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Agronomy Journal 94:851-859 (2002)
© 2002 American Society of Agronomy

LEGUMES

Evaluation of Rhizobial Inoculation Methods for Chickpea

Stephen Kyei-Boahena, Alfred E. Slinkardb and Fran L. Walley*,a

a Dep. of Soil Sci., Univ. of Saskatchewan, 51 Campus Dr., Saskatoon, SK, Canada S7N 5A8
b Crop Dev. Cent., Univ. of Saskatchewan, 51 Campus Dr., Saskatoon, SK, Canada S7N 5A8

* Corresponding author (walley{at}sask.usask.ca)

Received for publication April 18, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
Rhizobia inoculated onto legume seeds are often exposed to adverse environmental conditions, which can affect survival and subsequent effectiveness. Hence, soil-applied granular inoculants have received much attention recently. We examined the efficacy of various inoculation methods at four sites in Saskatchewan, Canada, in 1997 and 1998 using desi- and kabuli-type chickpea (Cicer arietinum L.). Seed inoculation treatments (liquid or peat-based powder) were compared with soil inoculation (granular inoculant) either placed in the seed furrow or side-banded (i.e., 2.5 cm to the side) at depths of either 2.5 or 8 cm below the seed. Nodule formation in the seed inoculation treatments was restricted to the crown region of the root system, whereas soil inoculation enhanced nodulation on the lateral roots. In 1997, granular inoculant placed below the seed increased kabuli seed yield by 36 and 14% over the liquid and peat-based inoculants, respectively, whereas desi seed yield increased 17 and 5%, respectively. Seed yield responses were inconsistent in 1998. Seed protein concentration, percentage N derived from the atmosphere (%Ndfa), and amount of N2 fixed were typically lower for the liquid inoculant than for the peat and granular inoculants, which did not differ. The dry weight of lateral-root nodules was highly correlated with yield parameters, suggesting that the lateral-root nodules contributed significantly to N2 fixation and yield. Although the peat and granular inoculants were equally effective in establishing successful symbiosis, placing granular inoculant 2.5 to 8.0 cm below the seed may improve yield and quality.

Abbreviations: %Ndfa, percentage nitrogen derived from the atmosphere


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
CHICKPEA (Cicer arietinum L.) is one of the world's most important grain legume crops and is grown in many semiarid regions, including the Indian subcontinent, Turkey, the Middle East, Australia, and Mexico. It has recently been introduced into Saskatchewan and currently is grown on nearly 140000 ha (Saskatchewan Agriculture and Food, 2000). Chickpea can obtain a significant portion of its N requirement through symbiotic N2 fixation when grown in association with effective and compatible Rhizobium strains. However, Saskatchewan soils may not contain the specific rhizobia to establish an effective association; hence, inoculation is essential to ensure that a large and effective rhizobial population is available in the rhizosphere of the plant (Hynes et al., 1995). The success of inoculation often is limited by several factors, including environmental conditions, the number of infective cells applied, the presence of competing indigenous rhizobia, and the inoculation method (Brockwell et al., 1995).

The most common method of inoculation involves treating the seed with a peat-based powder or liquid inoculant before planting. However, some studies have shown that a large majority of the rhizobia, applied to seed via conventional seed inoculation, die on the seed before seeding or shortly after placement in the soil due to exposure to seed treatment chemicals, seed-coat toxins, dehydration, or excessive heat (Brockwell et al., 1980; Roughley et al., 1993). Consequently, interest is growing in the use of granular inoculants because they are applied directly to the soil, avoid direct contact with seed-treated chemicals, and are better able to withstand adverse environmental conditions. Scudder (1975), using granular inoculant in the seed furrow, obtained a 38% yield increase over seed-applied inoculant in soybean [Glycine max (L.) Merr.] under hot and dry conditions in Florida. Similarly, Bezdicek et al. (1978), working with soybean, found that placing granular inoculant in the soil with the seed was superior to seed-applied inoculant. Brockwell et al. (1980) summarized the results of experiments with several legumes, including chickpea, where granular inoculant was used. They found that when conditions were unfavorable for the survival of rhizobia, or when germination was delayed due to environmental conditions, soil inoculation resulted in better nodulation and often better plant growth and yield than seed-applied inoculants. Other investigators working with soybean (Muldoon et al., 1980), faba bean (Vicia faba L.) (Dean and Clark, 1977), arrowleaf clover (Trifolium vesiculosum Savi) (Ocumpaugh and Smith, 1991), and alfalfa (Medicago sativa L.) (Rice and Oslen, 1992) have reported similar findings.

The depth of inoculum placement is an important factor that can influence the benefits of soil inoculation. It is well established that movement of rhizobia in the soil is limited (Madsen and Alexander, 1982). This finding is supported by reports that seed-applied inoculum or granular inoculum at the seeding depth results in nodulation predominantly in the crown region of the root system (Danso and Bowen, 1989; Hardarson et al., 1989; Danso et al., 1990). Using the acetylene reduction assay, McDermott and Graham (1989), Wolyn et al. (1989), and Vikman and Vessey (1992) showed that lateral-root nodules that form later are more active during seed formation and can provide significant fixed N during the reproductive stages of plant growth compared with crown nodules. Thus, inoculation strategies aimed at positioning the inoculant rhizobia to intercept lateral roots may improve nodulation of the lower part of the root system and, consequently, improve N2 fixation. Hardarson et al. (1989) and Wadisirisuk et al. (1989) demonstrated this in soybean by placing the inoculum below the seed. However, none of the studies examined the optimum placement depth for effective nodulation and N2 fixation. Therefore, the objectives of this study were to (i) assess the effect of seed and soil inoculation methods on nodulation, N2 fixation, and yield of chickpea; (ii) determine the optimum placement depth for granular inoculum; and (iii) examine the contribution of lateral-root nodules to N2 fixation and yield.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
Four field sites with no history of chickpea production were established in Saskatchewan near Elbow, Kenaston, Outlook, and Watrous during the 1997 growing season. Studies were repeated at Outlook and Watrous in 1998 in different areas of the same fields. The soils were analyzed for selected physical and chemical characteristics before seeding (Table 1). Chickpea was grown on samples of the soils obtained from each site (0–30 cm depth) but did not nodulate after 6 wk in a growth chamber pot experiment, suggesting the absence of rhizobia specific to chickpea.


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  		Table 1. Characteristics of the 0- to 30-cm soil layer of the four experimental sites in Saskatchewan at the initiation of the experiments in 1997 and 1998.

 
Each experiment consisted of 11 inoculation treatments arranged in a randomized complete block design with four replications using either desi-type chickpea (cv. Myles) or kabuli-type chickpea (cv. Sanford). In 1997, the desi chickpea was planted on 14 May at Elbow, Kenaston, and Outlook and on 20 May at Watrous. For the 1998 desi experiments, planting was on 9 and 20 May at Watrous and Outlook, respectively. The kabuli chickpea was planted on 14 and 20 May at Kenaston and Watrous, respectively, in 1997 and on 9 and 20 May at Watrous and Outlook, respectively, in 1998. Seeding was accomplished using a double disc press drill with separate discs for seed and fertilizer placement and consisted of seven rows [six chickpea rows and one flax (Linum usitatissimum L.) row] that were 12 m long and 15 cm apart.

Six different inoculants containing Rhizobium ciceri were applied each year at the recommended rate (Table 2). Eleven inoculation treatments were used: seed inoculation using two different peat inoculants (designated A or B) or two different liquid inoculants (A or B); soil inoculation, with two granular inoculants (A or B) placed in the furrow with the seed at planting or side-banded (i.e., 2.5 cm to the side) at depths of either 2.5 or 8 cm below the seed; and a noninoculated control. Inoculants with the same designation, e.g., A or B indicate that the identical rhizobial strain or strains were used in the different carriers. Inoculant A contained a single strain, CP39 (ICARDA, Aleppo, Syria; and kindly formulated by MicroBio RhizoGen Corp., Saskatoon, SK, Canada), whereas inoculant B contained a mixture of three strains: 27A2, 27A7, and 27A9 (kindly formulated by LiphaTech, Milwaukee, WI). The liquid formulation of inoculant B was not available in 1997; hence, an experimental liquid formulation (inoculant C), containing single strain 27A2 (Agrium Biologicals, Saskatoon, SK, Canada), was used.


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  		Table 2. Name, designation, manufacturer, and the rates of commercial inoculants used in 1997 and 1998.

 
Seed inoculation was performed by thoroughly mixing a measured amount of peat or liquid inoculant (i.e., according to the manufacturer's recommendations) and 5 mL of 1% (w/v) gum arabic solution (as a sticker) with 1.5 kg of seeds. Seeds were treated in plastic bags immediately before seeding. Seed inoculation supplied approximately 105 rhizobia cells seed-1. The granular inoculants were soil-applied either in the seed row (at a depth of 5 cm) or to the side of the seed row at different depths, using a second set of discs (adjusted for the various depths) to deliver approximately 1011 rhizobia per hectare. Triple superphosphate (0–45–0) was applied at planting in the seed row at the rate of 8.6 kg P ha-1. To minimize contamination, the noninoculated plots were planted first. In addition, all treatments with the same rhizobia strain(s) were planted consecutively before switching to other treatments to minimize the potential for inadvertently contaminating the treatments. Moreover, the planter was thoroughly cleaned with a vacuum cleaner and then disinfected with 70% ethanol (C2H5OH) after planting each treatment plot. Flax (cv. McGregor) was used as reference crop for assessing percentage N derived from the atmosphere (%Ndfa) because the rooting and growth patterns are similar to those of chickpea. The plants relied on natural precipitation throughout the growing season (Table 3). Weeds were controlled by hand-hoeing during the growing season.


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  		Table 3. Mean monthly precipitation during 1997–1998 growing season of chickpea at four Saskatchewan locations, and long-term averages.{dagger}

 
Sampling was performed by excavating the roots of five random plants from the central rows of each plot at the early pod-filling stage. A spade was used to collect an undisturbed soil sample (measuring approximately 20 cm deep, with a radius of approximately 12 cm extending out from the central stem) containing the roots. The undisturbed soil samples were wrapped in plastic and transported back to the lab where the soil was washed from the roots using gently running tap water. Care was taken to ensure that the roots and nodules were recovered intact. Nodules from the crown region and lateral roots subsequently were removed separately from the roots. The crown region was defined as that part of the root extending 3 cm in all directions from the stem base, whereas the lateral roots were defined as those parts of the root system extending beyond 3 cm from the stem base. The nodules were dried in an oven at 60°C for 7 d, and dry weights were determined.

At maturity, a 1-m2 area of unsampled center rows of each plot was hand-harvested. Whole-plant samples were dried at 60°C for 48 h before threshing with a stationary thresher. Seeds were cleaned and weighed, and seed yields were calculated on a per-hectare basis. The seeds were milled to a <2-mm particle size with a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA) and then finely ground by passing through a cyclone mill (Cyclotec 1093, Tecator, Höganäs, Sweden) equipped with a 0.4-mm sieve. Seeds of flax were ground with a mortar and pestle. Approximately 1-mg samples of ground seed were analyzed for total N and atom percent 15N excess with an isotope ratio mass spectrometer VG Micromass 602E (Isotech, Middlewich, England) (Bremer and van Kessel, 1990). Seed protein concentration was determined as total N x 6.25. Atom percent 15N excess was calculated with reference to the natural 15N abundance of the atmosphere (0.3663 atom % 15N).

Natural 15N abundance was calculated according to Bremer and van Kessel (1990):

where the standard is atmospheric N2 gas (0.3663 atom % 15N).

The %Ndfa was then calculated as follows:

where x is {delta}15N of seeds of plants deriving all their N from soil (in this case flax), y is the {delta}15N in chickpea seed, and c is {delta}15N of chickpea seeds from plants grown in an N-free medium. The c values for desi chickpea (cv. Myles) were determined experimentally, according to the method of Kohl and Shearer (1980), to be -0.9062 and -0.5475 for the single strain (CP39) and mixed strain (27A2, 27A7, and 27A9), respectively. The value for kabuli chickpea (cv. Sanford) and rhizobial strain combinations was -0.7644. The amount of seed N fixed was calculated as (%Ndfa x seed yield x seed N concentration)/100.

Data for each site were analyzed separately, using the general linear model procedure of SAS (SAS Inst., 1996). The error terms for each year were examined for homogeneity of variance (Steel et al., 1997) using Bartlett's test, and combined analyses were performed separately for the 1997 and 1998 experiments. Years were analyzed separately because fewer number of experiments were conducted in 1998 using desi chickpea and, for kabuli chickpea, one of the 1998 experiments was conducted at a location different from that of 1997. Also, liquid inoculant B was not available in 1997, so liquid inoculant C was used instead; thus, one of the treatments differed between years. Inoculation treatment was considered a fixed effect while location, blocks within location, and location x inoculation were considered random. The location x inoculation error was used as the error term for the combined analysis. Significant differences among treatment means were evaluated using LSD at the 5% probability level. Contrast statements were used to compare inoculant formulations and inoculant placement. The relationships between dry weight of nodules and yield parameters averaged across locations were evaluated using correlation analyses.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
SUMMARY AND CONCLUSIONS
 REFERENCES
 
Inoculation treatments produced similar results for both desi and kabuli chickpea at all locations, and location x inoculation interactions for most of the parameters measured were not significant, indicating that the inoculants performed similarly across locations. Therefore, genotype data were averaged across locations for each year.

Nodule Distribution and Dry Weight at the Early Pod-Filling Stage
Inoculum placement significantly influenced the distribution of nodules on the root system, and the distribution was consistent across locations in both chickpea types in 1997 and 1998. The peat and liquid seed-applied inoculants produced the majority of their nodules at the crown region, whereas the soil-applied (granular) inoculants formed most of the nodules in the lower part of the root system, i.e., on the lateral roots (Tables 4 and 5). Lateral-root nodules typically developed in relatively narrow zones consistent with the depth of inoculant placement, i.e., inoculants placed at depth resulted in nodules forming on lower parts of the root system. Inoculation with granular inoculant at the seeding depth enhanced lateral-root nodulation, and this proportion increased when the granular inoculant was placed below the seed. In the 1997 growing season, granular inoculant placed to the side and either 2.5 or 8.0 cm below the seed formed 78 to 93% of the nodules (on dry-weight basis) on the lateral roots compared with only 23 to 36% for the peat and liquid inoculants for the desi chickpea (Table 4). Similarly, 87 to 97% of the nodules formed by the granular inoculant placed below the seed in kabuli were located on the lateral roots compared with 27 to 47% for the peat and liquid inoculants. The 1998 results based on dry weight also indicated that granular inoculants placed to the side and below the seed produced 79 to 96% of their nodules on the lateral roots in both chickpea types compared with 21 to 39% in the peat inoculants (Table 5). There were no marked differences in nodulation pattern among inoculant strains in either chickpea type in both years, indicating that the pattern of nodule formation was due primarily to the depth of inoculant placement.


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  		Table 4. Nodule and plant dry weight from various inoculation treatments of Myles desi and Sanford kabuli chickpea at the early pod-filling stage, averaged across Elbow, Kenaston, Outlook, and Watrous, SK, and Kenaston and Watrous, respectively, 1997.

 

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  		Table 5. Nodule and plant dry weight from various inoculation treatments of Myles desi and Sanford kabuli chickpea at the early pod-filling stage, averaged across Outlook and Watrous, SK, 1998.

 
The nodulation pattern observed in this study is consistent with data from other studies in which deeper placement of rhizobial inoculants in the soil resulted in substantial nodule formation on the lower part of the root system (Danso and Bowen 1989; Hardarson et al., 1989; Wadisirisuk et al., 1989). The preponderance of nodulation in the root zone immediately below the position of inoculum placement was due largely to the limited migration of the rhizobia to other infection sites along the root. According to Madsen and Alexander (1982), Bradyrhizobium japonicum movement in the soil is restricted in the absence of infiltrating water. Therefore, nodulation of the entire root system requires that the roots encounter the inoculant rhizobia in the soil. The enhanced lateral-root nodulation from placement of the granular inoculant in the seed furrow, compared with the seed-applied inoculants, further indicates the poor mobility of rhizobia in the soil when placed on the seed.

Total nodule dry weights averaged across locations for both chickpea genotypes in 1997 and 1998 were lower for the liquid inoculants than for the peat and granular inoculants (Tables 4 and 5). The low nodulation in the liquid inoculant treatments could be due to a decline in the number of rhizobia resulting from desiccation during and after planting because it was generally dry and windy at planting. The poor nodulation in the liquid treatments was more pronounced at Elbow in 1997 and at Outlook in 1998 (data not shown), which were drier than the other sites (Tables 1 and 3). Bissonnette and Lalande (1988) observed that carrier material for inoculum affected survival of rhizobia during stress. Zdor and Pueppke (1990), working with liquid and peat inoculant carriers, indicated that a peat formulation may help protect rhizobial strains from adverse environmental effects that would reduce their populations. Thus, although both the liquid and the peat-based inoculants were applied to the seed, a peat carrier, in contrast to a liquid carrier, may increase strain survival by reducing desiccation or heat stress of the cells, a major factor involved in the establishment of rhizobia in soil (Hansen, 1994). Granular inoculants contain less moisture (approximately 32%) and provide a microhabitat, which may offer greater protection to rhizobia than seed-applied inoculants. However, orthogonal contrasts indicated no significant differences in total nodule dry weight between the peat and granular inoculants whether placed in the seed furrow or below the seed for desi in both years. In contrast, the total nodule dry weights for the peat inoculant were 98 and 57 mg higher than for the granular inoculant in 1997 and 1998, respectively, for kabuli (Tables 4 and 5). The reason for the greater nodule dry weight for the seed-applied peat treatment in the kabuli experiments but not in the desi experiments is not clear. Possibly the kabuli chickpea is more aggressive in nodulation during the early stage of growth favoring seed inoculation compared with a later stage when the roots have to grow and encounter the inoculant rhizobia in the soil. For both chickpea types, inoculating the soil with granular inoculant at the seeding level or below the seed resulted in no difference in total nodule dry weight, just as nodule weight with the 2.5 and 8.0 cm placement below the seed did not differ.

Some limited nodulation was observed on some of the noninoculated plants. However, nodulation in the control treatments was sparse compared with the inoculated treatments and likely had little or no significant effect on the results of this study.

Seed Yield
At final harvest in 1997, seed yields for both kabuli and desi types, averaged across locations, were significantly increased by inoculation (Table 6). In particular, granular inoculant placed below the seed and seed inoculated with peat-based inoculant A produced the highest yields. The maximum increase in seed yield over the noninoculated control averaged across locations in the kabuli chickpea was 633 kg ha-1 and occurred when granular inoculant A was placed below the seed followed by granular inoculant B placed below the seed (440 kg ha-1). Seed yield differences between the peat and granular inoculants were relatively low and insignificant. For example, for the kabuli chickpea, the average seed yield increases for granular inoculant below the seed were 151 (14%) and 320 kg ha-1 (36%) greater than for the peat and liquid inoculants, respectively. For the desi chickpea, seed yield increases for the granular inoculants placed below the seed were 64 and 209 kg ha-1 (5 and 17%, respectively) greater than for the peat and liquid inoculants, respectively. The limited yield increases associated with the granular inoculant placed below the seed may be due, in part, to better moisture conditions in this soil zone and extra protection from heat for the rhizobia and, subsequently, for the nodules, favoring N2 fixation. Placement of granular inoculant below the seed may delay nodulation, and it is possible that nodules that form later may fix proportionately more N2 during pod-fill (Hardarson, 1993), thereby contributing to the higher yields. The low yield for seed-applied liquid inoculant is a reflection of limited N supply due to poor nodulation, resulting in low N2 fixation.


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  		Table 6. Seed yield, seed protein concentration, percentage N derived from the atmosphere (%Ndfa) for the seed, and amount of seed N fixed for Myles desi chickpea, averaged across Elbow, Kenaston, Outlook, and Watrous, SK, and Sanford kabuli chickpea, averaged across Kenaston and Watrous, SK, 1997.

 
The difference in seed yield between granular inoculant placed in the seed furrow and placement to the side and below the seed was significant only for the desi chickpea (Table 6). In each instance, granular inoculant below the seed performed better than that placed with the seed. The plants inoculated with granular inoculant placed with the seed nodulated adequately both at the crown and the lower part of the root system, and the proportion and amount of N2 fixed for the seed were similar or higher than for the seed-applied inoculants. Hence, the cause of the generally lower seed yield (not significant statistically) for this treatment compared with the peat-based inoculants could not be adequately explained. In other studies, greater seed yield for granular inoculant applied to the seed row compared with seed-applied inoculant was reported for soybean (Bezdicek et al., 1978; Muldoon et al., 1980) and faba bean (Dean and Clark, 1977).

Maximum seed yield at the various locations in 1997 ranged from 1100 kg ha-1 at Outlook to 1898 kg ha-1 at Watrous for desi, whereas kabuli was 1305 kg ha-1 at Kenaston and 1505 kg ha-1 at Watrous (data not shown). Higher seed yields were also obtained for experiments at Watrous compared with Outlook in 1998, with the maximum yield at each site varying from 1504 to 2242 kg ha-1 for desi and 1075 to 1800 kg ha-1 for kabuli (data not shown). Soil moisture conditions at Watrous throughout the 1997 and 1998 growing seasons favored early seedling emergence and more vigorous plant growth than the other sites, and this apparently resulted in higher yields at Watrous. In 1998, seeding at Outlook was 11 d later than at Watrous due to drought conditions, but no rain occurred during this delay. The average precipitation at Outlook for May 1998 was 57% less than the long-term average (Table 3). As a result of the low soil moisture, seedling emergence was slow, and plant stand was low, particularly in treatments where granular inoculants were placed below the seed. The soil was very dry, and it was observed that the upper 30 cm was very hard and difficult to penetrate with a shovel. The resistance encountered by the disc openers for both the granular inoculant (i.e., 2.5 and 8.0 cm below seed placement) and the seed prevented the discs from penetrating to the desired depth. Hence, the seeds were deposited just below the soil surface where the soil moisture content apparently was too low for optimum germination, particularly for the large-seeded kabuli. As a result, seed yields in 1998 at Outlook were low, and the effect was most severe in the treatments where granular inoculants were placed below the seed in kabuli.

Averaged across locations, seed inoculation with peat and granular inoculants placed with the seed in the kabuli chickpea resulted in higher seed yields compared with the other treatments (Table 7). Granular inoculant placed with the seed produced 215 kg ha-1 more seed yield compared with granular inoculant placed below the seed and was significant at P = 0.02. However, the contrast of liquid or peat-based inoculant vs. granular inoculant was not significant for seed yield. Data on nodule dry weight and dry matter yield per plant at the early pod-filling stage for the granular inoculant treatments indicated that plant growth in these treatments was not affected despite the delayed germination. Hence, the low seed yield was primarily due to the low plant density and the shorter growing season. In Saskatchewan, the growing season is relatively short and sometimes exacerbated by terminal drought as in 1997 and 1998. Chickpea is a long-season crop; thus, any delay in plant establishment likely will reduce the length of time for N2 fixation, pod filling, and seed maturation. Desi seed yields averaged across locations were significantly higher for the inoculated plants than for the control (Table 7). On average, inoculating the soil with granular inoculants consistently increased desi seed yields compared with the seed-applied liquid inoculant, but the contrast of peat vs. granular indicated no significant difference. The desi seed yield for the peat inoculants was 274 kg ha-1 higher than that for the liquid inoculants.


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  		Table 7. Seed yield, seed protein concentration, percentage N derived from the atmosphere (%Ndfa) for the seed, and amount of seed N fixed for Myles desi and Sanford kabuli chickpea, averaged across Outlook and Watrous, SK, 1998.

 
Seed Protein Concentration and Dinitrogen Fixation
Seed protein and the proportion and amount of N2 fixed for the seed averaged across locations in 1997 and 1998 were higher for inoculated plants in both chickpea types (Tables 6 and 7). The highest seed protein concentration, %Ndfa, and amount of N2 fixed generally occurred for soil inoculation treatments, particularly granular inoculant placed to the side and below the seed, but contrasts of peat vs. granular indicated no significant differences in both years and chickpea types. As with the other parameters measured, the liquid inoculants were inferior to the other inoculation treatments in terms of seed protein concentration, %Ndfa, and amount of seed N fixed, presumably due to poor nodulation. Previous reports on soybean indicate that soil inoculation produced higher N2 fixation (Muldoon et al., 1980; Hardarson et al., 1989) and seed protein concentration (Muldoon et al., 1980) than seed inoculation. With increasing granular inoculant rate, higher concentrations of seed protein were obtained by Muldoon et al. (1980), indicating that poor nodulation and N2 fixation limited protein production. Consistent with the seed yield, placing the granular inoculants to the side and below the seed in the 1997 significantly increased desi seed protein concentration, %Ndfa, and N2 fixed for the seed compared with placement in the seed furrow, a reflection of the differences in nodulation pattern.

In 1998, the amount of seed N fixed for kabuli was lower for the granular inoculant placed 8.0 cm below the seed than for placement at 2.5 cm as a result of the low seed yield for the former. In the 1997 desi experiments, the highest N2 fixation (63 kg ha-1) occurred at Watrous for the granular inoculant A placed to the side and below the seed, whereas the highest protein concentration (227 g kg-1) occurred at Elbow and Outlook in treatments in which granular inoculant A or B was placed 2.5 cm to the side and below the seed (data not shown). A similar trend was observed for the kabuli and 1998 experiments, suggesting that good moisture conditions enhanced N2 fixation, whereas dry conditions resulted in improved seed protein concentration. In general, the N2 derived from fixation was higher in 1997 than 1998, whereas protein concentration was higher in 1998 than 1997 due to the favorable growth conditions in 1997. This supports the negative correlation between yield and protein concentration, which often occurs in grain legumes under favorable growing conditions (Williams and Nakkoul, 1983). Apparently, the decrease in seed yield due to moisture stress in 1998 was greater relative to seed N yield and resulted in a higher protein concentration. As was the case for the other yield parameters, no differences with Rhizobium strains were observed in either chickpea type.

The results of the present study indicate that differences in yield parameters among the inoculants (although not consistently significant between the granular and peat-based inoculants) may be partly attributed to the nodulation pattern. Correlation analyses were performed to determine whether the yield parameters were more closely related to the lateral-root nodule or crown nodule dry weight (Table 8). The data indicated that the dry weight of lateral-root nodules for both chickpea types in 1997 averaged across locations was strongly correlated with seed yield (r = 0.69, P = 0.018 for desi; r = 0.73, P = 0.010 for kabuli) and amount of seed N fixed (r = 0.78, P = 0.005 for desi; r = 0.80, P = 0.003 for kabuli). In contrast, much lower or no correlation existed between dry weight of the crown nodules and these traits. Similar trends were observed in 1998, except for the seed yield of kabuli chickpea, which showed a weak correlation with the dry weight of lateral-root nodules at the early pod-filling stage due primarily to the delayed germination and reduced plant stand at Outlook as previously described. In this case, the correlation between seed yield and the dry weight of crown nodules at early pod-filling was significant. A significant positive correlation between the yield parameters and the dry weight of lateral-root nodules indicates that increased lateral-root nodulation was associated with higher N2 fixation and yields.


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  		Table 8. Correlations between nodule dry weight at early pod filling and yield parameters of chickpea averaged across various locations in Saskatchewan, 1997 and 1998.

 
The importance of lateral-root nodulation in N2 fixation, particularly during the reproductive phase of legumes, has been reported for soybean (Hardarson et al., 1989; McDermott and Graham, 1989; Danso et al., 1990) and common bean (Phaseolus vulgaris L.) (Wolyn et al., 1989; Vikman and Vessey, 1992). This observation was associated with the age of the nodules, suggesting that nodules on the lower part of the root system or on lateral roots (often formed later than those at the crown region) remain active during the entire period of high N demand at pod filling and seed maturation. In contrast, the crown nodules often were formed during the early growth stages of the plant and could be in senescence stages during the reproductive phase when soil N uptake rate could be low due to the depletion of soil mineral N levels. Results of our experiments suggest that the nodules formed on the lower or lateral roots contributed greater amounts of fixed N to the plant during seed formation.


   SUMMARY AND CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
REFERENCES
 
Desi and kabuli chickpea responded similarly to rhizobial inoculation, and there was no evidence of host x strain interaction for any of the parameters measured. The method of inoculation and the depth of inoculant placement influenced the distribution of the nodules on the root system. Inoculating the seed resulted predominantly in crown nodulation due to the restriction of the rhizobia to the vicinity of the seed and the inability of the rhizobia to contact the younger roots at the lower part of the root system. In contrast, granular inoculant applied to the soil enhanced lateral-root nodulation, and this proportion increased with the depth of inoculant placement.

The inoculum in liquid formulation was inferior to the peat-based inoculant and the granular inoculant, whether placed with the seed or to the side and below the seed, in total nodule dry weight at the early pod-filling stage. However, nodule dry weight was similar or higher for the peat-based inoculant applied to the seed compared with the granular inoculant at early pod filling. Chickpea seed yield, seed protein concentration, and the amount of seed N derived from the atmosphere were generally lower for the seed-applied liquid inoculant than for the other inoculant treatments. The peat-based inoculant applied to the seed did not differ significantly from the granular inoculant in seed yield, seed protein concentration, and amount of seed N fixed. However, granular inoculant placed to the side and below the seed was superior in these traits compared with the granular inoculant placed with the seed and peat-based inoculant applied to the seed, except for the seed yield in 1998 when shallow planting occurred in the treatment where the granular inoculant was placed below the seed. Apart from the seed yield for the kabuli chickpea in 1998, there was a strong positive correlation between the dry weight of lateral-root nodules and the parameters measured, whereas the dry weight of crown nodules and these traits were only weakly correlated, at best.

Notwithstanding the limited yield advantage of soil inoculation over seed inoculation, inoculating the soil 2.5 to 8 cm below the seed will be more beneficial than inoculating the seed, particularly in a year with unfavorable weather conditions. At that soil depth, the inoculant is placed in a more favorable environment and physically separated from seed-treated pesticides. The young growing roots of the host plant are more likely to encounter the inoculant strains at that soil depth for infection and subsequent nodule formation. These later-formed nodules may be important in supplying fixed N2 to the plant at a period when the N requirement is at its maximum.


   ACKNOWLEDGMENTS
 
We thank the Saskatchewan Department of Agriculture and Food (Agriculture Development Fund) and the Saskatchewan Pulse Growers for financial support. We also thank MicroBio RhizoGen Corp., Saskatoon, Agrium Biologicals, Saskatoon, and LiphaTech, Milwaukee, WI, for providing the Rhizobium inoculants. The technical assistance of Saskatchewan Wheat Pool and Bev Miller are greatly appreciated. Contribution no. 880 of the Saskatchewan Centre for Soil Research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 




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