Chitosan application on agriculture

Research and Application of Chitosan OligoSaccharide (COS)
in Agriculture
Yuguang Du
Institute of process engineering, Chinese Academy of Sciences

Contents
中 心 第 一 批 启 动 的 项 目
Background
Function and mechanism of COS
Key technology in production of COS
Application of COS in agriculture

Contents
中 心 第 一 批 启 动 的 项 目
Background
Function and mechanism of COS
Key technology in production of COS
Application of COS in agriculture

The plant immune system &
中
plant
心 第
vaccine
一 批 启 动 的 项 目
• Plant responds to infection using an innate immune system
• Chitosan Oligosaccharide is a natural biomacromolecular that can significantly trigger plant innate
immune system, just like plant vaccine
Jonathan and Jeffery, Science, 2006(444): 323-328
Heng and Du, Carbohydrate, 2010(82):1-8

What is Chitosan OligoSaccharide
中 心 第
(COS)?
一 批 启 动 的 项 目
• COS is composed of 2-20 β-1,4 linked glucosamine group;
• COS mainly exists in fungal cell wall, crustacean shells and insect shells;
• COS is regarded as a key signal molecular when plant recognizes fungal diseases;
Nour Eddine El Gueddari et al., New phytologist, 2002(156): 103-112

What we have done?
中 心 第 一 批 启 动 的 项 目
Staring from 1997, with the support of several National
programs, we performed large amounts of experiments to
know the effects of COS on various plants and.
1985
1997
2010
2016,with the technical support of Chinese Academy of Science,
Rx-COS was industrially produced. The factory was built in
Suzhou, China. Annual production is over 300 tons.
2016
2018
1985,Prof. Peter Albersheim, founder of CCRC,
for the first time discovered the effects of
Oligosacchrins in regulating plant growth and
defense responses.
2010,we for the first time proposed
“oligosaccharin as an efficient plant vaccine”,
and made a monograph about plant vaccine.
Prof. Du’s plant vaccine program
was broadcasted at CCTV-7, and
obtained nationwide attentions.

Reference-library of COS
A library that collects more than 6000 reference about COS has been built and maintained

Our position in the researching field of COS
This database contains 6203 literatures related to chitosan oligosaccharides in China from 1967 to
December 2018
研 究 论 文
学 位 论 文
会 议 论 文
专 利
共 3608 篇
共 433 篇
共 170 篇
共 1992 项
2018 年 新 增 240 篇 2018 年 新 增 23 篇
2018 年 新 增 7 篇
2018 年 新 增 124 项

Contents
中 心 第 一 批 启 动 的 项 目
Background
Function and mechanism of COS
Key technology in production of COS
Application of COS in agriculture

COS protects tobacco from
中
tobacco
心 第 一
mosaic
批 启 动
virus
的 项
(TMV)
目
Results:
COS
CK
Optimum concentration for spraying: 50 ppm;
COS is better to be applied at least one day before the outbreak of
TMV, and its anti-virus activity will last no less than 9 days;
Under 50 ppm, the copy of TMV and long-distance move in
tobacco could be significantly inhibited;
COS increases the ability of tobacoo against TMV
COS/(mg·L -1 )
Disease
index
Decreasing rate of
disease index/(%)
Significant
difference (5%)
Time of
pretreatment
Disease
index
Decreasing rate of disease
index/(%)
Significant
difference (5%)
25 15.00 78.18 b
72h
CK
13.01
70.54
81.56 a
50 10.00 85.45 a
75 21.00 69.45 b
100 35.00 49.09 c
CK 68.75
48h
CK
24h
CK
12h
CK
17.52
88.53
15.75
68.75
53.75
81.254
80.21 a
77.09 a
33.8 b

Mechanism of COS in disease control
• COS induces virus resistance via activation of salicylic acid (SA) signaling pathway;
• COS, Jasmonic acid (JA) pathway, fungal diseases;
• COS, SA & JA pathway, bacterial diseases;
Jia et al.; Sci Rep;. 2016;
Meng et al.; Glycoconj J, 2013

COS protects plants from chilling
中 心 第
damage
一 批 启 动 的 项 目
CK
COS

Mechanism of COS in anti-freezing
A
B
14
low temperature stress
low temperature stress+75mg/L COS
16:0 fatty acid composition(%)
12
10
8
6
4
2
C
Cold resistance of Wheat Induced by COS
low temperature stress
low temperature stress+75mg/L COS
D
0
0 12 24 36 48 60
time(hour)
COS reduces the content of saturated fatty acids content
of saturated fatty acids
1.4
1.2
18:1 fatty acid composition(%)
1.0
0.8
0.6
0.4
0.2
0.0
0 12 24 36 48 60
time(hour)
COS increases the content of unsaturated fatty acids
COS increases proline content
Wang MY, Du YG et al., Field Crops Research, 2015(172): 11–20

Effect of COS in increasing yield
Rx-COS
CK
Rx-COS
CK
Yield
(Kg/mu)
Rx-COS 4365.7±86.8
Increasing rate
(%)
23.98%
CK 3521.3±121.5
Surveyed at May 31 Surveyed at Aug 21

Mechanism of COS in promoting plant growth
Study on the growth promoting
mechanism of COS
COS can regulate the synthesis,
metabolism and transportation of auxin,
thus promoting the growth of wheat roots

COS protects plants from pest
中 心 第 一 批 启 动 的 项 目
• Plants, when pretreated with COS, might liberate some secondary metabolites that are repellent for pests;
COS
SA
COS
SA
CK
COS
CK
3h
CK
5h
CK
COS
CK
Chemical
pesticide
COS

Effect of COS in degrading residual chemical pesticide

In summary of the mechanism of COS
• Based on our research, we for the first time proposed that chitosan oligosaccharide is a plant vaccine;
• Unsolved question: the COS-binding protein??
Oligosaccharide
s
Field Crop Res, 2015,172: 11–20; Int J Plant Sci, 2013, 174(4): 722-732
H 2 O
J Agri Food Chem, 2012, 60, 136−143; Ca 2+ 2
Carbohyd Polym, 2012, 90:491-495
Process Biochem, 2012, 47: 1559-1562… J Plant P, 2007, 89(1): 69-79…
Int J Biol Macromol,
ATP
2015, 81:1069-1073;
J Hort Sci Biotech,
2013, NO88(4): Ca 2+ 502-508
O
Carbohyd Polym,
H
NH
2 O Plant Physiol Biochem,
2
2013, 71: 49-56; 2014, 113:446-454;
NO
Carbohyd Polym,
+
Plant Cell Rep
2011,30: 1153-62; 2010, 82:136–142;
Carbohyd Polym, Plant Phys Biochem,
2011, 86(2):774-82; 2006, 44: 910–916…
2009, 75(4):612-617…
Carbohyd Polym, 2010, 82:1-8
Defense genes
E 《 糖 链 植 Transcript 物 疫 苗 factor 研 究 genes 与 应 用 》
《Research R TFProgress in Oligosaccharins》
NH 2
L-citrulline
+ H 3N COO -
Ca 2+
Ca 2+ K + Cl - Vacuole
NOS
H 2N
NH
NH 2
+
L-arginine
+ H 3N COO -

Contents
中 心 第 一 批 启 动 的 项 目
Background
Function and mechanism of COS
Key technology in production of COS
Application of COS in agriculture

Relationship between Activity and Structure
• Eliciting activity of oligosaccharins with different structure might differ a lot ;
• So, a very important point is how to prepare unique oligosaccharins with high activity;

How to prepare high-quality COS?
Raw
materials
Unique enzyme
catalyst
Stable
production
High-quality COS
Composition
Water solubility
Degree of polymerization
Degree of acetylation
Pattern of acetylation
Bioactivity
Salt selected

Structure-controllable COS
• We have developed a key technology by which COS with controllable structure can be prepared;
2
1.8
1.6
1.4
1.2
1
N-acetylglucosamine
Aminoglucose
COS 1.0 —— polymerization control is available at home and abroad
COS 2.0 —— no controlled polymerization and deacetylation at
home and abroad
COS 3.0 —— no controlled polymerization, controlled deacetylation
and controlled acetylation sites at home and abroad

Core technology
Ⅰ 型
18 家 族
CDA 4
CDA 1
Chitase
Ⅱ 型
Ⅲ 型
chitinase
19 家 族
CDA 5
CDA 2

Core technology
Name Opt. pH Opt. temp
(℃)
Thermal
tolerance
Yield
(mg/ L)
Sp. Act
(U/mL)
Substrate
specificity
Type
CSN 6.5 60 Stable at 80 ℃ 300 25.2 D-D; A-D Cho I
Patent numbers
BACSN 5.0 45 Stable at 50 ℃ 230 52.2 D-D Cho II 201711013149.3
BSCSN 5.6 55 Stable at 50 ℃ 300 84.5 D-D Cho II 201710629508.1
BCCSN 5.6 60 Stable at 50 ℃ 770 49.1 D-D; D-A Cho III 201711018444.8
TRCHI18 5.6 65 Stable at 50 ℃ 170 0.52 A-A; A-D GH18 201710630518.7
STCHI18 5.6 60 Stable at 80 ℃ 200 0.98 A-A; A-D GH18 201711018461.1
CPCHI19 5.6 75 Stable at 70 ℃ 180 0.32 A-A; A-D; D-A GH19 201710749572.3
STCHI19 5.6 60 Stable at 70 ℃ 300 0.58 A-A; A-D; D-A GH19 201711012737.5
Summary of characterization of seven expressed chitosan hydrolases and one existing chitosan hydrolase in laboratory (7 patents applied)
24

D3
D3
A3
D4
A1D3
D4
A3D1
A1D4
A3D2
A4D1
D5
A1D5
A2D4
A3D3
A4D2
A1D6
A2D5
D6
A4D3
A2D6
A4D4
D7
A5D3
A3D6
A5D4
D8
A6D4
Different structure, different activity
Differences of insect resistance induced by COS with different structures
Intens.[a.u.]
x10 4 523.690
1.0
COS-62-NP
* D -O N LY\0_J5\1\1R ef,"B aseline subt.",S m oothed
0.8
566.501
0.6
0.4
0.2
588.500
604.491
684.882 888.040
726.919
1049.171
1091.196
1210.300
1252.281
1413.381
1616.471
COS-MP-162
0.0
Intens.[a.u.]
x10 4
5 544.528
600 800 1000 1200 1400 1600 1800
m /z
649.857
COS-62-PA
* M -O N LY\0_I11\1\1R ef,"B aseline subt.",S m oothed
4
811.026 972.167
3
2
1
582.519
676.946
760.628
1014.199
1175.315
1539.539
1336.418
1133.258 1497.529 1700.624
1903.720
COS-62-NP
0
Intens.[a.u.]
523.946
600 800 1000 1200 1400 1600 1800
m /z
COS-MP-162
* 16-COS-500-2000\0 _ C 24\1 \1 Ref ,"Baselinesubt .",Sm oothed
3000
685.202
2000
846.432
Control
1000
0
539.935
701.189
727.266
862.412
888.473
1007.629
1023.621
1132.748
1168.827
1294.924
1330.002
500 600 700 800 900 1000 1100 1200 1300 1400
m /z
On this basis, it has applied for the support of the national 13th five year key research and
development plan - "development of high-end agricultural marine biological immune products"
Screening and obtaining specific structural chitooligosaccharides with high insect resistance induction activity (Patent No.:201810531128.9)

Generation 1 (1997) Generation 2 (2006) Third generation technology
industrialization (2017)
Upgrade of key technology
Dalian Kaifei Co.,Ltd Hainan Zhengye Co.,Ltd RunXin Co.,Ltd
Advanced Technology

危 害 程 度 (%)
危 害 程 度 (%)
Comparison of COS from different company
Harm degree of Spodoptera ramie treated with different concentration of sugar chain plant vaccine
60
RunXin Co.,Ltd
60
Shandong company B
50
50
40
40
Water solubility
30
20
10
0
ck 20 40 60 80 120 160 200
concentration(ppm)
chit8
The concentration difference is nearly 40 times, and the 0ppm activity is reduced 3 times!!
30
20
10
0
ck 250 500 1000.0 2000.0 3000.0 4000.0 5000.0
concentration(ppm)

Contents
中 心 第 一 批 启 动 的 项 目
Background
Function and mechanism of COS
Key technology in production of COS
Application of COS in agriculture

Application of COS on wheat
‣ COS has been widely applied on wheat in China, with an average increasing yield of 3-10%.
Effects of seed dressing with oligosaccharide immune inducer on Wheat Yield and structure (Shaanxi, 2010-2012)
Varieties
Treament
Ears Number
(10,000/Mu)
±CK% Grains Num per ear ±CK % Rice yield(kg/Mu) ±CK%
Xiaoyan 22
Zhongmai 349
Xinong9871
Zhongmai175
COS 43.77 2.1 32.28 4.2 647.14aA 7.0
CK 42.85 30.98 604.69bA
COS 42.30 -0.4 33.87 5.3 680.97aA 4.5
CK 42.48 32.17 651.53bB
COS 42.68 0.5 33.94 4.4 697.98aA 5.4
CK 42.48 32.51 662.24bB
COS 38.45 6.0 32.46 8.8 501.44aA 10.2
CK 36.27 29.83 455.03bB

Application of COS on wheat
The effect of marine oligosaccharide
compound preparation in wheat production is
obvious, with an increase of 3.1% - 10.2%. It is
suggested to increase production test and
demonstration efforts!

Application of COS on rice
Cost : less than 45 dollars per ha
COS
C.K
COS
Benefit :
1) more than 750 kg rice per ha;
2) Less amount of NPK fertilizers;
3) Less amount of chemical pesticides;
How to apply COS?
1、Seed soaking,promote root development, enhance its ability to resist diseases;
2、Leaf spraying at the early tillering stage,promote the efficiency of tiller;
3、Leaf spraying at the early booting stage,increase grain number per spike;
4、Leaf spraying at the full heading stage,maintain a higher boot leaf chlorophyll content, improve the inferior grains filling rate;
C.K

Application of COS on economic crops
COS
C.K
Less diseases;
Reduced chemical pesticides;
Increased yield;
30 tons per ha

Application of COS on economic crops
‣ COS has been widely applied on Apple in China;
‣ COS has good efficiency in protecting plants from being infected by various fungal diseases;
COS group grows well
C.K group fallen leaves seriously
Treament Total leaves fallen
leaves
sick leaves
fallen leaves
rate%
sick leaves
rate%
Sick index%
Control
effect%
COS 531 143 229 26.93 59.02 16.12 62.47
CK 654 498 155 76.15 99.36 42.90 —

Application of COS in China
‣ Large scale demonstration and application of oligosaccharide agricultural preparation have been realized
It has been popularized and applied to more than 30 crops in more than 20
provinces and cities in China, with a total of more than 85.8 million mu times

Application of COS in China
吃 “ 大 虾 ” 的 蔬 果

Application of COS in Tanwai

Application of COS in South America
Rx-COS increases the total amount of the extra-grade
tomato compared to the control.
Lab experiment performed in Argentina, 2017

Application of COS in Indonesia
‣ Cooperation with NU organization, Sahid Univ. and Djuanda Univ., Indonesia
‣ COS product output;
‣ Technical support;

Application of COS in Indonesia

COS has been widely used in organic agriculture, France
• Reduce pesticide use?
• A new kind of environmental friendly method for the
control of fungal diseases
• Syngenta (France) developed a product based on
chitosan oligosaccharide and pectin
oligosaccharide cos-oga;
• The product has achieved great success in France;
• In the past two years, more than 400000 hectares of wine
grapes have been used in France, i.e. half of the grape
bases in France are in use;

COS is a good candidate in organic agriculture, China

Tang Huajun, member of the Party group of the Ministry of agriculture, President of the Chinese Academy of Agricultural Sciences and academician of
the Chinese Academy of engineering, was invited to attend the fifth collective interview of the 19th National Congress of the Communist Party of China on
October 21, 2017, with the theme of "agricultural science and technology innovation".
Academician Tang focused on plant immunity. The concept of green immunity is used to protect crops against disease and increase production.
Controlling the quality of agricultural products and protecting the agricultural production environment from the source is a bright spot in the structural
reform of agricultural supply side.

Acknowledgements
Glyco bioengineering group
Main research cooperation team
• Prof. Bing Wang’, Charles Sturt University,
Australia
• Prof. Frederic A Troy, University of California
School of Medicine, Davis, USA.
• Prof. Chen DW Sichuan Agricultural
University
• Prof. Liang XM & prof. Lin BC Dalian
Institute of Chemical Physics, CAS
• Prof. Zhang Jinsong , Institute of Metal
Research, CAS
Supported by

Contact:Prof. Yuguang Du ; Prof. Yong Zhao
Tel:010-82545039;13910491944
E-mail: Fenix_tom@outlook.com
Add:No1, Beiertiao street, Haidian district, Beijing