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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
^^2^'•'^•^sr*:
DS1788
^7^CJ:>^I^^J^
^ -
y
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 .
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