Agriculture

Ecosystems &
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ELSEVIER Agriculture, Ecosystems and Environment 53 (1995) 71-81

Effects of ammonia and root-knot nematode on tomato

Mujeebur Rahman Khan*, M. Wajid Khan
Enviromnental Pollution Research Unit, Departmentof Botany, Aligarh Muslim University,Aligarh 202 002, India
Accepted 27 July 1994

Abstract

Tomato plants inoculated with 2000 juveniles (JJ2) of root-knot nematode (Meloidogyne incognita race 1) or intermittently
exposed to 152/zg NH3 m-3 exhibited significant suppression in growth, yield and leaf pigments compared with uninoculated
or unexposed plants. However, NH3 at 76/zg m -3 did not cause significant effects. The leaves of nematode-inoculated or
uninoculated plants exposed to NH3 ( 152/zg m-3) turned yellow in all treatments.
Root galling and reproduction efficiency of M. incognita increased at 76/~g NH3 m-3 but was suppressed at 152/zg m-3.
Fecundity, however, declined at both levels. The nematode and NH3 interacted synergistically at 76/~g m - 3 and antagonistically
at 152/xg m -3. Reductions in the plant growth, yield and photosynthetic pigments of the tomato were greatest in post- or
concomitant-inoculation treatments at 152/zg NH 3 m - 3 and concomitant inoculation treatment at 76/zg m- 3. Nitrogen content
of foliage and roots increased significantly at both levels of the gas, being greater in post- and concomitant-inoculation treatments
at 152/zg and 76/xg m - 3, respectively. Meloidogyne incognita or NH3 decreased the number and size of stomata but increased
the width of stomatal pores. Length and number of trichomes increased in the exposed plants, but remained unaffected in plants
inoculated with the nematode alone. A positive correlation mostly occurred between width of stomatal pore and percent reduction
in the plant growth, yield and leaf pigments of tomato.

Keywords: Ammonia; Nematode; Tomato growth; Tomato yield; Nitrogen; Stomata

1. Introduction foliage and soil surface in the form of fine particles.

Ammonia (NH3) is relatively little studied among
the air pollutants causing toxicity in plants. Atmos-
pheric NH3 originates from the use of ammonium fer-
tilisers, brick-kilns, ceramic and pottery industries,
refineries and cooling plants (Khan et al., 1991). The
major source of NH3 is, however, intense livestock
farming or animal slurry dumps (Van Dijk and Roelofs,
1988). Ammonia readily reacts with ambient SO2 and
forms ammonium sulphate which deposits on plant
* Corresponding author at: Department of Plant Protection, Institute
of Agriculture, Shaft House, Qila Road, Aligarh Muslim University,
Aligarh 202 002, India.
Accumulation of ammonium sulphate increases soil
acidity (Ryden et al., 1987). The NH 3 and NH4 com-
pounds are later converted into nitrite and nitrate
through nitrifying bacteria, Nitrosomonas and Nitro-
bacter.
Ammonia creates a serious nutritional imbalance in
soil by excessively increasing soil nitrogen (Nihlgard,
1985) and causes direct toxicity in plants. Yellowing
of needles and reduction in carotenoids and chloro-
phylls of Scot pine grown in an NH3 polluted area have
been reported (Van Dijk and Roelofs, 1988). Dueck
(1990) recorded 20% inhibition in seedling survival
of Calluna vulgaris exposed to 53 and 105 ~g NH 3
m - 3. The effect of NH3 on forest trees has been fairly

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72 M.R. Khan, 11/1.W. Khan/Agriculture, Ecosystems and Environment 53 (1995) 71-81
well studied, whereas the response of crop plants has
not been tested. Most of the research on NH 3 effects
has been carried out in the Netherlands, where NH 3
originating from stockbreeding is implicated directly
or indirectly in the general decline of forests (Van
Breeman and Van Dijk, 1988).
Forest decline is apparently a multipathogen-stress
consequence, involving interactions of pollutants and
pathogens. Air pollutants can influence host-parasite
relationships. Air pollution has been shown to affect
plant parasitic nematodes. Studies have shown that 03
at 490/zg m - 3 inhibited reproduction and development
of Heterodera glycines and Paratrichodorus minor,
while Belonolaimus longicaudatus and Aphelenchoi-
des fragariae remained unaffected (Weber et al.,
1979). Recently, Khan and Khan (1993) recorded
enhanced disease intensity caused by Meloidogyne
incognita on tomato exposed to 286/zg SO2 m-3.
Root-knot nematodes (Meloidogyne species), one
of the most important groups of plant parasitic nema-
todes, have an exceedingly wide host range and interact
with other plant pathogens. Vegetable crops are their
preferred hosts. Meloidogyne incognita is widespread
in the tropics and subtropics and estimated yield losses
in vegetable crops due to root-knot nematodes in dif-
ferent regions of tropics vary from 5 to 43% (Sasser,
1979). Root-knot nematodes induce development of
cancerous outgrowths or knots on roots resulting in
impaired absorption of water and minerals, transloca-
tion of assimilates etc. (Khan and Khan, 1987; Veech
and Dickson, 1987).
Response of vegetable crops to NH3 gas has been
rarely tested and there is no information on the inter-
action of NH 3 and plant parasitic nematodes. The pres-
ent study on effects of NH3 on root-knot nematode and
tomato was carried out to determine the following:
(i) effect of intermittent treatments of NH 3 at 76 and
152 /~g m -3 on plant growth, yield, photosynthetic
pigments and epidermal characters of tomato;
(ii) cumulative effect of NH 3 and M. incognita on
different growth parameters of tomato as in (i);
(iii) effect of intermittent exposures of NH3 on the
disease intensity and reproduction efficiency of the
nematode.
2. Materials and methods
2.1. Exposure system
Three exposure chambers, each of 90 c m X 9 0
cmX 120 cm dimensions were used. Two chambers
were used to expose the plants to NH 3 and one for
control (ambient air) set. The chambers were made of
transparent fibre glass with an exhaust duct at the top
and a double-walled bottom. The exposure chambers
have been described elsewhere (Khan and Khan,
1993). The generation of NH3 gas in a generator (Stan-
dard Appliances, Varanasi, India) was principally
based on heating of liquid ammonia. The degree of
heating of liquid NH 3 and rate of suction of the gas
from the heating unit determined the concentration of
NH 3 gas, which was controlled by a flow meter. The
outlet of the generator was connected to the blower
assembly of the chamber, which mixed incoming NH3
with ambient air and uniformly dispensed the mixture
in the chamber. The desired concentrations of NH3, i.e.
76 and 152/zg m -3 were obtained by calibrating the
flow meter of the generator after repeated ammonia
samplings inside the chamber with the help of a port-
able air sampler (Kimoto Electric, Osaka, Japan). For
the sampling, 20 ml diluted sulphuric acid (absorbing
medium for NH3) was poured into the impinger of the
air sampler. The sampler was placed inside the expo-
sure chamber and was run for 3 h. After sampling, the
medium was analysed colorimetrically to determine
exact concentration of ammonia inside the chamber
(Anonymous, 1986a). The blower of the chamber was
run at a constant air flow rate, i.e. 1.9 m s- ~. The air
(with NH3) inside the chamber was replaced approx-
imately eight times in 1 min. To reduce differences
among chambers in the microclimate, the plants were
rotated among the chambers at each exposure. For
example, if the treatment of 76 ~g m - 3 was given on
one occasion in chamber 2, the next time chamber 3
was used for the same treatment. The mean concentra-
tion of NH3 inside the chamber after completion of all
the exposures was 76+8.37 and 152+ 13.8/xg m -3.
The third chamber, used for exposure of the control
(without NH3) plants was run at the same air flow rate
(1.9m s - l ) .
 M.R. Khan, M.W. Khan/Agriculture, Ecosystemsand Environment53 (1995) 71-81
2.2. Treatments and plant culture
Three-week-old seedlings of tomato, Lycopersicon
esculentum Mill. cv. 'Pusa Ruby', grown in autoclaved
soil were transplanted on 31 October 1988, in 15 cm
diameter pots containing 1.5 kg steam-sterilised soil
(clay, sand, compost, 2 : 1 : 1 ) . In some treatments
freshly hatched second stage juveniles (JJ2) of root-
knot nematode, Meloidogyne incognita race 1 (Kofoid
and White) Chitwood, obtained from single egg mass
glasshouse-culture were added (2000 JJ2 per pot) to
small holes made in the soil around the roots of tomato
seedlings. Inoculation and exposure of the plants to
NH3 were done according to three modes of sequential
inoculation exposures. In pre-inoculation exposure,
NH 3 treatment started 1 week before (on 7 November
1988) nematode inoculation (on 15 November). In
post-inoculation exposure, the plants were inoculated
(on 7 November 1988) 1 week before NH3 treatment
( 15 November). In concomitant-inoculation exposure,
NH3 treatment and nematode inoculation were done
simultaneously (on 15 November). A separate and
independent set for each sequential treatment corre-
sponding to the date of inoculation was maintained. In
total there were four control (unexposed) sets: C1,
uninoculated; C2, C3 and C4, inoculated, correspond-
ing to pre-, post- and concomitant-inoculation expo-
sures, respectively. Similarly, there were four exposed
sets at each concentration of NH3: T1, uninoculated;
T2, T3 and T4, pre-, post- and concomitant-inoculation
exposures, respectively.
All treatments were replicated ten times and the pots
were placed on glasshouse benches at 25 +__2°C in a
complete randomised block design. The designated
plants were exposed to NH 3 at 76 and 152 /zg m -3
every third day for 3 h from 09:00 h for 75 days in
separate chambers. Pots of the control set (C1-C4)
were placed in the third chamber (air flow rate 1.9 m
s - J) for the same duration at the same time. This cham-
ber was not connected to the NH 3 generator and hence
received ambient air. The concentration of NH3 in
ambient air of the Botany Department, AMU was 7.18
/zg m 3. Each treatment received 27 NH3 exposures.
In pre- and concomitant-inoculation treatments, expo-
sures (27) were completed on 22 January 1989, and
on 29 January in the pre-inoculation treatment. Five
plants out of the ten replicates of each treatment were
73
harvested on 29 January 1989 and the following para-
meters were measured.
2.3. Plant growth, yield and root-knot disease
Tomato plants were examined regularly for visible
disorders attributable to NH3 or root-knot nematode.
The flower buds developing during the 3 month period
were counted. At termination, the number of fruits, per
plant were counted and weighed, The soil in each pot
was saturated with water to facilitate root recovery at
the time of uprooting the plants to determine lengths
and fresh weights of shoot and root. Shoots and roots
were dried in a hot air oven at 60°C for 48 h to determine
dry weights. Betbre drying the roots, galls and egg
masses were counted and numbers of galls and egg
masses per gram fresh weight of root were calculated.
For determining fecundity (number of eggs per egg
mass) 100 egg masses picked from the roots (20 egg
masses were from each of the five fresh roots of
tomato) were blended in 500 ml of 1% NaOCI solution
in a blender for 40 s. The suspension was sieved
through 100 and 400 mesh sieves. The retained eggs
were washed with water and transferred to a beaker
containing 100 m l of water. The number of eggs in the
suspension was determined with a counting dish under
a stereo-microscope and fecundity was calculated.
2.4. Photosynthetic pigments and nitrogen
Carotenoids, chlorophyll a, chlorophyll b and total
chlorophyll were determined by grinding 1 g fresh leaf
tissue from third leaflet of the third leaf of branches of
each plant separately in 80% acetone with a mortar and
pestle. The suspension was filtered through two What-
man filter papers (no. 1) in a Buchner funnel equipped
with a suction pump. The residue was again ground
with acetone and filtered. Final volume of the filtrate
was made to 100 ml and percent transmittance in the
spectrophotometer was read at 480 and 510 nm for
carotenoids (MacLachlan and Zalik, 1963) and 645
and 663 nm for chlorophylls (Mackinney, 1941).
Nitrogen content of leaves and roots was determined
by digesting dry powder (100 rag) in concentrated
H2SO4 (98%) and H202 (30%) in Kjeldahl flasks.
After digestion, sodium hydroxide and sodium silicate
solutions were added to neutralise the acidity. Later,
0.5 ml Nessler's reagent was added to 5 ml of the
74 M.R. Khan, M.W. Khan/Agriculture, Ecosystemsand Environment53 (1995) 71-81
solution and transmittance was read in a spectropho-
tometer at 525 nm (Linder, 1944).
2.5. Leaf epidermal characters
Pieces ( 1 cm 2) of fresh and mature leaves, fixed in
formaline-aceto-alcohol (FAA) and preserved in 70%
ethanol, were gently boiled in 40% HNO3 to separate
the epidermal peels. The peels were then stained with
iron alum and haematoxylin after washing in water
(Ghouse and Yunus, 1972), dehydrated in ethanol
series and mounted in DPX mountant. The numbers of
stomata and trichomes present on both the leaf surfaces
were counted and their sizes measured.
2.6. Statistical analysis
The means of observations for each treatment were
calculated. Data were subjected to analysis of variance
(ANOVA) for two-factor pot culture experiment, i.e.
NH3 (0, 76 and 152/zg m -3) and nematode (0, pre-,
post- and concomitant-treatments) and least significant
differences (LSD) were calculated at three probability
levels, i.e. P~<0.05, P~<0.01 and P<0.001 (Dospek-
hov, 1984). The experiment on effects of NH3 and
nematode on plant growth, yield, etc. had four controls
(C1, C2, C3, C4) and four exposed sets (T1, T2, T3,
T4). To test the individual effects ofNH 3and nematode
on plant growth etc. the means of uninoculated-unex-
posed plants (C 1) were compared with the inoculated-
unexposed treatments (C2, C3, C4) and uninoculated-
exposed (T1) plants. To test the combined effects, the
inoculated-exposed treatments (T2, T3, T4) were
compared with their respective controls (inoculated-
unexposed plants), i.e. C2, C3 and C4, respectively.
The data on nematode disease and reproduction were
subjected to a single factor ANOVA and this experi-
ment had three controls (C2, C3, C4) and three
exposed sets (T2, T3, T4). To test the significance of
differences between the treatments, inoculated-
exposed plants (T2, T3, T4) were compared with inoc-
ulated-unexposed plants (C2, C3, C4, respectively).
3. Results
3.1. Symptoms
Intermittent exposures of nematode inoculated or
uninoculated tomato plants to NH3 at 152 /xg m -3
caused yellowing of the leaf; NH3 at 76/xg m -3 did
not produce any visible injury. Root-knot nematodes
produced characteristic galls on roots of the inoculated
plants. There was no difference in NH3 injury on the
nematode inoculated or uninoculated plants. However,
the disease intensity (number of galls) was higher on
the plants exposed to 76/zg NH 3 m-3, regardless of
the mode of inoculation and exposure.
3.2. Plant growth
Meloidogyne incognita or intermittent exposures to
NH3 at 152/xg m - 3 caused significant decline in length
and fresh and dry weights of shoot and root, compared
with uninoculated and unexposed plants (Table 1).
The suppression of root growth due to 152/zg NH3 was
invariably significant at P ~<0.01. Nematode-inocu-
lated plants intermittently exposed to NH3 (T2, T3 and
T4) exhibited suppression in growth parameters in
comparison with their respective controls (C2, C3 and
C4). This effect was significant at P ~<0.01 for length
and at P ~<0.05 for dry weight of root in post and con-
comitant treatments (T3-T4) at 152/xg m -3 (Table
1). Corresponding values for pre-inoculation treatment
were significant at P ~<0.05 and P ~<0.01. Fresh weight
of root decreased significantly in pre-inoculation expo-
sure (T2) at both levels of NH3 and in post-inoculation
exposure (T3) at 152/zg m -3 (P~<0.05) compared
with C2 and C3, respectively. Overall, percent reduc-
tion in shoot was highest in concomitant treatment and
in root in post-inoculation treatment at 152/~g m -3.
According to the two-factor ANOVA, individual
effects (F value) of NH3 and nematode were signifi-
cant for all the considered plant growth parameters.
Interactive effect was, however, synergistically signif-
icant for fresh weight of root (Table 1).
3.3. ~eM
Intermittent exposures to NH3 at 152/xg m -3 and/
or M. incognita inhibited flowering and fruiting of
tomato. The decline in the number of fruits per plant
was significant in all the treatments of nematode
(P<0.01) or 152 /zg NH3 m -3 (P~<0.001). The
decline was significant at P ~<0.01 in infected-exposed
plants at 152/zg, and also at 76/xg m-3 in concomitant
treatment compared with their respective controls
(Table 2). NH3 at 76/zg m -3 significantly enhanced
 M.R. Khan, M. W. Khan/Agriculture, Ecosystems and Environment 53 (1995) 71--81 75

Table 1
Effects of NH3 and Meloidogyne incognito race 1 on the growth and dry matter production of tomato plants

Treatment NH3 Length (cm) Fresh weight (g) Dry weight (g)
(/zg m -3 )
Shoot Root Shoot Root Shoot Root

CI Uninoculated control 0 48.3 29.8 43.1 14.5 8.5 2.17

T1 Uninoculated 76 49.1 30.2 45.5 15.0 8.3 2.21
152 43.0* 24.8*** 40.0* 12.1"** 7.5* 1.71"**
C2 Inoculated control 0 41.9' 25.1"** 38.6** 12.8" 7.4** 1.83"*
T2 Pre-inoculation exposure 76 39.7 23.3 37.9 11.0" 7.1 1.72
152 37.9 22.1 * 37.0 10.8" 6.9 1.48**
C3 Inoculated control 0 40.6** 24.5*** 37.1"** 12.3"* 7.2** 1.88"
T3 Post-inoculation exposure 76 39.4 22.8 37.5 11.8 6.7 1.75
152 38.3 21.0'* 37.0 10.8" 6.3 1.61'
C4 Inoculated control 0 40.1'* 24.9*** 37.0*** 12.5" 7.2** 1.90"
T4 Concomitant-inoculation exposure 76 38.7 23.1 36.4 12.0 6.6 1.70
152 36.8 21.9"* 35.9 11.2 6.5 1.64"
F value NH3 (d.f.=2) 5.80S 13.60S 5.82S 11.46S 8.12S 16.41S
Nematode (d.f. = 3) 7.28S 9.15S 5.13S 17.50S 12.57S 23.17S
NH3× Nematode (d.f. = 6) NS NS NS 3.04S NS NS

Each value is the mean of five replicates. Asterisks indicate a significant difference from the respective control: *P_<0.05; **P_<0.01;
***P _<0.001 ; S, significant at P < 0.05; NS, not significant at P < 0.05.

Table 2
Effects of NH 3 and Meloidogyne incognita race 1 on the yield of tomato plants

Treatment NH 3 Number Fresh weight (g)

(/~g m -3)
Flowers Fruits Fruits per plant Mean fruit

CI Uninoculated control 0 14 13 426 32.8

TI Uninoculated 76 16" 13 404 31.1
152 13 10"** 283*** 28.3*
C2 Inoculated control 0 12" 10"** 279*** 27.9*
T2 Pre-inoculation exposure 76 13 9 229* 25.4
152 11 8** 198"** 24.7
C3 Inoculated control 0 13 11'* 298*** 27.1"*
T3 Post-inoculation exposure 76 12 10 239** 23.9
152 12 9** 211"** 23.5
C4 Inoculated control 0 12 11"* 294*** 26.7**
T4 Concomitant-inoculation exposure 76 12 9** 211"** 23.5
152 11 9** 209*** 23.2
F value NH3 (d.f. = 2) NS 10.71S 37.40S 4.72S
Nematode (d.f. = 3) NS 15.30S 21.47S 13.88S
NH3× Nematode (d.f. = 6) NS NS 3.29S NS

Each value is the mean of five replicates. Asterisks indicate a significant difference from the respective control: *P_<0.05; **P_<0.01;
***P_<0.001; S, significant at P_< 0.05; NS, not significant at P < 0.05.

f l o w e r p r o d u c t i o n . In o t h e r t r e a t m e n t s at 7 6 / z g m - 3 all the t r e a t m e n t s , b e i n g significant in p l a n t s e x p o s e d

the f l o w e r i n g w a s u n a f f e c t e d in c o m p a r i s o n with the to 1 5 2 / z g NH3 m - 3 (P~< 0.05) or i n o c u l a t e d w i t h the
control, but w h e n it w a s c o m p a r e d w i t h the c o r r e s p o n d - n e m a t o d e ( P ~ < 0 . 0 1 ) . The h i g h e s t d e c r e a s e in m e a n
ing fruiting, t h e r e w a s 18.7% ( a v e r a g e ) inhibition in fruit w e i g h t o c c u r r e d in the c o n c o m i t a n t - i n o c u l a t i o n
fruit setting. M e a n fruit w e i g h t o f t o m a t o d e c r e a s e d in t r e a t m e n t at 152/.*g m -3. T h e total w e i g h t o f fruits p e r
76 M.R. Khan, M.W. Khan/Agriculture, Ecosystemsand Environment53 (1995) 71-81

Table 3
Effects of NH3 and Meloidogyne incognitarace 1 on photosynthetic pigments (kLgg- 1 fresh weight of leaf) and nitrogen content of tomato
plants

Treatment NH3 CarotenoidsChlorophyll a Chlorophyllb Total Nitrogen(%)

(/zg m- 3) chlorophyll
Foliage Root

C1 Uninoculatedcontrol 0 6.81 843 329 1172 1.49 1.91

TI Uninoculated 76 6.75 819 315 1134 2.32*** 2.45***
152 6.20** 748** 305* 1054" 4.27*** 3.83***
C2 Inoculated control 0 6.04*** 773* 310 1084" 1.16" 2.20**
T2 Pre-inoculation 76 5.73 724 290 1015 3.41"** 2.59***
exposure 152 5.48** 692** 286* 979** 5.14"** 3.91"**
C3 Inoculatedcontrol 0 5.95*** 755** 306 1062"** 1.22 2.10"
T3 Post-inoculation 76 5.57 708 280* 989 3.23*** 2.60***
exposure 152 5.40** 680** 272** 953** 5.21"** 3.94***
C4 Inoculated control 0 5.95*** 763** 309 1073" 1.07" 2.18"*
T4 Concomitant-inoculation 76 5.60* 691" 295 987* 3.48*** 2.63***
exposure 152 5.38** 679** 280** 960** 5.01"** 3.88***
F value NH3 (d.f~=2) 18.30S 25.15S 11.63S 21.81S 127.46S 109.20S
Nematode (d.f.= 3) 31.72S 38.50S NS 35.60S 35.83S 43.17S
NH3 x Nematode (d.f. = 6) NS 4.39S 3.18S NS 12.26S 9.61S
Each value is the mean of five replicates. Asterisks indicate a signifcant difference from the respective control: *P<0.05; **P<0.01;
***P < 0.001; S, significant at P __.0.05; NS, not significant at P < 0.05.

plant significantly declined ( P ~<0.001 ) in all the treat-
ments of nematode a n d / o r 152/xg N H 3 m - 3 in com-
parison with the yield of their respective controls, being
lowest in the pre-inoculation treatment. At 7 6 / z g m - 3,
the decrease in inoculated plants was mostly significant
at P ~<0.01. Individual effects ( F value) of NH3 and
M. incognita were significant for the number of fruits,
mean fruit weight and total weight of fruits per plant.
The synergistic interaction was significant for weight
of fruits per plant (Table 2).
3.4. Photosynthetic pigments and nitrogen
NH3 inhibited the synthesis of carotenoids and chlo-
rophyll a, chlorophyll b and total chlorophyll in tomato
leaves. This inhibition was significant at 152/zg m -3
at P~<0.01 (Table 3). Meloidogyne incognita also
caused a significant decrease (P~<0.001) in the pig-
ments compared with the uninoculated plants ( C 1 ) .
Combined treatments o f the nematode and NH 3 at 152
/zg ( P ~<0.01 ) and 76/xg m - 3 ( p ~<0.05) significantly
suppressed the leaf pigments, compared with their
respective controls. Lowest carotenoids and chloro-
phyll a was recorded in concomitant treatments and
chlorophyll b and total chlorophyll in post-inoculation
treatment at 152 /zg m -3. In addition to individual
effects of NH3 and root-knot nematodes, their interac-
tive effects were also significant for chlorophyll a and
chlorophyll b (Table 3).
Foliage and roots of the exposed plants contained
significantly ( P ~<0.001) higher nitrogen compared
with their controls (Table 3). The nematode, however,
caused a significant ( P ~<0.05) decrease in leaf nitro-
gen, but root nitrogen increased ( P ~<0.01). Combined
treatments of NH 3 and M. incognita significantly
(P~<0.001) enhanced the nitrogen contents o f both
leaves and roots compared with their respective con-
trols. Highest nitrogen contents of leaves and roots
were recorded in post- and pre-inoculation exposures
at 152 /xg m -3, respectively. Interactive effects ( F
value) of NH3 and root-knot nematode were synergis-
tically significant for foliar nitrogen and antagonistic
for root nitrogen (Table 3).
3.5. Foliar epidermal characters
Root-knot nematode or NH 3 suppressed stomatal
numbers and their size as well as length of the stomatal
pores on both surfaces of the leaves (Table 4). These
effects were not significant at the lower level o f NH3.
Combined treatments of NH3 and root-knot nematode
in pre-inoculation-exposure at 152/xg m - 3 caused a
 M.R. Khan. M.W. Khan/Agriculture, Ecosystems and Environment 53 (1995) 71-81 77

Table 4
Effects of NH3 and Meloidogyne incognita race 1 on leaf epidermal characters of tomato plants

Treatment (NH3 Number (cm -2) Length of Stomata (/xm) Stomatal aperture
(/.Lg trichomes (/zm)
m -3) (/xm)
Stomata Trichomes Length Width Length Width

C1 Uninoculated control 0 U 19925 2207 158.3 28.27 18.34 13.18 5.61

L 35804 4823 171.8 29.57 18.51 13.26 5.57
TI Uninoculated 76 U 19815 2271 159.7 28.11 18.15 13.22 5.74
L 35712 4790 170.1 28.74 18.38 13.22 5.70
152 U 19160** 2473*** 165.2" 26.81" 17.03" 12.25" 6.81"**
L 34651** 5421"** 179.7' 26.19" 16.82" 12.19" 6.87***
C2 Inoculated control 0 U 17679*** 2115 157.4 27.29* 17.08" 12.48" 5.91"
L 29514*** 4691 169.5 27.58* 17.19" 12.33' 5.76
T2 Pre-inoculation exposure 76 U 17428 2297** 160.4 27.03 17.28 12.21 6.70***
L 28739 4785 172.8 27.31 16.99 12.17 6.81"
152 U 17393 2501"** 166.5"* 25.48* 16.62" 11.76" 6.82***
L 28156** 5498*** 178.6" 25.17" 16.41" 11.81" 6.78***
C3 Inoculated control 0 U 17523*** 2092 157.0 27.29* 17.18" 12.37" 5.84
L 29601*** 4571 169.8 27.66 17.29" 12.39" 5.84
T3 Post-inoculation exposure 76 U 17516 2208 161.0 27.13 17.13 12.08 6.65***
L 29640 4713 172.3 27.18 17.81 12.12 6.61"**
152 U 17074 2546*** 165.2" 25.30* 16.54" 11.71" 6.83***
L 28902 5513"** 170.5 25.39* 16.51" 11.58" 6.80***
C4 Inoculated control 0 U 17603* 2050 156.0 27.11" 17.13" 12.25" 5.71
L 29437* 4539 170.5 27.37* 17.29" 12.25" 5.66
T4 Concomitant-inoculation 76 U 17720 2296** 158.9 27.41 16.91 11.93 6.69***
exposure L 29753 4780** 172.8 27.15 17.07 11.98 6.58***
152 U 17281 2570*** 166.0"* 25.18' 16.73' 11.69" 6.67***
L 28907 5571"** 171.1 25.22* 16.39" 11.61" 6.73***
LSD P < 0.05 U 522 129 6.25 0.71 0.39 0.43 0.29
L 871 183 7.38 0.86 0.54 0.61 0.34
F value NH3 (d.f. = 2) U 8.13S 27.8S 6.81S 3.71S 41.50S 32.55S 19.51S
L 7.20S 31.6S 4.29S 3.42S 37.13S 23.70S 17.30S
Nematode (d.f.=3) U 22.50S NS NS 9.50S 16.55S 18.58S NS
L 19.57S NS NS 11.07S 22.81S 18.02S NS
NH3 × Nematode (d.f. = 6) U NS 3.25 NS NS NS NS 3.18
L 2.78 NS NS NS NS NS 2.71

u, upper leaf surface; L, lower leaf surface.

Each value is the mean of five replicates.
Asterisks indicate a significnt difference from the respective control. *P < 0.05; **P < 0.001; ***P< 0.01; S, significant at P < 0.05; NS, not
significant at P < 0.05.

s i g n i f i c a n t ( P ~< 0 . 0 1 ) d e c r e a s e in the n u m b e r s o f sto- at 1 5 2 / z g NH3 m - 3 ; NH3 at 1 5 2 / z g m - 3 a l o n e or w i t h

m a t a o n the l o w e r l e a f surface. S t o m a t a l size ( l e n g t h
a n d w i d t h ) , a n d l e n g t h s o f s t o m a t a l p o r e s w e r e signif-
icantly s u p p r e s s e d at P ~<0,001 in all the t r e a t m e n t s o f
NH 3 (152/zg m -3) and the nematode (T2-T4) com-
pared with their controls (C2-C4). Lowest numbers
o f s t o m a t a w e r e r e c o r d e d o n u p p e r a n d l o w e r l e a f sur-
f a c e s in p o s t - a n d p r e - i n o c u l a t i o n s t r e a t m e n t s at 152
/zg m - 3, r e s p e c t i v e l y . S m a l l e s t s t o m a t a were, h o w e v e r ,
observed on plants receiving pre-inoculation-exposure
M. incognita ( T 2 - T 4 ) i n d u c e d a s i g n i f i c a n t i n c r e a s e
( P ~<0 . 0 0 1 ) in the w i d t h o f s t o m a t a l p o r e s c o m p a r e d
w i t h their r e s p e c t i v e c o n t r o l s ( C 2 - C 4 ) , b e i n g w i d e s t
in the p o s t - i n o c u l a t i o n t r e a t m e n t . N e m a t o d e - i n o c u -
lated p l a n t s e x p o s e d to 7 6 p,g m - 3 also s h o w e d signif-
icantly w i d e r p o r e s o f s t o m a t a o n b o t h l e a f s u r f a c e s at
P ~<0.001. T h e i n c r e a s e in p o r e w i d t h in n e m a t o d e -
i n o c u l a t e d p l a n t s w a s not significant. T h e n u m b e r o f
t r i c h o m e s significantly ( P ~ 0 . 0 0 1 ) i n c r e a s e d o n the
78 M.R. Khan, M. W. Khan/Agriculture, Ecosystems and Environment 53 (1995) 71-81

Table 5
Effects of NH3 on disease intensity reproduction and fecundity ofMeloidogvne incognita race 1 on tomato plants

Treatment NH 3 Number of galls per Number of egg masses Number of

(/xg m -3) per eggs per egg
mass
Root g fresh Root g fresh (fecundity)
system weight of system weight of
root root

C2 Inoculated control 0 131 10.23 114 8.91 229

T2 Pre-inoculation exposure 76 145 13.18** 129 11.72* 211
152 95*** 8.79 63*** 5.83** 197"
C3 Inoculated control 0 138 11.22 113 9.19 237
T2 Post-inoculationexposure 76 158" 13.38' 135" 11.44* 206*
152 106"** 9.72 54*** 4.95*** 172"*
C4 Inoculated control 0 135 10.8 118 9.44 224
T4 Concomitant-inoculation 76 156" 13.0" 141* 11.75* 218
exposure 152 92*** 8.21"* 41"* 3.66*** 141"**
Each value is the mean of five replicates, Asterisks indicate a signfiicant difference from the respective control: *P < 0.05; **P < 0.01;
***P <0.001.

leaves of nematode-inoculated or inoculated plants 3.6. Nematode disease

exposed to NH3 at 152/xg m -3, being highest in the
Root galling caused by M. incognita in the nema-
concomitant-inoculation treatment. Similar effects of
tode-inoculated plants was enhanced significantly
7 6 / z g N H 3 m - 3 were obtained in the concomitant-
(P~<0.05) by exposure to 76 /xg N H 3 m - 3 (except
inoculation treatment on both leaf surfaces and in pre-
pre-inoculation exposure), compared with the controls.
inoculation treatment on the upper leaf surface
This effect was highest in post-inoculation treatment
compared with their respective controls at P~< 0.01.
(Table 5). Root galling was suppressed significantly
Trichome lengths were enhanced significantly
(P~<0.001) on plants exposed to 152/xg NH 3 m -3.
( P ~<0.05) in plants exposed to 152/zg m - 3 alone or
Lowest numbers of galls were recorded in plants o f
with the nematode in pre-inoculation NH3 exposure.
concomitant-inoculation treatment. A similar trend
Similar effects on trichome lengths were also recorded
occurred in numbers of galls per gram fresh weight of
on the upper leaf surface of plants which received post-
roots. Reproduction o f the nematode (numbers o f egg
inoculation ( P ~<0.05) and concomitant-inoculation
masses per root system or per gram fresh weight of
(P~<0.01) treatments at 152 /xg m -3. The longest
root) was enhanced at 76/zg m - 3 ( p ~<0.05) and sup-
trichomes were recorded when the uninoculated plants
pressed at 152/zg m - 3 ( p ~<0.001 ). Fecundity (num-
were exposed to NH 3 at 152 /xg NH 3 m -3. Similar
ber o f eggs per egg mass) o f the nematode was
increase in trichome lengths occurred in nematode-
significantly inhibited at P~< 0.001 in post- and con-
inoculated plants in pre-inoculation treatment at the
comitant-inoculation and at P ~<0.05 in pre-inoculation
same level o f NH3. According to A N O V A , individual
treatments at 152 /zg m -3 and at P~<0.05 in
effect o f NH3 was significant for all the considered
post-inoculation treatment at 76 /zg m - 3 compared
epidermal characters. The effect of M. incognita was
with the respective controls (Table 5). The lowest
not significant for number and length o f trichomes or
numbers of eggs were recorded in the egg masses
width o f stomatal pore. The interactive effect was syn-
obtained from the plants o f concomitant-inoculation
ergistically significant for width o f stomatal pore on
treatments at 152 ~ g NH 3 m -3.
both leaf surfaces and number of trichomes on the upper
leaf surface and antagonistically for the number of sto-
4. Discussion
mata on the lower leaf surface (Table 4).
A m m o n i a effects on photosynthetic pigments
appeared as a yellowing of tomato leaves. Gas ammo-
 M.R. Khan, M. W. Khan/Agriculture, Ecosystems and Environment 53 (1995) 7141
nia, diffusing through stomata may have inhibited syn-
thesis of carotenoids and chlorophylls. Breakdown or
denaturing of chlorophylls may have also occurred,
because NH 3 causes excessive loss of magnesium ions
from the foliage of exposed plants (Van Dijk and Roe-
lofs, 1988) as Mg + occupies a central position in the
tetrapyrol ring of the chlorophyll molecule. Suppres-
sion of leaf pigments in nematode-infected plants may
also have resulted from impaired absorption and trans-
location of nutrients from the roots due to pathogenic
effects of M. incognita (Bergeson, 1966). Khan and
Khan (1987) offered similar reasons for a decrease in
carotenoids and chlorophylls of tomato plants infected
with root-knot nematodes.
Conversion of ammonia into nitrogenous com-
pounds and their accumulation in leaves may account
for the significant increase in foliar nitrogen of the
exposed plants. Some nitrogen may have been derived
from the soil as well, as there was about a 63% and
170% increase in soil nitrogen in the pots exposed to
76/xg and 152 ~g NH 3 m -3, respectively (data not
presented in tabular form). Ammonia has been found
to increase nitrogen deposition in forest stands by as
much as 10-20 times the natural supply of 6-10 kg N
ha-1 year-~ (Anonymous, 1986b). In M. incognita
infected plants, the nematode-induced inhibition in the
upward movement of nutrients (Bergeson, 1966) was
probably responsible for greater nitrogen content of
roots and lower nitrogen content of leaves. In the com-
bined treatments ofNH 3 and root-knot nematode, foliar
nitrogen was synergistically enhanced, i.e. the increase
was greater than the sum of the increases caused by
NH 3 and nematode individually. This synergistic inter-
action possibly resulted from greater uptake of NH 3 by
the leaves. During the accelerated transpiration of
plants infected with M. incognita (Odihirin, 1971),
stomata may have opened wide, leading to greater dif-
fusion of NH 3 into the leaves, as we observed wider
pores of stomata in the nematode-infected plants. Inter-
action of NH3 and the nematode was antagonistic (neg-
ative) for root nitrogen, i.e. the sum of increases in
nitrogen caused by NH 3 and M. incognita individually
was greater than their combined effect. This antagonis-
tic effect may have been caused by the impaired func-
tioning of the nematode-infected roots in relation to
absorption of nutrients and downward translocation of
photosynthates (Bergeson, 1966).
79
The physiological disorders induced by M. incognita
were responsible for the suppressions in plant growth
and yield of tomato. Root-knot nematodes can reduce
the yield of vegetables by 5-43% depending upon the
species and geographical region (Sasser, 1979). The
extremely high levels of total nitrogen in the leaves and
roots of plants exposed to NH 3, especially at the higher
concentration, indicates that the plants may have suf-
fered from a severe nitrogen overload, resulting in sup-
pressed plant growth, yield and photosynthetic
pigments. Excessive accumulation of nitrogen in soil
and plant parts causes suppressions of plant growth and
yield (Krauss et al., 1986; Van Dijk and Roelofs,
1988).
The joint action of NH3 (at 76 /zg m -3) and M.
incognita was synergistic, leading to greater reduction
in plant growth, yield and leaf pigments of tomato.
Wider stomatal pores in the inoculated-exposed plants
than the inoculated or exposed plants suggests that NH 3
uptake by leaves may have been enhanced because of
extra opening of stomata resulting from the interaction
of NH3 (at 76/zg m -a) and M. incognita. The signif-
icant increase in root galling of plants exposed to the
lower level of NH3 shows that the increase in nitrogen
content of soil and root (63% and 36%, respectively)
favoured the root-knot nematode. Excess of nitrogen
in soil has been found to stimulate development of
nematode diseases (McClure and Viglierchio, 1966).
NH 3 at 152/zg m-3, and the nematode jointly caused
antagonistic reductions in growth, yield and leaf pig-
ments of tomato plant. Diffusion of NH3 may have been
greater as stomatal pores were widest in the infected
plants exposed to 152/zg NH3 m -3. Low root galling
and decline in production of the nematode indicates
that 170% increase in soil nitrogen and 103% increase
in root nitrogen were harmful for the nematode. Such
poor root galling by the nematode eventually resulted
in less suppressed plant growth, yield and photosyn-
thetic pigments. Concentration-dependent stimulatory
(268/zg m -3) and inhibitory (571 /xg m -3) effects
of SO2 on root-knot nematodes have been recently
demonstrated by Khan and Khan (1993).
Sedentary females of root-knot nematodes which are
responsible for parasitism obtain their nutrition from
giant cells which surround their head region. The quan-
tity and/or quality of nutrients in giant cells may have
been possibly reduced owing to poor health of the
plants exposed to 152/zg NH3 m-3. This poor nutrient
80 M.R. Khan, M.W. Khan/Agriculture, Ecosystems and Environment 53 (1995) 71-81
status of the giant cells would have resulted in inhibi-
tion of egg mass production. Decline in the numbers of
eggs per egg mass (fecundity) may have been caused
by the inadequate supply of nutrients to the females.
Egg mass production by root-knot nematodes is sup-
pressed in nutrient-deficient hosts (Oteifa, 1953).
Reproduction of Heterodera glycines was found to be
suppressed in soybean plants exposed to 490/zg 03
m -3 (Weber et al., 1979).
The decrease in the number and size of stomata
seems to be a morphological adaptation of tomato
plants to check the diffusion of gaseous air pollutants,
including NH 3. Similar epidermal adaptations devel-
oped in tomato plants in response to SO2 (Khan and
Khan, 1993). The increase in the length and number
of trichomes may be a response of the plants to the air
pollutants. Trichomes provide an outer line of defence
for plants against stresses imposed from the atmosphere
(Levin, 1973). Widening of the stomatal pores in
exposed and/or inoculated plants indicates that despite
the presence of these defences, they did not overcome
the adverse effects of the stress and thus damage was
sustained. The width of the stomatal pore mostly
showed a positive relationship with the percent reduc-
tion in different parameters, e.g. stomatal apertures
were widest in those treatments of nematode and NH3
(152 /zg m -3) that caused highest suppressions in
plant growth, yield, etc.
The present investigation revealed that NH 3, at 76
/xg m - 3, is not toxic to tomato but its favourable impact
on root-knot nematode is alarming. Thus, low levels of
NH3 may enhance the nematode population density
substantially under ambient conditions, with plants
being liable to sustain damage from these pests at levels
of the gas that are too low to have a direct impact on
the plant themselves.
Acknowledgements
The senior author wishes to dedicate the present
paper to his beloved father Abba Aziz, who left this
"Ryden, J.C., Whitehead, D.C., Lockyer, D.R., Thompson, R.B.,
world on 30 August 1993 and who was a constant
source of inspiration. The author also acknowledges
research associateship from C.S.I.R., New Delhi, India.
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