herbicidal properties of the antibiotic monensin

J Sci Food Agric 1996,70,373-379

Herbicidal Properties of the Antibiotic Monensin* Robert E Hoagland USDA, ARS, Southern Weed Science Laboratory, PO Box 350, Stoneville, MS 38776, USA (Received 1 February 1995; revised version received 20 July 1995; accepted 22 September 1995)

Abstract: The agricultural antibiotic monensin caused herbicidal injury to 1- to 2-week-old seedlings of seven weed and two crop species when applied at M as a foliar spray in the greenhouse. The non-ionic surfactant tergitol TMN (0.1 ml litre- ') aided foliar absorption of monensin and increased herbicidal injury. Fresh weight reductions ranged from 20 to 75% (compared to control seedings treated with tergitol alone) 65 h after spray application of M monensin with tergitol. When root-fed at lo-" M, monensin (without tergitol) caused death in all nine species within 24-72 h after treatment. Monensin supplied to roots at lo-' M caused fresh weight reductions in spurred anoda, velvetleaf and prickly sida of 43, 32 and 23%, respectively. Substantial growth effects also occurred in hemp sesbania and sicklepod (33 and 15% reductions in fresh weight, respectively); jimsonweed and johnsongrass had the least fresh weight reductions. Injury included chlorosis, necrosis, desiccation and leaf abscission. Cotton and okra were the most tolerant of all species tested; however, three malvaceous weeds were severely damaged by the compound. When root-fed at M, monensin caused limited injury. Monensin exhibited some degree of selectivity among these crop and weed species and caused greater injury in light-treated than dark- treated plant tissues.

Key words: antibiotic, herbicidal injury, monensin, phytotoxicity, weed control.

INTRODUCTION

Monensin is a carboxylic polyether antibiotic isolated from the mycelium of an actinomycete, Streptomyces cinnamonensis (Fig 1). The compound is an ionophore that binds potassium, sodium and hydrogen ions (Tartakoff 1983). The lipophilic character of the mol- ecule renders it soluble in lipid components of bio- logical membranes (Sutko et a1 1977; Basset et a1 1978), and it has been shown to induce changes in mycelial lipid and sterol composition (Fonvieille et al 1991).

Fig 1. Structure of the polyether antibiotic, monensin sodium.

* This article is a US Government work and, as such, is in the public domain in the USA.

Various studies have shown that monensin causes swelling, disruption and cytological changes in dictyosomes (Golgi apparatus) of animals (Tartakoff and Vassali 1978) and plants (Robinson 1981; Mollenhauer et a1 1982; Morre et a1 1983; Schnepf 1983). Due to the effects of monensin of Golgi apparatus function, this compound is a potent inhibitor of protein secretion in animals (Tartakoff 1983) and plants (Heupke and Robinson 1985; Melroy and Jones 1986; Sticker and Jones 1988).

Generally, there has been little work on the effects of monensin on plant growth. This fact, and the know- ledge that monensin is introduced into the environment via animal excreta from extensive use as an antibiotic in chickens (Ruff et al 1976) and a feed additive in cattle (Goodrich et al 1984), prompted a study of its effect on plant growth (Mollenhauer et al 1986). In that study, monensin was tested on only one plant species, ryegrass (Loliurn multijorum Lam), and found to inhibit germi- nation and growth when seeds were allowed to imbibe aqueous solutions containing M monensin. Mon- ensin has also been shown to inhibit synthesis of

373 J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

374 R E Hoagland

cellulose or matrix polysaccharides and to suppress auxin-induced elongation of pea (Pisium satiuum L) stem segments Brummel and Hull 1985; Kutachera and Briggs 1987). Monensin inhibited the incorporation of B-glucans into cellulose and callose of fibers of cotton ovules in uitro (Francey et a1 1989). Recently, elongation of rice coleoptiles was found to be supressed by monensin at very low concentrations M) (Hoson and Masuda 1991). This compound can also ameliorate galactose suppression of auxin-induced cell elongation in oats (Auena satiua L) (Yamamato et a1 1981; Yama- mato and Masuda 1984).

Comprehensive studies on the effects of monensin on plant growth are lacking, while at the same time various microbial products are continually being tested for potential use as bioherbicides or to provide new chem- istries for the development of synthetic herbicides (Hoagland 1990). Therefore, the objective of this research was to examine the effects of various concentrations of monensin on the growth of a variety of plant species, including crops and major agricultural weeds.

MATERIALS AND METHODS

Chemicals

Monensin sodium salt, 90-95% pure, and tergitol TMN-10 polyglycol ether (non-ionic surfactant) were purchased from Sigma Chemical Company (St Louis, MO, USA).

Seed sources and plant growth

Seeds of seven weed and two crop species were used. Seeds of cotton (Gossypium hirsutum L) and okra (Hibiscus esculentus L) were obtained from commercial sources. The weed seeds hemp sesbania (Sesbania exaltata (Raf) Rydb ex A W Hill, sicklepod (Cassia obtusvolia L), jimsonweed (Datura stramonium L), johnsongrass (Sorghum halapense (L) Pers), velvetleaf (Abutilon theophrasti Medicus), spurred anoda (Anoda cristata (L) Schlecht, and prickly sida (Sida spinosa L) were obtained from weed nursery plots at the Southern Weed Science Laboratory, Stoneville, MS. All seeds were planted in vermiculite/jiffy mix/peat (1 : 1 : 1, w/w/w) in 80 x 60 x 60 mm plastic trays and supplied with dilute NPK (10 : 10 : 10, w/w/w), micronutrients and sequestered iron. Plants were grown in a growth chamber for 10-14 days (14 : 10 h, 30 : 26°C light : dark; 300 pE m- ’ s- ’ photosynthetically active radiation; 80% RH) before treatment.

Plant treatment

Plants (10-14 days old) were root-fed or treated foliarly with monensin. Foliar applications were made using a

hand-held compressed air atomiser, and foliage was sprayed to the point of runoff. Preliminary tests showed that low concentrations (0.1 ml litre-’) of the non-ionic detergent Tergitol TMN- 10 enhanced entry of monensin into plant tissues, thereby increasing injury (data not presented). This low Tergitol concentration caused no effects on plant growth. Therefore, monensin spray solutions for all foliar-applied treatments contained Tergitol (0.1 ml litre- ’), and Tergitol was used as the control for these treatments. In the root-feeding experiments, water was used as the control treatment. Monensin concentrations of and were used for foliar spray applications and concentrations ranging from lop6 to

M were used for root-feeding experiments. Three trays of each species (consisting of 6-12 seedlings each, 10-14 days old) were used in the foliar treatments. Roots of 4-6 intact seedlings (10-14 days old) from each of three additional trays were washed free of soil and placed in tubes containing monensin solutions or water controls, and placed in a growth chamber under the conditions stated above. Herbicidal injury and growth effects were monitored over about three days following treatment. Studies of photodynamic effects of monensin were conducted using 20-day-old greenhouse- grown hemp sesbania (cotyledons and leaflets) and sicklepod (leaves). Excised tissues were placed on blotter paper moistened with water in Petri dishes, and 5 pl droplets of monensin (lop4 M) were pipetted onto the tissue surfaces. One set was placed in total darkness and the other set exposed to light (300 pE m-’ s-l). Visual damage in the two treatments was compared after 24 h. All experiments were repeated in time.

Statistical analysis

All experiments were based on a complete random design with a factorial structure. Data on plant elongation and fresh weight were subjected to ANOVA and mean comparisons performed using Fisher’s protected LSD.

RESULTS

Foliar spray applications of monensin

Visual symptomology of injury, treatment Because monensin at lo-’ M did not cause substantive damage of seedlings (data not shown), higher concentrations were used. When solutions of monensin M) containing Tergitol were applied to seedling foliage, cotton and okra exhibited little or no visible effects 65 h after treatment (data not shown). Jimsonweed and johnsongrass seedlings were slightly affected and had light- brown necrotic spots and red-brown necrotic spots with some chlorosis, respectively (data not shown). The

M , 65 h after

Monensin: herbicidal properties 375

malvaceous weeds, prickly sida, spurred anoda and velvetleaf were severely damaged (extensive light-brown necrosis, chlorosis and some desiccated areas), and prickly sida was the most affected of these three species (Fig 2A). The most severely damaged of the nine species tested were the leguminous weeds, hemp sesbania and sicklepod; ie, total collapse with desiccation, and necrosis and chlorosis with desiccation, respectively (Fig. 2B)

Eflects on plant growth Monensin applied at M in 0.1 ml litre-' Tergitol as a foliar spray produced a wide range of injury on shoot elongation among the species tested (Fig 3). Cotton was the least affected species tested, and there was no significant difference between control and treated plants. Okra was affected, but elongation of treated seedlings compared to control was less than 12%. Shoot elongation of the treated malvaceous weeds, spurred anoda, prickly sida and velvetleaf was reduced by 15, 36 and 39%, respectively. Elongation of johnsongrass, the only grass species tested, was also only slightly (- 7%) affected by this treatment. Hemp sesbania exhibited the highest degree of inhibition (49%) of shoot elongation.

M also caused a wide range of effects on fresh weight accumulation (Fig 4). As with shoot elongation, cotton was not signifi- cantly affected. Okra was the species least affected, with an 18% reduction in shoot fresh weight, while hemp sesbania was the most sensitive with a 75% reduction. Sicklepod, jimsonweed, velvetleaf, prickly sida and

Foliar application of monensin at SHOOT LENGTH, mm

Fig 3. Effects of foliar application of monensin M in 0.1 ml litre-' Tergitol) on shoot length of various weed and crop plant species 65 h after treatment. Solid bars: control seedlings (0.1 ml litre-' Tergitol in H,O); hatched bars: monensin-treated seedlings. 'Percent reduction compared to control seedling length caused by treatment within each species. Asterisks signify differences at the 95% confidence

level.

spurred anoda were affected dramatically, with reductions of 69, 55, 51, 46 and 34%, respectively, compared to control values.

Tests for possible photodynamic effects of monensin (applied in 5 p1 droplets) on excised leaflets and cotyledons of hemp sesbania and leaves of sicklepod indicated that more plant injury occurred in the light than in the dark (Fig 5). No visual differences were observed in leaflets and cotyledons exposed to the light or dark in the absence of monensin. However, more chlorosis was noted in the light-treated tissues of both species after 24 h.

Monensin applications via root feeding

Visual symptornology, M to M, 48 h after treatment

M) to Monensin at 10-6 caused very slight chlorosis in several species : johnsongrass and jimsonwood d sickle- Pod < hemp sesbania (Fig 6A). There was a somewhat greater effect at this concentration on prickly sida which

Fig 2. Effects of foliar application of monensin intact 14-day-old seedlings. (A) Excised leaves of the most severely affected malvaceous plants, 65 h after treatment. (B) Excised leaves of the most severely affected leguminous plants,

65 h after treatment.

376 R E Hoagland

I

% Reduction I SPURRED

ANODA

COTTON

OKRA

PRICKLY SlDA

VELVETLEAF

JOHNSON GRASS

JIMSONWEED

HEMP SESBANIA

SICKLEPOD

34 *

- 5

18 *

46 *

51 *

20 *

55 *

75 *

69 * I I I

0 1 2 3

FRESH WEIGHT, g/shoot Fig 4. Effects of foliar application of monensin M in 0.1 ml litre-' Tergitol) on the fresh weight of various weed and crop plant species 65 h after treatment. Solid bars: control seedlings (0.1 mi litre-' Tergitol in H,O); hatched bars: monensin-treated seedlings. 'Percent reduction compared to control seedling weight caused by treatment within each species. Asterisks signify statistical differences at the 95%

confidence level.

was greater than or equal to the effect on spurred anoda, and no effect was noted on cotton, okra or velvetleaf (Fig. 6B). Higher concentration of monensin (lo-' M) generally caused more intense symptoms such as chlorosis and some necrosis. The malvaceous species (Fig 6B) generally exhibited less visual symptomology

Fig 5. Photodynamic effects of monensin (loe4 M) 24 h after application to hemp sesbania leaflets (left) and cotyledons (right) and sicklepod leaf pairs. Five microlitre droplets were applied to the tissues followed by exposure to continuous light

(300 pE m-* s-l) or darkness.

than the other species (Fig 6A). The order of visual injury was: cotton and okra < prickly sida and velvetleaf < spurred anoda < johnsongrass < jimsonweed < sicklepod < hemp sesbania. When root-fed at

M, monensin (without Tergitol) caused death in all nine species within 24-72 h of treatment.

Effects on plant growth Root-feeding monensin at M caused death of all species 24-65 h after treatment (data not shown). Therefore, root-feeding experiments were done using lower monensin concentrations to assess the sensitivity of these species to the compound (Fig 7). Except for okra and johnsongrass, shoot fresh weight was signifi- cantly reduced by lo-' M monensin (without Tergitol). The malvaceous weeds, prickly sida, velvetleaf and spurred anoda were reduced by 29, 34 and 37%, respectively. The legumes, hemp sesbania and sicklepod were reduced by 23 and 25%, respectively, while johnsongrass was reduced by 33%. Monensin, root-fed at a lower concentration M), inhibited shoot fresh weight accumulation in about half of the species, but had no effect on the others compared to the control.

DISCUSSION

The use of microbes and microbial products as bioherbicides has achieved recent attention, and this search has produced several naturally-occurring phytotoxins with potential as new herbicides (Hoagland 1990). Nigericin is an antibiotic produced by Streptomyces hygroscopicus that has also been discovered to possess herbicidal activity (Heisey and Putnam 1986). This polyether compound is closely related to monensin and has been shown to be phytotoxic (pre- and post- emergence) to several weed and crop species (Heisey and Putnam 1990). It also inhibits photophosphorylation of isolated chloroplasts (Shavit and San Pietro 1967). Another antibiotic, laidlomycin, is also structurally related to monensin and nigericin and is produced by a Streptomyces spp (Kida and Shibai 1986). Laidlomycin and deoxy-laidlomycin have also been isolated from Streptoverticillium olivoreticuli (Grafe et al 1989), and laidlomycin has been shown to inhibit de novo starch synthesis and photosynthesis (Kida and Shibai 1986).

Velvetleaf was found to be very sensitive to foliarly- applied nigericin (Heisey and Putnam 1990). In the present study, the three malvaceous weeds, velvetleaf, spurred anoda and prickly sida were also found to be very sensitive to foliar application and root-feeding of monensin, but two malvaceous crops, cotton and okra, were monensin-tolerant.

M to seeds in soil caused over a 90% inhibition of emergence (Mollenhauer et al 1986). Ryegrass growth was also

In ryegrass, monensin applied at


Fig 6. Effects of root-feeding of monensin or M) to intact 14-day-old seedlings. (A) Excised leaves of grass and legume species 48 h after treatment. (B) Excised leaves of malvaceous plants 48 h after treatment.

severely inhibited by M monensin and only par- tially by a lo-, M concentration. Monensin had some, but not strong, phytotoxic effects on johnsongrass (the only monocot tested here). Nigericin, applied to foliage, damaged several monocots in the following order: barn- yardgrass (Echinochloa crus-galli L Beauv) < proso millet (Panicum mileacum L) c giant foxtail (Setaria faberi Herrm) c large crabgrass (Digitaria sanguinalis L, Scop). Laidlomycin was more phytotoxic to barnyard millet (Panicurn crus-galli) than to rice (Oryza sativa L); ie, inhibition of shoot elongation of I , , = 90 pm ml-' and I , , > p250 g ml-l, respectively, when applied to soil. However, this compound was not highly herbicidal to either rice or barnyard millet when applied to foliage even at a high concentrations (- 2000 ppm) (Kida and Shibai 1986). In Petri dish bioassays with germinating seeds treated with nigericin, garden cress (Lepidium sativum L), large crabgrass, pigweed (Amaranthus retrojlexus L), and tomato (Lycopersicon esculentum Mill) were the most sensitive of 10 species, and corn (Zea mays L) was the most tolerant species (Heisey and Putnam 1990). Few legumes have been tested for

growth inhibition by, or sensitivity to, any of these polyether antibiotics. In tests with nigericin, soya bean (a legume) was one of the most resistant of several monocot and non-leguminous dicot plants (Heisey and Putnam 1990). In contrast, the present studies showed that the two leguminous weeds, sicklepod (non-nodulating legume) and hemp sesbania, were very sensitive to root exposure and foliar application of monensin. Since these polyether compounds are antibiotics, one can spe- culate about their effects on rhizosphere organism inter- actions with roots of legumes (nodulating and non-nodulating) and non-leguminous plants when these compounds are excreted into or applied to rhizosphere soils in herbicidal tests. Thus far, no one has reported on this subject.

The above studies, including those reported here, generally suggest that absorption of monensin through foliage is more limited than root or seed absorption. The fact that addition of a surfactant (Tergitol) caused an increase of phytotoxic symptoms in some cases (data not shown) also adds support. Root absorption appears to be more rapid, with a concomitant expression of

378 R E Hoagland

% Reduction

O A 5.9 A

36.7 B

SPURRED ANODA

O A COTTON 0.7 A

5.0 B

OA OKRA 2.3 AB

5.0 B

O A PRICKLY SlDA 23.8 B

28.6 C

VELVETLEAF

JOHNSON GRASS

JIMSONWEED B F HEMP

SESBANIA

O A 11.4 B 34.1 C

O A 4.0 AB

10.0 B

O A 28.6 B 33.3 c

O A 16.4 B 23.0 C

O A 10.0 B 25.0 C

0 0.5 1 1.5 2

FRESH WEIGHT, g/shoot Fig 7. Effects of root-fed monensin (lo-’ and M without Tergitol) on fresh weight accumulation of various seedlings 72 h after treatment. Solid bars: control seedlings; hatched bars : M monensin-treated seedlings; open bars: lo-’ M monensin-treated seedlings. ‘Percent reduction compared to control seedling weight caused by treatment within each species. Values for each species followed by the same letter are

not statistically significant at the 95% confidence level.

phytotoxicity. Similar observations (ie, lack of foliar uptake; more rapid uptake in germinating seeds and whole seedling exposures) were noted in herbicidal tests with nigericin (Heisey and Putnam 1990). Thus, it is evident that either differential absorption and/or metabolism of monensin (and nigericin or other analogues) in various plant species is operative and plays a major role in the herbicidal selectivity of these compounds. Since the plants tested here have cuticles composed of varying types and amounts of lipid materials, the interaction of these compounds with plant cuticle constituents is of primary concern in their absorption. Another inter- esting point with regard to this polyether/cuticle interaction is that some of these compounds can inhibit lipid biosynthesis in fungi (Weete et a2 1989). Further in uitro and in uiuo comparisons of all of these polyether compounds and the effects of various surfactants will aid in the understanding of the herbicidal properties, including absorption and translocation, selectivity, metabolism, and the molecular mode of action of this relatively new family of natural products with novel chemistry from microbes.

ACKNOWLEDGEMENT

The author thanks Ms Velma Robertshaw for her excel- lent technical support during this project.

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herbicidal properties of the antibiotic monensin

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