Rust-Enhanced Allelopathy of Perennial Ryegrass against White Clover

Connolly, 1990). Crown rust is the most serious ryegrass fungal pathogen in these areas (Eagling and Clark, 1993), Perennial ryegrass (Lolium perenne L.) and white clover (Trifowith epidemics regularly occurring between spring and lium repens L.) are important pasture components in the higher rainfall areas of southeastern Australia. Crown rust (Puccinia coronata autumn. A preliminary study demonstrated that rust acCorda f.sp. lolii Brown) is the most serious ryegrass pathogen in these celerates senescence and reduces ryegrass yield by 56% areas. In a preliminary investigation, rust reduced ryegrass biomass (Mattner, 1998). Despite this, in ryegrass and clover mixby 56%. Yet, interference from rusted ryegrass suppressed the yield tures, interference from rusted ryegrass suppressed clover of neighboring clover plants more than interference from healthy biomass by up to 47% compared with interference from ryegrass. The role of allelopathy in this relationship was investigated healthy ryegrass. This suppression did not result from in a greenhouse study using two bioassays. Soil previously growing a direct effect of crown rust on clover, because rustrusted ryegrass suppressed clover biomass by 36% compared with inoculated and non-inoculated clover monocultures soil previously growing healthy ryegrass. Similarly, leachate from soil yielded the same, which was expected since clover is a surrounding rusted ryegrass suppressed clover biomass by 27% compared with that from healthy ryegrass. This is the first demonstration nonhost of crown rust. Similarly, clover suppression is that a pathogen may influence allelopathy between plants and that not explained by rust increasing ryegrass competitiverust may enhance ryegrass allelopathy against clover. Possible implicaness because, if this were so, the reduction in clover tions of this in pasture ecology and the evolution of mutualism are disyield would be greatest at high densities where resources cussed. are most limited. Instead, clover suppression was greatest at low densities, where competition for resources was minimal. Ryegrass allelopathy is well documented, particularly against clovers and medics (Gussin and P ryegrass and white clover predominate Lynch, 1981; Takahashi et al., 1988, 1991, 1993; Quigley improved pastures in the higher rainfall areas of et al., 1990; Chung and Miller, 1995). For these reasons, southeastern Australia. Their growth in mixtures withwe investigated the hypothesis that rusting increases out added nitrogen results in greater herbage yields than ryegrass allelopathic ability. otherwise can be achieved economically (Menchaca and Both Rice (1984) and Einhellig (1995) hypothesized that pathogens enhance their host’s allelopathic ability. S.W. Mattner, Agriculture Victoria, Institute for Horticultural DevelEvidence supporting this hypothesis occurs in at least opment, Private Bag 15, Scoresby Business Centre, Victoria 3176, Australia; D.G. Parbery, Institute of Land & Food Resources, The two forms. Firstly, pathogens stimulate phytoalexin (anUniv. of Melbourne, Parkville, Victoria 3052, Australia. Received 29 timicrobial compounds) production by their hosts Nov. 1999. *Corresponding author ([email protected]). (Smith, 1996), which can belong to similar chemical groups and are synthesized via the same biochemical Published in Agron. J. 93:54–59 (2001). MATTNER & PARBERY: RUST-ENHANCED ALLELOPATHY OF PERENNIAL RYEGRASS 55 total there were 108 pots, half with rusted ryegrass and half pathways as allelochemicals. For example, isoflavonoids with nonrusted ryegrass. Plants in rusted treatments were are important phytoalexins (Dakora and Phillips, 1996) spray inoculated (Villalta and Clarke, 1995) 52 d after sowing and allelochemicals (Tamura et al., 1967, 1969) from with an aqueous solution containing 1 3 10 urediniospores/ the Leguminosae. Indeed, many phytoalexins act as mL, 0.1% soft soap as a surfactant, and 0.5% gelatine. Plants allelochemicals against plants, inhibiting their germinain nonrusted treatments were sprayed with a similar solution tion (Chang et al., 1969), growth (Glazener and Vancontaining no spores. Following inoculation all plants were Etten, 1978), and cellular metabolism and function (Giplaced in plastic tents for 1 wk, which maintained humidity annini et al., 1990; Spessard et al., 1994). Secondly, under between 92 and 98%. Donor plants were grown for a total of some conditions, the mutualistic fungus Neotyphodium 100 d. lolii (Latch, Christensen and Samuels) Glenn, Bacon Soil for the bioassay was gathered directly from the pots and Hanlin, increases the allelopathic ability of some used to grow rusted and nonrusted donor ryegrass, all of which was removed by hand and then passed through a 5-mm sieve. ryegrass genotypes (Sutherlund and Hoglund, 1990; Soil from each source was bulked and thoroughly mixed prior Quigley et al., 1990). Despite this, no one has previously to further treatment. A fresh preparation of soil was also observed that diseased plants suppress the growth of included as a control. In addition, plus and minus nutrient neighboring species (Tang et al., 1995), even though the and steam-sterilization treatments were incorporated into the effect of pathogens on interference between host and design. This was done in an attempt to detect confounding nonhost plants has been extensively investigated (Bureffects caused by differences in soil microflora and nutrient don, 1987; Ayres and Paul, 1990). Since competition content in the soils resulting from prior rusted and nonrusted is the dominant process of plant interference (Tilman, ryegrass growth. Nutrient enrichment consisted of adding con1988), competition effects often obscure any allelopathic centrations of the nutrients listed previously, to ensure they effects in such experiments (Trenbath, 1974). This is were slightly in excess of growth requirements. The soils were particularly so at high plant densities where competition then placed into 12.5-cm-diam. pots and used to grow two clover receivers. At 70 d after sowing, receivers were washed is intense. While it is difficult to separate the processes free of soil and dried at 808C for 4 d before total biomass was of competition and allelopathy in the field, separation determined. Additionally, a nodulation index was determined can be achieved using bioassays. To date, however, reby bunching individual plant roots; removing three 2-cm secsearchers have not used bioassays to investigate the tions from the upper, central, and lower portion of the bunch; effects of pathogens on allelopathy. counting the nodules captured in each section; drying each The present investigation examined the hypothesis sample; and recording the number of nodules per gram of root. that rust enhances perennial ryegrass allelopathy against The bioassay was conducted as a randomized complete white clover using two bioassay techniques. factorial design with three blocks. There were four pots per treatment in each block. Factors consisted of soil source (three METHODS AND MATERIALS levels: soil previously growing rusted ryegrass, soil previously growing nonrusted ryegrass, and freshly prepared soil as a Pathogen and Plant Material control), nutrient application (two levels: plus and minus) and In the following bioassays, perennial ryegrass (cv. Victosoil sterilization (two levels: plus and minus). rian) was used as the donor species and white clover (cv. Tamar) was used as the receiver species. The perennial ryegrass cultivar Victorian is highly susceptible to crown rust Soil Leachate Bioassay (Critchett, 1991). Crown rust urediniospores were collected Pots (15 cm in diam.) containing the standard soil mix were with a side-arm flask collector from naturally infected ryegrass sown to contain eight ryegrass donor plants. Twenty days after at the Mt. Derrimut field station, 22 km west of Melbourne, sowing, irrigation lines were placed into individual pots and Australia (378479 S, 1448479 E) (Mattner, 1998). calibrated to deliver 250 mL of water per pot every day. Soil leachate was collected in plastic trays beneath the wire mesh Growing Conditions and Soil Type benches, bulked for each treatment, and thoroughly mixed Bioassays were conducted in temperature-controlled glassprior to application to receiver plants. Seventy days after sowhouses (20–238C during the day and 12–158C at night) at the ing, plants in the rusted treatment were inoculated with rust Mt. Derrimut field station. Pots were drip-irrigated at the rate as described previously. One-half of the 64 pots contained of 150 mL of water twice daily, which maintained soil at near rusted ryegrass and the other half nonrusted plants. field capacity. The soil mixture was three parts red-brown The investigation was conducted in two parts. In the first, earth topsoil to one part sand and two parts peat moss. The the pre-inoculation bioassay, the bioassay was made before following nutrients (g/L) were added to the mixture: 0.200 N, donor plant inoculation to ensure that there were no intrinsic 0.116 P, 0.140 K, 0.096 S, 0.652 Ca, 0.024 Cu, 0.012 Zn, 0.008 differences in the allelopathic potential of donor plants within Mn, 0.028 Fe, 0.012 Mo, 0.360 Mg, and 0.001 B. The mixture the rust treatments prior to inoculation. The second, the postwas then pasteurized by steam prior to planting. Following inoculation bioassay, was made after inoculation when rust pasteurization, soil used to grow white clover was inoculated symptoms had fully developed. The methodology of the biowith the appropriate strain of Rhizobium trifolii Dangeard. assays was the same. Pots (12.5 cm in diam.) filled with the This was performed by watering soil contained in individual standard soil mix were prepared containing two clover receivpots with 150 mL of an inoculum solution (10 g of commercial ers. Each was hand watered daily with 150 mL of the approinoculum per 10 L of water). priate leachate or with water in the case of the controls. Receivers were harvested and compared 50 d after sowing Soil Retrieval Bioassay by determining plant biomass, leaf area (with a planimeter, Paton Industries Pty. Ltd., South Australia), leaf number, stoPots (15 cm diam.) containing the standard soil mix were sown to contain an average of 10 ryegrass donor plants. In lon number, and nodulation index. Measurements were taken 56 AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001 Table 2. The effect of soil sterilization on the nodulation of Table 1. Summary of the levels of statistical significance for the various treatments and their interactions on white clover biowhite clover. mass and nodulation. No. nodules per Treatment gram of root LSD ( p , 0.05) Source of variation Dry biomass Nodulation Sterilized 554.55 Soil source (SS) *** NS† 191.95 Nutrient (N) NS NS Nonsterilized 752.12 Sterilization (St) NS ** SS 3 N ** NS SS 3 St NS NS N 3 St NS NS Although soil sterilization did not affect clover bioSS 3 N 3 St NS NS mass, it reduced root nodulation by 26% (see Table 2). ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level. Soil Leachate Bioassay † Not significant. The pre-inoculation bioassay detected no difference on one randomly selected plant per treatment in each block, in the growth of clover watered with leachate from ryeexcept for biomass where all plants were measured. grass in nonrusted or rusted treatments (see Table 3). The bioassay was conducted as a randomized complete This indicates that there was no intrinsic difference in block design. The treatments were the daily application of 150 the allelopathic ability of ryegrass plants assigned to the mL of leachate derived from either rusted ryegrass, nonrusted treatments prior to inoculation. Growth in the control, ryegrass, or irrigation water. There were eight blocks conhowever, was greater than that of plants receiving soil sisting of four pots per treatment. leachate from ryegrass. In the post-inoculation bioassay, the growth of clover Statistical Analysis receiving soil leachate from rusted ryegrass was less than Data was analyzed using analysis of variance (ANOVA) that of plants watered with leachate from nonrusted as performed on Minitab Version 12 (Minitab, 1998). Homoryegrass or the control, according to each parameter geneity of variance was determined by examining plots of measured (see Table 3). Plants exposed to leachate from fitted values versus residuals, while histograms of residuals nonrusted ryegrass did not differ from the control in assessed normality of distribution. any way. Nodulation did not vary between treatments. RESULTS DISCUSSION Soil Retrieval Bioassay Even though several authors have suggested that Table 1 presents statistical significance levels of main pathogens increase their host’s allelopathic ability (Rice, treatments and their interactions in influencing clover 1984; Einhellig, 1995), the present study is the first to biomass and nodulation. Sterilization of the soils used evaluate the pathogen effect on allelopathy between in this bioassay had no effect on the biomass of clover plants. Furthermore, no one has previously observed growing in them. As such, sterilization treatments have that diseased plants suppress the growth of neighboring been grouped to provide a clearer demonstration of the species (Tang et al., 1995). Despite this, a preliminary effects of the different soil source and nutrient treatinvestigation found that rusted ryegrass suppressed the ments (see Fig. 1). The biomass of clover grown in soil growth of neighboring clover plants by up to 47% comfrom rusted ryegrass was less than that in the control pared with when grown with healthy ryegrass (Mattor in soil from nonrusted ryegrass, with this effect being ner, 1998). particularly marked in nutrient treated soils. In contrast, In the present study, the soil retrieval bioassay demclover grown in soil from nonrusted ryegrass produced onstrated that, overall, soil previously growing ryegrass less than the control only when no nutrients were added. reduced the subsequent clover growth compared with Table 3. Production of white clover in response to leachate derived from rusted or nonrusted perennial ryegrass or a control of irrigation water. Bioassays were performed prior to and after the inoculation of ryegrass with rust. Leachate source LSD White clover Nonrusted Rusted growth parameter Control ryegrass ryegrass p , 0.05 p , 0.01 Pre-inoculation bioassay Total biomass (g) 1.10 0.76 0.78 0.16 0.22 Post-inoculation bioassay Total biomass (g) 2.86 3.11 2.28 0.53 0.75 Leaf area (cm2) 203.88 196.2 145.4 49.9 NS† Stolon number 4.71 5.00 4.00 0.76 NS Leaf number 36.5 34.12 25.5 8.78 NS Nodulation index Fig. 1. The biomass of white clover grown in soil from rusted or (no. nodules nonrusted perennial ryegrass. The control consisted of freshly preper gram root) 1222.81 1121.11 1143.78 NS NS pared soil. Nutrients were added at concentrations described in the text. † Not significant. MATTNER & PARBERY: RUST-ENHANCED ALLELOPATHY OF PERENNIAL RYEGRASS 57 a control of freshly prepared soil. The modification of chate only from mature ryegrass, starting at 60 d after sowing. soil microflora by ryegrass to contain species antagonisIn contrast to leachate from healthy ryegrass, plant tic to clover growth does not explain this result because age did not affect the phytotoxicity of leachate from soil sterilization had no effect on the relationship. In rusted ryegrass, where the leachate suppressed clover contrast, nutrient addition alleviated the suppressive efbiomass by 20% compared with the control and by 27% fect that soil previously growing healthy ryegrass had compared with nonrusted ryegrass. This is again in simion clover production. This suggests that the main effect lar proportions to the preliminary investigation (Mattof healthy ryegrass was to deplete soil nutrients and ner, 1998). Similar reductions occurred in all parameters thereby diminish clover growth. This was not the case measured in clover exposed to leachate from rusted for soil previously growing rusted ryegrass, however, ryegrass, strongly supporting the hypothesis that rust where nutrient application markedly increased its supincreases ryegrass allelopathic ability against clover. The pressive effect on clover growth. Under these condiresults also suggest that in addition to enhancing ryetions, soil previously growing rusted ryegrass suppressed grass allelopathic potential, rust prolongs allelopathy clover biomass by 36% compared with soil previously well into maturity. growing healthy ryegrass. This was in similar proporWhile results from each bioassay suggest that rust can tions to that in the preliminary experiment where the increase ryegrass allelopathy against clover, there was yield of clover grown in mixtures with rusted ryegrass no evidence of allelopathy acting against nodulation. fell by an average of 37% (Mattner, 1998). Nutrient Furthermore, the uniformity of size and pinkness of toxicity does not explain this result because twice the nodules from plants in all treatments suggested that concentration of nutrients applied to soil in the rusted allelopathy did not impair nodule nitrogen-fixing capactreatment had no effect on clover growth in the control. ity, although no measurement of this was made. The Instead, the result provides strong evidence that rust only treatment that affected nodulation was soil sterilincreases ryegrass allelopathic ability against clover. ization, which reduced it by 26%. This was probably Furthermore, the above results demonstrate a potential due to sterilization reducing the resident rhizobial popufor rusted ryegrass to release allelochemicals into soil lation in the soil prior to planting. at concentrations phytotoxic to clover. The ability of rust to increase ryegrass allelopathic Previous soil-based bioassays have not reported enpotential is not surprising given that infection by pathohanced allelopathy following nutrient addition (Buchgens can induce phytoalexin production by their hosts holtz, 1971). Despite this, there are at least three possi(Smith, 1996). Phytoalexins can belong to similar chemible explanations of how nutrients might enhance clover cal groups as allelochemicals. For example, Mayama suppression by soil previously growing rusted ryegrass: (1981, 1982) discovered that rust resistant oat (Avena (i) The presence of abundant nutrients in some way sativa L.) produced three phytoalexins belonging to the facilitates allelochemical uptake. phenolic acid group when challenged by crown rust. These phenolic acids inhibited rust spore germination (ii) Nutrient addition may initiate cationic exchange. at concentrations as low as 200 mg/L. Although these Here, nutrient addition in the form of cations chemicals were not shown to suppress plant growth, releases allelochemicals bound to anionic colloithey have a great potential to do so given that many dal or organic material into the soil solution, allelochemicals are phenolic acids. where they are absorbed by receiver plants. Another potential allelochemical source from rusted (iii) A reaction of allelochemicals with the added nuryegrass in the present experiment is from the rust itself. trients may increase their toxicity and/or staIt is well established that pathogens can produce a range bility. of phytotoxins that are important in pathogenesis (Daly Takahashi et al. (1988, 1991) established that leachate and Deverall, 1983). Despite this, a preliminary investifrom perennial ryegrass can be phytotoxic to white clogation demonstrated that clover monoculture inoculaver. In a similar manner, the present study showed that tion with crown rust had no effect on its growth (Mattsoil leachate from developing ryegrass (up to 70 d after ner, 1998). This suggests that the allelochemical source sowing) suppressed clover biomass by 30% compared from rusted ryegrass in the present experiment is more with the control (i.e., in the pre-inoculation bioassay). likely to be from ryegrass than from rust. It is important This highlights the potential for ryegrass to suppress to note, however, that irrespective of the phytotoxin clover through allelopathy. As healthy ryegrass develsource from rusted ryegrass, the relationship between oped, however, the phytotoxicity of its leachate diminrusted ryegrass and clover remains allelopathic. This is ished and was lost, suggesting that ryegrass allelopathic because rust is a biotrophic parasite of ryegrass and potential is greatest when it is young. Work on other because allelopathy is defined as the beneficial and detplant species has also shown a similar decrease in allelorimental chemical interaction among plant organisms, pathy as plants age (Koeppe et al., 1970; Woodhead, including microorganisms (Rice, 1984). 1981). The hypothesis that allelopathic potential deAn important next step in the confirmation of encreases with plant age may also explain why Newman hanced allelopathy by rusted ryegrass is to identify the and Rovira (1975) and Newman and Miller (1977) oballelochemicals involved. What is their source? Are served no clover yield depression following exposure to these compounds in higher concentrations in rusted plants or do rusted plants produce entirely different leachate from ryegrass. This is because they used lea58AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001 allelochemicals than healthy plants? In a related ques-CONCLUSIONStion, what is the effect of disease severity on ryegrass The bioassays of the present study showed that soilallelopathic ability? The average disease severity of and leachate from rusted ryegrass suppressed cloverrusted ryegrass at 42 d after inoculation in the soil regrowth compared with that from nonrusted ryegrass.trieval bioassay of the present experiment was 6.6% Together, these bioassays provide strong evidence for(as determined by a calibration method [Mattner and the hypothesis that rust enhances perennial ryegrassParbery, 1999]). If rust enhances allelopathy between allelopathic ability against white clover. This hypothesisryegrass and clover, heavily rusted plants should prowould explain why rusted ryegrass suppressed theduce more allelochemicals than lightly rusted plants. growth of neighboring clover plants in a preliminaryMoreover, the ryegrass cultivar used in the present exexperiment (Mattner, 1998).periment was highly susceptible to rust. 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