The Regulation of Microorganism on the Formation of TSNAs in Tobacco

Congming Zou

Yunnan Academy of Tobacco Agricultural SciencesKunming, China

Zhenyuan Xia, Liping Lei, Wei Huang, Gaokun. Zhao, Yan Jin, Qun Wu

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Yunnan TobaccoProduces around 40% tobacco in China, and 20% flue-cured tobacco in this world !

800000 tons/year

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Oriental Tobacco#1 China

Burley Tobaccowas # 1 China

Sun-cured Yellow Tobacco# 1 China

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Introduction We care about TSNAs…….

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Introduction 1、Tobacco TSNAs mainly form in curing and storage process

2、Tobacco TSNAs formation have a relationship with Microbes

3、Can we use tobacco endophytic bacteria and fungi to control TSNAs formation?

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1、Select microbes （tobacco endophytic bacteria and fungi） which could feed on nicotine

2、TSNAs substrate solution incubation study

3、Tobacco short air-curing study

4、Cigarette leaves incubation study

Experiment Design

5、Tobacco long air-curing study

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1、 Select microbes which could feed on nicotine

122/869 tobacco endophytic bacteria and fungi can successfully feed on nicotine and TSNAs

There microbes mainly belong to Firmicutes，Actinobacteria, Proteobacteria and Bacteroidetes.

16S rDNA sequence method

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2、TSNAs substrate solution incubation study

Treatment NNN(ng/g)

NAT(ng/g)

NAB(ng/g)

NNK(ng/g)

TSNAs(ng/g)

Compared with CK

(%)Ck 344 226 42 21 629 0J45 866 199 32 76 1174 +86.6 J54 1391 315 36 37 1779 +182.8 J70 729 458 41 12 1241 +97.3

05-5402 341 100 37 11 489 -22.3 05-101 381 69 33 13 496 -21.1

Solution from Burley tobacco

Inoculate different microbes

Incubate 3-5 days

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3、Tobacco short air-curing study

Burley tobacco

Half leaves method

Air-cured 7 days

Spray and not spray

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3、Burley tobacco curing study

Treatment NNN(ng/g)

NAT(ng/g)

NAB(ng/g)

NNK(ng/g)

TSNAs(ng/g)

Compared with CK

(%)J45ck 19.25 29.66 2.39 5.31 56.61 0J45 18.84 29.31 3.07 4.44 55.66 -1.7

J54ck 27.85 41.54 2.28 5.44 77.11 0J54 37.14 58.96 2.83 16.12 115.05 +49.2

J70ck 24.43 32.55 2.69 6.01 65.68 0J70 27.09 31.27 2.61 5.21 66.18 +0.8

05-5402ck 26.41 49.69 2.28 6.09 84.47 0 05-5402 21.19 43.38 1.09 4.18 69.84 -30.2 05-101ck 22.39 15.95 1.5 4.41 44.25 0 05-101 20.97 17.54 1.41 3.69 43.61 -1.4

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4、Cigarette leaves incubation study

Cigarette leave from Zhongnanhai

Inoculate different microbes

Incubate 5 days

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4、Cigarette leaves incubation study

Treatment NNN(ng/g)

NAT(ng/g)

NAB(ng/g)

NNK(ng/g)

TSNAs(ng/g)

Compared with CK(%)

CK 5148.31 1404.29 44.55 196.5 6793.65 0.0

J45 5361.76 1414.24 47.33 207.43 7030.82 +3.5

J54 5640.51 1403.84 52.04 209.9 7305.93 +7.5

J70 4824.2 1361.41 36.66 177.64 6399.76 -5.8

05-5402 4225.38 1268.09 39.37 185.63 5706.81 -16.0

05-101 5002.29 1436.27 45.08 211.89 6695.53 -1.4

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Summary

Class Study 1 Study 2 Study 3

J45 Pseudomonas +86.6 -1.7 +3.5

J54 Pseudomonas +182.8 +49.2 +7.5

J70 Pseudomonas +97.3 +0.8 -5.8

05-5402 Bacillus -22.3 -30.2 -16.0

05-101 Paenibacillus -21.1 -1.4 -1.4

Relatively stable microorganisms

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5、Tobacco long air-curing study

Air-cured 50 days

Spray and not spray

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5、Tobacco long air-curing study

Treatment NNN (ng/g)

NAT(ng/g)

NAB (ng/g)

NNK (ng/g)

TSNAs (ng/g)

Compared with CK(%)

CK 567.4 783.5 21.9 162.5 1535.3 0

J45 270.2 501.1 13.9 117.6 902.8 -41.2*J54 222.5 462.7 12.4 122.2 819.8 -46.6*

J70 393.8 654.2 18.8 132.6 1199.3 -21.9*

05-5402 294.1 584.0 16.5 138.9 1033.5 -32.7*

05-101 435.2 838.5 25.2 160.6 1459.4 -4.9

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Question?

Microbes which can successfully feed on nicotine and TSNAs

could potentially degrade the formation of TNSAs. However,

it might depend on microbe’s incubation time, substitute

substrate availability and environment conditions.

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6、Future action ( for flue-cured tobacco flue-curing)

Pool with selected bacteria Ready for flue-curing

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Acknowledgement

National Natural Science Foundation of China(No.41601330)

Yunnan Applied Basic Research Project( 2017FB074)

( 2016YN28, 2016YN31, 2017YN09)

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