PROJECT RESULTS

Improvements to the Inoculation of Pea and Lentil Crops in Southern Manitoba

Applicant: 

Dr. J. Kevin Vessey
Department of Plant Science
University of Manitoba
Winnipeg, Manitoba  R3T 2N2  Canada

Table of Contents:

• Background and Objectives
• Procedure and Project Activities
• Results and Discussion
• Conclusions

ARDI Project:

#98-021

Project Status:

Completed January, 2002

Background and Objectives:

Pea and lentil can obtain a large part of their nitrogen from the atmosphere. The bacterium, known as Rhizobium leguminosarum bv. viciae, that forms nodules on the roots of these crops is responsible for converting atmospheric nitrogen into a form that can be used by plants. For pea and lentil to take maximum advantage of their potential to harvest nitrogen from the atmosphere to produce a high yielding, high protein crop, they must be grown in the presence of an effective, efficient strain of Rhizobium leguminosarum bv. viciae. Hence, it is currently recommended that producers should always inoculate their pea and lentil crops with a commercial inoculant of superior quality. However, you may have heard reports of some producers who did not inoculate pea, or did not seed the whole field to inoculated pea, and yet appeared to realize reasonable yield at harvest. One may ask, ‘If I grew inoculated pea or lentil in my field just a few years ago, wouldn’t the bacteria introduced in the inoculant still be there? Is it really necessary to invest the time, effort, and money in inoculating pea and lentil at every planting time? Ideally, inoculation of pea or lentil is necessary if Rhizobium leguminosarum bv. viciae is absent in the soil, if its population is very low, or if it is less efficient in harvesting atmospheric nitrogen than a commercial inoculant. Hence, the relevant questions addressed in the project were:

1. How abundant is Rhizobium leguminosarum bv. viciae in southern Manitoba soils?
2. How efficient are these indigenous strains?
3. How diverse are these strains?
4. How competitive are these strains relative to an inoculant strain?

If Rhizobium leguminosarum bv. viciae is abundant in southern Manitoba soils, it may have come from commercial inoculants introduced in previous inoculations, native strains which nodulate native legumes such as vetch and Lathyrus species, or "hybrid" descendants of commercial and native strains.

Having large populations of Rhizobium leguminosarum bv. viciae strains does not guarantee that large amounts of nitrogen will be fixed because native strains may be effective in forming nodules, but inefficient in fixing atmospheric nitrogen. On the other hand, commercial strains introduced in previous inoculations may lose effectiveness after several years in the soil because of mutations or genetic exchange with native strains or with other soil microorganisms.

Locally adapted indigenous strains can be highly competitive for nodulation and can prevent the inoculant strain from occupying a significant proportion of pea or lentil root nodules. In case these strains are less effective than an inoculant strain, pea and lentil will not be able to take maximum advantage of their potential to fix nitrogen.

Procedure and Project Activities:

Field sites (21) were located throughout southern Manitoba in 1998 where the field history could identify whether pea or lentil had been grown in the previous ten years and which brands of commercial inoculants had been used in previous pea or lentil inoculations. The objective of this field survey was to "trap" rhizobia from a broad range of sites in southern Manitoba. These sites ranged from those that had been sown to pea in recent times to those that had never been cultivated. For detailed study, the number of field sites was reduced from 21 to only five in 1999 and 2000. These five sites were located at Carman, Letellier, Morden, Souris and Westbourne and had all been grown to pea or lentil in the previous one to four years. The cooperation of the landowners and the Manitoba Pulse Growers Association (MPGA) was essential in site selection.

Micro-plots (measuring 1 m by 1 m) of uninoculated pea, inoculated pea, and inoculated pea supplied with 100 Kg N/ha were established in the aforementioned sites for three years (1998-2000). Micro-plots of non-fixing mutant pea were included in the detailed study (1999 and 2000) to determine the amount of nitrogen fixed by inoculated and uninoculated pea. The level of nodulation exhibited by uninoculated plants compared to that exhibited by inoculated plants was used to indicate the abundance of Rhizobium leguminosarum bv. viciae. Pea cultivar "Express" and N-Prove commercial strain (Philom Bios, Saskatoon) were used in all the experiments. The rate of seed inoculation adopted was the one recommended by the manufacturer.

Pea plants were harvested at the early pod-filling stage and aboveground biomass dried and weighed. The aboveground samples were then finely ground and their nitrogen content determined. Nodules from root cores of pea plants were picked, counted, dried and weighed. The plants were allowed to grow to physiological maturity in 2000 and seed yield was determined at harvest. The seed samples were dried, finely ground and their nitrogen content determined. Statistical analysis was performed on all the parameters measured.

Nodules were collected from uninoculated plants grown in 20 sites (1998 field survey) and rhizobia isolated from them. These rhizobia were cultured and their DNA fingerprints made by using "plasmid profiling" and "polymerase chain reaction-restriction fragment length polymorphism" (PCR-RFLP) techniques. The resulting DNA fingerprints were compared to those of 16 inoculant strains (four of the major inoculant suppliers in western Canada cooperated by providing their strains) and to native strains collected from the nodules of naturally occurring Lathyrus species. This was done to establish the source(s) of the field strains and whether commercial strains introduced in previous inoculations remained genetically stable in southern Manitoba soils.

The Manitoba Pulse Growers Association and Philom Bios (Saskatoon, SK) provided financial assistance for this project, while the Canadian Commonwealth Scholarship and Fellowship Plan provided financial support to my PhD student, George Chemining’wa, who worked on the project.

Results and Discussion:

Uninoculated pea plants were well nodulated in cultivated sites (including sites that had not been grown to pea or lentil in over 15 years), but were poorly nodulated in the sites that had no history of cultivation. Cultivated soils had approximately 17 times more nodules per plant than virgin soils. In the detailed experiments (1999-2000), plants that were not inoculated had mostly the same number of nodules as the inoculated (without nitrogen fertilizer) plants. Since the number of nodules per plant is a good indicator of the population level of rhizobia, the present results show that Rhizobium leguminosarum bv. viciae is abundant in most cultivated soils of southern Manitoba.

More often than not, inoculated plants supplied with nitrogen had fewer and lighter nodules than those that were not fertilized. This observation was expected because many studies have shown that nitrogen interferes with the process of nodulation and, therefore, results in reduced nodule numbers.

Averaged across all sites, plants sampled from inoculated plots had 19.4 - 40% higher shoot dry matter than plants sampled from uninoculated plots in 1998 and in 2000, but inoculation had only a marginal effect in 1999. The superior plant growth exhibited by inoculated plants compared to uninoculated plants indicated that indigenous strains of Rhizobium leguminosarum bv. viciae may be less efficient at fixing atmospheric nitrogen than the inoculant strain used in the present study. In most cases, no differences in plant growth were observed among the plants grown in the inoculated plots (with or without nitrogen fertilizer). This observation supports the current recommendation that addition of nitrogen fertilizer is not necessary when pea is inoculated with a good quality commercial inoculant. Treatment effects on seed yield were tested in 2000 only. While inoculation improved shoot dry matter, it had no effect on seed yield in most of the sites. Inoculation affected seed yield only at Carman where inoculated plants (without nitrogen fertilizer) yielded 18% more than uninoculated plants. One would expect the superior plant growth of inoculated plants to translate into greater seed yield at all sites. In fact, we believe the lack of an inoculation effect on seed yield at four of the sites was actually an artifact, due to unavoidable damage to the shoots during the harvest of above ground biomass at the early pod-filling stage.

Inoculation increased biologically fixed shoot N only at Carman in both 1999 and 2000. Similarly, inoculation increased seed fixed N by approximately 65% at Carman.

Plasmid profiles (DNA fingerprints) of 230 isolates were established, out of which 77 were unique or distinct. This is considered a high level of genetic diversity. Huge diversity was also observed using the PCR-RFLP technique. Tremendous diversity among strains of rhizobia may not be good. In sites with diverse populations of Rhizobium leguminosarum bv. viciae, pea and lentil are likely to be exposed to strains of varying nitrogen fixing abilities. The strains that are highly competitive will occupy most of the nodules, but they may be poor nitrogen fixers compared to the inoculant strains. In such a scenario, pea and lentil crops may not reap maximum benefits from nitrogen fixation. In this study, the inoculant strain occupied only 14% of the nodules from inoculated plants at Morden 2 and did not occupy any of the nodules examined at Glenlea 1. This is a good indicator that indigenous strains in these two sites, and possibly in the others, may be more competitive than inoculant strains. In addition, we found that the inoculant strain was more prevalent in the nodules borne on the crown than in the nodules borne on lateral roots. This suggests that inoculant strains applied to the seed get poorly distributed in the soil.

The commercial strains that were introduced in previous inoculations of pea or lentil were rarely found in the field. Thus, the inoculant strains may not remain genetically static in the soil. The "new" strains that arise (by mutation or genetic exchange) may become less efficient than the original inoculant, thereby warranting frequent inoculations with a commercial strain.

Conclusions:

This research has shown that indigenous populations of Rhizobium leguminasorum bv. viciae are abundant in cultivated soils of southern Manitoba and appear to be less effective at fixing nitrogen than commercial inoculants. In addition, the commercial strains introduced in previous pea or lentil inoculations did not appear to remain genetically static. These findings, in conjunction with the increase in plant growth in inoculated plots in two of the three test years, support the current recommendation that producers should always inoculate their pea crop with a good quality inoculant. This study has also shown that there is no need to apply nitrogen fertilizer to pea when it is inoculated with a good quality inoculant.

Indigenous Rhizobium leguminasorum bv. viciae populations in southern Manitoba appear to be more competitive in forming nodules than inoculant strains. When a superior inoculant strain fails to occupy the majority of pea nodules, the pea crop can not obtain optimum nitrogen fixation benefits from inoculation. Hence, there is need to develop strategies that enhance the ability of inoculant strains to occupy nodules in the presence of highly competitive indigenous strains. One of the strategies is to adopt inoculation techniques (to complement or replace seed inoculation) that result in uniform distribution of the inoculant strain in the soil. (There have been some recent successes with granular inoculant to help achieve this goal in pea.) This would increase the proportion of inoculant strains occupying lateral root nodules which contribute significant amount of nitrogen during grain filling when most crown nodules would be senescing.

Acknowledgements:

This project was made possible due to funding from the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI), the Manitoba Pulse Growers Association, Philom Bios (Saskatoon), and The Canadian Commonwealth Scholarship Program for sponsoring George Chemining’wa’s PhD studies. We also thank the pea producers who cooperated with the on-farm research.

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