THE INFLUENCE OF SOIL AERATION ON THE EFFICIENCY OF VESICULAR-ARBUSCULAR MYCORRHIZAE

Transcription

1 Neu> Phytol. (1981) 88, THE INFLUENE OF SOIL AERATION ON THE EFFIIENY OF VESIULAR-ARBUSULAR MYORRHIZAE I. EFFET OF SOIL OXYGEN ON THE GROWTH AND MINERAL UPTAKE OF EUPA TORIUM ODOR A TUM L. INOULATED WITH GLOMUS MAROARPUS BY S. R. SAIF Institut fiir Tropischen und Subtropischen Pflanzenbau der Universitdt Gottingen, 3400 Gottingen, Germany {Accepted 12 December 1980) SUMMARY Oxygen concentrations in the soil atmosphere greatly influenced the growth and mineral uptake of Eupatorium odoratum inoculated with Glomus macrocarpus. Shoot and root dry weights and length ofmycorrhizal plants increased with O^ concentration up to 16 o- Mycorrhizal plants at "0 O2 or non-aerated controls were smaller than those at 12 and 16 "o Og. Non-mycorrhizal plants had lower shoot and root dry wts than mycorrhizal plants at all Og levels except at 0 o- Phosphorus concentration in mycorrhizal and non-mycorrhizal plants differed significantly but did not increase witb increasing Og. Mycorrhizal plants contained higher quantities of N, K, a and Mg than non-mycorrhizal and showed positive response in nutrient uptake to increase in soil OJ. Inoculation and increased soil Og resulted in higher concentrations of K and Mg but not of N and a. The development of Glomus macrocarpus exhibited quantitative and qualitative response to different soil Og levels. INTRODUTION The symbiotic relationship between host and VA mycorrhizal fungus is sensitive to a variety of ecological and physiological stresses. Many reports regarding these factors have been published (,see Mosse, 1973; Slankis, 1974; Sanders, Mosse and Tinker, 1975). No study has, however, dealt with the influence of soil Og on this symbiosis. As regards influence of soil aeration on root development, plant growth and mineral uptake in general, a large number of papers have been published (see Aung, 1974; Stolzy, 1974). In none of these reports was VA mycorrhizal association considered. However, there are some publications about the eflfect of soil O2 on ectomycorrhizae (Harley, 1969; Gadgil, 1972; Read and Armstrong, 1972; Theodorou, 1978) reporting positive effects of soil aeration on the development and efficiency of ectomycorrhizal fungi. It has been said that soil Og, as one of the factors of the chemical environment which directly influence root growth, may also drastically affect ion absorption (Lonergan, 1979). In view of the important role of VA mycorrhizal associations especially in phosphorus uptake and the large variation which occurs in soil Og content, a series of experiments on the influence of soil aeration on the efficiency and development of these symbiotic associations have been conducted. In this first paper the influence of soil O.^ on the growth and mineral uptake of mycorrhizal and non-mycorrhizal plants of Eupatorium odoratum and on the development of a VA mycorrhizal fungus, Glomus macrocarpus, in the host roots is reported X/81/ $02.00/ The New Phytologist

2 650 S. R. SAIF Fig. 1. Diagram of the experimental arrangement. A, Metal tube with manometers; B, Y-shaped plastic tubing;, wood plate; D, flow meter; E, hydrating bottle; F, gas distributor; G, stopcock; H, plastic pot; I, rubber tubing; J, rigid plastic mesh; K, pebble gravel; L, soil mixture; M, rubber stopper; N, rigid plastic piece; O, rubber stopper; P, test tube. MATERIAL AND METHODS A diagram of the equipment used for mixing N2 and O2, control of flow rate and distribution of the gas mixture is shown in Figure 1 which is largely self-explanatory. The hydrating bottle (E) served to moisten the gas and as a visual check on the flow. Another flow meter or bubbling technique (G) was used to control equal distribution of the gas mixture before it entered the pots. The pots (H) were of clear plastic with a size of 14 5 cm height x 110 cm diameter, holding 1 kg soil. The space between the plastic piece (N) and the pot walls was sealed with an inert material. The same substance was used as a thin layer to flll the space between the plant stem and the rubber stopper (M). The gas escaped from the pots through (i) the gas outlet tube dipped in water (P); (ii) through leakage primarily around the plant stem. The outward flow of gas around the plant stem prevented back diflfusion or flow of oxygen from the atmosphere. Oxygen concentrations used were 0, 2, 4, 8, 12, 16 and % by volume. The gas mixture was supplied at a rate of 1 litre h"^ to each pot throughout the experiment, which lasted 5 weeks. One treatment consisted of plants growing in open, unaerated pots along with the experimental plants which were designated as control (). The soil was acidic forest soil, containing very little available phosphate, mixed with river sand (grain size up to 8 mm) in the ratio 1:1. This soil mixture was used at ph 5 5 (in 001 M alg); the ph value at harvest was 6 2. The soil was steamed at 90 for 2h and air dried. In addition to a basic fertilization with 190 4mgNH4NO3kg-i (corresponding to 0 kg N ha'^), 25-4 mg Kl kg-' (40 kg K ha-i) and 29-7 mg K2SO4 kg-^ (40 kg K ha^^) phosphate was supplied as hydroxyapatite, a5(po4)3oh at a rate of 162mgkg"^ corresponding to 90 kg P ha-^ Eupatorium odoratum L., a broadleaved weed of warm regions requiring at optimum a rainfall in excess of 1500 mm, was used as host plant. In those regions, on roadsides, in old cultivation and waste places generally E. odoratum grows very

3 Aeration and VA Mycorrhizae 651 densely, forming thickets in which few other plants can grow (Ivens, 1974). Nothing is known about its ability to transfer O2 through the shoot to the roots. In this institute it has been found to respond to mycorrhizal fungi and to depend entirely on VA mycorrhiza for utilization of insoluble phosphate (hydroxyapatite). Seedlings were raised in plastic trays containing a sterilized soil/peat mixture. The seedling roots were inoculated with a suspension of small root pieces of.e. odoratum infected with Glomus macrocarpus (Gottingen strain). The infected root material was treated with 2 % chloramine for 3 min (Koch, 1961), cut into small pieces, washed with distilled water and suspended in water in a Petri dish. For inoculation the seedling roots were dipped in this suspension so that an adequate amount of infected root pieces adhered to the seedling roots. In the text and figures inoculated plants are designated as M (mycorrhizal), non-inoculated plants as NM (non-mycorrhizal). The experiment was conducted in a growth room with a light intensity of lx (daylight fiuorescent tube). The plants were given a daily 14 h light period. Temperatures ranged from 25 at night to 30 during the day. For each treatment three replicates and in each pot three plants were used. At the end of the experiment, the percentage of root segments (1 cm in length) and root length infected, and the number of vesicles were estimated as described by Saif (1977) by staining with trypan blue in lactophenol (Phillips and Hayman, 1970). Plant material (only shoots) was digested in H2SO4-Se. Phosphorus and N were determined colorimetrically and K, a and Mg with an atomic absorption spectrophotometer (Perkin-Elmer, Model 432). The statistical analysis of the data was done by using two-factor variance analysis. RESULTS Growth Inoculation with Glomus macrocarpus and supply of different O2 concentrations in the soil atmosphere had profound effects on the growth of Eupatorium plants. M plants showed significant increase in shoot length with increasing O2 up to 12 % (Fig. 2). M plants at % or non-aerated plants were smaller than the M plants at the 12 and 16% O2 level. Shoot length of NM plants increased slightly from 0 to 4 % O2 and did not respond to high O2 concentrations. Except at 0 %, the NM plants remained always significantly smaller than the M plants. The mean value (of all treatments) of M : N M ratio was (Table 1). The shoot dry wts of M plants increased with rising O2 content with a peak at 12 and 16 % O2 (Fig. 3). Similar to shoot length, shoot dry wt of M plants at % and also of control M plants was smaller than that of the M plants at 12 and 16 %. Shoot dry weight of NM plants increased slightly up to 4 % O2, and then remained constant at the same level as the control NM plants. The mean value of M : N M ratio was 2-5 (Table 1). The root dry weight of M plants also increased with rising soil O2 up to 16% (Fig. 4). Root dry wt of M plants at % and of non-aerated plants was significantly smaller than that of M plants at 12 and 16%. Root dry wt of NM plants increased considerably from 0 to 4 % O2 and showed a slight increase from 12 to 16%. The mean value of M : N M ratio was 1-9 (Table 1). Inoculation with the mycorrhizal fungus favoured the development of shoots more than of roots, leading to a significant increase in the shoot:root ratio except

4 652 S. R. SAIF NMM Fig. 2. Shoot length (cm) of M (#) and NM (O) plants grown at different concentrations of oxygen in the soil atmosphere., Unaerated control. T3 "o 2 in Fig. 3. As Figure 2, but for shoot dry wt (g per NMM pot). o> NMM Fig. 4. As Figure 2, but for root dry wt (g per pot).

5 Aeration and VA Mycorrhizae 653 Table J. The mycorrhizal.non-mycorrhizal ratios of growth parameters and mineral composition Percentage oxygen in soil atmosphere Shoot length Shoot dry wt Root dry wt a P b a Mineral c:ompositior N K a b a b a 1 b a Mg b Mean , Unaerated control; a, uptake (mg per pot); b, concentration (mg g ' dry wt). Table 2. Shoot/root ratio of dry wts of mycorrhizal {M) and non-mycorrhizal plants grown at different concentrations of oxygen in the soil atmosphere {NM) Percentage oxygen M NM L.S.D. 5 % L.S.D. 1 % , Unaerated control. at 2% O2 (Table 2). The shoot:root ratio of M plants did not differ significantly from each other except at 0 %. The same was true for most NM plants except at 2% with a value higher than the rest, and at 16 % with a value lower than at 12 %. Mineral composition The accumulation of various minerals in the shoots of Eupatorium plants during the study is shown in Figures 5 to 9. The data are presented on a concentration basis (mg g~^ dry wt) and as nutrient uptake (mg per pot). The P uptake [Fig. 5(a)] of NM plants increased very slightly from 0 to 4% O2 and remained at the same low level at the higher O2 concentrations. In M plants, the P uptake at 0% Og was as low as in NM plants but increased sharply at 2%, rising further up to 12%, and falling slightly at %. The small changes in P concentration [Fig. 5(b)] in NM plants at the lowest O2 levels probably are insignificant due to the low absolute values; at higher O2 concentrations they remained constant. In M plants the P concentration increased only from 0 to 2 %

6 654 S. R. SAIF I Q. 4 3 L.S.D. 5% ' I - 1% / (a) D Q. >^ cr c u co u X f// 7 ^ IO/ - L.S.D. 5% 1 /o I I / ' _^? r r1 o o ^ ^ r- - \< NMM Fig. 5. (a) Phosphorus uptake (mg per pot) and (b) phosphorus concentration (mg g ' dry wt) in shoots of M (#) and NM (O) plants grown at different concentrations of oxygen in the soil atmosphere., Unaerated control. s s s (b I O2 and then remained at the same level. This shows that growth followed closely P uptake. The mean values of the M: NM ratios of P uptake and P concentration were 61 and 2 4 (Table 1), considerably higher than those of the other elements. The N uptake [Fig. 6(a)] of NM plants increased considerably more than the P uptake with rising O2 content of the soil atmosphere up to 12% O2. Since the dry wt ofthe NM plants did not increase in a similar way with increasing O2 (Figs 2 and 3), N concentration [Fig. 6(b)] rose also up to 12% O2 and surpassed the values of the M plants considerably. In the M plants, N uptake followed closely the growth curve so that the N concentration showed little change. A consequence of the increasing N uptake by NM plants was that the M.NM ratio of the concentration (Table 1) remained below 1 at all O2 concentrations (mean value 07). The K uptake [Fig. 7(a)] of NM plants showed the same pattern as the N uptake (maximum at 12% O2) though at a considerably lower level. The K uptake of M plants was more strongly increased by rising O2 concentrations. The K concentration of the NM plants reached high levels at 8 and 12 % O2 [Fig. 7(b)] but the K concentration of M plants remained above the values of NM plants at all Oj levels. This is also shown by the figures ofthe M:NM ratios (Table 1). NM and M- plants (unaerated pots) had considerably lower K concentrations than the aerated plants at the optimum O2 levels. The a uptake of NM plants [Fig. 8(a)] was fairly high at all O^ levels except 0%. From 2% O2 onward it showed no influence of O2 supply, so that the a concentration [Fig. 8(b)] remained constant. In the M plants, a uptake rose with increasing O2 concentration up to 16%, and then dropped sharply at %. This pattern followed fairly closely the growth curve (except the peak at 16% O2), so that the a concentration remained, with some statistically insignificant fluctuations, at the same level from 2 to 16% O2.

7 Aeration and VA Mycorrhizae L.S.D. 5% 1% NMM Fig. 6. As Figure 5, but for nitrogen uptake and concentration. (a) o Q P 3 (b) 25 o c o u NMM Fig. 7. As Figure 5, but for potassium uptake and concentration. Uptake and concentration of Mg rose in NM plants with increasing O2 supply up to the highest O2 level [Fig. 9(a), (b)]. The course of Mg uptake by M plants closely paralleled the a uptake with a peak at 16 % and a drop at %. However, Mg uptake was more increased by the mycorrhiza than a uptake, with the consequence that Mg concentration was higher in M than in NM plants at most

8 6s6 S. R. SAIF uptake (mgkpot) O o $ X) T c u co u a L.S.D. 5% I - I/^ / e L.S.D. 5% I [ 1% (a) I y\ _/ \ X \ ^ \ 1% I I I 1 1 o -^ I I I Percentoge oxygen 1s \ s ss D) V > S s V s s s NMM Fig. 8. As Figure 5, but for calcium uptake and concentration. L.S.D. 5% 1% ^ 4 >^ L.S.D. 5% 1% (b) I I concn NMM Fig. 9. As Figure 5, but for magnesium uptake and concentration. O2 levels; this is also shown by the M:NM ratios in Table 1. Similar to K, the unaerated control M plants had a considerably lower Mg concentration than the M plants at 12 and 16% O2. A comparison of the effect of mycorrhiza and O2 content of the soil atmosphere on uptake of the five elements, as summarized in Table 1, shows the following.

9 Aeration and VA Mycorrhizae 657 At zero O2 there was no difference between M and NM plants. At 2 % O2 concentration mycorrhizal plants showed strongly increased uptake of all the elements. Higher O2 concentrations up to 12 % improved the effect of mycorrhiza on the uptake of the five elements further. Higher O2 concentrations (16 and %) led to better mycorrhiza efficiency only in the case of K ( %) and Mg (16%). Uptake of P was much more strongly improved by mycorrhiza than the uptake of other elements. The respective figures for the mean mineral uptake ratios (M.NM) were P, 61; K, 3-4; Mg, 30; a, ; and N, 1-8. This order is also reflected in the ratios of mean element concentrations: P, 24; K, 14; Mg, 12; a, 0-9; and N, 07. VA mycorrhizal infection The VA mycorrhizal infection was influenced quantitatively and qualitatively by different concentrations of O2 in the soil atmosphere. The percentage of root segments infected showed a smaller response to increasing soil O2 than the total percentage infection and the number of vesicles (Fig. 10). The difference between the percentage of root segments infected and the total percentage infection was greater at lower O2 concentrations and decreased with rising O2, with a minimum at 16%. The percentage of root segments and total infection increased with increasing O2 to an optimum, at 16%, of 95 and 91 %, respectively. The values at % and those of unaerated control M plants were slightly smaller than at 16 %. The vesicle number per 50 cm root also increased with rising soil O2, with values below 1000 at 2% to more than 5000 at % (Fig. 10) a> a. in 0) o in a> i6 SI Rl V Fig. 10. Percentage root segments (O) and percentage root lengtb infected ( ) and tbe number of vesicles (O) per 50 cm root of plants grown at different concentrations of oxygen in tbe soil atmospbere., Unaerated control; SI, segments infected; RI, root lengtb infected; V, vesicles. At the lower O2 concentrations in the soil atmosphere (2 and 4%) very few arbuscules were formed relative to the number of longitudinally running fungal hyphae in the roots. Also, very few entry points and a small amount of external mycelium were observed at these two levels. At 8 and 12% soil O2, increasing numbers of fully developed arbuscules, entry points and vesicles were found. At 16 and % soil O2, almost no arbuscules were observed. The root cortex at these levels was fully occupied by vesicles and fungal hyphae.

10 658 S. R. SAIF DISUSSION The host plant E. odoratum used in this experiment belongs to the group of plants which depend entirely on VA mycorrhiza for their P uptake and growth when phosphate is not readily available (effectivity group 2 after Graw, Moawad and Rehm, 1979). Therefore, with hydroxyapatite as P source NM plants showed little response to increasing soil O2 as compared to M plants. In NM plants growth was affected far more by phosphorus deficiency than by soil Og. The possibility of detrimental effects of Og on root activity did not exist in this experiment because O2 was continually removed by the flow of the gas mixture (O2 and Ng). M plants, on the other hand, obtained adequate amounts of P through the fungus (Fig. 5) and did show a large response in shoot and root growth (Figs 3 and 4) to increasing soil Og., The uptake of N and a by M plants increased with increasing Og concentration to about the same extent as the dry wt, the uptake of K and Mg slightly more, with the consequence that the concentration of the latter elements was somewhat increased at the higher Og levels. In NM plants too, raising Og concentration induced an increased uptake of N, K, a and Mg. Indeed, N and a uptake exceeded growth to such an extent that their concentration was higher in NM plants than in M plants. There is no indication that possible increased nitrification of NH4 at higher Og levels had an influence on nitrogen accumulation. Non-aerated () plants showed normal but lesser response to mycorrhiza than those in the 12 and 16 % Og treatments. This may have been due to the suboptimal soil Og or to a possible detrimental accumulation of Og or again, perhaps the physical and chemical soil conditions of aerated and non-aerated pots may have differed. Due to the full dependence of this host plant on mycorrhiza this experiment cannot answer the question whether nutrient uptake by mycorrhiza needs more or less Og than nutrient uptake by NM roots. To clarify this, as well as some other points, experiments using Sorghum and Guizotia, which are not so strongly dependent on mycorrhiza, have been conducted and will be reported in the following papers. As mentioned in the Introduction, ectomycorrhizae are sensitive to soil aeration; the present experiment conducted with Glomus macrocarpus showed that endomycorrhizae too need a fairly high concentration of soil Og for optimum development. There were qualitative and quantitative differences in the Og requirement for different processes, for instance hyphal infection was already fair but arbuscule development was very low at 2 %. The difference in arbuscule and vesicle number at different Og concentrations cannot be explained on the basis of the available data. Generally, Og concentration in the soil atmosphere had a stronger effect on the physiological activity of the mycorrhiza than on the degree of infection. The differing effects of Og percentage on the number of arbuscules, and on the phosphorus concentration in M plants, seem unusual since arbuscules are considered to be the main organs for P transfer from the fungus to the host plant. This problem and the question of relationship between fungal development and mycorrhizal efficiency will be dealt with in the following paper, on experiments with the same host plant but two other mycorrhizal endophytes.

11 Aeration and VA Mycorrhizae 659 AKNOWLEDGEMENT The author wishes to thank Professor Dr S. Rehm, Director of the Institute, for his interest in initiating this work and comments on a draft. REFERENES AUNG, L. H. (1974). Root-shoot relationship. In: The Plant Root and its Environment (Ed. hy.. W. arson), pp University Press of Virginia, harlottesville. GADGIL, P. D. (1972). Effect of waterlogging on mycorrhizas of radiata pine and Douglas fir. New Zealand Journal of Forestry Science, 2, GRAW. D., MOAWAD, M. & REHM, S. (1979). Untersuchungen zur Wirts- und Wirkungsspezifitat der VA-Mykorrhiza. Z. Acker- und Pflanzenbau, 148, HARLEY, J. L. (1969). The Biology of Mycorrhiza, 2nd edn, 334 pp. Leonard Hill, London. IvENS, G. W. (1974). The problem of Eupatorium odoratum L. in Nigeria. PANS,, KOH, H. (1961). Untersuchungen iiber die Mykorrhiza der Kulturpflanzen unter besonderer Berucksichtigung von Althea officinalis L., Atropa belladonna L., Helianthus annuus L. und Solanum lycopersicum L. Gartenbauwissenschaft, 26, LONERGAN, J. F. (1979). The interface in relation to root function and growth. In: The Soil-root Interface (Ed. by J. L. Harley & R. S. Russel), pp Academic Press, New York and London. MOSSE, B. (1973). Advances in the study of vesicular-arbuscular mycorrhiza. ^nwua/i?e^'^>^«o/p/^jfo/)a</^o/o^>', 11, PHILLIPS, J. M. & HAYMAN, D. S. (1970). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society, 55, READ, D. J. & ARMSTRONG, W. (1972). A relationship between oxygen transport and the formation of the ectomycorrhizal sheath in conifer seedlings. Netc Phytologist, 71, SAIE, S. R. (1977). The influence of stage of host development on vesicular-arbuscular mycorrhizae and endogonaceous spore population in field-grown vegetable crops. I. Summer grown crops. New Phytologist, 79, SANDERS, F. E., MOSSE, B. & TINKER, P. B. (1975). Endomycorrhizas, pp Academic Press, New York and London. SLANKIS, V. (1974). Soil factors influencing formation of mycorrhizae. Annual Review of Phytopathology, 12, STOLZY, L. H. (1974). Soil atmosphere. In: The Plant Root and its Environment (Ed. by E. W. arson), pp University Press of Virginia, harlottesville. THEODOROU,. (1978). Soil moisture and the mycorrhizal association oi Pinus radiata D. Don. Soil Biology and Biochemistry, 10, ANP 88

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