Sensitivity of some nitrogen fixers and the target pest Fusarium oxysporum to fungicide thiram

This study was carried out to investigate the toxic effects of the fungicide thiram (TMTD) against five nitrogen fixers and the thiram target pest Fusarium oxysporum under laboratory conditions. Nitrogen fixing bacteria Falvobacterium showed the highest values of LD50 and proved to be the most resistant to the fungicide followed by Fusarium oxysporum, while Pseudomonas aurentiaca was the most affected microorganism. LD50 values for these microorganisms were in 2–5 orders of magnitude lower in comparison with LD50 value for Fusarium oxysporum. Thiram was most toxic to Pseudomonas aurentiaca followed by Azospirillum. The lowest toxicity index was recorded for Fusarium oxysporum and Flavobacterium. The slope of the curve for Azomonas, Fusarium oxysporum and Flavobacterium is more steep than that of the other curves, suggesting that even a slight increase of the dose of the fungicide can cause a very strong negative effect. Thiram was more selective to Pseudomonas aurentiaca followed by Azospirillum, Rhizobium meliloti and Azomonas. The lowest selectivity index of the fungicide was recorded for Falvobacterium followed by Fusarium oxysporum. The highest safety coefficient of the fungicide was assigned for Flavobacterium, while Pseudomonas aurentiaca showed the lowest value.

disease, onion smut. It is also used as a seed disinfectant for many vegetables, fungal diseases on safflower, black root of sugar beet, grey mould of strawberries, Botrytis blight in tulips, Colletotrichum lint on flax, for protection of forest nursery seedlings against damping-off and as repellent against rabbit rodents, deer and blackbirds (Montegomery et al. 1936;Harrison, 1961;Muskett & Colhoun, 1940;Harrington, 1941;Newhall, 1945;Taylor & Ruppert, 1946;McKeen, 1950;Hildebrand et al. 1949;Hildreth & Brown, 1955). Horsfall (1956) reported that the relationship of thiram to enzyme systems has provided an area of fundamental investigation. He was the first to propose that the fungicidal effect of thiram was connected with its ability to form complexes with heavy metal ions. It was observed that fungitoxicity of TMTD was not reversed by addition of the trace metals Fe, Zn, Cu, Mn and Mo to the medium.

Introduction
Pesticides are used for the welfare of human beings but in time they will challenge us by showing their toxicity. We can be exposed to them directly, or indirectly through the food chain. Pesticides are toxic compounds to all living organisms, however the effects vary from species to species. Their excessive use causes serious damage to the ecosystem -terrestrial as well as aquatic, and consequently to the surrounding flora and fauna (Paliwal et al., 2009).
Thiram (Tetramethylthiuram disulfide) is a nonsystemic seed dressing fungicide that belongs to the ethylene bisdithiocarbamate (EBDC) chemical class. It is one of the most widely applied dithiocarbamate fungicides in modern agriculture for controlling, damping-off diseases, apple scab, brown rot of stone fruit, Botrytis rot, turf Sensitivity of nitrogen fi xers to TMTD ISSN: 1337-6853 (print version) | 1337-9569 (electronic version) resulting from infection by soil-borne pathogens (Schroth & Hildebrand, 1964). However, many fungicides are toxic to rhizobia (Diatloff, 1970;Hofer, 1958), and some reduce the amount of N 2 fixed (Fisher, 1976;Staphorst, & Strijdom, 1976). Thus, seed protection and seed inoculation are frequently incompatible. One way of allowing for successful infection of legume roots with Rhizobium after treatment of seeds with fungicides is to use a fungicide-resistant inoculant (Odeyemi & Alexander, 1977). Ogunseitan & Odeyemi (1985) suggested that in the chemical control of pests it is important to avoid serious injury to a great variety of microbes whose functions are vital to the crop-producing power of the soil. Odeyemi & Alexander (1977) reported that treatment of legume seeds with Thiram, Spergon and Phygon before rhizobial inoculation decreased the weight of plants and nitrogen fixation considerably. Lennox & Alexander (1981) reported that application of thiram to seeds inoculated with a thiramresistant strain of Rhizobium resulted in a significant increase in dry weight and nitrogen contents of plants compared with inoculation or thiram treatment alone.
The aim of this study was to evaluate the toxic effect of the fungicide thiram on some soil beneficial microbes with special emphasis on nitrogen fixers, besides testing the efficiency of the fungicide on controlling the target pest.

Nitrogen Fixing Bacteria and Fungi Studied
Azomonas sp, Azospirillum sp, Flavobacterium sp, Pseudomonas aurentiaca, and Rhizobium meliloti, were obtained from the microbiological collection of the Department of Biofertilization of the Environment and Natural Resources Research Institute (ENRRI, Sudan).
Fusarium oxysporum, was obtained from the microbiological collection of the Department of Biological Control of the Environment and Natural Resources Research Institute (ENRRI).

Culture Media Used
Two different media meat peptone agar and Czapek Dox agar, were prepared by dissolving the ingredients of each (g) in one liter of distilled water as follows (Tepper et al., 1993)

LD 50 Determination
The concentrations of the fungicide that caused 50% destruction of the cells of pure cultures of the microorganisms (LD 50 ) were calculated by log-dose/probit regression line method Finney (1971) using computer software (Biostat, 2008).
A preliminary experiment was conducted to determine thiram effective concentration limits (20-80%) for Azomonas sp, Azospirillum sp, Flavobacterium sp, Pseudomonas aurentiaca and Rhizobium meliloti as suggested by Zinchenko et al. (1974). Each bacterium strain was grown on meat peptone broth for 24 hours. The amount of 0.5 ml of this culture broth was transferred and used to inoculate plates of meat peptone agar supplemented with different thiram concentrations. The plates were incubated at 28 °C for 48 hours and then the colonies present were counted. A control set of MPA plates not supplemented with thiram was prepared for comparison. The inhibition index for each strain was calculated by subtracting the number of colonies counted for the thiram amended plates from the number of colonies recorded for the control plates. The inhibition index so obtained was used to calculate thiram LD 50 for each strain obtained.
For determining thiram effective concentration limits for Fusarium oxysporum, the fungus was grown onto CZA plates for one week and 1.1cm discs were then cut and seeded onto the surface of CZA plates supplemented with different thiram concentrations. A control set in which the fungal discs were seeded onto CZA plates not supplemented with thiram was included. Ten days later, the growth diameters in the treated and control plates were measured and recorded in cm (Shattock, 1988). The index of inhibition was calculated by subtracting the growth diameter recorded for thiram amended plates from those recorded for the control. The value was then used to calculate thiram LD 50 for Fusarium oxysporum.
The calculated LD 50 for each strain was used to determine the thiram selectivity Index (SI) and safety coefficient (SC) (Kruglov, 1991)  Toxicity index of thiram was determined according to Sun (1950).

Eff ects of Thiram on pure cultures of some N 2 fi xers and Fusarium oxysporum
The results of studying the influence of the fungicide thiram upon growth and development of pure cultures of soil bacteria (N 2 fixers) and Fusarium oxysporum are presented in Tables 1 and 2, Figures 1-3  oxysporum showed different resistance to thiram with selectivity indexes (SI) in the range of 1.496-7447.5 (Table 1).
The highest LD 50 (44.685) was recorded for Falvobacterium followed by Fusarium oxysporum, Azomonas and Rhizobium meliloti. Azospirillum and Pseudomonas aurentiaca were the most affected as they recorded the lowest LD 50 of 6.875 and 0.006 respectively. Table 1 shows the Index of Selectivity for the different organism tested. It seems quite evident that thiram is more selective to Pseudomonas aurentiaca, followed by Azospirillum, Rhizobium meliloti and Azomonas. The lowest Selectivity Index was recorded for Falvobacterium and Fusarium oxysporum. The highest safety coefficient 78134.289 was signed for the associated nitrogen fixing bacteria Flavobacterium, while Azomonas showed a low safety coefficient value ( Table 2). The toxicity index depending on LD 50 values of thiram on Azomonas, Flavobacterium, Rhizobium meliloti, Pseudomonas aurentiaca, Azospirillum and Fusarium oxysporum is shown in Table 2. Thiram was most toxic to Pseudomonas aurentiaca with toxicity index (100), followed by Azospirillum. The lowest toxicity index was recorded for Fusarium oxysporum (0.0201) and Flavobacterium (0.0134).

Discussion
The fungicide did not kill the target organism Fusarium oxysporum at the concentrations tested in the in vitro experiment, but it was most toxic to the fungus and significantly reduced its growth rate and final colony size at 10 ppm or greater concentrations compared to growth on an amended zapek Dox medium (fig 2, plate 1). This may be attributed to the fact that Fusarium oxysporum was isolated from a soil that had a history of repeated application of the pesticides particularly the fungicide thiram. Fravel et al. (2005) found that at concentrations of 10, 30, 50 or 100 ppm a.i. the fungicide thiram did not kill Fusarium oxysporum strain CS-20 in the in vitro experiment, but it was most toxic to the fungus and significantly reduced its growth rate and final colony size at 30 ppm or greater. Figures 1-3 show that for Azomonas, Azospirillum Flavobacterium, Fusarium oxysporum, Pseudomonas aurentiaca, and Rhizobium meliloti the dependence of the biological effect of the fungicide on its concentration is very similar as for the angle of inclination, and correspondingly, the rate of rise of the effect. At the same time, the slope of the curve for Azomonas, Fusarium oxysporum and Flavobacterium is more steep than that of the other curves, suggesting that even a slight increase of the dose of the fungicide can cause a very strong negative effect. Kalinin et al. (2002) found that the slope of the dose-reaction curve for Klebsiella planticola was more steep than that of the curves of Pseudomonas putida, Azotobacter chrococcum and Clostridium acetobutilicum. Kalinin et al. (2002) found that EC 50 values for Pseudomonas putida, Klebsiella planticola, Azotobacter chrococcum and Clostridium acetobutilicum were in 3-5 orders of magnitude higher in comparison with EC 50 values for different strains of Phytophthora infestans and thus proved to be more resistant to the fungicide azoxystrobin.
Depending on LD 50 values, thiram was most toxic to Pseudomonas aurentiaca with the toxicity index 100. Daoud et al. (1990) found that the fungicide benomyl was the most toxic compound against Alternaria sp followed by fluazifop and Decis (deltamethrin). Kalinin et al. (2002) found that the selectivity indexes of Pseudomonas putida, Klebsiella planticola, Azotobacter chrococcum, Clostridium acetobutilicum and  Phytophthora infestans were in the range of 13.5-20, indicating that Azoxystrobin had a strong selectivity ability. The safety coefficient refers to the possibility of the use of microorganisms under test with a specific concentration of the fungicide. From these results we conclude that thiram can be used without any limitations in association with microbial inoculants of biological nitrogen fixers for all the bacteria tested, except the genus Pseudomonas aurentiaca. Revellin et al. (1993) reported that thiram had a small or no effect on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybeans.