Maataloustieteellinen Aikakauskirja Vol. 56: I—7, 1984 A modified stairstep apparatus for studies of allelopathy and other phytotoxic effects J. V. LOVETT* and KARI JOKINEN Department of Plant Husbandry, University of Helsinki, SF-00710 HELSINKI 71, Finland Abstract. The characteristics and performance of a modified stairstep, nutrient solution recycling, apparatus are described. An experiment in which the allelopathic potential of Agropyron repens was examined showed the apparatus to be reliable and accurate in operation. Allelopathic activity by A. repens was confirmed. The apparatus is also suited to studies of phytochemicals produced in stubble retention reduced tillage systems, in green manuring and in cognate areas. 1. Introduction The presence of phytochemicals and the roles which they play in agricultural systems are receiving increasing attention in the lite- rature. Allelopathic phenomena (Rice 1974, 1979), whilst often difficult to distinguish among the interactions which take place between climate, soil, plants and other orga- nisms (Lovett 1982), occur commonly and demand better understanding than is present- ly the case. Phytotoxins are also of interest in reduced cultivation systems, especially where residues of crop and weed plants are retained • Professor of Agricultural Science, University of Tas- mania, G.P.O. Box 252C, Hobart, Tas. 7001, Austra- lia. and decompose at rates determined by am- bient climatic and edaphic conditions, by the activity of micro-organisms and by the method of cultivation (Lovett et al. 1982). In the agricultural systems ofFinland both allelochemicals and phytotoxins produced during the decomposition of plant residues in cropping systems seem likely to be of im- portance. Allelopathy has, for example, long been associated with Agropyron repens (L.) Beauv. (Aamisepp 1970) one of Finland’s most important weed species. Living Agro- pyron may produce allelochemicals but greater phytotoxicity is usually associated with decomposition of the rhizomes (Wel- bank 1963). Lynch et al. (1980) suggest that short chain aliphatic acids are produced Index words: Allelopathy, A. repens, method 1 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=COWBxxJaR1084hl5.qQ43dpZEYwGjG-TgCWjJzQ.8AE7PrgAPB7fBLwWt85nHtE1QJdDCxBOrvkaedUwenB4KYsyzIK9pj3IZ3oJjbR9E7w_YInx3-7rlEpiGEW6HXRsQ7y7YQwRIR2ouk1waFUDR4ClROYp522kqaRBA3t8HYCw9q57jo3YGwiXI4SDqmpTg3mabYcBId8A79eIy8qrJEiksyXPhseKnQ where decomposition is under anaerobic con- have all applied the stairstep apparatus to ditions. Such conditions also favour the pro- duction of similar substances, of proven phy- totoxicity, from crop residues (Lynch 1977). Edaphic conditions favourable to the activ- ity of facultatively anaerobic micro-orga- nisms frequently occur in the heavy soils of Finland, particularly in autumn and early in spring. The possibility of phytotoxin produc- tion from A. repens, crop and weed residues is high at that time of year. Wilson and Rice (1968), Bell and Koeppe (1972) and Walters and Gilmore (1976) studies of allelopathy. The technique seeks to distinguish between competition and allelo- pathy as components of interference (sensu Harper 1977) by providing a nutrient flow through a closed system in which the inter- fering plants are physically separate but sub- ject to the effects of phytochemicals released into the nutrient flow. In this paper we describe a modified stairstep apparatus which had wideapplication in studies of phy- tochemicals in agricultural systems. Table 1. Comparison of allelopathy experiments using the stairstep apparatus. Reference Wilson and Rice (1968) Bell and Koeppe (1972) Walters and Gilmore (1976) Allelopathy of; Helianlhus annuus L. with Setaria faberii Herrm. with Festuca arundinacea Screb. nine test species. Zea mays L. with Liquidamber styraciflua L. APPARATUS No. of lines No. of steps No. of replicates 5 4 4not specified 4 4 3 Gravity flow and recycle Gravity flow and recycle 4h/ Gravity flow and recycle 4h/ 4 h/day. Nutrient solu- day. Full strength Hoagland’s day. Half strength Hoagland’s tion replenished. Solution replenished. Solution replenished. Method pH and conductivity of solu- pH of solution monitored tion monitored. Environment Greenhouse. Greenhouse. 16 h photoperiod Greenhouse. 16 h photoperiod with supplementary light. with supplementary light. Temperature: 21 °C to 30°C. Quartz sand.Medium Quartz sand. Quartz sand. Test substances Live H. annuus plants. Live and dead Setaria material. Live and dead Festuca mate- rial. Quantity of tissue related to field conditions. Parameters monitored Dry weight of test species. Fresh weight and dry weight Height, dry weight and con- of test species. tent of N, P, K, Ca and Mg of test species. Duration Four weeks. Four weeks. Sixteen weeks. Observations Allelopathic effects on Allelopathy by substances from Allelopathy by substances pri- five species, including dead Setaria material. marily, from dead Festucamarily, from dead Festuca autotoxicity. tissue. Allelopathic Dry weight reduced. Reductions of height, fresh Reductions in dry weight, weight and dry weight. Possible effects on chloro-Possible effects on chloro- phyll, N and P content. effect Primary No information. No information. Possible effect on mineral uptake.mechanism Allelochemicals Chlorogenic and isochlo- No information, rogenic acids suspected. No information. 2 2. Material and Methods Development of the stairstep technique Martin and Rademacher (1960) reported an improvement to a system devised by Börner (1958) where in nutrient solution circulated through a series of pots in which plants of two species were planted, alternately. Vari- ous combinations of plants were tested by Martin and Rademacher (1960) who met »the most important technical requirement of the experiment», the prevention of deple- tion of nutrients in the system, by regular supplementation. There were some, appar- ently, allelopathic effects but no allelochem- icals were identified. The Martin and Rademacher apparatus does not appear to have followed the stair- step principle and we assume that this is the modification of the nutrient flow system to which Wilson and Rice (1968) refer. The salient features of their technique and those of Bell and Koeppe(l972) and Walters and Gilmore (1976) are summarised in Table 1. The Viikki apparatus A modified stairstep apparatus was design- ed and built at the Department of Plant Husbandry, University of Helsinki. Each nutrient solution recycling unit within the system (Figure 1) comprised five steps. From a storage tank at the top of the system (step one) solution flowed, by gravity, through a sequence of pots to a collector tank (step five) from which it was automatically recy- cled by an electric pump (Johnson L 100 Bilge Pump.). On step two of the apparatus was an incu- bation tray (55 x 37 x 18 cm) from which the circulating solution was channelled to test pots on each of the third and fourth steps. The test pots (15 cm diameter, 14 cm height) were replicated four times and were individually supplied with solution from 3 mm internal diameter tubing inserted into collecting pipes below the second and third steps. The incubation trays were lined with nylon mesh (0.6 mm aperture) on which was placed a layer of gravel (4.60—5.70 mm particle size, 15.4 kg dry weight). A second mesh, above the gravel base, was covered with sand (0.50—1.20 mm particle size, 18.0 kg dry weight) and the entire system thoroughly flushed with tap water. Individual test pots were filled in similar fashion and contained 770 g gravel and 2.3 kg sand. Three recycling units were set up in a greenhouse (mean temperature 21 °C ± 2°C). A 16 h photoperiod was used, supple- mentary illumination being provided through- out by A/S Bergen RS 400, Lucalox U 400/ 40 lamps. The lamps did not affect tempera- ture at the different step levels but there was a drop in light intensity from 370 /*Em~2 s~' to 200 /tEm~ 2 s ' between the third and fourth steps ('Top' and 'Base' sets of test pots). Fig. 1. The Viikki apparatus 3 The volume of 30 1 0.2 % ’Kemira Neste- mäinen Y-lannos’ complete nutrient solution (pH 7.2, N 140mg/1, P 20 mg/1, K 120 mg/1, Na 16 mg/1, S 14 mg/1, B 54 g.g/1, Mo 12 gg/ 1, Fe 18 gg/\, Mn 6 gg/\, Ca 4 g.g/1, Mg 1.2 gg/\, Cu 0.6 /tg/1, Zn 0.6 gg/\, Co 0.6 /ig/1), made up with tap water, in the storage tank was replenished on alternate days. Flow rate of solution through the sys- tem was adjusted to 23 1/h and the solution was cycled for 6 h/day. Application of the apparatus The modified stairstep apparatus was used in an assessment of the allelopathic potential of Agropyron repens (L.) Beauv. (couch- grass). Couchgrass was freshly harvested from the field at Viikki. Top growth was cut back to 3 cm height and the rhizomes thoroughly washed in tap water. Into the incubation tray of one unit was placed 1.2 kg (fresh weight) rhizomes with top growth, an amount which corresponded to that recovered from an equivalent volume of soil in a badly contami- nated field. A similar weight of material was finely chopped and incorporated into the sand at 10 cm depth in a second incubation tray, where it decayed. The third, control, tray received only nutrient solution. Each experiment in a series ran for one week. The height couchgrass foliage in the ’Live’ treatment was maintained at, or near, 10 cm height. Any shoots appearing from the ’Decay’ treatment were removed. The test species was Hordeum vulgare L. (barley) cv. Kustaa. Into each test pot were sown 14 graded seeds (2.5 to 2.8 mm diame- ter). Emergence was monitored from its commencement on Day 3 of each experi- mental run. On day 7 the plants were careful- ly washed from the test pots. Root loss was minimal. The height of the first leaf and length of the longest seminal root were deter- mined. The plants were divided into leaf, seed and root portions which were dried at 100°C for 48 h and weighed. It was possible to thoroughly clean and refill the test pots with fresh gravel and sand; to clean the tubes in the recycling system, and to commence the next run on the day of harvest. Data were analysed using a statistical pack- age available through the Helsinki School of Economics Computer Centre. Analysis of Variance was applied to untransformed data. Variability was small and there was, for example, no necessity to re-randomise pots within steps as the flow rate of nutrient solu- tion and the ambient conditions were uni- form. 3. Results and Discussion Previous examples of the use of the stair- step apparatus (Table 1) provide little infor- mation on performance. The Viikki appara- tus proved simple and reliable in operation. pH was monitored daily and did not vary from the initial value of 7.2. The NO s con- centration of the solution in the recycling units varied from 133 to 146 mg/1 as com- pared with 151 mg/1 in unused solution. Ger- mination tests, using solution pipetted from the recycling units whilst in operation, indi- cated no contamination by pathogens which might affect growth of the test species. Some algal growth was, however, observed. Developments from earlier versions (Table 1) include the provision of large incubation trays, as compared with pots of 12 cm dia- meter (Bell and Koeppe 1972) or 19 cm dia- meter (Walters and Gilmore 1976). The rel- ative homogeneity of phytotoxin-producing material, the amount of which was related to field conditions, and the possibility of main- taining the material for indefinite periods in large containers are significant advantages. They permit, for example, monitoring of phytotoxin production over time, and se- quential harvesting as compared with single harvests in earlier experiments, Table 1. A difference in light intensity between steps was observed by Walters and Gilmore (1976). This occurred also in our experiments 4 but there was no difference in ambient tem- perature. Statistically significant effects of position were recorded for shoot height at harvest (Table 2) and for shoot and total dry weight. Whilst data are here presented, primarily, in validation of the technique, they confirm the allelopathic potential of A. repens. Thus the presence of decaying or live couchgrass material affected the test species from the commencement of growth and was evidenced in morphological and physiological character- istics at harvest, Tables 3 and 4. The appar- ently progressive decline in total dry weight (Table 4) is a consequence of delayed sowing in Run 2 whilst the system was cleaned and of the removal of the second leaf of all plants in Run 3 for the determination of chlo- rophyll content. The primary causes of the effects and the possibility that different chemicals are pro- duced by live (Gabor and Veatch 1981) and decaying (Lynch 1977) material of A. repens are the subject of further investigations. In earlier experiments, alternate pots of phytotoxin-producing and test species have been employed (Borner 1958; Martin and Rademacher 1960; Wilson and Rice 1968; Bei i and Koeppe 1972; Martin and Gilmore 1976). Only Wilson and Rice (1968) used species which demonstrated that autotoxicity could occur in a stairstep apparatus. The type and amount of phytochemicals pro- duced by autotoxified plants may, of course, differ from that of non-toxified material. Table 2. Effect of position of test pots on height or barley shoots (means of four replicates). Position Mean shoot height (cm) Run 1 Run 2 Run 3 Top 18.58 19.37 18.05 Base 17.58 18.78 17.15 p < 0.001 < 0.01 < 0.001 LSD (t) p < 0.05 0.52 0.34 0.52 p < 0.01 0.71 0.47 0.71 Coefficient of Variation (%) 3.36 2.08 3.43 Table 3. Effect of couchgrass on length of barley roots (means of four replicates). Treatment Mean root length (cm) Run t Run 2 Run 3 Control 11.11 13.91 10.75 Decay 9.97 12.48 11.25 Live 9.71 11.25 12.42 p < 0.05 < 0.001 < 0.001 LSD (t) p < 0.05 1.01 0.84 0.72 p < 0.01 1.38 1.15 0.98 Coefficient of Variation (%) 9.32 6.35 5.95 Table 4. Effect of couchgrass on total dry weight of barley (means of four replicates). Treatment Mean total dry weight (mg) Run 1 Run 2 Run 3 Control 39.69 32.78 29.36 Decay 39.94 30.86 29.02 Live 39.34 30.64 27.38 p N.S. < 0.05 < 0.01 LSD (t) p < 0.05 1.66 1.21 p < 0.01 2.27 1.66 Coefficient of Variation (%) 5.58 5.02 4.02 The Viikki apparatus does not exclude the possibility of autotoxicity, however, output from the incubation trays is filtered through two series of test pots before recycling, reduc- ing the possibility of its occurring. This pro- position is supported by the interaction between position and treatments (Figure 2), uFig. 2. Position by treatment interaction in emergence6 of seedlings. Run 2. 5 in which an apparent inhibition by decaying couchgrass at the top position becomes a stimulation at the base. This effect implies a reduced concentration of phytochemicals, and the type of response noted by Lovett (1982). More detailed information on allelopathy by A. repens as manifest in the stairstep apparatus will be published elsewhere. The apparatus is equally suited to studies of phy- tochemical production during decomposition of crop and weed residues in reduced cultiva- tion systems or where green manuring is practised. Given that the growing season in Finland is short it is desirable to minimise the stresses to which crop and pasture plants are subject, particularly during early growth and devel- opment. Understanding phytochemical ef- fects is of potential significance in reducing stress and improving productivity in many agricultural systems. Acknowledgements; The Finnish Academy of Sciences generously funded the visit, organised by Professor Eero Varis, which made possible the participation of one of us (J. V. L.) in this programme. The equipment was skil- fully constructed by kenttämestari Reino Hakala. Our thanks for these several sources of support are gratefully recorded. References Aamisepp, A. 1970. Influence of plant extracts on growth of seedlings. Lantbr.högsk. Ann. 36: 153 178. Bell, D. T. and Koeppe, D. E. 1972. Noncompetitive effects of giant foxtail on the growth of corn. Agron. J. 64; 321—325. Börner, H, 1958. Experimentelle Untersuchungen zum Problem der gegenseitigen Beeinglussung von Kulturpflanzen und Unkräutern. Biol. Z.bl. 77: 310—328. Gabor, W. E. and Veatch, C. 1981. Isolation of a phy- totoxin from quackgrass (Agropyron repens) rhizo- mes. Weed Sci. 29; 155—159. Harper, J. L. 1977. Population Biology of Plants. Aca- demic Press. London. 892 p. Lovett, J. V. 1982. Allelopathy and self-defence in plants. Austr. Weeds 2: 33—36. Lovett, J. V., Hoult, E. H., Jessop, R. S. and Purvis, Christine E. 1982. Implications of stubble retention. Proc. Second Austr. Agron. Conf. p. 101—115. Lynch, J. M. 1977. Phytotoxicity of acetic acid pro- duced in the anaerobic decomposition ofwheat straw. J. Appi. Bact. 42: 81—87. Lynch, J. M., Hall, K. C., Anderson, H. A. and Hepburn, A. 1980. Organic acids from the anaerobic decomposition of Agropyron repens rhizomes. Phy- tochemistry 19: 1846—1847. Martin, P. and Rademacher, B. 1960. Studies on the mutual influences of weeds and crops. In »The Biol- ogy of Weeds» ed. J. L. Harper, Blackwell, Oxford, p. 143—152. Rice, E. L. 1974. Allelopathy. Academic Press, New York. 353 p. Rice, E. L, 1979. Allelopathy an update. Bot. Rev. 45: 15—109. Toai, T. V. and Linscott, D. L. 1979. Phytotoxic effects of decaying quackrass (Agropyron repens) residues. Weed Sci. 27: 595—598. Walters, D. T. and Gilmore, A. R. 1976. Allelopathic effects on the growth of sweetgum. J. Chem. Ecol. 2: 469—479. Welbank, P. J. 1963.Toxin production during decay of Agropyron repens (Couch Grass) and other species. Weed Res. 3: 205—214. Wilson, R. E. and Rice, E. L. 1968. Allelopathy as expressed by Helianthus annuus and its role in old-field succession. Bull. Torrey Bot. Club 95: 432—448. Ms received December 1, 1984 6 SELOSTUS Muunneltu uuttojärjestelmä allelopaattisten ja muiden fytotoksislen vaikutusten tutkimiseksi J. V. Lovett jaKari Jokinen Helsingin yliopisto kasvinviljelytieteen laitos, 00710 Helsinki 71 Tässä työssä kuvataan kiertävään liuosjärjestelmään perustuvan laitteiston ominaisuuksia jakäyttöä. Koe, jossa tutkittiin juolavehnän mahdollista allelopaattis- ta vaikutusta, osoitti, että kehitetty laitteisto on käy- tössä luotettava ja tarkka. Koe vahvisti myös käsityk- siä, joiden mukaan juolavehnäon allelopaattisten yli- disteiden tuottaja. Laite on sovellettavissa myös tutkimukseen, jossa sel- vitetään eri viljelymenetelmissä mahdollisesti esiinty- vien fytokemikaalien vaikutusta kasvien alkukehityk- seen. 7