The effect of two pesticides ( Vitavax-300 and Gaucho ) on rhizobia and on the nodulation of four legumes

The application of seed-protecting pesticides is often a prerequisite for raising legumes in the tropics. However, these chemicals may influence the development of root nodule symbiosis. In the present study, high concentrations of Gaucho insecticide (imidacloprid) and Vitavax-300 fungicide (carboxin and captan) clearly inhibited the growth of root nodule bacterium under laboratory conditions. However, they did not effect to the nodulation or biomass production of Arachis pintoi, Arachis hypogaea, Mucuna pruriens or Desmodium ovalifolium raised in a green house in eastern Costa Rica. Explanations for these results are discussed.

ntroduction Different types of fungi and insects cause great economic losses for the tropical agriculture. In particular, the nutrient-rich seeds of the legumes are frequently attacked. However, many pesticides which are used to reduce seed rot and seedling damping-off of the legumes, are also toxic to the rhizobia (Diatloff, 1970), the beneficial, nitrogen fixing root nodule bacterium of the leg-umes. Some pesticides reduce the amount of fixed N 2 (Fisher, 1976) and some may impair or even prevent the root nodule development (Staphorst and Strijdom, 1976). The goal of the present investigation was to study the influence of two commonly used Central-American pesticides on the growth of rhizobia under laboratory conditions. In addition, the sensitivity of the root nodule formation of Arachis pintoi Pinto, Arachis hypogaea L., Mucuna pruriens L. and Desmodium ovalifolium Wall, to these pesticides was studied.
Influence of the pesticides on the rhizobial isolate The pesticides (100 g) were poured into sterile water (200 ml) and centrifuged at 5000 rpm for 5 minutes to separate the inert compounds. The supernatant was dispensed into test tubes containing diluted (1:10) yeast-extract-mannitolbroth (YEM) described by Vincent (1970) to form a serial of increasing pesticide contents ( Fig. 1 and Fig. 2). The tubes were inoculated with rhizobial isolate from A. pintoi. The inocu-lum had been grown for 8 days in 10ml of YEMbroth, then it was diluted in 90 ml of sterile water and dispensed in 1 ml doses to the test tubes. The tubes were kept for 8 days in an incubation chamber at 32°C. The growth (cell division rate) of the isolate was determined by using a spectrophotometer Perkin-Elmer Junior Model 35. The microbial growth was expressed as absorbances using the sterile media as reference.

Influence of the pesticides on nodulation
The seeds of the legumes were surface sterilised by immersing them in concentrated H 20 2 for 1 minute and then rinsing them with sterile water three times. Then they were coated with Vitavax--300 (approximately 2 g/1 kg of the seeds), and Gaucho (approximately 5 g/1 kg of the seeds) and finally with peat inoculums. The seeds were sown in half-litre-containers, which had been filled with ordinary nursery soil collected from the riverside forest. Uncoated seeds served as controls, and non-inoculated seeds were used to check the occurrence of native rhizobia in the soil. The number of seeds per container varied from 4 to 10 at the beginning of the experiment, depending on the seed size of the species. The containers were arranged in a completely randomised setup (n = 4) and irrigated with tap water. The seedlings were raised in an open green   Vol. 5 (1996): 203-207. Table 1.Total dry weight of the plants and root nodules at the end of the 10-week-raising-period in a green house in Costa Rica, Turrialba. Seeds were coated with pestices Vitavax-300 and Gaucho and inoculated with peat inocula. Uncoated seeds served as controls. Standard errors are given in parenthesis, n = 4.

Legume
Total dry weight (g) Dry weight of root nodules (g) for 10 weeks. The plants were harvested, and weighed with theirroots at the end of the experiment. The root nodules were collected and dried separately. The total dry weight of the plants was determinedby drying them overnight at 80°C.

Results
High concentrations of Vitavax-300 clearly inhibited the growth of the rhizobia (Fig. 1). Three grams of Vitavax-300 in one litre of the nutrient broth was enough to halve the growth of the micro-organism. No growth was observed in the solutions in which the concentration of Vitavax--300 exceeded 30 g/1.
The influence of Gaucho on the rhizobia was not as clear (Fig. 2). The Gaucho concentrations from 0.7 to 17.9 g/1 did not inhibit the growth of the isolate. However, no clear growth was found in the tubes where the Gaucho concentration was 29 g/1 or more.
The treatment of the seeds with pesticides did not cause any notable changes in the dry mass production of the legumes (Table 1). The number and dry weight of the root nodules showed great variation in all treatments. Pesticides did not prevent nodulation.

Discussion
The low water-solubility of the investigated pesticides makes it very difficult to determine the exact concentrations of captan, carboxin or imidacloprid in the water-based experimental medium. However, it was clearly shown that high concentrations ofVitavax-300 and Gaucho were lethal to the rhizobial isolates in a liquid media under laboratory conditions. The deleterious influence of some pesticides to rhizobia has been reported earlier by Diatloff (1970) and Staphorst and Strijdom (1976). They have also found that strains of rhizobia from various origins differ in their relative sensitivity to pesticides.
Taking into consideration the clear effect of the pesticides on the rhizobia, it was quite surprising to notice that Vitavax-300 and Gaucho did not inhibit the nodulation of the investigated legumes. The concentrations of the pesticides inevitably diminishedas the roots elongated, and thus their influence did not reach the nodulation zone. In addition, the exudes from the seeds and roots of the legumes possibly decreased the toxicity of the pesticides, as reported by Richardson (1966), who noticed that the toxicity of thiram may be negated by exudes from the seeds and roots of soybeans. It is also possible that, as the pesticides moved into the soil, they enhanced the growth of rhizobia by killing soil protozoa, which normally feed on rhizobia, as reviewed Miettinen, P. & Echegoyen, RE.: Effect of twopesticides on rhizobia and nodulation offour legumes (Research Note) by Alexander (1985). Lennox and Alexander (1981) reported a thiram-resistant Rhizobium phaseoli-strain, which induced an increment in nitrogen content on beans when their seeds were treated with that pesticide. They also noticed that protozoa proliferated if thiram was not applied to the seed, but the numbers of ciliates and flagellated protozoa were deleteriously influenced by the pesticide. Diatloff (1970) also reported good nodulation of legumes after treating the seeds with pesticides.
The uninoculated legumes were all nodulated by the native rhizobia present in the greenhouse soil. In the case of Mucuna pruriens, this was not expected since it had not been cultivated in the region before. However, Faria et al. (1989) came to the conclusion that the effective rhizobia of several legumes are widely distributed outside the geographical occurrence of their hosts, since legumes introduced from Australia and Central and North America nodulated abundantly in these sites without previous rhizobial inoculation in Brazil. In such cases, inoculation with laboratory-grown rhizobia is unnecessary.