Survival and recovery of perennial forage ... - Wiley Online Library

New Phytol. (1998), 140, 439–449

Survival and recovery of perennial forage grasses under prolonged Mediterranean drought I. Growth, death, water relations and solute content in herbage and stubble B  F L O R E N C E V O L A I R E"*, H E N R Y T H O M A S#    F R A N C: O I S L E L I E V R E" " Institut National de Recherche Agronomique (INRA), LEPSE, 2 place Viala, 34060 Montpellier, Cedex 01, France # Institute for Grassland and Environmental Research, Plas Gogerddan, SY23 3EB, Ceredigion, UK (Received 10 December 1997 ; accepted 17 July 1998)  Swards of Dactylis glomerata cultivars (cvs) KM2 and Lutetia and of Lolium perenne cvs Aurora and Vigor were grown under full irrigation or prolonged summer drought (80 d) in a field experiment in the South of France. After irrigation was withheld, leaf extension rates of all cvs fell by 90 % within 9–12 d, and rapid scorching of laminae followed. Tiller mortality at the end of the drought was very different in the cocksfoot cvs (4 % for KM2 and 76 % for Lutetia) and intermediate (41 %) for both ryegrass cvs. Following re-watering, rates of herbage regrowth were closely correlated with tiller survival. Measured minerals contributed c. 0n52 MPa to osmotic potential in all treatments, whereas water-soluble carbohydrates (WSC) contributed 0n25 MPa under irrigation and 0n46 MPa during drought. There was no systematic difference between the two species for summer survival under severe drought, but large differences between the cocksfoot cvs. The traits most strongly associated with superior survival were : (a) a deep root system and greater water uptake at depth ; (b) low water and osmotic potentials in surviving laminae, i.e. better tolerance to dehydration ; (c) large pool-size of WSC reserves (fructans having degree of polymerization 4) in entire tiller bases (stubble) ; (d) low accumulation of proline in stubble ; (e) rapid nitrogen uptake after rewatering. Key words : Dactylis glomerata L. (cocksfoot), Lolium perenne L. (ryegrass), drought survival, water relations, solutes, carbohydrates.

 Drought is the major climatic factor limiting annual production of forages, cereals and other crops in temperate regions (Boyer, 1982). For most cultivated perennial forage grasses growing in Mediterranean rainfed areas, summer growth is inversely correlated with survival and persistence (Knight, 1973). Therefore the most important strategy is not maintenance of production during drought, but the ability to survive and recover rapidly after autumn rains * To whom correspondence should be addressed. E-mail : volaire!ensam.inra.fr

(Kemp & Culvenor, 1990). Since this aspect of drought resistance has received relatively little research attention, it is essential to understand the underlying mechanisms in order to develop perennial forage cvs suitable for sustainable grasslands in a Mediterranean climate. The aim of this study was to analyse the main developmental and physiological traits that might contribute to persistence of field-grown perennial forage grasses during prolonged Mediterranean drought. Promising traits included survival of leaves and tillers, deeper root profiles, maintenance of water status, and osmotic adjustment (Silsbury, 1964 ; McWilliam & Kramer, 1968 ; Biddiscombe,

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440

F. Volaire, H. Thomas and F. LelieZ vre

Rogers & Maller, 1977 ; White et al., 1992 ; Barker, Sullivan & Moser, 1993). Particular attention was paid to carbohydrate reserves, since there is strong evidence that these contribute to survival of cocksfoot (Dactylis glomerata L.) when photosynthetic leaf area is lost (Volaire, 1991, 1994, 1995). We also measured composition and contribution of solutes (minerals, amino acids, proline, sugars) to osmotic adjustment because no information was available for forage swards grown under such severe natural drought. We compared two important forage species, cocksfoot, which includes cvs adapted to the Mediterranean climate, and perennial ryegrass (Lolium perenne L.), which is generally more susceptible to drought. Two cvs of each species were chosen on the basis of their contrasting heading date, since early heading has been associated with drought survival in cocksfoot (Volaire, 1991 ; Volaire & Lelie' vre, 1997) and with herbage production during drought in perennial ryegrass (Thomas & Evans, 1990). This paper considers whole-plant responses, and deals with two tissue fractions : leaf laminae (approximately equivalent to herbage, the economically valuable part of a forage plant), and entire tiller bases (equivalent to the stubble remaining after defoliation).    Plant material The populations studied were : Dactylis glomerata L. (cocksfoot) cv. KM2 (synonym Medli), an earlyheading variety bred by INRA in the South of France ; D. glomerata cv. Lutetia, a late-heading variety bred by INRA in northwest France ; Lolium perenne (perennial ryegrass) cv. Aurora, an early variety from IGER based on populations collected in Swiss Alps ; and L. perenne cv. Vigor (synonym Melle), a late variety bred by RvP, Belgium. Experimental design and management The field experiment was carried out at the Mauguio Research Station of INRA, Montpellier (southern France). The soil was a loamy clay, with low organic matter content and pH (water) of 8n3 and was fertilized with 250 kg ha−" P O and K O before # & # sowing on 18 October 1994. The two treatments, summer drought and full irrigation, were imposed on close but separate areas. Each treatment area consisted of three blocks, each containing four 3-m# plots to which the cvs were randomly allocated. Each plot consisted of five drills 3 m long and 17 cm apart into which seed was sown at a rate of 3 g m−#. All plots were fertilized as usual for semi-intensive Mediterranean grasslands (210 kg N ha−" from December to June). Irrigated plants were further

supplied with 60 kg N ha−" on 7 July. The summer drought treatment was imposed from 1 July to 20 September (the ‘ drought period ’) by covering the plots with a clear polythene shelter as briefly as possible whenever rain was expected. The control treatment was irrigated every 2–4 d to maintain a calculated soil moisture deficit of 25 mm. From 20 September to 18 October (the ‘ recovery period ’) both treatments were fully irrigated. Between 28 April and 16 June 1995, the two earlyheading populations (‘ KM2 ’, ‘ Aurora ’) were cut to 5 cm three times, whereas the two late-heading populations (‘ Lutetia ’, ‘ Vigor ’) were cut only twice. In the irrigated treatment, plants were cut on 28 July, 4 September, and 28 September. All plots were cut on 18 October, the end of the recovery period. Sward structure and dynamics Samples of sward were taken on days 3, 26, 48 and 73 after the start of the drought. Control plants were sampled on days 26 and 73 only. After rewatering on day 82, plants were sampled on days 90 (or 96), 103, 109 and 113, depending of the variables to be measured. At each date, sward samples of area 1 dm#, consisting of the aerial parts and c. 3 cm of surface soil and roots, were dug from each plot, stored over ice, and taken to the laboratory. Numbers of live tillers, green herbage biomass, green leaf area and weight of stubble (tiller bases) were determined. During the drought period, lamina length was measured every 2–3 d on 8 mature tillers in each plot, a total of 24 tillers per cv. In the drought treatment, the length of dying (brown) lamina was measured very 3–4 d until no green laminae remained. Leaf extension and death rates (mm d−") and leaf appearance rates (leaves tiller−" d−") were calculated from these values. Root density and water content of soil On day 81, just before rewatering, a trench (depth 1n5 m, width 0n4 m, length 15 m) was dug along one side of the droughted plots with a mechanical digger. Soil samples were immediately collected at five depths, sealed, and taken to the laboratory for measurement of soil moisture content. The number of visible roots on the exposed face of the trench was counted using a vertical 0n2i0n1 m quadrat at 20-cm deep intervals under every plot. The maximum root depth was also recorded. Water relations Water potentials and osmotic potentials were measured on plants taken around dawn. Leaf water potential was measured on three detached green laminae from each plot, using a pressure chamber. These laminae were rapidly transferred to k20 mC


Drought survival in perennial forage grasses. I for subsequent determination of osmotic potential with a Wescor2 5100C osmometer or Wescor C52 chambers and a micro-voltmeter. Relative water content () were measured by weighing laminae tissue before and after rehydration overnight at 0 mC and after drying at 85 mC for 24 h. Because of rapid leaf mortality, it was not possible to sample sufficient living ryegrass laminae on days 48 and 73, or cocksfoot laminae on day 73. Solute concentrations Plant samples, taken as described above, were divided into two fractions of green tissue : herbage (laminae) and stubble (mature leaf sheaths and enclosed tissues), which were frozen and freezedried. Dead tissue was discarded, as were roots. One aliquot of dried tissue from each sample was extracted and partitioned in methanol-chloroformwater. Using the aqueous phase, total free amino acids and the imino acid proline were determined colorimetrically, and Na, Mg and Ca by flame photometry (Thomas, 1991). Another set of aliquots of dried tissue was extracted in 40 % ethanol at 85 mC, and purified with activated charcoal. Watersoluble carbohydrates (WSC) were quantified by HPLC using an Aminex HPX 42-C column and a differential refractometer calibrated against glucose, fructose, sucrose and inulin. Fructans having a degree of polymerization (DP) of 3 and 4 (‘ low DPfructans ’) and over 4 (‘ high DP-fructans ’) were quantified. Since the entire tiller bases (stubble) contain the bulk of reserve carbohydrates needed by grasses to survive drought when all photosynthetic leaf area is lost, we estimated the size of the WSC pool as the product of WSC concentrationiliving biomass of entire tiller bases per unit ground area. Contributions of solutes to bulk osmotic potential were estimated following Thomas (1991). Statistical analyses The data were analysed using the appropriate analysis of variance models in the SAS and Genstat statistical packages. Because of the experimental design and of different defoliation frequency in irrigated and droughted treatments, data from both treatments were analysed separately. It was not aimed to analyse the effect of drought per se but mainly to compare the responses to drought exhibited by the four populations.  Growth and growth components Leaf extension, death and appearance rates. In droughted plots, leaf extension rates (Fig. 1) declined rapidly, reaching 50 % of the initial rates 4–6 d after the last irrigation, and 10 % after 9–12 d. Leaf

441 extension stopped on 50 % of tillers after 9–13 d, and on 90 % of tillers by 14–19 d. Only 0n7–0n9 leaves emerged per tiller during drought, and there were no significant differences between cvs (data not shown). Following rewatering, the mean leaf extension rate of previously droughted tillers recovered rapidly to equal the rate in well irrigated tillers after 3 d, and subsequently exceeded it by c. 50 % (Fig. 1). Previously droughted plants produced leaves at the rate of 0n16 leaves tiller−" d−", c. 25 % faster than did continuously irrigated plants (data not shown). Tiller densities and herbage production (Table 1). At the beginning of the drought period, and in irrigated plots, mean tiller densities of ryegrasses were twice that of ‘ Lutetia ’, with ‘ KM2 ’ intermediate. In the irrigated treatment, cocksfoot grew faster than the ryegrass both during and after the drought period. During drought, ‘ KM2 ’ lost very few tillers (4 %), whilst about 76 % of ‘ Lutetia ’ tillers died. Following re-watering, ‘ KM2 ’ resumed tillering slowly, and after 4 wk exceeded pre-drought densities, whereas ‘ Lutetia ’, although it resumed tillering much more rapidly, achieved only about half of its original density. Death rates in the droughted plots were about half of the growth rates in the irrigated plots. The greatest difference between cvs was expressed after the droughted plots were rewatered, when ‘ KM2 ’ regrew more than three times faster than ‘ Lutetia ’. Tiller densities and regrowth rates of the ryegrasses were very similar to each other, and intermediate between those of the cocksfoot cvs. Water content of soil and root system Soil moisture at field capacity was 16 % at depths of 20 and 40 cm. After 80 d of drought the soil moisture fell to 5–7 % in the two top horizons for all populations (Fig. 2). Between 50 and 90 cm deep, soil moisture was significantly higher (P 0n05) under ‘ Lutetia ’ (10n5 %) than under the other populations (9n6 %). Water reserves located below a depth of 100 cm were not explored by the roots. The maximum root depth in ‘ Lutetia ’ was 70 cm, significantly shallower than in the other populations (93–106 cm). Root density of Lutetia at 50–90 cm was a half to a third that of the other cvs. Water relations Dawn water potentials (Ψw) in droughted laminae of all cvs were significantly less than zero only 3 d after the final irrigation, although RWC was near 100 % (Table 2). Solute potentials (Ψs) on this date were similar in all cvs at k1n2 to k1n3 MPa. We have few data on Ψw for the ryegrasses during drought because most leaves were scorched at the distal end, but measurements made on green parts of laminae on day 26 show that RWC had declined to about 70 % in all cvs. Comparisons can be made of cocksfoot


Rewatering


Leaf extension as proportion of control rate

442

2·5

2

1·5

1

0·5

0 0

10 20 30 Days after start of drought

80

110 90 100 Rewatering period

Figure 1. Leaf extension rate following cessation of irrigation on 30 June 1995, and following rewatering after 80 d of drought, expressed as a proportion of control rate, in Dactylis glomerata cvs KM2 (#——#) and Lutetia ( —— ), and Lolium perenne cv. Aurora (=– – –=) and Vigor (W– – –W).

Table 1. Herbage growth and senescence rates (HGR, g dry matter m−# d−") in plots of Dactylis glomerata cvs KM2 and Lutetia, and Lolium perenne cvs Aurora and Vigor during the drought period (30 June – 20 September 1995) and during the recovery period following rewatering (20 September – 18 October) HGR Treatment cultivar Irrigation ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ‘ Vigor ’ , cvs Drought during summer ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’ , cvs

Recovery period

Live tiller density (m−#) end of drought

3n02 2n83 2n56 2n21 1n04

6n84 5n89 4n78 4n45 0n24

6716 4645 11283 10884 265

k1n81 k1n41 k1n23 k0n86 0n10

7n23 2n19 4n62 4n07 0n48

6477 1134 6608 6441 504

Drought period

Tiller survival (%)

Live tiller density, end of recovery

96 24 59 59

7487 2685 10074 9957 596

Growth rates of irrigated plots during the drought period were calculated from three cuts (on 28 July and 4 and 28 Sept), and of both treatments during the recovery period from a single cut (on 19 Oct). Death rates of droughted plants were calculated from regression against time of mass of green herbage in subsamples taken on days 3, 26, 48 and 73 after final watering. Live tiller density (m−#) are given for the 4 cultivars at the end of the drought and recovery periods. Tiller survival (% of initial density). , standard error of the means.

water status until day 48 : in ‘ KM2 ’ laminae, Ψw, Ψs and RWC declined steadily, while in surviving plants of ‘ Lutetia ’ the change was much slighter, and Ψw and Ψs were not significantly different from pre-stress values. Water-soluble carbohydrates The main effect of drought on both herbage and stubble fractions was to increase WSC concentrations, at least over the first 45 d of drought (Fig.

3 a, f ). This was due to an increase in concentration of fructans having a high degree of polymerization (DP 4) (Fig. 3 b, g) and of sucrose (Fig. 3 d, i), although there was a decrease in concentration of low-DP fructans. On re-watering, total WSC, fructan and sucrose levels fell to levels similar to those of irrigated plants, whilst concentrations of monosaccharides increased, particularly in stubble (Fig. 3 e, j). There were no consistent differences between species, but the cvs did express a number of


Drought survival in perennial forage grasses. I 10

443

(b)

(a)

Soil depth (cm)

30 50 70 90 110 130 150 4

6

8 10 Soil moisture (%)

12

15 30 45 60 Number of roots × 10–2 m–2

0

75

Figure 2. (a) Soil moisture and (b) distribution of root density with soil depth after 80 d of drought, in two cvs of cocksfoot : ‘ KM2 ’ (#——#), ‘ Lutetia ’ ( —— ) and two cvs of ryegrass : ‘ Aurora ’ (=– – –=), ‘ Vigor ’ (W– – –W). Horizontal bars represent standard error of means, d.f. l 6.

Table 2. Water status at dawn on days 3, 26 and 48 after final watering date, of laminae of two cvs of cocksfoot (K, L) and two cvs of perennial ryegrass (A, V ) subjected to full irrigation in summer (means over days 3, 6 and 73) or to 80 d of drought under field conditions ΨW (kMPa)

Ψs (kMPa)

 (%)

Cultivars …

K

L

A

V

K

L

A

V

K

L

A

V

Irrigation  (cvs) Drought d3 d26 d48  (cvs)

0n41 0n11

0n28

0n40

0n31

1n58 0n10

1n44

1n50

1n55

95n9 2n9

92n4

94n1

88n4

1n13 2n40 3n05 0n26

0n93 1n40 1n43

0n73 n.d n.d

0n40 n.d n.d

1n19 2n40 3n42 0n16

1n19 1n40 1n64

1n31 n.d n.d

1n23 n.d n.d

99n9 66n2 69n7 5n3

98n6 69n3 65n1

96n0 71n9 n.d

93n5 68n1 n.d

Water potential, Ψw, P (cvs) l 0n05 ; osmotic potential, Ψs, P (cvs) l 0n02 ; relative water content, RWC, P (cvs) not significant ; n.d., not determined due to leaf scorch.

characteristic responses. The drought-resistant cocksfoot, ‘ KM2 ’, had a large, stable reserve pool of WSC in the stubble, whereas in the other cvs the pool size decreased during the later stages of drought (Fig. 4). Also, ‘ KM2 ’ accumulated less sucrose than the other cvs during drought (Fig. 3 i). In the drought-susceptible cocksfoot, ‘ Lutetia ’, the WSC pool declined rapidly during drought, and in both herbage and stubble the proportion of low-DP fructans was much higher (40–46 %) than in the other cvs (14–21 %). The ryegrass ‘ Aurora ’ generally had the highest WSC content of the four cvs, owing to greater concentrations of high-DP fructans and of sucrose. In ‘ Vigor ’, WSC contents were generally near average, except that large concentrations of monosaccharides accumulated in the stubble during regrowth.

Amino-acid and proline concentrations Contents of total free amino acids (AA) in herbage were fairly stable as drought progressed (Fig. 5 a),

and were significantly higher in ryegrass than in cocksfoot. In stubble (Fig. 5 b), AA contents followed a similar pattern to those in herbage, except that ‘ Lutetia ’ exhibited higher contents than ‘ KM2 ’ during drought. Proline contents of irrigated herbage were very low in all cvs, but increased 12-fold over the first 48 d of drought (Fig. 5 c). In both fractions under irrigation, the ryegrass contained more proline than the cocksfoot. In droughted stubble, proline contents increased most in ‘ Lutetia ’ and least in ‘ KM2 ’, with the ryegrasses intermediate (Fig. 5 d ). Mineral concentrations The most extreme effect of drought on mineral contents (Table 3) was to reduce concentrations of nitrate in both herbage and stubble to about 10 % of control values. K, Na and Mg were also reduced by drought, but less seriously. By the eighth day after rewatering and fertilizing, cation contents had returned to near control levels, whilst nitrate contents were generally extremely high. The main cultivar effect was that ‘ Lutetia ’ had the highest nitrate


444


Total WSC (mg g–1 DM)

600

(a)

(f )

(b)

(g)

(c)

(h)

(d )

(i)

(e)

(j)

500 400 300 200 100

High-DP fructans

500 400 300 200 100 0 150

Low-DP fructans

120 100 75 50 25 0 150

Sucrose

125 100 75 50 25 0

Monosaccharides

120 100 80 60 40 20 0 0

15

30

45

60

90 75 15 30 Days after final watering

45

60

75

90

Figure 3. Water-soluble carbohydrate (WSC) contents of herbage (a–e) and of stubble ( f–j) in two cvs of cocksfoot : ‘ KM2 ’ (#——#, $), ‘ Lutetia ’ ( —— , ) and two cvs of ryegrass : ‘ Aurora ’ (=– – –=, >), ‘ Vigor ’ (W– – –W, X) subjected to 80 d of drought followed by full rewatering. Droughted treatment : open symbols ; irrigated and rewatered treatments : solid symbols. (a, f ) Total WSC ; (b, g)) high-DP fructans ; (c, h) low-DP fructans ; (d, i) sucrose ; (e, j) monosaccharides. Vertical bars represent, from left to right for droughted, irrigated and recovery treatments (d.f. l 21, 14, 5, respectively for herbage, and 30, 13, 6, respectively for stubble).


445 Contribution of solutes to osmotic potential (Table 4)

120 100 Rewatering

WSC in live tiller bases (g m–2)

Drought survival in perennial forage grasses. I

80 60 40 20 0 0

15


90

Figure 4. Pool size (l biomassiconcentration, g WSC m−#) of total water soluble carbohydrate (WSC) in stubble of two cvs of cocksfoot : ‘ KM2 ’ (#——#, $), ‘ Lutetia ’ ( —— , ) and two cvs of ryegrass : ‘ Aurora ’ (=– – –=, >), ‘ Vigor ’ (W– – –W, X) subjected to 80 d of drought followed by full rewatering. Droughted treatment : open symbols ; irrigated and rewatered treatments : solid symbols. Vertical bars represent, from left to right,  for droughted, irrigated and recovery treatments (d.f. l 29, 13, 6, respectively).

Amino acids (µmol g–1 DM)

content when well irrigated and droughted, but did not recover well after rewatering. (Calcium and phosphate concentrations were generally below the detection threshold and are not presented.)

300

 We stated in the Introduction that the most important agronomic character for forages growing in areas subject to prolonged (c. 3 months) summer drought was not production during drought but the ability to survive and to recover rapidly. This is

(a)

(b)

(c)

(d )

250 200 150 100 50 0 120 100 80

Rewatering

Proline (µmol g–1 DM)

In irrigated and rewatered plants, measured minerals contributed 30–50 % to observed laminae osmotic potential, and carbohydrates 20 % (except in cocksfoot during recovery, where carbohydrates made up 39 %). Proline and amino acids made only a small contribution to total osmotic potential, but since they are concentrated in the cytoplasm, probably contributed a much greater osmotic potential to that compartment (see discussion). In droughted plants, the contribution of minerals to osmotic potential was similar to that in watered plants, whilst the contribution of sugars and proline approximately doubled. During the drought period we have a full set of data only for the cocksfoot cvs and there is a large discrepancy between calculated and observed values : osmotic potential of ‘ Lutetia ’ is underestimated by 14 %, but that of ‘ KM2 ’ by 187 %.

60 40 20 0 0

15


90

15


90

Figure 5. Amino acid (a, b) and proline (c, d ) contents in herbage (a, c) and stubble (b, d ) of two cvs of cocksfoot : ‘ KM2 ’ (#——#, $), ‘ Lutetia ’ ( —— , ) and two cvs of ryegrass : ‘ Aurora ’ (=– – –=, >), ‘ Vigor ’ (W– – –W, X) subjected to 80 d of drought followed by full rewatering. Droughted treatment : open symbols ; irrigated and rewatered treatments : solid symbols. Vertical bars represent, from left to right,  for droughted, irrigated and recovery treatments (d.f. l 30, 14, 6, respectively).


446


Table 3. Mineral contents (mg g−" dry matter) in laminae (herbage) and entire tiller bases (stubble) of two cvs of cocksfoot (‘ KM2 ’, ‘ Lutetia ’) and of two cvs of perennial ryegrass (‘ Aurora ’, ‘ Vigor ’) subjected to full irrigation in summer or to 80 d of drought followed by 8 d of rewatering (recovery) under field conditions Tiller bases

Laminae Treatment\ cultivar Irrigation ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’  cv. Drought ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’  cv. Recovery ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’  cv.

K+

Na+

Mg++

NO − $

K+

Na+

Mg++

NO − $

17n6 25n6 27n9 23n4 2n0***

1n6 3n8 2n5 3n1 0n2***

4n0 7n6 9n5 9n6 0n7***

0n97 2n56 1n00 0n52 0n37**

15n9 18n8 18n7 16n3 1n3 n.s.

1n9 2n3 1n9 2n2 0n1 n.s.

3n2 4n0 2n9 2n7 0n3*

1n11 2n15 1n38 1n03 0n39 n.s.

10n4 8n7 9n9 9n0 1n5 n.s.

0n9 2n9 0n8 1n4 0n3***

2n8 3n1 3n2 5n1 0n5*

0n02 0n04 0n03 0n02 0n01 n.s.

7n1 11n8 9n0 9n9 0n7***

1n0 1n9 1n1 1n8 0n1***

1n6 3n3 1n9 2n7 0n1*

0n06 0n21 0n09 0n05 0n07 n.s.

18n3 18n4 26n0 28n8 2n8 n.s.

1n8 3n1 2n9 4n1 0n3*

3n2 4n5 4n8 5n2 0n4 n.s.

4n90 0n69 4n37 — 1n49 n.s.

26n4 22n0 23n2 25n4 2n9 n.s.

3n0 2n9 3n3 3n3 0n3 n.s.

2n6 2n7 3n4 3n2 0n3 n.s.

6n86 1n21 2n57 5n46 1n20*

Contents of Ca and PO were generally below the measurement threshold (0n02 mg g−" dry matter) and are not shown. % Degrees of freedom l 14 (irrigation), 22 (drought, laminae), 30 (drought, entire tiller bases), 6 (recovery). Significance of cv. effect in  shown as : n.s., not significant ; *, **, *** significant at P 0n05, 0n01, 0n001.

Table 4. Components of bulk osmotic potential (MPa) in leaf laminae of two cvs of cocksfoot (‘ KM2 ’, ‘ Lutetia ’) and two cvs of perennial ryegrass (‘ Aurora ’, ‘ Vigor ’) subjected to full irrigation in summer or to 80 d of drought followed by 8 d of rewatering (recovery) under field conditions Estimated contribution to osmotic potential Treatment\ cultivar Irrigation ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’ Drought ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’ Recovery ‘ KM2 ’ ‘ Lutetia ’ ‘ Aurora ’ ‘ Vigor ’

Hf

Na+, K+ Mg++, NO − $

WSC

ProjAA

Total

Observed OP

3n13 3n15 3n57 4n15

k0n42 k0n70 k0n54 k0n42

k0n28 k0n28 k0n21 k0n21

k0n01 k0n02 k0n02 k0n02

k0n80 k1n00 k0n77 k0n65

k1n35 k1n33 k1n35 k1n34

2n21 2n07 2n26 3n26

k0n47 k0n68 k0n53 k0n48

k0n45 k0n47 k0n56 k0n36

k0n02 k0n02 k0n04 k0n03

k0n94 k1n17 k1n13 k0n87

k2n70 k1n48 n.d. n.d.

2n51 2n58 4n27 4n49

k0n54 k0n57 k0n44 k0n45

k0n49 k0n44 k0n26 k0n23

k0n02 k0n02 k0n06 k0n05

k1n05 k1n03 k0n76 k0n73

k1n20 k1n25 k1n14 k1n26

Osmotic potential (OP) of solutes was estimated from solute content (µmol g−" dry matter) and hydration of fresh tissue (Hf, g water g−" dry matter). ‘ ProjAA ’ l sum of proline and total amino acids ; n.d., not determined due to leaf scorch. Observed OP refer to the means of controls over days 3, 26 and 73 (irrigation) and days 3, 26, 48 and 73 (drought).

confirmed in the present study. Leaf growth was halved within 5 d of drought, and had almost ceased within 10 d, with only small differences between cvs (Fig. 1). On the other hand, the cvs differed greatly during the recovery phase, and there was a close

correlation between herbage growth rate on recovery (HGR, Table 1) and tiller survival (TS, Table 1) : HGR l 0n39j0n070TS, R# l 0n98. Detailed observations showed that, during the first week of recovery, all visible leaf growth was from


Drought survival in perennial forage grasses. I tillers that had been growing before drought started, and none from tillers developing from axillary buds. This discussion therefore concentrate on the analysis of traits likely to contribute to survival and regrowth of existing tiller during drought. The arrangement is based on the three strategies described by May & Milthorpe (1962) and developed by Ludlow (1989) : drought escape (or avoidance), delay of desiccation (maintenance of water status), and desiccation tolerance. Developmental traits and drought escape Unlike annual plants that can escape drought by maturing before stress becomes severe, perennial forages cannot escape drought completely by flowering early, but can at least avoid the damaging consequences of concurrent drought, flowering and defoliation (Thomas & Evans, 1990). In earlier work on cocksfoot we have shown that early flowering of contrasting ecotypes was related to their geographical origins, and correlated with ability to survive severe drought (Volaire & Lelie' vre, 1997). In the present study, early flowering was also associated with drought survival in cocksfoot, but had no effect on survival in ryegrass. In cocksfoot it is a developmental trait associated with the earliness of spring in Mediterranean environments, where the summers are, coincidentally, usually dry. Conversely, in the ryegrass cvs studied, the earliness of ‘ Aurora ’ can be associated with its alpine origin. Silsbury (1964) described diverse accessions of perennial ryegrass as dormant when they did not grow during drought, but survived to recover in autumn. Although the cvs studied here stopped growing soon after drought started, they grew well over the same period when well watered. Therefore, following the definition of Villiers (1975), they were quiescent rather than dormant. Consequently, we need to determine how nearly all ‘ KM2 ’ tillers survived quiescence, whilst most ‘ Lutetia ’ tillers died. Also, since the ryegrass cvs patterns were similar to each other, but intermediate between those of the cocksfoot cvs, we can seek traits which coincide with the observed rankings for survival. Desiccation delay Plants might reduce the rate of desiccation of perennating organs by reducing transpiration or by absorbing more water from the soil. Since leaf death progressed so rapidly in all cvs, it is likely that the main differences between them lay more in water uptake by roots than in reduction of transpiration. The drought-sensitive ‘ Lutetia ’ had fewest roots at all depths, and was least able to extract water from middle soil horizons, which probably caused its high tiller mortality. The importance of a deep root system (such as that of ‘ KM2 ’) in maintaining a

447 continuous, if small, supply of water during severe drought has been described in Phalaris aquatica, tall fescue and other major forage grasses (McWilliam & Kramer, 1968 ; Garwood & Sinclair, 1979) and in turfgrasses (Marcum et al., 1995 ; Carrow, 1996), and this trait has been the subject of selection programmes in Lolium rigidum (Bullitta, 1996). Desiccation tolerance Differences between populations in tolerance of desiccation are notoriously difficult to detect or analyse when control of water status also differs (Hanson & Hitz, 1982). The few tillers that remained in the susceptible cocksfoot (‘ Lutetia ’) were better hydrated than those of ‘ KM2 ’, which had lower osmotic potential and lower relative water content. This indicates that adaptation through desiccation tolerance in laminae is higher in ‘ KM2 ’ than in ‘ Lutetia ’. ‘ KM2 ’ consumed about the same volume of soil water as the ryegrasses, but maintained more living tillers, again indicating its better expression of desiccation tolerance. As most leaf laminae were dead after the first few weeks of drought, and had lost most of their WSC (data not shown), it is likely that most carbohydrates were reallocated to storage in the stubble. Total WSC contents varied greatly between cvs, and the ryegrass ‘ Aurora ’ exhibited particularly high concentrations, as reported previously under British conditions (Humphreys, 1989). This did not, however, improve its performance over that of ‘ Vigor ’. Increased WSC reserves, at least at the levels observed here (40–50 % at the end of drought) may not, per se, improve drought tolerance. In ‘ Lutetia ’ (and in other cocksfoot cvs, Volaire & Lelie' vre, 1997) a high proportion of low-DP fructans (or low proportion of high-DP fructans) in the stubble fraction seems to be associated with drought susceptibility. This trait, which is constitutive (expressed in both irrigated and droughted plants), might be due to slow assembly of fructans past the DP4 stage. It is unlikely to be due to inability of ‘ Lutetia ’ stubble to de-polymerize low-DP reserve fructans to sucrose, since sucrose contents were, if anything, higher than in ‘ KM2 ’ (Fig. 3 i). It is interesting to note that Thomas (1991) reported accumulation of DP3 fructans in droughted perennial ryegrass leaves just before they died. Proline accumulation in living stubble was greatest in the most sensitive cv., ‘ Lutetia ’ (Fig. 5 b), and there is a negative correlation (R# l 0n88) between mean proline content during drought and tiller survival. Turner (1990) and Thomas (1991) have postulated that proline accumulation might be both acclimatory in young tissues, and a symptom of impending death and remobilization of nitrogen in older tissues. Both of these mechanisms could, therefore, be represented in the stubble fraction,


448


which consists of tissues of all ages, but since mature leaf sheaths make up the bulk of the tissue, the over-riding response will be that associated with desiccation-induced death. In interpreting the contribution of osmolytes to osmotic potential, it is important to bear in mind that entire tissue fractions were homogenized. It is likely that both sugars and mineral ions are partitioned mainly to vacuoles (Mott & Stewart, 1982). During drought, the contribution of WSC to bulk osmotic potential doubled (Table 4), owing to both lower water content and increased WSC content (particularly sucrose). The contribution of minerals to osmotic potential was fairly constant, because reduced mineral content was balanced by reduced water status ; this might have been fortuitous, but was also beneficial to intracellular water relations. Nevertheless, the overall effect would have been to lower vacuole osmotic potential, leading to an imbalance in osmotic potential across the tonoplast. In this case, accumulation of proline in the cytoplasm would have maintained its isotonic balance with the vacuole. This mechanism could explain, at least in part, the coincidence of high contents of proline, minerals and low-DP fructans (which are more osmotically active than the same mass of high-DP fructans) observed in ‘ Lutetia ’. Autumn recovery The rate of regrowth following drought is a function of surviving tiller density, regrowth rate of surviving tillers, and (eventually) emergence and growth of new tillers from old or new tiller buds. We have already discussed the close correlation between recovery and tiller density. The rate of recovery of individual tillers after such severe drought was remarkable : after a lag of only 3 d, leaf growth was faster in the previously droughted than in the well irrigated plants (Fig. 1), and after 10 d more leaves (and hence sites for tiller production) had been produced by the previously droughted plants. The rapid regrowth of individual surviving tillers might have been due to the greater availability of reserves of WSC and N (in the form of proline) in stubble at the end of drought (Figs 3f, 5 d ), although 12 d after rewatering, concentrations of most solutes had returned to near-normal levels, whilst previously droughted tillers were still growing faster. Such ‘ complementary ’ growth has been reported before (Horst & Nelson, 1979 ; Thomas, 1991), but not for so prolonged a period, and might partly be the influence of previous drought on soil-mineral availability. Despite differences in tiller density at the end of drought, within 3 wk the ryegrasses had recovered their initial tiller densities. The drought-susceptible cocksfoot ‘ Lutetia ’ showed even greater powers of recovery, more than doubling its tiller density, which

alleviated the effect of severe mortality during drought. On the other hand, ‘ KM2 ’ was near its maximum tiller density even at the end of drought, and achieved its very rapid recovery principally through rapid leaf extension and appearance rates on existing surviving tillers. Conclusions Early flowering was not an effective drought-escape mechanism in the four cvs tested and especially in ryegrass. Although all exhibited responses that might have contributed to tolerance of desiccation, extreme responses were associated with susceptibility rather than tolerance. The functional significance of the balance between high- and low-DP fructans on tolerance in cocksfoot needs further exploration. The exceptional performance of the cocksfoot ‘ KM2 ’ over ‘ Lutetia ’ appears to be due, at least in part, to its greater ability to explore for water at depth, and over ryegrass to greater tolerance of desiccation. Events in unexpanded leaf bases, the main surviving shoot organs, are discussed in Volaire et al., 1998 b.                We thank N. Bertagne and P. Barrieu (Montpellier) and A. R. James (Aberystwyth) for technical assistance. H. Thomas thanks the BBSRC-INRA fellowship scheme for financial support for conducting the field work.

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