Perry E. Olsen

Biological contaminants in North American legume inoculants

Abstract A survey of 40 commercial North American rhizobial inoculants was made to determine the number of contaminant cells relative to rhizobial cells. All of the inoculants were manufactured for sale during the 1993 season and were made using non-sterile peat as carrier. Products from three major North American manufacturing companies were examined. All of the inoculant samples were collected as part of the Canadian Legume Inoculant and Pre-Inoculated Seed Product Testing Program. Only one of the inoculants contained more rhizobia (Rhizobium or Bradyrhizobium spp) than contaminants. Three others contained 1–2 times more contaminants than rhizobia, 21 of the samples contained 2–10 times more contaminants than rhizobia, 8 contained 10–100 times more contaminants than rhizobia, and 3 contained more than 1000 times more contaminants than rhizobia. Four of the samples contained too few rhizobia to be detected in the standard most-probable-number (MPN) test used by the Canadian regulatory inoculant testing program. The most common contaminants were bacteria (109–1010 g−1), actinomycetes (108–109 g−1), and fungi (105–107 g−1). Contaminant bacteria capable of inhibiting growth of R. leguminosarum bv. trifolii or R. meliloti were isolated from inoculant products of all three manufacturers.

Analysis and regulation of legume inoculants in Canada: The need for an increase in standards

Rhizobial inoculants for use in Canada are regulated and have been evaluated in a formal testing program since 1975. This program is carried out by Agriculture Canada under authority of the Fertilizers Act and involves inoculant strain and formulation registration (with Food Production and Inspection Branch) as well as analysis (by Research Branch) of approximately 220 inoculants and pre-inoculated seed products yearly. Inoculant evaluation is based upon the calculated number of viable rhizobia which would be provided per seed if the inoculant was applied at the manufacturers recommended rate. Current standards are 103, 104, and 105 viable rhizobia per seed, of the proper cross-inoculation group, for small, intermediate, and large seeded legumes, respectively. Application of these standards means that some inoculants are considered “satisfactory” even though they yield test results as low as 9.4 × 106 rhizobia per gram. No standards are currently applied relative to permissible levels of contaminants in inoculant products, despite the fact that some inoculants contain many more contaminating microorganisms than they doRhizobium cells. The demands of modern sustainable agriculture, taken together with advances in inoculant formulation technology, warrant an increase by a factor of ten in the minimum acceptable Canadian standards for legume inoculants and pre-inoculated seed products.

Rhizobial inoculant formulations and soil pH influence field pea nodulation and nitrogen fixation.

Crop production systems that include field pea (Pisum sativum L.) in rotation are important for sustainable agriculture on acid soils in northwestern Canada. Greenhouse experiments were conducted to compare the ability of liquid inoculant applied to the seed, powdered peat inoculant applied to the seed, and granular inoculant applied in a band with the seed to establish effective nodulation on field pea grown at soil pH(H2O) 4.4, 5.4 and 6.6. Plants were grown to the flat pod stage, and then total plant biomass dry weight, dry weight of nodules, number of nodules, plant nitrogen content, and proportion of plant nitrogen derived from the atmosphere (%Ndfa) were measured. Granular and powdered peat inoculants produced greater nodule numbers and weight, plant nitrogen content, %Ndfa and total biomass than liquid inoculant in at least two of the three experiments. Only granular inoculant was effective in establishing nodules at soil pH 4.4, but granular and powdered peat inoculants were effective at pH 5.4, a...

Co-culture of Rhizobium meliloti and a phosphorus-solubilizing fungus (Penicillium bilaii) in sterile peat

Abstract A biofertilizer containing Penicillium bilaii, which increases phosphate uptake of several field crops by solubilizing phosphorus, has been developed. Laboratory investigations were carried out to determine if Rhizobium meliloti and Penicillium bilaii can be co-cultured in a common sterile peat carrier. A combination inoculant was prepared by mixing equal proportions of yeast-extract mannitol broth (YEMB) cultures of both organisms with sterile (γ-irradiated) peat. The cured inoculant contained 6.1 × 109Rhizobium cfu g−1 and 1.1 × 107Penicillium cfu g−1. New inoculant of equal quality was obtained by suspending mature inoculant in YEMB, and adding the suspension to sterile peat at a rate of 10 mg g−1. Addition of 26 mg ml−1 sucrose to YEMB resulted in populations of 6.2 × 109Rhizobium cfu g−1 and 2.3 × 108Penicillium cfu g−1. The results showed that a common production and delivery system for R. meliloti and Penicillium bilaii is possible by co-culturing the organisms in sterile peat.

Symbiotic effectiveness of Rhizobium meliloti at low root temperature

Laboratory, growth chamber and field experiments were conducted to select among 226 isolates of Rhizobium meliloti for the ability to grow, nodulate alfalfa (Medicago sativa L.) and support N2-dependent plant growth between 9° and 12°C. There was wide variation in the abilities of R. meliloti isolates to grow and form nodules at 10°C. Culture doubling times (td) varied from 1 to 155h, and the number of nodules formed on alfalfa in growth pouches in 2 weeks varied from 0 to 3.8 nodules per plant. Nodulation occurred at 9°C, but there was no significant N2-dependent plant growth at this temperature. However, several isolates of R. meliloti had the ability to nodulate alfalfa and produce N2-dependent growth at root temperatures between 10° and 12°C root temperature than did 14 other isolates tested. In field experiments, inoculation with strain NRG-34 resulted in greater nodule numbers, nodule weight, proportion of nodules occupied by the inoculant strain and plant weight than did inoculation with a commercial strain (NRG-185). These results permitted selection of a strain with better low-temperature competitive abilities than the currently available commercial strains.

Field comparison of pre-inoculated alfalfa seed and traditional seed inoculation with inoculant prepared in sterile or non-sterile peat

Rhizobium inoculant products in North America are often prepared using non-sterile peat and may contain more contaminants than rhizobia. The effectiveness of sterile and non-sterile peat-based inoculants applied to alfalfa (Medicago sativa) seed using either traditional seed inoculation just before planting, or pre-inoculated and commercially coated, were evaluated for three years (eight harvests) at two sites in northwestern Alberta. Seeds inoculated just before planting had more rhizobia at planting time than pre-inoculated seed. When results from all the three years were combined, inoculation with Rhizobium, either seed-applied or pre-inoculated, significantly (P<0.05) increased nodule dry matter (DM) at both sites. However, the response of forage N and DM yields to inoculation were significantly higher with seed application than with seed coating at one site, and vice–versa at the other site. Differences resulting from the use of non-sterile peat or sterile peat were not significant.