1 McMillan and riedhart: hydrocarbonsphotosynthesis 15 LITERATURE CITED 1. Adams, D. F Recognition of the effects of fluorides on vegetation. Jour. Air. Pollution Control Asso ciation 13: Bellack, E. and P. J. Schouboe Rapid photo metric determination of fluoride in water. Anal. Chem. 30: Brewer, R. F., R. K. Creveling, F. B. Guillemet, and F. JL Sutherland The effects of hydrogen fluoride gas on seven citrus varieties. Proc. Am. Soc. Hort. Sci. 75 : Brewer, R. F., F. H. Sutherland, F. B. Guillemet, and R. K. Creveling Some effects of hydrogen fluoride gas on bearing Navel orange trees. Proc. Am. Soc. Hort. Sci. 76 : Halevy, A. H The influence of progressive in creases in soil moisture tension on growth and water balance of gladiolus leaves, and the development of physiological indicators for irrigation. Proc. Am. Soc. Hort. Sci. 76 : Thomas, M. D Air pollution with relation to agronomic crops: I. General status of research on the effects of air pollution on plants. Agron. Jour. 50: Umbreit, W. W., R. H. Burris, and J. F. Stauffer Manometric techniques. Burgess Publishing Co. 8. Wander, I. W. and J. J. McBride, Jr A chlo rosis produced by fluorine on citrus. Science 123: Woltz, S. S., R. O. Magie, and C. M. Geraldson Studies on leaf scorch of gladiolus. Proc. Fla. State Hort. Soc. 66: THE INFLUENCE OF HYDROCARBONS ON PHOTOSYNTHESIS OF CITRUS LEAVES R. T. McMillan and J. M. Riedhart University of Miami Coral Gables, Fla. Introduction The general effects of petroleum spray oils on photosynthesis and respiration in citrus have been reported by various workers (15, 26, 27, 36). Using iodine as a test for starch Knight et al. (15) measured the effect of saturated petroleum oils on photosynthesis and respiration of citrus leaves by painting oil on both surfaces of leaves that had been kept in the dark for 15 days. He found that those leaves treated with an oil having a viscosity of 106 seconds had not re turned to normal starch synthesis at the end of forty days. The leaves treated with an oil having a viscosity of 50 seconds returned to nor mal in four days. Using a carbon dioxide absorptiontitration method, the same workers found the respiration rate of leaves with oilpainted surfaces showed a significant stimulation; while the same oils applied as a spray demonstrated a similar effect, it was not to the same degree. Wedding et al. (37) using a manometric tech nique measured the effects of a 2% California mediumgrade emulsivetype spray oil on Wash ington Navel orange leaves and Eureka lemon leaves. Photosynthesis and respiration were sig nificantly depressed in orange leaves the day fol lowing the application of the oil, but recovery oc curred with time. Lemon leaves showed a similar effect for photosynthesis, but respiration was affected only slightly and the depression of the rate was not significant. Recovery of photosyn thesis occurred more rapidly in lemon plants than in orange plants. Riehl and Wedding (27) measured the effects of hydrocarbon composition and molecular size of various oils on inhibition of photosynthesis in Bearss lime and Eureka lemon leaves by the Warburg procedure. They noted that ultraviolet light was not an important factor in the effect of California lightmedium or medium grade spray oils on the photosynthesis of citrus leaves following applications at levels of oil necessary to control citrus pests. Recovery of photosyn thesis occurred in less time in plants sprayed with naphthenic oils than in those sprayed with paraffinic oils. In a later paper, Riehl and Wed ding (28) reported that the difference in rate of recovery of photosynthesis associated with the difference in paraffinicity in greater for oils of comparable viscosity than for those of com parable molecular weight. The present investigation was initiated in order to determine the effects of pure hydro carbons and a typical spray oil on photosynthesis and respiration of citrus leaves. Materials and Methods Leaves from threeyearold nurserygrown trees of Citrus sinensis, Osbeck, variety Valencia, were used as the source of plant material. The oils used were practical grade dodecane, tridecane, tetradecane, hexadecane, octadecane, one part octadecane to two parts dodecane mix ture, and a typical oil. The properties of the hydrocarbons used are listed in Table I. The oils were applied to the dorsal (lower) or ven tral (upper) surfaces of the leaves using a set tling tower with a shutter at the base to control the amount of oil (22). The amount of oil de posited and volatilized was determined gravimetrically using aluminum paper strips (20).
2 16 FLORIDA STATE HORTICULTURAL SOCIETY, 1964 AUTOSWITCH FLOAT VALVE DRYING COLUMN 3W A Y SOL N 0 I D S PURGE P UMP METERING TALVES LEAF CHAMBERS Figure 1. Diagram of the arrangement for multipoint sampling system with continuous purge. Determinations of photosynthesis and respira tion were made using continuous infrared CO2 analysis with a multipoint sampling system (21). Air from the atmosphere, regulated by fine metering valves at thirty liters per hour, was passed over leaves placed in water cooled plastic chambers, through a dririte drying column, a flow meter, and a Hartmann Braun URAS In frared CO2 analyzer. An automatic switching unit permitted sequential sampling from several leaf chambers or sampling points. A purge pump maintained a constant flow at thirty liters per hour over the leaves not being channelled through the analyzer, Figure 1. Apparent photosynthesis was measured by the amount of CO2 in ppm removed from the air stream in the light and respiration was meas ured by the amount of CO2 in ppm produced in the dark. The oil treatments and all experiments were conducted at ambient room temperature, while the gas from the test and control leaves was analyzed at 54 C. The results were recorded in ppm. The light source, artificial, was adjusted to obtain maximum apparent photosynthesis of each leaf. The leaves being analyzed were illuminated from 8:00 A.M. to 5:00 P.M. True photosyn thesis was not determined. All experiments were repeated several times with only minor variations in results which were not considered statistically significant.
3 McMillan and riedhart: hydrocarbonsphotosynthesis 17 LOU Air o oh^ ^t* S 225 : j o 225.7Z] D 0 D EC A N E TETRADECANE Control ' OCTADECANE Oil Air 225 : 225 TWO PARTS DODECANE ONE PART noctadecane TYPICAL OIL 6 10 Figure 2. Influence of various hydrocarbons and a typical spray oil on apparent photosynthesis when applied to the dorsal (lower) surface. Solid line represents evaporation of oil from aluminum paper strips. Mg/cm2. Lines have been extrapolated through the dark periods. Results C02 from tne air stream before and after the application of oil. Oil deposits are shown in Apparent photosynthesis of Valencia orange ^g/cm2. leaves as effected by various hydrocarbons is Figure 2 shows inhibition of apparent photoshown in figures 2 and 3. The degree of inhibition synthesis for leaves treated with hydrocarbons of apparent photosynthesis is expressed by a on the dorsal (lower) surface. Maximum decomparison of the ability of a leaf to remove pression occurred immediately after application
4 18 FLORIDA STATE HORTICULTURAL SOCIETY, Oil Air Control /\. / \ OCTA DECANE TYPICAL OIL Figure 3. Influence of a hydrocarbon and a typical spray oil on apparent photosynthesis when applied to the ventral (upper) surface. Solid line represents evaporation of oil from aluminum aper strips, iig/cm*. Lines have been extrapolated through the dark period. of dodecane, and tetradecane, while for hexadecane and octadecane a lag occurred between the time of application and maximum inhibition of photosynthesis. Inhibition also occurred im mediately after application of mixed oils (one part octadecane to two parts dodecane). In the case of the typical spray oil, the depression was preceded by a period of slight stimulation. The degree of inhibition of photosynthesis of the leaves treated with the typical spray oil was further increased by the application of dodecane. These leaves showed no signs of recovery through out the experimental period. The reason for the difference in the initial activity produced by the typical oil, and the de pression effect of an application of a lighter hydrocarbon following the application of a heavy hydrocarbon has not yet been investigated, al though it is probably a matter of rate of pene tration of the various fractions. Not shown by the figures, but worthy of mention, is the obser vation that oilsprayed leaves as compared with unsprayed leaves exhibited a lag attaining maxi mum photosynthesis upon illumination. This phenomenon occurred only with pure hydrocar bons of a distillation range of 419 to 487 F. The correlation of the recovery from the in itial depression of apparent photosynthesis with the dissipation of the various hydrocarbons from the leaves is obvious (Figs. 2 & 3). The slight discrepancies in the correlation can be accounted for by the fact that the dissipation of oil from leaves occurs more slowly than it does from aluminum paper strips. TABIE 1. Physical Properties of the Hydrocarbons and Oil. Material Distillation Unsulfonated Viscosity S.S.U. MdI. F Residue Gravity at 100 F Wt. Dodecane 1* Tridecane 1*55 181*. 36 Tetradecane 1* Hexadecane *3 Octadecane 602 Octadecane & Dodecane Mixture Typical Oil 66280U (1090*) O
5 McMillan and riedhart: hydrocarbonsphotosynthesis 19 2iL 00r ou Oil 300 i Test 300 Control OCTADECANE TYPICAL OIL * to "" Figure 4. Influence of hydrocarbons and a typical spray oil on respiration. Solid line represents evaporation of oil from aluminum paper strips, Mg/cm. Lines have been extrapolated through light periods. Inhibition of apparent photosynthesis was not obtained with the application of hydrocarbons on the ventral (upper) surface, Fig. 3. The effect of various hydrocarbons on respira tion is shown in Figure 4. Respiration is ex pressed as ppm CO2 evolved in the dark. Oil de posits are expressed in ^,g/cm2. The measure ments of respiration are from the same leaf samples that were utilized throughout the light period. All leaves treated on the dorsal (lower) surface demonstrated some change in respiration. Those leaves treated with the pure hydrocarbons having a distillation range from 419 to 487 F and the mixed oil (one part octadecane to two parts dodecane) showed a significant increase while those treated with the oils of a distillation range of 552 F or higher showed a definite in crease. This stimulation of oxygen uptake was also reported by Knight et ah (15), Green and Johnson (9), Green (10), and others. Oil penetration was determined on the basis of its ability to influence photosynthesis and respiration. Measured this way there was no penetration, throughout the experimental period, of the ventral surface of the leaves by the pure hydrocarbons and the typical oil. The only instance of ventral (upper) penetration was in an experiment, shown in Figure 3, where an excess of dodecane was applied to a leaf that previously had received an application of typical spray oil. The inhibition, however, was only temporary and the leaf recovered upon the dis sipation of the dodecane. The most obvious sur face to be penetrated by all of the hydrocarbons was the dorsal (lower) surface, Figure 2, where most of the stomata of citrus are located, (19). The pure hydrocarbons, having a distillation range of 419 to 487 F, exhibited a faster rate of penetration than those with a higher distilla tion range.
6 20 FLORIDA STATE HORTICULTURAL SOCIETY, 1964 In all instances the application of oils in these experiments showed no visual symptoms of leaf injury usually attributed to their use. Discussion The results of the present investigation indi cate unequivocally that the application of petro leum oil to a leaf, providing there is penetration, inhibits photosynthesis. This is in agreement with earlier studies of Green and Johnson (9) on beans, Green (10) on bean, apple and barley, Knight et al. (15) on citrus, Riedhart (20) on bananas and (22) on Bauhinia, Riehl (25) on lemon and lime, Riehl and Wedding (28) on lemon, and Wedding et al. (37) on citrus. Of added significance from the present investigation is the fact that inhibition was shown to occur with the application of minute amounts of oil and under normal concentration of CO2 in air. In regard to the magnitude and the duration of inhibition, the present investigation indicates a direct relationship to the amount of oil initially deposited and its eventual dissipation from the leaf. This is in agreement with the observations of Reidhart (20, 22), and Riehl and Wedding (27). The transitory stimulation of apparent photosynthesis obtained in the case of the typical spray oil can be attributed to solubilization phe nomenon, as suggested by Van Overbeek and Blondeau (36), because of the aromatics present in the oil. This observation assists in explaining some of the discrepancies between the results of earlier investigations. It is quite likely that had a time factor concerning penetration been considered in earlier studies the results would be in closer agreement. The data accumulated here in regard to the influence of petroleum on respiration is in par tial agreement with that of Green (10), Helson and Minshall (12), and Wedding et al. (37). Oils having. a distillation range of 487 F or lower brought a stimulation of respiration while those having a distillation range of 552 F or higher brought a decrease in respiration. As with apparent photosynthesis the minor discrepancies with previous results probably arise from con sideration of the time factor and hydrocarbon types. The contrast of the results obtained with the application of a hydrocarbon on the dorsal (lower) surface versus the ventral (upper) sur face suggests that penetration occurs only on the lower surface. Since corollary to this is the fact that stomata occur only on the dorsal (lower) surface in citrus, it can be postulated that, at the levels of oil used in these experiments, pene tration occurs through the stomata and not through the cuticle. Consideration of varia tions in the amount of hydrocarbons, variations in plant material, and physical properties of hydrocarbons, this postulation is consistent with the results of Calpouzos et al. (1), Calpouzos and Colberg (2), Crafts and Reiber (5), De Ong (6, 7), Griffiths and Janes (11), Helson and Minshall (12), Hoffman (13), Johnson and Hoskins (14), Minshall and Helson (17), Reidhart (20), Riehl and Wedding (27, 28), Schroeder (30), Tucker (35), and Wedding et ah (37). In regard to the mechanism of action of oil on apparent photosynthesis, the results corrobo rate the hypothesis that inhibition results from mechanical interference of gaseous exchange as reported by Helson and Minshall (12), Riedhart (20, 22), Riehl and Wedding (27, 28), Rohrbaugh (29) and Wedding et al. (37). It is of interest to note that a similar action has been hypothe sized in order to explain the lethal action of oil on insects (4), (6), (8), (16), (18), (23), (33), insect eggs (3), (18), (24), (31), and fungi (1), (34). The complete recovery from inhibition upon dissipation of the oil supports the postulation of Van Overbeek and Blondeau (36) in that no solubilization occurs with molecules as large as those of foliage spray oils. This is also in agree ment with the studies of Riehl and Wedding (27) and Riedhart (20, 22). Contributing to the selection and the more discriminate use of petroleum oils in agriculture, the data suggest that more attention be given to the distillation range and coincidentally to its evaporation rate. By placing more emphasis on the distillation range, oils may be more carefully selected in order that the deposit is just sufficient to effect control of the pest while remaining be low the level that produces plant injury, as sug gested by Smith (32). Phytotoxicity, as measured by the influence of petroleum oil on photosynthesis and respiration probably cannot be avoided; however, equilibrat ing this factor against the economic advantages of using petroleum oils to control plant pests, it can hardly be considered detrimental to their continued use. Acknowledgement Acknowledgement is made to the donors of The Petroleum Research Fund, administered by
7 SMITH, HRNCIAR, SCUDDER: PHOSPHATE EFFECTS ON SOIL 21 the American Chemical Society, for support of this research. LITERATURE CITED 1. Calpouzos, L., Delfel, N. E., Colberg, C., and Theis, T Viscosity of Naphthenic and Paraffinic Spray Oils in Relation to Phytoxicity and Sigtoka Disease Control on Banana Leaves. Phytopathology. 51: and Colberg, C Importance of Source of Spray Oils for Sigatoka Disease Control and Phytotoxicity to Banana Leaves. Phytopathology. 54 (2) : Chapman, P. J., Pearce, G. W., and Avens, A. W Relation of Composition to the Efficiency of Foliage or Summer Type Petroleum Fractions. J. Econ. Ent. 36: , Lienk, S. E., Avens, A. W. and White, R. W Selection of a Plant Spray Oil Combining Full Pesticidal Efficiency with Minimum Plant Injury Hazards. J. Econ. Ent. 55: Crafts, A. S. and Reiber, H. G Herbicidal Properties of Oils. Hilgardia. 18 (2): de Ong, E. R., Knight, H. and Chamberlin, J. G A preliminary Study of Petroleum Oil as an Insecti cide for Citrus Trees. Calif. Agr. Expt. Sta. Hilgardia. 2: Chemistry and Uses of Insecti cides. Reinhold Pub. Corp., New York, N.Y Chemistry and Uses of Pesticides. Reinhold Pub. Corp., New York, N.Y. 9. Green, J. R. and Johnson, A. G Effect of Petroleum Oils on the Respiration of Bean Leaves. Plant Physiology. 6 : Green, J. R Effect of Petroleum Oils on the Respiration of Bean Plants, Apple Twigs and Leaves, and Barley Seedlings. Plant Physiology. 11: Griffiths, A. E. and Janes, M. J Phytotoxic and Insecticidal Study of a Petroleum Isoparaffinic Fraction. Agr. Appl. of Petroleum Products. Am. Chem. Soc, Wash., D. C. 12. Helson, V. A. and Minshall, W. H Effects of Petroleum Oils on the Carbon Dioxide Output in Respiration of Parsnip and Mustard. Plant Physiology. 31 (1): Hoffman, M. B The Effects of Several Sum mer Oils in the Carbon Dioxide Assimilation by Apple Leaves. Proc. Am. Soc. Hort. Sci. 32: Johnson, C. M. and Hoskins, W. M The Re lation of Acids and Peroxides in Spray Oils to the Respira tion of Sprayed Bean Leaves and the Development of In jury. Plant Physiology. 27: Knight, H., Chamberlin, J. C, and Samuels, C. D On Some Limiting Factors in the Use of Saturated Petroleum Oils as Insecticides. Plant Physiology. 4: Madsen, H. F. and Marshall, J Dormant Sprays for the Control of the Pear Psylla, Psylla Pericola, in British Columbia. J. Econ. Ent. 54: Minshall, W. H. and Helson, V. A The Herbi cidal Action of Oils. Proc. Am. Soc. Hort. Sci. 53: Pearce, G. W. and Chapman, P. J Insecti cidal Efficiency of Petroleum Fractions and Synthetic Isoparaffins. Agri. Appl. of Petroleum Products, Am. Chem. Soc. Wash., D. C. 19. Reed, H. S. and Hirano, E The Density of Stomata.in Citrus Leaves. J. Agr. Research. 43 (3): Riedhart, J. M Influence of Petroleum Oil on Photosynthesis of Banana Leaves. Trop. Agr. Trin. 38 (1): Determinations of Phytotoxicity Using Continuous Infrared CO2 Analysis. Agr. Chem. 19 (3): 26, Influence of Hydrocarbons and Oil on Photosynthesis in the Orchid Tree. Proc. Am. Soc. Hort. Sci., in press. 23. Riehl, L. A. and La Due, J. P Evaluation of Petroleum Fractions Against California Red Scale and Citrus Red Mite. Agr. Appl. of Petroleum Products, Am. Chem. Soc, Wash., D.C. 24., La Due, J. P., and Rodriguez, J. L Evaluation of Representative California Spray Oils Against Citrus Red Mite and California Red Scale. J. Econ. Ent. 51 (2) : , Influence of Water Phase of Oil Spray on Photosynthesis in Eureka Lemon and Bearss Lime Leaves. J. Econ. Ent. 52 (1): , La Due, J. P., and Rodriguez, J. L., Jr Efficiency of Ethion in Oil Spray Against California Red Scale and Citrus Red Mite. J. Econ. Ent. 52 (2): and Wedding, R. T Relation of Oil Type Deposit and Soaking to Effects of Spray Oils on Photosynthesis in Citrus Leaves. J. Econ. Ent. 52: Effect of Naphthenic and Paraf finic Petroleum Composition at a Comparable Molecular Weight or Viscosity on Photosynthesis of Eureka Lemon Leaves. J. Econ. Ent. 52: Rohrbaugh, P. W Physiological Effects of Petroleum Oil Sprays on Citrus. J. Econ. Ent. 34 (6): Schroeder, R. A The Effect of Some Summer Oil Sprays Upon the Carbon Dioxide Absorption of Apple Leaves. Proc. Am. Soc. Hort. Sci. 33: Smith, E. H. and Pearce, C. W The Mode of Action of Petroleum Oils as Ovicides J. Econ. Ent. 41: Tree Spray Oils. Agri. Appl. of Petroleum Products, Am. Chem. Soc, Wash., D.C. 33. and Phillips, J. H Influence of Oil Viscosity and Timing of Treatment on Semidormant Control of European Fruit Lecanium. J. Econ. Ent. 54: Thompson, W. L Some Problems of Control of Scale Insects on Citrus. Proc. Fla. State Hort. Soc 55: Tucker, R. P Oil Sprays. Chemical Proper ties of Petroleum Unsaturates Causing Injury to Foliage. Ind. Eng. Chem. 28: Van Overbeek, J. and Blondeau, R Mode of Action of Phytotoxic Oils. Weeds. 3 (1): Wedding, R. T., Riehl, L. A., and Rhoads, W. A Effect of Petroleum Oil Spray on Photosynthesis and Respiration in Citrus Leaves. Plant Physiology. 27 (2): CHANGES IN SOIL COMPOSITION DURING TWENTY YEARS OF DIFFERENTIAL PHOSPHATE APPLICATION Paul F. Smith, G. Hrnciar, and G. K. Scudder, Jr.1 U. S. Department of Agriculture Agricultural Research Service Horticultural Field Station Orlando, Florida 1 Research Plant Physiologist, Physical Science Technician, and Agricultural Research Technician, respectively. The horticultural aspects of a longterm ex periment on phosphate rates were described last year (7). In brief, a tract of virgin land near Tavares was cleared and set with Pineapple orange on Rough lemon stock in Beginning with the first application of fertilizer, and con tinuing for 20 years, plots of 12 trees each were differentially fertilized with superphosphate and compensating amounts of gypsum. Each treat ment was randomized in 6 blocks with guard rows around each plot of 12 record trees. The experiment covered 8 acres of grove. One treat