efecto del glifosato sobre bacterias asociadas a la...

Efecto del glifosato sobre bacterias asociadas a la rizosfera

Judy Madelén Giraldo Duque

Universidad Nacional de Colombia

Facultad de Ciencias

Medellín, Colombia

2014

Glyphosate effect on plant rhizobacteria




Medellín, Colombia

2014

Glyphosate effect on plant rhizobacteria


Thesis submitted in partial fulfillment of the requirements for the degree

of:

Master of Science – Geomorphology and Soils

Director:

Ph.D, Juan Carlos Pérez Naranjo

Codirector:

Ph.D, Chantal Hamel



Medellín, Colombia

2014

(Dedication)

To my parents for their deep and unconditional love.

For all the sacrifices they have done to make me the

person I am today. They are my strength and the

biggest reason I have reached my dreams and I hope

giving to them the satisfaction of accomplishment.

To Keith Hanson for being there for me even through

the distance and for having made my days so happy

in an unknown country. Now I have the certainty of

having found a great person, a true friend.

Acknowledgments

Foremost, I would like to express my sincere gratitude to my co-director Professor

Chantal Hamel for the constant support during my research, for her patience, immense

motivation and especially for infecting me with her passion for microorganisms. To have

known her and to discover the amazing human being she is makes me feel really lucky.

I appreciate my advisor Professor Juan Carlos Perez Naranjo for giving me the

opportunity so that I could conduct my research in Canada at the Semiarid Prairie

Agricultural Research Centre in Swift Current, SK.

Thanks to the Emerging Leaders in the Americas Program (ELAP) for the scholarship that

allowed me to do this research.

Thanks to the Dr. Myriam Fernandez for setting up the field experiment

I am thankful to my labmates in soil microbiology at the Semiarid Prairie Agricultural

Research Centre in Swift Current, SK: Tony Yang and Mulan Dai for their help and

especially to technician Keith Hanson who was always my support in the moments when

there was no one to answer my queries.

A special thanks to Professors Guillermo Correa of Universidad Nacional de Colombia

and Jhon Stephen Brewer of University of Mississippi for their support and help with

statistics.

I would like express appreciation to my friends Margarita Sizquiarco, Cristina Valencia,

Andres Medina and Soraya Uribe Celis because they were there to support me spiritually

and to help me to feel hope again when my strength was exhausted.

Last but not the least, I thank to the Universidad Nacional de Colombia for its academic

training.

List of content

Abstract........................................................................................................................ VIII

List of figures................................................................................................................ XII

List of tables ................................................................................................................ XIII

1 Introduction ............................................................................................................. 1

1.1 Hypothesis ........................................................................................................ 2

1.2 Objective ........................................................................................................... 2

2 Literature review ..................................................................................................... 3

2.1 World wheat production .................................................................................... 3

2.2 Wheat production in Canada ............................................................................. 4

2.2.1 Production systems ....................................................................................... 5

2.2.2 Weed management ....................................................................................... 6

2.3 Glyphosate and its non-target effects on microorganisms ................................. 8

2.3.1 Effect of glyphosate on soil microorganisms .................................................. 9

2.3.2 Effect of glyphosate on rhizosphere microorganisms .................................. 10

2.4 Effect of tillage and crop rotation on the microbial community in wheat rhizosphere ................................................................................................................. 12

3 Dynamics of rhizosphere communities after glyphosate application to mustard and wheat ...................................................................................................................... 14

3.1 Abstract........................................................................................................... 14

3.2 Introduction ..................................................................................................... 14

3.3 Materials and methods .................................................................................... 16

3.3.1 Soil and experimental design ...................................................................... 16

3.3.2 Glyphosate application ................................................................................ 17

3.3.3 Sampling rhizosphere and counting of microorganisms ............................... 17

3.3.4 Data analysis .............................................................................................. 18

3.4 Results ............................................................................................................ 18

3.5 Discussion ...................................................................................................... 21

3.6 Conclusion ...................................................................................................... 23

4 Glyphosate-mediated changes in the microbial communities inhabiting the rhizosphere of field-grown wheat and tansy mustard ............................................... 25

4.1 Abstract........................................................................................................... 25

4.2 Introduction ..................................................................................................... 26

4.3 Materials and methods .................................................................................... 27

4.3.1 Field site and experimental design .............................................................. 27

4.3.2 Rhizosphere sampling and microbial counts ............................................... 28

4.3.3 Polymerase Chain Reaction (PCR) and sequencing ................................... 28

4.3.4 Data analysis .............................................................................................. 29

4.4 Results ............................................................................................................ 31

4.5 Discussion ...................................................................................................... 40

4.6 Conclusion ...................................................................................................... 44

References cited ........................................................................................................... 52

VIII

Abstract

Results obtained from several studies suggest that the pre-seeding application of the

widely used herbicide glyphosate can alter the microbial community of the rhizosphere of

non-target plants, as well as soil processes mediated by microorganisms. Although this

impact should be related to the response of weed plants to glyphosate application, little is

known on the changes taking place in the microbial community of weed plant rhizosphere.

A field and a greenhouse experiments were conducted in order to test the influence of

recommended doses of glyphosate on the rhizosphere community of the weeds mustard,

tansy mustard, and volunteer wheat. The greenhouse experiment determined the effect of

two recommended doses of glyphosate (450 g a.i. ha-1, 1800 g a.i. ha-1) on the fungi and

bacteria inhabiting the rhizosphere of the targeted volunteer wheat (Triticum aestivum L.

cv. AC Lillian) and mustard plants (Brassica juncea) as revealed by plate counts

conducted 24 h, 3 d and 7 d after application. Glyphosate shifted microbial community

size, increasing the rhizobacterial counts in a dose-dependent manner. This effect could

be direct, as glyphosate can be released by roots into the rhizosphere, or through

physiological changes experienced by dying plants after glyphosate application. In the

field experiment, the rhizosphere soil of wheat (Triticum aestivum L. cv. AC Lillian) and

tansy mustard plants (Descurainia pinnate (Walter) Britton) growing in the pea and the

wheat phases of a pea-wheat rotation system, was collected before and after glyphosate

application (450 g a.i. ha-1). The microbial communities were analyzed by plate counts

based on colony morphology. Bacterial morphotypes were identified based on 16S rDNA.

Glyphosate triggered no detectable effects on the rhizobacterial community of tansy

mustard or on fungi, but glyphosate influenced differently the rhizobacterial communities

of the wheat crops grown in both, the pea and the wheat stubble environments.

Glyphosate increased the abundance of the known triazine-s decomposer Arthrobacter

aurescens, and decreased the abundance of potentially plant-growth-promoting

Mesorhizobium loti and Variovorax paradoxus strains. It is concluded that pre-seeding

IX

applications of glyphosate may have undisclosed agronomic and environmental

implications.

Keywords: rhizosphere microbial community, herbicide, weeds, rotation system,

field recommended dose, glyphosate, wheat.

X

Resumen

Los resultados obtenidos desde numerosos estudios sugieren que la aplicación pre-

siembra del herbicida ampliamente usado “glifosato”, puede alterar la comunidad

microbial de la rizosfera de plantas que no son el objetivo de este herbicida, y por lo

tanto, los procesos del suelo que son mediados por los microorganismos. Aunque este

impacto debería estar relacionado con la respuesta de las malezas a la aplicación del

glifosato, poco es conocido sobre los cambios que toman lugar en la comunidad microbial

de la rizosfera de las malezas. Un experimento a nivel de campo y uno a nivel de

invernadero fueron conducidos con el fin de evaluar la influencia de dosis recomendadas

de glifosato en la comunidad de la rizosfera de malezas como mostaza, mostaza

tanaceto y trigo voluntario. En el experimento de invernadero el glifosato fue aplicado a

plantas de mostaza (Brassica juncea) y trigo voluntario (Triticum aestivum L. cv. AC

Lillian) a dos dosis recomendadas (450 g i.a. ha-1, 1800 g i.a. ha-1) y, su efecto sobre las

bacterias y hongos asociados a la rizosfera de estas plantas fue determinado a través de

conteos de placa realizados 1, 3 y 7 días después de la aplicación. El glifosato cambió el

tamaño de la comunidad microbial al incrementar los conteos rizobacteriales en una

manera dependiente de la dosis. Este efecto pudo ser directo ya que el glifosato puede

ser liberado desde las raíces a la rizosfera, o indirecto a través de los cambios

fisiológicos experimentados por las plantas que están muriendo como resultado de su

aplicación. En el experimento de campo, el suelo rizosférico fue colectado antes y

después de la aplicación de glifosato (450 g a.i. ha-1) desde plantas de trigo voluntario

(Triticum aestivum L. cv. AC Lillian) y mostaza tanaceto (Descurainia pinnate (Walter)

Britton) que crecieron en las fases de arveja y trigo de un sistema de rotación arveja-

trigo. Las comunidades microbiales fueron analizadas por conteos de placa basados en

características morfológicas de las colonias. Los morfotipos bacteriales fueron

identificados basados en la región 16S del ADNr. No hubo efectos detectables del

glifosato en la comunidad de bacterias u hongos de la rizosfera de mostaza tanaceto,

XI

pero influenció diferencialmente las comunidades rizobacteriales de los cultivos de trigo

voluntario que crecieron en entornos de residuos de arveja y trigo. El glifosato incrementó

la abundancia de Arthrobacter aurescens, un degradador de triazina-s, y decreció la

abundancia de cepas de Mesorhizobium loti y Variovorax paradoxus, potenciales

promotores del crecimiento de plantas. Se concluye que las aplicaciones de glifosato pre-

siembra pueden tener implicaciones ambientales y agronómicas no reveladas.

Palabras claves: comunidad microbial de la rizosfera, herbicida, malezas, sistema

de rotación, dosis de campo recomendadas, glifosato, trigo.

XII

List of figures

Figure 3-1: Effect of sampling time on the structure of the rhizosphere community of mustard and wheat. Vertical bars denote the standard error of the mean (n = 9). Means with different letters indicate significant differences within each microbial population according to permutational univariate analysis of variance (P ≤ 0.05). ............................ 19

Figure 3-2: Effect of plant species on the structure of the rhizosphere community at three sampling times. Vertical bars denote the standard error of the mean (n = 9). Means with different letters indicate significant differences within each microbial group according to permutational univariate analysis of variance (P ≤ 0.05). ................................................ 20

Figure 3-3: Effect of glyphosate dose on rhizosphere community structure. Vertical bars denote the standard error of the mean (n = 18). Means with different letters indicate significant differences within each microbial group according to permutational univariate analysis of variance (P ≤ 0.05). ...................................................................................... 21

Figure 4-1: Relative abundance (%) of the bacterial morphotypes contributing at least 70% of the differences in the rhizobacterial community structure from ‘volunteer’ wheat in durum wheat stubble as influenced by glyphosate use, according to SIMPER. Number are means ± standard error of the mean (n = 8). .................................................................. 33

Figure 4-2: Relative abundance (%) of the bacterial morphotypes contributing at least 70% of the differences in the rhizobacterial community structure from ‘volunteer’ wheat in pea stubble as influenced by glyphosate use, according to SIMPER. Number are means ± standard error of the mean (n = 8). ................................................................................. 34

Figure 4-3: Relative abundance (%) of fungal morphotypes contributing at least 70% of the differences in the rhizosphere of tansy mustard before and after glyphosate application, according to SIMPER. Numbers are means ± standard error of the mean (n = 8). ................................................................................................................................... 37

Figure 4-4: Relative abundance (%) of bacterial morphotypes contributing at least 70% of the difference in the structure of the rhizosbacterial community of tansy mustard found before and after glyphosate application in the pea stubble environment, according to SIMPER. Numbers are means ± standard error of the mean (n = 8). ............................. 38

XIII

List of tables

Table 2-1: Estimated areas, production and yield of different wheat crops in Canada for 2013. ................................................................................................................................ 4

Table 3-1: P-values of the effects of the factors plant species, sampling time, Glyphosate dose, and their interactions on the total numbers of bacteria and fungi of the rhizosphere, according to permutational multivariate analysis of variance (PerMANOVA). The analyses were conducted using 1000 permutations. ..................................................................... 19

Table 4-1: P-values of the effects of the factors rotation phase, glyphosate use, sampling time, and their interactions on bacterial and fungal community structure of wheat and tansy mustard rhizosphere, according to permutational multivariate analysis of variance (PerMANOVA). The analyses were conducted using 1000 permutations. ...................... 32

Table 4-2: Identity of the rhizobacterial morphotypes associated with wheat based on 16S rDNA sequence similarity, according to BLAST in NCBI. ................................................ 34

Table 4-2: (Continuation) ............................................................................................... 35

Table 4-3: Identity of the rhizobacterial morphotypes associated with tansy mustard based on 16S rDNA sequence similarity, according to BLAST in NCBI. ......................... 39

1

1 Introduction

Glyphosate is a highly effective and relatively inexpensive broad spectrum herbicide. The

genetic modification giving glyphosate resistance to major crop plants such as maize (Zea

mays L.), soybean (Glycine max [L.] Merr.), alfalfa (Medicago sativa L.) and canola

(Brassica napus L.) has greatly simplified weed control and contributed to make

glyphosate the most commonly used herbicide in agriculture worldwide (Mijangos et al.,

2009). It was first thought that glyphosate was environmentally safe (Duke and Powles,

2008), but many concerns have been raised recently. Non-target effects of glyphosate

might have been overlooked. Glyphosate has relatively low toxicity on mammals as they

do not possess the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that

is targetted by glyphosate (Lane et al., 2012b), and it has been suggested that when it is

used according to the manufacturer instructions, it should have no harmful effects on

human health (Williams et al., 2000). However, glyphosate can impact soil

microorganisms (Busse et al., 2001).

Soil microorganisms play a pivotal role in plant productivity (Neumann, 2007), and on the

maintenance of the chemical equilibrium of planet Earth, as they participate in the

biogeochemical cycles of carbon, nitrogen, sulfur, iron, and other elements (Madigan et

al., 2003, Sylvia, 2005). Several steps of nutrient cycling are performed exclusively by soil

microorganisms (Andrade, 2004). In many cases soil microorganisms are the unique

biological agents generating essential elements in forms usable by other organisms such

as the plants (Madigan et al., 2003). Thus, defining the impact of the widespread use of

glyphosate on the microorganisms inhabiting cropland is an important research task.

Several reports have shown that the enzyme EPSPS of many soil microorganisms can be

inactivated by glyphosate and that many soil microorganisms can be impacted by

glyphosate use. However, the results of most studies have little relevance since they were

2

conducted under artificial conditions and/or used unrealistically high rates of applications

(Roslycky, 1982, Wardle and Parkinson, 1990a, Haney et al., 2000, Busse et al., 2001,

Araújo et al., 2003, Ratcliff et al., 2006). Only a few studies have addressed the question

of the risk derived from glyphosate use in field situations (Busse et al., 2001, Means et al.,

2007, Weaver et al., 2007, Pereira et al., 2008, Hart et al., 2009, Kremer and Means,

2009, Lupwayi et al., 2009, Barriuso et al., 2010). Most of these studies suggest that

glyphosate applied at recommended field rates has no, or minimal and mainly transient

effects on the soil microbial community. Although the impact on soil microorganisms

should be at least partly mediated by the response of target weeds to glyphosate

application, these studies have been mainly focused on glyphosate-resistant plants and

little is known on the changes taking place in the rhizosphere community of weed plants

treated with glyphosate. It is necessary to understand the impact of realistic rates of

glyphosate application on the microbial community residing in the rhizosphere of weeds.

Thus, a greenhouse and a field experiment were undertaken to gain information on the

impact of glyphosate use. These experiments were designed to test the following

hypothesis:

1.1 Hypothesis

Pre-seeding glyphosate application at field recommended rates affects the microbial

community size residing in the rhizosphere of target weeds. Such effect would depend on

the rate of glyphosate used, the type of plant, and the time after herbicide application.

1.2 Objective

To document the influence of recommended field rates of glyphosate on the rhizosphere

microbial community of target weeds under controlled environment and field conditions, in

the context of wheat production.

3

2 Literature review

2.1 World wheat production

Globally, wheat is produced in a wide range of climatic, environmental and geographic

regions. It is the most widely cultivated food crop in the world. Its production is second

only to corn (USDA, 2014). Therefore, wheat is one of the most important crops for global

food security. This cereal is also important in human nutrition as direct consumption of

wheat contributes 20% and 22% of the calories and protein in the diet of humans,

respectively (Porter et al., 2007). In 2013, more than 218 million ha harvested produced

around 713 million tons of wheat with an average yield of around 3.3 tons ha-1 (FAO,

2013).

The regions of European Union, Southern Asia, Eastern Asia and Northern America are

the principal producers accounting for roughly 20.1%, 19.6%, 17.3% and 13.4% of the

total yield, respectively. Although European Union and Southern Asia have similar

production, the area used in wheat production in the European Union (EU) is much

smaller (approximately 26 vs 49 million ha, respectively) and thus, yields are two times

higher (FAO, 2013). China, India, United States of America and Russian Federation are

the principle producing countries (121.7, 93.5, 58 and 52.1 million tons, respectively)

contributing over 45% of the wheat produced in the world on 95.5 million ha (FAO, 2013).

Wheat can be classified as winter and spring type, based on the requirement of

vernalization. Durum wheat (Triticum durum or Triticum turgidum) accounts for

approximately 5% of global wheat production. Durum wheat is mainly produced in North

Africa and Western Asia (35%), North America (35%), and EU (30%) (He et al., 2013).

Almost all durum wheat grown in North America is spring-planted (spring type).

4

2.2 Wheat production in Canada

Wheat production in Canada is mostly located in the Prairie region that consists of the

provinces of Saskatchewan, Alberta, and Manitoba. These provinces produce

approximately 49%, 30% and 14% of the wheat produced in Canada, respectively.

Ontario has a small wheat production amounting to 6.4% of national production (Table 2-

1) (Statistics Canada, 2014). Spring wheat covers over 7.6 million ha and is the main type

of wheat produced accounting for 73% of Canadian wheat production, with durum wheat

accounting for 17%. Saskatchewan hosts 87% of the production of durum wheat. Winter

wheat production occupies around 0.8 million ha, mainly in Ontario where 60% of

Canadian winter wheat is produced.

Table 2-1: Estimated areas, production and yield of different wheat crops in Canada for

2013.

Geography Type of wheat crop Harvested area (million ha)

Production (million tons)

Yield (tons ha

-1)

Canada Wheat all 10.44 37.53 3.6

Durum wheat 2.00 6.50 3.3

Spring wheat 7.64 27.24 3.6

Winter wheat remaining 0.80 3.79 4.7

Ontario Wheat all 0.46 2.39 5.3



Prairie provinces Wheat all 9.88 34.76 3.5




Manitoba Wheat all 1.33 5.16 3.9

Durum wheat * * *



Saskatchewan Wheat all 5.68 18.30 3.2




Alberta Wheat all 2.87 11.30 3.9




*Not available. Statistics Canada (2014)

5

2.2.1 Production systems

Management practices such as crop rotation and tillage must be adapted to regional

conditions of soil and climate. For example, in the prairie region, soils are practically never

tilled because the region is characterized by low levels of precipitation and no-till

technology is used most of the time for seeding as a soil moisture conservation practice.

No-till or zero-till minimizes soil disturbance to a minimum. This protects the soil from

erosion while conserving soil moisture. Tillage, on the other hand, buries plant residues

and mixes topsoil leaving most of the soil surface bare. Thus, spring wheat is direct-

seeded in the stubble of the previous crop in the prairie province, while in Ontario,

conventional tillage system is preferred. Under subhumid climates, tillage helps soil

warming and drying in spring, allowing earlier seeding. Wheat-based rotation systems

include canola, pulse crops (eg. pea or lentil) and other cereal crops (eg. barley or oats).

Pulse crops are not dependent on N fertilization as they have the ability to form symbiotic

association with N2-fixing bacteria (Knight, 2012) and their inclusion in rotation with wheat

can improve the yield of wheat and substitute at least in part, the N fertilizer inputs (Soon

and Lupwayi, 2008, Danga et al., 2009) it is a sustainable alternative to wheat

monoculture. In central and Atlantic Canada, spring wheat is grown as a rotational crop in

a variety of cropping systems including small grain-oilseed rotations, forage-based

rotations, corn-soybean rotations and potato/vegetable-based rotations (AAFC, 2010a).

Although spring wheat tolerate a wide range of conditions, it grows best on well-drained

soils (AAFC, 2010a).

On the prairies, winter wheat is also grown in crop rotations involving canola, and other

spring cereal crops. Again, no-till seeder is normally used in the Prairie Provinces, but

outside of this region, winter wheat is predominantly grown under conventional tillage

systems. In central Canada, this crop is grown in corn-soybean-winter wheat rotations,

and in Atlantic Canada it is grown as a rotational crop in a variety of cropping systems

including small grain-oilseed rotations, forage-based rotations and potato/vegetable-

based rotations (AAFC, 2010b).

Winter wheat fields are usually left fallow after harvest until the following spring, but in

some cases the producers use cover crops immediately after the winter wheat harvest as

a soil conservation practice. Winter wheat prefers well-drained soils and can be

6

established in a wide range of soil moisture conditions, including very dry conditions

(AAFC, 2010b). Under subhumid climate wheat grain quality is reduced by the presence

of toxin from Fusarium head blight, and the wheat is mainly grown for straw and for the

soil structuring effect of the plant.

2.2.2 Weed management

Crop yield can be negatively affected by several factors including the presence of weeds

competing with the crop for available light, nutrients and moisture, and thus weeds may

be the greatest single cause of economic loss in absence of chemical weed control

(Alberta Ministry of Agriculture and Rural Development, 2011). In the case of wheat,

losses due to weed pressure are mainly attributable to a decrease in tiller production.

Annual and perennial grasses, annual and perennial broadleaf and volunteer crops are

the types of the weeds found in wheat fields of western Canada. Some of them can cause

particularly important economic losses. Downy brome (Bromus tectorum) and japanese

brome (Bromus japonicus), green foxtail (Setaria viridis), wild oats (Avena fatua), wild

mustard (Sinapsis arvensis and Brassica kaber), stinkweed (Thlaspi arvense), flixweed

(Descurainlia sophin), shepherd’s purse (Capsella bursa-pastoris), lamb’s-quarters

(Chenopodium album), quackgrass (Elytrigia repens), Canada thistle (Cirsium arvense),

sow thistle (Sonchus arvensis) are among the most noxious weed species in western

Canada (AAFC, 2010a, AAFC, 2010b, Alberta Ministry of Agriculture and Rural

Development, 2011).

It is important to note that volunteer crops (e.g: volunteer wheat, volunteer canola), which

grow from seeds that fell on the ground at harvest or through shattering losses, can affect

the quality of the next harvest. Volunteers act as a “green bridge” allowing diseases to

persist through the rotation phases of cropping systems (AAFC, 2010a) in addition to

competing with the crop for moisture and nutrients, as do weeds, and their control is

necessary.

An efficient weed control not only depends on the implementation of chemical practices,

but also requires the use of cultural practices. Integrated weed management is the most

successful alternative in weed control. Cultural practices such as crop rotation, banding

7

fertilizers, and tillage contribute to weed control (AAFC, 2010a, AAFC, 2010b). Crop

rotation can reduce weed pressures in creating variations in planting or maturation dates,

growth habit, competitive ability, associated cultural practices, and fertility requirements of

the crop in different phases of the rotation (Liebman and Dyck, 1993). Such variations

create conditions that may disrupt the growth and development of weedy species at

certain points in time of the rotation cycle. Additionally, crop rotation can aid in

establishing a rotation of herbicide groups preventing the development of herbicide

resistant weeds (AAFC, 2010a, AAFC, 2010b). It has been shown that soil tillage systems

influence the size, profile distribution and species density of weed seedbank (Grundy,

2003). Thus, tillage practices are a factor deserving consideration in the elaboration of a

weed control strategy. These practices change the weed seed depth in soil and then

affect the abundance of the weeds as much of the weed seedlings cannot emerge if

seeds are buried deeply (Mishra and Singh, 2012). On the other hand, the tillage also

modifies growth factors (temperature, moisture, aeration, nutrients) which affect weed

infestation (El Titi, 2003). Conservation tillage (minimum-tillage and no-till) has become

common practice in western Canada (Arshad et al., 2002, Zentner et al., 2002), and it has

caused an increase in the problems with perennial weeds as green foxtail (Setaria viridis)

and foxtail barley (Hordeum jabatum), which can be easily controlled with tillage practices

(AAFC, 2010a, AAFC, 2010b). Weed control is mainly chemical in the prairie, increasing a

dependence on herbicide use.

There is a wide variety of herbicides registered for weed management in Canadian wheat

crops, but the number of herbicide groups is limited, and in the case of weeds such as

downy brome (Bromus tectorum) and japanese brome (Bromus japonicus) there are few

herbicides available (AAFC, 2010a, AAFC, 2010b). There are herbicides that can be

applied in-crop, pre-seeding or after seeding but pre-emergence, pre-harvest and post-

harvest (AAFC 2010a, AAFC, 2010b). Pre-seeding and pre-emergence herbicide

treatments are very important as early control can reduce yield losses, and since no in-

crop chemical control may exist for some important perennial weeds. In this last case, it is

recommended choose fields with low weed population and do the control in the year

previous to wheat production. Glyphosate used alone or in combination with others

herbicide (2,4-D, Bromoxynil, Bromoxynil/MCPA, dicamba, Heat, MCPA, tribenuron) is the

pre-seeding and pre-emergence herbicide most used in to control weeds in wheat

(Saskatchewan Ministry of Agriculture, 2010). Special care must be taken how frequently

8

the same herbicide or group of herbicide is used as resistance to glyphosate has been

reported in several weeds including volunteer crops (AAFC, 2010a, AAFC, 2010b, Alberta

Ministry of Agriculture and Rural Development, 2011).

2.3 Glyphosate and its non-target effects on microorganisms

Currently, glyphosate [N-(phosphonomethyl)glycine] is the non-selective systemic

herbicide most commonly used in agricultural practices on a global scale (Mijangos et al.,

2009). Its herbicidal activity is based on inactivation of the enzyme 5-enolpyruvoyl-

shikimate-3-phosphate synthase (EPSPS) in the shikimate metabolic pathway, by

chelating Mn, a cofactor for this enzyme, and preventing the synthesis of aromatic amino

acids needed for plant growth and survival (Franz et al., 1997, Duke, 2005, Johal and

Huber, 2009). This pathway is absent in animals (Gomez et al., 2009) and thus,

glyphosate has been thought to have low toxicity to mammals, including humans (Duke

and Powles, 2008, Lane et al., 2012b).

Once glyphosate is absorbed by foliage, it is transported throughout the whole plant via

the phloem to growing tissues such as the meristems of shoots and roots. This

mechanism results in glyphosate being exuded into rhizosphere soil (Neumann et al.,

2006, Laitinen et al., 2007). In the soil, glyphosate can be inactivated or removed in

different ways. Glyphosate can be inactivated by strong and rapid adsorption to clay

mineral, soil oxides and hydroxides, and soil organic matter, limiting the movement and

leaching of the chemical (Veiga et al., 2001, Vereecken, 2005, Duke and Powles, 2008,

Coupe et al., 2012). Glyphosate can also be degraded by microorganisms (Anderson et

al., 1993, Haney et al., 2000), uptaken by plant roots (Laitinen et al., 2007) or leached into

groundwater (Vereecken, 2005). Sorption rapidly inactivates glyphosate, but increases its

persistence in soil (Veiga et al., 2001). Reports in the literature have shown that the half-

life of glyphosate in soil varies from a few days up to several months, and in some cases

its persistence has been much greater, even years (Veiga et al., 2001, Andréa et al.,

2003).

9

Glyphosate belongs to the group of synthetic organophosphonates characterized by the

presence of a covalent bond between carbon and phosphorus (C-P) (Ternan et al., 1998).

The C-P linkage is chemically and thermally very stable and therefore, this herbicide

presents a high resistance to chemical hydrolysis, thermal decomposition and photolysis

compared with analogous compounds (Ternan et al., 1998). Therefore, the main route of

removal of glyphosate in soil is microbial degradation (Krzyśko-Lupicka et al., 1997,

Shushkova et al., 2009). Two microbial degradation pathways of glyphosate have been

suggested (Pipke and Amrhein, 1988b, Dick and Quinn, 1995, Ternan et al., 1998). In the

first one, the C-N bond is cleaved with the formation of aminomethylphosphonic acid

(AMPA) in which the C-P bond is still conserved. In the second one, the C-P bond is

cleaved producing sarcosine and inorganic phosphate.

The rapid sorption and microbial degradation of glyphosate in soil are reasons why this

herbicide has been considered environmentally safe. However, environmental concerns

about its persistence in soil and non-target effects on soil microorganisms are increasing.

2.3.1 Effect of glyphosate on soil microorganisms

The impacts of glyphosate on soil microorganisms are sometimes considered marginal,

but despite the rapid sorption and microbial degradation in soil, several studies have

demonstrated that glyphosate impacts the soil microbial community. The EPSPS enzyme,

which is ubiquitous in plants, is also present in microorganisms, which are key

components in the process of nutrient cycling and soil fertility (Andréa et al., 2003, Powell

et al., 2009). Microbial EPSP enzymes have variations in sensitivity to glyphosate and

while certain soil microbial species with the glyphosate-resistant version of the enzyme

can gain a competitive advantage in metabolizing glyphosate (Schulz et al., 1985, Kremer

and Means, 2009), the microorganisms with a glyphosate-sensitive version of the enzyme

are negatively affected by the herbicide (Busse et al., 2001).

Conflicting results have been obtained from the numerous studies that evaluated the

effect of glyphosate on soil microbial communities. Glyphosate has been reported to

stimulate microbial respiration, total bacteria, total fungi, culturable bacteria, functional

diversity, and microbial activity (Roslycky, 1982, Wardle and Parkinson, 1990b, Wardle

and Parkinson, 1990a, Haney et al., 2000, Busse et al., 2001, Andréa et al., 2003, Araújo

10

et al., 2003, Ratcliff et al., 2006). However, these effects were usually detected when

glyphosate was applied at concentrations higher than those recommended for normal use

in the field. The stimulation of microorganisms by glyphosate was often attributed to the

use of the herbicide as a substrate for microbial metabolism and growth. Some authors

(Ratcliff et al., 2006, Weaver et al., 2007, Lane et al., 2012b) observe no change in

microbial community structure as a result of glyphosate application at recommended field

rates or in some cases, even at applications greater than recommended field rates

(Weaver et al., 2007).

Chakravarty and Chatarpaul (1990) found deleterious effects of glyphosate on the soil

microbial community. They reported that both fungal and bacterial populations shrank

after glyphosate application and that high concentrations strongly suppressed the growth

of some species of ectomycorrhizal fungi. There is consistent evidence of strong toxicity

of glyphosate on certain culturable bacteria and fungi (Busse et al., 2001). However,

glyphosate appears to select for fungal species able to use the herbicide as a substrate

for metabolism (Krzyśko-Łupicka and Orlik, 1997, Krzysko-Lupicka and Sudol, 2008).

It is difficult to draw conclusions from the numerous studies that have evaluated the effect

of glyphosate on soil microbial communities. Conflicting results obtained from these

studies can often be attributed to the use of methodological approaches irrelevant to field

conditions, such as the use of very high dose of glyphosate, and direct application to soil

rather to target plants. However, the glyphosate effect on microorganism appears to be

dose-dependent and transitory.

2.3.2 Effect of glyphosate on rhizosphere microorganisms

The rhizosphere is that narrow zone of soil directly surrounding the roots that is influenced

by roots exudates (Walker et al., 2003). These exudates include diverse compounds

acting as chemical attractants for a great number of soil microbial communities, which are

actively metabolizing the molecules released by plant roots (Walker et al., 2003).

Rhizobacteria are a group of bacteria adapted to life in the root environment (Schroth and

Hancock, 1982). Rhizobacteria are more versatile in transforming, mobilizing and

solubilizing nutrients than the bacteria living in bulk soil (Hayat et al., 2010). Thus, the

rhizobacteria are a major driving forces in the process of nutrient recycling in the

11

agroecosystems and thus, they are a very important determinant of soil fertility (Andréa et

al., 2003, Powell et al., 2009, Ahemad and Kibret, 2013a). Rhizobacteria can have

positive, as well as negative or neutral effects on plant growth, and depending of their

effect, they can be called deleterious rhizobacteria (DRB) or plant growth promoting

rhizobacteria (PGPR) (Nehl et al., 1997). The PGPR have raised much interest because

of their potential to increase crop yield, whereas the DRB have received less attention.

Some examples of PGPR are: Arthrobacter, Agrobacterium, Azospirillum, Bacillus,

Flavobacterium, Pseudomonas, Mesorhizobium, Bradyrhizobium, Rhizobium (Ahemad

and Kibret, 2013a). Rhizobacteria can promote plant growth through a variety of direct

mechanisms (promoting nutrient uptake, regulating plant hormone levels) or indierect

(acting as biocontrol agents) (Ahemad and Kibret, 2013b).

Glyphosate released into the rhizosphere changes the environment by the influx of

carbon, phosphorus and nitrogen from the herbicide itself and by the introduction of more

vegetative from the plants killed as a consequence of the post-emergent treatment (Hart

et al., 2009). It has been demonstrated that the glyphosate can impact the rhizosphere

community (Kremer and Means, 2009, Sheng et al., 2012) and, by extension, rhizosphere

processes (Ahemad and Kibret, 2013a). Most of the studies on the impact of glyphosate

on rhizosphere microorganisms have evaluated the effect of pre-seeding and in-crop use

of glyphosate on GR crops. In a study on the effect of the use of in-crop glyphosate on

GR maize, Barriuso et al. (2010) found that glyphosate changed the rhizobacterial

community, and particularly impacted the Actinobacteria. Pre-seeding glyphosate use was

shown to stimulate the growth of bacteria associated with non-GR crops and this effect

was modulated by the rotation system. The bacteria were increased in the rhizosphere of

pea, but no effect was detected in the rhizobacterial community of durum wheat or in

saprotrophic fungi (Sheng et al., 2012). In that study, Sheng et al. (2012) also reported

that glyphosate use modified the overall structure of the rhizosphere microbial community.

Likewise, the structure of the community of culturable bacteria associated with GR maize

or glyphosate-sensitive plants has changed as a result of glyphosate application

(Mijangos et al., 2009, Barriuso et al., 2011).

Conversely, glyphosate had no effect on microbial community structure in GR soybean,

according to fatty acid methyl esters (FAMEs) profiling, even when the herbicide was

applied at rates exceeding recommendations (Weaver et al., 2007). These results agree

12

with those reported by Hart et al. (2009), who found that the structure of denitrifying

bacteria or fungi in the rhizosphere of GR maize treated with glyphosate were not

different from that found when another herbicide was applied. These results were

consistent with results for non-GR maize. On the other hand, glyphosate applied to

glyphosate-sensitive plants had a stimulatory effect on the activity of the culturable soil

microbial community, 15 days after application (Mijangos et al., 2009). Similarly, Means et

al. (2007) found an increase in soil respiration and enzyme activity after in-crop

glyphosate application on GR soybean.

Certain studies have demonstrated that when glyphosate was applied at recommended

field doses, there was no effect on mycorrhizal or rhizobial symbioses in GR crops as

soybean, cotton and maize (Powell et al., 2009, Savin et al., 2009). In contrast, a negative

effect of glyphosate on rhizobial colonization was reported by Reddy and Zablotowicz

(2003). The differences were attributed to variability in glyphosate tolerance among GR

crop varieties and to the adjuvants and surfactants which differ among glyphosate

formulations and that may also cause toxic effects on the soil biota (Powell et al., 2009).

Other negative effects were reported by Zobiole et al. (2011) in GR soybean. In that study

it was found that glyphosate reduced fluorescent pseudomonads, Mn-reducing bacteria,

and indoleacetic acid-producing bacteria in the rhizosphere. Kremer and Means (2009),

Johal and Huber (2009) and Zobiole et al. (2011) report that glyphosate application

enhanced the colonization of roots by pathogenic fungi, which can have serious

consequences on the production of several crops.

2.4 Effect of tillage and crop rotation on the microbial community in wheat rhizosphere

Agricultural management practices such as tillage, fertilization, and cropping systems

influence soil microbial communities and processes through several mechanisms which

include: changes in the quantity and quality of plant residues returning to soil, fertilizer

inputs and physical changes (Christensen, 1996). Traditional management systems

involving conventional tillage decrease the level of soil organic matter, which influences

soil biological properties (Caravaca et al., 2002, Roldán et al., 2003). No-till can favor the

microbial activity and crop development by mulching the soil surface with crop residues,

13

which protect microorganisms from abrupt changes of temperature and moisture

(Andrade et al., 2003, Roldán et al., 2003).

While many studies have evaluated the effect of agricultural management practices on

the bulk soil microbial community, few studies have been focused on investigate the

microbial community associated to crop rhizosphere.

Yang et al. (2013) reported that the tillage system influences the microbial community of

winter wheat rhizophere, in a maize-winter wheat-soybean rotation system. No-till

stimulated the microbial biomass carbon and the total microbial community activity

compared to conventional tillage. Zero tillage increased the utilization of most carbon

sources by the rhizosphere microbial community compared to conventional tillage

management (Yang et al., 2013). Similarly, Lupwayi et al. (2010) found that tillage

influenced the soil microbial biomass, functional microbial diversity, and rhizobacterial

community structure in wheat growing after canola. Microbial biomass carbon was higher

under no-till than under conventional tillage whereas functional diversity decreased under

no-till. Similar results were reported by Lupwayi et al. (2007) in wheat rhizosphere under

rotation systems including different GR crop frequencies. They reported a greater

functional diversity in the wheat rhizosphere under conventional tillage than under low

soil-disturbance direct-seeding and the microbial biomass was greatest in wheat

rhizosphere under conventional tillage. Enzymatic activity was higher under low soil-

disturbance direct-seeding than under conventional tillage. Interestingly, it was also found

that the microbial biomass in wheat rhizosphere increases with GR crop frequency, with

negative impacts on enzymatic activity and modification of the soil bacterial community

structure (Lupwayi et al., 2007). Sheng et al. (2012) found a glyphosate-induced increase

in the abundance of rhizobacteria in pea but not in durum wheat, while no effect on fungal

biomass was observed. These effects were mitigated by tillage, perhaps by diluting in

bulk soil the glyphosate exuded in the rhizosphere of treated plants.

14

3 Dynamics of rhizosphere communities after glyphosate application to mustard and wheat

3.1 Abstract

Results obtained from several studies suggest that the widely used herbicide glyphosate

can has negative side effects on soil microbial community (non-target organisms) as well

as in soil processes mediated by microorganisms. There is limited information about the

impact of this herbicide on the rhizosphere of the sensitive target-weeds. I evaluated the

effects of recommended doses of glyphosate on rhizosphere community dynamics in a

microcosm experiment involving mustard (Brassica juncea) and wheat (Triticum aestivum

L.) under controlled conditions. Three doses of glyphosate, 0, 450, 1800 g ai ha-1 were

applied on the leaves of wheat and mustard to study the effect of glyphosate use on the

structure of the rhizosphere community. Rhizosphere soil samples were taken 24 h, 3 d

and 7 d after glyphosate application and the abundance of fungi and bacteria in

rhizosphere soil was determined by plate count. Glyphosate increased the number of

bacterial CFU per g of rhizosphere soil which rose about five times when the dose applied

changed from 450 g ha-1 to 1800 g ha-1. This effect could be direct as the glyphosate can

be released by roots into the rhizosphere or indirect through physiological changes

experienced by dying plants after glyphosate application. We conclude that glyphosate

applied at recommended doses change the microbial community structure of the

rhizosphere in a dose-dependent manner, and the bacteria are particularly influenced.

Apparently, this taxonomic group exhibits a greater responsiveness to changes in

rhizodeposition than fungi.

3.2 Introduction

Glyphosate [N-(phosphonomethyl)glycine] is the active ingredient of herbicide products

commercially sold as Roundup, Rodeo, and Glyphomax, among other brands.

15

Nowadays, it is the non-selective systemic herbicide most commonly used for agriculture,

on a global scale. Although this compound exhibits desirable qualities such as highly

effective control of a wide variety of competitive vegetation, rapid inactivation in soil, and

low mammalian toxicity (Andréa et al., 2003, Lane et al., 2012b), side-effects of

glyphosate on non-target organisms as soil microorganism have raised environmental

concern (Busse et al., 2001, Ratcliff et al., 2006, Gomez et al., 2009).

Glyphosate inhibits the enzymatic activity of 5-enolpyruvoyl-shikimate-3-phosphate

synthase (EPSPS) in the shikimate pathway, thus preventing the synthesis of aromatic

amino acids needed for plant growth and survival (Franz et al., 1997, Duke, 2005). Since

most living organisms, including animals, do not have this pathway, the risk of non-target

effect of glyphosate on humans is low (Gomez et al., 2009). Although, EPSPS is

characteristic of plants, this enzyme is also important in microorganisms, which play a key

role in nutrient cycling and soil fertility (Andréa et al., 2003, Powell et al., 2009). Variation

exists in the sensitivity of microbial EPSPS, and glyphosate use gives a competitive

advantage to glyphosate-resistant microbial species in the soil system (Schulz et al.,

1985, Kremer and Means, 2009).

The impacts of glyphosate on soil microorganisms is sometimes considered marginal, but

despite the rapid sorption (Duke and Powles, 2008, Lane et al., 2012b) and microbial

degradation of glyphosate in soil (Anderson et al., 1993, Haney et al., 2000), there is

evidence of glyphosate impacts in the rhizosphere microbial community (Kremer and

Means, 2009, Sheng et al., 2012) and, by extension, the rhizosphere processes. It is

difficult to draw conclusions from the numerous studies that have evaluated the effect of

glyphosate on rhizosphere microbial communities (Wardle and Parkinson, 1990b, Wardle

and Parkinson, 1992, Hart and Brookes, 1996, Haney et al., 2000, Busse et al., 2001,

Andréa et al., 2003, Ratcliff et al., 2006, Weaver et al., 2007, Hart et al., 2009, Kremer

and Means, 2009, Mijangos et al., 2009, Lane et al., 2012b). The conflicting results

obtained from these studies can often be attributed to the use of methodological

approaches irrelevant to field conditions, such as the use of very high dose of glyphosate,

direct application to soil rather to target plants.

Although the impacts of the recommended field doses of glyphosate on soil

microorganisms have been examined in short- and long-term studies (Wardle and

16

Parkinson, 1990b, Olson and Lindwall, 1991, Wardle and Parkinson, 1992, Haney et al.,

2000, Lupwayi et al., 2004, Ratcliff et al., 2006, Weaver et al., 2007), little is known on the

effects of field doses of this herbicide on the microorganisms of the rhizosphere. It is

possible that the glyphosate application affects the structure of the microbial community of

the rhizosphere. Such effect would depend on the rate of glyphosate used, the type of

plant, and the time after herbicide application.

This study defined the effects of recommended doses of glyphosate on rhizosphere

community dynamics in a microcosm experiment involving mustard (Brassica juncea) and

wheat (Triticum aestivum L.) under controlled conditions. Oriental mustard was used as

the broadleaf weed because there are a large number of weeds within mustard family

(Brassicaceae) causing problems in crop production (Callihan et al., 2000, Entz et al.,

2001).

3.3 Materials and methods

3.3.1 Soil and experimental design

The experiment had a complete randomized design with a 2 x 3 x 3 factorial arrangement

of treatments. Each treatment had three replicates. Thus, a total number of 54 pots were

used in this study. Treatments consisted in the factorial combinations of (i) two plant

species: mustard and wheat; (ii) three levels of glyphosate application: 0, 450, and 1800 g

of active ingredient ha-1; and (iii) three sampling times: 24 h, 3 d and 7 d after glyphosate

application. In order to simulate an agricultural situation, both doses of glyphosate

application are within the recommended range of field application rates suggested by the

manufacturer (Roundup WeatherMax®; Monsanto, St Louis, MO).

The soil was collected from the top layer (0-30 cm) of a field regularly cropped to cereals,

which was located 27 km northwest of Swift Current (latitude 50° 24’ N; longitude 108° 04’

W) (Saskatchewan, Canada). It was chosen because of its light texture (87% sand, 5.5%

silt, and 7.2% clay). The soil was classified as a Brown Chernozem (Mollisol). After

pasteurization at 80°C for 3 h, this soil had a loamy sand texture, a pH of 6.5, and an

electrical conductivity 0.48 mS cm-1; it contained 19.7 mg NH4-N kg-1 and 14.1 mg NO3-N

17

kg-1 extractible by KCl (Maynard and Karla, 1993), 21.3 mg P kg-1 and 324.5 mg K kg-1

extractible by sodium bicarbonate (Olsen et al., 1954), and 5.7 g organic C kg-1 and 0.8 g

total N kg-1, as determined on a CNS analyzer (Vario Microcube, Elementar Co., Hanau,

Germany). Once pasteurized, the soil was stored in an open bin for two years until being

used in this study. The dry soil was sieved through 2 mm and transferred into plastic pots

(535 g dry-weight per pot).

Four seeds of either wheat cv. “AC Lillian” or oriental mustard cv. “Cutlass” were planted

in each pot. One week later, the plants were thinned to two plants per pot. Plants were

grown in a growth chamber of the Semiarid Prairie Agricultural Research Centre of

Agriculture and Agri-Food Canada in Swift Current. Plants were subjected to a 14-h

photoperiod, a day/night temperature regime of 20°C/10°C, and were watered as

required. Wheat and mustard were used to simulate a field condition. These test plants

represent grassy and broadleaf weeds, respectively.

3.3.2 Glyphosate application

Three weeks after seeding, a glyphosate solution (540 g L-1) formulated as Roundup-

Weathermax was applied on the plants, which then had 3-4 leaves. A hand operated

spray boom was used to deliver the prescribed amount of glyphosate in order to

reproduce the coverage received by field plants. An experienced and licensed pesticide

applicator operated the equipment to ensure appropriate application. Control plants were

sprayed with water instead of glyphosate; this application corresponded to the rate 0 g ha-

1.

3.3.3 Sampling rhizosphere and counting of microorganisms

Sampling was made 24 h, 3 d and 7 d after glyphosate application by gently brushing off

the rhizosphere soil adhering to roots into polyethylene bags. Then, rhizosphere samples

were stored at 4°C and analyzed within one week. The microbial community structure of

the rhizosphere was described here by the counts of two taxonomic groups, bacteria and

fungi. For this purpose, dilution series up to 10-6 were made using 1 g of soil and a saline

solution (0.85 % NaCl). The first dilution was mixed for 5 min on a reciprocating shaker

operating at 280 oscillations per minute. All other dilutions were shaken vigorously by

hand for 10 seconds in a reciprocating fashion. For bacterial counts 25 µL of the 10-4, 10-

18

5, and 10-6 dilutions were transferred onto a nutrient-agar medium. For fungi, the same

amount of the 10-2, 10-3, and 10-4 dilutions were transferred on a starch-casein-nitrate-

agar medium with rose bengal. Details on the composition of the media used are shown

in Annex A. All dishes were incubated at 28°C and observed daily. Colony forming units

(CFU) were enumerated after 3, and 6 days for bacteria and fungi, respectively.

3.3.4 Data analysis

Normality test (Shapiro-Wilk) using the function shapiro.test of the package “stats” in R

version 2.14.0 (R Development Core Team, 2011) showed that the residuals for all

response variables were not normally distributed, even after applying several types of

transformation. Non-parametric analyses of variance were applied to test the significance

of treatment effects on bacteria and fungi revealed by plate counts. The main effects of

plant species, glyphosate levels, sampling times, and their interactive effects were tested

by PerMANOVA (Permutational multivariate analysis of variance) (Anderson, 2001a)

using the function Adonis of the package “vegan” (Oksanen et al., 2011) in R version

2.14.0 (R Development Core Team, 2011). This test was applied to each taxonomic group

separately (Anderson, 2001b). Two missing values were replaced with the mean of other

replicates (Ruxton and Colegrave, 2011). A new PerMANOVA was conducted to do

individual pair-wise comparisons between particular groups when treatment effects were

detected (Anderson, 2001a). For each PerMANOVA, Bray-Curtis method was used to

calculate pairwise distances.

3.4 Results

The plants that received glyphosate exhibited symptoms only after three days. The plant

tissues showed chlorosis 3 days and necrosis 7 days after glyphosate application. The

symptoms were more intense at the highest dose of glyphosate (1800 g ha-1) and mustard

plants appeared most affected by the herbicide.

The bacterial communities of wheat and mustard rhizosphere were dynamic and varied

with sampling time, as shown by a significant plant x time interaction (Table 3-1, Annex

B). An effect of this interaction was also detected on the fungal community.

19

Table 3-1: P-values of the effects of the factors plant species, sampling time, Glyphosate

dose, and their interactions on the total numbers of bacteria and fungi of the rhizosphere,

according to permutational multivariate analysis of variance (PerMANOVA). The analyses

were conducted using 1000 permutations.

Source of effect Bacteria Fungi

----------- Pr(>F) -------

Plant 0.002 ** 0.505

Time 0.001 *** 0.260

Glyphosate 0.014 * 0.160

Plant x Time 0.024 * 0.012 *

Plant x glyphosate 0.329 0.283

Time x Glyphosate 0.074 0.191

Plant x Time x Glyphosate 0.539 0.438

*** indicates significance at α = 0.001 level; ** at α = 0.01; * at α = 0.05

In mustard, rhizosphere bacteria were most abundant at day-7 (80.9x106 CFU per g),

when the number of CFU was 5.1 and 10.8 folds that found 24 h and 3 days after

glyphosate application, respectively (Figure 3-1). In wheat rhizosphere, the abundance of

bacteria was 41.6x106 CFU per g at 24 h, decreased on day-3 and then increased at day-

7 as compared to day-3. In the case of the fungi, the differences were only detected in the

rhizosphere of mustard, where the abundance of bacteria was 10.1x104 CFU per g at

day-7, reaching counts 1.6 and 1.3-fold higher than the counts obtained 24 h and 3 days

after glyphosate application.

Figure 3-1: Effect of sampling time on the structure of the rhizosphere community of

mustard and wheat. Vertical bars denote the standard error of the mean (n = 9). Means

with different letters indicate significant differences within each microbial population

according to permutational univariate analysis of variance (P ≤ 0.05).

ba

b a

bb

b a

aa

a a

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

Mustard Wheat Mustard Wheat

Bacteria Fungi

Log

CFU

g-1

dry

so

il

24 h 3 d 7 d

20

The initial number of bacteria in the rhizosphere of wheat (107.62) was 2.6 times higher

than that of mustard (107.20), and at day-3 and day-7 these differences disappeared

(Figure 3-2). On the other hand, the initial count of fungi decreased from 84x103 CFU per

g of wheat rhizosphere soil to 63x103 CFU per g of mustard rhizosphere soil. Conversely,

at day-7 the number of fungi was 1.3 times higher in the mustard rhizosphere than in that

of wheat. There was no effect of the plant species on fungal abundance at day-3.

Figure 3-2: Effect of plant species on the structure of the rhizosphere community at three

sampling times. Vertical bars denote the standard error of the mean (n = 9). Means with

different letters indicate significant differences within each microbial group according to

permutational univariate analysis of variance (P ≤ 0.05).

The dose of glyphosate influenced the abundance of rhizosphere bacteria (Table 3-1).

Thus, when the dose applied increased from 450 g ha-1 to 1800 g ha-1, the number of

bacteria CFU per g of rhizosphere soil rose about five times (Figure 3-3).

aa

a

a a b

ba

a

b a a

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

24 h 3 d 7 d 24 h 3 d 7 d

Bacteria Fungi

Log

CFU

g-1

dry

so

il

Wheat Mustard

21

Figure 3-3: Effect of glyphosate dose on rhizosphere community structure. Vertical bars

denote the standard error of the mean (n = 18). Means with different letters indicate

significant differences within each microbial group according to permutational univariate

analysis of variance (P ≤ 0.05).

3.5 Discussion

The results of this study indicate that rhizosphere community dynamics can be affected by

the application of recommended doses of the herbicide glyphosate on weeds. However,

when the herbicide is used within the spectrum of recommended doses the effect of

glyphosate on the size of rhizosphere communities appears to be relatively small. Our

observation concurs with earlier studies showing that low doses of glyphosate applied to

soil had little or no effect on soil microorganisms. Doses at the high end of the range of

recommended doses of glyphosate needed to be applied to trigger detectable effects on

soil rhizosphere microorganisms. A few studies found that the application of glyphosate to

soil had a stimulatory effect on bacteria and fungi, but these effects were all dose

dependent (Roslycky, 1982, Wardle and Parkinson, 1990a, Ratcliff et al., 2006).

Glyphosate in this study was sprayed onto plants and its effects on rhizosphere microbial

community would be mediated by the plant. The glyphosate applied on the plants is first

absorbed foliarly, translocated via the phloem to growing regions such as meristems of

roots, and then exuded into rhizosphere soil (Laitinen et al., 2007). Subsequently, the

glyphosate can be degraded by resident microorganisms (Kremer and Means, 2009).

abb

a

a a a

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

Gly 0 Gly 450 Gly 1800 Gly 0 Gly 450 Gly 1800

Bacteria Fungi

Log

CFU

g-1

dry

so

il

22

The highest glyphosate dose killed the plants and concurrently increased the abundance

of rhizobacteria. This increase was often attributed to the use of the herbicide as a

substrate for bacterial metabolism and growth (Ghisalba et al., 1987, Wardle and

Parkinson, 1990b, Haney et al., 2000, Ratcliff et al., 2006, Gomez et al., 2009). In this

study, it seems to be that bacteria were more responsive than fungi to changes in

rhizodeposition. Our results agree with the generalized concept that bacteria are favored

when labile substrates (glyphosate and cellular material, both release through cellular

disruption during herbicide-induced plant death) are available, whereas fungi are favored

when complex substrates dominate soil C (Ratcliff et al., 2006). In addition, fungi could

have been negatively impacted by glyphosate treatments more severely than bacteria and

thus less able to use the nutrient flush induced by plant death. Sheng et al. (2012)

suggested that glyphosate-tolerant enzyme 5-enolpyruvylshikamate-3-phophate synthase

(EPSPS) may be more common in soil bacteria than in soil fungi. However, this has not

been confirmed.

The rise in the abundance of bacteria observed seven days after glyphosate application in

both plant species may have three explanations. First, it is well known that damaged root

cells from dying plants can release glyphosate (Neumann et al., 2006) as well as cell

material, generating in this way nutritional resources for microbial growth (Wardle et al.,

1994). Increase in bacterial abundance could also be attributable to the selection by

glyphosate of bacterial populations capable of using the herbicide as a source of P, C or

N (Ermakova et al., 2008, Kremer and Means, 2009). A direct stimulation of bacterial

growth by glyphosate application has often been reported (Araújo et al., 2003, Ratcliff et

al., 2006, Zabaloy et al., 2008). Third, the stimulation of microbial growth may be

associated with shifts in the quality of rhizodeposition, which may favor certain microbial

groups (Keith et al., 1986, Steer and Harris, 2000). Since glyphosate application had no

effect on the abundance of fungi in rhizosphere soil, the increased nutrient release by

dying plants is the likely explanation for the increase over time of fungal biomass that was

observed in the rhizosphere of mustard plants (Figure 3-1).

Negative impacts of glyphosate on rhizosphere microorganisms were also reported

(Kremer et al., 2005, Kremer and Means, 2009, Zobiole et al., 2011, Lane et al., 2012a).

Certain bacteria are unable to tolerate glyphosate, which inhibits their enzyme EPSPS

and their ability to synthesize essential aromatic amino acids (Schulz et al., 1985, Franz

23

et al., 1997, Busse et al., 2001, Feng et al., 2005). In this study, a declining trend in the

abundance of rhizobacteria was observed between 24-h and three days after glyphosate

application when the herbicide was applied at 450 g ha-1. This may be the result of the

death of glyphosate-susceptible bacteria. Likely these dead cells became an additional

source of nutrients to new generation of bacteria (Roslycky, 1982), which may also

contribute to the rise in bacterial abundance between day-3 and day-7.

The differential effects of plant species on the structure of the soil microbial community

have been attributed to differences in plant root exudation patterns. Different amounts and

types of carbon compounds released from plants into the rhizosphere trigger specific

response in associated microbial communities (Grayston et al., 1998, Kowalchuk et al.,

2002, Motavalli et al., 2004, Garbeva et al., 2008, Hossain et al., 2010, Ladygina and

Hedlund, 2010). Plant exudation patterns are altered by glyphosate (Kremer et al., 2005,

Means et al., 2007). A possible consequence of the alteration of rhizobacterial

communities is the modification of the soil processes mediated by microorganisms.

Microbial processes may be beneficial (eg.: phytohormone production, fixation nitrogen,

nutrients solubilization such as P, promotion of mycorrhizal function, glyphosate

degradation) or deleterious (eg.: pathogenicity, N2O emissions) to agroecosystem

function (Nehl et al., 1997, Sturz and Christie, 2003, Ahemad and Kibret, 2013a).

Identification of the specific soil processes that may be modified as a result of increased

bacterial population size will requires examining functional gene expression, enzyme

activity, crop productivity and other indicators of soil function, concurrently with the

microbial community at the species level.

3.6 Conclusion

The results of the present work showed that spraying glyphosate on plants at

recommended doses changed the microbial community structure of their rhizosphere in a

dose-dependent manner. These effects could be direct as the glyphosate can be released

into the rhizosphere or indirect through physiological changes experienced by dying

plants after its application. Bacterial abundance was particularly influenced, seemingly

due to the greater responsiveness of this taxonomic group than fungi to changes in

rhizodeposition.

24

It appears that all bacteria are not similarly affected or resistant to the effects of

recommended doses of glyphosate. Glyphosate likely has specific impacts (eg.: to

population level) on both bacteria and fungi, but they are not revealed by measures of

abundance done at a broad taxonomic scale. This study show that it is essential to

develop more detailed approaches that consider soil microbial communities at the

functional level and with a finer taxonomic precision.

25

4 Glyphosate-mediated changes in the microbial communities inhabiting the rhizosphere of field-grown wheat and tansy mustard

4.1 Abstract

Numerous reports show that the structure of the microbial community in the rhizosphere

of crop plants can be altered by pre-seeding application of glyphosate. Although this

impact should be at least partly mediated by the response of weed plants to glyphosate

application, little is known on the changes taking place in the rhizosphere community of

weed plants treated with glyphosate in spring. We evaluated in 2011 the effect of

glyphosate on the microbial community in the rhizosphere of field-grown tansy mustard

(Descurainia pinnate (Walter) Britton) and wheat (Triticum aestivum L. cv. AC Lillian)

seedlings used as simulated grassy weeds and volunteer wheat. Rhizosphere soil was

collected three days after glyphosate application from plants growing in soil previously

planted with pea or wheat, in the plots of a long-term experiment established in 2006, in

southwest Saskatchewan, Canada. The microbial communities were analyzed by plate

counts based on morphology and bacterial morphotypes were identified based on 16S

rDNA. Glyphosate triggered no detectable effects on the rhizobacterial community of

tansy mustard and on fungi. Glyphosate influenced the rhizobacterial communities of

wheat grown in pea or wheat stubble differently. Glyphosate increased the abundance of

the known s-triazine decomposer Arthrobacter aurescens, and decreased the abundance

of potentially plant-growth-promoting Mesorhizobium loti and Variovorax paradoxus

strains. We conclude that pre-seeding applications of glyphosate may have previously

undisclosed agronomic and environmental implications.

26

4.2 Introduction

Glyphosate is the herbicide most commonly used in agricultural fields (Mijangos et al.,

2009). The success of glyphosate has been attributed to several reasons including its low

cost, effectiveness in weed control (Cerdeira and Duke, 2006), rapid inactivation in the

soil (Vereecken, 2005) and low mammalian toxicity (Duke and Powles, 2008, Lane et al.,

2012b). Glyphosate acts by blocking the biosynthesis of aromatic amino acids in the

plants through the inactivation of the enzyme 5-enolpyruvoyl-shikimate-3-phosphate

synthase (EPSPS) in the shikimate metabolic pathway (Franz et al., 1997, Duke, 2005,

Johal and Huber, 2009). Most living organisms do not have this pathway but it is

ubiquitous in microorganisms, which contribute importantly to the processes of nutrient

cycling and soil fertility (Andréa et al., 2003, Powell et al., 2009). Thus, the environmental

safety of glyphosate is being questioned. The effects of glyphosate on non-target

microorganisms should be defined and understood.

Numerous reports show that pre-seeding or in-crop applications of glyphosate positively

or negatively affect the soil microbial community of the rhizosphere of crop plants (Kremer

and Means, 2009, Mijangos et al., 2009, Barriuso et al., 2010, Barriuso et al., 2011).

Glyphosate influences microbial biomass and abundance, the activity and structure of

microbial communities, and the level of root colonization by symbionts and pathogens. It

was also found that cropping practices can modify the response of rhizosphere

microorganisms to glyphosate use (Sheng et al., 2012). Other studies have shown

contrasting results in which the microbial community of the rhizosphere and the

mycorrhizal or rhizobial symbioses were unaffected by glyphosate (Weaver et al., 2007,

Hart et al., 2009).

Although the impacts of glyphosate should be at least partly attributed to the response of

weed plants to glyphosate application, little is known on the changes taking place in the

rhizosphere community of weed plants treated with glyphosate. We tested in the field the

effect of pre-seeding glyphosate “burnoff” application on the bacteria and fungi inhabiting

the rhizosphere of tansy mustard (Descurainia pinnate (Walter) Britton) and simulated

volunteer wheat (Triticum aestivum L.) in the pea and durum wheat phase of a pea-durum

wheat rotation.

27

4.3 Materials and methods

4.3.1 Field site and experimental design

The experimental site was located in Swift Current, Saskatchewan, Canada (latitude:

50°17’N; longitude: 107°41′W; elevation: 825 m) in 20 m x 10 m wide plots. Briefly, the

soil was classified as a very gently sloping (1%) Brown Chernozem (Mollisol) of the Wood

Mountain soil association with a clay loam texture and a columnar prismatic to solod

profile. Prior to establishment of the experiment in the spring of 2006, the top soil layer

(0-15 cm) had (per kg of soil) 2.40 mg NO3-N and 9.88 mg PO4-P extractable by KCl

(Maynard and Karla, 1993) and sodium bicarbonate (Olsen et al., 1954), respectively.

We used a subset of 16 plots of a long-term field experiment established in 2006 to

conduct this study in 2011. The cropping system established in these research plots was

a pea (Pisum sativum L.) – durum wheat (Triticum turgidum L. var durum) rotation under

no-till management, with both phases of the rotation present each year. The four

treatments used were the factorial combinations of (i) rotation phase, which had two

levels i.e. durum wheat (cv. Strongfield) and pea (cv. Eclipse), and (ii) two glyphosate

levels: with and without. The experiment had a split-split-plot design in a randomized

complete block design. The rotation phase levels were assigned to main plots, and the

glyphosate levels were assigned to subplots. Experimental treatments were replicated in

four blocks. The rhizosphere of tansy mustard and wheat was sampled at two times:

before glyphosate application, to test the long-term effect of glyphosate use, and three

days later, to test the short-term effect of glyphosate. Thus, sampling time was included

in the design as a repeated measures component and treated as a split-split-plot factor.

The use of glyphosate as a pre-seeding treatment was the focus of this study, so spring

wheat was seeded early in the growing season to simulate grassy weeds and volunteer

wheat. In 2011, all plots were seeded on 7 May with a low rate of hard red spring wheat

cv. “AC Lillian” to simulate volunteer wheat. The tansy mustard plants included in this

study grew naturally in the plots. Glyphosate formulated as Roundup-Weathermax™ was

applied in the designated plots at the recommended dose of 450 g a.i. ha-1 on June 6,

when the plants were at the 3-4 leaf stage.

28

Each year, prior to seeding, all plots have received a mixture of 500 g glufosinate

ammonium ha-1 formulated as Liberty, 30 g clethodim ha-1 formulated as Select (Bayer

CropScience), with the adjuvant Amigo (Bayer CropScience) (0.5%, v/v) to remove the

standing vegetation from all plots. After the second herbicide application, the plots were

seeded. Crops were managed according to best management practices. The site and

the agronomic practices used to maintain the plots were described earlier (Sheng et al.,

2012).

4.3.2 Rhizosphere sampling and microbial counts

Samples were taken on 6 June, just before glyphosate application, and 9 June 2011, prior

to the second, non-glyphosate, pre-seeding herbicide application. Rhizosphere soil from

three plants of tansy mustard and wheat was collected by gently brushing off the soil

adhering to roots and passing through a 2mm sieve prior to storage in polyethylene bags.

A total of 32 samples were taken for each species and stored at 4°C until processing.

Both the bacterial and fungal communities were analyzed by plate count considering

different morphotypes and the number of individuals per morphotype. Differences in the

macroscopic appearance of the microorganisms in a growth medium were used to identify

the morphotypes (e.g.: configuration, margin, elevation). Bacterial morphotypes were

counted on nutrient-agar medium, and a starch-casein-nitrate-agar medium amended with

rose bengal was used to count fungal morphotypes. Details on the serial dilutions and

culture medium used can be found in the materials and methods section of Chapter 3.

4.3.3 Polymerase Chain Reaction (PCR) and sequencing

To identify the bacterial morphotypes, single bacterial colonies were picked from each

morphotype growing on isolation spread plates, and then transferred onto a nutrient-agar

medium. After the plates were incubated for 3 days at 28°C, the colonies from pure

culture were taken with the end of a bacteriological loop and immersed into MicroAmp

Fast Reaction Tubes (Applied Biosystems) containing a PCR mixture.

DNA from the colonies was subjected to PCR using the 16S rDNA-targeting primers 968f

/ 1401r to amplify an approximately 450 bp fragment. The mixture for PCR with a final

elution volume of 27 µl was prepared with 22.5µl of Platinum® PCR SuperMix (Cat. No.

http://www.bayercropscience.ca/English/LabelMSDS/273/File.ashx



29

11306-016, InvitrogenTM), 0.3 µl of each primer and 3.9 µl of ultrapure water. PCR was

conducted on a VeritiTM 96-well fast Thermal Cycler (Applied Biosystems) using the

following conditions: initial denaturation at 94 °C for 6 min followed by 30 cycles at 94 °C

for 45 s, annealing at 56 °C for 45 s and elongation at 72 °C for 1 min. Finally, the

samples had a 10 min period at 72° C before being held at 4 °C. PCR success was

evaluated using electrophoresis. Aliquots of 5 µl of PCR products were mixed with 2 µl of

Trackit Loading Buffer (InvitrogenTM) and loaded on a 0.8% agarose gel containing 14 µl

of SybrSafe nucleic acid stain (InvitrogenTM) at 80V, 65 mA current for 40 min. Clear

bands in the correct size range compared to a DNA ladder indicated successful

amplification. The concentration of the PCR product of each colony type was measured

using the Qubit system (InvitrogenTM). Samples with concentrations of 5 ng µl-1 were then

submitted for sequencing.

Sanger sequencing was conducted at the Plant Biotechnology Institute (National

Research Council, Saskatoon, SK). The sequences obtained were aligned using the

ClustalW Multiple Alignment tool of BioEdit software. Comparisons of the similarity of the

partial 16S rDNA sequences were performed using the National Centre for Biotechnology

Information online standard BLAST (Basic Local Alignment Search Tool) program

(http://www.ncbi.nlm.nih.gov/), and those sequences matching with an identity of 97% or

greater were used to identify the morphotypes.

The fungi inhabiting the rhizosphere of tansy mustard and ‘volunteer’ wheat were not

isolated and identified.

4.3.4 Data analysis

The significance of the rotation phase, long-term glyphosate use, immediate effect of

glyphosate application revealed by sampling time and the interactive effects of these

factors on the structure of the bacterial and fungal communities of tansy mustard and

wheat rhizosphere were evaluated by PerMANOVA (Permutational multivariate analysis

of variance) (Anderson, 2001b). In order to conduct this analysis in a split-split-plot

design, two centroid matrices based on Bray-Curtis distances were constructed, one for

the main-plot factor (grouping by block and rotation phase) and a second one for the split-

plot factor (grouping by block, rotation phase and glyphosate). These two reduced

http://www.ncbi.nlm.nih.gov/

30

matrices were considered as the dataset to run a PerMANOVA to evaluate the main plot

factor using the first matrix, and another PerMANOVA was run to evaluate the subplot

factor using the second matrix. In this second analysis, a new variable containing all block

by rotation phase combinations was included. The new variable and block effects

removed all the whole-plot variation from the residual error term. The F test for block and

the new variable is not interesting and was disregarded. To test the immediate effect of

glyphosate, i.e. sampling time, the original dataset was used in a third PerMANOVA in

which a new variable accounting for all block, rotation phase, and long-term glyphosate

use combinations was considered. The effects of interest evaluated with the residual error

term of this last analysis were sampling time, sampling time x rotation phase, time x

glyphosate use and sampling time x rotation phase x glyphosate use.

When significant interactions between the factors were detected by PerMANOVA, a new

PerMANOVA was employed to do individual pair-wise comparisons and identify the factor

combinations responsible for those differences (Anderson, 2001a).

To identify the morphotypes differentiating the rhizophere communities established under

different treatments, and to evaluate the contribution of these morphotypes to the

treatment effects, we conducted a similarity percentage test (SIMPER) based on the

decomposition of Bray-Curtis dissimilarity index (Clarke, 1993). Briefly, SIMPER allows

you to rank the contributions of morphotypes to the differences between two groups or

treatments. The results from SIMPER give the average abundance for each morphotype

relative to the total morphotype count for each treatment (relative abundance), as well as

the contribultion of each morphotype to the differences between treatments (contribution

to overall dissimilarity). Indicator Species Analysis (ISA) (Dufrêne and Legendre, 1997)

was also used to identify the most prominent morphotypes under one group as compared

to another. These morphotypes were identified with the indicator value ‘IndVal index’ and

the significance of this index was assessed by using 1000 permutations. The IndVal index

combines a species mean abundance and its frequency of occurrence in the groups. A

high indicator value is obtained by a combination of large mean abundance within a group

compared to the other groups (specificity) and presence in most sites of that group

(fidelity). IndVal is calculated by multiplying the relative frequency by the relative

abundance.

31

The bacterial and fungal communities of tansy mustard and those of ‘volunteer’ wheat

plants were analyzed separately in R version 3.0.2 (R Development Core Team, 2013).

The Betadisper, Adonis and SIMPER functions of the package “vegan” were employed to

create the centroid matrices, to run the PerMANOVA and conducted SIMPER,

respectively. The indicator species analysis was performed using the function Indval of

the “labdsv” package (Roberts, 2010). The Hellinger transformation was applied to the

data prior to analysis as prescribed by Legendre and Gallagher (2001).

4.4 Results

Microbial community of ‘volunteer’ wheat rhizosphere

Glyphosate use changed the structure of the bacterial community in ‘volunteer’ wheat

rhizosphere, but no immediate effects of glyphosate were detected on the fungal

community, three days after application of the herbicide (Table 4-1, Annex C). The effect

of glyphosate varied with the rotation phase, as shown by a significant glyphosate x

rotation phase interaction. According to Pair-wise PerMANOVA, the bacterial rhizosphere

communities of ‘volunteer’ wheat growing in both, pea and durum wheat stubbles, were

influenced by glyphosate use (P = 0.006 and P = 0.006, respectively).

The results from SIMPER revealed that 17 morphotypes contributed at least 70 % of the

dissimilarity in the structure of rhizobacterial community between glyphosate-treated and

non-treated plots in durum wheat stubbles. Of these, six morphotypes were more

abundant in plots receiving glyphosate than glyphosate-free plots, while seven

morphotypes decreased in abundance (Figure 4-1). Morphotypes B46 and B50, identified

as Pseudomonas and Flavobacterium (Table 4-2) were only present in plots receiving

glyphosate, while morphotypes B5 and B34, identified as Rhodococcus fascians and

Bacillus megaterium, were absent in these plots.

32

Table 4-1: P-values of the effects of the factors rotation phase, glyphosate use, sampling

time, and their interactions on bacterial and fungal community structure of wheat and

tansy mustard rhizosphere, according to permutational multivariate analysis of variance

(PerMANOVA). The analyses were conducted using 1000 permutations.

Source of effect Df Bacteria Fungi Bacteria Fungi

Wheat rhizosphere ---------- Pr(>F) --------

Tansy mustard rhizosphere ------------- Pr(>F) ----------

Block 3 0.393 0.852 0.649 0.231

Rotation phase 1 0.393 0.852 0.649 0.231

Residual (A) 3

Glyphosate use 1 0.001 *** 0.532 0.347 0.386

Glyphosate use x Rotation phase

1 0.001 *** 0.373 0.182 0.949

Residual (B) 6

Sampling time 1 0.189 0.838 0.430 0.004 **

Sampling time x Rotation phase

1 0.351 0.250 0.038 * 0.962

Sampling time x Glyphosate use

1 0.782 0.180 0.865 0.420

Sampling time x Rotation phase x Glyphosate use

1 0.522 0.808 0.766 0.066

Residual (C) 12

Total 31


Indicator Species Analysis (ISA) identified the morphotype B11, Mesorhizobium loti, as

the most prominent member of the rhizosphere community in absence of glyphosate

(IndVal = 64 %, P = 0.005), while morphotype B8, Arthrobacter aurensens, was identified

as the morphotype most selected by glyphosate use (IndVal = 57 %, P = 0.008).

33

Figure 4-1: Relative abundance (%) of the bacterial morphotypes contributing at least

70% of the differences in the rhizobacterial community structure from ‘volunteer’ wheat in

durum wheat stubble as influenced by glyphosate use, according to SIMPER. Number are

means ± standard error of the mean (n = 8).

Twelve morphotypes contributed at least 70 % of the dissimilarity between the structure of

rhizobacterial community in glyphosate-treated and non-treated plots in pea stubbles. The

application of glyphosate increased the abundance of four of these morphotypes and

decreased the abundance of eight (Figure 4-2). The morphotypes B44 and B7 were

identified by ISA as the morphotypes most closely related to the absence or use of

glyphosate, respectively (IndVal = 82 %, P = 0.003; IndVal = 68 %, P = 0.003). B44 was

identified as a Bacillus sp and B7 was identified as an Arthrobacter ramosus (Table 4-2).

34

Figure 4-2: Relative abundance (%) of the bacterial morphotypes contributing at least

70% of the differences in the rhizobacterial community structure from ‘volunteer’ wheat in

pea stubble as influenced by glyphosate use, according to SIMPER. Number are means ±

standard error of the mean (n = 8).

Table 4-2: Identity of the rhizobacterial morphotypes associated with wheat based on 16S

rDNA sequence similarity, according to BLAST in NCBI.

Morphotype Description from BLAST Accession Ident (%)

E-value

B1 Bacillus sp. PG-2010-18 FR746082.1 99 2.E-96

B2 NS* B3 Variovorax paradoxus strain 7bCT5 JN627864.1 100 1.E-101

B4 Bacillus muralis strain BCHCNZ314 GU188931.1 93 1.E-107

B5 Rhodococcus fascians strain KK25 JN638062.1 100 5.E-148

B6 Bacillus vallismortis BIT-33 EF646150.1 86 6.E-27

B7 Arthrobacter ramosus AB648984.1 100 5.E-148

35

Table 4-3: (Continuation)

B8 Arthrobacter aurescens TC1 strain TC1 NR_074272.1 100 5.E-148

B10 Aeromicrobium sp. PDD-24b-9 HQ256801.1 100 5.E-148

B11 Mesorhizobium loti MAFF303099 strain MAFF303099 NR_074162.1 100 2.E-146

B12 Arthrobacter sp. Ellin106 AF408948.1 100 5.E-148

B14 Pseudomonas sp. T28 EU111709.1 100 1.E-147

B15 Paenibacillus taohuashanense strain gs65 JQ694712.1 100 5.E-148

B16 Flavobacterium sp. AP5s2-M1ld KF561875.1 100 2.E-147

B17 NS* B19 Bacillus sp. 459-1 FN298326.1 99 8.E-100

B20 Bacillus sp. R-26223 HE586353.1 98 7.E-95

B21 Arthrobacter sp. TN222 JN800351.1 99 2.E-146

B23 Bacillus simplex strain Q1-S5 JX994099.1 100 2.E-146

B24 Bacillus simplex strain JP44SK32 JX144722.1 96 1.E-123

B25 Massilia namucuonensis strain 333-1-0411 JF799985.1 100 1.E-143

B26 Pseudomonas fluorescens strain Nfb4SF JN699420.1 100 2.E-95

B27 Bacillus horneckiae AB723495.1 100 2.E-146

B28 Pseudomonas kuykendallii strain H2 JF749828.1 100 2.E-147

B29 Bacillus sp. 5080 KC236645.1 100 2.E-146

B30 NS* B31 NS* B33 Rhodococcus sp. EG2 KF571869.1 100 5.E-148

B34 Bacillus megaterium strain Q2-R13 JX994130.1 100 2.E-146

B35 Clavibacter michiganensis subsp. Nebraskensis AM410697.1 100 5.E-148

B36 Pedobacter roseus strain CL-GP80 NR_043555.1 100 5.E-148

B37 Bacillus simplex strain NBP1 HQ256542.1 100 2.E-146

B39 Arthrobacter sp. TY33McD HQ406735.1 100 5.E-148

B40 Variovorax sp. 12S KC795991.1 100 8.E-146

B41 NS* B42 Bacillus simplex strain CCMM B622 JN208087.1 99 5.E-153

B43 Bacillus megaterium WSH-002 plasmid WSH-002_p1 CP003018.1 100 2.E-146

B44 Bacillus sp. LLR27 JF682089.1 99 2.E-96

B45 Methylobacterium sp. PK29_S1 JF274801.1 99 4.E-97

B46 Flavobacterium sp. T16F.07.C.CHS.SRW.W.Kidney.D JX287799.1 99 1.E-97

B47 NS* B48 Paenibacillus sp. IMP09 FR727721.1 99 3.E-98

B49 NS* B50 Pseudomonas sp. OK1_1_1b3_s5 JF274725.1 100 7.E-47

B52 Chryseobacterium piperi strain CTM NR_108294.1 99 7.E-100

B53 Pseudomonas sp. Nfb2AC JN699288.1 100 2.E-95

B55 Bacillus sp. UE-2011-A16 HE578130.1 98 7.E-95

B57 Arthrobacter sp. PL20g1b_S1 JF274911.1 100 5.E-148

B58 Paenibacillus sp. DJM2B5 JF753496.1 99 7.E-100

B59 Paenibacillus sp. M1-61ª DQ180952.1 89 2.E-102

B61 Microbacterium sp. PX16a_S2 JF274931.1 99 9.E-99

B62 Bacillus sp. RW-1 DQ869045.1 99 1.E-97

B63 Bacillus thuringiensis strain TSAD KF008237.1 99 4.E-97

B64 Lysobacter antibioticus FR822760.1 99 3.E-98

B67 Bacillus sp. Ult-816 GU733396.1 100 2.E-146

*Morphotypes not sequenced

Eliminado: 2

36

Microbial community of tansy mustard rhizosphere

No long-term effect of glyphosate use on the structure of the bacterial or fungal

communities inhabiting the rhizosphere of tansy mustard was detected. However,

glyphosate modified the structure of both communities within three days of application

(Table 4-1, Annex D).

Six fungal morphotypes contributed at least 70 % of the differences observed in the fungal

community structure before and after glyphosate application. Of these, only two

morphotypes decreased in abundance three days after glyphosate application (Figure 4-

3). An ISA revealed that before glyphosate application, the most prominent member of the

fungal community was the morphotype F4 (IndVal = 67 %, P = 0.001). However, after

glyphosate application, no morphotypes were closely related to this ecological condition,

and although some of them had high indicator values (> 30 %), these were not significant

at α = 0.05.

37

Figure 4-3: Relative abundance (%) of fungal morphotypes contributing at least 70% of

the differences in the rhizosphere of tansy mustard before and after glyphosate

application, according to SIMPER. Numbers are means ± standard error of the mean (n =

8).

The immediate effect of glyphosate application on tansy mustard rhizobacterial

community varied with the phase of the rotation, as indicated by a significant sampling

time x rotation phase interaction (Table 4-1, Annex D). This interaction reveals that the

structure of tansy mustard rhizobacterial community was affected by the sampling time

only in a pea stubble environment. This effect was only marginally significant, according

to Pair-wise PerMANOVA (P = 0.062). The SIMPER analyses identified 13 fungal

morphotypes contributing at least 70 % of the dissimilarity between the structure of tansy

mustard rhizosphere community before and after glyphosate application in pea stubble.

Of these, four morphotypes increased in abundance after glyphosate application and five

38

decreased in abundance (Figure 4-4). Three morphotypes were only found after

glyphosate application, whereas one morphotype had disappeared within three days of

glyphosate application. Before glyphosate application, the rhizobacterial community was

dominated (IndVal = 63 %) by the morphotype B4, an Arthrobacter nitroguajacolicus, while

B30, an Arthrobacter sp (Table 4-3), dominated (IndVal = 52 %) after glyphosate

application. However, these rhizobacteria only had marginally significant indicator values

(P = 0.068 and P = 0.062, respectively).

Figure 4-4: Relative abundance (%) of bacterial morphotypes contributing at least 70% of

the difference in the structure of the rhizosbacterial community of tansy mustard found

before and after glyphosate application in the pea stubble environment, according to

SIMPER. Numbers are means ± standard error of the mean (n = 8).

39

Table 4-4: Identity of the rhizobacterial morphotypes associated with tansy mustard

based on 16S rDNA sequence similarity, according to BLAST in NCBI.

Morphotype Description from BLAST Accession Ident (%) E-value

B1 Arthrobacter sp. SJCon Gq927310.2 100 4.E-148

B2 Paenibacillus agaridevorans R-42 FR682747.1 100 1.E-147

B3 Rhodococcs sp. BR2 JN196542.1 100 4.E-148

B4 Arthr. nitroguajacolicus BLN18 GQ181046.1 100 4.E-148

B5 Arthrobacter sp. SUK 1205 JQ312666.1 99 2.E-100

B6 Uncultured bacterium 1112842459949

HQ119549.1 100 1.E-147

B7 Pedobacter sp. Tb2-14-II AY599662.1 100 4.E-148

B8 Flavobacterium sp. R-41499 FR772077.1 99 6.E-146

B9 Uncult. Bacterium 07-164 JF820148.1 100 6.E-146

B10 Pseudomonas sp. Cantas8 JN609537.1 100 1.E-147

B11 Uncult. Bacterium 111286561613a HQ120587.1 100 1.E-147

B13 Pseudomonas brenneri KOPRI 25585

HQ824860.1 100 1.E-147

B14 Mycetocola sp. PX8c_S1 JF274939.1 99 2.E-100

B16 Agrobacterium sp. H13-3 CP002248.1 100 2.E-126

B17 Bacillus sp. PG-2010-18 FR746082.1 100 2.E-126

B20 Rhodococcus qingshengii KOPRI 25555

HQ824843.1 99 4.E-97

B21 Uncult. Bacterium PBH(21) HQ342141.1 100 1.E-147

B22 Mesorhizobium loti r6 HQ424937.1 99 1.E-142

B23 Uncult. Bacterium 1112863845235a HQ121194.1 100 2.E-146

B26 Rhodococcus sp. EG2 KF571869.1 100 5.E-148

B28 Aeromicrobium alkaiterrae KSL-107 NR_043207.1 100 4.E-148

B29 Uncult. Bacterium EH-11035 HM117744.1 100 2.E-146

B30 Arthrobacter sp. SAZ1-3 HQ236024.1 100 4.E-148

B31 Pseudomonas sp. SAP49_1 JN872540.1 100 1.E-147

B35 Pedobacter sp. MCC-Z JF279930.2 99 1.E-96

B36 Okibacterium sp. Asd M5-11B FM955871.1 81 3.E-54

B40 Strenotrophomonas rhizophila PCA_13

JF711015.1 100 4.E-148

B43 Bacillus simplex CCMM B622 JN208087.1 100 8.E-155

B44 Flavobacterium sp. L-111-12 FJ786049.1 99 7.E-151

B45 Arthrobacter sp. TY33McD HQ406735.1 99 2.E-151

B47 Arthrobacter sp. R-37013 FR691391.1 99 2.E-151

B48 Rhodococcus sp. BF1 3332 JN807761.1 99 9.E-155

B49 Sphingomonas aerolata R36940 FR691420.1 99 7.E-151

Morphotype identification

Tables 4-2 and 4-3 show the identities to the nearest BLAST match for bacterial

morphotypes isolated from the rhizosphere of wheat and tansy mustard during this study.

Some bacterial morphotypes could not be sequenced because their isolation into pure

culture failed, but they were included in all the statistic analyses.

Eliminado: 3

40

4.5 Discussion

The results show that glyphosate used at the recommended field dose can modify the

structure of the microbial community in the rhizosphere of a weed plant growing in pea

and wheat stubbles, in early spring of the Canadian prairie. This effect of glyphosate use

was detected in the bacterial community of wheat rhizosphere and in both, the bacterial

and fungal communities of tansy mustard rhizosphere.

This study concurs with other studies that revealed changes in the structure of the

culturable rhizobacterial community (Mijangos et al., 2009, Barriuso et al., 2011) as well

as in the whole rhizobacterial community (Barriuso et al., 2010, Barriuso and Mellado,

2012b) induced by glyphosate use. Barriuso and Mellado (2012a) also found shifts in the

rhizobacterial communities of the glyphosate-resistant cotton GHB614, but concluded that

glyphosate use has little effect on the rhizobacterial community structure in this

genetically modified plant. My results indicate on the contrary that glyphosate use may

have important effects. The effects found here on wheat rhizobacteria was not due to

plant death or any other immediate effect of glyphosate application, but rather to the long-

term influence of glyphosate use on the soil environment. After six years of glyphosate

use, the rhizobacterial community had changed compared to the control treatment. The

effect of glyphosate use on the soil environment was probably a direct effect of

glyphosate on soil microorganisms in this experiment, because weeds were well

controlled in all, glyphosate treated and untreated plots, with a blanket application of

Liberty link.

The glyphosate foliarly applied to target plants can be translocated throughout the plant

and released into the rhizosphere by root tissues (Neumann et al., 2006, Laitinen et al.,

2007), where it can influence the composition of the microbial community (Kremer et al.,

2005). The ability to use the glyphosate as a substrate for metabolism and growth is an

explanation often proposed to explain the increase in abundance of certain bacteria

sometimes seen in response to glyphosate use (Ghisalba et al., 1987, Wardle and

Parkinson, 1990b, Haney et al., 2000, Ratcliff et al., 2006, Gomez et al., 2009). In this

study, no significant shift in microbial community structure occurred within three days of

glyphosate application on ‘volunteer’ wheat, but in the rhizosphere of tansy mustard, a

structural shift in the fungal community was apparent, indicating an influence of the

41

physiology of the target weed species in the development of the immediate effects of

glyphosate application.

Certain rhizobacteria associated with ‘volunteer wheat’ were found to have higher

populations with glyphosate use. In the wheat rhizosphere, Arthrobacter aurescens strain

TC1 and Bacillus sp. 459-1 were more abundant in glyphosate treated plots in both

rotation phases. These results are consistent with the report that several species of

Bacillus can metabolize glyphosate and use it as a source of carbon or phosphorus

(Quinn et al., 1989, Fan et al., 2012). Although Arthrobacter aurescens strain TC1 does

not contain genes or pathways for the catabolism of glyphosate (Mongodin et al., 2006),

this bacteria possesses the enzymes amine flavoprotein dehydrogenases and oxidases,

which are thought to metabolize sarcosine, an intermediary product of glyphosate

degradation (Meskys et al., 2001, Shapir et al., 2007).

The increases of Bacillus sp. and A. aurescens associated with glyphosate use on

‘volunteer’ wheat may be beneficial to crop production. Bacillus species have been

reported to promote plant growth (Akhtar et al., 2012, Verma et al., 2013). In addition, A.

aurescens strain TC1 has a high potential for the bioremediation of agricultural soils

contaminated with s-triazine herbicides (Strong et al., 2002).

Glyphosate positively impacts some rhizobacterial species, but negatively impacts others,

as shown here and elsewhere (Kremer et al., 2005, Kremer and Means, 2009, Zobiole et

al., 2011, Lane et al., 2012a). Certain bacteria are unable to tolerate glyphosate, because

it inhibits their enzyme 5-enolpyruvylshikamate-3-phophate synthase (EPSPS) and thus

their ability to synthesize essential aromatic amino acids, which prevent their growth

(Schulz et al., 1985, Franz et al., 1997, Busse et al., 2001, Feng et al., 2005).

In our study, the abundance of the potentially beneficial Mesorhizobium loti MAFF303099,

Arthrobacter sp. Ellin106 and Variovorax paradoxus strain 7bCT5 were consistently

decreased in the rhizosphere of wheat growing in plots managed with glyphosate.

Previous studies conducted to evaluate the effect of glyphosate on several strains of

Mesorhizobium show that this herbicide can reduce the growth of some strains,

suggesting that glyphosate may adversely affect the process of symbiotic nitrogen-fixation

(Zabaloy and Gómez, 2005, Vercellino and Gómez, 2013). Species of Arthrobacter were

42

reported to metabolize glyphosate (Pipke et al., 1987, Pipke and Amrhein, 1988a).

Variations in response to glyphosate use appear to exist in Arthrobacter. We found that

the abundance of Arthrobacter sp. Ellin106 was negatively impacted by this herbicide, but

most of the Arthrobacter of ‘volunteer’ wheat and tansy mustard rhizosphere were more

abundant in plots receiving glyphosate.

Glyphosate use may also adversely impact the agroecosystem by inhibiting Variovorax

paradoxus. This rhizobacteria has frequently been associated with important

biodegradative processes in nature including the degradation of several pollutants

(Franzetti et al., 2012). Variovorax paradoxus has also been reported to be involved in

plant growth promotion (Satola et al., 2013). Insufficient information prevents the

assessment of possible variation in response to glyphosate among V. paradoxus strains.

Interestingly, Flavobacterium sp. T16F.07.C.CHS.SRW.W.Kidney.D and Pseudomonas

sp. OK1_1_1b3_s5 were only present in the rhizosphere of wheat growing after a durum

wheat crop where the herbicide was added, while Bacillus megaterium strain Q2-R13 and

Rhodococcus fascians strain KK25 disappeared. This is not surprising as some

Pseudomonas species possess a glyphosate-resistant EPSPS and are not inhibited by

glyphosate use (Schulz et al., 1985). It was also demonstrated that Pseudomonas sp.

strain PG2982 can use glyphosate as a nutrient source (Moore et al., 1983). Likewise,

Flavobacterium sp. was reported to degrade glyphosate (Balthazor and Hallas, 1986).

The reduction of the abundance of the potential phytopathogen Rhodococcus fascians, a

phytopathogenic bacterial species (Lechevalier, 1986), could be a positive effect of

glyphosphate use.

Some of the rhizobacteria which disappeared in response to glyphosate application in this

study were found to tolerate and even utilize glyphosate as P source in earlier studies

(Quinn et al., 1989, Al-Arfaj et al., 2013). This shows there may be some differences in

the bacterial strains between studies or possibly in the experimental conditions that may

lead to inconsistent results.

Other wheat rhizobacteria that were common to both rotation phases were not

consistently affected by glyphosate, and while some benefited from glyphosate in a

rotation phase, their abundance was decreased in the other. Clearly, pea and wheat

43

stubbles are different environments. The different conditions of the soil environment

under pea and wheat stubbles may modify the response of microorganisms to glyphosate.

For example, monovalent cations such as potassium and ammonium ions are known to

reduce the tolerance to glyphosate of certain bacterial EPSPS enzymes (Priestman et al.,

2005). Differential fertilization programs and different patterns of nutrient uptake by wheat

and pea may have modified the chemical environment of the soil and influenced the

competitive ability of the bacteria inhabiting the pea and wheat stubbles environments.

The absence of glyphosate effect on soil microorganisms in the wheat phase and the

presence of such effect in the pea phase confirm previous reports from this site (Sheng,

et al. 2012).

Overall, my results show that glyphosate is one of the factors that can influence the soil

environment and the structure of the bacterial community established in the rhizosphere

of wheat, in the Canadian prairie. Glyphosate use positively influences certain bacteria

and negatively impacts others, and this, in turn, may modify the functionality of the soil.

Similar to that reported by Sheng et al. (2012), we also found that cropping practices such

as rotation can modulate the glyphosate effects on rhizosphere microbial community.

The sampling of tansy mustard in my study shows that glyphosate may also have very

rapid effects on rhizosphere microorganisms, probably mediated by the death of target

plants. In tansy mustard, the change observed in the structure of both the bacterial and

fungal communities following the application of glyphosate on the weed plant may have

resulted from the flush of substrates derived from herbicide-induced cellular disruption in

dying plants (Wardle et al., 1994). These substrates could favor certain microorganisms

and shift competitive relationships resulting in the reduction of other microorganisms

(Figure 3, Figure 4). This is the most likely explanations of the effect of glyphosate on the

bacterial community of tansy mustard rhizosphere, since only an immediate effect of

glyphosate application was detected in these communities.

The genus Arthrobacter, which dominated the rhizobacterial community of tansy mustard,

was seemingly favored by the increase of soluble carbon and nitrogen released from

dying plants 3 days after glyphosate application. Similarly, Bacillus simplex CCMM B622

and Flavobacterium sp. L-111-12 were positively affected. Several species belonging to

44

these genera have plant growth promoting abilities (Belimov et al., 2005, Ahmad et al.,

2008, Banerjee et al., 2010, Akhtar et al., 2012, Verma et al., 2013). Conversely, a

deleterious immediate effect of glyphosate application was observed on Rhodococcus sp.

EG2 and Rhodococcus qingshengii KOPRI 25555. Rhodococcus sp. EG2 is potentially

beneficial due to its capacity of degrade RDX (hexahydro-1,3,5-trinitro-1,3,5-

triazine) (Andeer et al., 2013).

The impact of the changes taking place in the rhizosphere community of weed plants

treated with glyphosate on a field site depends on how much tansy mustard was there,

and subsequently, if plants were succumbing. The absence of immediate effect of

glyphosate in the rhizosphere of ‘volunteer’ wheat in this study shows that the identity of

the weed plants treated may also be a factor influencing the effect of glyphosate

application on rhizosphere microorganisms associated with weeds.

4.6 Conclusion

We conclude that pre-seeding applications of glyphosate impact weed rhizosphere

communities and may have undisclosed agronomic and environmental implications, as

this can positively or negatively influence certain bacteria which have a high potential in

bioremediation processes and/or plant growth promotion.

45

A. Annex: Preparation of culture media

Culture medium for fungi: Starch Casein Nitrate Agar (with rose Bengal)

Original compound Compound used

Amount 1000 mL-1 Amount 500 mL-1

Agar 10 g 5 g

Soluble starch 10 g 5 g K2HPO4 2 g 1 g KNO3 2 g 1 g NaCl 2 g 1 g Casein 0.3 g 0.15 g MgSO4.7H2O 0.05 g 0.025 g CaCO3 0.02 g 0.01 g FeSO4.7H2O 0.01 g Fe2(SO4)3 0.006 g Rose bengal 670 g (5 mL of solution) 335 g (2.5 mL of

solution containing 134 mg ml-1)

Add components to distilled/deionized water, except the agar and ajust pH to 7.0. Bring volume to 1 L and mix thoroughly. In order to obtain a highly clear pink plate medium, heat the medium in a water bath at 90°C for 15 min, filter through gauze and sterilize by autoclaving for 15 min at 15 psi pressure and 121°C. Let cool to 45 to 50°C and pour approximately 15 mL into sterile Petri dishes. Culture medium for bacteria: Nutrient agar medium (50%)

Compound

Amount 1000 mL-1

Amount 500 mL-

1 Nutrient broth 4 g 2 g

Agar 15 g 7.5 g

Add components to distilled/deionized water and bring volume to 1 L and mix thoroughly. Heat the medium in a water bath at 90°C for 15 min and sterilize by autoclaving for 15 min at 15 psi pressure and 121°C. Let cool to 45°C to 50°C and pour approximately 15 mL into sterile Petri dishes.

46

B. Annex: Significance of the effects of the factors Plant species, Sampling time, Glyphosate dose, and their interactions on the total numbers of bacteria and fungi of the rhizosphere, according to permutational multivariate analysis of variance (PerMANOVA). The analyses were conducted using 1000 permutations

Bacteria

Source of effect Df Sums of squares

MeanSqs F.Model R2 Pr(>F)

Plant 1 0.6756 0.67562 6.4053 0.07497 0.002 **

Time 2 1.854 0.92701 8.7887 0.20572 0.001 ***

Glyphosate 2 0.685 0.34251 3.2473 0.07601 0.014 *

Plant x Time 2 0.639 0.31948 3.0288 0.0709 0.024 *

Plant x Glyphosate 2 0.2408 0.12041 1.1416 0.02672 0.329

Time x Glyphosate 4 0.7516 0.1879 1.7814 0.0834 0.074

Plant x Time x Glyphosate 4 0.3691 0.09229 0.8749 0.04096 0.539

Residuals 36 3.7972 0.10548 0.42133

Total 53 9.0124 1


47

Fungi

Source of effect Df Sums of squares

MeanSqs F.Model R2 Pr(>F)

Plant 1 0.01952 0.019522 0.599 0.00926 0.505

Time 2 0.09363 0.046816 1.4365 0.0444 0.260

Glyphosate 2 0.12266 0.061329 1.8818 0.05817 0.160

Plant x Time 2 0.30079 0.150393 4.6147 0.14264 0.012 *

Plant x Glyphosate 2 0.08469 0.042344 1.2993 0.04016 0.283

Time x Glyphosate 4 0.18767 0.046918 1.4396 0.089 0.191

Plant x Time x Glyphosate 4 0.12653 0.031632 0.9706 0.06 0.438

Residuals 36 1.17324 0.03259 0.55637

Total 53 2.10872 1


48

C. Annex: Significance of the effects of the factors rotation phase, glyphosate, sampling time and their interactions on the structure of the bacterial and fungal communities in wheat rhizophere, according to permutational multivariate analysis of variance (PerMANOVA). The analyses were conducted using 1000 permutations

Whole plot factor

Source of effect Df

Bacteria Fungi

Sums

of Sqs MeanSqs

F.

Model R2 Pr(>F)

Sums

of Sqs MeanSqs

F.

Model R2 Pr(>F)

Block 3 0.088 0.029 0.964 0.419 0.393 0.102 0.034 0.892 0.427 0.852

Rotation phase 1 0.031 0.031 1.012 0.147 0.393 0.023 0.023 0.591 0.094 0.852

Residuals 3 0.092 0.031 0.435 0.115 0.038 0.479

Total 7 0.211 1 0.239 1


49

Split plot factor

Source of effect Df

Bacteria Fungi

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Block 3 0.177 0.059 1.263 0.186 0.001*** 0.204 0.068 0.870 0.186 0.632

Rotation.block 4 0.245 0.061 1.314 0.259 0.012* 0.274 0.069 0.876 0.250 0.790

Glyphosate 1 0.117 0.117 2.500 0.123 0.001*** 0.064 0.064 0.822 0.059 0.532

Glyphosate x rotation

phase 1 0.130 0.130 2.781 0.137 0.001*** 0.084 0.084 1.070 0.076 0.373

Residuals 6 0.280 0.047 0.295 0.469 0.078 0.428

Total 15 0.947 1 1.096 1


Within subjects effects (Split-split plot factor)

Source of effect Df

Bacteria Fungi

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Sums

of

Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Block 3 0.283 0.094 0.771 0.080 0.730 0.210 0.070 0.816 0.080 0.500

Rotation.block.glyphosate 12 1.303 0.109 0.886 0.367 0.775 1.054 0.088 1.022 0.403 0.484

Time 1 0.165 0.165 1.349 0.046 0.189 0.020 0.020 0.230 0.008 0.838

Time x rotation phase 1 0.135 0.135 1.099 0.038 0.351 0.122 0.122 1.414 0.046 0.250

Time x glyphosate 1 0.085 0.085 0.692 0.024 0.782 0.154 0.154 1.791 0.059 0.180

Time x rotation phase x

glyphosate 1 0.113 0.113 0.923 0.032 0.522 0.026 0.026 0.308 0.010 0.808

Residuals 12 1.470 0.123 0.414 1.031 0.086 0.394

Total 31 3.554 1.000 2.618 1.000


50

D. Annex: Significance of the effects of the factors rotation phase, glyphosate, sampling time and their interactions on the structure of the bacterial and fungal communities in tansy mustard rhizophere, according to permutational multivariate analysis of variance (PerMANOVA). The analyses were conducted using 1000 permutations

Whole plot factor

Source of effect Df

Bacteria Fungi

Sums

of Sqs MeanSqs

F.

Model R2 Pr(>F)

Sums

of Sqs MeanSqs

F.

Model R2 Pr(>F)

Block 3 0.154 0.051 0.797 0.380 0.649 0.089 0.030 0.950 0.359 0.231

Rotation phase 1 0.057 0.057 0.893 0.142 0.649 0.065 0.065 2.080 0.262 0.231

Residuals 3 0.193 0.064 0.477 0.094 0.031 0.378

Total 7 0.404 1 0.249 1


51

Split plot factor

Source of effect Df

Bacteria Fungi

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Block 3 0.307 0.102 1.001 0.184 0.156 0.179 0.060 0.739 0.162 0.684

Rotation.block 4 0.500 0.125 1.223 0.300 0.162 0.319 0.080 0.987 0.289 0.552

Glyphosate 1 0.114 0.114 1.110 0.068 0.347 0.083 0.083 1.031 0.075 0.386

Glyphosate x rotation

phase 1 0.134 0.134 1.307 0.080 0.182 0.040 0.040 0.490 0.036 0.949

Residuals 6 0.614 0.102 0.368 0.485 0.081 0.438

Total 15 1.669 1 1.105 1


Within subjects effects (Split-split plot factor)

Source of effect Df

Bacteria Fungi

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Sums

of Sqs

Mean

Sqs

F.

Model R2 Pr(>F)

Block 3 0.431 0.144 0.820 0.076 0.249 0.173 0.058 0.726 0.059 0.081

Rotation.block.glyphosate 12 2.407 0.201 1.146 0.424 0.196 1.213 0.101 1.274 0.413 0.171

Time 1 0.179 0.179 1.021 0.031 0.430 0.326 0.326 4.114 0.111 0.004**

Time x rotation phase 1 0.341 0.341 1.951 0.060 0.038* 0.001 0.001 0.013 0.000 0.962

Time x glyphosate 1 0.102 0.102 0.580 0.018 0.865 0.084 0.084 1.057 0.029 0.420

Time x rotation phase x

glyphosate 1 0.124 0.124 0.710 0.022 0.766 0.187 0.187 2.358 0.064 0.066

Residuals 12 2.100 0.175 0.369 0.952 0.079 0.324

Total 31 5.684 1 2.936 1


52

References cited

AGRICULTURE AND AGRI-FOOD CANADA (AAFC) 2010a. Crop profile for spring wheat in Canada.

AGRICULTURE AND AGRI-FOOD CANADA (AAFC) 2010b. Crop profile for winter wheat in Canada.

AHEMAD, M. & KIBRET, M. 2013a. Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective [Online]. Available: http://www.sciencedirect.com/science/article/pii/S1018364713000293 [Accessed 0.

AHEMAD, M. & KIBRET, M. 2013b. Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective [Online]. Available: http://www.sciencedirect.com/science/article/pii/S1018364713000293 [Accessed March 13 2014].

AHMAD, F., AHMAD, I. & KHAN, M. S. 2008. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiological Research, 163, 173-181.

AKHTAR, A., ROBAB, M. I. & SHARF, R. 2012. Plant growth promoting Rhizobacteria: An overview. Journal of Natural Product & Plant Resources, 2, 19-31.

AL-ARFAJ, A., ABDEL-MEGEED, A., ALI, H. M. & AL-SHAHRANI, O. 2013. Phyto-microbial degradation of glyphosate in Riyadh area. Journal of Pure and Applied Microbiology, 7, 1351-1365.

ALBERTA MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT. 2011. Effects of weeds on wheat [Online]. Available: http://www1.agric.gov.ab.ca/$Department/deptdocs.nsf/all/crop1280 [Accessed January 18 2014].

ANDEER, P., STAHL, D. A., LILLIS, L. & STRAND, S. E. 2013. Identification of microbial populations assimilating nitrogen from RDX in munitions contaminated military training range soils by high sensitivity stable isotope probing. Environmental Science and Technology, 47, 10356-10363.

ANDERSON, M. J. 2001a. A new method for non-parametric multivariate analysis of variance. Austral Ecology, 26, 32-46.

ANDERSON, M. J. 2001b. Permutation tests for univariate or multivariate analysis of variance and regression. Canadian Journal of Fisheries and Aquatic Sciences, 58, 626-639.

ANDERSON, T. A., GUTHRIE, E. A. & WALTON, B. T. 1993. Bioremediation in the rhizosphere. Environmental Science & Technology, 27, 2630-2636.

ANDRADE, D. S., COLOZZI-FILHO, A. & GILLER, K. E. 2003. The soil microbial community and soil tillage. Soil tillage in agroecosystems, 51-81.

ANDRADE, G. 2004. Role of functional groups of microorganisms on the rhizosphere microcosm dynamics. Plant surface microbiology. Springer.

http://www.sciencedirect.com/science/article/pii/S1018364713000293

http://www.sciencedirect.com/science/article/pii/S1018364713000293

http://www1.agric.gov.ab.ca/$Department/deptdocs.nsf/all/crop1280

53

ANDRÉA, M. M. D., PERES, T. B., LUCHINI, L. C., BAZARIN, S., PAPINI, S., MATALLO,

M. B. & SAVOY, V. L. T. 2003. Influence of repeated applications of glyphosate on its persistence and soil bioactivity. Pesquisa Agropecuária Brasileira, 38, 1329-1335.

ARAÚJO, A. S. F., MONTEIRO, R. T. R. & ABARKELI, R. B. 2003. Effect of glyphosate on the microbial activity of two Brazilian soils. Chemosphere, 52, 799-804.

ARSHAD, M. A., SOON, Y. K. & AZOOZ, R. H. 2002. Modified no-till and crop sequence effects on spring wheat production in northern Alberta, Canada. Soil and Tillage Research, 65, 29-36.

BALTHAZOR, T. M. & HALLAS, L. E. 1986. Glyphosate-degrading microorganisms from industrial activated sludge. Applied and environmental microbiology, 51, 432-434.

BANERJEE, S., PALIT, R., SENGUPTA, C. & STANDING, D. 2010. Stress induced phosphate solubilization by arthrobacter sp. and bacillus sp. isolated from tomato rhizosphere. Australian Journal of Crop Science, 4, 378-383.

BARRIUSO, J., MARÍN, S. & MELLADO, R. P. 2010. Effect of the herbicide glyphosate on glyphosate-tolerant maize rhizobacterial communities: A comparison with pre-emergency applied herbicide consisting of a combination of acetochlor and terbuthylazine. Environmental Microbiology, 12, 1021-1030.

BARRIUSO, J. & MELLADO, R. P. 2012a. Glyphosate affects the rhizobacterial communities in glyphosate-tolerant cotton. Applied Soil Ecology, 55, 20-26.

BARRIUSO, J. & MELLADO, R. P. 2012b. Relative effect of glyphosate on glyphosate-tolerant maize rhizobacterial communities is not altered by soil properties. Journal of Microbiology and Biotechnology, 22, 159-165.

BARRIUSO, J., VALVERDE, J. R. & MELLADO, R. P. 2011. Effect of the herbicide glyphosate on the culturable fraction of glyphosate-tolerant maize rhizobacterial communities using two different growth media. Microbes and Environments, 26, 332-338.

BELIMOV, A. A., HONTZEAS, N., SAFRONOVA, V. I., DEMCHINSKAYA, S. V., PILUZZA, G., BULLITTA, S. & GLICK, B. R. 2005. Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (< i> Brassica juncea</i> L. Czern.). Soil Biology and Biochemistry, 37, 241-250.

BUSSE, M. D., RATCLIFF, A. W., SHESTAK, C. J. & POWERS, R. F. 2001. Glyphosate toxicity and the effects of long-term vegetation control on soil microbial communities. Soil Biology and Biochemistry, 33, 1777-1789.

CALLIHAN, R. H., UNIVERSITY OF IDAHO. COOPERATIVE EXTENSION, S., WASHINGTON STATE UNIVERSITY. COOPERATIVE, E. & WESTERN RURAL DEVELOPMENT, C. 2000. Mustards in Mustards: Guide to Identification of Canola, Mustard, Rapeseed and Related Weeds, Cooperative Extension System.

CARAVACA, F., MASCIANDARO, G. & CECCANTI, B. 2002. Land use in relation to soil chemical and biochemical properties in a semiarid Mediterranean environment. Soil and Tillage Research, 68, 23-30.

CERDEIRA, A. L. & DUKE, S. O. 2006. The current status and environmental impacts of glyphosate-resistant crops: A review. Journal of Environmental Quality, 35, 1633-1658.

CHAKRAVARTY, P. & CHATARPAUL, L. 1990. Non-target effect of herbicides: I. effect of glyphosate and hexazinone on soil microbial activity. Microbial population, and in-vitro growth of ectomycorrhizal fungi. Pesticide Science, 28, 233-241.

CHRISTENSEN, B. 1996. Matching Measurable Soil Organic Matter Fractions with Conceptual Pools in Simulation Models of Carbon Turnover: Revision of Model

54

Structure. In: POWLSON, D., SMITH, P. & SMITH, J. (eds.) Evaluation of Soil Organic Matter Models. Springer Berlin Heidelberg.

CLARKE, K. R. 1993. Non‐parametric multivariate analyses of changes in community structure. Australian journal of ecology, 18, 117-143.

COUPE, R. H., KALKHOFF, S. J., CAPEL, P. D. & GREGOIRE, C. 2012. Fate and transport of glyphosate and aminomethylphosphonic acid in surface waters of agricultural basins. Pest Management Science, 68, 16-30.

DANGA, B. O., OUMA, J. P., WAKINDIKI, I. I. C. & BAR-TAL, A. 2009. Legume–wheat rotation effects on residual soil moisture, nitrogen and wheat yield in tropical regions. Advances in agronomy, 101, 315-349.

DICK, R. E. & QUINN, J. P. 1995. Glyphosate-degrading isolates from environmental samples: occurrence and pathways of degradation. Applied Microbiology and Biotechnology, 43, 545-550.

DUFRÊNE, M. & LEGENDRE, P. 1997. Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecological monographs, 67, 345-366.

DUKE, S. O. 2005. Taking stock of herbicide-resistant crops ten years after introduction. Pest Management Science, 61, 211-218.

DUKE, S. O. & POWLES, S. B. 2008. Glyphosate: a once-in-a-century herbicide. Pest Management Science, 64, 319-325.

EL TITI, A. 2003. Soil tillage in agroecosystems, CRC press. ENTZ, M. H., GUILFORD, R. & GULDEN, R. 2001. Crop yield and soil nutrient status on

14 organic farms in the eastern portion of the northern Great Plains. Canadian Journal of Plant Science, 81, 351-354.

ERMAKOVA, I. T., SHUSHKOVA, T. V. & LEONT’EVSKII, A. A. 2008. Microbial degradation of organophosphonates by soil bacteria. Microbiology, 77, 615-620.

FAN, J., YANG, G., ZHAO, H., SHI, G., GENG, Y., HOU, T. & TAO, K. 2012. Isolation, identification and characterization of a glyphosate-degrading bacterium, Bacillus cereus CB4, from soil. Journal of General and Applied Microbiology, 58, 263-271.

FENG, P. C. C., BALEY, G. J., CLINTON, W. P., BUNKERS, G. J., ALIBHAI, M. F., PAULITZ, T. C. & KIDWELL, K. K. 2005. Glyphosate inhibits rust diseases in glyphosate-resistant wheat and soybean. Proceedings of the National Academy of Sciences of the United States of America, 102, 17290-17295.

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO) 2013. FRANZ, J. E., MAO, M. K. & SIKORSKI, J. A. 1997. Glyphosate: a unique global

herbicide, American Chemical Society. FRANZETTI, A., GANDOLFI, I., RAIMONDI, C., BESTETTI, G., BANAT, I. M., SMYTH, T.

J., PAPACCHINI, M., CAVALLO, M. & FRACCHIA, L. 2012. Environmental fate, toxicity, characteristics and potential applications of novel bioemulsifiers produced by Variovorax paradoxus 7bCT5. Bioresource Technology, 108, 245-251.

GARBEVA, P., ELSAS, J. D. & VEEN, J. A. 2008. Rhizosphere microbial community and its response to plant species and soil history. Plant and Soil, 302, 19-32.

GHISALBA, O., KUENZI, M., RAMOS TOMBO, G. & SCHAR, H.-P. 1987. Microbial degradation and utilization of selected organophosphorus compounds - strategies and applications.: Chimia.

GOMEZ, E., FERRERAS, L., LOVOTTI, L. & FERNANDEZ, E. 2009. Impact of glyphosate application on microbial biomass and metabolic activity in a Vertic Argiudoll from Argentina. European Journal of Soil Biology, 45, 163-167.

GRAYSTON, S. J., WANG, S., CAMPBELL, C. D. & EDWARDS, A. C. 1998. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biology and Biochemistry, 30, 369-378.

55

GRUNDY, A. C. 2003. Predicting weed emergence: A review of approaches and future

challenges. Weed Research, 43, 1-11. HANEY, R. L., SENSEMAN, S. A., HONS, F. M. & ZUBERER, D. A. 2000. Effect of

glyphosate on soil microbial activity and biomass. Weed Science, 48, 89-93. HART, M. M., POWELL, J. R., GULDEN, R. H., DUNFIELD, K. E., PETER PAULS, K.,

SWANTON, C. J., KLIRONOMOS, J. N., ANTUNES, P. M., KOCH, A. M. & TREVORS, J. T. 2009. Separating the effect of crop from herbicide on soil microbial communities in glyphosate-resistant corn. Pedobiologia, 52, 253-262.

HART, M. R. & BROOKES, P. C. 1996. Soil microbial biomass and mineralisation of soil organic matter after 19 years of cumulative field applications of pesticides. Soil Biology and Biochemistry, 28, 1641-1649.

HAYAT, R., ALI, S., AMARA, U., KHALID, R. & AHMED, I. 2010. Soil beneficial bacteria and their role in plant growth promotion: A review. Annals of Microbiology, 60, 579-598.

HE, Z., JOSHI, A. K. & ZHANG, W. 2013. 2.06 - Climate Vulnerabilities and Wheat Production. In: PIELKE, R. A. (ed.) Climate Vulnerability. Oxford: Academic Press.

HOSSAIN, M. Z., OKUBO, A. & SUGIYAMA, S.-I. 2010. Effects of grassland species on decomposition of litter and soil microbial communities. Ecological Research, 25, 255-261.

JOHAL, G. S. & HUBER, D. M. 2009. Glyphosate effects on diseases of plants. European Journal of Agronomy, 31, 144-152.

KEITH, H., OADES, J. M. & MARTIN, J. K. 1986. Input of carbon to soil from wheat plants. Soil Biology and Biochemistry, 18, 445-449.

KNIGHT, J. D. 2012. Frequency of field pea in rotations impacts biological nitrogen fixation. Canadian Journal of Plant Science, 92, 1005-1011.

KOWALCHUK, G. A., BUMA, D., DE BOER, W., KLINKHAMER, P. L. & VAN VEEN, J. 2002. Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms. Antonie van Leeuwenhoek, 81, 509-520.

KREMER, R. J. & MEANS, N. E. 2009. Glyphosate and glyphosate-resistant crop interactions with rhizosphere microorganisms. European Journal of Agronomy, 31, 153-161.

KREMER, R. J., MEANS, N. E. & KIM, S. 2005. Glyphosate affects soybean root exudation and rhizosphere micro-organisms. International Journal of Environmental Analytical Chemistry, 85, 1165-1174.

KRZYSKO-LUPICKA, T. & SUDOL, T. 2008. Interactions between glyphosate and autochthonous soil fungi surviving in aqueous solution of glyphosate. Chemosphere, 71, 1386-1391.

KRZYŚKO-LUPICKA, T., STROF, W., KUBŚ, K., SKORUPA, M., WIECZOREK, P., LEJCZAK, B. & KAFARSKI, P. 1997. The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds. Applied microbiology and biotechnology, 48, 549-552.

KRZYŚKO-ŁUPICKA, T. & ORLIK, A. 1997. The use of glyphosate as the sole source of phosphorus or carbon for the selection of soil-borne fungal strains capable to degrade this herbicide. Chemosphere, 34, 2601-2605.

LADYGINA, N. & HEDLUND, K. 2010. Plant species influence microbial diversity and carbon allocation in the rhizosphere. Soil Biology and Biochemistry, 42, 162-168.

LAITINEN, P., RÄMÖ, S. & SIIMES, K. 2007. Glyphosate translocation from plants to soil–does this constitute a significant proportion of residues in soil? Plant and Soil, 300, 51-60.

56

LANE, M., LORENZ, N., SAXENA, J., RAMSIER, C. & DICK, R. P. 2012a. Microbial

activity, community structure and potassium dynamics in rhizosphere soil of soybean plants treated with glyphosate. Pedobiologia, 55, 153-159.

LANE, M., LORENZ, N., SAXENA, J., RAMSIER, C. & DICK, R. P. 2012b. The effect of glyphosate on soil microbial activity, microbial community structure, and soil potassium. Pedobiologia, 55, 335-342.

LECHEVALIER, H. A. 1986. Nocardioforms. Bergey's manual of systematic bacteriology, 2, 1458-1506.

LEGENDRE, P. & GALLAGHER, E. D. 2001. Ecologically meaningful transformations for ordination of species data. Oecologia, 129, 271-280.

LIEBMAN, M. & DYCK, E. 1993. Crop Rotation and Intercropping Strategies for Weed Management. Ecological Applications, 3, 92-122.

LUPWAYI, N. Z., GRANT, C. A., SOON, Y. K., CLAYTON, G. W., BITTMAN, S., MALHI, S. S. & ZEBARTH, B. J. 2010. Soil microbial community response to controlled-release urea fertilizer under zero tillage and conventional tillage. Applied Soil Ecology, 45, 254-261.

LUPWAYI, N. Z., HANSON, K. G., HARKER, K. N., CLAYTON, G. W., BLACKSHAW, R. E., O'DONOVAN, J. T., JOHNSON, E. N., GAN, Y., IRVINE, R. B. & MONREAL, M. A. 2007. Soil microbial biomass, functional diversity and enzyme activity in glyphosate-resistant wheat-canola rotations under low-disturbance direct seeding and conventional tillage. Soil Biology and Biochemistry, 39, 1418-1427.

LUPWAYI, N. Z., HARKER, K. N., CLAYTON, G. W., O’DONOVAN, J. T. & BLACKSHAW, R. E. 2009. Soil microbial response to herbicides applied to glyphosate-resistant canola. Agriculture, ecosystems & environment, 129, 171-176.

LUPWAYI, N. Z., HARKER, K. N., CLAYTON, G. W., TURKINGTON, T. K., RICE, W. A. & O’DONOVAN, J. T. 2004. Soil microbial biomass and diversity after herbicide application. Canadian Journal of Plant Science, 84, 677-685.

MADIGAN, M. T., MARTINKO, J. M. & PARKER, J. 2003. Brock Biology of Microorganisms, Prentice Hall/Pearson Education.

MAYNARD, D. G. & KARLA, Y. P. 1993. Nitrate and exchangeable ammonium nitrogen. In: M., C. (ed.) Soil sampling and methods of analysis. . U.S.A.: C.R.C. Press, Boca Raton.

MEANS, N. E., KREMER, R. J. & RAMSIER, C. 2007. Effects of glyphosate and foliar amendments on activity of microorganisms in the soybean rhizosphere. Journal of Environmental Science and Health Part B, 42, 125-132.

MESKYS, R., HARRIS, R. J., CASAITE, V., BASRAN, J. & SCRUTTON, N. S. 2001. Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp. European Journal of Biochemistry, 268, 3390-3398.

MIJANGOS, I., BECERRIL, J. M., ALBIZU, I., EPELDE, L. & GARBISU, C. 2009. Effects of glyphosate on rhizosphere soil microbial communities under two different plant compositions by cultivation-dependent and -independent methodologies. Soil Biology and Biochemistry, 41, 505-513.

MISHRA, J. S. & SINGH, V. P. 2012. Tillage and weed control effects on productivity of a dry seeded rice–wheat system on a Vertisol in Central India. Soil and Tillage Research, 123, 11-20.

MONGODIN, E. F., SHAPIR, N., DAUGHERTY, S. C., DEBOY, R. T., EMERSON, J. B., SHVARTZBEYN, A., RADUNE, D., VAMATHEVAN, J., RIGGS, F. & GRINBERG, V. 2006. Secrets of soil survival revealed by the genome sequence of Arthrobacter aurescens TC1. PLoS genetics, 2, e214.

57

MOORE, J. K., BRAYMER, H. D. & LARSON, A. D. 1983. Isolation of a Pseudomonas sp.

which utilizes the phosphonate herbicide glyphosate. Applied and environmental microbiology, 46, 316-320.

MOTAVALLI, P. P., KREMER, R. J., FANG, M. & MEANS, N. E. 2004. Impact of genetically modified crops and their management on soil microbially mediated plant nutrient transformations. Journal of Environmental Quality, 33, 816-824.

NEHL, D. B., ALLEN, S. J. & BROWN, J. F. 1997. Deleterious rhizosphere bacteria: an integrating perspective. Applied Soil Ecology, 5, 1-20.

NEUMANN, G. 2007. Root exudates and nutrient cycling. Nutrient Cycling in Terrestrial Ecosystems. Springer.

NEUMANN, G., KOHLS, S., LANDSBERG, E., STOCK-OLIVEIRA SOUZA, K., YAMADA, T. & RÖMHELD, V. 2006. Relevance of glyphosate transfer to non-target plants via the rhizosphere. Journal of Plant Diseases and Proctection, Supplement, 963-969.

OKSANEN, J., GUILLAUME BLANCHET, F., KINDT, R., LEGENDRE, P., MINCHIN, P. R., O'HARA, R. B., SIMPSON, G. L., SOLYMOS, P., STEVENS, M. H. H. & WAGNER, H. 2011. vegan: Community Ecology Package.

OLSEN, S. R., COLE, C. V. & DEAN, L. A. 1954. Estimation of available phosphorus in soils by extraction with sodium bicarbonate.: U.S. Department of Agriculture.

OLSON, B. M. & LINDWALL, C. W. 1991. Soil microbial activity under chemical fallow conditions: Effects oF 2,4-D and glyphosate. Soil Biology and Biochemistry, 23, 1071-1075.

PEREIRA, J. L., PICANÇO, M. C., SILVA, A. A., SANTOS, E. A., TOMÉ, H. V. V. & OLARTE, J. B. 2008. Effects of glyphosate and endosulfan on soil microorganisms in soybean crop. Planta Daninha, 26, 825-830.

PIPKE, R. & AMRHEIN, N. 1988a. Degradation of the phosphonate herbicide glyphosate by arthrobacter atrocyaneus ATCC 13752. Applied and Environmental Microbiology, 54, 1293-1296.

PIPKE, R. & AMRHEIN, N. 1988b. Isolation and characterization of a mutant of Arthrobacter sp. strain GLP-1 which utilizes the herbicide glyphosate as its sole source of phosphorus and nitrogen. Applied and environmental microbiology, 54, 2868-2870.

PIPKE, R., AMRHEIN, N., JACOB, G. S., SCHAEFER, J. & KISHORE, G. M. 1987. Metabolism of glyphosate in an Arthrobacter sp. GLP-1. European Journal of Biochemistry, 165, 267-273.

PORTER, J. R., JAMIESON, P. D. & GRACE, P. R. 2007. Wheat Production Systems and Global Climate Change. Terrestrial Ecosystems in a Changing World. Springer.

POWELL, J. R., CAMPBELL, R. G., DUNFIELD, K. E., GULDEN, R. H., HART, M. M., LEVY-BOOTH, D. J., KLIRONOMOS, J. N., PAULS, K. P., SWANTON, C. J., TREVORS, J. T. & ANTUNES, P. M. 2009. Effect of glyphosate on the tripartite symbiosis formed by Glomus intraradices, Bradyrhizobium japonicum, and genetically modified soybean. Applied Soil Ecology, 41, 128-136.

PRIESTMAN, M. A., FUNKE, T., SINGH, I. M., CRUPPER, S. S. & SCHÖNBRUNN, E. 2005. 5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate. FEBS Letters, 579, 728-732.

QUINN, J. P., PEDEN, J. M. M. & DICK, R. E. 1989. Carbon-phosphorus bond cleavage by Gram-positive and Gram-negative soil bacteria. Applied Microbiology and Biotechnology, 31, 283-287.

58

R DEVELOPMENT CORE TEAM 2011. R: A Language and Environment for Statistical

Computing. R Foundation for Statistical Computing,. Vienna, Austria,. R DEVELOPMENT CORE TEAM 2013. R: A Language and Environment for Statistical

Computing. R Foundation for Statistical Computing,. Vienna, Austria,. RATCLIFF, A. W., BUSSE, M. D. & SHESTAK, C. J. 2006. Changes in microbial

community structure following herbicide (glyphosate) additions to forest soils. Applied Soil Ecology, 34, 114-124.

REDDY, K. N. & ZABLOTOWICZ, R. M. 2003. Glyphosate-resistant soybean response to various salts of glyphosate and glyphosate accumulation in soybean nodules. Weed Science, 51, 496-502.

ROBERTS, D. W. 2010. labdsv: Ordination and Multivariate Analysis for Ecology. ROLDÁN, A., CARAVACA, F., HERNÁNDEZ, M. T., GARCÍA, C., SÁNCHEZ-BRITO, C.,

VELÁSQUEZ, M. & TISCAREÑO, M. 2003. No-tillage, crop residue additions, and legume cover cropping effects on soil quality characteristics under maize in Patzcuaro watershed (Mexico). Soil and Tillage Research, 72, 65-73.

ROSLYCKY, E. B. 1982. Glyphosate and the response of the soil microbiota. Soil Biology and Biochemistry, 14, 87-92.

RUXTON, G. D. & COLEGRAVE, N. 2011. Experimental design for the life sciences, Oxford; New York, Oxford University Press.

SASKATCHEWAN MINISTRY OF AGRICULTURE 2010. Guide to Crop Protection: Weeds, Plant Diseases, Insects. Saskatchewan Ministry of Agriculture Regina, SK.

SATOLA, B., WÜBBELER, J. H. & STEINBÜCHEL, A. 2013. Metabolic characteristics of the species Variovorax paradoxus. Applied Microbiology and Biotechnology, 97, 541-560.

SAVIN, M. C., PURCELL, L. C., DAIGH, A. & MANFREDINI, A. 2009. Response of mycorrhizal infection to glyphosate applications and P fertilization in glyphosate-tolerant soybean, Maize, and Cotton. Journal of Plant Nutrition, 32, 1702-1717.

SCHROTH, M. N. & HANCOCK, J. G. 1982. Disease-suppressive soil and root-colonizing bacteria. Science, 216, 1376-1381.

SCHULZ, A., KRÜPER, A. & AMRHEIN, N. 1985. Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate. FEMS Microbiology Letters, 28, 297-301.

SHAPIR, N., MONGODIN, E. F., SADOWSKY, M. J., DAUGHERTY, S. C., NELSON, K. E. & WACKETT, L. P. 2007. Evolution of catabolic pathways: genomic insights into microbial s-triazine metabolism. Journal of bacteriology, 189, 674-682.

SHENG, M., HAMEL, C. & FERNANDEZ, M. R. 2012. Cropping practices modulate the impact of glyphosate on arbuscular mycorrhizal fungi and rhizosphere bacteria in agroecosystems of the semiarid prairie. Canadian journal of microbiology, 58, 990-1001.

SHUSHKOVA, T. V., VASILIEVA, G. K., ERMAKOVA, I. T. & LEONTIEVSKY, A. A. 2009. Sorption and microbial degradation of glyphosate in soil suspensions. Applied biochemistry and microbiology, 45, 599-603.

SOON, Y. K. & LUPWAYI, N. Z. 2008. Influence of pea cultivar and inoculation on the nitrogen budget of a pea-wheat rotation in northwestern Canada. Canadian Journal of Plant Science, 88, 1-9.

STATISTICS CANADA. 2014. Table 001-0010 - Estimated areas, yield, production and average farm price of principal field crops, in metric units, annual [Online]. Available: http://www.statcan.gc.ca/start-debut-eng.html [Accessed February 23, 2014 2014].

http://www.statcan.gc.ca/start-debut-eng.html

59

STEER, J. & HARRIS, J. A. 2000. Shifts in the microbial community in rhizosphere and

non-rhizosphere soils during the growth of Agrostis stolonifera. Soil Biology and Biochemistry, 32, 869-878.

STRONG, L. C., ROSENDAHL, C., JOHNSON, G., SADOWSKY, M. J. & WACKETT, L. P. 2002. Arthrobacter aurescens TC1 metabolizes diverse s-triazine ring compounds. Applied and environmental microbiology, 68, 5973-5980.

STURZ, A. V. & CHRISTIE, B. R. 2003. Beneficial microbial allelopathies in the root zone: the management of soil quality and plant disease with rhizobacteria. Soil and Tillage Research, 72, 107-123.

SYLVIA, D. M. 2005. Principles and applications of soil microbiology, Pearson Prentice Hall.

TERNAN, N. G., MC GRATH, J. W., MC MULLAN, G. & QUINN, J. P. 1998. Review: Organophosphonates: Occurrence, synthesis and biodegradation by microorganisms. World Journal of Microbiology and Biotechnology, 14, 635-647.

UNITED STATES DEPARTMENT OF AGRICULTURE (USDA). 2014. World Agricultural Production [Online]. Available: http://www.fas.usda.gov/data/world-agricultural-production.

VEIGA, F., ZAPATA, J. M., FERNANDEZ MARCOS, M. L. & ALVAREZ, E. 2001. Dynamics of glyphosate and aminomethylphosphonic acid in a forest soil in Galicia, north-west Spain. Science of the Total Environment, 271, 135-144.

VERCELLINO, M. & GÓMEZ, M. A. 2013. Denitrifying capacity of rhizobial strains of Argentine soils and herbicide sensitivity. Annals of Microbiology, 1-8.

VEREECKEN, H. 2005. Mobility and leaching of glyphosate: A review. Pest Management Science, 61, 1139-1151.

VERMA, J. P., YADAV, J., TIWARI, K. N. & KUMAR, A. 2013. Effect of indigenous Mesorhizobium spp. and plant growth promoting rhizobacteria on yields and nutrients uptake of chickpea (Cicer arietinum L.) under sustainable agriculture. Ecological engineering, 51, 282-286.

WALKER, T. S., BAIS, H. P., GROTEWOLD, E. & VIVANCO, J. M. 2003. Root exudation and rhizosphere biology. Plant Physiology, 132, 44-51.

WARDLE, D. & PARKINSON, D. 1990a. Influence of the herbicide glyphosate on soil microbial community structure. Plant and Soil, 122, 29-37.

WARDLE, D. A., NICHOLSON, K. S. & RAHMAN, A. 1994. Influence of herbicide applications on the decomposition, microbial biomass, and microbial activity of pasture shoot and root litter. New Zealand Journal of Agricultural Research, 37, 29-39.

WARDLE, D. A. & PARKINSON, D. 1990b. Effects of three herbicides on soil microbial biomass and activity. Plant and Soil, 122, 21-28.

WARDLE, D. A. & PARKINSON, D. 1992. Influence of the herbicides 2,4-D and glyphosate on soil microbial biomass and activity: A field experiment. Soil Biology and Biochemistry, 24, 185-186.

WEAVER, M. A., KRUTZ, L. J., ZABLOTOWICZ, R. M. & REDDY, K. N. 2007. Effects of glyphosate on soil microbial communities and its mineralization in a Mississippi soil. Pest Management Science, 63, 388-393.

WILLIAMS, G. M., KROES, R. & MUNRO, I. C. 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regulatory Toxicology and Pharmacology, 31, 117-165.

YANG, Q., WANG, X. & SHEN, Y. 2013. Comparison of soil microbial community catabolic diversity between Rhizosphere and bulk soil induced by tillage or residue retention. Journal of Soil Science and Plant Nutrition, 13.

http://www.fas.usda.gov/data/world-agricultural-production

http://www.fas.usda.gov/data/world-agricultural-production

60

ZABALOY, M. C., GARLAND, J. L. & GÓMEZ, M. A. 2008. An integrated approach to

evaluate the impacts of the herbicides glyphosate, 2,4-D and metsulfuron-methyl on soil microbial communities in the Pampas region, Argentina. Applied Soil Ecology, 40, 1-12.

ZABALOY, M. C. & GÓMEZ, M. A. 2005. Diversity of rhizobia isolated from an agricultural soil in Argentina based on carbon utilization and effects of herbicides on growth. Biology and Fertility of Soils, 42, 83-88.

ZENTNER, R. P., LAFOND, G. P., DERKSEN, D. A. & CAMPBELL, C. A. 2002. Tillage method and crop diversification: Effect on economic returns and riskiness of cropping systems in a Thin Black Chernozem of the Canadian Prairies. Soil and Tillage Research, 67, 9-21.

ZOBIOLE, L. H. S., KREMER, R. J., OLIVEIRA, R. S. & CONSTANTIN, J. 2011.

Glyphosate affects micro‐organisms in rhizospheres of glyphosate‐resistant soybeans. Journal of applied microbiology, 110, 118-127.

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