Synergistic and additive interactions of Shewanella sp., Pseudomonas sp. and Thauera sp. with chlorantraniliprole and emamectin benzoate for controlling Spodoptera litura (Fabricius)

The imprudent use of insecticides causes the development of resistance in insect pest populations, contamination of the environment, biological imbalance and human intoxication. The use of microbial pathogens combined with insecticides has been proposed as an alternative strategy for insect pest management. This IPM approach may offer effective ways to control pests, in addition to lowering the risk of chemical residues in the environment. Spodoptera litura (Fabricius) is a major pest of many crops like cotton, maize, tobacco, cauliflower, cabbage, and fodder crops globally. Here, we evaluated the combined effects of new chemistry insecticides (chlorantraniliprole and emamectin benzoate) and entomopathogenic bacterial strains, Shewanella sp. (SS4), Thauera sp. (M9) and Pseudomonas sp. (EN4) against S. litura larvae inducing additive and synergistic interactions under laboratory conditions. Both insecticides produced higher larval mortality when applied in combination with bacterial isolates having maximum mortality of 98 and 96% with LC50 of chlorantraniliprole and emamectin benzoate in combination with LC50 of Pseudomonas sp. (EN4) respectively. The lower concentration (LC20) of both insecticides also induced synergism when combined with the above bacterial isolates providing a valuable approach for the management of insect pests. The genotoxic effect of both the insecticides was also evaluated by conducting comet assays. The insecticide treatments induced significant DNA damage in larval hemocytes that further increased in combination treatments. Our results indicated that combined treatments could be a successful approach for managing S. litura while reducing the inappropriate overuse of insecticides.

The imprudent use of insecticides causes the development of resistance in insect pest populations, contamination of the environment, biological imbalance and human intoxication.The use of microbial pathogens combined with insecticides has been proposed as an alternative strategy for insect pest management.This IPM approach may offer effective ways to control pests, in addition to lowering the risk of chemical residues in the environment.Spodoptera litura (Fabricius) is a major pest of many crops like cotton, maize, tobacco, cauliflower, cabbage, and fodder crops globally.Here, we evaluated the combined effects of new chemistry insecticides (chlorantraniliprole and emamectin benzoate) and entomopathogenic bacterial strains, Shewanella sp.(SS4), Thauera sp.(M9) and Pseudomonas sp.(EN4) against S. litura larvae inducing additive and synergistic interactions under laboratory conditions.Both insecticides produced higher larval mortality when applied in combination with bacterial isolates having maximum mortality of 98 and 96% with LC 50 of chlorantraniliprole and emamectin benzoate in combination with LC 50 of Pseudomonas sp.(EN4) respectively.The lower concentration (LC 20 ) of both insecticides also induced synergism when combined with the above bacterial isolates providing a valuable approach for the management of insect pests.The genotoxic effect of both the insecticides was also evaluated by conducting comet assays.The insecticide treatments induced significant DNA damage in larval hemocytes that further increased in combination treatments.Our results indicated that combined treatments could be a successful approach for managing S. litura while reducing the inappropriate overuse of insecticides.
The tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), is a notorious polyphagous pest of many field crops, including cotton, corn, groundnut, soybean, tobacco, and vegetables 1 .It is found throughout temperate and tropical Asia, Australasia, and Pacific Islands 2 .Early instar larvae are gregarious feeders while the later instars disperse and feed voraciously, causing complete defoliation of plants when present in abundance 3 .A variety of insecticides with different mechanisms of action are being used to manage S. litura.However, there are reports indicating the development of resistance in S. litura to many of the commonly used insecticides 4,5 .Although chemical insecticides are the most reliable tool in insect pest management but resistance to insecticides is a major problem associated with the chemical control of insect pests.Imprudent spraying and repeated use of insecticides including chlorinated hydrocarbons, carbamates, organophosphates, and pyrethroids not only led to the development of resistance in many insect pests but also posed a threat to beneficial creatures such lower concentration, all the concentrations induced significantly higher larval mortality over control.The mortality rate increased significantly from 24 to 96% in a dose-dependent manner (F (5, 24) = 115.67,p ≤ 0.05) (Fig. 1).The lower lethal and median lethal values of chlorantraniliprole against S. litura larvae after 72 h of treatment were found to be: LC 20 = 0.001 ppm (95% confidence interval 0.001-0.002ppm) and LC 50 = 0.011 ppm (95% confidence interval 0.008-0.017ppm).Similarly, with respect to control, all the concentrations of emamectin benzoate significantly increased the larval mortality except for the lower two concentrations (F (5, 24) = 58.79,p ≤ 0.05) (Fig. 2).The LC 20 and LC 50 value of emamectin benzoate against S. litura were 0.002 ppm (95% confidence interval: 0.000 -0.009 ppm) and 0.032 ppm (95% confidence interval: 0.006 -0.368 ppm) respectively

Combined effects of bacteria and insecticide against S. litura.
The studies revealed significant effect on larval mortality due to combined treatments when compared with individual treatments (Fig. 3E).The combination treatment of chlorantraniliprole with bacterial isolates i.e.SS4, M9 and EN4 induced higher mortality (78-88%) in S litura larvae used in the first experiment (F (6, 28) = 14.43, p ≤ 0.05).Additive interaction was found in all the combinations (χ 2 ˂ 3.84) (Table 1).In the second experiment, all the combination treatments increased the larval mortality in a synergistic way (F (6, 28) = 18.29, p ≤ 0.05) (χ 2 > 3.84) except for M9 culture (LC 50 Cg + LC 50 M9).When alternate insecticide and bacterial treatments were given to larvae in the third experiment, the mortality rate increased further.All the combination treatments exhibited synergistic impact indicating that each bacterial isolate and insecticide contributed to S. litura larval mortality (Table 1).The combined treatment, LC 50 Cg + LC 50 EN4 caused maximum larval mortality (98%) (χ 2 > 3.84) compared to  other groups (F (10, 44) = 16.88,p ≤ 0.05).The lower concentration (LC 20 ) of chlorantraniliprole when combined with bacteria also induced synergistic effect thus providing a valuable approach for the control of insect pests.
As for emamectin benzoate (Table 2), we observed an additive effect in all combinations of first experiment (χ 2 < 3.84; p ≤ 0.05).The results of second experiment indicated synergistic effect in the combination treatment, LC 50 Eb + LC 50 EN4 with χ 2 value of 4.79 (F (6, 28) = 23.32,p ≤ 0.05) while additive effects were recorded in other treatments (Table 2).Synergistic effect was observed across all the combination treatments in the third experiment except for LC 20 Eb + LC 50 SS4 and LC 20 Eb + LC 50 M9 treatments that exhibited additive effect (F (10, 44) = 32.63,p ≤ 0.05).Overall, these results indicated that combining insecticide with bacterial treatment increased the mortality rate of S. litura larvae.

Comet assay.
In the current study, treatment with chlorantraniliprole and emamectin benzoate induced genotoxic effects in S. litura larvae.The larvae treated with insecticide and bacterial cell suspension i.e. combination treatments, had much higher levels of damage in larval hemocytes as compared to control and individual treatments (Fig. 5).
The tail length was significantly increased in individual treatments of both the insecticides as compared to control, however, the increase in tail length was observed to be higher in combination treatment groups as compared to individual exposure groups.In case of chlorantraniliprole, it significantly increased from 10.73 μm in control (distilled water) to 12.76 μm and 16.63 μm in LC 20 and LC 50 concentrations, respectively.However, the tail length further increased in combination treatments with a maximum value of 35.69 μm in LC 50 Cg + LC 50 EN4 (F (9, 20) = 277.06,p ≤ 0.05).A similar pattern was also observed for tail length in larvae treated with emamectin benzoate with maximum increase of 43.73 μm in LC 50 Eb + LC 50 EN4 treatment (F (9, 20) = 572.04,p ≤ 0.05) (Table 3).With respect to control, the percent tail DNA values also increased significantly in both the individual insecticide (LC 20 and LC 50 ) and the combination treatment groups.Likewise, TM and OTM values were analyzed to be significantly and maximally increased in combined treatment groups of chlorantraniliprole and emamectin benzoate in comparison to their individual and control groups (Tables 3, 4).

Discussion
Bacterial isolates, Shewanella sp.(SS4), Thauera sp.(M9) and Pseudomonas sp.(EN4) were found to exhibit pathogenicity against S. litura in our previous experiments 23,24 .Shewanella inventionis HE 3 and a number of Pseudomonas species such as Pseudomonas chlororaphis, Pseudomonas taiwanensis, Pseudomonas fluorescens, Pseudomonas entomophila, Pseudomonas putida, and Pseudomonas paralactis have been documented for insecticidal properties against many insect pests [25][26][27][28][29] .Pathogenicity of Shewanella sp.(SS4), Thauera sp.(M9) and Pseudomonas sp.(EN4) may be attributed to various hydrolytic enzymes viz.catalases, proteases, chitinases, lipases, oxidases and phospholipases which have been reported to be produced by these bacterial isolates [30][31][32] .All these bacterial isolates were found to be compatible with chlorantraniliprole and emamectin benzoate.Different combination treatments carried out in the present studies induced higher larval mortality in S. litura than individual bacterial or insecticide treatments.www.nature.com/scientificreports/Our findings indicated that even single application of insecticides followed by bacterial treatment increased the larval mortality in an additive manner.However, more than one application of insecticide alternating with bacteria enhanced the mortality and the interactions of bacteria and insecticides turned out to be synergistic.Among the two insecticides, bacterial cultures combined with chlorantraniliprole were found to be more effective showing mostly the synergistic effects even at low concentration (LC 20 ) of insecticide.The increased mortality due to the additive effect as confirmed by Chi-square test, indicated that mortality observed in the combination treatments was caused by independent action of both bacterial isolates and insecticides whereas synergistic interaction demonstrated a significant interaction between two treatments 33 .
The combined use of bacteria with insecticides causes high mortality in pests, because chemical insecticides may act as stressor, weakening the immune response and increasing the susceptibility of insect to bacterial pathogens 34,35 .The anthranilic diamide, chlorantraniliprole is a new-generation insecticide and effective against lepidopteran insects that activate the ryanodine-sensitive intracellular calcium release channels (ryanodine receptor).The release of internal calcium stores leads to Ca 2+ depletion, feeding cessation, lethargy, muscle paralysis and finally insect death 36,37 .Emamectin benzoate is a semi-synthetic derivative of abamectin that act as chloride channel activator, decreasing the excitability of neurons of lepidopterans and dipterans 38 .The insect larvae stop feeding immediately following exposure, become irreversibly paralyzed, and die within 3-4 days 16 .
Our results on comet assay indicated that combination treatments further enhanced DNA damage in hemocytes of S. litura as a significant increase was detected in all the comet parameters.It suggests that combined treatments cause more stress which further enhance pathogenicity and mortality in S. litura larvae.The studies are in line with the previous reports indicating genotoxicity due to insecticide exposure to various insects [39][40][41] .Microbial control agents have also been reported to cause genotoxicity, although very few reports are there 42,43 .Different pesticides such as delmithrin, endosulfan, malathion, cypermethrin, paraquat and λ-cyhalothrin etc. have been reported to increase the activity of oxidative stress enzyme [44][45][46][47] .This oxidative stress lead to production of reactive oxygen species (ROS) that disrupts the cellular redox balance causing lipid and protein oxidation as well as DNA damage 48 .The formation or lengthening of a tail is an indicator of the apoptosis process, as cells undergoing apoptosis exhibit nuclear fragmentation/disintegration in the form of DNA tails 49 .As the hemocytes play a vital role in providing defensive functions, thus direct effect of insecticidal and bacterial treatment may  www.nature.com/scientificreports/affect the cellular immune response by changing the viability and number of hemocytes, causing stress and making the insect more vulnerable to pathogens as well as suppressing the growth and developmental process.
Recent studies by Uma et al. 50, documented sub-additive and synergistic effects of combination treatments of B. thuringiensis and chlorantraniliprole against Spodoptera frugiperda (J.E.Smith) larvae.An additive effect was observed between EPN species with LC 25 and LC 50 of emamectin benzoate on third instar larvae of cabbage white butterfy, Pieris rapae (Linneaus) after 3 days post-treatment 51 .Similar interactions were documented by other workers between entomopathogenic fungus and Bt against various insect pests [52][53][54] .Contrary to these studies, Amizadeh et al. 35 reported antagonistic effect of Bt and insecticides when Bt was applied immediately after the applications of chemicals against tomato leafminer Tuta absoluta (Meyrick).Morales-Rodriguez and Peck 55 also observed similar effect between Bt and neonicotinoid insecticides, imidacloprid and clothianidin against Amphimallon majale (Razoumowsky) and Popillia japonica (Newman), respectively.This antagonist effect may be due to incompatible nature of entomopathogenic fungi with chemical insecticides due to lower germination rate, decreased production of enzymes necessary for penetration of the insect's cuticle, and poor mycelium growth ratio 56 .However, none of the associations were antagonistic in the present study rather these were additive and synergistic.Thus, use of biocontrol agents in combination with insecticides would not only increase the efficacy of biocontrol agents but also help to decrease the number of insecticide applications and thus help to reduce the load of chemical insecticides on environment.This strategy would ultimately result in improved pest management by natural enemies and finally delays the emergence of insecticide resistance.

Rearing of insects.
To establish the culture of S. litura, egg masses and larvae were collected from cauliflower and cabbage fields in and around Amritsar (Punjab), India.Mass rearing was carried out in the laboratory as per the protocol of Thakur et al. 57 Larval rearing was carried out on fresh Ricinus communis leaves (Accession/ Voucher number: 7590, identified from Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar (Punjab), India) at controlled temperature of 25 ± 2 ο C and 65 ± 5% humidity conditions respectively.The pupae were shifted to pupation jars (15 cm × 15 cm) having moist and sterilized sand.To facilitate egg-laying process, the adults were transferred to oviposition jars (15 cm × 15 cm) lined with filter paper.The adults were fed on honey solution (1 part honey to 4 parts water) soaked on a cotton swab that had been refreshed daily.The culture of S. litura was raised for three generations in the laboratory before employing for experiments.) and Thauera humireducens SgZ-1 (GenBank accession number MK619795) [58][59][60] , were procured from Department of Microbiology, Guru Nanak Dev University, Amritsar (Punjab), India, were found to exhibit insecticidal activity in our previous experiments 23,24 .These cultures were maintained on Luria Bertani (LB) plates.The bacteria were inoculated in LB broth and incubated at room temperature for 48 h at 30 °C.After centrifugation, the pellet was suspended in 1 ml phosphate buffer solution (PBS) (pH 7.0).The bacterial density was then optimized at optical density (OD 600 ) at LC 50 values for SS4, M9 and EN4.

Bacterial cultures and preparation of bacterial stocks.
Insecticide formulation.The formulations of chlorantraniliprole (18.5% SC) and emamectin benzoate (5% SG) were purchased from FMC India Private Limited and Sinochem India Co. Private Limited respectively.

Estimation of sub-lethal concentrations of insecticides.
Preliminary bioassays with chlorantraniliprole and emamectin benzoate were performed to estimate the lethal concentrations killing second instar larvae of S. litura.A total of 50 larvae were used for each of the above mentioned five concentrations of both the insecticides.The stock solution (10 ppm) of each insecticide was prepared in 1000 ml distilled water and then serially diluted to prepare the five concentrations (0.0001 ppm, 0.001 ppm, 0.01 ppm, 0.1 ppm, and 1 ppm).Leaf dip method was adopted to conduct the experiment as per protocol of Sharma et al. 61 .The leaves treated with distilled water served as control.A single leaf disc (about 10 cm 2 ) dipped in each insecticide concentration was air dried, cut into small pieces and placed in rearing tube containing S. litura larva.Only one larva was kept in each rearing tube.Each insecticide concentration was repeated five times (10 larvae per replication).The leaves were changed after 48 h of treatment.The experiments were conducted at constant temperature and humidity conditions of 25 ± 2 °C and 65 ± 5% respectively.Larval mortality was recorded after 24, 48 and 72 h of larval exposure to insecticide.LC 20 and LC 50 values were calculated after 72 h of treatment by Probit analysis.Larvae were considered dead when no movement of appendages was seen upon touching with a brush.

Compatibility of bacteria and insecticide.
To determine the compatibility of bacterial cultures with chlorantraniliprole, shake flask and plate assays were conducted.In the shake flask assay, three different concentrations of the insecticide (0.016 ppm, 0.1 ppm and 1.0 ppm) were added to 100 ml Luria Bertani broth in 250 ml flasks.The culture medium without insecticide served as control.A single colony of each bacterial isolate was inoculated into each treatment and control LB broth and incubated for 48 h at 30 °C and 180 rpm.Bacterial cultures were then centrifuged at 10,000 rpm and 4 °C for 10 min to observe the bacterial pellet growth.In the plate assay, the bacterial suspension of each culture (100 µL) was layered over LB plates with the help of a spreader.Then wells were made in four quadrants of plate.PBS was added to one of the well that served as control and three different concentrations of chlorantraniliprole (0.016 ppm, 0.1 ppm and 1.0 ppm) were added to the other three wells and left to dry for overnight.These plates were then incubated for 48 h at 30 °C to check the growth of bacterial cell suspensions.Similar procedure was followed for checking the compatibility of bacteria with emamectin benzoate.The compatibility of insecticide against bacterial cultures was demonstrated by diameter of clear or halo zones around the wells in comparison to the control well 62 .
Effect of different concentrations of insecticide and bacteria against S. litura.For the individual treatments, the larvae were fed on sterile castor leaves treated with LC 50 and LC 20 concentrations of chlorantraniliprole and emamectin benzoate.The insecticide solutions were made in distilled water and experiment was carried out as mentioned above using leaf disc method.The leaves were changed after every 48 h till pupation.The larval mortality was observed on alternate days.Similarly for bacterial treatment, the larvae were fed on leaves treated with LC 50 concentrations of bacterial cell suspensions (1.59 × 10 9 , 1.21 × 10 9 and 1.67 × 10 9 cfu/ Three different experiments were conducted on the basis of number of insecticide treatments given to the larvae.Second instar larvae of S. litura were selected and experiments were conducted using the leaf dip method.The experiments were replicated five times with ten larvae per replicate and laboratory conditions were maintained at 25 ± 2 °C temperature and 65 ± 5% relative humidity. In the first experiment, larvae were treated with LC 50 concentration of insecticide on the first day of experiment and 24 h post insecticide application, the larvae were treated with LC 50 concentrations of bacterial cell suspensions (SS4, M9 and EN4).After that, only bacterial treatment was given on alternate days till pupation.Therefore, there was only one insecticide treatment.
In the second experiment, there were three insecticide treatments with alternate bacterial treatments.The first treatment with LC 50 concentration of insecticide was followed by bacterial treatment after 24 h.Then second and third insecticidal treatments were given after 48 and 96 h with bacterial treatment in between at 72 h.After that the larvae were only treated with bacterial cell suspension on alternate days till pupation.
In the third experiment, there were six combination treatments that include two sub lethal concentrations of the insecticide i.e., LC 20 and LC 50 .One set of larvae was fed on LC 20 and the other set on LC 50 concentration of insecticide.Insecticidal and bacterial treatments were given alternatively after every 24 h till pupation.

Comet assay.
Comet assay was done in alkaline conditions, using the protocol of Singh et al. 63 with slight modifications.For the individual chlorantraniliprole treatments, the third-instar S. litura larvae were fed on LC 20 concentration for 96 h.In the combination treatments of chlorantraniliprole, the larvae were fed on LC 20 concentration of insecticide in alternation with LC 50 concentrations of bacterial cell suspension (SS4, M9 and EN4) for 96 h.Similar procedure was followed for LC 50 concentrations of chlorantraniliprole alone and in combination with bacterial isolates.Likewise the comet assay was also performed with emamectin benzoate and bacterial isolates.The prolegs of third instar larvae were shrugged off and hemolymph (from ten larvae per treatment) was collected in eppendorf tubes containing phosphate buffer.The slides were coated with 1% normal melting point agarose (NMPA) and hemocytes were layered on coated slides and kept in a refrigerator at 4 °C to settle down.The slides were then immersed in the lysing solution (2.5 M NaCl, 100 mM EDTA, 0.25 M Tris aminomethane, 0.25 M NaOH, 1% Triton X-100, 10% DMSO, double distilled water, pH 10.0), which was kept overnight in the refrigerator.Electrophoresis was performed using an electrophoretic unit (25 V; 300 mA) containing electrophoretic buffer (1 mM EDTA, 300 mM NaOH, double distilled water, pH > 13) for 20 min.The slides were neutralized for 15 min in a neutralization buffer (0.4 M Tris amino methane, double distilled water, pH 7.5).The slides were stained with 50 g/ml ethidium bromide and then dried.These were examined under a Nikon fluorescence microscope.Three replicates were used for each treatment.Using Casplab, the tail length, the Olive Tail Moment and the percentage of tail DNA were computed.

Statistical analysis.
One-way analysis of variance (ANOVA) was used to analyze differences in mortality data means with Tukey's test at p ≤ 0.05.The statistical analysis was carried out using SPSS software for windows version 16.0 (SPSS Inc, Chicago).Probit analysis was used to calculate the lower and median lethal values.A chi square test was used to determine whether the insecticide and bacterial samples had antagonistic, additive, or synergistic effect 64 .The formula ME = MB + MI (1 − MB/100) was used to determine the expected mortality value for bacterial-insecticidal interactions, where MB and MI stand for the observed mortality percentage brought on by the entomopathogenic bacteria and insecticide alone, respectively.The results of the χ 2 test were compared with the χ 2 table value for 1 degree of freedom using the formula χ 2 = (MBI − ME) 2 /ME, where MBI is the observed mortality of the combination treatment.If the predicted value of χ 2 is greater than the value in the table, a synergistic or antagonistic action between the two agents was found while if the tabular value is more than the χ 2 value, an additive interaction was noticed.A significant interaction was observed to be synergistic if the difference between MBI and ME was positive; conversely, if it was negative, the interaction was deemed to

Figure 1 .
Figure 1.Screening of different concentrations of chlorantraniliprole for insecticidal potential against second instar S. litura larvae.Bars represent the Mean ± SE.Different letters above the bars represent significant differences at Tukey's test p ≤ 0.05.

Figure 2 .
Figure 2. Screening of different concentrations of emamectin benzoate for insecticidal potential against second instar S. litura larvae.Bars represent the Mean ± SE.Different letters above the bars represent significant differences at Tukey's test p ≤ 0.05.

Figure 3 .
Figure 3. Larval mortality due to treatment with insecticides and bacterial suspensions (A) Healthy larva, (B) Dead larvae due to insecticide treatments, (C,D) Dead larva due to bacterial infection and (E) Dead larvae due to combined treatments.

Table 1 .
Effect of combined treatments of chlorantraniliprole (Cg) and bacterial cell suspensions [Shewanella sp.(SS4), Pseudomonas sp.(EN4) and Thauera sp.(M9)] on cumulative larval mortality of S. litura.Number of insecticide treatments in combination with bacterial treatments in Combined Experiment 1 = 1, Combined Experiment 2 = 3 and Combined Experiment 3 = 6.Figures are Mean ± Standard Error for observed mortality.Means followed by different superscript letters within a column are significantly different.Tukey's test, p ≤ 0.05, **Significant at 1% 64 .Expected mortality ME = MB + MI (1 − MB), where MB and MI are the observed mortality percentage caused by bacteria and insecticide alone.Test for interaction based on χ 2 with 1 df, using the formula χ 2 = (MBI − ME) 2 /ME, wherein MBI is the observed mortality caused by bacteria + insecticide.

Table 2 .
Effect of combined treatments of emamectin benzoate (Eb) and bacterial cell suspensions [Shewanella sp.(SS4), Pseudomonas sp.(EN4) and Thauera sp.(M9)] on cumulative larval mortality of S. litura.Number of insecticide treatments in combination with bacterial treatments in Combined Experiment 1 = 1, Combined Experiment 2 = 3 and Combined Experiment 3 = 6.Figures are Mean ± Standard Error for observed mortality.Means followed by different superscript letters within a column are significantly different.Tukey's test, p ≤ 0.05, **Significant at 1% 64 .Expected mortality ME = MB + MI (1 − MB), where MB and MI are the observed mortality percentage caused by bacteria and insecticide alone.Test for interaction based on χ 2 with 1 df, using the formula χ 2 = (MBI − ME) 2 /ME, wherein MBI is the observed mortality caused by bacteria + insecticide.

Table 4 .
Effect of individual emamectin benzoate (Eb) treatments and its combination with bacterial cell suspensions [Shewanella sp.(SS4), Pseudomonas sp.(EN4) and Thauera sp.(M9)] on genotoxic parameters of S. litura after 96 h of treatment.Figures are Mean ± Standard Error.Means followed by different superscript letters within a column are significantly different.Tukey's test, p ≤ 0.05, **Significant at 1%