Soil Biology & Biochemistry 34 (2002) 181±188

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Response of lentil under controlled conditions to co-inoculation with

arbuscular mycorrhizal fungi and rhizobia varying in ef®cacy
L.J.C. Xavier a, J.J. Germida b,*
a
Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
b
Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Received 25 August 2000; received in revised form 31 July 2001; accepted 8 August 2001

Abstract
This study assessed the co-inoculation response of lentil (Lens culinaris cv. Laird) to arbuscular mycorrhizal fungi (AMF) and effective
and ineffective Rhizobium leguminosarum bv. viceae strains. Plants were inoculated with the AMF species Glomus clarum NT4 or G.
mosseae NT6 and/or nine Rhizobium strains varying in ef®cacy, and grown for 110 d in soil containing indigenous AMF and rhizobia. The
effectiveness of the Rhizobium strains on the N nutrition of 6-week-old lentil grown under gnotobiotic conditions was correlated (P , 0.045;
r ˆ 0.64) with the N nutrition of 110-d-old lentil grown in natural soil. The growth and yield responses of lentil to co-inoculation with AMF
and Rhizobium strains depended on the particular AMF-Rhizobium strain combination. In some cases, the productivity of lentil inoculated
with an effective Rhizobium strain was signi®cantly (P , 0.05) reduced by an apparently incompatible AMF species compared to the
Rhizobium treatment. In contrast, the yield of lentil inoculated with some ineffective Rhizobium strains was signi®cantly (P , 0.05) enhanced
by an apparently compatible AMF species compared to the Rhizobium treatment. However, maximum lentil productivity was achieved only
in treatments with effective Rhizobium strains or co-inoculation treatments with effective Rhizobium strains and a compatible AMF species.
Correlation analyses indicated that total shoot dry matter production was signi®cantly (P , 0.05) correlated with the total shoot N
(P , 0.0001; r ˆ 0.94) and P content (P , 0.0001; r ˆ 0.96), indicating that this response was mediated by enhanced N and P nutrition.
Furthermore, a (P , 0.05) positive relationship between AMF colonization of roots and total shoot dry matter production (P , 0.0187;
r ˆ 0.43), N (P , 0.0116; r ˆ 0.45) and P content (P , 0.0183; r ˆ 0.43) of shoots was also noted. Our results suggest that synergistic
interactions between AMF and Rhizobium strains can enhance lentil productivity. q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Arbuscular mycorrhizal fungi; Rhizobia; Interactions; Lentil; Yield

1. Introduction leguminous hosts to many different AMF species (Ianson

Arbuscular mycorrhizal fungi (AMF) and Rhizobium spp.
form an intimate association with leguminous plants, which
is often termed the `tripartite symbiosis'. Plants bene®t from
this association in many ways including enhanced plant
growth, yield, and nutrient content, especially N and P
(Pacovsky et al., 1986; Azcon et al., 1991; Ianson and
Linderman, 1993; Ahmad, 1995; Saxena et al., 1997), and
in other less well-de®ned ways (Azcon and El-Atrach,
1997). In addition, research reports suggest that plant bene-
®ts derived from the tripartite symbiosis are superior to that
of uninoculated control plants or that of plants inoculated
with either AMF or Rhizobium alone.
Many workers have assessed the inoculation response
of nodulated (i.e. with a single Rhizobium spp. strain)
* Corresponding author. Tel.: 11-306-966-6836; fax: 11-306-966-6881.
E-mail address: germida@sask.usask.ca (J.J. Germida).
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(01)00165-1
and Linderman, 1993; Saxena et al., 1997; Nwoko and
Sanginga, 1999; Houngnandan et al., 2000). These studies
indicate that plant response to AMF inoculation varies
signi®cantly with AMF species. Furthermore, some reports
suggest that these differences are mediated by speci®c inter-
endophyte interactions between the Rhizobium strain and
the different AMF species, which become evident under
N-de®cient conditions (Ianson and Linderman, 1993).
However, it may be dif®cult to predict the co-inoculation
response of a leguminous host with only one Rhizobium
strain. Relatively fewer workers have evaluated the effect
of mycorrhizal leguminous hosts (i.e. inoculated with a
single AMF species or colonized by indigenous AMF) to
different Rhizobium strains (Vejsadova et al., 1992;
Thiagarajan and Ahmad, 1993). These studies concluded
that Rhizobium strains had no speci®c interactions with
AMF species, and that all the nodulated plants bene®ted
from the AMF association.
182 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188
Table 1
Sources of the Rhizobium leguminosarum bv. viceae strains and isolates
used, and their effectiveness on lentil
Strain Effective a Source
92A1 No Nitragin Inoculants, Liphatech
Corporation, Milwaukee, WI
LE13 No ICARDA b, Syria
LX66 No Saskatchewan lentil ®elds c
RGL14 d Yes MicroBio Rhizogen
Corporation, Saskatoon, SK
PB101 d Yes Philom Bios Inc. Saskatoon, SK
LX72, LX77, LX81, LX84 Yes Saskatchewan lentil ®elds c
a
Effectiveness was determined based on the ability of the Rhizobium
strains to enhance lentil (cv. Laird) growth and N content after six weeks
of growth (Johnny, 1999). Germinated lentil seeds were inoculated with ca.
10 8 colony forming units per ml and grown in Leonard jars (Leonard, 1943)
containing an N-free nutrient solution.
b
ICARDA, International Center for Agricultural Research in Dry Areas.
c
All LX isolates were obtained from the nodules of lentil plants collected
from inoculated ®elds at Moose Jaw, Rosetown, Drinkwater or Allan,
Saskatchewan.
d
Commercial inoculant.
The effects of speci®c interactions between different
AMF species and Rhizobium strains on legume plants
have been examined (Azcon et al., 1991; Ahmad, 1995).
For example, Azcon and co-workers (1991) evaluated
the interactions between Glomus fasciculatum, G.
mosseae or G. caledonium and six R. meliloti strains,
and found that plant growth and yield were dependent
upon the speci®c combination of AMF species and
Rhizobium strain. A similar response was reported by
Ahmad (1995) for kidney bean. These results indicate
that AMF and Rhizobium interact intimately with the
host and therefore, in¯uence plant productivity. Further-
more, it has been demonstrated that this tripartite
symbiosis is not a random occurrence, but one that is
regulated by the legume host (Duc et al., 1989;
Bradbury et al., 1991).
Research reports clearly indicate that the response of
a legume host to Rhizobium can be modi®ed by the
AMF species involved in the tripartite association.
However, some important questions relating to the
ef®cacy of endosymbionts, i.e. AMF and the Rhizobium
strain, have not been previously addressed: for example
(1) can the effectiveness of an effective Rhizobium
strain on the host be reduced because of an incompati-
ble AMF association?; (2) can a compatible AMF
association with a legume host render an ineffective
Rhizobium strain effective?; and (3) how important is
the effectiveness of the Rhizobium symbiosis to the
legume host involved in the tripartite symbiosis?. This
study assessed the effects of interactions between
the AMF species G. clarum or G. mosseae and R.
leguminosarum bv. viceae strains varying in ef®cacy
on the growth, yield and nutrient content of lentil
grown in soil containing indigenous AMF and rhizobia.
2. Materials and methods
2.1. AMF inocula
Monospeci®c cultures of the AMF species Glomus
clarum Nicolson and Schenck (INVAM no. SA101) NT4
and G. mosseae (Nicolson and Gerdemann) Gerdemann &
Trappe (INVAM no. SA103) NT6 which are dominant in
Saskatchewan soils (Talukdar and Germida, 1993), were
produced in a (1:1) soil:sand mix using maize as host. The
AMF strain Glomus clarum NT4 has been recently renamed
(Kennedy et al., 1999); but the original nomenclature has
been retained here for cross-reference to our previous work.
The AMF inocula consisting of spores, external mycelium
and AMF-colonized roots were stored at 48C. At the time of
use, the G. clarum inoculum contained 330 propagules per
50 g and the G. mosseae inoculum contained 390 propa-
gules per 50 g, as determined using a most probable number
assay (Porter, 1979).
2.2. Rhizobium strains
The Rhizobium strains used in this study are listed in
Table 1. The Rhizobium strain LE13 was previously identi-
®ed as ineffective on lentil (Bremer et al., 1990; L.M.
Nelson, pers. comm.). Under gnotobiotic conditions, lentil
inoculated with strains 92A1 and LX66 exhibited symptoms
similar to that of LE13 (Johnny, 1999). Strains RGL14 and
PB101 are commercial inoculants widely used in Saskatch-
ewan. Strains RGL14, PB101, LX72, LX77, LX81 and
LX84 enhanced the growth and N content of 6-week-old
lentil plants grown under gnotobiotic conditions. For this
study, Rhizobium cultures were prepared by inoculating a
loopful of cells from a stock culture into 100 ml of yeast
extract mannitol broth and growing in a gyrotory shaker
(150 rev min 21) at 288C for 72 h. The Rhizobium cultures
contained ca. 10 8 colony forming units per ml (cfu ml 21).
2.3. Soil
The loamy sand soil used in this study was collected from
Bradwell, Saskatchewan. The soil was air-dried, sieved
(4 mm) and characteristics determined at the Enviro-Test
Laboratories, Saskatoon, Saskatchewan. The characteristics
of the soil were as follows: N, 10 mg g 21; P, 13 mg g 21;
K, 300 mg g 21; S, 9.4 mg g 21; B, 0.72 mg g 21; Cu,
0.56 mg g 21; Fe, 11.2 mg g 21; Mn, 9.6 mg g 21; Zn,
3.18 mg g 21; pH, 8.1; EC, 0.2 mS/cm; organic carbon,
1.1%; and organic matter, 1.9%. The soil was mixed 1:1
(w/w) with silica sand and two kilograms potted in 15-cm
diameter pots. To each pot, 200 ml of distilled water was
added and the soil:sand mix allowed to equilibrate for 10 d
before planting. The number of indigenous rhizobia and
AMF propagules in the soil:sand mixture were 5.5 per g
and 170 per 50 g respectively, as determined using the
most probable number assay of Somasegaran and Hoben
(1994) for rhizobia and Porter (1979) for AMF.
 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188 183

Table 2
Mean (n ˆ 4) shoot and root dry weight (nodule 1 root) of lentil plants inoculated with the AMF species G. clarum NT4 or G. mosseae NT6 and/or nine
Rhizobium strains and grown for 110 d in soil containing indigenous AMF and rhizobia. Treatment means were separated using the least signi®cant difference
(LSD) test at P , 0.05 [Mean (R) refers to the mean of the main effect of rhizobia; Mean (A) refers to the mean of the main effect of AMF]

Rhizobium treatment AMF treatment

Shoot dry weight a (g/pot) Root dry weight b (g/pot)

None NT4 NT6 Mean (R) None NT4 NT6 Mean (R)

None 1.40 3.63 3.49 2.84 1.55 2.38 2.51 2.14

92A1 3.48 3.53 3.11 3.37 1.74 1.22 1.18 1.38
LE13 2.77 2.61 3.49 2.96 2.41 1.51 0.80 1.57
LX66 2.44 2.85 3.61 2.96 2.59 1.63 1.84 2.02
RGL14 2.86 2.70 6.99 4.18 1.87 2.16 1.53 1.85
PB101 3.08 6.48 5.19 4.91 2.70 1.34 1.04 1.69
LX72 3.35 6.56 4.75 4.89 1.86 4.28 1.45 2.53
LX77 3.49 5.21 3.65 4.11 2.48 4.44 2.11 3.01
LX81 3.26 5.31 4.60 4.39 3.37 2.33 3.29 3.00
LX84 2.38 3.91 3.16 3.15 2.93 3.90 2.50 3.11
Mean (A) 2.85 4.28 4.20 2.35 2.52 1.82
a
LSD for AMF effect, 0.14 g; rhizobia effect, 0.25 g; AMF £ rhizobia interaction effect, 0.44 g.
b
LSD for AMF effect, 0.16 g; rhizobia effect, 0.29 g; AMF £ rhizobia interaction effect, 0.50 g.

2.4. Inoculation and plant growth
For all treatments, four lentil (Lens culinaris cv. Laird)
seeds were placed at 5-cm depth from the soil surface. For
the AMF treatments, 10 g of the appropriate AMF inoculum
(i.e. NT4 or NT6) was placed in soil over which seeds were
planted. For the Rhizobium treatments, 2.0 ml of Rhizobium
culture was added over seeds, as previously described by
Ahmad (1995), Ames et al. (1991), and Azcon et al. (1991).
For the AMF 1 Rhizobium treatments, 10 g of the AMF
inoculum was placed in soil over which seeds were planted
and 2.0 ml of Rhizobium culture was added. Plant count was
reduced to two per pot following seedling emergence.
Plants were grown in a growth chamber with the following
conditions: 258C, 16 h day and 208C, 8 h night and
375±400 mm m 22 s 21 of irradiance. Soil was maintained
at ,60% moisture holding capacity by periodically adding
water. Pots were randomized in the growth chamber and
repositioned once a week.
modi®ed gridline intersect method of Giovannetti and
Mosse (1980).
2.6. Statistics
The study was a 3 (control, NT4 and NT6) £ 10 (control
plus nine Rhizobium strains) factorial resulting in 30 treat-
ment combinations. Percentage values for AMF coloniza-
tion were subjected to angular transformation before
statistical analysis. All data were analyzed using the
ANOVA procedure, and means separated using Fisher's
protected least signi®cant difference (LSD) test. All statis-
tical procedures including Pearson's partial correlation
analysis were carried out using PC-SAS (SAS, 1997).
Unless otherwise indicated, all treatment means were tested
for signi®cant differences at P , 0.05.
3. Results
3.1. Dry matter production

2.5. Plant harvest and parameters
Plants were harvested at 110 d after planting. The above
ground plant material was separated, dried (658C; 48 h) and
weighed. Seeds were separated from the dried shoot
material and weighed. Harvest index (partitioning of dry
matter into seed) (HI) was expressed as a percentage of
the total aboveground dry matter. Shoot and seed material
was digested using an H2SO4 ±H2O2 mixture, and N and P
concentrations for shoot and seed material determined
separately (Thomas et al., 1967). However, results are
presented as total shoot (shoot 1 seed) N and P. The percen-
tage of AMF-colonized root length was determined using a
The effect of G. clarum NT4 or G. mosseae NT6
inoculation on the shoot growth of plants was not signi®-
cantly different from each other (Tables 2 and 6). However,
the shoot dry weight of lentil plants inoculated with NT4 or
NT6 was higher than that of the uninoculated control plants,
regardless of the Rhizobium strains (Table 2). In contrast,
there were signi®cant differences between Rhizobium
strains in their ability to enhance shoot growth, regardless
of the AMF treatment. Correlation analysis of lentil shoot
dry weight data after 6 weeks under gnotobiotic conditions
and after 110 d in non-sterile soil revealed a positive rela-
tionship (P , 0.09; r ˆ 0.59) for all Rhizobium strains
except strain LX84. Co-inoculation with AMF and rhizobia
184 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188

Table 3
Mean (n ˆ 4) seed yield and harvest index of lentil plants inoculated with the AMF species G. clarum NT4 or G. mosseae NT6 and/or nine Rhizobium strains
and grown for 110 d in soil containing indigenous AMF and rhizobia. Treatment means were separated using the least signi®cant difference (LSD) test at
P , 0.05 [Mean (R) refers to the mean of the main effect of rhizobia; Mean (A) refers to the mean of the main effect of AMF]

Rhizobium treatment AMF treatment

Seed yield a (g/pot) Harvest index b

None NT4 NT6 Mean (R) None NT4 NT6 Mean (R)

None 0.48 0.16 0.56 0.40 26 4 14 15

92A1 0.64 0.70 0.30 0.55 16 17 9 14
LE13 1.12 0.49 0.45 0.69 29 16 12 19
LX66 0.56 0.70 0.81 0.69 19 20 19 19
RGL14 1.42 1.16 0.55 1.04 34 29 7 23
PB101 1.15 0.45 0.58 0.72 27 7 10 15
LX72 1.49 1.53 0.47 1.16 31 19 9 20
LX77 1.82 1.84 0.72 1.46 34 26 17 26
LX81 1.21 0.71 1.50 1.14 27 12 25 21
LX84 0.58 1.48 1.04 1.03 20 27 25 24
Mean (A) 1.05 0.92 0.70 26 18 14
a
LSD for AMF effect, 0.07 g; rhizobia effect, 0.13 g; AMF £ rhizobia interaction effect, 0.23 g.
b
LSD for AMF effect, 1; rhizobia effect, 3; AMF £ rhizobia interaction effect, 5.

signi®cantly increased the shoot biomass of plants
compared to the uninoculated control. Furthermore, the
AMF species signi®cantly altered the effect of the same
Rhizobium strain on shoot growth (Table 2). Interactions
between the AMF G. clarum NT4 and ®ve of the six effec-
tive Rhizobium strains PB101, LX72, LX77, LX81 and
LX84 (i.e. under gnotobiotic conditions: Table 1) resulted
in superior growth compared to that co-inoculated with G.
mosseae NT6. In contrast, two of the three ineffective
Rhizobium strains LE13 and LX66 (i.e. under gnotobiotic
conditions: Table 1) were effective on lentil when combined
with the AMF G. mosseae NT6. The shoot growth response
of lentil to either AMF species and four of the six effective
Rhizobium strains was signi®cantly higher than that of lentil
inoculated with the Rhizobium strain alone, indicating that
the native AMF were ineffective. Alternatively, this
re¯ected the bene®t lentil derived from a compatible and
speci®c AMF-Rhizobium strain combination. The effective-
ness of an ineffective strain was enhanced by a compatible
AMF species for two of three strains (e.g. LE13 or
LX66 1 NT6). In contrast, the maximum bene®t potentially
obtainable from a compatible AMF-Rhizobium strain (e.g.
RGL14 1 NT6 or LX77 1 NT4) association was signi®-
cantly reduced in an incompatible AMF-Rhizobium strain
association (RGL14 1 NT4 or LX77 1 NT6), despite
inoculation with an effective Rhizobium strain (RGL14 or
LX77).
Statistical analysis revealed that the AMF species
G. clarum NT4 signi®cantly enhanced the total root dry
weight (nodule 1 root dry weight) of plants compared
with G. mosseae NT6-inoculated or control plants, irrespec-
tive of the Rhizobium strain in combination (Tables 2 and 6).
Similarly, irrespective of the AMF species, the total root dry
weight varied signi®cantly between plants inoculated with
the different rhizobia (Table 2). For three of nine Rhizobium
strains, co-inoculation with G. clarum NT4 signi®cantly
increased the total root dry weight of plants inoculated
with the same Rhizobium strain, compared to plants
inoculated only with the Rhizobium strain.
Analysis of the main AMF treatment effect on lentil yield
revealed that the native AMF community was more effec-
tive at enhancing lentil yield compared with G. clarum NT4
or G. mosseae NT6, regardless of the Rhizobium strains
(Tables 3 and 6). Of the two AMF species, G. clarum
NT4 was more effective than G. mosseae NT6, regardless
of the Rhizobium strain. The Rhizobium strains signi®cantly
altered seed yield, regardless of the AMF treatment
(Table 3). Inoculation of lentil with some of the Rhizobium
strains alone (e.g. RGL14, PB101 and LE13) produced the
highest seed yield compared to co-inoculation with NT4 or
NT6. In some other cases, co-inoculation of lentil with NT4
and the Rhizobium strains produced the highest yield (e.g.
92A1, LX72 and LX77). For strains LX66 and LX81, the
yield was highest when combined with G. mosseae NT6. In
contrast, it appeared that inoculation with either AMF
species enhanced the yield of LX84-inoculated lentil,
compared to the Rhizobium treatment alone. Furthermore,
an incompatible AMF species reduced the yield of lentil
inoculated with an effective Rhizobium strain (e.g.
LX81 1 NT4, LX72 or LX77 1 NT6). On the other hand,
seed yield from an ineffective Rhizobium-lentil association
was enhanced when combined with a compatible AMF
species (e.g. LX66 1 NT6).
The HI of control plants was signi®cantly higher than that
of plants inoculated with NT4 or NT6, regardless of the
Rhizobium strain (Tables 3 and 6). Irrespective of the
AMF species in combination, inoculation of lentil with
some Rhizobium strains increased the HI compared to others
 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188 185

Table 4
Mean (n ˆ 4) total shoot (shoot 1 seed) N and P of lentil plants inoculated with the AMF species G. clarum NT4 or G. mosseae NT6 and/or nine Rhizobium
strains and grown for 110 d in soil containing indigenous AMF and rhizobia. Treatment means were separated using the least signi®cant difference (LSD) test
at P , 0.05 [Mean (R) refers to the mean of the main effect of rhizobia; Mean (A) refers to the mean of the main effect of AMF]

Rhizobium treatment AMF treatment

Total shoot N a (mg/pot) Total shoot P b (mg/pot)

None NT4 NT6 Mean (R) None NT4 NT6 Mean (R)

None 22.62 40.29 46.05 36.32 5.22 7.29 8.59 7.03

92A1 43.25 48.63 34.59 42.16 9.14 10.61 7.10 8.95
LE13 50.29 35.56 39.16 41.67 10.22 7.85 9.20 9.09
LX66 33.80 36.70 53.42 41.31 5.97 7.60 9.74 7.77
RGL14 57.41 58.14 92.11 69.22 10.18 9.76 15.32 11.75
PB101 52.35 81.88 54.83 63.02 9.66 13.49 11.22 11.45
LX72 63.11 112.34 60.51 78.66 10.45 17.48 9.73 12.55
LX77 74.89 100.63 55.57 77.03 12.16 14.93 8.78 11.95
LX81 70.38 75.02 81.47 75.62 10.57 12.36 12.35 11.76
LX84 40.84 70.89 56.77 56.17 6.35 10.59 8.48 8.47
Mean (A) 50.89 66.00 57.45 8.99 11.20 10.05
a
LSD for AMF effect, 4.24 mg; rhizobia effect, 7.74 mg; AMF £ rhizobia interaction effect, 13.36 mg.
b
LSD for AMF effect, 0.64 mg; rhizobia effect, 1.17 mg; AMF £ rhizobia interaction effect, 2.02 mg.

(Tables 3 and 6). Inoculation of lentil with Rhizobium
strains and/or AMF signi®cantly altered the HI compared
to uninoculated plants. Inoculation of lentil with ®ve of nine
Rhizobium strains resulted in statistically similar or higher
HI compared to those co-inoculated with rhizobia and AMF
species. For example, lentil inoculated with the Rhizobium
strains PB101, LE13, LX72 or LX77 had higher HI
compared to plants co-inoculated with AMF and the
Rhizobium strains. Furthermore, in some instances, for the
same Rhizobium strain, co-inoculation with one AMF
species (e.g. 92A1 or RGL14 1 NT4) signi®cantly
enhanced the HI compared with the other AMF species.
There was no particular trend observed for the HI of co-
inoculated plants. However, co-inoculation of lentil with an
effective Rhizobium strain and an incompatible AMF
species signi®cantly reduced the HI of plants (e.g. RGL14
or LX77 1 NT6). The HI of lentil inoculated with ineffec-
tive Rhizobium strains was statistically comparable to that of
lentil inoculated with an effective Rhizobium strain and/or
AMF species. Analysis of the HI data revealed that parti-
tioning of assimilates into seed (i.e. HI) was signi®cantly
correlated with seed N (P , 0.0001; r ˆ 0.76) and P content
(P , 0.0001; r ˆ 0.81) of lentil.
3.2. Nutrient parameters
The shoot N content of lentil inoculated with the different
Rhizobium strains after six weeks under gnotobiotic condi-
tions was positively correlated (P , 0.045; r ˆ 0.64) with
the N content of lentil after 110 days' growth in natural soil.
Plants inoculated with G. clarum NT4 contained the highest
amount of total shoot N compared to uninoculated plants or
those co-inoculated with G. mosseae NT6, regardless of the
Rhizobium treatment (Tables 4 and 6). Uninoculated plants
and plants inoculated with the ineffective Rhizobium strains
92A1, LE13 and LX66 contained signi®cantly lower levels
of total shoot N compared to the other Rhizobium treatments
(except LX84), regardless of the AMF treatment (Tables 4
and 6). Lentil inoculated with LX72 and LX77 had the high-
est levels of total shoot N compared to the other effective
strains, regardless of the AMF treatment. Uninoculated
lentil plants had the least amount of total shoot N. The
indigenous Rhizobium community apparently had no speci-
®c interactions with NT4 or NT6, as there were no signi®-
cant differences between plants inoculated with NT4 or NT6
in the levels of total shoot N. For the same Rhizobium treat-
ment, co-inoculation with NT4 signi®cantly enhanced the
total shoot N of PB101, LX72, LX77 and LX84 compared to
the NT6 combination or the indigenous AMF combination.
In contrast, for the Rhizobium strains LX66 and RGL14, co-
inoculation with NT6 resulted in total shoot N enhance-
ments over the other two combinations.
The total shoot P content of lentil inoculated with G.
clarum NT4 was signi®cantly higher than G. mosseae
NT6 among the AMF treatments, regardless of the
Rhizobium treatment (Tables 4 and 6). There were signi®-
cant differences in the total shoot P content of lentil inocu-
lated with the different Rhizobium strains, regardless of the
AMF treatment. For example, lentil inoculated with the
effective Rhizobium strains (except LX84) had signi®cantly
higher levels of shoot P compared to the ineffective strains
or the uninoculated control (Table 4). The total shoot P
pattern for all effective AMF £ Rhizobium strain combina-
tions was similar to that of the total shoot N. For example,
lentil co-inoculated with G. clarum NT4 and the Rhizobium
strains PB101, LX72, LX77 and LX84 had signi®cantly
higher levels of shoot P than the NT4 or indigenous AMF £
Rhizobium strain combinations. In contrast, co-inoculation
186 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188

Table 5

Analysis of variance for the shoot and root dry weight, seed yield, harvest index, total plant N and P and AMF colonization of lentil inoculated with the AMF species G. clarum NT4 or G. mosseae NT6 and/or

AMF colonization (MS)

Mean (n ˆ 4) AMF-colonized root length of lentil plants inoculated with
the AMF G. clarum NT4 or G. mosseae NT6 and/or nine Rhizobium strains
and grown for 110 d in soil containing indigenous AMF and rhizobia.
Treatment means were separated using the least signi®cant difference
(LSD) test at P , 0.05 [Mean (R) refers to the mean of the main effect

249.81**
184.74**
255.38**
of rhizobia; Mean (A) refers to the mean of the main effect of AMF]

13.89
13.35
Rhizobium treatment AMF treatment

Total shoot P (MS)

AMF-colonized root length a (%)

None NT4 NT6 Mean (R)

48.64**
48.80**
20.21**
None 26 30 35 30

2.08
14.32
92A1 28 32 20 26
LE13 19 20 21 20
LX66 22 25 35 27

Total shoot N (MS)

RGL14 24 25 40 30
PB101 25 25 29 26
LX72 36 32 24 31

923.64**
2297.29**
3360.05**

91.03
16.41
LX77 33 48 18 33
LX81 30 27 15 24
LX84 31 43 22 32
Mean (A) 27 31 26

Harvest index (MS)

a
LSD for AMF effect, 2; rhizobia effect, 3; AMF £ rhizobia interaction

nine Rhizobium strains and grown for 110 d in soil containing indigenous AMF and rhizobia (MS, mean square; **P , 0.0001)
effect, 5.

0.146**
0.021**
0.020**
0.001
16.65
with G. mosseae NT6 and the Rhizobium strain LX66 and
RGL14 bene®ted lentil more than the NT4 or indigenous
AMF £ Rhizobium strain combinations.

Seed yield (MS)

1.257**
1.293**
0.626**
3.3. AMF colonization

0.026
18.09
Control plant roots were readily colonized by indigenous
AMF. Mycorrhizal colonization of plants inoculated with
Total root dry weight (MS)

NT4 was higher than uninoculated plants or those inocu-

lated with NT6, irrespective of the Rhizobium treatment
(Tables 5 and 6). The AMF colonization of lentil roots
was signi®cantly altered by the Rhizobium strains, irrespec-
tive of the AMF species (Tables 5 and 6). For example, the
5.250**
4.940**
2.490**

AMF colonization levels of lentil inoculated with the Rhizo-

0.125
15.87

bium strains LX72, LX77 or LX84 or colonized by the

native rhizobia were signi®cantly higher than plants inocu-
Shoot dry weight (MS)

lated with strains LX81 or LE13, indicating that interactions

between the native AMF community and the Rhizobium
strains were speci®c. In seven of nine cases, co-inoculation
resulted in signi®cant differences in the AMF colonization
25.824**
7.925**
4.330**

levels between the AMF species NT4 and NT6 for the same
0.098
8.30

Rhizobium treatment (Table 5). For example, plants co-

inoculated with the NT6 1 RGL14 combination had signif-
18
90
2
9
df

icantly higher levels of AMF colonization than the

NT4 1 RGL14 combination. Similarly, plants co-inocu-
Coef®cient of variation (%)

lated with the NT4 1 LX77 combination had a ca. 2.5-

fold increase in AMF colonization over that of the
NT6 1 LX77 combination. Correlation analyses revealed
that AMF colonization was signi®cantly correlated with
Rhizobia (R)

total shoot dry weight (P , 0.0187; r ˆ 0.43), total shoot

AMF (A)
Table 6

Source

N (P , 0.0116; r ˆ 0.45) and total shoot P (P , 0.0183;

A£R
Error

r ˆ 0.43) of plants.
 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188
4. Discussion
The tripartite association is relatively less well-
understood with respect to the factors that regulate it,
compared to the Rhizobium-legume symbiosis. For exam-
ple, the steps involved in the formation of a nodule and a
functional legume have been elucidated (Sprent, 1989;
Hirsch, 1992). However, literature presents no direct experi-
mental evidence, for instance, on the performance of a
legume inoculated with an effective Rhizobium strain in
the absence of compatible AMF associations. Our study
demonstrated that there were synergistic interactions
between compatible AMF species and Rhizobium strains
which were manifested as growth, yield and tissue N and
P increases compared with the uninoculated control. These
growth and yield increases were not restricted to the
effective AMF species or rhizobia, but was speci®c to the
particular AMF-Rhizobium combination(s).
Many workers have shown that interactions between
AMF species and rhizobia signi®cantly in¯uence the growth
and productivity of legumes (Ames et al., 1991; Azcon et
al., 1991; Vejsadova et al., 1992; Ianson and Linderman,
1993; Ruiz-Lozano and Azcon, 1993; Ahmad, 1995; Saxena
et al., 1997; Nwoko and Sanginga, 1999). These interactions
may result in enhanced legume productivity, reduction in
yield, or alternatively, there can be no response to inocula-
tion. In addition, research ®ndings suggest that enhanced or
reduced yields are noted in speci®c AMF-Rhizobium strain
combinations. For example, of the four Glomus species and
six R. meliloti strains they tested, Azcon et al. (1991) found
that alfalfa inoculated with G. fasciculatum and a speci®c
R. meliloti strain produced the highest shoot dry matter
compared with other AMF-Rhizobium strain combinations.
Similarly, in comparing the effects of interactions between
G. pallidum, G. aggregatum and Sclerocystis microcarpa
and four R. phaseoli strains on the growth and yield of
three kidney bean cultivars, Ahmad (1995) reported that
the symbiotic ef®ciency was `dependent on the particular
combination' of the AMF species, Rhizobium strain and the
host cultivar. Similar observations indicating that growth
and yield enhancements in lentil appeared to depend on
the particular AMF-Rhizobium strain combination were
noted in our study.
Field-grown legumes are often inoculated with effective
Rhizobium strains (Hynes et al., 1995), but legumes often
form tripartite associations with the ubiquitous AMF and
both indigenous and introduced Rhizobium strains. In
contrast to other studies, wherein only effective rhizobia
were included, ineffective Rhizobium strains were also
included in this study in order to understand the signi®cance
of the Rhizobium symbiosis. Results indicated that the ef®-
cacy of an effective Rhizobium strain on the host was
reduced signi®cantly when combined with an incompatible
AMF species (e.g. NT4 1 LX81). On the other hand, the
ef®cacy of lentil inoculated with a less effective Rhizobium
strain was enhanced when combined with a compatible
187
AMF species (e.g. NT6 1 LX66). However, this enhanced
productivity was still signi®cantly lower than that of
an effective Rhizobium strain or AMF-Rhizobium strain
combination.
The role of AMF in the tripartite symbiosis may be
signi®cant in soils with a low available P content (such as
the soil used in this study) as nitrogen ®xation is impaired by
an inadequate P supply. Research ®ndings suggest that
growth and yield increases of legumes inoculated with
AMF and rhizobia are generally associated with enhanced
N and/or P uptake (Manjunath et al., 1984; Pacovsky et al.,
1986; Kucey and Bonetti, 1988; Azcon et al., 1991;
Ruiz-Lozano and Azcon, 1993). We too found that the
increase in lentil productivity (shoot 1 seed) as a result of
co-inoculation with AMF and rhizobia was associated with
increased N and P content. This indicates that the yield
increase noted in compatible AMF 1 Rhizobium treatments
was probably due to enhanced nutrient uptake. Yield and P
content increases have been shown to be signi®cantly corre-
lated with increases in the AMF colonization levels
(Ibijbijen et al., 1996). In our study, lentil productivity
and total shoot N and P content increases noted were
associated AMF colonization levels.
This study demonstrated that by carefully co-selecting for
AMF and rhizobia which are compatible with each other
and with the host plant, the growth, yield and nutrition of
lentil can be signi®cantly enhanced even in non-sterile soil
containing indigenous AMF and rhizobia. Furthermore, this
study shows that in high yielding co-inoculation treatments,
the increased yield may be due to enhanced N and P nutri-
tion. We also noted that co-inoculation with one AMF
species signi®cantly increased plant productivity over the
other for the same Rhizobium strain or isolate, irrespective
of its effectiveness on the host. Legume seeds are invariably
treated with an effective Rhizobium inoculant before plant-
ing. These Rhizobium inoculants are available for commer-
cial use after considerable ®eld evaluations. Despite these
considerations, a Rhizobium inoculant may not deliver its
full potential on the host in a given soil (Nwoko and
Sanginga, 1999; Houngnandan et al., 2000). It is possible
that these problems are somewhat mediated by incompatible
AMF associations. Fluctuations in AMF community
dynamics may be the result of several agronomic practices
such as crop rotation that favours a less bene®cial AMF
community. Therefore, it is crucial to consider compatibility
between Rhizobium strains and the AMF community not
only for enhanced productivity, but also for the enhanced
survival and establishment of legumes growing in less than
ideal environmental conditions.
Acknowledgements
This study was supported by Natural Sciences and
Engineering Research Council of Canada, Western Grains
Research Foundation and Agriculture Development Fund.
188 L.J.C. Xavier, J.J. Germida / Soil Biology & Biochemistry 34 (2002) 181±188
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