107 N-( 2-PIRYDYLAMINO ) METHYLENEBISPHOSPHONIC ACID AS A SOLE SOURCE OF A FUNGI FUSARIUM STRAINS GROWING

The active substance of new herbicide generation is N-(2-pirydyloamino) methylenobisphosphonic acid. Two strains Fusarium culmorum CB3 and Fusarium oxysporum XVI, isolated from soil treated with examined substance, were chosen to investigation of using it as a sole source of phosphorus, nitrogen or carbon in the concentrations at 1.0 mM. Interactions between the dominating strains of fungi and N-(2-pirydyloamino) methylenobisphosphonic acid, mycelium growth rate, the spore germinate index, the level chemical changes of N-(2-pirydyloamino) methylenobisphosphonic acid and pH were investigated after cultivation in mineral medium and its modifications. The results of growth kinetics of the studied Fusarium fungi in the presence of N-(2-pirydyloamino) methylenobisphosphonic acid, were expressed by dry weight [g·dm-3]. Only Fusarium oxysporum XVI has been capable to grow in acidic medium (pH 4.0) using N-(2-pirydylamino) methylene-bisphosphonic acid as an alternative source of phosphorus. In these conditions the N-(2-pirydylamino) methylenebisphosphonic acid degradation was carried out to utilize phosphorus compounds as a source of nutrient components for this strain. The presence of N-(2-pirydylamino) methylenebisphosphonic acid receded a growth rate of mycelium but did not have an effect on spores of the tested fungi.

nutriment and also as energetic substrate, with herbicide glyphosate being the most intensively studied [Bujacz et al. 1995 The presence of N-(2-pirydylamino)methylenebisphosphonic acid in soil leads to temporary changes of soil microorganisms, particularly the elevated growth of phytotoxic strains, especially those of Fusarium species [Krzysko- Lupicka 2005].
The herbicides also affect the microorganisms development, what is caused by disturbances in cell metabolism, but not all of these changes lead to inhibition of microorganisms reproduction.
The goal of this study was to determine the influence of N-(2-pirydylamino) methylenebisphosphonic acid used as sole source of carbon, nitrogen, or phosphorus or both nitrogen and phosphorus on growth of Fusarium sp. and on spores germination rate index.The possibility of fungi Fusarium strains to biodegrade of this substance was also investigated.

MATERIALS AND METHODS
The Fusarium culmorum CB 3 and Fusarium oxysporum XVI strains as a potentially phytopatogenic fungi were chosen for further studies.Their growth intensities and possibility of production of stain, which are either associated with mycelium or diffuse to medium in dependence on their modifications, were similar.Earlier, these fungi were selected by observing their significant growth in presence of N-(2-pirydylamino)methylenebisphosphonic acid [Krzysko- Lupicka 2005].
The N-(2-pirydylamino)methylenebisphosphonic acid (B) was used, in the concentrations of 1.0 mM, as a sole source of carbon, or nitrogen, or phosphorus or both nitrogen and phosphorus.
The pure fungal cultures were growing in full (Cz) and mineral modification Czapek medium at 25°C through 1 -4 weeks.The medium modification consisted of addition of 1 mM of N-(2pirydylamino) methylenebisphosphonic acid (B) as an alternative source of following nutriments: carbon (Cz-C+B); nitrogen (Cz-N+B); phosphorus (Cz-P+B) or nitrogen and phosphorus (Cz-N-P+B).The growth of fungi in full mineral (pH 5.6) and acid (pH 4.0) media was treated as controls, whereas the growth of fungi in medium without nutriment -carbon (Cz-C), nitrogen (Cz-N), phosphorus (Cz-P) or nitrogen and phosphorus (Cz-N-P), was an unqualified control.The fungi population density applied to inoculate the medium was 2•10 6 cell•cm -3 .
After 1, 2, 3 and 4 weeks of culture in following parameters were determined: • kinetics of the growth of the studied Fusarium by determination of mycelium dry mass (g d.m. dm - The obtained data have been statistically processed using one factor variance analysis and multiple range Duncan test.In parallel, the macro-and microscopic changes of studied fungi, were observed.

RESULTS
N-(2-pirydylamino)methylenebisphosphonic acid, added to Fusarium culmorum CB3 and Fusarium oxysporum XVI cultures, as a sole source of nutriment (Cz-C+B, Cz-N+B, Cz-P+B, Cz-N-P+B) led to inhibition of fungal growth.Only in case of Fusarium oxysporum XVI a biomass increase in culture with N-(2pirydylamino)methylenebisphosphonic acid used as an alternatively source of phosphorus (Cz-P+B) was observed.However, it was lower when compared with the growth in full Czapek (pH 5.6) and acid (pH 4.0) media.It was, however, higher than the growth of the fungi in medium without phosphorus (Figure 1).
In parallel, the pH changes of tested strains after four weeks of incubation (II) in comparison with initial cultures were investigated and significant increase of pH in modified cultures, except Cz-N, Cz-N+B and Cz-N-P, were observed (Figure 2).
The changes of medium pH can be a chemical indicator of N-(2-pirydylamino)-methylenebisphosphonic acid biodegradation in presence of tested fungi or their metabolites.
The spectroscopic analysis showed, that absorption decreases only in case of Fusarium oxysporum XVI culture with N-(2-pirydylamino) methylenebisphosphonic acid used as an alternative source of phosphorus (Figure 3).
The growth rate index (ITW) showed that N-(2 -pirydyloamino) methylenobisphosphonic acid added to medium as an alternative source of nutriment led to a mycelium inhibition of F. oxysporum but in the case of F. culmorum this index was different depending on the availability of nutrients.There was no significant effect of the compound on the rate of growth in the medium with no readily available phosphorus compared to the substrate complete (Cz) (Table 1).The spores germination  rate index (INK) depended only on well assimilable alimentary components (spores of tested fungi were resistant to added acid) (Table 1).
The culture modifications led to formation of chlamydospores, to change in structure and colour of mycelium, and to elimination ability of stains diffusion to the medium.The stains are not only by-products of metabolism but they take part in enzymatic transmutations of fungi.The both strains did not produce stains in cultures without well assimilable sugar; in other modifications they formed stains coloured from yellow to violet.The colour was depended on fungi species.The environmental conditions may lead to a change of stain characteristics among the same strain.
The next studies ought to concentrate on analysis of by-products obtained as a result of N-

DISCUSSION
Like the glyphosate N-(2-pirydylamino) methylene-bisphosphonic acid has a selective effect on soil microorganisms favoring the development of i.e. potentially phytopathogenic Fusarium fungi [Lejczak et al. 1996, Forlani et 2005] proved the herbicidal activity of this acid, but there is no information about the degradative ability of autochthonous soil microorganisms yet.Among the autochthonic Fusarium soil species of only Fusarium oxysporum XVI was capable to grow in acidic medium (pH 4.0) using N-(2-pirydylamino)methylene-bisphosphonic acid as an alternative source of phosphorus.In these conditions the N-(2-pirydylamino)methylenebisphosphonic acid degradation was carried out to utilize phosphorus compounds as a source of nutrient components for this strain.It was observed that the presence of N-(2-pirydylamino) methylenebisphosphonic acid receded a growth rate of mycelium but did not have an effect on spores of the tested fungi.
The spectroscopic analysis suggests biodegradation of this acid results in obtaining new components which do not absorb in the analyzed region of spectra.Therefore, the biomass increase is connected with usage of phosphorus in different forms.In the other cases, the absorption changes were not observed (or a little increase in one case).

CONCLUSIONS
The research conducted on biodegradation capabilities of organophosphate active ingredients found in the new generation of herbicides, indicate the possibility of removal of these compounds on the basis of microbial potential of soil.Like in the case of glyphosate biodegradation, it is likely to be a feature of microbial strains.

Table 1 .
The influence of N-(2-pirydylamino)methylenebisphosphonic acid on spores germination rate index INK (%) and on growth rate index ITW of Fusarium fungi