~ Pergamon

0098-8472(95)00023-2

EnvironmentalandExpenmentalBotany, Vol. 35, No. 3, pp. 287 298, 1995 Copyright © 1995 Elsevier Science Ltd

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ROOT-ZONE TEMPERATURE AND SOYBEAN [GLYCINE MAX. (L.) MERR.] VESICULAR-ARBUSCULAR MYCORRHIZAE: DEVELOPMENT

AND INTERACTIONS WITH THE NITROGEN FIXING SYMBIOSIS

FENG ZHANG,* CHANTAL HAMEL,~ HORMOZDYAR KIANMEHR~ and DONALD L. SMITH*

*Department of Plant Science, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9; 1"Department of Natural Resources Science, Macdonald Campus of McGill University,

Ste. Anne de Bellevue, Quebec, Canada H9X 3V9; ++Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

(Received 15 December 1994; accepted in revised form 6 April 1995)

Zhang F., Hamel C., Kianmehr H., and Smith D. L. Root-zone temperature and soybean [Glycine max. (L.) Mar.] vesicular-arbuscular mycorrhizae." Development and interactions with the nitrogen fixing symbiosis. Environmental and Experimental Botany 35, 287-298, 1995.--Suboptimal root-zone temperature (RZT) has been shown to decrease soybean [Glycine max (L.) Merr.] nodulation and nitrogen fixation. However, there are few studies on the effects of suboptimal RZTs on vesicular-arbuscular (VA) mycorrhizal colonization of their host plants, and no investigation of those effects in soybean. In addition, there have been no investigations of the tripartite symbiotic association (VA mycorrhizal fungi, Bradyrhizobiumjaponicum and soybean) over a range of RZTs. A controlled-environment experi- ment was conducted to examine the effect of RZTs on VA mycorrhizal colonization, nutrient uptake, nodulation and nitrogen fixation, and plant growth and development. The experiment was organized as a randomized complete block split-plot design; the main-plot units consisted of four RZTs, 15, 18.2, 21.6 and 25°C; the four harvest stages and inoculation, or not, with mycorrhizal fungi formed the sub-plot units. The results of this study indicated that (1) the optimal RZT for mycorrhizal infection of Glomus versiforme was 21-22°C - - above and below this range, mycorrhizal colonization was inhibited; (2) mycorrhizal colonization increased until flowering (the third harvest), but decreased thereafter at all tested RZTs; (3) mycorrhizal colonization had a negative effect on nodule estab- lishment (nodule number) at lower RZTs, but a positive one at higher RZTs; and (4) the lower nodule number at lower RZTs was more than compensated for by increased mass per nodule so that mycorrhizal infection stimulated N2 fixation at lower RZTs.

Key wor&: B.japonicum, Glomus versiforme, Glycine max, root zone temperatures.

INTRODUCTION

Soybean [Glycine max. (L.) Merr.] is a subtropical legume, and requires root zone temperatures (RZT) in the 25 30°C range for optimal growth and pro-

duction. (23) Current soybean production in eastern Canada is at the northernmost North American geographic limit of the crop. In most areas of Canada, early vegetative growth of soybean occurs under elevated shoot temperatures but suboptimal

Abbreviations: LSD: least significant difference; RZT: root zone temperature; VA: vesicular-arbuscular. 287

288 FENG ZHANG et al.

RZTs. Average soil temperatures at 10 cm of depth during the first month of the growing season in Canadian soybean production areas are often below 15°C, and may be below 20°C until July. (29/

Under field conditions, soybean plants generally bear two types of symbiotic microorganisms on their roots. These symbionts, Bradyrhizobium spp. and ves- icular-arbuscular (VA) mycorrhizal fungi, are able to enhance plant growth by supplying nitrogen and otherwise poorly-mobile soil nutrients such as phos- phate, respectively, to their host plant. However, suboptimal temperature restricts the growth of N2- fixing soybean plants more than that of plants uti- lizing combined nitrogen. (26'~7) Studies of sub-opti- mal RZT effects on nitrogen fixation by soybean have concluded that suboptimal RZTs decrease nodulation and nodule function. (17'23'25'3°~ Recent studies have indicated that the time between inocu- lation and the onset of nitrogen fixation by soybean plants decreases 2-3 days per °C as RZT drops from 25°C to 17°C, and then decreases about 7 days per °C as RZTs decline below 17°C. (45'46)

Vesicular-arbuscular mycorrhizal fungi form a symbiotic relationship with most vascular plants./18/ Generally, plants benefit from the presence of these fungi which, by extending the plant root system, facilitate nutrient absorption. The extent of the mycorrhizal relationship depends not only on the susceptibility of the plant species to mycorrhizal infection and the infectivity of the fungal species,/1'33/ but also on soil environmental conditions such as temperature, moisture, nutrient levels, microbial competition, organic matter level and pH. (6'12'37) Compared with the data regarding suboptimal RZT influences on soybean nodulation and nitro- gen fixation, the effects of RZT on the mycorrhizal symbiosis are much less understood./19'37/ Inves- tigations regarding the influence of soil temperature have examined effects on spore germination ~38~ and growth of the fungus both pre- and post-infection./~4~ Smith and Bowen (4°) found that, in subterranean clover (Trifolium subterraneum) roots, an increase in soil temperature (from 20 to 25°C) was associated with a greater number of root entry points by VA mycorrhizal fungi sooner after inoculation. Graham eta/. (14) reported that an isolate of Glomusfasciculatus from a desert soil colonized a greater proportion of plant roots at 30°C than 25°C, even in P-deficient plants.

The coexistence of a bacterium and a fungus as

root endophytes of soybean establishes a tripartite symbiotic association. To date, there have been no investigations of RZT effects on the dynamic relationships between the soybean symbioses with Bradyrhizobium and Glomus, and no investigations of RZT effects on mycorrhizal development in soybean. Therefore, the present study was con- ducted to test the following hypotheses: (1) mycorrhizal infection is inhibited by the same RZT found to be suboptimal for soybean nodulation and nitrogen fi afion; and (2) mycorrhizal colonization affects soybean nodulation and N2 fixation dif- ferently at different RZTs.

MATERIALS AND METHODS

Plant materials Seeds of the soybean cultivar 'Maple Glen' were

surface-sterilized in sodium hypochlorite (2% solu- tion containing 4 ml 1 -l Tween 20), then rinsed several times with distilled water. 14) The cultivar 'Maple Glen' was selected because it was developed for production in, and yields well under, the short, cool season conditions of eastern Canada. The sur- face-sterilized seeds were planted in trays con- taining a sterilized Turface (Applied Industrial Materials Corp., Deerfield, IL 60015, U.S.A.) : sand (1:1, v/v) mixture. Two 7-day-old seedlings at the VC stage (unifoliolate leaves unrolled sufficiently that the edges were not touching) (ll~ were trans- planted into each of the two sub-divisions of each sterilized 13-cm diameter plastic pot containing the same medium and located on a Conviron growth bench (Model GB48, Controlled Environments Ltd., Winnipeg, Manitoba, Canada). The sub- divisions were formed by lining each side of the pot with a separate perforated plastic bag. The plastic bags allowed the plants on each side of the pot to be harvested separately with minimal root disturbance. Eight pots were sealed to the bottom of each of eight plastic tanks (68 x 42 cm) with a silicon sealant (GE Silicones Canada, Picketing, Ontario, Canada). Light (300/~E m -2 s -l) was provided by cool white fluorescent tubes. The day:night cycle was 16:8 hr. As this work attempted to isolate RZT effects, air temperature was held constant at 25°C. Root-zone temperatures (___ 0.5°C)were controlled by circulating cooled water around the pots, with eight pots in each tank. A hole drilled in the tank bottom below each pot allowed the pots to drain

DEVELOPMENT AND INTERACTIONS OF SOYBEAN 289

Table 1. Descriptions of the four harvest stages [see Fehr and Caviness IHI]

Harvest Growth time stage Description

1 V3 Three nodes on the main stem with fully developed leaves beginning with the unifoliolate nodes

R1 One open flower at any node on the main stern

R2 Open flowers at one of the two uppermost nodes on the main stem with a fully developed leaf.

R4 Pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf

Inoculation Plants to be colonized by VA mycorrhizal fungi

were inoculated with the fungus Glomus versiforme (Karsten) Berch. One gram of fresh leek root colon- ized by G. versiforme was placed at a depth of 5 cm in each mycorrhizal pot before transplanting the soybean seedling into the pot. Autoclaved roots from the same source were applied to non- mycorrhizal pots. One hundred grams commercial inoculum ofB.japonicum (LiphaTech Inc., Milwau- kee, WI 53029, U.S.A.) were mixed with 1 1 of sterilized water and then stirred with a magnetic stirrer for 2 hr in a laminar flow hood. Soybean seedlings were soaked in the resulting commercial inoculum solution of B. japonicum for 30 s before being transplanted.

when watered. Plants were then acclimatized for 24 h prior to inoculation. Plants were watered with a quarter-strength Hoagland's solution, (2°/in which the nitrogen and phosphorus were reduced to 2.0 m M (43/and 5.1 ~M, (15) respectively. Prior to each watering, the stock Hoagland's solution was diluted by mixing an appropriate amount of refrigerated water (4°C) and distilled water (25°C) in order to bring the solution to the same temperature as the treatment RZTs ( __ 0.5°C).

Experimental design The experiment was arranged in a randomized

complete block split-plot design with four repli- cations. The main-plot unit was R Z T at four levels: 25°C [optimal temperature for soybean nodulation and nitrogen fixation(l°'23i], 21.6°C [suboptimal temperature, but still above the normal spring soil temperature of Canada(28/], 18.2°C [sub-optimal temperature, but still above the critical 17°C, below which soybean nodulation and nitrogen fixation were strongly inhibited, although infection still occurred(29], or 15°C [at this RZT, soybean infec- tion and nodulation were strongly inhibited(29'45/]. The combinations of with or without mycorrhizal colonization and four harvest stages [V3, R1, R2, R4 (Table 1)] were arranged as the sub-plot units. The plant growth stages were not different (p_< 0.05) for the different RZTs since plant tissues above the ground were exposed to the same environmental conditions, such as air temperature and light density.

Harvest and data collection The following data were collected at each of the

harvests: leaf area (Delta-T area meter, Delta-T Devices Ltd., Cambridge, U.K.), leaf number, nodule number, nodule dry weight, nitrogen con- centration on a whole plant tissue basis [by colori- metry(2~/]. To determine the percentage of root colonization by the mycorrhizal fungus, samples were taken at random from the roots. The roots were cleared by immersion in 10% K O H and sub- sequently stained with acid fuchsin./5/Percentage of mycorrhizal colonization was determined by the grid line intersection method./~3/

Statistical analysis Results were analyzed statistically by analysis of

variance using the SAS system. (a6/When analysis of variance showed significant treatment effects, the least significant difference (LSD) test was applied to make comparisons among the means at the 0.05 level of significance."42)

R E S U L T S

Mycorrhizal colonization Mycorrhizal colonization was affected by R Z T

at each of the four harvests. For G. versiforme, the optimal R Z T for colonization of soybean was 21.6°C, with an average of 33% of roots being infected over all harvests, while at 15°C R Z T mycorrhizal infection was lowest at 7% (Fig. 1). The effects of RZTs on colonized root mass were similar to those observed for the percentage infection of

290 FENG ZHANG et al.

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RZT (°C) harvest stages RZT (°C) harvest stages Fig. 1. The main effects of suboptimal root zone temperatures and harvest stages on VA mycorrhizal infection percentages of root tissues (a), and colonized root masses and uncolonized root masses (b). In a and b, each bar represents the mean value from eight plants. The two bars (I) in the upper left and right of (a) represent the least significant difference values (0.05) for RZTs and harvest stages, respectively. In (b), empty bars and shaded bars represent the colonized and uncolonized root masses, respectively. Bars in the upper left represent least significant difference values (0.05) for colonized root mass (shorter) and uneolonized root mass (longer).

soybean roots (Fig. 1), except that the colonized root mass at 21.6°C R Z T was not significantly greater than that at 25°C RZT, a situation that occurred because total root dry matter was lower at 21.6°C RZT.

Mycorrhizal colonization of plant roots was detected at the first harvest, and had an average value across RZTs of 4%. This value then increased until the third harvest (about the flowering stage), when it reached 40%. Mycorrhizal colonization then declined to 24% by the last harvest (Fig. 1). Colonized root mass followed the same pattern as that of infection percentage (Fig. 1).

Nodulation and nitrogen fixation Soybean nodule formation was affected by

mycorrhizal inoculation at most RZTs. At lower RZTs, mycorrhizal colonization had a negative effect on soybean nodule number. The negative effect was significant at 18.2°C RZT, while at 15°C R Z T there was a trend towards a negative effect (p = 0.25). However, at higher RZTs (21.6 and 25°C) mycorrhizal plants generally had higher nod- ule numbers than non-mycorrhizal plants (Fig. 2).

At all suboptimal RZTs (<25°C) nodule mass showed an initial tendency to be decreased by

mycorrhizal infection (Fig. 3). However, the plants ultimately recovered from this and mycorrhizal infection eventually resulted in a stimulation of nod- ule mass at 15 and 21.6°C. Except at 25°C RZT, the weight per nodule was increased (p_<0.05) by mycorrhizal colonization at the last two harvests (Figs 2 and 3).

Even though mycorrhizal plants had lower nod- ule numbers at lower RZTs (15 and 18.2°C), nitro- gen concentrations were increased in mycorrhizal plants when compared with non-mycorrhizal plants at the last two harvest stages (Fig. 4). For the 18.2 and 15°C RZTs, the nitrogen concentration dif- ferences between mycorrhizal and non-mycorrhizal plants increased from the third to the fourth harvest (Fig. 4). Total nitrogen content per plant was higher for mycorrhizal plants than non-mycorrhizal plants, at the three suboptimal RZTs (15, 18.2 and 21.6°C) at the last two harvest stages, except for the last harvest of plants grown at 18.2°C R Z T (Fig. 5). Also, over the whole experiment, the tissue nitrogen concentrations of plants grown at 15 and 18.2°C RZTs showed a positive relationship with degree of mycorrhizal colonization; the correlation coef- ficients (r 2) were 0.63 (p < 0.05) and 0.68 (p_< 0.01), respectively. At 21.6°C RZT, even though the num-

DEVELOPMENT AND INTERACTIONS OF SOYBEAN 291

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Harvest stages Fig, 2. Changes in nodule numbers of both mycorrhizal and non-mycorrhizal plants across four harvests at four different root zone temperatures. The line with the symbol (0) indicates mycorrhizal plants and (O) indicates non-mycorrhizal plants. Each point

represents the mean (___ S.E. indicated by I) value of eight plants.

bers (Fig. 2) and weights (Fig. 3) of nodules of mycorrhizal plants were higher at the last two har- vests, the nitrogen concentration was lower than non-mycorrhizal plants; however, the nitrogen con- tent of mycorrhizal plants at this RZT had the highest rate of increase and was higher than those of non-mycorrhizal plants (Fig. 5).

Growth variables Leaf number and leaf area of mycorrhizal plants

were not different (p> 0.05) when compared with non-mycorrhizal plants, even though colonized plants had numerically higher leaf numbers and leaf areas across most harvest stages and RZTs (data not shown). Only at the RZT with the highest level ofmycorrhizal colonization (21.6°C) was total plant

dry matter of mycorrhizal plants greater (15.1%, p_< 0.01) than that of non-mycorrhizal plants over the four harvest stages. At the other three RZTs, mycorrhizal colonization generally did not change the plant shoot weights (p>0.05) relative to non- mycorrhizal plants (data not shown). At the final harvest, mycorrhizal plants had higher root weights (p_<0.05) than non-mycorrhizal plants at 15, 18.2 and 21.6°C RZTs (Fig. 6), whereas there was no difference between mycorrhizal and non-mycorrhi- zal plants at 25°C RZT.

DISCUSSION

The results of this experimentation indicate that the hypothesis that mycorrhizal infection is

292 FENG ZHANG et al.

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Fig. 3. Changes in nodule weights per plant of both mycorrhizal and non-mycorrhizal plants across four harvests at four different root zone temperatures. The line with the symbol (0) indicates mycorrhizal plants and (C)) indicates non-mycorrhizal plants.

Each point represents the mean (+ S.E. indicated by I) value of eight plants.

inhibited by the same RZT found to be suboptimal for soybean nodulation and N2 fixation can be rejected. Soybean, in symbiotic association with Bradyrhizobium, requires RZTs in the ranges of 25- 30°C for optimal nodulation and nitrogen fixation. (z3~ When the RZTs are below 25°C both nodulation and nitrogen fixation are inhibited. 145'46/ However, the highest mycorrhizal colonization occurred at 21.6°C RZT, which had an average infection rate, over all harvests, of 33% (Fig. 1). Root zone temperatures, above or below 21.6°C, decreased mycorrhizal colonization of soybean roots. This experiment also showed that mycorrhi- zal colonization varied among the harvest stages. Mycorrhizal colonization of plant roots was detected at the first harvest (V3), and increased

linearly until the flowering stage (R2), after which it decreased (Fig. 1). Since nitrogen and photo- synthate are mobilized and translocated from veg- etative tissues to reproductive tissues during the reproductive stage, the pool of nitrogen and photo- synthate in vegetative tissues is depleted at that time. The vegetative tissues eventually lose their physiological activity as the plants become 'self- destructive'. (39) The shift from vegetative to repro- ductive development has been shown to inhibit soybean nitrogen fixation in some soybean cul- tivars (4~ and our data suggested that mycorrhizal colonization is similarly inhibited.

The second hypothesis, that mycorrhizal colon- ization would affect soybean nodulation and N2 fixation differently at different RZTs, can be

DEVELOPMENT AND INTERACTIONS OF SOYBEAN 293

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Harvest stages Fig. 4. Changes in nitrogen concentrations of both mycorrhizal and non-mycorrhizal plants across four harvests at four different root zone temperatures. The line with the symbol (0) indicates mycorrhizal plants and (C)) indicates non-mycorrhizal plants.

Each point represents the mean (_ S.E. indicated by I) value of eight plants.

accepted in the case of nodulation, but rejected for N2 fixation. Vesicular-arbuscular mycorrhizal colonization was influenced considerably by RZT, and, in turn, mycorrhizal inoculation affected plant nodulation, nitrogen fixation and growth. At the lower RZTs, plant nodule number was negatively affected by mycorrhizal colonization. However, at higher RZTs (21.6 and 25°C), mycorrhizal plants had higher nodule numbers than non-mycorrhizal plants (Fig. 2). The process ofnodulation and nitro- gen fixation is very energetically expensive; photo- synthate is required for nodule development, maintenance, and nitrogen fixation. (~5) Mycorrhizal fungi also require substantial amounts of photo- synthate, which is rapidly translocated to the root systems of mycorrhizal plants and, in part, to intra-

cellular and extramatrical fungal structures./3'7~ Pang and Paul {34/found additional allocation of car- bon to mycorrhizal roots of Viciafaba, equivalent to about 10% of total photosynthate. Kucey and Paul (24/found that nitrogen-fixing nodules of non- mycorrhizal V.faba utilize 6% of total photosynthate and those of mycorrhizal plants 12%, and that the carbon fixation rate (g C g-~ shoot dry wt. hr-1) is increased in plants supporting both microbial symbionts, presumably to compensate for the additional symbiotic carbon utilization. The above suggests that competition for carbon sources could exist between Bradyrhizobium and Glomus. Millhollon and Williams (~2) reported that soybean photo- synthesis and transpiration rates decrease at subop- timal RZT. Therefore, competition for photo-

294 FENG ZHANG et al.

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Harvest stages Fig. 5. Changes in total nitrogen content per" plant of both mycorrhizal and non- mycorrhizal plants across four harvests at four different root zone temperatures. The line with the symbol (0) indicates mycorrhizal plants and (O) indicates non-mycorrhizal plants. Each point represents the mean (__ S.E. indicated by I) value of eight plants.

synthate between mycorrhizal fungi and nitrogen fixing bacteria is probably more intense at lower RZT, particularly during development of the sym- biosis, when extra carbon is required but the plant is not yet experiencing any benefits. This com- petition for carbon could explain the observed reduction in nodule number of mycorrhizal plants at lower RZTs (Fig. 2). In nitrogen-limited soybean plants, photosynthesis increases as soon as nitrogen fixation starts (45/so that the competition for carbon between nitrogen-fixing bacteria and mycorrhizal fungi should have decreased with increasing nitro- gen availability and subsequent CO2 fixation. Thus, the nodule number of mycorrhizal plants was higher than that of non-mycorrhizal plants at the second harvest for 25°C RZT and at the last two

harvest stages for 21.6°C RZT (Fig. 2). In most field experiments, mycorrhizal colonization stimulates the early development of nodules, resulting in higher nodule numbers and dry weights./16~

At all suboptimal temperatures (<25°C), mycorrhizal colonization tended to cause an initial decrease in nodule mass, which was overcome as the plants developed. Again, this argues that the competition for available photosynthates is intense and detrimental early in the establishment of the two symbioses, prior to the onset of nutritional advantages due to the symbioses. I24~ In two of the three suboptimal RZTs, mycorrhizal colonization eventually stimulated nodule mass and, presum- ably, this would have occurred eventually at the third suboptimal RZT (18.2°C). At lower RZTs,

DEVELOPMENT AND INTERACTIONS OF SOYBEAN 295

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Harvest stages Fig. 6. Changes in root weights of both mycorrhizal and non-mycorrhizal plants across four harvests at four different root zone temperatures. The line with the symbol (0) indicates mycorrhizal plants and (O) indicates non-mycorrhizal plants. Each point

represents the mean (+_ S.E. indicated by I) value of eight plants.

symbiotic events occurring before the Onset of nitro- gen fixation, such as early infection, may have been decreased by mycorrhizal colonization, possibly as a result of carbon limitation. Once nitrogen fixation started [indicated by a leaf colour change from light green to dark green(45~, the competition for photosynthate between mycorrhizal fungi and nitrogen-fixing bacteria decreased within each of the RZT, and later occurring N 2 fixation symbiotic events of mycorrhizal plants were increased com- pared with those ofnon-mycorrhizal plants, leading to larger weight per nodule values for mycorrhizal than non-mycorrhizal plants at all three suboptimal RZTs (Figs 2 and 3).

At the lowest RZT (15°C) used in this study, nodule numbers of mycorrhizal plants were not

significantly different when compared with non- mycorrhizal plants, while final nodule weights were increased when compared with non-mycorrhizal plants. The explanation for the lack of differences in nodule number between mycorrhizal and non- mycorrhizal plants at 15°C RZT might be that during early growth stages, this low RZT strongly inhibits soybean nodulation,/46) and lowers mycorrhizal colonization (Fig. 1) such that the com- petition for carbon between Bradyrhizobia and VA mycorrhizae was reduced. Since plant nutrient absorption was improved by mycorrhizal colon- ization at late growth stages, the nodule weights of mycorrhizal plants were increased compared to non-mycorrhizal plants.

Vejsadova et aL 1441 found that VA mycorrhizae-

296 FENG ZHANG et al.

colonized plants fix considerably more nitrogen than non-colonized plants. The general explanation is that VA mycorrhizae increase the uptake of a variety of nutrients and water, so that colonized hosts are more physiologically fit and capable of supporting more nitrogen fixation than non- mycorrhizal plants9 ~ Our study found that at lower RZTs (15 and 18.2°C), nitrogen concentration had a positive relationship with mycorrhizal infection, and at three of the four RZTs, 15, 18.2 and 21.6°C, the total plant nitrogen content of mycorrhizal plants was significantly (p_< 0.05) higher than that of non-mycorrhizal plants at the last two harvest stages. The exception to this was that the total plant nitrogen contents between mycorrhizal and non- mycorrhizal plants grown at 18.2°C were not dif- ferent at the last harvest stage (Fig. 5). Increased nitrogen concentrations have been reported in VA mycorrhizal plants. However, Harley and Smith ~18~ indicated that because of the symbiosis with N2 fixation bacteria, the increased nitrogen con- centration in VA mycorrhizal plants can be attri- buted to a secondarily induced increase in N2 fixation, e.g. by improving plant growth and min- eral nutrition uptake, rather than by direct uptake of nitrogen compounds from the soil./18~ Therefore, our observation that the total plant nitrogen con- tents increased at the low RZTs, probably rep- resents an increase in nitrogen fixation. These results agreed with previous findings, obtained under normal temperature conditions (25-30°C), that the amount of nodule tissue, the concentration of leghemoglobin, and the rates of acetylene reduction are increased by mycorrhizal infection in Phaseolus, Medicago and Arachis. (8'9"22'31'4°'44)

Mycorrhizae did not increase shoot weight at 15 and 18.2°C RZTs, but the root weight ofmycorrhi- zal plants at 15, 18.2 and 21.6°C RZTs were higher than those of non-mycorrhizal plants (Fig. 6). Since nodule initiation and development, nitrogen fixation, and mycorrhizal colonization all require carbon from host photosynthesis, (2/ this results in increased utilization of photosynthates in the root and hence relatively more photosynthetic products being transferred to, and metabolized in, the root. Over the whole experiment, the ratio of plant shoot weight to root weight for mycorrhizal plant s (3.74) was lower (p_< 0.05) than that for non-mycorrhizal plants (4.67).

In summary, the results of this controlled

environment experiment show that (1) the optimal RZT for mycorrhizal infection of G. versiforme was 21-22°C, while above and below this range, mycorrhizal colonization was inhibited; (2) mycorrhizal colonization increased until flowering (the third harvest), but decreased thereafter at all tested RZTs; (3) mycorrhizal colonization had a negative effect on nodule formation (nodule num- ber) at lower RZTs, but a positive one at higher RZTs; and (4) the lower nodule number at lower RZTs was offset by increased mass per nodule so that mycorrhizal infection stimulated N 2 fixation at the two lowest RZTs. Overall, these findings indi- cate that the effects of mycorrhizal colonization on host plant growth, nodulation and nitrogen fixation vary, depending on the plant growth RZT. At higher RZTs, mycorrhizal colonization increased nodulation, whereas at lower RZTs (18.2 and 15°C), it decreased nodule number but still increased N 2 fixation. This study also suggested that the optimal temperature for G. versiforme mycorrhi- zal colonization of soybean was 21-22°C RZT.

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2. BareaJ. M. and Azctn-Aguilar C. (1983) Mycorrhi- zas and their significance in nodulating nitrogen- fixing plants. Adv. Agron. 36, 1-54.

3. Bevege D. I., Bowen G. D. and Skinner M. F. (1975) Comparative carbohydrate physiology of ecto- and endo-mycorrhizas. Pages 149--174 in F. E. Sanders, B. Mosse, and P. B. Tinker, eds. Endomycorrhizas. Academic Press, London.

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