STUDIES ON P U N T PARASITIC NENATODES AND SOME FUNGI ASSOCIATED

WITH PUtK CROPS

PISSERTATION SUBMITTED FOR THE DEGREE OF

M^ittv of ^diloiB^optip IN

BOTANY

BY

NASE6R HUSSAIN SHAH

DEPARTMENT OF BOTANY ALIGARH M U S U M UNIVERSITY

ALI6ARH (INDIA)

1990

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DS1788

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He (Allah) taught man that which he knew not (The Quran 96.5)

Jt-^

T

A

E

Dr. Mohd. Farooq Azam M S( I'h n. (Al i ( i )

Genior Nematologist

D t l ' A R T M i NT OF ROrAfv).'

A L I G A R H MUSLIM iJNIVEHS

A! IGA-IH 202 001 f l N P l A i

IXif.v

CERTIFICATE

This is to cert ify t h a t the work embodied in th i s d i s s e r t a t i o n ent i t led

"STUDIES ON PLANT PARASITIC NEMATODES AND SOME FUNGI ASSOCIATED WITH

PULSE CROPS" i s the bonafide and o r i g i n a l r e s e a r c h v;ork c a r r i e d out by

Mr. Naseer Hussain Shah unde r my s u p e r v i s i o n and is s u i t a b l e for submission

to A.M.U. Al igarh for the cons ide ra t ion of the aware Df the degree of Master

Of Philosophy in Botany.

(Dr. Mohd. Farooq Azam)

ACKNOWLSDGSMgNT

I bow i n g r a t i t u d e t o the Almighty ' A l l a h ' who enabled

me t o achieve t h i s t a r g e t .

I was most f o r t una t e t o have a highly imag ina t ive ,

e n t e r p r i s i n g and accomodative r e sea rch guide Dr. M, Farooq Azam

Senior Nematologist, Department of Botany, A.M.U,, A l iga rh ,

who inspi red me t o i n i t i a t e the r e sea r ch work i n the f i e l d of

P lant Nematology, I deem p leasure i n express ing my deep sense

of g r a t i t u d e to him for sugges t ing a problem of v i t a l i n t e r e s t ,

s k i l f u l l guidence and sympathetic behaviour throughout t he

course of t h i s work.

Thanks a re due t o Prof. S. PC Saxena for h i s hea l thy

c r i t i c i s m and va luab le sugges t ions and for spa r ing h i s p rec ious

time i n going through t h i s manuscript c r i t i c a l l y .

I t g ives me immense p l easu re to thank Prof. A. K.M.Ghouse,

Chairman, Department of Botany for providing a l l l i b r a r y and

labora tory f a c i l i t i e s .

I t would be q u i t e u n j u s t i f i e d i f I d o ' n t extend my

s ince re thanks to my sen io r r e sea rch Col leagues Er , .Kansoor A .

S idd iqu i , Mr. S.A. T iyag i , Mr. M. Imran Khan, Mr. Hissamuddin,

Mr. Mahmooduzzafar, Mr. Munawar Faza l , Mr. Kamal Singh and

Mr. Nafees Ahmad for t h e i r t imely help and cons t an t encourage­

ment.

I a l so acknowledge with g r a t i t u d e the c o - o p e r a t i o n

accorded to me by my resea rch co l l eagues and f r i e n d s , Mr,Pirooq

A. Lone, Mr, Ab, Rashid Malik and Miss Roshan Ara Shah who

[ii]

shared their knowledge and offered constant encouragement

during the preparation of this dissertation, I find word*

inadequate to express my indebtness to them, I am also

thankful to my other colleagues Mr, Ashraf Hameed Khan and

Syed Iqbal Hussaln for their ideal suggestions and co-operation.

>iy cordial thanks are due to my friends M/s Zameer Makdoomi,

Showkat Ahmad, Ab, Hameed, Bashir Ahmad, Mohd, Ayub, Muataq Ahmad,

Asif Iqbal, Rashid Bhat and Shafat Qadri for their all out

help and manifold co-operation throughout the course of this

work.

Lastly, I would miss in my duty if I don't make a mention

of my parents whose sacrifices and blessings gave me impetus to

achieve this goal.

Naseer Hussain Shah

C O N T E N T S

1. Introducticm 1 - 4

2. Review of Li terature 5 - 3 1

3 . Plan of Work , . 3 2 - 3 4

4 . Materials and Methods 3 5 - 5 4

5 . References 5 5 - 6 9

INTRODUCTION

Pulses are important food crops from which a significant

part of the world population obtains much of its dietary protein

(17-A39i). Their cultivation is world wide from ancient times.

Pulse crops have been the mainstay of Indian Agriculture, enabling

land to turn out reasonable quantities of food grains. Pulses

are also rich in two essential micxvjnutrients, namely calcium and

iron. These crops are generally included in rotation in most of

the areas, because of their ability to keep soil alive and

productive.

Being leguminous crops possessing root nodules, they fix

and utilize atmospheric nitrogen and contribute substantially to

soil fertility. Some pulses are considered to be the excellent

forage and grain concentrates in the feed of large cattle

population of the country and some of them are excellent green

manure crops, which add much needed humus and major plant nutrients

to soil. Thus pulse crops are useful in improving the nitrogen

status of soil, drawing out plant nutrients from deeper soil

layers with the help of their root system and making them

available in the form of plant residues. They also provide in

many cases a protective ground cover against soil erosion.

Pulses as a whole have wide range of climatic requirements

and as such they are grown under various climatic conditions

throughout the country. In India the area under pulses is 22.5

2

million hectares with an average of 11.2 million tonnes of

production per year. The national average production of pulses

at present is 6-8 q/ha which is very low in comparison to other

countries of the world. The over all yield has been fluctuating

between 500-700 kg/ha but the potential yield from presently

grain cultivars is over 300 kg/ha

Unfortunately, production of pulse crops is subject to

several constraints including those of pests and disease which

play leading role in yield reduction.

It Is inherent in nature of all living organisms to

cooperate or compete with other when occupying a common niche

and specially if they have similar and overlapping food sources.

This is all the more true for soil which is a complex ecosystem

having a wide variety of life forms (including plants and animals).

These organisms often develop symbiotic, synergistic or anta­

gonistic relationship amongst themselves, which could primarily

be because of nutritional or spatial competition, The plants in

one form or the other are the direct or indirect source of food

for consumers of all trojfAiic levels, including plant pathogenic

organisms. It is well known that certain, fungi, bacteria and

viruses are important pathogens and often cause damage without

being influenced by other biotic agents. Plants are constantly

exposed to numerous pathogenic organisms many of which are

common components of soil biosphere. The nematodes, among the

soil biota form a separate group and constitute a significant

cc«nponent (about 12% of soil microflora and fauna) of the soil

ecosystem. We are usually unaware of their presence because of

their microscopic size and protective position with in the soil.

Plant parasitic nematodes are invariably found in the soil around

the roots of plants and may act as limiting factor in the crop

production. Plant parasitic nematodes themselves are quite

capable of causing serious plant diseases which are independent

of other micro-organisms,but economic damage often becomes more

destructive and high when they interact with other micro-organisms.

A large number of phytophagus nematodes have been reported

from India around the roots of pulse crops. In recent periods

Meloidogyne spp, Telotylenchus spp, and Rotylenchulus renlformls

have been found as potent parasites causing potential damage to

pulses. These damages are reflected in the form of yellowing,

stunting, poor plant growth and reduction in yield. The effect

of nematode is variable on different pulse crops of India.

It is not realistic to assume that a plant although

infected with one pathogen will not be affected by another. It

seems much more appropriate that because a host is infected by

one pathogen its response to additional invradors will be altered.

The alterations greatly influence the disease development within

a particular host and the epidemiology of the pathogen. The

study of disease- complex is as important as the study of

" monopathogenic" situation, because under field conditions there

is probably no soil borne plant disease that can be said to be

of monopathogenic origin.

Nematodes contribute to the disease complex by modifying

the physiology of the host plant and occasionally by mechanical

affects they exert on the host.

Recently several workers have reviewed the work on

interactions of plant parasitic nematodes with other pathogens

like nematode-fungus (Powell, 1971; Norton, 1978; Pitcher, 1978;

Dropkin, 1980; Dmowska, 1962), nematode-root nodule bacteria

(Pitcher, 1978; Norton, 1978; Dropkin, 1980), nematode-pathogenic

bacteria(Powell, 1971; Pitcher, 1978; Norton, 1978; Dropkin, 1980),

nematode-vlrus (Powell, 1971; Norton, 1978; Martelli, 1979;

Lamberti, 1981) and nematode-nematode (Powell, 1971; Norton,

1978; Dropkin, 1980). Although the Involvement of disease

complex situations Is quite frequent and widespread and the

literature Is quite extensive, but in the present treatment,

the discussion will be limited to the interactions involving

plant nematodes-fungi and root nodule bacteria.

RBVIBW OF LITSRATURS

ROLS OF NEMATODES IN FUNGAL DISEASES

Nematodes play an important role in the disease caused

by fungi. Commonly the disease is very severe when both the

organisms are present at one time.

1.1 NEMATODS-FUNGUS INTERACTIONS

1.1,1 Fusarium - Nematode Complexes

Atkinson in 1892 was the first to report a nematode

fungal interaction causing a disease complex. He found that

the infection of root-knot nematode, Meloidogyne spp,

increased the severity of Fusarium wilt in cotton, thus laying

the historical ground work for the problems considered in his

article. This is logically an important and striking interac­

tion existing between these two pathogens. This relationship

is specially important with cotton and toma'toes, although it

has been described on a number of other crop plants,

Singh et al, (19B1) reported that simultaneous inocula­

tions of Phaseolus vulgaris with M. incognita and Fusarium

oxysporum, F, solani or inoculation of plants with

M, incognita prior to Fusarium gave maximum percentage of

wilting, indicating that Fusarium wilt is increased in french

bean in presence of M, incognita. Chahal and Chhabra (1985)

reported that in combined inoculations pea cultivar caused

significantly greater reduction of the growth than by

M, incognita alone. When both pathogens were present there

was more damage than with either alone and earlier wilting

than with F, equiseti alone. Seedlings inoculated with

M. incognita alone wilted only temporarily during the heat

of day while as those with combined inoculations failed to

recover, ThakEtr et al. (1986) reported that Cjcer arietinum

plants line ICC-12275 (resistant to F, oxysporum f, ciceri)

showed no wilt when inoculated with fungus alone, but in

presence of M. incognita« fungus caused 50? mortality of

plants, Patel a^, (1986) reported the increase in the

incidence of wilt, v^en chickpea seedlings var, chaffa were

inoculated with 1000 larvae (I2) of M, incognita/or 50 ml

mycelial suspension (2 g mycelial mat) of Fusarium oxysporum

f. ciceri on the day of sowing and 10 days later. Root-knot

index was 3,0-3.1 in all treatments.

Mani and Sethi (1987) conducted pot experiments with

10 days old plants of chickpea (Cicer arietinum) CV, JG62

inoculated with M. incognita at 100 or 100© larvae/500 g, soil

and Fusarium oxysporum f, ciceri and F. solani at 1.0 and 2,0

mycelial mat respectively either singly or in combination.

All these organisms affected rhizobial nodulation. The

occurrence of M, incognita in combination with F, oxysporum

f. ciceri and F, solani not only increased the severity of

disease but also shortened the incubation period for disease.

Furthermore when Inoculation of nematodes preceded that of

fungi by one week, plant drying due to infection with

F, solani appeared early and root-knot index was also high.

The development and multiplication of M, incognita was

adversely affected by F. solani irrespective of time and

level of inoculum, F. oxysporum f. ciceri did not affect

the nematode population significantly,

Padilla et al, (1980) reported that the concomitant

inoculation of nematodes Meloidogyne spp, (M, Incognita •»•

M, hapla) and Fusarium oxysporum f, plsl at planting caused

death of pea plants (CV, little Marvel) under green house

conditions, The inoculation of Meloidogyne species at

planting caused a severe stunting of the plants, whereas

F, oxysporum alone at planting or 3 weeks later caused no

detrimental effects,

Kumar and Ahmad (1988) reported that inoculation of

M, incognita at 1000 Jp/plant or F, oxysporum f, ciceri at

1 g mycelial suspension/plant to 15 days old seedlings of

chickpea CV,JGr-62 reduced plant growth in pot experiments.

Highest reduction were obtained with simultaneous inoculation

of both pathogens, followed by the treatment in which nematode

inoculation preceded the fungus by 10 days and vice versa.

There was lower multiplication of nematodes and galling in

presence of the fungus.

8

Mousa and Hague (1967) exposed Armosy (wilt susceptible)

and Coll (wilt resistant) Varieties of Soyabean to Pusarium

oxysporum f. glycines in presence of the nematode. There was

a large increase in the amount of fungus in vascular tissues

of roots and stem, compared with the amoxmt found in plants

exposed to fungus alone. The fungus was Isolated from leaves

and seeds of wilt resistant cultivars only when the nematode

was present,

Mousa and Hague in 1968 again reported that the fungal

invasion caused destruction of giant cells when M. incognita

and wilt fxongus F, oxysporum f. glycines were inoculated on

the growing seedlings of soyabean cultivars ware and coll

(resistant to fungus). The overall effect was to reduce the

ntanber of females of M, incognita and increase the proportion

of males,

Goswami and Agarwal (1978) conducted pot experiments

to determine the relationship between M. incofgnita and

F, oxysporum, F, solani, F, graminearum and F, equiseti on

soyabean seedlings. The results showed an antagonistic

interaction of F, oxysporum and F. solani with M, incognita,

the fungus when established first suppressed nematode

multiplication while the nematode when established first

reduced the symptoms due to fungus. The interaction of

F, graminearum and F, equiseti with M. incognita was

synergistic, nematode infested plants being more seriously

damaged by the fungus than non infected.

Rushdi et al. (i960) studied the histological changes

caused by Meloidogyne javanica and Fusarium species in the

roots of faba bean and cowpea. Both pathogens caused much

damage to plant tissue. Giant cells induced by the nematode

were also infected by the hyphae and lost their cytoplasm.

Sakhuja and Sethi (1986) studied the effect of

M, javanica, F. solani and R. bataticola on Arachis hypogea

in 12 different combinations. It was found that both fungi

reduced galling significantly in all treatments, except where

F, solani succeeded nanatode after a week. M, javanica

multiplication reduction was maximum where one or both fungi

were inoculated simultaneously with the nematode. R. bataticola

exhibited greater antagonistic activity compared with F, solani.

Ibrahim and El-Saedy (1977) reported that fewer galls were

produced on the roots of groundnut CV-Giza 4, when infected

with M. javanica and either Fusariiun solani or Rhizoctonia

solani than when infected with nematode alone.

Nath and Dwivedi (19B1) reported that Cicer arietinum

plants exhibited wilt symptoms earlier, upon the combined

inoculation of M, .javanica and , oxysporum f. ciceri or

Meloidogyne and Rhizoctonia than with fungus alone. Inoculation

of soil with F. oxysporum f. ciceri + R. solani have shown low

wilt incidence, suggesting antagonism between the fungi and

its duration longer* Upadhyay and Dwivedi (1987) studied

the effect of inoculation of M. javanica alone or in combination

10

with F, oxysporum f. clcerl on chiclqpea seedlings var. Arodhi,

Plants inoculated with fungus alone showed slight wilting but

wilting was maximum and rapid in plants where nonatode inocula­

tion preceded the fungus, Upadhay and Dwivedi (l967b) again

conducted pot experiments with 500 freshly hatched larvae of

M. javanica/300 g soil and ^ (W/W) F, oxysporum f. ciceri,

inoculated alone, simultaneously or 10 days apart at the base

of test plant of chickpea CV. K-850. Wilt symptoms were

greatest in plants inoculated simultaneously with both

M, javanica and F, oxyspoxim f, ciceri followed in severity of

inoculations of the nematode preceding the fungus and the

fungus preceding the nematode. The maximum number of root-

knot galls (889) were recorded on roots inoculated with

nematode alone as compared to maximum number (20) on roots

where inoculation of the naiatode preceded that of the fungus.

The greatest reduction in nodules/plant were observed with

inoculations of nematode alone.

Salam and Khan (lSe6) reported that when seedlings of

Cajanus cajan were inoculated with 2 g. mycelium of F. udum

and 500 2nd stage juveniles of M. 3avanica« all cultivars

proved susceptible in varying degree to fungus. Cultivars viz.,

Pusa-33, Pusa-85 and Pusa-78 were resistant and Pusa-84 was

tolerant to M, javanica. The nematode suppressed nodulation

on tolerant and susceptible cultivars. Susceptibility of all

cultivars to F, udum increased when nematode and fungus were

11

present together. Highly susceptible cultivars wilted sooner

in presence of both pathogens than when inoculated with

F. udum alone. The two pathogens appear to act synergistically

on C, ca.jan though there is no previous report of such

interaction on this crop. Ribeiro and Feraz (1983) reported

that susceptibility of cultivars to the fungus was higher on

the inoculation with fungus + nematode. Vegetative growth and

conidial production by the fungus were greater in the media

containing extracts from bean plants infested with M. .javanica

than in those with extracts from healthy bean plants.

Sharma and Cerkauskas (1985) reported that when 3 days

old seedlings of Cicer arietinum were inoculated with 1000

M. incognita eggs/kg soil or 1 ml suspension of mycelia and

conidia of F. oxysporum f. ciceri. the reduction in plant

height was ^,^Q% with fungus alone 17.5396 with nematode alone

and 19.479 with fungus + nematode after 99 days of inoculation.

Reduction in fresh root weight was 3.7896, 30.61 and 49.30%

respectively. Combined infections caused the most damage to

plants. Goel and Gupta (l966) reported that the growth of

Cicer arietinum was reduced when both organisms were present

together irrespective whether they were inoculated simulta­

neously or with one week interval. Reduction in various growth

parameters was noticed when the nematode was inoculated 30 days

prior to the fungus.

12

Patel e t a l . (1985) reported tha t increasing the

inoculum leve l of F. solani to 2 g or 4 g mycelial mat/pot

decreased the days required for wil t ing i n groundnut

(Arachis hypogea). \Vhen F. so lan i was inoculated with 1000

or 2000 larvae of M arenaria , the wil t ing was fur ther reduced,

Edward and Singh (1979) reported t h a t vrtien pigeon pea

var ie ty type 21 was inoculated with H, ca.lani (50 c y s t s /

po t ) and Fusarium udum. Heterodera alone caused l e s s damage

than vrtien associated with P, udum. Transverse sect ions of

the roots inoculated with the nematode and fungus showed t h a t

only old syncyt ia l or non syncyt ia l regions were invaded by

the fungus,

Killbrew e t a l , (1988) reported t h a t r o o t - r o t symptoms

were most prevalent in presence of fxingus. Five Fusarium so lan i

i so la tes tes ted were pathogenic when inoculated on soyabean

and each was re i so la ted from inoculated symptomatic p l a n t s .

Isolates differed in vi rulence. Disease sever i ty was not

substant ia l ly changed. When F. so lani and soyabean cyst

nematode, H, glycines were inoculated on soyabean in combina-

nation as compared with F, so lani or soyabean cyst nematode

alone, the fungus reduced yield under f ie ld condi t ions . Disease

severity was grea ter with poor qual i ty seeds inoculated with

F, solani than with inoculated high qual i ty seeds. Roy _et a l ,

(1989) isolated two morphologically d i s t i n c t forms of F, solani

1 *- ^

designated FS-A and FS-B fJrom soyabean plants with symptoms of

sudden death syndrome. Dual inoculation of soyabean with

FS-A and soyabean cyst nematode (H. glycines) caused more

severe foliar symptoms than those caused by FS-A alone. It

is concluded that FS-A is primarily causal agent of sudden

death syndrome. FS-A caused reduction in plant height, root

and shoot dry weight, number of seeds/plant and seed yield,

but only in continuously irrigated plants as compared with

periodically irrigated plants,

Hutton et al, (1973) reported that percentage of dry

root-rot in kidney bean was greater when plants were inoculated

with Pratylenchus penetrans or mixture of Meloidogyne spp,

(including M. arenaria and M. javanica) than among nematode

free plants. At high levels of fungal inoculum all plants

whether nematode free or nematode infected become infected by

the fungus (Fusarium solani f, phaseoli).

Seinhorst and Kuniyasu (1971) reported that wilting

of pea var. Rondo, caused by Fusarium oxysporum f, pisi race 2

was enhanced by Pratylenchus penetrans at an initial density of

about 500 or more nematodes per 500 g soil. Some wilting

occurred in the absence of nematode but none at any nematode

density in the absence of the fungus, P, penetrans alone

possibly reduced stem and seed weight at initial densities

exceeding 30,000 nematodes per 5OO g, soil. P. penetrans +

Fusarium did not reduce stem weight but at 500 and more

14

nematodes per 500 gm soil, seed weight was reduced below that,

which occured at smaller initial nematode densities. Seed

weight in pods with P. penetrans •»• Fusarivmi at O-500 nematode/g

soil was about 80% of that at the same nematode densities in

pots without Fusarium. The presence of fungus probably reduced

the rate of multiplication of P. penetrans at all initial

densities of this nematode. Rates of multiplication indicate

that the attack by nematodes reduced the quality of food

available to them by damaging the roots. At low initial

densities the rate of multiplication of the nematode was reduced

because of under population. The rate of multiplication of

R. renlformis on pea (var. Rondo) was increased by the presence

of F. oxysporum f, pisi race 1.

Oyekan and Mitchell (1971) studied the effect of

P. penetrans on the resistance of pea var, Wisconsin perfection

to wilt disease caused by F, oxysporum f, pisi race 1 in

controlled growth chamber experiment. Plants growing in soil

inoculated with both pathogens started wilting 3 days after

inoculation, P, penetrans alone caused considerable reduction

of plant height. When the roots of plants were damaged with

sterile needle before inoculation with Fusarium oxysporum

their normal resistance to wilt was impaired. This is believed

to be the first report of breakdown of plant resistance to

wilt by the root lesion nematode.

15

Szerszen (1980) studied the effect of Pratvlenchus

penetrans on the course of Fusarium wil t of peas in g lass

house experiments using two d i f fe ren t s o i l types and two

v a r i e t i e s of pea differing in t h e i r su scep t i b i l i t y to

Fusarium. The presence of nematode increased the sever i ty

of wi l t symptoms, especially i n l i g h t s o i l s . The r e s i s t ance

of var . Alaska Ekspres was p a r t i a l l y broken by the addi t ion

of nematodes. In further experiments wi l t d isease i n the

susceptible var . Szlachetna 'Per la was more severe following

prolonged expos\are to P. penetrans than a f t e r only a shor t

exposure period with Alaska Ekspres, The course of disease

was the same af te r both long and short exposure per iods .

Sumner and Minton (198?) conducted experiments with

three f i e ld so i l s in te rna l grey to black and showed t h a t

stem discolouration and in te rve ina l chlorosis and necrosis

of leaves in Cobb var . of soyabean was most severe i n s o i l s

infested with F. oxysporum f. tracheiphilum race 1,

Belonolaimus longicaudatus and Pralylenchus brachyurus.

Wounding roots with a knife 18-20 days a f t e r p lant ing did not

increase wi l t or in ternal stem discolourat ion.

Hasan (1964) reported t h a t in pot experiments mixed

inoculations of Fusarium udum with Tylenchorhynchus vu lga r i s ,

Helicotylenchus indicus or Hoplolaimus indieus e i t h e r

simultaneously or sequentially gave no s ign i f i can t difference

in wil t development of C. cajan. When Heterodera ca.iani was

16

associated with fungus, there was s igni f icant increase in

wilting (93,6?^ p l an t s ) , occuring when seedlings were inoculated

with H. cajani 3 weeks pr ior to the fungus inocula t ion.

1,1,2 Rhizoctonia-neroatode complexes

Reddy e t a l , (1979) reported tha t the inocula t ion of

Phaseolus vulgar is with M. incognita alone, simultaneously

with R. solani or 10 days pr ior to fungal infec t ion , reduced

plant height and fresh weight and gave maximum root-knot

index. Simultaneous inoculations of both pathogens caused

greater damage then e i ther organism acting alone.

Varshney e t a l , (1987) reported tha t combined inocula t ions

of Vigna unguiculata (Cowpea) seedlings CV, Russian with

M. incognita and Rhlzoctenia solani caused a s ign i f i can t

reduction in the nodulation due to Rhizobium.

Costa and Huang (l986) observed no synerg i s t i c

pathological effects in pot t r i a l s using d i f fe ren t sequences

of infes ta t ion by the nematode and the fungus. Root branching

appeared to be stimulated by the nematode and growth of

infested plant was found to be improved suggesting some

protection against R. solani .

Goel and Gupta (1986) studied the effect of M. javanica

and R. batat icola alone and in combination on chickpea seedl ings .

He found that combined inoculations i r respec t ive of time,

reduced growth parameters, compared with when inoculated a lone .

17

Kanwar et , (1967) reported that when seedlings of

cowpea (Vigna unguiculata) were inoculated with 1000 juveniles

of M. javanica and 2 g mycelial mat of R, solani. the plant

shoot and root length and dry weight were decreased as

compared to the uninfested controls. Plant growth was better

in loamy sand which supported greatest number of nodules

than in sand and sandy loam. Fewer galls were formed on

plants inoculated simultaneously with both organisms than on

those infested with nematode alone,

Singh et al, (1986) reported that in combined infections

of cowpea (Vigna ungulculata) the nematode population

(M, javanica) was suppressed in the presence of R. solani.

Culture filtrates of the fungus significantly reduced hatching

of larvae.

Kanwar et a]., (1989) reported significant reduction in

the growth of cowpea (Vlgna unguiculata) CV. HFC 42-1^when

1000 juveniles of M. javanica were inoculated 3 weeks prior to

2 gm. mycelial mat of R, solani in pot experiments. The number

of nematode galls were significantly decreased in presence of

the fungus, and galls were minimum when both pathogens were

inoculated simultaneously. The number of bacterial nodules

were also significantly reduced in presence of both pathogens,

with the greatest reduction in nematode only treatments. Loamy

sand was more favourable for plant growth than sand or sandy

loam (greater sand content soil).

IS

Khan and Hussain (1968) reported tha t individual ly

Rhizoctonia solani was the most aggressive pathogen of cowpea

followed by M. incognita and R. renlformis, whi^e concomitance

of nematode and fungus was more damaging, than the assoc ia t ion

of both nematode species, but the assoc ia t ion of R. so lan i

with M. incognita caused d i s t i nc t ly grea te r p lan t growth

reduction than i t s associat ion with R. reniformis> I r r e spec t ive

of the pathogen,inoculated unbacterized p lan ts suffered g rea te r

damage than the bacterized ones. Inoculat ion of Rhizobium

15 days pr ior to any t e s t pathogen both singly or in various

combinations resul ted in s igni f icant ly reduced plant growth,

poor nematode mult ipl icat ion, low root-knot index and improved

nodulation as compared with i t s inoculat ion 15 days a f t e r the

t e s t pathogen/pathogens. There was no s ign i f i can t difference

in plant growth reduction of bacterized p lants whether

simultaneously inoculated with e i ther nematode species and

the fungus or when the nematode inoculat ion preceded fungus,

but on the other hand there was s ign i f ican t ly less reduct ion

in plant growth when fungus preceded nematode inocula t ion.

In sequential inoculations in teract ions were time dependent.

Rhizoctonia solani whether inoculated simultaneously or

sequentially reduced the rate of mul t ip l ica t ion of both the

nematode species as compared to the i r r a t e of mul t ip l i ca t ion

when present alone. Khan and Hussain again in 1969 reported

that the cowpea Vigna unguiculata (CV. r e s i s t a n t or moderately

r e s i s t an t against individual pathogens), l o s t t h e i r r e s i s t ance

IS

in the presence of other pathogens, except CV, IC-503

(moderately r e s i s t a n t to M. incogni ta) . CV-2-A88 ( r e s i s t a n t

to Rotylenchulus reniformis) did not remain r e s i s t a n t in the

presence of M. incognita though i t did not loose i t s

resistance in the presence of R, so lan i . Similarly the

resistance of CV, Co-4 to R, so lani broke down in the presence

of M. incognita but not in the presence of R. reni f ormis.

Both R. reniformis and M, incognita when present in assoc ia t ion

with R. solani broke the res is tance of CV, IC-244 agains t

R, solani . The moderately r e s i s t an t response of CV.RC-8 and

SC-4213A to R, reniformis and t h e i r complete r e s i s t ance agains t

R. solani broke down when simultaneously inoculated with both

the nematodes spp. or R, solani with e i the r R, reniformis or

M. incognita.

Walia and Gupta (l966a) reported tha t ne i ther H, ca jan i

nor R. solani had any effect on the growth of Vigna unguiculata

when inoculated separately, '.vhen R. solani was inoculated one

week prior to H. cajani there was a s igni f icant reduction in

the number of nematode cysts and larvae and when R. solani was

inoculated 2 weeks af te r H. ca jani there was a s ign i f i can t

reduction in the top growth of plants compared with controls

or any other treatment. Walia and Gupta (l986b) again observed

the inhibi t ion of mul t ip l icat ion of H. cajani r e su l t ing in

to t a l absence of cys ts , when R. bata t icola was inoculated

7 days prior to nematode in Vigna unguiculata (cowpea seedlin^eip).

Dave (1975) reported tha t damage caused by R, solanl

to soyabean in presence of nematodes vjas not more than

addi t ive, ft. solani inhibited nematode populat ion development.

However, H. glycines was the most suppressed and Tylenchorhynchus

martini the l eas t ,

1,1,3 Macrophomina-nematode complexes

Agarwal and Goswami (1974) conducted pot experiments

using inoculation of soyabean var ie ty Bragg with M. incognita

and/or the fungus Macrophomina phaseollna. The g r e a t e s t

reductions in the seedlings root and shoot weight and highest

mortality r^ te (3C^) were seen in plants inoculated with the

nematode followed 3 weeks l a t e r by the fungus. More severe

symptoms and elevated mortality r a t e (253 ) were a l so seen in

plants inoculated with the two pathogens simultaneously,

compared with when the nematode followed the fungus by 3 weeks

or e i ther pathogen was inoculated alone. Al-Hazmi (1985)

reported tha t in green house experiments sever i ty of

Macrophomina root - ro t of bean (Phaseolus vu lga r i s ) increased

when the nematode was introduced 2 weeks before the fungus,

compared with tha t caused by the fungus a lone. Nematode

infection and reproduction was reduced when the fungus was

introduced f i r s t . Inoculation with e i ther pathogen or with

both generally reduced root weight in both tes ted cu l t i va r s

and s ignif icant ly reduced pod weight in the c u l t i v a r Romano

21

I t a l i a n but not in GV, Harvester. Harvester was more to l e r an t

of both pathogens than Romano I t a l i a n .

Tiyagi e t a l . (1968) reported tha t combined inoculat ion

of M, javanica and M, phaseolina caused s ign i f ican t reduction

in plant growth including pod formation of l e n t i l (Lens

cu l lna r i s ) CV. L-912 In pot experiments. This damaging effect

was more pronounced in simultaneous than in sequent ial

inoculation of the pathogens a t an in te rva l of 10 days. There

was a negative in terac t ing co r re l a t ion between the organisms,

with the fungus being inhibi tory to the nematode, especia l ly

when inoculated 10 days previously.

1,1,4 Cyllndrocladium-Nematode Complexes

Diomande and Beute (1979) reported tha t the sever i ty

of black ro t caused by Cylindrocladium c ro ta l a r i ae on IC 3033

and f lo r ig ian t peanut cu l t iva rs increased in presence of

1,000 and 10,000 M. hapla eggs/pot, Macroposthonia ornata

increased disease severi ty of 10,000 larvae with 0.25 and 2,5

microsclerotia per pot on f lo r ig i an t but did not af fec t the

disease on NC 3033. Diomande e t ail. (1981) again reported

that in USA 2 populations of M, arenaria (i^ce 2 incompatible

on pea nut) enhanced development of Cylindrocladium black ro t

(CBR) on GBR r e s i s t a n t peanut CV. NC 3033 in greenhouse

fac tor ia l experiments. Nematode population 256 and A86

22

(0, lCy, 10^ eggs/l5 cm pot) were tested in all combinations

with C, crotalariae (0, 0.5, 5, 50) microsclerotia/cm-'^ soil.

Root-rot index increased in presence of either population.

Inoculum density disease curve were changed by both populations,

indicating increased efficiency of microclerotia when peanuts

were grown in presence of these nematodes. Although little or

no reproduction occurred with either population on NC 3033,

larvae of 256 and 486 penetrated roots. U86 did not induce

root galls and was not successful in establishing feeding sites,

while 256 produced a few small eliptical galls,

Fortnum and Lewis (1978) studied the effect of

M, incognita, Hoplolaimus columbus, Pratylenchus scribneri

singly or in combination to soyabean. Cylindrocladium

crotalariae was applied after 14 days sampling. Heterodera

columbus remain unaffected by other organism but number of

juveniles in the soil were larger in presence of €. crotalariae.

P. scribneri population were significantly lower after 75 days

in presence of H, columbus and C. crotalariae. Meloidogyne

incognita gall index was depressed by the presence of

H. columbus or C. crotalariae.

1.1,5 Other Fungal Diseases and Nematodes

Bell et al. (lS7l) studied the effect of A., f lav us and

M. arenaria separately and together on peanuts (Arachis

hypogea). It was observed that damage by M. arenaria did not

2i affect the incidence of A. f lavus, t o t a l fungi or a f lo toxin

CO ntamina t ion .

Patel et al^. (1966) reported that combined infec t ions

were synergis t ic in reducing shoot growth when an inoculum of

4 g fungal mycelium and 1000-2000 larvae of nematode were

used. There were fewer ga l l s /p lan t and the f ina l population

of the nematode was lower than when i t was inoculated alone.

Gupta et £ l . (1976) studied the effect of cyst nematode

(H. v ig in l ) and 7 fungal species on cowpea alone or in

combination, in r e l a t ion to root growth and nematode population.

I t was observed tha t fungi reduced the nematode i n f e s t a t i o n ,

the grea tes t reduction being with Penicillium cltrinura and

l eas t with Aspergillus t e r reus . The average root weight /plant

was s ignif icant ly reduced when fungus was present i n d i r e c t

proportion to the number of nematodes in the r o o t s . Difference

in sex r a t ios in the presence of fungi were a lso noted,

Adeniji e t a l . (1975) foiand the re la t ionsh ip of

Heterodera glycines and Phytopthora magasperma f, so.jae in

soyabean (Glycine max). Seedling disease was more severe

in the cu l t iva rs Corosoy and Dyer (both suscept ible to

P. magasperma) when both fungus and nematode were present than

in presence of fungus alone. Harosoy 63 ( r e s i s t an t to

P. magasperma failed to develop symptoms in presence of both

organisms. Infection with P, magasperma s ign i f ican t ly

24

suppressed the H. glycines population on roots of Corosoy

but did not break the resistance of Dyer to the nematode.

Campos and Schmitt (1S81) studied the effect of

P. magaspenaa var. sojae (race I) and/or H. glycines on

the soyabean cultivar Sssex (slightly resistant to _P.

ma^asperma var, sonae) and susceptible to (H. glycines),

Forrest (moderately resistant to Phytopthora and resistant

to Heterodera), They fotind that H. glycines did not

reproduce on Forrest and its population density declined on

Essex and Mack in presence of the fungus. Significant plant

mortality in microplots was caused by the fungus on Mack, and

by both pathogens alone or in combination on Forrest and Essex,

Book Binder and Bloom (1978) noticed that interaction

between M. incognita and Uromyces phaseoli reduced fresh

weight of shoot and root of a soyabean to a large extent

than caused by either pathogen alone. Gall formation was

reduced only when U, phaseoli was applied several days before

inoculation with M, incognita on the cultivar most susceptible

to IJ, phaseoli. The presence of U, phaseoli did not affect

hatching. Book Binder and Bloom (l980) again reported more

growth reduction of bean's shoot and root weights when

Keloidogyne incognita and Uromyces phaseoli were present.

The shoot weight was suppressed by 7, 9 and 15% and root weight

by 8, 16 and 23% respectively, suggesting that there was an

additive effect. Both the pathogens influenced the reproduction

25

of one ano the r , fungal uredia were reduced i n s i z e and spo ru -

l a t i o n c a p a c i t y . M. incogni ta produced fewer r o o t g a l l s and

fewer eggs/egg mass.

Garcia and Mi tche l l (1975) r epor t ed inc reased s e v e r i t y

of pod r o t of pea nut when pods were exposed to s o i l c o n t a i n i n g

combinations of Phythlum myriotylum with Fusarium s o l a n i and/

or M. a r ena r l a than when pods were exposed to P. myriotyltjm

a lone .

NEMATODB-ROOT NODULE EACTBRIA INTERACTIONS

Mil l e r (1951) was f i r s t t o observe i n h i b i t i o n of

nodula t ion on p l a n t roo t s in presence of r o o t - k n o t nematode,

i n f e c t i o n .

Later on s e v e r a l other workers have a l s o r e p o r t e d t h a t

roo t -knot nematode caused reduc t ion i n nodu la t i on of leguminous

p l a n t s , e . g . Meloidogyne hapla on ha i ry ve tch (Malek and

Jenk ins , 1964) and on soyabean (Balasubramanian, 1971);

M, javanica on white clover (Taha and Raski , 1969), on soyabean

(Balasubramanian, 1971) and on mung, Vigna r a d i a t a (Bopaiah

e t a l . , 1976); M. incogni ta on mung bean.Phaseolus aureus

(Plussaini and Seshadr i , 1975) and on soyabean ( te lasubramanian ,

1971; Sr ivas tava e t a_l., 1974; Hussey and Barker, 1974, 1976;

Baldwin e t a l . , 1975).

2b

Hussaini and Seshadri (1975) observed that M.incognita

was pathogenic to mung bean/green gram (Phaseolus aureus)

and hampered nitrogen fixation. The nematode caused significant

decrease in weight of plants (fresh and dry weight of roots

and shoot). Reduction in nitrogen content yas considered by

them to be due to the overall reduction in root nodulation,

anatomical changes in nodules and altered physiology of the

host.

All et al, (1981) studied the antagonistic Interaction

between M. incognita and railzoblum leguminosarum on cowpea.

They observed that M, incognita reduced nodulation and

inhibition of nitrogen fixation by about 639* in the nodular

tissue. Infected nodules contained different developmental

stages of the nematodes but the nematode did not alter the

structure of nodules. However, infected nodules deteriorated

earlier than the uninfected ones.

Raut and Sethi (1960 ) examined and found a progressive

decrease in the growth of soyabean plant and root nodulation

as the inoculum level of M» incognita increased. The nodule

reduction takes place in both the seasons, summer as well as

winter. Raut (1980 ) again pointed out that mung bean

inoculated with Meloidogyne incognita showed gradual reduction

in growth with an increase in initial inoculum level. The

nitrogen fixing capacity was also affected in mimgbean variety

" PIMS-I" .

27

Singh et aj., (1977) reported that there was a correspond­

ing decrease in chlorophyll content, number of nodules and

nitrogen content of grain with an increase in the inoculum

level of Meloidogyne incognita both in presence and absence

of Rhizobium phaseoli. The nematode galls were observed on

nodules when inoculum level of the nematode was 1000/pot.

Finally they said that M. incognita adversely affected the

symbiotic process of nitrogen fixation by Rhizobixan phaseoli

on miang (P, aureus).

Chahal and Chahal (1968) studied the effect of

interaction of Rhizobium and M. incognita on Np fixation and

translocation of iron (Fe) in the shoots of Vigna radiata

CV, ML-267 grown in pots containing sterilized river sand

supplied with 0,300 ppm Fe. They observed that M, incognita

infection reduced all growth characteristics, as well as Fe,

translocation in the shoots of nodulated and no nodulated

plants.

Chahal and Chahal (l989) again reported that

M, incognita reduced the leghaeraoglobin and bacteroid content

of Rhizobium nodules of mungbean (Vigna radiata ) CV. G65

irrespective of initial level of inoculum (250, 500, lOOO or

2000 2nd stage Juveniles/seedlings) thus adversely affecting

the functioning of nodules. Nitrogenase activity of nodules

decreased with an increase in the initial level of inoculum.

28

Verde jo et al, (l989) observed the invasion of

M. Incognita in the aseptic roots of pea and blackbean

(P, vulgaris). Females were developed in about 19 days.

Juvenile nematode invaded nodules initiated by Rhizobium,

nodules formed also on galls initiated by nematodes.

M. incognita suppressed root and nodule growth. However it

stimulated the initiation of nodules which remained

undeveloped, Sffective nodules have more nitrogenase activity

per gm, on plants with M, incognita than on those without

nematodes. M, Incognita increased the leghaemoglobin content

of nodules on pea and decreased it on P. vulgaris. All the

effects of nematode invasion were transient and 7 days

difference in invasion date altered the degree of effect

recorded at different harvest dates,

Sharraa and Sethi (1976) showed that Meloidogyne

incognita and Heterodera cajani. singly or in concomitant

inoculation significantly reduced the growth of cowpea,

Vigna sinensis. Addition of Rhizobium tended to reduce the

damage to some extent. Similarly both the nematodes affected

the nitrogen fixing capacity of the plant, M, incognita

reduced nitrogen content to a greater extent than H, ca.jani.

Root examination showed that both nematode species could thrive

well and complete their life cycle on the nodular tissue,

Srivastava et al. (l979) reported that the growth of

soyabean Glycine max was affected when rootr-knot nematode

M, .javanica was inoculated at different inoculum levels. Number

of nodules per plant were reduced significantly even at low

inoculum level (10 larvae/kg soil). When the inoculum level

was increased, there was a reduction in the growth. Pod

formation was also affected by increasing the inoculum level.

Bopaiah _et al. (l976) reported that inoculation of

Vigna radiata (mung) with J1. javanica had deleterious effect

on plant growth and this could be observed when inoculated with

nematode alone, simultaneously or 2-7 days preceding to

Rhizobium inoculation. The nematode inoculation prior to

Ethlzobium resulted in maximum reduction of nodules. The

infestation by the nematode interfered with nitrogen fixation

and reduced the nitrogen content of the plant,

Dhanger and Gupta (1983) observed the pathogenecity

of Meloidogyne .javanica to chickpea (Cicer arietinum) in

relation to soil types, Rhizobium treatment, size of pots

and time intervals. They found pathogenecity in all three

types of soil and at different time intervals. Similarly

Rhizobium treated seedlings showed better growth characters

than untreated seeds.

Taha and Kassab (1S60) observed the effect of simul­

taneous inoculations of M, javanica and Rotylenchulus renlformis

with Rhizobium on Vigna sinensis and indicated that their

association did not affect nodulation. Nodule formation was

30

hindered only when R, renlformls preceded rhizobial inoculation.

Nodules were formed on Meloidogyne javanica galls.

Ross (1969) reported that Heterodera glycines affected

the yield of non nodulating soyabean at different nitrogen

levels and he found that besides reducing nodulation and

nitrogen fisotion, H, glycines caused reduction in yield

inciting deterious host responses that increased with

nitrogen deficiency,

Lehman et al, (1971) examined three races of

Heterodera glycines (l, 2 and 4) and found that in combination

of race 1 of H. glycines plus Rhizobixxm 3aponicum there were

fewer nodules per gram of iroot tissue and N-fixing capacity

was less than with R. .japonicum alone. Similar inoculum

levels (100, 200 and 400 crushed cyst) of race 2 and 4 had no

effect. Nodule number and weight were inversely prt>portional

to the increasing densities of race 1,

Barker et al, (l971 ) reported that application of

nitrogen (N) as well as inoculation of soyabean with Rhizobium

japonicum gave an adverse effect on the activity of

Heterodera glycines. The application of NaNO, or NHi to soil

reduced nematode hatch, penetration and cyst development.

Simultaneous inoculations of nodulating and non-nodulating

isogenic lines of soyabean with Rhizobium and H, glycines

reduced the number of cyst development especia l ly on the

nodulating l i n e s .

Barker e t a l , (1972) again observed suf f ic ien t i nh ib i t i on

of nodules upon the simultaneous inoculat ion of H, glycines

and Rhizobium .japonicum on soyabean. However, 14 days delay

of H. glycines resul ted in only s l i gh t to moderate i nh ib i t i on

of nodulation.

Gupta and Yadav (1979) studied the pathogenecity of the

reniform nematode, Rotylenchulus reniformls to urd, Vigna mungo

and found s igni f icant reduction in plant height and shoot and

root weights, Nodulation was a lso affected.

Most of the invest igators have reported suppression or

inhibi t ion of nodulation by p lant p a r a s i t i c nematodes. However,

stimulation of nodulation on leguminous plants by pathogenic

nematodes was reported by Hussey and Barker (1976), They

studied the influence of Meloidogyne hapla, Pratylenchtis

penetrans and Belonolaimus longicaudatus on nodulation of

soyabean, M, hapla and P. penetrans stimulated nodule formation,

while Belonolaimus longicaudatus s l i gh t ly inhibi ted the

nodulation.

32

Future Plan of Work

The above l i t e r a t u r e shows tha t much a t t en t ion has been

given to the in terac t ions between nematodes - fungi, nematodes -

root nodule bacteria and nematode-nematode separate ly , though

these micro-organisms inh ib i t a common niche. Therefore, i t

was considered desirable to invest igate the effect of plant

pa r a s i t i c nematodes, fungi and root-nodule bacteria on some

leguminous p l an t s .

The following experiments have been planned for de ta i led

study : -

1. Effect of d i f ferent inoculum levels of root-knot nematode,

Meloidogyne incognita and roo t - ro t fungus, Rhizoctonla solani

alone and in combination on the root-knot and roo t - ro t

development and plant growth of french bean (Phaseolus

vu lgar i s ) in the presence and absence of Rhizobium,

2. Effect of di f ferent inoculum levels of root-knot nematode,

Meloidogyne incognita and roo t - ro t fungus, Rhizoctonia solani

alone and in combination on the root-knot and roo t - ro t

development and plant growth of mung bean (Phaseolus aureus)

in the presence and absence of Wiizobium.

3 . Histological changes induced by M, incognita and R. solani

alone and in combination on french bean (Phaseolus vu lgar i s )

in the presence and absence of Rhizobium.

33

4. Histological changes induced by M. incognita and R, so lan i

alone and in combination on mung bean (Phaseolus aureus )

in the presence and absence of Rhizobium.

5. Effect of M. incognita and R, solani alone and in combination

on leghaemoglobin bacteroids produced by Rhizobium on

french bean (Phaseolus vu lga r i s ) .

6. Effect of M, incognita and R, solani alone and in combination

on the leghaemoglobin bacteroids produced by Rhizobium on

mung bean (Phaseolus aureus).

7 . Effect of Carbofui^n (neaaticide) and/or Bavistin (fungicide)

on leghaemoglobin bacteroids produced by Rhizobium on french

bean (Phaseolus vulgar is) in the presence of M. incognita

and R, so lan i .

8. Effect of Carbofuran (nematicide) and/or Bavistin (fungicide)

on leghaemoglobin bacteroids produced by Rhizobium on mung

bean (Phaseolus aureus) in the presence of M. incognita

and R. so lan i ,

9. Effect of different ra t ios of sand and clay on the development

of root-knot, root- rot and nodulation on french bean

(Phaseolus vulgaris ).

10. Effect of different ra t ios of sand and clay on the development

of root-knot, root-rot and nodulation on mung bean

(Phaseolus aureus).

34

11, Effect of different sources and levels of nitrogenous

f e r t i l i z e r s on the development of root-knot, r o o t - r o t

and nodulation on french bean (Phaseolus v u l g a r i s ) .

12, Sffect of different sources and levels of nitrogenous

f e r t i l i z e r s on the development of root-knot, r o o t - r o t

and nodulation on mung bean (Phaseolus aureus) .

13, Effect of M, incognita and R, solani alone and in combination

on NPK contents in french bean (Phaseolus vu lga r i s )

in the presence and absence of Rhizobim.

14, Sffect of M, incognita and R. solani alone and in combina-

nation on NPK contents in mung bean (Phaseolus aureus) in

the presence and absence of Rhizobium,

15

MATBRULS AND METHODS

The different materials to be used and the methods to

be employed during the course of proposed experimental programme

are generalised as follows :

2.1 Selection of test materials :

In the proposed plan of work the root-knot nematode,

Meloidogyne incognita (Kofoid and White) Chitwood, and the

root-rot fungus, Rhizoctonia solani Kuhn. have been selected

which will serve as test pathogens for the studies. French

bean, Phaseolus vulgaris L, will be used as a test plant

during winter season and green gram/mung bean, Phaseolus

aureus Roxb. during summer season. Respective rhizobia of

these test plants will also be used as and when required.

2«2 Preparation of jpoculum of the nematodes :

Pure cultures of the root-knot nematode will be

maintained on tomato in microplots. Species of root-knot

nematode and races will be identified on the basis of

characteristic of perineal patterns and differential host

test.

A single egg-mass, obtained from tomato roots infected

with root-knot nematode, Heloidogyne incognita, will be

3C

surface sterilized in 1:500 solution of chlorox (Calcium

hypochlorite) for five minutes and will be washed thrice in

sterilized water. The egg-nass will be allowed to hatch in

sterilized distilled water at 27°C, Tomato seedlings raised

in autoclaved soil will be inoculated with the larvae thus

obtained. Later more plants will be inoculated at regular

intervals with a view to have a regular supply of inoculum.

Every time prior to inoculation the egg-masses will be

removed fiX)m the infected roots of tomato plant and will be

allowed to hatch (Stemerding, 1963). For larval count one ml

of larval suspension thus obtained will be pipetted in

Hawksly counting slide and the number of larvae will be counted

under a sterioscopic microscope. The process will be repeated

thrice and the number of larvae present per ml of the larval

suspension will be standarized. Calculations will be made for

the total suspension to be used for inoculation.

2.3 Preparation of the inoculxaa of the fungi :

Pure cultures of the root-rot fungus, Rhizoctonia

solani will be maintained in the culture tubes containing

potato - dextrose agar (PDA) which will be prepared from the

following constituents.

Potato 200.00 g

Dextrose 20.00 g

Agar 20.00 g

Distilled water 1000.00 ml

37

The inoculum of the roo t - ro t fungus, Rhlzoctonla solani

wil l be raised on Richard's l iquid medium (Riker and Riker,

1936) having the following composition;

Potassium n i t r a t e 10,00 g

Potassium dihydrogen

phosphate 5.00 g

Magnesium sulphate 2,50 g

Ferric chloride 0,02 g

Sucrose 50.00 g

Distilled water 1000,00 ml

The fungi will be incubated for I5 days in an incubator

running at 28 + 2^C temperature.

After the incubation period the mycelial mats will be

removed, washed in distilled water to remove the traces of

the medium and then it will be gently pressed between sterile

blotting papers to remove the excess amount of water. Inoculum

will be prepared by making 10 g fungal mycelium in 100 ml of

sterilised distilled water and blending it for 30 seconds in

a waring blender (Stemerding, 1963). In this way each 10 ml

of this homogenate will contain 1 g of the fungus.

2.4 Maintalnence of test plants :

Sandy loam soil, which is commonly found in Aligarh

will be collected from a fallow field and will be passed

through a course sieve (I mm pore size) to remove stone

3S

particles and debris etc. Compost manure at the rate of 4; 1

(soil : compost) will be added and thoroughly mixed with soil.

Six cm. clay pots will be filled with 1 kg of such soil -

compost mixture and then these pots will be autoclaved and will

be used for all studies. Four or five surface sterilized

seeds of French bean, (Phaseolus vulgaris) and green gram/

mung bean, (Phaseolus aureus) will be sown in these pots. Surface

sterilization of seeds will be done by treating them in 0,1%

solution of mercuric chloride for 2 minutes. Then these seeds

will be washed thrice with sterilized distilled water to remove

the traces of Mercuric chloride. After emergence the seedlings

will be thinned and only one seedling will be allowed to grow

in one pot. Plants at three leaf stage will be inoculated with

nematode and the fungus, as per schedule of the experiments.

In the case of root-nodule bacteria the seeds will be

bacterized before sowing with their respective rhizobia.

Sucrose solution (^) will be used as Sticker and it will be

mixed with the rhizobial culture. Seeds will be mixed with

this mixture in such a manner that a uniform coating be formed

on their surface. These bacterized seeds will be dried at room

temperature and then sown.

The pots will be placed on a green house bench in a

randomised manner. Necessary weeding and watering will be done

as and when required. Special requirements of different

experiments, if any, are described in the appropriate columns.

3S

2.5 Inoculation Technique :

(a) Inoculation with nematodes :

For the inoculation of plants with nematodes, appro­

priate amount of nematode suspension (according to the inoculum

level) with the help of pipette will be poured around the

roots which will be exposed by removing small amount of soil.

After inoculation the exposed roots will be covered by levelling

the soil properly.

(b) Inoculation with fungi :

For the inoculation of plants with fungi, homogenate

of fungal mycelium will be prepared as described in (2,3). It

will be taken out in such an amount so that the required

iiwcultam level is fulfilled. For inoculation the soil will

be removed from around the roots of the test plants to expose

the roots and then the required amount of fungal suspension

will be poured near the roots, which will be later covered by

levelling the soil properly,

2.6 Determination of inoculum threshold :

In order to determine the inoculum threshold of the

selected nematode species, capable of causing significant

damage, the seedlings of both the plants French bean/mung bean

will be inoculated with different inoculum levels viz., 10, 100,

1000 and 10,000 of test nematode species M. incognita.

4b

Similarly for the determination of fungal inoculum threshold

the seedlings will be inoculated with 0.5, 1.0, 2.0 and 3.0 g

of the fungus per plant. There will be 5 replicates for each

inoculum level. Separate sets will be maintained for

bacterized as well as non bacterlzed plants.

2.7 Studies on Interactions of different inoculum levels of

test pathogens : (Nematode fungus interactions)

Only one inoculum level will be selected after

analysing the data obtained in experiment (determination of

inoculum threshold). Plants will be raised according to the

procedure described in 2.4 for both bacterized as well as for

non bacterized conditions and the plants will be inoculated

according to following schedule.

1. Uninoculated (control)

2. Nematode alone

3. Fungus alone

4. Nematode and fungus simultaneously

5. Nematode one week prior to fungus

6. Nematode two weeks prior to fungus

7. Nematode one week after the fungus

8. Nematode two weeks after the fungus

This inoculation procedure will be according to method

described in 2. 5. Then final recording of the data will be

done after 2 months of inoculation.

4i

EXPSRIMEffTS

3.1 Histopathologlcal studies :

Inoculated seedlings will be uprooted carefully of the

soil from the first until the sixth day, then every three days

until the 30th day and finally on the Oth day. The roots

will be washed gently, but thoroughly to remove all soil

particles adhering to them. Roots of healthy and infected

plants will be cut into one cm long pieces and processed as

follows :

3.1.1 Fixation :

The roots collected from experimental pots wi l l be fixed

in FAA (Johansen, 19A0), FAA (Formalin-aceto-alcohol) w i l l be

prepared as follows :

Bthanol 5096 90 ml

Formaline 379 5 ml

Glacial ace t ic acid 5 ml

The roots wil l be placed in the f ixa t ive for a minimum

period of 24 hr to several days, depending on i t s th ickness .

I teterial may also be stored in the f ixat ive inde f in i t e ly .

3.1.2 Dehydration :

Dehydration is accomplished by moving the roots stepwise

through increasingly higher concentrations of alcohols. Tertiary

4^

butyl-alcohol (TBA) dehydration schedule (Table 1) will be

followed (johansen, 1940),

3.1.3 Infiltration ;

In this step, alcohols in the tissues will be replaced

by paraffin so that the tissue is saturated with a pure solution

of paraffin, '^en the TBA. dehydration schedule is followed,

the 10C^ TBA solution in step 8 will be replaced with a 1:1

mixture of 100^ TBA and paraffin oil. The tissue will be

allowed to remain in this solution for 1 hr or more, depending

on its thickness. Shortly before the next step, another

container will be filled 3/4th of its volume with melted paraffin

and allowed to solidify slightly. The roots from the TBA-

paraffin oil mixture will then be placed on top of the solidified

paraffin oil solution. This container will be placed uncovered

in an oven set at slightly above the melting point of the

paraffin. After 1-3 hr, the TBA - paraffin oil mixture will

be poured off and replaced with pure melted paraffin wax, and

kept in oven for about 3 hr. This step will be repeated at

least once more,

3.1.A Embedding :

The roots will be placed in metal base molds or folded

paper. Molds will first be coated with a thin layer of

glycerine and then liquid paraffin will be poured. Roots will

be placed into the mold with heated forceps and additional

r -3

TABLS - I

Step % Alcohol Time D i s t i l l e d 95% IOO96 100% water Ethanol S thano l TM (ml) (ml) (ml) (ml)

1.

2.

3.

4.

5.

*6.

7.

8.

50

70

85

95

100

100

100

100

2 hr or more

Overnight

1 - 2 hr

1 - 2 hr

1 - 3 hr

1- 3 hr

1 - 3 hr

Overnight

50

30

15

0

0

0

0

0

AO

50

50

45

0

0

0

0

0

0

0

0

25

0

0

0

10

20

35

35

75

100

100

100

* TEA changes must be c a r r i e d out i n a warm p l ace as i t

s o l i d i f i e s a t 25.5 C.

4^

melted paraffin will be added to fill the mold. Once the

paraffin begins to solidify over the top of the mold, the mold

will be plunged into ice water and left there until solidi­

fication. After hardening, it will be cut into smaller blocks

and trimmed. The blocks will be mounted on block holders.

3.1.5 Sectioning

Transverse and longitudinal sections of 8-12 Mn thickness

of the roots will be cut serially with the help of rotary

microtome. The paraffin ribbon thus obtained will be mounted

on a clean glass slide with an amount of albumin and glycerine

dissolved in water. The slides will be left overnight In an

incubator at 40°G to allow water to evaporate. These slides

will then be kept at 60°C for one hr to melt the paraffin.

3.1.6 Staining :

The process of s ta ining wi l l remove the paraff in from

the sections and increase the con t ra s t in the t i s s u e s . Staining

wil l be done with Safranin and F^st Green combination (Table 2)

(Sass, 1951). Then, s l ides wi l l be removed from the xylene,

the final step in a s taining procedures and la id on a f l a t ,

absorbant surface. The mounting medium wil l be applied to the

surface of the s l ide before evaporation of the xylene, and

cover-slip will be lowered gradually over the s l i d e .

4ii

TABLE - 2

Step

1.

2 .

3 .

A.

5.

6 .

7 .

8.

9 .

10.

11 .

12.

13.

14.

15.

16.

17. 18.

Solu t ion

xylene

abso lu te e thano l

9^ e thanol

70?6 e thanol

509^ e thanol

30% e thanol

196 aqueous s a f r an in 0

r i n s e in t a p water

309^ e thanol

5056 e thanol

703^ e thanol

9596 e thanol

0.194 f a s t green FCF i n 955 e thanol

abso lu te e thanol

abso lu te e thanol

xy lene-absolu te e thanol

xylene

xylene

Time

5 min

5 min

5 rain

5 min

5 m±n

5 min

1-12 h r

3 rain

3 min

3 min

3 min

5-30 sec

15 sec

3 min

5 min

5 min

5 min or longer

46

Finished slides will be left to dry for at least 24 hr

at room temperature. The medium will harden better if the

slides are held on a 60 C warming tray overnight. The slides

will be examined under Laborlux - K compound microscope.

Necessary photographs will be taken.

3.2 Studies on the effect of different nitrogen levels

3.2.1 Preparation of different levels of nitrogen from different

sources :

The different nitrogen sources will be nitrate of soda

(16% N), Ammonium sulphate {2^% N), Ammonium nitrate (34% N),

Ammonium sulphate nitrate (26% N), Ammonium chloride (26% N),

Anhydrous Ammonia (99%N), Urea, biuret =1.5% (46% N) Urea

coated, (45%.N), calcium ara ionium nitrate (25% N), calcium

ammonium nitrate (26% N) and calcium ammonium nitrate (28% N).

Different nitrogen levels viz., -N (No Nitrogen), 1/2 N

(0.5 g), 1 N (1.0 g) and 2 N (2.0 g) per kg of soil will be

calculated from each source and will be mixed with the soil

before sowing the seeds of test plant.

3.3 Studies on the effect of different ratios of sand and clay :

For this experiment sand soil mixtures containing

different proportions of clay and sand will be prepared in

following manner. These have been designated as soil types.

47

Sand percent

100

75

50

25

0

Clay percent

0

25

50

75

100

Soil type

1

2

3

A

5

These mixture wi l l be analysed for organic matter , pH and

pa r t i c l e s ize . Then 15 cm pots wi l l be f i l l e d with one

kilogram of each so i l type. These pots wi l l be steam

s te r i l i zed in an autoclave and a f t e r t h i s A-5 seeds of each

t e s t plant wi l l be sown in each pot . One week a f t e r sowing

thining wil l be done to keep one plant per pot . Freshly

hatched second stage larvae of M. incognita and r o o t - r o t fungus^

R, so Ian i alone and in combination wi l l be added around the

exposed roots in the presence and absence of Rhizobium spp.

a t the three leaf stage. Two months a f t e r inocula t ion a l l

these pots wil l be depotted and then observations w i l l be

recorded on the root and shoot length, fresh and dry weight

of root and shoot, number of nodules and g a l l s per p lan t .

Finally a l l these observations wil l be presented in the t a b l e .

3.4 Studies on the effect of nematiclde/fungicide on the leghaemoglobin contents :

For th i s experiment both the t e s t p lants wi l l be

treated with e i ther nematicide (carbofuran) and/or fungicide

(Bavistin) or both in addition to root-knot nematode (M.incognita)

4S

and root-rot fungus (R. solanl) in the presence and absence of

Rhlzobium spp. There will be another set of untreated plants

in which no nematicide or fungicide will be added.

The leghaemoglobin content will be estimated from

both treated and untreated plants in the following manner.

3.4,1 Estimation of leghaemoglobin from nodules :

Benzidine hydrogen per oxide method (Proctor, 1963) will

be used for the estimation of leghaemoglobin. Nodules will

be picked up from the roots and washed throughly with double

distilled water. Fresh nodular tissue weighing 500 mg will be

crushed and homogenized in 9 ml of trisacetic acid buffer

(0,1 M acetic acid plus 0,2M tris (hydroxyImethyl) amino

methane to obtain a pH of 4,0). The homogenate will be kept

overnight in a deep freezer to get complete extraction of

leghaemoglobin. The homogenate will be shaken from time to

time to facilitate extraction. The volume of homogenate will

be made upto 10 ml by adding tris acetic acid buffer and

centrifuged at 3000 rpm for 20 minutes. The supernatent will

be collected in a test tube. An aliquot of 0.5 ml of super­

natant will be diluted to a final volume of 4.0 ml with tris

acetic acid buffer and 2 ml of freshly prepared benzidine

reagent (lOO mg benzidine with 0.5 ml of hydrogen peroxide of

100 percent by volume added to 50 ml absolute alcohol) will

be added to it. The colour density will be measured at 660 nm

on spectrophotometer. The results will be expressed in bovine

haemoglobin equilents.

42

3.5 ANALYSIS OF NPK FROM LBAVBS AND ROOTS :

The plants will be uprooted after 60 days of inoculation

and shoot and root system will be washed gently with double

distilled water. Fully mature healthy leaves will be picked

up and some part of the root system from inoculated and o

uninoculated replicates, will be dried in an oven at 78+2 G

separately for 24 hrs. The dried material will be grounded

into fine powder and passed through a 72 mesh sieve. The powder

will be kept in desiccator so as to ensure the availability

of perfectly dry material.

The leaf and root powders will be digested for determining

the NPK contents by the method of Linder (1944) that is briefly

described below.

From each sample, 100 mg of the dried leaf and root powder

will be weighed and carefully transferred to a 50.0 ml Kjeldahl

flask and 2.0 ml of chemically pure sulphuric acid will be

added. The flask will be kept for digestion for about 2 hrs.

to allow complete reduction of nitrates present in the plant

material, giving off dense white fumes untill the contents

turn black.

Flask will then be allowed to cool down for about 15

minutes after which 0.5 ml of 3C^ hydrogen peroxide will be

added dropwise and the solution will be heated again till its

colour changes from black, to light yellow. Heating will be

5a

continued for about 30 min. The flask wil l be cooled for 10

min. and an addi t ional amount of 3-A drops of 30% hydrogen

peroxide wi l l be added followed by gentle heating for another

15 min. to get a c lea r and colourless ex t rac t . At t h i s stage

care will be taken in the addition of Hydrogen peroxide because

i f i t i s added in excess there i s poss ib i l i ty t h a t i t would

oxidise the ammonia in the absence of organic matter . The

sulphuric acid peroxide digested material w i l l be d i lu ted with

double d i s t i l l e d water and will be t ransferred with 3 or A

washings to a 100 ml volumetric f lask and f i na l ly the volume

wil l be made upto mark. For the analysis of ni t rogen,

phosphorus and potassium al iquots wi l l be taken from these

digested samples, the methods employed for t h i s ana lys is are

brief ly summerized below :

3.5.1 Nitrogen estimation :

Nitrogen content of the sample will be estimated

according to the method o: Linder (1944), A 10.0 ml a l iquot

of digested material wil l be taken«in a 50 ml volumetric

f lask. The excess of acid wil l be p a r t i a l l y neutral ized with

2.0 ml of 2.5 N sodium hydroxide and 1.0 ml of 10% sodium

s i l i c a t e wil l be added to prevent tu rb id i ty . Finally the

volume wil l be made upto the mark. A 5.0 ml a l iquo t of th i s

solution wi l l be taken in a 10 ml graduated t e s t tube and

0.5 ml of Nessler 's reagent wi l l be added drop by drop, mixing

51

thoroughly a f te r each instalment. The volume wi l l again be

made upto the mark with the help of d i s t i l l e d water and the

contents wil l be allowed to stand for 5 min. for maximum

colour development. The solut ion wi l l be t ransfer red to a

colorimetric tube and i t s op t ica l density wi l l be measured,

a t 525 nm on a " spectronic 20" colorimeter, A blank wi l l

be run with each se t . The amount of nitrogen in the a l iquot

w i l l be read from a ca l ibra t ion curve, obtained by using known

d i lu t ions of a standard ammonium sulphate so lu t ion ,

3 .5 .2 Phosphorvts estimation :

Total phosphorous in the sulphuric acid peroxide

digested solut ion wil l be determined by following the method

of Fiske and Subba Row (1925). A 5.0 ml of a l i quo t wi l l be

taken in a 10,0 ml graduated tube and 1,0 ml molybdic acid

(2,5% ammonium molybdate in 10 N H2S0^) wil l be careful ly

added, followed by 0,4 ml of 1,2,4-amino-napthol-sulphonic

acid, which wil l turn the contents blue. D i s t i l l ed water

wi l l be added to make up the volume upto 10,0 ml and the

solut ion wil l be allowed to stand for about 5 minutes a f t e r

mixing throughly. I t wi l l be then transferred to a c o l o r i ­

metric tube and the opt ical density will be read a t 620 nm

on a colorimeter, A blank wi l l be run side by s ide . A

ca l ib ra t ion curve wil l be prepared by using known d i lu t ion of

a standard monobasic potassium phosphate so lu t ion .

3.5.3 Potassixim estimation :

Potassium wil l be estimated using a flame photometer.

A 1,0 ml al iquot wi l l be taken and read a t 768 nm with the

help of potassium f i l t e r . A blank wi l l be run for each

determination. The reading wi l l be compared with a c a l i b r a t i on

curve plotted for different d i lu t ions of a standard potassium

sulphate solut ion.

3.6 Recording of data :

3.6.1 Plant grovrbh determination ;

For determining the plant growth, p lan t s wi l l be

uprooted a f te r 60 days of inoculation and the roo ts w i l l be

thoroughly and gently washed. The length ( in cm) and f resh/

dry weights (in g ) of shoot and root wi l l be taken separa te ly .

Before taking the fresh weight, excess amount of water wi l l be

removed by putting root and shoot between b lo t t ing sheets ,

while for determining the dry weight shoot and root wi l l be

dried in an oven a t 60^C and then separately weighed,

3.6.2 Determination of root~knot index :

The degree of root infect ion caused by root-knot

nematode will be assessed according to the ra t ing scale of

Taylor and Sasser (1978) for the presence of root ga l l s and

egg masses on roots as under :

Gall index (GI)

2gg mass index (EI)

1 = No g a l l , no egg-masses

2 = 1-10 ga l l s / egg-masses

3 = 11-30 ga l l s / egg-masses

4 = 31-100 ga l l s /egg-masses

5 = 101 & above ga l l s / egg-masses

3 ,6 ,3 Determination of root nodule index :

On the bas i s of v i s u a l o b s e r v a t i o n , t h e r o o t nodule

index w i l l be r a t ed on the fol lowing s c a l e .

1 » no nodulation

2 » light nodulation

3 = moderate nodulation

4 = heavy nodulation

5 = very heavy nodulation

3 , 6 , 4 Determination of popu la t ion o f nematodes :

The popula t ion of nematodes i n the r o o t s w i l l be

determined by mascerat ing the r o o t - p i e c e s i n waring b lender

for few seconds and then counting t h e i r numbers. The nematodes

p resen t i n the s o i l w i l l be i s o l a t e d by Cobb's s i e v i n g and

decanting method.

54

3.6.5 Determination of roo t - ro t Index ;

The r e l a t i v e amount of R. solani damage wi l l be determined

by scoring the extent of disease on the following sca l e .

1 = no root - ro t

2 = 25% roo t - ro t

3 = 50% roo t - ro t

A » 75% roo t - ro t

5 = 100% root-rot( severe roo t - ro t )

3.6.6 S ta t i s t i ca l analysis :

The data observed will be analysed s t a t i s t i c a l l y .

5; REFERENCES

Adeni j i , M.O.; Edwards, D . I . ; S i n c l a i r , J . B . and Malek, R. B. (1975) .

I n t e r r e l a t i o n s h i p of Heterodera g l y c i n e s and Phytopthora

ma^asperma v a r , sopae i n soyabean. Phytopathology 6 ^ ( 6 ) :

722-725.

Agarwal, D.K, and Goswami, B.K. (1974). I n t e r r e l a t i o n s h i p between

a fungus. Macrophomina phaseo l ina (Maubl,) Ashby, and

roo t -kno t nematode, M. i n c o g n i t a (KiDfoid and White)

Chitwood, i n soyabean (Glycine max L. ) M e r i l l . Proceedings

of Ind ian Nat ional Science Academy B. ^(6): 701-704.

Al-Hazmi, A.S, (1965). I n t e r a c t i o n of M. i ncogn i t a and

Macrophomlna phaseo l ina i n a r o o t - r o t d i s e a s e complex of

french bean. Phytopa thologlsche Z e i t s c h r i f t . 113(A);

311-316.

A l i , M.A,; T r a b u l s i , J .V. and Abd-Elsamea, M.S. (1981). An tagon i s t i c

i n t e r a c t i o n between M, i n c o g n i t a . Rhizobium leguminosarum

on cowpea. P l a n t Dis.« 6 5 ; 432-435.

Atkinson, G.F. (1892). Some d i s e a s e s of c o t t o n . B u l l . Ala . Agr ic .

Exp. S t a . 41 ; 65 P.

Balasubramanian, M. ( l 9 7 l ) . Root-knot nematodes and b a c t e r i a l

nodula t ion i n soyabean. Cur r . S c i . , 40(3) : 6 9 - 7 1 .

Baldwin, J . G . , Barker, K.R. and Nelson, L.A. ( l 975 ) . I n t e r a c t i o n

of Meloidogyne incogn i t a with Rhizobium .laponicum on

soyabean. J . Nematol, , J^i 319 (Abstr, ) .

56

Barker, K.R., Huislngh, D. and Johnson, S.A. (1972). A n t a g o n i s t i c

i n t e r a c t i o n between Heterodera g l y c i n e s and Rhlzobium

japonicum on soyabean. Phytopathology, 6_2(10): 1201-1205.

Barker, K.R., Lehman, P .S . and Huis ingh, D. (1971). In f luence of

n i t r o g e n and Rhizobiua japonicum on t h e a c t i v i t y of

Heterodera g l y c i n e s . Nematologica. 17: 377-385.

B e l l , D.K., Mlnton, N.A. and Doupnik, J r . B. (1971). E f fec t of

Meloidogyne a r e n a r i a , A s p e r g i l l u s f l avus and cu r ing t ime

on i n f e c t i o n of peanut pods by A s p e r g i l l u s f l a v u s .

Phytopathology 61 (8 ); 1038-1059.

Book Binder, M.G, and Bloom, J . R , (1978). An i n t e r a c t i o n of

Uromyces phaseo l l and Meloidogyne Incogn i t a on bean.

Proceedings of the American Fhytopa tho log ica l S o c i e t y .

4: 183-184.

Book Binder, M.G. and Bloom, J .R . (1980). I n t e r a c t i o n of

Uromyces phaseo l l and M. Incogni ta on bean, J , Nematol.

12(5) : 177-182.

Bopaiah, B.M., P a t i l , R.B. and Reddy, D.D.R. ( l 9 7 6 ) . Ef fec t of

Meloidogyne navanica on nodu la t ion and s y n e r g i s t i c

n i t rogen f i x a t i o n in mung Vlgna r a d l a t a . Ind ian J . Nematol. ,

6 ( 2 ) : 12A-130.

Campos, V.P. and Schmitt , D.P. (1981). Nematode popu la t i on ,

soyabean emergence and developnent as a r e s u l t of s imu l ­

taneous i nocu l a t i on with H. g l y c i n e s and P, magasperma

F i topa to log ia B r a s i l a i r a . 6 ( 3 ) : 489-496.

5

Chahal, P.P.K. and Chahal, V.P.S. (1988). In te rac t ion of

Rhlzoblum and Meloido^ne IncoKnlta on symbiotic ni trogen

f ixat ion in mxingbean under d i f ferent concentrat ions of

iron. Plant nu t r i t i on . 2 I (6 -11) : 821-828.

Chahal, P.P.K, and Chahal, V.P.S. (1989). Effect of MeloidoRyne

incognita on leghaemoglobin bacteroids and nitrogenase

ac t iv i ty of nodules produced by FUiizobium spp, on

Mungbean (Vigna r a d i a t a ) . Pakistan J . Nematol., 2(1)»

21-25.

Chahal, P.P.K, and Chhabra, H.K. (1985). Effect of M. incognita

and Fusarium equise t i on grovrth characters and wi l t ing of

pea seedl ings. Journal of Research, Punjab Agr icul tura l

University, 22(4): 68A-686.

Costa Manso, E,S,B.G. and Haxmg, C.S, (1986), In te rac t ion

between Meloidogyne incognita (race 4) and i s o l a t e s of

Rhizoctonia solani on Biaseolus vulgar is CV. Rico 23.

Fitopatologia Bras i l^ i ra . r [ ( 4 ) : 857-864,

Dave, G.S. (1975). In te r re la t ionship od Rhizoctonia solani with

Heterodera glycines, Pratylenchus sc r ibne r i and

Xylenehorhynchus martini on d a r k 63 soyabean.

Disser tat ion Abstracts In te rna t iona l . 36B; 1991-1992.

Dhanger, D.S. and Gupta, D.C. (1985). Pathogenicity of

Meloidogyne .lavanica to chickpea (Cicer ar iet inum) in

re la t ion to so i l types, Rhizobium treatment, s ize of

pots and time in te rva l s . Indian J . Nematol.. 13 (2); 161-170.

58

Diomande, M, and Beute, M. K. (1S79). Influence of M. hapla and

and Macroposthonla omata on the effectiveness of

microsclerotia inocula of Cyllndrocladlum crotalariae in

causing black rot (CHl) on peanuts. Phytopathology

69(1 )il - A3

Diomande, M., Black, M.C., Beute, M.K.and Barker, K.R, (1981).

Enhancement of Cylindrocladiim crotalariae rot-root hy

Meloidogyne arenarja (race 2) on peanut cultivar

resistant to both pathogens. J, Nematol. 2^(3): 321-327.

Dmowska, E. (1982). Interaction of soil nematodes and fungi

and bacteria responsible for plant disease, (interakeje

podmiedzy nincieniami glebowymi a grazybami I bakteriami

powodujacymi choroby roslin), Wiadmosci Ekologiczne.

28(3/4): 219-226.

Dropkin, V.H. (1980). Interactions of nematodes and other

organisms in plant disease. In " Introduction to plant

Nematology" . John Wiley and Sons, New York. 217-230.

Edward, J.C. and Singh, K.P. (1979). Interaction between

H. cajani and Fusariun udum on pigeon pea, Allahabad

farmer. ( l ) : 23-24,

Fiske, C.H, and Subba Row, Y, (1925). The colorimeteric deter­

mination of phosphorous. J. Biol. Chem. 66; 575-400.

5b

Fortnum, B.A, and Lewis, S.A. (1978), I n t e r a c t i o n between

Cylindrocladium r o o t - r o t and po ly spec i f i c nematode

popu la t ion on soyabean, J . Ngmatol. J_0(A): 287.

Garcia , R. and M i t c h e l l , D.J . (1975). I n t e r a c t i o n of Phythium

myriotylum with seve ra l fungi and with M. a r e n a r i a i n

peanut pod r o t and pre-emergence damping off . Phvtopatho~

logy. 6 5 ( 7 ) : 832-333.

Goel, S.R. and Gupta, D.C. (l9e6a). I n t e r a c t i o n of M. .iavanica

and F.oxysporum f. c i c e r i on chickpea, Ind ian Phytopathology

^ ( 1 ) : 112-114.

Goel, S.R. and Gupta,G.C. (1986b). I n t e r a c t i o n of M. navanica

and Rhizoctonia b a t a t i c o l a on chickpea. Ind ian £ .

Nematol. J 6 ( 1 ) : 133-134.

Goswami, B.K. and Agarwal, D.K. ( l978) . I n t e r r e l a t i o n s h i p between

spec ie s of Fusarium and r o o t - k n o t nematode M* i n c o g n i t a on

soyabean. Nematologia Medi terranea. 6^(1): 125-128.

Gupta, D.C. and Yadav, B.S. ( l979) . S tud ies on p a t h o g e n i c i t y

of reniform nematode, Rotylenchulus r en i fo rmis t o xirad,

Vlgna mungo L. Wilczek. Indian J . Nematol. , 9 ( 1 ) : 4 8 - 5 0 .

Gupta, P . , Singh, K.P. and Edward, J .C . (1976). S t u d i e s on t h e

e f f e c t of some s o i l borne fungi on the development of

Heterodera vipcnl on cowpea. Indian J . Nematol. ^ ( 1 ) : 132-135.

B(J

Hasan, A. (1964). Synergism between Heterodera ca.ianl and

Fusarium udum a t t ack ing Cajanus ca jan . Nematologia

Mediterranea. ^ 2 ( 1 ) : 159-162.

Hussa in i , S.S. and Seshadr i , A.R, (1975). I n t e r r e l a t i o n s h i p

between Meloidogyne incop^nita and Rhizobium s p e c i e s on

mung bean, Fhaseolus aureus^ Indian o[» Nematol . . ^ ( 2 ) :

189-199.

Hussey, R.S, and Barker, K,R, ( l 9 7 4 ) . Effec t of nematodes with

d i f f e r e n t feeding hab i t s on nodu la t ion b a c t e r i a .

J . Nematol., 6 ( 4 ) : 143 ( A b s t r . ) .

Hussey, R.S. and Barker, K.R, (1976), Inf luence of nematodes

and l i g h t sources on growth and nodu la t ion of soyabean.

J . Nematol. 8 ( 1 ) : 84-52.

Hutton, D.G., Wilkinson, R.S. and Mai, W.F. (1973) . Ef fec t of

two p l a n t p a r a s i t i c nematodes on Fusarium dry r o o t - r o t

of beans. Phytopathology. 63 ; 6 ) : 749-751.

Ibrahim, I.K,A. and El-Saedy, M.A. ( l977) . P a t h o g e n i c i t y of

M. javanica and c e r t a i n fungi on p e a n u t s . Alexandria

Journal of Agr i cu l tu ra l Research. 2 4 ( 2 ) : 411-415,

Johansen, D.A. (1940). In " P lan t Micro tehanique" . McGraw-

H i l l Book Co. New York pp. 523.

6i

Kanwar, R.S.» Gupta, D.C. and Walia, K, K. ( l 9 8 7 ) . I n t e r a c t i o n

of M. javanica and Rhizoctonla s o l a n l on cowpea,

Nematologia Mediterranea. 1 5 ( 2 ) : 385-586.

Kanwar, R . S . , Gupta, D.C. and Walia, K. K. (1989). E f f ec t

of nematode i n o c u l a t i o n a t d i f f e r e n t i n t e r v a l s and s o i l

types on i n t e r a c t i o n between Keloidogyne .javanica and

Rhizoctonia s o l a n l on cowpea, Indian J . Nematol, 18 (1 ) :

112-113.

Khan, T.A, and Hussain, S . I . (1988). Ef fec t of i n d i v i d u a l ,

concomitant and s e q u e n t i a l i n o c u l a t i o n s of Rhlzoblum,

Rotylenchulus r en i fo rmis , M. Incogni ta and Rhizoc tonia

s o l a n l on cowpea p l a n t growth, d i s e a s e development and

nematode m u l t i p l i c a t i o n . Ind ian J . Nematol. 18 ( 2 ) ; 232-238.

Khan, T.A. and Hussain, S . I . (1989). R e l a t i v e r e s i s t a n c e of

s i x cowpea c u l t i v a r s a s a f f e c t e d by the concomitance of

two nematodes and a fungus. Nematologia Medi te r ranea .

17(1) : 39-41

Killebrew, J . F . , Roy, K.W., Lawrence, G.W., Mclean, K.S, and

Hodges, H.H. ( lS88) . Green house and f i e l d e v a l u a t i o n

of Fusarium s o l a n l pathogeneci ty t o soyabean s e e d l i n g s .

Plant P i s . 22 (12 ) : 1067-1070.

KujTiar, R., Ahmad, S. and Saxena, S.K, (1988). Disease complex i n

chickpea involving M. Incognita and Fusarium oxysporum.

I n t e r n a t i o n a l Nematology Network Newsle t t e r . 5 ( 3 ) : 12-14.

6 o

Lamberti , F, (1981). Combating nematode v e c t o r s of p l a n t v i r u s e s ,

P lan t P i s . 6^: 113-117.

Lehman, P . S , , Huisingh, D. and Barker, K.R. (1971). The

in f luence of races of Heterodera g lyc ines on n o d u l a t i o n

and nitix>gen f ix ing capac i ty of soyabean. Phytopathology,

61,(10): 1239-1244.

Lander, R.C. ( l 944 ) . Rapid a n a l y t i c a l methods fo r some of t h e

more common inorganic c o n s t i t u e n t s of p l a n t t i s s u e .

P l an t Phys io l . 1 9 : 76-89.

Malek, R.B. and Jenk ins , W.R. ( l 9 6 4 ) . Aspects of t he hos t

p a r a s i t e r e l a t i o n s h i p of nematodes and ha i ry v e t c h . Bu l l .

N. J . Agric . Bxp. S t a . No. 813 , P. 3 1 .

Mani, A. and S e t h i , C.L, (l9B7). I n t e r a c t i o n of r o o t - k n o t

nematode H. incogni ta with Fusarium oyysporum f. s p .

c i c e r i and Fusarium s o l a n i on chickpea. Ind ian J .

Nematol. 17(1) : 1-6.

M a r t e l l i , G.P, (1979). Nematode borne-v i rus of g rapev ine ,

t h e i r epidemiology and c o n t r o l , Nematol. Medi t . , 6 :

1-27.

M i l l e r , L . I . (1951). A r epo r t on the e f f ec t of e thy lene bromide

s o i l t r ea tment on roo t -kno t c o n t r o l , nodu la t ion and

y i e ld of pea. Va. J . Sc i . 2: 109-112.

6b

Moussa,S.M. and Haugue, N.G.M. Cl987). The in f luence of r o o t -

knot nematode M. incogni ta on the development of

F, oxysporum f. sp , g lyc ines in w i l t r e s i s t a n t and w i l t

s u s c e p t i b l e soyabean c u l t i v a r s , Mededelingen van de

F a c u l t e i t landbouwwetenschappen R i j k s s u n i v e r s i t e l t Gent.

22{2\>h 571-575.

Moussa, E.M. and Hague, N.G.M. (1988). Inf luence of Fusarium

oxysporum f, sp , g lyc ines on the invas ion and development

of r o o t - k n o t nematode. Revue de Nengttologie. 11 (4 ) ;437-439 .

Nath, R. and Dwivedi, R.P. (1981), Effect of r o o t - k n o t nematode

on development of gram caused by Fusarlum oxysporum f.

c l c e r l and r o o t - r o t by Rhlzoctonla spp . Ind ian Jo\ j rnal

of Mycology and P lan t Pathology. 11 ( 1 ) : 6 ^ 4 9 .

Norton, D.C. ( l978) . "Ecology of p l a n t p a r a s i t i c nematodes"

John Wiley and Sons, New York.

Oyekan, P.O. and Mi t che l l , J . S . (1971). Effect of P ra ty lenchus

penet rans on the r e s i s t a n c e of pea v a r i e t y t o Fusarium

w i l t . P I . P i s . Reptr . J5^(l1): 1032-1035.

P a d i l l a , C . , Lopez, R. and Vargas, E. ( l980) . I n t e r a c t i o n between

Meloidogyne spp. and Fusarium oxysporum f. p i s i on pea.

Agronomia Cos ta r r i cense . 4 ( 1 ) : 55-60.

P a t e l , H.R., Thakar, N.A., I ^ t e l , B. K. and P a t e l , C.C. (1967).

I n t e r a c t i o n between M.incognita . Fusarium oxysporum f. s p .

c iceJ ' i on chickpea v a r i e t y Chaffa. Indian J . Nematol. 17 (1 ) :

124.

64

Patel,H.R., Vaishnav, M.U. and Dhruj, I.U. (l985). Interaction

of M. arenarja and Fusarium solanl on groundnut. Indian

J. Nematol. 1^(1): 98-99.

Patel, H.R., Vaishnav, M.U. and Dhruj, I.U. (1986). Effect of

interaction between M. arenaria and Aspergillus f lav us

on groundnut, Indian Journal of Mycology and Plant

pathology. J6(2): 10>107.

Pitcher, R.S. (1978), Interaction of nematodes with other

pathogens. In " Plant Nematology" (Ed, J,F. Southey),

Tech. Bull, No. 7 Min. Agric. Fish and Food. H.M,S,0,

London, 63-77,

Powell, N,T. (1971). Interaction of plant parasitic nematodes

with other disease causing agents. In " Plant Parasitic

Nematodes" . Vol. II (Eds.- B.M. Zuckerman, W.F. Mai and

R.A, Rohde), Academic Press, New York, 119-136,

Proctor, M.H. (1963), A note on haemoglobin estimation. jj. .J.

Sci. 6: 60-63,

Raut, S.P. (1980). Effect of initial inoculum levels of

Meloidogyne incognita on plant growth and Rhizobial

nodulation of mungbean, Indian Phytopath.. 33 (2);351-353,

Raut, S.P. and Sethi, C.L. (1980). Studies on pathogenecity

of Meloidogyne incognita on soyabean. Indian J, Nematol.,

10(2): 166-174.

Reddy, P . P . , Singh, D. B. and Sharma, S.R. (1979) . I n t e r a c t i o n

of M. incogni ta and _R, s o l a n i i n r o o t - r o t d i s e a s e complex

of french bean. Indian Phytopathology. ^ ( 4 ) : 651-652.

fUbeiro, C.A.G. and Feraz , S. (1983). S tud ies on t h e i n t e r a c t i o n

between M. ^^avanica and Fusarium oxysporum f. s p . phaseo l i

on bean (Phaseolus v u l g a r i s ) . F i t o p a t o l o g i a B r a s i l e i r a .

8 ( 3 ) : 439-4A6.

Riker , A.J . and Riker , R,S. (1936), I n t r o d u c t i o n t o r e sea rch on

p l an t d i s e a s e . J o h n ' s Swift Co, I n c . , S t a . Louis ,Chicago

New York, Indiana p o l e s ,

Ross, J . P , (1969), E f fec t of Heterodera g l y c i n e s on t he y i e l d

of non nodula t ing soyabean grown a t v a r i o u s n i t r o g e n l e v e l s ,

J . Nematol.. 1.(1): 40-42.

Roy, K.W., Lawrence, G.W,, Hodges, H.H,, Mclean, K.S. and

Killebrew J . F . ( l989) . Sudden death syndrome of soyabean.

Fusarium s o l a n i as i n c i t a n t and r e l a t i o n of Heterodera

g lyc ines t o d i s ea se s e v e r i t y . Phytopathology. 79(2 ) ;191-197 .

Rushdi, M.H., Sellam, M.A., Abd-Elrazik, A., Allam, A,D. and

Salem,A. ( l980) . H i s t o l o g i c a l changes induced by

M. javanica and Fusarium s p e c i e s on r o o t s of s e l e c t e d

leguminous p l a n t s . Egyptian Jou rna l of Phytopathology.

12(1 /2) : 43-47.

66

Sakhuja, P. K. and S e t h i , C.L. (1986). M u l t i p l i c a t i o n of

M. .javanica as a f fec ted by Fusarium s o l a n i and Rhizoctonia

b a t a t i c o l a on groundnut. Indian J , Nematol. JI6_(1): 1-3.

Salam, M.A. and Khan, M.W, ( l986) . React ion of some c u l t i v a r s of

pigeon pea a g a i n s t Fusarium udum and M. j a v a n i c a .

I n t e r n a t i o n a l Nematology Network n e w s l e t t e r . 3 (A) :16-17 .

Sass , J . E . (1951). In " Botan ica l Mic ro - t echan ique" Iowa S t a t e

co l l ege P r e s s , Ames, Iowa, pp . 228,

Se inho r s t , J.W, and Kuniyasu, K. (1971). I n t e r a c t i o n of

Pratylenchus penet rans and Fusarium oxysporiam f, p i s i

race 2 and of Rotylenchulus r en i fo rmis and Fusarium

oxysporum f. p i s i race I on p e a s . Nematologica. 17 (3 ) ;

44A-452.

Sharma, N.K. and S e t h i , C.L. ( l 9 7 6 ) . I n t e r r e l a t i o n s h i p between

Meloidogyne i n c o g n i t a , Heterodera ca.iani and Rhizoctonia

s o l a n i and Rhizobium spec ies ^n cowpea Vigna s i n e n s i s L.

Savi . Indian J . Nematol. , 6 ( 2 ) : 117-123.

Sharma, R.D, and Cerkauskas, R.F. ( l965) . I n t e r a c t i o n between

M. javanica and F, oxysporum f. s p . c i c e r i on chickpea .

Nematologia B r a s i l e i r a . 9 ( l ) : 113-121.

Singh, D.B., Reddy, P .P . and Sharma, S.R. (1981 ) . Ef fec t of r o o t -

knot nematode M.incognita on Fusarium w i l t of french beans.

Indian J . Nematol. 1J_(1): 8 ^ 8 5 .

Singh, I . , Chahal, V . P . S . , Sakhuja, P.K, and Chohan,J .S . (1977) .

Effec t of d i f f e r e n t l e v e l s of Meloidogyne i n presence or

absence of Rhlzobium phaseo l i on Phaseolus a u r e u s . Indian

J . Nematol. X^^): 172-174.

Singh, R., Gupta, D.C. and Tfelia, K.K. (1986). S f f e c t of

Rhizoctonia s o l a n i c u l t u r e f i l t r a t e s on the hatching of

M. javanica l a r v a e . Indian Phytopathology. 39(4) :624-625 .

S r i v a s t a v a , A.K., Upadhyay, K.D. and Singh, G. (1974). Ef fec t of

roo t -kno t nematode M. i ncogn i t a on gram c rop . Ind ian J .

Nematol. , 4 ( 2 ) : 2/i8-251.

S r ivas t ava , A .S . , Upadhyay, K.D. and Singh, B.P. (1979). E f f ec t of

roo t -kno t nematode Meloidogyne .javanica on t h e growth of

soyabean. Glycine ma c. Ind ian J . Nematol., _9(l) : 38-40.

Stemerding, S. (1963), Sen mixe r^wa l l en f l i t e r methode on

Vri jbeweegl i jke e n d o p a r a s i t i a r e nematoden wi t wor le t s to

Verzamelen. V e r s l . P l z i e k t . Diens t . 141; 170-175.

Sumner, D.R,, and Minton, N.A. ( l 987 ) . I n t e r a c t i o n of Fusarium

wi l t and nematodes i n Cobb soyabean. P lant P i s . 71 (1 );

20-23.

Szerszen, J . (1980). Influenceof F. oxysporum f . s p . p i s i a lone

and in combination with PratylerKihus penet rans on peas .

Ekolozia Polksa. 28(4) : 615-631.

B8

Taha, A.H.Y. and Kassab, A.S. (1980). I n t e r r e l a t i o n s h i p between

Meloido^yne .javanica, Rotylenchulus ren i fo rmis and

Rhizobium s p e c i e s on Vigna s i n e n s i s . J . Nematol . , 12 (1 ) :

57-62.

Taha, A.H.Y. and Raski , D.J , (1969), I n t e r r e l a t i o n s h i p between

roo t -nodule b a c t e r i a , p l a n t p a r a s i t i c nematodes and t h e i r

leguminous h o s t . J . Nematol. 2^0): 201-211.

Taylor , D.P. and Sas se r , J.N. ( l978) . Biology, i d e n t i f i c a t i o n

and c o n t r o l of roo t -kno t nematodes (Meloidogyne spp , )

Coop. Pub. Pep. Plant P a t h o j . . North Carol ina S t a t e Univ.

and U.S. Agency I n t . Dev. Releigh, N.C. 111 P.

Thakar, N.A., P a t e l , H.R., P a t e l , B.K. and P a t e l , C.C. (1966) .

Breaking of r e s i s t a n c e t o Fusarium w i l t i n r e s i s t a n t

chickpea by roo t -kno t nematode. Madras A g r i c u l t u r a l

J o u r n a l . 7 1 ( 7 ) : 410-411.

T i y a ^ i , S.A., Za ld i , S .B . I , and Alam, M.M. (1988). I n t e r a c t i o n

between M. navanica and Macrophomina phaseol lna on l e n t i l .

Nematologia Medi terranea. ^ ( 2 ) : 221-222.

Upadhyay, K.D. and Dwivedi, B.K. (19873). Root-knot nematode

M. javanica breaks w i l t r e s i s t a n c e in chickpea v a r i e t y

Aurodhi. Current Science. ^ ( 1 7 ) : 915-916.

Upadhyay, K.D. and Dwivedi, B.K. ( l987b). Ef fec t of i n t e r a c t i o n

between M. javanica and Fusarium oxysporum f. sp . c i c e r i

on chickpea. Indian J . Nematol. J 2 ( l ) : 145-146.

RS

Varshpey, V . P . , Khan, A.M. and Saxena, S.K. (1987). I n t e r a c t i o n

between M. Incogn i t a , Rhizoc ten ia s o l a n i and Rhizobium

spec ies on cowpea. I n t e r n a t i o n a l Nematology Network

Newsle t te r . 4 ( 3 ) : 11-14.

Verdojo, S . , Green, C.D. and Fodder, A. K. (1969). In f luence of

Meloidogyne Incogni ta on nodula t ion and growth of pea and

black bean. Nematologica 3_4(1): 88-97.

Walla, K.K. and Gupta, D.C. ( l986a ) . Antagonism between

Heterodera c a j a n i and R, s o l a n i on cowpea. I n d i a n J .

Nematol. ^ ( 1 ) : 41-43.

Vfalia, K.K, and Gupta, D.C. ( l986b) . Effect of fungus R.

b a t a t i c o l a on the popula t ion development of pigeonpea c y s t

nematode, H. ca.lani on cowpea (Vigna u n g u i c u l a t a ) . Ind ian

J . Nematol. 1^(1): 131-132.