# Effect of Glyphosate and Mancozeb on the Rhizobia Isolated from Nodules ofVicia faba L. and on Their N2-Fixation, North Showa, Amhara Regional State, Ethiopia

Authors: Birhan Aynalem; Fassil Assefa
Journal: Advances in Biology
 (2017)

---

## Abstract

This study was designed to assess the effect of glyphosate and mancozeb on growth ofVicia faba rhizobia isolates in vitro and on their N2-fixation performance. Hence, ten isolates were isolated using plant-soil trap method from soil samples collected from farm lands. Those isolates were morphologically characterized using YEMA medium and authenticated as nodulating rhizobia using sand culture. These isolates were treated with 100, 150, and 200 μg a.e. L−1 glyphosate, 100, 150, and 200 mg L−1 mancozeb, and their combinations. The result showed that almost all isolates were affected (only 4–10% survival) at lower (100 mg L−1) concentration of mancozeb. However, 80% of isolates treated with higher concentration (200 μg a.e. L−1) of glyphosate for 72 h formed colonies on YEMA medium. Moderate (40%) isolates also showed better (31–50% and 17–45%) survival within 100 : 100 and 150 : 150 combinations of glyphosate and mancozeb, respectively. For in vivo experiment, faba bean seedlings in sand culture were inoculated with four relatively in vitro test resistant and one sensitive isolates. The inoculated isolates were treated with field recommended concentration of glyphosate, mancozeb, and combinations. Thus, experimental plants almost all showed normal (61–124 nodule plant−1) nodulation and N2-fixation (90–109%) performance as compared to the control.


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## Body

## 1. Introduction

The Ethiopian agriculture is mainly characterized by small-holding low-input traditional production system that feeds up on more than 85% of the population in the country [1]. The system includes crop rotation, mixed cropping, and agroforestry components with little or no agricultural inputs. Leguminous plants are important components of such systems for improving the soil fertility of the agroecosystem through biological nitrogen fixation.Faba bean (Vicia faba L.) is one of the cool season leguminous crops which is widely grown in Ethiopia. It covers 370,000 hectares with an annual production of 450,000 tones [2]. Faba bean contains high protein (24%) and appreciable amount of minerals and vitamins, and thus it is part of the various popular Ethiopian dishes [3]. In addition, faba bean is one of the most effective nitrogen fixing plants by forming symbiotic association withRhizobium leguminosarum bv.viceae and increases the nitrogen content in the soil [4]. Because of this, it is commonly integrated within crop rotation of traditional mixed low-input agricultural system in the highlands of the country.Currently, to manage soil runoff in the highland part of the ecosystem, there is an interest in no-tillage agriculture in the country. This is very important initiative to minimize erosion and avoid soil organism disturbance through tillage. Under this circumstance, there is a tendency to use herbicides and fungicides to enhance crop production even under low-input agricultural set-up. For this situation, farmers might be initiated to use the economical and broad spectrum type of agrichemicals.Glyphosate (N-phosphonomethyl glycine) is one of the nonselective, broad spectrum type herbicides which is mostly applicable on no-tillage agricultural practice for cleaning up of the weeds from the farm land before sowing [5]. Likewise, mancozeb is also another commonly used fungicide to inhibit fungal pathogens and farmers apply it for pathogen control through spraying over plant vegetative part after seedling or by dressing seeds using recommended concentration before sowing [6].Normally, glyphosate causes crop injury when it was applied directly on the broad leaved crops likeVicia faba. This is because of its inhibition effect on an enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that is involved in shikimate pathway [7]. Such EPSPS inhibition disrupts the shikimate pathway and leads to general metabolic disruption and several associated metabolic disturbances, including the arrest of protein production and prevention of subsequent product formation like hormones, vitamins, lignins, alkaloids, and phenolics in plants and microorganism [8]. The shikimate pathway is also found in fungi and bacteria so that herbicides and fungicides may implicitly have a tremendous damage on nontarget soil microflora [9].Furthermore, herbicides and fungicide adversely affect biological N2-fixation including the host, the endosymbiont rhizobia, and the fixation process. Moorman [10] showed that herbicides directly affect the rhizobial symbiont and exert indirect effects on the physiology of the host plant. There are also reports concerning different fungicides such as captan, thiram, mancozeb, and darosal; those inhibit the number of rhizobial strains, even though the effect varies from species to species and chemical concentrations [11–13]. In addition, Mubeen et al. [12] also reported that the use of fungicides causes tremendous damage on legumes when they were used in conjunction with microbial inoculants with negative effect on root infection, nodule formation, and bacterial growth hormone production.Thus, knowledge on the effect of herbicides and fungicides on rhizobia and symbiotic N2-fixation has paramount importance in selecting resistant endosymbiont as microbial inocula for low-input, no-tillage agricultural application with the presence of toxic agrochemicals. Therefore, this study was targeted to assess the effects of glyphosate and mancozeb on the growth of indigenous faba bean (Vicia faba L.) nodulating rhizobial isolates in the laboratory and their N2-fixation performance under greenhouse condition.


## 2. Materials and Methods

### 2.1. Sample Collection

Soil samples were collected from ten farm fields growing faba bean near the town of Checki, North Showa, Amhara Regional State, Ethiopia. From each study site, faba bean plants were selected and soil from 20 to 30 cm depth of the root was pooled and collected in alcohol sterilized (70%) plastic bags in September 2011. The soil samples were taken to Microbiology Laboratory, Departments of Microbial, Cellular and Molecular Biology, Addis Ababa University (AAU), for further work.


### 2.2. Isolation of Rhizobium from Nodules

Rhizobia were entrapped from collected soil samples by plant-soil trap method in pots [14] using Wolki variety of faba bean under greenhouse. After 45 days of plantation, the plants were uprooted and yellowish nodules were selectively picked, surface-sterilized, and crushed to release the rhizobia and 1 mL of suspensions was inoculated on the Yeast Extract Mannitol Agar (YEMA) medium (DIFCO) and incubated at 28°C for 5 to 7 days [15]. Those isolates were subsequently retransferred onto YEMA for purification. Then, purified isolates were preserved in YEMA slants, containing 0.3% (w/v) CaCO3 and stored at 4°C of refrigerator [14]. For every experiment, isolates were routinely cultured at room temperature on rotary shaker operating at 120 rpm (revolution per minute) to standardize the inoculums (size of 108 cells mL−1) for 72 h, unless stated otherwise.


### 2.3. Characterization of Isolates

The isolates were characterized on the basis of their morphological, cultural, and physiological characteristics [16] and authenticated as root nodule bacteria upon inoculating them into faba bean seedlings growing on sterilized sand pot culture irrigated with N2-free medium under greenhouse [17]. The purified and authenticated isolates were then designated as AAUW with numbers (Table 1).Table 1
Resistance evaluation of rhizobial isolates towards various concentrations of glyphosate.
Isolates
Control
Inhibition effect of different concentrations of glyphosate (µg a.e. L−1) on rhizobial isolates against control (OD 620 nm)
100µg a.e. L−1
%S
150µg a.e. L−1
%S
200µg a.e. L−1
%S
AAUW-1
0.417c
0.091a
22
0.082ab
20
0.043c
10
AAUW-2
0.377fe
0.051c
14
0.044d
12
0.032d
8
AAUW-3
0.457b
0.062b
14
0.058c
13
0.044bcd
10
AAUW-4
0.456b
0.057b
12
0.041d
9
0.026d
6
AAUW-5
0.343f
0.064b
19
0.057c
17
0.054bc
16
AAUW-6
0.467a
0.094a
20
0.090a
19
0.085a
8
AAUW-7
0.385d
0.071b
18
0.068b
7
0.038d
10
AAUW-8
0.411c
0.017d
4
0.012f
2
0.008e
2
AAUW-9
0.373f
0.045c
12
0.032e
3
0.030d
8
AAUW-10
0.414c
0.092a
22
0.086a
21
0.077a
19
Numbers in the same column followed by different letters are significantly different atp=0.05 confidence level (Tukey’s HSD). Letters in the columns (a, b, c, d, e, f, g, and h) are ranks of the mean; %S = percentage of survival; AAUW = Addis Ababa University Wolki variety.


### 2.4. Effect of Glyphosate on Rhizobial Isolates

Liquid glyphosate (MONSANTO) was taken in full strength concentration according to the specification of the company and filtrated by using 0.45μm pore size filter paper [12]. Filtered 100, 150, and 200 μg a.e. L−1 of glyphosate were separately mixed with sterilized 100 mL of YEM broth using 250 mL Erlenmeyer flasks. Then efficient 1 mL (108 cells mL−1) of each isolate was inoculated into broth medium prepared with different concentrations of glyphosate and incubated on the shaker at room temperature for 72 h.


### 2.5. Effect of Mancozeb on Rhizobial Isolates

The stock solutions of mancozeb (Limin Chemical Co. Ltd.) were prepared by adding 10 g of mancozeb powder into 1000 mL of distilled water and filtrated by using 0.45μm pore size filter paper [12]. Then 100, 150, and 200 mg L−1 of filtered mancozeb were separately added to sterilized 100 mL of YEM broth in 250 mL Erlenmeyer flasks. Then activated 1 mL (108 cells mL−1) of each isolate was added to each concentration and incubated on shaker at room temperature for 72 h.


### 2.6. Effect of (Glyphosate + Mancozeb) on Rhizobial Isolates

In order to see the combination effect of both agrochemicals on isolates, three treatments, such as Treatment-1 (100μg a.e. L−1 + 100 mg L−1), Treatment-2 (150 μg a.e. L−1 + 150 mg L−1), and Treatment-3 (200 μg a.e. L−1 + 200 mg L−1), were prepared in 100 mL YEM broth using 250 mL Erlenmeyer flasks. The sizes of inoculum and incubation method were the same as the above experiments.Growth of isolates in different treatments was monitored through optical density measurement by using UV-7804C spectrophotometer at 620 nm and compared with isolates grown on agrochemical-free control.(1)%I=OD  of  control-OD  of  treatedOD  of  control×100,where OD is optical density and %I is percentage of inhibition.


### 2.7. Cell Viability Test

The viability of the isolates in 100 and 200μg a.e. L−1 glyphosate, 100 and 200 mgL−1 mancozeb, and their combinations is determined by culturing serially diluted suspensions of 72 h old culture onto YEMA plates. The 100 μL dilution (10−5 and 10−6) from each treatment was transferred into the YEMA medium in duplicate, using Miles and Misra drop plate method [15], and incubated at 28°C for 48 h for plate counting.


### 2.8. Pot Experiments and Agrochemical Application

The pot experiments were conducted in greenhouse, College of Natural and Computational Sciences, AAU. For this purpose, thoroughly washed and autoclaved river sand was used and alcohol sterilized (70%); plastic pots were filled with 3 Kg of sterilized sand. The moisture content was adjusted to 75% through watering.


#### 2.8.1. Treatment with Glyphosate

In the case of greenhouse application, 0.2 mL/m2 or 2 × 0.036 g a.e. mL−1/m2 (0.072 g a.e. mL−1/m2) of glyphosate was diluted in 40 mL of water per square meter (sqm) [18]. Pots containing the sand were arranged in 1 sqm outside the greenhouse and diluted glyphosates were sprayed on the surface of sand filed within pots by using Tobor N-1 sprayer nuzzle and returned back to greenhouse and water was supplied to maintain the moisture at normal level.Five sterilized seeds per pot were sown after 2 days of spraying to minimize direct contact of seeds with glyphosate. Then four (AAUW-3, AAUW-5, AAUW-6, and AAUW-10) relatively in vitro test resistant and one (AAUW-8) sensitive isolates were selected. Those isolates were activated and adjusted to inoculum size of 108 cells mL−1 and inoculated into each seedling after germination and thinned down into three.The experimental control containsRhizobium inoculated seedling but free from agrochemicals. Then all plants were fertilized with 100 mL pot−1 of N2-free nutrients once a week [15] and supplied with 1000 mL pot−1 tap water every two days.


#### 2.8.2. Treatment with Mancozeb

Faba been seedlings were inoculated with 1 mL (108 cells mL−1 plant−1) of five selected isolates as previous experiment. Then, after 25 days of growth, rhizobium inoculated faba bean plants were sprayed with field recommended concentration (0.15 g mancozeb diluted with 75 mL of distilled water per sqm) of mancozeb using Tobor N-1 nuzzle.


#### 2.8.3. Treatments with Glyphosate + Mancozeb

The combination treatment, that is, glyphosate treatments, is done at the beginning and 0.15 g of mancozeb sqm−1 was sprayed after 25 days of planting by taking pots out from the greenhouse.Then all experimental pots with two replicates were arranged as a complete randomized block design and a photoperiod was 12/12 h with an average temperature of 25/18°C day per night. After 45 days of their planting, the whole plants were uprooted in order to determine nodule number, nodule dry weight, and shoot dry weight. N2-fixation performances (fixation effectiveness) of the plants were checked by using the following formula:(2)%NFE=SDWoftreatedSDWofcontrol×100,where NFE is N2-fixation effectiveness and SDW is shoot dry weight.


### 2.9. Data Analysis

Data analyses were made by one way of variance (ANOVA) using version 20 SPSS statistical program. Mean separation was calculated using Tukey’s HSD test when the value was significant atp=0.05.


## 2.1. Sample Collection

Soil samples were collected from ten farm fields growing faba bean near the town of Checki, North Showa, Amhara Regional State, Ethiopia. From each study site, faba bean plants were selected and soil from 20 to 30 cm depth of the root was pooled and collected in alcohol sterilized (70%) plastic bags in September 2011. The soil samples were taken to Microbiology Laboratory, Departments of Microbial, Cellular and Molecular Biology, Addis Ababa University (AAU), for further work.


## 2.2. Isolation of Rhizobium from Nodules

Rhizobia were entrapped from collected soil samples by plant-soil trap method in pots [14] using Wolki variety of faba bean under greenhouse. After 45 days of plantation, the plants were uprooted and yellowish nodules were selectively picked, surface-sterilized, and crushed to release the rhizobia and 1 mL of suspensions was inoculated on the Yeast Extract Mannitol Agar (YEMA) medium (DIFCO) and incubated at 28°C for 5 to 7 days [15]. Those isolates were subsequently retransferred onto YEMA for purification. Then, purified isolates were preserved in YEMA slants, containing 0.3% (w/v) CaCO3 and stored at 4°C of refrigerator [14]. For every experiment, isolates were routinely cultured at room temperature on rotary shaker operating at 120 rpm (revolution per minute) to standardize the inoculums (size of 108 cells mL−1) for 72 h, unless stated otherwise.


## 2.3. Characterization of Isolates

The isolates were characterized on the basis of their morphological, cultural, and physiological characteristics [16] and authenticated as root nodule bacteria upon inoculating them into faba bean seedlings growing on sterilized sand pot culture irrigated with N2-free medium under greenhouse [17]. The purified and authenticated isolates were then designated as AAUW with numbers (Table 1).Table 1
Resistance evaluation of rhizobial isolates towards various concentrations of glyphosate.
Isolates
Control
Inhibition effect of different concentrations of glyphosate (µg a.e. L−1) on rhizobial isolates against control (OD 620 nm)
100µg a.e. L−1
%S
150µg a.e. L−1
%S
200µg a.e. L−1
%S
AAUW-1
0.417c
0.091a
22
0.082ab
20
0.043c
10
AAUW-2
0.377fe
0.051c
14
0.044d
12
0.032d
8
AAUW-3
0.457b
0.062b
14
0.058c
13
0.044bcd
10
AAUW-4
0.456b
0.057b
12
0.041d
9
0.026d
6
AAUW-5
0.343f
0.064b
19
0.057c
17
0.054bc
16
AAUW-6
0.467a
0.094a
20
0.090a
19
0.085a
8
AAUW-7
0.385d
0.071b
18
0.068b
7
0.038d
10
AAUW-8
0.411c
0.017d
4
0.012f
2
0.008e
2
AAUW-9
0.373f
0.045c
12
0.032e
3
0.030d
8
AAUW-10
0.414c
0.092a
22
0.086a
21
0.077a
19
Numbers in the same column followed by different letters are significantly different atp=0.05 confidence level (Tukey’s HSD). Letters in the columns (a, b, c, d, e, f, g, and h) are ranks of the mean; %S = percentage of survival; AAUW = Addis Ababa University Wolki variety.


## 2.4. Effect of Glyphosate on Rhizobial Isolates

Liquid glyphosate (MONSANTO) was taken in full strength concentration according to the specification of the company and filtrated by using 0.45μm pore size filter paper [12]. Filtered 100, 150, and 200 μg a.e. L−1 of glyphosate were separately mixed with sterilized 100 mL of YEM broth using 250 mL Erlenmeyer flasks. Then efficient 1 mL (108 cells mL−1) of each isolate was inoculated into broth medium prepared with different concentrations of glyphosate and incubated on the shaker at room temperature for 72 h.


## 2.5. Effect of Mancozeb on Rhizobial Isolates

The stock solutions of mancozeb (Limin Chemical Co. Ltd.) were prepared by adding 10 g of mancozeb powder into 1000 mL of distilled water and filtrated by using 0.45μm pore size filter paper [12]. Then 100, 150, and 200 mg L−1 of filtered mancozeb were separately added to sterilized 100 mL of YEM broth in 250 mL Erlenmeyer flasks. Then activated 1 mL (108 cells mL−1) of each isolate was added to each concentration and incubated on shaker at room temperature for 72 h.


## 2.6. Effect of (Glyphosate + Mancozeb) on Rhizobial Isolates

In order to see the combination effect of both agrochemicals on isolates, three treatments, such as Treatment-1 (100μg a.e. L−1 + 100 mg L−1), Treatment-2 (150 μg a.e. L−1 + 150 mg L−1), and Treatment-3 (200 μg a.e. L−1 + 200 mg L−1), were prepared in 100 mL YEM broth using 250 mL Erlenmeyer flasks. The sizes of inoculum and incubation method were the same as the above experiments.Growth of isolates in different treatments was monitored through optical density measurement by using UV-7804C spectrophotometer at 620 nm and compared with isolates grown on agrochemical-free control.(1)%I=OD  of  control-OD  of  treatedOD  of  control×100,where OD is optical density and %I is percentage of inhibition.


## 2.7. Cell Viability Test

The viability of the isolates in 100 and 200μg a.e. L−1 glyphosate, 100 and 200 mgL−1 mancozeb, and their combinations is determined by culturing serially diluted suspensions of 72 h old culture onto YEMA plates. The 100 μL dilution (10−5 and 10−6) from each treatment was transferred into the YEMA medium in duplicate, using Miles and Misra drop plate method [15], and incubated at 28°C for 48 h for plate counting.


## 2.8. Pot Experiments and Agrochemical Application

The pot experiments were conducted in greenhouse, College of Natural and Computational Sciences, AAU. For this purpose, thoroughly washed and autoclaved river sand was used and alcohol sterilized (70%); plastic pots were filled with 3 Kg of sterilized sand. The moisture content was adjusted to 75% through watering.


### 2.8.1. Treatment with Glyphosate

In the case of greenhouse application, 0.2 mL/m2 or 2 × 0.036 g a.e. mL−1/m2 (0.072 g a.e. mL−1/m2) of glyphosate was diluted in 40 mL of water per square meter (sqm) [18]. Pots containing the sand were arranged in 1 sqm outside the greenhouse and diluted glyphosates were sprayed on the surface of sand filed within pots by using Tobor N-1 sprayer nuzzle and returned back to greenhouse and water was supplied to maintain the moisture at normal level.Five sterilized seeds per pot were sown after 2 days of spraying to minimize direct contact of seeds with glyphosate. Then four (AAUW-3, AAUW-5, AAUW-6, and AAUW-10) relatively in vitro test resistant and one (AAUW-8) sensitive isolates were selected. Those isolates were activated and adjusted to inoculum size of 108 cells mL−1 and inoculated into each seedling after germination and thinned down into three.The experimental control containsRhizobium inoculated seedling but free from agrochemicals. Then all plants were fertilized with 100 mL pot−1 of N2-free nutrients once a week [15] and supplied with 1000 mL pot−1 tap water every two days.


### 2.8.2. Treatment with Mancozeb

Faba been seedlings were inoculated with 1 mL (108 cells mL−1 plant−1) of five selected isolates as previous experiment. Then, after 25 days of growth, rhizobium inoculated faba bean plants were sprayed with field recommended concentration (0.15 g mancozeb diluted with 75 mL of distilled water per sqm) of mancozeb using Tobor N-1 nuzzle.


### 2.8.3. Treatments with Glyphosate + Mancozeb

The combination treatment, that is, glyphosate treatments, is done at the beginning and 0.15 g of mancozeb sqm−1 was sprayed after 25 days of planting by taking pots out from the greenhouse.Then all experimental pots with two replicates were arranged as a complete randomized block design and a photoperiod was 12/12 h with an average temperature of 25/18°C day per night. After 45 days of their planting, the whole plants were uprooted in order to determine nodule number, nodule dry weight, and shoot dry weight. N2-fixation performances (fixation effectiveness) of the plants were checked by using the following formula:(2)%NFE=SDWoftreatedSDWofcontrol×100,where NFE is N2-fixation effectiveness and SDW is shoot dry weight.


## 2.8.1. Treatment with Glyphosate

In the case of greenhouse application, 0.2 mL/m2 or 2 × 0.036 g a.e. mL−1/m2 (0.072 g a.e. mL−1/m2) of glyphosate was diluted in 40 mL of water per square meter (sqm) [18]. Pots containing the sand were arranged in 1 sqm outside the greenhouse and diluted glyphosates were sprayed on the surface of sand filed within pots by using Tobor N-1 sprayer nuzzle and returned back to greenhouse and water was supplied to maintain the moisture at normal level.Five sterilized seeds per pot were sown after 2 days of spraying to minimize direct contact of seeds with glyphosate. Then four (AAUW-3, AAUW-5, AAUW-6, and AAUW-10) relatively in vitro test resistant and one (AAUW-8) sensitive isolates were selected. Those isolates were activated and adjusted to inoculum size of 108 cells mL−1 and inoculated into each seedling after germination and thinned down into three.The experimental control containsRhizobium inoculated seedling but free from agrochemicals. Then all plants were fertilized with 100 mL pot−1 of N2-free nutrients once a week [15] and supplied with 1000 mL pot−1 tap water every two days.


## 2.8.2. Treatment with Mancozeb

Faba been seedlings were inoculated with 1 mL (108 cells mL−1 plant−1) of five selected isolates as previous experiment. Then, after 25 days of growth, rhizobium inoculated faba bean plants were sprayed with field recommended concentration (0.15 g mancozeb diluted with 75 mL of distilled water per sqm) of mancozeb using Tobor N-1 nuzzle.


## 2.8.3. Treatments with Glyphosate + Mancozeb

The combination treatment, that is, glyphosate treatments, is done at the beginning and 0.15 g of mancozeb sqm−1 was sprayed after 25 days of planting by taking pots out from the greenhouse.Then all experimental pots with two replicates were arranged as a complete randomized block design and a photoperiod was 12/12 h with an average temperature of 25/18°C day per night. After 45 days of their planting, the whole plants were uprooted in order to determine nodule number, nodule dry weight, and shoot dry weight. N2-fixation performances (fixation effectiveness) of the plants were checked by using the following formula:(2)%NFE=SDWoftreatedSDWofcontrol×100,where NFE is N2-fixation effectiveness and SDW is shoot dry weight.


## 2.9. Data Analysis

Data analyses were made by one way of variance (ANOVA) using version 20 SPSS statistical program. Mean separation was calculated using Tukey’s HSD test when the value was significant atp=0.05.


## 3. Result and Discussion

All ten isolates were Gram’s negative, rod shaped bacteria, similar in colony appearance with circular, transparent, raised, large mucoid, and fast growing nature with diameters between 2.5 and 4.8 mm after 3–5 days of growth on YEMA media (data not shown). They also changed BTB-YEMA medium into yellow to confirm isolates, and they were authenticated as nodulating rhizobia by inoculating them into faba bean seedlings grown within sand culture under greenhouse condition [16].


### 3.1. Effect of Agrochemicals on Rhizobial Isolates

All isolates were grown on YEM broth containing different concentrations of glyphosate and showed progressive reduction ofOD(620nm) as compared to control through spectrophotometer. Isolates AAUW-1, AAUW-5, AAUW-6, and AAUW-10 showed relative resistance with 19–22% survival rate at 100 μg a.e. L−1 and 150 μg a.e. L−1 glyphosate concentrations as compared to the other isolates (Table 1).Isolates AAUW-5 and AAUW-10 were maintained at 16% and 19% survival at 200μg a.e. L−1 of glyphosate, respectively (Table 1). Other reports showed up to 87.5% reduction ofBradyrhizobiumpopulation at lower concentration of 43.2 μg a.e. L−1 [19] and 67% reduction at 450 μg glyphosate disk−1 [13] indicating that the local isolates were relatively resistant to the agrochemicals. However, isolates AAUW-7 and AAUW-8 were completely inhibited by 100 μg a.e. L−1 of glyphosate as determined from cell viability test (Table 4). Likewise, Zablotowicz and Reddy [20] reported that USDA 110, 123, and 138B. japonicum strains were completely inhibited at 5 mM and caused rapid cell death at 10 mM of glyphosate. This indicates that the sensitivity difference of rhizobia is associated with the inherent chemical resistance of the isolates and geographical areas from where they were isolated.The in vitro inhibition tests of mancozeb also showed that isolates were very sensitive with drastic cell reduction of (90–96%) even at the low concentration (100 mg L−1) as compared to the control (Table 2). This is similar to the reports of Cevheri et al. [21] that rhizobial isolate (V5) fromVicia palaestina was inhibited by 98% at field recommended rate of mancozeb in vitro. Even the 50% reduction of two rhizobial isolates (isolate USDA 3187 and one strain from peanut) at lowest (0.2 mg L−1) concentration of mancozeb [11] indicates the sensitivity of root nodule bacteria to the fungicide mancozeb.Table 2
Effect of mancozeb on the growth of rhizobial isolates.
Isolates
Control
Inhibition effect of different concentrations of mancozeb (mg L−1) on rhizobial isolates against control (OD 620 nm)
100 mg L−1
%S
150 mg L−1
%S
200 mg L−1
%S
AAUW-1
0.417c
0.032d
8
0.013dc
3
0.011e
3
AAUW-2
0.377f
0.036ab
9
0.028ab
7
0.022b
6
AAUW-3
0.457b
0.041a
9
0.031a
7
0.026a
6
AAUW-4
0.456b
0.027ef
6
0.015c
3
0.012ef
3
AAUW-5
0.343h
0.032e
9
0.019bc
5
0.014de
4
AAUW-6
0.467a
0.023f
5
0.023b
5
0.020bc
4
AAUW-7
0.385d
0.030e
8
0.021bc
5
0.018c
5
AAUW-8
0.411e
0.018g
4
0.012cd
3
0.010f
2
AAUW-9
0.373g
0.036bcd
10
0.023bc
6
0.019bc
5
AAUW-10
0.414c
0.036b
9
0.026bc
6
0.022b
5
Numbers in the same column followed by different letters are significantly different atp=0.05 confidence level (Tukey’s HSD). Letters in the columns (a, b, c, d, e, f, g, and h) are ranks of the mean; %S = percentage of survival; AAUW = Addis Ababa University Wolki variety.However, it is interesting to note that isolates were more resistant to the mixture of the two chemicals than they were to the individual ones. Many of the isolates (70%) were resistant to Treatment-1 (100 : 100) combinations with survival rates between 21 and 50% where isolate AAUW-4 was the most resistant (50% survival) (Table3). The isolates, AAUW-1 and AAUW-3, are consistent in their resistance for all tested combined concentrations. This is contrary to the report of Cevheri et al. [21] that showed a 97% inhibition ofRhizobium isolate (V5) fromVicia palaestina exposed to a mixture of mancozeb and carbendazim in a field recommended concentration. The resistance of isolates to the combined chemicals may be attributed to the buffering effect of chemical reactions between each other. Furthermore, one or both chemicals may add the nutritional value for the rhizobial isolates. In fact, glyphosate was reported as the sole source of phosphorus under P-limiting conditions to grow several members of Rhizobiaceae includingRhizobium spp. andAgrobacterium spp. in liquid culture [22]. Moreover, the cell viability plate count data confirmed the existence of several colonies on the combined chemical treatments when they are transferred into YEMA medium (Table 4).Table 3
The combination effect of glyphosate plus mancozeb on the growth of rhizobial isolates.
Isolates
Control
O
D
620
n
m of pesticide combinations of glyphosate plus mancozeb in deferent concentration in YEMB (μg a.e. L−1) + (mg L−1)
100 : 100
%S
150 : 150
%S
200 : 200
%S
AAUW-1
0.417
c
0.155
c
37
0.147
a
35
0.098
a
23
AAUW-2
0.377
f
0.112
f
30
0.103
c
b
27
0.029
c
8
AAUW-3
0.457
b
0.186
b
41
0.113
b
25
0.049
b
13
AAUW-4
0.456
b
0.230
a
50
0.079
d
17
0.018
d
4
AAUW-5
0.343
h
0.113
e
33
0.016
g
h
5
0.011
e
3
AAUW-6
0.467
a
0.147
d
31
0.030
e
6
0.021
d
4
AAUW-7
0.385
d
0.043
f
11
0.022
f
6
0.022
d
6
AAUW-8
0.411
e
0.036
f
g
9
0.035
e
8
0.013
e
3
AAUW-9
0.373
g
0.031
g
8
0.015
h
4
0.012
e
d
3
AAUW-10
0.414
c
0.088
f
21
0.034
e
8
0.011
e
3
Numbers in the same column followed by different letters are significantly different atp=0.05 confidence level (Tukey’s HSD). Letters in the columns (a, b, c, d, e, f, g, and h) are ranks of the mean; %S = percentage of survival; AAUW = Addis Ababa University Wolki variety.Table 4
Cell viability of isolates after exposure to agrochemicals.
Isolates
Control
100μg a.e. L−1glyphosate
200μg a.e. L−1glyphosate
Combination 100 : 100
Combination 200 : 200
Log
Log
%cfu
Log
%cfu
Log
%cfu
Log
%cfu
AAUW-1
2.56
0.50
20
0.39
15
1.23
48
0.67
26
AAUW-2
2.80
0.41
17
0.33
12
1.60
57
1.23
44
AAUW-3
2.42
0.45
19
0.33
14
1.43
59
0.72
30
AAUW-4
2.56
0
0
0
0
1.29
50
0.26
10
AAUW-5
3.01
0.41
18
0.37
12
1.33
44
0.48
16
AAUW-6
3.22
0.45
19
0.41
13
0.97
30
0.45
14
AAUW-7
3.05
0.09
0
0
0
0.50
16
0.42
14
AAUW-8
1.58
0.08
0
0
0
0.22
10
0.16
10
AAUW-9
1.73
0.19
11
0
0
0.28
17
0.21
12
AAUW-10
2.58
0.41
16
0.35
14
0.34
13
0.27
11
cfu = colony forming units; Log = logarithmic growth of isolates.Conversely, isolates treated with mancozeb only do not form any colony when they were transferred onto YEMA (data not shown). Similarly, Fawole et al. [23] experienced significant reduction of rhizobial cfu at 2.34 mg L−1 of mancozeb. Drouin et al. [13] also reported that mancozeb at 450μg disk−1 concentration affects cfu ofMesorhizobium andBradyrhizobium strains by 67% and 91%, respectively. Although 60% of the isolates showed 16–20% and 12–15% cfu growth at 100 μg a.e. L−1 and 200 μg a.e. L−1 of glyphosate, respectively, the highest (30–59%) cfu growth was observed at a combination (100 : 100) concentration of the two agrochemicals. The isolates AAUW-1, AAUW-2, and AAUW-3 also showed the further resistance towards the combination concentration (200 : 200) by maintaining their cfu of 26%–44% (Table 4).


### 3.2. Effect of Glyphosate, Mancozeb, and Their Combinations on N2-Fixation Performance of Isolates under Greenhouse

Based on the findings from in vitro test, four relatively resistant (AAUW-3, AAUW-5, AAUW-6, and AAUW-10) and one sensitive (AAUW-8) isolates were selected for in vivo experiment. The isolates inoculated plants showed significant difference on their parameters with better record of SDW with agrochemical treatments.All isolates except AAUW-5 showed highest nodulation range (72–113 NN p−1) with their SDW between 1.63 and 2.95 g plant−1. The experimental plants showed unaffected height and green leave character without any effect. They also showed effective (65%) to highly effective (100–109%) symbiotic N2-fixation performance with filed recommended concentration of all glyphosate, mancozeb, and combinations over the control (Table 5). Similarly, reports are indicating that glyphosate in field experiment with 1.12 and 3.36 kg a.e. ha−1 [24] and mancozeb in field recommended concentration [11] has no significant effect on shoot and nodule dry mass and nodule numbers. For instance, soya bean plants were relatively well nodulated and nodule biomass was relatively unaffected by glyphosate with two applications of 2.52 kg ha−1 as compared to the control [25]. The normal nodulation and better nitrogen fixation performance of isolates may result by which agrochemicals might lose their toxicity to isolates through adsorption or degradation [26]. In addition, isolates and/or faba bean plant might get the nutritional value from glyphosate and mancozeb degraded substrate components for growth as well as nutrient exudates for microorganism to activate and change their physiological function [27, 28].Table 5
Effect of glyphosate, mancozeb, and their combination on the N2-fixation performance of isolates in greenhouse condition.
Treatment
Isolates
Parameters
%NFE
Score
NN p−1
NDW g p−1
SDW g p−1
GBRI
AUWR-3
86
a
b
0.107
a
1.63
d
e
65
E
AUWR-5
23
c
0.04
b
1.78
d
106
VE
AUWR-6
113
a
0.066
b
2.95
a
108
VE
AUWR-8
95
a
b
0.106
a
2.84
b
107
VE
AUWR-10
72
b
0.119
a
2.80
c
108
VE
MARI
AUWR-3
79
a
0.81
b
2.6
a
104
VE
AUWR-5
61
c
0.07
c
1.64
c
98
VE
AUWR-6
75
b
0.05
c
2.55
b
93
VE
AUWR-8
68
b
0.05
c
2.65
a
100
VE
AUWR-10
76
a
b
0.94
a
2.73
a
105
VE
GBMARI
AUWR-3
76
a
0.13
a
1.98
b
79
E
AUWR-5
65
a
0.10
a
1.73
b
103
VE
AUWR-6
87
a
0.07
b
2.73
a
100
VE
AUWR-8
56
a
0.11
a
2.68
a
101
VE
AUWR-10
59
a
0.12
a
2.65
a
b
102
VE
Control
AUWR-3
103
a
0.07
b
2.50
b
AUWR-5
64
c
0.08
a
1.68
c
AUWR-6
102
a
b
0.06
c
2.73
a
AUWR-8
91
b
0.05
c
d
2.65
a
b
AUWR-10
102
a
b
0.06
c
2.60
a
b
GBRI = glyphosate application before isolates inoculation; MARI = mancozeb application after isolates inoculation; GBMARI = combination of glyphosate application before isolates inoculation and mancozeb after inoculation; NN p−1 = nodule number per plant; NDW = nodule dry weight; SDW = shoot dry weight; numbers in the table are the mean of two plants per pot; g p−1 = gram per plant; E = effective; VE = very effective; NFE = N2-fixation effectiveness.


## 3.1. Effect of Agrochemicals on Rhizobial Isolates

All isolates were grown on YEM broth containing different concentrations of glyphosate and showed progressive reduction ofOD(620nm) as compared to control through spectrophotometer. Isolates AAUW-1, AAUW-5, AAUW-6, and AAUW-10 showed relative resistance with 19–22% survival rate at 100 μg a.e. L−1 and 150 μg a.e. L−1 glyphosate concentrations as compared to the other isolates (Table 1).Isolates AAUW-5 and AAUW-10 were maintained at 16% and 19% survival at 200μg a.e. L−1 of glyphosate, respectively (Table 1). Other reports showed up to 87.5% reduction ofBradyrhizobiumpopulation at lower concentration of 43.2 μg a.e. L−1 [19] and 67% reduction at 450 μg glyphosate disk−1 [13] indicating that the local isolates were relatively resistant to the agrochemicals. However, isolates AAUW-7 and AAUW-8 were completely inhibited by 100 μg a.e. L−1 of glyphosate as determined from cell viability test (Table 4). Likewise, Zablotowicz and Reddy [20] reported that USDA 110, 123, and 138B. japonicum strains were completely inhibited at 5 mM and caused rapid cell death at 10 mM of glyphosate. This indicates that the sensitivity difference of rhizobia is associated with the inherent chemical resistance of the isolates and geographical areas from where they were isolated.The in vitro inhibition tests of mancozeb also showed that isolates were very sensitive with drastic cell reduction of (90–96%) even at the low concentration (100 mg L−1) as compared to the control (Table 2). This is similar to the reports of Cevheri et al. [21] that rhizobial isolate (V5) fromVicia palaestina was inhibited by 98% at field recommended rate of mancozeb in vitro. Even the 50% reduction of two rhizobial isolates (isolate USDA 3187 and one strain from peanut) at lowest (0.2 mg L−1) concentration of mancozeb [11] indicates the sensitivity of root nodule bacteria to the fungicide mancozeb.Table 2
Effect of mancozeb on the growth of rhizobial isolates.
Isolates
Control
Inhibition effect of different concentrations of mancozeb (mg L−1) on rhizobial isolates against control (OD 620 nm)
100 mg L−1
%S
150 mg L−1
%S
200 mg L−1
%S
AAUW-1
0.417c
0.032d
8
0.013dc
3
0.011e
3
AAUW-2
0.377f
0.036ab
9
0.028ab
7
0.022b
6
AAUW-3
0.457b
0.041a
9
0.031a
7
0.026a
6
AAUW-4
0.456b
0.027ef
6
0.015c
3
0.012ef
3
AAUW-5
0.343h
0.032e
9
0.019bc
5
0.014de
4
AAUW-6
0.467a
0.023f
5
0.023b
5
0.020bc
4
AAUW-7
0.385d
0.030e
8
0.021bc
5
0.018c
5
AAUW-8
0.411e
0.018g
4
0.012cd
3
0.010f
2
AAUW-9
0.373g
0.036bcd
10
0.023bc
6
0.019bc
5
AAUW-10
0.414c
0.036b
9
0.026bc
6
0.022b
5
Numbers in the same column followed by different letters are significantly different atp=0.05 confidence level (Tukey’s HSD). Letters in the columns (a, b, c, d, e, f, g, and h) are ranks of the mean; %S = percentage of survival; AAUW = Addis Ababa University Wolki variety.However, it is interesting to note that isolates were more resistant to the mixture of the two chemicals than they were to the individual ones. Many of the isolates (70%) were resistant to Treatment-1 (100 : 100) combinations with survival rates between 21 and 50% where isolate AAUW-4 was the most resistant (50% survival) (Table3). The isolates, AAUW-1 and AAUW-3, are consistent in their resistance for all tested combined concentrations. This is contrary to the report of Cevheri et al. [21] that showed a 97% inhibition ofRhizobium isolate (V5) fromVicia palaestina exposed to a mixture of mancozeb and carbendazim in a field recommended concentration. The resistance of isolates to the combined chemicals may be attributed to the buffering effect of chemical reactions between each other. Furthermore, one or both chemicals may add the nutritional value for the rhizobial isolates. In fact, glyphosate was reported as the sole source of phosphorus under P-limiting conditions to grow several members of Rhizobiaceae includingRhizobium spp. andAgrobacterium spp. in liquid culture [22]. Moreover, the cell viability plate count data confirmed the existence of several colonies on the combined chemical treatments when they are transferred into YEMA medium (Table 4).Table 3
The combination effect of glyphosate plus mancozeb on the growth of rhizobial isolates.
Isolates
Control
O
D
620
n
m of pesticide combinations of glyphosate plus mancozeb in deferent concentration in YEMB (μg a.e. L−1) + (mg L−1)
100 : 100
%S
150 : 150
%S
200 : 200
%S
AAUW-1
0.417
c
0.155
c
37
0.147
a
35
0.098
a
23
AAUW-2
0.377
f
0.112
f
30
0.103
c
b
27
0.029
c
8
AAUW-3
0.457
b
0.186
b
41
0.113
b
25
0.049
b
13
AAUW-4
0.456
b
0.230
a
50
0.079
d
17
0.018
d
4
AAUW-5
0.343
h
0.113
e
33
0.016
g
h
5
0.011
e
3
AAUW-6
0.467
a
0.147
d
31
0.030
e
6
0.021
d
4
AAUW-7
0.385
d
0.043
f
11
0.022
f
6
0.022
d
6
AAUW-8
0.411
e
0.036
f
g
9
0.035
e
8
0.013
e
3
AAUW-9
0.373
g
0.031
g
8
0.015
h
4
0.012
e
d
3
AAUW-10
0.414
c
0.088
f
21
0.034
e
8
0.011
e
3
Numbers in the same column followed by different letters are significantly different atp=0.05 confidence level (Tukey’s HSD). Letters in the columns (a, b, c, d, e, f, g, and h) are ranks of the mean; %S = percentage of survival; AAUW = Addis Ababa University Wolki variety.Table 4
Cell viability of isolates after exposure to agrochemicals.
Isolates
Control
100μg a.e. L−1glyphosate
200μg a.e. L−1glyphosate
Combination 100 : 100
Combination 200 : 200
Log
Log
%cfu
Log
%cfu
Log
%cfu
Log
%cfu
AAUW-1
2.56
0.50
20
0.39
15
1.23
48
0.67
26
AAUW-2
2.80
0.41
17
0.33
12
1.60
57
1.23
44
AAUW-3
2.42
0.45
19
0.33
14
1.43
59
0.72
30
AAUW-4
2.56
0
0
0
0
1.29
50
0.26
10
AAUW-5
3.01
0.41
18
0.37
12
1.33
44
0.48
16
AAUW-6
3.22
0.45
19
0.41
13
0.97
30
0.45
14
AAUW-7
3.05
0.09
0
0
0
0.50
16
0.42
14
AAUW-8
1.58
0.08
0
0
0
0.22
10
0.16
10
AAUW-9
1.73
0.19
11
0
0
0.28
17
0.21
12
AAUW-10
2.58
0.41
16
0.35
14
0.34
13
0.27
11
cfu = colony forming units; Log = logarithmic growth of isolates.Conversely, isolates treated with mancozeb only do not form any colony when they were transferred onto YEMA (data not shown). Similarly, Fawole et al. [23] experienced significant reduction of rhizobial cfu at 2.34 mg L−1 of mancozeb. Drouin et al. [13] also reported that mancozeb at 450μg disk−1 concentration affects cfu ofMesorhizobium andBradyrhizobium strains by 67% and 91%, respectively. Although 60% of the isolates showed 16–20% and 12–15% cfu growth at 100 μg a.e. L−1 and 200 μg a.e. L−1 of glyphosate, respectively, the highest (30–59%) cfu growth was observed at a combination (100 : 100) concentration of the two agrochemicals. The isolates AAUW-1, AAUW-2, and AAUW-3 also showed the further resistance towards the combination concentration (200 : 200) by maintaining their cfu of 26%–44% (Table 4).


## 3.2. Effect of Glyphosate, Mancozeb, and Their Combinations on N2-Fixation Performance of Isolates under Greenhouse

Based on the findings from in vitro test, four relatively resistant (AAUW-3, AAUW-5, AAUW-6, and AAUW-10) and one sensitive (AAUW-8) isolates were selected for in vivo experiment. The isolates inoculated plants showed significant difference on their parameters with better record of SDW with agrochemical treatments.All isolates except AAUW-5 showed highest nodulation range (72–113 NN p−1) with their SDW between 1.63 and 2.95 g plant−1. The experimental plants showed unaffected height and green leave character without any effect. They also showed effective (65%) to highly effective (100–109%) symbiotic N2-fixation performance with filed recommended concentration of all glyphosate, mancozeb, and combinations over the control (Table 5). Similarly, reports are indicating that glyphosate in field experiment with 1.12 and 3.36 kg a.e. ha−1 [24] and mancozeb in field recommended concentration [11] has no significant effect on shoot and nodule dry mass and nodule numbers. For instance, soya bean plants were relatively well nodulated and nodule biomass was relatively unaffected by glyphosate with two applications of 2.52 kg ha−1 as compared to the control [25]. The normal nodulation and better nitrogen fixation performance of isolates may result by which agrochemicals might lose their toxicity to isolates through adsorption or degradation [26]. In addition, isolates and/or faba bean plant might get the nutritional value from glyphosate and mancozeb degraded substrate components for growth as well as nutrient exudates for microorganism to activate and change their physiological function [27, 28].Table 5
Effect of glyphosate, mancozeb, and their combination on the N2-fixation performance of isolates in greenhouse condition.
Treatment
Isolates
Parameters
%NFE
Score
NN p−1
NDW g p−1
SDW g p−1
GBRI
AUWR-3
86
a
b
0.107
a
1.63
d
e
65
E
AUWR-5
23
c
0.04
b
1.78
d
106
VE
AUWR-6
113
a
0.066
b
2.95
a
108
VE
AUWR-8
95
a
b
0.106
a
2.84
b
107
VE
AUWR-10
72
b
0.119
a
2.80
c
108
VE
MARI
AUWR-3
79
a
0.81
b
2.6
a
104
VE
AUWR-5
61
c
0.07
c
1.64
c
98
VE
AUWR-6
75
b
0.05
c
2.55
b
93
VE
AUWR-8
68
b
0.05
c
2.65
a
100
VE
AUWR-10
76
a
b
0.94
a
2.73
a
105
VE
GBMARI
AUWR-3
76
a
0.13
a
1.98
b
79
E
AUWR-5
65
a
0.10
a
1.73
b
103
VE
AUWR-6
87
a
0.07
b
2.73
a
100
VE
AUWR-8
56
a
0.11
a
2.68
a
101
VE
AUWR-10
59
a
0.12
a
2.65
a
b
102
VE
Control
AUWR-3
103
a
0.07
b
2.50
b
AUWR-5
64
c
0.08
a
1.68
c
AUWR-6
102
a
b
0.06
c
2.73
a
AUWR-8
91
b
0.05
c
d
2.65
a
b
AUWR-10
102
a
b
0.06
c
2.60
a
b
GBRI = glyphosate application before isolates inoculation; MARI = mancozeb application after isolates inoculation; GBMARI = combination of glyphosate application before isolates inoculation and mancozeb after inoculation; NN p−1 = nodule number per plant; NDW = nodule dry weight; SDW = shoot dry weight; numbers in the table are the mean of two plants per pot; g p−1 = gram per plant; E = effective; VE = very effective; NFE = N2-fixation effectiveness.


## 4. Conclusion

From this study, it can be concluded that the results obtained under in vitro experiment and in vivo investigation were negatively correlated with each other. The isolates treated with glyphosate and mancozeb under in vitro experiment showed reduction of cell population in respect to concentration. This is the evident that accumulations of high agrochemical concentration are more destructive to beneficial microbes as compared to less concentration. Some agrochemicals like mancozeb also affectRhizobium even at lower concentration. On the other hand, isolates which are treated with combination of the two agrochemicals showed better cell survival. This may have resulted because of both chemicals buffering ability with each other and minimizing the toxicity or both/one of chemicals may have nutritional value for rhizobial isolates. Furthermore, the results under in vivo investigation showed unaffected nodulation, plant growth, and N2-fixation performance of faba bean inoculated withRhizobium isolates although treated with field recommended concentration of glyphosate, mancozeb, and their combinations in greenhouse condition as compared to control (Table 6). It is expected that the applied agrochemicals may be absorbed by plants/sand or degraded by isolates.Table 6
Summery on N2-fixation performance of isolates with agrochemical stress.
Isolates
Control
GBRI
MARI
GBMARI
SDW
SDW
SP%
SDW
SP%
SDW
SP%
AUWR-3
2.50
b
1.63
d
e
65
2.60
a
104
1.98
b
79
AUWR-5
1.68
c
1.78
d
106
1.64
c
109
1.73
b
c
105
AUWR-6
2.73
a
2.95
a
108
2.55
b
93
2.73
a
100
AUWR-8
2.65
a
b
2.84
b
107
2.65
a
100
2.68
a
101
AUWR-10
2.60
a
b
2.80
c
108
2.73
a
105
2.65
a
b
102
%SP = symbiotic performance of isolates; SDW = shoot dry weight in gram/plant.This is promising result to identify and obtain agrochemical resistant inoculum for more N2-harvest under no-tillage agriculture system, but further actual field experiment will be required.



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*Source: 5864598-2017-06-13.xml*