Germida, J.J.

McKercher, R.B.

Olson, B.M.

English

eCommons@USASK

Effect of trifluralin on growth, nodulation, and nitrogen fixation of peas

University of Saskatchewan Research Archive

- 177 -Effect of Trifluralin en Growth, Nodulaticn, am Nitrogen Fixaticn of Peas J .J. Germida, B.M. Olson and R.B. McKercher Department of Soil Science, University of Saskatchewan Saskatoon, Saskatchewan, S7N c:ml A variety of herbicides are used in Saskatchewan in order to protect crops and increase yields. This is of particular importance when herbicides are used with legume crops because these chemicals may not only control plant growth, but may also affect the Rhizobium-legume interaction or the population of rhizobia in the soil. The consequences of herbicides affecting such non-target systems might result in a decrease in nitrogen fixation and thus affect soil ferti-lity. Soil-incorporated herbicides such as trifluralin should be of special concern as they contact the root system where nodulation and nitrogen fixation processes occur. OUr previoos studies (Germida et al., 19831 Olson et al., in press) have shown that the soil incorporated herbicide trifluralin does not significantly affect soil microorganisms or decomposition processes in situ in soil; however, excessive rates (i.e., > 25,000 ppm) did affect the growth of soil microorganisms, including rhizobia, as pure cultures on laboratory media. Investigations by other workers (Brock, 1972; Dunigan et al., 1972; Kurst and Struckmeyer, 1971; DeRosa et al., 1978) have shown that trifluralin causes a reduction in the nodulation of legumes such as soybean and clover. A limited study by Harvey and Gritton (1977) has examined the effects of this herbi-cide on the growth of two pea cultivars. With this in mind, the objective of the present study was to investigate the effects of trifluralin on the growth, nodulation, and nitrogen fixation of pea plants grqwn in a greenhouse or in the field. Growth Chaniler Study Two separate experiments were conducted using a Dark Brown Cherno-zemic soil (Bradwell loam) • Basically, pea seeds were subjected to three types of inoculation (see below) and then seeded into soil treated with four rates of trifluralin (see below). Each experiment consisted of 4 replicates per treatment in a completely randomized block design. The characteristics of the Bradwell loam have been described previously (Germida et al., 1983). For herbicide incorporation, 1560 g of air-dried, sieved (2 nm) soil received 140 ml of distilled water containing a commercial preparation of trifluralin (Treflan EC) suffi-cient to provide the appropriate soil concentration (0, 1 ppm, 2 ppm, 3 ppm), 100 ml of a ·nutrient stock solution (0.0121 M K2HP04 and 0.0047 M K2so4) and then mixed in a large polyethylene oag. The control so1l was treated with the appropriate amount of nutrient solution and distilled water. All treated soils were allowed to equilibrate for 24 h before potting in 6" plastic pots. - 178 -Pisum sativum L. (var. Hanesteader) seeds were surface sterilized in a 20% javex bleach solution for 5 min and then rinsed iri sterile distilled water 7 times. One set of seeds was treated with a canmer-cia! inoculum (Nitragin Co.) according to the instructions. . Another set was inoculated (0.5 ml/seed} with an unwashed 72 h broth culture of Rhizobium leguminosarum strain 128CS2. This rhizobium strain was grown in 50 m1 of YEM. broth (Vincent, 1970) on a gyrotory shaker at 27C. Control seeds were surface sterilized but received no inoculum. Initially, ten seeds were planted in each pot (3/4" depth) and the soil covered with styrofoam beads (4 nm dia.) to protect against water loSSf after germination the number of seedlings was thinned to three. All pots were placed in a growth chamber with controlled temperature, lighting, and humidity as described previously {Germida et al., 1983). All pots were weighed and the soil maintained at field roisture-hold-ing capacity {about 20% roisture) during the 5-week growth period. At harvest, the plants and soil were dumped from the pot and the bulk soil carefully removed to prevent excessive root loss. The shoot and root systems were separated. Shoot material was oven dried (65 C), weighed, and analyzed for total nitrogen by the Kjeldahl proce-dure. Root systems and nodules were subjected to the nitrogenase as-say in order to measure their nitrogen fixation potential., For this assay, three unwashed root segments (and clinging soil) were :i.mnediately placed in 455 ml glass mason jars with lids fitted with rubber septums. Twenty-five cc of the atrrosphere was replaced with C2H2, and duplicate samples of the head space were collected after 30 min for determination of C H as described previously (Nelson and Child, 1981) • Samples (0. ~ &) were analyzed on a Hewlett Packard 5730A gas chromatograph fitted with a 1.5 m x 3 mm column of Poropak R (column tenp was 90 C) using N2 carrier gas and a H2 flame ionization detector at 200 C. Peak heights were measured using a Hewlett-Packard 3373B integrator. After analysis, roots were recovered, washed, number of nodules counted and root fresh weight and tap root measurements taken. Field Sony In order to assess the effects of trifluralin on peas grown under field conditions, a preliminary experiment was set up on a Dark Brown Chernozemic soil {Sutherland clay loam) at the University of Saskatchewan Kernen Farm (for soil characteristics see Germida et al., 1983). This study consisted of a canplete randomized block with split plot design on a 10 M x 45 M field plot. For herbicide incorporation, one pass of an 80 inch swath ( 4 nozzles set at 18 inches above the ground at 3 mph) for the recommended field rate {1.1 kg/ha) and 2 passes for 2 x recammended rate was made. Pisum sativum L. (var. Trapper) seeds (4.6 seeds/ft or 125 kg/ha) were sown at a depth of 2 l/2n using a Press drill (6" spacing) • Seeds were either inoculated with a canmercial ir..oculum of R. leguminosarum or else un-inoculated. No fertilizer was applied to the field. Plots were examined at various time intervals and observations made on the growth of pea plants and nodulation of representative plants from all plots. No attenpt was made to recover root systems and count nodules or determine nitrogenase activity; however, after - 179 -plani: mtur i i:y, seeds were colleci:ed and analyzed for total nitrogen content as described above. :msJI:rS AND DISO:ESICB Growth ChaniJer Study Effect of Herbicide on Pea Plant Growth. In general, there was no effect of trifluralin on the ger.mination of pea seeds in the Bradwell loam soil. Ot,her studies (not reported) have shown sane toxicity of trifluralin on seed ger.mination in sandy (Asquith) soil. Plants in trifluralin treated soil (Bradwell loam) exhibited slower growth than plants in non-treated soil, and the shoot growth of plants in soil receiving 3 ppm of trifluralin showed visible signs of damage (e.g., curled leaves and leaf-tip browning). These results are consistent with previously reported studies on various legumes (Brock, 1972; Dunigan et al., 1972; Harvey and Gritton, 1977). The effects of trifluralin on the growth of pea plants is shown in Table 1. There was significant reduction of plant root fresh weight and shoot dry weight due to 3 ppm trifluralin. This was the case regardless of whether the plants had been nodulated by rhizobia. Lower concentrations of trifluralin caused significant but less severe effects. There was, however, no significant effect or clear-cut trend on tap root length, or on shoot nitrogen content by any of the triflu-ralin concentrations tested. Visual observations indicated that lat-eral root development was extensive on all plants, although it was sanewhat stunted on plants in soil containing 3 ppm trifluralin. Sim-ilar effects; i.e., reduction in shoot and root weight due to triflu-ralin, have been reported for several Trifolium (clover) species Table 1. Effect of trifluralin on the growth of pea plants after 5 weeks in a growth chamber. Inoculum Trifluralin Root fresh wt Tap root Shoot dry Shoot-N concentration per pot (g) length per wt per per plant (em) plant (g) plant (%) None Control 13.5Sa* 26a 1.17a 3.1Sa None 1 ppm lO.SSbc 26a 1.20a 3.20a None 2 ppm 11. 75ab 29a 0.88b 2.89a None 3 ppm 8.97c 32a 0.58c 3.30a Ccmnercial Control 12.02a 29a 1.08a 3.14a Carmercial 1 ppm 10.39a 26a 1.09a 3.03a carmercial 2 ppm 10.84a 24a 0.88b 3.0Sa Ccmnercial 3 ppm 6.4Sb 33a 0.4Sc 3.58b Strain C52 Control 11.22a 26a !.lOa 3.46a Strain C52 1 ppm 7.62b 20a 0.88b 3.4la Strain C52 2ppm 9.37ab 26a 0.92ab 3.09a Strain C52 3 ppm 4.18c 26a 0.36c 3.67a * Means of four replicates; values followed by the same letter within each column for each inoculum do not differ at the 95% level as determined by Duncan's multiple range test. - 180 -(Brock, 1972) and for Glycine~ (soybeans) (Dunigan et al., 1972)., Harvey and Gritton (1977) found that trifluralin at a rate of 1.68 kgjha (about 0.8 ppm) caused a reduction in root fresh weight of greenhouse grown "Alaska" and "Perfection" pea cultivars. Effect of Herbicide on Nodulation of Plants. Pea plants grown in this Bradwell soil were not nodulated unless they had been inoculated with an appropriate rhizobilml strain. The effect of trifluralin on nodulation and nitrogenase activity of inoculated pea plants is shown in Table 2. There was a highly significant correlation (r = .95) between a decrease in root nodulation or nitrogenase activity, with increasing herbicide concentration. Note that 2 ppm and 3 ppm triflu-ralin caused a significant reduction in the number of nodules per g of root fresh weight and in the nitrogenase activity of these roots e These results were essentially the same regardless of the rhizobium inoculum used. Harvey and Gritton (1977) did not examine the effect of trifluralin on nodulation and nitrogenase activity of their pea cultivars. Other investigators, however, have shown that trifluralin (at rates carparable to those in the present study) causes a reduction in the number and size of nodules on various legumes (Brock, 1972~ Dunigan et al. , 1972; Kurst and Struckmeyer, 1971) • Because there is a direct relationship between the number of nodules and nitrogenase activity (Fig. 1}, it would appear that the main effect of trifluralin was a reduction in nodulation which then resulted in a corresponding decrease in nitrogenase activity. It has been suggested that the rea-son for reduced nodulation of legumes in trifluralin treated soils is Table 2. Effect of trifluralin on the number of nodules and nitrogenase activity of pea plants after 5 weeks in a growth chamber Inoculum Trifluralin No nodules Nitrogenase concentration (per g root activity fresh wt) ~les c2Hf g per h None Control 0 0 None 1 ppm 0 0 None 2 ppm 0 0 None 3 ppm 0 0 Ccmnercial Control 7 .9a* O.l54a Camnercial lpfm 7.lab O.l23ab Ccmnercial 2 ppm 3.8bc 0.035bc Ccmnercial 3pfm l.Oc 0.002c Strain C52 Control 9.3a 0.16la Strain C52 1 ppm S.Sab 0.062ab Strain C52 2pfm 3.0bc 0 .049bc Strain C52 3ppm O.Sbc O.OOSbc * Means of four replicates; values followed by the same letter within each column for each inoculum do not differ at the 95% level as determined by Duncan's multiple range test. ... 181 -due to the abnormal root growth caused by this herbicide (Brock, 1972) • We also. detected abnormal root growth and our results would seem to support this hypothesis. The results obtained were s:i.milar for both of the growth chamber studies. Field Study General Observations. The field study plots were seeded on June 2, 1983 ana were damaged approximately 3 weeks later due to a heavy rain. At 1 ronth of age, the pea plants on trifluralin treated plots, especi.ally the 2 x field rate plot, were visibly stunted canpared to the plants on the control plots. After 5 weeks of growth, plants were selected at randan fran all plots and examined for nodulation. All plants examined, even control uninoculated plants, exhibited good to heavy nodulation. By harvest, all plants appeared to have recovered fran the earlier effects of trifluralin. Based on an analysis of the pea seeds from plants on the various treated plots, there was no ef-fect of trifluralin on the nitrogen content of seeds. J="-2 1.6 ./ >- 1.5 1--:1.4 r = 0.97 _ ..... :1."=' >• •;;;J- ..s::: :1.2 1- :1.:1. / U.-t <(I :1.0 3: '9 LlJ '+- 0 en e> o.;;.o <( 7 z (IJ 6 • LlJ QJ C) .- 5 0 0 4 a:: e ./ • ~--~ 3 -- 2 z 1. 0 E0 i. 2 3 4 s 6 7 8 9 NO. NODULES (gfw-1) Fig. 2 Relationship between nodulation ~ nitrogenase activity of pea plants~ note y-axis scale E - 2 = 10-In general, trifluralin reduced pea plant shoot and root growth, and caused a reduction in plant nodulation under growth chamber con-ditions. Under field conditions, trifluralin did not appear to cause major effects on pea plant growth and concentrations up to 2 x field rate were not a problem. On the basis of this short-term study, it - 182 = would appear that recamnended rates of trifluralin would not inpair legume nodulation and growth in the field. Nevertheless, based on the growth chamber results, caution should be exercised when using tri-fluralin with legumes, as carelessness and accidental spills may have significant effects on legume growth and nodulation. The consequence of less effective nitrogen-fixing leguines could affect soil nitrogen fertility. We extend thanks to B.. Drew and L. Mills of the U. of S. Crop Developnent Center for setting up the field plots, and also to L. Nelson and F. Chalifour of the Plant Biotechnology Institute for advice and gas chromatography facilities for the nitrogenase assay. Brock, J .L. 1972. Effect of the herbicides trifluralin and carbetamide on nodulation and growth of legume seedlings. Weed Res. 12: 150-154. DeRosa, F., D. Haber, C. Williams and L. Margulis. 1978. Inhibitory effects of the herbicide trifluralin on the establishment of the clover root nooule symbiosis. Cytobios 21: 37-43. Dunigan, E.P., J.P. Frey, L.D. Allen, Jr., and A. McMahon. 1972. Herbicidal effects on the nodulation of Glycine Max (L.) Merrilo Agronany J. 64: 806-808. Germida, J .J., B .M. Olson and R.B. McKercher. 1983. Soil microor-ganisms and the herbicide trifluralin. Proceedings of the 1983 Soils and Crops Workshop, 223-236, Publication No. 482, Extension Division, University of Saskatchewan. Harvey, R.G. and E.T. Gritton.. 1977. Susceptibility of pea cultivars to trifluralin. Weed Science 25: 23-26. Kurst, C.A. and B.E. Stuckmeyer. 1971. Effect of trifluralin on growth, nodulation, and anatany of soybeans. Weed Science 19: 147-152. Nelson, L.M and J .J. Child. 1981. Nitrogen fixation and hydrogen netabolism by Rhizobium leguminosarum isolates in pea root nodules. Can. J. M~crobiol. Z7: 1028-1034. Olson, B.M., R.B. McKercher and J .J. Germida. 1984. Microbial populations in trifluralin-treated soil. Plant and Soil (in press). Vincent, J .M. 1970. A manual for the practical study of root-nodule bacteria. I.B.P. Handbook No. 15. Blackwell Scientific Publications Ltd., OXford. 

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Effect of trifluralin on growth, nodulation, and nitrogen fixation of peas

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