Tropical Grasslands (1999) Volume 33,

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1 Tropical Grasslands (1999) Volume 33, Effects of leachates from swards of Bothriochloa pertusa and Urochloa mosambicensis on the growth of four test species, B. pertusa, U. mosambicensis, Stylosanthes hamata cv. Verano and S. scabra cv. Seca and an assessment of the endophyte status of the grasses F.D. HU AND R.J. JONES CSIRO Tropical Agriculture, Townsville, Queensland, Australia Nixon rules out the possibility that these are involved in the poorer performance of Verano grown with Bowen in the field. Abstract In a previous grazing trial, the percentage of Stylosanthes hamata cv. Verano was much lower when grown with Bothriochloa pertusa cv. Bowen than when grown with Urochloa mosambicensis cv. Nixon. Reasons for this depression in the presence of Bowen have been investigated. In this study, the possibility of allelopathic effects on Verano and on Stylosanthes scabra cv. Seca from leachates of the grasses Bowen and Nixon was examined. Leachates from soil-filled boxes with or without Bowen or Nixon were used to water seedlings of Verano, Seca, Bowen and Nixon grown in pots along with a control treatment watered with demineralised water. Plant height, tiller or shoot number, leaf number and top and root weights were measured over a 12-week period to harvest. With Verano, there were no significant adverse effects of leachates from swards of the two grasses compared with the water control. Leachates from Bowen actually gave the highest yields of Verano. These results give no support to the hypothesis that Bowen had allelopathic effects on Verano. With Seca, lowest yield and plant height occurred with leachates from Bowen while the highest yield and plant height were obtained with leachates from bare soil. This indicates Seca is more susceptible to adverse effects from some factor in Bowen. Leachates from bare soil had no allelopathic effects on the growth of the four species. Failure to detect endophytes in Bowen or Correspondence: Dr R.J. Jones, CSIRO Tropical Agriculture, Davies Laboratory, PMB, PO Aitkenvale, QLD 4814, Australia. Raymond.Jones@tag.csiro.au Introduction In a grazing experiment at the CSIRO Pasture Research Station, Lansdown, near Townsville, north Queensland, the percentage contribution of the tropical legume Stylosanthes hamata cv. Verano declined with the ingress of the naturalised grass Bothriochloa pertusa cv. Bowen (Jones and Kerr 1993). Associated paddocks of Urochloa mosambicensis cv. Nixon-Verano retained a good balance of Verano. Possible reasons for the lower percentage of Verano associated with Bowen include: allelopathic effects from the grass associate, as suggested by Hussain et al. (1982; 1987); the presence of fungal endophyte in the grass (Sutherland and Hoglund 1989); competition for nutrients, water and light; differential response to defoliation; and selective grazing. In this paper, we report studies to investigate the potential effects of allelopathy and endophyte status of the associate grasses. Other papers will relate to the other factors listed. Materials and methods Leachate study The experiment was carried out in a glasshouse at the CSIRO Davies Laboratory, Townsville, Queensland, between March and August Donor swards were grown to provide leachate for watering pots growing seedlings of 4 acceptor species to assess any allelopathy (details given below). The soil used for the experiment was a yellow earth/podzolic intergrade from the area at

2 Leachates from grass and plant growth 123 the CSIRO Lansdown Research Station where the decline in Verano in association with Bowen had been measured. Top soil ( cm) was passed through a mm sieve and well mixed. The donor species, Bowen and Nixon, were grown in boxes of soil (donor boxes) to provide leachates. Leachate was also obtained from boxes of bare soil. There were 4 replicates of these 3 treatments. Collection of leachates from donor boxes commenced 4 months after planting, by watering and then collecting the drainage water which had passed over litter on the soil surface and through the soil. Donor boxes measured 7 cm 37 cm 21 cm. Twenty kg of pebbles (diameter mm) were placed on the bottom of each box and 22 kg of soil were placed on top of the pebbles. A mesh screen separated the soil and the pebbles to prevent soil loss. Each box had an outlet pipe on the bottom, fitted with a tap, for collection of leachate. Tillers of Bowen and Nixon were transferred into boxes on March 29, 1993 at a density of 2 tillers/m 2. Four grams of superphosphate, equivalent to kg/ha, were applied to each box on June 16, The soil surface in the bare soil boxes was covered by white polythene beads in order to reduce evaporation from the soil surface and to inhibit the growth of fungus. At flowering, the grasses were cut once and the cut material was put back into the same box before leachates were collected. The acceptor species used were: Verano, Seca, Bowen and Nixon. Bowen and Nixon were included to assess any effect of their leachates which could reduce yield and therefore competitive ability. All species were grown from seed in polythene pots, 14 cm tall with a top diameter of cm and a bottom diameter of cm. A plastic bag was placed in each pot before the soil was added to stop water running through the pot. Each pot was filled with 1. kg of soil. The four acceptor species were sown on June 1, 1993, and 3 weeks later, seedlings from each pot were thinned to 8. Rhizobium was applied to the legumes in acceptor pots (CB16 for Verano; CB82 for Seca) 3 weeks after sowing. The 64 acceptor pots for growing the 4 test species were set up as 2 blocks (legume and grass) on an automatic watering machine (Andrew and Cowper 1973) which moved the pots continuously during the day and automatically watered them to field capacity. Each block was a 2 species 4 treatments factorial design with 4 replicates. The 4 watering treatments were leachates from Bowen, Nixon and bare soil and a control watered with demineralised water. Artificial lighting from 17 h 2 h was used to simulate long days to prevent early flowering of Verano and Seca. Leachate collection and watering of acceptor pots commenced on June 21, 1993, 3 months after planting the donor species. All replicate leachates from the same treatment were mixed together before acceptor pots were watered. Each acceptor pot was given increasing volumes of leachate as the plants grew: ml leachate/ day during Weeks 4 and ; ml/d during Weeks 6, 7 and 8; and 3 ml/d during Weeks 9,, 11 and 12. All acceptor pots were watered twice a day with leachates. The pots were then adjusted to field capacity by the automatic watering system daily. Detailed plant measurements were made to detect treatment effects over time. These measurements started 4 weeks after sowing. Two plants in each pot were labelled and the height and number of leaves and tillers (shoots) were measured weekly for 8 weeks and again at harvest 12 weeks after sowing when tops were cut to ground level. The height and tiller number of all 8 plants in each pot were also measured. All pots were washed and sieved to recover roots after harvest. Roots and tops were dried for 3 days at 8 C, and the dry weights recorded. Data for the legume and grass treatments were analysed separately as a fully randomised design with 4 replicates. Analysis of variance was performed on untransformed data. Assessment of endophyte status Twelve green plant samples of Bowen were collected from the roadside at 4 km intervals between CSIRO Lansdown Research Station and the Davies Laboratory, Townsville. Three green plant samples of Nixon were also collected from 3 sites (Lansdown, Davies Laboratory and the roadside between Lansdown and Davies Laboratory). A method based on that described by Latch et al. (1984) was used for detection of endophytes. A leaf sheath was removed from the plant, and the adaxial epidermis was cut with a scalpel and then stripped from the sheath with forceps. The epidermal tissue was mounted in.1% cotton blue in lacto-phenol and the slide warmed for a few seconds over a flame to accelerate staining of mycelium. The slide was

3 124 F.D. Hu and R.J. Jones examined under a microscope for the presence of mycelium. Four leaf sheaths from 2 plants of each sample were examined. Results Leachate study legume growth For Verano, there were no significant differences (P>.) between treatments in shoot and root dry matter yield or in root : shoot ratio. With Seca, the differences between leachates from Bowen and soil were apparent in total DM and shoot DM but not in root DM yield. This resulted in a significant interaction of species treatment (P<.). Overall, Seca produced less root and shoot yield (P<.1) than Verano but both species had similar root : shoot ratios (Table 1). There were no significant effects of watering treatment on Verano plant height at 12 weeks (mean 17.3 cm) or shoot number (mean 1.9 shoots/plant). For Seca, leachates from Bowen reduced plant height (. cm) relative to leachate from bare soil (13.3 cm) (Figure 1). After Week 7, Seca plants receiving leachates from Bowen or Nixon grew more slowly (P<.) than those receiving leachate from bare soil. Verano treated with leachates from Bowen had more leaves than the other treatments after 6 weeks. These differences increased with time and were significant (P<.) after weeks (Figure 1). Seca leaf numbers were not affected significantly by treatment (P>.). During the 12- weeks growth period, Verano produced more leaves than Seca, partly because Verano developed lateral shoots whereas Seca did not. Leaf numbers/plant Plant height (cm) l.s.d.(p<.) l.s.d.(p<.) Figure 1. The effect of leachates from the water control on: leaf numbers of S. hamata cv. Verano; and height of S. scabra cv. Seca. Leachate study grass growth Watering treatments had only small effects on grass yields. There were no significant differences (P>.) between watering treatments in Table 1. Dry matter (shoot and root) yield and root : shoot ratio of S. hamata cv. Verano and S. scabra cv. Seca watered with leachates from B. pertusa cv. Bowen, U. mosambicensis cv. Nixon or bare soil or with demineralised water. Treatments (leachates) Total DM Shoot DM Root DM Root : shoot Verano Seca Verano Seca Verano Seca Verano Seca (g/pot) Bowen a a Nixon ab ab Soil b b Water control ab ab Mean 4.7A 2.B 3.9A 1.69B.98A.6B Within columns, treatment values followed by different lower case letters are different (P<.); within measurements, mean values followed by different upper case letters are different (P<.1).

4 Leachates from grass and plant growth 1 total yields or yields of shoots and roots for Bowen (Table 2). However, the root : shoot ratio was highest for the water control and lowest for plants grown with leachate from bare soil (P<.1). There was no significant difference (P>.) between watering treatments in total yield, shoot yield or the root:shoot ratio of Nixon. The root yield of Nixon was lowest when treated with leachates from Nixon and highest with leachates from bare soil (P<.) (Table 2). At the final harvest, there were no significant watering treatment effects on the heights of Bowen (mean 26.8 cm) or Nixon (mean.7 cm). For both Bowen and Nixon, heights increased similarly with time for all treatments. Leaf numbers and tillers at the final harvest for Bowen and Nixon were similar for all treatments. After Week 6, tiller numbers were higher for Bowen than Nixon (Figure 3) as were leaf numbers (P<.1) (Figure 2). Leaf numbers/plant 3 l.s.d.(p<.) l.s.d.(p<.) Assessment of endophyte status No endophyte was found on any leaf sheath epidermis of Bowen or Nixon. Discussion Leachates from Bowen and Nixon The absence of any negative effects of leachates from swards of both grasses on Verano growth indicate that allelopathy is most unlikely to have any major role in modifying the grass : legume balance of swards of this species. With Seca, there was some suggestion of a reduction in plant height and shoot and root weight. It is known that allelochemicals are specific in their effects (Rice Figure 2. The effect of leachates from the water control on the number of leaves produced by: Bowen; Nixon. 1984; Putnam and Tang 1986; Horsley 1991) and the trend in this experiment is similar to that described for the effects of extracts of plant material on seed germination and seedling growth of Verano and Seca (Hu and Jones 1997). The difference between our results and those of Table 2. Dry matter (shoot and root) yield and root : shoot ratio of B. pertusa cv. Bowen and U. mosambicensis cv. Nixon watered with leachates from Bowen, Nixon or bare soil or with demineralised water. Treatments (leachates) Total DM Top DM Root DM Root : shoot Bowen Nixon Bowen Nixon Bowen Nixon Bowen Nixon (g/pot) Bowen ab 1.4ab.38 Nixon a.38ab.3 Soil b.33a.41 Water control ab.47b.37 Mean Within columns, values followed by different letters are significantly different (P<.).

5 126 F.D. Hu and R.J. Jones Tiller numbers/plant l.s.d.(p<.) l.s.d.(p<.) Figure 3. The effect of leachates from the water control on the number of tillers produced by: Bowen; Nixon. Hussain et al. (1982; 1987), who alleged strong allelopathic effects of B. pertusa, may well be due to the different species used as acceptor plants. In addition, the effects of competition were confounded in some of their work as the effect of allelopathy could not be separated from resource competition. At least for Verano, the data presented here give no explanation for the field result which generated the study. However, it does appear that there may well be some allelopathic interference on Seca growth by leachates from swards of Bowen. Soil leachates Leachates from bare soil had no adverse effects on plant growth and actually stimulated plant growth. This may be explained by nutrients in the leachates from bare soil. It is well known that N application increases top growth and reduces the root : shoot ratio (Viets 196; Black 1968) which is expressed in greater plant height, tiller or shoot number and in dry weight of tops and roots. Therefore, the soil can be ruled out as an inhibiting factor containing allelochemicals adversely affecting growth of any of the species tested. Endophyte status In the absence of endophyte infection in both grasses, endophytes can not be implicated in the poor growth of Verano in association with Bowen compared with its growth with Nixon. Acknowledgements We thank the Meat Research Corporation (MRC) for providing part of the research funding for this project. References ANDREW, C.S. and COWPER, J.L. (1973) Automatic plant pot watering machine. Laboratory Practices, 22, BLACK, C.A. (1968) Soil-Plant Relationship. (Wiley: New York). HORSLEY, S.B. (1991) Allelopathy. In: Avery, M.E., Cannell, M.R. and Ong, C.K. (eds) Biophysical research for Asian agroforestry. pp (Winrock International: USA; Asia Books: South Asia). HU, F.D. and JONES, R.J. (1997) Effects of plant extracts of Bothriochloa pertusa and Urochloa mosambicensis on seed germination and seedling growth of Stylosanthes hamata cv. Verano and Stylosanthes scabra cv. Seca. Australian Journal of Agricultural Research, 48, HUSSAIN, F., NAQVI, H. and ILAHI, I. (1982) Interference exhibited by Cenchrus ciliaris (L.) and Bothriochloa pertusa (L.) A. Camus. Bulletin of the Torrey Botanical Club, 9, HUSSAIN, F., MUBARAK, B. and ILAHI, I. (1987) Allelopathic effects of Pakistani weed Bothriochloa pertusa. Pakistan Journal of Agricultural Research, 8, JONES, R.J. and KERR, A. (1993) Bothriochloa pertusa a useful grazing-tolerant grass for the seasonally dry tropics? Proceedings of the XVII International Grassland Congress, New Zealand and Australia, pp LATCH, G.C.M., CHRISTENSEN, M.J. and SAMUELS, G.J. (1984) Five endophytes of Lolium and Festuca in New Zealand. Mycotaxon,, 3. PUTNAM, A.R. and TANG, C.S. (1986) The Science of Allelopathy. (Wiley: New York). RICE, E.L. (1984) Allelopathy. 2nd Edn. (Academic Press: Orlando). SUTHERLAND, B.L. and HOGLUND, J.H. (1989) Effect of ryegrass containing the endophyte Acremonium lolii on the performance of associated white clover and subsequent crops. Proceedings of the New Zealand Grassland Association,, VIETS, F.G. (196) The plant s need for and use of nitrogen. In: Bartholomew, W.V. and Clark, F.E. (eds) Soil Nitrogen. (American Society of Agronomy Inc.: Madison, Wisconsin, USA). (Received for publication August 14, 1998; accepted January 6, 1999)