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Revista de la ciencia del suelo y nutrición vegetal

versión On-line ISSN 0718-2791

R.C. Suelo Nutr. Veg. v.10 n.2 Temuco  2010 

R.C. Suelo Nutr. Veg. 10(2): 115 -125 (2010)




Daniela Alarcón1, Fernando Ortega2, Fernando Perich3, Fernando Pardo3, Leonardo Parra1 and Andrés Quiroz1'3'4*

1Laboratorio de Ecología Química, Universidad de La Frontera, Temuco, Chile.
2Instituto de Investigaciones Agropecuarias, INIA Carillanca, Chile. Casilla postal 58-D, Temuco, Chile.
3Departamento de Ciencias Químicas, Universidad de La Frontera, Temuco, Chile.
4Center of Chemical Ecology of Terrestrial and Aquatic Systems (CETAS), BIOREN, Universidad de La Frontera, Temuco, Chile. Corresponding author:


Red clover (Trifolium pratense L.) is a valuable forage legume grown alone or in mixture with grasses in temperate regions of the world. Red clover is adapted to a wide range of soil types, pH levels, environmental and management conditions. However, the main limitation of this species is the lack of persistence related to the high mortality of plants. In Chile the main biotic factor affecting survival of plants is the root borer Hylastinus obscurus (Marsham) (Coleóptera; Scolytidae). In 1989, a red clover (T. pratense L.) breeding program was started at Carillanca Research Center of the Institute of Agricultural Research (INIA), Chile, with the main objectives of improving the survival of plants, forage yield and persistence. In 2002 a research line to study the interaction between the root borer and the plant was started. This paper describes briefly the improvement obtained in survival of young plants after twenty years of breeding and the importance of root borer population regarding forage yield. Experiments were conducted at Carillanca Research Center under irrigated conditions, comparing under cutting the dry matter yield of new synthetic lines and Redqueli-INIA with Quinequeli-INIA. The experimental lines Syn Int IV, Syn Pre III, Syn Int V and Syn Int VI were more productive than the cultivars Redqueli-INIA and Quiñequeli-INIA at the second season. The evaluation of the biological parameter allowed identifying a tolerant line, Syn Pre I, and three potential new varieties: Syn Pre III, Syn Int V and Syn Int VI. This is the first report showing an inverse relation between dry matter yield of red clover and root borer population (P≤0.05). Moreover, we report the first evidence that H. obscurus start the colonization of red clover plant of 6-month-old.

Keywords: Hylastinus obscurus, red clover, plant population, breeding, dry matter yield



Red clover (Trifolium pratense L.) is an  important forage legume grown on  approximately 4 million hectares in temperate regions of the world and serves a multiple purpose in agricultural  rotations (Riday, 2009). In Chile is a valuable resource for animal production and for the seed industry with exports in the last decade from 700 to 1700 tons year-1 and 100,000 ha cultivated, corresponding to 20% of the total sown pastures (Ortega, 2009). This is a legume that is used primarily in short-rotation pastures (Catrileo and Rojas, 1987), grazing and cutting (hay, silage and soiling). Although red clover is botanically considered a perennial plant, production levels decline dramatically two years after sowing (Cuevas and Balocchi, 1983; Leath, 1985; Steiner and Alderman, 2003), determined by the high mortality of plants (Ortega, 1996; Rhodes and Ortega, 1997).

Factors involved in mortality of red clover plants include root rot and infestations by the root borer Hylastinus obscurus Marsham (Coleóptera: Scolytidae) (Graham and Newton, 1959; Carrillo and Mundaca, 1974). This scolytid is the most important pests of red clover (T. pratense) throughout the world (Steiner and Alderman, 2003). Both larvae and adults bore and feed into the roots causing a significant reduction in production levels and persistence of red clover stands within two years after sowing (Cuevas and Balocchi, 1983; Steiner and Alderman, 2003). The root damage affects the carbohydrate reserves, the development of stems and leaves, resulting in a total destruction of the plant (Graham and Newton, 1959; Koehler et al, 1961; Carrillo and Mundaca, 1974; Matamala, 1976). The intensity of the attack can reach between 70 and 100% of plants affected the second and third year of clover established (Aguilera, 1995). In 1989, a red clover breeding program was launched at Carillanca Research Center of the Institute of Agricultural Research (INIA-Chile), with the main objectives of improving the survival of plants, forage yield and persistence compared to the Chilean diploid and double cut cultivar Quiñequeli-INIA released in 1962. The first new synthetic developed was Redqueli-INIA, cultivar released in 1997.

Previous studies have shown that H. obscurus respond to chemical stimulus from the plant (Quiroz et al, 2005; Tapia et al, 2005; Tapia et al, 2007), evidencing that this insect is able to recognize their host by detecting chemical compounds released from both aerial and root parts of red clover plant. These results suggested that the insect is strongly associated to the red clover roots because of the chemical compounds released from roots.

There are not studies about the population dynamic of H. obscurus and their potential influence on selecting new red clover cultivars. This research aims to relate H. obscurus population with conventional agronomical parameters evaluated in red clover breeding, such as dry matter yield, forage cover and plant density. Moreover, crown diameter of red clover plant was incorporated in these evaluation, because its relation with the oviposition behavior of H. obscurus. The research was performed studying two cultivars, Redqueli-INIA and Quiñequeli-INIA, and seven experimental lines proposed by INIA Carillanca.



Location of the research and experimental design

A trial was established in spring of 2007 at INIA Carillanca Research Center, located in the Araucania Region, Cautín Province, 38° 41' south latitude and 72° 25' west longitude and 200 m.s.n.m. Quiñequeli-INIA and Redqueli-INIA cultivars (Avendaño, 1965; Ortega et al, 2003) and 7 experimental lines under evaluation (Syn Pre I, Syn IV, Syn Pre II, Syn Int IV, Syn Pre III, Syn Int V and Syn Int VI) were distributed in an experimental design of ran complete blocks with three replicates. Plot size was 1.8 x 7 m each.

Agroclimatic characteristics of the research site

The climate is characterized by annual average temperatures of 10°C, with a maximum mean monthly value of 21.5°C January and a minimum of 2.3°C in July. Average annual precipitation is close to 1,400 mm. There is an annual sum of 2,346 hours of cold and thermal summation base temperature of 5°C of 1.394 day. The frost-free period is between January and February.

The soil is an Andisol, belonging to the series Vilcún, Temuco family, classified as Medial, Music, Entice, Dystrandept. Agriculture has depth of 80 cm and a flat topography with a slope of 0-2%.

Trial establishment and management

Cultivars and experimental lines were sown in previously prepared soil on September 12, using a Planet Junior manual seeder. Seedlings were in rows with a separation of 20 cm each other. The seed dosage was 15 kg ha-1 using rhizobium inoculated seed.

Fertilizers were applied annually according to soil analysis; fertilization at sowing consisted of 140 kg P205 ha-1, 151 kg K20 ha-1 , 5 kg S ha-1; 1 kg Mg ha-1 ; 65 kg Na ha-1; 153 kg CaO ha-1; maintenance fertilization the second season was 140 kg P205 ha-1 and 200 kg KzOha-1.

The trial was irrigated five times in the first season (from January to march of 2008) and eight times during the second season (from November of 2008 to March of 2009. More prominent weeds were removed by hand but no herbicide was applied.

Evaluation of Hylastinus obscuvus (Marsham) in red clover roots

Insect were sampled monthly from November 2007 to May 2009. An average of 12 plants of each cultivar and experimental line were randomly chosen from the three replicates. Plants were extracted with both aerial and radical parts, and they were put into a paper bag and transferred to the Chemical Ecology Laboratory of the Universidad de La Frontera. The radical part was separated from the aerial part and it was carefully cut with a scalpel for evaluating the presence of adult and larvae of H. obscurus inside red clover roots because these stages cause weakening and death of red clover plants (Aguilera et ah, 1996). These surveys were started when the plants were 4-month-old.

Crown diameter and length of the main root

Crown diameter and length of the main root was evaluated in the same plants used for H. obscurus evaluation. Diameter was measured with a Vernier caliper and root length with a metallic rule. Evaluations were performed with the total number of plants collected in the field.

Evaluation of agronomic parameters

Seven cuts were carried out for determining dry matter (DM) yield. Three cuts were performed in the first growing season (2007-2008), and four in the second season (2008-2009). The aged of the plants in the first season were 5 (January 2 2008), 6 (February 20 2008) and 7 (March 31 2008) months, while for the second season the aged of the plants were 14 (October 16 2008), 16 (December 11 2008), 18 (February 9 2009) and 21 (May 5 2009) months.

Because red clover is a perennial, temperate climate species, the beginning of the second growing season cycle was considered as spring 2008. Cuts were made when the clover was found with 5 to 10% of flowering or when it reached an altitude of 40 to 50 cm. Aerial growth was mowed leaving a residue of approximately 5 cm from the soil. A central section of each (1.2 m2) with two quadrants of 0.6 m2 each, was used by determining the agronomical parameters: a) plant population was determined as number of plants per m2; b) the foliage cover was evaluated determining the percentage of empty spaces of at least 10 cm between plants found in 1.2 m2; c) dry matter (DM) was evaluated from a subsample of 150 to 200 grams dried in an oven at 65°C for 48 hours, for obtaining the yield in t DM ha-1.

Statistical Analysis

The normality of the data was determined by Shapiro Wilk test. As the nmumber of insects did not correspond to normal data, it was performed the non parametric Friedman test followed of Conover (P≤0.05) test. Accumulative number of larvae and adult of H. obscurus were used for statistical analysis. As agronomical parameters correspond to normal data, ANOVA (P≤0.05) analysis was carried out and the separation of the groups was determined by LSD (P≤0.05). Correlation between parameters was tested with Kendall's rank and Spearman's rank tests (P≤0.05).



Total dry matter yield of red clover in two seasons is shown in Figure 1. The results showed that there were not significant differences among cultivare and experimental lines in the first season.

However, the experimental lines Syn Int IV, Syn Pre III, Syn Int V and Syn Int VI were more productive than the cultivars Redqueli-INIA and Quiñequeli-INIA at the second season. Ortega et al. (2003) reported 17.40 and 15.00 t DM ha-1 for Redqueli-INIA and Quiñequeli-INIA respectively for the total yield of the first two seasons. In this study, both cultivars were roughly less productive, 15.25 and 14.00 t DM ha-1, respectively. However, DM yield from the experimental line Syn Int VI (20.32 t DM ha-1) at second season was comparable to the forage yield of Redqueli (21.10 t DM ha-1) reported by Ortega et al. (2003) after three seasons.

There were not significant differences of forage yield among cultivars and experimental lines in the three cuts at the first season (Figure 2), and plants did not show infestation by H. obscurus at the first cut performed when plants were 5-month-old. However, the beginning of H. obscurus colonization started in all cultivars and experimental lines when plants were six months old. This differs from report of González (1989), indicating that this scolitid does not attack red clover during the first year of establishment. Our results indicated that the insects were found at the end of February, after the fly period and when the insect deposit the eggs in the base of the crown of red clover plants (Matamala, 1976; Aguilera, 1989).

Considering the presence of H. obscurus, there were not significant differences among cultivar and experimental lines at the second and third cuts, which could be due to percentage of damaged roots represented on average only the 26% of the roots sampled in each cultivar and experimental line evaluated. Nevertheless, a trend for bearing a higher amount of H. obscurus per plant was observed on the Redqueli-INIA cultivar at second and third cuts, with 1.78 and 1.18 insects per plant respectively.



During the second season, significant differences were observed on both forage yield and insect population (Figure 3). In the second (December 2008) and third (February 2009) cuts of the second season, Syn Int VI, Syn Int V and Syn Pre III were more productive than the cultivars Requeli-INIA and Quinequeli-INIA. The low forage yield shown by all the cultivars and experimental lines at the fourth cut, 1.8 t DM ha-1, can be explained because this evaluation was carried out during the autumn (May 2009).

The decrease in dry matter production is common during the winter according to data reported by Demanet (2008), indicating that Quiñequeli-INIA and Redqueli-INIA produce only between 9 to 11% of the total annual production of dry matter, and the maximum production is obtained during the summer. Significant differences in the population of H. obscurus between cultivars and experimental lines were only observed in the second season (Figure 3).

The negative association between H. obscurus population and DM yield is shown in Figure 4. The experimental lines Syn Pre III, Syn Int V and Syn Int VI showed significantly higher forage yields and the lowest H. obscurus populations. The same effect was observed when the insect population was related to plant density (Figure 5). A correlation analysis of both parameters showed that H. obscurus population is negatively associated to plant density (Kendal's rank test, P0.05; Spearman's rank test, P≤0.05)








Because of this association, these experimental lines could be considered with a higher partial resistance to the root borer attack. On the other hand, Syn Pre I showed forage yield similar to the synthetic lines mentioned above, but with a significantly higher load of insects (Figure 4). Moreover, Syn Pre I showed a similar plant density, foliage cover and plant population to resistant lines Syn Pre III, Syn Int V and Syn Int VI (Figures 5 and 6). These data suggest that Syn Pre I could be considered as tolerant to H. obscurus, according to the definition reported by Rosenthal and Agrawal (1994) and Strauss and Agrawal (1999),and can be evaluated under both artificial and natural environments (Tiffin and Inouye, 2000).

The results shown in Figure 4 allow distinguishing two groups: a) one formed by the partially resistant lines Syn Pre III, Syn Int V and Syn Int VI, and b) Redqueli-INIA and Quiñequeli-INIA and the rest of experimental lines, including the tolerant line, Syn Pre I. The second group is significantly more attacked by H. obscurus than the first group. Redqueli-INIA and Quiñequeli-INIA showed an increase growing pattern of the root crown diameter, in comparison to the resistant lines (Figure 7).


This finding is in accordance to the existence of a positive correlation coefficient between the crown diameter of red clover plants and the number of individuals of H. obscurus founded inside the roots (Matamala, 1976).



Our study reports the first evidence that H. obscurus start the colonization of red clover plant of 6-month-old. Moreover, we report a complementary new biological tool for selecting new varieties of red clover, such as the evaluation ofH. obscurus population. The results showed an inverse relation between forage yield and H. obscurus population.

The evaluation of the biological parameter allowed identifying a tolerant line, Syn Pre I, and three potential new varieties: Syn Pre III, Syn Int V and Syn Int VI. These experimental lines showed the lowest population of H. obscurus and the highest forage yield of red clover under the experimental conditions.



Financial support for this research was provided by FONDECYT grant 1070270 and INIA grant 500302-70.



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