II.

GIỚI THIỆU TỔNG QUAN (PROJECT DESCRIPTION):

Giới thiệu tóm tắt về đề tài (nêu vấn đề, mục tiêu và phương pháp nghiên cứu, không quá 300
từ) (Brief introduction of project (context, objectives and research methodology) maximum
300 words).
Rice (Oryza sativa) is among the most economically important crops in many countries
including Vietnam (Khanh et al., 2021). However, production of rice in Vietnam and other
countries is threatened by pests and diseases causing huge losses (Khanh et al., 2021),
particularly, bacterial leaf blight, blast and sheath blight are considered as major constraints in
rice cultivation. In modern society, farmers usually use agrochemicals such as chemical
fertilisers, herbicides, and pesticides to gain higher productivity and prevent pathogens and
weeds (Elahi et al., 2019). Applying agrochemicals in paddies is considered an easy solution to
control diseases and maintain high yield; however, the overuse of these chemicals might cause
tremendous impact on environment, public health, and non-target organisms due to the high
levels of toxic residues (Ali et al., 2017). At the same time, prolonged agrochemical exposure
of paddies also reduces their effectiveness and might increase the number of insecticide-
resistant strains (Fitri et al., 2020). In Vietnam, during the period of 1995 – 2018, the positive
correlations between the amount of rice production and the use of pesticides indicated the
intractable routines of farmers in using of pesticides for rice production. Moreover, the improper
use of pesticides by farmers (e.g., too high dosages, cocktailing of pesticides, inadequate pre-
harvest intervals etc.) leads to the heavy dependence of pesticides in rice production, especially
in poorer areas where farmers have to largely rely on cheap but often with old and more toxic
pesticides. This problem need to be urgently addressed because it causes significant adverse
impacts on food safety, soil, water environment, and human health (Pham et al., 2013 ; Le and
Tran, 2020). Thus, alternative solutions are being developed to alleviate the dependence of
agrochemicals. Beneficial endophytic bacteria (EB) have been studied as an alternative measure
to improve crop yield, grain quality, and to prevent pathogens; at the same time, the use of
endophytes does not cause serious damage to the crops and ecosystem (Moronta-Barrios et al.,
2018 ; Fitri et al., 2020).
EB colonise internal plant tissues without causing infection or damage (Schulz and
Boyle, 2006). EB are found in many plant crops including rice (Sen and Chandrasekhar, 2014 ;
Shen et al., 2019 ; Singh et al., 2019), and their relationships with host plants have been widely
studied (Reinhold-Hurek and Hurek, 2011). These interactions have been shown to benefit the
hosts, namely, promoting growth via phytohormone biosynthesis, nitrogen fixation, nutrient
solubilisation, and phytopathogen suppression through antibiotic production and/or cell wall-
degrading enzymes like matrix metalloproteinases (Inoue et al., 2007; Olanrewaju et al., 2017).
These beneficial properties make EB promising candidates for biocontrol agents (Olanrewaju
et al., 2017).
Taking advantage from my own previous studies, we propose further evaluating the
antifungal activity of rice root endophytic bacteria against Magnaporthe oryzea (Mo) causing
rice blast disease on rice. The Mo strains were isolated from infected rice in the North of
Vietnam during the main season of 2018. Paralelly, we also would like to screen the
antifungal activity of rice root EB against Fusarium spp. causing wilt and root rot diseases on
different crops.

3
III. TỔNG QUAN TÌNH HÌNH NGHIÊN CỨU VÀ SỰ CẦN THIẾT TIẾN HÀNH
NGHIÊN CỨU (STATE OF THE ART AND NECESSITY TO CONDUCT RESEARCH)
3.1. Tình hình nghiên cứu trong và ngoài nước (Research context in Vietnam and
abroad):
- Nêu rõ những nghiên cứu do các nhà khoa học nước ngoài, trong nước tiến hành theo hướng
nghiên cứu của đề tài và các kết quả đạt được, đồng thời nhận xét về những kết quả đã nêu
(Indicate the research framework in Vietnam and abroad of the research project, achieved
results and comments on these results).
In recent years, the interest of scientists around the world has been focused on
researching and finding rhizobacteria that are able to promote plant growth and/or inhibit plant
pathogens. These studies mainly focused on exploring the mechanism of action of Plant Growth
Promotion Rhizobacteria (PGPR) on plants in a direct or indirect manner. In the direct manner,
PGPR can have the ability to produce ammonia, phytohormones, siderophore, etc. that are
directly served as nutrition for the plant development. Besides that, some PGPR are able to
produce antibiotics and/or hydrolytic enzymes that inhibit the growth of some plant pathogens
(Kumar et al., 2011). Rice endophytes, such as the strains of Burkholderia sp., Enterobacter
sp., Pantoea sp., Pseudomonas sp., and Sphingomonas sp., have exhibited the ability to produce
phytohormones such as indole-3- acetic acid (IAA) and siderophore (Wang et al., 2016).
In 2004, Kloepper et al. used Bacilli strains such as B. amyloliquefaciens, B. subtilis, Bacillus
pasteurii, Bacillus cereus, Bacillus pumilus, Bacillus mycoides, and B. sphaericus to study the
antagonistic activity against pathogens on various crops. These Bacilli strains are able to induce
systemic resistance on tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.),
maskmelon (Cucumis melo L.), watermelon (Citrulus lanatus [Thunb.] Matsum. and Nakai),
suger beet (Beta vulgaris L.), tobaco (Nicotiana tabacum L.), Arabidopsis spp., and cucumber
(Cucumis sativus L.) against the diseases caused by fungi, bacteria, viruses, and nematodes.
This research indicated that most of the studied strains have both the ability to induce sytemic
resistance and promote the plant growth.
Although there are many studies on PGPR worldwide, they mostly focuses on
vegetables but rarely on the rice. In 2011, Chithrashree et al. investigated 7 Bacilli strains on
the rice. The results showed that these strains possess PGP properties and antibacterial activity
against Xanthomonas oryzea pv. oryzea (Xoo) causing the leaf blight disease (Chithrashree et
al., 2011).
In vitro experiments have shown that endophytic bacteria including strains of Bacillus,
Klebsiella, and Streptomyces could effectively suppress the growth of some phytopathogenic
bacteria and/or fungi such as Xoo, Burkholderia glumae, F. oxysporum, Rhizoctonia solani,
Magnaporthe oryzea (Naik et al., 2009; Ikeda et al., 2010; Ji et al., 2014; Chung et al., 2015).
In 2013 and 2014, Nagendran et al., reported that the bacterial endophytes Bacillus subtilis var.
amyloliquefaciens (FZB24) isolated from different plant sources showed their efficacy against
Xoo and Rhizoctonia solani inciting sheath blight in rice with a maximum 31.36 % and 36%
inhibition of Xoo and R.solani, respectively (Nagendran et al., 2014).
In 2016, Bertani and her colleagues implemented a research on rice microbiome in
greenhouse in Italia. Twenty five bacterial species isolated from three developmental stages
(tillering, boosting and maturation) were identified. Among these strains, some are able to
promote the growth of rice in nethouse conditions (Bertani et al., 2016).

4
 In 2020, Kumar et al. investigated 32 bacterial endophytes isolated from four plant
tissues (root, stem, leaf, and grain) of six rice varieties cultivated in central-eastern and
northeastern states of India. They found that, these bacterial isolates belonging to phyla
Proteobacteria and Firmicutes. Out of 32 isolates, 17 (53.1%) were able to produce indole
acetic acid, 9 (28.1%) siderophore and 18 (56.2%) phosphate solubilization activities indicating
the plant growth-promoting (PGP) ability. The antagonistic activity of identified bacterial
endophytes was determined against bacterial leaf blight disease-causing pathogen, Xoo (8
isolates) and soilborne fungal pathogens viz, Rhizoctonia solani (17 isolates), Fusarium
verticillioides (15 isolates) and Sclerotium rolfsii (9 isolates) were exhibiting antagonistic
activities against tested pathogens (Kumar et al., 2020).
Despite the numerous beneficial effects of endophytic bacteria on plant growth
promotion and antagonistic activities, the EB diversity in important rice varieties grown in
Vietnam in general and in the North of Vietnam, in particular had rarely been discovered.
However, for the last decades, the studies on rhizospheric microbiome in order to reveal
potential PGPR were increasing. Although, scientists also focus on other crops rather than rice.
Studies have been reported on isolation and characterization of endophytic bacteria for their
PGP properties. In 2015, Cao Ngoc Diep and Nguyen Thi Mong Huyen isolated 36 endophytic
bacteria from sweet potato (Ipomea batatas) culvivated in Hon Dat, Kien Giang. These isolates
were characterized for PGP properties such as nitrogen fixation, phosphate solubilization and
IAA production (Cao Ngọc Điệp and Nguyễn Thị Mộng Huyền, 2015). In another research,
endophytic bacteria from ananas (Ananas comosus) cultivated in Vinh Thuan, Kien Giang were
also isolated and characterized for PGP properties. In 2014, Van Thi Phuong Nhu and Cao Ngoc
Diep isolated and characterized the PGP properties of rice endophytic bacteria in Phu Yen
province (Van Thi Phuong Nhu and Cao Ngoc Diep, 2014). The especial attention was
dedicated to the studies of Duong Van Hop et al. (2014) for their evaluating the effects of
Streptomycetes, Pseudomonas stutzeri A15 and strains on the growth of rice seedlings in net
house conditions and against Xoo (Dương Văn Hợp et al., 2014). Especially, from 2017 – 2021,
Nguyen Van Phuong et al., have in vitro characterized the rice root endophytic bacteria isolated
from submerged and upland paddies in the North of Vietnam for their PGP properties and
antagonistic activities against Xoo, Magnaporthe oryzea (Mo) and Fusarium oxysporum (Fo)
(Nguyen Van Phuong et al., 2020; Ta Thi Thuy Linh et al., 2020; Nguyen Van Phuong et al.,
2021).
Briefly, in the recent decades, a huge attention was focusing on the studies of endophytic
bacteria in order to develop an eco-friendly sustainable agriculture worldwide. Though, the
studies on rice endophytic bacteria are limited in the world as well as in Vietnam. Moreover,
Vietnam is an agricultural country with diversity of agro-ecosystems from the north to the south
of more than two thousands kilometers. The diversity of agro-ecosystems not only leads to the
biodiversity of rice species, from upland to the submerged but also leads to the biodiversity of
plant-associated microbiome and phytopathogens. Therefore, more deep comprehension of the
existence, biodiversity and interaction of rice endophytic bacteria with their host will serve as
active tools to manage the plant growth and biocontrol against pathogens without using or less
dependance of agrochemicals.
- Nêu mức độ cập nhật của các nghiên cứu trong nước so với các nghiên cứu mà các nhà khoa
học của các nước tiên tiến đang tiến hành theo cùng hướng nghiên cứu. Những tổ chức khoa

5
học đang tiến hành những nghiên cứu liên quan (Indicate a comparison of the project with
respect to Vietnamese and foreign context).
Endophytic bacteria have been reported to possess different efficient beneficial traits
that not only induce denfense genes of host plants (Gond et al., 2015), but also may suppress
fungal diseases by resource competition and antibiosis of pathogens (Verma and White, 2018).
For the last decades, majority of studies focused on isolation and characterization of endophytic
bacteria for PGP traits and antagonistic activities by plate assays. Only few studies analyzed
further the molecular mechanisms of antagonism at level of genes or secondary metabolites
produced by endophytes. A wide variety of bioactive compounds involved in pathogen control
have been identified for endophytes. These include siderophores, hydrogen cyanide, 2,4-
diacetylphloroglucinol, pyrrolnitrin, pyoluteorin, phenazines, 2,5-dialkylresorcinol,
quinolones, gluconic acid, rhamnolipids, and various structurally diverse lipopeptides (Gross
and Loper, 2009; D’aes et al., 2014; Van Der Voort et al., 2015).
Recently, studies on endophytic bacteria also attracted the attention of different research
groups of Vietnamese scientists. However, their studies are limited to the isolation and
identification of bacteria but rarely implement to identify which bioactive compounds involved
in antagonism. Therefore, in this project, we want to detect different genes encoding for
different lipopeptides that have been reported to be involved in biocontrol against pathogens.
- Nêu những kết quả nghiên cứu của chủ nhiệm đề tài đã đạt được theo hướng nghiên cứu của
đề tài (Indicate achieved results of the PI with the same orientation of the project).
During the period of 2017-2021, I was a PI of a research project on the search for
efficient plant growth promoting and biocontrol bacteria from rice roots in the North of
Vietnam. This project was funded by GUST, VAST and was successfully finished. In the frame
of this project, I have isolated 80 and 150 rice root endophytic bacteria from submerged and
upland paddies, respectively. These strains have been characterized by in vitro qualitative
assays for plant growth promoting traits and biocontrol activities against Xanthomonas oryzea
pv. oryzea, Magnaporthe oryzea and Fusarium oxysporum. The obtained data have been
published in the 02 international papers (01 Scopus, SCIE and 01 ISSN), 01 national paper
(VAST2), 01 full article and 01 poster in the proceedings of the National Conference of
Biotechnology 2020. Surely, these results will be served as the basic database for further studies
on isolated bacteria by quantitative assays for the most important PGP traits and antifungal
activities against popular fungal pathogens.
3.2. Sự cần thiết tiến hành nghiên cứu (Necessity to conduct research):
Nêu rõ vấn đề mà đề tài tập trung giải quyết; phân tích tính mới, tính thời sự, ý nghĩa khoa học
và sự cần thiết của vấn đề cần nghiên cứu (Indicate issues that the project focuses on; analyze
the novelty, timeline, scientific value and necessity of the research issues).
The global population has been forecasted to reach nine billion by 2050. However, the
global cereal crop production is predicted to be suppled only maximum for half of the
population by 2050, therefore it needs to increase by 50% to meet the demand of the world
population at that time. To keep this development in crop production, the farmers are increasing
the application of chemical fertilizers and pesticides in agriculture. This manner usually is
uncontrolled in the poor and developing countries including Vietnam leading to different effects
not only on the quality of agricultural products but also on human health and ecosystems. These
unsustainable agricultural practices have brought us to the situation that there is an urgent need

6
to have a revolution in agricultural production to ensure future sustainability and food security.
This should take us to come back to the nature and rely on the indigenous living things – plant
and/or soil-associated microorganisms that possess different efficient beneficial traits for plant
growth and development and inhibition of phytopathogens. Colonizing by these microbes, crops
can grow well and maintain productivity under nutrient-poor environments.

Indole-3-acetic acid (IAA), a plant growth hormone, functions as an important signalling

molecule in regulating plant development (Mockaitis and Estelle, 2008). Different factors affect
the IAA production from plant growth-promoting (PGP) rhizobacteria, such as types of species
and strains, culture conditions, growth stage, and substrate sources (Tan et al., 2014). The
inoculation of IAA-producing EB could be considered a promising way to enhance plant
biomass, root length, root tip number, and root surface area (Chen et al., 2017)
Iron is a micronutrient needed for the development of plants and microorganisms.
However, it is scarce in neutral to alkaline soils, which makes plants iron deficient (Rout and
Sahoo, 2015). Siderophores are chelating agents with low molecular weights (200–2,000 Da)
and high Fe3+ chelating affinity for facilitating iron uptake (Singh et al., 2019). Plants and other
microorganisms benefit from siderophore-producing EB that can solubilise and chelate iron
from insoluble organic and inorganic sources in the soil (Singh et al., 2019). Siderophore-
producing bacterial genera include Pseudomonas, Azotobacter, Bacillus, Rhizobium,
Agrobacterium, Staphylococcus, Burkholderia, Erwinia, Serratia, Alcaligenes, and
Enterobacter (Sayyed et al., 2012 ; Gupta et al., 2015). Thus, siderophores not only promote
plant growth but also aid in plant defence against phytopathogens that are inhibited by iron
starvation or competitive exclusion. Furthermore, EB have been widely known for their
antagonistic activities such as competing for substrates or producing inhibitory compounds. The
latter is critical for inducing systemic resistance in plants (Gupta et al., 2015).

Phosphorus is one of the essential macronutrients required for plant growth and
development (Dong et al., 2019). Phosphorus normally exists in forms of organic and inorganic
phosphates, which are either insoluble or very poorly soluble. The total available phosphates
for plants in soil are very low since most occur as metal phosphates with iron, calcium, and
aluminium, which cannot be taken up by plants (Kirkby and Johnston, 2008). Therefore,
phosphorus fertilisers are usually applied to croplands (Walia et al., 2017). Different
microorganisms, dominated by bacteria, can produce soluble phosphorus via acidification,
chelation, and exchange reactions. Acinetobacter, Azotobacter, Enterobacter, Bacillus,
Burkholderia, Paenibacillus, Pseudomonas, Rhizobium, Mesorhizobium, Flavobacterium,
Klebsiella, Erwinia, and Micrococcus have been reported as efficient phosphate solubilisers
(Villegas and Fortin, 2002; Paul and Sinha, 2013).

IV. MỤC TIÊU CỦA ĐỀ TÀI (OBJECTIVES)

Nêu rõ mục tiêu cần đạt được của đề tài, làm cơ sở xác định nội dung nghiên cứu và kế hoạch
triển khai (Indicate main objectives, research contents and implementation plan).
In this project, first, we will investigate further PGP properties such as Indole Acetic
Acid (IAA) and siderophore production, phosphate solubilization by quantitative assays on 80
rice root endophytic bacteria (EB) isolated from submerged paddy in Hai Duong province. Then
the antifungal activity will be tested in vitro against Magnaporthe oryzea (10 strains), Fusarium
oxysporum (1 strains) and Fusarium oxysporum f.sp.cubense (2 strains). Finally, the endophytic
7
bacterial strains showing strong antifungal activity will be analyzed further to detect the
presence of lipopeptide genes such as surfactin, Iturin A, bacillomycin D and fengycin.
Feasibility of success. Taking advantage of previous project, I have a lot of experience
in these experiments. I have the capability to manage the project to be finished on time and
achieved all main proposed objectives. Moreover, in our team, all members have been working
for a long time in microbiology, molecular biology, plant physiology, ect. We have been
working together for several years; therefore, we will encourage each other and share our
contributions properly. In recent years, Dr. Nguyen Quang Huy and Dr. Pham Hoang Nam who
spent much of time on microbial research and achieved much success in this field, will actively
contribute their role in the work packages 1 and 2. While Dr. Mai Thi Phuong Nga and Dr. Le
Thi Van Anh who have good background and skills in molecular biology and plant physiology,
will actively contribute in work package 3 and also collaborate with the others in two previous
packages.
Our project will be carried out at the laboratories of USTH, where the research
equipment and all needed facilities have been intensively equipped for the last few years.
Therefore, taken together of all factors above, I strongly believe that the capacity for
success of this project is feasible.

V. CÁCH TIẾP CẬN & PHƯƠNG PHÁP NGHIÊN CỨU (APPROACHES &
RESEARCH METHODS)
Mô tả chi tiết cách tiếp cận, phương pháp nghiên cứu, kỹ thuật sử dụng phù hợp với từng nội
dung của đề tài nhằm đạt được mục tiêu của đề tài (Detailed description of approaches,
research methods, techniques of each research content).
5.1. Quantitative assays for PGP properties: IAA and siderophore production, and
phosphate solubilization.
For these tests, the bacterial isolates will be pre-cultured under constant agitation with
the aid of an orbital shaker at 160 rpm for 24 h at 28 ± 2 °C in 10 mL of 1/6 Tryptic Soy Broth
(TSB). The optical density (OD) at 600 nm of all bacterial suspensions will be adjusted to 0.1
by dilution with saline solution sterile (0.85%).
The IAA production of rice root endophytic bacteria will be quantitatively determined
by colorimetric technique described by Glickmann and Dessaux (1995) with some
modifications. Briefly, 100 µL of pre-cultured suspension will be added to 10 mL of 1/6TSB
supplemented with 5 mM L-Tryptophan. The test tubes will be incubated on an orbital shaker
at 160 rpm for 5 days at 28 ± 2 °C. The broth will be centrifuged at 10,000 rpm for 15 min. One
mL of sepernatant will be collected and added an equal volume of Salkowski reagent (1 ml of
0.5M FeCl3 in 49 ml of 35% perchloric acid (HClO4)). The mixture will be throughly mixed
and incubated in the dark for 30 min. The absorbance will be obsevered at 530 nm by UV-1800
UV-VIS Spectrophotometer (Shimadzu, Tokyo, Japan). The amount of IAA will be estimated
using a standard curve with its linear regression line made by IAA standard (10-100 µg/mL)
(Gordon and Weber, 1951; Glickmann and Dessaux, 1995).
The siderophore production by rice root endophytic bacteria will be quantitatively
estimated by CAS-shuttle assay (Schwyn and Neilands, 1987; Alexander and Zuberer, 1991).
The strains will be grown in 1/6TSB and incubated for 7 days at 28 ± 2 °C with constant shaking
at 160 rpm on rotator shaking incubator. After incubation the fermented broth will be taken and
centrifuged at 10,000 rpm in a cooling centrifuge at 4 °C for 10 min and cell-free supernatant
8
will be mixed with 0.5 ml CAS solution. The color obtained will be determined using the UV-
1800 UV-VIS Spectrophotometer at absorbance 630 nm after 20 minutes of incubation with
reference containing 0.5 ml CAS solution with 0.5ml uninoculated 1/6TSB medium. The
percentage of siderophore units was estimated as the proportion of CAS color shifted using the
formula
𝐴𝑟 − 𝐴𝑠
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑆𝑖𝑑𝑒𝑟𝑜𝑝ℎ𝑜𝑟𝑒 𝑈𝑛𝑖𝑡 = [ ] × 100
𝐴𝑟
where Ar is the Absorbance of reference (CAS assay solution + uninoculated media) and As is
the Absorbance of the sample (CAS assay solution + cell-free supernatant).
The quantitative analysis of phosphate solubilization potential of EB isolates will be
measured in vitro by determining available soluble phosphate in the liquid medium of National
Botanical Research Institute’s Phosphate (NBRIP) supplemented with 0.5% of tricalcium
phosphate (Nautiyal, 1999). The medium will be inoculated in triplicate with phosphate
solubilizing bacterial strains. The flasks will be incubated at 28 ± 2 °C for 5 days on rotary
shaker at 160 rpm and centrifuged at 10,000 rpm for 10 min. Phosphomolybdate method will
be used for determination of available soluble phosphate in culture supernatant (Murphy and
Riley, 1962). The pH of the broth medium was also measured with a digital pH meter (Jenway
3510) after regular intervals.
5.2. Antagonistic test against soil-borne fungal pathogens.
Rice root endophytic bacteria will be tested in vitro for their antagonistic activity toward
the soil-borne plant pathogenic fungi Magnaporthe oryzea, Fusarium oxysporum and Fusarium
oxysporum f. sp. cubense using a dual culture approach. These pathogenic fungal strains will
be provided by Agricultural Genetics Institute and Vietnam National University of Agriculture.
A 6 mm mycelial disk of each fungal pathogen from 5-day old culture will be placed on the
center of a potato dextrose agar (PDA) plate and each endophytic bacterial isolate will be
streaked at distance of 20 mm from fungal disk. The plates with only fungal culture will be
served as control. The PDA plates will be incubated at 28 ± 2°C for 5 days or till fungal growth
reaches the edge of the plates in the control. The antagonistic activities of the isolates will be
indicated by the inhibition zones formed between the endophytic bacterial and fungal isolates
(Edi-Premono et al., 1996). Percent inhibition of fungal growth will be compared with control
using the formula
𝐶−𝑇
𝐼=[ ] × 100
𝐶
where I is the percent inhibition, C and T are the radial growth of the pathogen in control and
treatment, respectively. EB isolates that showed more than 50% inhibition against any of the
three pathogens will be further analyzed (Dinesh et al., 2015; Nguyen et al., 2020). All dual
culture assays will be carried out in triplicates.
5.3. Detection of lipopetide genes using PCR.
It has been reported that different endophytic taxa produce molecules of lipopeptides
(LPs) molecules that play a crucial role in biocontrol acting directly as antimicrobial/antifungal
compounds and as ISR elicitors in plant hosts. These amphiphilic compounds are formed by a
short cyclic oligopeptide linked to a lipid tail (Pérez-García et al., 2011; Nigris et al., 2018).
The most widely studied LPs belong to the surfactin, iturin and fengycin families, according to
their chemical structure. Surfactins are heptapeptides interlinked with a β-hydroxy fatty acid to
form a cyclic lactone ring; due to their strong bio-surfactant activity, these molecules can readily
9
associate and anchor themselves to the double layer of phospholipids interfering with membrane
integrity. Iturins are heptapeptides bound to a β-amino fatty acid chain 14–17 carbons long.
Fengycins are lipodecapeptides with an internal lactonering in the peptidic moiety and a β-
hydroxy fatty acid chain (C14-C18) that may be saturated or unsaturated (Ongena et al., 2007).
These molecules, according to their chemical characteristics, fight bacteria, fungi, mycoplasmas
and viruses. Due to their strong surfactant power, LPs enable and favor plant colonization by
the producer strain hindering pathogenic tissue infection. Bacteria synthesize these families of
lipopeptides in a non-ribosomal way through large enzymatic complexes, namely, lipopeptide
synthetases. These mega-enzymes are organized in iterative modules that catalyze reactions for
lipopeptide production (Nigris et al., 2018).
The rice root endophytic bacterial isolates showing strong antifungal activity will be
screened further to detect the presence of lipopeptide genes such as surfactin (Sfp and Srf C),
iturin A (ItuD), bacillomycin D (Bam C), and fengycin (Fen D). The five pairs of gene-specific
primers previously reported by Gond et al., (2015) and Kumar et al., (2020). PCR amplification
of these genes will be performed with the reaction condition of initial denaturation at 95 °C for
5 min followed by denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension
at 72 °C for 1 min for 35 cycles and a final extension at 72 °C for 7 min.

Collection of 80
submerged rice root
i endophytic bacteria
WP1 WP2
Quantitative Dual antifungal assays
characterization of PGP against
traits
Auxin production Magnaporthe oryzea

| Siderophore production Fusarium oxysporum

P solubilization Fusarium oxysporum f.

sp. cubense

WP3 /
Detection of lipopeptide
genes

fn
Identification of effective EB for further
inplanta assays

Figure 1. Work flow of project.

10
 The rice root endophytic bacterial isolates showing strong antifungal activity will be
screened further to detect the presence of lipopeptide genes such as surfactin (Sfp and Srf C ),
iturin A ( ItuD ) , bacillomycin D ( Bam Q , and fengycin ( Fen D). The five pairs of gene specific
-
primers were previously reported by Gond et al., (2015) and Kumar et al., (2020). PCR
amplification of these genes will be performed with the reaction condition of initial denaturation
at 95 °C for 5 min followed by denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min
and extension at 72 °C for 1 min for 35 cycles and a final extension at 72 °C for 7 min.
5.4. Evaluation of antifungal activities in planta
The potential EB strains obtained from above experiments will be evaluated their antifungal
activities in planta. This experiment will focus only on the rice leaf blast fungal strains. The
rice varieties DH40, Jasmine 85, and BC15 which sentitive with tested fungal strains in this
study will be used for this experiment. The steps of this experiment are described in the Figure
2.

Surface sterilized rice seeds

i
Germination on water agar 0.8%

1
3-day old rice seedlings
Incubation
Potential EB strains
for 60 min
I
Rice seedlings on pots in nethouse

1 Spray on
30-day old rice plants Fungal spore suspention
on pots in nethouse the leaf 105 spores/mL

1
Estimation of disease symptom
at 7 days post infection

Figure 2. Flow chart of evaluation of antifungal activities in rice plant.

VI. NQI DUNG NGHIEN CtfU VA TIEN DO THUfC HIEN (RESEARCH CONTENTS
AND IMPLEMENTATION TIME)
6.1 Noi dung nghien cifu (Mo ta chi tiet rihung noi dung nghien cicu cua de tai/ ....) (Research
contents (Detaileddescription about research contents/ ...))
This project contains three work packages (WP) including in vitro quantitative assays for
PGP properties (Auxin and siderophore production, and phosphate solubilizaton) (WP1);
antagonistic tests against soil-borne fungal pathogens (WP2); detection of lipopeptide genes
using PCR ( WP3), and evaluation of antifungal activities of potential EB isolates against fungal
pathogens in planta ( WP4) (Figure 1 ) .
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 The WP1 and WP2 can be independently paralelly carried out. While WP2 and WP3 have
a strong connection. In the WP3, experiments will be performed only for those strains showing
antifungal activities against at least one tested pathogenic fungi. Afterward, based on the results
obtained from previous three packeages, the WP4 will be carried out inplanta with the potential
EB strains.
WP1. In vitro quantitative assays for PGP traits: IAA and siderophore production, and
phosphate solubilisation
In this work package, eighty EB isolates will be applied for in vitro quantitative assays for IAA
and siderophore production and phosphate solubilisation (For more details, see 5.1).
WP2. Antagonistic test against soil - borne fungal pathogens
This work package will be paralelly carried out with the WP1. Eighty rice root endophytic
bacterial isolates will be tested in vitro for their antagonistic activity against 05 strains of
Magnaporthe oryzea, 01 strains of Fusarium oxysporum, and 02 strains of Fusarium oxysporum
f. sp. cubense using a dual culture approach. The potential antifungal strains will be considered
when they will show the inhibition ratio 50%.
WP3. Detection of lipopeptide genes using PCR
This work package will be carried out with the potential EB strains obtained from previous
work packages. The potential EB strains should be possess all PGP traits tested in the WP1&2
and show their antifungal activities against at least one tested fungal strains.
WP4. Evaluation of antifungal activities in planta
The potential EB strains obtained in the WP2 will be evaluated their antifungal activites against
M. oryzea. We will focus on the rice leaf blast fungi in this experiment. Three rice varieties
which are sensitive with blast disease will be tested. The disease symptoms will be estimated
following the Standard Evaluation System for Rice (SES, IRRI, 2013).
6.2 Tien do thirc hien ( Implementation time)

Thoi gian
Cac noi dung, cong ( bat dau -ket
N° viec thyc hien
San pham thuc ) Nguoi thuc hien
(Contents)
(Outcomes) Implementation (Implementor)
time
-
(Start complete )
WP1 . In vitro - A list of strains 03/2022 - Nguyen Van Phuong
quantitative assays possessing ability to 09/2022
for PGP properties: tLe Thi Van Anh
produce IAA
IAA and siderophore - A list of strains Mai Thi Phuong Nga
production, and possessing ability to
phosphate produce siderophore Pham Hoang Nafri
solubilization - A list of strains Nguyen Quang Huy
possessing ability to i
solubilize tricalcium
phosphate

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2 WP2. Antagonistic - A list of strains 10/2022 - Nguyen Van Phuong
tests against soil possessing ability to 04/2023
Le Thi Van Anh
borne fungal inhibit the growth of
pathogens Magnaporthe oryzea Mai Thi Phuong Nga
- A list of strains !
Pham Hoang Nam
possessing ability to f
inhibit the growth of Nguyen Quang Huy 2
Fusarium oxysporum.
- A list of strains
I
possessing ability to
I
inhibit the growth of i
Fusarium oxysporum f.
sp. cubense.

3 WP3. Detection of - A list of strains carrying 05/2023 - Nguyen Van Phuong

lipopeptide genes the lipopeptide gene(s) 06/2023
Le Thi Van Anh
using PCR
Mai Thi Phuong Nga
Pham Hoang Nam
Nguyen Quang Huy

4 WP4. Evaluation of - A list of potential EB 06/2023 - Nguyen Van Phuong

antifungal activities strains possessing 12/2023
Le Thi Van Anh
in planta antifungal activity
against M. oryzea Mai Thi Phuong Nga
Pham Hoang Nam
Nguyen Quang Huy

5 Writing and submit Submitted manuscript (s) 06/2023 - Nguyen Van Phuong
papers 12/2023
Le Thi Van Anh
Mai Thi Phuong Nga
Pham Hoang Nam
Nguyen Quang Huy

6 Writing final report A final report 01/2024 - Nguyen Van Phuong

02/2024
Le Thi Van Anh
Mai Thi Phuong Nga
Pham Hoang Nam
Nguyen Quang Huy

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