Abstract

Media summary

Key words

Saponins, Alfalfa, Alfalfa weevil resistance

Introduction

Some secondary metabolites may be used by the plant as self-defense mechanisms against insects. Saponins are found in more than 100 plant families including legumes such as alfalfa. They have high molecular weights and are located in vacuoles (Hostettman and Marston 1995). Their quantity in plants varies with genotype, plant age, physiological, environmental and agronomic factors (Fenwick et al. 1991). Two effects of alfalfa saponins are: (1) they are poisonous against insects, and (2) they are harmful by causing the production of gases in the intestines of ruminants (Mazahery-Laghab 1997). It has been shown that saponins present in alfalfa weevil are poisonous to Tribolium castaneum (Fenwick et al. 1991). It has also been found that there are different types of saponins present in alfalfa, some of which produce low amounts of gas in ruminants and have useful toxic effects on insects and fungi (Mazahery-Laghab 1997).

The alfalfa weevil (Hypera postica Gyll.) is a pest of legume crops. Adults and larvae of this species feed on alfalfa foliage causing skeletonization of leaves, reducing leaf area and reducing yield. The aim of this study is twofold: (1) to determine total amounts of saponins in alfalfa varieties, and (2) to find the types of saponins and their relationships with alfalfa weevil resistance.

Materials and Methods

a) Cultivars used

Fifteen Iranian and seven foreign alfalfa cultivars and lines, which had previously been studied for their resistance against alfalfa weevil (Mazahary-Laghab and Yazdi – Samadi 1991), were used (Table 1).

b) Saponin extraction and determination

Aerial tissues of alfalfa, which had already been taken and frozen in liquid nitrogen, were used for extraction in duplicate samples (2 replications). Saponins were extracted using the method of Massiot et al. (1992 & 1988). Total saponin amounts were determined by spectrophotometry (Ebrahimzadeh and Niknan 1998). The different kinds of saponins were determined using thin layer chromatography (TLC).

c) Statistical analysis

Data analysis was done for total saponins according to a randomized complete block design with two replications, and mean comparisons by Duncan’s Multiple Range Test. Relationships between each kind of saponin with weevil larval damage explored using Pearson’s Correlation Method.

Results and discussion

Cultivars and lines showed significant differences for the total amounts of saponins; Bam cultivar with the highest and Gooran Teleghan with the lowest amounts (Table 2).

Qualitative analysis of saponins, using the TLC method, showed eight banding patterns. Comparison of bands with standard saponin patterns revealed that band number 3 with Rf = 0.23 was Medicoside J (Rf = the distance of band movement from origin / the distance of solvent movement from origin). Saponin no.4 with Rf = 0.31 was brown in color and recognized as Soyasaponin I and band no.7, being light green, with RF = 0.59, was Medicoside A. The other five bands were not recognized.

The relations between scoring of the eight banding pattern for the alfalfa varieties under study with alfalfa weevil larval damage were found (Table 3). Table 3 shows that there is no significant correlation between total saponin and larval damage (equation 1). Saponins 1, 2 and 8 showed significant negative correlations with weevil larval damage (equations 2, 3 and 9) i.e., they have positive relation with weevil resistance. Saponins 3 and 4 showed significant positive correlations with larval damage (equations 4 and 5), meaning that these saponins reduce weevil resistance. However, saponins 5,6 and 7 showed no significant correlations with larval damage (equations 6,7 and 8). Figure 1 shows the diagrams of linear regression between different saponins and weevil larval damage.

Table 1. Mean larval damage of alfalfa varieties used in the experiment

Table 2. Total amount of saponins in alfalfa varieties (g/kg fresh weight)

Table 3. Regression equations and correlation coefficients for total saponins and weevil damage

+ Y, amount larval damage; X, saponin scores; r, correlation coefficient between banding pattern and larval damage; n.s., non-significant; *,** significant at. 05 and .01 levels, respectively.

Figure 1. Linear regressions between different saponins and weevil larval damage.

References

Ebrahimzadeh H, and Niknam V(1998). A revised spectrophotometric method for determination of triterpenoid saponins. Indian Drugs 35, 379-381.

Fenwick GR, Price KR, Tsukamoto C and Okubo K (1991). Saponins in toxic substances in crop plants. Royal Society of Chemistry, Cambridge, 285-327.

Hostettman K and Marston A(1995). Saponins. Cambridge University Press (Cambridge, U.K.).

Massiot, Lavaud GC, Benkhaled M and Men-Oliver LL (1992). Soyasaponin A, new maltol conjugate from alfalfa and soybean. J. Nat. Prod. 55, 1339-1342.

Massiot, Lavaud GC, Guillaume D and Men-Oliver LL(1988). Reinvestigation of the sapogenins and prosapogenins from alfalfa (Medicago sativa L.). J. Agri. Food Chem. 36,902-909.

Mazahery – Laghab H (1997). Endogeneous resistance to insect pests in alfalfa engineering for enhanced resistance. Ph.D. thesis, Durhan University, U.K.

Mazahery-Laghab H, and Yazdi-Samadi B(1991). Resistance of alfalfa cultivars to alfalfa weevil. Iranian J. Agric. Sciences 25, 11-17 (in Farsi).