STUDIES ON PLANT PARASITIC NEMATODES AND OTHER MICROORGANISM ASSOCIATED

WITH LEGUMINOUS CROPS

Disgertatfon SUBMITTED FOR THE DEGREE OF

jUa^iter of ^IjtloiBiopii? IN

BOTANT

BY

MOHD. N4 FEES

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

1990

. ^ , . _ - t,^^-^^

••''^•V-:

I DS1785

D E D I D I C A T E D

TO MY

LOVING PARENTS

Dr. Mohd. Farooq Azam M.Sc., Ph. D.(Alig)

c g a t X f x c A t i

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH 202 001 (INDIA)

Dated

Thia is to 9W%itf '^t tilt vork tii)io4it4 In this

m> Oram wmtoomunm ASSOCXATBD vzza jMmmm3m enova** is a MnafU* and «riglfial vaatavali woHt aairltd out tgr

11^ MM* laJMS wdMr mf vvfifvriMim vaA is suitablt for

MMUttlOK t t A.ll»ir* All«aifi fiir tlio oonoidoratiOB of tbo

mmrd of tfao dogvit of Mtstvr of fbilaoop^ in Botaiqr.

^ L UK^L^

(Dr# IMid. ikupooq AiHi)

ACKNOWIEDGSMENTS

I was most fortunate t o have a highly imaginative,

enterpr is ing and accomodative research guide Dr. M. Farooq Azam,

Senior Nematologist, Department of Botany, A.M.U., Aligarh,

who inspired me t o i n i t i a t e the research work in tha field of

Plant Pathology, I deem pleasure i n expressing my deep sense

of grat i tude to him for suggesting a problem of v i t a l i n t e re s t ,

sk i l fu l guidence and sympathetic behaviour throughout the

course of work.

Thanks are due t o Prof. S, K, Saxena for his healthy

c r i t i c i sm and valuable suggest ion and for sparing his precious

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

I t gives me immense pleasure t o thank Prof, A, K,M,Ghouse,

Chairman, Department of Botany for providing a l l l ibrary and

laboratory f a c i l i t i e s .

I t would be quite un jus t i f i ed i f I don ' t extend my sincere

thanks to my senior research colleague Dr. Mansoor Ahmad SiddiquJ

and Dr, Syed Rais Haider for t h e i r help and encouragement,

I also acknowledge with g ra t i t ude the co-operation

accorded to me by my research colleagues and friends

M/S Saiyed Iqbal Husain, Mohd. F&rhan Farooqui, Zalieer Ahmad

Malik " Babur" , Ateeq Ahmad, Mohd, Rashid, Naseer H. Shah,

[u]

Ashraf Hameed Khan, Khawaja Mohd, As i f and Amir Haseeb, who

share t h e i r knowledge and o f fe red c o n s t a n t encouragement dur ing

the p repara t ion of t h i s d i s s e r t a t i o n .

F ina l ly , I beg t o e x p r e s s my g r a t i t u d e thanks , a p p r e c i a ­

t i o n and indebtedness t o t h e " ALMIGHTY GOD" fo r providing and

guid ing a l l the channels t o work i n cohes ion and coord ina t ion

t o make the study p o s s i b l e .

(HOtmT NACCES)

C O N T E N T S

1. Introduction 1 - 4

2 . Review of Literatxire 5 - 2 9

3. Plan of work 30-31

4. Materials and Methods 3 2 - 5 0

3* References 5 1 - 6 5

INTRDDUCTION

Leguminous pu l s e s occupy an important p o s i t i o n in human

dietary and are very good source of vegetable prote ins and

supplement to cerea l based d i e t . Their cu l t iva t ion i s world

wide frcwtt ancient t imes. Pulse crop have been the main-stay

of Indian Agricul ture, enabling land to turn out reasonable

qua l i t i e s of food g ra ins . They provide essen t ia l aminoacids

and they are a lso r e l a t ive ly r i ch in two essen t ia l micro-

nut r ien ts namely jCalcium and i ron . They have four times the

r ibof lavin and ten times the thiamin present in most of the

tarsals^ Generally included in ro ta t ion in most of the a reas ,

because of t h e i r a b i l i t y to keep s o i l al ive and productive.

Root nodules f ix and u t i l i z e atmospheric ni trogen and

contribute subs tan t i a l ly to s o i l f e r t i l i t y . Some legumes are

considered to be excel lant forage and grain concentrates in

the feed of large scale ca t t l e population of the country and

some of them are excel lant green manure crops, which add much

needed humus and major plant nu t r i en t s to s o i l . These pulse

crops are useful in improving the nitrogen s ta tus of s o i l ,

drawing out p lant nu t r ien ts from deeper so i l layers with the

help of the i r root system and making then available in the

form of plant res idues . They a lso provide in many cases a

protective ground cover against s o i l erosion.

Pulses as a whole have wide range of cl imatic requirements

and as such they are grown under varioxis cl imatic conditions

throughout the country. In India the area planted annually to

pulses have varied in recent years between 20-24 mil l ion hectares,

The annvial production i s about 12 mil l ion tonnes but our r equ i re ­

ment i s about 17 million tonnes indicat ing a deficiency of over

30 percent . The average production of pulses a t present i s

6-8 q/ha which i s very low in comparison to other countr ies of

the world.

Unfortunately, the pulse crop production suffer from

severe constrants of which pes ts and diseases have t h e i r

leading r o l e .

I t i s inherent in nature of a l l l iving organisms to

cooperate or compete with other when occupying a common niche

and special ly i f they have s imilar and overlapping food sources .

This i s a l l the more true for s o i l which i s a complex ecosystem

having a wide var ie ty of l i fe forms (including p lan t s and

animals). These organisms often develop symbiotic, synerg i s t i c

or antagonis t ic relat ionship amongst themselves, which could

primarily be because of nu t r i t iona l or spa t i a l competition.

The plant in one form or the other are the d i rec t and i nd i r ec t

source of food for consumers of a l l t ropic l eve l s , including

p lant pathogenic organisms. I t i s well known tha t c e r t a i n

fungi, bacteria and viruses are important pathogens and often

cause damage without being influenced by other b io t i c agents .

Plants are constantly exposed to numerous pathogenic organisms

many of which are common components of s o i l biosphere. The

nematodes, among the s o i l biota form a separate group and

const i tute a s igni f icant component (about 1296 of s o i l microflora

and fauna) of the s o i l ecosystem. We are usually unaware of

t he i r presence because of t he i r microscopic size and protect ive

posi t ion with the s o i l . Plant p a r a s i t i c nematodes are invariably

found in s o i l around the roots of p lan ts and may ac t as l imit ing

factor in the crop production. Plant p a r a s i t i c nematodes

themselves are quite capable of causing ser ious p lan t diseases

are independent of other microorganisms, but economic damage

often becomes more destinictive and high when they in t e r ac t with

other microorganisms,

A large number of phytophagus nematodes have been reported

from India around the roots of pulse crops. Recently Meloidogyne

spp, Telotylenchus spp, and Rotylenchulus reniformls have been

found as potent paras i tes causing p o t e n t i a l damage to pulses .

The damage i s reflected in the form of yellowing, s tunt ing , poor

plant growth and induction in the y i e ld . The e f fec t of nematode

i s variable in different pulse crops in India.

I t i s not r e a l i s t i c to assume tha t a plant although

infected with one pathogen wi l l not be affected by another. I t

seems much more appropriate tha t because a host i s infected by

one pathogen, i t s response to add i t iona l invaders wi l l be

altered. The alterations greatly influence the disease

development within a particular host andthe epidemiology of

the pathogen. The study of disease complex is as important

as the study of " monopathogenic" situation, because under

field condition there is probably no soil borne plant diseases

that can be said to be of monopathogenic origin. Nematodes

contribute to the disease complex by modifying the l hysiology

of the host plant and occassionally by mechanical affects they

exerts on the hosts.

Recently several workers have reviewed the work on

interactions of plant parasitic nematodes with other micro­

organisms (Powell, 1971; Norton, 1978; Pitcher, 1978; Martelli,

1979; Dropkin, 1980; Khan, 1981; lamberti, 1981 and 1982).

Although the involvement of disease complex situation

is quite frequent and widespred 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.

REVIEW OF LITERATURE

ROUS 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 NEMATODE-FUNGUS INTERACTIONS

1,1.1 Fusarium - Nematode Complexes

Atkinson as early as 1892 noticed that the infection

of iroot-knot nematode, Meloidogyne spp, considerably increased

the severity of Fusarium wilt in cotton. This is the first

report of an interaction between fungus and nematode, Singh

Si jai* (1561) reported that when seedlings of PhaSeolus

vulgaris inoculated simultaneously with M, Incognita and

Fusarium oxysporum. _F, solani or plants inoculated 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 (l985) reported that in combined

inoculations pea cultivar caused significantly greater reduc­

tion in growth than by M, incognita alone. When inoculated with

both the pathogens damage was more severe than with either of

the pathogen alone and plants wilted earlier in combination

than with F. e q u i s e t l a lone. Seedlings inoculated with

M. incognita alone wilted only temporarily a t noon while those

with combined inoculat ions fa i led t o recover. Thakar e t a l .

(1966) reported tha t Cicer arietinum plants l ine ICC-12275

( r e s i s t an t t o F, oxysporum f. c i c e r i ) showed no wi l t when

inoculated with fungus alone, but in presence of M. incognita ,

fungus caused 509 mortal i ty of p l an t s . Pate l e t a l . (1986)

reported an increase in the incidence of wi l t of chickpea

seedlings var . Chaffa when inoculated with 1000 larvae (I2) of

M. incogni ta /or 50 ml mycelial suspension (2g mycelial mat) of

Fusarium oxyspomjn f. c i c e r i .

Mani and Sethi (l967) conducted pot experiments with

10 days old p lants of chickpea (Cicer ar iet inum) cv. JG 62

inoculated with M. incognita a t 100 or 1000 larvae/500g, s o i l

and Fusarium oxysporum f, c i c e r i and F, so lan i a t 1,0 and 2.0g

mycelial mat respect ively e i t h e r singly or in combination. All

these organisms affected rh izob ia l nodulation. The occurrence

of M. incognita incombination with F, oxysporum f. c i c e r i and

F, solani not only increased the sever i ty of disease but a l so

shortened the incubation period for d i sease . Furthermore when

inoculat ion of nematodes preceded tha t of fungi by one week,

p lant drying due to infec t ion with F, so lani appeared ear ly

and root-knot index was a lso high. The development and mul t i ­

p l i c a t i o n of M. incognita was adversely affected by _F. solani

i r r e spec t ive of time and l eve l of inoculum. _F. oxysporum f.

c i c e r i did not af fec t the nematode population s ign i f i can t ly .

Padil la e t a l , (1980) reported t h a t the concomitant

inoculat ion of nematodes Meloidogyne spp. (M, incognita +

M. hapla) and Fusarium oxysporum f . p i s l a t p lant ing caused

death of pea p lan ts (C.V. l i t t l e Marvel) under green house

condi t ions . The inoculat ion of Meloidogyne species a t plant ing

caused a severe s tunt ing of the p l an t s , where as F, oxysporum

alone a t plant ing or 3 weeks l a t e r caused no detr imental

e f f e c t s .

Kumar e t a l . (1968) reported tha t inoculat ion of 15

days old seedlings of chickpea C.V. JG--62 with M« incognita

with 1000 J2 /p lan t or F, oxysporum f. c i c e r i with 1g mycelial

suspension/plant reduced p lant growth in pot experiments.

Highest reduction were obtained with simultaneous inoculat ion

of both pathogens, followed by the treatment in which nematode

inoculat ion preceded the fungus by 10 days and v ice-versa .

However, there was lower mul t ip l i ca t ion of 'nematodes and

ga l l ing in presence of the fungus.

Mousa and Pfeuge (1987) exposed Armosy (wil t suscept ib le)

and Coll (wil t r e s i s t a n t ) v a r i e t i e s of soyabean to Fusarium

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

a large increase in the amount of fungus in vascular t i s sues

of root and stem, compared with the amount found in plants

exposed to fungus alone. The fungus was i so la ted from leaves

and seeds of wi l t r e s i s t a n t c u l t i v a r s only when the nematode

was present .

8

Mousa and Hague further reported t h a t the fungal invasion

caused des t ruc t ion of g iant c e l l s when M, incognita and wi l t

fungus F. ox7Sporum f. glycines were inoculated on the growing

seedlings of soyabean c u l t i v a r s Ware and Coll ( r e s i s t a n t t o

fungus). The overa l l e f fec t was to reduce the number of

females of M, incognita and increase the proport ion of males.

Goswami and Agarwal (1978) conducted pot experiments to

determine the re la t ionsh ip between M, incognita and F.oxysporum.

F, so l an i . F, graminearum and F, egu i s e t i on soybean seedl ings .

The I n s u l t s showed an antagonis t ic i n t e r ac t i on of F, oxysporum

and F. solani with M. incognita« The fungus when es tabl ished

f i r s t suppressed nematode mul t ip l i ca t ion while the nematode

when es tabl ished f i r s t reduced the symptoms due to fungus.

The i n t e r ac t i on of _F, graminearum and F, e guise t i with

M, incognita was syne rg i s t i c , nematode infected plants damaged

by the fungus than non infected, Rushdi e t a l . (1980) studied

the h i s to log ica l changes caused by Meloidogyne .javanica and

Fusarium species in the roots of faba bean and cowpea.Presence

of e i t h e r of the fungus or the two fungi brought about optimal

reduction. Both pathogens caused much damage to plant t i s s u e .

Giant c e l l s induced by the nematode were a l so infected by the

hyphae and l o s t t h e i r cytoplasm,

Sakhuja and Sethi (1986) studied the e f fec t of M.javanica,

F. solani and R. ba ta t ico la on Arachis hypogea in 12 d i f fe ren t

combinations. I t was found t h a t both fungi reduced ga l l ing

significantly in all treatments, except where F, solani

succeeded nematodes after a week. The reduction in multi­

plication of M. .lavanica 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 Fusarium solani or

Rhjzoctenia solani. than when infected with nematode alone.

Nath and Dwivedi (1981) reported that Cicer arietinum

plants exhibited wilt symptoms earlier, when inoculated with

M, .javanica and F, oxvsporum f, cicerl or Meloidogyne and

Rhizoctonia than with either fungus alone. Inoculation of

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

wilt incidence, suggesting antagonism between the fungi,

Upadhyay and Dwivedi (1987) studied the effect of inoculation

of M, javanica alone or in combination with F, oxysponjm f.

ciceri on chickpea seedlings var. Arodhi, Plants inoculated

with fungus alone showed slight wilting but wilting was maximum

and rapid in plants where nematode inoculation preceded the

fxmgus. Upadhyay and Dwivedi (l987b) again conducted pot

experiment with 500 freshly hatched larvae of M. .lavanica/500g

soil and 3% (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

10

inoculated simultaneously with both M. javanlca and F, oxysporum

f, c i c e r i followed in sever i ty of inoculat ion of the nematode

preceding the fungus and the fungus preceding the nematode. The

maximum number of root-knot g a l l s (889) were recorded on roots

inoculated with nematode alone as compared to maximum number

(20) on roots where inoculat ion of the nematode preceded tha t

of the fungus. The g rea t e s t reduct ion i n nodules/plant were

observed with inoculat ions of nematode alone,

Salam and Khan (1986) reported tha t when seedlings of

Cajanus cajan were inoculated with 2g mycelium of F, udum and

500 2nd stage juveni les of M, javanlca, a l l c u l t i v a r s proved

suscept ible to a varying degree despite the facts cu l t i va r s

v i z , , Pusa-33f Pusa-85 and Pusa-7B were r e s i s t a n t and Pusa-8A

was t o l e r an t t o M, javanica. The nematode suppressed nodulation

on to l e ran t and susceptible c u l t i v a r s . Suscep t ib i l i t y of a l l

cu l t i va r s to F, udum increased when nematode and fungus were

present together . Highly suscept ible c u l t i v a r s wilted sooner

in presence of both pathogens than when inoculated with F. udum

alone. The two pathogens appear to ac t syne rg i s t i ca l l y on

C. cajan though there i s no previous repor t of such in t e r ac t ion

on t h i s crop, Ribeiro and Peraz (1983) reported t h a t suscep t i ­

b i l i t y of c u l t i v a r s to the fungus was higher on inoculat ion with

fungus + nematode. Vegetative growth and conid ia l production

by the fungus were g rea te r in the media containing ex t rac t s

from bean p lan ts infested with M. javanica than in those with

ex t r ac t s from healthy bean p l an t s .

11

Sharma and Cerkauskas (1985) reported t h a t when 3 weeks

old seedlings of Clcer arietinum were inoculated with 1000

M. incognita eggs/kg s o i l or 1 ml suspension of mycelia and

conidia of F, oxvsporum f, c i c e r i . the reduction in plant

height was 1,189^ with fungus alone 17,53 percent with nematode

alone and 19,477 with fungus + nematode a f t e r 90 days of

inocula t ion. Reduction in fresh root weight was 3.78%, 30,61

and 49,3096 respec t ive ly . Combined infect ions caused damage to

p l an t s , Goel and Gupta (1986) reported tha t the growth of

Clcer arietinum was reduced when both organisms were present

together i r respec t ive whether they were inoculated simultaneously

or with one week i n t e r v a l . Reduction in various growth para­

meters was noticed when the nematode was inoculated 30 days

p r io r to the fungus.

Pate l e_t a l . (1965) reported tha t increasing the inoculum

leve l of F» solani to 2g or 4g mycelial mat/pot decreased the

days required for wil t ing in groundnut (Arachis hypogea). When

F. solani was inoculated with 1000 or 2000 larvae of M,

a renar ia , the wil t ing was further reduced,

Edward and Singh (1979) reported tha t when pigeon pea

var ie ty type 21 was inoculated with H. ca.jani {,50 cys t s /po t )

and Fusarium udum, Heterodera alone caused less damage than

when associated with F, udum. Transverse sect ions of the roots

inoculated with the nematode and fungus showed tha t only old

syncyt ia l or non syncyt ia l regions were invaded by the fungus.

12

Killbrew e t ^ l . (1988) reported tha t r oo t - ro t symptoms

were most prevalent in presence of fungus. Five Fusarium solani

i so l a t e s tes ted were pathogenic when inoculated on soyabean and

each was re i so la ted from inoculated symptomatic p l an t s . I so la t e s

differed in v i ru lence . Disease sever i ty was not subs tan t i a l ly

changed when F, solani and soyabean cyst nematode, H. glycines

wei^ inoculated on soyabean in combination as compared with

F, solani or soyabean cyst nematode alone, the fungus reduced

y ie ld under f ie ld condi t ions . Disease sever i ty was g rea te r with

poor qual i ty of seeds inoculated with F, so lani than with inocu­

la ted with high qual i ty seeds, Roy e t a l . (lSe9) i so la ted two

morphologically d i s t i n c t forms of F, so lani designated FS-A

and FS-B ft'om soyabean p lants with symptoms of sudden death

syndrome. Combined inoculat ion of soyabean with FS-A and

soyabean cyst nematode (H. g lycines) caused more severe f o l i a r

symptoms than those caused by FS-A alone.

Hutton £ t £ l . (1973) reported tha t percentage of dry

roo t - ro t in kidney bean was grea te r when p lants were inoculated

with Pratylenchuus penetrans or mixture of I^eloidogyne spp.

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

p l an t s . At high leve ls of fungal inoculum a l l p lants whether

nematode free or nematode infected become infected by the fungus

(Fusarium solani f. phaseo l i ) .

Oyekan and Mitchell (1971 ) studied the ef fec t of

P, penetrans on the res i s tance of pea var . Wisconsin Fterfection

of wi l t disease caused by F. oxvsporum f. p i s i race 1 in

13

controlled growth chamber experiment. Plants growing in soil

inociilated 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,

Szerszen (1980) studied the effect of Pratylenchus

penetrans on the course of Fusarium wilt of peas in glass house

experiments using two different soil types and two varieties of

pea differing in their susceptibility to Fusarium. The presence

of nematode increased the severity of wilt symptoms, especially

in light soils. The resistance of var, Alaska Ekaspres was

partially broken by the addition of nematodes. In further

experiments wilt disease in the susceptible var, Szlachetna

•Perla was more severe following prolonged exposure of P.

penetrans than often only a short exposure period with Alaska

Ekspres. The course of disease was the same after both long

and short exposure periods.

Sumner and Minton (1987) conducted experiments with three

field soils internal grey to black and showed that stem dis­

colouration and inverveinal chlorosis and necrosis of leaves

in Cobb var, of soyabean was most severe in soils infested with

F, oxysporum f, tracheiphilum race 1, Belonolaimus longicaudatus

1A

and Pratylenchus brachyurus. Wounding roots with a knife 18-20

days a f t e r plant ing did not increase wi l t or i n t e rna l stem

discoloura t ion.

Hasan (1969) reported tha t in pot experiments mixed

inoculat ions of Fusarium udum with Tylenchorhynchus vu lga r i s ,

Helicotylenchus indieus or Hoplolaimus indieus e i t h e r simul­

taneously or sequent ia l ly gave no s ign i f i can t difference in

wi l t development of C, can an. When Heterodera ca.1ani was

associated with fungus, there was s ign i f i can t increase in wil t ing

(93.69& p l a n t s ) , occuring when seedlings were inoculated with

H, cajanl 3 weeks p r io r to the fungus inocula t ion,

1,1,2 Rhlzoctonla-nematode Complexes :

Reddy e t a l . (1979) reported tha t the inoculat ion of

PhasePlus vulgar i s with M. incognita alone, simultaneously with

R, solanl or 10 days p r io r to fungal in fec t ion , reduced plant

height and fresh weight and gave maximum root-knot index.

Simultaneous inoculat ion of both pathogens caused grea te r

damage than e i t h e r organism act ing alone.

Varshney e t a_l. (1987) reported tha t combined inoculat ions

of seedlings of Vigna unguiculata CV, Russian with M. incognita

and Rhizoctonia so lan! caused a s ign i f i can t reduction in the

nodulation due to Rhizobium.

15

Costa and Haung (1986) observed in pot t ^ J l s using

d i f ferent sequences of i n f e s t a t i on ty the nematode and the

fungus, no synerg is t ic pathological e f fec ts were observed.

Root branching appeared to be stimulated by the root-knot

nematode, M, incognita and growth of infested plants to be

improved suggesting some protec t ion against Rhizoctonia so l an i ,

Goel and Gupta (1986) studied the e f fec t of M. javanica

and R, ba ta t ico la alone and in combination on chickpea seedl ings .

He found tha t combined inoculat ion i r respec t ive of time,

reduced growth parameter, compared with when inoculated alone.

Kanwar ejb al.. (1567) reported tha t when seedlings of

cowpea (Vigna unguiculata) were inoculated with 10CX) Juveniles

of M. t')avanica and 2g mycelial mat of R, so l an i . the plant

shoot and root length and dry weight were decreased as compared

to the uninfested con t ro l s . Plant growth was be t t e r in sandy

loam which supported g rea tes t number of nodules than in sand

and sandy loam, Ffewer g a l l s were formed on p lan ts inoculated

simultaneously with both organisms than on than on those infested

with nematode alone.

Singh e_t a l . (lS66) reported tha t in combined infect ions

of cowpea (Vigna unguiculata) the nematode population

(M. .iavanica) was suppressed in the presence of R, so lan i .

Culture f i l t e r a t e s of the fungus s ign i f i can t ly reduced hatching

of la rvae .

16

Kanwar e^ al., (1989) reported s ign i f i can t reduction in

the growth of cowpea (Vigna unguiculata) CV. HPC 42-1, when 1000

juveni les of M, .lavanica were inoculated with 3 weeks p r io r to

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

of nematode g a l l s were s ign i f i can t ly decreased in presence of

the fungus, and ga l l s were minimiM when both pathogens were

inoculated simultaneously, The number of bac t e r i a l nodules were

a l so s ign i f i can t ly reduced in presence of both pathogens, with

the g rea te s t reduction in nematode only. Sandy loam s o i l was

more favourable for plant growth than sand or sandy loam s o i l

(greater sand content s o i l ) .

Khan and Hussain (1968) reported tha t indiv idual ly ,

Rhizoctonia solani was the most aggressive pathogen of cowpea

followed by M, lncojB:nita and R, reniformis. while 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, solani with

M, incognita caused d i s t i n c t l y g rea te r plant growth reduction

than i t s assoc ia t ion with R, reniformis. I r respec t ive of the

pathogen, inoculated unbacterized plants suffered grea ter

damage than the bacterized ones. Inoculation of Rhizobium

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

combinations resul ted in s ign i f i can t ly reduced plant growth,

poor nematode mul t ip l i ca t ion , low root-knot index and improved

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

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

in p lant growth reduction of bacterized plants whether simul­

taneously inoculated with e i t h e r nematode speices and the fungus

17

or when the nematode inoculat ion preceded fungus, but on the

other hand there was s ign i f i can t ly l ess reduction in plant

growth when fungus preceded nematode inocula t ion. In sequent ia l

inoculations in te rac t ions were time dependent. Rhizoctonia

solanl whether inoculated simultaneously or sequent ia l ly reduced

the ra te of mul t ip l i ca t ion of both the nematode species as

compared t o t h e i r r a t e of mul t ip l i ca t ion when present alone.

Khan and Hussain again in 1989 reported tha t the cowpea Vi^na

unguiculata (CV r e s i s t a n t or moderately r e s i s t a n t against

individual pathogens), l o s t t h e i r res i s tance in the presence of

other pathogens, except CV. IC-5O3 (moderately r e s i s t a n t to

M, incogni ta) CV-2-A88 ( r e s i s t an t to Rotylenchulus reni fonnis)

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 res i s tance in the presence of R. jsolani.

Similarly the res is tance of CV. CO-4 to R, so lani broke down in

the presence of M. incognita but not in the presence of

R, reniformis. Both R. renifonnis and M. incognita when present

in assoc ia t ion with R. so lani broke the res i s tance of CV, IC-244

against R« so l an i . The moderately r e s i s t a n t response of CV.

RC-8 and £C-4213 A to R. reniformis and t h e i r complete res i s tance

against R. so lani broke down when simultaneously inoculated with

both the nematode spp. or R. so lani with e i t h e r R. reniformis

or M, incogni ta .

Walia and Gupta (l986a) reported tha t ne i ther H. ca.iani

nor R. so lani had any ef fec t on the growth of Vigna unguiculata

when inoculated separa te ly . When R. so lani was inoculated one

18

week p r io r to H. ca.ianl there was a s ign i f i can t reduction in

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

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

reduction in the top growth of p lan ts compared with controls

or any other t reatment . Walia and Gupta (lSe6b) again

observed the inh ib i t ion of mul t ip l i ca t ion of H, ca.jani r e s u l t ­

ing in t o t a l absence of cys t s , when R, ba ta t i co la was inoculated

7 days p r io r to nematode in Vigna unguiculata (cowpea seedl ings) .

Dave (1975) reported t h a t damage caused by R. so lani to

soyabean in. presence of nematodes was not more than add i t i ve .

R, so lani inhibi ted nematode population development, however,

H. gjlycine was the most suppressed and Tylenchorhynchus mart ini

the l e a s t .

1 .1 ,3 Macrophomina - nematode complexes :

Agarwal and Goswami (1974) reported t h a t in pot exper i ­

ments, when seedlings of soyabean va r ie ty Bragy were inoculated

with M. incognita and/or the fungus Macrophomina phaseolina.

The g r e a t e s t reductions in the seedlings root and shoot weight

and highest mortal i ty r a t e (30^) were seen in p lan ts inoculated

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

severe symptoms and elevated morta l i ty ra te (259 ) were also

seen in p lan ts inoculated with the two pathogens simultaneously,

compared with when the nematode followed the fungus by 3 weeks

or e i t h e r pathogens was inoculated alone. Al-Hazmi (19B5)

19

reported t h a t in greenhouse experiments sever i ty of Macrophomina

roo t - 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 alone. Nematode infect ion and

reproduction was reduced when the fungus was introduced f i r s t .

Inoculation with e i t h e r pathogen or with both generally reduced

root weight in both t es ted c u l t i v a r s and s ign i f i can t ly reduced

pod weight in the c u l t i v a r Romano I t a l i a n but not in CV

Harvester. Harvester was more to le ran t of both pathogens than

Romano I t a l i a n .

Tiyagi £ t a l . (1S68) reported tha t combined inocula t ion

of M, javanica and M. phaseollna caused s ign i f i can t reduction

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

c u l i n a r l s ) CV I J -912 in pot experiments. This daonaging e f fec t

was more pronounced in simultaneous than in sequent ia l inociila-

t ion of the pathogens a t an in t e rva l of 10 days. There was a

negative in t e rac t ing co r re l a t ion between the organisms, with

the fungus being inhib i tory to the nematode, espec ia l ly when

inoculated 10 days previously.

1.1,4 Cylindrocladlum-Nematode Complexes :

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

of black r o t caused by Cylindrocladium c r o t a l a r i a e on NC 3033

and Elor igiant peanut c u l t i v a r s increased in presence of 1,000

and 10,000 M, hapla eggs/pot , Macroposthonla ornata increased

disease sever i ty of 10,000 larvae with 0,25 and 2,5

20

microsclerot ia per pot on Flor ig iant but did not a f fec t the

disease on NC 3033. Diomande et_ a l . (1981) again reported

tha t in USA 2 populations of M, arenar ia (race 2 incompatible

on peanut) enhanced development of Cyllndrocladium black r o t

(CBR) on CBR 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 (O, 10^, 10^

eggs/15 cm pot) were tes ted in a l l combinations with C,

c ro t a l a r l ae (O, 0.5, 5, 50) microsclerotia/cm^ s o i l . Root-rot

index increased in presence of e i t h e r populat ion. Inoculum

density disease curve were changed by both populat ions, ind ica t ­

ing increased efficiency of microclerot ia when peanuts were

grown in presence of these nematodes. Although l i t t l e or no

reproduction occured with e i t h e r population on NC 3033, larvae

of 256 and A86 penetrated r o o t s . 256 did not induce root g a l l s

and was not successful in es tab l i sh ing feeding s i t e s , while

U86 produced a few small e l i p t i c a l g a l l s .

Fortnum and Lewis (l978) studied the e f fec t of

M. incognita . Hoplolaimus columbu_s« Pratvlenchus sc r ibne r i and

Cylindrocladium c ro t a l a r i ae singly or in combination to

soyabean, Cylindrocladium c ro ta l a r i ae was applied a f t e r 14

days sampling. Heterodera columbus remain unaffected by other

organism but number of Juveniles in the s o i l were la rger in

presence of C. c r o t a l a r i a e . P. s c r ibne r i population was

s ign i f i can t ly lower a f t e r 75 days in presence of H. columbus

and C, c ro ta la r iae^ Meloidogyne incognita g a l l index was

depressed by the presence of H, columbus or C, c r o t a l a r i a e .

21

1,1.5 Other Fungal Diseases.and Nematodes :

Bell e t a l . (1971 ) studied the e f fec t of A. flavus and

M. arenar la seperately and together on peanuts (Arachls

hvpogea). I t was observed tha t damage by M. arenarla did not

affect the incidence of A, f lavus. t o t a l fungi or af lotoxin

contamination,

Patel e^ a l . (1966) reported tha t combined infect ions

of Meloidogyne arenaria and Aspergillus flavus were synerg is t i c

in reducing shoot growth of ground nut plant when an inoculum

of Ug fungal mycelium and 1000-2000 larvae of nematode were

used. There were fewer g a l l s / p l a n t and the f ina l population

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

Gupta £ t a l . (1976) studied the e f fec t of cyst nematode

(H, v i g l n l ) and 7 fungal species on cowpea alone or in combi­

nation, in r e l a t i o n to root growth and nematode population.

I t was observed tha t fungi reduced the nematode in fes t a t ion ,

the g rea te s t reduction being with Penicill ium citrinum and

l e a s t with Aspergil lus t e r r e u s . The average root weight/plant

was s ign i f i can t ly reduced when fungus was present in d i r ec t

proportion to the number of nematodes in the roo t s . Difference

in sex r a t i o in the presence of fungi was a lso noted.

Adeniji e_t a l . (1975) found the r e l a t ionsh ip of

Heterodera glycines and Phytopthora megaspenna f. sojae in

soyabean (Glycine max)« Seedlings disease was more severe in

22

the cultivars Corosoy and Dyer (both susceptible to P.

megasperma) when both fungus and nematode were present than

in presence of fungus alone, Harosoy 63 (resistant to

P, megasperma failed to develop symptom in presence of both

organisms. Infection with P. megasperma significantly

suppressed the H. glycines population on roots of Corosoy but

did not break the resistance of Dyer to the nematode,

Campos and Schmitt (1988) studied the effect of

P. megaspoerma f, so.jae (race 1) and/or H. glycines on the

soyabean cultivar Essex (slightly resistant to P. megasperma

f, sojae) and susceptible to (H. glycines), Forrest

(moderately resistant to Fhytopthora and resistant to Heterodera),

They found 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) reported that infection of

M. incognita and Uromyces phaseoli reduced bean's fresh weight

of shoot and root to a larger extent than infection by pathogen

alone, Sporulation and egg formation were reduced on hosts

infected with both pathogens. Gall formation was reduced only

when U, phaseoli was applied several days before inoculation

with M, incognita on the cultivar most susceptible to

U. phaseoli. The presence of U. phaseoli did not affect

23

h a t c h i n g . Book Binder and Bloom (1980) aga in repor ted the

r e d u c t i o n i n growth of b e a n ' s shoot and r o o t weights when

Melo ido^ne i n c o g n i t a and Uromyces p h a s e o l i were p r e s e n t .

The shoot weight was suppressed by 7 , 9 and 159 and roo t weight

by 8, 16 and 239^ r e s p e c t i v e l y , sugges t ing t h a t t h e r e was an

a d d i t i v e e f f e c t . Both the pathogens inf luenced the r e p r o d u c t i o n

of an ano the r fungal u r ed i a were reduced i n s i z e and s p o r u l a -

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

fewer eggs/egg mass.

Garcia and M i t c h e l l (1975) r epo r t ed inc reased s e v e r i t y

of pod r o t of peanut when pods were exposed t o s o i l con t a in ing

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

M, a r e n a r i a than when pods were exposed t o P. myriotylum a l o n e .

1.2 NEMATODE BOOT NODUIJS BACTERIA INTERACTIONS

M i l l e r (1951) was the f i r s t t o obseirve i n h i b i t i o n of

nodu la t ion on p l a n t r o o t s i n 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 o the r workers have a l s o r epo r t ed t h a t

r o o t - k n o t nematode caused r educ t i on in nodu la t i on of leguminous

p l a n t s , e . g . Meloidogyne hapla on h a i r y ve tch (Malik and

J e n k i n s , 1964) and or soyabean (Balasubramanian, 1971)

M. j a v a n i c a in white c l o v e r (Taha and Raski , 1969), on soyabean

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

e t a l . . 1976). M. i n c o g n i t a on mung bean, Phaseolus aureus

24

(Hussainl and Seshadri, 1975) and on soyabean (Balasubramanian,

1971; Srlvastava e t a l . , 1974, Hussey and Barker, 1974, 1S76,

Baldwin e t a l . , 1975). Hussaini and Seshadri (1975) observed

tha t M, incognita was pathogenic to raung bean/green gram

(PhasePlus aureus) and hampered nitrogen f ixa t ion . The nematode

caused s ign i f i can t decrease in weight of p lants (fx^esh and dry

weight of roots and shoot) . Reduction in ni t rogen content was

considered by them to be due to the overa l l reduction in root

nodulation, anatomical changes in nodules and a l t e red

physiology of the host.

Jamal (1976), while working with M. incognita and

Rhizobiuro phaseoli on Vigna aureus found t h a t Rhizobium

inoculat ion mitigated the i l l e f fec t of root-knot nematode.

Giant c e l l s produced on nodular t i s sues contained few females,

however, such noduler t i s s u e s had few bac te r iods . Further

nematode inoculat ion resul ted in s ign i f i can t increase in

leghaemoglobin. These s tudies led Jamal (1976) to conclude

tha t nematode infect ion did not adversely af fect N fixing

capaci ty.

All e t _al. (1981) studied the antagonis t ic i n t e rac t ion

between M. incognita and Rhizobium legxjminosarum on cowpea.

They observed tha t M. Incognita reduced nodulation and i n h i b i ­

t ion of ni trogen f ixat ion by about 63% in the nodular t i s s u e .

Infected nodules contained d i f fe ren t developmental stages of

25

the nematodes but the nematode did not a l t e r the s t ructure of

nodules. However, infected nodules de te r io ra ted e a r l i e r than

the uninfected ones.

Pfeut 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 t ha t mung bean inoculated

with Meloidogyne Incognita showed gradual reduction in growth

with an increase in i n i t i a l inoculum l eve l . The nitrogen

fixing capacity was a lso affected in mungbean var ie ty

" PIMS-I" ,

Singh e t al, (1977) reported tha t there was a correspond­

ing decrease in chlorophyll content, number of nodules and

nitrogen content of gram with an increase in the inoculum leve l

of Meloidogyne incognita both in presence and absence of

Rhizobium phaseol i . The nematode g a l l s were observed on nodules

when inoculum level of the nematode was 10(X)/pot. Finally they

said tha t M. incognita adversely affected the symbiolic process

of ni trogen f ixa t ion by Rhizobium phaseoli on mung (P. aureus) .

Chahal and Chahal (1989) again reported tha t M. incognita

reduced the leghaemoglobin and bacteroid content of Rhizobium

nodules of mungbean (Vigna r a d i a t e ) CV. G-65 i r respec t ive of

i n i t i a l l eve l of inoculum (250, 500, 1000 or 2000 2nd stage

juven i les / seed l ings ) thus adversely affect ing the functioning

26

of nodules, Nitrogenase activity of nodules decreased with

an increase in the initial level of inoculum.

Verde Jo et al. (1989) 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.

Effective nodules have more nitrogenase activity per gm on plants

with M, incognita than on those without nematodes, Meloidogyne

Incognita increased the leg haemoglobin 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.

Sharma 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. cajani. Root examination showed

that both nematode species could thrive well and complete their

life cycle on the nodular tissue.

27

Srivastava e t a l . (1979) reported tha t the growth of

soyabean Glycine max was affected when the root-knot nematode

M. javanlca was inoculated a t d i f fe ren t inoculum l e v e l s .

Number of nodules per plant were reduced s ign i f i can t ly even at

low inoculum leve l (10 larvae/kg s o i l ) . When the inoculum

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

formation was a l so affected by increasing the inoculum l eve l ,

Bopaiah ejb a l . (1976) reported tha t inoculat ion of

Vigna rad ia ta (mung) with M. javanica had de le te r ious ef fect

on plant growth and t h i s could be observed when inoculated with

nematode alone, simultaneously or 2-7 days preceding to

Rhizobium inocula t ion. The nematode inoculat ion p r io r to

Rhizobium resu l ted in maximum reduction of nodules. The

in fe s t a t ion by the nematode in ter fered with ni trogen f ixa t ion

and reduced the ni trogen content of the p l an t .

Dhanger and Gupta (1983) observed the pathogenecity of

Meloidogyne .javanica to chickpea (Cjcer ar iet inum) in r e l a t i o n

to s o i l types, Rhizobium treatments , s ize of pots and time

i n t e r v a l s . They found pathogenicity in a l l three types of s o i l

and a t d i f fe ren t time i n t e r v a l s . Similarly Rhizobium tes ted

seedlings showed be t t e r growth characters than untreated seeds.

Taha and Kassab (1980) observed the ef fec t of s imulta­

neous inoculat ion of M. javanica and Rotylenchulus reniformis

with Rhizobium on Vigna s inensis and indicated tha t t h e i r

associa t ion did not affect nodulation. Nodule formation was

28

hindered only when R, renlformis preceded rh izobla l inoculat ion.

Nodules were formed on Meloidogyne ^javanica g a l l s .

Ross (1969) reported t h a t Heterodera glycines affected

the yie ld of non-nodulating soyabean a t d i f f e ren t nitrogen

levels and he found tha t besides reducing nodulation and

nitrogen f ixa t ion , H. glycines caused reduction in yield

inc i t i ng deter ious host responses tha t increased with ni trogen

deficiency,

Lehman e t a_l. (1971) examined three races of Heterodera

glycines ( l , 2 and 4) and found tha t in combination of race 1

of H, glycines plus Rhizobium japonicum there were fewer nodules

per gram of root t i s sue and N-fixing capacity was less than

with R. japonicum alone. Similar inoculum leve ls (100, 200

and 400 crushed cys t ) of race 2 and 4 had no e f f ec t . Nodule

number and weight were inversely proport ional to the increasing

dens i t i e s of race 1.

Barker £ t ^ . (1971 ) reported tha t appl ica t ion of

ni trogen (N) as well as Inoculat ion of soyabean with Rhizobium

japonicum gave an adverse e f fec t on the a c t i v i t y of Heterodera

g lycines . The appl ica t ion of NaNO, or NH to s o i l reduced

nematode hatch, penet ra t ion and cyst development. Simultaneous

inoculat ion of nodulating and non nodulating isogenic l ines of

soyabean with Rhizobium and H, glycine reduced the number of

cyst development espec ia l ly on the nodulating l i n e s .

29

Barker e t a l . (1972) again observed su f f i c ien t i nh ib i t ion

of nodules upon the simultaneous inoculat ion of H. glycines and

Rhizobiuro japonicum on soyabean. However, 1A days delay of

H,glycines resul ted in only s l i g h t to moderate inh ib i t ion of

nodulation,

Gupta and Yadav (1979) studied the pathogenecity of

reniform nematode, Rotylenchulus reniformis to Urd, Vigna mungo

and found s ign i f ican t reduction in plant height and shoot, root

weights, Nodulation was a l so affected.

Most of the inves t iga t ions have reported suppression or

inh ib i t ion of nodulation by plant p a r a s i t i c nematodes. However,

s t imulat ion of nodulation on leguminous plants by pathogenic

nematodes was reported by Hussay and Barker (1976). They

studied the influence of I>feloidogyne hap l a , Pratylenchxxs

penetrans and Belonolaimus longicaudatus on nodulation of

soyabean. M, hapla and P, penetrans st imulated nodule formation,

while Belonolaimus longicaudatus s l i g h t l y inhibi ted the

nodulation.

30

Future Plan of Work

The above literature shows that in the case of

leguminous crops much attention has been given to the interac­

tions between nematodes - fungi and nematodes - root nodule

bacteria separately, though these microorganisms inhibit a

common niche. Therefore it was considered desirable to

investigate the effect of plant parasitic nematodes, fungi

root nodule bacteria on some leguminous plants.

The following experiments have been planned for detailed

study :-

1. Effect of different inoculum levels of reniform nematode,

Rotylenchulus reniformis and wilt fungus, Fusarium solani

alone and in combination on the nematode population wilt

development and plant growth of french bean (P, vulgaris)

and mung bean (P. aureus) in the presence and absence of

Rhizobium.

2. Histological changes induced by R, reniformis and R, solani

alone and in combination on french bean (P, vulgaris) and

mung bean (P, aureus) in the presence and absence of

Rhizobium,

3. Effect of R. reniformis and _F. solani alone and in combina­

tion on leghaemoglobin bacteroids produced by Rhizobium on

french bean (P, vulgaris) and mung bean (P. aureus).

31

A, Sffect of carbofuran (nematlcide) and/or Bavist in (Fungicide)

on leghaemoglobin bacteroids produced by Rhlzobium on french

bean (P, vu lga r i s ) and mung bean (P, aureus) in the presence

of R. reniformis and F, s o l a n i .

5, Effect of d i f fe ren t r a t i o s of sand and clay on the population

of reniform nematode wi l t and nodulation on french bean

(p. vu lga r i s ) and mung bean (P, aureus) .

6, Effect of d i f fe ren t sources and leve ls of nitrogenous

f e r t i l i z e r s on the development of nematode, wi l t and

nodulation on french bean (P, vu lga r i s ) and mung bean

(p. aureus) .

7, Effect of R, reniformis and F, so lani alone and in combina­

t i on on NPK contents in french bean (P. vu lga r i s ) and

mung bean (P, aurevts) in "the presence and absence of

Rhizobium.

M/ TERIALS AND METHODS

The d i f f e r e n t m a t e r i a l s t o be used and the methods t o

be employed dur ing the course of proposed expe r imen ta l

programme are g e n e r a l used a s fo l lows :

2.1 S e l e c t i o n of t e s t mater ia l s ;

In the proposed p l an of work the reni form nematode,

Rotylenchulus r en i fo rmi s and the w i l t fungus, Fusarium

s o l a n i , have been s e l e c t e d which w i l l serve a s t e s t pathogens

fo r the s t u d i e s , French bean. Phaseolus v u l g a r i s L. w i l l be

used as a t e s t p l a n t dur ing w in t e r season and green gram/

mung bean Phase olus aureus Roxb. dur ing summer season .

Respect ive r h i z o b i a of these t e s t p l a n t s w i l l a l s o be used as

and when r e q u i r e d ,

2 .2 Preparation of the inoculum of fungi ;

Pure c u l t u r e s of the w i l t fungus, Fusarium s o l a n i w i l l

be maintained i n the c u l t u r e tubes con ta in ing p o t a t o dex t rose

aga r (PDA) which w i l l be prepared from the fol lowing c o n s t i ­

t u e n t s .

Po t a to 200.00 g

Dextrose 20,00 g

Agar 20,00 g

D i s t i l l e d water 1000.00 ml

33

The inoculum of the wi l t fungus Fusarium solani wi l l

be ra ised on Richard's l iquid medium (Riker and Riker, 1936)

having the following composition:

Potassium n i t r a t e

Potassium dihydrogen phosphate

Magnesium sulphate

Ferric chloride

Sucrose

D i s t i l l e d water

10.00 g

5.00 g

2.50 g

0.02 g

50.00 g

1000.00 ml

The fungi wi l l be incubated for 15 days in an incubator

running a t 28+2°C temperature.

After the incubation period the mycelial mats wil l be

removed, washed in d i s t i l l e d water to remove the t races of

the medium and then i t w i l l be gently pressed between s t e r i l e

b lo t t ing papers to remove the excess amount of water.

Inoculum wi l l be prepared by making 10 g fungal mycelium in

100 ml of s t e r i l i s e d d i s t i l l e d water and blending i t for

30 seconds in a waring blender (Stemerding, 1963). In t h i s

way each 10 ml of t h i s homogenate wi l l contain 1 g of the

fungus,

2,3 Malntalnence of t e s t p l an t s ;

Sandy loam s o i l , which i s commonly found in Aligarh wi l l

be col lected from a fallow f ie ld and wi l l be passed through

a course sieve (1 mm pore s i z e ) to remove stone p a r t i c l e s and

34

debris e t c . Compost manure a t the ra te of 4:1 ( so i l : compost)

wi l l be added and thoroughly mixed with s o i l . Six cm. clay-

pots wi l l be f i l l ed with 1 kg of such s o i l - compost mixture

and then these pots wi l l be autoclaved and wi l l be used for

a l l s t ud i e s . Four or five surface s t e r i l i z e d seeds of

F^nch bean, (Fhaseolus vu lga r i s ) and green gram/mung bean

(PhasePlus aureus) wi l l be sown in these po t s . Surface

s t e r i l i z a t i o n of seeds wi l l be done by t r e a t i n g them in 0.1%

solut ion of mercuric chloride for 2 minutes, ^hen these

seeds wi l l be washed th r ice with s t e r i l i z e d d i s t i l l e d water

t o remove the t races of Mercuric ch lor ide . After emergence,

the seedlings wi l l be thinned and only one seedling wil l be

allowed t o grow in one pot . Plants of thr^e leaf stage wi l l

be inoculated with nematode and the fungus, as per schedule

of the experiments.

In the case of root-nodule bacter ia the seeds wi l l be

bacterized before sowing with t h e i r respect ive rhizobia .

Sucrose so lu t ion (596) wi l l be used as s t i c k e r and i t wi l l be

mixed with the rh izobia l cu l t u r e . Seeds wi l l be mixed with

t h i s mixture in such a manner tha t a uniform coating be

formed on t h e i r surface. These bacterized seeds wi l l be

dried a t room temperature and then sown.

The pots wi l l be placed on a green house bench in a

randomised manner. Necessary weeding and watering wi l l be

done as and when required. Special requirements of d i f fe ren t

experiments , i f any, are described in the appropr ia te , columns.

35

2.4 Inocxilation Techniques :

(a) Inoculation with nematodest

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 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 inoculat ion of p lan t s with fungi, homogenate

of fungal mycellium wi l l be prepared. I t w i l l be taken out

in such an amount so tha t the required inoculum leve l i s

f u l f i l l e d . For Inoculat ion the s o i l w i l l be removed from

around the roots of the t e s t p lants to expose the roots and

then the required amount of fungal suspension wi l l be poured

near the roo t s , which wi l l be l a t e r covered by leve l l ing the

s o i l properly,

2.5 Determination of inoculum threshold;

In order to determine the inoculum threshold of the

selected nematode species , capable of causing s ign i f ican t

damage the seedlings of both the p lan ts French bean/mung bean

wi l l be inoculated with d i f fe ren t inoculum leve ls v i z . , 10,

1CX) and 10,000 of t e s t nematode species Rotylenchulus

reniformis. Similarly for the determination of fungal inoculum

56

threshold the seedlings wi l l be inoculated with 0 ,5 , 1.0, 2,0

and 3.0 g of the fungus per p l an t . There wi l l be 5 r e p l i c a t e s

for each inoculum l e v e l . Seperate se t s wi l l be maintained

for bacterized as well as non bacterized p l a n t s .

2,6 Studies on interaction of different inoculum leve l s of t e s t

pathogens : (Nematode fungus interactions)

Only one inoculum leve l wi l l be se lected a f t e r

analysing the 'da ta obtained in experiment (determination of

inoculum th resho ld) . Plants wi l l be ra ised according to

the procedure described in 2,4 for both bacterized as well as

for non bacter ized conditions and the p lan ts w i l l be inoculated

according to following schedule,

1. Uninoculated (control)

2 . Nematode alone

3 . Fungus alone

4. Nematode and Fungus simultaneously

5. Nematode one week pr ior to fungus

6. Nematode two weeks p r i o r t o fungus

7 . Nematode one week a f t e r the fungus

8. Nematode two weeks a f t e r the fungus

This inoculat ion procedure w i l l be according to method

described in 2 , 5 , Then f ina l recording of the data wi l l be

done a f t e r 2 months of inocula t ion.

37

EXPERIMENTS

3.1 Histopathologlcal studies :

Inoculated seedlings wi l l be uprooted careful ly of the

s o i l from the f i r s t u n t i l the s ix th day, then every three

days u n t i l the 30th day and f ina l ly on the AOth day. The

roots wi l l be washed gent ly, but thoroughly to remove a l l

s o i l p a r t i c l e s adhering to them. Roots of healthy and infected

p lants w i l l be cut in to one cm long pieces and processed as

follows:

3,1#1 Fixation;

The roots col lected from experimental pots wi l l be

fixed in FAA (Johansen, 19A0), FAA (Formalin-aceto-alcohol)

wi l l be prepared as follows:

Sthanol 509« 90 ml -^.r-^^'" "" - %

Formaline 37« 5 ml .J. £)S/^RSr |

Glacial acetic acid 5 ml

The roots will be placed in the fixative for a minimum

period of 24 hr to several days, depending on its thickness.

Material may also be stored in the fixative indefinitely,

3.1.2 Dehydration :

Dehydration is accomplished by moving the roots stepwise

through increasingly higher concentrations of alcohols. Tertiary

38

butyl-alcohol (TBA) dehydration schedule (Table 1) wi l l be

followed (Johansen, 1940).

3.1,3 Infiltration :

In t h i s s t ep , alcohols in the t i s s u e s w i l l be replaced

by paraff in so t ha t the t i s sue i s sa tura ted with a pure

solut ion of paraf f in . When the TEA dehydration schedule i s

followed, the 1009< TBA solut ion in s tep 8 w i l l be replaced

with a 1:1 mixture of 1009< TBA and paraff in o i l . The t i s sue

wi l l be allowed to remain in t h i s so lu t ion for 1 hr or more,

depending on i t s th ickness . Shortly before the next s t ep ,

another container w i l l be f i l l e d 3/4th of i t s volume with

melted paraff in and allowed to so l id i fy s l i g h t l y . The roots

from the TBA paraff in o i l mixture wi l l then be placed on top

of the so l id i f i ed paraff in o i l so lu t ion . This continer wi l l

be placed uncovered in an oven se t a t s l i g h t l y above the melt­

ing point of the paraf f in . After 1-3 hr, the TBA-paraffin

o i l mixture w i l l be poured off and replaced with pure melted

paraff in wax, and kept in oven for about 3 h rs . This step wi l l

be repeated a t l e a s t once more.

3.1.A Embed4lpf;

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.

TABIE - 1

39

Step % Alcohol Time D i s t i l l e d 93% 100% 1009^ water E thanol Ethanol TEA (ml) (ml) (ml) (ml)

1.

2.

3.

4.

5.

6.

7.

8.

# TEA

at

50

70

85

95

100

100

100

100

changes

25.5°C.

2 hr or more

Overnight

1 - 2 hr

1 - 2 hr

1 - 3 hr

1 - 3 hr

1 - 3 hr

Overnight

must be carried

50

30

15

0

0

0

0

0

out in a

AO

50

50

45

0

0

0

0

warm place

0

0

0

0

25

0

0

0

as it

10

20

35

35

75

100

100

100

solidifies

AO

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

solidification. After hardening, it will cut into smaller

blocks and trimmed. The blocks will be mounted on block

holders.

5.1.5 Sectioning :

Transverse and longitudinal sections of 8-12 Wn

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 C 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 in ing wi l l remove the paraff in from

the sect ions and incirease the cont ras t in the t i s s u e s . Staining

wi l l 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 f ina l s tep in a s ta in ing procedures and la id on a f l a t ,

absorbent surface. The mounting medium wil l be applied to

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

and cover -s l ip wi l l be lowered gradually over the s l i d e .

41

TABUE - 2

S tep

1.

2 .

3 .

A.

5.

6 .

7 .

8 .

9 .

10.

1 1 .

12 .

13.

14.

15.

16.

17.

18.

S o l u t i o n

xylene

abso lu t e e t h a n o l

959^ e t h a n o l

70?^ e t h a n o l

509i e t h a n o l

309^ e t h a n o l

^% aqueous s a f r a n i n 0

r i n s e i n t ap water

309^ e t h a n o l

509^ e t h a n o l

709^ e t h a n o l

959< e t h a n o l

0.196 f a s t g reen PCF i n 93% e t h a n o l

a b s o l u t e e t h a n o l

a b s o l u t e e t h a n o l

xy lene -abso lu t e e t h a n o l

xylene

xylene

Time

5 min

5 min

5 min

5 min

5 win

5 min

1-12 hr

3 min

3 min

3 min

3 min

5-30 sec

15 sec

3 min

5 min

5 min

5 min or longer

42

Finished s l i des wi l l be l e f t t o dry for a t l ea s t 24 hr

a t room temperature. The medium wi l l harden be t t e r i f the

s l ides are held on a 60 C warming t ray overnight. The s l i des

wi l l be examined under Laborlux - K compound microscope.

Necessary photographs wi l l be taken.

3,2 Studies on the e f fec t of d i f fe ren t ni t rogen leve ls :

3 ,2.1 Preparation of d i f fe ren t l eve ls of ni trogen from d i f fe ren t sources :

The d i f fe ren t ni trogen sources wi l l be n i t r a t e of soda

(1696 N) Ammonium sulphate (2156 N) Ammonium n i t r a t e (34% N)

Ammonium sulphate n i t r a t e (2696 N) Ammonium chloride (26% N)

Anhydrous Ammonia (99% N) Urea, b iure t = 1 , 5 % (^% N) Urea

coated (45% N) Calcium ammonium n i t r a t e (25% N), Calcium

ammonium n i t r a t e (26% N) and Calcium ammonium n i t r a t e (28% N).

Different ni trogen leve ls v i z . , -N (No Nitrogen), 1/2 N

(0.5 g ) , 1 N (1.0 g) and 2 N (2.0 g) per kg of s o i l wi l l be

calculated from each source and wi l l be mixed with the s o i l

before sowing the seeds of t e s t p l an t s ,

3.3 SttKJies on the e f fec t of d i f fe ren t r a t i o s of sand and clay :

For t h i s experiment sand s o i l mixture containing

d i f fe i^n t proportions of clay and sand wi l l be prepared in

following manner. These have been designated as s o i l types .

43

Soil type Sand percent Clay percent

1 100 0

2 75 25

3 50 50

4 25 75

5 0 100

These mixture w i l l be analysed for o rganic ma t t e r ,

pH and p a r t i c l e s i z e . Then 15 cm p o t s w i l l be f i l l e d with

one kilogram of each s o i l t y p e . These p o t s w i l l be s t e r i l i z e d

in an au toc l ave and a f t e r t h i s V-5 seeds of each t e s t p l a n t

w i l l be sown i n each p o t . One week a f t e r sowing t h i n i n g

w i l l be done t o keep one p l a n t per p o t . Immature females

(4th s t a g e ) of Rotylenchulus r en i fo rmls and w i l t fungus

Fusarium s o l a n l a lone and i n combinat ion w i l l be added around

the exposed r o o t s i n the presence and absence of Rhizobium spp .

a t the t h r ee l e a f s t a g e . Two months a f t e r i n o c u l a t i o n a l l

these p o t s w i l l be depot ted and then obse rva t i ons w i l l be

recorded on the r o o t and shoot l e n g t h , f resh and dry weight

of the r o o t and shoot, number of nodules and no. of females

per p l a n t . F i n a l l y a l l t hese obse rva t ions w i l l be p resen ted

on the t a b l e .

3 . 4 S tud ie s on the e f f e c t of nema t l c ide / fung ic ide on the leghaemoglobln c o n t e n t s :

For t h i s experiment both the t e s t p l a n t w i l l be

t r e a t e d with e i t h e r nemat ic ide (carbofuran) and /o r fungic ide

44

(Bavistin) or both in addi t ion to reniform nematode

(Rotylenchulus reniformis) and wi l t fungus (Fusariuro so lan i )

in the presence and absence of Rhizobium spp. These wi l l

be another s e t of untreated plants in which no neraaticide or

fungicide wi l l be added.

3.4,1 Estimation of leghaemoglobin

The leghaemoglobin content w i l l be estimated from both

t rea ted and untreated p lan ts in the following manner.

Benzidine hydrogen peroxide method (Proctor, 1963) wi l l

be used for the est imation of leghaemoglobin. Nodules wi l l be

picked up from the roots and washed throughly with double

d i s t i l l e d water. Fresh nodular t i s sue weighing 500 mg wi l l be

crushed and homogenized in 9 ml of t r i s a c e t i c acid buffer

(0,1M ace t i c acid plus 0,2M t r i s (hydroxylraethyl) amino methane

to obtain a pH of 4 .0 ) . The homogenate wi l l be kept overnight

in a deep freezer to get complete ex t rac t ion of leghaenoglobin.

The homogenate w i l l be shaken from time to time to f a c i l i t a t e

ex t rac t ion . The volume of the homogenate w i l l be made upto

10 ml by adding t r i s ace t ic acid buffer and centrifuged a t

3000 rpn for 20 minutes. The supematent wi l l be col lected

in a t e s t tube. An a l iquot of 0.5 ml of supernatant wi l l be

d i lu ted to a f ina l volume of 4.0 ml with t r i s ace t ic ac id .

Buffer and 2 ml of freshly prepared benzidine reagent (100 mg

benzidine with 0,5 ml of hydrogen per oxide of 100 percent

by volume added to 50 ml absolute alcohol) w i l l be added to i t .

45

The colour density wi l l be measured a t 660 run on spectrophoto­

meter. The r e s u l t s wi l l be expressed in bovine haemoglobin

equ i l en t s ,

3 . 5 ANALYSIS OF NPK FROM LEAVES AND ROOTS :

The plants wi l l be uprooted a f te r 60 days of inoculat ion

and shoot and root systan wi l l be washed gently with double

d i s t i l l e d water. Fully mature healthy leaves wi l l be picked

up and some par t of the root system from inoculated and unino-

culated r e p l i c a t e s , wi l l be dried in an oven a t 78j^2°C

separately for 24 h r s . The dried mater ia l wi l l be grounded

in to fine powder and passed through a 12 mesh s ieve . The

powder wi l l be kept in des icca tor so as to ensure the

a v a i l a b i l i t y of perfect ly dry mate r ia l .

The leaf and root powders wi l l be digested for de te r ­

mining the NPK contents by the method of Linder (1944) t ha t i s

br ief ly described below.

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

powder wi l l be weighed and careful ly t ransfer red to a 50.0 ml

Kjeldahl f lask and 2.0 ml of chemically pure sulphuric acid

wi l l be added. The flask wi l l be kept for d iges t ion for about

2 hrs to allow complete reduction of n i t r a t e s present in the

plant mater ia l , giving off dense white fumes u n t i l l the contents

turn black.

A6

Flask wi l l then be allowed to cool down for about 15

minutes a f t e r which 0 .5 ml of 3096 hydrogen peroxide wi l l be

added dropwise and the solut ion wi l l be heated again t i l l i t s

colour changes from black to l i g h t yellow. Heating wi l l be

continued for about.30 minutes. The f lask wi l l be cooled for

10 min and an add i t iona l amount of 3-A drops of 30% hydrogen

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

15 min t o get a c lea r and colour less e x t r a c t . At t h i s stage

care wi l l be taken in the addi t ion of Hydrogen peroxide

because i f i t i s added in excess there i s p o s s i b i l i t y tha t i t

would oxidise the ammonia in the absence of organic matter .

The sulphuric acid peroxide digested mater ia l wi l l be d i lu ted

with double d i s t i l l e d water and wi l l be t ransfer red with 3 or

4 washings to a 100 ml volumetric f lask and f ina l ly the volume

wi l l be made up to mark. For the analys is of ni trogen,

phosphorus and potassium a l iquo ts wi l l be taken from these

digested samples, the methods employed for t h i s analys is aJ?e

briefly summerized below :

3.5.1 Nitrogen estimation :

Nitrogen content of the sample wi l l be estimated

according to the method of Linder ( l944), A 10.0 ml a l iquot

of digested mater ia l w i l l be taken in a 50 ml voltmietric f lask

The excess of acid wi l l be p a r t i a l l y neutra l ized with 2.0 ml

of 2.5 N sodium hydroxide and 1,0 ml of ^0% sodium s i l i c a t e

wi l l be added to prevent tu rb id i ty . Finally the volume wil l be

47

made up t o the mark, A 5.0 ml a l iquo t of t h i s so lu t ion wi l l

be taken in a 10 ml graduated t e s t tube and 0.5 ml of Ness le r ' s

reagent wi l l be added drop by drop, mixing thoroughly a f t e r

each instalment . The volume wi l l again be made up to the

mark with the help of d i s t i l l e d water and the contents wi l l

be allowed to stand for 5 rain for maximum colour development.

The solut ion wi l l be t ransferred to a color imetr ic 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 s e t . The amount of nitrogen in the a l iquo t wi l l be read

from a ca l i b r a t i on curve obtained by using known d i lu t ions

of a standard ammonium sulphate so lu t ion ,

3 .5 .2 Phosphorous estimation

Total phosphorous in the sulphuric acid peroxide

digested so lu t ion wi l l be determined by following the method

of Fiske and Subba Rao (1925). A 5.0 ml of a l iquo t wi l l be

taken in a 10,0 ml graduated tube and 1.0 ml molybdic acid

(2,3% ammonium molybdate in 10 NI^SO^) wi l l be careful ly

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

which wi l l turn the contents blue. Di 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 wi l l

be allowed to stand for about 5 minutes a f t e r mixing throughly.

I t w i l l be then t ransferred to a color imeter ic tube and the

op t ica l density wi l l be read a t 620 nm on a colorimeter, A

blank wi l l be run side by s ide , A ca l i b r a t i on curve wi l l be

u&

prepared by using known dilution of a standard monobasic

potassium phosphate solution,

3.5.3 Potassium estimation :

PotassiiM will be estimated using a flame photometer.

A 1.0 ml aliquot will be taken and read at 768 nm with the help

of potassium filter. A blank will be run for each determina­

tion. The reading will be compared with a calibration curve

plotted for different dilutions of a standard potassium

sulphate solution.

3.6 Recording of data

3.6.1 Plant growth determination

For determining the plant growth, p lan ts wi l l be

uprooted after 60 days of inoculat ion and the roots wi l l be

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

dry weights ( in g) of shoots and roots w i l l be taken

separa te ly . Before taking the fresh weight, excess amount of

water wi l l be removed by put t ing root and shoot between

b lo t t ing shee ts , 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-nodule index

On the basis of v i sua l observation, the root nodule

index wi l l be rated on the following sca l e .

49

1 = no nodulation

2 = light nodulation

3 = moderate nodulation

A = heavy nodulation

5 = very heavy nodulation

3.6.3 Determination of population of nematodes

The population of nematodes in the roots will be

determined by mascerating the root-pieces in waring blender

for four seconds and then counting their numbers as males;

females and immature specimens separately determine sex ratio.

Reproduction factor (R) of the nematode will be calculated

by the formula of Oostenbrink (1956) as follows :-

R = Pf/Pi

where Pf represents the final population and Pi represents the

initial population of the nematodes.

Wilting Index Method

The degree of chlorosis of the tomato leaves and wilting

will be quantified as follows.

0 = no symptoms,

1 = light

2 « moderate,

3 " heavy and

A = severe symptom or dead.

50

For each plant a disease index will be calculated as

follows i-

„. . , Sum of leaf rating per plant Disease index « ^ ^ *

Total no. of leaves per plant

Wilting index will be determined after 15, 30, 45, 60 days

of inoculation of F, solani.

REFERENCES

Adeniji , M.O.; Edward, D . I , ; S inc l a i r , J .B . and Malek, R.B.

(1975). I n t e r r e l a t i onsh ip of Heterodera glycines and

Phvtopthora magasperma var , sojae in soyabean. Phytopa­

thology 6^(6): 722-725.

Agax^al, D,K, and Goswami, B.K. (1974). In t e r r e l a t ionsh ip between

a fungus, Macrophomina phaseolina (Maubl.) Ashby, and

root-knot nematode, M» incognita (Kofoid and White) Chitwood

in soyabean (Glycine max L, ) Mer i l l . Proceedings of

Indian National Science Academy B. 59(6): 701-704.

Al-Hazmi, A.S, (1985). In te rac t ion of M. incognita and

Macrophomina phaseolina in a r o o t - r o t disease complex of

french bean, Phvtopathologische Z e i t s c h r i f t . 113(4):

311-316,

Al i , M,A.; Trabuls i , J.V. and Abd-Elsamea, M.E, (1981),

Antagonistic i n t e r ac t i on between M, incogni ta . Rhizobium

leguminosarum on cowpea. Plant P i s . , 6^: 432-435.

Atkinson, G.F, (1892), Some diseases of cot ton. Bull . Ala.

Agric. Bxp. St a. 41: 65 P.

Balasubramanian, M. (1971). Root-knot nematodes and bac t e r i a l

nodulation in soyabean. Curr. S c i , , 40(3): 69-71.

52

Baldwin, J .G , , Barker, K,R« and Nelson, L.A. (1975). In te rac t ion

of Meloidogyne incognita with Rhizobium .japonicum on

soyabean, J . Nematol., Ji 319 (Abst r , ) ,

Barker, K.R,, Huisingh, D, and Johnson, S.A. (1972). Antagohistic

i n t e rac t ion between Heterodera glycines and Rhizobium

japonicum on soyabean. Phytopathology. 62(10: 1201-1205.

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

ni trogen and Rhizobium .japonicum on the a c t i v i t y of

Heterodera g lycines . Nematologica. 1^: 377-385.

Bell , O.K., Mlnton, N.A. and Doupnik, J r . B. (1971) Effect of

Meloidogyne arenar ia , Aspergil lus flavxxs and curing time

on infec t ion of peanut pods by Aspergil lus f lavus .

Phytopathology 61,(8): 1038-1039.

Book Binder, M.G, and Bloom, J.R. (1978). An in t e r ac t ion of

Uromyces phaseoli and Meloidogyne incognita on bean.

Proceedings of the American Phytopathological Society.

4: 183-184.

Book Binder, M.G. and Bloom, J.R. (19B0). In te rac t ion of

Uromyces phaseoli and M. incognita on bean. J , Nematol .

12(3) : 177-182.

Bopaiah, B.M., P a t i l , R.B. and Reddy, D.D.R. ( l976) . Effect of

Meloidogyne .jayanica on nodulation and synerg i s t i c ni t rogen

f ixa t ion in mung Vigna r a d i a t a . Indian J . Nematol.. 6 (2 ) :

12A-130.

53

Campos, V.P, and Schmitt, D.P. (1961). Nematode population,

soyabean emergence and development as a r e s u l t of simul­

taneous inoculat ion with H, glycines and P. magasperma.

Fitopatologia Bras i l a i r a . 6 (3 ) : ^ 9 - 4 9 6 .

Chahal, P.P.K. and Chahal, V.P.S, (1968). In te rac t ion of

Rhizobium and Meloldogyne incognita on symbiotic nitrogen

f ixa t ion in mungbean under d i f fe ren t concentrat ions of

i ron . Plant n u t r i t i o n . r i ( 6 - 1 1 ) : 821-828.

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

incognita on leghaemoglobin bacteroids and nitrogenase

a c t i v i t y of nodules produced by FUiizobium spp. on

Mungbean (Vigna £adiata)E Pakistan J . Nematol., ^ ( 1 ) :

21-25.

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

and Fusarium equ i se t i on growth characters and wil t ing of

pea seedl ings . Journal of Research. Pun;}ab Agr icu l tura l

Universi ty, 22(4): 684-686.

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

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

Rhizoctonia solani on Phaseolus vulgar is CV. Rico 23.

Fi topatologia Bras i l e i r a . 21^^): 857-864.

Dave, G.S. (1975). In t e r r e l a t ionsh ip of Rhizoctonia solani with

Heterodera glycines , Pratylenchus s c r ibne r i and

Tylenchorhynchus mart ini on d a r k 63 soyabean.

Disser ta t ion Abstracts In t e rna t iona l . ^ B : 1991-1992.

54

Dhanger, D.S. and Gupta, D.C. (1983). Pathogenicity of

Meloidogyne .javanica to chickpea (Cjcer arietinum) in

relation, to soil types, FUiizobium treatment, size of

pots and time intervals, Indian J. Nematol., 13(2);

161-170.

Diomande, M. and Beute, M,K. (l979). Influence of M, hapla and

Macroposthonia omata on the effectiveness of mlcro-

sclerotla inocula of Cylindrocladium crotalariae in

causing black rot (CBR) on peanuts. Phytopathology

69(1): 1-A3.

Diomande, M., Black, M.C., Beute, M.K. and Barker, K.R. (1961).

Enhancement of Cylindrocladium crotalariae rot-root by

Meloidogyne arenaria (race 2) on peanut cultivar

resistant to both pathogens. J, Nematol. ( 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 choroty roslin), Wiadmosci Ekologiczne,

28(3/4): 219-226.

Dropkln, 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. (l979). Interaction between

H. cajani and Fusarium udum on pigeon pea. Allahabad

farmer. ^ ( 1 ) : 23-24.

55

Flske , C.H. and Subba Row, Y. (1925) . The c o l o r i m e t e r l c d e t e r ­

minat ion of phosphorous, J . B i o l . Chem. 66 : 375-AOO.

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 p o l y s p e c i f i c nematode

popu la t i on on soyabean, J , Nematol, _22(A): 287,

Garc ia , 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 s e v e r a 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 . Phytopatho­

logy . 6 ^ ( 7 ) : 832-833 .

Goel, S.R, and Gupta, D.G. ( l9B6a) , I n t e r a c t i o n of M. j a v a n i c a

and F, oxysporum f, c i c e r i on chiclqpea. Ind ian Phytopa­

thology 3 9 ( 1 ) ; 112-114.

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

and Rhizoctonia b a t a t i c o l a on ch ickpea . Ind ian J .

Nematol, ^ ( l ) : 1 3 > 1 3 4 ,

Goswami, B, K. and Agarwal, D,K, (1978). I n t e r r e l a t i o n s h i p

between s p e c i e 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 te r ranea . 6 ( 1 ) :

125-128.

Gupta, D.G, and Yadav, B.S, ( l 9 7 9 ) . S tud ies on p a t h o g e n i c i t y

of i*eniform nematode, Rotylenchulus r e n i f o r m i s t o urad ,

Vigna mungo L. Wilczek, Ind ian J , Nematol. 9 ( l ) : 4 9 - 5 0 .

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

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

Hater ode r a v i g n i on cowpea, Ind ian J , Nematol. ^ ( l ) : 132-135,

56

Hasan, A, (1984) Synergism betvveen Heterodera cajani and

Fusarium urum at tacking Cajanus ca.ian.Nematologia

Mediterranea. 22(1) : 15^162 .

Hussaini, S.S, and Seshadri, A.R. (1975). I n t e r r e l a t i onsh ip

between Meloidogyne incognita and Rhizobium species on

mung bean. Phaseolus aureus. Indian J , Nematol.. ^ ( 2 ) :

189-199.

Hussey, R.S. and Barker, K, R, (1974). Effect of nematodes with

d i f fe ren t feeding habits on nodulation bac te r i a . J .

Nematol.. 6 (4 ) : 143 (Abstr. ) .

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

and l i g h t sources on growth and nodulation of soyabean.

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

Hutton, D.G., Wilkinson, R.E. and Mai, W. F. ( l973). Effect of

two plant p a r a s i t i c nematodes on Fusarium dry roo t - ro t

of beans. Phytopathology. 6^(6) : 749-751.

Ibrahim, I.K.A. and El-Saedy, M.A. (l977). Pathogenicity of

M. javanica and c e r t a i n fungi on peanuts. Alexandria

journal of Agricul tural Research. 24(2): 411-415.

Jamal, A. ( l976). Studies on the i n t e r ac t ion of root nodule

bacter ia and root-knot nematode, Meloidogyne incognita

(Kofoid & White, 1919) Chitwood, 1949, on ce r t a in

cu l t iva ted legumes. Ph.D. Thesis , Botany Dept t . , Aligarh

Muslim Universi ty, Aligarh.

57

Johanson, D.A, (1940) . In "Plant Microtehanique". McGraw-

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

Kanwar, R.S- , Gv^jta, D.C. and Walia, K.K, (1987). I n t e r a c t i o n

of M. lavanica and Rhizoctonia solani on cowpea.

Nematoloqia Mediterranea. ^5.(2) : 385-386,

Kanwar, R.S- , Gupta, D.C. and Walia, K.K. (1989). Effect of

nematode inocu la t ion at d i f fe ren t i n t e r v a l s and so i l

types on i n t e r a c t i o n between Meloidoqyne lavanica and

Rhizoctonia so lan i on cowpea. Indian J . Nematol. i 8 ( l ) :

112-113.

Khan, A.M* (1981). Certain b i o t i c f ac to r s inf luencing Patho-

genecity in Nanatodes. Indian Phytopath. 34_(2) :143-158.

Khan, T.A. and Hussain, S . I . (1988). Effect of i nd iv idua l ,

concomitant and sequential inocula t ions of Rhizobium,

Rotvlenchxilus reni fo imis , M» incognita and Rhizoctonia

solani on cowpea p lan t growth, d isease development and

nematode m u l t i p l i c a t i o n . Indian J . Nematol. ;8(2):232-238,

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

s ix cowpea c u l t i v a r s as affected by the concomitance of

two nematodes and a fungus. Nonatoloqia Medi terranea.

12(1) : 39 -41 .

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

Hodges, H.H- (1988). Green house and f i e l d eva lua t ion

of Fusarium so lan i pathogenecity t o soyabean s e e d l i n g s .

P lant ^ i s . 72(12) s 1067-1070.

58

Kumar, R,, Ahmad, S. and Saxena, S.K, (1968). Disease complex

in chiclqjea involving M. incognita and Fusarium oxysporuro.

In te rna t iona l Nematology Network Newsletter. ^ ( 3 ) : 12-14.

Lamberti, F, (1981), Combating nematode vectors of plant v i ru se s .

Plant P i s . 6^: 113-117.

Lehman, P .S . , Huising, D. and Barker, K.R. ( l 97 l ) . The influence

of races of tfeterodera glycines on nodulation and ni trogen

fixing capacity of soyabean. Phytopathology. 6l (10):

1239-1244.

Linder, R.C. (1944). Rapid a n a l y t i c a l methods for some of the

moi?e common inorganic cons t i tuen ts of p lant t i s s u e .

Plant Physiol. 19- 76-89.

Malek, R.B. and Jenkins , W. R. ( l964) . Aspects of the host

Paras i te r e l a t ionsh ip of nematodes and hairy vetch. Bull ,

N. J . Agric. Exp. Sta. No. 813, P. 31 .

Mani, A.and Se th i , C,L. (1987). In te rac t ion of root-knot

nematode M. incognita with Fusarium oxysporuro f . sp .

c i c e r i and Fusarium solani on chickpea, Indian J .

Nematol. I j d ) : 1-6.

Mar te l l i , G.P. (1979). Nematode borne-virus of grapevine,

t h e i r epidemiology and con t ro l . Nematol. Medit., 6_:

1-27,

59

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

s o i l t r e a t m e n t on r o o t - k n o t c o n t r o l , n o d u l a t i o n and y i e l d

of pea . Va, J . S c i . 2: 1 0 ^ 1 1 2 .

Moussa, B.M. and Haungue, N.G.M, (1987). The in f luence of r o o t -

knot nematode M. i n c o g n i t a on the development of

F, oxyspoi^un f. s p . g l y c i n e s i n 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&cul te i t Landbouwwetenschappen R i , 1 k s s u n v i e r s i t e i t Gent.

^ ( 2 b ) : 571-575.

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

pxysporum f. s p , g l y c i n e s on the i nvas ion and development

of r o o t - k n o t nematode. Revue de Nematologie. 11 (4):A37-A39.

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

on development of gram caused by Fusarium oxysporum f.

c i c e r l and r o o t ^ r o t by Rhizoctonia spp . Ind ian J o u r n a l of

Mycology and P lan t Pathologv. 11 ( l ) : 6/4-49.

Norton, D.C. (197B). •• Ecology of p l a n t p a r a s i t i c nematodes"

John Wiley and Sons, New York.

Oyeken, P.O. and M i t c h e l l , J . E . (1971). E f f ec t of Pra ty lenchus

pene t 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 . D i s . Rept r . ^ ( 1 1 ) : 1032-1035.

P a d i l l a , C, , Lopez, R, and Vargas, E. ( l9B0) . I n t e r a c t i o n

between Meloidogyne spp . and Fusarium oxysporum f. p l s i

on pea , Agronomia C o s t a r r i c e n s e . hil )i 55-60.

60

Patel, H.R., Thakar, N.A. ,Patel, B.K. and Patel, C.C. (19B7).

Interaction between M. incognita, Fusarium oxysporum f,

sp. ciceri on chickpea variety Ghaffa. Indian J. Nematol.

12(1): 124.

Patel, H.R., Vaishnav, M.U. and Dhrvg, I.U. (1965). Interaction

of M. arenaria and Fusarium solani on groundnut. Indian

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

Patel, H.R., Vaishnav, M.U. and Dhruj, I.U. (l9B6). Effect of

interaction between M, arenaria and Aspergillus flavus

on groundnut. Indian Journal of Mycology and Plant

pathology. J6(2): 103-107.

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

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

Tech. Bull. No. ? Min. Agric. Fish and Food. H.M.S.O.

London, 63-77.

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

with other disease causing agents. In " Plant Parasitic

Nematodes" . Vol, II (Sds.- 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. N.^.J.

Sol. 6: 60-63.

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

Meloidogyne incognita on plant growth and Rhizobial

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

61

Raut, S.P. and Se th i , C.L. (1980). Studies on pathogenicity

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

10(2): 166-174.

Reddy, P.P. , Singh, D.B, and Sharma, S.R. ( l979) . In te rac t ion

of M. incognita and R. so lani in roo t - ro t disease complex

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

Ribeiro, C.A.C. and Fteraz, S. (l983). Studies on the in t e rac t ion

between M, .jayanica and Fusarium oxysporum f, sp. phaseoli

on bean (Phaseolus y u l g a r i s ) . Fitopatologia Bras i l e i r a ,

8 (3 ) : 439-446.

Riker, A.J, and Riker, R.S, (1936). Introduct ion to research on

plant d i sease , John ' s Swift Co, Inc , , Sta , Louis. Chicago

New York, Indiana poles ,

Ross, J , P . (1969). Effect of Heterodera glycines on the yield

of non nodulating soyabean grown a t yarious ni trogen

l e v e l s . J , Nematol., 1.(1): AO-42.

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

Killebrew, J , F . (1989). Sudden death syndrome of soyabean,

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

glycines to disease seve r i ty . Phytopathology, 79(2) :

191-197.

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

Salem, A, (1980). His to logica l changes induced by

M.,1avanica and Fusarium species on roots of selected

leguminous p l an t s , S ^ t i a n Journal of Phytopathology.

12(1/2): 43-47.

62

Sakhuja, P.K. and Sethi,C.L. (1966). Mul t ip l ica t ion of

M. .javanica as affected by Fusarium so lan i and Rhizoctonia

ba ta t ico la on groundnut, Indian J . Nematol. 1^(1): 1-3.

Salam,M.A, and Khan, M.W. ( l9e6) . Reaction of some cu l t i va r s of

pigeon pea against Fusarium udum and M, .javanica.

In te rna t iona l Nematology Network Newsletter. ^ (4) :16-17.

Sass, J .E . (1951). In "Bo tan i ca l Micro-techanique" Iowa State

college Press , Ames, Iowa, pp. 228,

Sharma, N.K, and Seth i , C.L, (1976). In t e r r e l a t i onsh ip between

Meloldogyne incognita , Heterodera cajani and Rhizoctonia

solani and Rhizobium species on cowpea Vigna s inensis L.

Savi, Indian J . Nematol., 6 (2) : 117-123.

Sharma, R.D, and Cerkauskas, R, F, ( l985). In te rac t ion between

M, pavanica and F, oyysporum f, sp, c i c e r i on chickpea.

Nematologia Bras i l e i r a . 9 ( l ) : 113-121.

Sidhu, G, and Vfebster, J.M. (1977 ) . Predisposi t ion of tomato to

the wi l t fungus (Fusarium oyysporum lycope r s i c i ) by roo t -

knot nematode, ^teloidogyne incognita . Nematologica, 2^t

436-442.

Singh, D.B,, Reddy, P.P. and Sharma, S,R. (1981). Effect of

root-knot nematode M. incognita on Fusarium wil t of fr^nch

beans, Indian J , Nematol. Vli^)', 8A-85,

63

Singh, I . , Ghahal, V.P.S. , Sakliuja, P. K. and Chohan, J . S . (1977).

Effect of d i f fe ren t leve ls of Meloidofiyne in presence or

absence of Rhlzobium phaseoli on Phaseolus aureus. Indian

J . Nematol. 2 ( 2 ) : 172-174.

Singh, R., Gupta, D.C. and Walia, K. K. (1986). Effect of

Rhizoctonia solani cul ture f i l t r a t e s on the hatching of

U* .iavanica l a rvae . Indian Phytopathology. 39(4);62A-625.

Srlvastava, A.K., Upadhyay, K.D. and Singh, G. (197A). Effect of

root-knot nematode M. incognita on gram crop.Indian J .

Nematol. 4(2) : 2A8-251.

Srivastava, A.S. , Upadhyay, K.D. and Singh, B. P. (1979). Effect

of root-knot nematode Meloidogyne .iavanica on the growth

of soyabean. Glycine max. Indian J , Nematol.. ^ ( l ): 38-40.

Stemerding, S. (1963). Sen mixer-wal lenf i l te r raethode on

Vrijbeweeglijkg endoparasi t iare nematoden wit worlets to

Verzamelen. Versl . P lz iek t .Diens t . 141: 170-175.

Sumner, D.R,, and Minton, N.A. (1967). In te rac t ion of Fusarium

wil t and nematodes in Cobb soyabean.Plant P i s . 71 (l )t

20-23.

Szerszen,J . (1980). Influence of F, oxysporum f. sp. p i s i alone

and in combination with Pratylenchus penetrans on peas.

Skolozia Polksa. 28(4): 615-631.

64

Taha, A.H.Y, and Kassab, A.^, (1980), I n t e r r e l a t i onsh ip between

Meloldogyne .javanica. Rotylenchulus reniformis and

Rhizobium species on Vi^na 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 onsh ip between

root-nodule bacter ia , p lant p a r a s i t i c nematodes and t h e i r

leguminous host. J . Nematol. j ( 3 ) : 201-211.

Taylor, D, P. and Sasser, J.N. (1978). Biology, i d e n t i f i c a t i o n

and cont ro l of root-knot nematodes (Meloidogyne spp. )

Coop. Pub. Pep. Plant F&thol., North Carolina State Univ.

and U.S. Agency In t . Dev. Releigh, N.C. 111 P.

Thakar, N.A., Pa te l , H.R., F&tel, B.K. and Pa te l , C.C. (1986).

Breaking of res i s tance to Fusarium wil t in r e s i s t a n t

chickpea by root-knot nematode. Madras Agr icul tura l

Journal . 21(7) : 410-/411.

Tlyagi, S.A., Zaidi , S.B.I, and Alam, M.M. ( l988). In te rac t ion

between M. .javanica and Macrophomlna phaseolina on l e n t i l .

Nematologia Mediterranea. ^ ( 2 ) : 221-222.

Upadhyay, K,D, and Dwivedi, B.K. (l967a). Root-knot nematode

M. .javanica breaks wi l t res is tance in chickpea var ie ty

Aurodhi. Current Science. ^ ( 1 7 ) : 915-916.

65

Upadhyay, K.D. and Dwivedi, B. K. ( l9B7b). E f f e c t of i n t e r a c t i o n

between M. . javanlca and Fusarium oxysporum f. sp , c i c e r i

on chickpea, Ind ian J . Nematol. J ]^(1) : 145-1A6.

Varshney, V .P . , Khan, A.M. and Saxena, S.K. (1967). I n t e r a c t i o n

between M, i n c o g n i t a . FUiizoctonia s o l a n i and Rhizobium

s p e c i e s on cowpea. I n t e r n a t i o n a l Nematology Network

News le t t e r . 4 ( 3 ) : 11-14.

Verdojo, S . , Green, C.D. and Podder, A.K. (1969). Inf luence of

Meloidogyne Incogn i ta on nodu la t ion and growth of pea and

black bean, Nematologica 3 4 ( l ) ; 88-97 .

Walia, K.K. and Gupta, D.C. ( l 986a ) . Antagonism between

Heterodera ca. iani and R, s o l a n i on cowpea. Ind ian J .

Nematol. I 6 ( l ) t 41-43 .

Walia, K.K. and Gupta, D.C. ( l986b) . E f f ec t of fungus R.

b a t a t i c o l a on the p o p u l a t i o n development of pigeonpea

c y s t nematode, H, ca.jani on cowpea (Vigna u n g u i c u l a t a ) .

Ind ian J . Nematol. ^ ( l ) : 131-132.