Chemical inhibition of fire-prone grasses by fire-sensitive shrub, Conradina canescens

Journal of Chemical Ecology, VoI. 15, No. 5, 1989

CHEMICAL INHIBITION OF FIRE-PRONE GRASSES BY FIRE-SENSITIVE SHRUB, Conradina canescens

G . B R U C E W I L L I A M S O N , 1 N I K O L A U S H . F I S C H E R , 2

D O N A L D R . R I C H A R D S O N , 3 a n d A N A D E L A P E l q A 2

~Department of Botany

2Department of Chemistry, Louisiana State University Baton Rouge, Louisiana 708003

3Department of Biology, University of South Florida Tampa, Florida 33620

(Received February 16, 1988; accepted July 26, 1988)

Abs t rac t - - In an investigation of potential chemical activity of fire-sensitive shrubs in Florida's sand pine scrub community, bioassays of foliar washes of Conradina canescens showed significant inhibitory activity on three native grasses that are known to fuel frequent surface fires; inhibition was concentrated seasonally in spring and summer . Application of runoff from Conra- dina leaves to one of the grasses caused a 50% reduction in growth over a 20-week period. Isolation of the biologically active fractions from the fresh leaves of C. canescens yielded numerous monoterpenes, a number of which were identified from a GC-MS reference library and/or MS comparison to authentic compounds: 11 from the diethyl ether extract, 11 from steam distillation, and four from the foliar leaf wash. Numerous other monoterpenes present in the extractions were unknown. The terpenoid fraction completely inhibited seed germination of one of the native grasses and of lettuce. Satu- rated aqueous solutions of nine of the monoterpenes inhibited germination and radicle growth of two native grasses. SEM views of the leaf surfaces of Conradina reveal secretory trichomes that appear to be the source of the monoterpenes as well as the triterpene, ursolic acid. The biological activity of C. canescens as a fire-sensitive component of the scrub community is reviewed in light of the chemical evidence.

Key Words--Allelopathy, fire, monoterpenes, trichomes, Conradina canescens, Pinus, Schizachyrium scoparium.

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1568 WILLIAMSON ET AL.

INTRODUCTION

Two different plant communities exist on the upland, well-drained sands of Florida: the sand pine scrub characterized by sand pine [Pinus clausa (Chapm. ex Engelm.) Vasey ex Sarg.] with a dense shrub cover but no herbaceous ground cover, and the sandhill or high pineland dominated by longleaf pine (P. pal- ustris Mill.) with a complete graminoid ground cover but few shrubs (Chapman, 1932; Laessle, 1958; Garren, 1943). Fire is a frequent feature of the sandhill, burning through the deciduous surface fuels every three to eight years (William- son and Black, 1981). However, healthy scrub, which is evergreen, burns infre- quently, about once in 50 years or once per generation of the dominant sand pine, which regenerates via serotinous cones (Harper, 1915; Richardson, 1977). As scrub and sandhill communities both occur on upland sandy soils, they are intermingled and contiguous throughout the Gulf Coastal Plain. The difference ifffire susceptibility is remarkable in that fires sweep through the sandhills until they encounter patches or strands of scrub, where often they are extinguished by changes in the fuel and live vegetation. Webber (1933) called the scrub "a fire-fighting machine," although recognition of the differences between scrub and sandhill trace back to the last century. Nash (1895) noted that "The scrub flora is entirely different from that of the high pine land, hardly a single plant being common to both; in fact these two floras are natural enemies and appear to be constantly fighting each other . . . . . " and Whitney (1898) reiterated that "i t is an impressive sight to stand at the border line between the scrub and the high pine land and notice the difference in the character of the vegetation."

In the absence of surface fires in the sandhills, the woody species from the scrub will colonize (Laessle, 1958; Veno, 1976). They grow well in the sandhills, often faster than in the scrub, unless a surface fire sweeps through the graminoid ground cover and kills them (Veno, 1976; Hebb, 1982). Large shrubs and especially groups of shrubs may escape mortality, if graminoid growth around them has been suppressed. Therefore, we have been investigating the hypothesis that shrubs from the scrub produce chemicals that inhibit the growth of the grasses and perhaps pines, which provide the fuel for surface fires that otherwise would kill the shrubs. In particular, we are focusing on the early colonizers of the scrub community.

One such species is Conradina canescens (Torr. & Gray) (Lamiaceae), a small, evergreen mint, that is endemic to the sand pine scrub community. Its distribution is highly disjunct with two major populations--one on the Gulf coast from Horn Island, Mississippi, to just south of Tallahassee, Florida, and the other concentrated on Florida's Central Ridge in Highlands and Polk coun- ties. In both locations the species is a relatively common shrub, reaching 2 m in height where scrub vegetation is colonizing and in full sunlight, but it dimin- ishes in abundance under the canopy of sand pine and the subcanopy of oaks,

CHEMICAL INHIBITION OF FIRE-PRONE GRASSES 1569

characteristic of mature scrub. The small (2 x 10 mm), recurred leaves of Conradina release a strong mintlike or terpenoid odor when crushed, whereas undamaged leaves emit no odor.

M E T H O D S A N D M A T E R I A L S

Foliage from several Conradina shrubs was collected monthly from a site near Sun Ray, Florida. Care was taken to cut the stems so as not to tear the leaves in order to prevent the release of internally bound compounds. In the laboratory, 50 g of foliage was placed in 500 ml of distilled water for 24 hr under refrigeration at 8~ The resulting leaf wash was filtered, and 5 ml was added to 30 target seeds placed on a sheet of Whatman No. 1 filter paper in a 10-cm-diameter Petri dish (Richardson, 1985; Williamson et al., 1988). Seeds of three native sandhill grasses, Schizachyrium scoparium (Michx.) Nash, Andropogon gyrans Ashe, and Leptochloa dubia (HBK.) Nees, were used as target species, each in four replicate dishes kept in the dark at 26~ Control dishes given distilled water were not adjusted for pH or osmolarity because the Conradina leachates were within ranges (5-7 pH, 2-9 m0sm) that did not influ- ence seed germination and growth.

Results of the treatment means (T) were compared to the control means (C) each month in a one-way analysis of variance (ANOVA) with four samples per monthly treatment. The magnitude of inhibition or stimulation is reported as RI, a response index that gives the percentage inhibition of the treatment relative to the control (a negative RI) or the percentage inhibition of the control relative to the treatment (a positive RI, when stimulation occurs). RI values allowed parametric comparisons (Williamson and Richardson, 1988).

A second bioassay was performed by misting water (6 mm/hr) over potted Conradina shrubs in a growth chamber and collecting the foliar runoff in glass funnels. Then the runoff was used to water flats (5 x 25 x 60 cm) of sandhill soil sown with 300 seeds of Andropogon gyrans. Control flats were given equal amounts of water. Flats were fertilized to field capacity every 18 days with Peters 20 :20 :20 (N/P/K). Throughout the experiment the seedlings were thinned only when a plant's leaves reached beyond the stems of its neighbors, so thinning occurred when plants reached a common size, but not necessarily at the same time. After 140 days, each remaining plant was removed from the flat and oven-dried at 105 ~ for three days, and the weights of leaves and roots were recorded.

Plant material for chemical analysis was collected from Dauphin Island, Alabama, on October 8, 1983, and again from Perdido Key, Alabama, on June 15, 1984. The sites are within 50 km of each other and probably are part of the same population.


Crude extracts of air-dried stems and of air-dried leaves were obtained by sequential use of petroleum ether (PE), diethyl ether (EE), and ethyl alcohol (EtOH) as follows: plant material was soaked in the solvent for 48 hr at 8~ then filtered by suction and dried before soaking in the next solvent (de la Pefia, 1985). Thereafter, the plant material was washed with 60~ ethyl alcohol and finally EtOH-Soxhlet extracted. Solvents were rotary evaporated below 50~

Saturated aqueous solutions were prepared from each of the five residues of the crude extracts of both stems and leaves and then tested for biological activity with Petri dish bioassays on Schizachyrium scoparium, the same native grass employed in the monthly bioassays. Replication varied from two to four dishes of 30 seeds. In addition, parallel bioassays were performed with com- mercial lettuce (Lactuca sativa).

Inhibitory activity was concentrated predominantly in the EE fraction from the leaves, so further chemical analysis was concentrated there. Silica gel column chromatography of the EE fraction yielded 80 fractions, which were combined according to results of thin-layer chromoatography (TLC) into 11 fractions for subsequent bioassays. Biological activity was again concentrated in one sample, EE 6-7, which caused nearly complete inhibition of seed germination. Fractionation of EE 6-7 on a silica gel column yielded 59 fractions, which were combined according to TLC results to give 19 fractions for bioassays; from these, one sample, EE 6.16-6.24 was as inhibitory as EE 6-7. The EE 6.16- 6.24 was analyzed using gas chromotography-mass spectral analysis (GC-MS) to identify the components of the mixture. The GC-MS analyses were performed on a Hewlett-Packard model 5985 instrument. A 30-m bonded silica capillary column was used for the GC-MS analysis, under the following con- ditions: injection temperature 250~ column temperature program 60 ~ for 1 min., followed by 5~ increases to 210 ~ temperature retained at 210 ~ for 5 min. Compounds were identified by using the computerized MS spectral search system from Hewlett-Packard: Flavor-Fragrance, 59817D National Bureau of Standards Subset Library. Identities of some compounds were further corrob- orated by comparison of retention times and mass spectra to known standards.

Because many of the compounds in the EE active fraction appeared to be volatile organics, a steam distillation of Conradina foliage was undertaken. Fresh leaves in distilled water were boiled with the vapors trapped in an attached condenser. The distillate was extracted with diethyl ether, dried with anhydrous magnesium sulfate, filtered, and concentrated in a rotatory evaporator. The residue, labeled SD (steam distillation), was analyzed (GC-MS) for identification of compounds.

In order to determine compounds that might leach from foliage in precipitation, fresh leaves of Conradina were extracted with distilled water as follows: 360 g were twice soaked in 3 liters of distilled water for 3 days at 8 ~ and the aqueous extract reextracted with ethyl acetate; the organic layer was dried with


anhydrous magnesium sulfate, filtered, and the solvent rotary evaporated. The 0.6-g residue, labeled Soak, was analyzed (GC-MS) to identify components of the mixture.

Nine of the monoterpenes, identified from the active fractions of Conra- dina, were tested for biological activity on two native grasses, Leptochloa and Schizachyrium, in triplicate Petri dish bioassays, as described earlier, 30 seeds per dish. Most of these monoterpenes are low-molecular-weight, nonpolar en- tities, whose solubilities in water have never been determined, so saturated aqueous solutions were prepared for the bioassays here.

Scanning electron micrographs of the surface of the leaves were examined for possible trichomes, as sources of production of active compounds. Both fresh leaves and fresh leaves dipped in an organic solvent, dichloromethane (DCM), were examined.

RESULTS AND DISCUSSION

The monthly bioassays of Conradina leaf washes showed similar effects on the three sandhill grasses, with Leptochloa experiencing the most inhibition, Andropogon the least, and Schizachyrium intermediate (Table 1). Simple ANO- VAs of each month' s data produced significant inhibition of germination in 52 % of the monthly tests--in 88 % for Leptochloa, 44 % for Schizachyrium, and 25 % for Andropogon. Inhibition of radicle length was about half as frequent--in 28 % of the monthly tests overall, 50 % for Leptochloa, 22 % for Schizachyrium, and 12% for Andropogon. The frequency of the significant results suggested that inhibition occurred regularly, but not every month.

Species differences in the magnitude of the monthly responses followed

TABLE ][. GERMINATION AND RADICLE LENGTH RESPONSES OF THREE SANDHILL GRASSES TO

LEAF WASHES OF Conradina canescens RELATIVE TO WATER CONTROLS

Leptochloa Schizachyrium Andropogon Total

Months tested 8 9 8 25 Significant inhibition

Of germination 7 4 2 13 Of radicle length 4 2 1 7

Mean monthly response (RI) Of germination -0 .34 _+ 0.17 a -0 .17 + 0.37 -0 .09 + 0.17 -0 .22 + 0.28 a Of radicle length -0 .13 _+ 0.14 b -0 .10 _+ 0.22 +0.03 _+ 0.39 -0 .08 _+ 0.25 b

aTreatment was significantly different from the control at P -< 0.01. bTreatment was significantly different from the control at P <- 0.05.


closely their differences in the frequency of significant inhibitions. Average percent inhibition o f germination was 22 % relative to the control overall--34 % for Leptochloa, 17% for Schizachyrium, and 9% for Andropogon (Table 1). Inhibition o f radicle length was only 8% of the control overall--13% for Lep- tochloa and 10% for Schizachyrium, whereas the control was 3 % less than the treatment (stimulation) with Andropogon (Table 1). For the three species combined, monthly inhibition averaged 22 % for germination and 8 % for radicle length.

Monthly variation in bioassay effects was large but by no means random. Inhibition of germination of the three grasses rose to a sharp peak in June and was distinctly absent in the winter months (Figure 1). Inhibition of radicle length seemed to be concentrated in the spring, with less predictable seasonal variation the rest of the year. The monthly germination responses (RI) were negatively correlated with 30-year averages for precipitation (r = - 0 . 7 4 , P = 0.01) and temperature (r = - 0 . 7 2 , P = 0.01), so inhibition of germination was concentrated in the warm, wet months--exactly the growing season in central Florida (Figure 1). Radicle length responses were not correlated with precipitation (r = - 0 . 1 4 , P = 0.70) or temperature (r = - 0 . 4 9 , P = 0.15).

Application of leaf mist collected from Conradina to seedlings of Andro- pogon gyrans, growing in flats of sandhill sand, caused significant (P = 0.05)

25-

20-

T (~ 15-

lO- PCPT (c~) 5-

k /~

~,\\/ , /~176 ! o'~//'\ f I 1A I i ,, s ; \ -0-- / /'\~

\ / /

\ / \ /

+0.4

+0.2

0 RI

- 0 . 2

- - 0 4

- - 0 . 6

FIG. 1. Dashed lines show the mean monthly responses (RI) of three sandhill grasses treated with leaf washes of Conradina canescens relative to grasses given water controls. Negative RI values are inhibition and positive RI values are stimulation for germination (circles) and radicle length (squares). Solid lines show the 30-year monthly precipitation mean (dots) and the 30-year monthly temperature mean (boxes) near the site (NOAA 1983).


inhibition of growth at the end of the 140-day experiment. Seedlings watered with Conradina mist weighed only 48% of the control plants (2.4 +_ 0.2 g versus 4.0 +_ 0.6 g). Root weights from the Conradina treatment were not different from the control (1.0 + 0.3 g versus 0.9 + 0.1 g), but leaf weights in the treatment were only 43% of the control (1.4 + 0.3 g versus 3.1 + 0.5 g). Height of the grasses given the treatment was only half that of the control plants (43 +_ 4 cm versus 86 • 7 cm). Thus, inhibition of growth was quite strong in the mist test but was not apparent in the Petri dish bioassays with Andropogan.

Two possible mechanisms could account for the difference. Growth in the Petri dish tests was limited to one week, but in the mist experiment growth was continued for 20 weeks; therefore, inhibition of Andropogan may be limited to later in development. Second, in the Petri dish bioassays, the Conradina leach- ate was applied only once at the beginning of the bioassay, which requires seven days for germination followed by seven days for growth, but in the mist experiment the leaf rinse was applied every three days. If the active compounds are volatiles, as is suggested below, then they may have dissipated from the Petri dishes before radicle growth commenced. The dishes were not sealed, nor were any of the bioassays performed in limited gas volumes, as has been the case in other bioassay studies of volatiles (Muller et al., 1964; Muller, 1965; Muller and del Moral, 1966; Fischer, 1986).

Chemical analysis of Conradina foliage revealed a variety of monoterpenes, as is the case with other allelopathic mints (Muller et al., 1964; Muller, 1965; Muller and del Moral, 1966; Fischer, 1986). Many of them were known compounds found in the GC-MS reference library and then confirmed by comparison to standards (Table 2). The diethyl ether (EE) extract produced 11 known monoterpenes, many of them, such as 1,8-cineole, camphor, bomeol, and p-cymene, inhibitors of seed germination and plant growth (Fischer, 1986). The steam distillation produced 11 known monoterpenes, and the leaf soak produced four known monoterpenes (Table 2). Some (seven of 11) of the known monoterpenes from steam distillation and all of them from the leaf soak (four of four) were also present in the EE extract. The GC traces of the steam distillation and the leaf soak revealed a large number of suspected monoterpenes (lower-molecular-weight compounds) that were not known from the reference library--an indication that Conradina canescens contains a highly unique complement of volatile compounds.

Petri dish bioassays of nine of the monoterpenes, applied to Leptochloa and Schizachyrium, revealed complete inhibition of germination by five compounds and statistically significant inhibition of germination in each case with one exception--cineole did not inhibit Leptochloa (Table 2). Where germination did occur, there was subsequent evidence for inhibition of radical growth in most cases, although sample sizes were limited by the small numbers of


TABLE 2. MONOTERPENES IN ACTIVE DIETHYL ETHER FRACTION (EL), STEAM

DISTILLATION EXTRACT (SD), AND LEAF SOAK (SOAK) OF Conradina, WITH MS

CONFIDENCE LEVEL, MS CONFIRMATION TO AUTHENTIC COMPOUND AND PERCENT

INHIBITION OF GERMINATION AND RADICLE LENGTHS OF Leptochloa AND Schizachyrium AS PERCENT OF DISTILLED WATER CONTROLS

Compound

Leptochloa Schizachyrium

EE SD Soak MS Confirmed Germ. Rad. Germ. Rad.

1,8-Cineole + + + 95 yes 100 Camphor + + + 89 yes 12 ~ Pinocarveol + + - 96 no Borneol + - + 91 yes 0 a Pinocamphone + + - 97 no

Myrtenal + + - 95 yes 24 a-Terpineol + + + 98 yes 04 Myrtenol + + - 97 yes 0 a Carveol + - - 95 yes 0 a Carvone + - - 91 yes 0 ~

Citroneliol + - - 90 no 0 ~ p-Cymene - + - 98 no Sabinene - + - 94 no 1,6-Dihyrosylvestone - + - 94 no Caryophyllene - + - 97 no

97 20 a 26 ~ 34 a 16 a 61

8 ~ 47 b

5 a 8 a 13 ~ 0 a 0 a 0 n

0 ~

0 .

"Treatment was significantly different from the control at P <_ 0.01. bTreatment was significantly different from the control at P <__ 0.05.

ge rmina t ing seeds (Table 2). Thus , saturated aqueous solut ions o f mos t o f the

m o n o t e r p e n e s tes ted p rov ided s t rong phyto toxic i ty . In these tests the Petri d ishes

were not sea led , and vola t i l iza t ion o f the m o n o t e r p e n e s was quite ev iden t f rom

thei r odors in the tes t room, espec ia l ly the first f ew days o f the expe r imen t .

Exac t ly h o w such c o m p o u n d s are t r ansmi t t ed to the soil solut ion in nature

r emains an en igma , a l though in chaparra l volat i les appear to sett le and adsorb

on l ipophi l ic clay par t ic les and s eed / s eed l i ng m e m b r a n e s (Mul le r and del Mora l

1966).

F l o r i d a ' s sandy soils are cons ide rab ly less l ipophi l ic , but m o n o t e r p e n e s

may reach the soil in w a t e r runoff . One l ine o f our research sugges ts that m o n -

o te rpenes may be assoc ia ted wi th b io logica l surfactants that reduce the i r vola-

t i l i ty and inc rease the i r solubi l i ty in wa te r (F i scher , 1986; F i s ch e r et al . , 1988).

In the case o f Conradina, a k n o w n t r i te rpene , ursol ic acid , c o m p o s e d 6 % o f

the dry w e i g h t o f the p lant and 50% o f the c rude E E extract (de la Pefia, 1985).

In acr id ine dye tes ts , ursol ic acid p r o d u c e d mice l l es (de la Pefia, 1985; Wil -

l i amson et al . , 1988). The re fo re , the b ioassays wi th m o n o t e r p e n e s a lone (Table


2) may underestimate their activities in nature if they form micelles with ursolic acid or other organic compounds.

Scanning electron micrographs of the leaf surfaces of Conradina showed numerous nonglandular, filamentous trichomes as well as fewer glandular, spherical ones. The latter were surrounded and covered by the former, and cross-sections through the latter revealed secretory cells under a fold of the cuticle that harbored the secretion (Figure 2). Similar glandular and nonglandular trichomes are found in some, but not all, mint species (Metcalfe and Chalk, 1965). Dipping the leaves in a polar solvent such as diethyl ether or dichloromethane (DCM) dissolved the fold of cuticle and the underlying secre- tions, whereas the filamentous trichomes remained intact. The resulting DCM extract contained monoterpenes as well as ursolic acid.

In summary, the preliminary results presented here indicate that aqueous washes of foliage of Conradina canescens seasonally inhibit germination of

FIG. 2. At left is a cross-section through the leaf and through a glandular trichome on the leaf lower surface; trichome consists of the fold of cuticle, the pocket that contained the secretion, and the underlying secretory cells. At fight is the upper leaf surface show- ing the nonglandular trichomes covering a spherical glandular trichome. Line in the lower right of each micrograph is 50/xm.


na t ive g rasses . T h e w a s h e s c o n t a i n m o n o t e r p e n e s tha t are p r o b a b l y the source

o f the an t ib ios i s . T h e m o n o t e r p e n e s are p a c k a g e d on the l ea f surface wi th ursol ic

ac id as s ec re t ions o f t r i c h o m e s . T h e sec re t ions are u n d e r the cut ic le , bu t r i n s ing

the l eaves g ives a l e acha t e tha t con t a in s the m o n o t e r p e n e s , so the l eaves a p p e a r

to p r o v i d e a l e a c h a b l e sou rce o f p h y t o t o x i n s . T h u s , a l l e lopa thy m a y func t i on

to p r e v e n t the a c c u m u l a t i o n o f f ine fuel (grass l i t ter) a r o u n d C o n r a d i n a sh rubs ,

w h i c h o t h e r w i s e suffer mor t a l i t y in sur face fires. T h e effects o f the a l l e lopa th ic

i n h i b i t i o n re l a t ive to o t h e r k inds o f b io log i ca l i n t e rac t ions such as c o m p e t i t i o n

and h e r b i v o r y h a v e no t ye t b e e n inves t iga t ed , so its i m p o r t a n c e in na tu re is still

u n r e s o l v e d .

Acknowledgments--This material is based upon work supported by the U.S. Department of Agriculture, Competitive Research Grants Program for Forest and Rangeland Renewable Resources under agreement No. 88-33520-4077. SEM photos were taken by Sharon Mathews.

REFERENCES

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FISCHER, N.H., TANR1SEVER, N., and WILLIAMSON, G.B. 1988. Allelopathy in the Florida scrub community as a model for natural herbicide actions, pp. 232-249 in H. Cutler (ed.). Biolog- ically Active Natural Products: Potential Use in Agriculture. ACS Symposium Series No. 380. American Chemical Society, Washington, D.C.

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NOAA. 1983. Climatological Data, Annual Summary, Florida 1983. National Oceanic and Atmo- spheric Administration, National Climatic Center, Ashville, North Carolina.

RICHARDSON, D.R. 1977. Vegetation of the Atlantic Coastal Ridge of Palm Beach County, Florida. Fla. Sci. 40:281-330.

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Chemical inhibition of fire-prone grasses by fire-sensitive shrub, Conradina canescens

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