Inoculation of arbuscular mycorrhizal fungi to improve soybean growth under drought in the context of climate change

VIETNAM NATIONAL UNIVERSITY, HANOI
VIETNAM JAPAN UNIVERSITY

NGUYEN DUC TAM

INOCULATION OF ARBUSCULAR
MYCORRHIZAL FUNGI TO IMPROVE
SOYBEAN GROWTH UNDER DROUHGT IN
THE CONTEXT OF CLIMATE CHANGE

MASTER’S THESIS


VIETNAM NATIONAL UNIVERSITY, HANOI
VIETNAM JAPAN UNIVERSITY

NGUYEN DUC TAM

INOCULATION OF ARBUSCULAR
MYCORRHIZAL FUNGI TO IMPROVE
SOYBEAN GROWTH UNDER DROUHGT IN
THE CONTEXT OF CLIMATE CHANGE
MAJOR: CLIMATE CHANGE AND DEVELOPMENT
CODE: 8900201.02QTD

RESEARCH SUPERVISOR:
DR. HOANG THI THU DUYEN
DR. DANG THANH TU

Hanoi, 2021

PLEDGE
I assure that this thesis is original and has not been published. The use of results
of other research and other documents must comply with regulations. The
citations and references to documents, books, research papers, and websites must
be in the list of references of the thesis.

Author of the thesis

Nguyen Duc Tam


TABLE OF CONTENT
LIST OF TABLES .......................................................................................................... i
LIST OF FIGURES ........................................................................................................ ii
LIST OF ABBREVIATIONS ....................................................................................... iii
ACKNOWLEDGMENT ............................................................................................... iv
CHAPTER 1. INTRODUCTION ....................................................................................1
1.1 Background and motivation of the study ..............................................................1
1.2. The necessity of the research ................................................................................2
1.2.1. Drought risk in Quang Nam province ...........................................................2
1.2.2. Soybean in crop system in Quang Nam province .........................................3
1.2.3. Mechanisms of soybean and arbuscular Mycorrhiza Fungi (AMF)
symbiosis .................................................................................................................5
1.2.4. Arbuscular Mycorrhiza Fungi (AMF) enhance phosphorus uptake by plants
under drought...........................................................................................................6
1.3. Research Framework: ...........................................................................................8
1.4. The Research questions and hypotheses ............................................................10
1.5. Objectives and scope of the research..................................................................10
CHAPTER 2. MATERIALS AND METHODOLOGIES ............................................11

2.1. Methodology and experimental setup ................................................................11
2.1.1. Collecting data base and analysis ................................................................11
2.1.2. Fieldwork and soil sampling .......................................................................11
2.1.3. Soil property measurement ..........................................................................11
2.1.4. Defining water holding capacity (WHC) ....................................................12
2.1.5. Experimental setup ......................................................................................12
2.2. Root, shoot biomass and root mycorrhiza inoculation .......................................14
2.3. Microbial biomass phosphorus (MBP) ...............................................................14
2.4. Microbial respiration ..........................................................................................15
2.5. Statistical analysis ..............................................................................................16
CHAPTER 3: RESULTS AND DISCUSSION ............................................................17
3.1. Characteristics of drought condition at the study site ........................................17
3.2. AMF inoculation ................................................................................................20
3.3. Microbial Biomass Phosphorus (MBP) ..............................................................21
3.4. Soil respiration ....................................................................................................22
3.5. Root – Shoot characteristics ...............................................................................23
CHAPTER 4. CONCLUSIONS AND RECOMMENDATIONS ................................26
4.1. Conclusions ........................................................................................................26
4.2. Recommendations for future research ................................................................26


REFERENCES ..............................................................................................................28


LIST OF TABLES

Table 1. Methodologies to analyze soil physic-chemical properties.............................11

i

LIST OF FIGURES

Figure 1.1. Effects of drought on plant growth ...............................................................2
Figure 1.2. Research framework .....................................................................................9
Figure 2.1. Determine Location of sampling area in Quang Nam province using
Google Maps application ...............................................................................................11
Figure 2.2. Soil was packed in total 16 transparent rhizoboxes ....................................13
Figure 2.3. Microbial biomass P was measured in 96-wells microplates: a) the
calibration was prepared for different concentrations of P and b) the microbial biomass
was calculated based on the difference between fumigation and non-fumigation
treatments.......................................................................................................................15
Figure 3.1. Average monthly sunshine hours (2000 – 2019) in Quang Nam ...............18
Figure 3.2. Average monthly temperature (2000 – 2019) in Quang Nam ....................18
Figure 3.3. Average monthly percipitation (2000 – 2019) in Quang Nam ...................19
Figure 3.4. Average monthly evaporation (2000 – 2019) in Quang Nam ....................20
Figure 3.5. The fungal structures ...................................................................................20
Figure 3.6. Microbial Biomass Phosphorus analysis results (μg P/ g soil) ...................21
Figure 3.7. Soil respiration ............................................................................................23
Figure 3.8. The root nodules on the main roots .............................................................23
Figure 3.9. Average dry weight Root – Shoot (g) experimental box ............................24
Figure 3.10. Result Root – Shoot length (cm) ...............................................................25

ii


LIST OF ABBREVIATIONS

C
IPCC

N

Carbon
Intergovernmental Panel on Climate Change
Nitrogen

TC

Total carbon

TN

Total nitrogen

WHC
MBP

Water holding capacity
Microbial Biomass Phosphorus

AMF

Arbuscular Mycorrhiza Fungi

iii


ACKNOWLEDGMENT

To complete this thesis, I would like to thank the lecturers and staff of the

Program on Climate Change and Development, Vietnam Japan University, Hanoi
National University and the lecturers of Experimental center, Faculty of Forestry of
the Vietnam National University of Forestry for their supports. My research cannot be
successful without their advices and collaboration.
First and foremost, I would like to express my gratitude and sincere thanks to Dr.
Hoang Thi Thu Duyen, who directly supervised my research implementation, for her
enthusiastic instruction and dedication to orient my research topic, sampling soil,
conducting lab experiment, data processing, and analysis.
My deepest and most heartful thanks to my sub supervisor - Dr. Dang Thanh Tu,
my advisors - Dr. Kotera Akihiko, Dr. Nguyen Van Quang, MS. Hoa for their
dedication and valuable advice to the thesis.
In addition, the author also highly appreciates the financial support of the VNU
Project (QG.20.63), without this support, the implementation of the thesis would not
be possible.
Finally, I would like to dedicate this thesis to my parents, my wife, my children
and my friends as a gesture of thanks for their support and putting their trust in me. In
the process of studying, researching and implementing the topic, I have also received a
lot of valuable attention, suggestions and support from teachers, colleagues, friends
from Vietnam Japan University.

iv


CHAPTER 1. INTRODUCTION

1.1. Background and motivation of the study
Climate change (CC) is a natural process however anthropogenic activities have
fosterred this process and worsened its impacts (IPCC, 2012). The increase in CO2
concentration in the atmosphere over the past centuries has caused global warming and
led to a series of unpredictable weather phenomena. Prolonged drought that becomes

more severe in high-risk areas is one of the consequences of climate change (IPCC,
2019). Global temperature is expected to increase by 1.5 to 2°C between 2081 and
2100 (Collins et al., 2013). Each increase in the air temperature results in an increase
in the air humidity by 7% (Bui et al., 2019). Therefore, rain becomes more
concentrated during the year and the dry season is longer. In arid sensitive regions
such as the Mediterranean, Northeast Asia, West Asia, parts of South America, and
much of Africa (IPCC, 2019), global warming tend to intensify. During the years 1999
to 2018, the Climate Risk Indicators (CRI) (Eckstein et al., 2019) showed that Vietnam
ranked 6th out of the 10 countries most affected by extreme weather events especially
an increase in the level of drought. The severity of drought is threatening agricultural
production in these regions. Along with global warming, natural drought is one of the
global challenges to agriculture to meet the food demand for the growing world
population. Droughts occur when the effective amount of water in the soil drops while
the air humidity is low, causing the water to continuously evaporate or releasing to the
air. Many researches have demonstrated that droughts inhibit the expansion and
development of plant cells (Shao et al., 2008), reducing dry and fresh biomass
accumulation in plants (Farooq et al., 2009). Especially when a drought occurs during
the development of cereal crab triggering the decrease of grain yield (Kamara et al.,
2003; Monneveux et al., 2006). In general, drought seriously affects the growth of
plants (Fig.1).
Quang Nam is located in the South Central region of Vietnam with its diverse
topography and harsh climate, strongly influenced by drought. According to the report
by the People's Committee of Quang Nam province (2010), prolonged drought caused
losses of 3.841 out of 4.500 hectares of summer-autumn rice crop. In addition, there
1


were more than 3.000 hectares of rice that could not be cultivated due to drought, in
parallel with 5.000 hectares of crops lacked irrigation water in mountainous areas of
Quang Nam province. Right from the beginning of the summer-autumn crop in 2019,

the weather was very unusual with prolonged hot weather that took place continuously
for many days. The amount of irrigation water reduced by 20-60% compared to the
average of many years, of which many dams and lakes were completely dry (EVN,
2019). Soybean is one of the main crops in Vietnam as it has been one of the main
sources of plant protein for the people for decades.. Soybean area in Quang Nam
province tends to expand (report by the People's Committee of Quang Nam province,
2010) but unfavorable weather condition like drought is threatening this expansion.

DROUGHT

Reduce seed
growth

Limiting the
growth of
cell size

Decreased cell
division

Limiting the growth of
plants

Figure 1.1. Effects of drought on plant growth (Jaleel et al., year 2009)
1.2. The necessity of the research
1.2.1. Drought risk in Quang Nam province
Global warming, natural disasters, and drought are global challenges of
agriculture to meet the food needs of the growing world population. Drought occurs
when the amount of water in the soil decreases, and the moisture in the soil is low,
2

causing water to continuously evaporate. An area is called drought when it is
characterized by severe water shortages, to the point of hindering or preventing the
growth and development of plants and animals. An increase in temperature from 1 to
2oC can change an area from semi-arid to arid, resulting in a decrease in agricultural
land. In addition, changes in land use affect soil water preservation and cause drought.
Drought limits plant cell growth and reduces the accumulation of dry and fresh
biomass in plants. Especially, when drought occurs during the grain development
stage, grain size and yield will decrease. In general, drought seriously affects the
development of crops (Fig. 1). Unfortunately, drought is predicted to increase due to
climate change impact in Quang Nam Province.
Annual average temperature in Quang Nam is about 26oC, the average total
number of sunny hours is about 1,850 – 2,250h/year. Quang Nam is located in the
tropical climate zone, a South Central region of Vietnam. Its weather condition was
divided into 2 seasons: the rainy season from September to December and the dry
season from January to August. The average rainfall is about 2000 to 2500 mm/year
and unevenly distributed throughout the year. The rainfall concentrates in the rainy
season and accounts for 82% of the annual rainfall (Vu et al., 2015). In recent years,
due to the impacts of climate change, rainfall in the dry season tends to decrease,
leading to more severe drought so the weather in Quang Nam is more erratic.
Frequently prolonged hot weather and high temperature seriously affect agricultural
production and productivity. Prolonged drought has damaged hundreds of hectares of
crops in the province. Meanwhile, agricultural land in Quang Nam province mainly
receives irrigation water from Thu Bon and Vu Gia basins but recently water resources
in these rivers and other reservoirs continally declines to low and very low levels
especially from March to August annually Joost BUURMAN et al, 2015, causing
saline intrusion which greatly affects water supply for production. Importantly, the
drought occurrence is predicted to increase in Quang Nam in future which challenges
all agricultural plan scenarios by the provincial governors.

1.2.2. Soybean cultivation in Quang Nam province
In Vietnam, soybeans, peanuts, and green beans are common and strategic
crops to be developed by the Government in recent years to meet the needs of crop
3


restructuring in some regions (Decision 3367/QĐ-BNN-TT dated July 31, 2014), and
also for domestic use and export demand of oil vegetable (Worldbank, 2016). The
government has implemented scientific and technological research programs for the
development and expansion of these crops which are input sources of food production
and organic husbandry. Being characterized with the average annual temperature of
26oC and the total average hours of sunshine about 1,850 - 2,250 hours/year (Report
on the situation of agricultural production in Quang Nam province in 2017), Quang
Nam is a good region to grow soybean.
Soybean is a short-term agricultural crop with a wide consumption market and
high processing capacity. Harvested soybean seeds can be processed into a variety of
foods for humans and animals. With the advantage of seed quality, locally grown
soybean products are assessed to be superior in quality compared to products made
from imported soybeans. Recognizing the competitive advantages of domestic soybean
products, the Government has taken positive steps to develop arable land and increase
domestic soybean yield and production. Even so, Vietnam is still facing a decline in
soybean acreage coupled with low crop yields (only about 50% of the worldwide
soybean average yield). Domestic soybean production currently only meets 7% of
actual demand, leading to an increase in imports to meet processing or demand
production and consumption needs (General Statistics Office (GSO), Ministry of
Agriculture and Rural Development, 2018). On the other hand, worldwide researches
have demonstrated that drought is one of the main constrains for soybean productivity
(Manavalan et al., 2009; Wang et al., 2020). Therefore, many studies have been
carried out to improve soybean varieties, increasing yield and its tolerance to extreme
climatic conditions, but the replication of research results is still facing many

difficulties. On the other hand, many of these studies focus on the invention of new
gene-modified species, requiring complex techniques (Nguyen Van Manh et al., 2017)
which takes many years of laboratory testing before being applied in the field and
officially brought to the market (Nguyen Khac Anh et al., 2008; Mai Quang Vinh et
al., 2010, 2012). Meanwhile, the adjustment of fertilizer use and taking use of plant
symbiotic microorganisms can strongly enhance plant nutrient acquisition, and hence,
improve drought resistance capacity.
4


In the crop system of Quang Nam province, soybean is one of the key crops.
According to the socio-economic report in 2018 of Quang Nam province, the soybean
area was 116 hectares. Soybean is also grown in an intercropping model with upland
rice and maize on slope land, acidic soils, medium to poor fertility (Hoang Minh Tam
et al., 2013). According to agricultural experts, soil conditions and the central coastal
climate are very suitable for concentrated commodity-oriented soybean production.
However, soybean production in Quang Nam province is very limited, around 18000
kg to 19000 kg per ha from 2010 to 2014 (Nguyen Van Tuc, 2015). In Quang Nam
province, soybeans are grown in winter-spring and summer-autumn crops on nutrientpoor riverine and hilly alluvial soil. These are mainly fields of 3 crops/year. Soybean
varieties planted by farmers include DTDH.10, DT2008, MTD176, DN29L, DT12,
Dt84, etc. have a short growth period of 90 days, average yield is about 20 quintals/ha.
The summer-autumn soybean growing areas often lack water due to drought.
effectively cope with drought conditions, prolonged drought. Local officials have
actively developed plans for epidemic prevention and control, guiding farmers to
restructure crops in rice-growing areas with difficulties in irrigation water, poor soil
nutrients, and low rice yield. short-term farming (especially soybean). Based on that,
the author selected the research object of soybean in Dai Son commune, Dai Loc
district, Quang Nam province in the dry season to clarify and evaluate the potential of
co-inoculation use (AMF and rhizobium bacteria) and soybean activity to deal with
drought. Research results will be an important scientific basis for making climate

change adaptation strategies in local area.
1.2.3. Mechanisms of soybean and arbuscular Mycorrhiza Fungi (AMF) symbiosis
Soybean (Glycine max L.) is a leguminous species, of Fabaceae family. This
species is able to adapt to various soil properties and climatic conditions so it is
cultivated on large scale worldwide. Although soybean was first introduced in
Southeast-Asia as a source of protein thousands of years ago (Pratap et al., 2016), the
USA and Brazil are leading countries in the world for growing and exporting soybean
to global market for soybean meal and seed oil production. The world demand for
soybean-derived products is increasing as people tend to reduce animal-derived protein

5


and replace with meatlike products, whipped topping, frozen desserts, protein drinks
made of soybean.
During growth, soybeans are cappable of making symbiosis with rhizobium
bacteria and arbuscular mycorrhizal fungi which support the plant to fix nitrogen and
the ability to mobilize nutrients. Arbuscular mycorrhizal fungi faciliate the plant
growth in different ways, such as: i) stabilizing soil aggregates by their mycelium
network (Rillig, 2004), ii) increasing the uptake of inorganic nutrients, mainly P
(Neumann and George, 2010), iii) improving resistance to water limitation (Garg and
Chandel, 2010) and iv) protecting plant from pathogens (Jung et al., 2012). By
extending the mycelium, the plants can make connection with surrounding soil
microhabitats and enlarge the absorption surface for nutrients.
The mechanism behind the symbiosis between AMF and soybean lines in root
exudates. Prior to direct contact, both AMF and soybean release diffusible molecules
playing as signaling molecules and cellular transmission to attract each other. The host
plants exude labile organic compounds to stumulate fungal metabolism and hyphal
branching. The mechanism on the interaction between AMF and soybean was clearly
described by Choi et al. (2018). In details, after receiving reciprocal recognition from

plants, AMF formulates hyphopodia on root surface, which develop on isolated cell
wall fragments of the host. Following epidermal penetration, the fungal mycelium
manupulate inter- and intracellularly through the outer cortical layer via the formation
of PPA. After that, the AMF reach the inner cortex of plant roots, then forms their
branching structure.
1.2.4. Arbuscular Mycorrhiza Fungi (AMF) enhance phosphorus uptake by plants
under drought
Phosphorus (P) is one of the essential elements in plant nutrition and plays a
crucial role in plant processes, such as photosynthesis, root elongation, signal
transduction and nitrogen fixation (Korir et al. 2017). P fertilizer is necessary to
improve drought resitance of plants. Although P accounts for 0.2% of dry plant
biomass, it is the element that is difficult to be absorbed by plants (Smith et al., 2011).
P uptake is more difficult under drought conditions which greatly reduce its mobility,
6


limit root-to-stem P transport rate due to delayed plant evaporation and membrane
permeability. Droughts are exacerbated in subtropical regions (Abrams and Hock,
2006), significantly reducing agricultural crop yields. When the soil moisture drops to
a point that plants cannot absorb water. At the same time, the plant's metabolism along
with the cell structure is completely disrupted, postponing the enzymatic metabolism
of the plant (Smirnoff, 1993; Jaleel, 2007). In soybean, drought reduces stem length
(Specht, 2001; Zhang et al., 2004), slowing leaf growth and narrowing leaf area
(Zhang et al., 2004), leading to a decrease in plant biomass. In addition, soybean yield
was also reduced by 30% due to drought, as shown by a limited number of pods and a
limited seed to fruit ratio (Specht et al., 2001). However, most plants have mechanisms
to adapt to drought conditions, but the degree of adaptation depends on the plant
species (Jaleel et al., 2009). By forming symbiosis with rhizobium bacteria, soybean
can fix nitrogen (N2) from atmosphere to withstand drought conditions and increase
the ability to absorb P. Additionally, soybean is able to make symbiotic relationship

with Arbuscular Mycorrhiza Fungi (AMF) (Shi et al., 2017) which is a group of
microorganisms capable of forming a symbiotic relationship with over 90% of various
higher plant species, especially legumes. In this symbiotic relationship, plants provide
a source of carbon (C) through the fungal root zone secretions to synthesize their
biomass (Smith and Read, 2008; Smith et al., 2011). Meanwhile, mycorrhiza helps to
enhance the mobilization of nutrient P, trace elements, produce more glomalin and
antibiotics, contributes to improving soil structure, increasing resistance of host plants
to disease and water stress (Sanchez-Diaz et al., 1990). The smaller size of
mycorrhizal mycelium (< 1 µm) than roots (Wu et al., 2011) enable their spread to
several centimeters and easily access to various P resources in soil. Hence, P is
transported directly and rapidly into root cell wall (Bolan, 1991). The degree of
symbiosis between fungi and roots is highly dependent on the fungus strain, cultivar
species, and environmental conditions (Shi et al., 2017). Root morphology is one of
the important criteria to evaluate the impact of infertility and fertility factors on plant
growth. For legumes, in the absence of P or low soil moisture, the root morphology is
often altered to adapt to environmental conditions such as more branching formation
(Ruyter-Spira et al., 2011), or the formation of more broom roots (Nuruzzaman et al.,
2006). Furthermore, the symbiosis with mycorrhiza increased the length of the roots,
7


increasing the total root surface area but decreasing the root diameter (Wu et al.,
2011). However, the variation of root morphology in symbiosis with fungi under
drought conditions has not been clarified.
Glomus mosseae (G. Mossea) is a crucial AM fungus species in agricultural
system (Benedetto et al., 2005) that is ubiquitous in worldwide ecosystems. In
Vietnam, G. Mossea was found in Hưng Yên (Hoàng Kim Chi, 2020), Nghe An
province (Nguyễn Thị Kim Liên & nnc., 2012), Mekong delta (Võ Thị Tú Trinh và
Dương Minh, 2017) which means that G. Mossea has a wide range of distribution.
Abdelmoneim et al. (2014) demonstrated the increase of plant P uptake by 88.34 94.91% in G. Mossea inoculated plants under stress condition. Consequently,

mycorrhiza plants synthesize higher biomass and grain yields than non-host plants
affected by drought (Al-Karaki et al., 2004). Therefore, this research chose G. Mossea
as an inoculant to test resistance of soybean to drought.
The mycelium of endosymbiotic mycorrhizal fungi can accumulate nutrients in
the soil faster than the roots and form small branched "suction" hyphae in the humus.
It absorbs nutrients in the same form as the roots, but the mycelium has a better
absorption capacity when phosphorus is in a less soluble form. As a result,
mycorrhizal fungi can access to a form of nutrients that the plant cannot use directly,
and these nutrients are transported to the roots. Therefore, the host plant can better
absorb nutrients from the outside (especially under drought) by binding to the
mycelium and providing the mycelium with the necessary nutrients from the plant
photosynthesis.
1.3. Research framework
In this study, the author will develop a detailed survey and field plan at the
research site. Conduct field surveys in Quang Nam province to collect the following
information: Climatic conditions, weather, drought and water shortage for agricultural
production, losses in agricultural production caused by drought, soil types and annual
cropping status, areas of concentrated soybean cultivation, crop and soybean planting
status. Then proceed to take soil samples for soybean cultivation in the study area, take
samples of topsoil (from 0 to 30cm)/ Number of samples taken: 16 samples.
8


Experimental arrangement under laboratory conditions, soil sample treatment
and seed sterilization. Study on morphology and biomass of soybean roots in dry humid conditions, measure root biomass. Study on the role and mechanism of action
of soybean symbiotic mycorrhizal fungi on phosphorus metabolism in soil under
drought conditions in Quang Nam province.

CLIMATE CHANGE
Precipitation

Temperature

Drought

AMF

Plant root –
microorganisms
Rhizosphere

AMF

Microbial

Soil

Root,

Root, shoot

innoculum

Biomass

respiration

shoot

length

biomass

Phosphorus

Improve

Plant resistance to climate change

Figure 1.2. Research framework
9


1.4. The Research questions and hypotheses
The research was conducted to answer to three questions:
1) How is the drought occurrence in Quang Nam province?
2) How does AMF inoculation improve plant growth under drought effects?
3) How do rhizosphere microbial activities (microbial respiration and microbial
biomass phosphorus) alter as response to drought effects in AMF-host plants?
The hypotheses are:
i) Due to the intense of climate change, drought tends to happen more frequently in
Quang Nam province in future;
ii) AMF inoculation induces plants to increase root biomass and length under drought
effect;
iii) Microbial characteristics as microbial biomass and microbial respiration were
improved by AMF inoculum under drought condition.
1.5. Objectives and scope of the research
Research objectives:
- Providing an overview on drought situation in Quang Nam province;
- Identifying and clarifying the mechanisms and effectiveness of mycorrhiza
(G. Mosseae) in soybean growth under drought condition in Quang Nam province.

Scope of research:
The sample was collected in Dai Son commune, Dai Loc district, Quang Nam
province. The arbuscular mycorrhiza fungi was selected for this research was Glomus
Mosseae (G. Mosseae) and Soybean (Glycine max L.) was utilized for this experiment.
The research mainly focused on some biochemical characteristics of soil collected
within rhizosphere such as microbial respiration, microbial biomass and AMF
inoculation to demonstrate role of AMF to drought resistance of the soybean.
10


CHAPTER 2. MATERIALS AND METHODOLOGIES

2.1. Methods and experimental setup
2.1.1. Collecting secondary data and analysis
Weather information including precipitation, temperature, evaporation, drought
was collected from two meteorological stations that fully observe meteorological
factors for a long time (starting in 1976) namely Tam Ky and Tra My stations. Other
information on population, agricultural production and productivity were collected
from the Provincial People's Committee of Quang Nam and other research materials.
2.1.2. Study site and soil sampling
Surface loam soil (0-30 cm) was taken in soybean-cultivated areas in Dai Loc
district, Quang Nam province (15o49’11’’N, 108o09’77’’E) (Fig 3) to serve for the
experimental arrangement and analysis of soil physical and chemical properties.
2.1.3. Soil property measurement
The characteristics of soil samples including: pH, soil texture, total P, N and C
were measured based on TCVN as mentioned below:

Figure 2.1. Determine Location of sampling area in Quang Nam province using
Google Maps application

11


Table 2.1. Methods to analyze soil physic-chemical properties
Criteria

Methods

pHH2O

TCVN 5979 : 2007

Soil texture

TCVN 8567 : 2010

Total C

TCVN 6642 : 2000

Total N

TCVN 7373 : 2004

Total P

TCVN 7374 : 2004

2.1.4. Defining water holding capacity (WHC)
Water holding capacity (WHC) was determined by modifying methods

proposed by Naeth et al. (1991). Accordingly, 30g soil after sieve of 2mm was placed
in a 100 cm3 cylinder. The cylinder was kept on 20cm sand layer within a big
container, which was then saturated with water for at least 24 hours. After that, water
was drained out of the big container for 24 hours. Finally, soil in the cylinder will be
dried in an oven overnight at 105 oC. WHC was calculated as below:
(Saturated soil Weight – Dried soil Weight)∗100
WHC (%) =
Dried soil Weight
2.1.5. Experimental setup
Soil samples (fresh soil) were sieved through a 2 mm mesh and preserved at 4
o

C until experiment. The soil was mixed with sterilized sand to create a sandy soil

(0.32% C, 0.1% N, 0.19% P2O5) of 72% sand, 16.37% limon and 11.63% clay to avoid
soil cracking during conditional drought. Soil was packed in total 16 transparent
rhizoboxes (20 × 20 × 3 cm) to make bulk density of soil in each box of 1 g cm-3. The
experiment consisted of 4 treatments, including 4 replications (Fig. 4 Soil was packed
in total 16 transparent rhizoboxes):

12


Figure 2.2. Soil was packed in total 16 transparent rhizoboxes
Treatment 1: Soybean with AMF at 60% WHC;
Treatment 2: Soybean no AMF at 60% WHC;
Treatment 3: Soybean with AMF at 20-30% WHC;
Treatment 4: Soybean no AMF at 20-30% WHC.
Glomus mosseae inoculum was provided by Division of Microbiology, Soil and
Fertilizers Research Institute, Vietnam. We choose this fungal species because Glomus

is the major fungal species of arbuscular mycorrhiza which tremendously induces P
uptakes (Tran Van Mao, 2004). Firstly, 20 g of mycorrhiza inoculum (Glomus
mosseae, > 100 cell g-1) (Ruiz-Lozano et al., 2001) was added to 8 rhizoboxes.
Soybean (Glycine max L.) seeds were sterilized respectively with ethanol 70%,
hydropeoxit 10% and rinsed again 3 times with distilled water (Ibiang et al., 2017).
These sterilized seeds were germinated on wet filter paper in a petri dish for 3 days
before transplanting in the rhizoboxes. All the rhizoboxes were kept incline at 45o with
the open side like a door facing down in a greenhouse chamber at 20-22 oC, 12 hours
per day at light intensity (controlled by automatic on/off meter) of 300 mmol m-2 s-1.
The emplacement of the rhizoboxes guarantees root growth along the lower side of the
boxes (Razavi et al., 2016). The rhizoboxes were covered with aluminum foil to
reduce evaporation and prevent algae growth. Soil moisture of each rhizobox was
gravimetrically checked every two days to ensure 60% WHC during the first two
months of the experiment by adding distilled water if necessary. After two months of
optimal condition, soil moisture of 8 rhizoboxes will be reduced to 20-30% WHC and
13


maintained at drought condition for 2 weeks, the remaining boxes were still
maintained at 60% WHC till harvest. Briefly, the experiment took place for two and a
half months.
2.2. Root, shoot biomass and root mycorrhiza inoculation
After the plants were harvested, roots and shoots were separately collected to
measure biomass and length. To collect plant roots, one side of the rhizobox was
opened and the plant was lifted off the box and shaken to remove bulk soil.
Rhizosphere soil samples were collected from root-attached soil after shaking. Roots
were detached from soil, washed under tape water and cut in pieces of 0.5 cm for
staining with ink and vinegar (Vierheilig et al., 1998). Roots were cleared by boiling in
10% (wt/vol) KOH for 5 minutes and then rinsed several times with tap water. As
roots were clear, they are continuously boiled for 3 min in a 5% ink-vinegar solution

with pure white household vinegar (5% acetic acid). Then, roots were rinse in tap
water added with a few drops of vinegar to destain the ink for 20 min. The destained
roots were kept in tap water at room temperature until visualized under 4x
magnification of a light microscopy.
For each experimental treatment, 4 boxes were permanently marked to monitor
the following growth parameters:
Root length (cm): measured from the base with a tape measure
Shoot length (cm): each shoot, mark and track the fixed shoots distributed, each
shoot, measure the length from the location of the shoot to the beginning of the shoot.
2.3. Microbial biomass phosphorus (MBP)
By the end of the experiment, soil attaching to roots after shaking was collected
for further measurement. Microbial biomass phosphorus was determined by
chloroform fumigation-extraction using anion exchange membrane (Kouno et al.,
1995; Yevdokimov and Blagodatskaya, 2014; Maharjan et al., 2018). To prepare for
the experiment, anion exchange membrane (AEM) strips were shaken in 200 mL 0.5
mol L−1 NaHCO3 for 24 h, then washed and kept in deionized water until use.
Subsequently, each 50-mL centrifuge tube contains the same sample equivalent to 3 g
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dry soil was filled with 30 mL of deionized water. The fumigated tube was added with
300 μl of chloroform to solubilize microbial biomass P, followed by placing one anion
exchange membrane (AEM) strip in both fumigated and non-fumigated tubes. These
tubes were shaken for 24 h continuously to induce the recovery of inorganic P from
the soil extract. After shaking, AEM strips were lifted out of the tubes and gently
washed again in deionized water prior to being submerged into another centrifuge tube
filled with 45 mL of 0.25 M H2SO4. Tubes containing membranes and 0.25 M H2SO4
were shaken for three hours to release membrane-fixed P back to the solution. 150 μL
acid extract was mixed with 30 μl Reagent 1 (prepared with 14.2 mmol L-1 ammonium
molybdate tetrahydrate in H2SO4 3.1 M) and Reagent 2 (prepared with 3.5 g L-1

aqueous polyvinyl alcohol reagent and malachite green) (D'Angelo et al., 2001) in
microplates. These microplates were exposed to 40 oC for 30 – 40 min in a dryer
(thermostat) and read at 630 nm in CLARIOstar plus (BMG LABTECH, Germany).

(a)

(b)

Figure 2.3. Microbial biomass P was measured in 96-wells microplates: a) the
calibration was prepared for different concentrations of P and b) the microbial biomass
was calculated based on the difference between fumigation and non-fumigation
treatments
2.4. Microbial respiration
Microbial respiration was measured using MicroRespTM method. A volume of
soil with a dry weight of 30gwas weighted in deepwell plate and covered with an agar
layer. The plates were incubated for one week and measured on microplate reader
connected to computer. The CO2 rate is calculated by converting the 6h % CO2 to
μg/g/h CO2-C using gas constants and constants for headspace volume in the well (945
μl), fresh weight of soil per well (g), incubation time (h) and soil sample % dry weight.
15


(

)
(

)
(

(

)

(

(

))

)

Note: - Fwt: fresh weight incubation time
- Dwt: dry weight incubation time
- vol: headspace volume
2.5. Statistical analysis
The significant differences of microbial biomass P and microbial respiration
between droughts vs control with AMF vs without AMF were confirmed by Two-Way
ANOVA after checking normality and homogeneity of values. A probability level of p
< 0.05 indicated the significance in comparison. Error bars demonstrated the standard
error of the means.

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