Breeding red clover for improved persistence in Chile: a review

Fernando OrtegaA,C, Leonardo ParraB, and Andrés QuirozB

AInstituto de Investigaciones Agropecuarias, INIA Carillanca, km 10 camino Cajón-Vilcún s/n,Región de La Araucanía, Chile.

BUniversidad de La Frontera, Laboratorio de Química Ecológica, Departamento de Ciencias Químicas y RecursosNaturales, Temuco, Chile.

CCorresponding author. Email: fortega@inia.cl

Abstract. The main limitation of red clover (Trifolium pratense L.) worldwide including in Chile is the lack ofpersistence related to the high mortality of plants due to a complex of biotic and abiotic factors. We have demonstrated invarious trials in Chile that red clover plant population is highly correlated with forage yield once the plant populationhas dropped to a certain level, from the second or third season onward, depending on the environment of evaluation. Wehave also found that in the south of Chile, among the biotic and abiotic factors affecting red clover survival, thecurculionid Hylastinus obscurus (Marsham) is the main deleterious factor. However, because persistence is a complextrait, we have used a practical approach in our breeding program. We selected for general adaptability under fieldconditions and used a modified among and within half-family selection methodology, evaluating at the same timefamilies as swards and spaced plants. This breeding methodology and strategy have yielded reasonable genetic gainssince we started our breeding program in 1989 at INIA Carillanca, Chile. Since then, we have conducted five cyclesof recurrent selection, and two cultivars have been released to replace the old cultivar, Quiñequeli INIA. These areRedqueli INIA and, more recently, Superqueli INIA. Depending on location and trial, average forage yield of thenewest cultivar Superqueli INIA has been 23–69% higher than Quiñequeli INIA and 5–36% higher than Redqueli INIA;this difference increases in the third and fourth seasons. Superqueli INIA had four times the yield of Quiñequeli INIA inthe fourth season. Therefore, the average realised genetic gain has been 0.4–2.6% per year, depending on location,showing the effectiveness of the breeding methodology and approach used.

Additional keywords: abiotic stress, breeding strategies, genetic improvement, plant breeding, plant–insect interactions,selection programs.

Received 13 September 2013, accepted 6 February 2014, published online 19 March 2014

Introduction

Red clover (Trifolium pratense L.) is an important forage speciesin temperate regions of the world. Grown alone or in mixturewith grasses, it is adapted to a wide range of soil types, pH levels,and environmental and management conditions. It is a valuableresource in Chile for animal production and the seed industry,representing nearly 15% of the sown pastures and 60% of theforage seed exports (Ortega and Levío 2011a).

The main limitation of this species worldwide is lack ofpersistence related to the high mortality of plants due to acomplex of biotic and abiotic factors, determining a life spanof two or three seasons (Cuevas and Balocchi 1983; Ortega1996; Taylor and Quesenberry 1996; Rhodes and Ortega1996; Rhodes and Ortega 1997; Steiner and Alderman 2003;Herrmann et al. 2007; Hejduk and Knot 2010; Marshall et al.2011;Ortega et al. 2012a, 2012b). Persistence is an economicallyimportant trait that may be defined as sustained forage yieldover several years (Conaghan and Casler 2011). In red clover, itis dependent on population and yield ability (Ortega 1996).

There are many biotic (pests, diseases, competition andmorpho-physiology of the red clover plant itself), abiotic (pH,soil fertility and climatic conditions) andmanagement (frequencyand intensity of cutting and/or grazing) factors that are importantin determining the survival of red clover plants. These factorsinteract, acting as a complex, and the importance of each onevaries depending on the environment. The effect of the frequencyof cutting, grazing and mixture with other species on plantmorphology and its interaction with plant survival has beenreported by different authors (Cosgrove and Brougham 1985;Sheldrick et al. 1986; Ussher 1986; Frame 1990; Ortega 1996;Ford and Barrett 2011). Ortega (1996) did not find differencesin plant populations after 3 years of evaluation when comparinginfrequently cut and sheep-grazed swards; however, plantsunder grazing were much smaller in terms of crown diameterand root weight. Belzile (1987) also studied the effect ofmixture on red clover plant survival, finding an interactionwith winter severity; mixtures can also affect the phytosanitaryperformance of red clover (Lewis et al. 1985). Among the biotic

Journal compilation � CSIRO 2014 www.publish.csiro.au/journals/cp

CSIRO PUBLISHING

Crop & Pasture Science Reviewhttp://dx.doi.org/10.1071/CP13323

factors associated with red clover mortality in Chile, the mainfactor is the root borer Hylastinus obscurus (Marsham)(Carrillo and Mundaca 1974; Quiroz et al. 2005; Tapia et al.2005; Tapia et al. 2007; Alarcón et al. 2010; Palma et al. 2012).

Because of the importance of red clover in Chile, a red cloverbreeding program was started at INIA Carillanca ResearchCenter, Chile, in 1989, with the main objective of improvingthe survival of plants, forage yield and persistence. Since then,two cultivars have been released to replace the old cultivar,Quiñequeli INIA (Avendaño 1965); the first was RedqueliINIA (Ortega et al. 2003), and the more recent, SuperqueliINIA (Ortega and Levío 2011b).

This paper summarises and discusses the main results of thebreeding program and research conducted by our team over a20-year period. We discuss the effectiveness of the breedingmethodology and experimental approaches that we have used.

Breeding methodology

We have conducted five cycles of polycross and half-sib family(HSF) evaluation since 1989, developing 10 synthetics, two ofwhich have become registered varieties. Polycrosses and HSFevaluation is conducted at one site (INIA Carillanca, Chile;388410S; 728250W), and the evaluation of the experimental

synthetics is conducted in different locations in the south ofChile as described in the next section. In the first cycle, wefollowed a genotypic selection method to develop our first newcultivar (Redqueli INIA), and from the second cycle onward,we have adapted and performed a geno-phenotypic selectionmethod (Taylor 1987). This method has been described andstudied as ‘among and within half-sib family selection’ (Caslerand Brummer 2008; Conaghan and Casler 2011).

Figure 1 shows a diagram of the breeding methodology andTable 1 gives details of each breeding cycle. The evaluation ofprogenies simultaneously as swards and spaced plants allowsthe selection of the best and most homogenous plants fromspaced plants of the best progenies evaluated in plots. Aftereach cycle, selected plants are organised in a new polycross tostart the next cycle, and the best of the selected plants are alsopolycrossed to create new synthetics.

Each of our selection cycles started with the polycross of30–55 plants to produce HSFs (Table 1). From the second cycleonward, HSFs have been evaluated for forage yield and survivalof plants in swards infrequently cut (three or four times/season)and, at the same time, in a nursery of spaced plants formorphological characters, flowering date, vigour and survival.Nurseries are evaluated at 60-cm spacing and usually cut twiceduring the season, once after flowering evaluation and again

POLYCROSS

HARVEST SEED OF HSF

AMONG AND WITHIN HSFSELECTION FROM SPACEDPLANTS

POLYCROSS FORSYNTHETICFORMATION

POLYCROSS TOBEGIN NEXT CYCLE

HSF EVALUATED AS SPACED PLANTS INONE ENVIRONMENT (3-seasons)

HSF EVALUATED IN SAWRDS INTWO ENVIRONMENTS (3-harvestseasons)

Fig. 1. Diagram of the breeding methodology conducted since 1989. HSF, Half-sib family.

Table 1. Number of genotypes and origin in each polycross cycle and number of plants established in each half-sib family(HSF) nursery

Polycross No. ofgenotypes

Origin of genotypes No. of plants established/HSFin the nursery as spaced plants

I 55 Selected from Quiñequeli INIA, introducedcultivars and common seed provenance

None

II 38 Nine HSF from first cycle, four to five genotypes/HSF 30 (three reps of 10 plants each)III 30 Five HSF from first cycle, six genotypes/HSF 30 (three reps of 10 plants each)IV 30 Five HSF from first cycle, six genotypes/HSF 30 (three reps of 10 plants each)V 30 Six HSF from first cycle, five genotypes/HSF 45 (three reps of 15 plants each)

B Crop & Pasture Science F. Ortega et al.

at the end of the season. After 3 years of progeny evaluation,selection among HSF is decided according to sward evaluation,but plants are selected within HSF evaluated in the nursery ofspaced plants. On average, for each cycle, the among-HSFselection intensity has been 1/6 (9 of 55 for cycle II, 6 of 30for cycle V), and the within-HSF selection intensity has alsobeen 1/6, with a combined selection intensity of ~2.8% (Table 1).

The effectiveness of the breeding methodology and approachused is detailed in the results of field trials to evaluate theexperimental synthetics developed by the program.

Field trials for agronomic evaluation of experimentalsynthetics and realised forage genetic gain

We analysed seven experiments conducted in three differentlocations and four environments. Five experiments wereconducted at INIA Carillanca Research Center in twoenvironments (irrigated; non-irrigated), and the other twoexperiments were conducted at INIA Humán (irrigated)(378260S, 728140W) and INIA Remehue (non-irrigated)(408520S, 738120W). The experiments at INIA Carillanca weremanaged under irrigated conditions for sowing years 2005,2007 and 2008 and non-irrigated conditions for sowing years2006 and 2008. Trials were evaluated under infrequent cutting(three or four cuts/season); Carillanca (irrigated) trials wereevaluated for four seasons, and the other trials were evaluatedfor three seasons.

The trials considered the Chilean cultivars (Quiñequeli INIA,Redqueli INIA and Superqueli INIA) and seven experimentalsynthetics. Sowing rate was 15 kg/ha in rows separated by 20 cm;the design was complete randomised blocks with three or fourreplicates and plots of 1mby5mor 1.8mby7meach, dependingon the trial. Among the variables evaluated, in this paper wedescribe plant population (no. of plants/m2) and forage yield(dry matter (DM), kg/ha). Plant population was evaluated bynon-destructive counting at the beginning and end of eachseason by counting 1m of three central rows of each plot;forage yield was evaluated by sampling two quadrats of 0.6mby 1m each of the central part of each plot at 10% flowering,or when clover was 40–60 cm high. Results of each trial wereanalysed by ANOVA and means were separated by Duncan’stest (P = 0.05). Additionally, annual yield of all trials wasanalysed by a combined analysis of variance using the GLMprocedure. All the statistical analyses were done using theSAS software ver. 9.3 (SAS Institute 2013). The relationshipbetween plant population and forage yield of trials sown atINIA Carillanca was studied by linear Pearson correlations foreach trial and season. The realised genetic gain was estimatedby comparing the forage yield per site of cvv. Redqueli INIAand Superqueli INIA and dividing by the 14-year intervalbetween the creating of the two cultivars.

Typical yield and plant population patterns of red clovertrials sown under irrigated conditions at INIA Carillanca areshown in Fig. 2a. For spring sowing, maximum yield isobtained in the second season, with average red clover plantpopulation >100 plants/m2. From the third season onward,average yield declines together with plant population. Non-irrigated trials are usually sown in autumn, and maximumaverage forage yield is obtained in the first season; both yield

and plant population decline sharply from the second seasononward (Fig. 2b).

Despite the general tendency described above, there areimportant differences between cultivars and synthetics in theirbehaviour. Under irrigated conditions, results of trials sown in2005 showed that in the first season, there were no significantdifferences between cultivars, and from the second seasononward, there were significant differences; the tendency was toincrease the gap between the oldest cultivar, Quiñequeli INIA,and Redqueli INIA and the newest cultivar, Superqueli INIA(Fig. 3a). In the fourth season, Superqueli INIA reached aconsiderable yield, close to 10 000DMkg/ha, more than twicethe yield of Quiñequeli INIA. In the same trial, the SuperqueliINIA plant population was significantly higher than bothRedqueli INIA and Quiñequeli INIA in the third and fourthseasons (Fig. 4a). In addition, non-irrigated trials sown in2006 showed significant superiority in forage yield ofSuperqueli INIA in the second and third seasons, with nodifferences between Redqueli INIA and Quiñequeli INIA(Fig. 3b). The plant population results were similar (Fig. 4b),with Superqueli INIA superior to Quiñequeli INIA from thesecond season onward and superior to Redqueli INIA in thethird season.

The relationship between forage yield and plant populationin three irrigated trials at INIA Carillanca is summarised in

0Season 1

Season 1

Season 2

Season 2

Season 3

Season 3

Season 4

2000

4000

6000

8000

10 000

12 000

14 000 (a)

(b)

3244

104

242

6318

9528

11 542

Dry (kg/ha) No. of plants/m2

7593

0

50

100

150

200

250

300

No.

of p

lant

s/m

2

2000

4000

6000

8000

10 000

12 000

14 000

235

18

542649

10 090

11 538

DM

(kg

/ha)

0

50

100

150

200

250

300

Fig. 2. Average forage dry matter (DM) yield and plant population of redclover synthetics and cultivars: (a) INIA Carillanca, irrigated trial sown in2005; (b) INIA Carillanca, non-irrigated trial sown in 2006.

Advances in red clover persistence improvement by breeding Crop & Pasture Science C

Table 2. In the three reported trials, significant correlations wereobtained from the third season onward when the average plantpopulation was 44–54 plants/m2 and the range of the populationswas 17–70 plants/m2. However, in the trial sown in 2005, a weakbut significant correlation was obtained in the second season.Conversely, in both trials performed under non-irrigatedconditions, highly significant correlations were obtained in thethird season,with an average plant population of 19–26 plants/m2

and a population range of 6–41 plants/m2 (Table 3). The samelevel of population that gave significant correlations underirrigated conditions did not yield significant correlations innon-irrigated trials. Figure 5 shows the relationship betweenforage yield and plant population of the three cultivars and

0

2000

4000

6000

8000

10 000

12 000

14 000

16 000

e

cdde

c

c

aabc

a

a

Superqueli INIA Redqueli INIA Quiñequeli INIA

0

2000

4000

6000

8000

10 000

12 000

14 000

16 000

ab

a

ab

cdbcd

a

d

cd

ab

DM

(kg

/ha)

(a)

(b)

Season 1 Season 2 Season 3 Season 4

Season 1 Season 2 Season 3

Fig. 3. Red clover dry matter (DM) seasonal yield of cultivars at INIACarillanca, Chile: (a) Irrigated trial sown in 2005; (b) non-irrigated trialsown in 2006. Means with no letters or the same letter in the same season arenot significantly different according toANOVAandDuncan’s test (P= 0.05).

0

10

20

30

40

50

60

70

d

c

a

e

cd

Superqueli INIA Redqueli INIA Quiñequeli INIA

a

0

10

20

30

40

50

60

70

c

bc

a

d

cd

abc

No.

of p

lant

s/m

2

Season 2 Season 3

Season 3 Season 4

(a)

(b)

Fig. 4. Average plant population of red clover cultivars at INIA Carillanca,Chile: (a) Irrigated trial sown in 2005, third and fourth seasons; (b) irrigatedtrial sown in 2006, second and third seasons. Means with the same letter inthe same season are not significantly different according to ANOVA andDuncan’s test (P= 0.05).

Table 2. Correlations (r) between average red clover plant population (no. of plants/m2) and forage dry matter seasonal yield (kg/ha) during fourseasons in three irrigated trials at INIA Carillanca, average population and range of population

*P< 0.05; **P< 0.01; n.s., not significant (P> 0.05)

Season Trial 2005 Trial 2007 Trial 2008r Av. pop. Range r Av. pop. Range r Av. pop. Range

Y1 –0.15n.s. 242 174–320 –0.08n.s. 286 233–364 0.48n.s. 163 114–227Y2 0.68* 104 72–129 0.63n.s. 110 74–131 0.66n.s. 74 39–94Y3 0.88** 44 24–58 0.86** 46 17–70 0.83** 54 20–70Y4 0.89** 32 14–48 0.86** 31 9–52 0.89** 35 9–54

D Crop & Pasture Science F. Ortega et al.

experimental synthetics in the irrigated trial sown in 2005;there is a consistent improvement in both forage yield andplant population from the oldest cultivar Quiñequeli INIA tothe newest cultivar Superqueli INIA, with Redqueli INIA inbetween. A similar tendency is observed under non-irrigatedconditions (Fig. 5), but in this environment, the performanceof Redqueli INIA and Superqueli INIA was closer.

Figure 6 provides a summary of the forage yield of seventrials (24 seasons of evaluation in total) and the result ofa combined analysis of variance by environment. Theenvironment where the maximum difference between the threecultivars was expressed was Carillanca (irrigated), and Humánwas the site where differences were lowest, even though theywere also significant. At Carillanca (irrigated), the averageforage yield of Superqueli INIA was 69% greater than that ofQuiñequeli INIA, and at the Humán site, it was 23% greater.Comparing the two cultivars released by our breeding program,Superqueli INIA outyielded Quiñequeli INIA by 36% atCarillanca (irrigated) and by 5% at Humán. The time betweenthe generation of the cultivars was 14 years, representing arealised genetic gain for forage produced over 3–4 years of2.6% per year for Carillanca (irrigated), 0.8% per year forCarillanca (non-irrigated), 0.7% per year for Remehue and0.4% per year for Humán.

Studies of plant and root borer interactions

We reviewed general aspects of research conducted by our teamin eight publications to date (Tapia et al. 2005, 2007;Quiroz et al.2005; Alarcón et al. 2010; Manosalva et al. 2011; Palma et al.2012, 2013; Parra et al. 2013).

The effect of the presence ofH. obscurus in the production ofred clover forageyieldwasdemonstratedbyAlarcón et al. (2010).Figure 7 shows significant variation between the accessions, bothin insect population and forage yield, over 2 years. Theexperimental lines Syn Pre III, Syn Int V and Superqueli INIA

Table 3. Correlations (r) between average red clover plant population(no. of plants/m2) and forage dry matter seasonal yield (kg/ha) duringthree seasons in two non-irrigated trials at INIA Carillanca, average

population and range of population*P< 0.05; **P< 0.01; n.s., not significant (P> 0.05)

Season Trial 2006 Trial 2008r Av. pop. Range r Av. pop. Range

Y1 0.06n.s. 235 196–282 –0.16n.s. 161 110–217Y2 0.48n.s. 54 41–64 0.68* 38 25–53Y3 0.94** 19 6–29 0.94** 26 15–41

00 10 20 30 40 50 60

2000

4000

6000

8000

10 000

12 000

14 000

Non-irrigated

r = 0.88**

Quiñequeli INIA

Redqueli INIASuperqueli INIA

Redqueli INIA

Superqueli INIA

DM

(kg

/ha)

Quiñequeli INIA

r = 0.94**

Irrigated

No. of plants/m2

Fig. 5. Relationship between plant population and forage dry matter(DM) yield of Chilean red clover cultivars and synthetics in the thirdseason at INIA Carillanca, Chile: (a) irrigated trial sown in 2005; (b) non-irrigated trial sown in 2006.

0Carillanca I Carillanca D Humán Remuhue

2000

4000

6000

8000

10 000

12 000

124%

136%

117%123%

118%131%

124%

169%

ab

ab

bc

ab

a

a

d

cd

cdc

DM

(kg

/ha.

year

)

Superqueli INIA Redqueli INIA Quiñequeli INIAa

Fig. 6. Average forage dry matter (DM) yield of different cultivars of redclover in different seasons and environments: Carillanca irrigated (I): threetrials, 12 seasons; Carillanca dryland (D): two trials, six seasons; Humán: onetrial, three seasons; Remehue: one trial, three seasons. Means with the sameletter are not significantly different by combined analysis of variance(Duncan’s test, P= 0.05).

Red

quel

i IN

IA

Qui

ñequ

eli I

NIA

Syn

PR

E I

Syn

IV

Syn

PR

E II

Syn

Int I

V

Syn

PR

E II

I

Syn

Int V

Sup

erqu

eli I

NIA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

a

ababab

b

c

ab

c

bc

BC

AB ABAB

A

ABC

C

C

H. obscurus Dry matter

No.

of i

nsec

ts/p

lant

C

10

12

14

16

18

20

22

24

Dry

mat

ter

yiel

d (t

/ha)

Fig. 7. Total forage yield of red clover from two seasons and the number ofindividuals of Hylastinus obscurus per plant at INIA Carillanca, Chile.Irrigated trial sown in 2007. Means with the same letter are notsignificantly different based on ANOVA and l.s.d. test (forage yield) andFriedman and Conover-Inman non-parametric tests (number of individuals)(P= 0.05). Source: Alarcón et al. (2010).

Advances in red clover persistence improvement by breeding Crop & Pasture Science E

showed the lowest H. obscurus populations and the highestforage yield.

Detailed research of the semiochemical interaction betweenplants and insects has been reported by Tapia et al. (2007) andManosalva et al. (2011). Plant volatiles were identified, andthe interaction with the insect was studied using olfactometricbioassays (Tapia et al. 2007) to test three different concentrations(0.1, 1 and 10mg/mL) of two synthetic compounds. E-2-Hexenalattracted insects at concentrations >0.4mg/mL and limoneneshowed repellent activity to borers at concentrations >0.4mg/mL (Fig. 8). E-2-Hexenal concentration decreased from 2.12 to0.76mg/mL, and limonene concentration increased significantlyfrom 0.26 to 0.68mg/mL when 1.5- and 2.5-year-old plantswere compared. Thus, when plants were 1.5 years old, theconcentration of E-2-hexenal was more attractive than in 2.5-year-old plants, whereas limonene repellent activity increasedwith age (Fig. 8).

The relationship between the insect and root extracts andpure fatty acids was reported by Manosalva et al. (2011). Inolfactometer bioassays, the extract (10mg/mL) from9-month-oldred clover roots was attractive to both sexes of the borers(Table 4). Hexane solutions of lauric acid, palmitic acid andoleic acid significantly attracted female H. obscurus (Table 4).By contrast, no significant male responses were observed withregard to attraction to any of the substances compared with thecontrol. Thus, despite their low volatility, lauric, palmitic andoleic acid elicited a significant attractive olfactory response fromfemale H. obscurus.

Contact bioassays showed that the root extract waspreferred significantly by both male and female ofH. obscurus compared with the control (Table 4). Both the

synthetic pure fatty acids and the blend showed significanteffects on female H. obscurus behaviour (Table 4). Thepalmitic and oleic acid treatments had similar significanteffects on the preference of male H. obscurus, whereas lauricor stearic acids and the blend of fatty acids showed nosignificant effects on male behaviour.

In electroantennography experiments, root volatiles suchas E-2-hexenal, hexanal, 3-octanone, limonene and a-pinene

0.4

–1.0 –0.5 0.0 0.5 1.0

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

b

a

a

Olfa

tom

etric

T/C

inde

x

Log concentration (µg/mL) of active compounds

Limonene

E-2-hexenal

attractive (T/C > 1)

repellent (T/C < 1)

b

Fig. 8. Effect of the concentration of semiochemicals on the olfactometricresponse of Hylastinus obscurus, where T is the time spent on treatmentarms and C is the time spent on control arms. Compounds in 1.5-year-oldroot extracts are marked with an arrow and ‘a’, and those in 2.5-year-oldroot extracts are marked with an arrow and ‘b’. Source: Tapia et al. (2007).

Table 4. Biological activity of extracts and pure compounds extracted from red clover on H. obscurusEAG, Electroantennography

Chemical extract/pure compound

Olfactometricactivity

Contact activity EAG response(yes/no)

Field activity Reference

Ethanolic extracts from healthy roots Attraction Tapia et al. 2005Ethanolic extract from fungi infected roots Attraction Tapia et al. 2005Volatiles released from 1.5-year-old roots Attraction Tapia et al. 2007E-2-Hexenal Attraction Yes Attraction Tapia et al. 2007;

Palma et al. 2013;Parra et al. 2013

Methyl benzoate Attraction Tapia et al. 2007Limonene Repellent Tapia et al. 2007Dichloromethane root extract

(9-month-old)Attraction Attraction Manosalva et al. 2011

Lauric acid Attraction (female) Attraction (female) Manosalva et al. 2011Palmitic acid Attraction (female) Attraction (male) Manosalva et al. 2011Oleic acid Attraction (female) Attraction (male) Manosalva et al. 2011Stearic acid No response Attraction (female) Manosalva et al. 2011Fatty acid blend No response Attraction (female) Manosalva et al. 2011Leaves essential oil (2-year-old) Attraction Quiroz et al. 20051.5-year-old leaves extract Attraction Quiroz et al. 20052.5-year-old leaves extract Attraction Quiroz et al. 2005Hexanal Repellent Yes Palma et al. 20133-Octanone Repellent Yes Palma et al. 2013R-Limonene Repellent Yes Palma et al. 2013S-Limonene repellent Yes Palma et al. 2013Limonene (racemic mixture) No response Parra et al. 2013

F Crop & Pasture Science F. Ortega et al.

were tested. The results indicated that all of the testedcompounds were perceived by H. obscurus, but with mixedresponses in olfactometric bioassays. For females, hexanal, 3-octanone, R-limonene and S-limonene were repellent, and E-2-hexenal was attractive to both sexes (Palma et al. 2012). Palmaet al. (2013) determined that olfactory receptors, or sensilla, ableto perceive these compounds correspond to four different types.Chaetica and basiconica were the most abundant sensilla types.The field validation experiment showed that H. obscurus wasfound in significantly higher numbers on plant from areastreated with E-2-hexenal (dispensers) than from areas treatedwith limonene. These results provide the first evidence underfield conditions for the potential use of E-2-hexenal as anattractant for H. obscurus (Parra et al. 2013).

Discussion

A sharp decline in red clover plant populations, as observedin our field trials, has also been recorded by other authors.Frame (1990) reports that a density of 200 plants/m2 in autumnof the establishment year is suitable. Other trials show thatat the beginning of the second year, plant populationsalready dropped to an average of 63 plants/m2 (Frame et al.1976). Investigations conducted by Sheldrick et al. (1986),using a sowing rate for red clover of 14 kg, reportedpopulations of 187 and 119 plants/m2 at the beginning ofthe first winter, in monoculture and mixture, respectively. Bythe end of the first and third years, numbers decreased inboth monoculture and mixture, to 109 and 37 plants/m2,respectively. In Canada, Choo et al. (1987) foundpopulation levels of 22–44 plants/m2 after the secondwinter. Furthermore, Ortega (1996) evaluated eight cultivarsin swards in monoculture and mixture under infrequentand frequent cutting and reported an average plantpopulation in the establishment year of 459 plants/m2 and areduction to 85 plants/m2 at the end of the secondproduction year.

There are few studies of red clover plant densities and theirrelationships with forage yield because of the difficulty inquantification. However, the relationship will depend on theenvironment, management and morpho-physiology of thegenetic material. In pure stands, Jewis (1993) reported aminimum population of 30 plants/m2 to sustain an economicproduction; in mixtures with grasses, Frame et al. (1976) andSheldrick et al. (1986) showed that a population density of~100 plants/m2 is low enough to affect yield.

The results of our plot trials demonstrate the relationshipbetween red clover plant populations and forage yield.However, the numerical relationship depends on theinteraction between plants and the environment. In fact, inour studies, plots sown in monoculture, irrigated and cutinfrequently showed significant correlations between plantpopulation and forage yield with an average of ~50 plants/m2. However, at the same site but without irrigation, plantpopulations that gave a significant correlation were lower. Thereason for the different pattern between irrigated and non-irrigated conditions could be that hydric stress interacts withbiotic factors that affect root growth such as the root borer, asplants with weaker roots are unable to withstand hydric deficit

and die sooner, thereby determining a faster plant populationdecline under non-irrigated conditions. Nevertheless, withoutirrigation, each plant and the pasture have less forage yieldpotential; therefore, the sward may withstand lower plantpopulations before forage yield is affected. At the INIACarillanca site, hydric stress is produced when pastures aregrowing, from November to March, with an average rainfall ofjust 253 mm; depending on the year, irrigated trials are irrigatedfive–eight times during this period (each irrigation: ~30–45mmwater).

Because of the crown and taproot morphology of red clover,any factor that affects either of them will decrease the survivaland persistence of plants. In that sense, high mortality of plantswas reported as a consequence of grazing and crowndeteriorationby disease (Skipp and Christensen 1990) or internal breakdown(Zeiders et al. 1971). In Chile, the research conducted byAlarcónet al. (2010) demonstrated the importance of the root borer in thepersistence of red clover. This is in agreement with previousreports (Graham and Newton 1959; Carrillo and Mundaca 1974;Quiroz et al. 2005; Tapia et al. 2005). This insect, in its larval andadult stages, feeds on roots, causing weakening and subsequentdeath of clover plants. Chemical control of H. obscurus is notfeasible and management of the insect by crop rotation has beensuggested (Aguilera et al. 1996). Furthermore, the achievementsof the INIA’s red clover breeding program in terms of plantsurvival and forage yield (persistence) demonstrate that breedingis the main strategy to solve the problem. On the other hand, thestudies on semiochemicals and insect interactions may enhancethe genetic gain for persistence.

Red clover breeding programsmust consider the environmentandmanagement, because plant behaviour, especially persistence,is the result of the interaction of genotype and biotic and abioticfactors of the environment. Ortega (1996) studied the effect ofdifferent environments on plant morphology, survival of plants(plant mortality) and forage yield. In spaced plants, the crowndiameter, total weight of roots and weight of the main root werehighly correlated with survival of plants, and when managementintensified towards frequently cut swards or grazed swards,these same morphological characters were not correlated. Aftera relatively long period of trial evaluation (3 years), plantmortality of spaced plants was significantly correlated withmortality of swards infrequently cut in both mixture andmonoculture, as well as swards frequently cut in monoculture.However, in all cases where shorter periods were studied andwhere swards were frequently cut in mixture with grass or grazedwith sheep, correlations were non-significant (Ortega 1996).Ford and Barrett (2011) reported improvements in adaptationsto sheep grazing and found that most of the traits evaluatedunder grazing in rows with plants spaced 10 cm apart wereweakly to moderately correlated with performance in mixedswards.

Genetic gains in breeding programs for forage yield havebeen reported by some authors as low to non-existent (Casler andBrummer 2008). However, important breeding achievements inpersistence of red clover are described in the current paper, andhave been reported by other authors (Herrmann et al. 2007;Marshall et al. 2011). Therefore, the use of efficient breedingmethods and strategies, as well as the environment of screening,is fundamental to make use of additive genetic variance and

Advances in red clover persistence improvement by breeding Crop & Pasture Science G

achieve genetic gain (Casler and Brummer 2008; Conaghan andCasler 2011).

Selection for agronomic traits in swards could be performedusing spaced plants if they are correlated (Conaghan and Casler2011). Casler and Brummer (2008) and Conaghan and Casler(2011) compared theoretical expected genetic gains of differentbreeding methods in perennial forages and reported thatdifferences between breeding methods will depend mainly onheritability of the traits, number of traits to be considered,correlation between traits, selection intensity and selectioncycle time. Casler and Brummer (2008) concluded thatamong- and within-family selection is better than or equal tofamily selection providing that the within-family selectioncriterion (X or Y) is heritable and has a positive geneticcorrelation with the desired trait (Y). Ortega et al. (1997)reported narrow-sense heritability for plant mortality in redclover of 20%. Because of the low h2, a progeny test method(genotypic selection) would be more advisable; however, thehigh mortality of plants makes it difficult and expensive tomaintain parental clones. By contrast, yield and red cloverplant population have significant correlations in trialsevaluated over an extended period as reported in this paper;also, Ortega (1996) reported significant correlations betweensurvival of red clover spaced plants and infrequently cutswards when long evaluation periods were considered. Thus,based on the complexity of persistence as a character to beimproved and the main findings of our studies and previousreports, our breeding program has used a practical approach.The combination of recurrent selection of half-sib families, theamong-family selection based on sward performance, and,finally, the within-family selection based on spaced plantsevaluated simultaneously over 3 years has been successful interms of combining survival of plants, forage yield andpersistence. Depending on location, we have obtained arealised genetic gain in forage yield over several years(persistence) of 0.4–2.6% per year.

Conclusions

The complexity of the character persistence and the positive andhighly significant correlation between plant population andforage yield demonstrates the importance of selecting plantsfor survival under field conditions with the final aim ofimproving the persistence of red clover. The improvedperformance of the newest cultivars and experimental linesselected is a demonstration of the successful breeding strategyused, consisting of a modified among and within half-sib familyrecurrent selection method, evaluating at the same time familiesas swards and as spaced plants. Depending on location, wehave obtained a realised genetic gain in forage yield overseveral years (persistence) of 0.4–2.6% per year. Our researchon plant semiochemicals and their interaction with the rootborer may enhance our breeding program in the future butalways considering that persistence is a complex trait thatdepends on the interaction of many biotic and abiotic factors.

Acknowledgements

This work was supported by INIA grant 500302-70, FONDECYT grants1020297, 1070270, 1100812 and 11130715.

References

Aguilera A, Cisternas E, Gerding M, Norambuena H (1996) Plagas de laspraderas. In ‘Praderas para Chile’. (Ed. I Ruiz) pp. 309–339. (Instituto deInvestigaciones Agropecuarias: Santiago, Chile)

Alarcón D, Ortega F, Perich F, Pardo F, Parra L, Quiroz A (2010)Relationship between radical infestation of Hylastinus obscurus(Marsham) and the yield of cultivars and experimental lines of redclover (Trifolium pratense L.). Revista de la ciencia del suelo ynutrición vegetal 10, 115–125. doi:10.4067/S0718-27912010000200003

Avendaño R (1965) La variedad Quiñequeli y su evaluación con respecto aalgunos tréboles rosados corrientes. Agricultura Técnica 25, 167–171.

Belzile L (1987) Effet de la présence de la fleole des prés sur la survie a l’hiverdu trèfle rouge. Canadian Journal of Plant Science 67, 1101–1103.doi:10.4141/cjps87-148

Carrillo R, Mundaca N (1974) Biología de Hylastinus obscurus (Marsham)(Coleoptera: Scolytidae). Agricultura Técnica 24, 29–35.

Casler M, Brummer C (2008) Theoretical expected genetic gains for among-and-within-family selection methods in perennial forage crops. CropScience 48, 890–902. doi:10.2135/cropsci2007.09.0499

Choo TM, Connolly BJ, Langille JE, Drapeau R, Coulman B, Walton RB,Bubar JS, Goguen B, Madill J, Proulx JG, Fairey DT (1987) Marino redclover. Forage Notes, Canada, No. 31, pp. 64–65.

Conaghan P, Casler M (2011) A theoretical and practical analysis of theoptimum breeding system for perennial ryegrass. Irish Journal ofAgricultural and Food Research 50, 47–63.

Cosgrove GP, Brougham RW (1985) Grazing management influences onseasonality and performance of ryegrass and red clover in a mixture. In‘Proceedings of the NZ Grassland Association’ Vol. 46, 71–76. (NZGrassland Association Inc.: Mosgiel, New Zealand)

Cuevas E, Balocchi O (1983) ‘Producción de forraje.’ Serie B7. (Instituto deProducción Animal, Universidad Austral de Chile: Valdivia, Chile)

Ford JL,BarrettBA (2011) Improving red clover persistence under grazing. In‘Proceedings of theNZGrasslandAssociation’Vol. 73, pp. 119–124. (NZGrassland Association Inc.: Mosgiel, New Zealand)

FrameJ (1990)The role of red clover inUnitedKingdompastures. In ‘Outlookon agriculture’. Vol. 19(1), pp. 49–55. (The West of Scotland College:Glasgow, Scotland)

Frame J, Harkess R, Hunt I (1976) The influence of date of sowing andseed rate on the production of pure sown red clover. Journal of theBritish Grassland Society 31, 117–122. doi:10.1111/j.1365-2494.1976.tb00783.x

Graham JH, Newton PC (1959) Relationship between root feeding insectsand incidence of crown and root rot in red clover. Plant Disease Reporter43, 1114–1116.

Hejduk S, Knot P (2010) Effect of provenance and ploidity of red clovervarieties on productivity, persistence and growth pattern in mixturewith grasses. Plant, Soil and Environment 56, 111–119.

Herrmann D, Boller B, Studer B, Widmer F, Kölliger R (2007)Characterization of persistence in red clover (Trifolium pratense L.).In ‘Proceedings of the XXVII EUCARPIA Symposium: Improvementof Fodder Crops and Amenity Grasses’ Copenhagen. pp. 71–76.(EUCARPIA)

Jewis OR (1993) Shoot development and number. In ‘Sward measurementhandbook’ 2nd edn (Eds A Davies, RD Baker, SA Grant and ASLaidlaw) pp. 99–120. (British Grassland Society: Kenilworth, UK)

Lewis GC, Heard AJ, Gutteridge RA, Plumb RT, Gibson RW (1985) Theeffects of mixing italian ryegrass (Lolium multiflorum) with perennialryegrass (L. perenne) or red clover (Trifolium pratense) on the incidenceof viruses. Annals of Applied Biology 106, 483–488. doi:10.1111/j.1744-7348.1985.tb03139.x

Manosalva L, Pardo F, Perich F,Mutis A, Parra L, Ortega F, Isaacs R, QuirozA (2011) Behavioral responses of clover root borer to long-chain fattyacids from young red clover (Trifolium pratense) roots. EnvironmentalEntomology 40, 399–404. doi:10.1603/EN10008

H Crop & Pasture Science F. Ortega et al.

Marshall AH, Lowe M, Vale J (2011) Improved persistence of red clover(Trifolium pretense L.) varieties in mixed swards. In ‘Grassland in achanging world’. Grassland Science in Europe, Vol. 16, pp. 73–75.(European Grassland Federation)

Ortega F (1996) Variation in mortality, yield and persistence of red clover(Trifolium pratense L.). PhD thesis. University of Wales, Aberystwyth,UK.

Ortega F, Levío J (2011a) SuperQueli INIA, Nuevo Cultivar Chileno deTrébol Rosado (Trifolium pratense L.). I- Origen y descripciónmorfológica. In ‘Libro de resúmenes del XXXVI Congreso Anual dela Sociedad Chilena de Producción Animal (SOCHIPA)’. Punta Arenas,Chile. pp. 195–196. (SOCHIPA A.G.)

Ortega F, Levío J (2011b) SuperQueli INIA, Nuevo Cultivar Chileno deTrébol Rosado (Trifolium pratense L.). II. Comportamiento Agronómicoen La Araucanía, Chile. In ‘Libro de resúmenes del XXXVI Congresoanual de la Sociedad Chilena de Producción Animal (SOCHIPA)’. PuntaArenas, Chile. pp. 193–194. (SOCHIPA A.G.)

Ortega F, Galdames R, Aguilera A (1997) Fitomejoramiento de trébol rosado(Trifolium pratense L.) II- Comportamiento de una prueba de progeniesy sus relaciones con el bloque de policruzamiento. Agricultura Técnica57(2), 79–86.

OrtegaF,GaldamesR,AguileraA,RomeroO,Ruiz I, Soto P, TorresA (2003)Redqueli-INIA, Nuevo cultivar de trébol rosado sintético. AgriculturaTécnica 63(2), 207–211.

Ortega F, Galdames R, Soto P, Teuber N, Torres A, Levío J (2012a) Avancesen mejoramiento genético de trébol rosado (Trifolium pratense L.) enChile. In ‘638CongresoAgronómico, Contribuyendo a la SustentabilidadAlimentaria’. Temuco, Chile, 322.

Ortega F, Quiroz A, Parra L, Levío J (2012b) Relaciones entre población deplantas, rendimiento de forraje y persistencia productiva en trébol rosado(Trifolium pratense L.). In ‘638 Congreso Agronómico, Contribuyendo ala Sustentabilidad Alimentaria’. Temuco, Chile, 325.

Palma R, Mutis A, Manosalva L, Ceballos R, Quiroz A (2012) Behavioraland electrophysiological responses of Hylastinus obscurus to volatilesreleased from the roots of Trifolium pratense (L.). Journal of Soil Scienceand Nutrition 12, 183–193.

Palma R, Mutis A, Isaacs R, Quiroz A (2013) Type and distribution ofsensilla in the antennae of the red clover root borer,Hylastinus obscurus.Journal of Insect Science 13, 1–10.

Parra L, Mutis A, Ortega F, Quiroz A (2013) Field response of Hylastinusobscurus Marsham (Coleoptera: Curculionidae) to E-2-hexenal andlimonene, two host-derived semiochemicals. Ciencia e InvestigaciónAgraria 40, 645–650.

Quiroz A, Ortega F, Ramirez C, Wadhams L, Pinilla K (2005) Response ofthe beetle Hylastinus obscurusMarsham (Coleoptera: Scolytidae) to redclover (Trifolium pratense L.) volatiles in a laboratory. EnvironmentalEntomology 34, 690–695. doi:10.1603/0046-225X-34.3.690

Rhodes I, Ortega F (1996) Progress in ForageLegumeBreeding. In ‘Legumesin sustainable farming system’. (Ed. D Younie) pp. 62–71. BritishGrassland Society, Occasional Symposium No. 30, Aberdeen,Scotland. : (British Grassland Society: Kenilworth, UK)

Rhodes I, Ortega F (1997) Plant breeding achievements and prospects,Forage Legumes. In ‘Seeds of Progress. (Ed. JR Weddell) pp. 15–27.British Grassland Society, Occasional Symposium No. 31, Aberdeen,Scotland. (British Grassland Society: Kenilworth, UK)

SAS Institute (2013) ‘SAS 9.3.’ (SAS Institute: Cary, NC, USA)Sheldrick RD, Lavander RH, Tewson VJ (1986) The effects of frequency of

defoliation, date of first cut and heading date of a perennial ryegrasscompanion on the yield, quality and persistence of diploid andtetraploid broad red clover. Grass and Forage Science 41, 137–149.doi:10.1111/j.1365-2494.1986.tb01798.x

Skipp RA, Christensen MJ (1990) Selection for persistence in red clover:influence of root disease and stem nematode. New Zealand Journalof Agricultural Research 33, 319–333. doi:10.1080/00288233.1990.10428425

Steiner JJ, Alderman SC (2003) Red clover seed production: IV. Effect andeconomics of soil pH adjusted by lime application. Crop Science 43,624–630. doi:10.2135/cropsci2003.0624

Tapia S, Pardo F, Perich F, Quiroz A (2005) Clover root borer Hylastinusobscurus (Marsham) (Coleoptera: Scolytidae) has no preference forvolatiles from root extracts of disease infected red clover. ActaAgriculturae Scandinavica, Sect. B Soil & Plant Science 55, 158–160.

Tapia T, Perich F, Pardo F, Palma G, Quiroz A (2007) Identification ofvolatiles from differently aged red clover (Trifolium pratense) rootextracts and behavioural responses of clover root borer (Hylastinusobscurus) (Marsham) (Coleoptera: Scolytidae) to them. BiochemicalSystematics and Ecology 35, 61–67. doi:10.1016/j.bse.2006.05.020

Taylor NL (1987) Forage legumes. In ‘Principles of cultivar development.II. Crop species’. (Ed. WR Fehr) pp. 209–248. (Macmillan PublishingCompany: New York)

Taylor NL, Quesenberry KH (1996) ‘Red clover science’. Current PlantScience and Biology in Agriculture Vol. 28. (Springer: Berlin,Heidelberg)

Ussher G (1986) Case study information reflecting farmer use of Pawerared clover in northern northland. In ‘Proceedings of the NZ GrasslandAssociation’ Vol. 47, pp. 179–182. (NZ Grassland Association Inc.:Mosgiel, New Zealand)

Zeiders KE, Graham JH, Sprague VG, Wilkinson SR (1971) Internalbreakdown of red clover (Trifolium pratense L.) in relation toenvironmental, cultural, and genetic factors. Agricultural ResearchService, Plat Science Research Division, US Department ofAgriculture 34–126. 24 p.

Advances in red clover persistence improvement by breeding Crop & Pasture Science I

www.publish.csiro.au/journals/cp