JOURNAL OF PLANT NUTRITION, 25(1), 75-92 (2002)
EFFECT OF RHIZOBIUM INOCULATION
AND PHOSPHORUS APPLICATION
ON NATIVE TEXAS LEGUMES GROWN
IN LOCAL SOIL
Ezedeen Osman,! James P. MUir/'· and
Anjo Elgersma J
'Ministry of Agriculture, PO. Box 1045, Asmara, Eritrea
2Texas Agricultural Experiment Station, 1229 North US
Hwy 281, Stephenville, TX 76401
3Department of Crop and Weed Ecology, Wageningen
University, The Netherlands
I
I
I
11
ABSTRACT
Greenhouse experiments were conducted to detennine the effects of Rhizobium inoculation and addition of phosphorus to soil on the productivity and quality of the Texas range legumes Des manthus iIIinoiensis (Michx.) MacMill. ex B.L. Rob. & Fernald, Desmanthus velutinus Scheele, Desmanthus virgatus (L.) Willd., and Neptunia lutea (Leavenw.) Benth. Phosphorus (P) fertiliza tion was applied at 0, 40, 80, and 120 kg P ha- I in Experiment I with half the pots also receiving D522-1/2 (a Desmanthus iso late). Experiment 2 consisted only the Desmanthus species and tested four different inoculant treatments (uninoculated control,
"Corresponding author. Fax: 254-965-3759; E-mail:
[email protected] 75 Copyright
©
2002 by Marcel Dekker, Inc.
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76
OSMAN, MUlR, AND ELGERSMA
inoculated with commercial cowpea Rhizobia, D522-1/2 from Desmanthus isolates, and Ll145, a Leucaena isolate). Two soil treatments were used in Experiment 2, gamma radiation sterilized and unsterilized soil, to detennine whether introduced Rhizobia were as effective as native populations. Shoot dry matter (DM) yield, root DM weight, and shoot nitrogen (N) and P concen tration increased with increasing P application levels up to 80kgPha- 1 • Rhizobium strain D522-1/2 was an effective inocu lant for all legumes when applied in combination with P fertilizer (Experiment I). In sterilized P-deficient soil (Experiment 2), inoculation improved the parameters measured. In unsterilized soil, however, only D522-1/2 and Lll45 improved shoot nitro gen concentration while the cowpea Rhizobium strain was detri mental in the case of D. velutinus. The low concentration of available soil P and the scarcity of active Rhizobium in the soil might be the cause of the slow establishment of native legume species in disturbed soils without native Rhizobia populations.
INTRODUCTION
Native North American warm-season legumes, despite their adaptation to local climate, soil, pest and disease pressures, and their ability to retain high quality forage under stress, are not being widely seeded in rangelands and cultivated pastures to compliment native and introduced grasses (1). For wildlife, these native forbs offer food and protection (2); they can also increase forage quality for livestock during the warm season, since nitrogen is usually a limiting factor for animal production from warm-season grasses (3-5). One of the reasons for the under-utilization of native warm-season legumes is the lack of infonnation about establishment and nutrient requirements of these plants. Deficient soil nutrient supply «(r...8) and ineffective rhizobial symbioses (9,10) may limit utilization. The latter may result from lack of inoculant, the use of poor quality inoculants, or Rhizobium strains that are incompatible with local soil and/or climatic conditions. Dear and Virgona (II) have suggested some strategies for developing legumes specifically for low input cultivation. These include selection of more persistent cultivars, selection of legumes that are P efficient and function at low P level, and evaluation of the ability of legume species to add nitrogen to the system. Numerous studies have shown that P is the most important nutrient for establishment and growth of legumes, although species and lines vary in their P requirement (12). Phosphorus deficiencies can adversely affect host plant growth, symbiotic rhizobia! establishment, and nodule function in legumes (13,14). An
77
NATIVE TEXAS LEGUMES
association of suitable strains of Rhizobia with legumes enhances the establish ment and growth of legumes (9-17). However, our knowledge of the nutritional requirements of native North American range legumes and the influence of soil nutrients on legume-Rhizobium interaction is inadequate to suggest practical recommendations of both chemical and biological amendments. The objective of this study was to examine the effects of P supply and Rhizobium inoculation on growth, N, and P accumulation of selected native Texas legumes. MATERIALS AND METHODS
Two greenhouse experiments were conducted simultaneously from Septem ber, 1999 to January, 2000 at the Texas A&M University Research and Extension Center, 2 kIn north of Stephenville, Texas (32° 13'38.8"N, 98° 13'9.I"W, and elevation of399 m), USA. Temperatures were maintained at a minimum 20°C, pots were watered (taking care not to splash soil) to field capacity every 4 days and artificial lighting was used to extend day-length starting in November.
Experiment One This experiment was designed to investigate the effect of P supply on the growth of native legumes as well as nodulation and N fixation with and without inoculation of seeds with a commercial strain of specific Rhizobia. The objective was to determine whether P fertilization is beneficial to native legumes when grown in low-P soils. Seeds of the four range legumes native to Texas were either collected in North Texas [Desmanthus velutinus Scheele, Desmanthus illinoiensis (Michx.) MacMil1. ex RL. Rob & Fernald, and Neptunia lutea (Leavenw.) Benth.] or acquired from South Texas [Desmanthus virgatus (L.) Willd.]. Topsoil to a 150cm depth was collected from a field of Windthorst fine sandy loam (fine, mixed, thermic Udic Paleustalf) where D. illinoiensis growth was abundant. Soil was never air-dried or exposed to high temperatures. Baseline laboratory analysis showed that the soil had an average pH of 8.3 and 13 ppm phosphorus (P; Texas A&M extractant). Plastic pots 15 cm in diameter were filled with 1.68 kg sieved (I cm 2) soil and placed in a greenhouse. Phosphorus (triple super phosphate) was mixed into pots at 0, 40, 80, and 120 kg P ha - 1 and two inoculum treatments (uninoculated control, inoculated by mixing into the soil 0.5 g peat poe I commercial strain of Desmanthus 522-1/2 marketed by the Urban Laboratories, St. Joseph, Missouri, USA) were applied. Seeds were mechanically scarified (18) and then sown directly in pots at a depth of 2 cm (9). Seedlings were thinned to two per pot. Pots were watered to field capacity every other day and maintained free of weeds throughout the experiment.
78
OSMAN, MUIR, AND ELGERSMA
The experimental design was a split-split plot with legume species as the main plots, P as the subplot, and inoculation as the sub-subplot. Treatments were replicated four times and each experimental unit consisted of two pots. The pots were arranged on greenhouse benches in a randomized complete block design. Plants were harvested by pot 105 days after sowing (before reproductive activity) and separated into root and aerial portions at the soil level. Roots of all samples were harvested by washing the soil over a fine sieve and separating all visible nodules. Shoots, roots, and nodules were separated and oven-dried at 55°C for 48 hours. Following weighing, all aboveground plant material was batched by experimental unit and ground in a Wiley mill through a 2-mm screen. To determine P and N, samples were digested using a modification of the aluminum block digestion procedure of Gallaher et al. (19). Sample weight was 1.0 g, digest used was 5 g of 33 : I : I K2 S04 : CUS04 : Ti0 2 , and digestion was conducted for up to 2 h at 400°C using 17 ml of H2S04, Phosphorus and N in the digestate were determined by semiautomated colorimetry (20) using a Technicon Autoanalyzer II. Shoot, root, and nodule dry matter (DM) yield as well as N and P concentrations of herbage were determined. Nitrogen and P yields were obtained by multiplying their concentration by plant DM yield.
Experiment Two This experiment was designed to investigate the effect of three commercially available rhizobial strains (including a specific strain isolated from Desmanthus spp.) on growth, nodulation, and nitrogen accumulation of selected Desmanthus species collected in North Texas grown in the presence of absence of indigenous, native Rhizobia. The objective was to determine the need for inoculation of Desmanthus spp. with Rhizobia specific for the genus. Treatments consisted of three Desmanthus species (D. illinoiensis, D. velutinus, and D. virgatus), 4 inoculant treatments (uninoculated control; inoculated with commercial broad-spectrum cowpea Rhizobia; inoculated with commercial Rhizobium strains of D522-1/2, D. illinoiensis isolates; and inoculated with Rhizobium strain L I 145, a Leucaena isolate marketed by Urbana Laboratories, St. Joseph, MO), and two types of soils (gamma radiation sterilized and unsterilized). Care was taken to avoid air-drying the soil or exposing it to high temperatures. Pots were arranged in the greenhouse in a randomized complete block design (RCBD) with four replications. Each experimental unit consisted of two pots with two plants per pot. Soil type and source were the same as in Experiment I. Half of the soil collected was sterilized by gamma radiation (minimum dose 27.8 kGy and maximum dose 54.3 kGy) by SteriGenenics International Co., Fort Worth, TX, while the other half was unsterilized. Ganuna rays emitted from the Cobalt 60
79
'IYE TEXAS LEGUMES
)pe destroyed all soil microorganisms, including the Rhizobium population. methodology was similar to that followed in Experiment 1 except that ) fertilizer was applied to the soils. Plants were harvested at 125 days after sowing. Harvest and sample ysis procedures were identical to those of Experiment I. Shoot, root, and lie DM yields as well as Nand P concentrations of the aboveground material : determined. Nitrogen and P yields were obtained by multiplying their :entration by plant DM yield. All data in both experiments were analyzed using a general linear model M) technique of SPSS for Windows (21). When the effects of treatment were ificant at the 0.05 probability level, least significant differences (LSDo.os) es were calculated and used for comparison of treatment means. ~equent
RESULTS AND DISCUSSION Results from Experiment I, which did not show three-way interactions ng treatments, are presented in graphic form in the following sub-sections. \Use of significant three-way interactions, results from Experiment 2 are ~nted in table form.
Shoot DM Yield :riment One There was a species x P interaction (p < 0.05) in shoot yield. All species mded with higher shoot DM when P fertilizer was applied and achieved mum shoot DM yield at either 80 or 120 kg P ha - 1 (Fig. I). Similar results been reported for a number of legumes (16,22,23). A study in India with mpea (Cajanus cajan) showed that P application up to 40 kg P ha- 1 ficantly increased shoot DM production (24). Ahlawat and Saraf (25) mined that increases in shoot DM yield in a similar study involving legumes due to increased soil water and nutrient extraction efficiency. Phosphorus iency disturbs metabolic function associated with growth and impairs ation of available non-structural carbohydrate in plants (26). Andrew and ns (27) showed that plant species differ in their response to added P. In study, Stylosanthes humilis produced its maximum DM production at a : P ha- I application whereas Lotononis bainesii and Medicago sativa red a lower application of P to obtain maximum DM production. mtrast, Macroptilium atropurpureum and Desmodium intortum required :g Pha- I to achieve maximum herbage production.
80
OSMAN, MUIR, AND ELGERSMA D. ill: y=0.14h-0.Ol; RJ:.o.36; P=G.OOI
D. vel: y=O.7Ox+O.10; RJ:.o.67; P=G.OOI D. vir: y=O.179J:+0.16; RJ:.o.39; P=G.OOI N. luI: y=O.177s+O.07; RJ:.o.32; P=G.Ol
0.9
R
0.8
1.
0.7
i"
0.6
;
O.S
Q
0.4
i
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0.3
CIl
0.2
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/
.....
./
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-------~
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0.1
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/
o
40
80
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--D. ill ...... D.vel -B-D. vir -N.lul
120
Fertilizer P kg ba"
Figure 1. Shoot DM yield response of D. illinoiensis (D. ill), D. velutinus (D. vel), D. virgatus (D. vir), and N. II/tea (N. lut) to P application rates (p x species interaction p < 0.05; LSD o05 0.17).
=
There was no significant species x inoculant x P application interaction for shoot yield. However, there was a significant (p < 0.05) interaction between Rhizobium inoculation and the level of P application in mean shoot DM production for all four species (Fig. 2). Inoculation with D522-1/2 had no significant effect at the lowest P levels, 0 and 40 kg/ha, but resulted in higher shoot DM at the highest P level compared to the uninoculated control.
Experiment Two There was a species x soil treatment x inoculation interaction for shoot yield (Table I). Only inoculation with Rhizobium strains D522-1/2 increased shoot DM weight in D. virgatus and D. velutinus on the sterilized soil when compared with the unsterilized control. In contrast, D. illinoiensis produced greater shoot yields when inoculated with either cowpea or D522-1/2 inoculant over the sterilized, uninoculated control. In the unsterilized soil, D522-1/2 was consistently effective in all species. Rhizobium strain cowpea also resulted in a significant increase of shoot DM for D. velutinus in unsterilized soil. The plant genotype significantly modified the performance of Rhizobium strain, i.e. a highly effective strain in one species could be rated as low or moderately efficient in another species. Thus, this interaction between plant species and Rhizobium strain indicates the need for evaluating specific strains for individual plant species. There are also advantages, however, to identifYing general strains with broad
ATIVE TEXAS LEGUMES
81
0.7 0.6
.
'i.
.
0.5
~
Clo
0.4
~
Q
....'8
'"
0.3 0.2 0.1 0
o
40 80 Fertilizer P kg ba- I
120
igure 2. Effects of inoculation and phosphorus application on total shoot DM yield leraged for D. illinoiensis, D. velutinus, D. virgarns, and N. lutea (P x inoculant Lteraetion p < 0.05; LSD o.05 = 0.12).
)mmercial application. On average, all species produced 29% higher shoot DM the sterilized soil than the unsterilized soil. Soil pathogens may have depleted lant energies in the unsterilized soil.
I
Root DM Yield xperiment One The effect ofP application on root DM weight plant- I varied with the plant Jecies and inoculation. A significant (p < 0.05) interaction between species and P )plication was obtained for root DM production. All species except D. velutinus lcreased root weight with increased P (Fig. 3). Application of more than J kg P ha- I failed to significantly increase root DM production in all species. There was also a significant (p < 0.05) interaction between Rhizobium loculation x species in root DM yield. Inoculation increased root DM in all the Jecies except D. virgatus (Table 2). xperiment Two An interaction (p < 0.0 I) among species x soil treatment x inoculation eatment was observed in root DM production (Table I). In the sterilized soil, hizobium strains cowpea and D522-1/2 significantly increased root DM ."Oduction in all species, but D522-1 /2 increased root OM yield of D. illinoiensis lore than did all other inoculants Rhizobium strain Lll45 affected root yields
82
OSMAN, MUIR, AND ELGERSMA
Table 1. Effect of Inoculation and Soil Treannent on Shoot OM Yield, Nodule OM, Shoot Nitrogen %, Shoot N OM Yield, and Root OM Weight of D. iIIinoiensis, D. velutinus, and D. virgatus (Experiment 2)
Parameter Mean Values
Soil Treannent
Shoot N Root OM Nodule Shoot N OM Yield Weight Rhizobium Shoot OM Inoculant (g/plant) OM (mg/plant) (% shoot) (mg/plant) (g/plant)
Desmanthus illinoiensis Unsterilized Control Cowpea 0522·1/2 Lll45
0.24 0.28 0.38 0.20
12.98 12.43 14.66 5.92
1.83 1.66 1.87 1.54
4.35 4.61 7.01 4.29
0.27 0.37 0.26 0.27
Control Cowpea 0522·1/2 Lll45
0.29 0.40 0.60 0.33
1.56 22.31 26.94 15.59
1.46 2.23 2.24 2.19
4.20 8.81 13.52 7.13
0.30 0.44 0.52 0.36
Desmanthus velutinus Unsterilized Control Cowpea 0522·1/2 Lll45
0.12 0.19 0.18 0.15
6.83 7.77 9.57 10.36
1.61 1.37 1.59 1.65
1.93 2.63 2.91 2.47
0.06 0.15 0.15 0.16
Control Cowpea 0522·1/2 Lll45
0.20 0.23 0.32 0.22
1.08 2.12 27.45 23.58
1.36 2.08 2.06 2.09
2.76 4.83 6.53 4.57
0.15 0.29 0.26 0.23
Desmanthus virgatus Unsterilized Control Cowpea 0522-1/2 Lll45
0.31 0.32 0.36 0.33
5.14 6.81 16.41 24.26
1.54 1.56 1.90 1.81
4.71 5.06 6.90 6.05
0.26 0.24 0.31 0.24
Control Cowpea 0522-1/2 Lll45
0.34 0.31 0.47 0.36
0.32 12.40 26.33 11.53
1.57 2.06 2.28 1.96
5.30 6.34 10.64 7.13
0.27 0.45 0.51 0.26
p value
0.04 0.05
Sterilized
Sterilized
Sterilized
LSOo.os
0.008 7.6
0.0001 0.16
0.001 0.96
0.01 0.07
83
UIVE TEXAS LEGUMES D. ill: y=(l.09OI+O.II: R'=O.52: P=O.OOI D. vel: y=(l.045I+O.12: R'=O.J6: P=O.OOI D. vir: y=(l. 1401+0. 10; R'=O.49; P=O.OOI N. lut: y=(l.092I+O.18: R'=O.42; P=O.OOI 0.7 ~
.. " .., co
15. ':;'
0.6 0.5 ...... D. ill 0.4 +--------7~~~~=--=.-__i D. vel
.....
:l:
0.3
l
0.2
-D.vir
Q
.... N.lut
0.1 0
o
80
40
120
P fertilizer levels kg ha"
'gure 3. Root dry matter yield response of Desmanthus illinoiensis (D. ill), esmanthus velutinus (D. vel), D. virgatus (D. vir), and N. lutea (N. lut) to P application Ie (species x P rate interaction p < 0.05; LSD o05 ::: 0.10). rble 2. I.
Effects of Inoculation on Root DM Yield and Shoot P Yield of D. illinoiensis ill), D. velutinus (D. vel), D. virgatus (D. vir), and N. lurea (N. lut) (Experiment 1) Root DM Yield (g/plant)
Phosphorus DM Yield (mg/plant)
oculant
D. ill
D. vel
D. vir
N.lutt
D. ill
D. vel
D. vir
N.lutt
)ntrol 522
0.30 0.37
0.58 1.12
0.22 0.25
0.53 0.86
0.39 0.50
1.29 2.20
0.32 0.50
1.02 2.44
value ;;Do. 05
0.047 0.07
0.002 0.41
lly in D. velutinus in the sterilized and unsterilized soil when compared to the mtrol. In general, all species produced 47% higher root DM in the sterilized soil an in the unsterilized soil. Nodule DM Yield ,,-periment One
2
Phosphorus application increased nodulation linearly (y = 8.68x+1.62; in all the species whether inoculated or not (data not
= 0.97; p = 0.003)
84
OSMAN, MUIR, AND ELGERSMA
shown). The addition of P increased (p < 0.01) nodule OM weight from 9.30 mgplanC 1 at OkgPha- I to 37.34mgplanC I at 1200 kg Pha- I • Gates (22), in his study on the effect of P in Stylosanthes humilis grown in a greenhouse, found that P had a beneficial effect on the initiation of nodule formation. In that study, nodules were observed three days earlier in high P than in low P plants. Nodules' relative growth rates were stimulated from 0.3 g day- I at low P treatment to 0.7 g/day at high P treatment over days 23-26. In another study, nodule O 2 permeability has been shown to vary with P nutrition (28,29). According to Vadez et al. (29), P deficiency caused O 2 limitation in the nodule by increasing the rate of O2 consumption. Thus, P deficiency might have a direct negative effect on nodule formation and function. Inoculation had no significant effect on nodule OM accumulation but a difference (p < 0.0 I) in nodule OM weight between species was observed. Desmanthus illinoiensis had the lowest nodule OM weight (15.4 mg planC 1) while N. lutea yielded the highest (37.3 mg planC 1).
Experiment Two Nodulation showed response interactions (p = 0.008) among species x soil treatment x inoculation treatments (Table I). Inoculated plants of all species had increased nodule OM weight in the sterilized soil when compared to the uninoculated control. However, inoculation with Rhizobium 0522-1/2 resulted in a higher nodule OM yield than the other inoculants. There were no significant (p> 0.05) responses to any inoculation treatment in the unsterilized soil for D. velutinus whereas both Rhizobium strains Lll45 and 0522-1/2 increased nodule DM in D. virgatus in the unsterilized soil.
Shoot Phosphorus Concentration Experiment One There was a (p < 0.05) species x P level interaction effect on shoot P concentration because N. lutea showed a progressive increase in P concentration over the full range of P treatments (Fig. 4). The other species showed a response in shoot P concentration only at the highest level of P. Increasing the P level to 120 kg P ha- I increased P concentrations by approximately 57, 69, 92, and 107% plant- 1 for D. illinoiensis, D. velutinus, D. virgatus, and N. lutea, respectively, compared to the respective controls.
85
TIVE TEXAS LEGUMES D. iU: y=O.26l
