The toxicity of azadirachtin, plant extract of Azadirachta indica and captan, active ingredient of fungicide for seed treatment (Bralin 48 SC), to Fusarium graminearum Schwabe, isolated from maize root, was researched. The experiments were carried out in vitro, on potato dextrose agar (PDA) with incorporated different concentrations of azadirachtin or captan. The experiment was carried out in two series, with 5 concentrations, in 5 repetitions.

The obtained data were processed by linear functional model of probate regression, method of maximal probability, and obtained experimental data were evaluated by c²-test.

It was proved that the extract of Azadirachta indica manifested higher toxic effect (over 6000 times) to the mycelium F. graminearum (EC₅₀ = 40.76 mg/l), then captan (EC₅₀ = 26140.57 mg/l). These results are good basis for further investigations and eventually for introducing azadirachtin for the control of F. graminearum on maize by seed treatment.

The coefficient b for captan was 0.74 and for azadirachtin 0.76, which means that F. graminearum is relatively equally susceptible to both compounds. This was also confirmed on the basis of the parallel relationship between their regression lines (ec-p lines).

Key words: azadirachtin, captan, toxicity, F. graminearum, maize, seed treatment

Seed blight, root and stalk rot and lodging can be caused by several fungi and bacteria, such as Diplodia zeae, Fusarium moniliforme, Fusarium graminearum (sexual stage Gibberrela zeae) (Hooker, 1977; McGee, 1988; Ivanović, 1992). Fusarium graminearum is an important plant pathogen that attacks wide range of plant species such as maize, wheat, barley, etc. According to Neergard (1979), Fusarium graminearum is one of the six most dangerous pathogens that attack corn.

In most of the corn-growing states in the U.S.A., the losses are frequently higher than 7.5 %; sometimes they are reported to be as high as 10-20% (Christensen et Wilcoxson, 1966; Roane, 1950; Wiliams et Scchmitthenner, 1963, cit. Neergard, 1979).

According to Smiljaković and Penčić (cit. Anonymous, 1986, 1977) root and stalk rot are the most dangerous diseases of corn in Yugoslavia, where 30% of stalks can be infected with total yield loss of up to 11%. Spreading of corn hybrids brought 90% of lodging of corn stalks in Yugoslavia in some years (cit. Anonymus, 1977).

Damage caused by the fungus is expressed, among other symptoms, in the loss of germinability, reduced emergence and postemergence blight of seedlings. Within the general control measures that should prevent seed-borne and seed-transmitted diseases, seed treatment is one of the measures that can not be avoided in the actual agricultural practice (Neergard, 1979). At present, seven fungicides (active ingredients and mixtures) are registered in Yugoslavia, and captan is one of them (Mitić, 1998).

Bioneem is eco-friendly product based on need seed kernels, which contains many triterpenoids including azadirachtin. This chemical is registered under the Insecticide act 1968, from Central Insecticide Board of the govt. of India (Anonymous*). Azadirachtin acts as an insect repelent, anti-feedant and insect growth regulator. Compared with unrestricted use of pesticides, integrated pest management (IPM) has many advantages and bioneem (azadirachtin) is well suited for this concept. Up to nowadays there has been little information about fungicidal activity of this oil.

A need for further improvement of efficacy of chemical control and requirements of integrated pest management made us decide to seek for an alternative in chemical control of plant pathogens.

This experiment was conducted with two prepararations: plant extract of Azadirachta indica (Bioneem with 0.03% of azadirachtin) and captan, active ingredient of fungicide for seed treatment (Bralin 48 SC with 480 g/l of captan), and fungus Fusarium graminearum Schwabe. Isolates were taken from maize root on PDA and monosporial culture was isolated on CLA. The experiment was carried out in vitro, on potato dextrose agar (PDA) (Leroux and Grdt, 1972) in Petri-dishes, with different incorporated concentrations of azadirachtin or captan. The incorporated concentrations were inoculated with disks from seven-day-old colonies of Fusarium graminearum , and incubated at 25°C, in dark for 3 days. The trial was carried out in two series, with 5 concentrations (7-210 mg/l for azadirachtin and 100-1.000.000 mg/l for captan), in 5 repetitions.

The experiment was assessed after 24, 48 and 72 hours, by measuring the diameter of colonial growth. The data obtained after 48 hours were processed by linear functional model of probity regression, method of maximal probability (Finney, 1964; Lakić et al., 1991), and obtained experimental data were evaluated by c²-test (Spiegel, 1961).

All concentrations of the investigated active ingredients significantly inhibit the colonial growth of Fusarium graminearum. The inhibition from 4.6 to 86.8% (arithmetic mean) was obtained with captan, and from 30.45 to 69.6% with azadirachtin.

After processing raw data by linear functional model of probate regression, the best adapted probate regression lines for captan and azadirachtin were calculated as in table 1.

Common coefficient of relative susceptibility for these two chemicals was calculated and the value of 0.75 was obtained. The fact that the coefficients of relative susceptibility and common coefficient of relative susceptibility were similar gave us the information about parallel relationship, which can also be seen in graph 1.

Graph 1. EC-P lines for captan and azadirachtin

Calculated data were tested by the chi-square test. There were no statistically significant differences for each compound because the calculated chi-square values were zero (much lower then table chi-square value which is 7.82).

Seed treatment is an old and well-known method for preventing diseases as well as for insect control. However, combination of fungicide with insecticide is not always appropriate regarding seed-phytotoxity. There were several indications about negative influence of fungicide-insecticide combinations on seed germination, germination energy, total length of seedlings stalk and roots in Yugoslavia (Milinčić et Šinžar, 1968 cit. Bača et al. 1989; Šinžar et Kljajić, 1969 cit. Bača et al. 1989; Bača et al. 1989). Azadirachtin has already been proved as good insect repelent, an anti-feedant and insect growth regulator (Anonymous*), but since nowadays there has been few information about fungicidal activity of azadirachtin.

Our results proved that the extract of Azadirachta indica manifested higher toxic effect (over 6000 times) to the mycelium F. graminearum (EC₅₀ = 40,76 mg/l), then captan (EC₅₀ = 26140.57 mg/l), standardly used pesticide for maize seed dressing.

The coefficients b for captan were 0.74 and for azadirachtin 0.76, which means that F. graminearum is relatively equally susceptible to both compounds, which also can indicate similar way or mode of action. This was also confirmed on the basis of common coefficient of relative susceptibility (0.75) and the parallel relationship between their regression lines (ec-p lines).

These results on azadirachtin fungicidal effect, as well as already known pest control effects, are good bases for further investigations. Level and spectrum of activity to plant pathogens, type of formulation, field performances etc., should be investigated. Finally, a need for eventual introducing azadirachtin for the control of F. graminearum (another pathogens and insects too) on maize by seed treatment is supported, also, by the fact that bioneem is fully degradable, environment friendly and does not induce resistance in insects. These performances give potential opportunities for azadirachtin in IPM strategy.

The extract of Azadirachta indica manifests higher toxic effect (over 6000 times) to the mycelium of F. graminearum then captan.

The obtained performances give potential opportunities to azadirachtin for maize seed treatment in IPM strategy.

These results are not final and they are good basis for further investigations

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