pb, Cr, As, Ti, Zn, Cd,

Ni, Sr and Sb. In all the cases LC^Q 'values for 1-4 days of

exposure were determined. It was found that the metals Pb, Cr

and Cd had the lowest LCgQ at 96 h (0.06 mg/L) with C.

elegans while Sr had the highest LC^Q (465 mg/L) . All the

metals except As had a significantly lower LC^Q (20 - 15,000

times lower) at 96 h than at 24 h. Comparison with other

invertebrates indicated that C. elegans was more sensitive to

Pb, Cr and Be and less sensitive to As. This type of study

may be valuable in determining its usefulness in identifying

C. elegans as an aquatic test species (Williams & Dusenbery,

1990) . Again they carried out a similar study on C. elegans

to assess mammalian acute lethality to 8 metallic salts, viz.

HgCl2, BeS04.4H20, A1(N03)3. 9H2O, CUCI2.2H2O, ZnCl2,

Pb(N03)2, CdCl2 and Sr NO3. C. elegans was found to be a

predictor of mammalian acute lethality, having Lc^Qg parallel

to rat and mouse LD^Q^ (Williams & Dusenbery, 1988). Toxicity

of cadmium has also been studied in C. elegans (Popham &

Webster, 1979).

In a recent study made by Van Kessel et al. (1989), it

was found that the growth of C. elegans was significantly

reduced from a level of 1 uM CdCl2. Reproduction of the

nematodes was also reduced at 1 uM exposure level. The growth

was retarded at the early juvenile stages at levels of 160

and 320 juM and did not reach the adult stage and therefore

did not reproduce.

15

The toxicity of seven heavy metals, viz. nickel,

cadmium, lead, chromium, mercury, copper and zinc was studied

on a free-living nematode, Panagrellus silusiae. Acute lethal

toxicity values (LCCQ values) were obtained for each heavy

metal and Panagrellus was found to be highly resistant.

Juvenile worms were slightly more sensitive than adults

but this trend was not significant. It was found that copper,

chromium and cadmium effectively blocked growth at all stages

of development. The highest soluble concentrations of lead

and nickel were without any effect on growth of the worms,

high concentration of zinc (>500 mg/1) partially blocked

growth and mercury was either lethal at 20 mg/1 or

ineffective in blocking growth at 10 mg/1 (Haight et al. ,

1982) .

The importance of cadmium as a pollutant of the marine

environment has been recognised for sometime. It is a highly

toxic metal and introduced to the sea as a by-product of

several industrial processes. It is accumulative and may be

concentrated by organisms to levels well in excess of those

in the environment (Douglas - Wilson, 1972; Eisler, 1981;

Wright, 1978) .

Cadmium has been used to study the movement of the

metal through the marine nematode, Enoplus brevis. When the

nematode was exposed to filtered seawater containing 109 Cd

as cadmium chloride, the metal first appeared in association

with cuticle polyacrylamide gel electrophoresis showed that

16

the metal was being bound in the cuticle by a protein having

a molecular weight of 45,000 Daltons. Thereafter, it was

detected in the hypodermis and muscle layers found to be

associated with a protein of M.W. of 28,000 Daltons and

finally it appeared to accumulate in the gut, associated

with a protein of M.W. of 200,000 Daltons.

Howell & Smith (1983) found that the proteins in the

marine nematode Enoplus brevis were associated with the

binding of copper and cadmium from seawater. The molecular

weights of the proteins were estimated to be 29,000 and

63,000 Daltons under denaturing conditions while they were

found to be 28,000 and 450,000 Daltons under non-denaturing

conditions. It has been found that the initial uptake of

metals by marine invertebrates may occur by binding to a

variety of proteins (Decleir at al. , 1978; Jennings et al. ,

1979; George & Pirie, 1980). Recently several workers have

used living nematodes to study the toxic effects of heavy

metals (Popham & Webster, 1979, 1982; Haight et al., 1982;

Mudry et al., 1982).

Cadmium has been found to be toxic against saprophytic

and plant parasitic nematode also. The metal has nematicidal

fraction of the molecule and its efficacy depends upon water

solubility and method of application. In vitro studies have

shown that mixed population of Rhahditis and Panagrellus

was affected by soluble cadmium compounds (e.g. sulfate,

nitrate, chloride, bromide, acetate) and were directly

17

related with concentrations and exposure time. Three

insoluble compounds, cadmium acetyl acetonate (CAA) and the

oxide and carbonate, showed less activity. CAA was lethal

after one week at 200 ppm and others showed a varying degree

of toxicity (Feldmesser & Rebois, 1966).

PLANT PARASITIC NEMATODE (TABLE 5)

During the last decade, research on plant parasitic

nematodes in ecological and agricultural processes has

gained more attention. Their large numbers in soils and

water and their ubiquitous nature ask for this attention. In

terrestrial ecosystems, nematode populations can reach

densities of 30-55 million individuals per square meter. They

can be dominated by one single species, but various species

may also be present (Yeats, 1979, 1981) . There are various

reports available on the effects of air pollutants on plant

parasitic nematodes, and comparatively less account regarding

the effect of heavy metals on plant parasitic nematodes is

found. The two most common air pollutants, viz. SO2 and 0-.

have been discussed at length regarding their effects on

nematodes and diseases caused by them.

Weber et al. (1979) reported the effects of sulfur

dioxide, ozone and charcoal filtered air (control) on

reproduction and host-parasite relationships of some plant

parasitic nematodes. The reproduction and development of

Heterodera glycines and Paratrichodorus (Nanidorus) minor was

inhibited when infected soybean plants were exposed to O3 +

18

SO2 and charcoal filtered air (control). The nematode

Belonolaimus longicaudatus, however, remained uneffected by

the pollutant exposure. Host plants when exposed to SO2

enhanced the reproduction of Pratylenchus penetrans than

those which were exposed to the charcoal filtered air

control or O3. The same was true with begonia infected with

Aphelenchoides fragariae. Leaves pre-exposed to O3 or a

mixture of O3+SO2 resulted in greater suppressive effects

before leaves were inoculated with A. fragariae than after

the leaves were inoculated with nematodes. The growth of

nematode-infested soybean and the mixture of O3+SO2 caused

reduction in nodulation of soybean plants inoculated with B.

longicaudatus and P. minor. The maximum inhibition of

nodulation of soybean was causd by H. glycines while

pollutants had no further detectable effect.

Shew et al. (1982) made an investigation to see the

response of ozone and sulfur dioxide on tomatoes in the

presence of Pratylenchus penetrans. There was a decrease in

excised plant parts exposed to 0.2 ul O3/I of air and the

more decrease in dry weight was caused by exposure to SO2 at

0.2 Jul SO2/I of air. But exposed to a mixture of 0.2 ul O3

+ 0.8 ^1 SO4/I of air, they were antagonistic to each other

and caused less change in dry weight of leaf and shoot than

when the pollutants were used individually. The presence of

P. penetrans on root stimulated the negative effects of

O3+SO2 on leaf growth (dry weight), but suppressed the

inihibitory effects of O3+SO2 on the dry weight of axillary

19

Shoots. Concentration of SO2 at 0.8 ul SO2/I of air reduced

the weight of tomato fruit but it was antagonised in the

presence of 0^.

Bisessar & Palmer (1984) investigated the effects of O3

on Meloidogyne hapla. Seedlings of tobacco cv. Virginia - 115

were transplanted in the fields receiving O3 at 800 ppm

concentration and the effect of pollutant was monitored in

the absence and presence of the nematode. Some seedlings were

sprayed with an anti-oxidant (EDUC-ethylene diurea). Growth

and yield of tobacco decreased in the presence of O3,

irrespective of that whether plants were inoculated with M.

hapla or not. However, the inoculated plants were subjected

to more O3 injury than uninoculated ones. Galling was also

redcuced in the plants which were inoculated with M. hapla

and received sprayings of EDU than non-sprayed plants.

Shriner (1978) observed reduction in root infection and

reproduction of M. hapla on red kidney beans when plants were

treated three times weekly with simulated acid rain at pH 6.0

or 3.2.

Singh (1989) found the suppressive effects of SO2 at

0.1 and 0.2 ppm on eggmass production, root galling and

juvenile hatching of Meloidogyne incognita and M. javanica on

chickpea and lentil. Suppressive effects of SO2 (0.1 and 0.2

ppm) and nematodes were also noted on growth and yield of

plants and this was more pronounced at 0.2 ppm concentration

of SO2. Exposure to O3 also gave similar responses as SO2 but

20

it was found to be more deleterious than SO2 • Their combined

effect (O3+SO2) was more than that of their individual

effects. The effects of O3+SO2 mixture were more suppressive

at 0.2 + 0.2 ppm than 0.1 + 0.1 ppm.

Khem & Khan (1993) investigated the interactive effects

of SO2 and root-knot nematode, Meloidogyne incognita (Mi) on

tomato. Plants exposed intermittently to different

concentrations of SO2 induced disease symptoms and the

intensity of symptoms was found to be directly related with

the concentration of the pollutant. There was an increase in

the disease incidence in the SO2 - exposed plants infected

with M. incognita, particularly in post- and concomitant-

inolculation exposures. Both Mi and SO2, alone or in

combination significantly reduced different plant growth

parameters.

Particulate materials, such as carbon and a variety of

dusts, form undesirable deposits on vegetation. It includes

cement dust, lim.e dust, coal dust, fly ash etc. Extensive

utilisation of coal as an energy has created problems

asociated with the disposal of combustion residues (e.g. fly

ash, bottom ash). Fly ash is that portion of the residue from

coal combustion which enters the gas stream (Adriano et al.,

1980) . This material is significant due to its large

quantities of production. Approximately 70 million MT of fly

ash containing Cd, Co, Cu, Fe, Al, Mn, Mo, Ni and Zn is

produced annually (Furr et al., 1977; Elseewi & Page, 1984).

21

Fly ash at low dosages has been found to improve plant

growth and yield (Adriano et al., 1980; Tolle et al., 1982),

because it also contains essential nutrients. But higher

dosages have been found harmful to plants as well as the

associated microbes (Wong & Wong, 1986). Probably the heavy

metal content of fly ash, though very small, may be

responsible for the toxic effect when used in bulk. Haq et

al. (1985) have found reduced infestation of root-knot

nematode on tomato when soil was treated with fly-ash.

Besides air pollutants, there are many metallic ions

which are reported to have toxic effects on nematodes and

thereby effecting the diseases caused by them. Heavy metals

are common environmental contaminants arising from metal

minings and numerous other industrial,urban and agricultural

activities (Foy et al.,1978) and are being continuously added

to the soil.

Toxicity of Cu to various nematodes is well documented

(Hafkenschied, 1971; Pitcher & McNamara, 1972). Hafkenscheid

(1971) observed that during virus transmission many specimens

of Trichodorus pachydermus lost their motility when they were

extracted from soil by Oostenbrink (1960) method. The reason

attributed to this was that the copper supported sieves

released copper ions which were toxic to the nematodes and

caused death of nematodes.

Alphey & Brown (1987) also studied the effects of

pollutants on plant parasitic nematodes. The nematodes used

22

for studies were Xiphinema divers icaudatim, Longidorus

elongatus, Trichodorus primitivus and Pratylenchus

pachydermus. They found that the presence of DBP

(Dibutylphthalate) - contaminated glazing strip in the soil

did not significantly decrease the numbers of migratory plant

parasitic nematodes. They also observed the mean numbers of

nematodes surviving after two months in Cu watered soil and

found the different nematode counts as Xiphinema in fresh

tap-water (34.1), Cu tap water (21.8), CuSO^ solution

(13 .7) , Longri dor us 233, 189.3 and 202.5 and trichodorids

148.6, 141.7, 117.1 respectively.

Sodium hypochlorite was also found to effect the egg

pathogens of M. incognita and Heterodera schachtii (Owino et

al. , 1992). They found that egg development and parasitism

decreased with an increase in the duration of treatment and

the concentration of sodium hypochlorite. Available data also

reveal that NaOCl and enzymes (pectinase-cellulase enzyme)

can effect the physiology of eggs and juvenile mortality

(Barker, 1985; Galper et al., 1990).

Bisessar et al. (1983) studied the effects of heavy

metals and Meloidogyne hapla on celery which was grown on

organic soil in the vicinity of a nickel refinery. The

treatment consisted of nickel at 7,500 ppm, copper at 800 ppm

and cobalt at 100 ppm. It was found that nematode alone was

responsible for an average shoot weight 12% less than the

controls while heavy metals alone resulted in shoot weight

23

79% less than the controls. Their combined treatment (heavy

metal + nematode) caused a shoot weight 85% less than the

controls. Root galls were also much pronounced in the plants

which received the treatment of nematode and heavy metal in

comparison to those received the treatment of nematode only.

The plausible explanation attributed to this was that heavy

metals, primarily nickel, predisposed celery to greater

attack by M. hapla.

Sturhan (1986) studied the influence of heavy metals

and other elements on soil nematodes. Twelve elements, Be,

Cd, Hg,. Sn, Pb, V, Cr, Ni, Se, F, Br and As were applied to

the soil at two different concentrations. In case of Vanadium

(V), at higher concentration, there was significant reduction

in nematode population. Fluorine (F) and Vanadium (V)

appeared to be toxic to certain nematodes even at the lower

concentration; criconematids, certain dolichodorids,

trichodorids, plectids and mononchids seemed to be especially

susceptible. Trichodorus priwitivus was affected by seven of

the twelve elements, Anaplectus gr "ar!uIosus and Scutylenchus

tartuensis by four, Criconemoides informis by at least three

and Pratylenchus spp. by two, while members of Aphelenchoides

spp. and Aphelenchus avenae were not markedly reduced in any

of the contaminated soils. Related species such as

Tylenchorhynchus dubius, Scutylenchus tartuensis and

Merlinius microdorus reacted differently to the same

elements. The studies revealed that the soil nematode fauna

can be affected by heavy and other elements and certain

24

nematode taxa may be specially susceptible and different

nematode taxa may react to particular elements in different

ways. Nematodes may be suitable as bio-indicators for certain

environmental chemicals.

The interactions of nematode and fungal diseases and

the disease response in the presence of pollutants has also

been studied by some scientists. Jabri et al. (1989) worked

upon the nematode and fungal disease of spinach in the

presence of air pollutants. They found that Rhizoctonia

solani, M. incognita and Rotylenchulus reniformis caused

significant plant-growth reductions but smoke pollution

increased the disease intensity by 2-3 times. Pollutants also

reduced root galling and multiplication of M. incognita and

R. reniformis.

Khan & Salam (1990) observed the effects of nickel and

cobalt on the interactions of Meloidogyne javanica, Fusarium

udum and Rhizobium on pigoenpea. They found that M. javanica

caused a reduction in plant growth, particularly at higher

inoculum levels, i.e. 1000 and 10,000 J2. Wilting symptoms

were induced in the p- -esence of Fusarium udum and at higher

concentrations of Ni and Co, plant growth was reduced.

Interaction of M. javanica and F. udum increased the severity

of wilting symptoms in comparison to that of single

inoculation of F. udum. Nickel caused reductions in plant

growth and nodulation and complete inhibition of wilting. M.

javanica could not cause galling in the presence of Ni but

nodulation was reduced. Cobalt also reduced wilting and

25

nodulation but in the presence of M. javianica caused

increased root galling and more reduced plant growth than

found in the individual treatments.

Table 1. Effect of pollutants on fungi.

26

Pollutant

SO, (large dose)

Fungus

Aspergillus niger, Altemana brassicicola. Didvmellina maaospora

Effect

Fungal hyphae remained unaffected

Reference

Mc Callan & Weedon, 1940

SO, (90 ppm) Penicillium spp Growth of fungal hyphae slightly stimulated

Saunders, 1966

SO, Cronartiunu Cdeosponunx Melampsora, Pendermiunx Puccinia

Deaease in parasitism Scheffer &

Hedgcock, 1955

SO, Armillaria mellea Inwease in fungal disease Donaubauer,1968, incidence on trees Jancank, 1961.

Kudela & Novakova, 1962

Oj (high dose) Botrytis gladiolorum Inhibited the develqjment Magie, 1960,1963 of disease in petals of Chrysanthemum

0, (Small dose) Also inhibits obligate Heagle, 1970 parasitism

O,

O,

Alternaria solani

Colletotrichum

lindemuthianum

Comdia gprmuiated while

they were still attached

to conidiophore

Caused loss of pigmenta

tion and formation of

hght refractive globules

Rich & Tomlmson,

1968

Treshow et al,

1969

Contd

Ta le 1. Contd.

27

Pollutant Fungus Effect Reference

Tnchoderma viridae. Botrytis allii, Penicilhum egyptiacum. P expansum. Hivtophthora cactonum. CoUrtotnchum Imdemuthianum, Alternana oleracea, Helnunthosponum sativum

Caused suppression of aerial hyphae

Watson, 1942, Hibben & S t o t z k y , 1 9 6 9 , Treshow et a] , 1969

NaF (m nutrient agar) Verticillium alboatrum. Inhibited colony growth Treshow, 1965 more amount Helnunthosponum sativum of fungi

NaF Pythium debarvanum Caused inhibition in Treshow, 1965 (less amount rec[uired) colony growth of the

fungus

NaF (low cone ) Botrytis cinerea. Colletotnchum spp

Colony growth of fungi

was slightly stimulated Treshow, 1965

NaF (in nutrient agar)

Aspergillus terreus Decreased sugar utilisation and formation of itaconic acid

Lai & Bhargava, 1962

Uromyces phaseoli Ensvphe polygoni. Altenana solani

Decreased foliar infection and invasion of the pathogen

Treshow, 1965

Table 2. Effect of pollutants on bacteria.

28

Pollutant Bacterium Effect Reference

SO, Bacteria Reduced the numbers of

bacteria in bacterial population

Mrkva & Grunda,

1969

HF

NO,

Pseudonxyiasphaseolicola Decreased the disease incidence

Pseudomonas aeruginosa Inhibited active transport uptake of glucose

McCune et a] (c f, Heagle, 1973) Rowe et aj 1979

Table 3. Effect oi pollutants on viruses.

29

Pollutant Virus Effect Reference

O, Tobaooo Mosaic Virus (IMV)

Increased virus multiplication and infectivity of existing virus particles

Brennan&Leone, 1970

O3 and F TMV Either predispose pinto bean leaves to TMV iirfec-tion or increase the infectivity of the virus particles

Treshow, 1965, Treshow,e t aj , 1967

F(100-300ppm) TMV

F(maEthan500pf8n) TMV

Increased the number of

TMV lesions on the host

Decreased the number of

TMV lesions

Treshow, 1965

Treshow, et a]

1967

Treshow, 1965

Table 4. Effect of pollutants on marine/free living nematodes

30

Pollutant Nematode Effect Reference

Ag.Hg,Cu,Be,Al,Fb, G,As,Ti,Zh,Cd,NB, Sr& Sb

Caenorhabditis elegans

Ni, Cd, Pb, Cr, Hg, Cu, Zn

Cu, Cr, Cd

Panagrellus silusiae

P silusiae

Ft Or&Cd-LoswstlxSO at 96-h(0 06mg/L)S^-h]ghest Lc50 (465 mg/L) All the metals except As had lower Lc50 (20-15,000 times lower) at 96-h than at 24-h

Nematode was highly resistant

Effectively blocked growth at all stages of development

Williams & Dusenbery,1990

Haight et al , 1982

Haight et al , 1982

Pb. Ni P silusiae Did not effect the growth Haight et a] of worms 1982

Zn (>500 mg/1) P silusiae Partially blocked growth Haight et al of juveniles 1982

Hg (20 mg/1) P silusiae Was lethal mbkrkmggrowth Haight et al 1982

Hg (10 mg/1) P silusiae Was meflfective m blocking Haight et al growth (10 mg/1) 1982

Contd.

Table 4. Contd.

31

Pollutant Nematode Effect Reference

CdCl (IfiM)

Cd

C elegans

CdCl2(160-320MM) C elegans

Enoplus brevis

Growth and reproduction of the nematode was reduced significantly

Growth was retarded at the early juvenile stages and did not reach the adult stage

Proteins in the nametode were associated with the

Kessel et a\, 1989

Kessel et al , 1989

Howell & Smith 1983

binding of copper and cadmium from seawater

Cd Rhabditis , (soluble cadmium PanaRrellus compounds, e g sulfate, nitrate, chloride, bromide, acetate)

Three insoluble conqxxinds cadmium acetyl acetonate, (CAA), oxide & carbonate were less toxic to the nematode and CAA was lethal after one week at 200 ppm and others showed a varying degree of toxicity

Feldmesser & Rebois,1966

Table 5. Effect of pollutants on plant parasitic nematodes.

32

Pollutant Nematode Effect/Host Reference

O, Pratvlenchus penetrans

Aphelenchoides fraganae

Less effective than SOj in increasing the reproduction of the nematode

Caused greater suppressive effects in inoculated leaves of begonia pre-exposed to O3 prior to inoculation than inoculated leaves which were exposed

Weber et al , 1979

Weber et al , 1979

to O, after inoculation

O, Aphelenchoides fragariae Caused more inhibition in the growth of nematode infested soybean plants and leaves of begonia than similar controls which were grown in the presence of nematodes and charcoal filtered air

Weber et al 1979

O,

S0,+0,

Belonolaimus longicaudatus. Pratvlenchus penetrans

A fragariae

Caused reduction in nodulation of soybean plants

Had greater suppressive effects when leaves were pre-exposed to the

mixture of SO^+O, than after the leaves were inoculated with nematodes

Weber et al . 1979

Weber et al , 1979

Contd.

Table 5. Contd.

33


O,, S0,+0^ B longicaudatus. P minor

Caused reduction in nodu- Weber et al., lation of soybean plants. 1979

Heterodera glycines Caused maximum inhibition of nodulation of soybean.

O (800 ppm) Meloidogyne hapla Growth and yield of tobacco cv. Virginia-115 were decreased irrespective of the presence of nematode.

Bisessar & Palmer, 1984.

However, more injury was caused in the inoculated plants.

O, (0 2 nl/1) Pratylenchus penetrans Caused reduction in excised plant parts.

Shew etal., 1982

SO (0 2nl/l) P penetrans Resulted in more decrease in dry weight of plant

SO, P penetrans Host plants when exposed Weber et al , to SO, enhanced the 1979 reproduction of nematode than charcoa! filtered air or O,

SO, (0 8^1/l) P penetrans Caused reduction in the weight of tomato fruit but it was antagonised in the presence of Oj.

Weber et al . 1979

Contd.

Table 5. Contd.

34


SOj Meloidogyne incognita. (01and02ppm) M javanica

O, ,0,+ SOj Heterodera glycines. Paratrichodorus (Nanidorus) minor

0 , , 0,+SOj Belonolaimus longicaudatus. Pratylenchus minor

0,,0,+SO Aphelenchoides fragariae

O, + SO, Pratylenchus penetrans (0 2^1 + 0 8 nl/1)

O, + SO. P penetrans

Suppressed eggmass Singh, 1989 production, root galling and juvenile hatching on chick-pea and lentil.

Caused inhibition in the Weber et al.,

reproduction and 1979 development of these nematodes in soybean plants.

Remained unaffected by pollutant exposure.

Caused greater suppressive effects when leaves were pre-exposed to pollutants than after the leaves were inoculated with nematodes

Became antagonistic to each other and caused

Weber et al 1979

Weber et al 1979

Weber et al 1979

less change in dry wt of leaf and shoot than when the pollutants were used individually

The presence of P penetrans on roots stimulated the negative effects of pollutants on leaf growth(dry wt ) but

suppressed

the inhibitory effects of of pollutants on the dry

wt of axilliary shoots

Weber et al , 197Q

Contd

35

Table 5. Contd.


Simulated acid M hapla rain (pH 6 0 or

3 2)

Reduced the root infection and reproduction on red kidney beans when plants were treated three times weekly

Shriner,1978

O, + SO, M incognita. Pollutants were more suppressive at 0 2 + 0 2ppm than 0 1+0 1 ppm

Singh, 1989

Cu Tnchodorus pachydermus Found to be toxic to the nematodes and caused death of nematodes

Hafkenscheid, 1971

NaOCl M incognita. Egg development and p a rasitism decreased with an increase in the duration of treatment and the cone of sodium hypo-chlorite

Barker, 1985, Galper et al , 1990

Nickel C 500 ppm) Copper (800 ppm) Cobalt (100 ppm)

M hapla When tested individually, Bisessar, 1983 on celery heavy metals resulted in more reduction in shoot weight than nematodes (heavy metal + nematode) However, their combined treatment resulted in a further reduction in shoot weight Nickel was found to prediqx)se coloity to greater attack by M hapla Root

galls were also pronounced in the plants receiving the treatment of nematode and

heavy metal simultaneously

Contd.

36

Table 5. Contd.

Pollutant

Be,Cd,Hg,Sn, V, Cr,Ni,Se,F, Br, As

SO,, CO, CO,

Nematode

Tnchodonis pnmiti vus — > Anaplectus granulosus — > Scutvlenchus tartuinsis — > Cnconemoides informis — > Pratvlenchus spp — > Aphelenchoides spp — > Aphelenchus arenae — > Tvlenchorhvchus dubius — >

Menhnius microdorus — >

M incoKnitaj Rotylenchulus reniformis

Effect/Host

Affected by 7 elements 4 4 3 2

Not affected by pollutants

n »» »? >>

Reacted differently to the

7 elements

11 »» 51 71

Effects were seen on spinach where pollutants caused significant growth

Reference

Sturhan, 1986

Jabn et a l , 1989

reduction and smoke pollution increased the disease intensity by 2-3 times R solani and M incognita caused maximum plant growth reduction than R solani but higher than that caused by R reniformis

Niand Co Meloidogyne lavanica Nematode reduced plant growth

Khan & Salam 1990

37

3. MATERIALS AND METHODS

3.1. Selection of test materials:

The most widely occurring and economically important

nematode species of the area, viz. the root-knot nematode,

Meloidogyne incognita (Kofoid & White) Chitwood and an

economically important vegetable host, viz. tomato

{Lycopersicon esculentum Mill.) cv. Pusa R\iby were selected

for the present study. The experiments embodying the thesis

were conducted in the presence and absence of heavy

metals. For this purpose, the two heavy metals, viz. lead

(Pb) and cadmium (Cd) were selected. Both the heavy metals

were used in the form of nitrate salts.

For the experiments pertaining to the effects of heavy

metals on the efficiency of biocontrol agent against the test

nematode, a common soil inhabiting fungus, Paecilomyces

lilacinus (Thorn.) Samson was selected.

The experiments relating to the efficacy of

organic amendments in the presence and absence of heavy

metals were conducted using oil-seed cake and leaf of neem

{Azadirachta indica A.Juss.) and castor {Ricinus communis

Linn.) and rice polish. For comparing the results, inorganic

fertilisers in the form of urea, superphosphate and murate

of potash were also included in the study.

38

3.2. Maintenance of nematode culture:

In case of root-knot nematode a single eggmass was

collected from the infected root of tomato with the help of

forcep and placed on a small coarse sieve (1 mm pore size)

lined with moist tissue paper which was then placed in a

Petri dish (10 cm diameter) containing distilled water. After

hatching, second stage juveniles {J2) were collected

alongwith the water from the Petri dishes after every 24 h.

Fresh water was added to the Petri dishes after each

collection. These juveniles were used as the initial

inoculum to inoculate the seedlings of tomato raised in

sterilised soil manure mixture in clay pots. Two months

after inoculation, the plants were uprooted carefully, roots

washed gently and the eggmasses were removed. A suspension

of J2 was obtained in a similar manner as described above

and these were used to raise the nematode culture on tomato

in big pots containing autoclaved soil mannure mixture.

Eggplant (Solanum melongena Linn.) was used as an alternate

crop in absence of tomato. The species of the root-knot

nematode was ascertained by close examination of the

perineal pattern and morphometries of the juveniles.

Separate aqueous suspensions of the nematode were

stirred gently and then 5 ml suspension was transferred to

a Doncaster's counting dish (Southey, 1986) and the nematodes

in each sample were counted under a stereoscopic microscope.

An average of five counts was made in each case to determine

39

the density of nematodes per unit volume of the suspension.

Appropriate quantities of nematode suspensions were used to

inoculate plants at different inocula in different

experiments.

3.3. Maint«*"*"f'* of fungal culture :

The test fungus, Paecilomyces lilacinus (Thorn.) Samson,

obtained from the Division of Mycology and Plant Pathology,

Indian Agricultural Research Institute (lARI), New Delhi,

was maintained on PDA* (potato dextrose agar) in culture

tubes. It was later transferred to sterilised Petri dishes

containing PDA under a laminar flow and incubated at 25 C.

This inoculum was used in the experiment concerning the

growth of the fungus in relation to heavy metals.

3.4. Preparation of stock solutions of heavy metals for experiments concerning nematode bio-control fungus. Paecilomyces lilacinus :

Stock solution (1200 ppm) of cadmium was prepared

using cadmium nitrate {(Cd N03)2.4H20} considering atomic

weight of cadmuim (112.4) and molecular weight of the salt

(308.47). Similarly for lead, the stock solution was

prepared from lead nitrate [Pb(N03)2] taking into account

atomic weight of lead (207.2) and molecular weight of the

salt (331.2). Appropriate amounts of stock solutions were

added to 100 ml liquid medium contained in Erlenmeyer flasks

''!?__''f_ "" ^ ^° ^^ ^° 5^^ desired concentrations of the

* PDA: Potato=250 g, Dextrose=17 "gr""AgarV20 "g' Distilled water=1000 ml

40

metals in the medium, e.g. 600, 400, 300, 200, 150, 100, 75

50 and 25 ppm.

3.5. Preparation of stock solutions of heavy mealts for experiments concerning nematode hatching / mortality:

Stock solutions of cadmium and lead (120 ppm) were

prepared in the same manner as described above. Desired

dilutions of 60.0, 30.0, 15.0 and 7.5 ppm were obtained by

adding appropriate quantities of the stock soltuion to the

nematode suspension.

3.6. Preparation of stock solutions of heavy metals for pot experiments:

Stock solutions of different concentrations, viz. 75,

150, 300 and 600 ppm of test metals were prepared in the same

manner as described above in 3.4. One hundred ml solution of

each concentration was added to 1 kg autoclaved pot soil so

as to get 7.5, 15.0, 30.0 and 60.0 ppm concentration (on dry

weight basis) of the metals in relation to the known quantity

of soil used per pot. In order to maintain an equal level of

nitrogen in different pots, appropriate quantities of NaN03

were added considering the amount of NO3 present in different

doses of Cd(N03)2.4H20 and Pb(N03). The calculations were

made by taking into account the mol. wts. of Cd(N03)2.4H 0,

Pb(N03)2, NaN03 and NO3 being 308.48, 331.2, 85 and 62

respectively.

41

3.7. To study the in vitro growth of nematode biocontrol fungus,Paecilomyces lilaclnus in the presence of heavy metals;

One hundred ml of Richard's liquid culture medium was

poured into 250 ml Erlenmeyer flasks and different amounts

of cadmium nitrate/lead nitrate were added into the flasks so

as to obtain the different concentrations of Cd/Pb, viz. 25,

50, 75, 100, 150, 200, 300, 400 and 600 ppm as per procedure

described earlier in 3.4. The flasks which did not receive

heavy metal served as control. There were three replicates

for each treatment. The flasks were then autoclaved at the

pressure of 15 lb/in . After cooling, the flasks were

inoculated with the test fungus, Paecilomyces lilaclnus under

a laminar flow and incubated at 25°C for 15 days. On the

basis of visual observation, sporulation of the fungus was

rated on a 0-5 scale-. 0= no sporulation, 1= scanty

sporulation, 2= light sporulation, 3= moderate sporulation,

4= heavy sporulation and 5= severe sporulation. Thereafter,

the mycelial mats, which developed in the flasks, were

taken out and kept in an oven running at 50°C temp, and dry

mycelial weight was noted.

* Potassium nitrate = 10.Og, Potassium dihydrogen phosphate = 5.0g, Magnesium sulphate = 2.5g, Ferric chloride = 0 02g Sucrose = 50.Og and Distilled Water = 1/OOOna

42

3 .8 . To study the e f f e c t of heavy metals on the hatching and morta l i ty of the root-knot nematode. Meloidpqyne incogni ta ;

Different concentrations of cadmium and lead were

prepared in distilled water at five levels, e.g. 0, 7.5,

15.0, 30.0 and 60.0 ppm using their nitrate salts as per

procedure described earlier in 3.4 and 3.5. The different

experiments were carried out as follows :

3.8.1. Juvenile hatching:

Five freshly picked eggmasses of average size were

transferred to Petri dishes (40 mm diameter) containing 5 ml

solutions of different concentrations of cadmium and lead.

All the treatments were replicated three times including

distilled water control. The Petri dishes were incubated at

25'-'c and the total number of hatched juveniles were counted

after 5 days with the help of Doncaster's counting dish.

3.8.2. Juvenile mortality :

About 100 freshly hatched second stage juveniles (J2)

of the nematode were transferred to Petri dishes (40 mm

diameter) containing 5 ml solution of different

concentrations of Cd and Pb following the procedure

described by Alam (1985). An aqueous suspension containing

about 100 specimens of J2 was poured over a metallic sieve of

350 meshes/linear inch (1.5 cm diam. and 1.0 cm height,

fitted with a flat handle of 5 cm) . Thus the nematode

juveniles remained over the mesh and the water drained off.

Then the sieve was inverted over a Petri dish and the

43

juveniles were washed down with 5 ml solution of the heavy-

metal. In this way, the juveniles were transferred to the

solution without changing its concentration. The same

operation was repeated for differnt concentrations using

washed sieve. Each treatment including distilled water

control was replicated three times. Number of immobile

juveniles was counted after 12, 24, 48, 72 and 96 h. Death

of nematodes was ascertained by transferring them into plain

water and then percent mortality was determined.

3.9. To study the effect of heavy metals on the root-knot development caused by MeloidoQYê incoonita and plant growth parameters of tomato:

Seedlings of tomato (Lycopersicon esculentum) cv. Pusa

Ruby were raised in clay pots (25 cm diameter) containing

autoclaved mixture of soil -. cow dung manure (3:1). Three-

week old seedlings were transplanted singly in 15 cm diameter

clay pots, containing 1 kg autoclaved soil manure mixture

lined with polyethene sheets to obstruct the absorption and

movement of heavy metals through the pots. Appropriate

quantities of cadmium and lead nitrates alongwith sodium

nitrate were added to the pots so as to get differnt levels

of heavy metals, e.g. 7.5, 15.0, 30.0 and 60.0 ppm as per

procedure described earlier(3.6). The plants were later

inoculated with freshly hatched second stage juveniles of the

root-knot nematode, at 5000 J2/pot. Untreated pots served as

control. Each treatment was replicated three times. The

schedule of the treatments was as follows:

44

1. Untreated uninoculated 2. Untreated inoculated (5000 J2) 3. Cd/Pb 7.5 ppm uninoculated 4. Cd/Pb 7.5 ppm inoculated (5000 J2) 5. Cd/Pb 15.0 ppm uninoculated 6. Cd/Pb 15.0 ppm inoculated (5000 J2) 7. Cd/Pb 3 0.0 ppm uninoculated 8. Cd/Pb 30.0 ppm inoculated (5000 J2) 9. Cd/Pb 60.0 ppm uninoculated

10. Cd/Pb 60.0 ppm inoculated (5000 J2)

Aftercare of the plants including watering was done as

and when required. The experiment was terminated after three

months. The plants were carefully uprooted and the roots

washed thoroughly and gently. The plant growth and other

parameters and root-knot index were assessed as under:

3.9.1. Plant growth-.

Fresh and dry weights (g) and lengths (cm) of shoots

and rooLs were taken separately. The plants were pressed

between blotting sheets to remove excess amount of water

prior they were weighed fresh. For determining the dry

weights, shoots and roots were first dried in an oven running

at 60°C and then weighed separately.

3.9.2. Root-knot development:

The degree of root-knot infection caused by the root-

knot nematode was assessed according to the rating scale

(0-5) of Taylor & Sasser (Sasser et al.,1984).

Root-knot index No. of galls/root system

0 0 1 1-2 2 3-10 3 11-30 4 31-100 ^ > 100

45

3.9.3. Water absorption :

Water absorption capability of plant roots was assessed

prior to the recording of plant weights by using the method

of Alam et al. (1974) . Erlenmeyer flasks of 250 ml capacity

were filled with water and weighed. Plants from different

treatments were placed in each flask singly. Flasks

containing water without seedlings were kept as control.

After 24h the plants were taken out of the flasks and the

amount of water loss was determined. Amount of water lost

from the control flasks was deducted from the amount of

water lost from the flasks having plants. The difference

gave the actual amount of water absorbed by the roots.

3.9.4. Estimation of chlorophyll content:

The chlorophyll content of leaves was determined by the

method described by Hiscox & Israelstam (1979) . One hundred

mg of leaf tissue in small pieces was placed in a vial

containing 7 ml DMSO (Dimethyl-Sulfoxide - BDH, Chemical

Ltd., Toronto) and chlorophyll was extracted into the fluid

without grinding at 65°C by inrnbating for about an hour.

The extract was transferred to a graduated tube and made up

to a total volume of 10 ml with DMSO assayed immediately or

transferred to vials and stored at 0-4°C until required for

analysis. A 3 .0 ml sample of chlorophyll extract was

transferred to cuvette and the OD (optical density) at 645

and 663 nm were read in Beckman DBG Spectrophotometer

against a DMSO blank.Chlorophyll contents were calculated by

the following formulae :

46

Chi.a =[12.7(D663)]-[2.69(D645)] x V x W 1000

Chl.b =I22.9(D645)]-[4.68(D663)] x V x W 1000

Where,

V = Volume of the extract in ml

w = Fresh wt. of the sample in g

3.10. To study the effect of heavy metals on the penetration of second stage juveniles of Meloidocrvne incognita into the roots of tomato:

The experiment was conducted as per procedure described

by Ismail & Alam (1975) . Seedlings of tomato {Lycopersicon

esculentum) cv. Pusa Ruby were raised in 25 cm diam. clay

pots containing autoclaved soil manure mixture as described

above in 3.9. Seedlings when 3-week old, were taken out and

their roots washed thoroughly and gently to remove soil

particles adhering to it. The seedlings were then separately

placed in beakers containing solutions of 7.5 and 15.0 ppm

of cadium and lead for different durations, i.e. 12, 24 and

4 8 h. The seedlings were placed in such a way that only their

root systems were dipped in the solution.After that, the

seedlings were taken out, their roots washed thoroughly and

gently and then transferred to thermocole cups (5 cm diam.)

containing acid-leached and thoroughly washed river sand.

Each treatment, including un-dipped control, was replicated

three times. The seedlings were inoculated with 1000 freshly

hatched second stage juveniles (J2) of Meloidogyne incognita.

Care was taken to keep the sand moist for 5 days by gently

47

adding water with the help of pipette along the rim of the

cup. After 5 days, the cup was placed in a bucket and washed

gently to take out the seedlings. The roots as well as cups

were washed properly in the bucket and then taken out from

the bucket. The bucket was filled with water and the

contents were stirred thoroughly and left for about 30

seconds. After that, the supernatant was passed through a set

of 300 mesh sieves and the catch transferred into a beaker.

The operation was repeated for three times and (catches) were

put in the same beaker. Number of juveniles were counted in

each sample and deducted from the number of initial

inoculum, i.e. 1000. Thus the number of juveniles penetrated

into the roots was determined.

3.11. To study the effect of heavy metals on the growth of females of the root-knot nematode. Afeloidogvne incoQxii ta on tomato:

The experiment was conducted on the same lines as

described in 3.9. After termination of the experiment, the

galled roots were fixed in FAA and then stained in 0.01%

cotton blue in lactophenol to differentiate nematode

females from the root tissue. The females were excised

from the roots with the help of needles under a stereoscopic

microscope and then mounted in lactophenol on cavity slides.

Cross-sectional area of the females was determined following

*FAA: 95% Ethanol=20ml, For-malin (40% formaldehyde) = 6ml, Glacial acetic acid = Iml, Distilled water = 40 ml.

**Lactophenol: Phenol (liquid) =500ml, Lactic ar.id=500 ml, Glycerol=1000 ml. Distilled water=500 ml.

48

the method of Bird (1959) . Outlines of the females were

drawn on a paper of uniform thickness using camera lucida.

The paper was cut along these outlines and the cut-out areas

were weighed (W ) . Similarly, a 10° sq ^ of same paper,

obtained by drawing this area by the above method using a

micrometer slide, was also weighed (W2). The cross-sectional

area of the females from different treatments was calculated

by the following formula:

Cross-sectional area of * = W- / W2 X 10° sq p

3.12. To study the effect of heavy metals on the efficacy of nematode biocontrol ftmcnis,PaeciIofliyces lilacinus on the root-knot development caused by Meloidocryne incognita on tomato:

The experiment was conducted on same line as

described in 3.9. However, only one concentration of heavy

metal (15.0) ppm was used. After transplantation of 3-wk old

seedlings of tomato cv. Pusa Ruby, the pots were inoculated

with second stage juveniles of Meloidogyne incognita at the

rate of 5000 J2/pot and /or one gram inoculum of

Paecilomyces lilacinus as per the following schedule :

1. Uninoculated + Untreated (Control) 2. Uninoculated + 15.0 ppm Cd/Pb 3. Meloidogyne incognita alone 4. M. incognita +15.0 ppm Cd/Pb 5. Paecilomyces lilacinus alone 6. P.lilacinus +15.0 ppm Cd/Pb 7. M. incognita + P. lilacinus 8. M. incognita + P. lilacinus +15.0 ppm Cd/Pb

Each of the above treatments was replicated three

times. Usual aftercare such as watering was done when

49

required. The experiment was terminated after 3 months.

Readings for different paraimeters were recorded on the same

lines as described in 3.9.

The fungal inoculum was prepared in Erlenmeyer flasks

containing Richard's medium as described in 3.7. After 15

days, the mycelial mats were removed and then gently pressed

between sterile blotting sheets to remove the excess amount

of liquid. The inoculum was prepared by mixing 10 g fungal

mycelium in 100 ml of sterilised distilled water in a waring

blender for few seconds (Stemerding, 1964). In this way, each

1 ml of this homogenate contained 1 g of fungal mycelium.

3.13. To study the response of some selected accessions/ cultivars of tomato to the root-knot nematode, Afeloidoq-yne incotmita in the presence of heavy metals:

The accessions/cultivars selected for the experiment

were Arka vikas, Damyanti, F-, Hybrid, Hybrid Dipti,

Hybrid Padmarag, Hybrid Tripti, Marudam, Punjab Chhoara,

Pusa Early Dwarf, Punjab Kesari, P.K.M. -1, Roma and Pusa

Ruby. Seedlings were raised and transplanted singly in 15

cm diam. clay pots containing 1 kg autoclaved soil manure

mixture in the same manner as described earlier in 3.9. The

pots were later treated with an appropriate quantity of

cadmium and lead nitrates alongwith sodium nitrate so as to

get the heavy metal concentration of 15.0 ppm as per

procedure described earlier. The plants were inoculated at

5000 J2 of M. incognita. Untreated pots served as control.

Due care and watering of plants was done according to the

50

requirement. The experiment was run for 3 months and

terminated thereafter. Plant length and weight (fresh and

dry) and root-knot index were determined in the same manner

as described in 3.9.1 and 3.9.2.

3.14. To study the efficacy of oraamic amendements on the root-taiot develoiament caused by Afeloidogyne incognita ar< pi*Tit qrowt>» p^Twmeters of tomato in the presence of heavy metals:

The organic amendments included in the present study

were: rice polish (a byproduct of rice mills which is rich in

nitrogenous substances and riboflavin-Vit.B6) , oil seed cakes

and leaves of neem (Azadirachta indica) and castor {Ricinus

communis). These materials were added (@ Ig N/kg soil) to 15

cm clay pots containing 1 kg autoclaved soil and with

polythene sheets. In addition to untreated control, another

control was also maintained with in organic fertilizers in

the form of urea (@ Ig N/kg soil), superphosphate (@ 0.5 g

P/kg soil) and murate of potash (@ 0.5 g K/kg soil) .

Appropriate quantities of cadmium / lead nitrate alongwith

NaN03 were also added to the pots in order to obtain the

concentration of 7.5 ppm of heavy metal in s-oil {en dry

weight basis) as per procedure described earlier. The

different treatments, each replicated three times, were as

follows:

1. Untreated - Uninoculated 2. " " - Inoculated 3. Inorg.ferti. alone - Uninoculated 4. " " " - Inoculated 5. " " + Cd/Pb 7.5 ppm - Uninoculated 6 " " + .. " _ Inoculated 7. Neem cake alone - Uninoculated 8. " " " - Inoculated

51

9. 10. 11 . 12 . 13 . 14 . 15 . 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26

Neem cake + Cd/Pb7.5 ppm

Castor cake alone

" + Cd/Pb 7.5 ppm II M

Neem leaf alone II II

II

II M

Castor leaf alone

+ Cd/Pb 7.5 ppm

II

" + Cd/Pb 7.5 ppm II II M

Rice polish alone II II

" + Cd/Pb 7.5 ppm

Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated Uninoculated Inoculated

N.B.== Inoculation was made with 5000 J2 of M. incognita/Tpot.

The contents of the pots were kept moist for one week

to facilitate proper decomposition of the additives.

Thereafter, 3-wk old seedlings of tomato (Lycopersicon

esculentum) cv.Pusa Ruby raised in autoclaved soil manure

mixture, were transplanted ssingly to the pots. The plants

were then inoculated with freshly hatched second stage

juveniles of the root knot nematode at 5000 J2/pot. Untreated

pots served as control. Each treatment was replicated three

times. Aftercare of plants including watering was done as

and when required. The experiment was terminated after three

months and the data were recorded as described in 3.9.1 and

3.9.2.

3.15. Estimation of heavy metals by Atomic Absorption Spectrophotometry (AAS)in tomato plants infected with root- knot nemetode, Afeloidocyvne incognita :

The experiment was conducted on the same lines as given

in 3.9. After terminatioin, the plants were uprooted and the

52

roots were cleaned from the adhering soild particles. The

plant samples for analysis of heavy metals were prepared by

washing them several times with double distilled water.

These were dried at 105°C for 24 h. The dried plant material

was ground in a pestle and mortar. Five gram of the powdered

material was weighed and digested in 20 ml boiling Analar

HNO3 -"- ^° ^-^ Kjeldhal flask. Digestion usually completed

within about half an hour. The digests were made up to 25 ml

by adding required quantity of HNO3. The prepared solution

was used for the determination of heavy metals by using

Atomic Absorption Spectrophotometer (Harding & Whitton,

1981).

For determination of cadmium in plant samples, a stock

solution of 100 ppm Cd was prepared by using cadmium oxide

and different concentrations within the range of 0.2-1.5 ppm

were prepared, e.g. 0.2, 0.5, 0.8, 1.1 and 1.5 ppm.

Wavelength was fixed at 228.8 nm. Readings of Standard

solutions of known concentrations were noted and then

readings for plant samples of unknown concentrations were

calibrated.

For determination of lead in plant samples, a stock

solution of 100 ppm Pb was prepared by using lead sheet and

then Standard solutions within the range of 2.0-20.0 ppm

were prepared, e.g. 2.0, 5.0, 8.0, 11.0, 15.0 and 18.0 ppm.

The wavelength was fixed at 217 nm. Observations were taken

in a similar way as in case of cadmium.

53

3.16. Estimation of Cu. Zn and Mn by Atomic Asorption Spectrophotometry (AAS)in tomato plants infected with root-knot nematode, AfeloidocTyne incocroita :

For determining the amounts of micronutrients in plants

treated with cadmium and lead, Standards of each were

prepared by using pure metals. The range of 1-5 ppm was

used. For the estimation of copper Standard solutions, e.g.

1, 2, 3, 4 and 5 ppm were prepared from 100 ppm stock

solution. Readings were noted for these known concentrations

of the Standard solutions and then readings of plant samples

of unknown concentrations were calibrated. Wavelength for

absorption of copper was fixed at 324.7 nm.

In case of zinc, range of 0.4-1.5 ppm was used.

Standards, viz. 0.4, 0.6, 0.8, 1.0 and 1.5 ppm were prepared

from 100 ppm stock solution. Wavelength was fixed at 213.9

nm. Readings were taken in a similar way as described

above. For manganese, range of 1-4 ppm was used. Standards,

viz. 1, 2, and 4 ppm were prepared from 100 ppm stock

solution. Wavelength was fixed at 279.8 nm. Observations

were taken in similar way as above.

3.17 .Estimation of Na, K and Ca by Flame Photometry in tomato plants infected with root-knot nematode. Afeloidogyne incognita:


given in 3.9. After terminatioin, the plants were uprooted

and the roots were cleaned off to remove the adhering soil

particles. Estimation was done by Flame Spectroscopy or Flame

54

Emission Spectroscopy (FES). Flame Spectroscopy i s an

a n a l y t i c a l t echnique used for the q u a l i t a t i v e and

quant i ta t ive determinatioin of an element in a sample . In

th i s method, the sample, in the form of a homogeneous

l i q u i d , i s in t roduced i n to a flame where thermal and

'chemical^ r e a c t i o n s c r ea t e ' f r e e ' atoms capable of

absorbing, e m i t t i n g or f l u o r e s c i n g a t c h a r a c t e r i s t i c

wavelengths. The emitted radia t ion i s proport ional to the

concentration of atoms present . For determining the amount of

Na, K and Ca in plant samples by Flame Photometer, Standards

of each were prepared in the following manner:

For Na, Standards, v iz . 50, 100, 200, 300, 400 and 500

ppm were prepared from 1000 ppm stock so lu t ion . Readings for

these Standards of known concentrations were noted by put t ing

the Standards in Flame Photometer. Readings thus obtained

were termed as Instrument Reading (IR) . P lan t samples

containing unknown amount of Na, were passed through Flame

Photometer and IR was noted in a s imi lar manner. Standard

curve was p lo t t ed by the IR of known concentrations of

Standard s o l u t i o n s and then IR of unknown samples was

c a l i b r a t e d wi th Standard curve so as to get the

concen t ra t ion of Na in p lan t samples in ppm. From t h i s

concentration the amount of sodium in ug/g was calculated.For

K and Ca, Standards were prepared in the same manner as in

case of sodium and readings were a lso taken in a s imilar way.

55

3.18. To study the effect of heavy metals on protein profile of females of the root-knot nematode, Afeloidocrvme incocTni ta and infected roots of tomato. LYCopersicon esculentum cv. Pusa Ruby :


described in 3.9 , however, only two levels of the heavy

metals, viz. 7.5 and 15.0 ppm were used during the course of

investigation. After termination of the experiment fresh

root samples and excised mature females of the root-knot

nematode, Meloidogyne incognita were obtained. The material

was stored in glass vials in a freezer at the temperature

between 0-4°C. The material was used for the analysis of

polypeptide profile with the help of Gradient SDS-

Polyacrylamide Gel Electrophoresis (PAGE) following the

method of Laemmli (1970) with some modifications, using a

complete vertical slab gel electrophoresis assembly, gradient

maker at power supply 30-40 mA (milli ampere) separating 7-

15% gradient slab gel and 5% stacking gel was used on a

vertical slab gel system (Pharmacia LKB, Sweden).

3.18.1. Gel preparacion:

The separating gel of linear gradient 7-15% was

prepared as follows:

Ingradient 7% 15%

Acrylamide 3.736 ml 8.000 ml Double distilled water 9.780 ml 5.520 ml Tris-HCl buffer pH 8.9 2.480 ml 2.480 ml 10% APS 80.000 1 BO.OOO^ml

56

The stacking gel of gradient 5% was prepared as follows

Ingradient 5%

Acrylamide 2.50 ml Double distilled water 10.30 ml Stacking gel buffer 2.00 ml 10% APS 100.00 ul

All the solutions were mixed thoroughly and freshly

prepared 10% APS solution was added. The gel mixture was then

poured to the glass plate moulds of size 14x14 cm prepared

by using 1 mm thick spacers. Once the gel solution was

poured, it was carefully overlaid with a few drops of double

distilled water and allowed to polymerise at room

temperature for 45 min. After polymerisation, the water was

carefully removed from the gel surface and 5% stacking gel

solution was poured into the mould and the desired comb was

gently inserted and gel solution was allowed to polymerise at

room temperature for 30 min.

3.18.2. Sample preparation:

The soluble protein sample containing about 100 ug

protein was mixed with equal volume of Laemmli's sample

buffer, 0.625 M Tris-HCl, pH 6.8, containing 5% (v/v)

mercaptoethanol, 20% (w/v)SDS, 10% (v/v) glycerol and 0.05%

aqueous bromophenol blue as the marker dye and then it was

boiled for 8 min. at 100°C in a boiling water bath. Similarly

a standard molecular weight protein markers, ranging from 2 9

KD to 205 KD (LMW kit; LKB Pharmacia) was also prepared. The

mixture of protein marker contained carbonic anhydrase 29KD,

57

ovalbumin 45 KD, BSA 66 KD, phosphorylase 97.4 KD,p-Y-

galactosidase 116 KD and myosin 205 KD. A total of 50 ugm of

protein samples and standard protein markers were carefully

loaded with a five micro-sample applicator syringe into the

separate well and electrophoresis was carried out at 6°C

using a multitemp. II, thermostatic circular by applying 30

mA/slab gel constant current until the marker tracking dye

reached 1 cm before the end of the gel. The gel was removed

from the mould after the power was disconnected and used for

staining procedure.

3.18.3. Staining of gel:

The gels were fixed in 10% (v/v) acetic acid, 45% (v/v)

methanol for Ih and then gels were stained in 0.25% commassie

brilliant blue (CBBR-250) prepared in fixing solution. The

stained gels were destained in high destaining solution

(HDS) consisting of a mixture of methanol (5%) and acetic

acid (7%) and again cleaned in low destaining solution

(LDS) , a mixture of 7% (v/v) acetic acid and 5% (v/v)

methanol in double distilled water until the background

became clear finally gels were stored in 7% (v/v) acetic

acid in double distilled water.

3.18.4. Determination of molecular weights:

The molecular weights for each polypeptide was

determined by the method of Weber & Osborn (1969) . The

relative mobility (Rf) of each polypeptide was calculated as

follows:

58

Distance of polypeptide migration Rf =

Distance of tracking dye migration

Distance of differnt polypeptides were recorded

including those for standards and Rf values were determined.

From the standard curve, the molecular weights of unknown

polypeptides were calculated.

59

4. RESULTS

4.1 To Study the effect of cadmium (Cd) on the hatching of the root-knot nematode, Ifeloidogyne incocynita in vitro-.

The results as presented in Table 1, clearly indicate

that cadmium adversely affected the juvenile hatching of the

root-knot nematode, Meloidogfyne incognita. Hatching decreased

with an increase in the concentration of the heavy metal

(Fig. 1) . The inhibition in the hatching relative to the

control was 90.7, 88.7, 78.6 and 74.4 % in 60.0, 30.0, 15.0

and 7.5 ppm concentrations of cadmium respectively (Table

1). Inhibition in juvenile hatching over control at all the

concentrations of cadmium was statistically significant at P

=0.05 and P= 0.01. However, 30.0 and 60.0 ppm concentrations

were almost equally effective.

4-2 To study the effect of lead (Pb) on the hatching of the root-knot nematode, Meloidocrviie incoonita in vitro-.

It is quite evident from the results presented in

Table 2 that lead had more pernicious effect on juvenile

hatching than cadmium. In case of Pb also it was noticed that

the juvenile hatching gradually decreased with an increase

in the concentration (Fig. 2) . As compared to cadmium, the

juvenile hatching was completely checked at 60.0 ppm

concentration of the heavy metal. The inhibition in juvenile

hatching relative to the control was 100, 95.6, 86.8 and

77.6 % in 60.0, 30,0, 15.0 and 7.5 ppm concentrations of Pb

60

Table 1. Effect of cadmium on the hatching of the root-knot nematode, Meloidogyne incognita in vitro.

Treatment (ppm)

Control

07.5

15.0

No.of juveniles hatched/ eggmass after 5 days

580.0

148.0

124.0

% inhibition over control

-

74.4

78.6

30.0 065.0 88.7

60.0 053.6 90.7

CD. (P=0.05) CD (P=0.01)

018.88 027.47

Each value is an average of 3 replicates.

61

600 n

500

t 400 x:

&5 300

200 ©

100

DW 75 15.0 30.0 Concentration of Cd(ppm)

60.0

Fig.l. Effect of cadniiuni(Cd) on the hatching of the root-knot nematode Meloidogyne incognita in vitro.

62

Table 2. Effect of lead on the hatching of the root-knot nematode, Meloidogyne incognita in vitro.

Treatment No. of juveniles hatched/ (ppm) eggmass after 5 days

% inhibition over control

Control 580.0

7.5 029.6 077.6

15.0 076.0 086.8

30.0 025.3 095.6

60.0 000.0 100.0

CD. (£=0.05) 014.57 CD. (P=0.01) 021.20

Each value is an average of 3 replicates.

600 n

500

63

X!

400-

5S ^

^-g 300

© 200-

100-

^ ^

DW 7.5 15.0 30.0

Concentration of Pb (ppm)

60.0

Fig.2. Effect of lead (Pb) on the hatching of the root-knot nematode Meioidogyne incognita in vitro.

64

respectively (Table 2) . Inhibition in juvenile hatching due

to different test concentrations of lead was statistically-

significant at P = 0.05 and P= 0.01, both relative to control

as well as different concentrations.

4.3 To studv the effect of cadmium (Cd) on the mortality of second staoe -juveniles (J2) of the root-knot nematode^ Meloidooyne incognita in vitro:

The results as presented in Table 3 clearly indicate

that nematode mortality increased with an increase in the

concentration of cadmium and exposure period. There was a

linear relationship between mortality and concentration of

the heavy metal (Fig. 3). Percent mortality was found as 1.6,

1.8, 3.6 and 10.9 % for 7.5, 15.0, 30.0 and 60.0 ppm when

nematode juveniles were exposed to the heavy metal for 12 h.

Rate of nematode mortality increased with an increase in the

exposure period and was found to be 8.6, 11.5, 15.7 and

18.9 % after 24 h; 24.5, 26.5, 29.2 and 32.8% after 48 h;

28.0, 30.3, 32.1 and 38.3 % after 72 h, and 31.1, 35.3, 40.0

and 50.0 % after 96 h in 7.5, 15.0, 30.0 and 60.0 ppm

concentrations of Cd respectively.

Regression line was also drawn for all the

concentrations, and with the help of regression line EC^Q

value for cadmium was calculated and it was found to be

50.8 ppm at 96 h of exposure (Fig. 3).

4.4 To studv the effect of lead (Pb) on the mortality of second stage juveniles (J2) of the root-knot nematode. Afeloidocrvne incognita in vitro:

Lead was found to be more toxic to the nematode than

cadmium. The rate of nematode mortality increased with an

65

Table 3. Effect of cadmium (Cd) on the mortality of second stage juveniles (J2) of the root-knot nematode, Meloidogyne incognita in vitro.

Exposure Period (h)

12

24

48

72

96

Percent mortality in different cone, of cadmium (ppm)

0

0(-1.14)

0(1.96)

0(8.54)

0(9.60)

0(9.50)

7.5

01.6(01.22)

08.6(06,45)

24.5(15.57)

28.0(17.67)

31.1(20.39)

15.0

01.8(03.58)

11.5(10.94)

26.5(22.60)

30.3(25.74)

35.3(31.28)

30.0

03.6(05.94)

15.7(15.43)

29.2(29.63)

32.1(33.81)

40.0(42.17)

60.0

10.9(08.30)

18.9(19.92)

32.8(36.66)

38.3(41.88)

50.0(53.06)

Regression

Y=03.58+02.36(X-2.00)

Y=10.94+04.49(X-2.00)

Y=22.60+07.03(X-2.00)

Y=25.74+08.07(X-2.00)

Y=31.28+10.89(X-2.00)

Each value is an average of 3 replicates. In parentheses are given calculated values. Temp. =25°C.

66

60

60

s too o ; j

o >% •^^ «

O

E -«>> c &>

0.

40

30

20

10

-10

Y = 3.58 + 2.36 (X-2.00) 12 h Y = 10.94 + 4.49 (X-2.00) 24 h Y = 22.60 + 7.03 (X-2.00) 48 h Y = 25.74 + 8.07 (X-2.00) 72 h Y = 31.28 + 10.89 (X-2.00) 96 h

0 7.5 15.0 30.0

Concentration of Cd(ppm)

12 * 24 - ^ - 4 8 - - ^ 7 2 -

60.0

96h

Fig.3. Regression lines showing linear relationship between different concentrations of cadniiuni(Cd) and percent mortality of second stage juveniles (Jj) of the root-knot nematode, Meloidogyne incognita in vitro.

67

Table 4. Effect of lead (Pb) on the mortality of second stage juveniles (J2) of the root-knot nematode, Meloidogyne incognita in vitro.

Exposure Period (h)

12

24

48

72

96

Percent

0

0(-0.66)

0(-1 94)

0(7.24)

0(9.12)

0(9.22)

mortality in different cone, of

7.5

00.0(00.00)

05.0(03.37)

28.5(17.70)

30.0(20.12)

44.5(30.44)

15.0

00.0(00.66)

04.3(08.68)

29.8(28.16)

36.0(31.12)

53.8(51.66)

lead (ppm)

30.0

00.0(01.31)

10.2(13.99)

31.9(38.62)

40.0(42,12)

61.1(72.88)

60.0

03.3(01.97)

23.7(19.30)

50.6(49.08)

50.3(53.12)

98.1(94.10)

Regression

Y=00.66+00.66(X-2.00)

Y=08.68+05.3ip<-2.00)

Y=28.16+10.46(X-2.00)

Y=31.12+11.00(X-2.00)

Y=51.66+21.22(X-2.00)

Each value is an average of 3 replicates. In parentheses are given calculated values. Temp =25° C

68

100

80

s ft*

i 60

©

S: 40

o

0.

Y = 0.66 + 0.66 (X-2.00) 12 h Y = 8.68 + 5.31 (X-2.00) 24 h Y = 28.16 + 10.46 ( X-2.00) 48 h Y = 31.12 + 11.00 (X-2.00) 72 h Y = 51.66 + 22.20 (X-2.00) 96 h

20 J ^

0

-20 0 7.5 15.0 30.0 60.0

Concentration of Pb(ppni)

12 ^ 24 - ^ ~ 4 8 -^^72 " - ^ 96h

Fig.4. Regression lines showing linear relationship between different concentrations of lead(Pb) and percent mortality of second stage juveniles (J^) of the root-knot nematode, Meloidogyne incognita in vitro.

69

increase in the concentration of the heavy metal and the

exposure period (Table 4) . There was a linear relationship

between mortality and concentration of the heavy metal (Fig.

4). The nematode mortality was found to be 0.0, 0.0, 0.0

and 3.3 % after 12 h; 5.0, 4.3, 10.2 and 23.7 % after 24 h;

28.5, 29.8, 31.9 and 50.6 % after 48 h; 30.0, 36.0, 40.0 and

50.3% after 72 h, and 44.5, 53.8, 61.1 and 98.1% after 96 h

respectively in 7.5, 15.0, 3.0.0 and 60.0 ppm concentrations

of Pb (Table 4) . More than 50.0 % mortality occurred after

48 h and it increased to 98.0 % after 96 h in 60.0 ppm

concentration of lead. At this exposure period, even 15.0

ppm concentration caused more than 50.0 % mortality of the

juveniles of M. incognita. Regression line was drawn for all

the concentrations and EC^Q value for Pb was calculated, and

it was found to be 13.9 ppm at 96 h of exposure and 53.5 ppm

at 72 h (Fig. 4).

4.5 To study the response of some selected cultivars of tomato, Lvcopersicon esculentum to the root-knot nematode. Meloidocryne incocmita in the presence of cadmium (Cd) :

All the test cultivars of tomato were found to be

infected with the root-knot nematode but to a varying extent

(Table 5). Plant growth parameters were reduced accordingly.

Minimum reduction in fresh weight was observed in cv. Punjab

Kesari (10.4 %) , closely followed by Arka Vikas (12.5 %) ,

Roma (14.3 %) and F^ Hybrid (17.5 %) . Highest reduction in

fresh weight was observed in cv. Damayanti (42.3 %) . The

plant growth was further reduced in all the cultivars

70

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excepting Arka Vikas when plants were âlso treated with

cadmium at 15.0 ppm and inoculated with the nematode (Table

5) . As far as root-knot development is concerned, lowest

root-knot index was observed in cv. Arka Vikas (0.6) and

maximum in cv. Pusa Ruby (4.0). The heavy metal treatment

significantly reduced the root-knot development further

(Table 5) . Thus the results indicate that susceptibility of

the cultivars was greatly influenced by the treatment of the

heavy metal.

4.6 To Study the response of some selected cultivars of tomato jLycoversicon esculenfcum to the root-knot nematode, Afeloidogyne incognita in the presence of lead iPbl:

The test cultivars of tomato showed varying response

to the root-knot nematode, Meloidogyne incognita (Table 6).

Plant growth parameters were reduced accordingly. The cv.

Punjab Kesari showed the minimum reduction in fresh weight

as 10.4 %, followed by Arka Vikas (12.5 %) , Roma (14.3 ^),F^

Hybrid (17.5 %) and the maximum reduction was observed in cv.

Damayanti (42.3 %) . There was further reduction in plant

growth in all the cultivars except cv. Arka Vikas when the

plants received the treatment of lead at 15.0 ppm and

nematode at 5000 J2/pot as well (Table 6) . The impact of

heavy metal was quite obvious on the root-knot development.

In the untreated set the lowest root-knot index was

observed in cv. Arka Vikas (0.6) and the maximum was found in

cv. Pusa Ruby (4.0). In the treated sets, the heavy metal

treatment further reduced the root-knot development

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significantly (Table 6). The above findings also reveal that

susceptibility of the cultivars was greatly influenced by the

treatment of the heavy metal. When the results with respect

to Cd and Pb were compared (Tables 5, 6), it was observed

that Pb was more inhibitory to the root-knot development as

well as plant growth.

4.7 To study the the effect of cadmium (Cd) on the penetration of second stage juveniles (J2)of the root-knot nematode, Meloidogyne incocjnita into the roots of tomato. Lycopergicon esculentum cv. Pusa Ruby:

It is evident from Table 7 that cadmium had an

inhibitory effect on the penetration of second stage

juveniles (J2) of the nematode when seedlings were kept in

different concentrations of the heavy metal (7.5 and 15.0

ppm) for different durations (12, 24 and 48 h) . However, the

minimum number of nematodes which could succeed to penetrate

into the root system was 690.4 after 48 h at 15.0 ppm

concentration of the heavy metal while the maximum number was

observed as 879.4 after 12 h at 7.5 ppm concentration of

cadmium as compared to 891.7 in control (Table 7, Fig.5).

4.8 To study the effect of lead (Pb) on the penetration of second stage juveniles (J2) of the root-knot nematode, Meloidogyne incoQziita into the roots of tomato Lycopersicon esculentum cv. Pusa Ruby:

It is quite indicative from Tables 7 and 8 that lead

was more inhibitory than cadmium for root penetration of

second stage juveniles (J2) of the nematode. The minimum

number of juveniles which could penetrate into the root

system was 630.0 after 48 h dip duration at the 15.0 ppm

80

Table 7. Effect of cadmium (Cd) on the penetration of second stage juveniles(J2) of the root-knot nematode, Meloidogyne incognita into the roots of tomato, Lycopersicon esculentum cv. Pusa Ruby.

Root-dip No.of J2 penetrated/plant at different root dip duration(h) Treatment (Cd) Undipped 12 24 48

CD.

P=0.05 P=0.01

Control

7.5 ppm

15.0 ppm

CD. (P=0.05) CD. (P=0.01)

891.7

879.4(1.37)

870.4 (2.38)

080.61 133.69

778.7(12.67) 715.0(19.81) 84.20 139.65

762.0(14.54) 690.4(22.5) 73.44 121.80

075.03 124.44

066.18 109.76

Each value is an average of 3 replicates. In parentheses are given the values of percent reduction over control.

81

1000 n

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c a

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200 ©

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12 h root-dip

48 h root-dip

24 h root-dip

Fig.5. Effect of cadniium(Cd) on the penetration of second stage juveniles (J ) of the root-knot nematode, Meloidogyne incognita into the roots of tomato {Lycopersicon esculentum) cv. Pusa Rubv.

82

Table 8. Effect of lead (Pb) on the penetration of second stage juveniles (J2) of the root-knot nematode, Meloidogyne incognita into the roots of {oma\o,Lycopersi(X)n esculentum cv. Pusa Ruby.

Root-dip Treatment (Pb)

No. of J2 penetrated/plant at different root-dip duration(h) CD.

Undipped 12 24 48 P=0.05 P=0.01

Control

7.5 ppm

15 0 ppm

CD. (P=0.05) CD. (P=0.01)

891.7

862.4 (3.28)

830.4 (6.87)

077.50 128.53

768.4(13.82) 683.4(23.35) 78.94 130.92

753.7(15.47) 630.0(29.34) 70.50 116.92

073.43 121.78

065.29 108.28

Eacti value is an average of 3 replicates. In parenttieses aregiven the values of percent reduction over control.

83

1000 1

s

in

© 800 o u u a

4 B

a

©

600

4 0 0 -

^ 2 0 0 -

7.5 15.0

Concentrations of Pb(ppni)

] 12 h root-dip

i 48 h root-dip

:d 24 h root-dip

Fig.6. Effect of lead(Pb) on the penetration of sec -ond stage juveniles (J ) of the root-knot nematode, Meloidogyne incognita into the roots of tomato (Lycopersicon esculentum) cv. Pusa Ruby.

84

concentration of the heavy metal, while the maximum number

was found as 862.4 after 12 h dip duration at 7.5 ppm

concentration of lead as compared to 891.7 in control (Table

8, Fig. 6).

4.9 To study the effect of cadmitim (Cd)on the root-knot development caused by the root-knot nematode, Jtfeloidoqyne incognifca and plant growth parameters of tomato, Lycoperaicon eaculentvm cv. Pusa Rxiby:

There was an increase in retardation of plant growth

with an increase in the level of cadmium and that was

further ameliorated significantly when plants were inoculated

with the root-knot nematode, Meloidogyne incognita (Table

9a, Fig. 7a,b). A comparison between uninoculated and

inoculated sets in the cibsence of cadmium revealed a

reduction in fresh weight from 48.5 to 33.8 g and in dry

weight from 26.3 to 18.3 g. In the treated - uninoculated

set, percent redcution in fresh weight of plants was found

to be 32.3, 40.3, 52.8 and 57.7 % for 7.5, 15.0, 30.0 and

60.0 ppm concentrations of the heavy metal respectively over

untreated-uninoculated control. The reduction in fresh plant

weight was also noted to be positively correlated with the

increase in the concentration of the heavy metal in the

presence of the nematode. In such plants, the percent

reduction in fresh weights was observed to be 8.1, 16.2, 23.9

and 25.4 % for 7.5, 15.0, 30.0 and 60.0 ppm concentrations of

the heavy metal respectively over untreated-inoculated

control (Table 9a, Fig. 7a) . The same trend was observed

with respect to the reduction of dry weight of plants (Table

9a, Fig 7b).

85

The chlorophyll (Chl.b and Chi.a) content of tomato

leaves decreased in the treated plants both in presence and

absence of the nematodes. In both the case?, reduction was

minimum in the lowest concentration of cadmium (7.5 ppm) and

it increased with the increase in the concentration of the

metal. In the uninoculated set, the percent reduction in the

Chl.b content over control in different treatments (Cd 7.5-

60.0 ppm) ranged between 24.4 - 58.8 % and for chl.a the

percent reduction ranged between 40.7 - 61.4 %, whereas, the

range in the inoculated plants for chl.b and a was between

12.9 - 53.2% and 14.5 - 46.8 % respectively (Table 9b, Fig.

7d,e).

Water absorption capacity of roots significantly

decreased in the inoculated plants as compared to the

uninoculated ones. In the treated-uninoculated sets, the

percent reduction in water absorption capacity over

untreated-uninoculated control was 11.57, 30.05, 46.63 and

55.61 % at 7.5, 15.0, 30.0 and 60.0 ppm concentration of Cd

respectively while the corresponding figures in the

inoculated plants were 7.2, 16.0, 33.5 and 44.9 % at

different levels of treatment over untreated-inoculated

control (Table 9c, Fig. 7f) .

Root-knot index was highest as 4.1 in untreated

control and it was further reduced significantly in the

plants treated with the heavy metal. At 7.5 ppm

concentration, the root-knot index was reduced to 1.8 with a

86

Table 9a Effect of cadmium (Cd) on the root-knot development caused by the root-knot nematode, Meloidogyne incognita and growth parameters of tomato, Lycopersicon esculenfum c\t Pusa Ruby

Treat

(ppiii

0

75

150

30 0

60 0

CD CD

ment in soil)

Fresh Weight (g)

UN IN

48 5 - 33 8 -

42 6 (32 3) 30 7 (08 1)

39 5(40 3)28 3(16 2)

33 5 (52 8) 25 7 (23 9)

25 6 (57 7) 25 2 (25 4)

(P=0 05) 04 33 02 67 (P=0 01) 06 30 03 88

CD

0 05

4 64

3 88

3 41

3 19

2 44

0 01

10 70

08 95

07 87

07 36

05 63

Dry Weight (g)

UN IN

26 3 - 18 3 -

17 8 (32 3)17 2(06 0)

15 7 (40 3)13 4(26 7)

12 4 (52 8)10 2(44 2)

11 1 (57 7)06 8(62 8)

02 52 01 56 03 67 02 27

CD

0 05

2 70

1 52

1 40

1 27

1 15

0 01

6 23

3 50

3 23

2 93

2 65

0 56 0 93

Root-knot Index (0-5 Scale)

41

1 8 (056 0)

0 8 (080 0)

0 0(100 0)

0 0(100 0)

Each value is an average of 3 replicates. In parentheses are given the values of percent reduction over control UN=Uninoculated IN=lnocuiated with 5000 J2 of M incognita per plant

87

Table 9b Effect of cadmium (Cd) on the chlorophyll content of tomato, Lycopersicon esculentum cv Pusa Ruby in the presence of the root-knot nematode, Meloidogyne incognita

Treatment Chlorophyll b (mg/g) CD Chlorophyll a (mg/g) C D (ppm

0

07 5

15 0

30 0

60 0

C D C D

in

(E= {P=

soil)

=0 05) =0 01)

UN

0 90

0 68 (24 4)

0 63 (30 0)

0 54 (40 0)

0 37 (58 8)

0 52 0 76

IN 0 05

0 77 - 0 28

0 67 (12 9)0 11

0 62 (19 4)0 09

0 51 (33 7)0 14

0 36 (53 2)0 16

0 49 0 71

0 01

0 65

0 25

0 21

0 37

0 37

UN IN

1 40 - 0 96 -

0 83 (40 7)0 82 (14 5)

0 73 (47 8) 0 73 (23 9)

0 63 (55 0) 0 62 (35 4)

0 54 (61 4) 0 51 (46 8)

0 89 0 68 1 29 0 99

00 05

00 50

00 17

00 10

00 12

00 13

0 01

1 15

0 39

0 23

0 28

0 30

Each value is an average of 3 replicates In parentheses are given the values of percent reduction over control UN = Unionculated IN = Inoculated with 5000 J2 of M incognita per plant

88

Table 9c Effect of cadmium (Cd) on water absorption capacity of tomato, Lycopersicon esculentum cv Pusa Ruby in the presence of root-knot nematode, Meloidogyne incognita

Treatment Amount of water absorbed / plant (g) C ^

0

7 5

15 0

30 0

60 0

C D

C D (P= (P=

=0 05) =0 01)

UN

57 9

51 2 (11 57)

40 5 (30 05)

30 9 (46 63)

25 7 (55 61)

04 99

07 26

IN

38 7

35 9

32 5

25 7

21 3

03 70 05 38

-

(07 2)

(16 0)

(33 5)

(44 9)

0 05

5 34

4 51

3 65

3 04

2 38

00 01

12 32

10 40

08 42

07 01

05 49

Each value is an average of 3 replicates In parentheses are given the values of percent reduction over control UN=Unmoculated IN=lnoculated with 5000 J2 of M incognita per plant

89

50

40

« 30 a

J0£

^ 20

10

> • • • • • •

••*••*

li

« • • • * *

, •••••• •••••• ••••••

* • » * • •

* • « • • •

^ * « * * * * » • • * • • •

0.0 7.5 15.0 30.0 Concentrations of Cd(ppni)

Uninoculated

60.0

Inoculated

Fig.Va.Effect of cadmiuni(Cd) on fresh weight of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

90

30

25

U)

B

20

: 15

'Z

I 10

•

0 0.0 7.5 15.0 30.0

Concentrations of C(i(ppm)

Uninoculated

60.0

Inoculated

Fig.Tb.Effect of cadmiuni(Cd) on dry weight of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

91

e

o c I

'^ o o X

2 -

0.0 7.5 15.0 30.0

Concentrations of Cd(ppm) 60.0

Fig.7c. Effect of cadmium(Cd) on root-knot indices of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

92

E

Si

a. o

« * • o

e 3 o E <

0.8

0.6

0.4-

0.2

»*«*« • >•*«#«

» • • • • • *

«•«• •« ««*«•<

0.0 7.5 15.0 30.0

Concentrations of Cd(ppm)

Z] Uninoculated

60.0

Inoculated

Fig.Td.Effect of cadmium(Cd) on chlorophyll b content of tomato (Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

1.6

93

1.4

I 1.2 H

0)

a o o

3 o E <

0.8-

"5 0 .6 -

0.4

0.2

0

' • « • • • •

•••**••

0.0 7.5 16.0 30.0


Inoculated

60.0

J Uninoculated

Fig.7e. Effect of cadniium(Cd) on chlorophyll a of tomato (Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

94

60

60

B

t3 JO u o

JS

u

4 0 -

30

- 20 i s o E

<

10

• * • • • • «

*•*••* •••••• •••••• «••«•• •••••• *•••*• •••••• ••••••

• » • • • • •••••* •• • » • • •*•••• •••••• •*•••• •••••• •••••• •*•••• ••*••• •*••** •••••• ••••••

•••••• •••*•• »••••* •••••• •••••• *••••• •* • * « • •••••• • • • • * • 4 ••••••^ • •••** 4

»••*••« *•••** 4

•

.

.

.

•

* • • « • • • « • • • •

• • » » • • •••••• ••••*• •••**• •••••• • « • • * • • » » * • •

•••••• '•••••• •••••• «••**« •••**• '•••••• '•»•* • • ' * • • « • • '•••••• •••••• «*•••« •**••• •••••• *••••• • • • • « • «••••* •••••* ••••*•

• • i ^ ^ ^ j ,

«••••*) I -, .-

^tt«::

••*••••

0.0 7.5 15.0 30.0 60.0


inoculated Uninoculated

Fig.7f. Effect of cadmium(Cd) on water absorption capacity of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

95

reduction of 56.0 % over control, and at 15.0 ppm it came

down as low as 0.8 with 80.0 % reduction over control (Table

9a, Fig. 7c) . The galls failed to develop at all the

remaining higher concentrations tested, viz. 30.0 and 60.0

ppm of the heavy metal showing a 100.0 % redution over

control (Table 9a, Fig. 7c).

The results of the experiments clearly reveal that

cadmium is highly injurious to tomato plants at all the

concentrations tested for. The inhibitory effect on plant

growth and other parameters (fresh and dry weights of plant,

chlorophyll content of leaves, water absorption capability

of roots) significantly increased with an increase in the

concentration of the metal. It was further ameliorated in

the presence of the nematode. However, it was interesting to

note that plant damage in terms of percentage was

comparatively less in the nematode inoculated plants than

uninoculated ones (Table 9a, Fig. 7a,b). These treatments

also inhibited root penetration by the nematode juveniles as

well as the subsequent development of root galls. The toxic

effect of cadmium on plant growth of tomato was, however,

found to be less severe than lead (Tables 9a, 10a, Fig.

7a,b, 8a,b).

4.10 To study the effect of lead (Pb) on the root-knot development caused by the root-knot nematode, MeloidoayTie incognita and plant growth parameters of tomato, Lvcoversicon esculentwn cv. Pusa Ruby:

Lead (Pb) was found to be more toxic to tomato plant

and plant growth decreased with an increase in the level of

96

lead and the inoculation of plants with the root-knot

nematode, Meloidogyne incognita caused further deterioration

in plant growth parameters signifinantly (Table 10a, Fig.

8a,b). After inoculation with M. incognita, there was a

reduction in fresh weight from 48.5 to 33.8 g and in dry-

weight from 26.3 to 18.3 g in plants receiving no treatment

of the heavy metal. In the treated-uninoculated set, percent

reduction in fresh weight of plants was found to be 9.0,

14.4, 19.3, 48.4 for 7.5, 15.0, 30.0 and 60.0 ppm

concentrations of the heavy metal respectively over

untreated-uninoculated control. There was an increase in the

percent reduction of plants which received the treatment of

heavy metal and inoculated with nematodes as well. In such

sets, the percent reduction in fresh weight was found as 4.1,

7.1, 25.4 and 44.6 at 7.5, 15.0, 30.0 and 60.0 ppm

concentration of the heavy metal respectively over

untreated-inoculated control (Table 10a, Fig. 8a). A similar

trend was observed with respect to the reduction of dry

weight of plants (Table 10a, Fig. 8b).

The chlorophyll (Chl.b and Chi.a) contents of leaves

decreased in the treated plants both in presence and absence

of the nematodes. Reduction was found to be minimum in the

lowest concentration of lead (7.5 ppm) and it increased with

the increase in the concentration of the metal. In both the

cases, reduction was minimum in the lowest concentration of

lead (7.5 ppm) and maximum in the highest concentration

(60.0 ppm). In the uninoculated set, the percent reduction

97

in the Chi.b content over control in different treatments (Pb

7.5 - 60.0 ppm) ranged between 32.2 - 66.6 % and for Chi.a,

it was found between 55.7 - 90.7 %. In the inoculated set,

the range of percent reduction was found between 35.0 -

92.2 % for Chl.b and 46.8 - 80.2 % for Chi.a (Table 10b, Fig.

8e[,e) .

Water absorption capacity of roots decreased in the

inoculated plants in comparison to that of uninoculated ones.

In the treated -uninoculated sets, the percent reduction in

water absorption capacity was 37.1, 44.3, 50.7 and 62.8 at

7.5, 15.0, 30.0 and 60.0 ppm concentration of lead

respectively over untreated-uninoculated control. In the

inoculated plants, the percent reduction was found as 25.5,

38.7, 45.2 and 50.9 at different levels of treatment over

untreated-inoculated control (Table 10c, Fig. 8f).

The root-knot index also showed a declining trend with

increasing treatment of the metal. Galls failed to develop

at 30.0 and 60.0 ppm concentration of the metal and were

formed only in plants which received the lower doses of lead

(7.5 and 15.0 ppm). The root-knot index was rated as 1.3

showing a percent reduction of 68.2 over control for 7.5

ppm and 0.3 for 15.0 ppm (% reduction of 92.6) against 4.1

in control (Table lOa, Fig. 8c).

The results clearly indicate the toxic effect of the

metal on different plant growth parameters (fresh and dry

weight, cholorophyll content, water absorption capacity) and

98

Table 10a Effect of lead (Pb) on the root-knot develoment caused by the root-knot nematode, Meloidogyneincognlta and growth parameters of tomato, Lycopersicon esculentum cv Pusa Ruby

Treat

(ppm

0

07 5

150

30 0

60 0

CD CD

merit in soil)

(P=0 05;

Fresh Weight (g)

UN IN

48 5 - 33 8 -

44 1 (09 0)32 4 (04 1)

41 5(14 4)31 4 (07 1)

39 1 (19 3)25 2(25 4)

25 0(48 4) 18 7(44 6)

) 04 53 02 86 (P=0 01)06 59 04 16

CD

0 05

4 65

3 97

3 66

3 30

2 59

0 01

10 73

09 16

08 44

07 61

05 97

Dry Weight (g)

UN IN

26 3 - 18 3 -

19 1 (27 3) 17 0(07 1)

18 2(30 7) 16 5(09 8)

16 5(37 2) 11 2(38 7)

10 4(60 4) 08 4(54 0)

02 44 01 78 03 55 02 52

CD.

0.05

2.73

1.58

1 49

1 40

1 12

0.01

6 30

3 64

3 44

3 23

2 58

Root-knot

Index (0-5 Scale)

41 -

1 3 (068 2)

0 3 (092 6)

0 0(100 0)

0 0(100 0)

0 37 0 54

Each value is an average of 3 replicates In parentheses are given the values of percent reduction over control UN=UninoGulated IN=lnoculated v ith 5000 J2 of M incognita per plant

99

Table 10b. Effect of lead (Pb) on the chlorophyll content of tomato, Lycopersicon

esculentum cv. Pusa Ruby in the presence of the root-knot nematode,

Meloidogyne incognita.

Treatment (ppm in soil)

0

07.5

15.0

30.0

60.0

CD. CD

(P=0.05) (P=0.01)

Chlorophyll b (mg/g)

UN IN

0.90 - 0.77 -

0.61 (32.2) 0.50 (35.0)

0.57 (36.6)0.48 (37.0)

0 56 (37.7)0.28 (63.6)

0.30 (66.6)0.06 (92.2)

0.39 0.45 0.57 0.65

CD.

0.05

0.12

0.08

0.06

0.17

0.11

0.01

0.28

0.18

0.14

0.39

0.25

Chlorophyll a (mg/g)

UN

1.40 -

0.62 (55.7)

0.36 (74.2)

0.35 (75.0)

0.13(90.7)

0.83 1.21

IN

0.96 -

0.51 (46.8)

0.34 (64.5)

0.32 (66.6)

0.19(80.2)

0.55 0.80

CD

0.05

0.49

0.10

0.08

0.06

0.09

i_

0.01

1.13

0.23

0.18

0.14

0.21

Each value is an average of 3 replicates. In parentheses are given the values of percent reduction over control. UN=Unionculated, IN=lnoculated with 5000 J2 of M. incognita per plant.

100

Table 10c Effectof lead (Pb) on water absorption capacity of tomato, Lycopersicon esculentum cv. Pusa Ruby in the presence of the Root-knot nematode, Meloidogyne incognita

Treatme (ppm in

0

7 5

15 0

30 0

60 0

C D (P= C D (P=

int soil)

=0 05) =0 01)

Amount of water absorbed / plant (g)

UN

57 9 -

36 4 (37 1)

32 2 (44 3)

28 5 (50 7)

21 5 (62 8)

04 84 07 04

IN

38.7

28.8 (25.5)

23.7 (38.7)

21.2 (45.2)

19 0 (50 9)

03.22 04.68

CD,

0.05

5.34

3.87

3.50

2 84

2 02

0 01

12 31

08 93

08.07

06 55

04 66

Each value is an average of 3 replicates In parentheses are given the values of percent reduction over control UN=Uninoculated, IN=lnoculated with 5000 J2 of M incognita per plant

1 0 1

50 n

40

bX)

e

^ 3 0 O

'Z

. . . . . . *

20

* • • • » •

1 0 1

0.0 7.5 15.0 30.0 Concentration of Pb(ppni)

60.0

Uninocuiated iiihl inoculated

Fig.Sa.Effect of lead(Pb) on fresh weight of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

102

30

25

5 20 e ct:

* 15

'Z

t 10

•••••*

0.0 7.5 15.0 30.0

Concentration of Pb(ppm)

Uninoculated

60.0

Inoculated

Fig.Sb.Effect of !ead(Pb) on dry weight of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

5n

103

3 -

B

O B

• ^ ^

O O X

0.0 7.5 16.0 30.0


60.0

Fig.Sc. Effect of lead (Pb) on root-knot indices of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

'5b E

9i

104

0.8

0.6-

& o u s

0.4-

s 3 O

0.2

;.0 7.5 15.0 30.0

Concentration of Pb(ppni)

Un inoculated

60.0

inoculated

Fig.8d.£ffect of lead(Pb) on chlorophyll b content of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

-5f

B

1.6

1.4

B 1.2

105

a o

o

0.8 -

0.6

e 3 o S

0.4

0.2

0 0.0 7.5 15.0 30.0


SHiB Inoculated

60.0

Un inoculated

Fig.8e. Effect of lead(Pb) on chlorophyll a content of tomato {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

106

60

6 0 -bc

e es

I 40 «u

.Q u o

es u 30 es

- 20 s o E <

10 • • « • • c ^

# * * * • r 1

•

•

•

0.0 7.5 15.0 30.0


Uninocuiated

60.0

inoculated

Fig.Sf. Effect of lead(Pb) on water absorption capacity of tomnto {Lycopersicon esculentum) cv. Pusa Ruby in the presence of the root-knot nematode Meloidogyne incognita.

107

the toxicity was further ameliorated in the presence of the

nematode. Lead was found to be more toxic than cadmium

(Tables 9a,10a, Fig. 7a,b, 8a,b).

4.11 To study the effect of cadmium (Cd) on the growth of females of the root-knot nematode^ Meloidoovne incooni ta on tomato iLycopersicon esculeatup' T" PIIHA Ruby:

An experiment (similar to experment No. 4.9) was

conducted to study the effect of cadmium on the growth of

females of the root-knot nematode, Meloidogyne incognita. The

results indicate that all the test doses of the heavy metal

(7.5 - 60.0 ppm) were highly injurious to the growth of the

females. As compared with 203,611.70 sq u average cross-

sectional area of the females in the untreated control, the

lowest concentration of Cd (7.5 ppm) reduced the cross-

sectional area of the females to 158,942.40 sq u and in the

highest concentration (60.0 ppm) to 51, 586.10 sq u (Table

11) . The reductions in the growth of nematode females were

21.94, 36.92, 60.11, and 74.67 % in 7.5, 15.0, 30.0 and 60.0

ppm concentrations of cadmium respectively (Table 11, Fig.

9a, b) .

4.12 To study the effect of lead (Pb) on the growth of females of the root-knot nematode, MeloidooYne incoonita on tomato , LocYPsrsicon esculentum cv. Pusa Ruby:

As in experiment No. 4.10, the effect of lead was

studied on the growth of females of the root-knot nematode,

Meloidogyne incognita. All the test doses of lead (7.5-60.0

108

Table 11 Effect of cadmium (Cd) on the growth of females of root-knot nematode, Meloidogyne incognita on tomato, Lycopersicon esculentum cv Pusa Ruby

Treatment (ppm)

Untreated

7 5

15 0

30 0

60 0

C D (P=0 05) C D (P=0 01)

Cross sectional area of nematode female (Sq p)

2,03 611 70

1,58,942 40

1,28 442 20

0,81,225 40

0,51,586 10

0,23,617 39 0,34 360 95

(2,01,115 20)

(1,62,938 38)

(1,24,761 56)

(0,86,584 74)

(0 48,407 92)

% reduction over control

-

21 94

36 92

60 11

74 67

Each value is an average of 30 females from 3 replicates Regression value Y=124761 56 - 38176 82 (X-2 00) In parentheses are given the calculated values

1 0 9

O

o I

"D O

O O n

I

CJ ^-c

E 4 -o

o in

I

(J

IT)

I T3 U

E o

"5 E 0/

>

c (/)

cx Of

••-O

i_ +-c

110

Thousands 250

s:

s:

200

Y-124761.66 - 38176.82(X-2.00)

150

« cr

^ ^ 100 IS e o

e u 50

0 Untreated 7.5 15.0 30.0


60.0

Fig.9b. Regression lines showing linear relationship between different concentrations of cadmium(Cd) and cross sectional area of females of the root-knot nematode, Meloidogyne incognita.

I l l

Table12. Effect of lead (Pb) on the growth of females of root-knot nematode, Meloidogyne incognita on tomato, Lycopersicon esculentum cv. Pusa Ruby.

Treatment (ppm)

Untreated

7.5

15.0

30.0

60.0

CD. (P=0.05) CD. (P=0.01)

Cross sectional area of nematode female (Sq [i)

2,03,611.70

1,44,500.30

0,98,705.90

0,62,129.10

0,43,981.70

0,20,167.51 0,29,341.72

(1,90,911.98)

(1,50,748.86)

(1,10,585.74)

(0,70,422.62)

(0,30,259.50)

%reduction over control

-

29.03

51.52

69.49

78.40

Each value is an average of 30 females from 3 replicates. Regression value Y=110585.74-40163.12(X-2.00). In parentheses are given the calculated values.

112

113

Thousands 200 n

93

E 150

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^ 100

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CQ s

^©

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60

Untreated 7.5 15.0 30.0


60.0

Fig.lOb.Regression lines showing linear relationship between different concentrations of lead(Pb) and cross sectional area of females of the root-knot nematode, Meloidogyne incognita.

114

ppm) were found to be more toxic to the growth of the

females than cadmium. As compared with 203,611.70 sq p.

average cross-sectional area of the females in the untreated

control, the lowest concentration of Pb (7.5 ppm) caused the

reduction in cross-sectional area of the females to

144,500.30 sq and in the highest concentration (60.0 ppm)

to 43,981.70 sq fx (Table 12). The reductions in the growth

of nematode females were 29.03, 51.52, 69.49 and 78.40 % in

7.5, 15.0, 30.0 and 60.0 ppm concentrations of lead

respectively (Table 12, Fig. 10a, b).

4.13 To study the effect of cadmium (Cd) on the protein profile of females of the root-knot nematode, MeloidoOYiie incoonita:

Effect of cadmium on the protein profile of females of

the root-knot nematode, Meloidogyne incgonita was assessed

(Table 13 , Fig. 11) . It was observed that the number of

polypeptides gradually decreased with an increase in the

concentration of the heavy metal. Total number of

polypeptides in M. incognita females was found to be 19 in

the untreated control. The number of polypeptides decreased

to 13 in the treatment with 7.5 ppm and 11 with 15.0 ppm of

cadmium in soil. It was interesting to note that number of

polypeptides of both higher molecular weight (>205 KD) as

well as low molecular weight (<29 KD) reduced at 7.5 ppm and

it totally disappeared at 15.0 ppm Cd. In the untreated

control, there were two bands of polypeptides of >205 KD mol.

wt. and four bands of < 29 KD mol.wt. and this reduced to

one band of > 205 KD mol.wt. and three bands of <29 KD mol.

115

Table 13. Effect of cadmium (Cd) on the protien profile of females of root-knot nematode, Meloidogyne incognita.

s. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Total

Mol.Wt . (KD)* of polypetides detected in females of M. incognita in different concentrations** of Cd,

no. of Polypeptides

Untreated

>205 >205 143 -134 122 118 109 -105 67 -60 -51 --44 39 --35 -31 <29 <29 <29 <29

19

7.5 ppm

*

>205 -142 134 -118 -107 --66 -52 --47 ---36 -33 -<29 <29 <29 -

13

15.0 ppm

_ -143 -134 -118 -107 --66 -52 -48 ---38 --33 -<29 <29 --

11

* KD = Kilo Dalton ** Cd applied in pot soil at 7.5 and 15.0 ppm on w/w basis.

116

f

200-

190 •

180"

170 •

160 •

150 '

lifO •

130 -

i?n

110 • 1 1 W

100-

90 •

80 -

70 -

fin .

50 .

kO •

or\ 30 -

A-Untreated,

B«Cd-7-5 ppm.

C = Cd-15-0 ppm,

B

Fig.11. Protein profile of females ot the root-knot nematode, Meloidogyne incognita from cadTr\ium-stressed Plonts of tomato, Lycopersicon tsculentum cv- PusaRuby-

117

wt. at 7.5 ppm Cd and further reduced to two bands of

polypeptides of <29 KD mol. wt. and none of >205 KD mol. wt.

at 15.0 ppm level of application of cadmium. Some additional

polypeptides of relatively lower molecular weights appeared

in the presence of heavy metal as compared with those present

in the untreated control. Fragmentation of polypeptides of

higher molecular weights may be the plausible explanation for

the appearance of low molecular weight polypeptides.

4.14 To study the effect of lead (Pb) on the the protein profile of females of root-knot nematode, Afeloidoqyne incocroi ta:

Effect of lead on the protein profile of females of the

root-knot nematode, Meloidogyne incognita was assessed (Table

14, Fig.12). Lead appeared to be more injurious than cadmium,

as in the presence of lead, the total number of polypeptides

decreased to 13 and 6 at 7.5 and 15.0 ppm concentrations

respectively as compared to 19 in the untreated control.

Moreover, only one band of polypeptide of >205 KD mol. wt.

and none of <29 KD mol. wt. was found at 7.5 ppm level of

application of lead. The polypeptide of >205 KD mol. wt. did

not appear at all and only one band of <29 KD mol. wt.

appeared at 15.0 ppm level of Pb application. Besides, the

polypeptides of 143, 134, 118 and 109 KD mol. wt. did not

appear at all in 15.0 ppm level of Pb application (Table 14,

Fig. 12). It was also observed that some additional

polypeptides of low molecular weights appeared in the heavy

metal treatment and a polypeptide of 86 KD mol. wt. appeared

118

Table14 Effect of lead (Pb) on the protein profile of females of root-knot nematode, Meloidogyne incognita.

S Mol Wt (KD)* of polypetides detected in females of No M incognita in diffrent concentrations** of Pb

Untreated 7.5 ppm 15 0 ppm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

>205 >205 143 134 122 118 109 -

105 -

67 -

60 --

51 -

44 39 -

35 --

31 <29 <29 <29 <29

->205 143 134 -

118 -

107 ---

66 -

54 52 51 47 --

36 -

33 32 -----

122

105 86

60

39

<29

Total no of 19 13 Polypeptides

* KD = Kilo Dalton ** Pb applied in pot soil at 7 5 and 15 0 ppm on ww/basis.

119

+ 200 H

190

180-

170 -

160-

150 -

130-

120 -,

110

100-

90 -

80 -

70

60

50 4

kO -

30 -

A= Untreated

B-Pb-7-5 ppm

C=Pb-150PPnn

A B C

Fig 12. Protein profile of females ot the root-k/iot nomatode Meloidogyne incognita from Lead-stressed plants of

tamoto. Lycopersicon "iiculentum cv- Pusa Ruby.

120

at 15.0 ppm level of lead, probably as a result of

fragmentation of polypeptides of higher molecular weights.

4.15 To study the efficacy of orqemic amendments on the root-knot development caused by Mel oidogyne incognita emd its resultant effect on pT*"h growth parameters of tomato/Lycopersi con esculentiun cv. Pusa Ruby in the presence of cadmium (Cd) :

Efficacy of organic amendments against the root-knot

nematode, Meloidogyne incognita attacking tomato,

Lycopersicon esculent urn cv. Pusa Ruby was compared in

presence and absence of cadmium. The results of the

experiment are presented in Table 15. It was clearly revealed

that the nematode caused significant reductions in the

growth parameters of tomato plants. In the untreated-

inoculated control, the reductions in length and fresh and

dry weights of plants were to the extent of 53.0, 30.7 and

61.7 % respectively. The root-knot index was recorded as

4.3. All the organic materials included in the present study

(neem and castor cakes and leaves, rice polish) as well as

inorganic fertilisers improved plant growth because of their

nematicidal/manurial effects (Table 15, Fig. I3a-d).

Amongst the organic additives, neem cake caused

greatest improvement in plant growth parameters followed by

castor cake, neem leaf, castor leaf and rice polish. Both

the cakes gave better results than inorganic fertilisers.

However, the leaf and rice polish amendments were found to be

more or less equal to the inorganic fertilisers. In all these

treatments, though the nematode caused reductions in plant

121

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L' = Untreated - Uninoculated I = Untreated + Inoculated 1 = Amendment alone ( " ) 2 = Amendment alone ( " ) 3 = Amendment + Cd ( " ) 4 = Amendment + Cd ( " ) A = Inorganic fertilizer, B = Neem cake, C = Neem leaf D = Castor cake, E = Castor leaf, F = Rice polish

20

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A B C D E F Treatment

Shoot Root

Fig. 13a. Efficacy of organic amendments in the presence of cadmiiun(Cd) on plant growth parameter (length) of tomato {Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

124

70

l' = Untreated - Uninoculated I = Untreated + Inoculated 1 = Amendment alone ( " ) 2 = Amendment alone ( " ) 3 = Amendment + Cd ( " ) 4 = Amendment + Cd ( " ) A = Inorganic fertilizer, B = Neem cake, C = Neem leaf D = Castor cake, E = Castor leaf, F = Rice polish

60

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Fig.l3b. Efficacy of organic amendments in the presence of cadmium (Cd) on plant growth parameter (fresh weight) of tomato (Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

125

U = Untreated - Uninoculated I = Untreated + Inoculated 1 = Amendment alone ( " ) 2 = Amendment alone ( " ) 3 = Amendment + Cd ( " ) 4 = Amendment + Cd ( " ) A = Inorganic fertilizer, B = Neem cake, C = Neem leaf D = Castor cake, E = Castor leaf, F = Rice polish

2 -

Ul 1234 A

1234 B

1234 1234 c D Treatment

Shoot H Root

1234 E

1234 F

Fig. 13c. Efficacy of organic amendments in the presence of cadmium (Cd) on plant growth parameter (dry weight) of tomato (Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

126

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A B C D E F G H I Treatment

J K L M

Fig. 13d. Efficacy of organic amendments in the presence of cadmium (Cd) on the root-knot development of tomato (Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

127

growth parameters to some extent but the rate of reduction

was much less than in untreated-inoculated controls. These

treatments also brought about reductions in root-knot index

which ranged between 2.1 - 3.9 (Table 15, Fig. 13 d).

The test dose of cadmium (7.5 ppm) was found to be

injurious to tomato as evident from the reduction in the

growth parameters (Table 15) . And this reduction was more

than that caused by the nematode in all the treatments

(inorganic fertiliser, as well as organic amendments) . The

combined effect of cadmium and the nematode was much higher

than caused by either of them, and in all the cases, the

reductions in the growth parameters of tomato was more than

the sum total of the reductions caused by cadmium and the

nematode alone, excepting neem and castor cake (Fig. 13a-c).

4.16 To Study the efficacy of organic amendments on the root-knot development caused by Meloidocryne incoonita and its resultant effect on plant growth parameters of tomato, Lycopersicon esculenttm ev. Pusa Rxiby in the presence of lead (Pb):

In this experiment also, the efficacy of organic

amendments against the root-knot nematode, Meloidogyne

incognita was adjudged in presence and absence of lead. The

results of the experiment are presented in Table 16. Here

also, it was found that nematode caused significant

reductions in the growth parameters of tomato plants. In the

untreated-inoculated plants, there was 53.0, 30.7 and 61.7 %

reductions in length, fresh weight and dry weight

respectively over untreated-uninoculated control. The root-

128

knot index was 4.3 m the untreated-inoculated control (Fig.

I4d). The organic materials as well as inorganic fertilisers

played a positive role in the improvement of plant growth

parameters because of their nematicidal/manurial effects.

As far as the efficacy is concerned, neem cake was

found to be most effective in improving plant growth

parameters followed by castor cake, neem leaf, castor leaf

and rice polish. Neem and castor cakes, both proved to be

more congenial for improving soil condition and suppressing

nematode activity as compared to inorganic fertilisers.

However, rest of the organic amendments (neem and castor

leaf and rice polish) were comparable with inorganic

fertilisers in terms of their efficacy. Plant growth was

reduced due to the nematode infection but the reduction was

much less in the presence of amendments. There was further

reduction in plant growth when both nematode and the heavy

metal were present together.

The test dose of lead (7.5 ppm) had toxic effects on

the growth of tomato plant as it is quite evident from the

reductions in the growth parameters (Table 16, Fig. 14a-c).

This reduction wa;: more than that caused by the nematode in

all the amendments (inorganic as well as organic). The

treatments receiving the heavy metal and the nematode both

resulted in more damage than caused by either of them

singly. In all the cases, the reduction in the growth

parameters of tomato exceeded the sum total of the

reductions caused by lead and the nematode alone, excepting

neem and castor cakes.

129

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U = Untreated - Uninoculated I = Untreated + Inoculated 1 = Amendment alone ( " ) 2 = Amendment alone ( " ) 3 = Amendment + Pb ( " ) 4 = Amendment + Pb ( " ) A = Inorganic fertilizer, B = Neem cake, C = Neem leaf D = Castor cake, E = Castor leaf, F = Rice polish

i 60

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Treatment

Shoot H Root

1234 F

Fig.l4a. Efficacy of organic amendments in the presence of lead (Pb) on plant growth parameter (length) of tomato {Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

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) 2 = Amendment alone ( " ) ) 4 = Amendment + Pb ( " ) B = Neem cake, C = Neem leaf E = Castor leaf, F = Rice polish

I UM234 1234 1234 1234 1234 1234


Shoot Root

Fig. 14b. Efficacy of organic amendments in the presence of lead (Pb) on plant growth parameter (fresh weight) of tomato {Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

133

1

II = Untreated - Uninoculated I = Untreated + Inoculated 1 = Amendment alone ( " ) 2 = Amendment alone ( " ) 3 = Amendment + Pb ( " ) 4 = Amendment + Pb ( " ) A = Inorganic fertilizer, B = Neem cake, C = Neem leaf D = Castor cake, E = Castor leaf, F = Rice polish

3-

0£

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Shoot Root

Fig.l4c. Efficacy of organic amendments in the presence of lead (Pb) on plant growth parameter (dry weight) of tomato {Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

134

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A B C D E F G H I Treatment

J K L M

Fig,14d. Efficacy of organic amendments in the presence of lead (Pb) on the root-knot development of tomato {Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

135

4.17 To study the in vitro growth of nematode biocontrol fungus, Paecilomyces lilacinus in presence of r/trm-iiim (Cd) :

The dry weight of the fungus decreased with an increase

in the concentration of the heavy metal (Table 17) . The dry

weight of mycelium was maximum in the control flasks

containing no heavy metal and just by addition of a little

amount of Cd (i.e. at 25 ppm concentration), the weight was

reduced by 17.2 % and it was statistically significant at P =

0.05. With further increase in the concentration of

cadmium its toxicity to the fungus increased as was proved by

the reduction in its dry weight by 40.3 % at 600 ppm

concentration over control. The same trend was followed in

the sporulation too. In the control flasks, sporulation

rating was 5.0 and it decreased by 8.6 % at the lowest

concentration of Cd (25 ppm) . However, statistically

significant (£ = 0.05) reduction in the sporulation was

observed at 75 ppm concentration onwards. At the highest

concentration (600 ppm) sporulation rating was decreased by

76.6 % over control (Table 17).Regression line was also drawn

for all the concentrations to see the correlation between

concentration of the metal and mycelial growth and

sporulation of the fungus. There was found a linear relation

between the concentration of cadmium and mycelial growth and

sporulation of the fungus (Fig. 15 a,b) .

4.18 To study the in vitro growth of nematode biocontrol fungus, Paecilomvces lilacinus in presence of lead iPbl:

It is quite evident from Tables 17 and 18 that lead

was more toxic to the growth of fungus than cadmium. The dry

136

Table17 In vitro growth of nematode biocontrol fungus, Paecilomyces lilacmus in the presence of cadmium (Cd)

Treatment (ppm)

0 25 50 75 100 150 200 300 400 600

C D C D

(P= (P= =0

Mycelial weight

2 519 2 084 2 077 2 055 2 046 2 038 1 960 1 941 1 865 1 502

05) 0 048 =0 01) 0 066

(g)

(2 3264801) (2 2558623)

(2 18524^5) (2 1146267) (2 0440089) (1 9733911) (1 9027733) (1 8321555) (1 7615377)

(1 6909199)


_

17 268 17 546 18 420 18 777 19 094

22 191 22 945

25 962 40 373

Sporul ation (0-5 scale)

5 000 4 567 4 567 4 000 3 500 3 000

2 500 2 000 1 500 1 167

0 73

1 00

(5 1928542) (4 7455755) (4 2982968) (3 8510180) (3 4037393)

(2 9564607)

(2 5009200) (2 0619032) (1 6146245) (1 1673458)

%reduction over Control

_

08 6 08 6 20 0 30 0

40 0

50 0

60 0 70 0 76 6

Each value is an average of 3 replicates Regression value for Mycelial weight Y= 2 0087-0 0701780(X-4 50) Regression value for Sporuiation Y= 3 1801-0 4472787(X-4 50) In parentheses are given the calculated values

137

2.6 n

2 -

^1.5

'Z

.2 "Z o

0.5

Y-2.0087-0.070178(X-4.50)

25 50 75 100 150 200 300 400 600


Fig. 15a. Regression line showing the effect of cadmium (Cd) on the mycelial growth of biocontrol fungus, Paecilomyces lilacinus in vitro.

138

6n

(J

e 3 , e o

s o

Y-3.1801-0.4472787(X-4.50)

0 0 26 50 75 100 150 200 300 400 600


Fig.lSb. Regression line showing the effect of cadmium(Cd) on the sporulation of biocontrol fungus, Paecilomyces 1:1^^:^..

139

Table18 In vitro growth of nematode biocontrol fungus, Paecilomyces lilacinus in the presence of lead (Pb).

Treatment (ppm)

Mycelial weight (g)


Sporuiation (0-5 scale)


0 25 50 75 100 150 200 300 400 600

C D C D

(P=0 05) {P=0 01)

2 772 1 085 1 080 1 046 1 039 1 031 1 029 1 027 0 948 0 209

0 047 0 064

(2 0319925) (1 8307940) (1 6295957) (1 4283974) (1 2271991) (1 0260009) (0 8248026) (0 6236043) (0 4224060) (0 2212077)

-60 874 61 024 62 253 62 509 62 823 62 861 62 959 65 783 92 460

5.000 4.533 4.533 3 067 2 500 2 500 2 067 1 533 1 500 0 500

0 661 0 90O

(4.9477815) (4.4645634) (3.9813453) (3.4981272) (3.0149090) (2.5316910) (2.0484729) (1 5652547) (1 0820366) (0 5988185)

-09 21 09 21 38 66 50 00 50 00 58 66 69 34 70 00 90 00

Each value is an average of 3 replicat_es Regression value for Mycelial weight Y= 1 1266-0 2011983(X-4 50) Regression value for Sporuiation Y= 2 7733-0 4832181 (X-4 50) In parentheses are given the calculated values Temp = 25°C

140

2.5

^ 1.5

Y-1.1266-0.2011983(X-4.60)

.2 (J

0.5

0 25 50 75 100 150 200 300 400 600


Fig. 16a. Regression line showing the effect of lead (Pb) on the mycelial growth of biocontrol fungus, PaecUomyces lilacinus in vitro.

1 4 1

I

©

e o

s c

-J

J

Y-2.7733-0.4832181(X-4.50)

0 0 25 50 75 100 150 200 300 400 600


Fig.]6b. Regression line showing the effect of lead(Pb) on the sporulation of biocontrol fungus^ Paecilomyces lilacinus in vitro.

142

weight of mycelium in the control flask (no treatment of

heavy metal) was 2.772 g and it was reduced by 60.874 % at 25

ppm concentration of lead in the culture medium and it was

statistically significant at P=0.05 (Table 18). There was

gradual reduction in the weight of fungal mycelium by

increase in the concentration of the heavy metal and maximum

reduction came down as low as 92.460 % at 600 ppm

concentration of lead over control. Similar trend was

followed in sporulation also. Sporulation was rated as 5.0

in the control flasks and it was decreased by 9.216 % at the

lowest concentration of Pb (25 ppm) and it was drastically

decreased by 90.0 % at 600 ppm concentration of lead over

control (Table 18) . The regression line showed a linear

relation between the cone, of lead and mycelial growth and

sporulation of the fungus (fig. 16a,b).

4.19 To study the efficacy of nematode biocontrol fungus, Paecilomyces lilacinus on the root-knot development caused by Jfeloidogyne incognita and its resultant effect on plant growth parameters of tomato^ Lycopersicon esculentum cv. Pusa Rxiby in presence of cadmium (Cd) :

Efficacy of the nematode biocontrol fungus,

Paecilomyces lilacinus was assessed against the root-knot

nematode, Meloidogyne incognita in presence of cadmium. In

the untreated set, the root-knot nematode caused significant

reduction in plant growth parameters (Table 19). Simultaneous

inoculation of Paecilomyces lilacinus with Meloidogyne

incognita, however mitigated the plant damage caused by the

nematode. The reduction in fresh weight was 8.0 % as

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compared to 22.6 % in the absence of the fungus. These

resutls also revealed a -positive correlation with the

reduction in root-knot index which reduced from 4.0 to 0.6

when fungus was also inoculated alongwith the nematode. In

other words, the fungus caused a reduction of 85.0 % in the

root-knot development (Table 19). The treatment with cadmium

caused significant reduction in all plant growth parameters

and this reduction was more in presence of the nematode.

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nematode was quite evident in the treated sets but it was not

at par to that found in untreated sets. It indicates

clearly that the efficacy of the fungus was lessened by the

treatment of heavy metal.

4.20 To study the efficacy of nematode biocontrol fungus, Paecilomvces lilacinus on the root-knot development caused by Jfeioidoffyng incoonita and its resultant effect on plant growth parameters of tomato, Lycopersicon esculentum cv. Pusa Ruby in presence of lead (Pb) :

The nematode biocontrol fungus, Paecilomyces lilacinus,

as an efficient control agent against the root-knot nematode,

Meloiodcfyne incognita was studied in the presence of lead.

Plant growth was severely hampered in the cases where

plants were infected with the root-knot nematode alone

(Table 20). The plant damage was however, reduced when the

plant received both the treatments of Paecilomyces

lilacinus and Meloidogyne incognita at a time. The reduction

in fresh weight was 8.0 % as compared to that of 22.6 in the

absence of the fungus. There was a positive correlation

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between the root-knot index and the impact of P. lilacinus.

The root-knot index reduced from 4.0 to 0.6 when the

nematode inoculated plants received the treatment of the

fungus (Table 20). The treatment with lead caused

significant reduction in all plant growth parameters and

reduction was further ameliorated in the presence of the

nematode. Here also, the fungus was quite efficient in

reducing the plant damage due to the nematode in the treated

plants but not to the extent of that found in untreated

sets. The above finding implies the toxic effect of heavy-

metal on the fungus and thus reducing its efficiency.

4.21 To determine the levels of cadmium (Cd)bv Atomic Absorption Spectrophotometry (AAS) in tomato. Lycopersicon esculenturn cv. Pusa Ruby infected with the root-knot nematode. AfeloidocTyne incocTnita :

Level of cadmium was assessed in tomato plants by

Atomic Absorption Spectrophotometry (AAS) on dry weight

basis. The amounts of cadmium in the uninoculated controls

were recorded as 1.5, 4.0, 12.0, 15.0 g/g in shoot and 4.0,

11.0, 12.5, 12.5 ^g/g in roots for 7.5, 15.0, 30.0 and 60.0

ppm levels of the heavy metal in soil. However, in the

inoculated sets, the concentration of the heavy metal

increased and was recorded as 5.0, 10.0, 12.0, 12.5 )xg/g in

shoot and 6.0, 13.0, 14.5, 15.0 /ig/g in root at the above

levels of the heavy metal in soil, respectively (Table

21) .

147

Table 21 Level of cadmium (Cd) estimated by Atomic Absorption Spectrophotometry (AAS) in tomato, Lycopersicon esculentum cv Pusa Ruby infectedwith the root- knot nematode, Meloidogyne incognita


Amount of cadmium m plant material on dry weight basis (pg/g) Uninoculated Inoculated

Shoot Root Average Shoot Root Average

0

7 5

15 0

30 0

60 0

01 5 04 0 02 75

04 0 11 0 07 50

12 0 12 5 12 25

15 0 12 5 13 75

C D (P=0 05) 01 36 01 49 C D (P=0 01) 01 98 02 17

05 0 06 0 05 50

10 0 13 0 11 50

12 0 14 5 13 25

12 5 15 0 13 75

01 28 01 51 01 86 02 20

Each value is an average of 3 replicates inocuium level = 5000 J2 of M incognita per pot

148

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7.5 16.0 30.0


Unlnoculated Shoot ~^ Unlnoculated Root

Inoculated Shoot -^^ Inoculated Root

60.0

Fig. 17. Level of cadiniuin(Cd) estimated by Atomic Absorption Spectrophotometry (AAS) in tomato {Lycopersicon esculentum) cv. Pusa Ruby in fected with the root-knot nematode, Meloidogyne incognita.

149

It is quite apparent from the results that in the

absence of the nematode, movement of cadmium was a bit

restricted than in the inoculated ones. In the inoculated

sets, it appears that the damage caused by the nematode,

weakened the plants and paved the way for increased up-take

of cadmium and thus causing more damage to plants. (Fig.

It) .

4.22 To de termine the l e v e l s of l e a d (Pb) by Atomic Absorpt ion Spectrophotometry (AAS) in tomato^ LYCopersicon esculentum cv. Pusa Ruby i n f e c t e d with the root-knot nematode, Afeloidogyne incoemita:

Accumulation of l ead in tomato p l a n t s was a s ses sed by

Atomic A b s o r p t i o n S p e c t r o p h t o m e t r y (AAS) on d r y weight

b a s i s . I t was i n t e r e s t i n g to note tha t l ead was absorbed by

the p l a n t s i n a l e s s e r amount as compared t o cadmium

(Tables 21, 22) .

The amounts of lead in uninoculated sets were recorded

as 0.0, 0.0, 5.0 and 9.0 yug/g in shoot at 7.5, 15.0, 30.0 and

60.0 ppm concentration of the heavy metal in soil

respectively, and 5.0, 9.0, 10.0 and 7.0 /ig/g in root at the

same levels of application of the heeivy metal (Table 22,

Fig. 18) . In the inoculated set, the amount of lead absorbed

increased a bit as the plants were weakened in the presence

of nematode and the same amount of the heavy metal became

more toxic to plant growth. In such case, the amount of

lead accumulated in plants was found as 0.0, 0.0, 4.5 and

11.0 g/g in shoot and 7.5, 10.0, 11.0 and 12.5 jug/g in root

at 7.5, 15.0, 30.0 and 60.0 ppm concentrations of the metal

respectively.

150

Table22 Level of lead (Pb) estimated by AtomicAbsorption Spectrophotometry (MS) m tomato, Lycopersicon esculentum cv Pusa Ruby infected with the root-knot nematods, Meloidogyne incognita

Treatmf (ppm in

0

7 5

15 0

30 0

60 0

C D (P= C D (P=

3nt soil)

= 0 05) =0 01)

Amou nt of cadmium in Uninoculated

Shoot Root Average

-

-

5 0

9 0

0 52 0 76

05 0

09 0

10 0

07 0

00 87 01 27

2 50

4 50

7 50

8 00

plant material on dry weight basis (pg/g)

Shoot

-

-

04 5

11 0

00 64 00 93

Inocul Root

07 5

10 0

11 0

12 5

01 05 01 53

ated Average

03 75

05 0

07 75

11 75

Each value is an average of 3 replicates Inoculum level =5000 J2 of M incognita per pot

151

O

3 W)

C 3 O £ <

-B-

0.0 7.5 15.0 30.0


Unlnoculated Shoot -^^ Uninoculated Root

Inoculated Shoot -^^ Inoculated Root

60.0

Fig. 18. Level of lead(Pb) estimated by Atomic Absorption Spectrophotometry (AAS) in tomato {Lycopersicon esculentum) cv. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

152

4.23 To determine the levels of micronutrients, viz. Cu , Zn and MP by Atomic Absorption Spectrophotometry (AAS) in cadmium (Cd) stressed tomato LvcoperBicon eBCulentwn cv. Pusa Ruby infected with the root-knot nematode. Meloidoiryne incoemi fca:

Effect of cadmium on the aibsorption of micronutrients,

viz. Cu , Zn and Mn was studied in tomato infected with the

root-knot nematode. In controls where no treatment of the

heavy metal was given, Cu could not be traced in shoot and

root irrespective of the presence or absence of the nematode

while Zn and Mn were recorded as 5.0 and 5.0 }ag/g

respectively in shoots, and 6.5 and 15.5 yug/g respectively

in roots in the uninoculated sets while their absorption

was reduced a bit in the inoculated sets and it was 4.0 and

0.0 ;ug/g respectively in shoot, and 3.5 and 5.0 yug/g

respectively in root (Table 23). The level of Cu , Zn and Mn

was found to be 0.0, 3.5 and 10.0 jug/g respectively in

shoot, 5.0, 3.0 and 15.0 jag/g respectively in root in

uninoculated sets at 7.5 ppm concentration of heavy metal

while in the inoculated ones, the amount of Cu, Zn and Mn

was found as 0.0, 3.5 and 5.0 jug/g respectively in shoot and

5.0, 7.5 and 10.0 pc:/g respectively in root at the same

concentration. The absorption of micronutrients was,

however, a bit facilitated at higher levels of application of

the heavy metal. At 15.0 ppm concentration of cadmium, the

amount of these micronutrients was found as 5.0, 7.0 and

10.0 g/g respectively in shoot and 5,0, 7.5, and 10.0 ;jg/g

respectively in root in uninoculated sets while in the

inoculated sets it was 5.0, 4.5 and 5.0 jug/g respectively in

153

Table 23 Level of micronutrients Cu, Zn and Mn estimated by Atomic Absorption Spectrophotometry (AAS) in cadmium stressed tomato, Lycopersicon esculentum cv Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita


Control

7 5

15 0

30 0

60 0

C D (P=0 05) C D (P=0 01)

Controi

7 5

15 0

30 0

60 0

C D (P=0 05) C D (P=0 01)

Amou

Shoot

0 0

0 0

5 0

9 0

7 0

0 63 0 92

0 0

0 0

5 0

6 5

6 0

0 58 0 84

nt of micronutrients

Cu Root

0 0

5 0

5 0

6 5

6 0

0 57 0 83

0 0

5 0

5 0

5 5

5 0

0 55 G 80

Average

0 00

2 50

5 00

7 75

6 50

0 00

2 50

5 00

6 00

5 50

in plant materials on dry weight basis i

Shoot Zn Root

Uninoculated

5 0

3 5

7 0

7 0

5 0

0 66 0 96

Inocu

4 0

3 5

4 5

2 5

1 0

0 43 0 63

6 5

3 0

7 5

6 5

5 5

0 69 1 00

lated

3 5

7 5

7 0

6 0

4 0

0 56 0 81

Average

5 75

3 25

7 25

6 75

5 25

3 75

5 50

5 75

4 25

2 50

Shoot

05 0

10 0

10 0

06 0

04 5

00 94 01 37

00 0

05 0

05 0

04 5

02 5

00 49 00 71

[\iQ'Q)

Mn Root

15 0

15 0

10 0

07 5

05 0

01 35 01 96

05 0

10 0

10 0

10 0

05 0

00 77 01 12

Average

10 00

07 50

10 00

06 75

04 75

02 50

07 50

07 50

07 25

03 75

Each value is an average of 3 repLcates

154

UNINOCULATED

u o

••-> e ^«

i

3 O

£ <

Control 7.5 15.0 30.0


Cu In Shoot * Cu In Root -^ Zn In Shoot

Zn in Root -^ Mn In Shoot —^ Mn In Root

60.0

Fig.l9a.Level of micronutrients, viz. Cu, Zn and Mn estimated by Atomic Absorption Spectrophotometry in cadmium(Cd) stressed tomato {Lycopersicon esculentum)c\. Pusa Ruby.

155

INOCULATED

73

Xi u o

JO cs er

e .2

"s ^ c ^

e 3 o

A .

Control 7.6 16.0 30.0 60.0


Cu In Shoot * Cu In Root - ^ Zn In Shoot

Zn In Root -^ Mn In Shoot - + - Mn In Root

Fig.l9b.LeveI of micronutrients, viz. Cu, Zn and Mn estimated by Atomic Absorption Spectrophotometry in cadmium(Cd) stressed tomMo {Lycopersicon esculentum)c\. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

156

shoot and 5.0, 7.0 and 10.0 ;ag/g respectively in root in the

inoculated ones (Table 23, Fig. 19a,b).

Maximum absorption of copper was 9.0 /jg/g in the

shoots of uninoculated tomato plants at 30.0 ppm while the

minimum amount (5.0 /ig/g) was found in root at 7.5 ppm and

shoot and root of uninoculated plants of 15.0 ppm

concentration of the heavy metal. The same was also found in

the shoot and root of inoculated plants at the same level of

application of the heavy metal. At 60.0 ppm also, the same

amount of copper ( 5.0 ^g/g) was detected in the root of

inoculated plants.

The amount of zinc increased at 15.0 and 30.0 ppm level

of application of the heavy metal. It was found as 7.0 ^g/g

in the shoot of uninoculated plants, 7.5 and 6.5 /ig/g at 15.0

and 30.0 ppm concentration of the heavy metal respectively in

the root of uninoculated plants. The amount of zinc absorbed

by the plants decreased to as low as 4.5 and 2.5 jjg/g (in

shoot) and 7.0 and 6.0 yug/g (in root) at 15.0 and 30.0 ppm

concentrations respectively (Table 23) .

Absorption of manganese decreased with an increase in

the concentration of the heavy metal. There was further

decrease in its level in the inoculated plants.

4.24 To determine the levels of micronutrients. viz. Cu , Zn and Mn by Atomic Absorption Spectrophotometry (AAS) in lead (Pb) stressed tomato. Lycopersicon esculentum cv. Pusa Ruby infected with the root-knot nematode, MeloidoQYue incoonita:

Effect of lead on absorption of micronutrients, viz. Cu,

Zn and Mn was studied in tomato infected with the root-knot

157

Table 24 Level of micronutrients Cu, Zn and Mn estimated by Atomic Absorption Spectrophotometry(AAS) in lead stressed tomato, Lycopersicon esculentum cv Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita


Control

7 5

15 0

30 0

60 0

C D (P=0 05)

C D (P=0 01)

Control

7 5

15 0

30 0

60 0

C D (P=0 05) C D (P=0 01)

Amount of

Shoot

0 0

2 0

0 0

0 0

0 0

0 02

0 03

0 0

0 0

0 0

0 0

0 0

0 00 0 00

Cu Root

0 0

0 0

GO

0 0

0 0

0 0

0 0

0 0

0 0

5 0

4 5

0 0

0 08 0 12

micronutrients

Average

0 00

1 00

0 00

0 00

0 00

0 00

0 00

2 50

2 25

0 00

, in plant materials on dry

Shoot Zn Root

Uninoculated

5 0

3 5

3 0

2 5

0 0

0 37

0 54

4 5

4 0

5 0

4 5

2 5

0 49

0 71

Inoculated

3 5

2 5

8 0

2 5

2 0

0 46 0 67

3 5

2 5

3 5

3 0

1 0

0 31 0 45

Average

4 75

3 50

4 00

3 50

1 25

3 50

2 50

5 75

2 75

1 50

weight basis (pg/g)

Shoot

05 0

03 5

04 5

03 0

02 5

00 44

00 64

00 0

02 0

15 0

02 5

01 5

00 50 00 73

Mn Root

6 0

6 0

5 5

4 0

2 5

0 54

0 79

4 5

4 5

4 0

3 0

1 5

0 39 0 57

Average

5 50

4 75

5 50

3 50

2 50

2 25

3 25

9 50

2 75

1 50

Each value is an average of 3 replicates

158

UNINOCULATED

6 -t3

Xi

O Vi

• ^ *

C .^

t =

c E

<

0 •M- 4- ^ %

Control 7.5 15.0 30.0


Cu in Shoot * Cu In Root - ^ Zn in Shoot

Zn In Root - ^ Mn in Shoot — ^ Mn in Root

60.0

Fig,20a.Level of micronutrients, viz, Cu, Zn and Mn estimated by Atomic Absorption Spectrophotometry in lead(Pb) stressed iiim2i\xs{J.ycopeTsicon esculentum)c\. Pusa Ruby.

159

INOCULATED

Control 7.6 16.0 30.0 Concentration of Pb(ppm)

Cu In Shoot * Cu In Root - ^ Zn In Shoot

Zn in Root -^ Mn in Shoot —^ Mn in Root

60.0

Fig.20b.Level of micronutrients, viz. Cu, Zn and Mn estimated by Atomic Absorption Spectrophotometry in Iead(Pb) stressed tomsito {Lycopersicon esculentum)c\. Pusa Ruby infected with the root-knot nematode, Meloidogyne incognita.

160

nematode. In the absence of Pb, no copper could be traced in

plants. It was found to be 2.0 pg/g in the shoot of

uninoculated set only at 7.5 ppm level of Pb in soil. In the

inoculated set, copper was recorded as 5.0 and 4.5 /ig/g in

roots respectively at 15.0 and 30.0 ppm concentration of

lead applied in soil (Table 24) . Level of Zn reduced in

shoots of uninoculated plants with an increase in the

concentration of the heavy metal (Pb) in soil, while in

roots it increased a bit at 15.0 ppm concentration of Pb in

soil and then again reduced with further increase in the

concentration of the heavy metal (Pb) . The amount of Zn was

also increased in the shoot and root of inoculated plants at

15.0 ppm Pb and decreased with further increase in the

concentration of the heavy metal. Amount of Mn also increased

at 15.0 ppm concentration of Pb in soil both in the

uninoculated and inoculated shoot, and reduced with further

increase in the concentration of lead (Table 24, Fig. 20a,b),

161

5. DISCUSSION

Man is aware of the problems of maintaining himself and

his descendants on this planet of fixed size. Not only is the

problem of food supply becoming critical, but the waste and

end products of man's existence must be disposed off or

utilised in such a way that the quality of his environment is

not impaired for this or future generations. However, lavish

life-styles and comforts have been achieved at the cost of

many environmental problems, the pollution being the

foremost. Pollution may be defined as an undesirable

accumulation of residues of various organic or inorganic

materials in soils and water as a result of man's activities.

The problem of pollution would appear to have three phases

: inadvertent or uncontrolled release of materials into

ecosystems; release of pollutant materials from disposal

systems because of improper or insufficient treatment or

lack of knowledge concerning possible hazards;and use of

materials for specific purposes on land and vegetation which

may have unrecognised pollutant hazards due to other

properties of the materials.

Heavy metals like lead, cadmium, chromium, nickel,

etc. have significantly been detected in soils due to

industrial effluents, organic wastes, refuse burning ,

transport, power generation, smoke release from domestic and

industrial chimneys, etc. and also found to depress the plant

162

growth and yield at their different levels of application.

Similarly micro-organisms including nematodes may also be

greatly influenced by heavy metal contamination in the soil.

Infoirmation with respect to the effect of heavy metals Oii

plant parasitic nematodes is, however, meagre. The focal

theme of the present study is to assess the impact of heavy

metals, viz. lead and cadmium on the root-knot nematode,

Meloidogyne incognita and its host tomato Lycopersicon

esculentum. The results are being discussed in detail in the

present chapter.

5.1 Effect of the heavy metals on the hatching and mortality of Meloidogyne incognita :

In vitro studies were made to assess the effect of the

test metals, Cd and Pb, on the hatching and mortality of M.

incognita. It is quite evident from the results that both

the metals were highly inhibitory to the nematode hatching ,

lead being more inhibitory than cadmium. (Tables 1,2).

Studies with cyst nematodes (Heterodera spp.)

indicated that several inorganic ions, including nickel (Ni)

stimulated hatching of eggs. Calcium (Ca) and inagnesium (Mg)

caused moderate hatching of Heterodera schachtii

(Wallace, 1956) at a concentration of 3mM, but potassium (K)

and sodium (Na) were inactive. It has been found that Ca,

Mg, Na and K did not effect the hatching of Globodera

rostochiensis (Ellenby & Gilbert, 1958). Robinson & Neal

(1959) reported that adding microgramme amounts of zinc

sulphate and cadmium chloride to dithizone-extracted potato

163

root diffusate inhibited hatching of G. rostochiensis and

they suggested that the metal ions were responsible for the

inhibition. Cadmium chloride was found to be weakly active

at 0.2-0.6 mM but at greater concentrations inhibited hatch

to below that in water.

The experiment conducted to find out the effect of test

metals on the mortality of second stage juveniles (J2) of M.

incognita revealed that the mortality increased with an

increase in the concentration of the metals and the duration

of exposure (Tables 3, 4). However, lead was found to be

more toxic to the juveniles than cadmium and this was

determined on the basis of EC^Q value. Determination of EC^Q

is a convenient parameter for comparing the relative toxicity

of a substance. Acute toxicity studies with heavy metals on

nematodes are rare. In earlier studies, it has been shown

that cadmium salts when tested on the mixed populations of

Panagrellus and Rhabditis, the 48h-Lc5Q ranged between 35-40

mg/1 and this value compares favourably with the mean adult

Lc^Q value of 26.3 mg/1 for P. silusiae (Feldmesser & Rebois,

1966) .

Various concnetrations of CdCl2 when tested for

Caenorhabditis elegans, revealed that the growth and

reproduction was significantly reduced from a level of liiM

CdCl2. Growth was retarded at the early juvenile stages at

levels of 160 and 320yuM and the organisms did not reach the

adult stage and therefore could not reproduce (Van Kessel et

al., 1989) .

164

Apart from the above studies, the information

regarding the ECcn of toxic metals for plant parasitic

nematodes has not been fetched so far. The present study

unravels quite revealing information regarding the ECcn

values of the heavy metals for a plant parasitic nematode,

e.g. Meloidogyne incognita. The results clearly indicated a

direct relationship between mortality and concentration of

the heavy metals. Nematode mortality increased with an

increase in the cone, of the heavy metals as well as the

exposure period. EC^Q for cadmium was found to be 508.00 ppm

at 96h of exposure duration while for lead it was 139.0 and

535.0 ppm at 96 and 72h of exposure respectively (Fig.3,4).

The death of nematodes may be due to the blockage caused by

heavy metals in the uptake and / or assimilation of nutrients

resulting in the death of nematodes (Popham & Webster, 1979).

Hafkenschied (1971) and Pitcher & Mc Namara (1972)

demonstrated the toxic effects of Cu ions on plant parasitic

nematodes. It appears from the survey of literature that the

effect of heavy metals, Cd and Pb, on larval hatching and

mortality of M. incognita has been investigated for the

first time.

5.2 Effect of the heavy metals on larval penetration of Meloidogyne inc<7onita into the roots of tomato:

After assessing the effects of the heavy metals on the

hatching and mortality of the root-knot nematode, an

experiment was conducted to see their effects on the

penetration of second stage juveniles (J2) of M. incognita

165

into the roots of tomato cv. Pusa Ruby and subsequent gall

formation on the roots. The inhibition in larval penetration

increased with an increase in the concentration of the heavy

metals as well as an increase in the dip duration of host

roots (Tables 7, 8) . However, lead was proved to be more

inhibitory than cadmium. The above results have thus shown a

similar trend as has been noticed in the earlier experiments

concerning hatching and mortality. The studies pertaining to

the effects of heavy metal on larval penetration of M.

incognita have been conducted for the first time.

5.3 Response of tomato cultivars to Afeloidog-yne incoonita in relation to the heavy metals:

In nature, performance of pathogens/parasites vary

with changes in the host as well as environmental conditions,

including the pollutants. There are several reports

available on the interaction of pathogens/parasites with

air/soil pollutants (Heagle, 1973). In such situations,

where plants are screened for resistance to a pathogenic

disease, misleading interpretations may come up where host

plants are subjected to pollutants. Crop cultivars, in

addition to possessing gradations in resistance to

parasitic/pathogenic agencies may also differ in their

reaction to the pollutants. So, it was considered worthwhile

to work out that whether the observed disease response of a

particular variety was a matter of resitance to the pathogen

or to the pollutant or both.

166

Keeping in view the above facts, thirteen cultivars of

tomato were screened against the root-knot menatode,

Meloidogyne incognita in presence and absence of heavy-

metals, e.g. lead and cadmium. All the cultivars showed

susceptibility to the root-knot nematode but the gravity of

infection varied from cultivar to cultivar. These results are

in conformity with those of earlier workers (Khan et

al.l975;Patel et al., 1979). In presence of the heavy metals,

the growth parameters of tomato were further reduced,

showing an additive effect of the heavy metal and the

nematode. It was interesting to note that plant damage

after adding the heavy metal was on the same pattern as in

case of the condition when nematode was given alone. This

kind of study has been carried out for the first time and may

help,in a way, in selecting a suitable cultivar in fields

infested with the root-knot nematode and polluted with the

heavy metals. Since, cv. Pusa Ruby was found to be most

susceptible to M. incognita, it was selected as the test

cultivar for further studies.

5.4 Effect of the heavy metals on the root-knot development and plant growth parameters of tomato:

An experiment was conducted to assess the influence of

cadmium/lead on the root-knot development caused by the root-

knot nematode, Meloidogyne incognita and plant growth

parameters of tomato (Lycopersicon esculentum) cv. Pusa

Ruby. Plant growth parameters showed a declining trend both

when treated with the nematode and / or the heavy metals.

167

'• ant weight (fresh and dry) and chlorophyll content

- creased in the inoculated sets and was further decreased

ith the treatment of the heavy metals.

It is well known that heavy metals when present in the

J 11 environment cause a hinderance in the absorption of

'^cro- and macro-nutrients available in the soil. The

i*osorption of these nutrients may be hampered as heavy metals

. '-e known to form complexes with them and therefore are not

?adily absorbed through root cells and consequently causing

c êduction in plant growth. The heavy metals after entering

the plant system may suppress the activity of enzymes

resulting in reduced vital activities and thus causing

reduction in plant growth. These influences of the heavy

metals were confirmed in later studies, where the reduction

m the accumulation of micronutrients is clearly indicated

due to the application of cadmium and lead (Tables 23,24).

The lowest concentration of the heavy metals (7.5 ppm)

was not much toxic to plant growth but the plants inoculated

with the nematodes (5000 J2/pot) showed a remarkable

reduction in fresh weight because this concentration did not

hinder the survival of the nematodes which caused gall

formation and other related symptoms on the plants. Hence,

resulting a reduction in plant growth. It may also be

possible that adsorption of the small quantities of

pollutants by soil particles decreased the toxic effect of

these materials upon the soil inhabiting nematodes (Alphey &

Brown, 1987). The same may be applicable for plant growth.

168

There was slight gall formation at 15.0 ppm concentration

of the heavy metals and it was completely checked in the

presence of higher doses, viz. 30.0 and 60.0 ppm. These

doses were highly detrimental to plant growth and lethal to

the nematode. Similar results have also been reported by

Alphey & Brown (1987).

The results of the study made by Howell (1982), suggest

that the property of mucus for binding heavy metals may

greatly influence the uptake and loss of metals. Mucus

secreted at the anterior end of the body is thought to form

a thin covering over the cuticle (L. Smith, c.f.

Howell,1982) which could then result in a high concentration

of metal ions (relative to the environmental concentration)

at the cuticular surface (Korringa, 1952) . This could lead to

an enhanced rate of heavy metal uptake by the nematode

through the cuticle and could result in the apparent

anomalies observed in relating hea\ ' metal concentrations

measured in the animals to those in their immediate

environment (Howell, 1982a) . This might be one of the

plausible explanation for the anomalous development and

activity of nematodes due to the heavy metals as has been

confirmed in later experiments (Tables 5,6,11,12). The

heavy metal application suppressed larval penetration of

roots which gradually increased with an increase in the

concentration of the heavy metals. This resulted in poor

gall formation at the lower doses. It was completely checked

at the higher concentrations, viz. 30.0 and 60.0 ppm. The

169

slight root galling at the lower doses might be due to the

survival of the nematodes at these concentrations. One

probable reason of the survival of nematodes at lower

concentration of the heavy metal may be the decreased

seretion of mucus at the anterior end of the nematode body

resulting in low concentration of metal ions (Howell, 1982).

Decreased larval penetration of the roots followed by

suppression in the root-knot development was found to be

positively correlated in later experiments with the

anomalous development of the nematode (Fig. 9a,10a) as has

been proved by the reduced cross-sectional area of nematode

females (Table 11,12) and fragmentation of polypeptides of

higher molecular weight proteins as well (Table 13,14).

The water absorption capacity of plants also provides

an exposition that though the nematode population was highly

affected by the increased concentration of heavy metal (i.e.

15.0, 30.0 and 60.0 ppm), the further decrease in the water

absorption capacity of roots in the inoculated plants might

be due to plant damage caused by nematodes during initial

stages. However, in due course of time, the nematodes could

not survive.

In earlier studies, Alam et al. (1974) and Ismail &

Alam (1975) found that water absorption efficiency of

tomato roots was adversely affected by the infection of the

root-knot nematode, M. incognita. Subramaniam & Saraswathi-

Devi (1959) reported that poor water absorption by diseased

170

plants might be due to root injury caused by bacterial and

viral infection or due to deformation, choking and

disturbances in the arrangement of tracheary elements. These

possibilities can not be ruled out in case of plants infected

with the root-knot nematode as Endo (1971) observed various

abnormalities in root tissues due to hypertrophic and

hyperplastic development of cells. These findings were also

supported by Swami & Krishnamurthy (1971) and Siddiqui et al.

(1974) who have noted mechanical damage of cells, disruption

in the arrangement of tracheary elements, deformation and

blockage of vessels in tissues due to the infection of the

root-knot nematodes, M. javanica and M. incognita

respectively. Alam et al. (1974) and Ismail & Alam (1975)

have concluded that deformed tracheary elements in root-knot

nematode infected tissues might be responsible for poor

water absorption by the roots. Ismail & Alam (1975) also

gave the reason that the root-knot infection brought about

reduction in root weight or in other words total surface

area of the roots resulting in poor water absorption.

Moreover, reduction in shoot weight might have caused a check

in the transpiration pull and that would have made them

indirectly responsible for adversely affecting water

absorption.

It is well known that heavy metals may enter the xylem

cells where they may formcomplexes with the elements of

xylem cell sap and get deposited on the cell walls causing a

hinderance in supply of water to the aerial parts of the

171

plants. In the present study, poor water absorption by roots

might also be attributed to the reasons given above which

have been caused by the nematode infection and/or the heavy

metals. This kind of study has been carried out for the first

time.

5.5 Effect of heavy metals on the growth of females of the root-knot nematode. Meloidoeryne incognita on tomato :

The experiment, conducted to study the effect of heavy

metal on the growth of Meloidogyne incognita female, showed

that the test heavy metals, i.e. Pb and Cd not only retarded

significantly the growth of the females but also caused

deformities in their shape and size (Fig. 9a,b, 10a,b) .The

growth of females as measured in their cross - sectional area

showed a gradual decline with a corresponding increase in

the concentration of heavy metal (Tables 11,12,Fig.9,10) .

Lead was found to be more injurious to the growth of the

females as compared to cadmium. These findings can be

corroborated with the results of the experiment concerning

the effect of heavy metals on protein profile of females of

the root - knot nematode, M. incognita. There also, it was

found that the number of polypeptides of high molecular

weight was reduced with the increase in the dose of heavy

metals (Tables 13,14). Moreover, the heavy metals also caused

possible fragmentation of polypeptides of high molecular

weight (Tables 13,14). These aberrations in the protein

profile of females and other physiological disturbances would

have caused an overall retardation in the growth of females.

172

Survey of literature clearly reveals that this type of study

has been carried out for the first time.

5.6 Effect of the heavy metals on the protein profile of Meloidocryne incognita females:

The microslab gel electrophoresis provides opportunity

for qualitative analysis of biomolecules that are likely to

provide the basis of meaningful investigation of nematodes

thriving in polluted soils. Plant parasitic nematodes, like

many other obligate parasites, are capable of altering host

metabolism. But they themselves are encountered by so many

environmental constraints in general and pollutants in

particular. In the present study, protein profile of females

of the root-knot nematode, Meloidogyne incognita was

assessed.

Ko ell Sc Smith (1983) conducted an experiment on the

binding of heavy metals with protein of a marine nematode,

Enoplus brevis. Recently several workers have used free-

living nematodes to study the toxic effects of heavy metals

(Popham Sc Webster, 1979, 1982; Haight et al. , 1982; Mudry et

al. , 1982) but the biochemical mechanisms mediating these

effects are not known. Some marine nematodes can apparently

withstand high concentration of pollutant metals in the

environment.

In so far, no attempt has been made to see the impact

of heavy metals on the protein pattern of root-knot nematode

females. It was found that the number of poylpeptides

173

gradually decreased with an increase in the concentration of

the hea\Y metal (Tables 13, 14) . The total number of

polypeptides was found to be 19 in the females of M.

incognita taken from untreated plants. In cadmium treated

plants, the number of polypeptides decreased to 13 in the

treatment with 7.5 ppm and 11 with 15.0 ppm in the soil. The

corresponding figures for lead were 13 and 6 respectively.

The decrease in the number of polypeptides in lead

treated females was to a greater extent than in cadmium

treated females. Moreover, it was found that both the heavy

metals caused reduction in the frequency of high molecular

weight polypeptides and these were replaced by low

molecular weight ones, which were caused with all the

probability due to fragmentation of the proteins of high

molecular weight. Appearance of lesser number of polypeptides

at higher doses of lead is also quite indicative towards the

fact that lead was more toxic to nematode survival. This

finding is also in consonance with the results presented in

Table 10 where more plant damage was recorded with lead as

compared to cadmium treated plants.

Though, it is still premature to give exact reason for

reduced penetrability of J2 (Tables 7, 8) and further

development of the nematode (Tables 11,12) and its

pathogenic potential (Tables 9,10), but the analysis of

protein profile certainly gives a cue that heavy metals are

definitely nemato-toxic, affecting the biochemical changes

and disturbing their metabolic activities. This kind of study

174

related to the root-knot nematode was conducted for the

first time.

5.7 Accumulation of the heavy metals in tomato in relation to Afeloidocrvne incognita:

Levels of the heavy metals, i.e. lead and cadmium were

determined by Atomic Absorption Spectrophotometry (AAS) in

tomato {Lycopersicon esculentuw) cv. Pusa Ruby infected with

the root-knot nematode, Meloidogyne incognita. The amount

of heavy metals in plants was estimated on dry weight basis.

It was quite interesting to note that lead was absorbed by

plants in a much lesser amount than cadmium (Tables 21, 22) .

However, the concentration of the heavy metal absorbed was

more in the inoculated sets than that found in the

uninoculated sets. There was a positive relationship between

the concentration of the hea\.y metals in the soil and that in

the plant.

Relatively little is known aobut the factors which

control the availability of metals at the root/soil

interface. Koeppe (1977) has reviev/ed the uptake and

translocation of Pb in plants. He found that translocation of

heavy metals is highly dependent on the physiological status

of plant.

It was also noticed that rate of increase in metal

accumulation in the plants due to increase in the dose was

more at lower doses than at higher ones, and it was more

pronounced in case of cadmium. It is understandable because

175

lower doses are known to casue lesser damage to physiological

process as well as the plant tissues.

Role of nematode was more at low concentrations as the

toxicity of the metal was at its minium at these

concentrations (i.e. 7.5 and 15.0 ppm) and potentiality and

vitality of nematodes was retained; conversely, at high

concentrations, nematodes themselves were killed or

inhibited affecting the accumulation of heavy metals. The

findings are in accordance with those of Bisessar (1983),

who found that nickel contents of stalks and leaves of

plants from the heavy metal soils infested with the root-knot

nematodes/ Meloidogyne hapla were significantly higher than

that m stalks and leaves of plants from the heavy metal

soil without nematodes. It indicates that nematode

infestation resulted m an enhancement of metal uptake. They

also observed the pattern of heavy metal concentration in

celery and found them m the order of root > leaf > stalk.

This has also been observed by other researchers (Agarwala

et al., 1977; Cataldo et al. , 1978). In our results also it

was found that accumulation of heavy metal was more m roots

than in shoots (Tables 21, 22) and may be represented as

root> shoot.

5.8 Micronutrient status of the heavy metal-stressed tomato infected with Meloidocrviie incognita. :

The uptake/accumulation of heav;/ metals had a great

impact on the translocation of other micronutrients present

176

in the soil. The three important micronutrients, viz. Cu, Zn

and Mn were studied in the heavy metal-stressed tomato

plants. It was found that Mn was absorbed in the maximum

amount as compared to other micronutrients in both the

inoculated as well as uninoculated sets in heavy metal-

stressed plants.

It was quite interesting to note that the

micronutrients absorbed in the inoculated plants was in a

much lesser amount than in the uninoculated ones. It

indicates towards an inverse relation between the amount of

heavy metals absorbed in the inoculated sets and the

absorption of micronutrients (Cu, Zn and Mn) . It has already

been pointed out that the accumulation of heavy metals was

more in the inoculated plants, which might have resulted in

decreased uptake of m.icornutrients as compared to that in the

uninoculated sets. Therefore, the decreased uptake of

micronutrients may be attributed to the accumulation of

heavy metals as well as nematode infection.

Khan & Khan (1983) also found that Pb and Cd

application to soil decreased the uptake of most of the

nutrients, viz. Mn, Zn, Fe, Cu and Na in eggplant and Zn in

tomato. They also stressed that higher availability of Pb

and Cd results in a decrease of the selectivity of these

nutrients and thereby reducing their concentration in plants

(Frausto da-Silva & Williams, 1976).

177

5.9 Efficacy of organic eunendments against Afeloidoqyne incocTnita in relation to the heavy metals on tomato;

It is well known that organic amendments are good

suppressants of plant parasitic nematodes and the disease

they cause. Neem cake in particular has consistently shown

its efficacy against a variety of nematodes on different

crops (Singh & Sitaramaiah, 1970; Muller & Gooch, 1982; Alam,

1993; Akhtar & Alam, 1993). Its efficiency has been

attributed to many factors including the presence of

allelochemicals such as azadirachtin, nimbin, nimbidin,

nimbidic acid, kaempferol, quercetin, thionemone etc. (Khan

et al., 1974; Siddiqui & Alam, 1988, 1989a, b; Alam, 1993).

Similarly, castor cake is also known to have great

nematicidal potential (Singh & Sitaramaiah, 1970, Muller &

Gooch, 1982; Akhtar & Alam, 1993). In addition to neem and

castor cakes, their leaves and rice polish have also been

included in the present study. All these materials have

proved to be effective significantly at P = 0.05 and 0.01

against the root-knot nematode, Meloidogyne incognita

attacking tomato, Lycopersicon esculentum cv. Pusa Ruby

(Tables 15, 16). These results are in consonance with those

of the early reports (Singh & Sitaramaiah, 1970; Muller &

Gooch 1982; Akhtar & Alam, 1993). In case of rice polish,

presence of pyridoxins (Vitamin B6) might have played some

role in masking the ill effects of the root-knot nematode.

These results could be correlated, in a way, with the

findings of Samiullah et al. (1988) who have found enhanced

plant growth with the treatment of vitamin B6.

178

However, such kind of studies have not been noticed in case

of nematode infected plants particularly in relation to heavy

metal pollution.

Efficacy of the organic additives was also evaluated in

presence of the heavy metals, e.g. Cd and Pb. As expected

both the metals were more damaging than M. incognita

irrespective of the treatments. It was also noted that

heavy metal and the nematode together caused more plant

damage than that caused by either of them alone. It is

interesting to note that barring the oil-cakes of castor and

neem, all the treatments could not check the synergistic

effect of the metal-nematode combine. Therefore, it is safe

to conclude that these oil-cakes are benefic (i\l to tomato

plants under the attack of root-knot nematode even in the

soils polluted with the heavy metals.

This kind of study has been carried out for the first

time and it may go a long way in evloving a suitable nematode

control strategy- in heavy metal polluted soils.

5.10 Efficacy of biocontrol fiincrus, Paecilomyces lilacinus against Meloidoayne incocmita in relation to the heavy metal on tomato:

Paecilomyces lilacinus, a common soil fungus is highly

antagonistic to the nematodes (Jatala, 1986; Alam, 1990).

There are also reports of its antagonism against plant

pathogenic fungi (Shahzad & Ghaffar, 1988) . In order to have

a complete understanding of biocontrol process of plant

179

pathogens and parasites, it is necessary to know the effect

of environmental factors including pollutants on natural

antagonists present in the soil. Therefore, it was considered

worthwhile to study the effect of lead and cadmium, the two

important heavy metal pollutants, on P. lilacinus.

In vitro studies have shown that the mycelial weight of

the fungus decreased with an increase in the concent^ration

of the heavy metal (Table 17,18). Maximum reduction in dry

weight was 40.30 % at 60.0 ppm concentration of Cd over

control. Similar trend was also observed in sporulation. Lead

was proved to be more toxic as the maximum reduction in dry

weight was as high as 92.64 % at 60.0 ppm concentration.

The results have been found in consonance with the findings

of Singh et al. (1992). They examined five heavy metals, viz.

nickel, cobalt, chromium, lead and copper and found that all

these heavy metals suppressed the growth of the fungus P.

lilacinus irrespective of the fact whether the fungus was

raised in liquid or solid media. The extent of suppression

was however related with the concentration of the heavy

metal, reaching to the same conclusion that the soils

polluted with these heavy metals may hamper the growth of

this efficient bio-control agent and thereby reducing its

potential in nematode control.

Based upon in vitro studies, a pot experiment was

designed to assess the efficacy of nematode biocontrol

fungus, P. lilacinus on the root-knot development caused by

M. incognita and its resultant effect on plant growth

180

parameters of tomato L. esculentum cv. Pusa Ruby in presence

of lead and cadmium. The study revelaed some interesting

results related with the root-knot development and plant

growth parameters. Plant growth improved and the root-knot

development decreased when the plants received the treatment

of the fungus only. This indicated that P. lilacinus was

quite efficacious in controlling the menace of the root-knot

nematode. This confirms and extends the earlier studies

(Alam, 1990) . However, the plant receiving the treatment of

the heavy metals, i.e. lead and cadmium showed a reduction in

their growth parameters and this reduction was further

increased in the presence of M. incognita, suggesting the

cumulative/interactive effects of the heavy metals and the

nematode. In the presence of the fungus, however, the growth

parameters were improved to some extent but as it has been

established from in vitro experiment that the metals were

toxic to the growth of the fungus, subsequently effecting its

efficiency as a biocontrol agent. Nordgren et al. (1985)

working on soil microfungi, found that the fungal community

was strongly affected by the heavy metal pollution. Tatsuyama

et al. (1975) found that P. lilacinus was resistant to

cadmium. However, in the present study, cadmium was found to

be toxic to the growth of P. lilacinus. The variation in

response could be due to the strain of the test organism.

This type of study is of great relevance in assessing

the biocontrol measures implied to control the menace of

root-knot nematodes. It further implicates that the soils

181

heavily polluted with heavy metals put a serious question

mark on the applicability of bio-control agents such as P.

lilacinus against root-knot nematodes.

182

6. SUMMARY

The present study deals with the impact of two

important heavy metal pollutants, viz. cadmuim and lead on

the pathogenicity and control of the root-knot nematode,

Meloidogyne incognita on tomato, Lycopericon esculentum.

Aspectwise summary of the results is presented below:

In vitro studies were made to assess the toxic nature

of the heavy metals and the results clearly indicate that

both the heavy metals, Cd and Pb adversely affected the

hatching and survial of M. incognita. Not only the 3uvenile

hatching of the nematode was inhibited but the heavy metals

also caused significant moitality of the nematode juveniles

(J2) . Both, hatching and mortality were found to be directly

related with the dose of pollutant and exposure duration as

well Inhibition m the juvenile hatching was 74.4 % at the

lowest dose (7 5 ppm) and increased to 90.7 % at the high

dose (60.0 ppm) for Cd. It was 77.6 % at 7.5 ppm and 100 0 %

at 60.0 ppm for Pb. It also indicates the higher mnibitory

nature of the latter. Similarly, juvenile mortality increased

with an increase m the concentration of the heavy metals as

well as the exposure period. Ec ^Q value for Cd was 50.8 ppm

at 96 h exposure whereas for Pb the values were 13.9 ppm at

96 h and 53.5 ppm at 72 h exposure.

After ascertaining the toxic nature of the heavy

metals, an expreriment was conducted on the influence of the

183

heavy metals on penetration of second stage juveniles (J2) of

the root-knot nematode into the roots of highly susceptible

cultivar of tomato, viz. Pusa Ruby. Both the heavy metals,

viz. Cd and Pb adversely influenced penetratibility of second

stage juveniles (J2) of M. incognita and lead caused more

inhibition in Larval penetration than cadmium (690.0 and

630.0 J2 per plant after 48 h root dip duration at 15.0 ppm

of Cd and Pb respectively).

These toxic effects of the heavy metals on the

nematode, ultimately had an adverse effect on the

pathogenesis and development of the disease. It does not

indicate that the poor development of the disease resulted in

good plant growth, but on the contrairy the plant damage

wasincreased in the presence of both the nematode and heavy

metal as well. It was also manifested in the form of poor

root-knot development. The nematode females failed to attain

their normal size and shape. As compared with 203611.70 sq n

average cross-sectional area of the nematode females in the

untreated control, the lowest concentration of the heavy

metals (7.5 ppm) brought down the figure to 158942.40 sq u

and 144500.30 sq ji respectively in case of Cd and Pb

treatments respectively. In the higher dose (60.0 pm) of the

heavy metals, the size of the females further reduced and

figures for cross-sectional area of the females were 51586.90

sq p and 43981.70 sq yu in Cd and Pb respectively. A great

deal of change was observed in the shsipe of females obtained

from plants treated with the heavy metals indicating

interference in the development of the females.

184

The above findings were further confirmed by the

results of the experiment conducted to study the protein

profile of the females of the nematode. Both the heavy metals

caused significant reductions in the number of polypeptides

which showed a declining trend with increasing concentration

of the heavy metals. Again, lead brought about more reduction

m the number of polypeptides than cadmium. As compared to 19

polypeptides, in the nematode females obtained from the

untreated control, the number decreased to 13 and 11 when

plants were treat ed with 7.5 and 15.0 ppm of Cd in soil

respectively. The corresponding figure for Pb were 13 and 6

respectively. These results are again in conformity with the

earlier findings indicating more injurious nature of Pb as

compared to Cd. In addition to the reductions in total number

of polypeptides, it was interesting to note that there was

disappearence of many polypeptides of high molecular weights

and appearance of those having low molecular weights

suggesting a possible fragmentation of long chain

polypeptides. This kind of influence of heavy metals on

polypeptides was more in the presence of lead than cadmium.

The results concerning the uptake/assimilation of heavy

metals and their subsequent effect on the absorption of

micronutrients (Cu,Zn,Mn) also showed quite interesting

trends. There was a tendency of more accumulation of the

heavy metals with the increasing doses of the heavy metals.

The uptake/accumulation of the heavy metals was further

increased m the plants inoculated with the nematode. This

185

trend in the absorption of the heavy metals, probably had

direct bearing on the uptake/accumulation of micronutrients,

viz. Cu, Zn and Mn. These micronutrients were absorbed in a

much lesser amount in nematode-inoculated plants than the

uninoculated ones. This indicates an inverse relationship

between the amount of heavy metals absorbed and the uptake of

the micronutrients.

Both the heavy metals as well as the nematode had

injurious effects on plants, the former one being more-

harmful. The nematode, though themselves weakend by the heavy

metals, caused considerable damage to the otherwise weakend

plants. Similar results were also found in the experiment

related with the response of different cultivars of tomato to

the root - knot nematode. Though the different cultivars

exhibited varied responses,these responses remained by and

large on the same pattern even in the presence of the heavy

metals. Here again / lead was found to be more toxic than

cadmium.

Both the heavy metals, viz. Cd and Pb also interfered

with the control measures of the nematode. All the organic

amendments, viz. oil-seed cakes and leaves of neem and castor

and rice polish were found to be more effective against the

nematode in absence of heavy metals than in their presence.

However, an important observation was made that all the

organic materials especially oil-seeds cakes of neem and

castor were able to mask the ill effects of the heavy metals

to great extent.

100

The bio-control agent, Paecilomyces lilacinus which is

efficacious against nematode was also adversely effected by

both the heavy metals. In vitro studies clearly revealed that

both the heavy metals, significantly suppressed the dry

mycelial weight as well as sporulation of the fungus. Lead

caused more inhibition as compared to cadmium. The efficacy

of P.lilacinus against the rootknot nematode was found to be

significantly reduced when the heavy metals were also added

to the soil.

187

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Bottor of ^liilasiopl)? · 2018. 1. 4. · dose (60.00 ppm) were 51586.90 squ and 43981.70 squ respectively. Thus the test doeses (7.5-60.0 ppm) of the heavy metals caused reductions