USDA Forest Service Phytotoxic Grass Residues Research Paper RM-153

Reduce Germination and September 1975 Initial Root Growth of Rocky Mountain Forest and Range Experiment Station Ponderosa Pine Forest Service W. J. Rietveld U.S. Department of Agriculture Fort Collins, Colorado 80521 Abstract

Rietveld, W. J. 1975. Phytotoxic grass residues reduce germination and initial root growth of ponderosa pine. USDA For. Serv. Res. Pap. RM-153, 15 p. Rocky Mt. For. and Range Exp. Stn., Fort Collins, Colo. 80521

Extracts of green foliage of Arizona fescue and mountain muhly significantly reduced germination of ponderosa pine seeds, and retarded speed of elongation and mean radicle length. Three possible routes of release of the inhibitor were investigated: (1) leaching from live foliage, (2) root exudation, and (3) overwinter leaching from dead residues. The principal route remains uncertain. The ecological implications of the inhibitory substance are discussed.

Keywords: (Nutt.) Hitchc., arizonica Vasey, Laws., allelopathy, phytotoxic substances, competition, chemical ecology.

COVER: An example of the ponderosa pine- bunchgrass community of northern Arizona. Clearcut logging in the early 1920's allowed the su bcl imax bunchgrasses, Festuca arizonica and Muhlenbergia montana, to capture and dominate the site in nearly pure stands for over 50 years. USDA Forest Service Research Paper RM-153 September 1975

Phytotoxic Grass Residues Reduce Germination and Initial Root Growth of Ponderosa Pine

W. J. Rietveld, Plant Physiologist Rocky Mountain Forest and Range Experiment Station'

'central headquarters is maintained at Fort Collins in cooperation with Colorado State Univer- sity; research reported here was conducted at the Station's Research Work Unit at Flagstaff, in cooperation with Northern Arizona University. CONTENTS Page Introduction ...... 1 Chemical Ecology: Chemical Interactions among Species ...... 1 Experimental Studies and Results ...... : ...... 2 Germination of Seeds and Growth of Seedlings in Aqueous Extracts of Grass Residues ...... 2 Materials and Methods ...... 2 Results and Discussion ...... 3 Growth of Various Species Associations in Sand Culture ...... 6 Materials and Methods ...... 6 Results and Discussion ...... 8 Seed Germination in Leachates from Grass Residues ...... 10 Materials and Methods ...... 10 Results and Discussion ...... 10 Overwintering of Pine Seeds under Various Plant Residues ...... 10 Materials and Methods ...... 11 Results and Discussion ...... 11 Discussion of Ecological Significance ...... 12 Conclusions ...... 13 Literature Cited ...... 14 Phytotoxic Grass Residues Reduce Germination and Initial Root Growth of Ponderosa Pine

W. J. Rietveld

INTRODUCTION CHEMICAL ECOLOGY: CHEMICAL INTERACTIONS AMONG SPECIES The widespread pine-bunchgrass community of northern ~rizonaconsists of ponderosa pine (Pinus Many interactions between can be attributed ponderosa), Arizona fescue (Festuca arizonica), and to such specific factors as competition for light, water, mountain muhly (Muhlenbergia montana) as princi- nutrients, and growing space; susceptibility or im- pal species. Typically, the ponderosa pine forest munity to insects and diseases; and the differential supports a grass understory of varying density which effects of other environmental stresses. However, it extends through parklike landscapes. Best develop- has become increasingly apparent that there are some ment of +the community occurs on better soils where interactions involving chemical substances released the pine overstory is less than 20 square feet basal area from one plant which negatively influence another per acre, with frequent larger openings. plant. The study of these interactions is the subject of In parklike stands and openings created by fire or the new and rapidly expanding field of chemical logging, the bunchgrasses promptly capture the site, ecology (Sondheimer and Simeone 1970). Many terms and develop into dense, exclusive communities have been applied to the toxic chemicals and chemical seemingly impenetrable by other species. Numerous effects of one species on another, but the term examples are found near Flagstaff where the ponder- allelopathy (meaning mutual harm) is most commonly osa pine climax has not returned in 50 to 100 years used. Whittaker (1970, p. 44) defined allelopathic after fire or timber harvesting (Schubert 1974). Seeds substances as "chemicals that are released from from surrounding trees repeatedly fall in these higher plants (directly or by way of decay processes) openings, but few seedlings emerge. Pine seedlings that inhibit the germination, growth, or occurrence of transplanted directly into the grasses typically fail to other plants." survive or grow poorly. Even where dense grass cover Evidence regarding the occurrence and ecological is mechanically removed, survival and growth of roles of allelopathy is voluminous, and has been planted trees may be depressed. reviewed repeatedly and comprehensively.2 Allelo- In addition to competition for available water, pathic effects have been reported for agricultural and nutrients, and growing space, chemical interactions wild species of most growth forms and habitats. (allelopathy) may play an important role in plant The routes by which allelopathic substances reach competition. Jameson (1961, 1968) tested for the the soil from aboveground plants include adsorption presence of growth inhibitors in Arizona fescue and of volatile materials on soil particles, rain washing of other northern Arizona native s~ecies.and found that soluble materials from living plant surfaces, leaching an aqueous extract of fescue f;iage inhibited radicle of materials from plant litter (or seedcoats) on the soil elongation of squirreltail (Sitanion hystrix), blue gra- surface, and release of toxic compounds during ma (), and ponderosa pine. Elonga- microbial decomposition of litter. Routes of release tion of pine radicles in fescue extract was only 40 per- within the soil include root exudation, leaching from cent of that in water. In a small pilot test, ponderosa living and dead roots, and decomposition of dead

Lnine seed overwintered under Arizona fescue litter roots and incorporated residues (Whittaker 1971). were found to have reduced germination the following Liberation of phytotoxic substances from grasses summer (USDA-FS 1957). Seeds subjected to fescue and grasslike plants capable of inhibiting germination leachings had only 63 percent germination, compared to 90 percent for uncovered seed. 2~ublishedreviews include those by Bonner (1950), Borner (1960), Evenari (1949), Garb (1961), McCalla and This Paper reports research to determine (1) if Haskins (1964), Muller (1966), National Academy of various grass residues contain substances phytotoxic Sciences (1971), Rice (1974), Sondheimer and Simeone to ponderosa pine, and (2) their route of release. (1970), Whittaker and Feeny (1971), and Woods (7960). and/or growth of another species appears to be a Of 20 species involved in the old-field successions, widespread phenomenon.3 The nature of the allelo- 16 species had some and 10 considerable inhibitory pathics, route of release, and mode of action in most activity against nitrogen-fixing and nitrifying bacteria cases is species specific, that is, the allelopathic (Abdul-Wahab and Rice 1967; Rice 1964, 1965; Rice mechanism has evolved to fulfill a particular eco- and Parenti 1967). logical role. Barley, for example, inhibits all other There is considerable potential for chronic and plants around it. Hordeum spontaneum grows in the indirect allelopathic effects. Relative allelopathic , wild state in almost pure stands (Went 1970), and the effects vary with the levels of phytotoxic substances, cultivated species Hordeum sativum successfully the kind of soil and other environmental conditions, excludes weeds from fields by excretion of inhibitors and the sensitivity of different plant species. It is from its roots (Overland 1966). Quackgrass (Agro- reasonable to postulate that the observed cases of pyron repens), a widespread weed, inhibits growth of allelopathy stand out from a background of more cultivated plants by release of inhibitors from widespread, less conspicuous effects on plant growth decaying roots which interfere with mineral nutrition and plant distribution. The combined influence of (Griimmer 1961). The tillage practice of stubble allelopathy.and competition could explain why certain mulching, which places residues of oat, wheat, corn, species quickly capture a site following disturbance, or sorghum on the soil surface, has resulted in re- and persist for so long in almost pure stands before duced growth of subsequent crops. Liberation of phe- the climax vegetation is ultimately restored. nolic acids to the soil during microbial decomposition of the residues was found to be responsible. Bacterial metabolism of these compounds in the soil resulted in EXPERIMENTAL STUDIES AND RESULTS the formation of other toxic substances (Guenzi and McCalla 1966). Germination of Seeds and Growth of Seedlings in Allelopathic effects also may be a significant Aqueous Extracts of Grass Residues influence in the sequence and timing of succession of plant communities. A dominant species may, by A laboratory experiment was conducted to deter- allelopathic suppression, speed its invasion of a mine the phytotoxic effects of various residues of community and delay its replacement by other species fescue and muhly on seed germination and primary (Whittaker 1970, 1971). Studies on old-field suc- root elongation of ponderosa pine. cession in Oklahoma (Rice 1971, 1972; Wilson and Rice 1968) showed that sunflower (Helianthus an- nuus), one of the dominants of the first stage of Materials and Methods succession, produces substances inhibitory to most other species of the stage. Threeawn (Aristida Samples of fresh green foliage (including seed- oligantha), an annual grass, quickly invades because stalks), new litter (standing dead grass litter), old it is tolerant of the toxic substances as well as low soil litter (matted dead litter accumulated in the center of nitrogen levels. Threeawn maintains dominance for the clump), and roots (live roots clipped from growing several years by allelopathic suppression of nitrogen- grass plants) of fescue and muhly were collected in fixing bacteria and blue-green algae, thertby keeping mid-September on the Fort Valley Experimental soil nitrogen at levels too low to support species from Forest near Flagstaff, Arizona. Excess soil was shaken later stages of succession. Eventually, broomsedge from the grass roots, but they were left unwashed to (Andropogo~zvirginicus), which is also tolerant of low- avoid losing any excreted phytotoxins. The residues fertility soils, replaces threeawn by means of were air-dried for 26 days, then ground in a mill to chemical inhibition and competition, and for many pass a 20-mesh screen. Powdered residues were refrig- years delays replacement by its successor, little blue- erated in sealed metal cans until use. stem (Andropogon scoparius). Each extract was prepared by homogenizing 20 g of powdered residue with 200 ml of sterile distilled water in a blender at low speed for 15 minutes. The extracts 3~heoccurrence of toxic substances has been reported were centrifuged (5,600 rpm for 10 minutes) and in western wheatgrass (Jameson 1961), squirreltail filtered, using suction to remove suspended matter. (Jameson 7961, 1968), cheatgrass (Jameson 1961), The control "extract" consisted of sterile distilled Johnsongrass (Hoveland 1964), Bermudagrass (Hoveland 1964), blue grama (Jameson 1968), Sudangrass (Patrick, water homogenized for 15 minutes. The pH and Toussoun, and Snyder 1963), quackgrass (Griimmer osmotic potential (by cryoscopy, Harris and Gortner 1961), timothy (Patrick and Koch 1958), rye (Holm 1969, 1914) of the freshly prepared extracts were measured. Patrick and Koch 1958; Patrick, Toussoun, and Snyder Extracts used in the germination tests were not 1963), false flax (Grummer 1960), brome (Myers and Anderson 1942), as well as oat, wheat, and barley sterilized since heat might affect stability of inhibitory (Guenzi, McCalla, and Norstadt 1967; Holm 1969; substances in the extracts, and the chemical sterilants Patrick, Toussoun, and Snyder 1963). sodium benzoate and sodium hypochlorite were found to inhibit pine seed germination. To reduce fungal effects is apparent in figure 1 in which speed of contamination, seeds were surface-sterilized by soak- germination of seeds and speed of elongation of ing in 30 percent hydrogen peroxide for 15 minutes radicles in the,extracts are expressed as percentages and rinsed with sterile distilled water, and all of the control values. A few inconsistent, less glassware and glass filter paper were sterilized by significant effects appear in the data: MR extract autoclaving. The interior of the growth chamber was inhibited mean radicle length and FOL increased washed with sterilizing chemicals. Five ml of unsteril- percentage germination and speed of germination. ized extract was added to each petri dish only once; These results could be reverse responses to low sterile distilled water was added when additional concentration cf a growth substance or due to water was needed. Control dishes were wetted with 5 secondary metabolites in the extracts. However, the ml of sterile distilled water. variance for mean radicle length was high, and these The experiment was conducted in a controlled results may simply be chance events. environment chamber held at a constant temperature Retarded pine germinants tended to have a stocky of 75°F and a photoperiod of 16 hours of combined appearance with a shortened, stubby radicle and fluorescent and incandescent lighting daily. The short hypocotyl. Other symptoms were an enlarged incandescent lights were turned off 15 minutes after root collar and dark brown sheath covering both the the fluorescent lights to expose the seeds to light in root collar and radicle tip. In the most toxic extracts both the red and far-red regions at the end of each the radicle tip eventually died. day, as in the natural condition. Relative humidity The pH and osmotic potential of aqueous extracts was maintained above 80 percent. The arrangement of grass residues were as follows: of petri dishes, four per treatment combination, in the growth chamber was completely randomized; Source of Osmotic locations were shifted daily to distribute the effects of extract potential microclimatic variation. (atm) Seeds were considered germinated when the rad- FF -2.60 icle length equaled the length of the seed. The FNL -0.50 number of germinated seeds was recorded daily for FOL -0.30 25 days. 'Radicle length (in millimeters) from the FR -0.60 seedcoat or cotyledons to radicle tip, of the first 10 MF -1.60 seeds to germinate in each petri dish, was measured MNL -0.60 daily until the seedlings began to decline due to MOL -0.30 exhaustion of the endosperm and growth factors MR -0.60 contained in the extracts. Fungal contamination was Control 0.00 not serious until the last days of the experiment, but 90 percent of the germination had occurred by that The pH of the control "extract" was slightly acidic time. due to the incorporation of carbon dioxide by the Percentage germination, speed of germination blender. Although no tests were run, pH values are (Maguire 1962), time for 50 percent germination, not considered to be low enough to significantly speed of elongation (adapted from Maguire 1962), affect seed germination or initial growth. Moreover, and mean radicle length before decline were calcu- the pH values of the extracts that induced the lated and tested for significant differences by factorial strongest responses (FF, FNL, MF) are similar to the analysis of variance. Speed of germination and control. elongation take both the promptness and complete- Extract osmotic potential decreased with increas- ness of the respective processes into account. ing age and decomposition of residues. Extracts that significantly inhibited germination and initial de- velopment also had the lowest osmotic potentials, Results and Discussion especially FF and MF. Larson and Schubert (1969a) reported that moderately low osmotic potentials ~xtractsprepared from live foliage of fescue (PF) (down to -3 bars) benefit germination of ponderosa and muhly (MF) by far produced the most dominant pine seeds, but the reason for this response, as well effects (fig. 1). With few exceptions, FF and MF as the effects on speed of germination, and speed of extracts significantly (a= 0.01) reduced percentage elongation of pine, are unknown. germination and speed of germination, increased The separate effects of extract osmotic potential time for 50 percent germination, and reduced speed were tested in a followup experiment in which pine of elongation and mean radicle length. FNL similarly seeds were germinated in solutions of polyethylene inhibited seed germination, speed of elongation, and glycol (PEG, molecular weight 20,000) with osmotic mean radicle length, but to a lesser degree than potentials of -1, -2, -4, and -8 atmospheres. extracts from live foliage. The magnitude of these Percentage germination, speed of germination, and Speed of germination Type of Speed of elongation residue ,

FNL * FOL FR MF MNL MOL MR

Percent of control Percent of control

Mean speed Mean t ime Mean speed Mean Mean o f for 50 percent of radicle germination qerrnination qermination- elongation length (Percent) (No./day) (Days 1 fm/day ) (m) FF---FESCUE FOLIAGE FNL--FESCUE NEW LITTER FOL--FESCUE OLD LITTER FR---FESCUE ROOTS MF---MUHLY FOLIAGE MNL--MUHLY NEW LITTER MOL--MUHLY OLD LITTER MR---MUHLY ROOTS

0.01 96 4.1418 1.0805 Variance (5') 1.0805 38.0467 14.0 2.0 1.4 Least significant difference (5%) 1.0 6.2 18.7 2.7 1.8 Least significant difference (1% 1.4 8.2

;'Differs significantly from control at 5% level. *;':Differs significantly from control at 1% level. Figure 1.-Germination and growth responses of ponderosa pine to extracts prepared from grass residues.

speed of elongation were measured under the same degree. Speed of elongation of pine radicles (fig. 2C) experimental conditions described previously. The was found to be quite sensitive to osmotic potential PEG solutions and distilled water control were at all potentials tested below -1 atmosphere. Overall, changed daily to minimize the increase in concentra- in the range 0 to -3 atmospheres, which includes the tion of the solutions by seed imbibition of water. osmotic potential of the extracts, osmotic potential Percentage germination of pine seeds (fig. 2A) is should not interfere significantly with percentage or enhanced by low levels of osmotic potential, beyond speed of germination of pine seeds. Speed of which germination drops off gradually. This pattern elongation of pine is sensitive to the osmotic potential is in general agreement with the results of Larson of the wetting solution. and Schubert (1969a). Speed of germination of pine Thus, extract osmotic potential was a principal seeds (fig. 2B) followed a trend similar to percentage confounding factor which inconsistently accounted germination within the range tested, but to a lesser for part of the observed responses to the extracts. C .2 50 - LR o Control .-C - w2 40 ~3 FNL" MF*" 30 - ;F* * 20 I I I I

Control 1' .;R

01 ' I I I I I I I I 0 -1 -2 -3 -4 -5 -6 -7 -8 Osmotic potential (atmospheres)

Figure 2.-Effects of different osmotic poten- tials imposed by solutions of polyethylene glycol (PEG) on: A, percentage germination; B, speed of germination; and C, speed of elongation of ponderosa pine.

When extract osmotic potential is taken into ac- count, the inhibitory effects of FF, FNL, and MF extracts on percentage germination and speed of germination (figs. 3A and 3B) would be greater, rather than lesser, compared to the control values. - 0 -I - 2 -3 - 4 Speed of elongation of pine (fig. 3C) radicles would Osmotic potential (atrnospheres) likely not be significantly different from the re- spective controls when the separate effects of extract osmotic 'potential are taken into account. If the Figure 3.-Comparisons of: A, percentage ger- controls consisted of PEG solutions with osmotic mination; B, speed of germination; and C, potentials equivalent to the extracts, the actual speed of elongation of ponderosa pine in depression of germination of pine seeds by FF polyethylene glycol (PEG) solutions and extract, for example, would be 61.4 percent (fig. 3A) grass residue extracts of different osmotic of the control compared to 44.2 percent calculated potentials. One asterisk indicates the dif- from the tabulation in figure 1. Speed of germination ference between the extract and control of pine seeds was depressed 50 percent of the control values is significant at the 0.05 probability value in figure 1, and 64.3 percent if extract osmotic level; two asterisks indicate significance at potential is taken into account (fig. 3B). However, the 0.01 probability level. for speed of elongation of pine radicIes, the actual of release of the inhibitory substance. Paired com- depression is 16.5 percent (fig. 3C) compared to 36 binations of pine, fescue, and muhly were grown in percent calculated from the tabulation in figure 1. intimate contact to (1) determine if inhibitory Thus, elongation of pine radicles appears to be much substances are excreted by grass roots, and (2) more sensitive to the osmotic potential of the wetting measure their relative toxicity to an associated pine solution than is seed germination. or grass plant. The extract experiment also revealed that pine seeds germinate much better under conditions of limited water than under wet conditions. Apparently Materials and Methods the improved aeration and small negative water potential afforded by minimum wetting stimulated Pine, fescue, and muhly seeds were germinated in germination. This is illustrated also by the ability of petri dishes. Pairs of plants in all possible combina- ponderosa pine seeds to withstand air-drying during tions of the three species were then planted in 4%- germination (Larson and Davault 1975). inch-diameter by 10-inch deep metaI pots. The pots The low percentage germination of 49.3 for the were filled with an equal weight of number 60 silica pine control treatment (fig. 1) is attributed to sand and a 1-inch layer of Perlite on the surface to overwetting (5 ml water per 9 cm petri dish). Also, reduce evaporation. Group A consisted of 36 pots of fungal contamination may have interfered. To test plants with simultaneous planting times; Group B these possibilities, a small experiment was performed contained 36 pots of plants with age differing by 3 to test the effects of sterilized FF and MF extracts, 3 months. ml per petri dish, on germination of surface- The experiment was conducted in a controlled sterilized pine seeds. All glassware was similarly environment chamber programed for two periods of sterilized by autoclaving. The test was run in a small plant growth separated by a 10-week cool period. germinator githout light at 75°F with high humidity. The warm environment consisted of a thermoperiod Germination in the control treatment was much of 75°F during the day and 60°F during the night, higher, 84.8 percent, than in the original experiment and photoperiod of 16 hours of combined fluorescent (fig. 4), but the differences in germination between and incandescent lighting. The transition period of the extracts and the control persisted: temperature changes coincided with light changes, and a 15-minute period of twilight was provided at Source of Germination day end as described previously. After 4 months. extract (Percent) (No./day) under the warm environment, the growth chamber FF 62.3 11.9 was set for a cool environment of 10 weeks' duration, MF 60.8 11.8 consisting of a thermoperiod of 50°F during the day Control 84.8 17.9 (8 hours) and 35'F at night. Following the cool period, the warm environment was resumed for an As a consequence of higher percentage germination, additional 3 months. speed of germination was correspondingly higher A watering system based on the relationship than in the original experiment. However, the between matric potential and water content was treatment differences are consistent. developed which regulated soil moisture potential Another followup experiment was run to determine within the range of -0.3 atmosphere (field capacity) the effects of extract added after germination. Fescue and -3.0 atmospheres by a corresponding range in foliage extract, 1 or 2 ml per 7 cm petri dish, was pot weight. The plants were watered with one-half added to pine germinants with radicles % or 1 inch strength Hoagland solution at 2- to 4-day intervals long. Response was evaluated during a 3-day incuba- during the entire experiment to maintain water and tion period. The results were similar regardless of nutrients at adequate levels. Leaching of the pots at initial radicle length: 2 ml of extract per dish monthly intervals was necessary to remove accumu- retarded radicle elongation by 30 percent. Thus, it lated salts. Each pot was leached with 2,000 ml of appears that the inhibitory substance retards both water. Since phytotoxins excreted from roots could germination and initial growth processes, rather than be lost in the water leached from the pots, the first the latter being a consequence of the effects upon 250 ml of Ieachate from each pot in Group A was germination. collected and tested for toxicity to yellow sweetclover (Melilotus ofJicinalis) seed germination.4 A 25 m1

Growth of Various Species Associations in Sand Culture 4~erminationand initial growth of yellow sweetclover were found to be extremely sensitive to the phytotoxins produced by Arizona fescue and mountain rnuhly, and The sand culture experiment was one of three therefore provided a quick bioassay for the presence of experiments conducted to investigate possible routes the inhibitor(s). STERI LIZED EXTRACT CONTROL

FESCUE

JE NEW LI

iLY FOLI

Figure 4,-Germination of surface-sterilized pine seeds in sterilized extracts compared with the control. aliquot of the leachate from each pot was evaporated tion developed as early as 3 months into the to dryness, rewetted with 3 ml of distilled water and experiment and intensified as the experiment pro- added to 100 clover seeds in a petri dish; distilled gressed. Because of competition, it was very difficult water only was used as a control. The osmotic to establish the second species in the later planting, potential of the leachates was not determined. especially where large fescue and muhly plants were Foliage height (distance from cotyledons to shoot present. Toward the end of the experiment, the pots tip) of pine, and total height and basal diameter of were distinctly crowded by large grass plants (fig. S), the two grasses, were measured monthly for the and most pine seedlings planted 3 months after the 9-month duration of the experiment. At the end of grass were suppressed or dead. This awesome the experiment, dry weight of the top and roots of demonstration of the competitive power of fescue and individual plants was determined. These data and muhly is in accord with the results of Larson and calculated top/root weight ratios and plant volumes Schubert (1969b), especially with regard to planting (grasses) were analyzed for significant differences by pines in an established grass stand. Seedlings that analysis of variance. died were assigned a value of zero for their corresponding data. The data presented in table 1 for Group A show mainly the effects of plant competition, or lack of it, Results and Discussion on various growth parameters. Fescue plants made superior growth in terms of top and root dry weight Though the experiment was designed to test for and top volume when associated with pine compared toxic root excretions in the absence of competition to another fescue or muhly plant. The lack of for light, water, and nutrients, strong plant competi- competition from the associated pine is undoubtedly

Figure 5.-The pine-muhly species combina- tion, planted simultaneously (Group A), at age 9 months. Competition for growing space occurred as early as 3 months into the experiment. Table 1.--Comparative growth of ponderosa pine, Arizona fescue, and mountain muhly

Species Species Mean Mean Mean Mean Pine mean Grass Group evaluated combination total top dry root dry , top/root foliage mean top height weight weight weight ratio height volume 3 em g g em em GROUP A' Ponderosa pine Pi ne/P i ne 20.0 0.75 0.26 3.96 17.5 Pine/Fescue 16.8 .49 .I4 3.79 14.3 Pine/Muhly 16.7 .55 -24 3.27 14.2 Arizona fescue Fescue/Fescue 67.8 8.82 7.07 1.52 259.5 Fescue/Pine 64.0 13.605 11.09" 1.56 376.9" Fescue/Muhly 61.2 7.79 4.18 1.98 216.2 Mountain muhly Muhly/Muhly 57-05" 18.38 7.31 4.43 275.1 Muhly/Pine 69.3 35.47 9.93 3.95 585. o* Muhl y/Fescue 68.2 32.73 7.14 5.25 460.5 GROUP B* Ponderosa pine Pine/Pine 19.5 a, 1.34 a 0.60 a 2.26 17.0 a Pine/Fescue 19.6 a 1.55 a .68 a 2.30 17.1 aj Pine/Muhly 24.2a 1.80a .94a 1.99 21.7 ai Pine/Pine 7.2 be .I2 b .06 b 3.13 e 5.3 be Fescue/Pine 4.2 bf .02 b .O1 b 1.75 f 2.9 bf Muhly/Pine .O bf .OOb .00b .oo f .O bf Arizona fescue Fescue/Pine 67.8 a 18.86 a 21.65 a .98 a 447.5 a Fescue/Fescue 63.0 a 20.35 a 25.63 a .89 a 526.5 a Fescue/Muhly 68.2 a 20.78 a 18.70 a 1.46 a 503.9 a Pine/Fescue 37.7 beg .39 b .66 b .63 b 3.4 b Fescue/Fescue .O bf .OOb .OOb .OO b .O b Muhl y/Fescue -0 bfh .OO b .a0 b -00 b .O b Mountain muhly Muhly/Pine 71.4 a 40.42 a 11.42 a 3.67 a 695.5 a Muhl y/Fescue 73.9 a 43.80 a 13.66 a 3.52 a 713.5 a Muhl y/Muhl y 75.6 a 42.71 a 13.66 a 3.91 a 709.2 Pine/~uhly 23.4beg .80b .35b 2.00bg 8.4 b Fescue/Muhl y -0 bfh .OO b .OO b .OO bh .O b Muhly/Muhly .O bf .OOb .ODb .OO b .O b * Differs significantly from other two members of group at 5 percent level. ** Differs significantly from other two members of group at 2.5 percent level. 'Three species planted simultaneously in various combinations; plants grown for 9 months in controlled environment. 'Three species planted at different times in various combinations; second species planted 3 months after first. Species left of / planted early; species right of / planted 3 months later. Compa r i son Interpretation a,a,a vs. b,b,b Collectively, early-planted species combinations (a,a,a) differ significantly from late-planted species combinations (b,b,b) . e vs. f,f Pine or grass species planted late with an existing pine (e) differs significantly from the same species planted date with an existing grass plant, considered collectively (f ,f) . ~VS.h Fescue or muhly planted late with an existing pine (g) differs significantly from fescue or muhly planted late with the alternate grass species (h). i VS. j Pine planted early with muhly (i) differs significantly from pine planted early with fescue (j). related to the difference in growth. Muhly plants Materials and Methods were significantly shorter when associated with another muhly and had only half as much top Samples of fescue and muhly live grass blades and volume after 9 months compared to muhly plants seedstalks were collected in mid-September on the associated with pines. Fort Valley Experimental Forest. Fescue stalks had Data for Group B (table 1) and interpretation of begun to dry when collected, whereas muhly stalks significant specific comparisons show that the re- were still green; blades of both species were green. In sponses generally, are the same as in Group A. In all the laboratory, the samples were separated into seed-' cases, except mean top/root weight ratio of pine, stalks and blades. A 50 g sample (fresh weight) of plants of the species evaluated established early each of the four residue types was soaked in 100 ml (a,a,a) collectively differed significantly from plants of distilled water in a large beaker for either 5 established 3 months later (b,b,b). This is an minutes or 12 hours. Each was agitated for 5 minutes expected difference due to the difference in age of to wet all of the material. At the end of the soaking the plants. The top/root weight ratio of the grasses periods the eight leachates were drained off and apparently changes with age, as indicated by the refrigerated. difference between early- (a,a, a) and late-planted Surface-sterilized pine seeds were used to test the (b,b ,b) seedlings. Pine or grass seedlings established leachates for phytotoxicity. Ten ml of leachate were late with an existing pine seedling (e) demonstrated transferred to each of three 9 cm petri dishes, superior height growth compared to seedlings evaporated to dryness, autoclaved, and coded. In a planted late with the two grasses considered col- separate test, it was found that autoclaving grass lectively (f,f). residue extracts did not alter their growth-inhibiting Considering the grasses independently, fescue or potential. Three ml of sterile distilled water and 100 muhly seedlings planted late with an existing pine (g) unstratified surface-sterilized seeds were added to grew taller .than when planted late with the other each petri dish. Germination tests were conducted in grass species (h). Although the significant (e) vs (f,f) darkness in a small Mangelsdorf germinator with and (g) vs (h) comparisons might be considered temperature set at 7S°F and high humidity. Other meaningless because of almost complete mortality of procedures and germination criteria were the same as seedlings planted late with an existing grass plant, described previously. The number of germinated they are important in that they depict the intense seeds was recorded daily for 11 days. From these vegetative competition expressed by an established data, percentage germination and speed of germina- grass plant. Finally, pines planted early with muhly tion were. calculated and tested for significant (i) had significantly greater foliage height than when differences by analysis of variance. planted early with 'fescue (j). A similar difference developed in mean total height of pines, but is not significant due to high variation. In all cases, the Results and Discussion observed effects can be ascribed to the lack of competition because of the presence of a pine None of the leachates affected percentage germina- seedling, or conversely, due to intense competition tion or speed of germination of pine seed. Seeds from an existing grass plant. Muhly appears to be germinating in the 12-hour leachates exhibited the slightly less competitive toward the pines than fescue. same appearance symptoms as in the extract experi- There were no significant differences in germina- ment. The dark brown sheaths at the root collar and tion of yellow sweetclover seeds incubated either in radicle tip may simply be the result of staining by the leachate from pots representing the species combina- extract or leachate. tions in Group A or distilled water, indicating no detectable amount of inhibitory substance arising from grass roots was leached from the pots. Overwintering of Pine Seeds Under Various Plant Residues Seed Germination in Leachates From Grass Residues Newly fallen pine seeds normally lie on the ground in the presence of grass and pine residues until the following July, 8 months or more, before environ- The results of the extract experiment indicated mental conditions are right for germination. In this that a phytotoxic substance is contained in the experiment, samples of ponderosa pine seed were residues of grass plants, and is particularly concen- overwintered under various plant residues in differ- trated in live foliage. In this experiment, leaching ent stages of decomposition to determine if continu- from live foliage was explored as a possible route of ous exposure to leachates of plant residues over the release of the inhibitor. natural period would adversely affect seed viability. Materials and Methods screen to remove extraneous materials. One hundred seeds from each replication were germinated in a 9 Fescue, muhly, and pine residues were collected on cm petri dish under the same conditions as the the Fort Valley Experimental Forest in mid-Novem- extract experiment. The seeds were wetted with a ber. The grasses had entered dormancy by that time, minimum amount of sterile distilled water. Germina- so the residues collected were FNL, FOL, FR, MNL, tion was recorded daily; seeds were considered MOL, MR (all described in the extract experiment), germinated when radicle length equaled the length of plus freshly fallen pine needles (PNL) and partially the seed. Locations of dishes in the growth chambhr decomposed pine needles (POL). All of the residues were randomized after each germination count. After were chopped into smaller pieces so they would form 18 days the ungerminated seeds were cut to deter- a dense mat over the seeds. mine soundness. Percentage germination data, based The experiment was conducted at the Fort Valley on sound seeds, were tested for significant differ- Experimental Nursery, which is surrounded by a ences by analysis of variance. rodentproof fence (fig. 6). The area utilized had been kept free of vegetation, including conifers, for several years. The soil surface was leveled and covered with Results and Discussion approximately 2 inches of washed sand to eliminate interference from substances in the soil. Thirty-six The winter of 1969-70 was an "open winter" with metal retaining rings 12 inches in diameter and 2 only 9.74 inches of precipitation during the period inches deep were arranged in a grid pattern on the November 20 to June 5; 7.14 inches of this total fell sand with 2 feet between rings. The rings were during March. Consequently, during most of the randomly assigned to treatment. winter there was only minimal moisture available for Approximately 225 ponderosa pine seeds, mea- leaching. sured by weight, were evenly distributed on top of the There were no significant differences in percentage sand in the retaining rings and covered with the pre- germination of pine seeds overwintered under the determined type of residue to the top of the ring. The different plant residues: control treatment consisted of rings filled with washed sand. Each ring was covered with a square of Type of residue Percentage germination %-inch hardware cloth for further protection. FNL 89.0 On June 5 the seeds were recovered from the FOL 85.4 individual retaining rings. They were not washed FR 85.7 before the germination test, but were shaken on a MNL 88.5 MOL 89.1 MR 79.7 Figure 6.-Pine seeds were overwintered under PNL 90.5 various residues of pine, fescue, and muhly. POL 88.4 The seeds were recovered and germinated Control 92.6 the following summer. The germination in each case was quite high, fescue and muhly were not toxic to germinating pine indicating no general loss of viability due to the seeds, however. To fully understand the role of treatments. leaching as a route of release, research is needed on These results do not agree with findings from an the presence, toxicity, and leachability of the inhibi- informal study conducted in the winter of 1956-57 tor in live and dead grass residues during the growth (USDA-FS 1957). Pine seeds overwintered under period of the receiver species. Arizona fescue litter yielded 63 percent germination A potential mechanism of release of the inhibitor the following summer, compared to 85 percent for which was not studied is the formation of phytotoxic pine needle litter and 87 percent for no cover. substances during microbial decomposition of dead Leaching conditions were somewhat better during the residues on or in the soil. The liberation of period November to June 1956-57, with 12.27 inches substances toxic to living plants during decomposi- of precipitation, of which 6.17 inches fell during tion of plant residues appears to be a widespread January. Furthermore, seeds were placed directly on phenomenon (McCalla and Haskins 1964, Patrick the surface of a clay soil, which potentially would 1971, Patrick and Koch 1958, Patrick, Toussoun, allow some accumulation of leached substances. and Koch 1964). Soil toxicity due to decomposition Such accumulation would be unlikely in the sand of organic constituents is most frequently associated layer used in the present experiment. with heavy, poorly aerated, or waterlogged soils. However, localized pockets of anaerobiosis are wide- spread in the soil and may prevail for varying periods DISCUSSION OF ECOLOGICAL SIGNIFICANCE of time following rainfall (Patrick 1971). Conditions that lead to the formation of high concentrations of phytotoxic decomposition products may, therefore, The route of release of the inhibitory substance be more common than is generally realized, and not remains uncertain, in spite of several efforts to necessarily confined to waterlogged soils. Additional identify a pathway. Release of the inhibitor under- research is needed on microbial decomposition of ground as a root excretion can probably be ruled out, dead residues on or in the soil as a possible judging from the lack of any evidence pointing to mechanism of release of the inhibitor. chemical effects in the sand culture experiment, and There is evidence to suggest that the inhibitor since an extraction of fescue and muhly roots was not must be present at a time of physiological activity inhibitory in the extract experiment. The inhibitory (germination and initial development) of the receiver. substance is apparently present in aboveground grass species in order to be effective. Extracts prepared residues and most concentrated in live, green foliage. from FF, FNL, and MF residues significantly The question reduces to: how is the inhibitor depressed germination of ponderosa pine seeds in the released to the external environment, and how does it extract experiment (fig. I), whereas similar resi- enter the receiver species? Since little microbial dues collected 2 months later had no effect on decomposition occurs in standing dead residues, the germination of pine seeds in the overwintering principal mechanisms of breakdown would be bio- experiment. The newer grass residues employed in chemical degradation during senescence, and normal the overwintering experimelit must have contained a weathering processes. Any inhibitory substance in considerable amount of the inhibitor,s but over- the standing residues at the time senescence begins winter exposure of dormant pine seeds to the would subsequently (1) undergo biochemical degra- inhibitory substance leached from the residues did dation, (2) oxidize as a result of weathering pro- not have any effect on germination in distilled water cesses, or (3) remain intact for some indefinite the following summer. period. The principal processes for removal of The results of the followup experiment in which a breakdown products or an intact inhibitor would be postgermination addition of extract from fescue translocation, volatilization, or leaching. foliage retarded radicle elongation of pine seedlings Overwinter leaching does not appear to be the further supports this suggestion. This point could be route of release, unless there is accumulation in the clarified by (1) a study on low-temperature soaking soil up to and during the time of germination and of seeds in fescue foliage extracts and subsequent growth of the receiver species. Considering the high germination (after rinsing) in distilled water at a water solubility and probable subjectivity to mi- higher temperature, and (2) a study to determine if crobial decomposition, it is unlikely that the inhibi- the effects of the inhibitor can be reversed. tor could persist, even in clay soils, until the following July. Leaching should not be ruled out, but it appears that leaching at or near the time of seed 5~~~ in the extract experiment had been dead nearly germination and initial growth of the receiver species 12 months, whereas the residue designated FNL in the is more likely to be effective. Leachates prepared overwinter leaching experiment had been dead only 2 from mid-September live foliage and new litter of months. An important question that should be considered that the existing severe competition for water, is the relation between the amounts of the residues nptrients, and growing space (Larson and Schubert used to prepare the extracts in the experiments and 1969b) is the near-total mechanism. the amounts that occur in nature in an arbitrary area If allelopathy' occurs in the ponderosa pine - of influence around a single seed. The amount of bunchgrass community of northern Arizona, what is herbage production depends upon the extent of tree its ecological role? The grasses are superior competi- cover. Arnold (1956) estimated that herbage pro- tors for available water and nutrients (Larson an$ duction would vary from 1,000 pounds dry weight Schubert 1969b), and the sand culture experiment per acre per year in forest openings to 50 pounds per indicated they also compete successfully for growing acre per year under dense forest canopies. In these space. These characteristics alone would seem to openings, assuming no grass utilization, the potential enable the grasses to dominate the site. But they do addition of residues to the soil each year is 1,000 not explain the observed phenomenon where bunch- pounds dry weight per acre. If 1 square inch is grasses have persisted on a site for 100 years or more conservatively estimated to be the area of influence (Schubert 1974), encompassing numerous seed crops around a single pine seed, then an average of 0.072 g and moist periods that are suitable for the establish- of grass residue would be introduced into this area ment of pines. But no new pines emerge unless the each year. In the extract experiment, 5 ml of extract grass cover is disturbed. The combined effects of an obtained from 0.67 g of dry residue (extract yield allelopathic mechanism and plant competition is a from 20 g of residue and 200 ml of water was 150 ml) more feasible explanation for the long-term exclusion was sufficient to produce the reported effects in 100 of new pine seedlings and other species from the seeds, which is equivalent to 0.3067 g of residue per community. Therefore, allelopathy may prolong the seed. The followup experiment utilizing sterilized bunchgrass successional stage and forestall the extracts found that 0.004 g of residue per seed was ponderosa pine climax. inhibitory: Dividing the yearly production of 0.072 g of residue per 1 square inch by the calculated experimental rate per seed shows that 10.8 times as CONCLUSIONS much residue is added to the area of influence per year than was necessary to induce the observed A growth-inhibitory substance is produced pre- effects in the extract experiment. dominantly in live foliage, and to a lesser extent in These calculations serve only to illustrate that the dead residues, of Arizona fescue and mountain amount of the residues used to prepare the extracts is muhly in the 'northern Arizona ponderosa pine - conservative compared to the amounts that occur in bunchgrass community. The inhibitor is capable of nature. The primary difference between the two cases substantially reducing total germination and retard- is the concentration of inhibitor around a seed at any ing germination rate and initial radicle development one time. In the extract experiment, the inhibitory of ponderosa pine. The osmotic potential of the substance contained in the residues was almost extracts confounds to some extent their phytotoxic completely released into solution at one time, effects. whereas in nature the phytotoxins are undoubtedly The route of release of the inhibitor remains released more slowly. Under natural conditions, even uncertain, although three possible routes were though greater amounts of residues may occur, the studied. Neither winter-long leaching of dead resi- concentration of inhibitor at a particular time may dues nor root excretion appears to be the principal not approach that of the extract experiment. The nel route of release. Since dormant seeds and seedlings influence of plant sensitivity to the inhibitor and the are insensitive to the inhibitor, it was suggested that effect of interacting environmental variables that leaching from live grass tissues and release of affect the concentration of inhibitor present would substances by microbial decomposition of dead determine the extent of allelopathy. At this time we residues during the growing season are potentially can only state that there is potential for allelopathy significant routes of release. Further research is in the ponderosa pine - bunchgrass community. recommended on these pathways. Allelopathic effects would be conditioned by a host Further research is also needed to clarify the of factors, including the route and rate of release of ecological significance of the inhibitor. On the basis the inhibitor, timing of release with growth processes of the concentrations found toxic in laboratory of the receiving species, persistence of the inhibitor experiments, there is considerable potential for toxic in the soil, distribution of seeds and residues, and concentrations to occur under natural conditions. weather conditions. The outcome of interactions Actual inhibitory effects would be conditioned by a among these environmental factors could vary within number of environmental variables, including those broad limits, ranging from striking examples of which (1) affect the rates of leaching and residue allelopathy to fluctuations or chronic expressions, or decomposition, and (2) influence the persistence or no effect at all. Furthermore, it is entirely possible rate of degradation of the free inhibitory substance in the soil. Coincidence of toxic levels of the inhibitor Hoveland, C. S. with initial growth processes appears to be necessary. 1964. Germination and seedling vigor of clovers as affected by grass root extracts. Crop Sci. 4~211-213. Jameson, Donald A. 1961. Growth inhibitors in native plants of north- LITERATURE CITED ern Arizona. U.S. Dep. Agric., For. Serv., Rocky Mt. For and Range Exp. Stn., Res. Note Abdul-Wahab, A. S., and E. L. Rice. 61, 2 p. Fort Collins, Colo. 1967. Plant inhibition by Johnson grass and its Jameson, Donald A. possible significance in old-field succession. 1968. Species interactions of growth inhibitors in Bull. Torrey Bot. Club 94:486-497. native plants of northern Arizona. U.S. For. Arnold, Joseph F. Serv. Res. Note RM-113, 2 p. Rocky Mt. For. 1956. Conversion of poor and non-commercial and Range Exp. Stn., Fort Collins, Colo. pine stands to grasslands. p. 90-99. In Recov- Larson, M..M., and Michael Davault. ering Rainfall, Part 11, Ariz. Watershed Pro- 1975. Pine seeds withstand severe drying before, gram, 218 p., Univ. of Ariz., Tucson. after germination: seedling drought tolerance may be reduced. Tree Planters' Notes 26(1):22- Bonner, J. 1950. The role of toxic substances in the inter- 23. action of higher plants. Bot. Rev. 16:51-65. Larson, M. M., and Gilbert H. Schubert. 1969a. Effect of osmotic water stress on germina- Borner, H. tion and initial development of ponderosa pine 1960. Liberation of organic substances from higher seedlings. For. Sci. 1530-36. plants and their role in the soil sickness prob- lem. Bot. Rev. 26:393-424. Larson, M. M., and Gilbert H. Schubert. Evenari, M. 1969b. Root competition between ponderosa pine 1949. Germination inhibitors. Bot. Rev. 15153- seedlings and grass. USDA For. Sew. Res. Pap. 194. RM-54, 12 p. Rocky Mt. For. and Range Exp. Stn., Fort Collins, Colo. Garb, S. Maguire, James D. 1961. Differential growth inhibitors produced by 1962. Speed of germination - aid in selection and plants. Bot. Rev. 26~422-433. evaluation for seedling emergence and vigor. Griimmer, G. Crop Sci. 2:176-177. 1960. The influence exerted by species of Camelina McCalla, T. M., and F. A. Haskins. on flax by means of toxic substances. p. 153- 1964. Phytotoxic substances from soil micro- 157. In Biology of weeds. Blackwell Sci. Publ., organisms and crop residues. Bacteriol. Res. Oxford. 28:181-207. Griimmer, G. Myers, H. E., and K. L. Anderson. 1961. The role of toxic substances in the inter- 1942. Bromegrass toxicity vs nitrogen starvation. relationships between higher plants. Soc. Expl. J. Am. Soc. Agron. 34:770-773. Biol. Symp. 15219-228. Muller, C. H. Guenzi, W. D., and T. M. McCalla. 1966. The role of chemical inhibition (allelopathy) 1966. Phenolic acids in oat, wheat, sorghum, and in vegetational composition. Bull. Torrey Bot. corn residues and their phytotoxicity. Agron. J. Club 93:332-351. 58:303-304. National Academy of Sciences. Guenzi, W. D., T. M. McCalla, and Fred A. Nor- 1971. Biochemical interactions among plants. stadt. Wash., D. C., 134 p. 1967. Presence and persistence of phytotoxic sub- Overland, L. stances in wheat, oat, corn, and sorghum resi- 1966. The role of allelopathic substances in the dues. Agron. J. 59: 163-165. "smother crop" barley. Am. J. Bot. 53:423-432. Harris, J. A., and R. A. Gortner. Patrick, Z. A. 1914. Notes on the calculation of the osmotic pres- 1971. Phytotoxic substances associated with the sure of expressed vegetable saps from the decomposition in soil of plant residues. Soil Sci. depression of the freezing point with a table for 111:13-18. the values of P for A=O.OO1° to A=2.99g0. Am. Patrick, Z. A., and L. W. Koch. J. Bot. 1:75-78. 1958. Inhibition of respiration, germination and Holm, LeRoy. growth by substances arising during the decom- 1969. Chemical interactions between plants on position of certain plant residues in the soil.. agricultural lands. Down to Earth 25!1):16-22. Can. J. Bot. 36:621-747. Patrick, Z. A., T. A. Toussoun, and L. W. Koch. Schubert, Gilbert H. 1964. Effects of crop residue decomposition pro- 1974. Silviculture of southwestern ponderosa pine: ducts on plant roots. Ann. Rev. Phytopath. the status of our knowledge. USDA For. Serv. 2:267-292. Res. Pap. RM-123, 71 p. Rocky Mt. For. and Patrick, Z. A., T. A. Toussoun, and William C. Range Exp. Stn., Fort Collins, Colo. Snyder. Sondheimer, Ernest, and John B. Simeone, eds. 1963. Phytotoxic substances in arable soils associ- 1970. Chemical ecology. Academic Press, New ated with decomposition of plant residues. York, 336 p. Phytopathology 53:152-161. U.S. Department of ~griculture.Forest Service. 1957. Leachings from certain litter damage pon- Rice, E. L. derosa pine seed. p. 33-34. In Rocky Mt. For. 1964. ~nhibitionof nitrogen-fixing and nitrifying and Range Exp. Stn. 1957 Annu. Rep. Fort bacteria in seed plants. Ecology 45824-837. Collins, Colo. Went, F. W. Rice, E. L. 1970. Plants and the chemical environment. p. 71- 1965. Inhibition of nitrogen-fixing and nitrifying 82. In Chemical ecology. Ernest Sondheimer bacteria in seed plants. 11. Characterization and and John B. Simeone, eds. Academic Press, identification of inhibitors. Physiol. Plant. New York. 18:255-268. Whittaker, R. H. Rice, Elroy L. 1970. The biochemical ecology of higher plants. p. 1971. Some possible roles of inhibitors in old- 43-70, In Chemical ecology. Ernest Sondheimer field succession. p. 128-132. In Biochemical and John B. Simeone, eds. Academic Press, interactions among plants. Nat. Acad. Sci., Nat. New York. Res. Council, Wash., D.C. Whittaker, R. H. Rice, Elroy L. 1971. The chemistry of communities. p. 10-18, In 1972. Allelopathic effects of Andropogon virginicus Biochemical interactions among plants. Nat. and its persistence in old-fields. Am. J. Bot. Acad. Sci., Nat. Res. Council, Wash., D.C. 59: 752-755. Whittaker, R. H., and P. 0.Feeney. 1971. Allelochemics: chemical interactions be- Rice, Elroy L. tween species. Science 171:757-770. 1974. Allelopathy. Academic Press, New York, Wilson, R. E., and E. L. Rice. 353 p. 1968. Allelopathy as expressed by Helianthus Rice, E. L., and R. L. Parenti. annuus and its role in old-field succession. Bull. 1967. Inhibition of nitrogen-fixing and nitrifying Torrey Bot. Club 95432-448. bacteria by seed plants. V. Inhibitors produced Woods, F. W. by Bromus japonicus Thumb. Southwest. Nat. 1960. Biological antagonisms due to phytotoxic 12:97-103. root exudates. Bot. Rev. 26546-569.

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