In the present study, fungal strain JASA1 was isolated from sugarcane field soil sample using enrichment technique and three morphologically different strains were isolated on Czapek Dox agar plate containing malathion. Malathion gradient plate assay was applied to screen the isolates for highest tolerance to malathion. The growth performance was recorded as the length of fungal growth in centimeters across the malathion gradient. Among all the three strains JASA1 showed a growth of above 8 cm across the gradient plates, which was further confirmed using broth assay. The MIC of malathion was performed for JASA1 strain which could tolerate upto 500 mg L−1 and had a confluent growth at 400 mg L−1 of malathion. In a similar study, Aspergillus oryzae ARIFCC 1054 and Aspergillus terreus JAS1 showed growth upto 900 and 400 mg L−1 of organophosphorus pesticides, respectively [14, 15].
The molecular characterization of 18S rRNA sequence and BLAST results exhibited close relationship and 99% similarity to that F. oxysporum. Multiple sequence alignments and phylogenetic tree (Figure 1) revealed the strain JASA1 cluster with Fusarium sp. Therefore, JASA1 isolate was designated as F. oxysporum JASA1 and the sequence result was submitted to GenBank NCBI database and accession number KF175514 was obtained.
Growth kinetics of F. oxysporum JASA1 in the presence and absence of malathion as a function of time is presented in Figure 2. The metabolism of malathion by F. oxysporum JASA1 was assessed by the increase in mycelial growth. Initially, the growth was found to be suppressed in presence of malathion, but after acclimatization to malathion, the culture was capable of growing rapidly exhibiting high growth rate. In later stage, the amount of biomass produced in the medium containing pesticide was much higher than the growth in the absence of malathion (control). This could be because of the availability of additional carbon and sulfur upon degradation of malathion in the medium. Growth kinetic study was done to learn the patterns of growth of the efficient JASA1 strain. Czapek Dox broth was spiked with malathion as sole carbon source and the JASA1 strain grew luxuriantly in it. While comparing the patterns with test and control, there was increased biomass in the test conditions.
HPLC was used to monitor the degradation of malathion and the results are presented in Figure 3. Recovery experiment was conducted in the M1 medium and soil for the study on extraction efficiency of the methods established. Different known concentrations of malathion were spiked in 50 mL of the M1 medium (100, 200, 300, and 400 mg L−1) and 50 g of soil (100, 200, 300, and 400 mg kg−1). Average recoveries of malathion from the M1 medium at levels of 100, 200, 300, and 400 mg L−1 were measured to be 96.2 ± 4.3, 97.6 ± 4.8, 98.4 ± 3.2, and 97.6 ± 1.2 %, respectively. The corresponding recoveries from the soil at levels of 100, 200, 300, and 400 mg kg−1 were 94.7 ± 3.6, 96.7 ± 3.2, 95.3 ± 2.9, and 94.2 ± 0.6 %, respectively. These data indicate that HPLC for malathion determination has a high accuracy, and the extraction procedures are efficient in extracting the malathion residues from the M1 medium and soil. The HPLC analysis revealed that JASA1 strain was capable of growing in M1 medium containing malathion as the sole source of carbon and energy, and confirmed the degradation of malathion. F. oxysporum degraded malathion in the aqueous medium to an undetectable level in 5 days (Figure 3b), which was compared with the HPLC peaks for the malathion standard (Figure 3a). The degradation dynamics of malathion in the soil are presented in Figure 4. The strain JASA1 was inoculated in soil with 400 mg kg−1 of malathion and nutrients (carbon, nitrogen, and phosphorous) were amended, after 8 days of incubation it showed 100% degradation of malathion (Figure 4a). There was no appreciable difference in the soil inoculated with JASA1 strain in the absence of nutrients and the 100% degradation was recorded after 9 days (Figure 4b). In previous study , it was reported that, malathion was degraded after 7 days of incubation by strain KB2 which degraded 72.20% of malaoxon and 36.22% of malathion, whereas strain PU degraded 87.40% of malaxon and 41.30% of malathion. Singh et al.  demonstrated degradation of malathion by B. thuringiensis/cereus bacteria by performing two systems, soil slurry system and soil box system. After 4 days of incubation, it was found that 65.87% of malaoxon and 30.93% of malathion was degraded and in case of soil box system 74.75% of malaoxon and 26.12% of malathion was degraded. These results indicated that degradative pathway of malathion might be facilitated by the activity of esterase enzyme . In few studies the gene encoding carboxylesterase was cloned and the recombinant protein was expressed [18, 19]. The carboxylesterase family comprises a group of esterases hydrolyzing carboxylic ester bonds, which is present in malathion, with relatively broad substrate specificity. They show a high degree of sequence similarity and are believed to be involved in the detoxification of many xenobiotics .