Abstract: The allelopathic effects of three plant tissues of Conyza albida (stems, leaves and inflorescences) on oat growth were further investigated using in vivo tests. Oat growth (fresh and dry weights of above and underground parts) was significantly inhibited from phytotoxic activity of upper leaves and inflorescence tissues of C. albida in pot experiments. The inhibition was significantly higher than in the case of stems. The inhibiting action of crude extracts and volatile compounds from young plants (rosette) were examined using two bioassay methods: (a) seed germination and radicle growth of oat and (b) fresh weight of duckweed plants. Both bioassayed species exhibited great phytotoxic response from the young plants, collected in winter, confirming the results of previous studies.
INTRODUCTION
Horseweed (Conyza albida Willd. ex Sprengel) is an annual-biennial herb originating from South America. It is a widespread species found in many parts of the world. In Greece it is an well known species growing mainly in urban habitats and known since 1976. According to our observations it also presents a vigorous growth causing it to become a persistent weed problem in vineyards and orchards and recently vegetable gardens in many parts of Greece. It is a difficult noxious weed to control because it produces dense stands and can tolerate a variety of habitats and environmental conditions (Economou et al., 2002). Despite its invasiveness, the biology and ecology of this species is poorly documented (Thebaud and Abbott, 1995).
Allelopathy is one of the predominant forces in the development of plant communities and spatial patterns therein (Rice, 1984). Very few studies reported to date have assessed the allelopathic potential of C. albida (Economou et al., 2002). However, studies on species of the related genus Erigeron showed that cisdehydromatricaria ester and cis- and trans-matricaria esters were released (Putnam, 1988). These C10 polyacetylenes were discovered in soils in concentrations inhibitory to test plants and are probably allelopathic substances of ecological importance. Indeed, the potential for undesirable environmental contamination from herbicides is relatively high and these create a need for environmentally safe herbicides that are equally or more effective and selective than currently available synthetic herbicides (Putnam et al., 1983).
This study reports a preliminary investigation into the allelochemical characteristics of C. albida, supplementary of the study of Economou et al. (2002). The objective of this study was to evaluate the allelopathic activity: (a) of different horseweed tissues on oat seedling growth (b) of plant extracts and estimate the dose-response of oat radicle growth to varying concentrations and (c) to access the inhibitory action of volatile compounds on growth of oat radicle and duckweed fresh weight.
MATERIALS AND METHODS
Pot Experiment
Plant material was collected from a natural population established in the
Benaki Phytopathological Institute field in Kifisia, a suburb of Athens, at
the reproductive stage during (1998, 1999). For the purposes of allelopathic
experiments three tissue types were used: (i) inflorescences (ii) leaves and
(iii) stems. Bioassay experiments were used to determine the inhibitory potential
of each of these tissues on oat above- and underground growth in glasshouse
pot studies. Oat (Avena sativa) was included in this study since it has
been used extensively in allelopathy research as the receiver plant to test
compounds released by a donor plant. Oat seeds germinate evenly, resulting in
a uniform and rapid plant growth that enables qualification of biological response
in plants. In addition, oat biotest is considered as a sensitive and easily
facilitated method (Rice, 1984). Six seeds of oat were placed and grown in 10
cm diametric plastic pots containing 20 g freeze tissues per 200 g of vermiculite.
The seeds were placed 2 cm deep in 20 test pots. The pots were watered daily
with equal volumes of deionised water. All pots were watered to maintain adequate
moisture and artificial light was supplied. Temperatures ranged from 18 to 25°C
and daylength averaged 15 h. The upper and underground growth of oat were measured
after two weeks and used as an index of allelochemical activity. Seedlings were
collected two weeks after planting and the average shoot fresh and dry weight
per pot was determined. The experimental design was a randomized block with
four replicates for each treatment and control.
Plant Extracts Bioassay
The phytotoxicity of plant extracts was quantified with an Avena sativa
seed bioassay. The plant material was collected from the University of Athens
Campus at the rosette (February) stage during 1998. The air-dried aerial parts
were cut into small pieces and extracted successively with methanol. Aqueous
dilutions of the dried residues of crude extracts were bioassayed on filter
paper in plastic Petri plates and the two most effective dilutions were identified
and further investigated. Six oat seeds were placed onto two layers of 9 cm
filter paper in Petri plates treated with 3 mL of test solution, exposed to
vapour the methanol, wetted with 6 mL of distilled water, covered and incubated
at 25°C in the dark.
The experimental design was a randomized block with four replicate plates for each treatment. Control solutions were prepared using mannitol-water adjusted to correspond to the osmotic potentials of the different extracts (Bell, 1974). Inhibitory concentrations were calculated after 7 days and used as an index of allelochemical activity. An analysis was conducted according to Finney (1962). A 5 mm radicle length was considered germinated. Growth was quantified by measuring the radicle length of germinated oat.
Allelopathic Characteristics of Volatile Compounds
The volatile compounds from the two most effective oil fractions of the
fresh aerial parts of the above sample (rosette) were obtained by steam distillation
for 3 h using a modified Clevenger apparatus (Hellenic Pharmacopoeia, 1989).
The oils were dispersed as an emulsion in water using Tween 20. Seven concentrations
were used. Dilutions were made with distilled water. Oat seeds were placed on
filter paper in conical tapped vials (six seeds per vial) and were soaked with
3 mL of the tested water dilutions. The vials were wetted with distilled water
and incubated in darkness at 25°C. Radicle elongation of oat was measured
at 7 days, using the same approach and that outlined above for the germination
bioassay with corresponding controls. Seeds that did not germinate were considered
to have a radicle length at 0 mm. The experiment was repeated four times as
described previously. Data were expressed as a percentage of radicle elongation
in control vials. The dose needed to inhibit oat radicle growth to 50% of control
radicle growth (hereafter called the I50 value) was determined from
dose-response bioassays (Finney, 1962; 1978). For all the measurements SPSS
software was used (SPSS, 1997).
The same dilutions from the two oils were tested via another bioassay using as test plant a species of duckweed (Spirodella polyrhiza L.) and measuring the decrease of its fresh weight. This plant indicator has been used in several allelopathic studies, since the bioassay is sensitive and reliable especially at the first steps of a screening procedure. Moreover, duckweed species are highly sensitive to chemicals that inhibit the function of Photosystem II and their response by chlorosis is readily measurable through the drastic decrease in their fresh weight (Leather and Einhellig, 1985; Olofsdotter et al., 1995).
RESULTS AND DISCUSSION
Pot Experiment
The response of oat bioassay to the horseweed (Conyza albida) debris
varied among the three types. The leaves and inflorescences showed a higher
phytotoxic effect on the oat growth. The fresh and dry weight accumulation was
significantly inhibited, more than the stems, when they were incorporated on
the vermiculite surface (Table 1). Fresh and dry weights of
above and underground oat parts were significantly inhibited from phytotoxins
derived from horseweed leaves, such as in the measurements of Economou et
al. (2002). Strongly inhibitory substances were released from horseweed
debris derived from inflorescence tissues, too. The biological activity of compounds
derived of Conyza species flowers has been also reported elsewhere (Peterson
et al., 1989). The same plant tissues caused a greater inhibitory effect
on underground oat growth reducing its biomass to a significant degree. These
findings are in accordance with those of Putnam and Duke (1978), Rice (1984)
and Economou et al. (2002) whose studies showed that quantities of allelochemicals
within plants vary with plant tissue. Allelochemicals may be synthesized and
stored in other tissues and then transported into new leaves and inflorescence.
Alternatively, it may indicate transport of allelochemicals from root via phloem
of the inner bark to the developing leaves (Heisey, 1990). Furthermore, as the
radicles of newly germinated seeds are very susceptible to phytotoxins, it is
possible that the wide distribution of C. albida is due to allelopathic
potential of the species.
Plants Extract Bioassays
The tissue debris of horseweed was found to have an inhibitory effect on
the oat growth bioassay and the crude plant extracts also demonstrated inhibition
in oat seedling growth. In Table 2, it is shown the inhibitory
effects of the two most effective samples (Ca2 and Ca7) on oat growth. There
is a strong inhibition response of the two samples, as the I50 estimates
(189 and 96 μg mL-1) were about two to three times lower than
the average I50 estimate of the rosette stage (324 μg mL-1)
and three to five times lower than the average I50 estimate (524
μg mL-1) derived from the samples at the mature stage (Economou
et al., 2002). The strong inhibitory action of Sample Ca7 was true even
from the concentration of 153 μg mL-1, as long as the oat radicle
length was 73 % lower than the control.
| Table 1: | Response of oat growth to vermiculite-incorporated plant tissues of Conyza albida debris |
| Means followed by the same letter(s) within a row are not significantly different at p = 0.05 Fisher’s least significant difference test | |
| Table 2: | Response of oat radicle elongation to the allelopathic component from Conyza albida most effective crude extracts |
| Numbers in parentheses indicate percent inhibition. | |
| Table 3: | Response of oat radicle elongation and duckweed fresh weight to the most effective volatile compounds obtained from Conyza albida tissues in vegetative stage (rosette) |
| Numbers in parentheses indicate percent inhibition | |
Even if Sample Ca7 was significantly more effective than Ca2, for both samples, radicle elongation was reduced with increasing extract concentrations (Table 2). Indeed, it is well known that the magnitude of phytotoxic activity is dependent upon the concentration and chemical stability of the active compounds (Einhellig, 1986).
Allelopathic Characteristics Of Volatile Compounds
Placing emulsions of the oils obtained from the potted vial with germinating
oat seeds resulted in a considerable decrease in seedling growth compared to
the control seedling. The most effective samples (Cawoil8 and Cawoil10) were
identified and isolated (Table 3). The same results are in
accordance with the duckweed bioassay, as long as the emulsions of the oils
obtained were tested for its influences on the duckweed fresh weight as well.
The oil obtained from the most effective winter samples of our study caused
a strong inhibition response, as the I50 estimates (47 and 26 μg
mL-1) were more than five times lower than the I50 estimate
(316 μg mL-1) derived from the samples at the mature stage (Economou
et al., 2002). It is also noticeable that only 69 μg mL-1 of
the most effective oil sample of the rosette stage (Cawoil10) could totally
inhibit oat growth (100 %), while the corresponding value reported by Economou
et al. (2002) for the mature stage of C. albida was 3166 μg
mL-1 (Table 3).
CONCLUSIONS
Present results support the assumption of Economou et al. (2002) that phytotoxicity from horseweed in the natural ecosystems and agroecosystems could occur from compounds that escape from the plant by volatility. Nowadays, allelopathy is important in research involving sustainable agriculture, also referred to as organic, low input, biodynamic or resource concerning. The allelopathic plant products are known to offer a vast array of secondary compounds which have the potential role of use directly as herbicide substitute or as structural leads for new synthetic herbicides. In order to try for a sustainable agriculture, the need for new herbicides is inevitable and the contribution of allelopathic plants on that could be very important.