Comparison of Early Development of Three Grasses: Lolium perenne, Agrostis stolonifera and Poa pratensis

Abstract

• Background and Aims To improve the management of grass communities, early plant development was compared in three species with contrasting growth forms, a caespitose (Lolium perenne), a rhizomatous (Poa pratensis) and a caespitose–stoloniferous species (Agrostis stolonifera).

• Methods Isolated seedlings were grown in a glasshouse without trophic constraints for 37 d (761 °Cd). The appearance of leaves and their location on tillers were recorded. Leaf appearance rate (LAR) on the tillers and site-filling were calculated. Tillering was modelled based on the assumption that tiller number increases with the number of leaves produced on the seedling main stem. Above- and below-ground parts were harvested to compare biomass.

• Key Results Lolium perenne and A. stolonifera expressed similar bunch-type developments. However, root biomass was approx. 30 % lower in A. stolonifera than in L. perenne. Poa pratensis was rhizomatous. Nevertheless, the ratio of above-ground : below-ground biomass of P. pratensis was similar to that of L. perenne. LAR was approximately equal to 0·30 leaf d⁻¹ in L. perenne, and on the main stem and first primary tillers of A. stolonifera. LAR on the other tillers of A. stolonifera was 30 % higher than on L. perenne. For P. pratensis, LAR was 30 % lower than on L. perenne, but the interval between the appearance of two successive shoots from rhizomes was 30 % higher than the interval between two successive leaf stages on the main stem. Above-ground parts of P. pratensis first grew slower than in the other species to the benefit of the rhizomes, whose development enhanced tiller production.

• Conclusions Lolium perenne had the fastest tiller production at the earliest stages of seedling development. Agrostis stolonifera and P. pratensis compensated almost completely for the delay due to higher LAR on tillers or ramets compared with L. perenne. This study provides a basis for modelling plant development.

INTRODUCTION

Perennial grass communities such as natural meadows or turves, are usually a mixture of different species. To keep compact long-lasting canopies, their management requires the relationships between growth strategies, environmental constraints, interplant competition and cultural practices to be taken into account. However, Graminae are clonal plants with a modular structure, composed of tillers or ramets that may express contrasting growth forms (Harper, 1977; Moulia et al., 1999a). First, bunch or caespitose types are highly branched with closely packed tillers, and ramets are formed close to the older shoots. Tiller emergence is intravaginal, i.e. tillers arise through the entire length of the sheath (Cattani and Struik, 2001). Secondly, many Gramineae species can grow from rooting plagiotropic shoots like rhizomes or stolons that arise through the sheath tissue by extravaginal emergence. These plants are able to spread radially to form large, clonal stands (Harper, 1977). Grasses with different growth forms respond differently to interplant competition, environmental constraints and human practices, and their presence in mixtures makes it hard to control canopy cover. Consequently, to improve the management of plurispecific communities, a greater knowledge of growth patterns encountered in grasses is required.

Some studies mainly conducted by agronomists have looked at the development of isolated plants. They have focused on a small number of Gramineae species of agronomic importance, such as maize (Moulia et al., 1999a, b), wheat (Triticum aestivum; Masle-Meynard and Sébillotte, 1981a, b), barley (Hordeum sativum; Fletcher and Dale, 1977; Kirby et al., 1985), Festuca spp. (Sugiyama, 1995) or perennial ryegrass (Lolium perenne; Richards et al., 1988; Neuteboom and Lantinga, 1989; Yang et al., 1998; Gautier et al., 1999). Plant development is described and quantified using the phyllochron, i.e. the number of growing degree days between the emergence of leaf number n and leaf number n + 1 (Klepper et al., 1982; Frank and Bauer, 1995). Thus, tillering models (also called ‘tillering patterns’) have been built either from the phyllochron of the main stem (MS), i.e. the first shoot emerged from the seed, or from the values of the leaf emergence rate (Masle-Meynard and Sébillotte, 1981a; Kirby et al., 1985; Bos and Neuteboom, 1998). These models are based on the assumption that tiller number increases with the successive leaf stages of the seedling MS. They may be used either to represent the theoretical maximal development of a plant, or the structure of the typical ‘mean’ individual. Furthermore, as leaf appearance rate (LAR) is related to the production rate of axillary buds, it determines the production rate of potential tillering sites. Thus, more recently, plant development has been described using the LAR and the site-filling ratio (Fs), which is a measure of tiller bud activity and occupancy of existing tillering sites (Davies, 1974; Neuteboom and Lantinga, 1989). Tillering activity has also been measured as site use (Skinner and Nelson, 1992) or nodal probability (Matthew et al., 1998).

The development patterns of isolated plants provide the first key elements towards a better understanding of tillering response of different growth forms of grasses to environmental conditions, interplant interactions and cultural practices. The description of growth patterns provides basic information to model isolated plants and also plurispecific canopies. Most relevant information is on caespitose types. Few attempts have been made to investigate the development of stoloniferous and rhizomatous types. Some studies on stolon development have been conducted, mainly with creeping bentgrass (Mueller and Richards, 1986; Cattani and Struik, 2001; Cattani et al. 2002). Therefore, it was intended to compare early development, particularly tillering dynamics of rhizome and stolon types with that of bunch types. For this purpose, three grass species likely to be found together in the same canopies, and known to express different growth strategies, were chosen for the study: the caespitose perennial ryegrass L. perenne, the caespitose–stoloniferous creeping bentgrass Agrostis stolonifera and the rhizomatous kentucky bluegrass Poa pratensis.

MATERIALS AND METHODS

Plant material and growth conditions

Species were selected based on their contrasting growth strategies and on the similarity of their ecological requirements. Thus, they could be grown under the same conditions. Three turfgrass cultivars were considered: Lolium perenne L. ‘Rival’, Agrostis stolonifera L. ‘Penneagle’ and Poa pratensis L. ‘Entopper’. Caryopses were sown in February on a moist artificial substrate (50 % sand and 50 % white peat), in a warmed glasshouse in Angers (0·33°E, 47·28°N, France). On average, air temperature was 18·9 ± 0·9 °C and relative humidity was around 60 ± 6·6 %. Twenty days after sowing, plants had formed their coleoptile and three leaves without any primary tiller. Twelve individuals of each cultivar were then selected, based on their homogeneous development, for a 37-d experiment (beginning mid-March). Each isolated plant was randomly pricked out in the middle of one of 36 culture trays placed on the tables described below. From the beginning until the end of the experiment, day length increased from 12 h to 13·5 h.

Transplanted seedlings were grown on two sub-irrigation tables (2 m × 4 m), in the glasshouse described earlier. Each table was covered with an absorbent felt cloth (Aquanappe®) to water and feed the plants by capillarity. The felt tablecloth was flooded three times a day, for 15 min, with a nutrient solution mixed in a 300-dm³ tank (Sevital® solution: N-P-K 12 : 4 : 6; 1 % ; 11 % urea; dilution 0·5 % during the first fortnight, and then 1 %, renewed weekly). After watering, the excess of solution percolated into a plastic gutter located in the middle of the table and back into the tank. The Aquanappe® was covered with a black plastic film punched with small holes, to limit solution evaporation and avoid algal development. Thirty-six bottomless culture trays made of a rectangular plastic frame (polyvinyl chloride, 60 cm long, 40 cm wide and 10 cm high), were laid out in rows on the sub-irrigation tables. Each table could support two rows of nine trays. Within a row, trays were adjacent by their longest edges. As a consequence, plants were 40 cm from their neighbours and 140 cm from seedlings of the other row; there was no above- nor below-ground interplant competition. Each tray was filled with 21 dm³ of substrate (sand, white peat and pouzzolana in equal proportions; pH 6·75 adjusted with CaCO₃ Recalcit®). Glasshouse conditions (nutrient solution, water, increasing photoperiod, temperature) were designed to enhance growth and to minimize stress.

Data collection

Tillering was monitored 5 d per week up to 37 days after transplanting (DAT). Leaf emergence was recorded as follows: a new leaf was scored when its tip had appeared above the level of the next older leaf sheath. After ligule development, a leaf was considered as fully emerged and was marked with a thin plastic ring bearing a code giving its location on the plant, and the location of its ‘parent’ phytomer. The seedling stem was called the MS. The first leaf to emerge on the MS (coleoptile), and the first leaf on a tiller (called the prophyll) were numbered 0. Ramifications on the MS (tillers) were named after their ‘parent’ leaf; e.g. T₃₁ was the secondary tiller arising at the axil of the 1st true leaf of the primary tiller, itself borne at the axil of the 3rd true leaf on MS (Fig. 1). When present, above-ground plagiotropic shoots (stolons, St) and below-ground shoots (rhizomes, Rh) can produce new rooted orthotropic shoots called S and R, respectively. Ramifications of S and R were also named after their ‘parent’ leaf; for example, R1₂ was the primary tiller borne at the axil of leaf 2 of R1, which was itself the first orthotropic shoot on a rhizome.

Fig. 1.

Different growth strategies in grasses: (A) bunch-type, (B) rhizomatous-type, and (C) stoloniferous-type. MS indicates seedling main stem, T, seedling ramifications; S, orthotropic shoots on stolon (St); R, shoots on rhizomes (Rh). Coleoptile leaf on the MS (closed circles) and prophylls (small open circles) are called leaf 0 on tillers. True leaves are large open circles. Code used for tillers: e.g. T₃ is the primary tiller arising at the axil of the 3rd true leaf of MS, T₃₁ is the secondary tiller arising at the axil of the 1st true leaf on T₃.

At 38 DAT, plant height was measured above the substrate surface. Horizontal spread of each plant was estimated by placing a wired fence with a 4-cm mesh over the tray, and by counting the number of threads crossed by the above-ground organs. Six of the 12 plants of each cultivar were harvested (remaining plants were kept for another experiment). Each plant was sorted into different samples: (a) MS pooled with R and S shoots when present, (b) primary tillers including St; (c) secondary tillers; (d) tertiary tillers; (e) roots; and (f) rhizomes. Each sample was dried up to constant weight in a forced-air drier at 60 °C, before biomass measurements.

Data analysis

The numbers of leaves (or phytomers) and tillers produced per plant were calculated as a function of thermal time (i.e. growing degree days, °Cd, base temperature 0 °C; McMaster and Wilhem, 1997). The phyllochron of the MS was estimated as the inverse of the slope of the regression of the MS phytomer number on thermal time. The number of fully emerged leaves on ‘parent’ tillers was counted when the first ‘daughter’ tiller emerged (Kirby et al., 1985). The rate of leaf appearance, i.e. the number of leaves per tiller per day was calculated for each order of tiller in each species. For this calculation, only true leaves were considered because prophylls expressed different behaviours, either appearing at the same time as the first true leaf or later. Site-filling (Fs) was obtained at successive leaf stages of MS, from the natural logarithm of the increase in tiller number during each leaf appearance interval (Neuteboom and Lantinga, 1989). The ratio of the number of new leaves and new tillers (ΔL/ΔT) at successive leaf stages of MS was also calculated. So as not to underestimate tillering potential, coleoptile leaves and prophylls were taken into account in the site-filling calculation (Neuteboom and Lantinga, 1989). Biomass of tillers, below-ground parts (roots and Rh), and the ratios of above-ground : below-ground parts and above-ground parts : roots were also compared among species.

Statistical analyses were performed with Systat 10 software (Systat 10, 2000). As standard deviations were not equal across samples, non-parametric Kruskal–Wallis and unpaired Mann–Whitney tests, based on median equality, were used to compare species. Equations of regressions were compared from Jerrold (1998). Tillering models (i.e. the structure of a typical ‘mean’ individual) combined the present experimental results and models given by Masle-Meynard and Sébillotte (1981a) and Kirby et al. (1985).

RESULTS

Spatial colonization and branching

During the course of the experiment, no plant or leaf died except coleoptiles. Plant height was significantly larger for P. pratensis with a mean height of 7·8 cm, compared with L. perenne (5·4 cm) and A. stolonifera (4·5 cm; P < 0·01, Table 1). However, P. pratensis clearly produced fewer leaves (around 29) than L. perenne (around 106) and A. stolonifera (around 86; Fig. 2A and B), and some of the phytomers were produced by new tillers on rhizomes (Fig. 2B). Horizontal spread was less important for P. pratensis; at the end of the experiment, 24 % of the trays were occupied, while L. perenne and A. stolonifera colonized 49 % and 60 % of the available area, respectively (Table 1). Growth patterns were as expected with one exception regarding A. stolonifera; L. perenne was caespitose and P. pratensis was rhizomatous with three to five R tillers per plant. Unfortunately, no S and only one extravaginal St was observed for A. stolonifera. In that species, some primary tillers with intravaginal development began to express a plagiotropic development. As they were not orthotropic, they were not considered as St for A. stolonifera.

Table 1.

Spatial development of isolated plants of L. perenne, A. stolonifera and P. pratensis grown in a greenhouse for 38 DAT

	L. perenne	A. stolonifera	P. pratensis	P rows
Plant height (cm)	5·4 ± 0·3b	4·5 ± 0·5c	7·8 ± 0·5a	0·0030
Colonized threads (%)	49·1 ± 4·4b	59·6 ± 6·1a	24·1 ± 3·1c	0·0033

Data are means ± standard error of six plants.

P values are given according to Kruskal–Wallis. Letters abc indicate significant differences among species in rows.

‘Colonized threads’ expresses the percentage of threads of a wired fence placed over the culture trays, crossed by above-ground parts of the plants at 38 DAT.

Fig. 2.

Evolution of the leaf number (N) as a function of thermal time (T_th) in L. perenne (open triangles), A. stolonifera (open squares), P. pratensis (open circles) and R of P. pratensis (closed circles). (A) Whole plants of L. perenne and A. stolonifera, (B) whole plants of P. pratensis; (C) only the MS for the three species. Data are from 12 plants grown in a greenhouse for 37 DAT. Equations: (A) N_{L. perenne} = 3·6045e^0·0048Tth (R² = 0·94) and N_{A. stolonifera} = 2·4659e^0·0048Tth (R² = 0·89); (B) N_{P. pratensis} = 3·3026e^0·0032Tth (R² = 0·88), N_{R. P. pratensis} = 0·0123T_th – 1·3295 (R² = 0·66); (C) N_{MS L. perenne} = 0·0106T_th + 4·0997 (R² = 0·92), N_{MS A. stolonifera} = 0·011T_th + 3·2443 (R² = 0·94), N_{MS P. pratensis} = 0·0086T_th + 3·5643 (R² = 0·93).

Generally, above-ground parts of L. perenne and A. stolonifera expressed the same development patterns with very few differences over the 37 DAT. First, in both species, the exponential curves that fitted the increase in leaf number on thermal time differed only by their constants (Fig. 2A). After log transformation, the y-axis intercepts of the regression lines were found to be significantly different (P < 0·0001), while the slopes were not (P > 0·05). Secondly, there was no significant difference between the slopes of the regression lines modelling MS leaf number on thermal time for the two bunch species (P > 0·05; Fig. 2C). This result indicates MS phyllochron similarity, with 94·3 °Cd leaf⁻¹ for L. perenne and 90·9 °Cd leaf⁻¹ for A. stolonifera. Thirdly, both species were characterized by a similar increase of the numbers of primary and secondary tillers with about 12 tillers at 761 °Cd (Fig. 3A, and B). Their development differed markedly from that of P. pratensis, in which primary tillers occurred at approx. 345 °Cd (i.e. about 150 °Cd later than in the two other species), and their number did not exceed 6 at 37 DAT (Fig. 3C). Tertiary tillers appeared at about 478 °Cd in L. perenne and only at about 618 °Cd in A. stolonifera. Nevertheless, the two species showed similar increases in phytomer (or leaf) numbers across time (Fig. 3D and E), and at the end of the experiment, no statistical difference could be found between the numbers of phytomers on primary (around 45), secondary (around 32) and tertiary tillers (around 5). In contrast, the number of phytomers per plant of P. pratensis remained low (Fig. 3F).

Fig. 3.

Increase in the number of tillers and phytomers in relation to thermal time. Evolution of the number of MS and new shoots on rhizomes (closed circles), primary (open circles), secondary (filled squares) and tertiary tillers (open triangles) in (A) L. perenne, (B) A. stolonifera and (C) P. pratensis. Numbers of phytomers on primary (open circles), secondary (filled squares) and tertiary tillers (open triangles) in (D) L. perenne, (E) A. stolonifera and (F) P. pratensis. Data are means and standard errors from 12 plants grown in a greenhouse for 37 DAT.

Biomass partitioning

Dry weights of above-ground samples were not significantly different between L. perenne and A. stolonifera (P > 0·05) except for primary tillers, which had a larger biomass in L. perenne (P = 0·0126; Fig. 4A). In both species, there was approx. 70–80 % less dry matter by tiller for secondary and tertiary tillers compared with MS and primary tillers (P < 0·001; Table 2). Furthermore, dry matter allocated by tiller to MS, primary, secondary and tertiary ramifications was not different between the two species (P > 0·05; Table 2). In contrast, biomass of primary and secondary tillers was significantly lower in P. pratensis than in the other two species (P < 0·0001; Fig. 4A). Biomass by tiller was also clearly lower in P. pratensis than in the other species for primary shoots (P < 0·0001) as well as for samples in which MS and R were pooled (P < 0·005; Table 2).

Fig. 4.

Biomass of (A) above-ground parts, (B) below-ground parts and (C) ratio of above-ground : below-ground parts (Ab./(Bel.) in L. perenne (open columns), A. stolonifera (grey columns) and P. pratensis (black columns), sampled after 37 DAT. MS indicates the seedling main stem, T is used for seedling ramifications (primary, T I- tillers; secondary, -T II- tillers; tertiary, -T III-tillers), and R is used for shoots from rhizomes (Rh). Below-ground parts include roots and rhizomes. Means and standard error are given for six plants.

Table 2.

Mean dry weight (mg) allocated by tiller in L. perenne, A. stolonifera and P. pratensis

Kinds of tillers	L. perenne	A. stolonifera	P. pratensis	P rows
MS + R	159·83 ± 20·62aA	126·33 ± 22·51aA	64·42 ± 11·84aB	0·0043
T I	143·55 ± 12·24aA	118·28 ± 11·35aA	41·91 ± 7·20aB	<0·0001
T II	51·92 ± 4·52bA	32·12 ± 5·04bAB	33·00 ± 4·42aB	0·0072
T III	20·20 ± 1·87cA	15·30 ± 2·34cA	–	n.s.
P columns	0·0006	0·0007	n.s.

MS, Seedling main stem; R, shoots from rhizome; T I, primary, T II, secondary and T III, tertiary tillers.

Data are means ± standard error of six plants grown in a greenhouse for 37 DAT. A, B and C indicate significant differences between medians in rows and a, b and c significant differences between medians in columns. P values are given according to Kruskal–Wallis test. n.s., Non-significant (P > 0·05).

Lolium perenne allocated significantly more biomass to below-ground parts than the other two grasses (P < 0·0001; Fig. 4B). Despite an efficient branching, the ratio of above-ground parts/roots was clearly lower in L. perenne (2·5) than in A. stolonifera (5·3) and P. pratensis (4·6; P < 0·001; Fig. 4C). In the rhizomatous P. pratensis, below-ground biomass was distributed between rhizomes and roots (Fig. 4B). Unexpectedly, when rhizomes were taken into account, no difference could be found between L. perenne and P. pratensis in the ratio of above-ground : below-ground biomass (P > 0·05; Fig. 4C).

Leaf appearance rate, tiller location and site-filling

Across all species and tillers, between 0·14 and 0·43 leaf emerged per day (Table 3). In L. perenne, the LAR reached 0·29 leaf d⁻¹ on MS, and there was no significant difference among the different ramifications (P > 0·05; Table 3). In P. pratensis, there was no significant difference among the LAR on MS, T₂, T₃ and T₄. However, LAR on R was about 30–50 % higher than on the other shoots (P < 0·05; Table 3). In A. stolonifera, LAR on T₅ and on secondary tillers was almost 30 % higher than on MS and on all other primary tillers (P < 0·05; Table 3). Leaf emergence rate on MS did not differ significantly between L. perenne and A. stolonifera, while in P. pratensis, it was approx. 30 % lower (P < 0·05; Table 3).

Table 3.

Mean leaf appearance rate on tillers (leaf d⁻¹) in L. perenne, A. stolonifera and P. pratensis (prophylls excluded)

Shoot location	L. perenne	A. stolonifera	P. pratensis	P rows
MS	0·29 ± 0·01aA	0·30 ± 0·02aA	0·20 ± 0·02aB	0·0042
Primary tiller emerged at:
leaf 1 axil on MS (T1)	0·29 ± 0·05aA	0·29 ± 0·04aA	–	n.s.
leaf 2 axil on MS (T2)	0·26 ± 0·03aA	0·30 ± 0·03aA	0·16 ± 0·01aB	0·0013
leaf 3 axil on MS (T3)	0·24 ± 0·04aA	0·33 ± 0·04abA	0·16 ± 0·02aB	0·0030
leaf 4 axil on MS (T4)	0·27 ± 0·03aA	0·35 ± 0·01aB	0·14 ± 0·03aC	<0·0001
leaf 5 axil on MS (T5)	0·33 ± 0·02aA	0·40 ± 0·01bcB		0·0043
Secondary tillers	0·31 ± 0·04aA	0·43 ± 0·03cB	–	0·0253
R	–	–	0·28 ± 0·02c	–
P columns	n.s.	0·0185	0·0020
P MS and R	–	–	0·0405

MS, Seedling main stem; R, shoots from rhizome.

Data are means ± standard error of 12 plants grown in a greenhouse during 37 DAT.

A, B, C indicate significant differences between medians in rows and a, b, c significant differences between medians in columns. P values are given according to Kruskal–Wallis test. n.s., Non-significant (P > 0·05).

In the three grass species, MS began to produce primary tillers after the appearance of five leaves (with the coleoptile included; Table 4). In L. perenne, T₁ was the first primary tiller to emerge in 66·7 % of the seedlings, and T₂ in the others (results not presented). In A. stolonifera, T₁ was the first primary tiller to appear in 58·3 % of the plants, and T₂ in the others. In both species, secondary and tertiary tillers emerged at the axil of leaf 1 and appeared when the ‘parent’ tiller showed four leaves (with the prophyll included; Table 4). In contrast, in P. pratensis, T₂ was always the first primary tiller. ‘Daughter’ tillers began to emerge when ‘parent’ tillers had four leaves (with the prophyll included; Table 4). In P. pratensis, R1 appeared when MS had a mean of 6·3 ± 0·3 leaves (Table 4). The interval between two successive R emerging was approx, 33 % higher than the interval between two successive leaf stages on MS (ΔMS = 2/3 ΔR; Table 4).

Table 4.

Leaf number on a ‘parent’ shoot when the first ‘daughter’ tiller emerged in L. perenne, A. stolonifera and P. pratensis (coleoptile leaves and prophylls included)

	Phytomer number on the ‘parent’ shoot at first ‘daughter’ tiller emergence
	L. perenne	A. stolonifera	P. pratensis
Leaf number on MS at growth of:
1st T I	5·7 ± 0·2a	5·3 ± 0·1a	5·8 ± 0·2a
R1	–	–	6·3 ± 0·3b
R2	–	–	6·7 ± 0·3bc
R3	–	–	7·4 ± 0·3c
R4	–	–	8·2 ± 0·2d
R5	–	–	9·3 ± 0·2e
Leaf number on T I at growth of:
1st T II on T1	4·7 ± 0·2b	4·1 ± 0·2b	–
1st T II on T2	4·3 ± 0·1b	4·2 ± 0·1b	4·0 ± 0f
1st T II on T3	4·1 ± 0·1b	4·6 ± 0·3ab	4·0 ± 0f
1st T II on T4	4·0 ± 0·2b	4·5 ± 0·3ab	–
1st T II on T5	4·3 ± 0·3b	–	–
Leaf number on T II at growth of 1st TIII	4·5 ± 0·3b	4·5 ± 0·8ab	–
P columns	<0·0001	<0·0001	<0·0001

MS, Seedling main stem; R, shoots from rhizome; T I, primary, T II, secondary and T III, tertiary tillers.

Data are means ± standard error of 12 plants grown in a greenhouse for 37 DAT.

P values are given according to Kruskal–Wallis. a, b and c indicate significant differences between medians in columns.

In the present study, no primary tiller emerged from the coleoptile. Only 3–5 % of the buds at the axil of a prophyll developed. There was no significant difference among species in the increase in total tiller numbers at the successive leaf stages (values were between 1·45 and 1·76; P > 0·05; Table 5), nor in the ratio of number of new leaves over number of new tillers (approximately four for all the species; P > 0·05; Table 5). As a consequence, site-filling did not differ significantly among the three species, and was between 0·523 and 0·606 tiller tiller⁻¹ LAR⁻¹ (P > 0·05; Table 5).

Table 5.

Increase in the total number of tillers (T), site-filling (Fs) and ratio of the number of new leaves over the number of new tillers (dL/dT) at the successive leaf stages of the main stem in L. perenne, A. stolonifera and P. pratensis

	L. perenne	A. stolonifera	P. pratensis	P (rows)
Increase in T (= exp Fs)
Case 1	1·62 ± 0·06	1·63 ± 0·16	–	n.s.
Case 2	1·76 ± 0·11	1·64 ± 0·06	1·45 ± 0·66	n.s.
Site-filling (Fs)
Case 1	0·523 ± 0·02	0·545 ± 0·02	–	n.s.
Case 2	0·557 ± 0·02	0·598 ± 0·02	0·606 ± 0·04	n.s.
dL/dT
Case 1	3·42 ± 0·18	4·00 ± 0·18	–	n.s.
Case 2	3·47 ± 0·27	4·24 ± 0·33	4·30 ± 0·34	n.s.

Values are means ± standard error of 12 seedlings grown in a greenhouse for 37 DAT.

In case 1, the first tiller emerged at the axil of leaf 1. In case 2, the first tiller appeared at the axil of leaf 2.

n.s., Non-significant according to Kruskal–Wallis test (P > 0·05).

Tillering models

Tillering models were constructed for each species based on (a) mean LAR on the different tillers, as presented in Table 3, (b) mean leaf number on ‘parent’ tiller when either the first ‘daughter’ tiller or the first R emerged, as shown in Table 4 and (c) location of the first ‘daughter’ tiller on the ‘parent’ tiller, as observed in a majority of experimental plants for each species. Thus, equal values for LAR were assumed on all tillers in L. perenne, and on most shoots of A. stolonifera (except for T₅ and secondary tillers). It was also considered that leaf 1 and the prophyll (leaf 0) emerged at the same time on tillers. In P. pratensis, the LAR on MS was one-third lower than on L. perenne. For L. perenne and A. stolonifera, it was decided that the first primary tiller would appear at the axil of leaf 1, and of leaf 2 for P. pratensis. R appearance rate in P. pratensis was chosen to be one-third higher than the LAR of MS of L. perenne. LAR on R was also considered to be one-third higher than on ‘mother plant’ tillers.

The values obtained from the tillering models were close to the observed data. In the diagrams, when the MS presents 11 leaves, the number of shoots reaches 31 in L. perenne and 27 in A. stolonifera (Table 6A and B). After 37 DAT, 29·17 ± 2·6 s.e.) shoot plant⁻¹ were obtained experimentally when there were 10·7 ± 0·2 (standard error) leaves on MS for L. perenne, and 20·25 ± 4·29 shoot plant⁻¹ when the MS had 11·2 ± 0·4 leaves for A. stolonifera. Poa pratensis produced 8·5 ± 0·89 leaves on the MS during the experiment and on average 7·5 ± 0·63 shoots plant⁻¹ while the tillering model gives a theoretical amount of 12 tillers when MS has eight leaves. From extrapolation, the theoretical number of tillers produced by a plant of P. pratensis at stage 11 leaves on MS is approx. 30 and 50 % higher than the shoot number obtained at the same plant stage in A. stolonifera and L. perenne, respectively.

Table 6.

Emergence pattern of the successive tillers in (A) L. perenne, (B) A. stolonifera and (C) P. pratensis

NpLp	Ramification emergence	Shoot number
(A) Lolium perenne when the 1st primary tiller emerges at the axil of leaf 1
1	MS	1
5	T1	2
6	T2	3
7	T3 – T11	5
8	T4 – T12 – T21	8
9	T5 – T13 – T22 – T31 – T111	13
10	T6 – T14 – T23 – T32 – T41 – T112 – T211	20
11	T7 – T15 – T24 – T33 – T42 – T51 – T113 – T212 – T221 – T311 – T1111	31
End of observations
12	T8 – T16 – T25 – T34 – T43 – T52 – T61 – T114 – T213 – T222 – T231 – T312 – T321 – T411 – T1112 – T1121 – T2111	48
13	T9 – T17 – T26 – T35 – T44 – T53 – T62 – T71 – T115 – T214 – T223 – T232 – T241 – T313 – T322 – T331 – T412 – T421– T511 – T1113 – T1122 – T1221 – T2113 – T2211 – T3111 – T11111 – T21111	75

NpLp	NpAs	Ramification emergence	Shoot number
(B) Agrostis stolonifera when the 1st primary tiller emerges at the axil of leaf 1
1	1	MS	1
5	5	T1	2
6	6	T2	3
7	7	T3 – T11	5
8	8	T4 – T12 – T21	8
9	9	T5 – T13 – T22 – T31	12
10	10	T6 – T14 – T23 – T32 – T41 – T111	18
11	11	T7 – T15 – T24 – T33 – T42 – T51 – T112 – T121 – T211	27
End of observations
12	12	T8 – T16 – T25 – T34 – T43 – T52 – T61 – T113 – T114 – T122 – T212 – T311 – T321 – T1111	41
13	13	T9 – T17 – T26 – T35 – T44 – T53 – T54 – T62 – T71 – T115 – T123 – T124 – T112 – T213 – T214 – T312 – T322– T411 – T511 – T1112 – T1121 – T1211 – T2111	64

NpLp	NpPp	Ramification emergence	Shoot number
(C) Poa pratensis when the 1st primary tiller emerges at the axil of leaf 2
1	1	MS	1
6	4
–	5	T2	2
9	6	T3 – R1	4
–	7	T4 – T22 – R2 – R3	8
12	8	T5 – T23 – T32 – R4	12
End of observations
–	9	T6 – T24 – T33 – T42 – T222 – R13 – R5 – R6	20
15	10	T7 – T25 – T34 – T43 – T52- T223 – T232 – T322 – R7 – R14 – R15 – R23 – R33	33
–	11	T8 – T26 – T35 – T44 – T53 – T62 – T224 – T233 – T323 – T332 – T422 – T2222 – R8 – R9 – R16 – R24 – R25 – R34 – R35 – R43 – R133	54

MS, Seedling main stem; T, seedling ramifications; R, shoots from rhizomes.

Np gives the number of phyllochrons on the MS, for L. perenne (NpLp), A. stolonifera (NpAs) and P. pratensis (NpPp). For an easier comparison between the three grasses, NpLp is presented as reference, left of the values of NpAs in (B) and left of the values of NpPp in (C). Subscripts indicate ‘parent’ leaves of tillers. Tillering models were calculated from values given in Tables 3 and 4.

DISCUSSION

Previous studies have described tiller dynamics in Lolium perenne (Mitchell, 1953; Richards et al., 1988; Neuteboom and Lantinga, 1989; Yang et al., 1998; Gautier et al., 1999). The present results on phyllochron value and total number of tillers at 37 DAT are consistent with those of Gautier et al. (1999). Taking into account the tiller prophyll in their model, Neuteboom and Lantinga (1989) obtained a maximum value for site-filling equal to 0·693; in that case, total tiller number would double at successive leaf stages. In the present experiment, all primary tillers emerged from the bud located immediately above the previous tiller. However, the plants showed a consistent lack of tiller emergence at the axil of the coleoptile and an irregular development of tillers at the axil of the prophylls. As a consequence, the site-filling value for L. perenne (and for the other species studied) seems low for isolated plants grown without trophic constraints. Nevertheless, the site-filling values were higher than 0·481, the maximum site-filling value expected for L. perenne when neither coleoptile nor prophyll tillers are produced (Davies, 1974).

The site-filling found was close to the value obtained by Bahmani et al. (2000) who studied seedlings within a population and demonstrated that shade drastically reduced the number of tillers produced by plants of L. perenne, with no change of the phyllochron. Cattani and Struick (2001), who conducted a similar short experiment with creeping bentgrass under long days (16 h) at 20 °C/15 °C, did not observe either the stolons they expected under low light intensity. Several authors have shown that a decrease of the red : far red ratio or an increase in blue radiation have the same effects (Deregibus et al., 1983; Casal et al., 1986; Warringa and Kreuzer, 1996; Gautier et al., 1999). On the other hand, in barley, Fletcher and Dale (1977) demonstrated that LAR (and phyllochron) was unaffected by shading.

In Angers, mean light intensity tends to increase from 430µmolm⁻²s⁻¹ at the beginning of March to 690µmolm⁻²s⁻¹ in mid-April (data collected over 25 years by INRA). It is likely that in the present experiment, tillering was reduced by a low level of photosynthetic active radiation, while warm temperatures enhanced leaf and bud productions. From the results published by Cao and Mos (1989) in wheat and barley, it appears that 12–13·5 h day length is not short enough to have a major effect on phyllochron. A longer day length (16 h) would have certainly enhanced initiation of the coleoptile tiller (Kirby et al., 1985), as has also been reported for A. stolonifera (Cattani et al., 2002). This may explain why the values of ΔL/ΔT were about 20 % greater than those obtained by Neuteboom and Lantinga (1989).

As had been observed in ryegrass by Mitchell (1953), the LAR in L. perenne was similar among all tillers, so that the tillering models of Masle-Meynard and Sébillotte (1981b) and Kirby et al. (1985) for wheat and barley were easy to transpose to this species. Kirby et al. (1985) proposed a model where the first primary shoot emerges at the axil of the coleptile leaf when the MS presents three fully expanded leaves (coleoptile included). In Masle-Meynard and Sébillote's experiments (Masle-Meynard and Sébillote, 1981b), T₁ was the first primary shoot and appeared when the MS had five fully expanded leaves. It was observed that in L. perenne and A. stolonifera, T₁ was the first primary shoot in most cases. On about 35 % of the plants of each species, the first primary shoot emerged at the axil of leaf 2. In the three species, primary and secondary first ‘daughter’ tillers emerged one bud earlier than on the MS. The variability was greater in A. stolonifera than in L. perenne and this might be partly due to the fact that A. stolonifera is an obligate outcrosser (Cattani et al., 2002).

Above-ground parts of L. perenne and A. stolonifera expressed similar bunch-type behaviours with the same MS phyllochron values. Their development was difficult to discriminate statistically. In the two species, the number of shoots increased exponentially with time. They both reached the fourth level of ramification at 37 DAT, and allocated similar amounts of dry matter to the different ramifications. Even though tiller dynamics and distribution appeared globally similar in both species, the production of the first ‘daughter’ tiller in A. stolonifera was delayed by one leaf interval on secondary axes and on the upper primary axes compared with L. perenne. By extrapolation from the tillering models, the highest LAR found on secondary tillers compared with the MS and primary tillers would not compensate for the delay in phytomer production. These differences are important to discriminate growth strategies of the two bunch types. They might be linked to the morphology of the phytomers; L. perenne is known to form compact tussocks with large numbers of long leaf blades, while A. stolonifera produces shorter leaf blades with larger and longer internodes useful for horizontal creeping (around 1 cm longer than in L. perenne). Because the production of few large tillers requires more carbon available within the plant than the production of many small tillers (Sugiyama, 1995), these morphological differences might partly explain the higher shoot : root ratio measured in A. stolonifera compared with L. perenne (50 % higher). Lolium perenne is a compact plant that actually colonizes both soil and air vertically, and new ramets are produced very close to the MS (Grime et al., 1988). By contrast, stolons of A. stolonifera provide large amounts of nitrogen and soluble carbohydrates to new ramets rooting at substantial distances from the seedling MS, especially in the later stages of plant development.

Olff et al. (1990) stressed that the combination of light intensity and nutrient supply is important for shoot : root ratios in several grasses. The present values seem to be in agreement with results obtained with L. perenne in comparable nutrient, light intensity and temperature conditions (Olff et al., 1990). Lolium perenne is known to be a good competitor for both light and nutrients. Lolium perenne is also known to contain large amounts of nitrogen compounds and water-soluble carbohydrates useful to support ramification (Ourry et al., 1988; Warringa and Kreuzer, 1996; Santos et al., 2002). Surprisingly, despite its intense root development, previous authors have shown that L. perenne has less ability to use N for regrowth of laminae after defoliation than other growth forms, even when N is supplied. Sink/source relationships in grasses have been described under successive defoliations in L. perenne (Ourry et al. 1988), and in different growth forms such as the caespitose–rhizomatous Poa trivialis, the caespitose–stoloniferous Agrostis castellana, and the rhizomatous Festuca rubra (Thornton et al., 1994).

In P. pratensis, biomass allocation to MS (+ R), primary and secondary tillers was much lower than in the other species. Rhizome growth seemed to be favoured compared with above-ground development. In this species, rhizomes are important sinks of nutrients for new ramets (rooted R and ramifications). However, on the MS and tillers, LAR as well as the biomass allocated to above-ground parts were 60 % lower in P. pratensis than in L. perenne. As a consequence, root : shoot ratios were similar between the two species even though they expressed opposite growth strategies. For example, >75 % of below-ground parts in P. pratensis were rhizomes, i.e. storage organs useful for the production of ramets far from the MS (Grime et al., 1988). While MS development was 30 % slower in P. pratensis than in the other species, the interval between the emergence of two successive rhizomes was approximately equal to a phyllochron on the MS of L. perenne and A. stolonifera. In addition, these new shoots had similar LAR than the rate observed on the MS of the other two species.

In conclusion, even though L. perenne had the fastest tiller production at the earliest stages of seedling development, A. stolonifera and P. pratensis compensated almost completely for the delay. No significant differences were observed in the rate of emergence of the successive tillers and in site-filling values on the MS of the three species, despite their contrasted growth forms. This study provides a basis for modelling plant development. However, changes in morphogenesis of these species with contrasting growth tactics are expected under different growth conditions, such as interplant competition, and longer studies on the development of rhizomes in P. pratensis and of stolons in A. stolonifera are required.