EFFECT OF VESICULAR-ARBUSCULAR MYCORRHIZA ON CITRUS JAMBHIRI WATER RELATIONS

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1 ^eu; Phytol. (19S0) S5, EFFECT OF VESICULAR-ARBUSCULAR MYCORRHIZA ON CITRUS JAMBHIRI WATER RELATIONS BY YOSEPH LEVY* AND JAMES KRIKUNf Division of Citriculture* and of Plant Pathology,^ Agricultural Research Organization, Gilat Regional Experiment Station, Mobile Post Negev, Israel {Accepted 22 August 1979) SUMMARY Recovery from water stress was studied on similarly sized VA mycorrhizal and non-mycorrhizal rough lemon seedlings {Citrus jambhiri Lush). VA mycorrhiza affected stom.atal conductance, photosynthesis and proline accumulation, but not leaf water potential, suggesting that most of the effect of the mycorrhizal association is on stomatal regulation rather than on root resistance. INTRODUCTION Research in recent years has established that vesicular-arbuscular (VA) mycorrhizae can improve citrus seedlings' growth through increased uptake of phosphorus and microelements such as Cu and Zn, especially in sterilized soil or non fertile desert soils (Daft and Nicolson, 1965; Hattingh and Gerdemann, 1975; Timmer and Leyden, 1978; Menge, Lambright and Johnson, 1977). Safir, Boyer and Gerdemann (1972) reported that root resistance to waterflowwas reduced by 40 % in mycorrhizal soybean in comparison with non-mycorrhizal plants. However, in their experiment, the mycorrhizal plants grew more rapidly and the changes attributed to mycorrhiza may have been caused by other factors related to rapid shoot growth. In recent work (Krikun and Levy, in prep.) we have succeeded in obtaining non-mycorrhizal citrus plants that did not differ in their growth rate from mycorrhizal plants. This study was made to investigate whether the water relations of citrus seedlings are affected by VA mycorrhizas. The soil plant-atmosphere model described by van den Honert (1948) provides a useful theoretical basis for examining soil-plant interactions. According to this model, under steady state conditions, water flux from the soil through the plant to the atmosphere can be expressed as a series of gradients and resistances. Flux = ^«"il~^^o t ^ froot-^leaf ^ ^leaf^air y T V ' soil-to-root ' root-to-leaf leaf-to-air where i/r = water potential and r = resistance. Water B.ow through the plant is driven by water potential gradients which result from water stress in the leaves, generated by transpiration. Flow of water through the system is controlled mainly by ractors influencing water vapour movement from the leaf to the atmosphere since the resistance in the leaf-air interface is very much greater than that of the plant or,jv X/8O/O5OO25-1-O / The New Phytologist

2 26 Y. LEVY AND J. K R I K U N plant-soil interface (Weatherley, 1976); thus, it is the main factor controlling transpiration flux. The leaf-air resistance comprises two components: a leaf (mainly stomatal) resistance and an air (boundary layer) resistance. When stomatal resistances and wind speeds are relatively high, and leaves are small, the air diffusion resistance represents only a minor component of the total diffusion resistance (Elfving, Kaufmann and Hall, 1972). Under these conditions transpirational flux density [the last term in eqn (1)] can also be expressed as: flux = (A, absolute humidity) x (leaf conductance). (2) Elfving et al. (1972) rearranged eqn (1) to describe leaf water potential as a function of soil water potential, flux and resistance to flow in the liquid phase (soil-to-leaf): ^leaf = V^soil~(flux) (r.^a-tcleaf) (3) According to this equation, ip-^^^^ should decrease when transpirational flux density increases, but this relationship changes depending upon ^g^ii ^^^ the liquid phase resistance to flow (Elfving et al, 1972; Kaufmann, 1977; Kaufmann and Levy, 1976). Results for citrus trees in the field iowowtd these predictions, but seedlings grown in a greenhouse with no history of water stress apparently had much lower resistance to flow of water and ^i^af remained constant over a broad range of transpiration rate (Camacho-B Kaufmann and Hall, 1974). Thus leaf conductance and leaf-to-air difference in absolute humidity controlled transpiration but had little effect on ^j^^f (except at high transpiration rates). M A T E R I A L S AND M E T H O D S Inoculation method VA mycorrhiza were obtained from the mycorrhizal roots of a very productive and healthy orchard of 20-year-old rough lemon rootstock {Citrus jambhiri Lush) budded to grapefruit (C. paradisi Macf.) at the Gilat Experiment Station, in the Negev region (Southern Israel). The original rootstocks had been grown from seed at Gilat and care was taken during the first years of development to prevent importation of soil or rooted plants from the main citriculture area of the country, it is possible that the VA myccorhiza developed in this orchard are different from those encountered in other citrus growing areas in the country. Samples of feeder roots were collected from the orchard; spores and external mycelium were mixed with 7-sterilized soil in 11 pots in which Sudangrass [Sorghmn vulgare var. Sudanese (Piper) Hitch] was then planted. Sundagrass roots infected with VA mycorrhiza were used to inoculate rough lemon seedlings. Rough lemon seeds were germinated in autoclaved vermiculate and the small seedlings then transplanted into 11 plastic pots with 7-irradiated sandy ioam soil. Ten ml of a homogenized mixture of Sudangrass roots and soil was placed in contact with the citrus seedling roots. Estimate of citrus root infection Mycorrhizal status oi inoculated axid lion-moculated plants was momtored during the experiment as IOWOWB: fifteen 1 cm )ong pieces of root were sampled at random from each plant, 3 and 6 months after inoculation. The roots were fixed in FAA (26 parts formaldehyde, 1 part glacial acetic acid, 40 parts 50% ethanol), cleared

3 Endomycorrhiza and citrus water relations 27 in 10% KOH and stained with Tryphan blue (Phillips and Hayman, 1970). Percentage infection, determined by measuring the length of mycorrhizal root in the 15 cm lengths, was found to be 40 to 60%. Only uniformly inoculated and non-inoculated plants were used for the experiment. The VA fungus remains unclassified, but it is probably Glomus fasdculatus (Thaxter) Gerd & Trappe. Environmental measurements These included air temperature and relative humidity (from a wet and dry bulb Assman psychrometer) and photosynthetically active radiation (PAR, nm wavelength) measured near the leaf surface with a quantum sensor (Model LI 190S, Ll-Cor Co., Lincoln, Nebraska, U.S.A.) held horizontally; PAR was 43 to 130/^E Cultural and experimental conditions Experiments were performed on 8-month-old inoculated and non-inoculated seedlings, grown in y-sterilized soil in a controlled-environment greenhouse under 30% shade and watered daily with 0-1 % (by weight) of a commercial nutrient solution ( microelements). Preliminary work had shown that at this high rate of P application, seedling growth rate did not respond to mycorrhizal inoculation. However, it was found (Krikun and Levy, in prep.) that mycorrhizal establishment was not suppressed as reported by Menge et al. (1978). Plants were transferred to a growth room two weeks before initiation of water stress treatment. Day and night temperatures were 27 C and 18 C respectively. Light was provided by 'Power groove' daylight fluorescent lamps (General Electric Co.) and tungsten incandescent lamps; the photoperiod was 12 h. Plants were watered daily with nutrient solution during the preconditioning period; then for 4 days water was withheld and finally, seedlings were irrigated daily during the recovery period. Plant measurements No stomata are present on the upper surface of citrus leaves. Lower leaf surface conductance was measured with a modified diffusive resistance meter (Model LI 20S, LI-COR Co., Lincoln, Nebraska, U.S.A.), following the precautions of Elfving et al. (1972), Stigter, Birnie and Lammers (1973), and Stigter and Visscher (1975). Leaf temperature, measured with a leaf-to-air thermocouple clamp, was recorded to determine whether stomatal cycling occurred during the study period (Levy and Kaufmann, 1976). No cycling was detected, probably because of the low light intensity and relatively high humidity. The absolute humidity difference {/ig cm~^) between leaf to air was estimated from leaf temperature and the external temperature and humidity, assuming that the air in the leaf was saturated with water. The product of this difference and the conductance (cm %~^) was used as an estimate of transpirational flux density (/^g cm-^ g-i). Photosynthesis was estimated according to Shimshi (1969) hy exposing a small portion of leaf to air containing a known amount of -"^^COa; the amount of ^*C taken up was measured after 20 s. Xylem pressure potential was measured with a pressure cha.mhtr (Kaufmann, 1968). Proline, used as an indicator of water stress, was estimated according to Bates \i973). Two 9 mm discs, taken from the leaf that was used for the xylem prsssure potential measurement, were placed in 2 ml cold 3 % sulfosalicylic acid and frozen overnight at 18 C; the discs were extracted by shaking in the same solution for

4 28 Y. LEVY AND J. KRIKUN 1 h at 30 C. One ml aliquots were placed in 1 ml glacial acetic acid and 1 ml acid ninhydrin and the colour developed. RESULTS AND DISCUSSION The effect of mycorrhizal association on the development and recovery of plants from water stress is shown in Figure 1. As indicated by leaf xylem pressure potential and proline content a slight, though non-significant difference, is indicated during the first day of water stress, but it disappears as water stress developed. Fig. 1. Effect of mycorrhizal association on (a) xylem pressure potential and (b) proline content during development and recovery from water stress imposed by withholding water for 4 days (, mycorrhizal; O, non mycorrhizal plants). Arrow denotes time of rewatering. As seen in Figure 2, stomatal conductance and calculated transpirational flux density were slightly, but not significantly, influenced by the mycorrhiza during the water withdrawal period. During the recovery period, however, leaf conductance, transpirational flux density and photosynthesis were consistantly higher in VA seedlings. During this period, leaf conductance and photosynthesis remained lower than pre-stress levels for several days, but xylem water potential returned to normal values in one day (Fig. 1) as described by Kaufman and Levy (1976). The after-effect Days 8

5 Endomycorrhiza and citrus water relations 29 of water stress on stomatal conductance and photosynthesis was much more pronounced in plants lacking mycorrhiza. According to Safir et al. (1972), the main factor in soybean influenced by mycorrhiza is root resistance. To check this the relationship between ^leaf and transpirational flux density was plotted both during the drying phase and after recovery (Fig. 3). No difference was observed between mycorrhizal and non-mycorrhizal CA 0-4 I? 0 2 o 008 E u 0 04 o C o i 0 8 o 0 4 o a Fig. 2. Effect of mycorrhizal association on (b) stomatal conductance, calculated (a) transpiration and (c) photosynthesis during development and recovery from water stress. Treatments described in Figure 1. plants, indicating that root resistances were similar for the two groups of plants [eqn (3); Kaufmann and Hall, 1974; Kaufmann, 1977]. Transpiration rates were low in this study, however. It is possible that at higher fluxes effects on resistances and on xylem pressure potential might be greater. Clearly, however, leaf conductances after the drying cycle were different for the two groups of plants. It is known that during water, osmotic or salinity stress, changes occur in the levels of endogenous hormones; root cytokinins generally decrease and leaf content of ABA Days

6 Y. LEVY AND J. KRIKUN increases (Vaadia, 1976). It may be postulated that mycorrhizal association affects plant water relations mainly through its effects on root-shoot hormonal balance. Kaufmann and Levy (1976) showed that C. jambhiri seedlings subjected to periods of water stress had a lower leaf conductance than plants that had not been previously stressed; in C. jambhiri lack of mycorrhiza had an effect similar to that of several cycles of water stress Transpirational flux density (^g cnrf^ s-') 006 Fig. 3. Relationship betw^een leaf xylem pressure potential and transpirational flux density during the drying (arrows from upper right-hand to lower left-hand corner) and recovery cycles in mycorrhizal (#) and non-mycorrhizal (O) plants. ACKNOWLEDGEMENTS Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, no. 104 E, 1979 Series. This work was supported by a grant from the Vinnik Institute, Tel Aviv. We thank Dr M. R. Kaufmann for his helpful criticism of the manuscript. REFERENCES BATES, L. S. (1973). Rapid determination of free proline for water stress studies. Plant and Soil, 39, 205. CAMACHO-B, S. E., KAUFMANN, M. R. & HALL, A. E. (1974). Leaf water potential response to transpiration by citrus. Physiologia Plantarum, 31, 101. DAFT, M. J. & NICOLSON, T. H. (1965). Effect oi Endogene mycorrhiza on plant growth. II. Influence of soluble phosphate on endophyte and host in maize. Nezv Phytologist, 68, 945. ELFVING, D. C, KAUFMANN, M. R. & HALL, A. E. (1972). Interpreting leaf water potential measurements with a model of soil-plant-atmosphere continuum. Physiologia Plantarum, 271, 161, HATTINGH, M. J. & GERDEMANN, J. W. (1975). Inoculation of Brazilian sour orange seed with endomycorrhizal fungus. Phytopathology, 65, VAN DEN HoNBRT, T. H. (1948). Water transport in plants as a catenary process. Discussions of the Faraday Society, London, 3, 146. KAUFMANN, M. R. (1968). Evaluation of the pressure chamber method for measurement of water stress in Citrus. Proceeding of the American Society for Horticultural Science, 93, 186. KAUFMANN, M. R. (1977). Citrus - a case study of' environmental effects on plant water relations Proceedings of the International Society of Citriculture, 1, 57. KAUFMANN, M. R. & HALL, A. E. (1974). Plant water balance and its relationship to atmospheric and edaphic conditions. Agricultural Meteorology, 14, 85.

7 Endomycorrhiza and citrus water relations 31 KAUFMANN, M. R. & LEVY, Y. (1976). Stomatal response of Citrus jamhhiri to water stress and humidity. Physiologia Plantarum, 38, 105. LEVY, Y. & KAUFMANN, M. R. (1976). Cycling of leaf conductance in citrus exposed to natural and controlled environments. Canadian Journal of Botany, 54, MENGE, J. A., LAMBRIGHT, H. & JOHNSON, E. L. V. (1977). Utilization of mycorrhizal fungi in citrus nurseries Proceeding of the International Society of Citriculture, 1, 129. MENGE, J. A., STEIRTE, D., BAGYARAJ, D. J., JOHNSON, E. L. V. & LEONARD, R. T. (1978). Phosphorus concentrations in plant responsible for inhibition of mycorrhizal infection. New Phytologist, 80, 575. 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. Transaction of the British Mycological Society, 55, 158. SAFIR, G. P., BOYER, J. J. & GERDEMANN, J. W. (1972). Nutrient status and mycorrhizal enhancement of water transport in soybean. Plant Physiology, 49, 700. SHIMSI, D. (1969). A rapid field method for measuring photosynthesis with labelled carbon dioxide. Journal of Experimental Botany, 20, 381. STIGTER, C. J., BIRNIE, J. & LAMMERS, B. (1973). Leaf diffusion resistance to water vapour and its direct measurement. II. Design, calibration and pertinent theory of an improved leaf diffusion resistance meter. Mededelingen Landbouwhoogeschool, Wageningen, STIGTER, C. L. & VISSCHER, C. J. W. (1975). Application of a new calibration method to an unventilated dynamic diffusion porometer. Netherlands Journal of Agricultural Science, 23, 303. TIMMER, L. W. & LAYDEN, R. F. (1978). Stunting of citrus seedlings in fumigated soils in Texas and its correction by phosphorus fertilization and inoculation with mycorrhizal fungi. Journal of the American Society for Horticultural Science, 103, 533. VAADIA, Y. (1976). Plant hormones and water stress. Philosophical Transaction of the Royal Society London, B, 273, 513. WEATHERLEY, P. E. (1976). Introduction: Water movement through Plants. Philosophical Transactions of the Royal Society London, B, 273, 435.

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

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