EFFECTS OF DROUGHT STRESS ON GROWTH RESPONSE IN CORN, SUDAN GRASS, AND BIG BLUESTEM TO GLOMUS ETUNICA TUM*

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1 New Phytol. (\9S7), 15, A2^\ 4O3 EFFECTS OF DROUGHT STRESS ON GROWTH RESPONSE IN CORN, SUDAN GRASS, AND BIG BLUESTEM TO GLOMUS ETUNICA TUM* BY B. A. DANIELS HETRICK, D. GERSCHEFSKE KITT AND G. THOMPSON WILSON Department of Plant Pathology, Kansas State University, Manhattan, KS 6656, USA {Accepted 24 September 1986) SUMMARY The responses of mycorrhizal corn (Zea mays L.), sudan grass [Sorghum vulgare (Piper) Hitch.], and big bluestem (Andropogon gerardii Vitman) under drought stress were compared. Although growth of each of the plant species benefited from the mycorrhizal fungus under adequately watered conditions, inoculation had no effect on the growth of corn or sudan grass when cyclic drought stress was imposed on these plants. In contrast, growth of mycorrhizal big bluestem was significantly greater than non-mycorrhizal big bluestem, even under severe drought stress. Drought-stressed mycorrhizal plants without phosphorus amendment were not larger than drought-stressed, non-inoculated, fertilized (15mgkg~^P) plants, suggesting no increased drought tolerance. The ability of Glomus etunicatum Becker & Hall to benefit plant growth under drought stress was apparently plant-mediated and possibly related to the dependency of the plant on this mycorrhizal fungus. Under adequately watered conditions, inoculated corn and sudan grass were respectively 1-23 and 1-13 times larger than non-inoculated plants, while inoculated big bluestem was 6-56-fold larger than non-inoculated control plants. Key words: Vesicular-arbuscular mycorrhizas, drought tolerance, Glomus etunicatum. INTRODUCTION Mycorrhizal fungi are believed to improve the water relations of host plants, by increasing hydraulic conductivity (Boyer, 1969; Safir, Boyer & Gerdemann, 1971, 1972; Hardie & Leyton, 1981), increasing transpiration rate and lowering stomatal resistance (Sanders & Tinker, 1973; Christensen & Allen, 1979, 198; Levy & Krikun, 198), or by altering the balance of plant hormones (Levy & Krikun, 198; Allen, Moore & Christensen, 1982). Nelsen & Safir (1982) proposed that such physiological changes might be explained by the improved phosphorus nutrition of mycorrhizal plants exposed to drought stress. In their experiments, non-mycorrhizal plants were fertilized so that they equalled the size of mycorrhizal plants. When this fertilizer level was applied to plants exposed to cyclic drought stress, mycorrhizal growth of onions {Allium cepa L.) was significantly greater than growth of fertilized, non-mycorrhizal onions, implying that drought tolerance was improved by mycorrhizal colonization (Nelsen & Safir, 1982). Using a similar method, Hetrick, Hetrick & Bloom (1984) were unable to demonstrate such drought tolerance in corn {Zea mays L.). In fact, mycorrhizal growth response was eliminated under drought stress. Hetrick et al. (1984) X/87/ $3./ 1987 The New Phytologist

2 4O4 B. A. D A N I E L S H E T R I C K e^ a/. suggested that their contradictory results might be attributed to the more severe drought stress they imposed on corn, difficulties in regulating water stress with such large plants in the greenhouse, differences in water relations between soil and sand, or to innate differences in plant response to drought stress. In later studies with big bluestem {Andropogon gerardii Vitman), a native prairie plant adapted to drought stress, mycorrhizal growth response but not drought tolerance was evident under cyclic drought stress (Hetrick, Kitt & Wilson, 1986). Because fertilized control plants were not included, only the magnitude of growth response between adequately watered and drought-stressed plants could be compared to assess drought tolerance. This paper examines more closely the relationship between mycorrhizal growth response and drought tolerance in corn and sudan grass as compared with big bluestem. MATERIALS AND M E T H O D S Growth and inoculation of plants To determine whether the failure of mycorrhizas to improve drought tolerance in corn was attributable to difficulties in regulating water-stress regimes in sand (Hetrick et al, 1984) instead of soil, seeds of Z. mays (O's Gold SX55OOA) were germinated in vermiculite. After 1 week, a seedling was transplanted into each 25 cm (diameter) pot containing 3-1 kg (dry weight) steamed TuUy silty clay loam, a fine mixed mesic, pachic, argiustoll of ph 6-2, and 5 mg kg-^ plant-available P. Plants were inoculated with 5 spores of Glomus etunicatum Becker & Hall obtained from sudan grass [Sorghum vulgare var. sudanense (Piper) Hitch.] pot cultures, or they remained non-inoculated as controls. Half of the non-inoculated pots received 5 mg kg-i P as KHgPO^, so that growth of these plants would exceed the mycorrhizal plants. There were six replicates for each treatment. Plants were watered daily to field capacity for 4 weeks. Thereafter, half were exposed to drought stress, i.e. watered to field capacity every 4 to 5 d when severe symptoms of stress (leaf curling) were evident at sunrise and leaf water potentials ranged from -1-7 to -1-9 MPa. The remaining plants were watered daily as during the first 4 weeks of the experiment and were maintained at average leaf water potentials of --4 to --6 MPa (Hetrick et al., 1984). To determine whether the failure of mycorrhizas to improve corn plant growth under drought stress was attributable to the difficulties imposed by working in the greenhouse with a large crop or to the severity of the stress (Hetrick et al., 1984), sudan grass, a smaller species of cultivated grass, was used in place of corn. Seeds of Sudan grass were germinated in vermiculite, and a seedling was transplanted after 1 week to each 12 5 cm (diameter) plastic pot containing M kg (dry weight) steamed fine-loamy, mixed mesic, udic argiustoll of ph 8-3, and 13-5mgkg~i plant-available P. This soil was chosen because it differed from the Tully silty clay loam used in the previous experiment and because mycorrhizal growth response in big bluestem was obtained previously in this soil under drought-stressed conditions by Hetrick et al. (1986). Plants were inoculated with species of G. etunicatum as already described, or they remained non-inoculated as controls. Since the ability of mycorrhizas to increase plant growth under drought stress was to be studied in this experiment, the P level of non-inoculated plants was not varied. To maintain plant growth in all treatments, however, it was necessary to add 8 m g k g - i p as KH2PO4 to each pot as a solution of 'Miracle Grow' fertilizer

3 Drought stress and mycorrhizal grasses 45 (Stein's Nurseries, Inc., Geneva & New York). All the pots were watered daily to field capacity for 6 weeks. Thereafter, one-third of the pots were moderately droughted, i.e. watered to field capacity every 3 to 4 d when leaf curling symptoms were first evident at sunrise and leaf water potentials ranged from 1-9 to 2-8 MPa. Another third of the plants were severely droughted, watered to field capacity after 4 to 5 d when leaf curling symptoms were severe at sunrise and leaf water potentials ranged from 3-2 to 4-3 MPa. The remaining plants were watered to field capacity ( -3 to -7 MPa) daily, as during the first 6 weeks of the experiment. There were five replicates of each treatment. To determine whether drought tolerance might be evident in big bluestem if plants of equal size were compared (Nelsen & Safir, 1982), big bluestem seeds were germinated in vermiculite, and a seedling was transplanted into a 6 x 25 cm plastic pot containing 4 g (dry weight) Tully silty clay loam. As before, seedlings were inoculated with 5 spores of G. etunicatum, or they remained non-inoculated as controls. Half of the non-inoculated controls received 15 mg kg"^ P as KHgPO^, while the other half received no additional P. Each treatment had five replicates. All the plants were watered daily to field capacity for 9 weeks. Thereafter, half of the plants were droughted, i.e. watered to field capacity every 4 to 5 d when leaf curling symptoms were severe and water potentials ranged from 2-5 to 4 MPa at sunrise. The other half were watered daily to field capacity ( -5 to *8 MPa), as during the first 9 weeks of the experiment. Experiment design and maintenance Pot size, soil volume, experiment duration, and P level varied in the three experiments because of differences in plant species, growth habits, and maturity rates. All the experiments were arranged in a randomized, complete block design and fertilized biweekly with Peters 'No-Phos Special Fertilizer' (Peter's Fertilizer Products, Fogelsville, Pennsylvania); and plants were grown in a greenhouse at 15 to 25 C. The experiments with corn, sudan grass, and big bluestem were harvested after 1, 14, and 15 weeks, respectively. A Scholander pressure bomb (constructed by Physics Shop, Kansas State University), modified for flat leaf material, was used in each experiment to determine leaf water potentials on 1 cm lengths of excised leaf tips when the necessity of watering was indicated by symptoms of drought stress in plants. A different block of the experiment was used for each determination of leaf water potential. The leaf tips were dried and added to final plant dry weights obtained at harvest. After drying, sub-samples of the root system were randomly removed, stained in trypan blue (Phillips & Hayman, 197), and examined microscopically to determine percentage root colonization and colonization intensity (Kormanik & McGraw, 1982). A simple analysis of variance (P = '5) was performed on plant dry weights using Duncan's multiple range test for mean separation. R E S U L T S AND D I S C U S S I O N When the impact of mycorrhizal colonization of corn grown in soil was assessed under adequately watered conditions, a typical mycorrhizal growth response was evident (Table 1), i.e. growth of inoculated, non-fertilized plants was significantly greater than that of non-inoculated plants without additional fertilizer. Fertilization of non-inoculated or inoculated plants resulted in growth significantly exceeding that of non-fertilized, inoculated plants.

4 4o6 B. A. DANIELS HETRICK ^f a/. Drought-stressed plants were significantly smaller than adequately watered plants (Table 1). Droughted plants amended with P fertilizer were significantly larger than non-inoculated, non-amended plants but not significantly different from inoculated, non-amended plants. No mycorrhizal response was evident. Although the fertilized mycorrhizal plants exceeded the growth of non-amended Table 1. The influence of inoculation with Glomus etunicatum and phosphorus fertilization on plant growth of corn (Zea mays) in silty clay loam under adequately watered and drought-stressed conditions PhosDhoru"? amendment Inoculated^ Non-amended 5 mg kg-i P Non-inoculated Non-amended 5 mg kg-i P Plant dry weight (g)* Adequately watered 11-2b 1216a 821c a Drought-stressed 51-5 de 56-5 d 42-7 e 59-1 d Adequately watered 4/3 2/1 Root colonization! Drought-Stressed 3/3 VI * Means followed by the same letter are not significantly (P = -5) different, as determined by Duncan's multiple range test. t Root colonization was rated on two criteria. Percentage colonization (first number); 1 = to 5 %, 2 = 6 to 25 %, 3 = 26 to 5%, 4 = 41 to 75 %, 5 = 76 to 1% colonization. Root colonization intensity (second number): = no colonization; 1 = small or widely scattered colonization sites; 2 = larger or uniformly distributed colonization sites; 3 = feeder roots almost entirely colonized. X 5 spores of Glomus etunicatum added. Phosphorus added as KH2PO4. non-inoculated plants, this is probably more a fertilizer response than a mycorrhizal response, considering the low level of root colonization in the droughted, fertilized mycorrhizal plants (Table 1). Colonization of roots was reduced following the addition of P fertilizer. It is well-known that high P levels can inhibit the activity and reduce colonization by mycorrhizal fungi under adequately watered conditions (Menge et al, 1978). However, Bolgiano, Safir & Warncke (1983) demonstrated that mycorrhizal colonization of roots of onions was sustained at higher P levels in soil with low water availability, presumably because the amount of P in soil solution available for uptake by plants or mycorrhizas decreases as soil water stress increases. Thus, it is surprising that in our experiments roots were colonized least in drought-stressed corn. These results are similar to those previously reported for corn in sand culture (Hetrick et al., 1984). Thus, the use of a field soil in the present experiment did not alter the response of corn to mycorrhizal inoculation and drought stress, and differences in growth medium alone do not explain the lack of response in growth or drought tolerance in mycorrhizal corn. In this first experiment, the choice of a fertilizer application which improved plant growth more than did mycorrhizal inoculation was intentional. Hetrick et al. (1984) reported a trend toward mycorrhizal improvement of droughted plant growth at higher P levels, when dry weight of droughted plants was considered as a percentage of that of adequately watered plants. That trend was absent at lower concentrations but evident when plants were amended with 22*5 and 3- mg kg~^ P, the highest concentrations tested. In the present study using an even higher P amendment, 5 mg kg~^ P in a soil already containing 5 mg kg~^

5 Drought stress and mycorrhizal grasses 47 plant-available P, no such trend was evident. Inoculated, fertilized, droughtstressed plants comprised 46% of the dry weight of inoculated, fertilized, adequately watered plants, while non-inoculated, fertilized, droughted plants comprised 48 % of the dry weight of adequately watered, fertilized plants. Thus, it appears that growth of corn does not benefit from mycorrhizal inoculation when severely drought-stressed. To determine whether the lack of benefit from mycorrhizas in drought-stressed corn reflected difficulties imposed by working in the greenhouse with a large crop or the severity of the stress level, as suggested previously (Hetrick et al., 1984), sudan grass was substituted in the next experiment. Under adequately watered conditions, mycorrhizal response was again significant, but this response was entirely eliminated under both moderately or severely stressed conditions (Table 2). However, a drought response was evident, since plant growth decreased as Table 2. The infiuence of Glomus etunicatum inoculation on growth of sudan grass (Sorgum vulgare) under three water regimes Plant dry weight* Root colonizationf Watering reginie Inoculated Non-inoculated Inoculated Non-inoculated Adequately watered Moderately stressed Severely stressed 31-9a 191c 12 5 d 28- b 19-6c 132d 2/2 3/2 2/2 * Means followed by the same letter are not significantly (P 5) different as determined by Duncan's multiple range test. t Root colonization was rated on two criteria. Percent colonization (first number); 1 = to 5%, 2 = 6 to 25 %, 3 = 26 to 5%, 4 = 41 to 75 %, 5 = 76 to 1% colonization. Root colonization intensity (second number): = no colonization; 1 = small or widely scattered colonization sites; 2 = larger or uniformly distributed colonization sites; 3 = feeder roots almost entirely colonized. X Adequately watered plants were watered to field capacity daily and maintained leaf water potentials of 3 to 7 MPa. Moderately stressed and severely stressed plants were watered to field capacity every 3 to 4 or 4 to 5 d when leaf water potentials were - 19 to -2-8 and -3 2 to 4 3 MPa, respectively. 5 Glomus etunicatum spores added. drought stress increased. Since the soil used in this experiment was previously conducive to a growth response to mycorrhizas in big bluestem, even under drought stress (Hetrick et al., 1986), it appears that severity of stress, plant size, or soil type cannot explain the lack of mycorrhizal benefit in corn and sudan grass under conditions of drought stress. In contrast, big bluestem, a native tallgrass prairie plant, has been reported to benefit from mycorrhizal colonization under conditions of drought stress (Hetrick et al., 1986), although drought tolerance was not observed when the magnitude of mycorrhizal response between drought-stressed and adequately watered plants was compared. Using the method of Nelsen & Safir (1982), a P addition was determined in preliminary experiments, which produced non-mycorrhizal plants of similar size to mycorrhizal plants. Addition of 15mgkg"^P to adequately watered, non-inoculated plants resulted in plant growth similar to that of adequately watered, non-amended mycorrhizal plants (Table 3). Droughtstressed, inoculated plants were smaller than adequately watered, inoculated plants, regardless of whether or not they were fertilized. By contrast, noninoculated plants were not significantly affected by drought unless they were fertilized, in which case drought-stressed plants were significantly smaller than

6 B. A. DANIELS HETRICK e^ a/. Table 3. The influence of Glomus etunicatum inoculation and P fertilization on growth of big bluestem (Andropogon gerardis) under adequately watered and drought-stressed conditions Plant dry weight* Root colonization! amendment Adequately watered J Drought-stressed Adequately watered Drought-stressed Inoculated Non-amended 15 mg kg~ipfl Non-inoculated Non-amended 15mgkg-» P 5-45 ab 6-48 a -83 d 5-14ab 2-48 c 3-95 be -39 d 3-29 c 2/2 1/1 3/3 2/2 * Means followed by the same letter are not significantly (P = 5) different, as determined by Duncan's multiple range test. t Root colonization was rated on two criteria. Percentage colonization (first number); 1 = to 5 %, 2 = 6 to 25 %, 3 = 26 to 5%, 4 = 41 to 75 %, 5 = 76 to 1% colonization. Root colonization intensity (second number): = no colonization; 1 = small or widely scattered colonization sites; 2 = larger or uniformly distributed colonization sites; 3 = feeder roots almost entirely colonized. X Adequately watered plants were irrigated daily to field capacity and had leaf water potentials of 5 to -8 MPa, while drought-stressed plants were watered every 4 to 5 d when leaf water potentials reached -2-5 to -4- MPa. 5 spores of Glomus etunicatum added. fl Phosphorus added as KH2PO4. adequately watered plants. Colonization of roots was greater in drought-stressed than in adequately watered plants, although P amendment reduced colonization under both water regimes. Unlike corn and sudan grass under drought stress, a mycorrhizal response was evident in big bluestem. Non-amended, inoculated plants were significantly larger than non-amended, non-inoculated control plants. However, drought-stressed, mycorrhizal plants without additional P were not larger than drought-stressed, non-inoculated, fertilized plants, suggesting no drought tolerance, as described by Nelsen & Safir (1982). In this experiment, the soil was the same as that used in the corn experiment. Since growth response under drought conditions occurred in this soil with big bluestem but not with corn, innate plant differences rather than soil differences appear to be responsible for the lack of mycorrhizal response in corn. That the ability of mycorrhizal fungi to benefit plant growth under drought stress is plant-mediated is further supported by the results obtained with sudan grass, which like corn was not affected by mycorrhizas under drought stress. It is interesting to note that drought stress decreases mycorrhizal colonization in corn and sudan grass but increases colonization in big bluestem. Other tallgrass prairie plants, such as Baptisia leucophaea Nutt. and Liatris aspera Miehx., also benefit from mycorrhizal colonization under drought stress, but drought tolerance as defined by Nelsen & Safir (1982) has not been observed (Zajicek, pers. comm.). Similarly, Johnson & Hummel (1985) reported that citrus benefits from mycorrhizas under drought stress, but they demonstrated no increased drought tolerance from mycorrhizas. Why certain crops benefit from mycorrhizas under drought stress is unclear. It is possible that only extremely dependent host plants respond to mycorrhizas under drought stress. Certainly, big bluestem, which shows a 6'6-fold increase in

7 Drought stress and mycorrhizal grasses 49 growth in response to mycorrhizal inoculation under adequately watered conditions, is highly dependent on these fungi. Other tallgrass prairie plants are similarly dependent (Hetrick et al., 1986; Zajicek, pers. comm.). The dependence of citrus on mycorrhizas has also been demonstrated many times (Menge, 1983). In contrast, cultivated grasses such as corn and sudan grass are probably less dependent on mycorrhizas, since inoculated plants were only 1-23 and 1*13 times larger than non-inoculated plants under adequately watered conditions, respectively. Thus, the dependency of the host plant may determine whether growth response from mycorrhizas will be evident under drought stress. The possibility that many field crops have been bred for reduced dependence on mycorrhizal fungi cannot be overlooked. A trend toward greater dependency of native prairie plants on mycorrhizas, as compared with field crops now grown on prairie soils, has been observed (Hetrick & Bloom, 1983; Hetrick et al., 1984; Liberta & Anderson, 1986). As previously mentioned, no drought tolerance as defined by Nelsen & Safir (1982) was observed, either with mycorrhiza-dependent crops like big bluestem or with relatively independent crops like corn and sudan grass. Apparently, either a mycorrhizal growth response occurs under drought stress or it does not, depending on the host plant, but no evidence exists in these mycorrhizal plants for an increase in drought tolerance, as measured by dry weight differences. The impact of physiological differences between mycorrhizal and non-mycorrhizal plants (altered transpiration rate, stomatal aperture, hormonal balance, hydraulic conductivity) may be too subtle to be measured by gross dry weight comparisons. The impact of such changes could, however, be significant on plant maturation, nutrient allocation, growth habit, etc., depending on a plant's response to drought stress (Fischer & Turner, 1978). Using dry weight comparisons to determine mycorrhizal drought tolerance may therefore be misleading, except in limited species where drought tolerance is measurable by dry weight. REFERENCES & CHRISTENSEN, M. (1982). Phytohormone changes in Bouteloua gracilis infected by vesicular-arbuscular mycorrhiza. I. Cytokinin increases in the host plant. Canadian Journal of Botany, 58, BoLGiANO, N. C, SAFIR, G. R. & WARNCKE, D. D. (1983). Mycorrhizal infection and growth of onion in the field in relation to phorphorus and water availability. Journal of the American Society for Horticultural Science, 18, BoYER, J. S. (1969). Free-energy transfer in plants. Science, 163, CHRISTENSEN, M. & ALLEN, M. F. (1979). Effects of VA Mycorrhizae on Water Stress Tolerance and Hormone Balance in Native Western Plant Species Final Report of the Rocky Mountain Institute of Energy and the Environment. CHRISTENSEN, M. & ALLEN, M. F. (198). Effects of VA Mycorrhizae on Water Stress Tolerance and Hormone Balance in Native Western Plant Species Final Report of the Rocky Mountain Institute of Energy and the Environment. FISCHER, R. A. & TURNER, N. C. (1978). Plant productivity in the arid and semiarid zones. Annual Review of Plant Physiology, 29, HARDIE, K. & LEYTON, L. (1981). The influence of vesicular-arbuscular mycorrhiza on growth and water relations of red clover. I. In phosphate deficient soil. New Phytologist, 89, HETRICK, B. A. DANIELS & BLOOM, J. (1983). Vesicular-arbuscular mycorrhizal fungi associated with native tallgrass prairie in comparison to cultivated winter wheat. Canadian Journal of Botany, 61, HETRICK, B. A. DANIELS, HETRICK, J. A. & BLOOM, J. (1984). Interaction of mycorrhizal infection, phosphorus level and moisture stress in growth of field corn. Canadian Journal of Botany, 62, HETRICK, B. A. DANIELS, KITT, D., GERSCHEFSKE, K. & WILSON, G. THOMPSON (1986). The influence of phosphorus fertilization, drought, fungal species and soil microorganisms on mycorrhizal growth response in tallgrass prairie plants. Canadian Journal of Botany, 64, ALLEN, M. F., MOORE, JR, T. S.

8 4IO B. A. DANIELS HETRICK e^ a/. JOHNSON, C. R. & HUMMEL, R. L. (1985). Influence of mycorrhizae and drought stress on growth oiponcirus X citrus seedlings. Hortscience, 2, KoRMANiK, p. p. & MCGRAW, A.-C. (1982). Quantification of vesicular-arbuscular mycorrhizae in plant roots. In: Methods and Principles of Mycorrhizal Research (Ed. by N. C. Schenck), pp The American Phytopathological Society, St Paul, Minnesota. LEVY, Y. & KRIKUN, J. (198). Effect of vesicular-arbuscular mycorrhizae on Citrus jambhiri water relations. New Phytologist, 85, 25-3L LiBERTA, A. E. & ANDERSON, R. C. (1986). Comparison of VAM species composition, spore abundance and inoculum in an Illinois prairie and selected agricultural sites. Bulletin of the Torrey Botanical Society 113, MENGE, J. A. (1983). Utilization of vesicular-arbuscular mycorrhizal fungi in agriculture. Canadian Journal of Botany, 61, MENGE, J. A., LABANAUSKAS, C. K., JOHNSON, E. L. V. & PLATT, R. G. (1978). Partial substitution of mycorrhizal fungi for phosphorus fertilization in the greenhouse culture of citrus. Soil Science of America Journal, 42, NELSON, C. E. & SAFIR, G. R. (1982). Increased drought tolerance of mycorrhizal onion plants caused by improved phosphorus nutrition. Planta, 154, PHILLIPS, J. M. & HAYMAN, D. S. (197). 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, SAFIR, G. R., BOYER, J. S. & GERDEMANN, J. W. (1971). Mycorrhizal enhancement of water transport in soybean. Science, 172, SAFIR, G. R., BOYER, J. S. & GERDEMANN, J. W. (1972). Nutrient status and mycorrhizal enhancement of water transport in soybean. Plant Physiology, 49, SANDERS, F. E. & TINKER, P. B. (1973). Phosphate flow into mycorrhizal roots. Pesticide Science, 4,

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