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Original Research Article

Microbial degradation of the fungicide Benomyl in soil as influenced by addition of NPK

Randa H. Elsalahi1*, Adil A. Elhussein2, Awad G. Osman1 and Ashraf M. Sherif1

1Biofertilization Department, Environment and Natural Resource and Desertification Research Institute (ENDRI), National Center for Research, Khartoum, Sudan

2Botany Department, Faculty of Science, University of Khartoum, Khartoum, Sudan

*Corresponding author

A B S T R A C T

Introduction

Continuous use of pesticides encompasses accumulation of appreciable quantities of them and their degradation products in the ecosystem. Prevailing data showed that only 2- 3% of the applied chemical pesticides reach their targets, while the rest remains in the soil (US.EPA 2005). This raises great alarm about the heavy contamination burden the soil is carrying and consequently poses great risk on soil microbes, hence interfering

with elements cycles and entering food chains.

Among pesticides used in Sudan, fungicides rank third after insecticides and herbicides in terms of quantities used. One of the recently introduced fungicides in Sudan is Benlate which is the commercial name for the active ingredient Benomyl or, Methyl 1-(butylcarbamoyl) benzimidazole-2-

ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 756-771 http://www.ijcmas.com

Benomyl is a broad spectrum fungicide with a long environmental persistence. It is a suspected human carcinogen and is highly toxic to aquatic and soil dwelling organisms. A laboratory experiment was carried out to determine the effect of Benomyl on the main groups of soil microorganisms and to elucidate their role in the fungicide degradation as affected by NPK addition. NPK was added as 375 mg N, 187.5 mg P and 187.5 mg K per 600 g soil. Benomyl was applied in three concentrations; 0.032, 3.2 and 8.0 mg/g soil. Microbial counts of the main groups of soil microorganisms and Benomyl residue were determined at different intervals for a period of 360 days. Benomyl has a remarkable stimulating effect on the growth and multiplication of different microorganisms. The highest percent increments in microbial counts have always been recorded in Pseudomonas sp. followed by organic nitrogen users and fungi. Addition of NPK has increased microbial counts substantially which in turn has been reflected positively on Benomyl degradation rates. Degradation of more than 90% of Benomyl at 8.0 mg/g soil was attained in the control and in the NPK amended soil after 360 and 60 days, respectively.

K e y w o r d s

Biodegradation. Benomyl. Soil microorganisms. NPK

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ylcarbamate. It belongs to the benzimidazole class; a member of the carbamate group (Marrs and Ballantyne 2004). Benomyl is an endocrine disrupter chemical (European Commission 2000) a suspected mutagen, a possible human carcinogen (U.S. EPA 2002) and a teratogen (Pesticide Action Network 2002).

It is selectively toxic to microorganisms and to invertebrates (Kamrin, 2010) and is a systemic broad spectrum, protective, curative and iradicant fungicide (Troy and Beringer 2005; Wang et al. 2009). Benomyl is used for the control of post-harvest rots (Lopez-Garcia et al. 2003; Valiuskaite et al. 2006) and many plant fungal diseases such as powdery mildews, Botrytis rot, Fusarium basal rot, black spot and blossom rot (Marrs and Ballantyne 2004). In Sudan it is used for the treatment of powdery mildews mainly in cucurbits and other vegetables such as okra, tomatoes and auberjines.

Pesticides in the environment may be degraded in soil through chemical, physical and biological mechanisms (Ortiz-Hernández et al. 2011). Biodegradation of pesticides in soil is affected by soil properties (structure, moisture, temperature, pH and organic matter content) and the presence of the degrading microbes (Castillo and Torstensson 2007; Vischetti et al. 2008 and Chowdhury et al. 2008).

Benomyl degrades rapidly by hydrolysis to Carbendazim (Methyl-2-benzimidazole carbamate), or MBC, which is also registered as an active ingredient (US.EPA 2002). It has a long environmental persistence where residues in soil might be detected even after four years of application (Lewandowska and Walorczyk 2010). Benomyl has a half-life of 3 to 6 months and when applied to bare soil the half-life is 6 to 12 months (Zhang et al. 2005).

Nutrients like carbon, nitrogen, and phosphorus stimulate microbes to produce the essential enzymes for the break down contaminants (Vidali 2001). Low nitrogen or phosphorus availability can inhibit or delay the degradation of toxic materials (Cosgrove 2010). Addition of mineral fertilizers (NPK) has been reported to increase the biodegradation of Oxyfluorfen and Chlorpyrifos in soils (Mohammed et al. 2011; Tortella et al. 2010). As microbes are very helpful to remediate the contaminated environment, the objective of this study was to assess the microbial degradation of the fungicide Benomyl in soil as influenced by addition of NPK.

Materials and Methods

Effect of Benomyl on the major groups of soil microorganisms

Soil samples were collected from Al Geraif (Blue Nile bank) area North Khartoum where there is no history of pesticide application. Samples were randomly taken with an auger from the top 10 cm from different parts in the selected site. Large clods were crushed to a uniform size, mixed thoroughly to make a composite sample and air dried at room temperature. Some characteristics of the soil were determined according to the methods described by Rayan et al. (1996).

Soil was then divided into six 600 g lots and each was transferred into 1000 ml jar. Three lot sets were prepared for each of the two soil treatments (NPK and control). Benomyl was then added to each of the soil sets in the concentrations of 0.032, 3.2 or 8.0 (mg/g soil). Soil in each jar was thoroughly mixed with Benomyl. NPK was added in a separate set; soil was then wetted with water to 60% field capacity and incubated in the dark at temperature 28°C for 360 days.

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Nitrogen was added in the form of urea at a rate of 375 mg per 600 g of Benomyl treated soil (1437 kg/ha) and potassium was added in the form of KCl at a rate of 187.5 mg/600g soil (718 kg/ha). Phosphate in the form of super phosphate was added at a rate of 187.5 mg/600g soil (718 kg/ha). At zero time and then after 7, 15, 30, 60, 120, 150, 180, 240 and 360 days, soil samples were taken for the determination of total viable counts on each of five different media.

The media used were: Czapek Dox Agar (CZA), Meat Peptone Agar (MPA), Starch Ammonium Agar (SAA) and Nitrate Agar (NA); prepared according to the method described by Tepper et al. (1993) and Pseudomonas (CFC) Selective Agar Base according to (ISO 2000). Viable count determinations were carried out according to the procedure of Tepper et al. (1993).

Determination of Benomyl residues in soil

Benomyl residue was determined by HPLC (Sycam, Germany) according to the method of Zweig and Gao (1983) into a chromatograph with UV detector, column: Li-chromosorb RPC18 (25 cm x 3.9-mm-ID stainless steel), column temperature 28°C, mobile phase: 70% acetonitrile: 30% water. Injection volume: 20 l, Mobile phase flow rate 0.7ml/min, detection: UV light at 286 nm (1-cm cell), retention time: 4.3 min. Residues of Benomyl were determined at zero time and then after 7, 15, 30, 60 120, 150, 180, 240 and 360 days.

Results and Discussion

Effect of different Benomyl concentrations on soil microorganisms

A reasonable increment in Pseudomonas counts was observed up to two to three weeks of incubation in both NPK and the control. Pseudomonas counts have gradually

declined, due to addition of NPK, reaching the inhibition zone after 120 days of incubation at all Benomyl concentrations Fig. 1 and 2.

Effect of different Benomyl concentrations on soil microorganisms growing on meat peptone agar

Counts of organic nitrogen users at the control exhibited a general trend of increasing during the first two months then decreasing towards the end of the incubation period with the exception of the highest concentration (8.0 mg Benomyl/g soil) where populations continued to increase up to the fourth month Fig. 3. Addition of NPK showed the highest increments at 180 and 360 days of incubation Fig. 4.

Effect of different Benomyl concentrations on soil microorganisms growing on Czapek Dox

Fungal counts after the first week underwent inhibition up to the end of incubation period. Fig. 5. Increment peak was noticed at day 60 due to NPK addition Fig. 6. Inhibition of 52% was noticed only at day 7 interval, with the highest increment percentage (2169.23%) appearing after 60 days of incubation.

Effect of different Benomyl concentrations on soil microorganisms using Mineral Nitrogen

Numbers of mineral nitrogen users showed increment peaks at 60 and 180 days of incubation for the control and NPK treated soil at all Benomyl concentrations. Actinomycetes counts showed a contradicting pattern with the other types of inorganic nitrogen users and for some intervals they went in the same direction Table 1.

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Effect of different Benomyl concentrations on soil microorganisms growing on Poor Medium

Numbers of microorganisms capable of growing on poor medium increased remarkably due to the addition of NPK to soil treated with Benomyl at seven and 15 days of incubation while the rest of the counts showed fluctuating increments and inhibitions compared to control Table 2. However, at 360 day interval a drastic decrease in the total count was observed.

Benomyl degradation in soil

In soils treated with 0.032 mg Benomyl/g soil the control showed higher values (73%) since the 60 days (Fig. 7). interval while with NPK, degradation started at lower rates (34%) (Fig. 8). At day 120 degradation percentage obtained by the control was 75% at the same time intensive degradation percentages of 83% for NPK then the maximum degradation took place at 360 (Fig. 8). At 3.2 mg Benomyl/g soil, Benomyl degradation started at lower rates and gradually increased reaching a maximum of 97.7% at 180 days interval for the control (Fig. 7). Whereas for NPK fertilization a gradual increase in degradation percentage, and maximum degradation (99.4%) was attained at 180 days interval (Fig. 8). After 15 days of incubation at 8.0 mg/g soil Benomyl degradation was only 13% in the control, while with the NPK 76% was degraded, NPK fertilization at this Benomyl concentration seems to enhance earlier intensive degradation where maximum degradation (98.5%) was attained at 150 days interval (Fig. 8) compared to (99.9%) at 360 days interval for the control (Fig. 7).

Counts of Pseudomonas sp. and microbes growing on meat peptone agar were higher

at the beginning of the incubation period but completely disappeared after 120 days of incubation. This coincided with a remarkable increase in degradation % of up to 80% recorded after 60 days. This result suggested that Pseudomonas sp. play a role in Benomyl degradation. In line with this, Fuchs and De Vries (1978) detected high numbers of fluorescent Pseudomonas strains in a mineral medium containing Benomyl as the sole source of carbon.

Comparatively, very low increment percentages in numbers of organic nitrogen users were observed in NPK amended soil at all Benomyl concentrations up to 150 days. This could be attributed to the suppression of such microbes by inorganic nitrogen addition. Das and Debnath (2006) reported the stimulatory effect of Oxyfluorfen herbicide on the growth of non-symbiotic N2- fixing bacteria in clay soil treated with NPK fertilizers. Mohammed et al. (2011) found that addition of NPK fertilizers increased the population of organic nitrogen users in Oxyfluorfen treated soils at all concentrations tested.

On the contrary, Banerjee and Dey (1992) recorded a significant reduction in the total number of non-symbiotic N2- fixing bacteria after 90 days in the rhizosphere of jute after application of Basalin herbicide and NPK fertilizers.

Organic nitrogen users might be outcompeted by the inorganic nitrogen users that could have been enhanced by addition of NPK. Addition of NPK to the fungicide treated soil produced remarkable increment in the numbers of fungi up to 180 days of incubation which indicates a widespread resistance to Benomy after which remarkable decline was observed up to the end of the incubation period.

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Table.1 Effect of Benomyl on Soil Microorganisms Capable of Growing on Mineral Nitrogen Medium (Starch Ammonium Agar) in clay soil amended with NPK, incubated at room temp.

(28ºC)

Benomyl doses (mg/g soil)

0.032 3.2 8.0 Counts (108)

Control NPK Control NPK Control NPK

Total (X108) 10.35 256 46.5 16629 81 250.5

% Inhibition - Increment 0.0 +2373.43 0.0 +35661.3 0.0 +209.26

Actinomycetes (X108) 4.75 0.0 6.45 0.0 1.5 81

% Inhibition - Increment 0.0 100 0.0 100 0.0 5300

% Actinomycetes to total 45.89 0.0 13.87 0.0 1.85 32.34

Total (X108) 15.6 107.5 13.1 57.6 340.45 1997

% Inhibition - Increment 0.0 +589.1 0.0 +339.69 0.0 +486.58

Actinomycetes (X108) 10.05 0.5 7.05 1.1 1.95 0.0

% Inhibition - Increment 0.0 -95.12 0.0 -84.4 0.0 -100

% Actinomycetes to total 64.42 0.47 53.82 1.91 0.57 0.0

Total (X108) 1571.51 635.51 5.56 6820.01 1099.06 10839.51

% Inhibition - Increment 0.0 -59.56 0.0 +122662.05 0.0 +886.25

Actinomycetes (X108) 0.01 1.01 0.0 12.01 0.01 140.51

% Inhibition - Increment 0.0 +10000 0.0 +120000 0.0 +1405000

% Actinomycetes to total 0.000064 0.159 0.0 0.18 0.00091 1.3

Total (X108) 216.85 330 291.35 423.5 11.8 198

% Inhibition - Increment 0.0 +52.18 0.0 +45.36 0.0 +1578

Actinomycetes (X108) 1.5 1.0 0.05 15.5 0.5 41.5

% Inhibition - Increment 0.0 -33.33 0.0 +30900 0.0 +8200

% Actinomycetes to total 0.69 0.3 0.017 3.66 4.24 20.96

Total (X108) 3.5 2901.5 1950.95 17287.5 2529.05 2588

% Inhibition - Increment 0.0 +82800 0.0 +986.11 0.0 +2.33

Actinomycetes (X108) 0.05 32 0.25 350 2.5 11996.5

% Inhibition - Increment 0.0 +63900 0.0 +139900 0.0 +47660

% Actinomycetes to total 1.43 1.1 0.013 2.02 0.099 46.23

Total (X108) 39550 949.5 149805 80 205850.01 318

% Inhibition - Increment 0.0 -97.6 0.0 -99.95 0.0 -99.85

Actinomycetes (X108) 0.01 57.5 5 2 0.01 27.5

% Inhibition - Increment 0.0 +574500 0.0 -60 0.0 +274900

% Actinomycetes to total 0.000025 6.06 0.0033 2.5 0.0000048 8.65

Total (X108) 9326.5 5032 2517.51 2520.16 11319.01 4288.51

% Inhibition - Increment 0.0 -46.05 0.0 +0.1 0.0 -62.11

Actinomycetes (X108) 60 19.5 0.01 45 0.01 0.01

% Inhibition - Increment 0.0 -67.5 0.0 +449900 0.0 0.0

% Actinomycetes to total 0.64 0.39 0.0004 1.79 0.0001 0.00023

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Continue:

Benomyl doses (mg/g soil)

0.032 3.2 8.0 Counts (108)

Control NPK Control

NPK Control

NPK

Total (X108) 3379 14979.15 1834 13812.5 10144 11114

% Inhibition - Increment 0.0 +343.3 0.0 +653.14 0.0 +9.56

Actinomycetes (X108) 2437 2.15 8 13.5 5 47

% Inhibition - Increment 0.0 -99.91 0.0 +68.75 0.0 +840

% Actinomycetes to total 72.12 0.014 0.44 0.091 0.049 0.42

Total (X108) 9214.01 27.01 605.01 3701 71.5 16211

% Inhibition - Increment 0.0 -99.71 0.0 +511.73 0.0 +22572.73

Actinomycetes (X108) 0.01 2.5 0.01 16.5 5 27

% Inhibition - Increment 0.0 +24900 0.0 +164900 0.0 +440

% Actinomycetes to total 0.00011 9.26 0.0017 0.45 6.99 0.17

Total (X108) 333 737.01 422 6508 642.51 117

% Inhibition - Increment 0.0 -121.32 0.0 +1442.12 0.0 -81.79

Actinomycetes (X108) 15 0.01 0.0 0.0 0.01 0.5

% Inhibition - Increment 0.0 9.93 0.0 0.0 0.0 +4900

% Actinomycetes to total 4.5 0.0014 0.0024 0.0015 0.0016 0.43

(%): Percentage of Inhibition (-) and Increment (+)

Table.2 Effect of Benomyl on soil microorganisms capable of growing on poor medium (Nitate Agar) in clay soil amended with NPK, incubated at room temp (28ºC)

All microorganisms Mycobacterium Micromonospora Nocardia Arthrobacter Bactoderma Benomyl

Doses mg/g soil

N. Cells/g dry soil (X108)

% N. Cells/g dry soil (X108)

% N. Cells/g dry soil (X108)

% N. Cells/g dry soil (X108)

% N. Cells/g dry soil (X108)

% N. Cells/g dry soil (X108)

%

At zero time Control 313.56 100 118.46 37.78 170.56 54.4 12.35 3.94 12.22 3.9 0.0 0.0

0.032 159.9 51 81.9 51.22 0.0 0.0 0.0 0.0 78 48.78 0.0 0.0

Control 668.33 100 616.33 92.22 13.39 2 14.95 2.24 23.66 3.54 0.0 0.0

3.2 1.3 0.19 1.3 100 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Control 74.49 100 40.3 54.1 4.55 6.11 9.75 13.09 19.76 26.53 0.13 0.17

8.0 269.1 361.3 0.0 0.0 0.0 0.0 9.1 3.38 0.0 0.0 260 96.62

At After 7 days Control 21.32 100 7.93 37.2 0.39 1.83 1.69 7.93 11.18 52.44 0.13 0.61

0.032 193.7 908.5 78.1 44.97 0.0 0.0 52 26.85 54.6 28.19 0.0 0.0

Control 98.93 100 15.86 16.03 0.13 0.131 54.73 55.32 28.21 28.52 0.0 0.0

3.2 61.1 61.8 16.9 27.66 9.1 14.89 3.9 6.38 31.2 51.06 0.0 0.0

Control 184.47 100 161.72 87.67 1.3 0.7 18.85 10.22 2.6 1.41 0.0 0.0

8.0 728 394.6 169 23.21 0.0 0.0 78 10.71 481 66.07 0.0 0.0

After 15 days Control 108.55 100 26.65 24.55 31.2 28.74 33.8 31.14 16.9 15.57 0.0 0.0

0.032 189.8 174.9 123.5 65.07 0.0 0.0 27.3 14.38 39 20.54 0.0 0.0

Control 155.35 100 40.04 25.77 0.0 0.0 29.51 19 85.8 55.23 0.0 0.0

3.2 633.1 407.5 500.5 79.06 1.3 0.21 41.6 6.57 89.7 14.17 0.0 0.0

Control 461.76 100 352.56 76.35 10.66 2.31 52.39 11.35 46.15 9.99 0.0 0.0

8.0 390 84.5 200.2 51.33 6.5 1.67 7.8 2 175.5 45 0.0 0.0

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After 30 days

Control 49.79 100 43.42 87.21 1.3 2.61 3.25 6.53 1.82 3.66 0.0 0.0

0.032 2109.9 4237.6 547.3 25.94 131.3 6.22 288.6 13.68 97.5 4.26 1045.2 49.54

Control 85.41 100 32.63 38.2 0.0 0.0 11.7 13.7 41.08 48.1 0.0 0.0

3.2 271.7 318.1 68.9 25.36 39 14.35 57.2 21.05 49.4 18.18 57.2 21.05

Control 12.35 100 4.16 33.68 0.52 4.21 1.17 9.47 6.5 52.63 0.0 0.0

8.0 393.9 3189.5 213.2 54.13 50.7 12.87 59.8 15.18 35.1 8.91 35.1 8.91

After 60 days Control 66.69 100 41.99 62.96 0.0 0.0 20.02 30.02 4.68 7.02 0.0 0.0

0.032 32041.1 48044.8 109.2 0.34 30899.7 96.44 40.3 0.13 1.3 0.004 990.6 3.09

Control 36.66 100 18.46 50.35 0.0 0.0 5.33 14.54 12.87 35.11 0.0 0.0

3.2 228.8 624.1 54.6 23.86 49.4 21.59 54.6 23.86 70.2 30.68 0.0 0.0

Control 47.45 100 42.64 89.86 0.0 0.0 2.08 4.32 2.73 5.75 0.0 0.0

8.0 540.8 1139.7 241.8 44.71 96.2 17.79 70.2 12.98 132.6 24.52 0.0 0.0

After 120 days Control 2398.5 100 1051.7 43.48 182 7.59 0.0 0.0 1060.8 44.23 104 4.34

0.032 262.6 10.9 91 34.65 26 9.9 1.3 0.5 79.3 30.2 65 24.75

Control 3764.8 100 2294.5 60.95 46.8 1.24 187.2 4.97 1050.4 27.9 185.9 4.94

3.2 5614.7 149.1 5465.2 97.34 9.1 0.16 27.3 0.49 71.5 1.27 41.6 0.74

Control 709.8 100 275.6 38.83 422.5 59.52 1.3 0.183 10.4 1.47 0.0 0.0

8.0 3118.7 439.4 2035.8 65.28 468 15.01 84.5 2.71 455 14.59 75.4 2.42

Continued:

All microorganisms Mycobacterium Micromonospora Nocardia Arthrobacter Bactoderma Benomyl

Doses mg/g soil

N. Cells/g dry soil (X108)

%

N. Cells/g dry soil (X108)

%

N. Cells/g dry soil (X108)

%

N. Cells/g dry soil (X108)

%

N. Cells/g

dry soil

(X108)

%

N. Cells/g dry soil (X108)

%

After 150 days Control 25333.1 100 24151.4 95.34 1181.7 4.66 0.0 0.0 0.0 0.0 0.0 0.0

0.032 1684.8 6.7 894.4 53.09 135.2 8.02 169 10.03 457.6 27.16 28.6 1.7

Control 3191.5 100 2493.4 78.13 113.1 3.54 559 17.52 26 0.81 0.0 0.0

3.2 867.1 27.2 471.9 54.42 57.2 6.6 81.9 9.45 230.1 26.54 26 3.06

Control 672.1 100 516.1 76.79 429 6.38 49.4 7.35 63.7 9.48 0.0 0.0

8.0 2164.5 284 1041.3 48.11 5.2 0.24 793 36.64 283.4 13.09 41.6 1.92

After 180 days Control 1106.43 100 245.7 22.21 0.0 0.0 839.93 75.91 20.8 1.88 0.0 0.0

0.032 90896 8215.3 61.1 0.067 26 0.029 26 0.029 10.4 0.011 90772.5 99.86

Control 150.15 100 114.4 76.19 27.3 18.18 7.15 4.76 1.3 0.866 0.0 0.0

3.2 88791.3 59135.1 88714.6 99.91 2.6 0.0029 5.2 0.0059 63.7 0.072 5.2 0.0059

Control 20.8 100 1.3 6.25 3.9 18.75 9.1 43.75 6.5 31.25 0.0 0.0

8.0 57345.5 275699.5 57048 99.5 6.5 0.01 68.9 0.12 170.1 0.3 52 0.091

After 240 days Control 15.6 100 0.0 0.0 0.0 0.0 13 83.33 2.6 16.67 0.0 0.0

0.032 2870.4 18400 2631.2 91.67 83.2 2.9 49.4 1.72 78 2.72 28.6 0.996

Control 304.2 100 2.6 0.855 1.3 0.43 27.3 8.97 13 4.27 260 85.47

3.2 10648.3 3500.4 9128.6 85.73 26 0.24 2.6 0.024 325 3.05 1166.1 10.95

Control 193.7 100 44.2 22.82 39 20.13 27.3 14.09 83.2 42.95 0.0 0.0

8.0 67941.9 35075.8 67834 99.84 52 0.077 1.3 0.0019 44.2 0.065 10.4 0.015

After 360 days Control 1328.6 100 704.6 53.03 0.0 0.0 546 41.1 78 5.87 0.0 0.0

0.032 5929.3 446.3 5816.2 98.09 0.0 0.0 48.1 0.81 65 1.1 0.0 0.0

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Control 40.3 100 14.3 35.48 13 32.26 0.0 0.0 13 32.26 0.0 0.0

3.2 1831.7 4545.2 1237.6 67.57 26 1.42 28.6 1.56 513.5 28.03 26 1.42

Control 275.6 100 133.9 48.58 26 9.43 0.0 0.0 115.7 41.98 0.0 0.0

8.0 68.9 25 1.3 1.89 0.0 0.0 0.0 0.0 41.6 60.38 26 37.74

Fig.1 Inhibition or increment percentages of Pseudomonas sp in Al Geraif soil incubated at 28ºC

by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g soil)

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Fig.2 Inhibition or increment percentages of Pseudomonas sp in Al Geraif soil amended with NPK and incubated at 28ºC by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and

d.8.0 mg/g soil)

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Fig.3 Inhibition or increment percentages of organic nitrogen users in Al Geraif soil amended

with NPK and incubated at 28ºC by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g soil)

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Fig.4 Inhibition or increment percentages of soil fungi in Al Geraif soil incubated at 28ºC by

different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g soil)

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Fig.5 Inhibition or increment percentages of soil fungi in Al Geraif soil amended with NPK and incubated at 28ºC by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g

soil)

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Fig.6 Benomyl degradation rates in Al Geraif soil incubated at 28°C

Fig.8 Benomyl degradation rates in Al Geraif soil amended with NPK and incubated at 28°C

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Generally, higher fungal counts were observed at higher Benomyl concentrations. In the same context, Iqbal et al. (2001) found that application of Monocrotophos and Menthamidophos insecticides with NPK fertilizers increased the fungal population in clay loam soil. Whereas, Banerjee and Dey (1992) found that Basalin herbicide in conjunction with NPK fertilizers decreased the total number of fungi in sandy clay soil.

Counts of microorganisms using inorganic nitrogen underwent a general suppression at the first two concentrations as a result of adding NPK to Benomyl treated soil. In contrast, Jones and Alexander (1988) found that adding nitrogen and phosphorus to the soil enhances microbial growth. Mineral fertilization of arable land positively affects and increases the biological productivity of various ecosystems as well as the microbial activity in soil (Barabasz et al. 2002). However, Mohammed et al. (2011) reported that the addition of NPK fertilizers to Oxyfluorfen treated soil stimulated the growth of inorganic nitrogen bacteria incubated at 40ºC while actinomycetes were suppressed at the end of incubation period (45 days).

Generally NPK stimulated growth of microorganisms capable of growing in poor medium at higher Benomyl concentrations. Mycobacterium was absolutely dominant followed by Arthrobacter. Mohammed (2009) found that the addition of NPK fertilizers clearly increased the number of microorganisms at all Oxyfluorfen concentrations tested and Mycobacterium was the dominant species throughout the incubation periods. Elsaid et al. (2009) found that application of Endosulfan insecticide and urea + phosphate fertilizers increased the number of inorganic nitrogen using microbes in soils after 45 days of incubation

Effect of NPK amendments on benomyl degradation

Higher Benomyl degradation rates were obvious at higher Benomyl concentrations. In line with this, Wang et al. (2009) reported higher degradation rates of Carbendazim at higher than at lower concentrations. The biodegradation of organic compounds is often slow because one or more of inorganic nutrients needed for microbial growth are in low concentrations in natural environments (Toolan et al. 1991; Coveney and Wetzel 1992). It was suggested that the addition of N and P may increase the biodegradation of organic compounds (Pritchard and Costa 1991). Results of Mohammed et al. (2011) indicated that the degradation of Oxyfluorfen in soils fertilized with NPK was higher than in non-fertilized soil

The most obvious characteristic was that with the addition of NPK the degradation percentage for Benomyl was high since after the first month of incubation for the higher fungicide concentrations. Degradation was 71% and 94 % for 3.2 and 8.0 mg Benomyl/g soil, respectively at 60 days interval. Addition of NPK up to 0.5% increased Chlorpyrifos degradation during the first days of incubation (Tortella et al. 2010).

Addition of NPK has increased microbial counts substantially which in turn has been reflected positively on Benomyl degradation rates. Benomyl has a remarkable stimulating effect (despite the considerable variations), on the growth and multiplication of different microorganisms. The highest percent increments in microbial counts have always been recorded for Pseudomonas sp. followed by organic nitrogen users and fungi. Maximum degradation percentage in soil was 99.9% at 8.0 mg Benomyl/g soil after 360 days of incubation for the control

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and 99.4% for NPK at 3.2 mg Benomyl/g soil after 180 days of incubation.

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