Enhanced biodegradation of phenoxyacetate and triazine herbicides by plant-microbial rhizoplane associations and adapted soil microorganisms

Strategies for the enhanced biological degradation of pesticides were examined in this research project. In one approach, the concept of the plant—microbial rhizosphere association was investigated as a laboratory model using the herbicide 2,4-D as a test compound. In another, an enhanced degradation of the recalcitrant herbicide atrazine was shown. Here, two microbial populations each capable of rapid atrazine metabolism were studied. The metabolism of 2,4-D by bacteria associated with the root system of wheat and canola seedlings was demonstrated in this study using a hydroponic system as well as a solid medium of sand and gravel. Significant and rapid 2,4-D metabolism (near 100% within 24h) was found in all hydroponic systems where the 2,4-D degrading microorganisms, Acinetobacter baumannii pJP4 transconjugant, Alcaligenes eutrophus and Azospirillum brasilense pIP4 transconjugant were associated with the roots. The metabolism of 2,4-D by Azospirillum brasilense pJP4 transconjugant associated with wheat was less rapid than associations with the other 2,4-D degrading bacteria. There was little difference in the rates of degradation between the hydroponic system and the sand/gravel mixture. The colonisation of the roots of seedlings by microbes was studied by both fluorescence and laser scanning confocal microscopy. Colonisation was often prolific without favoured areas of attachment on the root. A pre-treatment of seedlings with a synthetic auxin which formed para-nodular structures had little effect on the nature of colonisation. Counts of colony forming units, however, established that there was an increase of an order in magnitude of cells per root system when the plants were pretreated with this synthetic auxin. An average of 5.5 x 106 viable cells of 2,4-D degrading Acinetobacter baumannii were counted on para-modulated wheat root systems. It was demonstrated that the colonisation of the rhizosphere by suitable microbes could protect canola seedlings against phytotoxic effects of the applied herbicide. Whether this bio-safening effect will be seen in solid media or in field situations with these nonleguminous plants was not investigated. Significant rates of atrazine degradation either in the laboratory or in the field have rarely been reported. Attempts were therefore made to obtain microbes capable of such metabolism. These attempts had the ultimate goal of providing microbes for application in the model plant microbial rhizosphere association. Two microbial cultures, each capable of rapid atrazine metabolism, were obtained and studies of the metabolic processes were conducted. Rhodococcus sp. NI86/21 metabolised atrazine within l44h to two N—dealkylated products, desisopropylatrazine and desethylatrazine. Mineralisation of the ethyl-14C labelled sidechain to 14CO; was demonstrated, accounting for 25% of the total applied label in the broth culture. Desisopropylatrazine was shown to be the major metabolite. Desethylatrazine was shown as a terminal metabolite in the degradation of atrazine by Rhodococcus sp. N186/21, accumulating in the broth. In other studies using it as the substrate, no firrther metabolism was found. Desisopropylatrazine was also indicated to have been a terminal metabolite as it too accumulated in the broth. The metabolism of prometryn, a thio-ester (non-chlorinated) s-triazine was also investigated in these studies. Although Rhodococcus Sp. N186/21 did not rapidly degrade prometryn under similar conditions to those used with the atrazine assays, a mono-N-dealkylated metabolite was identified by mass-spectrometry. Possibly, prometryn was toxic to the Rhodococcus at the concentration used (lOug mL'l), as only minimal growth of the bacteria in the broth was observed by increased absorbance. Such a toxic effect was likely to have inhibited the N-dealkylation metabolism of the prometryn. In another approach, soil from a pesticide sullage site on a farm in northern NSW was assayed for its atrazine metabolising ability. Although there was no initial activity, after 30 months of perfirsion of the soil with a concentrated solution of the herbicide, it had acquired the ability to rapidly mineralise atrazine. A rapid conversion of the three carbons in the s—triazine ring to C02 was demonstrated using radiolabelled atrazine. Also, the labelled carbon in the ethyl sidechain of atrazine was rapidly metabolised to CO2. The sidechain 14C label was mineralised to 14C02 at a slower rate than the carbons in the ring. It was demonstrated that there was a likelihood of the sidechain carbon being incorporated into an unextractable intermediate metabolite, which was subsequently also less susceptible to attack by the microorganisms. There were no significant metabolites of atrazine accumulated in the broth. The likely presence of hydroxyatrazine was noted in the assay using uniformly ring—labelled [14C] atrazine. Hydroxyatrazine was also identified in the assay with ethyl-sidechain labelled [14C] atrazine, however, the amount detected was less. The presence of atrazine at 25pg mL-l inhibited nitrification reactions in the soil, however, at a saturating concentration of SOug mL-l there was some ammonia oxidation noted. Attempts to isolate single bacterial colonies capable of the metabolism of atrazine were unsuccessful. Although there was insufficient time to utilise these microbial cultures in the plant microbial rhizosphere associations, studies on the metabolism of atrazine have sufficiently characterised the nature of biodegradation to suggest that plant-microbial associations can be confidently tested in fiiture experiments