3.1. Effect on growth and biochemical composition
During five days of incubation, each strain showed continuous day dependent growth, which comparatively slowed down after 4 days (Fig. 1). The growth during first four days were near linear in all cases with a higher slope value of the tolerant type both in respect of wet wt and dry wt (m = 0.943 and 0.118 for wet wt and dry wt, respectively) indicating that the treated tolerant type grew better than the untreated control in the presence of malathion (500 µM) whereas the growth of the treated wild type was significantly low (LSD = 0.064 and 0.011 for wet wt and dry wt, respectively after 5 days; p = 0.05). The difference of wet and dry wt between the untreated control and treated tolerant strain was only about 4% after 5 days of incubation but this difference was found significant (LSD = 2.19%; p = 0.05). This indicated that after 5 days of malathion treatment the growth of the tolerant strain was significantly higher than of untreated control which may be due to the metabolic consumption of malathion by the tolerant strain. Further it was observed that on any day of observation, the growth of the tolerant strain was remarkably higher than of the wild type on treatment with 500 µM malathion. After 5 days of incubation, wet wt and dry wt of treated wild type were 57% and 55% of the treated tolerant strain, respectively (LSD = 2.28% and 2.09%, respectively; p = 0.05) showing a remarkable difference between growth performance of the strains in the presence of malathion. Abraham and Srujana (2014) have observed that all three isolated strains of Aspergillus could grow well in malathion (300µM) containing medium but Aspergillus sp MF1 was found to be most efficient to use malathion as a nutrient source. A. terreus have also been reported to perform enhanced growth at 1.2 mM of malathion (Silambarason and Abraham 2013). Similarly, other fungus like Rhizopus oryzae (Chatterjee et al. 2010) and Fusarium oxysporum JASA I (Peter et al. 2015) also had enhanced growth in the presence of malathion. In the presence of other OP insecticides like dimethoate more aggressive mycelial growth of Aspergillus isolate have also been observed (Nkang and Sanyaolu 2018).
Corresponding to the growth, the protein and carbohydrate contents of the mycelia of the tolerant type were significantly higher than of the wild type in the presence of the insecticide (Fig. 2). After five days of incubation the carbohydrate and protein contents of the treated tolerant strain were respectively 3.70% and 1.03%, on dry wt. basis, higher than of the untreated control whereas the respective contents of the treated wild type were 20% and 5% lower than of untreated control. (LSD = 3.09% and 1.86% for protein and carbohydrate, respectively, p = 0.05).
3.2. Activities of carbohydrate enzymes
Three carbohydrate enzymes viz. amylase, cellulase and invertase were measured after 5 days of treatment with 500 µM of malathion (Fig. 3). All the three enzymes had marginally higher activity (0.3–2%) in the treated tolerant strain than the activities measured in the untreated control, though such increase was not found significant. Mohapatra (2019) observed that there is no significant variation in the activities of carbohydrate enzymes of the tolerant strain when grown in the presence or absence of malathion. On the other hand, significant inhibition of the activities of these enzymes was observed in the treated wild type. The rate of activity of amylases, cellulase, and invertase in the treated wild type were 77%, 52%, and 90% of the untreated control indicating that cellulase activity was the most inhibited among the carbohydrate enzymes with malathion treatment. Dehydrogenase activity was also found to be inhibited by malathion treatment of the wild type whereas in the tolerant type there was no reduction of dehydrogenase activity. On the other hand, the enzyme activity of the treated tolerant type was 2.7% higher than of the untreated control, though such difference was found insignificant. (LSD = 3.15 nM TPF/g; p = 0.05) (Fig. 4). The enzyme activity of the treated wild type was 67% and 65% of the untreated control and treated tolerant strain respectively. The variations in the growth performance and enzyme activities among the control and the treated cultures (wild type and tolerant strain) were always found significant (Table 2).
Table 2
F value of one-way ANOVA among the control and treated (wild type and tolerant) cultures after 5 days of incubation under malathion (500 µM) treatment.
Parameter
|
F value
|
Wet weight
|
27.51 **
|
Dry Weight
|
103.55 **
|
Protein content
|
80.02 **
|
Carbohydrate content
|
13.27 *
|
Cellulase activity
|
9.83*
|
Invertase activity
|
5.98
|
Amylase activity
|
7.28*
|
Dehydrogenase activity
|
34.59 **
|
Alkaline Phospatase activity
|
44.43 **
|
Acid Phospatase activity
|
66.56 **
|
Total Phospatase activity
|
63.69 **
|
Esterase activity
|
223.03 **
|
Note: Significant difference at *P = 0.05 and **P = 0.01
The carbohydrate enzymes and dehydrogenase are good indicators of metabolic performance of fungi. In the present study significant difference, with respect to the activities of these enzymes, between the wild type and tolerant strains, after malathion treatment showed that the latter is efficient to grow in the presence of malathion and remained metabolically active like that of the untreated control whereas the metabolic performance of the wild type was severely affected by malathion at the applied concentration. Further the cellulase activity was more affected than of other two carbohydrate enzymes by malathion treatment in the wild type.
3.3. Activities of phosphatases and esterases
The activity of various oxidoreductase (oxygenase, laccases, peroxidases), hydrolases (lipase, cellulase, esterase), phytases and phosphatases have been measured as the indicators of the performance of fungi to metabolise OP insecticides (Mostafa et al. 1972; Ramadevi et al. 2012; Ahn et al. 2018; Meng et al. 2019). Shah et al. (2017) observed that the tolerance of A. nizer to chlorpyrifos, monocrotophos, and methyl parathion was due to the over-expression of phytases. In this study the activity of alkaline, acid, and total phosphatases and esterase were measured. For the purpose four different OP insecticides viz- malathion, parathion, chlorpyrifos, and dimethoate were taken. With each insecticide treatment, there was enhancement of the activities of phosphatases both in the wild type and tolerant strains (Fig. 5). Malathion treatment caused enhancement of total phosphatase activity by 25% and 59% in wild type and tolerant strains, respectively clearly indicating the difference between the strains with respect to the enzyme activity (Fig. 5A). There was also corresponding increase in the activities of alkaline and acid phosphatases in both the strains. On the other hand, there was no significant enhancement of total phosphatase activity of wild type with parathion treatment, but quite high activity (26% higher than that of untreated control) was observed in the tolerant strain (Fig. 5B). In chlorpyrifos and dimethoate treated cultures there was also enhancement of the activities of phosphatases in both the strains and as expected the activity enhancement was significantly high in the tolerant strain than in the wild type (Fig. 5C and D). Further it was also found that the activity of total phosphatases was the highest with malathion treatment (59%) followed by treatment with dimethoate (57%), chlorpyrifos (41%) and parathion (26%) in the tolerant type. The corresponding increase in the wild type were only 20%, 19%, 13% and 1%, respectively. In all cases the activity of alkaline phosphatases was more than of acid phosphatases. Enhanced alkaline phosphatase activities with malathion treatment have been reported in other species of Aspergillus but such induction with parathion treatment have not been recorded (Hasan 1999; Abraham and Srujana 2014).
Different species of Aspergillus, Fussarium, and Penicillium have shown enhanced expression of phosphatase in the presence of OP pesticides, like malathion and dimethoate (Hasan 1999; Xu et al. 2004). Xu et al. (2004) have reported that by enhanced phosphatase activities A. niger could use dimethoate as the only source of phosphorus. A. sydowii and A. flavus showed stimulated alkaline phosphatase activities at low concnetrations (≤ 90 mm) of malathion and release phosphate to the medium (Hasan 1999; Abraham and Srujana 2014). Quite a number of other insecticides stress with enhanced activities of phosphatases have also been reported (Kumar et al. 2019).
Irrespective of the insecticide, treatment caused enhancement of esterase activities in both wild type and tolerant strain (Fig. 6). In malathion treatment of wild type, the enzyme activity was enhanced by 38% when compared to untreated control whereas in the tolerant type the activity enhancement was 163% of the untreated control. Parathion treatment caused the least enhancement of esterase activity both in the wild type and tolerant strains, the activity being enhanced by 18% and 83%, respectively of untreated control, but such increased expression of esterases was also significant. Chlorpyrifos treatment enhanced esterase activity by 33% and 140% in wild type and tolerant strain, respectively. Dimethoate treatment caused 34% and 156% enhancement of esterase activity in wild type and tolerant strains, respectively. Comparison among the wild type cultures treated with different insecticide showed that the insecticide dependent variation in the activities was only 15% whereas in the tolerant strains the variation was 48% indicating more or less an insecticide specific expression of enzyme activity in the strain. However, in both the strains, the variation in the activities of esterase among different insecticides treatment were significant (F = 8.42 and 21.63; for wild type and tolerant strain respectively, n = 24).
Various hydrolases (lipase, cellulase, esterase, paraoxanase, phosphotriesterases) are known of been expressed (or over expressed) in the presence of OP insecticides (Liu et al. 2001). Liu et al. (2001) observed that a dimethoate tolerant A. niger strain had enhanced expression of esterase in the presence of dimethoate, malathion, and formathion but no expression reported in the presence of parathion and dichlorvos. In contrast we observed a significant enhancement of esterase activity (83%) as compared to untreated control of malathion tolerant strain in the presence of parathion but this was the least among the four insecticide tested. Similarly, enhancement of esterase activity of A. niger have been observed in the presence of insecticide like chlorpyrifos (Mukherjee and Gopal, 1996), monocrotophos (Pandey et al. 2014) and methyl parathion (Shah et al. 2017).
3.4. Removal of insecticides
In order to estimate the mycelial accumulation of insecticides and their removal, the fungal strains were cultured in the presence of insecticides and observation were taken each day. The mycelial accumulation of the insecticides increased with the days of incubation in both the strains and such time dependant increase of mycelial accumulation were always significant (Fig. 7, and Table 3). The mycelial concentrations of malathion, chlorpyrifos, and dimethoate of the tolerant strain continuously increased with days of incubation up to 4 days after which there was a decrease. In wild type strain, on the other hand, a continuous time dependent increase in the mycelial concentration of the insecticides was observed. With parathion treatment, the mycelial concentration showed continuous increase in both the stains during the observation period indicating continuous accumulation of insecticides in the mycelia (Fig. 7B). In all cases the day dependent variation of mycelial concentration of insecticide showed significant quadratic relationship (Table 3). For each insecticide, comparison of mycelial concentration between the wild type and the tolerant strains showed that the strains are different in accumulating the toxicants and the wild type strain has a significantly higher mycelial accumulation than the tolerant strain (Table 4).
Table 3: The equations of the quadratic relationship of the mycelial concentration of the insecticides and rate of removal (%) with days after incubation.
Strain type
|
Insecticide
|
R2
|
Equation
|
Mycelia concentration (mg/g dry wt)
|
tolerant
|
malathion
|
0.967
|
y = -0.535x3 + 3.897x2 − 3.913x + 0.459
|
wild type
|
0.983
|
y = -0.545x3 + 4.559x2 − 4.283x + 0.601
|
tolerant
|
parathion
|
0.994
|
y = -0.391x3 + 2.992x2 − 1.781x + 0.247
|
wild type
|
0.990
|
y = -0.586x3 + 4.716x2 − 3.524x + 0.460
|
tolerant
|
chlorpyrifos
|
0.983
|
y = -0.328x3 + 2.452x2 − 2.127x + 0.261
|
wild type
|
0.990
|
y = -0.335x3 + 2.967x2 − 2.382x + 0.352
|
tolerant
|
dimethoate
|
0.970
|
y = -0.503x3 + 3.716x2 − 3.641x + 0.446
|
wild type
|
0.976
|
y = -0.671x3 + 5.413x2 − 5.399x + 0.728
|
Insecticide removal (%)
|
tolerant
|
malathion
|
0.989
|
y = 3.143x2 − 1.777x + 0.324
|
wild type
|
0.987
|
y = 1.671x2 − 2.855x + 1.038
|
tolerant
|
parathion
|
0.987
|
y = 1.264x2 + 0.765x − 0.595
|
wild type
|
0.995
|
y = 0.598x2 − 0.058x + 0.139
|
tolerant
|
chlorpyrifos
|
0.989
|
y = 2.125x2 + 0.072x − 0.143
|
wild type
|
0.988
|
y = 0.447x2 + 1.891x − 0.467
|
tolerant
|
dimethoate
|
0.977
|
y = 2.515x2 + 0.773x − 0.903
|
wild type
|
0.978
|
y = 1.012x2 − 0.163x + 0.37
|
Note: - All R2 values are significant at P = 0.01; n = 18. |
Table 4
The t value of comparison between the treated wild and tolerant type cultures after 5 days of incubation under insecticides treatment.
Parameter
|
Malathion
|
Parathion
|
Chlorpyrifos
|
Dimethoate
|
Mycelia concentration
|
27.02
|
16.11
|
22.09
|
31.27
|
Insecticide removal (%)
|
33.16
|
17.19
|
31.04
|
26.42
|
Note: All the calculated t-values are significant at P = 0.001. |
As expected the insecticides were removed more efficiently by the tolerant strain than by the wild type. The removal of malathion from the medium containing 500µM of the insecticides was 68% by the tolerant strain and 29% by the wild type during 5 days of incubation (Fig. 8A). Similarly, removal of parathion, chlorpyrifos, and dimethoate were 33%, 54%, and 63% respectively by the tolerant strain and 15%, 19%, and 26% respectively by the wild type strain during 5 days (Fig. 8). This indicated that both the tolerant strain and the wild type of A. niger more efficiently remove malathion followed by dimethoate, chlorpyrifos, and the least of parathion. The analysis of the trend of the relationship between the rate of insecticide removal and the time after treatment was quadratic in case of both the strains and were found always significant (Table 3). With each insecticide treatment, significant difference between the wild type and tolerant strain was observed with respect to their ability to remove the OP chemical from the medium (Table 4).
The selection of appropriate strain for biodegradation of insecticides considered not only the potential of biodegradation but also the rate of growth (Mrozik and Piotrowska-Seget 2010). Previous studies have shown that many OP insecticides- dimethoate, chlorpyrifos, malathion, parathion, fenitrothion, etc. are metabolically degraded by a variety of other fungi (Gao et al. 2012; Mohapatra et al. 2018). Gao et al. (2012) and Bisht et al. (2019) have reported that the hydrolytic enzymes induced in Cladosporium cladosporioides on exposure to chlorpyrifos were highly effective in degrading many other phosphorothoates of structural similarity. The malathion tolerant strain A. niger MRU1 showed its efficiency not only to degrade malathion but also the other three insecticides quite effectively. The mycelial accumulation of the insecticides in the tolerant strain was significantly low as compared to that observed with the wild type strain because of its high efficiency of degradation. Taking A. niger, Sasikala et al. (2012) observed about 15% degradation of chlorpyrifos at 500 mg/l after 15 days whereas Liu et al. (2001) achieved 87% and 78% degradation of dimethoate and malathion, respectively by the dimethoate tolerant A. niger strain.
With other Aspergillus species (A. viridinutans and A. terrusi), Abdel Wareth and El-Hamid (2016) reported about 50% removal of chlorpyrifos whereas Alvarenga et al. (2015) could achieve 37% removal of chlorpyrifos in 10 days. Derbalah et al. (2020) observed that degradation rate of malathion by A. flavus increased as a function of the initial concentration at ≤ 5 mg/l but the rate of removal was low at high concentrations. Effective degradation of malathion was also achieved by A. fimigatus and A. terrus (Malik et al. 2014). The malathion tolerant A. niger MRU01 strain, developed in the present study grew very efficiently in the presence of malathion whereas the wild type was significantly inhibited by the same concentration of the insecticides. The tolerant strain not only grew efficiently in the presence of malathion but also metabolized the malathion as well as dimethoate quite effectively. More than half (54%) of the applied chlorpyrifos was also removed by the fungus indicating that the tolerant strain has a broad spectrum of tolerance against structurally similar OP chemicals. Unlike previous reports, the tolerant strain could also able to degrade parathion as about a third of the applied concentration could be removed by the fungus during five days.